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ABSTRACT: The nucleoside reverse transcriptase inhibitor zidovudine (3'-azido-3'-dexoythymidine, AZT) can be incorporated into DNA and cause DNA damage. Previously, we determined that the human hepatocellular carcinoma HepG2 cells are more susceptible to AZT-induced toxicities than the immortalized normal human liver THLE2 cells and the nucleotide excision repair (NER) pathway plays an essential role in the response to AZT-induced DNA damage. We have now investigated if the effects of AZT treatment on the expression levels of genes related to DNA damage and repair pathways contribute to the differences in sensitivity to AZT treatment between HepG2 cells and THLE2 cells. Of total 84 genes related to DNA damage and repair, two, five, and six genes were up-regulated more than 1.5-fold at 50, 500, and 2,500 µM AZT groups compared with that of control THLE2 cells. Seven genes showed a decreased expression of more than 1.5-fold following the 2,500 µM AZT treatment. Two-sided multivariate analysis of variance indicated that the change in expression of genes involved in apoptosis, cell cycle, and DNA repair pathways was significant only at 2,500 µM AZT. Statistically significant dose-related increases were identified in XPC gene expression and GTF2H1 protein level after the AZT treatments, which implicated the NER pathway in response to the DNA damage induced by AZT. In contrast, AZT treatment did not alter significantly the expression of the APE1 gene or the levels of APE1 protein. These results indicate that the NER repair pathway is involved in AZT-induced DNA damage response in immortalized human hepatic THLE2 cells.
International journal of biomedical science: IJBS. 03/2013; 9(1):18-25.
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ABSTRACT: Humans at all ages are continually exposed to triclosan (TCS), a widely used antimicrobial agent that can be found in many daily hygiene products, such as toothpastes and shampoos; however, the toxicological and biological effects of TCS in the human body after long-term and low-concentration exposure are far from being well understood. In the current study, we investigated the effects of TCS on the differentiation of human mesenchymal stem cells (hMSCs) by measuring the cytotoxicity, morphological changes, lipid accumulation, and the expression of adipocyte differentiation biomarkers during 21-day adipogenesis. Significant cytotoxicity was observed in un-induced hMSCs treated with high-concentration TCS (≥ 5.0 μM TCS), but not with low-concentration treatments (≤ 2.5 μM TCS). TCS inhibited adipocyte differentiation of hMSCs in a concentration-dependent manner in the 0.156 to 2.5 μM range as indicated by morphological changes with Oil Red O staining, which is an index of lipid accumulation. The inhibitory effect was confirmed by a decrease in gene expression of specific adipocyte differentiation biomarkers including adipocyte protein 2, lipoprotein lipase, and adiponectin. Our study demonstrates that TCS inhibits adipocyte differentiation of hMSCs under concentrations that are not cytotoxic and in the range observed in human blood.
Toxicology and Applied Pharmacology 07/2012; 262(2):117-23. · 4.45 Impact Factor
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ABSTRACT: Sertraline, a selective serotonin reuptake inhibitor, has been used for the treatment of depression. Although it is generally considered safe, cases of sertraline-associated liver injury have been documented; however, the possible mechanism of sertraline-associated hepatotoxicity is entirely unknown. Here, we report that mitochondrial impairment may play an important role in liver injury induced by sertraline. In mitochondria isolated from rat liver, sertraline uncoupled mitochondrial oxidative phosphorylation and inhibited the activities of oxidative phosphorylation complexes I and V. Additionally, sertraline induced Ca(2+)-mediated mitochondrial permeability transition (MPT), and the induction was prevented by bongkrekic acid (BA), a specific MPT inhibitor targeting adenine nucleotide translocator (ANT), implying that the MPT induction is mediated by ANT. In freshly isolated rat primary hepatocytes, sertraline rapidly depleted cellular adenosine triphosphate (ATP) and subsequently induced lactate dehydrogenase leakage; both were attenuated by BA. Our results, including ATP depletion, induction of MPT, inhibition of mitochondrial respiration complexes, and uncoupling oxidative phosphorylation, indicate that sertraline-associated liver toxicity is possibly via mitochondrial dysfunction.
Toxicological Sciences 03/2012; 127(2):582-91. · 4.65 Impact Factor
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ABSTRACT: Kava is a plant traditionally used for making beverages in Pacific Basin countries and has been used for the treatment of nervous disorders in the United States. The pharmacological activity of kava is achieved through kavalactones in kava extract, which include kawain, 7,8-dihydrokawain, yangonin, 5,6-dehydrokawain, methysticin, and 7,8-dihydromethysticin. Recent studies have shown that kava extract induces hepatic CYP1A1 enzyme; however, the mechanisms of CYP1A1 induction have not been elucidated, and the kavalactones responsible for CYP1A1 induction have not yet been identified. Using a combination of biochemical assays and molecular docking tools, we determined the functions of kava extract and kavalactones and delineated the underlying mechanisms involved in CYP1A1 induction. The results showed that kava extract displayed a concentration-dependent effect on CYP1A1 induction. Among the six major kavalactones, methysticin triggered the most profound inducing effect on CYP1A1 followed by 7,8-dihydromethysticin. The other four kavalactones (yangonin, 5,6-dehydrokawain, kawain, and 7,8-dihydrokawain) did not show significant effects on CYP1A1. Consistent with the experimental results, in silico molecular docking studies based on the aryl hydrocarbon receptor (AhR)-ligand binding domain homology model also revealed favorable binding to AhR for methysticin and 7,8-dihydromethysticin compared with the remaining kavalactones. Additionally, results from a luciferase gene reporter assay suggested that kava extract, methysticin, and 7,8-dihydromethysticin were able to activate the AhR signaling pathway. Moreover, kava extract-, methysticin-, and 7,8-dihydromethysticin-mediated CYP1A1 induction was blocked by an AhR antagonist and abolished in AhR-deficient cells. These findings suggest that kava extract induces the expression of CYP1A1 via an AhR-dependent mechanism and that methysticin and 7,8-dihydromethysticin contribute to CYP1A1 induction. The induction of CYP1A1 indicates a potential interaction between kava or kavalactones and CYP1A1-mediated chemical carcinogenesis.
Toxicological Sciences 09/2011; 124(2):388-99. · 4.65 Impact Factor
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ABSTRACT: In addition to primary human hepatocytes, hepatoma cell lines, and transfected nonhepatoma, hepatic cell lines have been used for pharmacological and toxicological studies. However, a systematic evaluation and a general report of the gene expression spectra of drug-metabolizing enzymes and transporters (DMETs) in these in vitro systems are not currently available. To fill this information gap and to provide references for future studies, we systematically characterized the basal gene expression profiles of 251 drug-metabolizing enzymes in untreated primary human hepatocytes from six donors, four commonly used hepatoma cell lines (HepG2, Huh7, SK-Hep-1, and Hep3B), and one transfected human liver epithelial cell line. A large variation in DMET expression spectra was observed between hepatic cell lines and primary hepatocytes, with the complete absence or much lower abundance of certain DMETs in hepatic cell lines. Furthermore, the basal DMET expression spectra of five hepatic cell lines are summarized, providing references for researchers to choose carefully appropriate in vitro models for their studies of drug metabolism and toxicity, especially for studies with drugs in which toxicities are mediated through the formation of reactive metabolites.
Drug metabolism and disposition: the biological fate of chemicals 03/2011; 39(3):528-38. · 3.74 Impact Factor
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ABSTRACT: Zidovudine (3'-azido-3'-dexoythymidine, AZT), a nucleoside reverse transcriptase inhibitor, can be incorporated into DNA and cause DNA damage. The mechanisms underlying the repair of AZT-induced DNA damage are unknown. To investigate the pathways involved in the recognition and repair of AZT-induced DNA damage, human hepatoma HepG2 cells were incubated with AZT for 2 weeks and the expression of DNA damage signaling pathways was determined using a pathway-based real-time PCR array. Compared to control cultures, damaged DNA binding and nucleotide excision repair (NER) pathways showed significantly increased gene expression. Further analysis indicated that AZT treatment increased the expression of genes associated with NER, including XPC, XPA, RPA1, GTF2H1, and ERCC1. Western blot analysis demonstrated that the protein levels of XPC and GTF2H1 were also significantly up-regulated. To explore further the function of XPC in the repair of AZT-induced DNA damage, XPC expression was stably knocked down by 71% using short hairpin RNA interference. In the XPC knocked-down cells, 100 μM AZT treatment significantly increased [³H]AZT incorporation into DNA, decreased the total number of viable cells, increased the release of lactate dehydrogenase, induced apoptosis, and caused a more extensive G2/M cell cycle arrest when compared to non-transfected HepG2 cells or HepG2 cells transfected with a scrambled short hairpin RNA sequence. Overall, these data indicate that XPC plays an essential role in the NER repair of AZT-induced DNA damage.
Toxicology and Applied Pharmacology 03/2011; 251(2):155-62. · 4.45 Impact Factor
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ABSTRACT: Triclosan has broad-spectrum anti-microbial activity against most gram-negative and gram-positive bacteria. It is widely used in personal care products, household items, medical devices, and clinical settings. Due to its extensive use, there is potential for humans in all age groups to receive life-time exposures to triclosan, and, indeed, triclosan has been detected in human tissues and the environment. Data gaps exist regarding the chronic dermal toxicity and carcinogenicity of triclosan, which is needed for the risk assessment of triclosan. The US Food and Drug Administration (FDA) nominated triclosan to the National Toxicology Program (NTP) for toxicological evaluations. Currently, the NTP is conducting several dermal toxicological studies to determine the carcinogenic potential of triclosan, evaluate its endocrine and developmental-reproductive effects, and investigate the potential UV-induced dermal formation of chlorinated phenols and dioxins of triclosan. This paper reviews data on the human exposure, environmental fate, efficacy of anti-microbial activity, absorption, distribution, metabolism and elimination, endocrine disrupting effects, and toxicity of triclosan.
Journal of Environmental Science and Health Part C Environmental Carcinogenesis & Ecotoxicology Reviews 07/2010; 28(3):147-71. · 3.23 Impact Factor
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ABSTRACT: Zidovudine (3'-azido-3'-deoxythymidine; AZT), which is currently used in the treatment of acquired immunodeficiency syndrome, has been shown to have anticancer properties. In the present study, we examined the mechanisms contributing to increased sensitivity of cancer cells to the growth-inhibitory effects of AZT. This was accomplished by incubating a hepatoma cell line (HepG2) and a normal liver cell line (THLE2) with AZT in continuous culture for up to 4 weeks and evaluating the number of viable and necrotic cells, the induction of apoptosis, cell cycle alterations, and telomerase activity. In HepG2 cells, AZT (2-100 microM) caused significant dose-dependent decreases in the number of viable cells at exposures > 24 h. During a 1-week recover period, there was only a slight increase in the number of viable cells treated with AZT. The decrease in viable cells was associated with an induction of apoptosis, a decrease in telomerase activity, and S and G2/M phase arrest of the cell cycle. During the recovery period, the extent of apoptosis and telomerase activity returned to control levels, whereas the disruption of cell cycle progression persisted. Western blot analysis indicated that AZT caused a decrease in checkpoint kinase 1 (Chk1) and kinase 2 (Chk2) and an increase in phosphorylated Chk1 (Ser345) and Chk2 (Thr68). Similar effects, to lesser extent, were observed in THLE2 cells given much higher concentrations of AZT (50-2500 microM). These data show that HepG2 cells are much more sensitive than THLE2 cells to AZT. They also indicate that a combination of a delay of cell cycle progression, an induction of apoptosis, and a decrease in telomerase activity is contributing to the decrease in the number of viable cells from AZT treatment, and that checkpoint enzymes Chk1 and Chk2 may play an important role in the delay of cell cycle progression.
Toxicological Sciences 06/2009; 111(1):120-30. · 4.65 Impact Factor
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ABSTRACT: Zidovudine (3'-azido-3'-deoxythymidine; AZT) and lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC] are nucleoside reverse transcriptase inhibitors used to treat and prevent human immunodeficiency virus-1 infections. In short-term incubations (<48 h), AZT, but not 3TC, has been shown to interfere with cell cycle progression. In the present study, we examined if these alterations persist during long-term incubations in which cells were exposed to AZT (0-1000 microM) or 3TC (0-500 microM) in continuous culture for up to 5 weeks. In addition, we investigated the reversibility of these effects upon removal of the drugs. Both drugs caused concentration- and time-dependent decreases in the number of viable cells, with the effect being more pronounced with AZT. There was only a slight increase in the number of viable cells treated with AZT for 5 weeks and then allowed a 1-week recovery period; cell viability in cells treated with 3TC returned to control levels during the recovery period. The decrease in viable cells was not due to apoptotic or necrotic cell death, but rather was associated with S and G2/M phase cell cycle arrest. Western blot analysis indicated that AZT treatment caused a decrease in checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) at all time points. Cyclin-dependent kinase 1 was decreased at later time points, while cyclin A was increased at early times. These data indicate that AZT and, to a lesser extent, 3TC interfere with cell growth by slowing cell cycle progression and that checkpoint proteins Chk1 and Chk2 may play an important role in this delay.
Mutagenesis 10/2008; 24(2):133-41. · 3.18 Impact Factor
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ABSTRACT: UDP-glucuronosyltransferase 1A10 (UGT1A10) is an extrahepatic enzyme expressed in aerodigestive tract tissues that exhibits significant glucuronidation activity against the important procarcinogenic benzo(a)pyrene (BaP) metabolite, BaP-trans-7,8-dihydrodiol (BPD), and the UGT1A10 codon 139 (Glu>Lys) polymorphism was previously implicated in risk for orolaryngeal cancer by Elahi et al. in their 2003 study. To better assess the potential role of UGT1A10 in risk for tobacco-related cancers, the glucuronidation activity of UGT1A10 was compared with that of other known UGT enzymes against selected polycyclic aromatic hydrocarbons, and the effects of the codon 139 polymorphism on UGT1A10 function were examined in vitro. UGT1A10 exhibited considerably more glucuronidation activity as determined by Vmax/Km against 3-hydroxy (OH)-BaP, 7-OH-BaP, 9-OH-BaP, and 1-OH-pyrene than any other UGT1A family member. Although a kinetic comparison using Vmax could not be performed against family 2B UGTs, UGT1A10 exhibited a 1.7- to 254-fold lower Km than active family 2B UGTs against 3-OH-BaP, 7-OH-BaP, and 1-OH-pyrene. A significantly (p < 0.01) higher Vmax/Km was observed for homogenates from wild-type UGT1A10139Glu-overexpressing cells against all four BaP metabolites tested (3-OH-BaP, 7-OH-BaP, 9-OH-BaP, and BPD). A similarly significant (p < 0.05) increase in Vmax/Km was observed for homogenates from wild-type UGT1A10139Glu-overexpressing cells against 1-OH-pyrene. Significant differences in Km were observed for homogenates from wild-type UGT1A10139Glu-overexpressing cells against 1-OH-pyrene (p < 0.05) and 3-OH-BaP (p < 0.01). Reverse transcription-polymerase chain reaction of total lung RNA showed low levels of UGT1A10 expression in human lung tissue. Together, these studies implicate UGT1A10 as an important detoxifier of polycyclic aromatic hydrocarbons in humans and that the UGT1A10 codon 139 polymorphism may be an important determinant in risk for tobacco-related cancers.
Drug Metabolism and Disposition 07/2006; 34(6):943-9. · 3.73 Impact Factor
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ABSTRACT: Tamoxifen (TAM) is an antiestrogen widely used in the treatment and prevention of breast cancer in women. One of the major mechanisms of metabolism of TAM and one of its major active metabolites, 4-hydroxytamoxifen (4-OH-TAM), is via glucuronidation. In the present study, the glucuronidating activities of three common variant isoforms encoded by the human UDP-glucuronosyltransferase (UGT) 1A4 gene were examined against TAM, trans-4-OH-TAM and cis-4-OH-TAM.
HPLC was used to detect glucuronide conjugates in microsomes from UGT1A4-overexpressing HK293 cells. The UGT1A4 wild-type cDNA was synthesized by RT-PCR using normal human liver total RNA. The UGT1A424Thr/48Leu and UGT1A424Pro/48Val variants were generated by site-directed mutagenesis of the pcDNA3.1/V5-His-TOPO plasmid expressing wild-type UGT1A424Pro/48Leu. Levels of UGT1A4 expression in UGT-overexpressing cell lines were measured by western blot analysis.
Microsomes from wild-type UGT1A424Pro/48Leu-overexpressing HK293 cells exhibited significant levels of activity against TAM, trans-4-OH-TAM and cis-4-OH-TAM, forming exclusively the tamoxifen quaternary ammonium glucuronide (TAM-N+-glucuronide) and the 4-hydroxytamoxifen quaternary ammonium glucuronides (trans-4-OH-TAM-N+-glucuronide and cis-4-OH-TAM-N+-glucuronide) with apparent Km values of 2.0 microM, 2.2 microM, and 2.1 microM, respectively. Higher glucuronidation activities were found by kinetic analysis for microsomes from the variant UGT1A424Pro/48Val-overexpressing cell line as compared with microsomes from wild-type UGT1A424Pro/48Leu-overexpressing cells against TAM and against both the trans and cis isomers of 4-OH-TAM. A significantly (P < 0.02) lower Km value (approximately 1.6-fold to 1.8-fold) was observed for both 4-OH-TAM isomers, while a near-significant (P = 0.053) decrease in Km was observed for TAM for the UGT1A424Pro/48Val variant as compared with wild-type UGT1A4. The Vmax/Km ratio for the UGT1A424Pro/48Val variant was significantly (P < or = 0.005) higher than that observed for the wild-type UGT1A4 isoform for both the trans and cis isomers of 4-OH-TAM after normalization for UGT1A4 expression by western blotting. No significant effect on enzyme kinetics was observed for the UGT1A424Thr/48Leu variant against either isomer of 4-OH-TAM or with TAM.
These data suggest that the UGT1A4 codon 48 Leu>Val polymorphism significantly alters glucuronidation rates against TAM and its active hydroxylated metabolites, and that this polymorphism may play an important role in individual pharmacological response to TAM therapy.
Breast cancer research: BCR 01/2006; 8(4):R50. · 5.24 Impact Factor
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ABSTRACT: Urethane is a carcinogen to which there is widespread exposure through the consumption of fermented foods and alcoholic beverages. In this study, we have assessed the carcinogenicity of urethane in combination with ethanol. Male and female B6C3F(1) mice (48 mice per sex per group) were exposed to 0, 10, 30, or 90 ppm urethane in the presence of 0%, 2.5%, or 5% ethanol in drinking water ad libitum for two years, at which time the extent of tumorigenesis was assessed. Additional mice (four per sex per group) received the same doses for four weeks to assess serum levels of urethane and ethanol, DNA adduct formation, and the induction of microsomal cytochromes P450, cell proliferation, and apoptosis. Urethane decreased cell replication in the livers of female, but not male, mice, decreased cell replication in the lungs of both sexes, and induced cytochrome P450 2E1 in the livers of female mice. Hepatic levels of the DNA adduct 1,N(6)-ethenodeoxyadenosine were increased by exposure to urethane and decreased by treatment with ethanol. Animal weights and survival were not affected by ethanol; in contrast, urethane administration decreased body weights and survival. Urethane caused dose-dependent increases in liver, lung, and harderian gland adenoma or carcinoma and hemangiosarcoma of the liver and heart in both sexes, mammary gland and ovarian tumors in females, and squamous cell papilloma or carcinoma of the skin and forestomach in males. The increase in hepatocellular tumors occurred in a relatively linear manner and was attributed to the formation of 1,N(6)-ethenodeoxyadenosine in hepatic DNA coupled with an increase in cell replication. Hemangiosarcomas were observed only at the 90 ppm urethane dose and were probably a result of high-dose urethane-induced toxicity. Lung alveolar/bronchiolar and harderian gland adenoma or carcinoma increased in a relatively linear manner, suggestive of a genotoxic mechanism for tumor induction. Ethanol induced a dose-dependent trend in hepatocellular adenoma or carcinoma in male mice, with the incidence being marginally increased at the highest dose. In female mice administered 10 ppm and 90 ppm urethane, ethanol caused dose-related increases in alveolar/bronchiolar adenoma or carcinoma and hemangiosarcoma of the heart, respectively. This may be due to ethanol decreasing the first-pass clearance of urethane, thus, increasing systemic distribution. In male mice a different relationship was observed: ethanol caused a dose-related decrease in alveolar/bronchiolar and harderian gland adenoma or carcinoma in mice administered 30 ppm urethane.
Food and Chemical Toxicology 02/2005; 43(1):1-19. · 3.00 Impact Factor
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Taghreed Almahmeed,
Jay O Boyle,
Erik G Cohen,
John F Carew,
Baoheng Du,
Nasser K Altorki,
Levy Kopelovich, Jia-Long Fang,
Philip Lazarus,
Kotha Subbaramaiah,
Andrew J Dannenberg
Carcinogenesis 06/2004; 25(5):793-9. · 5.70 Impact Factor
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ABSTRACT: The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke, and glucuronidation of its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), is an important mechanism for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone detoxification. Substantial interindividual variability in urinary NNAL glucuronide formation has been observed in smokers and tobacco chewers. To determine whether genetic variations may play a role in this interindividual variability, NNAL-glucuronidating activities were analyzed in 78 human liver microsomal specimens and compared with the prevalence of missense polymorphisms in the two major NNAL-glucuronidating enzymes UGT1A4 and UGT2B7. In vitro assays using liver microsomal specimens from individual subjects demonstrated a 70- and 50-fold variability in NNAL-N-Gluc and NNAL-O-Gluc formation, respectively, and a 20-fold variability in the ratio of NNAL-N-Gluc:NNAL-O-Gluc formation. Microsomes from subjects with a homozygous polymorphic UGT1A4(24Thr)/UGT1A4(24Thr) genotype exhibited a significantly higher (P < 0.05) level of NNAL-N-Gluc activity compared with microsomes from subjects with the wild-type UGT1A4(24Pro)/UGT1A4(24Pro) genotype, and a significantly higher (P < 0.05) number of subjects with liver microsomes having high NNAL-N-Gluc formation activity contained the UGT1A4(24Thr)/UGT1A4(24Thr) genotype. Microsomes from subjects with the homozygous polymorphic UGT2B7(268Tyr)/UGT2B7(268Tyr) genotype exhibited a significantly lower level (P < 0.025) of NNAL-O-Gluc activity when compared with microsomes from subjects with the wild-type UGT2B7(268His)/UGT2B7(268His) genotype, and a significantly (P < 0.05) higher number of subjects with liver microsomes having low NNAL-O-Gluc formation activity contained the UGT2B7(268Tyr)/UGT2B7(268Tyr) genotype. These data suggest that the UGT1A4 codon 24 and UGT2B7 codon 268 polymorphisms may be associated with altered rates glucuronidation and detoxification of NNAL in vivo.
Cancer Research 03/2004; 64(3):1190-6. · 7.86 Impact Factor
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ABSTRACT: The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke and is considered to be a causative agent for several tobacco-related cancers. Glucuronidation of the major metabolite of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), has been implicated as an important mechanism for NNK detoxification. To characterize NNAL metabolism by N-glucuronidation in humans, high-pressure liquid chromatography was used to detect glucuronide conjugates of NNAL formed in human liver microsomes in vitro. In addition to peaks corresponding to the O-glucuronides of NNAL (NNAL-O-Gluc), a second series of peaks were observed in human liver microsomes that were identified by liquid chromatography-mass spectrometry to be NNAL N-glucuronides (NNAL-N-Gluc). Microsomes prepared from liver specimens from individual subjects (n = 42) exhibited substantial variability in the levels of NNAL-N-Gluc (49-fold variability) and NNAL-O-Gluc (49-fold variability) formed in vitro. This variability was likely not due to differences in tissue quality, as substantial variability (5-fold) was also observed in the ratio of NNAL-N-Gluc/NNAL-O-Gluc formation, with a mean ratio of 1.7 in the 42 specimens. Liver microsomes from smokers (n = 14) exhibited no significant difference in the levels of either NNAL-N-Gluc or NNAL-O-Gluc formation, or in the ratio of NNAL-N-Gluc/NNAL-O-Gluc formation, as compared with liver microsomes from never smokers (n = 28). Overexpressed UDP-glucuronosyltransferase (UGT) 1A4 exhibited significant levels of N-glucuronidating activity (V(max)/K(m) = 3.11 microl. min(-1). g(-1)) in vitro; no NNAL-N-glucuronide formation was detected for the 11 other overexpressed UGT enzymes tested in these studies. These results demonstrate the importance of N-glucuronidation in the metabolism of NNAL and the role of UGT1A4 in this pathway.
Drug Metabolism and Disposition 02/2004; 32(1):72-9. · 3.73 Impact Factor
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ABSTRACT: Of the hepatic UDP-glucuronosyltransferases (UGTs), only UGT1A1 and UGT1A9 exhibit activity against benzo(a)pyrene-trans-7R,8R-dihydrodiol [BPD(-)], precursor to the highly mutagenic anti-(+)-benzo(a)pyrene-7R,8S-dihydrodiol-9S,10R-epoxide. The UGT1A1*28 allelic variant contains an additional (TA) dinucleotide repeat in the "TATAA" box [(TA)(6)>(TA)(7)] of the UGT1A1 promoter that has been linked to decreased expression of the UGT1A1 gene and decreased bilirubin conjugation, leading to the relatively nondebilitating condition known as Gilbert's syndrome. To determine whether the UGT1A1 TATAA box polymorphism may play a role in the overall glucuronidation of BPD(-) in humans, we compared UGT1A1 TATAA box genotype with BPD(-) glucuronidating activity in normal liver microsomes. Significant decreases in UGT1A1 protein (P < 0.005) and bilirubin conjugation activity (P < 0.001) were observed in liver microsomes from subjects homozygous for the UGT1A1*28 allelic variant compared with subjects homozygous for the wild-type UGT1A1*1 allele. Significant decreases in BPD(-) glucuronidation activity (P < 0.02) were observed in subjects with the UGT1A1(*28/*28) genotype compared with subjects having the wild-type UGT1A1(*1/*1) genotype in assays of liver microsomes that included 0.1 mM alpha-naphthylamine, a competitive inhibitor of UGT1A9 and not UGT1A1. Similar phenotype:genotype correlations were observed when we compared subjects with the UGT1A1(*28/*28) genotype with subjects having the UGT1A1(*1/*28) genotype. In assays with alpha-naphthylamine, the K(m) of liver microsomes against BPD(-) was similar to that reported for UGT1A1-overexpressing baculosomes (319 micro M versus 290 micro M; Fang et al., Cancer Res., 62: 1978-1986, 2002). These data suggest that the UGT1A1 TATAA box polymorphism plays a role in an individual's overall ability to detoxify benzo(a)pyrene and in cancer risk.
Cancer Epidemiology Biomarkers & Prevention 01/2004; 13(1):102-9. · 4.12 Impact Factor
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ABSTRACT: UDP-glucuronosyltransferases (UGTs) have been implicated as important detoxifying enzymes for several major tobacco carcinogens. Because the aerodigestive tract is a primary target for exposure to tobacco smoke carcinogens, the major goal of the present study was to determine whether aerodigestive tract tissues exhibit glucuronidating activity against metabolites of benzo[a]pyrene (BaP) and to explore the pattern of expression of UGT genes in a series of aerodigestive tract tissue specimens. Glucuronidation of the phenolic BaP metabolites 3-, 7-, and 9-hydroxy-BaP was observed in all upper aerodigestive tract tissue microsome specimens tested, as determined by high-pressure liquid chromatography analysis. Glucuronidating activity toward the procarcinogenic BaP metabolite trans-BaP-7,8-dihydrodiol(+/-) was also detected in aerodigestive tract tissues. By semiquantitative duplex reverse transcription-polymerase chain reaction analysis, UGT1A7 and UGT1A10 were shown to be well expressed in all aerodigestive tract tissues examined, including tongue, tonsil, floor of mouth, larynx, and esophagus. UGT1A8 and UGT1A6 were expressed primarily in larynx; no expression was observed for UGTs 1A1, 1A3, 1A4, 1A5, 1A9. Of the family 2B UGTs, only UGT2B4 and UGT2B17 exhibited significant levels of expression in aerodigestive tract tissues. Of the aerodigestive tract-expressing UGTs, only UGTs 1A7, 1A8, and 1A10 exhibited glucuronidating activity against 7-hydroxy-BaP, with UGT1A10 exhibiting the highest affinity as determined by kinetic analysis (K(m) = 49 microM). No UGT expression or glucuronidating activity was observed for any of the lung specimens analyzed in this study. These results suggest that several family 1 UGTs may potentially play an important role in BaP detoxification in the aerodigestive tract.
Drug Metabolism and Disposition 05/2002; 30(4):397-403. · 3.73 Impact Factor
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ABSTRACT: UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of benzo(a)pyrene-trans-7,8-dihydrodiol (BPD), precursor to the potent mutagen benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, may be an important pathway in the detoxification of benzo(a)pyrene. To better characterize this pathway in humans, high-pressure liquid chromatography (HPLC) was used to detect glucuronide conjugates of BPD formed in vitro. Three peaks were detected by HPLC after incubation of racemic BPD with human liver microsomes; these were identified as monoglucuronides by liquid chromatography-mass spectrometry analysis. Proton nuclear magnetic resonance spectroscopy of isolated fractions, combined with HPLC analysis of the glucuronide products from human liver microsomal incubations with purified benzo(a)pyrene-trans-7S,8S-dihydrodiol [(+)-BPD] and benzo(a)pyrene-trans-7R,8R-dihydrodiol [(-)-BPD] forms of BPD, indicated that peak 1 contained the 7-glucuronide of 7S,8S-BPD (BPD-7S-Gluc), peak 2 was a mixture of the 7-glucuronide of 7R,8R-BPD (BPD-7R-Gluc) and the 8-glucuronide of 7S,8S-BPD (BPD-8S-Gluc), and peak 3 contained the 8-glucuronide of 7R, 8R-BPD (BPD-8R-Gluc). In liver microsomes, peak 1 (BPD-7S-Gluc) was the largest peak observed, whereas in microsomes from aerodigestive tract tissues, peak 2 (both BPD-7R-Gluc and BPD-8S-Gluc) was the largest HPLC peak observed. The liver enzymes UGT1A1 and UGT2B7 formed BPD-7S-Gluc as the major diastereomer, whereas UGT1A8 and UGT1A10, extrahepatic enzymes present in the aerodigestive tract, preferentially formed both BPD-7R-Gluc and BPD-8S-Gluc. In addition, both UGT1A9 and UGT1A7 preferentially formed BPD-7R-Gluc. No detectable glucuronidating activity against BPD was observed by UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B15, or UGT2B17. The affinity of individual UGT enzymes as determined by K(m) analysis was UGT1A10 > UGT1A9 > UGT1A1 > UGT1A7 for (-)-BPD and UGT1A10 > UGT1A9 > UGT2B7 approximately UGT1A1 > UGT1A7 for (+)-BPD. These results suggest that several UGTs may play an important role in the overall glucuronidation of BPD in humans, with UGT1A1, UGT1A7, UGT1A9, UGT1A10 and potentially UGT1A8 playing an important role in the glucuronidation of the procarcinogenic (-)-BPD enantiomer, and that the stereospecific activity exhibited by different UGTs against BPD is consistent with tissue-specific patterns of BPD glucuronide diastereomer formation and UGT expression.
Cancer Research 04/2002; 62(7):1978-86. · 7.86 Impact Factor
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ABSTRACT: Usnic acid is a prominent secondary lichen metabolite that has been used for various purposes worldwide. Crude extracts of usnic acid or pure usnic acid have been marketed in the United States as dietary supplements to aid in weight loss. The US Food and Drug Administration (FDA) received 21 reports of liver toxicity related to the ingestion of dietary supplements that contain usnic acid. This prompted the FDA to issue a warning about one such supplement, LipoKinetix, in 2001 (http://www.cfsan.fda.gov/~dms/ds-lipo.html). Subsequently, usnic acid and Usnea barbata lichen were nominated by the National Toxicology Program (NTP) for toxicity evaluations. At present, a toxicological evaluation of usnic acid is being conducted by the NTP. This review focuses on the recent findings of usnic acid-induced toxicities and their underlying mechanisms of action.
Journal of Environmental Science and Health Part C Environmental Carcinogenesis & Ecotoxicology Reviews 26(4):317-38. · 3.23 Impact Factor
