Article

Development of a human lymphoblastoid cell line constitutively expressing human CYP1B1 cDNA: Substrate specificity with model substrates and promutagens

April 1997
Mutagenesis 12(2):83-9

April 1997
12(2):83-9

DOI:10.1093/mutage/12.2.83

Source
PubMed

Authors:

An AHH-1 TK+/- cell derivative was developed that stably expresses human cytochrome P4501B1 (CYP1B1) cDNA in an extrachromosomal vector which confers resistance to 1-histidinol and co-expresses NADPH cytochrome P450 oxidoreductase (OR). The CYP1B1-expressing cell line was designated h1B1/OR. Microsomes prepared from CYP1B1 cDNA expressing cells exhibit elevated levels of 7-ethoxy-resorufin deethylase (EROD), 7-ethoxy-4-trifluoromethyl-coumarin deethylase (EFCD), benzo(alpha)pyrene hydroxylase (BPH), bufuralol 1'-hydroxylase, testosterone hydroxylase activities and spectrally quantifiable cytochrome P450. CYP1B1-containing microsomes did not contain detectable coumarin 7-hydroxylase, p-nitrophenol hydroxylase, lauric acid hydroxylase, (S)-mephenytoin 4'-hydroxylase or diclofenac 4'-hydroxylase activities. Kinetic parameters for selected substrates were compared among CYP1B1 and the two additional members of the CYP1 family, CYP1A1 and CYP1A2. For BPH and EFCD, the rank order of rates of substrate metabolism were CYP1A1 > CYP1B1 > CYP1A2. For EROD, the rank order of substrate metabolism was CYP1A1 > CYP1A2 > CYP1B1. For both EROD and EFCD the apparent K(m) values for CYP1B1 were more similar to CYP1A1 than to CYP1A2. In order to begin to characterize the promutagen activating ability of CYP1B1, the mutagenicity of selected chemicals was examined in h1B1/OR cells; there was increased sensitivity (CYP1B1-expressing relative to control cells) to the mutagenicity of benzo(a)pyrene, cyclopenta(c,d)pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and aflatoxin B1 (AFB). CYP1B1, expressed in this system, appears to be particularly efficient at activating AFB.

Human CYP1B1 enzyme-mediated, AhR enhanced activation of aflatoxin B1 for its genotoxicity in human cells

Article

Nov 2022
TOXICOL LETT

Aflatoxin B1 (AFB1) is a human procarcinogen known to be activated by cytochrome P450 (CYP) 1A2 and 3A4. In a previous study AFB1 caused chromosomal rearrangement in a yeast strain genetically engineered for stably expressing human CYP1B1. Yet, further verification of the effect of AFB1 in human cells, a potential role of the aryl hydrocarbon receptor (AhR), and CYP1B1-catalyzed AFB1 metabolism remain unidentified. In this study, a human hepatocyte (L-02) line and a human lymphoblastoid (TK6) cell line were genetically engineered for the expression of human CYP1B1, producing L-02-hCYP1B1 and TK6-hCYP1B1, respectively. They were exposed to AFB1 and analyzed for the formation of micronucleus and elevation of γ-H2AX (indicating double-strand DNA breaks); the metabolites formed by CYP1B1 from AFB1 after incubation of AFB1 with human CYP1B1 isoenzyme microsomes were determined by LC-MS. The results showed significantly more potent induction of micronucleus by AFB1 in L-02-hCYP1B1 and TK6-hCYP1B1 than in the parental (L-02 and TK6) cells, and the effects were reduced by (E)-2,3',4,5'-tetramethoxystilbene, a specific CYP1B1 inhibitor. In the AFB1- CYP1B1 microsomes incubations AFM1, a known stable metabolite of AFB1, was detected. Moreover, in L-02 and TK6 cells, AFB1 apparently increased the protein levels of AhR, ANRT and CYP1B1, and caused the nuclear translocation of AhR and ARNT, the latter effect being blocked by BAY-218 (an inhibitor of AhR). In conclusion, this study indicates that human CYP1B1 is capable of metabolically activating AFB1 through the AhR signaling pathway.

Protective effect of gomisin N on benzyl butyl phthalate-induced dysfunction of testosterone production in TM3 Leydig cells

Article

Full-text available

Jan 2024
MOL BIOL REP

Background Exposure to benzyl butyl phthalate (BBP) may induce disorders in the male reproductive system. However, the molecular mechanisms remain unknown. Here we investigated the effect of BBP on testosterone production and its molecular mechanisms. Furthermore, we also investigated the role of gomisin N (GN) from Schisandra chinensis (S. chinensis) in testosterone synthesis in TM3 Leydig cells. Method and results First, we examined the effects of BBP on expression levels of testosterone biosynthesis-related genes (StAR, CYP11α1, CYP17α1, 3βHSD, and 17βHSD) and attenuation-related genes (CYP1β1, CYP19α1, and Srd5α1–3). Although testosterone biosynthesis-related genes did not change, attenuation-related genes such as CYP1β1 and CYP19α1 were upregulated with ROS generation and testosterone level attenuation in the presence of 50 µM of BBP. However, the compound with the highest ROS and ONOO⁻ scavenging activity from S. chinensis, GN, significantly reversed the expression of BBP-induced testosterone attenuation-related gene to normal levels. Subsequently, GN improved the testosterone production levels in TM3 Leydig cells. These events may be regulated by the antioxidant effect of GN. Conclusions On conclusion, our study suggests, for the first time, that BBP impairs testosterone synthesis by the modulation of CYP1β1 and CYP19α1 expression in TM3 cells; GN could potentially minimize the BBP-induced dysfunction of TM3 cells to produce testosterone by suppressing CYP19α1 expression.

Protective effect of gomisin N on BBP-induced dysfunction of testosterone production in TM3 Leydig cells

Preprint

Full-text available

May 2023

Background Exposure to benzyl butyl phthalate (BBP) may induce disorders in the male reproductive system. However, the molecular mechanisms remain unknown. Here we investigated the effect of BBP on testosterone production and its molecular mechanisms. Furthermore, we also investigated the role of gomisin N (GN) from Schisandra chinensis (S. chinensis) in testosterone synthesis in TM3 Leydig cells. Method and results First, we examined the effects of BBP on expression levels of testosterone biosynthesis-related genes (StAR, CYP11α1, CYP17α1, 3βHSD, and 17βHSD) and attenuation-related genes (CYP1β1, CYP19α1, and Srd5α1-3). Although testosterone biosynthesis-related genes did not change, attenuation-related genes such as CYP1β1 and CYP19α1 were upregulated with ROS generation and testosterone level attenuation in the presence of 50 μM of BBP. However, the compound with the highest ROS and ONOO⁻ scavenging activity from S. chinensis, GN, significantly reversed the expression of BBP-induced testosterone attenuation-related gene to normal levels. Subsequently, GN improved the testosterone production levels in TM3 Leydig cells. These events may be regulated by the antioxidant effect of GN. Conclusions In conclusion, our study suggests, for the first time, that BBP impairs testosterone synthesis by the modulation of CYP1β1 and CYP19α1 expression in TM3 cells; GN could potentially minimize the BBP-induced dysfunction of TM3 cells to produce testosterone by suppressing CYP19α1 expression.

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates

Article

Full-text available

May 2022

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity towards typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolised to a greater extent by certain younger ancestors and extant forms, suggesting that activity towards FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the haemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

Testosterone Metabolite 6β‐Hydroxytestosterone Contributes to Angiotensin II‐Induced Abdominal Aortic Aneurysms in Apoe –/– Male Mice

Article

Full-text available

Mar 2021

Background Sex is a prominent risk factor for abdominal aortic aneurysms (AAAs), and angiotensin II (Ang II) induces AAA formation to a greater degree in male than in female mice. We previously reported that cytochrome P450 1B1 contributes to the development of hypertension, as well as AAAs, in male mice. We also found that a cytochrome P450 1B1‐generated metabolite of testosterone, 6β‐hydroxytestosterone (6β‐OHT), contributes to Ang II‐induced hypertension and associated cardiovascular and renal pathogenesis in male mice. The current study was conducted to determine the contribution of 6β‐OHT to Ang II‐induced AAA development in Apoe –/– male mice. Methods and Results Intact or castrated Apoe –/– /Cyp1b1 +/+ and Apoe –/– /Cyp1b1 –/– male mice were infused with Ang II or its vehicle for 28 days, and administered 6β‐OHT every third day for the duration of the experiment. Abdominal aortas were then evaluated for development of AAAs. We observed a significant increase in the incidence and severity of AAAs in intact Ang II‐infused Apoe –/– /Cyp1b1 +/+ mice, compared with vehicle‐treated mice, which were minimized in castrated Apoe –/– /Cyp1b1 +/+ and intact Apoe –/– /Cyp1b1 –/– mice infused with Ang II. Treatment with 6β‐OHT significantly restored the incidence and severity of AAAs in Ang II‐infused castrated Apoe –/– /Cyp1b1 +/+ and intact Apoe –/– /Cyp1b1 –/– mice. However, administration of testosterone failed to increase AAA incidence and severity in Ang II‐infused intact Apoe –/– /Cyp1b1 –/– mice. Conclusions Our results indicate that the testosterone‐cytochrome P450 1B1‐generated metabolite 6β‐OHT contributes to Ang II‐induced AAA development in Apoe –/– male mice.

Human Family 1-4 cytochrome P450 enzymes involved in the metabolic activation of xenobiotic and physiological chemicals: an update**

Article

Full-text available

Jan 2021
ARCH TOXICOL

This is an overview of the metabolic activation of drugs, natural products, physiological compounds, and general chemicals by the catalytic activity of cytochrome P450 enzymes belonging to Families 1–4. The data were collected from > 5152 references. The total number of data entries of reactions catalyzed by P450s Families 1–4 was 7696 of which 1121 (~ 15%) were defined as bioactivation reactions of different degrees. The data were divided into groups of General Chemicals, Drugs, Natural Products, and Physiological Compounds, presented in tabular form. The metabolism and bioactivation of selected examples of each group are discussed. In most of the cases, the metabolites are directly toxic chemicals reacting with cell macromolecules, but in some cases the metabolites formed are not direct toxicants but participate as substrates in succeeding metabolic reactions (e.g., conjugation reactions), the products of which are final toxicants. We identified a high level of activation for three groups of compounds (General Chemicals, Drugs, and Natural Products) yielding activated metabolites and the generally low participation of Physiological Compounds in bioactivation reactions. In the group of General Chemicals, P450 enzymes 1A1, 1A2, and 1B1 dominate in the formation of activated metabolites. Drugs are mostly activated by the enzyme P450 3A4, and Natural Products by P450s 1A2, 2E1, and 3A4. Physiological Compounds showed no clearly dominant enzyme, but the highest numbers of activations are attributed to P450 1A, 1B1, and 3A enzymes. The results thus show, perhaps not surprisingly, that Physiological Compounds are infrequent substrates in bioactivation reactions catalyzed by P450 enzyme Families 1–4, with the exception of estrogens and arachidonic acid. The results thus provide information on the enzymes that activate specific groups of chemicals to toxic metabolites.

Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability

Article

Full-text available

Mar 2020

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable with T50 values ~30–40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared to related extant forms, but also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. While extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to their thermostability.

Coumarins and P450s, Studies Reported to-Date

Article

Full-text available

Apr 2019
MOLECULES

Cytochrome P450 enzymes (CYPs) are important phase I enzymes involved in the metabolism of endogenous and xenobiotic compounds mainly through mono-oxygenation reactions into more polar and easier to excrete species. In addition to their role in detoxification, they play important roles in the biosynthesis of endogenous compounds and the bioactivation of xenobiotics. Coumarins, phytochemicals abundant in food and commonly used in fragrances and cosmetics, have been shown to interact with P450 enzymes as substrates and/or inhibitors. In this review, these interactions and their significance in pharmacology and toxicology are discussed in detail.

Dihydrotestosterone induces minor transcriptional alterations in genital skin fibroblasts of children with and without androgen insensitivity

Article

Feb 2019

Endogenous and exogenous androgens induce masculinization of external genitalia through binding to the androgen receptor (AR). The target genes of androgens in external genitalia remain to be determined, although previous studies have shown that the apolipoprotein D gene (APOD) was significantly upregulated by dihydrotestosterone (DHT), the most potent androgen in humans. In the present study, we performed microarray analysis for genital skin fibroblasts obtained from four boys with buried penis (the control individuals) and a patient with partial androgen insensitivity syndrome (PAIS) due to a hypomorphic mutation in AR (the PAIS patient). We identified 24 transcripts that were upregulated or downregulated by DHT in all samples of control individuals and, to a lesser extent, in the sample of the PAIS patient. Differences between DHT-treated and -untreated samples were small; the results of 24 transcripts did not reach statistical significance. The 24 transcripts included CYP1B1, a gene possibly involved in the development of genital tubercle in mice, and APOD, as well as several genes that have been reported as androgen targets in prostate or other tissues. The results of this study indicate that androgen-mediated masculinization of external genitalia is unlikely to depend on massive transcriptional changes in specific AR target genes. Rather, minor transcriptional changes of several genes, and/or a complex molecular network may play a major role in penile development. Importantly, our data suggest the possible involvement of CYP1B1 in human genital development and confirm the clinical importance of APOD as a biomarker for AR function.

Cancer chemoprevention revisited: Cytochrome P450 family 1B1 as a target in the tumor and the microenvironment

Article

Nov 2017
CANCER TREAT REV

Cancer chemoprevention is the use of synthetic, natural or biological agents to prevent or delay the development or progression of malignancies. Intriguingly, many phytochemicals with anti-inflammatory and anti-angiogenic effects, recently proposed as chemoprevention strategies, are inhibitors of Cytochrome P450 family 1B1 (CYP1B1), an enzyme overexpressed in a wide variety of tumors and associated with angiogenesis. In turn, pro-inflammatory cytokines were reported to boost CYP1B1 expression, suggesting a key role of CYP1B1 in a positive loop of inflammatory angiogenesis. Other well-known pro-tumorigenic activities of CYP1B1 rely on metabolic bioactivation of xenobiotics and steroid hormones into their carcinogenic derivatives. In contrast to initial in vitro observations, in vivo studies demonstrated a protecting role against cancer for the other CYP1 family members (CYP1A1 and CYP1A2), suggesting that the specificity of CYP1 family inhibitors should be carefully taken into account for developing potential chemoprevention strategies. Recent studies also proposed a role of CYP1B1 in multiple cell types found within the tumor microenvironment, including fibroblasts, endothelial and immune cells. Overall, our review of the current literature suggests a positive loop between inflammatory cytokines and CYP1B1, which in turn may play a key role in cancer angiogenesis, acting on both cancer cells and the tumor microenvironment. Strategies aiming at specific CYP1B1 inhibition in multiple cell types may translate into clinical chemoprevention and angioprevention approaches.

Crofts FG, Sutter TR, Strickland PTMetabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human cytochrome P4501A1, P4501A2 and P4501B1. Carcinogenesis (London) 19: 1969-1973

Article

Nov 1998
CARCINOGENESIS

F G Crofts

While the metabolic activation of 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PhIP) by N-hydroxylation has been well documented, the relative roles of the human cytochrome P450 (CYP) enzymes that catalyze this reaction have not been established. Previous studies indicated that the mutagenic activation product, 2-hydroxyamino-PhIP (N 2 -OH-PhIP), is produced primarily by CYP1A2, and to a lesser extent by CYP1A1. We recently reported that human CYP1B1 also produces N 2 -OH-PhIP (Carcinogenesis, 18, 1793‐1798, 1997). In the present study, we examined PhIP metabolism by microsomes containing recombinant human CYP1A1, 1A2 or 1B1 expressed in Sf9 insect cells and compared the kinetic values for PhIP metabolite formation. PhIP metabolites were analyzed by high pressure liquid chromatography with fluorescence and absorbance detection. Vmax values for N 2 -OH-PhIP formation were 90, 16 and 0.2 nmol/min/nmol P450, and the apparent Km values were 79, 5.1 and 4.5 μM for human CYP1A2, 1A1 and 1B1, respectively. The non-mutagenic metabolite, 49-hydroxy-PhIP, was also formed by all three CYP enzymes with Vmax values of 1.5, 7.8 and 0.3 nmol/ min/nmol P450 and apparent Km values of 43, 8.2 and 2.2 μM for human CYP1A2, 1A1 and 1B1, respectively. Although the Vmax for N 2 -OH-PhIP production was highest for CYP1A2, the catalytic efficiency (Vmax/Km) of CYP1A1 was greater than that of CYP1A2. These results suggest that, for humans, extrahepatic CYP1A1 may be more important than previously thought for the metabolic activation of the dietary carcinogen PhIP.

Expresión del citocromo P-450 en modelos manipulados genéticamente

Chapter

Full-text available

Jan 2004

1. RESUMEN El CYP humano catalíticamente activo obtenido por expresión re-combinante es en la actualidad modelo biológico de uso generalizado en el contexto fármaco-toxicológico. Diferentes organismos, incluyendo bac-terias, levaduras, células de insecto y líneas celulares de origen mamífe-ro, pueden ser manipulados genéticamente para la expresión heteróloga —transitoria o estable— de diferentes isoformas CYP humanas. En muy pocos años, los diferentes sistemas han sido extraordinariamente optimi-zados en relación a los requerimientos específicos para la producción con alto rendimiento del enzima humano en su conformación nativa y plena-mente funcional. En la actualidad cualquiera de ellos representa una fuen-te constante, definida y reproducible de CYPs individuales adecuada a la caracterización estructural y funcional del CYP humano, y de aplicación a múltiples áreas de estudio. Cada modelo presenta ventajas y limitacio-nes, dictadas por factores técnicos (complejidad en la transformación) y por características inherentes al organismo hospedador: el modelo de elección depende en última instancia del objetivo experimental de cada estudio particular. Este capítulo presenta una revisión extensa y actuali-zada de los diferentes modelos de expresión heteróloga del CYP huma-no disponibles. Su primera parte introduce los principios metodológicos y requerimientos básicos para la transformación y producción del enzima recombinante en el hospedador, así como las ventajas y limitaciones par-ticulares de los diferentes sistemas. Finalmente, analizaremos las áreas de 149 aplicación experimental en las que la disponibilidad actual de sistemas de expresión funcional del CYP humano ha jugado un papel especialmente relevante, con un énfasis particular en su aplicación a estudios "in vitro" orientados a la identificación temprana de candidatos con propiedades far-macocinéticas óptimas, la predicción de potenciales interacciones medi-camentosas, y la implicación del polimorfismo genético como factor de variabilidad el metabolismo in vivo en el organismo humano.

Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[

Article

Full-text available

Jan 1999

Brain Testosterone-CYP1B1 (Cytochrome P450 1B1) Generated Metabolite 6β-Hydroxytestosterone Promotes Neurogenic Hypertension and Inflammation

Article

Full-text available

Aug 2020

Previously, we showed that peripheral administration of 6β-hydroxytestosterone, a CYP1B1 (cytochrome P450 1B1)-generated metabolite of testosterone, promotes angiotensin II-induced hypertension in male mice. However, the site of action and the underlying mechanism by which 6β-hydroxytestosterone contributes to angiotensin II-induced hypertension is not known. Angiotensin II increases blood pressure by its central action, and CYP1B1 is expressed in the brain. This study was conducted to determine whether testosterone-CYP1B1 generated metabolite 6β-hydroxytestosterone locally in the brain promotes the effect of systemic angiotensin II to produce hypertension in male mice. Central CYP1B1 knockdown in wild-type (Cyp1b1+/+) mice by intracerebroventricular-adenovirus-GFP (green fluorescence protein)-CYP1B1-short hairpin (sh)RNA attenuated, whereas reconstitution of CYP1B1 by adenovirus-GFP-CYP1B1-DNA in the paraventricular nucleus but not in subfornical organ in Cyp1b1-/- mice restored angiotensin II-induced increase in systolic blood pressure measured by tail-cuff. Intracerebroventricular-testosterone in orchidectomized (Orchi)-Cyp1b1+/+ but not in Orchi-Cyp1b1-/-, and intracerebroventricular-6β-hydroxytestosterone in the Orchi-Cyp1b1-/- mice restored the angiotensin II-induced: (1) increase in mean arterial pressure measured by radiotelemetry, and autonomic imbalance; (2) reactive oxygen species production in the subfornical organ and paraventricular nucleus; (3) activation of microglia and astrocyte, and neuroinflammation in the paraventricular nucleus. The effect of intracerebroventricular-6β-hydroxytestosterone to restore the angiotensin II-induced increase in mean arterial pressure and autonomic imbalance in Orchi-Cyp1b1-/- mice was inhibited by intracerebroventricular-small interfering (si)RNA-androgen receptor (AR) and GPRC6A (G protein-coupled receptor C6A). These data suggest that testosterone-CYP1B1-generated metabolite 6β-hydroxytestosterone, most likely in the paraventricular nucleus via AR and GPRC6A, contributes to angiotensin II-induced hypertension and neuroinflammation in male mice.

Insights into Aflatoxin B1 Toxicity in Cattle: An In Vitro Whole-Transcriptomic Approach

Article

Full-text available

Jun 2020

Aflatoxins, and particularly aflatoxin B1 (AFB1), are toxic mycotoxins to humans and farm animal species, resulting in acute and chronic toxicities. At present, AFB1 is still considered a global concern with negative impacts on health, the economy, and social life. In farm animals, exposure to AFB1-contaminated feed may cause several untoward effects, liver damage being one of the most devastating ones. In the present study, we assessed in vitro the transcriptional changes caused by AFB1 in a bovine fetal hepatocyte-derived cell line (BFH12). To boost the cellular response to AFB1, cells were pre-treated with the co-planar PCB 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a known aryl hydrocarbon receptor agonist. Three experimental groups were considered: cells exposed to the vehicle only, to PCB126, and to PCB126 and AFB1. A total of nine RNA-seq libraries (three replicates/group) were constructed and sequenced. The differential expression analysis showed that PCB126 induced only small transcriptional changes. On the contrary, AFB1 deeply affected the cell transcriptome, the majority of significant genes being associated with cancer, cellular damage and apoptosis, inflammation, bioactivation, and detoxification pathways. Investigating mRNA perturbations induced by AFB1 in cattle BFH12 cells will help us to better understand AFB1 toxicodynamics in this susceptible and economically important food-producing species.

Human CYP1B1-dependent genotoxicity of dioxin-like polychlorinated biphenyls in mammalian cells

Article

Nov 2019
TOXICOLOGY

Polychlorinated biphenyls (PCBs) are persistent organic pollutants and human carcinogens. It was reported that rat CYP1A1 and catfish CYP1A can hydroxylate 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77), while potential roles of other CYP1 enzymes in the metabolism of dioxin-like (DL) PCBs remain unconfirmed. In this study, three representative DL-PCBs, i.e., PCB 77, PCB 126, and 3,4,4',5-tetrachlorobiphenyl (PCB 81), were investigated on their genotoxicity in Chinese hamster V79-derived cell lines genetically engineered for the expression of human CYP1A1, 1A2 and 1B1, and in the human hepatoma C3A cell line, which endogenously expresses various CYPs. Under both 6 h/18 h and 18 h/6 h (exposure/recovery) regimes, PCB 77 and 81 induced micronuclei in V79-hCYP1B1 cells at micromolar levels, with slightly higher potency in the latter regime, while they were inactive in the parental V79-Mz cells and the V79-derived cell lines expressing human CYP1A1 and 1A2. However, PCB 126 was negative in each cell line. Likewise, PCB 77 and 81 induced micronuclei formation in C3A cells, which expressed CYP1B1. This effect was blocked by co-exposure to tetramethoxystilbene (30 nM), a selective CYP1B1 inhibitor. Immuno-fluorescent staining of centromere protein B in the micronuclei in PCB-treated cultures showed a predominance of centromere-negative micronuclei, which indicated a clastogenic effect. Moreover, all three PCBs elevated the level of γ-H2AX protein (indicating DNA double-strand breaks) in C3A cells, and these effects were blocked by tetramethoxystilbene (10 nM). This study demonstrates that some DL-PCBs are clastogenic in mammalian cells following metabolic activation by human CYP1B1.

Interleukin-6 selectively induces drug metabolism to potentiate the genotoxicity of dietary carcinogens in mammary cells

Article

Full-text available

Oct 2019
ARCH TOXICOL

Breast cancer is the most commonly diagnosed malignancy in females, the etiology being multifactorial and includes the role of lifestyle exposure to DNA-damaging chemicals such as dietary carcinogens benzo (a) pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP). Both compounds require cytochrome P450 (CYP)-mediated metabolic activation to DNA-damaging species, and both induce transcriptional responses through the nuclear receptors Aryl hydrocarbon receptor (AhR) and estrogen receptor α (ERα). BaP and PhIP are mammary carcinogens in rodents. Clinically, circulating IL-6 expression is linked with poor prognosis of cancer and 35% of the deaths in breast cancer are linked with inflammation. The objective of this work was to investigate the molecular toxicology and local activation of BaP and PhIP in the presence of IL-6. Our laboratory has previously reported that miR27b can regulate CYP1B1 expression in colorectal cells, here we have investigated if this mechanism is working in mammary cell models, MCF-7 and MDA-MB-231 cells. Treatment (24 h) of cells with BaP (10 nM-10 µM) and PhIP (100 nM-100 µM) significantly induced genetic damage (micronuclei formation) in a dose-dependent manner in both cell lines. This effect was potentiated in the presence of human IL-6 at concentrations reported to be expressed in clinical breast cancer. On its own, IL-6 treatment failed to induce micronuclei frequency above the control levels in these cells. Compared to BaP or PhIP treatment alone, IL-6 plus BaP or PhIP selectively induced CYP1B1 significantly in both cell lines. Additionally, miR27b expression was downregulated by IL-6 treatments and transfection with miR27b inhibitor confirmed that miR27b is a regulator of CYP1B1 in both cell lines. These data show that BaP- and PhIP-induced DNA damage in mammary cells is potentiated by the inflammatory cytokine IL-6 and that inflammation-induced CYP expression, specifically CYP1B1 via miR27b, is responsible for this effect.

Abstract 041: Brain Cytochrome P450 1B1-Testosterone Generated Metabolite 6Beta-Hydroxytestosterone Contributes to Angiotensin II-Induced Increased Sympathetic Outflow and Blood Pressure

Conference Paper

Full-text available

Sep 2018

Cytochrome P450 1B1 (CYP1B1)-generated testosterone (T) metabolite 6β-hydroxytestosterone (6β-OHT) contributes to angiotensin (Ang) II-induced hypertension in the male mice. However, the site of action of CYP1B1-testosterone metabolite 6β-OHT in Ang II-induced hypertension is unknown. The demonstration that Ang II increases blood pressure by its action in the subfornical organ and the presence of CYP1B1 in the brain led us to hypothesize that T contributes to Ang II-induced hypertension via its metabolism to 6β-OHT in the brain. To test this hypothesis, we examined the effect of intracerebroventricularly (ICV) administered T in castrated (Cas) wild-type (Cyp1b1+/+) and Cas Cyp1b1-/- mice on the action of systemic Ang II (700 ng/kg/min) for 14 days. T (3 μg/2μL/injection/every 2nd day) or its vehicle (2-Hydroxypropyl-β-cyclodextrin dissolved in artificial CSF) was injected via a cannula implanted in the brain. Mean arterial blood pressure (MAP, mmHg) was measured by radiotelemetry (n=4-5). Ang II increased MAP in T but not its vehicle-injected Cas Cyp1b1+/+ mice on Day 12 (159±2 vs. 98±2, P<0.05). In Cas Cyp1b1-/- mice Ang II increased MAP in ICV injected 6β-OHT (1.5 μg/2μL/injection every 2nd day) but not T (151±3 vs. 123±5, P<0.05). Power spectral analysis of the data on day 12 showed that Ang II increased the low to high-frequency ratio of heart rate variability, index of sympathetic outflow modulation in ICV T injected Cas Cyp1b1+/+ but not Cas Cyp1b1-/- mice (3.8±0.0.4 vs. 1.9±0.0); whereas ICV 6β-OHT in the later mice increased this ratio (4.7±0.6). Ganglionic blocker hexamethonium (30 mg/Kg, ip) on day 14 of Ang II infusion resulted in greater reduction in MAP (P<0.05) in centrally 6β-OHT injected Cas Cyp1b1-/-, and T-injected Cas Cyp1b1+/+ (Δ81±11 and Δ87±10) than in T injected Cas Cyp1b1-/- mice (Δ50±8). Furthermore, in intact Cyp1b1-/-, but not Cas Cyp1b1-/- mice transduction with adenovirus (Ad) of CYP1B1-DNA (ICV 2μL of 1.0 X 1012 particles/mL) increased Ang II-induced systolic blood pressure (P<0.05, n=5/group) measured by tail-cuff on Day12 (176±11 vs. 120±2, mm Hg). These data suggest that T contributes to Ang II-induced hypertension most likely via generation by the brain CYP1B1 to 6β-OHT, which increases sympathetic outflow in male mice.

Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic chalcones: Overcoming cisplatin resistance in CYP1B1-overexpressing lines

Article

Feb 2017

The structure of alpha-napthoflavone (ANF), a potent inhibitor of CYP1A1 and CYP1B1, mimics the structure of chalcones. Two potent CYP1B1 inhibitors 7k (DMU2105) and 6j (DMU2139) have been identified from two series of synthetic pyridylchalcones. They inhibit human CYP1B1 enzyme bound to yeast-derived microsomes (Sacchrosomes™) with IC50 values of 10 and 9 nM, respectively, and show a very high level of selectivity towards CYP1B1 with respect to the IC50 values obtained with CYP1A1, CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19 Sacchrosomes™. Both compounds also potently inhibit CYP1B1 expressed within 'live' recombinant yeast and human HEK293 kidney cells with IC50 values of 63, 65, and 4, 4 nM, respectively. Furthermore, the synthesized pyridylchalcones possess better solubility and lipophilicity values than ANF. Both compounds overcome cisplatin-resistance in HEK293 and A2780 cells which results from CYP1B1 overexpression. These potent cell-permeable and water-soluble CYP1B1 inhibitors are likely to have useful roles in the treatment of cancer, glaucoma, ischemia and obesity.

Associations of CYP1A2 Polymorphisms with the Risk Haplotypes in Lung Cancer in the Slovak Population

Article

Mar 2016
ADV EXP MED BIOL

Phase I enzymes, including cytochrome P450, family 1, subfamily A, and polypeptide 2 (CYP1A2), are involved in the activation of carcinogens to reactive intermediates that are capable of binding covalently to DNA to form DNA adducts, potentially initiating the carcinogenic process. The aim of present study was to investigate the association of CYP1A2 gene polymorphisms and haplotypes with lung cancer risk. A case-control study was carried out on 105 lung cancer patients and 189 controls. To investigate three CYP1A2 polymorphisms: rs2472299, rs2470890, rs11072508 we used a high resolution melting analysis. We found significant allele associations (rs2470890 and rs2422299) with lung cancer risk. We searched for meaningful associations for all variants in the dominant, recessive, and additive genetic models. Genotype associations in the recessive model were of marginal significance for the same single nucleotide polymorphisms. A haplotype analysis included five variants with the frequency higher than 1 %. The haplotype "acc", present with the highest frequency, was associated with increased lung cancer risk (38.7 % vs. 31.5 %; OR 1.38; 95 %CI 0.95-2.01). On the contrary, rare haplotype "gtc" was significantly associated with decreased lung cancer risk in the Slovak population. In conclusion, the present study identified the risk alleles and haploid genotype associations of the CYP1A2 gene in lung cancer.

6β-Hydroxytestosterone, a Cytochrome P450 1B1-Testosterone–Metabolite, Mediates Angiotensin II–Induced Renal Dysfunction in Male Mice

Article

Feb 2016

6β-Hydroxytestosterone, a cytochrome P450 1B1-derived metabolite of testosterone, contributes to the development of angiotensin II-induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the maintenance of renal homeostasis, development of hypertension, and end-organ damage, this study was conducted to determine the contribution of 6β-hydroxytestosterone to angiotensin II actions on water consumption and renal function in male Cyp1b1(+/+) and Cyp1b1(-/-) mice. Castration of Cyp1b1(+/+) mice or Cyp1b1(-/-) gene disruption minimized the angiotensin II-induced increase in water consumption, urine output, proteinuria, and sodium excretion and decreases in urine osmolality. 6β-Hydroxytestosterone did not alter angiotensin II-induced increases in water intake, urine output, proteinuria, and sodium excretion or decreases in osmolality in Cyp1b1(+/+) mice, but restored these effects of angiotensin II in Cyp1b1(-/-) or castrated Cyp1b1(+/+) mice. Cyp1b1 gene disruption or castration prevented angiotensin II-induced renal fibrosis, oxidative stress, inflammation, urinary excretion of angiotensinogen, expression of angiotensin II type 1 receptor, and angiotensin-converting enzyme. 6β-Hydroxytestosterone did not alter angiotensin II-induced renal fibrosis, inflammation, oxidative stress, urinary excretion of angiotensinogen, expression of angiotensin II type 1 receptor, or angiotensin-converting enzyme in Cyp1b1(+/+) mice. However, in Cyp1b1(-/-) or castrated Cyp1b1(+/+) mice, it restored these effects of angiotensin II. These data indicate that 6β-hydroxytestosterone contributes to increased thirst, impairment of renal function, and end-organ injury associated with angiotensin II-induced hypertension in male mice and that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension in male mice.

Biyomarker Olarak Sitokrom P450 Ekspresyonunun Değerlendirilmesi

Article

Full-text available

Mar 2014

Cytochrome P450 (CYP) enzymes are responsible for the metabolism of endogenous compounds and xenobiotics, including metabolic activation of numerous enviromental carcinogens and toxic chemicals. Genes coding for CYP1A1, CYP1A2, CYP1B1, and CYP2E1 are among the most responsible for the biotransformation of chemicals, especially for the metabolic activation of pre-carcinogens. There is evidence of association between gene polymorphism and cancer susceptibility. The association of CYP polymorphisms and human cancer risk due to hazardous enviromental toxicants, have attracted increasing attention

6 -Hydroxytestosterone, A Cytochrome P450 1B1 Metabolite of Testosterone, Contributes to Angiotensin II-Induced Hypertension and Its Pathogenesis in Male Mice

Article

Full-text available

Apr 2015
Hypertension

Previously, we showed that Cyp1b1 gene disruption minimizes angiotensin II-induced hypertension and associated pathophysiological changes in male mice. This study was conducted to test the hypothesis that cytochrome P450 1B1-generated metabolites of testosterone, 6β-hydroxytestosterone and 16α-hydroxytestosterone, contribute to angiotensin II-induced hypertension and its pathogenesis. Angiotensin II infusion for 2 weeks increased cardiac cytochrome P450 1B1 activity and plasma levels of 6β-hydroxytestosterone, but not 16α-hydroxytestosterone, in Cyp1b1(+/+) mice without altering Cyp1b1 gene expression; these effects of angiotensin II were not observed in Cyp1b1(-/-) mice. Angiotensin II-induced increase in systolic blood pressure and associated cardiac hypertrophy, and fibrosis, measured by intracardiac accumulation of α-smooth muscle actin, collagen, and transforming growth factor-β, and increased nicotinamide adenine dinucleotide phosphate oxidase activity and production of reactive oxygen species; these changes were minimized in Cyp1b1(-/-) or castrated Cyp1b1(+/+) mice, and restored by treatment with 6β-hydroxytestoterone. In Cyp1b1(+/+) mice, 6β-hydroxytestosterone did not alter the angiotensin II-induced increase in systolic blood pressure; the basal systolic blood pressure was also not affected by this agent in either genotype. Angiotensin II or castration did not alter cardiac, angiotensin II type 1 receptor, angiotensin-converting enzyme, Mas receptor, or androgen receptor mRNA levels in Cyp1b1(+/+) or in Cyp1b1(-/-) mice. These data suggest that the testosterone metabolite, 6β-hydroxytestosterone, contributes to angiotensin II-induced hypertension and associated cardiac pathogenesis in male mice, most probably by acting as a permissive factor. Moreover, cytochrome P450 1B1 could serve as a novel target for developing agents for treating renin-angiotensin and testosterone-dependent hypertension and associated pathogenesis in males.

Identification of a Novel Mammary-Restricted Cytochrome P450, CYP4Z1, with Overexpression in Breast Carcinoma

Article

Apr 2004

Michael A. Rieger

By screening a transcriptome database for expressed sequence tags that are specifically expressed in mammary gland and breast carcinoma, we identified a new human cytochrome P450 (CYP), termed CYP4Z1. The cDNA was cloned from the breast carcinoma line SK-BR-3 and codes for a protein of 505 amino acids. Moreover, a transcribed pseudogene CYP4Z2P that codes for a truncated CYP protein (340 amino acids) with 96% identity to CYP4Z1 was found in SK-BR-3. CYP4Z1 and CYP4Z2P genes consisting of 12 exons are localized in head-to-head orientation on chromosome 1p33. Tissue-specific expression was investigated using real-time reverse transcription PCR with normalized cDNA from 18 different human tissues. CYP4Z1 mRNA was preferentially detected in breast carcinoma tissue and mammary gland, whereas only marginal expression was found in all other tested tissues. Investigation of cDNA pairs from tumor/normal tissues obtained from 241 patients, including 50 breast carcinomas, confirmed the breast-restricted expression and showed a clear overexpression in 52% of breast cancer samples. The expression profile of CYP4Z2P was similar to that of CYP4Z1 with preference in breast carcinoma and mammary gland but a lower expression level in general. Immunoblot analyses with a specific antiserum for CYP4Z1 clearly demonstrated protein expression in mammary gland and breast carcinoma tissue specimens as well as in CYP4Z1-transduced cell lines. Confocal laser-scanning microscopy of MCF-7 cells transfected with a fluorescent fusion protein CYP4Z1-enhanced green fluorescent protein and a subcellular fractionation showed localization to the endoplasmic reticulum as an integral membrane protein concordant for microsomal CYP enzymes.

CYP1B1: A Unique Gene with Unique Characteristics

Article

Feb 2015

CYP1B1, a recently described dioxin inducible oxidoreductase, is a member of the cytochrome P450 superfamily involved in the metabolism of estradiol, retinol, benzo[a]pyrene, tamoxifen, melatonin, sterols etc. It plays important roles in numerous physiological processes and is expressed at mRNA level in many tissues and anatomical compartments. CYP1B1 has been implicated in scores of disorders. Analyses of the recent studies suggest that CYP1B1 can serve as a universal/ideal cancer marker and a candidate gene for predictive diagnosis. There is plethora of literature available about certain aspects of CYP1B1 that has not been interpreted, discussed and philosophized upon. The present analysis examines CYP1B1 as a peculiar gene with certain distinctive characteristics like the uniqueness in its chromosomal location, gene structure and organization, involvement in developmentally important disorders, tissue specific, not only expression, but splicing, potential as a universal cancer marker due to its involvement in key aspects of cellular metabolism, use in diagnosis and predictive diagnosis of various diseases and the importance and function of CYP1B1 mRNA in addition to the regular translation. Also CYP1B1 is very difficult to express in heterologous expression systems, thereby, halting its functional studies. Here we review and analyze these exceptional and startling characteristics of CYP1B1 with inputs from our own experiences in order to get a better insight into its molecular biology in health and disease. This may help to further understand the etiopathomechanistic aspects of CYP1B1 mediated diseases paving way for better research strategies and improved clinical management.

Baseline and genotoxic compound induced gene expression profiles in HepG2 and HepaRG compared to primary human hepatocytes

Article

Jul 2013

Increase of Human CYP1B1 Activities by Acidic Phospholipids and Kinetic Deuterium Isotope Effects on CYP1B1 Substrate Oxidation.

Article

Aug 2012
J BIOCHEM

The effect of phospholipids on the kinetic parameters of three substrates, 7-ethoxy-4- (trifluoromethyl)coumarin (7-EFC), 7-ethoxycoumarin (7-EC), and 17β-estradiol (E(2)), of human CYP1B1 was studied. In general, anionic phospholipids, phosphatidic acid and cardiolipin, increased catalytic efficiency by increasing k(cat) values or decreasing K(m) values. The advantages of using the 7-EFC as a substrate over 7-EC and E(2) include high k(cat), low K(m), and high catalytic efficiency. Spectral binding titrations indicated that the binding affinity of 7-EFC to CYP1B1 in the presence or absence of phospholipids is higher than that of 7-EC or E(2). Furthermore, phosphatidylcholine increased the binding affinity of the substrates to the CYP1B1. High noncompetitive intermolecular kinetic deuterium isotope effects (values 5.4-12) were observed for O-deethylation of 7-EFC and 7-EC with deuterium substitution at the ethoxy group, indicating that the C-H bondbreaking step makes a major contribution to the rate of these CYP1B1-catalyzed reactions. However, the intermolecular kinetic deuterium isotope effect is ~2 for the E(2) 4-hydroxylation reaction, indicating that the C-H bond-breaking step contributes only partially to the rate of this CYP1B1-catalyzed reaction. These results indicate that the reaction mechanism of CYP1B1-catalyzed reactions is distinct for each substrate.

Induction of cytochrome P450 family 1 mRNAs and activities in a cell line from the frog Xenopus laevis

Article

Mar 2012

Cytochrome P450 family 1 (CYP1) includes four subfamilies of enzymes: CYP1A, CYP1B, CYP1C, and CYP1D. In many vertebrates, CYP1A, 1B, and 1C expression is induced by agonists of the aryl hydrocarbon receptor, including toxic contaminants such as chlorinated dioxins, coplanar chlorinated biphenyls, and polynuclear aromatic hydrocarbons. Assessed at the level of mRNA, protein, or enzyme activity, CYP1s (especially CYP1As) represent potent and popular biomarkers of contaminant exposure in aquatic vertebrates. Alkylated resorufins are synthetic substrates used to detect, quantify, and describe catalytic activities of cytochrome P450s. The ability to oxidize specific resorufin-based substrates can distinguish the catalytic activities of individual CYP1s. Xenopus laevis, the African clawed frog, is the most widely employed amphibian model in aquatic toxicology, yet the number, inducibility, and activities of CYP1s have not been systematically characterized in this species. Here we report the cloning of cDNAs encoding two new CYP1 family members, X. laevis CYP1B and CYP1C, along with an integrated assessment of the induction of alkyloxyuresorufin-O-dealkylase (AROD) activities and mRNA expression of four known X. laevis CYP1s: CYP1A6, CYP1A7, CYP1B, and CYP1C. Using XLK-WG, an X. laevis kidney epithelial cell line, we determined that EROD (ethoxyresorufin substrate) and MROD (methoxyresorufin) were both induced 3000- to 5000-fold following 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) exposure up to 250 nM, while BROD (benzyloxyresorufin) and PROD (pentyloxyresorufin) activity was not detectable regardless of TCDD treatment. TCDD induced CYP1A6 and CYP1A7 mRNAs by 2-3 orders of magnitude, while CYP1B and CYP1C were unchanged. The more potent AHR agonist, FICZ (6-formylindolo[3,2-b]carbazole), induced CYP1B up to 10-fold at concentrations between 0.1 and 250 nM, while CYP1C induction was less than 3-fold. CYP1B mRNA showed the highest constitutive mRNA expression, 5- to 75-fold greater than the other CYP1 transcripts. Taken together, these results suggest that CYP1A6 and CYP1A7 perform the bulk of EROD and MROD activities we observed in these cells. The ability of each X. laevis CYP1 to catalyze oxidation of individual resorufin substrates remains to be determined. Correlating CYP1 mRNA and induced AROD activity is a significant step toward clarifying the biochemical meaning of these biomarkers and the roles of CYP1 enzymes in X. laevis. The cell culture approach represents an important complement to the long standing use of frog embryos and tadpoles in toxicological studies, providing a well suited model system for determining the molecular mechanisms underlying the regulation of these important biomarkers of contaminant exposure.

Chemoprevention of dibenzo[a,l]pyrene transplacental carcinogenesis in mice born to mothers administered green tea: Primary role of caffeine

Article

Aug 2008
CARCINOGENESIS

Our laboratory recently developed a mouse model of transplacental induction of lymphoma, lung and liver cancer by the polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene (DBP). Pregnant B6129SF1 females, bred to 129S1/SvIm males, were treated on day 17 of gestation with an oral dose of 15 mg/kg DBP. Beginning on day 0 of gestation, dams were given (ad lib) buffered water, 0.5% green tea, 0.5% decaffeinated green tea, caffeine or epigallocatechin-3-gallate (EGCG) (both at equivalent concentrations found in tea). The concentration of the teas (and corresponding caffeine and EGCG) was increased to 1.0% upon entering the second trimester, 1.5% at onset of the third trimester and continued at 1.5% until pups were weaned at 21 days of age. Offspring were raised with normal drinking water and AIN93G diet. Beginning at 2 months of age, offspring experienced significant mortalities due to an aggressive T-cell lymphoma as seen in our previous studies. Ingestion of caffeinated, but not decaffeinated, green tea provided modest but significant protection (P = 0.03) against mortality. Caffeine provided a more robust (P = 0.006) protection, but EGCG was without effect. Offspring also developed DBP-dependent lung adenomas. All treatments significantly reduced lung tumor multiplicity relative to controls (P < 0.02). EGCG was most effective at decreasing tumor burden (P = 0.005) by on average over 40% compared with controls. Induction of Cytochrome P450 (Cyp)1b1 in maternal liver may reduce bioavailability of DBP to the fetus as a mechanism of chemoprevention. This is the first demonstration that maternal ingestion of green tea, during pregnancy and nursing, provides protection against transplacental carcinogenesis.

Augmentation of 3-methylcholanthrene-induced bioactivation in the human hepatoma cell line HepG2 by the calcium channel blocker nicardipine

Article

Mar 2010
CANCER SCI

(Cancer Sci 2010; 101: 652–657) The abilities of the dihydropyridine calcium channel blocker nicardipine (Nic) to induce cytochrome P450 1 family enzymes (CYP1s) and to enhance the 3-methylcholanthrene (MC)-mediated induction of CYP1s and formation of MC-DNA adduct were examined in the human hepatoma cell line HepG2. The results from real time RT-PCR analysis demonstrated that Nic could induce CYP1 mRNAs and enhance the MC-mediated induction of the CYP1 mRNAs. The luciferase-reporter gene assay using the HepG2-A10 cell line, which has been previously established for the screening of aryl hydrocarbon receptor (AhR) activators, also indicated the augmentation of MC-mediated activation of AhR (induction of luciferase) by Nic, although Nic showed limited capacity for the activation of AhR. Furthermore, the results from the Western blot analysis of CYP1s, the enzyme activity assay, and the assay for MC-DNA adduct formation indicated that Nic could enhance the MC-mediated induction of CYP1s, especially CYP1A1. Furthermore, the intracellular accumulation level of [3H]MC after treatment of HepG2 cells with [3H]MC significantly increased in the presence of Nic. The present findings demonstrate that Nic can enhance the MC-mediated induction of CYP1s, especially CYP1A1, and the formation of MC-DNA adduct in HepG2 cells. Furthermore, the augmentation of the MC-mediated bioactivation by Nic is demonstrated to occur mainly through an increase in intracellular accumulation of MC.

Cytochromes P450: Role in Carcinogenesis and Relevance to Cancers

Article

Mar 2022

s Cancer is a leading factor of mortality globally. Cytochrome P450 (CYP) enzymes play a pivotal role in the biotransformation of both endogenous and exogenous compounds. Evidence from numerous epidemiological, animal, and clinical studies points to instrumental role of CYPs in cancer initiation, metastasis, and prevention. Substantial research has found that CYPs are involved in activating different carcinogenic chemicals in the environment, such as polycyclic aromatic hydrocarbons and tobacco-related nitrosamines. Electrophilic intermediates produced from these chemicals can covalently bind to DNA, inducing mutation and cellular transformation that collectively result in cancer development. While bioactivation of procarcinogens and promutagens by CYPs has long been established, the role of CYP-derived endobiotics in carcinogenesis has emerged in recent years. Eicosanoids derived from arachidonic acid via CYP oxidative pathways have been implicated in tumorigenesis, cancer progression and metastasis. The purpose of this review is to update on the current state of knowledge about the cancer molecular mechanism involving CYPs with focus on the biochemical and biotransformation mechanisms in the various CYP-mediated carcinogenesis, and the role of CYP-derived reactive metabolites, from both external and endogenous sources, on cancer growth and tumour formation.

CYP1B1 converts procarcinogens into genotoxins in Saccharomyces cerevisiae

Article

Jan 2022
MUTAT RES-GEN TOX EN

CYP1B1 activates many chemical carcinogens into potent genotoxins, and allelic variants are risk factors in lung, breast, and prostate cancer. However, few eukaryotic genetic instability endpoints have been directly measured for CYP1B1-activated metabolites. In this study, we expressed human CYP1B1 in yeast strains that measure DNA damage-associated toxicity and frequencies of chromosomal translocations. DNA damage-associated toxicity was measured in a rad4 rad51 strain, defective in both DNA excision and recombinational repair. Frequencies of chromosomal translocations were measured in diploid yeast strains containing two his3 fragments. These strains were exposed to benzo[a]pyrene-7, 8-dihydrodiol (BaP-DHD), aflatoxin B1 (AFB1), and the heterocyclic aromatic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). We observed that AFB1, BaP-DHD, IQ, and MeIQx conferred toxicity in the DNA repair mutant expressing CYP1B1. Translocation frequencies increased eight-fold and three-fold after exposure to 50 μM AFB1 and 33 μM BaP-DHD respectively. A DNA damage response was observed after AFB1 exposure, as measured by the induction of the small subunit of ribonucleotide reductase, Rnr3. While CYP1B1-mediated activation of BaP-DHD and heterocyclic aromatic amines was expected, activation of AFB1 to become a potent recombinagen was not expected. These studies demonstrate that chromosomal rearrangement is a useful genotoxic endpoint for CYP1B1-mediated carcinogen activation.

Human Cytochrome P450 Enzymes in Drug Metabolism and Chemical Activation

Thesis

Jan 2019

Aaron Bart

The cytochrome P450 superfamily are heme-containing monooxygenases found ubiquitously in living organisms. In humans, P450 enzymes are essential in the metabolism of drugs, pollutants, and foods, as well as in endogenous biosynthetic pathways for steroids, fatty acids, and vitamins. Thus, these enzymes can be sources of deleterious effects in humans, with issues relating to drug safety and efficacy, metabolism of compounds into carcinogens or toxins, and disruptions of signaling pathways. Prediction of P450 metabolism and design of selective P450 inhibitors are of great importance for human health. Furthermore, leveraging the diversity in P450 catalysis holds promise as biocatalysts for production of synthetically difficult chemicals and green chemistry. In both health and as biocatalysts, limited understanding of P450 structure and conformational flexibility hinders the rational pursuit of these goals. Understanding P450 structure and flexibility is complicated by the diverse compounds P450 enzymes bind and metabolize, and by structural changes that may occur when P450 enzymes interact with modulatory protein partners. This dissertation explores structural features allowing human P450 enzymes to interact with a protein partner, bind and metabolize diverse substrates, and structural interactions which contribute to a thermostable P450 biocatalyst. To understand how the catalytic modulator protein cytochrome b5 exerts variable effects on P450 catalysis, b5 complexes with different human drug-metabolizing P450 enzymes were characterized using NMR. These experiments revealed that P450 enzymes bind shared and distinct surfaces on b5 and have varied complex affinity. Mutating key residues on b5 confirmed the importance of individual b5 amino acids and functional experiments helped link complex formation to b5 modulation of P450 function. The aspect of P450 flexibility in response to diverse ligands they interact with was probed through X-ray structures of the human P450 1A1, which has prominent roles in carcinogen and toxin activation. This work demonstrated that CYP1A1 undergoes conformational changes in the active site roof when bound to structurally diverse ligands, and also revealed channels allowing CYP1A1 to bind larger and nonplanar ligands. Increased understanding of CYP1A1 structural conformations provides a valuable resource for computational prediction of the binding, metabolism, and activation of drugs and chemicals. For utilization of mammalian P450 enzymes as efficient biocatalysts, obstacles such as poor thermostability and activity need to be resolved. The discovery that reconstructed ancestors of mammalian P450 enzymes have significantly improved thermostability provides a promising route for using these enzymes as biocatalysts, but structural contributors to this enhanced thermostability are unknown. Both structure and ligand binding of the mammalian ancestor of human CYP1B1 were characterized to examine sources for increased thermostability. The structures of the ancestor CYP1B1 display an open active site conformation deviating significantly in the roof elements in comparison to the closed structure of human CYP1B1. Substrate binding and activity reveal similar substrate profiles but altered regioselectivity for the metabolites. Comparison of the ancestral and human CYP1B1 structure suggest that gains in thermostability may occur through increased electrostatic and aromatic/cation stacking interactions between distinct secondary structure elements. The combined results of this dissertation provide valuable insight into P450 structure and conformational flexibility for improvements in drug design, predictions of metabolic outcomes, and development of thermostable P450 enzymes in biotechnology.

Substrate Selectivity of Coumarin Derivatives by Human CYP1 Enzymes: In Vitro Enzyme Kinetics and In Silico Modeling

Article

Full-text available

Apr 2021

Of the three enzymes in the human cytochrome P450 family 1, CYP1A2 is an important enzyme mediating metabolism of xenobiotics including drugs in the liver, while CYP1A1 and CYP1B1 are expressed in extrahepatic tissues. Currently used CYP substrates, such as 7-ethoxycoumarin and 7-ethoxyresorufin, are oxidized by all individual CYP1 forms. The main aim of this study was to find profluorescent coumarin substrates that are more selective for the individual CYP1 forms. Eleven 3-phenylcoumarin derivatives were synthetized, their enzyme kinetic parameters were determined, and their interactions in the active sites of CYP1 enzymes were analyzed by docking and molecular dynamic simulations. All coumarin derivatives and 7-ethoxyresorufin and 7-pentoxyresorufin were oxidized by at least one CYP1 enzyme. 3-(3-Methoxyphenyl)-6-methoxycoumarin (19) was 7-O-demethylated by similar high efficiency [21–30 ML/(min·mol CYP)] by all CYP1 forms and displayed similar binding in the enzyme active sites. 3-(3-Fluoro-4-acetoxyphenyl)coumarin (14) was selectively 7-O-demethylated by CYP1A1, but with low efficiency [0.16 ML/(min mol)]. This was explained by better orientation and stronger H-bond interactions in the active site of CYP1A1 than that of CYP1A2 and CYP1B1. 3-(4-Acetoxyphenyl)-6-chlorocoumarin (20) was 7-O-demethylated most efficiently by CYP1B1 [53 ML/(min·mol CYP)], followed by CYP1A1 [16 ML/(min·mol CYP)] and CYP1A2 [0.6 ML/(min·mol CYP)]. Variations in stabilities of complexes between 20 and the individual CYP enzymes explained these differences. Compounds 14, 19, and 20 are candidates to replace traditional substrates in measuring activity of human CYP1 enzymes.

How the AHR Became Important in Cancer: The Role of Chronically Active AHR in Cancer Aggression

Article

Full-text available

Dec 2020
INT J MOL SCI

For decades, the aryl hydrocarbon receptor (AHR) was studied for its role in environmental chemical toxicity i.e., as a quirk of nature and a mediator of unintended consequences of human pollution. During that period, it was not certain that the AHR had a “normal” physiological function. However, the ongoing accumulation of data from an ever-expanding variety of studies on cancer, cancer immunity, autoimmunity, organ development, and other areas bears witness to a staggering array of AHR-controlled normal and pathological activities. The objective of this review is to discuss how the AHR has gone from a likely contributor to genotoxic environmental carcinogen-induced cancer to a master regulator of malignant cell progression and cancer aggression. Particular focus is placed on the association between AHR activity and poor cancer outcomes, feedback loops that control chronic AHR activity in cancer, and the role of chronically active AHR in driving cancer cell invasion, migration, cancer stem cell characteristics, and survival.

Sensing the Generation of Intracellular Free Electrons Using the Inactive Catalytic Subunit of Cytochrome P450s as a Sink

Article

Full-text available

Jul 2020
SENSORS-BASEL

Cytochrome P450 reductase (CPR) abstracts electrons from Nicotinamide adenine dinucleotide phosphate H (NADPH), transferring them to an active Cytochrome P450 (CYP) site to provide a functional CYP. In the present study, a yeast strain was genetically engineered to delete the endogenous CPR gene. A human CYP expressed in a CPR-null (yRD−) strain was inactive. It was queried if Bax—which induces apoptosis in yeast and human cells by generating reactive oxygen species (ROS)—substituted for the absence of CPR. Since Bax-generated ROS stems from an initial release of electrons, is it possible for these released electrons to be captured by an inactive CYP to make it active once again? In this study, yeast cells that did not contain any CPR activity (i.e., because the yeasts’ CPR gene was completely deleted) were used to show that (a) human CYPs produced within CPR-null (yRD-) yeast cells were inactive and (b) low levels of the pro-apoptotic human Bax protein could activate inactive human CYPs within this yeast cells. Surprisingly, Bax activated three inactive CYP proteins, confirming that it could compensate for CPR’s absence within yeast cells. These findings could be useful in research, development of bioassays, bioreactors, biosensors, and disease diagnosis, among others.

CHAPTER 11. The Micronucleus (MN) Assay Using Human-derived Cells

Chapter

Jul 2019

Concerns about the adverse health effects of chemicals and radiation present in the environment and at workplaces have created the need for better detection systems to assess their potential to cause DNA damage in humans and other organisms across ecosystems. The Micronucleus Assay in Toxicology is the first comprehensive volume concerning the use of micronucleus assays in genetic toxicology. It succinctly explains the mechanisms by which genotoxins cause micronucleus formation and its relation to diseases. Furthermore, it describes the methods which are currently used for the analyses of micronuclei in different types of cells in human in vivo biomonitoring studies, routine in vivo tests with rodents, in vitro studies with human and mammalian cells, environmental monitoring with invertebrates and vertebrates such as molluscs, fish and, also, in plant bioassays. Moreover, this book also focuses on the use of the micronucleus technique in other research areas, including the detection of DNA damage caused by important groups of genotoxic carcinogens (heavy metals, industrial chemicals, cytotoxic drugs, pesticides, ionising radiation, etc.) as well as study designs, statistical analyses, international regulatory guidelines, and the development of automated scoring devices for this assay. This book will serve as both, a reference and a guide to students, and investigators in biomedical, biochemical and pharmaceutical sciences interested in gaining a better understanding of the biology of micronuclei and their application in measuring DNA damage caused by natural or man-made genotoxins.

Toxicogenomic analysis and identification of estrogen responsive genes of di (n-ethylhexyl) phthalate in MCF-7 cells

Article

Sep 2005

Di (n-ethylhexyl) phthalate (DEHP) is thought to mimic estrogens in their action, and are called endocrine disrupting chemicals. DEHP is used in numerous consumer products, especially those made of flexible polyvinyl chloride and have been reported to be weakly estrogenic. In this study, DEHP were tested for estrogenic properties in vitro models and with microarray analysis. First, the E-screen assay was used to measure the proliferation of DEHP in MCF-7 cells, a human breast cancer cell line. DEHP induced an increase in MCF-7 cell proliferation at concentration of 10(-4) M. Second, we carried out a microarray analysis of MCF-7 cells treated with DEHP using human c-DNA microarray including 401 endocrine system related genes. Of the genes analyzed, 60 genes were identified showing significant changes in gene expression resulting from DEHP. Especially, 4 genes were repressed and 4 genes were induced by DEHP compared to 17 beta-estradiol. Among these genes, trefoil factor 3 (intestinal), breast cancer 1, early onset and CYP1B1 are involved in estrogen metabolism and regulation. Therefore it suggests that these genes may be associated with estrogenic effect of the DEHP on transcriptional level. The rationale is that, as gene expression is a sensitive end-point, alterations of these genes may act as useful biomarkers to define more precisely the nature and level of exposure to kinds of phthalates.

Trans-resveratrol modulates the catalytic activity and mRNA expression of the procarcinogen-activating human cytochrome P4501B1

Article

Nov 2000
CAN J PHYSIOL PHARM

The present study was performed to determine if trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) modulates the catalytic activity and gene expression of cytochrome P450 1B1 (CYP1B1). In vitro, trans-resveratrol decreased human recombinant CYP1B1-catalyzed 7-ethoxyresorufin O-dealkylation activity, with an IC50 value of 1.4 +/- 0.2 muM (mean +/- SEM). Enzyme kinetic analysis indicated that trans-resveratrol inhibited CYP1B1 enzyme activity by a mixed-type inhibition and the apparent K-i was 0.75 +/- 0.06 muM. To determine if trans-resveratrol modulates constitutive CYP1B1 gene expression, cultured MCF-7 human breast carcinoma cells were treated with trans-resveratrol. As indicated by RT-PCR analysis, treatment of MCF-7 cells with 10 muM trans-resveratrol decreased relative CYP1B1 mRNA levels after 5 h, but not after 1.5 or 3 h, of exposure. trans-Resveratrol treatment at 5, 7.5, 10, or 20 muM for 5 h produced a concentration-dependent decrease in CYP1B1 mRNA levels. The extent of suppression was similar to 50% at 20 muM concentration. The suppressive effect was not a consequence of a toxic response to the compound as assessed by a cell proliferation assay. Overall, our novel finding that trans-resveratrol inhibits the catalytic activity and suppresses the constitutive gene expression of CYP1B1 leads to the possibility that this nutraceutical confers protection against toxicity and carcinogenicity induced by compounds that undergo CYP1B1-catalyzed bioactivation.

Metabolism and Toxicokinetics of Xenobiotics

Chapter

Aug 2001

Xenobiotics entering biological systems undergo the following processes: (1) absorption: oral, dermal, inhalation; (2) tissue distribution (disposition); (3) protein and tissue binding; (4) enzymatic and nonenzymatic chemical transformation; and (5) excretion. Pharmacokinetics are defined as the rates of all metabolic processes related to the expression of pharmacology. Toxicokinetics are defined as the rates of all metabolic processes related to the expression of toxicologic end points. The purpose of pharmacokinetics or toxicokinetics is (1) to allow predictions of body burdens, both time to maximum and amount; (2) to allow prediction of duration in body after exposure terminated; (3) to determine percent absorbed; and (4) to determine schedule of drug dosage.

Contributions of Human Enzymes in Carcinogen Metabolism

Article

Full-text available

Apr 2012

Considerable support exists for the roles of metabolism in modulating the carcinogenic properties of chemicals. In particular, many of these compounds are pro-carcinogens that require activation to electrophilic forms to exert genotoxic effects. We systematically analyzed the existing literature on the metabolism of carcinogens by human enzymes, which has been developed largely in the past 25 years. The metabolism and especially bioactivation of carcinogens are dominated by cytochrome P450 enzymes (66% of bioactivations). Within this group, six P450s--1A1, 1A2, 1B1, 2A6, 2E1, and 3A4--accounted for 77% of the P450 activation reactions. The roles of these P450s can be compared with those estimated for drug metabolism and should be considered in issues involving enzyme induction, chemoprevention, molecular epidemiology, interindividual variations, and risk assessment.

Cytochrome P450 in Laboratory Animal Species

Chapter

Sep 2003

Effects of zearalenone on mRNA expression and activity of cytochrome P450 1A1 and 1B1 in MCF-7 cells

Article

Jun 2004

Zengli Yu

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous pollutant and promoter of carcinogenesis with both estrogenic and antiestrogenic effects in mammal epithelium. Zearalenone (ZEA) is a naturally occurring estrogenic contaminant of moldy feeds and is present in high concentrations in dairy products and cereals. Numerous studies describe a possible correlation between xenoestrogens and breast cancer risk. A potential mechanism for the etiology of breast cancer involves altered cytochrome P450 (CYP) enzymes. Since cocontamination of multiple compounds in our environmental and occupational circumstances likely happens and since few studies have addressed the molecular consequences of combinations of contaminants, we decided to investigate the effects of ZEA on basal and TCDD-induced mRNA expression and enzymic activity of CYP1A1 and CYP1B1 in human breast cancer MCF-7 cells. CYP1A1 enzyme activity was measured by the CYP1A1-referential activity assay, ethoxyresorufin O-deethylase (EROD), in MCF-7 cells. To investigate CYP1B1 activity, we employed the microsomal EROD assay prepared from baculovirus-infected insect cells expressing human cDNA CYP1B1. Reverse transcription-polymerase chain reaction was used to detect mRNA expression of CYP1A1 and CYP1B1 in MCF-7 cells. The results demonstrated that 10 nM TCDD could readily induce a significant increase in the enzyme activity and mRNA expression of CYP1A1 in MCF-7 cells and 5 nM estradiol (E2) significantly reduced both basal and TCDD-induced activity and mRNA expression in MCF-7 cells. The same pattern was observed with 50 nM ZEA. The estrogen receptor antagonist 4-hydroxytamoxifen could attenuate these inhibitive effects of both E2 and ZEA. Interestingly, Both E2 and ZEA could promote basal and TCDD-induced CYP1B1 activity but with no effect on CYP1B1 mRNA expression. These results suggest that the effect of ZEA on the TCDD-induced CYP1A1 activity and gene expression involved the estrogen receptor pathway and that the increase in the CYP1B1/CYP1A1 ratio underlying the basal or TCDD-treated condition might constitute one of the mechanisms underlying the synergic carcinogenic action of these compounds.

Chapter 28. Predictive Toxicology: An Overview

Article

Dec 1998

Joseph F Sina

Cytochrome P450 CYP1B1 protein expression

Article

Jul 2001
BIOCHEM PHARMACOL

The overexpression of human cytochrome P450 CYP1B1 has been observed in a wide variety of malignant tumours, but the protein is undetectable in normal tissues. A number of cytochrome P450 enzymes are known to metabolise a variety of anticancer drugs, and the consequence of cytochrome P450 metabolism is usually detoxification of the drug, although bioactivation occurs in some cases. In this study, a Chinese hamster ovary cell line expressing human cytochrome P450 CYP1B1 was used to evaluate the cytotoxic profile of several anticancer drugs (docetaxel, paclitaxel, cyclophosphamide, doxorubicin, 5-fluorouracil, cisplatin, and carboplatin) commonly used clinically in the treatment of cancer. The MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to determine the levels of cytotoxicity. The key finding of this study was that on exposure to docetaxel, a significant decrease in sensitivity towards the cytotoxic effects of docetaxel was observed in the cell line expressing CYP1B1 compared to the parental cell line (P = 0.03). Moreover, this difference in cytotoxicity was reversed by co-incubation of the cells with both docetaxel and the cytochrome P450 CYP1 inhibitor alpha-naphthoflavone. This study is the first to indicate that the presence of CYP1B1 in cells decreases their sensitivity to the cytotoxic effects of a specific anticancer drug.

ith Prostate Cancer in non-Hispanic and Hispanic Caucasians

Article

Full-text available

Stable Expression of Human Cytochrome P450 1B1 in V79 Chinese Hamster Cells and Metabolically Catalyzed DNA Adduct Formation of Dibenzo[a,l]pyrene

Article

May 1998

Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (−)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE−DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE−DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 μM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 μM (−)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (−)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (−)-anti-(11R,12S,13S,14R)-DB[a,l]PDE metabolite.

Biomarkers for Prostate Cancer Detection: Family-Based Linkage Analysis and Case–Control Association Studies

Chapter

Jan 2009

For decades, it has been well-recognized that genetics plays a critical role in the development of prostate cancer. Numerous epidemiological and molecular biological studies have shown evidence for a significant but heterogeneous hereditary component in prostate cancer susceptibility. Linkage analysis in twin and family-based study designs provided targeted candidate regions for prostate cancer risk and cancer aggressiveness. Subsequent mapping efforts and mutation screening yielded several strong candidate genes. More recent tools allow investigation of gene–gene and gene–environment interactions in population-based designs, such as case–control or cohort studies. These analyses have identified associations between single nucleotide polymorphisms within candidate genes and prostate cancer susceptibility. Understanding the role of these genes may help in defining heterogeneity in prostate cancer etiology and eventually lead to better detection, treatment, and, ultimately, prevention of prostate cancer. Key WordsCandidate gene-Prostate cancer risk-Association-Linkage-Single nucleotide polymorphism (SNP)-Disease aggressiveness

Xenobiotic Metabolizing Cytochrome P450 in Pig, a Promising Animal Model

Article

Full-text available

Apr 2011
CURR DRUG METAB

The pig has been used as an important animal model for human studies because of its similarity in size, physiology and disease development. However, in contrast to the extensive data available on the cytochrome P450 (CYP) system for humans and rodents, the data related to pig are limited because of, among others, the presence of intra-species differences (domestic pigs and minipigs). The knowledge of the CYP superfamily in a given experimental animal is crucial for pharmacological and toxicological tests in developing drugs and for understanding the metabolic pathways of toxicants and carcinogens. In addition, information on the CYP system in pigs is important since it plays a dominant role in the metabolism of veterinary drugs, whose residues remain in the porcine tissues which are food for humans. The aim of the present review is to examine - in the liver and extrahepatic tissues of pig - our current knowledge of the xenobiotic-metabolizing CYPs belonging to families 1-4, in terms of drug metabolism, substrate specificity, inhibition, gene expression and receptor-driven regulation, in comparison with human data. It is hoped, furthermore, that this review may stimulate research on the porcine drug-metabolizing enzymes in order to evaluate the hypothesis whereby pig data may better reflect human drug metabolism and toxicity than those obtained from the traditional non-rodent models.

Structure, function, regulation and polymorphism and the clinical significance of human cytochrome P450 1A2

Article

Dec 2009
DRUG METAB REV

Human CYP1A2 is one of the major CYPs in human liver and metabolizes a number of clinical drugs (e.g., clozapine, tacrine, tizanidine, and theophylline; n > 110), a number of procarcinogens (e.g., benzo[a]pyrene and aromatic amines), and several important endogenous compounds (e.g., steroids). CYP1A2 is subject to reversible and/or irreversible inhibition by a number of drugs, natural substances, and other compounds. The CYP1A gene cluster has been mapped on to chromosome 15q24.1, with close link between CYP1A1 and 1A2 sharing a common 5'-flanking region. The human CYP1A2 gene spans almost 7.8 kb comprising seven exons and six introns and codes a 515-residue protein with a molecular mass of 58,294 Da. The recently resolved CYP1A2 structure has a relatively compact, planar active site cavity that is highly adapted for the size and shape of its substrates. The architecture of the active site of 1A2 is characterized by multiple residues on helices F and I that constitutes two parallel substrate binding platforms on either side of the cavity. A large interindividual variability in the expression and activity of CYP1A2 has been observed, which is largely caused by genetic, epigenetic and environmental factors (e.g., smoking). CYP1A2 is primarily regulated by the aromatic hydrocarbon receptor (AhR) and CYP1A2 is induced through AhR-mediated transactivation following ligand binding and nuclear translocation. Induction or inhibition of CYP1A2 may provide partial explanation for some clinical drug interactions. To date, more than 15 variant alleles and a series of subvariants of the CYP1A2 gene have been identified and some of them have been associated with altered drug clearance and response and disease susceptibility. Further studies are warranted to explore the clinical and toxicological significance of altered CYP1A2 expression and activity caused by genetic, epigenetic, and environmental factors.

Substrate inducible rnicrosomal aryl hydrocarbon hydroxylase in mammalian cell culture.

Article

Full-text available

Dec 1968

The aryl hydroxylase enzyme system is inducible in hamster fetus cell cultures. The enzyme system is localized in the 105,000 × g pellet (“microsomal fraction”), has an absolute requirement for NADPH and molecular O2, a pH optimum of pH 7.5, and a partial requirement for divalent cations. Exposure to carbon monoxide reduces the enzyme activity. Treatment with ethylenediaminetetraacetate or trypsin completely prevents enzymatic activity. The Km for the hydroxylase is approximately 0.6 µm with benzo[a]pyrene as substrate. Benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, dibenz[a,h]anthracene, and dibenz[a,c]anthracene are also substrates for the enzyme system. The spectrophotofluorometric determination of hydroxylated benzo[a]pyrene products is sufficiently sensitive to detect 10⁻¹² mole per ml and, hence, has great utility in measuring the hydroxylase activity of cells grown in culture. This mammalian cell culture system is advantageous for the study of the mechanism of microsomal enzyme induction and the related areas of carcinogenesis and drug and steroid metabolism.

Protein measurement with the folin Phenol Reagent

Article

Full-text available

Nov 1951

Characteristics of a microsomal cytochrome P-448-mediated reaction. Ethoxyresorufin O-de-ethylation

Article

Full-text available

Jan 1977

Certain characteristics of ethoxyresorufin O de ethylation, as catalyzed by microsomes of liver, lung, and intestine of control and pretreated rats and hamsters, were studied. The results support previous suggestions that the reaction is catalyzed primarily by a 3 methyl cholanthrene (MC) inducible mono oxygenase which has a 448 nm absorption maximum in the reduced CO difference spectrum. Ethoxyresorufin exhibited a type I binding spectrum with liver microsomes from MC induced rats, but there was no clear interaction with microsomes from control or phenobarbital (PB) induced rats. Maximum MC induction of type I binding and de ethylase activity coincided with the appearance of a cytochrome P 450 spectrum whose absorption maximum was shifted to 448 nm. Low concentrations of α naphthoflavone (ANF) or benzo[α]pyrene (BP) inhibited the de ethylation with liver microsomes of MC treated rats (I50 = 10-9 M) but not those of control rats. A kinetic analysis of BP inhibition of this reaction showed it to be competitive. Inhibition of the MC induced liver microsomal reaction by low concentrations of BP or ANF diminished rapidly with time. MC induced rat liver microsomal de ethylation of ethoxyresorufin was less sensitive than the PB induced reaction to inhibition by metyrapone or SKF 525 A (I50 = 10-6-10-4 M). However, microsomes from rat liver, lung, and intestine had very low constitutive activities (<0.1 nmol/min/mg of protein). MC greatly induced the de ethylation reaction in liver (200x), intestine (40x) and lung (10x). De ethylation of ethoxyresorufin in microsomes from control and MC induced rat lung was inhibited by low concentrations of either ANF or BP (I50 = 10-8 M). Control hamster liver microsomes were several times more active in de ethylation than control rat liver microsomes, but MC induction of hamster liver was only 1/10 of that in rat liver. Control hamster lung activity was similar to that of control rat lung, but was not appreciably induced by MC.

Electrophoretic Transfer of Proteins From Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications

Article

Full-text available

Oct 1979

A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

Quantitation of Microsomal α-Hydroxylation of the Tobacco-specific Nitrosamine, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

Article

Full-text available

Nov 1991

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to DNA alkylating species via two different alpha-hydroxylation pathways. Methylene hydroxylation leads to DNA methylation, whereas methyl hydroxylation yields DNA pyridyloxobutylation. We have developed a high-pressure liquid chromatography assay utilizing radiochemical detection that permits the determination of the extent of metabolism through each pathway in microsomal preparations. Levels of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) were used to measure the extent of methyl hydroxylation, whereas levels of the aldehyde, 4-oxo-1-(3-pyridyl)-1-butanone (OPB), were used to quantify the extent of methylene hydroxylation. Incubations of [5-3H]NNK with microsomes and cofactors were conducted in the presence of 5 mM sodium bisulfite to trap the reactive OPB. The inclusion of bisulfite did not affect the rate of NNK metabolism. Trapping the aldehyde also inhibited its further oxidation to the corresponding acid or reduction to HPB. Furthermore, the conversion of HPB to OPB made only a minor contribution to the OPB levels under our incubation conditions. Analysis of incubation mixtures containing [5-3H]NNK, cofactors, and either A/J mouse liver or lung microsomes demonstrated that OPB was a significant metabolite of NNK. The OPB:HPB ratio was greater in liver (1.5) than in lung (0.2-1) microsomal preparations. Apparent Km values for OPB and HPB formation in lung microsomes were 23.7 and 3.6 microM, respectively, whereas the corresponding values for liver microsomes were 19.1 and 73.8 microM, respectively. These data are consistent with the involvement of more than one cytochrome P-450 isozyme in the activation of NNK to DNA reactive species.

Evidence for involvement of multiple forms of cytochrome P-450 in Aflatoxin B1 metabolism in human liver

Article

Full-text available

Dec 1990

Liver cancer is a major cause of premature death in many areas of Africa and Asia and its incidence is strongly correlated with exposure to aflatoxin B1 (AFB1). Because AFB1 requires metabolic activation to achieve a biological response, there is a need for detailed knowledge of the mechanism of activation to assess individual risk. We have carried out an extensive study using a total of 19 human liver samples to determine the individual variability in the metabolism of the toxin to mutagenic or detoxification products and to identify the specific cytochrome P-450 forms involved in these processes. Metabolism to the toxic 8,9-epoxide or to products mutagenic in the Ames test was found to exhibit very large individual variation. The rates of metabolic activation were highly correlated with both the level of proteins of the P450IIIA gene family and with the total cytochrome P-450 content of the microsomes. In agreement with this, antibodies reacting with P450IIIA proteins were strong inhibitors of both the metabolism and mutagenicity in the majority of the samples. However, the inhibition varied between 50% and 100%. The expression of a protein in the P450IIC gene family also correlated with AFB1 metabolism and mutagenicity. This result therefore indicated the involvement of cytochromes other than P450IIIA in the activation of AFB1 by human liver microsomes. This hypothesis was strongly supported by the finding that antibodies to P450IA2 and P450IIA1 were also effective inhibitors of metabolism in many of the samples. These data demonstrate that, although P450IIIA probably plays an important role in AFB1 activation, several other cytochrome P-450 forms have the capacity to activate the toxin. Similar considerations apply to detoxifying metabolism to aflatoxin Q1 and aflatoxin M1. The levels of expression of many of the forms of cytochrome P-450 involved in AFB1 metabolism are known to be highly sensitive to environmental factors. This indicates that such factors will be an important determinant in individual susceptibility to the tumorigenic action of AFB1.

Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

Article

Full-text available

Jul 1990

Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B1 to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B1 to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B1 to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, IIIA5, and IVB1, did not significantly activate aflatoxin B1 as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B1 activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B1 in human liver involves the contribution of multiple forms of P450.

A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog

Article

Full-text available

Jun 1988
BIOCHEM PHARMACOL

The microsomal O-deethylation of a novel coumarin analog, 7-ethoxy-4-trifluoromethylcoumarin (EFC), to a fluorescent product was characterized. Results indicate that this analog provides a rapid, convenient and highly sensitive means to assay cytochrome P-450-mediated metabolism. Like microsomal 7-ethoxycoumarin (7-EC) O-deethylation, EFC O-deethylation responded to both phenobarbital was greater than that seen with 7-EC (5- to 6-fold over control after 50 mg/kg/day for 4 days in Sprague-Dawley rats compared to approximately 2-fold for 7-EC). Since the reaction was monitored by direct fluorometry of the product, any departures from linearity under a particular set of reaction conditions (e.g. with highly induced samples) were immediately apparent. In the absence of an NADPH-regenerating system, background drift was very low (less than 0.01 fluorescent units), so the sensitivity of the assay was limited primarily by that of the fluorometer employed. This makes the assay particularly useful in situations where test material is limited, e.g. when measuring activity in cultured hepatocytes. Its simplicity, reproducibility, and response to a variety of inducing agents also make it suitable for a rapid screening assay for cytochrome P-450 induction.

Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2

Article

Full-text available

Jun 1994

Previously, levels of a novel human mRNA, detected by a recombinant cDNA designated clone 1, were shown to be increased 50-fold in response to treatment of a keratinocyte cell line with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in part as a function of increased rates of gene transcription (Sutter, T.R., Guzman, K., Dold, K.M., and Greenlee, W.F. (1991) Science 254, 415-418). Here we report the complete corresponding 5.1-kilobase cDNA sequence. A single open reading frame that predicts a protein of 543 amino acid residues was determined by computer-assisted analysis of the cDNA sequence. This predicted protein identifies a new gene subfamily of cytochrome P450, cytochrome P4501B1 (CYP1B1), that maps to human chromosome 2. Southern blot analysis of genomic DNA indicates that the human CYP1B subfamily is likely to contain only this single gene. Northern blot analysis of RNA isolated from primary cultures of normal human epidermal keratinocytes showed approximately 100-fold increased levels of the CYP1B1 mRNA after treatment with 10 nM TCDD for 24 h. Low levels of constitutive CYP1B1 mRNA were detected in 15 different human tissue samples. These results indicate that CYP1B1 is expressed in many normal human tissues and advance our understanding of the complexity of a gene family of cytochromes P450 whose expression is altered by TCDD.

Mouse cytochrome P-450EF, representative of a new 1B subfamily of cytochrome P-450s. Cloning, sequence determination, and tissue expression

Article

Full-text available

May 1994

A novel benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible cytochrome P-450 (P450EF), which is very active in polycyclic aromatic hydrocarbon metabolism, has been purified from C3H10T1/2 mouse embryo fibroblasts (Pottenger, L. H., Christou, M., and Jefcoate, C. R. (1991) Arch. Biochem. Biophys. 286, 488-497). P450EF was shown to be immunologically unrelated to the major known P-450 families. A 4.9-kilobase (kb) cDNA encoding P450EF has been isolated from a lambda ZAP cDNA expression library generated from mRNA of TCDD-induced C3H10T1/2 cells. This cDNA comprises 175-base pair (bp) 5'-noncoding, 1629-bp open reading, and about 3100-bp 3'-noncoding sequence. A SmaI fragment of the 4.9-kb cDNA hybridized to a 5.2-kb mRNA species equally induced by benz[a]anthracene (10 microM) and TCDD (10 nM) in C3H10T1/2 cells, consistent with the involvement of the Ah receptor in this induction process. The deduced amino acid sequence (543 amino acids), the longest of any known cytochrome P-450, exhibits 41 and 38% identity to mouse CYP1A1 and CYP1A2, respectively, and less but substantial similarity (30-33% identity) to many members of the CYP2 family. There are five extended regions of > or = 50% identity to CYP1A1 as follows: (a) 51-118; (b) 199-222; (c) 326-343 (I-helix, O2-binding threonine); (d) 357-430; and (e) 460-487 (heme-binding cysteine). These sequence relationships suggest that P450EF is a member of a new CYP1B subfamily (mouse CYP1B1). Hybridization of mRNA and immunoblot analyses of microsomes both demonstrated beta-naphthoflavone (beta-NF) inducibility of Cyp1b-1 expression in C3H mouse lung, liver, and uterus although at lower levels relative to Cyp1a-1. The mobility of the beta-NF-inducible immunoreactive liver protein was significantly higher than that of the CYP1B1 protein detected in mouse lung, uterus, and C3H10T1/2 cells. Compared with the beta-NF-induced uterus, polycyclic aromatic hydrocarbon-induced uterine fibroblasts exhibited 10-20-fold higher levels of CYP1B1, suggesting that stromal fibroblasts are a major source of the protein.

Activation of Chemically Diverse Procarcinogens by Human Cytochrome P-450 IBI1

Article

Full-text available

Aug 1996

A human cytochrome P-450 (P450) IBI cDNA was expressed in Sat-- charomyces cerevisiae, and the microsomes containing P450 IBI were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage. We also determined and compared these activities of P450 IBI with those catalyzed by recombinant human P450s 1A1 and 1A2, which were purified from membranes of Escherichia coli. The carcinogenic chemicals tested included 27 polycyclic aromatic hydro carbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons, iV-nitrosodimethylamine, vinyl carbamate, and acrylonitrile. Among the three P450 enzymes examined here, P450 IBI was found to have the highest catalytic activities for the activation of ll,12-dihydroxy-ll,12- dihydrodibenzo(a,/)pyrene, l,2-dihydroxy-l,2-dihydro-5-methylchrysene, (+)-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, ll,12-dihydroxy-ll,12-di- hydrobenzo(g)chrysene, 3,4-dihydroxy-3,4-dihydrobenzo(c)phenanthrene, 3-amino-l,4-dimcthyl-5/f-pyrido|4,3-A)mdole. 2-aminoanthracene, 3-meth- oxy-4-aminoazobenzene, and 2-nitropyrene. P450 IBI also catalyzed the activation of 2-amino-3,5-dirnethylimidazo(4,5-/)quinoline, 2-amino-3,K-di- mothyliniida/o(4,5-/)(|uinoxaline. 2-amino-3-methyUmidazo(4^-/|quinoline, 2-aminofluorene, 6-aminochrysene and its 1^-dihydrodiol, (â€")-7,8-dihy- drtixy-7,8-dihydrohenzo|Â«Jpyreiie, l,2-dihydroxy-l,2-dihydroohrysene, 1,2- diliydroxy-1.2-dihydro-5.6-dimi'thylchrysene. 2,3-dihydroxy-2,3-dihydroflu- oranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylhenz(a)anthracene, and 6-nitrochrysene to appreciable extents. However, P450 IBI did not produce genotoxic products from benzo(a)pyrene, irans-3,4-dihydroxy-3,4-dihydro- benzo(a)anthracene,

Hepatic Triphosphopyridine Nucleotide-Cytochrome c Reductase: Isolation, Characterization, and Kinetic Studies

Article

Full-text available

Sep 1962

Protein measurement with the Folin phenol reagent

Article

Nov 1950

Comparison of substrate metabolism by wild type CYP2D6 protein and a variant containing methionine, not valine, at position 374

Article

Aug 1995
PHARMACOGENET GENOM

We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a threefold lower turnover rate for (R)(+)-bufuraIol 1[spacing acute]-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to [alpha]-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6- Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1[spacing acute]-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates. (C) Lippincott-Raven Publishers.

An improved assay of 7-ethoxycoumarin O-deethylase activity: Induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin

Article

Jul 1978

An improved in vitro fluorometric assay of the O-deethylation of 7-ethoxycoumarin which is at least 10 times more sensitive than existing methods is described. Nearly quantitative recovery of the product which is essentially free of fluorescent impurities is obtained by a simple two-step extraction procedure. In C57BL/6J mice O-deethylating activity was induced 3-fold by 3-methylcholanthrene and 7-fold by phenobarbital. Administration of phenobarbital resulted in a 10-fold stimulation of enzyme activity in DBA/2J mice. Kinetic analysis indicated that at least two and possibly more components are involved in the metabolism of 7-ethoxycoumarin. Differential stimulation of the maximal activities associated with these components was observed after the administration of phenobarbital, 3-methylcholanthrene, or 2,3,7,8-tetrachlorodibenzo-p-dioxin. The ED50 values for the induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities in C57BL/6J mice were almost identical and approximately 10-fold less than in DBA/2J mice similarly treated.

Characterization of human lung microsomal cytochrome P-450 1A1 and its role in the oxidation of chemical carcinogens

Article

Jun 1992

Rat and human lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic activities towards several xenobiotic chemicals, including procarcinogens, in different microsomal preparations. Rat lung microsomal P-450s were more active than the human P-450s in catalyzing most of the monooxygenation reactions. Human lung microsomal P-450 was solubilized and purified. Human lung microsomes contain approximately 10 pmol of P-450/mg of protein, on the basis of Fe2+.CO versus Fe2+ difference spectra of the eluates obtained from an octylamino-agarose column. The partially purified P-450 preparations from two human lung microsomal samples showed high activities for the conversion of both (+)- and (-)-isomers of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to genotoxic products. After DEAE-cellulose column chromatography, a partially purified P-450 fraction containing polypeptides of Mr 52,000 and 58,000 was obtained from the early fraction of the octylamino-agarose column eluate, and an electrophoretically homogeneous protein having a molecular weight of approximately 52,000 was recovered from a latter fraction. The amino-terminal amino acid sequences of the two peptides in the earlier fraction were determined; neither polypeptide appears to resemble any known P-450 protein. The protein from the latter octylamino-agarose fraction was immunoreactive with anti-rat P-450 1A2 and anti-human P-450 1A2 but not with antibodies raised against other P-450 enzymes or autoimmune antibodies that specifically recognize human P-450 1A2. A tryptic peptide was isolated from the preparation, and the amino acid sequence matched that of human P-450 1A1 perfectly (residues 31-48) but not that of human P-450 1A2. All of nine human lung microsomal samples examined contained proteins that were immunoreactive with rabbit anti-rat P-450 1A2 and catalyzed the activation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene. The activities could be inhibited by rabbit anti-rat P-450 1A2 and, to a lesser extent, by anti-rat P-450 1A1. The addition of 7,8-benzoflavone caused inhibition or stimulation, depending upon the particular human lung microsomal preparation. Thus, this work clearly shows that human lung microsomes contain at least two major P-450 enzymes; human P-450 1A1 is present in lungs and can actually catalyze the activation of environmental procarcinogens, including polycyclic aromatic hydrocarbons.

A Novel Adrenocorticotropin-Inducible Cytochrome P450 from Rat Adrenal Microsomes Catalyzes Polycylic Aromatic Hydrocarbon Metabolism

Article

Sep 1991

7,12-Dimethylbenz(a)anthracene (DMBA) causes massive ACTH-dependent necrosis of the rat adrenal cortex. This may be related to an ACTH-inducible adrenal microsomal cytochrome P450 that metabolizes polycyclic aromatic hydrocarbons (PAH). The proportions of major monooxygenated products of rat adrenal microsomal DMBA metabolism (DMBA-8,9-diol, DMBA-3,4-diol, and DMBA-phenols) differ significantly from that of P450IA1, the most active PAH-metabolizing P450 in rat liver microsomes. After hypophysectomy, both DMBA metabolic activity and a 57K protein which is distinct from P450XXI disappear from rat adrenal microsomes. ACTH restores both 57K protein and DMBA metabolic activity in hypophysectomized rats almost to the levels in intact untreated rats, but not to levels in ACTH-induced intact rats. The 57K protein has been partially purified from solubilized microsomes in a single step, using detergent elution from a new HPLC matrix consisting of monolayers of phosphatidylcholine covalently bound to a silica support. The resulting P450 preparation contains a single major (57K) band, constituting approximately 70% of the total protein (specific content, 2 nmol P450/mg protein; turnover number, 1.5 nmol DMBA min-1. A rabbit polyclonal antibody raised against this preparation also recognizes a single ACTH-inducible 57K rat adrenal microsomal protein on immunoblots and dose-dependently inhibits DMBA metabolism in solubilized reconstituted rat adrenal microsomes. This 57K P450 is immunochemically distinct from rat P450s of the I, II, III, XVII, and XXI families, but it is immunochemically closely related to a 55K benz(a)anthracene-inducible P450 in the 10T1/2 mouse embryo fibroblast cell line.

A metabolically competent human cell line expressing five cDNAs encoding procarcinogen-activating enzymes: Application to mutagenicity testing

Article

Sep 1991

A human B-lymphoblastoid cell line, designated MCL-5, constitutively expressing human cytochrome P-450 CYP1A1 and also expressing five transfected human cDNAs encoding drug-metabolizing enzymes, has been developed. cDNAs encoding CYP1A2, CYP2A6, and microsomal epoxide hydrolase (mEH) were introduced by using a vector conferring hygromycin B resistance, and cDNAs encoding CYP2E1 and CYP3A4 were introduced by using a vector conferring resistance to 1-histidinol. MCL-5 cells stably expressed all five cDNAs and the native CYP1A1 as determined by measurement of form-specific enzyme activity levels. The mutagenicity of seven model procarcinogens to MCL-5 cells was examined at the hypoxanthine guanine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Exposure to benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), aflatoxin B1, (AFB1), 2-(acetylamino)fluorene (AAF), or benzidine (BZD) induced a statistically significant increase in mutant frequency. Linear interpolation of the concentration of procarcinogen necessary to produce a doubling of the mutant fraction at the hprt locus in MCL-5 cells and the parent AHH-1 cell line revealed that, for each of the chemicals examined, except BZD, MCL-5 cells were significantly more sensitive than the parent AHH-1 cells. The increase in sensitivity to mutagenicity ranged from 3-fold for AAF to greater than 40,000-fold for NDMA. MCL-5 cells have great potential as a screening system for the analysis of human procarcinogen/promutagen activation.

The development of a human cell line stably expressing human CYP3A4: Role in the metabolic activation of aflatoxin B1 and comparison to CYP1A2 and CYP2A3

Article

Mar 1991

We have developed a human lymphoblastoid cell line, designated 3A4/HoI, which stably expresses human CYP3A4 cDNA. This cell line exhibited testosterone 6β-hydroxylase activity, produced immunologically detectable CYP3A4 protein and was more sensitive to the cytotoxicity and mutagenicity of the carcinogenic mycotoxin aflatoxin B1 (AFB1 than was the parent cell line. The concentration-response for AFB1 cytotoxicity and mutagemcity in 3A4/Hol cells was compared to the responses of isogenic cell lines expressing comparable levels of human CYP1A2 (1A2/Hyg cells) and human CYP2A3 (2A3/Hyg cells). 1A2/Hyg cells were 3- to 6-fold more sensitive than 3A4/Hol cells to AFB1-induced mutation. 3A4/Hol cells were 10- to 15-fold more sensitive to AFB mutation than 2A3/Hyg cells. The differences in mutagemcity were supported by the relative binding of [3H]AFB1 to cellular DNA.

A tobacco smoke-derived nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiple human cytochrome P450s including the polymorphic human cytochrome P4502D6

Article

Aug 1991

We have developed a human B-lymphoblastoid cell line, designated 2D6/Hol, which stably expresses human cytochrome P450 CYP2D6 cDNA. This cell line exhibits bufuralol 1'-hydroxylase activity and immunologically detectable CYP2D6 protein. The specific activity of (+)-bufuralol 1'-hydroxylase in microsomes from 2D6/Hol cells was comparable to that observed in human liver microsomes. This cell line was used to examine the mutagenicity activation of three tobacco smoke-derived nitrosamines, N-nitrosonornicotine (NNN), 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal) (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2D6. Exposure of 2D6/Hol cells to NNK concentrations of 30-90 micrograms/ml induced a concentration-dependent decrease in relative survival and increase in mutant fraction at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus. In contrast, NNK was non-mutagenic and non-cytotoxic to control cells at exposure concentrations up to 150 micrograms/ml. NNK mutagenicity in 2D6/Hol cells was compared to the responses observed in isogenic cell lines expressing human CYP1A2 (1A2/Hol), human CYP2A3 (2A3/Hol) and human CYP2E1 (2E1/Hol). These three additional human cytochrome P450-expressing cell lines were also found to be sensitive to NNK-induced mutagenicity and cytotoxicity. We found no evidence for CYP2D6-mediated activation of NNN or NNA. NNN was non-cytotoxic and non-mutagenic to both control and 2D6/Hol cells. NNA was equally cytotoxic and mutagenic to control cells and 2D6/Hol cells. The activation of NNA to a mutagen may have been carried out by P450 native to the AHH-1 TK +/- cell line. The 2D6/Hol cell line, in conjunction with the control cell line and other isogenic cell lines expressing other human cytochrome P450 cDNAs provides a useful system for the examination of the role of the polymorphic CYP2D6 in human procarcinogen activation and drug metabolism.

Stable expression of human cytochrome P450IA2 cDNA in a human lymphoblastoid cell line: Role of the enzyme in the metabolic activation of aflatoxin B1

Article

Jan 1990

A human lymphoblastoid cell line stably expressing a human cytochrome P450IA2 cDNA was developed. This recombinant cell line displayed P450IA2 protein and estradiol 2-hydroxylase activity, neither of which was detected in the parental cell line. The recombinant cell line was also approximately 1000-fold more sensitive to the cytotoxicity and mutagenicity of the carcinogenic mycotoxin aflatoxin B1 than was the parent cell line. The increase in mutagenicity was supported by a corresponding increase in the level of aflatoxin B1 binding to DNA in cells expressing P450IA2 relative to control cells.

Human cytochrome P450IIA3: cDNA sequence role of the enzyme in the metabolic of promutagens comparison to nitrosamine activation by human cytochrome P450IIE1

Article

Sep 1990

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.

The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes

Article

Mar 1990

Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a lambda gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu160----His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a lambda max at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation (turnover number of 15 min-1) and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIA mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. In addition, kinetics of coumarin metabolism in two livers were monophasic over the substrate concentration tested. Km values obtained from human liver (2.3 microM) were similar to those obtained from lysates of hepatoma cells expressing IIA3 (3.6-7.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression and regulation of members of the cytochrome P450IA gene subfamily in human tissues

Article

Aug 1990

Cigarette smoking increases phenacetin O-deethylase (POD) activity in both the liver and placenta in man, but aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity is increased only in the placenta. Whilst there was no correlation between hepatic POD and AHH activities (rs = 0.42, P greater than 0.1), there was a highly significant correlation between these two activities in placenta (rs = 0.76, P less than 0.02). On Western blotting of human liver samples with an antibody specific to cytochrome P450IA2 in the rat, only the orthologue of P450IA2 could be detected. This antibody inhibited greater than 70% of hepatic high-affinity POD activity but had no effect on the placental activity. Furafylline, a methylxanthine that acts as a highly specific inhibitor of P450IA2-dependent activities in man, inhibited all of the high-affinity component of POD activity in human liver, but was at least three orders of magnitude less potent an inhibitor of placental POD and of both hepatic and placental AHH activities. As previously shown in the rat, exposure of man to polycyclic aromatic hydrocarbons, present in cigarette smoke, differentially induces P450IA2 in the liver and P450IA1 in extrahepatic tissues, at least in the placenta. Again, as in the rat, POD activity in the liver is catalysed by P450IA2, but in the placenta of women exposed to polycyclic aromatic hydrocarbons in cigarette smoke POD activity is catalysed by another isoenzyme, most likely P450IA1. Thus, tissue-dependent induction and substrate specificity of members of the P450IA family in man, at least in the placenta, appear to be the same as previously shown in the rat.

Human Cytochrome P-450PA (P-450IA2), the Phenacetin O-deethylase, is Primarily Responsible for the Hepatic 3-demethylation of Caffeine and N-oxidation of Carcinogenic Arylamines

Article

Nov 1989

Aromatic amines are well known as occupational carcinogens and are found in cooked foods, tobacco smoke, synthetic fuels, and agricultural chemicals. For the primary arylamines, metabolic N-oxidation by hepatic cytochromes P-450 is generally regarded as an initial activation step leading to carcinogenesis. The metabolic activation of 4-aminobiphenyl, 2-naphthylamine, and several heterocyclic amines has been shown recently to be catalyzed by rat cytochrome P-450ISF-G and by its human ortholog, cytochrome P-450PA. We now report that human hepatic microsomal caffeine 3-demethylation, the initial major step in caffeine biotransformation in humans, is selectively catalyzed by cytochrome P-450PA. Caffeine 3-demethylation was highly correlated with 4-aminobiphenyl N-oxidation (r = 0.99; P less than 0.0005) in hepatic microsomal preparations obtained from 22 human organ donors, and both activities were similarly decreased by the selective inhibitor, 7,8-benzoflavone. The rates of microsomal caffeine 3-demethylation, 4-aminobiphenyl N-oxidation, and phenacetin O-deethylation were also significantly correlated with each other and with the levels of immunoreactive human cytochrome P-450PA. Moreover, a rabbit polyclonal antibody raised to human cytochrome P-450PA was shown to inhibit strongly all three of these activities and to inhibit the N-oxidation of the carcinogen 2-naphthylamine and the heterocyclic amines, 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole and 2-amino-3-methylimidazo[4,5-f]-quinoline. Human liver cytochrome P-450PA was also shown to catalyze caffeine 3-demethylation, 4-aminobiphenyl N-oxidation, and phenacetin O-deethylation. Thus, estimation of caffeine 3-demethylation activity in humans may be useful in the characterization of arylamine N-oxidation phenotypes and in the assessment of whether or not the hepatic levels of cytochrome P-450PA, as affected by environmental or genetic factors, contribute to interindividual differences in susceptibility to arylamine-induced cancers.

The molecular biology of cytochrome P450s

Article

Jan 1989

Juan F. Martinez González

Several authors have reviewed the molecular biology of P450s during the past few years. Adesnik and Atchison nicely summarize the early work on the isolation and sequencing of the first P450 cDNAs and genes and critically review experiments on the mechanism of regulation of P450 genes. A more recent review describes the P450 gene superfamily and integrates the new recommended P450, gene nomenclature system. The mechanism of regulation of the Ah locus'-associated P450 genes has been reviewed in detail. The evolution and structural comparison of P450s have also been considered. In the remaining portion of this article, the structure and evolution of the P450 genes will be examined, with emphasis on the catabolic P450s. The molecular biology of the anabolic steroid-synthesizing P450s has been covered in earlier reviews. Recent work on cDNA-direct expression, P450 gene regulation, and human P450 catalytic activities, structure, and polymorphisms will be discussed in more detail.

Roles of cytochrome P-450 enzymes in chemical carcinogenesis and cancer chemotherapy

Article

Jul 1988

F. Peter Guengerich

High-performance liquid chromatographic assays for bufuralol 1′-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver

Article

May 1987

Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (-)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1'-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 micrograms of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (-)-bufuralol 1'-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (less than 1 mM) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (-)-bufuralol and dextromethorphan.

Analysis of human lymphoblast mutation assays by using historical negative control data bases

Article

Jan 1987

We describe here historical negative control data bases for gene locus mutation assays at the thymidine kinase locus and hypoxanthine-guanine phosphoribosyl transferase locus in TK6 and AHH-1 human lymphoblasts, respectively. Protocols have been designed which minimize the variability among independent experiments and thus facilitate the use of historical negative control data bases for assay analysis. The historical negative control data bases for both cell lines can be accurately modeled as gamma or Poisson distributions; confidence limits can be calculated from these distributions. We describe and justify a mutagenicity assay analysis procedure which uses a comparison to the concurrent negative control cultures via a t-test in conjunction with a comparison to the historical negative control data base. The incorporation of a comparison to the historical negative control data base allows the use of a higher confidence level without substantially sacrificing the sensitivity of the mutation assays. The analysis of the mutagenicity of saccharin in TK6 cells is presented as an example; saccharin was found to be nonmutagenic under the conditions tested.

The induction of aneuploidy by nitrogen mustard in a human lymphoblastoid cell line

Article

Feb 1987
MUTAT RES-FUND MOL M

We report that the presence of an extra Y chromosome can be used as a marker for the induction of aneuploidy (mitotic non-disjunction) in a human lymphoblastoid cell line. This endpoint is easily visualized in metaphase chromosome preparations after staining with quinacrine mustard. The induction of cells with two Y chromosomes by nitrogen mustard (NM) was examined. Exposure to 150 ng/ml nitrogen mustard induced a 6-fold increase in aneuploid frequency relative to untreated control levels; maximal induction of aneuploidy was observed 2 days after treatment. Lower concentrations of nitrogen mustard (36 and 75 ng/ml) induced smaller increases in aneuploid frequency, with maximal induction observed 1 day after treatment. This system has the potential to be used as an assay for the induction of aneuploidy in cultured human cells.

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line

Article

Jun 1985
CHEM-BIOL INTERACT

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF).

Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and Properties of Induced Enzyme

Article

Jan 1969

Cleavage Of Structural Proteins During Assembly Of Head Of Bacteriophage-T4

Article

Sep 1970

Ulrich K. Laemmli

Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.

Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism

Article

Oct 1984
MUTAT RES-FUND MOL M

A novel quantitative gene-locus mutation assay has been developed using a line of human lymphoblast cells, designated AHH-1, competent in oxidative xenobiotic metabolism. AHH-1 cells are sensitive to the mutagenic action of both chemically reactive mutagens and mutagens which require oxidative metabolism to exert their mutagenicity. These cells are readily mutated by direct exposure to ethyl methanesulfonate, ICR-191, 2-acetoaminofluorene, aflatoxin B1, benzo[a]pyrene (BP), cyclopenta[c, d]pyrene, dimethylnitrosamine, lasiocarpine, and 1-methylphenanthrene.

Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes

Article

Nov 1994

In vitro techniques have been utilized to investigate the microsomal enzymes involved in the metabolism of lauric acid and to establish conditions in which it can be used as a model substrate for both cytochrome P450 4A and cytochrome P450 2E1 in human liver microsomes. Studies of enzyme kinetics of lauric acid omega-hydroxylation in human liver microsomes indicated the involvement of more than one enzyme in this pathway, a relatively low Km enzyme with a Km of 22 microM +/- 12 (n = 8) and a high Km enzyme with a Km an order of magnitude higher (550 microM +/- 310, n = 7). The apparent Vmax for this component correlated with the rate of cyclosporin metabolism and was highly sensitive to ketoconazole inhibition. These results indicated that this enzyme was a member of the 3A subfamily. The activity associated with the low Km enzyme (P450 4A) did not correlate with P450 1A2, 2A6, 2C9/8, 2C19, 2D6, 2E1, or 3A activities in a bank of human liver microsomes and was not appreciably inhibited by ketoconazole, furafylline, quinidine, sulfaphenazole, or diethyldithiocarbamate (DDC). Lauric acid omega-1 hydroxylation demonstrated simple Michaelis-Menten kinetics in each of the human liver microsomal samples examined, with a Km of 130 microM +/- 42 (n = 8). This activity was highly correlated with chlorzoxazone 6-hydroxylation in human liver microsomes (r = 0.98, n = 14, p < 0.001) and was inhibited by both DDC and chlorzoxazone. Additionally, rats treated with the P450 2E1 inducer isoniazid demonstrated a 3-fold increase in lauric acid omega-1 hydroxylation relative to the control group. Thus, the lauric acid hydroxylation assay, at a substrate concentration of 20 microM, appears to be an effective and specific P450 model substrate capable of determining simultaneously P450 4A and P450 2E1 related activities in hepatic microsomal samples.

cDNA Cloning, Sequence Analysis, and Induction by Aryl Hydrocarbons of a Murine Cytochrome P450 Gene, Cyp1b1

Article

Aug 1994

C3H mouse embryo fibroblast cells, designated 10T1/2, can be transformed by physical and chemical agents including polycyclic aromatic hydrocarbons. In a previous report (Shen et al., Proc. Natl. Acad. Sci. USA 90, 11483-11487, 1993), we identified a cytochrome P450 gene induced by polycyclic aromatic hydrocarbons (PAHs) that is different from 1A1 or 1A2, and which we tentatively named P450CMEF. Here, we report the entire cDNA sequence of P450CMEF (5,128 bp) and the amino acid sequence deduced from it (543 residues). A comparison of the latter sequence with known cytochrome P450s indicates that P450CMEF is in a new subfamily of family 1 of the P450 superfamily. Accordingly, the Committee on Standardized Cytochrome P450 Nomenclature designated the gene Cyp1b1. Exposure to various aryl hydrocarbons (2.5 hr) induced Cyp1b1 mRNA in 10T1/2 cells to different degrees: 2,3,7,8-tetrachlorodibenzo-p-dioxin, 7,12-dimethylbenz[a]anthracene, benz[a]anthracene, benzo[a]pyrene, and beta-naphthoflavone were strong inducers; alpha-naphthoflavone and 3-methylcholanthrene, were moderate inducers; and benzo[e]pyrene was a weak inducer.

Rat CYP1B1: An adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals

Article

Jul 1995

The broad spectrum of biological responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is believed to be due to the alteration in expression of TCDD-inducible genes. The aim of this study was to investigate the effects of TCDD on the in vivo tissue-specific expression of the recently identified TCDD-inducible cytochrome P450 CYP1B1 [Sutter et al. (1994) J. Biol. Chem., 269, 13092-13099] in Sprague-Dawley rats. We cloned the 5.0 kb rat homolog of CYP1B1 from a TCDD-treated rat liver cDNA library and showed that the rat and human CYP1B1 predicted amino acid sequences are 80% identical. RNA hybridization analysis showed that CYP1B1 is constitutively expressed in the adrenal glands and also in the testes of untreated rats. This tissue distribution suggests that CYP1B1 may be a physiological steroid hydroxylase. Seventy-two hours post-administration of 25 micrograms/kg body wt TCDD by gavage, steady-state levels of the 5.1 kb CYP1B1 RNA were increased > 50-fold in liver, and to a lesser extent in kidneys, lung, heart and ovaries. Average CYP1B1 RNA levels were significantly higher in the kidneys and livers of TCDD-treated females than in those from similarly treated males. In contrast, no significant sex-difference was observed in the levels of CYP1A1 in these tissues in TCDD-treated animals. In Sprague-Dawley rats, TCDD is a more potent hepatocarcinogen in females than in males. The induction of CYP1B1 in TCDD rat liver may be a contributing factor to the carcinogenic action of this persistent environmental pollutant.

Spink DC, Hayes CL, Young NR, Christou M, Sutter TR, Jefcoate CR, Gierthy JFThe effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: evidence for induction of a novel 17-estradiol 4-hydroxylase. J Steroid Biochem Mol Biol 51: 251-258

Article

Jan 1995
J STEROID BIOCHEM

Rates of microsomal 17 beta-estradiol (E2) hydroxylation at the C-2, -4, -6 alpha, and -15 alpha positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 alpha and -15 alpha positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low Km 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E2 catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low Km hydroxylation of E2 at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE2) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE2 formation predominantly over 4-MeOE2. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E2 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily.

Development of a human lymphoblastoid cell line constitutively expressing human CYP1A1 cDNA: Substrate specificity with model substrates and promutagens

Article

Oct 1994

AHH-1 TK+/−cell derivatives were developed that stably express human CYP1A1 cDNA, and an AHH-1 TK+/−derivative expressing higher levels of CYP1A2 cDNA in extrachromosomal vectors which confer resistance to I-histidinol. The CYP1A1-expressing cell lines, designated h1A1 and h1A1v2, differ by containing one and two CYP1A1 cDNA expression units per vector. The CYP1A2-expressing cell line, designated h1A2v2, also has two CYP1A2 cDNA expression units per vector. Microsomes prepared from CYP1A1 cDNA expressing cells exhibit high, constitutive levels of 7-ethoxyresorufin deethylase (EROD), 7-ethoxycoumarin deethylase (ECD), 7-ethoxy-4-trifluoromethylcoumarin deethylase (EFCD), benzo[a]-pyrene hydroxylase (BPH) activities and spectrally quantifiable cytochrome P450. Kinetic comparisons between cDNA-expressed CYP1A1 and CYP1A2 indicate that CYP1A1 is more active than CYP1A2 for EROD, ECD, EFCD and BPH. CYP1A2 was more active than CYP1A1 for acetanilide hydroxylation and activation of aflatoxin B1 (AFB1). The mutagenicity of selected promutagens were examined in h1A1 cells and control cells. Relative to control cells, the h1A1 cell line exhibits increased sensitivity to the mutagenicity of benzo[a]pyrene, cyclopenta[c,d]pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and AFB1.

Role of Human Cytochromes P450 in the Metabolic Activation of Chemical Carcinogens and Toxins

Article

Feb 1994

Evaluation of Triacetyloleandomycin, α-Nasymphthoflavone and Diethyldithiocarbamate as Selective Chemical Probes for Inhibition of Human Cytochromes P450

Article

Jul 1994

A variety of chemicals, including triacetyloleandomycin (TAO), alpha-naphthoflavone (ANF), and diethyldithiocarbamate (DDC), are widely used as inhibitory probes for select individual human cytochrome P450 (CYP) enzymes, despite the fact that the selectivity of these inhibitors has not been rigorously evaluated. In the present study we take advantage of recent advances in cDNA-directed human P450 expression to evaluate directly the P450 form selectivity of TAO, ANF, and DDC, using a panel of 10 individual cDNA-expressed human P450s. Under experimental conditions known to yield maximal TAO complexation with P450 hemoproteins, TAO (20 microM) inhibited the catalytic activity of expressed CYPs 3A3, 3A4, and 3A5, whereas it did not affect CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, or 2E1 activity. ANF inhibited not only CYPs 1A1 and 1A2 (IC50 = 0.4-0.5 microM), but it was also similarly effective against CYPs 2C8 and 2C9. Increasing the concentration of ANF to 10 microM led to inhibition of CYP2A6 and CYP2B6. Although a previous study suggested that DDC is a selective inhibitor of CYP2E1, the present investigation shows that at concentrations required to inhibit CYP2E1 (IC50 approximately 125 microM when preincubated with NADPH), DDC also inhibited CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 3A3, and 3A4. Decreasing the concentration of DDC to 10 microM, however, led to inhibition of CYP2A6 (65% inhibition) and CYP2B6 (50% inhibition), but none of the other P450s examined, including CYP2E1. Overall, these results establish that (a) TAO is a selective inhibitor of the human CYP3A subfamily; (b) ANF potently inhibits CYP2C8 and CYP2C9, in addition to CYPs 1A1 and 1A2; and (c) DDC cannot be employed as a diagnostic inhibitory probe for CYP2E1.

Isolation and Characterization of Human Liver Cytochrome P450 2C19: Correlation between 2C19 and S-Mephenytoin 4′-Hydroxylation

Article

Nov 1993

In an effort to identify and characterize minor forms of human liver cytochrome P450, immunoblot analyses of microsome samples were developed with antibodies to various P450s that recognized multiple human P450s. Four P450s were recognized in immunoblot analyses of human liver microsome samples developed with an antibody previously demonstrated to specifically recognize rat 2B1/2. Three of these P450s were identified as 2A6, 2C9/10, and 2E1 and the fourth was termed P450UK. A monoclonal antibody to 2C9/10 recognized P450UK in addition to 2C9/10. In order to identify P450UK, it was purified and subjected to amino-terminal amino acid analysis. The amino-terminal sequence obtained for P450UK was identical to the sequence deduced from a cDNA encoding CYP2C19, thus identifying P450UK as 2C19. The relative levels of 2C19 were determined in 14 human liver microsome samples by quantitative immunoblot analyses developed with the anti-2C9/10 antibody. These analyses demonstrated that 2C19 was not detected in one sample and its levels varied 10.5-fold in the remaining samples. The levels of 2C19 were compared to the relative levels and catalytic activities of multiple human liver P450s. The levels of 2C19 and the ability of the samples to 4'-hydroxylate S-mephenytoin were found to strongly correlate (r2 = 0.79). In summary, this is the first demonstration of the expression of 2C19 at the enzyme level, and the correlation studies suggest that 2C19 plays a role in the 4'-hydroxylation of S-mephenytoin.

Micronucleus assays using cytochalasin-blocked MCL-5 cells, a proprietary human cell line expressing five human cytochromes P-450 and microsomal epoxide hydrolase

Article

Aug 1993

The MCL-5 cell line is a human lymphoblastoid TK+/- cell line that constitutively expresses a relatively high level of native CYP1A1, four other human cytochromes (CYP1A2, CYP2A6, CYP3A4 and CYP2E1) and microsomal epoxide hydrolase, carried as cDNAs in plasmids. The aim of this study was to evaluate this cell line for its suitability for detecting chromosomal anomalies, employing micronucleus formation in cells blocked at cytokinesis as the indicator of clastogenicity. Results from two laboratories ('ICR' and 'Swansea') using different protocols are reported. In the ICR protocol, aflatoxin B1, sterigmatocystin, benzo[a]pyrene, dibenz[a,h]anthracene, 3-methylcholanthrene, cyclophosphamide, N-nitrosodimethylamine, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline, benzidine, 2-aminofluorene, benzene, tamoxifen and omeprazole were tested and gave positive results. Anthracene, phenanthrene and pyrene were negative. In the Swansea protocol, AHH-1 cells, the parent line which constitutively expresses CYP1A1, but does not contain the genetically engineered human cytochromes or epoxide hydrolase, were tested in parallel with MCL-5 cells. Aflatoxin B1, sterigmatocystin, benzo[a]pyrene, N-nitrosodiethylamine, 2-acetylaminofluorene, benzene, omeprazole and tamoxifen were tested and gave positive results. Of these, only benzo[a]pyrene was equally potent in both cell lines. Assays of tamoxifen and omeprazole included kinetochore staining. Omeprazole, but not tamoxifen, induced a significant level of kinetochore-positive micronuclei. The detection of micronucleus formation in these genetically engineered cells appears to be a rapid, eclectic and sensitive method for screening for genotoxic activity in vitro.

Cytochrome P45OTB (CYP2C): A major monooxigenase catalyzing diclofenac 4′-hydroxylation in human liver

Article

Feb 1993
LIFE SCI

The nature of the enzyme(s) catalyzing the major metabolic pathway of diclofenac, 4'-hydroxylation, was investigated in human liver microsomes. Inhibition studies were performed with tolbutamide and sulfaphenazole (respectively the prototype substrate and a selective inhibitor of cytochrome P450TB--CYP2C subfamily), and with phenytoin and (+/-)-warfarin, other proposed substrates of P450TB. Diclofenac 4'-hydroxylation displayed single enzyme Michaelis-Menten kinetics and was similar in microsomes from one poor and five extensive metabolizers of debrisoquin (CYP2D6), with a Km of 5.6 +/- 1.5 microM (mean +/- sd) and a Vmax of 60.6 +/- 23.5 nmol/mgP/h. Inhibition by tolbutamide, sulfaphenazole, phenytoin and (+/-)-warfarin was comparable in all livers, with values predicted from their Km or Ki for cytochrome P450TB determined in separate studies and a competitive inhibition model. Sulfaphenazole competitively inhibited diclofenac 4'-hydroxylation (Ki = 0.11 +/- 0.08 microM, n = 3). Diclofenac 4'-hydroxylation is predominantly catalyzed by a cytochrome P450 isozyme of the CYP2C subfamily, most likely CYP2C9. This particular isozyme therefore appears to be responsible for the oxidation of polar acidic substances such as non-steroidal anti-inflammatory drugs from different chemical classes. It also constitutes a common site for drug interactions involving these compounds, as well as tolbutamide, phenytoin and warfarin.

Characterization of a human cell line expressing high levels of cDNA-derived CYP2D6

Article

Mar 1993
Pharmacogenetics

We have developed a human B-lymphoblastoid cell, designated h2D6v2, which expresses high levels of CYP2D6 cDNA. Microsomal P450 contents of 160 pmol mg-1 protein were observed. NADPH-fortified microsomes exhibited a substantial capacity to hydroxylate the prototype CYP2D6 substrates bufuralol and debrisoquine. Kinetic parameters, apparent Km, turnover number, Ki for quinidine inhibition and stereospecificity of bufuralol hydroxylation, observed with the human lymphoblast expressed enzyme were similar to those observed in human liver microsomes or purified liver CYP2D6 proteins. Therefore, the human lymphoblast expressed material appears to faithfully reflect the authentic protein. Relative to control cells, h2D6v2 cells were more sensitive to the cytotoxicity and mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), supporting our previous observation with a cell line expressing lower levels of CYP2D6. h2D6v2 microsomes were capable of metabolizing NNK and NNK metabolism and mutagenicity were markedly inhibited by the addition of quinidine, a CYP2D6 inhibitor. h2D6v2 cells coupled with control cells, represent a useful in vitro system for studying xenobiotic metabolism by the clinically important, polymorphic CYP2D6. The human lymphoblast system offers the desirable ability to couple metabolic transformation studies with toxicological endpoints such as cytotoxicity and mutagenicity.

Comparison of substrate metabolism by wild type CYP2D6 protein and a variant containing methionine, not valine, at position 374

Article

Sep 1995
Pharmacogenetics

We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a three-fold lower turnover rate for (R)(+)-bufuralol 1'-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to alpha-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6-Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1'-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates.

Role of cytochrome P4501A2 in chemical carcinogenesis: implications for human variability in expression and enzyme activity

Article

Nov 1995
Pharmacogenetics

Cytochrome P4501A2 (CYP1A2) has been identified as a key factor in the metabolic activation of numerous chemical carcinogens, including aflatoxin B1, various heterocyclic and aromatic amines, and certain nitroaromatic compounds. In addition, CYP1A2 contributes to the inactivation of several common drugs and dietary constituents, including acetaminophen and caffeine. Two xenobiotic-responsive-element (XRE)-like sequences and an antioxidant response element (ARE) have been identified in the regulatory region of the CYP1A2 gene; however, the functionality of the ARE remains to be demonstrated. Based on in vivo phenotyping assays, substantial interindividual variability in CYP1A2 activity has been reported. Some population-based studies have reported either bi- or tri-modal distributions in CYP1A2 phenotype, suggesting a genetic basis for the large interindividual differences in CYP1A2 activity. However, despite the polymodal distributions reported for CYP1A2 activity, a distinct functional genetic polymorphism in the gene has not been identified. Potential mechanisms contributing to the large interindividual variability in CYP1A2 activity are discussed. A thorough understanding of the functions and regulation of the CYP1A2 gene may ultimately lead to new methods for preventing or intervening in the development of certain chemically-related human cancers.

Kinetic Analysis of the Activation of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone by Heterologously Expressed Human P450 Enzymes and the Effect of P450- Specific Chemical Inhibitors on This Activation in Human Liver Microsomes

Article

Oct 1996

The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the alpha-methyl and alpha-methylene groups, leading to the formation of reactive species which can pyridyloxobutylate and methylate DNA, respectively. The present study examined the kinetic parameters of NNK-derived keto alcohol (alpha-methyl hydroxylation), and keto aldehyde (alpha-methylene hydroxylation) formation catalyzed by human P450s heterologously expressed by either the baculovirus-insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane preparations of the expressed P450s catalyzed the alpha-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol. Human P450 2A6 showed the lowest KM (118 microM) for the formation of keto aldehyde. A similar KM was observed for keto alcohol formation by expressed P450 2A6, but the kcat was lower than the value obtained for keto aldehyde formation. The addition of exogenous b5 increased the expressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both alpha-hydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capacity for keto alcohol formation; however, their KM values for this reaction were in the millimolar range. Expressed human P450 3A4 oxidized NNK to keto aldehyde also with a high KM. Ten human liver microsomal samples were each shown to activate NNK to keto aldehyde and keto alcohol. A positive correlation coefficient of 0.74 was found between keto aldehyde formation and both coumarin 7-hydroxylation (P450 2A6) and 6 beta-testosterone hydroxylation (3A4) activity in characterized human liver microsomes. Keto alcohol formation showed a significant correlation with ethoxyresorufin O-dealkylation (P450 1A2) in human liver microsomes. Both coumarin and troleandomycin, specific inhibitors of P450 2A6 and 3A4, respectively, inhibited the formation of keto aldehyde, but inhibited the formation of keto alcohol only slightly in human liver microsomes. Both furafylline, a P450 1A2 inhibitor, and N-nitrosodimethylamine, a P450 2E1 substrate, inhibited the formation of keto alcohol but not keto aldehyde in human liver microsomes. Quinidine, a specific inhibitor of P450 2D6, was not an effective inhibitor of NNK metabolism. These results demonstrate that P450s 2A6 and 3A4 may be important P450s for the activation of NNK to a DNA-methylating agent and keto aldehyde via the alpha-methylene hydroxylation pathway. P450s 1A2, 2E1, and 2D6 are shown to be selective for alpha-methyl hydroxylation of NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent.

The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties

Article

Aug 1964

Protein Measurement With Folin Fenol Reagent

Article

Dec 1951

Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.

Development of a human lymphoblastoid cell line constitutively expressing human CYP1B1 cDNA: Substrate specificity with model substrates and promutagens

Abstract

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