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Sulforaphane induces glutathione S‐transferase isozymes which detoxify aflatoxin B1‐8,9‐epoxide in AML 12 cells
Abstract
The aflatoxin B(1)-8,9-epoxide (AFBO) is hepatocarcinogenic intermediate of aflatoxin B(1) (AFB(1)) and is detoxified by glutathione S-transferases (GSTs). In this study, we investigated whether sulforaphane (SFN) could increase the rate of conjugation between AFBO and glutathione (GSH) as well as which of the GST isozymes were involved in the conjugation reaction. The conjugation potential was inhibited dose dependently with curcumin, an inhibitor of GSTs. SFN induced the expression of GST A3, GST A4, GST M1, GST P1, and GST T1 in alpha mouse line (AML) 12 cells. The cells treated with SFN (10 microM) for 12 h showed a 35-fold increase in conjugation potential of AFBO with GSH compared with the vehicle-treated cell. The conjugation potential was blocked partially by transfection of cells with siRNAs against each of the GST isozymes. The activity of GST A3 had the strongest effect on the conjugation potential. SFN treatment also increased total GST activity detected with 1-chloro-2,4-dinitrobenzene (CDNB) up to 4.3-fold. The induction fold was much lower than that detected with AFBO. These results suggest that the chemopreventive effect of SFN on the decomposition of AFBO is related to the upregulation of several GST isozymes genes. The increase of GST activity by SFN was extremely specific toward the conjugation reaction of AFBO compared with CDNB. Therefore, this system for detecting GST activity seems to be an excellent method for screening chemopreventive compounds toward AFB(1) toxicity.
... • The third aim of our trial was to give suggestions for expedient combinations of the single additives in order to maximise the impact on performance, microbial eubiosis, and on direct and indirect antioxidant effects. As the reference substances for the examination of indirect antioxidant effects and for the examination of xenobiotic enzyme induction we used a broccoli sprouts extract and turmeric oil, both having a proven impact on these parameters [15,[26][27][28]]. ...
... In this context experiments in which broilers were infected with Eimeria tenella or fed aflatoxin containing diets, the essential oil of oregano or turmeric powder counteracted the depressed feed intake and growth [49][50][51]. Accordingly some other studies with rats as model animals and tissue cultures have demonstrated the beneficial effects of thyme, rosemary and of sulforaphane on mycotoxin detoxification [28,[52][53][54][55]. ...
Objective: Since the ban of antibiotics as growth promoting feed additives in the EU in 2006 research in alternatives has gained importance. Phytogenic feed additives represent a heterogenous class of different plant derived substances that are discussed to improve the health of farm animals by direct and indirect antioxidant effects and by influencing microbial eubiosis in the gastrointestinal tract. Consequently our study aimed to investigate the influence of broccoli extract and the essential oils of tur- meric, oregano, thyme and rosemary, as selected individual additives, on intestinal and faecal microflora, on xenobiotic enzymes, and on the antioxidant system of piglets. Methods: 48 four weeks old male weaned piglets were assigned to 6 groups of 8. The piglets were housed individually in stainless steel pens with slatted floor. The control group (Con) was fed a diet without an additive for 4 weeks. The diet of group BE contained 0.15 g/kg sulforaphane in form of a broccoli extract. 535, 282, 373 and 476 mg/kg of the essential oils of turmeric (Cuo), oregano (Oo), thyme (To) and rosemary (Ro) were added to the diets of the remaining 4 groups to stan-dardise supplementation to 150 mg/kg of the oils’ key terpene compounds ar-turmerone, carvacrol, thymol and 1,8-cineole. The composition of bacterial microflora was examined by cultivating samples of jejeunal and colonic mucosa and of faeces under specific conditions. The mRNA expression of xenobiotic and antioxidant enzymes was determined by reversing transcrip- tase real time detection PCR (RT-PCR). Total antioxidant status was assayed using the Trolox Equivalent Antioxidant Capacity (TEAC), and lipid peroxidation was determined by measuring thiobarbioturic acid reactive substances (TBA- RS). Results: Compared to Con piglets all additives positively influenced weight gain and feed conversion in week 1. Over the whole trial period no significant differences in performance parameters existed between the experimental groups. Compared to group Con performance of Ro piglets was, however, slightly impaired. Com- pared to Con piglets Cuo, Oo and To increased the ratio of Lactobacilli:E. coli attached to the jejunal mucosa, whereas BE and Ro impaired this ratio slightly. In contrast in colonic mucosa Ro improved Lactobacilli:E. coli ratio. In faecal samples an improvement of Lactobacilli:E. coli ratio could be analysed for To and Ro. Ro was the only additive that reduced the incidence rate of piglets tested positive for enterotoxic E. coli (ETEC). All additives significantly increased jejunal TEAC and reduced TBA-RS. In the liver BE, Cuo, Oo and To increased TEAC in tendency and Ro significantly. Liver TBA-RS were slightly reduced by all additives compared to Con piglets. Whereas the influence of BE, To and Ro on jejunal TEAC mainly was derived from the induction of xenobiotic and antioxidant enzymes (indirect antioxidant effects), Cuo and Oo influenced TEAC by direct antioxidant effects. Discussion and Conclusions: Our results have shown: That within the labiatae oils Oo and To have the potential to improve performance slightly. That phytogenic substances have a small but not sig- nificant influence on intestinal microflora. That phytogenic feed additives up-regulate the anti- oxidant system of piglets either by direct or by indirect antioxidant effects and that they may thereby improve health status. That within the labiatae oils Oo has a high direct antioxidant potential whereas Ro potently induces xenobiotic and antioxidant enzymes. That broccoli extract is an attractive new phytogenic additive, improving antioxidant status by indirect antioxidant effects. That defined combinations of selected phytogenic substances may produce additive effects. That health promoting effects of phytogenic additives in the future should be studied systematically under the challenge with pathogenic microorganisms or food derived toxins.
... They can convert aflatoxins to less toxic compounds or small molecules and eventually reduce or eliminate aflatoxin damage. Several effective AFB1 degradation enzymes have been found, such as laccase [107], aflatoxin-detoxifying enzyme [135], glutathione S-transferases [136,137], manganese peroxidase [115,138], and co-expression of human glutathione S-transferases (GSTs) with GSTA1-1 or GSTP1-1 in Salmonella typhimurium strains [139]. Other enzymes, such as β-naphthoflavone (BNF), are inducers of various detoxification enzymes. ...
Aspergillus flavus is a ubiquitous pathogen that can infect many foods and grains, and it produces large amounts of aflatoxins during their storage. Aflatoxins are considered highly toxic and carcinogenic to humans, and they cause great damage to crop production, food security, and human health. Thus, controlling A. flavus and aflatoxins in grains presents a great challenge to humans worldwide. Over the past decade, many strategies have been demonstrated to be useful in controlling A. flavus and aflatoxins during food storage. These methods involve physical agents, chemical agents, biological agents, etc. Some of these methods are currently used in actual production. In this review, we summarize the reported methods for controlling A. flavus and aflatoxins during food storage in the past ten years and elucidate their advantages and disadvantages. The methods discussed include irradiation technology; low oxygen atmospheres; chemical fungicides (benzalkonium chloride, iodine, ammonium bicarbonate, and phenolic and azole compounds); biological agents from plants, animals, and micro-organisms; and aflatoxin elimination methods. We expect that this review will promote the applications of current strategies and be useful for the development of novel technologies to prevent or eliminate A. flavus and aflatoxins in food and feed during storage.
... Besides, in rat hepatocytes, SFN alone has also proved to significantly enhance the GSTA1 mRNA level in a dose-dependent manner, while co-treatment of SFN with β-naphthoflavone leads to a substantial increase in NQO1 activity and a marked decrease in CYP1A1, CYP2B, and CYP3A4 expression, thus exerting its chemopreventive activity [102]. In addition, the chemopreventive effect of SFN on detoxication of the aflatoxin B1-8,9-epoxide in alpha mouse liver (AML) 12 cells has been reported to be associated with the upregulation of several GST isozyme genes [103]. Except SFN, addition of PEITC to rats at all dietary doses could markedly elevate the quinone reductase in liver tissues and stimulate the activity of hepatic GSTs (Table 2) [91]. ...
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for about 90% of cases. Sorafenib, lenvatinib, and the combination of atezolizumab and bevacizumab are considered first-line treatments for advanced HCC. However, clinical application of these drugs has also caused some adverse reactions such as hypertension, elevated aspartate aminotransferases, and proteinuria. At present, natural products and their derivatives have drawn more and more attention due to less side effects as cancer treatments. Isothiocyanates (ITCs) are one type of hydrolysis products from glucosinolates (GLSs), secondary plant metabolites found exclusively in cruciferous vegetables. Accumulating evidence from encouraging in vitro and in vivo animal models has demonstrated that ITCs have multiple biological activities, especially their potentially health-promoting activities (antibacterial, antioxidant, and anticarcinogenic effects). In this review, we aim to comprehensively summarize the chemopreventive, anticancer, and chemosensitizative effects of ITCs on HCC, and explain the underlying molecular mechanisms.
... In addition, SFN has been demonstrated as an important agent in the regulation of functionalizing (phase I) and conjugating (phase II) xenobiotic biotransformation enzymes [21]. Among phase II enzymes, GSTs are known to be induced by SFN through the activation of the antioxidant responsive elements axis thanks its sulphur interaction with thiol groups of the Keap1 cysteine residues [22]. In addition, GSTs, and in particular the GSTM1 isoform, play an important role in enzymatic formation and cleavage of the GSH conjugates of isothiocyanates, contributing to SFN pharmacokinetics [23][24][25]. ...
Interferonopathies are rare genetic conditions defined by systemic inflammatory episodes caused by innate immune system activation in the absence of pathogens. Currently, no targeted drugs are authorized for clinical use in these diseases. In this work, we studied the contribution of sulforaphane (SFN), a cruciferous-derived bioactive molecule, in the modulation of interferon-driven inflammation in an immortalized human hepatocytes (IHH) line and in two healthy volunteers, focusing on STING, a key-component player in interferon pathway, interferon signature modulation, and GSTM1 expression and genotype, which contributes to SFN metabolism and excretion. In vitro, SFN exposure reduced STING expression as well as interferon signature in the presence of the pro-inflammatory stimulus cGAMP (cGAMP 3 h vs. SFN+cGAMP 3 h p value < 0.0001; cGAMP 6 h vs. SFN+cGAMP 6 h p < 0.001, one way ANOVA), restoring STING expression to the level of unstimulated cells. In preliminary experiments on healthy volunteers, no appreciable variations in interferon signature were identified after SFN assumption, while only in one of them, presenting the GSTM1 wild type genotype related to reduced SFN excretion, could a downregulation of STING be recorded. This study confirmed that SFN inhibits STING-mediated inflammation and interferon-stimulated genes expression in vitro. However, only a trend towards the downregulation of STING could be reproduced in vivo. Results obtained have to be confirmed in a larger group of healthy individuals and in patients with type I interferonopathies to define if the assumption of SFN could be useful as supportive therapy.
... In animals and humans, the metabolite of aflatoxins responsible for their carcinogenic properties is the short-lived AFB-2,3-epoxide, currently known as AFB1-8,9-epoxide (AFBO), which has the capacity to form adducts with DNA and proteins and results in mutations [29,59]. Cytochromes P-450 3A4 and 1A2 are major liver enzymes that are responsible for converting AFB1 to AFBO. ...
Mycotoxins are produced by fungi and are known to be toxic to humans and animals. Common mycotoxins include aflatoxins, ochratoxins, zearalenone, patulin, sterigmatocystin, citrinin, ergot alkaloids, deoxynivalenol, fumonisins, trichothecenes, Alternaria toxins, tremorgenic mycotoxins, fusarins, 3-nitropropionic acid, cyclochlorotine, sporidesmin, etc. These mycotoxins can pose several health risks to both animals and humans, including death. As several mycotoxins simultaneously occur in nature, especially in foods and feeds, the detoxification and/or total removal of mycotoxins remains challenging. Moreover, given that the volume of scientific literature regarding mycotoxins is steadily on the rise, there is need for continuous synthesis of the body of knowledge. To supplement existing information, knowledge of mycotoxins affecting animals, foods, humans, and plants, with more focus on types, toxicity, and prevention measures, including strategies employed in detoxification and removal, were revisited in this work. Our synthesis revealed that mycotoxin decontamination, control, and detoxification strategies cut across pre-and post-harvest preventive measures. In particular, pre-harvest measures can include good agricultural practices, fertilization/irrigation, crop rotation, using resistant varieties of crops, avoiding insect damage, early harvesting, maintaining adequate humidity, and removing debris from the preceding harvests. On the other hand, post-harvest measures can include processing, chemical, biological, and physical measures. Additionally, chemical-based methods and other emerging strategies for mycotoxin detoxification can involve the usage of chitosan, ozone, nanoparticles, and plant extracts.
... The physical processes include three steps: separation of the contaminated items, removal of mycotoxins, and decreasing the content of mycotoxins by exposing under sunlight, γ-radiation or microwave heating [34]. Chemical processes utilize ozone [35], ammonia [36], and citric acid [22], etc., while natural phytochemical processes employ chlorophyllins [33], sulforaphane [37], isoimperatorin isolated from Poncirustrifoliata Raf [38], etc. However, these physical and chemical processes may diminish the appearance and taste of foods. ...
Aflatoxins are toxic secondary metabolic products, which exert great hazards to human and animal health. Decontaminating aflatoxins from food ingredients to a threshold level is a prime concern for avoiding risks to the consumers. Biological decontamination processes of aflatoxins have received widespread attention due to their mild and environmental-friendly nature. Many reports have been published on the decontamination of aflatoxins by microorganisms, especially lactic acid bacteria (LAB), a well-explored probiotic and generally recognized as safe. The present review aims at updating the decontamination of produced aflatoxins using LAB, with an emphasis on the decontamination mechanism and influence factors for decontamination. This comprehensive analysis provides insights into the binding mechanisms between LAB and aflatoxins, facilitating the theoretical and practical application of LAB for decontaminating hazardous substances in food and agriculture.
... Both BITC and PEITC were found to prevent nicotine (BITC and PEITC at 25 μM and 3 μM respectively) and tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (BITC and PEITC at 1 μM and 0.3 μM, respectively) metabolism by inhibiting the activation of CYP2A6 and CYP2A13, respectively, in an NADPH-dependent manner [86]. Moreover, SFN (10 μM) increased the activity of various GSTs (A3, A4, M1, P1 and T1) in order to inactivate aflatoxin B1-8,9-epoxide (AFBO) in alpha mouse liver 12 (AML12) cells [87]. Oral administration of allyl isothiocyanate (AITC) (40 μmol/kg/day) also increased the activity of both quinone reductase (QR) and GSTs in urinary bladder, thus indicating a reduced risk of bladder cancer after frequent exposure to ITCs [88]. ...
Many studies have shown evidence in support of the beneficial effects of phytochemicals in preventing chronic diseases, including cancer. Among such phytochemicals, sulphur-containing compounds (e.g., isothiocyanates (ITCs)) have raised scientific interest by exerting unique chemo-preventive properties against cancer pathogenesis. ITCs are the major biologically active compounds capable of mediating the anticancer effect of cruciferous vegetables. Recently, many studies have shown that a higher intake of cruciferous vegetables is associated with reduced risk of developing various forms of cancers primarily due to a plurality of effects, including (i) metabolic activation and detoxification, (ii) inflammation, (iii) angiogenesis, (iv) metastasis and (v) regulation of the epigenetic machinery. In the context of human malignant melanoma, a number of studies suggest that ITCs can cause cell cycle growth arrest and also induce apoptosis in human malignant melanoma cells. On such basis, ITCs could serve as promising chemo-therapeutic agents that could be used in the clinical setting to potentiate the efficacy of existing therapies.
... It is abundant in several cruciferous vegetables, especially broccoli and can protect against tumors (Beecher, 1994;Zhang, Talalay, Cho, & Posner, 1992). Gao, Chen, Zhu, Choi and Kim (2010) used alpha mouse line (AML)-12 cells to identify the effects of SFN on GST isozymes, which are responsible for degradation of AFB1. Results showed that the conjugation potential of AFBO with GSH was 35 times higher after treatment of 10 μM SFN for 12 h. ...
... Due to sulforaphane's lipophilic profile and small molecular size, it is passively absorbed via enterocytes in the liver from the entero-portal system [30,31]. Sulforaphane is conjugated in the liver with glutathione by the action of glutathione-stransferase and excreted in the urine [32]. Investigators have reported that high cellular accumulation of sulforaphane occurs in mammalian cells noting cellular concentrations of 4.4-13.3 ...
... In addition, sulforaphane derived from cruciferous plants, such as broccoli, Brussel sprouts or cabbages, confers a protective effect against AFB1-mediated genotoxicity in human hepatocytes [63]. Sulforaphane effectively induces hepatic total GST activity and attenuates hepatic AFB1-DNA adducts in AFB1-exposed rats [64,65]. In addition, the thyme and calendula extracts alone or in combination ameliorate aflatoxin-induced oxidative stress and genotoxicity mechanistically demonstrated the alterative expression of p53, bax and bcl2 gene expression [66]. ...
Medicinal herbs have been increasingly used for therapeutic purposes against a diverse range of human diseases worldwide. Moreover, the health benefits of spices have been extensively recognized in recent studies. However, inevitable contaminants, including mycotoxins, in medicinal herbs and spices can cause serious problems for humans in spite of their health benefits. Along with the different nation-based occurrences of mycotoxins, the ultimate exposure and toxicities can be diversely influenced by the endogenous food components in different commodities of the medicinal herbs and spices. The phytochemicals in these food stuffs can influence mold growth, mycotoxin production and biological action of the mycotoxins in exposed crops, as well as in animal and human bodies. The present review focuses on the occurrence of mycotoxins in medicinal herbs and spices and the biological interaction between mold, mycotoxin and herbal components. These networks will provide insights into the methods of mycotoxin reduction and toxicological risk assessment of mycotoxin-contaminated medicinal food components in the environment and biological organisms.
... This conjugation provides protection against cellular/organ toxicity [245]. GSTs are induced by diverse chemical compounds, such as isothiocyanates (such as benzylisothiocyanate, allylisothiocyanate, and sulforaphane), phenobarbital, 1,4bis[2-(3,5-dichloropyridyloxy)]benzene, pregnenolone-16α-carbonitrile, 3-methylcholanthrene, 2,3,7,8-tetrachloro-dibenzo-p-dioxin, BNF, butylated hydroxyanisole, ethoxyquin, oltipraz, fumaric acid, coumarin, dexamethasone, thiazolidinediones 1-[2cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole, 12-O-tetradecanoylphorbol-13-acetate, glyphosate, citrus triterpenoids (nomilin, isoobacunoic acid, and deacetyl nomilin), rambo (0.6% permethrin), cinnamon, cardamom, protocatechuic acid, myristicin, and so on [246][247][248][249][250][251][252][253][254][255]. The gene expression of GSTs is mediated by multiple receptors, viz, constitutive androstane receptor (CAR), PXR, AhR, nuclear factor E2 related factor (Nrf2), peroxisome proliferator-activated receptor-α/γ (PPAR α/γ), and CAATT/enhancer-binding protein (C/EBP) β [249,256,257]. ...
Drug-metabolizing enzymes (DMEs) are primarily expressed in the liver but their role in the extrahepatic tissues such as gastrointestinal tract (GIT), pulmonary, excretory, nervous, cardiovascular system, and skin cannot be neglected. Generally, the expression of DMEs in extrahepatic tissues is quantitatively lower than that in the liver, but there are a few enzymes such as CYP1A1, CYP1B1, CYP2F1, and CYP2U1 that are more abundant in extrahepatic organs. As many extrahepatic organs are portals for administered drugs, DMEs expressed in these organs can be responsible for significant metabolism, leading to first-pass effects and lower bioavailability. Extrahepatic DMEs are also involved in bioactivation of prodrugs and formation of reactive metabolites that may interact with cellular components, resulting in organ-specific toxicity. Activity and expression of extrahepatic DMEs is often altered by coadministered drugs, leading to drug–drug interactions. Expression of DMEs in living beings affected by a host of environmental and genetic factors such as genetic polymorphism, age, gender, pathophysiological conditions, inborn errors in metabolism, food habits, and environmental pollutants, contributing to varied drug effects and idiosyncratic toxicities.
... Oxidative stress has been associated with the pathogenesis of different human diseases, including Alzheimer, diabetes, cardiovascular diseases, and cancer (7). To reduce damage, cells have a range of antioxidants encoding genes, such as NAD(P)H:quinone oxidorreductase 1 (NQO1) (8), hemeoxigenase 1 (HMOX1) (9), and several detoxificant genes such as glutathione S-transferase (GST) (10) and UDP-glucuronosiltransferase (UGT) (11). Most of these genes are regulated at the transcriptional level through the activation of a cis-acting element in their promoter region called antioxidant response element (ARE) (12)(13)(14). ...
Chronic myeloid leukaemia (CML) is one of the most frequent hematological neoplasia worldwide. The abnormal accumulation of reactive oxygen species may be an important factor in CML development. The transcription factor NRF2 can regulate the transcription of a battery of antioxidant and detoxificant genes after heterodimerizing with small-Maf proteins. Although the participation of NRF2 in the development of chronic-degenerative diseases has been thoroughly studied, the role of small-Maf genes has not been documented. We have identified polymorphisms in the 3 MAF genes (F, G and K) and assessed their association with CML. Over 260 subjects with CML and 399 unrelated healthy donors have been studied. After sequencing each MAF gene by Sanger technology, we found 17 variants in MAFF gene, 8 in MAFG and 7 in MAFK. In the case-control study, the homozygote genotype CC for the rs9610915 SNP of MAFF was significantly associated with CML. The frequency of the ACC haplotype from MAFK was significantly lower than controls. After stratification by gender, the ACC and GTG haplotypes were associated only with males with CML. These novel data suggest an association between MAFF and MAFG and the development of CML. This article is protected by copyright. All rights reserved.
Skin cancer incidence is rapidly growing over the last decades and is generally divided into malignant melanoma and non-melanoma (NMSC) with the latter being subdivided into squamous (SCC) and basal cell carcinoma (BCC). Among them, melanoma is the most aggressive type with high mortality rates. On the other hand, aberrant gene expression is a critical step towards malignant transformation. To this end, epigenetic modifications like changes in DNA methylation patterns and miRNA expression profile as well as histone modifications are all capable of inducing an altered gene expression profile involved in various cellular cascades including cell cycle, proliferation and apoptosis. In general, there is an interest about the beneficiary effect of various phytochemicals in the prevention and treatment of skin malignancies. Among them, glucosinolates are an important type of compounds, abundant in cruciferous vegetables, which are hydrolysed by an endogenous enzyme called myrosinase to a range of bioactive compounds including isothiocyanates (ITCs). These are the major biologically active products capable of mediating the anti-cancer effect of cruciferous vegetables. Their chemo-preventive action is mainly attributed to a plurality of anti-cancer properties including regulation of the epigenetic machinery. Current evidence supports the view that ITCs are potent compounds in interacting with the epigenome in order to restore the normal epigenetic landscape in malignant cells. This review article summarizes the current state of knowledge on the epigenetic modifications that lead to malignant transformation and the role of ITCs with respect to their ability to restore the epigenetic landscape that contributes to skin carcinogenesis.
The aim of this study was to analyse the effect of sulforaphane (SFN) in cultures of adult cardiomyocytes, evaluating oxidative stress at different times. Cells were isolated, cultured, and divided into 4 groups: Control, SFN (5μM), H2O2 (5μM), and SFN+H2O2 (5μM both), and subdivided into groups undergoing 1 or 24 h of SFN incubation. After 1 h of incubation, reactive oxygen species production was 40% lower in the SFN group than the Control, and lipid peroxidation was 63% higher in the H2O2 group than the Control, and it was reduced in both of the SFN groups. The SOD activity was 59% higher in groups incubated for 24 h than in those incubated for 1 h. Protein expression of SOD-1 and SOD-2 was higher in the 24-h groups compared to the 1-h groups (55% and 24%, respectively). The Nrf2 protein expression in the 1-h groups was 17% higher than in the 24-h groups, and the SFN + H2O2 group had 40% more Nrf2 than the Control in the 1-h groups. Unlike Nrf2, the PGC-1α expression was 69% higher in the 24-h groups in relation to the 1-h groups. Regarding the 24-h groups, the SFN and SFN+H2O2 groups were higher than the Control (32% and 33%, respectively), and the SFN+H2O2 group was increased (21%) compared to H2O2. SFN had a protective action against oxidative damage, but had no effect on the antioxidant enzymes analyzed. The different responses in the expression of Nrf2 and PGC-1α in relation to the incubation times, draws attention to the importance of establishing a timeline of the action of SFN, since there appears to be a temporal difference in its mechanism in adult cardiomyocytes.
Over 500 mycotoxins are currently known, and their number continues to rise. Mycotoxins are secondary metabolites produced by fungi that contaminate agricultural products as a result of fungal spoilage, and they may be produced before, during, or after harvest, or at any stage during the food chain. As a group, mycotoxins are difficult to classify, and they stand out by the wealth of molecular mechanisms and cellular targets that can mediate their biological effects. Some of the challenging aspects related to the pathogenesis of mycotoxins-caused illnesses are that one fungal species may generate more than one mycotoxin, and several fungal species may be concomitantly present in food products. Of particular importance is the emerging concept of “hidden mycotoxins,” which are products that are generated from mycotoxins as a result of plant metabolism, and because their presence can be underestimated by physicochemical analytical tests, the harm that they pose for animals and humans can easily be underestimated. This chapter discusses some of the main mycotoxins, reviews their mechanisms of action, and highlights key considerations for preventing and controlling the presence of mycotoxins in commodities.
Objective: To investigate the change in CYP3A4 activity and its significance in hepatocarcinogenesis induced by aflatoxin B1 (AFB1) in rats. Methods: Four-week old male Wistar rats were randomly divided into AFB1 and control groups. Rats in the AFB1 groups were intraperitoneally injected with AFB1 and dimethyl sulfoxide. During hepatocarcinogenesis, liver biopsies were performed on all animals on the 13th, 23rd, 33rd, 43rd, 53rd, and 63rd week of the experiment. The animals were sacrificed on the 73rd week and liver tissues were collected. Using the hepatic microsomal mixed-function oxidase enzyme system, the CYP3A4 activity was dynamically examined in liver samples via quantitative fluorescence spectrophotometry. Results: The hepatocellular carcinoma incidence in the AFB1 group (58.8%, 10/17) was significantly higher than in the control group (0.0%, 0/16), with P=0.001. Both groups exhibited different levels of changes in CYP3A4 activity. The CYP3A4 activity in both groups gradually increased from the 13th week of the experiment, with a peak value on the 23rd week. The activity then gradually decreased, and again increased on the 43rd week, forming double peaks. The CYP3A4 activities in the AFB1 group significantly decreased during the 13rd and 53rd weeks compared with those of the control group (P=0.000). The level of CYP3A4 activity in the AFB1 group was close but not statistically significant to that of the control group on the 63rd week (P=0.5086). Conclusion: The CYP3A4 activity was inhibited in rats during hepatocarcinogenesis. This inhibition may be due to the decreased carcinogen activation in cells and the change in protein expression caused by genetic polymorphisms.
Isothiocyanates (ITCs) are phytochemicals produced from the hydrolysis of glucosinolates, which are found at high concentrations in cruciferous vegetables. Vegetables of the Cruciferae family include, among others, broccoli, cauliflower, gardencress, watercress, and cabbage. A number of studies using animal models have suggested that certain ITCs are capable of preventing breast, lung, and prostate carcinogenesis. Additionally, certain ITCs such as sulforaphane (SFN), benzyl (BITC), and phenethyl (PEITC) isothiocyanate have been shown to elicit strong chemotherapeutic properties. SFN, BITC, and PEITC are suggested to target several cellular pathways that inhibit growth, induce apoptosis, and prevent migration, and are presently being investigated for their therapeutic potential. Work on ITCs is progressing quickly from bench to beside, and currently there are several ongoing clinical trials. One study is investigating PEITC's ability to inhibit lung carcinogenesis, while another trial is investigating how PEITC affects lymphoproliferative disorders, specifically in patients who have received the chemotherapeutic drug, fludarabine. Additionally, a Phase II clinical trial is investigating whether SFN can modulate the level of prostate specific antigen in patients with recurrent prostate cancer. This chapter will give an overview of the previously mentioned ITCs, and their reported ability to inhibit carcinogenesis in vivo and in vitro at three stages: initiation, promotion, and progression.
Glutathione (GSH), γ-Glu-Cys-Gly, is one of the most abundant small non-protein thiol molecules in mammalian tissues, particularly
in the liver. Although glutathione is present in thiol-reduced (GSH) and disulfide oxidized (GSSG) forms, the predominant
form is GSH and its content can exceed 10 mmol/L in liver cells. As an important intracellular reductant, GSH has many biological
functions in cells. Its major function is as an anti-oxidant as it can protect proteins from oxidation by reversible posttranslational
modification (glutathionylation) and decrease reactive oxygen species-mediated damage. However, it does have numerous other
functions, including to chelate metal irons; enhance the absorption of iron, selenium and calcium; participate in lipid and
insulin metabolism; regulate cellular events such as gene expression, DNA and protein synthesis, cell proliferation and apoptosis,
redox-dependent signal transduction pathways, cytokine production and the immune response; and control protein glutathionylation.
Therefore, GSH plays important roles in cell survival and health, and an imbalance in the GSH level can lead to many diseases.
In this review, we provide an overview of the function of GSH in mammalian cells and discuss future research of GSH.
Keywordsglutathione–GSSG–ROS–glutathionylation
The chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies.
Here we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not.
Although in vivo study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.
Hepatocellular carcinoma (HCC) is a common human cancer with high mortality, and currently, there is no effective chemoprevention or systematic treatment. Recent evidence suggests that cyclooxygenase-2 (COX-2)–derived PGE2 and Wnt/β-catenin signaling pathways are implicated in hepatocarcinogenesis. Here, we report that ω-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) inhibit HCC growth through simultaneously inhibition of COX-2 and β-catenin. DHA and EPA treatment resulted in a dose-dependent reduction of cell viability with cleavage of poly ADP ribose polymerase, caspase-3, and caspase-9 in three human HCC cell lines (Hep3B, Huh-7, HepG2). In contrast, AA, a ω-6 PUFA, exhibited no significant effect. DHA and EPA treatment caused dephosphorylation and thus activation of GSK-3β, leading to β-catenin degradation in Hep3B cells. The GSK-3β inhibitor, LiCl, partially prevented DHA-induced β-catenin protein degradation and apoptosis. Additionally, DHA induced the formation of β-catenin/Axin/GSK-3β binding complex, which serves as a parallel mechanism for β-catenin degradation. Furthermore, DHA inhibited PGE2 signaling through downregulation of COX-2 and upregulation of the COX-2 antagonist, 15-hydroxyprostaglandin dehydrogenase. Finally, the growth of HCC in vivo was significantly reduced when mouse HCCs (Hepa1-6) were inoculated into the Fat-1 transgenic mice, which express a Caenorhabditis elegans desaturase converting ω-6 to ω-3 PUFAs endogenously. These findings provide important preclinical evidence and molecular insight for utilization of ω-3 PUFAs for the chemoprevention and treatment of human HCC. [Mol Cancer Ther 2009;8(11):3046–55]
Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to the constitutively expressed rat liver Yc1 subunit. The significance of the fact that both mouse Yc and rat Yc2 exhibit high catalytic activity towards AFB1-8,9-epoxide, whereas rat Yc1 possesses little activity towards this compound, is discussed in terms of structure/function.
Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed.
The harmful effects of Aflatoxin B1 (AFB1) are a consequence of it being metabolized to AFB1-8,9-epoxide, a compound that serves as an alkylating agent and mutagen. The toxicity of AFB1 towards different cells varies substantially; sensitivity can change significantly during development, can be modulated by treatment with xenobiotics and is decreased markedly in preneoplastic lesions as well as in tumors. Three types of resistance, namely intrinsic, inducible and acquired, can be identified. The potential resistance mechanisms include low capacity to form AFB1-8,9-epoxide, high detoxification activity, increase in AFB1 efflux from cells and high DNA repair capacity. Circumstantial evidence exists that amongst these mechanisms the glutathione S-transferases, through their ability to detoxify AFB1-8,9-epoxide, play a major role in determining the sensitivity of cells to AFB1.
The biotransformation of the potential human carcinogen aflatoxin B1 (AFB1) was studied using hepatic microsomes from the rat, mouse, monkey, and human. Initial rates of AFB1 oxidation to aflatoxins Q1, M1, and P1, as well as the reactive intermediate AFB1-8,9-epoxide, were determined using a high performance liquid chromatography assay. The rates of generation of these AFB1 metabolites were investigated at low substrate concentrations (more representative of environmental exposures) and also at high ("saturating") concentrations commonly utilized in studies in vitro. Striking differences in ratios of the metabolites were observed. At an AFB1 concentration of 124 microM, mouse and monkey microsomes had the highest rates of AFB1-8,9-epoxide formation. Primate liver microsomes formed aflatoxin Q1 in large amounts but failed to produce detectable aflatoxin P1. Determination of the rates of formation over initial AFB1 concentrations ranging from 15 to 475 microM revealed that the proportion converted to AFB1-8,9-epoxide increased at lower substrate concentrations in the case of the rat and human microsomes but not with mouse or monkey microsomes. The differences in patterns of metabolite formation with varying concentrations have implications for interspecies comparisons of carcinogenic potency of AFB1.
Aflatoxin B1 (AFB1) has been postulated to be a hepatocarcinogen in humans, possibly by causing p53 mutations at codon 249. AFB1 is metabolized via the phase I and II detoxification pathways; hence, genetic variation at those loci may predict susceptibility to the effects of AFB1. To test this hypothesis, genetic variation in two AFB1 detoxification genes, epoxide hydrolase (EPHX) and glutathione S-transferase M1 (GSTM1), was contrasted with the presence of serum AFB1-albumin adducts, the presence of hepatocellular carcinoma (HCC), and with p53 codon 249 mutations. Mutant alleles at both loci were significantly overrepresented in individuals with serum AFB1-albumin adducts in a cross-sectional study. Mutant alleles of EPHX were significantly overrepresented in persons with HCC, also in a case-control study. The relationship of EPHX to HCC varied by hepatitis B surface antigen status and indicated that a synergistic effect may exist. p53 codon 249 mutations were observed only among HCC patients with one or both high-risk genotypes. These results indicate that individuals with mutant genotypes at EPHX and GSTM1 may be at greater risk of developing AFB1 adducts, p53 mutations, and HCC when exposed to AFB1. Hepatitis B carriers with the high-risk genotypes may be an even greater risk than carriers with low-risk genotypes. These findings support the existence of genetic susceptibility in humans to the environmental carcinogen AFB1 and indicate that there is a synergistic increase in risk of HCC with the combination of hepatitis B virus infection and susceptible genotype.
Much progress has been made in elucidating the biochemical and molecular mechanisms that underlie aflatoxin carcinogenesis. In humans, biotransformation of AFB1 to the putative carcinogenic intermediate. AFB-8,9-exo-epoxide, occurs predominantly by cytochromes P450 1A2 and 3A4, with the relative importance of each dependent upon the relative magnitude of expression of the respective enzymes in liver. Genetic variability in the expression of these and other cytochromes P450 may result in substantial interindividual differences in susceptibility to the carcinogenic effects of aflatoxins. Detoxification of AFB-8,9-epoxide by a specific alpha class glutathione S-transferase is an important protective mechanism in mice, and it accounts for the resistance of this species to the carcinogenic effects of AFB. This particular form of GST is expressed constitutively only at low levels in rats, but it is inducible by antioxidants such as ethoxyquin, and it accounts for much of the chemoprotective effects of a variety of substances, including natural dietary components that putatively act via an "antioxidant response element" (ARE). In humans, the constitutively expressed GSTs have very little activity toward AFB1-8,9-exo-epoxide, suggesting that--on a biochemical basis--humans should be quite sensitive to the genotoxic effects of aflatoxins. If a gene encoding a high aflatoxin-active form of GST is present in the human genome, but is not constitutively expressed, and is inducible by dietary antioxidants (as occurs in rats), then chemo- and/or dietary intervention measures aimed at inducing this enzyme could be highly effective. However, as it is possible that human CYP 1A2 may also be inducible by these same chemicals (because of the possible presence of an ARE in this gene), the ultimate consequence of dietary treatment with chemicals that induce biotransformation enzymes via an ARE is uncertain. The balance of the rate of activation (exo-epoxide production) to inactivation (GST conjugation plus other P450-mediated non-epoxide oxidations) may be a strong indicator of individual and species susceptibility to aflatoxin carcinogenesis, if the experimental conditions are reflective of true dietary exposures. There is strong evidence that AFB-8,9-exo-epoxide binds to G:C rich regions of DNA, forming an adduct at the N7-position of guanine. Substantial evidence demonstrates that AFB1-8,9-epoxide can induce activating mutations in the ras oncogene in experimental animals, primarily at codon 12.(ABSTRACT TRUNCATED AT 400 WORDS)
Cabbage, broccoli, Brussels sprouts, and other members of the genus Brassica have been widely regarded as potentially cancer preventative. This view is often based on both experimental testing of crude extracts and epidemiological data. The experimental evidence that provides support for this possibility is reviewed for the commonly consumed varieties of Brassica oleracea. In a majority of cases the biological activities seen in testing crude extracts may be directly related to specific chemicals that have been reported to be isolated from one of these closely related species, thus the chemical evidence further supports the data from testing extracts and epidemiology.
The metabolism of the carcinogenic mycotoxin aflatoxin B1 (AFB1) was examined in microsomes derived from human lymphoblastoid cell lines expressing transfected CYP1A2 or CYP3A4 complementary DNAs and in microsomes prepared from human liver donors (n = 4). Lymphoblast microsomes expressing only CYP1A2 activated AFB1 to AFB1-8,9-epoxide (AFB1-8,9-epoxide trapped as the glutathione, conjugate) at both 16 microM and 128 microM AFB1 concentrations, whereas activation of AFB1 to the epoxide in lymphoblast microsomes expressing only CYP3A4 was detected only at high substrate concentrations (128 microM AFB1). AFB1 epoxidation was strongly inhibited in CYP1A2 but not CYP3A4 lymphoblast microsomes pretreated with furafylline, a specific mechanism-based CYP1A2 inhibitor, whereas troleandomycin (TAO), a specific CYP3A inhibitor, strongly inhibited AFB1 epoxidation in CYP3A4 but not CYP1A2 microsomes. Formation of the hydroxylated metabolite aflatoxin M1 (AFM1) was observed only in the CYP1A2 microsomes whereas aflatoxin Q1 (AFQ1) production was observed exclusively in the CYP3A4 microsomes. Treatment of individual human liver microsomes (HLM) with TAO resulted in an average 20% inhibition of AFB1-8,9-epoxide formation at 16 microM AFB1, whereas incubation of HLM with furafylline at 16 microM AFB1 resulted in an average 72% inhibition of AFB1-8,9-epoxide formation at 16 microM AFB1. TAO was slightly more effective than furafylline in inhibiting AFB1 epoxidation at 128 microM AFB1 (46% inhibition by TAO, 32% inhibition by furafylline) in HLM. AFB1-8,9-epoxide formation was inhibited by 89% at low substrate concentration and 85% at high substrate concentrations when HLM were inhibited with a furafylline/TAO mixture. AFM1 formation was strongly inhibited by furafylline, whereas AFQ1 formation was strongly inhibited by TAO, in all HLM regardless of substrate concentration. Analysis of R-6- and R-10-hydroxywarfarin activities (respective markers of CYP1A2 and CYP3A4 activities) in the complementary DNA-expressed microsomes demonstrated that TAO was less effective than furafylline as a selective P450 isoenzyme inhibitor (60% inhibition of CYP3A4 by TAO as compared to 99% inhibition of CYP1A2 by furafylline). The rates of AFB1 epoxidation and AFQ1 formation in HLM were increased 7- and 18-fold, respectively, at high versus low substrate concentrations. These results are consistent with the hypothesis that CYP1A2 is the high-affinity P450 enzyme principally responsible for the bioactivation of AFB1 at low substrate concentrations associated with dietary exposure. CYP3A4 appears to have a relatively low affinity for AFB1 epoxidation and is primarily involved in AFB1 detoxification through AFQ1 formation in HLM.(ABSTRACT TRUNCATED AT 400 WORDS)
Two cDNA species, aggst1-5 and aggst1-6, comprising the entire coding region of two distinct glutathione S-transferases (GSTs) have been isolated from a 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) resistant strain (ZANDS) of Anopheles gambiae. The nucleotide sequences of these cDNA species share 80.2% identity and their derived amino acid sequences are 82.3% similar. They have been classified as insect class I GSTs on the basis of their high sequence similarity to class I GSTs from Drosophila melanogaster and Musca domestica and they are localized to a region of an An. gambiae chromosome known to contain further class I GSTs. The genes aggst1-5 and aggst1-6 were expressed at high levels in Escherichia coli and the recombinant GSTs were purified by affinity chromatography and characterized. Both agGST1-5 and agGST1-6 showed high activity with the substrates 1-chloro-2,4-dinitrobenzene and 1, 2-dichloro-4-nitrobenzene but negligible activity with the mammalian theta class substrates, 1,2-epoxy-3-(4-nitrophenoxy)propane and p-nitrophenyl bromide. Despite their high level of sequence identity, agGST1-5 and agGST1-6 displayed different kinetic properties. Both enzymes were able to metabolize DDT and were localized to a subset of GSTs that, from earlier biochemical studies, are known to be involved in insecticide resistance in An. gambiae. This subset of enzymes is one of three in which the DDT metabolism levels are elevated in resistant insects.
Cytosolic glutathione S-transferase (GST) isoenzymes from brain, heart, lung, liver, kidney and gonads of male and female CD-1 mice were identified and quantified with a combination of affinity purification, electrospray ionization MS, Edman microsequencing, Western blot analysis and reverse-phase HPLC. The three principal hepatic GST subunits, mGSTA3 (25271 Da), mGSTP1 (23478 Da), and mGSTM1 (25839 Da), were isolated from liver, lung, kidney, testes and female heart, whereas brain, ovaries and male heart contained mGSTM1 and mGSTP1. Additional isoenzymes were detected in tissues, including mu class subunits mGSTM2 (25580 Da) and mGSTM3 (25570 Da), an N-terminally blocked Alpha subunit (25480 Da) assigned as mGSTA4, and proteins of molecular masses 25490, 22540, 24493, 24378 and 25383 Da. Distinct gender differences in expression of GST subunits were observed for liver, heart, kidney and gonads, whereas GST expression was similar in brain and lung for both genders. In contrast with patterns of expression in liver (high ratio of mGSTA3 to mGSTP1 in females relative to males), mGSTP1 was the most abundant subunit in female gonads, whereas mGSTA3 was not present in detectable quantities. The profile of GST expression in kidney was similar to that in liver; however, male kidneys expressed 30% more soluble GST than female kidneys. A striking gender-related difference in GST expression was found in cardiac tissue, where female animals expressed 50% more soluble GST than male tissues, and the GST isoenzyme with the greatest documented activity towards lipid hydroperoxides, mGSTA3, was present in female tissue yet absent from male tissue. These results point to complex gender- and tissue-dependent expression of individual mouse GST isoenzymes.
The mycotoxin aflatoxin B(1) (AFB(1)) is a hepatocarcinogen in many animal models and probably a human carcinogen. Besides being a dietary carcinogen, AFB(1) has been detected in dusts generated in the processing and transportation of AFB(1)-contaminated products. Inhalation of grain dusts contaminated with AFB(1) may be a risk factor in human lung cancer. Aflatoxin B(1) requires cytochrome P-450 (CYP)-mediated activation to form cytotoxic and DNA-reactive intermediates, and this activation in human liver is mediated by the CYP 1A2 and 3A4 isoforms. Which isoforms are important in AFB(1) activation in human lung is not well understood. To investigate whether these CYPs can activate AFB(1) at low, environmentally relevant concentrations in human lung cells, SV40 immortalized human bronchial epithelial cells (BEAS-2B) that were transfected with cDNA for CYPs 3A4 (B3A4) or 1A2 (B-CMV1A2) were used. B-CMV1A2 cultured in 15 nM AFB(1) produced the AFB(1)-glutathione conjugate (AFB(1)-GSH) and aflatoxin M(1) (AFM(1)), while B3A4 cells produced only aflatoxin Q(1) (AFQ(1)) at 0.15 microM AFB(1). Nontransfected BEAS-2B cells produced no metabolites, even at 1.5 mM AFB(1). Microsomes prepared from B-CMV1A2 and B3A4 cells activated AFB(1) to AFB(1) 8,9-epoxide (AFBO), while those from BEAS-2B cells did not produce AFBO. Cytosol from all three cell types was ineffective at glutathione S-transferase (GST)-mediated trapping of enzymatically generated AFB(1) 8,9-epoxide. B-CMV1A2 cells were 100-fold more sensitive to AFB(1) compared to B3A4 cells, and were 6000-fold more sensitive than control BEAS-2B cells. Western immunoblots confirmed that only B-CMV1A2 cells expressed CYP 1A2 protein, while CYP 3A4 was only in B3A4 cells. B-CMV1A2 cells were the most sensitive to AFB(1), followed by B3A4 cells. CYP 3A4, which has been predicted to activate AFB(1) primarily at higher AFB(1) concentrations, was also responsible for significant AFB(1) toxicity at low concentrations. These data indicate that human lung cells expressing these CYP isoforms are capable of activating AFB(1), even at environmentally relevant concentrations.
Hepatocellular carcinoma (HCC) is one of the major cancers in the world. There is a striking variation in HCC incidence rates between various countries, with a highest-to-lowest ratio of 112.5 for males and 54.7 for females. The high-risk populations are clustered in sub-Saharan Africa and eastern Asia. The male-to-female ratio for HCC ranges from < 1 to 6.4 and mostly from 2 to 4. There exist significant variations in the incidence of HCC among different ethnic groups living in the same area and among migrants of the same ethnic groups living in different areas. The age curves of HCC are significantly different in various countries, suggesting variability in exposure to risk factors. Chronic carriers of hepatitis B and C viruses (HBV and HCV, respectively) have an increased risk of HCC. The relative and attributable HCC risk of HBV and HCV carrier status varies in different countries. There exists a synergistic interaction on HCC between the two viruses. Aflatoxin exposure, cigarette smoking, heavy alcohol consumption, low vegetable intake, inorganic arsenic ingestion, radioactive thorium dioxide exposure, iron overload and the use of oral contraceptives and anabolic steroids have been documented as HCC risk factors. Recent molecular epidemiological studies have shown that low serum retinol levels as well as elevated serum levels of testosterone, neu oncoprotein and aflatoxin B1-albumin adduct are associated with an increased HCC risk. There is a synergistic interaction on HCC between chronic HBV infection and aflatoxin exposure. Familial aggregation of HCC exists and a major susceptibility gene of HCC has been hypothesized. Patients of some genetic diseases are at an increased risk of HCC. The genetic polymorphisms of cytochrome P450 2E1 and 2D6 and arylamine N-acetyltransferase 2 are associated with the development of HCC. A dose-response relationship between aflatoxin exposure and HCC has been observed among chronic HBV carriers who have null genotypes of glutathione S-transferase M1 or T1, but not among those who have non-null genotypes. Human hepatocarcinogenesis is a multistage process with the involvement of a multifactorial aetiology. Gene-environment interactions are involved in the development of HCC in humans.
Large species differences exist in sensitivity to aflatoxin B, (AFB 1 )-induced liver cancer. Mice are resistant to AFB 1 -induced liver cancer because they express an alpha-class GST (mGSTA3-3) that has high activity toward the reactive intermediate aflatoxin B 1 -8,9-epoxide (AFBO). Rats constitutively express only small amounts of a GST with high AFBO activity (rGSTA5-5) and thus are sensitive to AFB 1 -induced hepatocarcinogenesis, although induction of rGSTA5-5 can confer resistance in rats. In contrast to rodents, constitutively expressed human hepatic alpha-class GSTs have little or no AFBO detoxifying activity. Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), has significant constitutive hepatic GST activity toward AFBO and most of this activity belongs to mu-class GSTs. To determine if any alpha-class GSTs in Mf liver have AFBO activity, a cDNA library from a male Mf liver was constructed and screened using the human alpha-class GstAl cDNA as a probe. Three different cDNA clones with full-length open reading frames were identified from the Mf hepatic cDNA library. Analyses of the cDNA deduced protein sequences indicated that these three alpha-class cDNA clones were 97-98% homologous with each other, and shared 93, 95, and 95% identity with human GSTA1, and were named mfaGSTAl, mfaGSTA2, and mfaGSTA3, respectively. Bacterially expressed mfaGSTA1-1 recombinant protein had similar activities toward classic GST substrates such as DCNB, CHP, and ECA, but slightly lower CDNB conjugating activity relative to human GSTA1-1. However, similar to hGSTA1-1, mfaGSTA1-1 had no AFBO conjugating activity. In addition, similar to human GSTA1 gene, cDNA-derived amino acid sequence analyses demonstrated that all of these Mf alpha-class GSTs genes (mfaGSTA1, mfaGSTA2, and mfaGSTA3) had none of the six critical residues that were identified previously to confer high AFBO activity in mouse alpha-class GSTA3-3. Thus, in contrast to rodents but similar to humans, alpha-class GSTs from the nonhuman primate, Mf, have little conjugating activity toward AFBO.
Aflatoxins have been extensively studied with respect to their mechanisms of toxicity. An understanding of metabolism, DNA adduct induction, mutagenicity and carcinogenicity has been paralleled by the development of biomarkers of aflatoxin exposure and biological effects (e.g. mutations) applied to human populations. The improvements in exposure assessment and their application in prospective epidemiological studies and the demonstration of a specific mutation in the TP53 gene in hepatocellular carcinomas from areas of high aflatoxin exposure have contributed significantly to the classification of aflatoxins as human carcinogens. In addition to establishing the carcinogenicity of aflatoxins in humans, understanding molecular mechanisms of action has provided the scientific rationale for prevention strategies, including primary and chemoprevention approaches. Overall, integrated, multidisciplinary research on aflatoxins has provided the platform on which to base decisions regarding acceptable exposures and priorities for interventions to reduce human risk in a public health context.
Glucoraphanin in Brassica vegetables breaks down to either sulforaphane or sulforaphane nitrile depending on the conditions, and sulforaphane can be further conjugated with glutathione. Using a high-throughput microtitre plate assay and TaqMan real time quantitative RT-PCR to measure mRNA, we show that sulforaphane and its glutathione conjugate, but not the nitrile, increased significantly (P < 0.05) both UGT1A1 and GSTA1 mRNA levels in HepG2 and HT29 cells. These changes were accompanied by an increase in UGT1A1 protein, as assessed by immunoblotting, and a 2‐8-fold increase in bilirubin glucuronidation. When treated together, the nitrile derivative did not affect sulforaphane induction. The induction of UGT1A1 and GSTA1 mRNA by sulforaphane was time and concentration dependent. The results show a functional induction of glucuronidation by sulforaphane but not sulforaphane nitrile, and show that the pathway of metabolism of glucosinolates in Brassica vegetables is important in determining the resulting biological and anticarcinogenic activities.
Isothiocyanates (ITCs) are a group of naturally occurring compounds that occur as thioglucoside conjugates, termed glucosinolates, in plants and cruciferous vegetables such as watercress, Brussels sprouts, broccoli, cabbage, kai choi, kale, horseradish, radish and turnip. ITCs inhibit the development of tumors in many of the experimental models investigated, and are being investigated as possible chemopreventive agents for specific human cancers. The goal of this review is to provide a mechanistic understanding for the biological activities of ITCs and to relate the metabolism of ITCs to their action as chemopreventive agents. In vivo animal studies have been conducted to address issues of tissue disposition, pharmacokinetics, and metabolism of ITCs. Methods for analysis of ITCs and their metabolites in urine and plasma have been developed. The metabolism of several naturally occurring ITCs as constituents of foodstuffs or as drugs has also been investigated in human studies. Finally, based on recent epidemiological studies, the role of dietary consumption of vegetables containing ITCs in prevention of human cancers and human cancer susceptibility is discussed.
Aflatoxin Q1 8,9-oxide was synthesized and found to yield lower levels of N7-guanyl adducts than obtained from aflatoxin B1 8,9-oxide when mixed with calf thymus DNA or Salmonella typhimurium TA 98 cells. However, when S. typhimurium TA 98 was treated with the (analogous) epoxides of aflatoxin B1, aflatoxin G1, aflatoxin Q1, or sterigmatocystin, the ratios of revertants to N7-guanyl DNA adducts were similar. Aflatoxin Q1 and aflatoxin B1 8,9-oxide (trapped here as the glutathione conjugate) are the major oxidative products formed from aflatoxin B1 at all substrate concentrations in human liver microsomes, and cytochrome P-450 (P-450) 3A4 appears to be the dominant enzyme involved in both oxidations, as judged by studies involving correlation of activities in different liver samples, chemical inhibition, immunoinhibition, and reconstitution with purified hepatic and yeast recombinant P-450 3A4. Aflatoxin Q1 is not appreciably oxidized in human liver microsomes and is not very genotoxic. The postulated formation of both aflatoxin Q1 and aflatoxin 8,9-oxide from aflatoxin B1 can be rationalized by a model in which P-450 3A4 binds the substrate in either of two different configurations. This is further demonstrated by the dichotomous effect of 7,8-benzoflavone--this flavone stimulates 8,9-epoxidation while inhibiting the 3 alpha-hydroxylation reaction to form aflatoxin Q1. Thus, the 3 alpha-hydroxylation of aflatoxin B1 to aflatoxin Q1 is viewed as a potentially significant detoxication pathway.
Hepatocellular carcinoma (HCC) is one of the major cancers in the world. There is a striking variation in HCC incidence rates between various countries, with a highest‐to‐lowest ratio of 112.5 for males and 54.7 for females. The high‐risk populations are clustered in sub‐Saharan Africa and eastern Asia. The male‐to‐female ratio for HCC ranges from < 1 to 6.4 and mostly from 2 to 4. There exist significant variations in the incidence of HCC among different ethnic groups living in the same area and among migrants of the same ethnic groups living in different areas. The age curves of HCC are significantly different in various countries, suggesting variability in exposure to risk factors. Chronic carriers of hepatitis B and C viruses (HBV and HCV, respectively) have an increased risk of HCC. The relative and attributable HCC risk of HBV and HCV carrier status varies in different countries. There exists a synergistic interaction on HCC between the two viruses. Aflatoxin exposure, cigarette smoking, heavy alcohol consumption, low vegetable intake, inorganic arsenic ingestion, radioactive thorium dioxide exposure, iron overload and the use of oral contraceptives and anabolic steroids have been documented as HCC risk factors. Recent molecular epidemiological studies have shown that low serum retinol levels as well as elevated serum levels of testosterone, neu oncoprotein and aflatoxin B1‐albumin adduct are associated with an increased HCC risk. There is a synergistic interaction on HCC between chronic HBV infection and aflatoxin exposure. Familial aggregation of HCC exists and a major susceptibility gene of HCC has been hypothesized. Patients of some genetic diseases are at an increased risk of HCC. The genetic polymorphisms of cytochrome P450 2E1 and 2D6 and arylamine N‐acetyltransferase 2 are associated with the development of HCC. A doseresponse relationship between aflatoxin exposure and HCC has been observed among chronic HBV carriers who have null genotypes of glutathione S‐transferase M1 or T1, but not among those who have non‐null genotypes. Human hepatocarcinogenesis is a multistage process with the involvement of a multifactorial aetiology. Gene‐environment interactions are involved in the development of HCC in humans.
Based on our previous observations (H. S. Ramsdell and D. L. Eaton, 1990, Cancer Res., 50, 615–620) that the proportion of aflatoxin B1 (AFB1) converted to the highly reactive AFB1-8,9-epoxide in microsomal incubations varies with substrate concentration, we have examined the hypothesis of T. Shimada and F. P. Guengerich (1989, Proc. Natl. Acad. Sci. USA, 86, 462–465) that cytochrome P450 IIIA4 is principally responsible for the activation (epoxidation) of AFB1 by human liver microsomes. The initial rates of formation of AFB1-8,9-epoxide and hydroxylated AFB1 metabolites were determined in microsomes prepared from livers of organ donors (n = 14) at AFB1 concentrations of 124 and 16 μm. Microsomal oxidation of nifedipine, catalyzed primarily by P450 IIIA enzymes, was also determined by HPLC. Rates of formation of AFB1 metabolites and nifedipine oxidation were poorly correlated at either AFB1 concentration (r2 = 0.13–0.41). A somewhat better correlation between AFB1 epoxidation and nifedipine oxidation was observed at 124 μm AFB1 (r2 = 0.41) than at 16 μm AFB1 (r2 = 0.26). Treatment of pooled microsomes with troleandomycin, an apparently specific inhibitor of P450 IIIA enzymes, resulted in 35% inhibition of AFB1-8,9-epoxide formation at the high AFB1 level but had little effect at 16 μm AFB1. An antibody against rat cytochrome P450 IIIA1 significantly inhibited AFB1 epoxidation at high, but not low, AFB1 concentrations, whereas AFQ1 formation was strongly inhibited at all substrate levels examined. These results are consistent with the hypothesis that cytochrome P450 IIIA enzyme(s) can form AFB1-8,9-epoxide, but are effective at only relatively high substrate concentrations. Another P450 enzyme(s) appears to be principally responsible for AFB1-8,9-epoxide formation at the low AFB1 levels that would be typical for dietary exposures.
Mice constitutively express glutathioneS-transferase mGSTA3-3 in liver. This isoform possesses uniquely high conjugating activity toward aflatoxin B1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B1-induced hepatocarcinogenicity. In contrast, rats constitutively express a closely related GST isoenzyme, rGSTA3-3, with low AFBO activity and, therefore, are sensitive to aflatoxin B1exposure. Although the two GSTs share 86% sequence identity and have similar catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approximately 1000-fold difference in catalytic activity toward AFBO. To identify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site. Twenty-one mutant rGSTA3-3 enzymes were generated by site-directed mutagenesis using combinations of nine different residues. Except for the E208D mutant, single mutations of rGSTA3-3 produced enzymes with no detectable AFBO activity. Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the six site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the highest AFBO activity (40 nmol/mg/min) of all the mutant enzymes. When this mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2.8 nmol/mg/min. Although wild-type mGSTA3-3 AFBO conjugation activity (265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we were able to identify six mGSTA3-3 residues; Ile104, Tyr108, His111, Phe207, Asp208, and Lys217that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjugation activity toward AFBO.
Lung cancer, a leading cause of cancer death, is associated with exposure to inhalation carcinogens, most commonly those found in tobacco smoke. We investigated the in vivo effect of dietary supplementation with a nutrient mixture containing lysine, proline, arginine, ascorbic acid, green tea extract, N-acetyl cysteine, selenium, copper and manganese on the development of urethane-induced lung tumors in male A/J mice.
After one week of isolation, seven-week-old male A/J mice (n = 25) weighing 17-19 g were randomly divided into three groups: group A (n = 5), group B (n = 10), and group C (n = 10). Mice in groups B and C were each given a single intraperitoneal injection of urethane (1 mg/g body weight) in saline, whereas group A mice received an injection of saline alone. Groups A and B were fed a regular diet, whereas group C was fed the same diet supplemented with 0.5% nutrient mixture. After 20 weeks, mice were sacrificed, lungs were excised and weighed, and tumors were counted and processed for histology.
Urethane-challenged mice developed tumors. However, the mean number of tumors and the mean lung weights in the mice on the supplemented diet were significantly reduced, by 49% (P < 0.0001) and 18% (P = 0.0025), respectively, compared to mice on the control diet. We observed neither significant differences in body weight gains nor in diet consumption among the mice. Pulmonary lesions were morphologically similar for both the groups (adenomas), but lesions were smaller in the test group.
The results suggest that nutrient mixture has inhibitory potential on the development of mouse lung tumors induced by urethane.
Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B 1 (AFB 1 ) because they constitutively express an alpha -class glutathione S -transferase (mGSTA3-3) that has high (∼200,000 pmol/min/mg) activity toward aflatoxin B 1 -8,9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB 1 -induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha- class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (∼300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, Mf GSTs were purified from liver tissue and characterized and, Mf mu- class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu -class GST cDNAs, mfaGSTM1 (GenBank accession # AF200709) and mfaGSTM2 (GenBank accession # AF200710), were generated by RT-PCR. CDNA-derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu -class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo - and endo -AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu -class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis .
On a global basis, primary liver cancer (PLC) is a very prevalent form of cancer. Wide variation of PLC incidence in different areas of the world suggests the involvement of environmental factors in its etiology. Two major classes of risk factors have been identified. Extensive evidence indicates the importance of infection by the hepatitis B virus as a major risk factor for PLC. Because many organic chemicals induce liver cancer in experimental animals, those to which human exposure is known to occur are also of interest with respect to their possible involvement as risk factors for PLC. Particular emphasis has been placed on aflatoxins because of the frequency with which they occur as food contaminants, together with their potency as liver carcinogens for a large number of experimental animals, including subhuman primates. Other mycotoxins, notably sterigmatocystin and fumonisin, also are relatively potent carcinogens for the liver of animals, but little is known about human exposure to them. Epidemiological surveys carried out over the past 25 years in Asia and Africa have revealed a strong statistical association between aflatoxin ingestion and PLC incidence. The combined experimental and epidemiological evidence has led to designation of aflatoxins as human carcinogens according to International Agency for Cancer Research criteria. Collectively, current evidence strongly suggests that PLC is of multifactorial origin, with probable interactions between viral and chemical agents in populations concurrently exposed to both classes of risk factors. Recently developed methods that permit individual monitoring of aflatoxin exposure, hepatitis B virus infection, and genetic damage caused by these agents are being applied in the design of molecular and biochemical epidemiological studies of the etiology of the disease. Application of this methodology may contribute to elucidation of the relative importance of interacting etiological agents in different populations.
Much evidence supports the view that the rate of conjugation of glutathione (GSH) with aflatoxin B1 (AFB1) exo-epoxide is an important factor in determining the species variation in risk to aflatoxins and that induction of GSH S-transferases can yield a significant protective effect. An assay has been developed in which the enzymatic formation of the conjugates of GSH and AFB1 exo-epoxide and the recently described AFB1 endo-epoxide is measured directly. 1H NMR spectra are reported for both the AFB1 exo- and endo-epoxide-GSH conjugates. Structural assignments were made by comparison with AFB1 exo- and endo-epoxide-ethanethiol conjugates, for which nuclear Overhauser effects were measured to establish relative configurations. The endo-epoxide was found to be a good substrate for GSH conjugate formation in rat liver cytosol while mouse liver cytosol conjugated the exo-epoxide almost exclusively. Human liver cytosol conjugated both epoxide isomers to much lower extents than did cytosols prepared from rats or mice. Purified rat GSH S-transferases catalyzed the formation of the AFB1 exo-epoxide-GSH conjugate in the order 1-1 approximately 4-4 approximately 3-3 greater than 2-2 greater than 4-6 (7-7 and 8-8 did not form the exo-epoxide-GSH conjugate at levels above the nonenzymatic rate). The only rat GSH S-transferases that conjugated the endo-epoxide were 4-4 and 4-6, with 4-4 being the more active.(ABSTRACT TRUNCATED AT 250 WORDS)
The complementary DNAs of rat glutathione S-transferase (GST, EC 2.5.1.18) Yc1 and of mouse Yc were expressed from a prokaryotic expression vector in E. coli. The purified proteins were analyzed for their activity toward aflatoxin B1-8,9-epoxide (AFBO), the reactive intermediate of the fungal mycotoxin aflatoxin B1 (AFB). The mouse Yc isozyme had about 50-fold higher conjugating activity toward AFBO than the rat Yc1 isozyme (144 nmol/mg/min versus 3.3 nmol/mg/min). The rat Yc1 isozyme had specific activities toward 1-chloro-2,4-dinitrobenzene, cumene hydroperoxide and ethacrynic acid of 10.7, 0.98 and 0.92 mumol/mg/min, respectively, whereas the mouse Yc isozyme had specific activities of 5.7, 2.1 and 0.1 mumol/mg/min for these substrates, respectively. These data provide further support for the hypothesis that the constitutive presence of the alpha class GST Yc isozyme in mouse liver protects mice from the hepatocarcinogenic effects of aflatoxin B1.
Consumption of vegetables, especially crucifers, reduces the risk of developing cancer. Although the mechanisms of this protection are unclear, feeding of vegetables induces enzymes of xenobiotic metabolism and thereby accelerates the metabolic disposal of xenobiotics. Induction of phase II detoxication enzymes, such as quinone reductase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] and glutathione S-transferases (EC 2.5.1.18) in rodent tissues affords protection against carcinogens and other toxic electrophiles. To determine whether enzyme induction is responsible for the protective properties of vegetables in humans requires isolation of enzyme inducers from these sources. By monitoring quinone reductase induction in cultured murine hepatoma cells as the biological assay, we have isolated and identified (-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane [CH3-SO-(CH2)4-NCS, sulforaphane] as a major and very potent phase II enzyme inducer in SAGA broccoli (Brassica oleracea italica). Sulforaphane is a monofunctional inducer, like other anticarcinogenic isothiocyanates, and induces phase II enzymes selectively without the induction of aryl hydrocarbon receptor-dependent cytochromes P-450 (phase I enzymes). To elucidate the structural features responsible for the high inducer potency of sulforaphane, we synthesized racemic sulforaphane and analogues differing in the oxidation state of sulfur and the number of methylene groups: CH3-SOm-(CH2)n-NCS, where m = 0, 1, or 2 and n = 3, 4, or 5, and measured their inducer potencies in murine hepatoma cells. Sulforaphane is the most potent inducer, and the presence of oxygen on sulfur enhances potency. Sulforaphane and its sulfide and sulfone analogues induced both quinone reductase and glutathione transferase activities in several mouse tissues. The induction of detoxication enzymes by sulforaphane may be a significant component of the anticarcinogenic action of broccoli.
As part of the studies of the biochemical basis for species differences in biotransformation of the carcinogen aflatoxin B1 (AFB1) and its modulation by phenolic antioxidants, we have investigated the role of mouse liver glutathione S-transferase (GST) isoenzymes in the conjugation of AFB1-8,9-epoxide. Isoenzymes of GST were purified to electrophoretic homogeneity from Swiss-Webster mouse liver cytosol by affinity chromatography and chromatofocusing. The isoenzyme fractions were characterized in terms of activity toward surrogate substrates and immunologic cross-reactivity with antisera to rat GSTs. The major isoenzymes were identified as SW 4-4, SW 3-3, and SW 1-1. The specific activity of SW 4-4 toward AFB1-8,9-epoxide was at least 50- and 150-fold greater than that of SW 3-3 and SW 1-1, respectively. Relatively high activity toward another epoxide carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, was observed with both SW 4-4 and SW 3-3. SW 1-1 had the highest activity toward 1-chloro-2,4-dinitrobenzene (CDNB) whereas SW 4-4 had relatively low CDNB activity. Following pretreatment with 0.75% butylated hydroxyanisole in the diet, the fraction of total GST contributed by SW 1-1 appeared to increase dramatically, whereas in control mice SW 3-3 constituted the predominant isoenzyme. The high GST activity of mouse liver cytosol toward AFB1-8,9-epoxide is apparently due to an isoenzyme that contributes little to the overall cytosolic CDNB activity.
Free radicals have a number of toxic effects on the cell including the initiation of lipid peroxidation (Pryor, 1976) and the induction of potentially mutagenic lesions in DNA (Ames and Saul, 1986). Current knowledge suggests that some of the lesions in DNA may be peroxides. Irradiation of free thymidine gives a substantial yield of thymine hydroperoxides (Schulte Frohlinde and von Sonntag, 1985) and the incubation of irradiated DNA with GSH and tissue extracts containing GSH peroxidase activity brings about the catalytic oxidation of GSH to GSSG which is assumed to be due to the reduction of hydroperoxide moieties in DNA by GSH (Christopherson, 1969).
Rat and hamster liver cytosolic glutathione (GSH) S-transferases purified by GSH-affinity chromatography have been examined for their effects on the microsome mediated binding of aflatoxin B1 (AFB1) to DNA and on the conjugation of AFB1-2,3-epoxide with GSH. Like previous studies with cytosolic preparations (Raj et al. (1984) Carcinogenesis 5, 879), our present study with purified GSH S-transferases showed 2-3-fold more inhibitory activity of AFB1-DNA binding with hamster than that with the rat. Concomitant with the inhibition of AFB1-DNA binding, increase in AFB1-GSH conjugation occurred. Subunit compositions of GSH S-transferases indicate preponderance of Yb and Ya subunits in the hamster and rat, respectively. The role of GSH S-transferases in modulating AFB1-DNA binding and AFB1 induced hepatocarcinogenesis is discussed.
Glutathione-S-transferase (GST) activity has been examined in liver cytosol fractions from guinea pigs, mice, control fed rats or rats with pre-neoplastic nodular liver lesions. The levels of activity in unfractionated cytosols have been assayed using the model substrates 1-chloro-2,4-dinitrobenzene (CDNB), 3,4-dichloronitrobenzene (DCNB) and monobromobimane (mBrB) with reduced glutathione (GSH). The order of activities in the various liver fractions using CDNB as substrate were: mouse greater than pre-neoplastic nodular rat greater than guinea pig greater than control rat and paralleled the capacities of the cytosols to catalyse the formation of aflatoxin B1-GSH from microsomally-activated aflatoxin B1 (AFB1) and GSH. Quantitative differences between the activities of the cytosols using the three model substrates were observed. In the mouse fractionation of GST activity by isoelectric focusing (I.E.F.) on preparative granular gels showed that the most basic component (isoelectric point pH 9.4) with the highest conjugating activity with respect to microsomally-activated AFB1 did not correspond with the peak of most activity for conjugating CDNB. In the pre-neoplastic nodular rat liver the CDNB conjugating activities of all fractions separated on granular I.E.F. gels, were higher than the corresponding fractions isolated from control rat liver, with particular enhancement of the peak containing the 3:3 isoenzyme. In contrast to control rat liver the 7:7 isoenzyme was detected in pre-neoplastic nodular liver preparations. These isoenzymes (3:3 and 7:7) did not contribute significantly to the enhanced level of AFB1-GSH formation catalysed by cytosol fractions prepared from pre-neoplastic nodular rat liver. The microsomally-activated AFB1-conjugating activity of unfractionated rat liver cytosols was increased to a relatively greater extent than CDNB conjugating activity during the induction of pre-neoplastic nodular liver lesions, and the elevated level of the activated AFB1-conjugating activity was found to be associated with the most basic fraction (isoelectric point pH 9.0). Analytical isoelectric focusing gels using mBrB as substrate demonstrated the presence of a basic GST isoenzyme in the pre-neoplastic nodular rat liver, not detected in preparations from the livers of control rats. The low level of activated AFB1-conjugating activity present in unfractionated guinea-pig cytosol was found to correspond with the fraction containing the peak of CDNB conjugating activity on preparative isoelectric focusing (isoelectric point pH 7.5). The lack of correlation between the conjugation of model substrates and the conjugation of xenobiotics could be of import
The glutathione S-transferases (GSTs) of hepatic cytosol of rats, mice, guinea pigs, rabbits and hamsters were simultaneously investigated with respect to substrate specificity, subunit composition and elution profile of GSTs. The activity towards 1-chloro-2,4-dinitrobenzene was the highest in hamsters, followed by rabbits, guinea pigs, mice and rats. On the analysis of SDS-PAGE, Ya subunit band of GST was detected in only rats, not others. There was also seen a marked species difference in elution profile of GST activity on S-sepharose column. The elution patterns from rats, mice and rabbits were quite different from hamsters and guinea pigs.
Aflatoxin B1 (AFB1)-8,9-oxide, the proposed ultimate carcinogen is conjugated enzymically with glutathione (GSH) to give 8-S-glutathionyl)-9-hydroxy-8,9-dihydro
AFB1 (AFB1-SG). The GSH conjugate isolated from rat bile was shown, on the basis of 1 H n. m. r. to be identical to AFB1 -SG. Of the seven soluble rat liver GSH transferases 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 5-5 (see reference 1 for the new system
of nomenclature), only the first three were active with microsomally generated AFB1 -88, 9-oxide, their rates of conjugation being 1.1, 0.61, and 0.64 nmol/min/mg enzyme, respectively. AFB1 -SG is a thioacetal, but it was not formed from the incubation of the hemiacetal, AFB1 -8,9-dihydrodiiol, with GSH or GSH plus GSH transferase 1-1 plus 1-2. The covalent binding of in vitro microsomally activated AFB1 to DNA and the formation of AFB1-SG were linearly related to AFB1concentration in range of 0.2–2μg/ml. DNA binding was decreased by 38% by the competing formation of AFB1-SG throughout this range of concentrations. These results are in accord with the observation of Scott Appleton et al. (Cances Res., 42, 3629-3662) that, in the rat in vivo, there is no evident threshold for the binding of AGB1 to DNA. These findings are also consistent with further observation, reported in this paper that GSH and GSH transferases
have no effectn on the mutagenicity of microsomally activated AFB1 to Salmonella typhimurium TA 100.
The formation of an aflatoxin B1-reduced glutathione (AFB1-GSH) conjugate in in vitro systems has been examined. AFB1 was activated by a chicken liver microsomal system and factors affecting the subsequent conversion to the AFB1-dihydrodiol or conjugation with GSH were investigated by HPLC. A requirement for glutathione S-transferase in the formation of the AFB1-GSH conjugate was observed. Studies using CM-cellulose columns showed the fractions containing glutathione S-transferase B activity were the most effective in catalysing the formation of the AFB1-GSH conjugate. The possibility of changes in the level of AFB1-GSH conjugate production in the liver during carcinogenesis by AFB1 has been examined. It has been found, using freshly isolated rat hepatocytes, that low level feeding with AFB1 in vivo increases the production of the conjugate in vitro. Further increases in the production of the conjugate by hepatocytes in vitro, accompanying increases in the preneoplastic lesions, are achieved by partially hepatectomising the AFB1-fed animals. Partial hepatectomy of control-fed animals yielded no similar changes. The AFB1/partial hepatectomy treatment resulted in increased levels of all the glutathione S-transferase activities fractionated on CM-cellulose. Macromolecular binding of AFB1 and/or of its metabolites was detected in the fractions containing glutathione S-transferase activity, but there was no evidence for a greater binding in the glutathione S-transferase B/ligandin containing fractions. Furthermore fractionation on Sephadex G-75 indicated a predominance of binding of AFB1 to proteins of a higher molecular weight than the glutathione S-transferases, although some binding in the molecular weight range of the latter was observed.
Ethoxyquin (EQ), a widely used antioxidant, inhibits the carcinogenic effects of polycyclic aromatic hydrocarbons. The aim of the present study was to determine whether EQ modifies the hepatocarcinogenic effects of aflatoxin B1 (AFB1) in rats. Both compounds were administered in the diet. Rats were fed either EQ for 2 weeks and then AFB1 for 6 weeks, EQ and AFB1 simultaneously or EQ following the cessation of AFB1 treatment. The results indicate that EQ can inhibit the hepatocarcinogenic effects of AFB1 and that the most effective inhibition is obtained when EQ and AFB1 are given simultaneously.
The separation of aflatoxin B2a, and 2,3-dihydroxy-2,3-dihydro-aflatoxin B1 (AFB1-dhd) and the Tris complexes of these compouds by high-performance liquid chromatography are described. Examination of the metabolism of aflatoxin B1 by microsomes isolated from rat, mouse, guinea pig, and chicken livers has shown that the previously reported production of AFB2a was due to a misidentification of AFB1-dhd. The relative production of this metabolite by microsomes from the various species parallels their in vivo susceptibilities to acute aflatoxin B1 poisoning. AFB1-dhd is shown to be a potent inhibitor of protein synthesis in an in vitro system and this may be a mechanism relevant to the acutely toxic action of aflatoxin B1.
The importance of thiol-mediated detoxification of anticancer drugs that produce toxic electrophiles has been of considerable interest to many investigators. Glutathione and glutathione S-transferases (GST) are the focus of much attention in characterizing drug resistant cells. However, ambiguous and sometimes conflicting data have complicated the field. This article attempts to clarify some of the confusion. The following observations are well established: (a) tumors express high levels of GST, especially GST psi, although the isozyme components vary quite markedly between tissues and the isozymes are inducible; (b) nitrogen mustards are good substrates for the GST alpha family of isozymes which are frequently overexpressed in cells with acquired resistance to these drugs; (c) most drugs of the multidrug-resistant phenotype have not been shown to be GST substrates and although GST psi is frequently overexpressed in multidrug-resistant cells, most indications are that this is an accompaniment to, rather than a cause of, the resistant phenotype; (d) transfection of GST complementary DNAs has produced some lines with increased resistance to alkylating agents. Most studies of the relationships between GST and resistance have overlooked the potential importance of other enzymes involved in the maintenance of cellular glutathione homeostasis, and this has complicated data interpretation. Translational research aimed at applying our knowledge of glutathione pathways has produced preclinical and clinical testing of some glutathione and GST inhibitors, with some encouraging preliminary results. In brief, GSTs are important determinants of drug response for some, not all, anticancer drugs. Caution should be encouraged in assessing cause/effect relationships between GST overexpression and resistance mechanisms.
Mammalian cells have evolved elaborate mechanisms for protection against the toxic and neoplastic effects of electrophilic metabolites of carcinogens and reactive oxygen species. Phase 2 enzymes (e.g. glutathione transferase, NAD(P)H:quinone reductase, UDP-glucuronosyltransferases) and high intracellular levels of glutathione play a prominent role in providing such protection. Phase 2 enzymes are transcriptionally induced by low concentrations of a wide variety of chemical agents and such induction blocks chemical carcinogenesis. The inducers belong to many chemical classes including phenolic antioxidants. Michael reaction acceptors, isothiocyanates, 1,2-dithiole-3-thiones, trivalent arsenicals, HgCl2 and organomercurials, hydroperoxides, and vicinal dimercaptans. Induction by all classes of inducers involves the antioxidant/electrophile response element (ARE/EpRE). Inducers are widely, but unequally, distributed among edible plants. Search for such inducer activity in broccoli led to the isolation of sulforaphane, an isothiocyanate that is a very potent Phase 2 enzyme inducer and blocks mammary tumor formation in rats.
The combined presence of CYP1A2 and 3A4, both of which oxidize aflatoxin B1 (AFB1) to the reactive aflatoxin B1-8,9-epoxide (AFBO) and to hydroxylated inactivation products aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), substantially complicates the kinetic analysis of AFB1 oxidation in human liver microsomes. In the present study, we examine the reaction kinetics of AFB1 oxidation in human liver microsomes (HLMs, N = 3) and in human CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the purpose of identifying the CYP isoform(s) responsible for AFB1 oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis-Menten kinetics (Km = 41 microM, Vmax = 2.63 nmol/min/nmol P450). Furthermore, the portion of AFBO formed in HLMs which was eliminated by furafylline, a specific mechanism-based inhibitor of CYP1A2, also followed Michaelis-Menten kinetics (Km = 32-47 microM, Vmax = 0.36-0.69 nmol/min/nmol P450). The formation of AFBO (activation product) and AFQ1 (detoxification product) in cDNA-expressed human CYP3A4 microsomes was sigmoidal and consistent with the kinetics of substrate activation. Accordingly, application of a sigmoid Vmax model equivalent to the Hill equation produced excellent fits to the cDNA-expressed CYP3A4 data and also to the data from HLMs pretreated with furafylline to remove CYP1A2. The Hill model predicted that two substrate binding sites are involved in CYP3A4-mediated AFB1 catalysis and that the average affinity of AFB1 for the two sites was 140-180 microM. Vmax values for AFQ1 formation were 10-fold greater than those for AFBO, and total substrate turnover to both was 67 nmol/min/nmol CYP3A4. Using the derived kinetic parameters for CYP1A2 and 3A4 to model the in vitro rates of AFB activation at low substrate concentrations, it was predicted that CYP1A2 contributes to over 95% of AFB activation in human liver microsomes at 0.1 microM AFB. The important role of CYP1A2 in the in vitro activation of AFB at low substrate concentrations was supported by DNA binding studies. AFB1-DNA binding in control HLMs (reflecting the contribution of CYP1A2 and CYP3A4) and furafylline-pretreated microsomes (reflecting the contribution of CYP3A4 only) catalyzed the binding of 1.71 and 0.085 pmol equivalents of AFB1 to DNA, respectively, indicating that CYP1A2 was responsible for 95% of AFB1-DNA adduct formation at 0.133 microM AFB. These results demonstrate that CYP1A2 dominates the activation of AFB in human liver microsomes in vitro at submicromolar concentrations and support the hypothesis that CYP1A2 is the predominant enzyme responsible for AFBO activation in human liver in vivo at the relatively low dietary concentrations encountered in the human diet, even in high AFB exposure regions of the world. However, because the actual concentrations of AFB in liver in vivo following dietary exposures are uncertain, additional studies in exposed human populations are needed. Quantitative data on the relative rates of AFM1 and AFQ1 excretion (potential biomarkers for CYP1A2 and 3A4 activity, respectively) in humans would be useful to validate the actual contributions of these two enzymes to AFB1 oxidation in vivo.
Several dietary compounds have been demonstrated to reduce gastrointestinal cancer rates in both humans and animals. We showed that high human gastrointestinal tissue levels of glutathione S-transferase (GST), a family of detoxification enzymes consisting of class Alpha, Mu, Pi and Theta isoforms, were inversely correlated with cancer risk. We now investigated whether the sulforaphane analog compound 30, indole-3-carbinol, D-limonene or relafen, supplemented in the diet for two weeks at 1450, 250, 10,000, and 200 ppm, respectively, influenced (i) GST activity, (ii) GST isoenzyme levels, (iii) GSH levels, or (iv) glutathione peroxidase (GPx) activity in the gastrointestinal tract of male Wistar rats. Sulforaphane analog compound 30 enhanced GST activity in all organs studied (1.2-2.4 x). It induced GST Alpha levels in small intestine and liver, GST Mu levels in stomach and small intestine, GST Pi levels in stomach and small and large intestine, and GSH levels in stomach and proximal and middle small intestine. Indole-3-carbinol induced gastric GST Mu and hepatic GST Alpha levels. D-limonene induced hepatic GST Alpha, colonic GST Pi levels and proximal small intestinal GST enzyme activity and GST Pi levels. Relafen induced hepatic GST Alpha levels, distal small intestinal and gastric GST Pi levels, and oesophageal and proximal small intestinal GSH levels. GPx activity was enhanced by relafen in oesophagus, and in distal small intestine by sulforaphane analog compound 30. Enhancement of GSTs and to a lesser extent GPx and GSH, resulting in a more efficient detoxification, may explain at least in part the anticarcinogenic properties of sulforaphane analog compound 30, and to a much lesser extent of indole-3-carbinol and D-limonene.
Cross-resistance between different cytostatic agents which are structurally and functionally dissimilar is a common phenomenon called multidrug resistance (MDR). The best characterized mechanism of MDR involves P-glycoprotein. However, this does not completely explain MDR. Within the last few years, two new genes that can confer MDR have been identified (MRP and LRP). Furthermore, topoisomerase II has been associated with a special form of MDR. During the past several years, considerable interest has been shown in strategies to reverse MDR by using pharmacological compounds, monoclonal antibodies, immunotoxins, bispecific antibodies, antisense oligodeoxynucleotides, ribozymes, and albumin-conjugated drugs in in vitro and in vivo assays. All these experimental assays demonstrated that MDR can be circumvented. Two agents that have received the most attention in the clinic are verapamil and cyclosporin A. Despite some promising results (especially in hematological malignancies), the results obtained in the treatment of solid tumors with modulators have so far been quite disappointing. This may be explained by the fact that the MDR phenotype alone does not completely account for the resistance of human cancer. Several other resistance-related proteins (e.g., glutathione S-transferase, metallothionein, O6-alkylguanine-DNA-alkyltransferase, thymidylate synthase, dihydrofolate reductase, heat shock proteins) can be also expressed in resistant tumors. Additionally, cell proliferation, vascularization and apoptosis are involved in resistance.
Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B(1) (AFB(1)) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (approximately 200,000 pmol/min/mg) activity toward aflatoxin B(1)-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB(1)-induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha-class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (approximately 300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, MF: GSTs were purified from liver tissue and characterized and, Mf mu-class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu-class GST cDNAs, mfaGSTM1 (GenBank accession # AF200709) and mfaGSTM2 (GenBank accession # AF200710), were generated by RT-PCR. CDNA-derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu-class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo- and endo-AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis.
Genetic and biochemical evidence has demonstrated that glutathione and glutathione-dependent enzymes play a central role in cellular defence against toxic environmental agents. Modulation of cellular glutathione homeostasis can also have a profound effect on the sensitivity of cancer cells to a wide range of drugs used in chemotherapy. These effects are produced by multifactorial mechanisms that involve inactivation of toxic electrophiles by conjugation, modulation of cellular redox state, activation of drug transporter systems and regulation of cell signalling and repair pathways. New data demonstrating the importance of these pathways in cytoprotection and greater understanding of the mechanisms which regulate their function reveal a number of new targets for novel anti-cancer agents. It is critical, however, if these targets are to be exploited correctly that the dynamics of glutathione regulation are taken into account. Copyright 1999 Harcourt Publishers Ltd.
It is now evident that most, if not all, of the remarkable species differences in susceptibility to AFB hepatocarcinogenesis is due in large part, if not exclusively, to differences in biotransformation. Certainly the relative rate of oxidative formation of the proximate carcinogen, AFB-8,9-exo-epoxide, is an important determinant of species and interindividual differences in susceptibility to AFB. However, mice produce relatively large amounts of exo-AFBO, yet are highly resistant to AFB-hepatocarcinogenesis because they express a particular form of GST with remarkably high catalytic activity toward the exo-epoxide of AFB. Rats, which are highly susceptible to AFB hepatocarcinogenesis,can be made resistant through dietary induction of an orthologous form of GST that is normally expressed in only very small amounts. Based on these findings in laboratory animal models, there is great interest in identifying chemicals and/or specific dietary constituents that could offer protection against AFB-hepatocarcinogenesis to humans. Current experimental strategies have focused on the antiparasitic drug, oltipraz, which induces protection in rats and has also shown some promise in humans. The mechanism of protection in rats appears to be via induction of an alpha class GST with high catalytic activity toward AFBO (rGSTA5-5). vet human alpha class GST proteins that are constitutively expressed in the liver (hGSTA1 and hGSTA2) have little, if any activity toward AFBO. Rather, it appears that mu class GSTs may be responsible for the very low, but potentially significant, detoxification activity toward AFBO. Oltipraz and certain dietary constituents may induce mu class GSTs in human liver, and this could afford some protection against the genotoxic effects of AFBO. However, it also appears that oltipraz, and perhaps certain dietary constituents, act as competitive inhibitors of human CYP1A2. As CYP1A2 appears to mediate most of the activation of AFB to exo-AFBO in human liver at low dietary concentrations of AFB encountered in the human diet, much of the putative protective effects of oltipraz could be mediated via inhibition of CYP1A2 rather than induction of GSTs. There is now evidence that human microsomal epoxide hydrolase (mEH) could play a role in protecting human DNA from the genotoxic effects of AFB, although the importance of this detoxification pathway, relative to mu class GSTs, remains to be elucidated. Oltipraz is an effective inducer of mEH in rats (Lamb Franklin, 2000), and thus induction of this pathway in humans could also potentially contribute to the protective effects of this drug toward AFB genotoxicity. Because the dihydrodiol of AFB may contribute indirectly to the carcinogenic effects of AFB via protein adduction and subsequent hepatotoxicity, the recently characterized human aflatoxin aldehyde reductase (AFAR) may also offer some protection against AFB-induced carcinogenicity in humans. Current and future dietary and/or chemointervention strategies aimed at reducing the carcinogenic effects of AFB in humans should consider all of the possible mechanistic approaches for modifying AFB-induced genotoxicity.