Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells

Cytochrome P450 (CYP) 2A13 is mainly expressed in the respiratory system and has the ability to metabolize aflatoxin B(1) (AFB(1)). However, the role of CYP2A13-mediated AFB(1) metabolism and its consequences in human lung epithelial cell is not clear. Therefore, the objectives of this study were to investigate the significance of CYP2A13 in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis. To achieve these objectives, CYP2A13 was stably over-expressed in immortalized human bronchial epithelial BEAS-2B cells (B-2A13) and its significance in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis was compared to cells with stably expression of CYP1A2 (B-1A2), the predominant AFB(1) metabolizing enzyme in liver, as well as CYP2A6 (B-2A6) as controls. AFB(1) induced B-2A13 cytotoxicity and apoptosis in a dose- and time-dependent manner. The cytotoxic and apoptotic effects of AFB(1) were significantly remarkable in B-2A13 cells than those of B-1A2 and B-2A6 cells. The increased expression of pro-apoptotic proteins, such as C-PARP, C-caspase-3, and Bax, and decreased expression of anti-apoptotic proteins, such as caspase-3, Bcl-2, and p-Bad further confirmed the data of AFB(1)-induced cytotoxicity and apoptosis. Furthermore, increased DNA adduct was observed in B-2A13 after AFB(1) treatment as compared to B-1A2 cells and B-2A6 cells. Finally, treatment with nicotine, a competitor of AFB(1), and 8-methoxypsoralen (8-MOP), an inhibitor of CYP enzyme, further confirm the critical role of CYP2A13 in AFB(1)-induced cytotoxicity and apoptosis. Collectively, these findings suggest adverse effects of AFB(1) in respiratory diseases mediated by CYP2A13.     

Mixed inhibition of P450 3A4 as a chemoprotective mechanism against aflatoxin B1-induced cytotoxicity with cis-terpenones

We recently reported the protective effect of 2-hydroxy- cis-terpenone (HCT) against aflatoxin B 1 (AFB1)-induced cytotoxicity in human HepG2 liver cells ( Zhou et al. Chem. Res. Toxicol. 2006, 19, 1415-1419 ); however, the mechanism was not clear. In this paper, the chemoprotective mechanism was investigated with liver microsomes and purified P450 3A4 enzyme. HCT showed effective inhibition of the metabolic conversion of AFB1 in liver microsomes at 40 microM, and more importantly, the inhibition of the carcinogenic exo-AFB1-epoxide formation from AFB1. Further study indicated the direct inhibition of purified P450 3A4 enzyme activity by HCT with an IC 50 value of 20 microM. Under aqueous conditions, HCT was slowly converted to an oxidized product OHCT, which exhibits similar inhibitory effects on both P450 3A4 and the metabolic conversion and carcinogenic activation of AFB1 with liver microsomes as those of HCT. Enzyme mechanism studies revealed that OHCT acted as a mixed inhibitor of P450 3A4 with K i and K i' at 17.6 +/- 5.6 and 7.6 +/- 1.5 microM, respectively. Finally, OHCT showed no cytotoxicity at 60 microM in HepG2 liver cells and effective chemoprotection at 40 and 60 microM against AFB1 (2 microM) induced cytotoxicity. In contrast, ketoconazole alone exhibited 20% cell mortality at 20 microM, while chemoprotection with ketoconazole against 2 microM AFB1 in HepG2 was observed at 10 and 20 microM, which was much higher than the 1 microM concentration used in the inhibitory assays of P450 3A4 activity and AFB1 metabolism with liver microsomes.     

Zinc inhibits aflatoxin B1-induced cytotoxicity and genotoxicity in human hepatocytes (HepG2 cells)

Aflatoxin B1 (AFB1) has strong carcinogenicity. Consumption of AFB1-contaminated agricultural products and the occurrence of hepatocellular carcinoma have received widespread attention. The aim of this paper was to investigate whether zinc supplementation could inhibit AFB1-induced cytotoxicity and genotoxicity in HepG2 cells and the mechanism of this inhibition. Our data suggest that zinc sources can relieve a certain degree of AFB1-induced cytotoxicity and genotoxicity by protecting against apoptotic body formation and DNA strand breaks, affecting S phase cell cycle arrest, reducing 8-OHdG formation, inhibiting global DNA hypomethylation and regulating gene expression in antioxidation, zinc-association and apoptosis processes. Consequently, zinc stabilizes the integrity of DNA and improves cell survival.These data provides new insights into the protective role of zinc in alleviating AFB1-induced cytotoxicity and mediating epigenetic changes in hepatocytes, demonstrating that zinc sources have detoxification properties in mycotoxin-induced toxicity.     

Inhibition of glycyrrhizic acid on aflatoxin B1-induced cytotoxicity in hepatoma cells

Aflatoxin B(1) (AFB(1)) causes oxidative stress and ROS formation via metabolic activation of AFB(1). Glycyrrhizic acid (GA) has been reported to have antioxidative properties. The present study was to investigate the effect of GA, a major component of licorice on AFB(1)-induced cytotoxicity in human hepatoma cell line (HepG2). GA displayed protective effects against AFB(1) treatment. Both CYP1A1, and glutathione S-transferase (GST) activities were increased in cells after treatment with the GA, while CYP1A2 did not seem to be affected by GA. For cells without GA pre-treatment, cell injury was implicated as indicated by the decrease in cell viability. The time-course study of GA showed pretreatment of cells with GA for 24 h was effective. The treatment of cells with GA and AFB(1) enhanced the detoxifying enzyme activity. The pre-treatment of cells with GA provides protective effects in terms of the enzyme activity and increase in cell viability. The results suggest that GA protects against aflatoxin-induced oxidative stress. This may contribute to its anticarcinogenic capability. The protective effect is likely due to its capacity to inhibit the metabolic activation of hepato-toxin, a critical factor in the pathogenesis of chemical-induced carcinogenicity.     

Protection against aflatoxin B1-induced cytotoxicity by expression of the cloned aflatoxin B1-aldehyde reductases rat AKR7A1 and human AKR7A3

The reduction of the aflatoxin B 1 (AFB 1) dialdehyde metabolite to its corresponding mono and dialcohols, catalyzed by aflatoxin B 1-aldehyde reductase (AFAR, rat AKR7A1, and human AKR7A3), is greatly increased in livers of rats treated with numerous chemoprotective agents. Recombinant human AKR7A3 has been shown to reduce the AFB 1-dialdehyde at rates greater than those of the rat AKR7A1. The activity of AKR7A1 or AKR7A3 may detoxify the AFB 1-dialdehyde, which reacts with proteins, and thereby inhibits AFB 1-induced toxicity; however, direct experimental evidence of this hypothesis was lacking. Two human B lymphoblastoid cell lines, designated pMF6/1A2/AKR7A1 and pMF6/1A2, were genetically engineered to stably express AKR7A1 and/or cytochrome P4501A2 (1A2). The pMF6/1A2/AKR7A1 cells were refractory to the cytotoxic effects of 3 ng/mL AFB 1, in comparison to pM6/1A2 cells, which were more sensitive. Diminished protection occurred at higher concentrations of AFB 1 in pMF6/1A2/AKR7A1 cells, suggesting that additional factors were influencing cell survival. COS-7 cells were transfected with either vector control, rat AKR7A1, or human AKR7A3, and the cells were treated with AFB 1-dialdehyde. There was a 6-fold increase in the dialdehyde LC 50, from 66 microM in vector-transfected cells to 400 microM in AKR7A1-transfected cells, and an 8.5-fold increase from 35 microM in vector-transfected cells to 300 microM in AKR7A3-transfected cells. In both cases, this protective effect of the AFAR enzyme was accompanied by a marked decrease in protein adducts. Fractionation of the cellular protein showed that the mitochondria/nuclei and microsomal fractions contained the highest concentration of protein adducts. The levels of human AKR7A3 and AKR7A2 were measured in 12 human liver samples. The expression of AKR7A3 was detectable in all livers and lower than those of AKR7A2 in 11 of the 12 samples. Overall, these results provide the first direct evidence of a role for rat AKR7A1 and human AKR7A3 in protection against AFB 1-induced cytotoxicity and protein adduct formation.     

Cytochrome P450-mediated metabolism and cytotoxicity of aflatoxin B(1) in bovine hepatocytes.

Aflatoxin B(1) (AFB(1)) biotransformation comprises cytochrome P450-mediated reactions resulting in hydroxylated and demethylated metabolites as well as AFB(1) epoxides. As the latter are highly nucleophilic, the species-specific rate of epoxidation and the ability for rapid conjugation to glutathione by glutathione S-transferase determines the individual susceptibility to AFB(1). Here we show the time- and dose-dependent rate of AFB(1)-metabolism in bovine hepatocytes. Aflatoxin M(1) (AFM(1)) is the most prominent metabolite formed within the first 2-8 hr of incubation, whereas AFB(1)-dhd is detectable in medium mainly after a prolonged incubation period. The delayed formation of AFB(1)-dhd corresponds to the cytotoxicity demonstrated by the MTT assay. alpha-Naphthoflavone and ketoconazole, inhibitors of CYP1A and CYP3A, respectively in humans, were used to evaluate the contribution of specific P450 isoenzymes in bovine biotransformation of AFB(1). Initial experiments confirmed that alpha-naphthoflavone and ketoconazole inhibited ethoxyresorufin O-deethylation and testosterone 6beta-hydroxylation also in bovine hepatocytes. Both inhibitors reduced AFM(1) and AFB(1)-dhd formation concentration dependently, suggesting that both enzyme groups contribute to the formation of these metabolites. However, the formation of AFM(1) was less inhibited by both compounds than the formation of AFB(1)-dhd.     

Effects of polybrominated diphenyl ethers on basal and TCDD-induced ethoxyresorufin activity and cytochrome P450-1A1 expression in MCF-7, HepG2, and H4IIE cells.

Polybrominated diphenylethers (PBDEs) are used as additive flame-retardants in consumer products to reduce the chances of ignition and burning. Levels of certain PBDE congeners have been increasing in fish, wildlife, and human tissues during the last decades. Some PBDEs are lipophilic and persistent, resulting in bioaccumulation in the environment. The structural similarity of PBDEs to other polyhalogenated aromatic hydrocarbons such as PCBs, has raised concerns that PBDEs might act as agonists for the aryl hydrocarbon receptor (AhR). To study the possible AhR-mediated effects of the environmentally relevant PBDEs (BDE47, 77, 99, 100, 153, 154, 183, 209), the induction of cytochrome P450-1A1 (CYP1A1) was studied in human breast carcinoma (MCF-7), human hepatocellular carcinoma (HepG2), and rat hepatoma (H4IIE) cells. 7-Ethoxyresorufin-O-deethylase (EROD) was used as a marker for CYP1A1 activity. Cells were exposed for 72 h to various PBDE concentrations (0.01-10 microM). Positive controls were 2,3,7,8-TCDD (0.001-2.5 nM) and PCB126 (0.01-10 nM). None of these PBDEs was capable of inducing EROD activity; this was confirmed by real time RT-PCR for CYP1A1 mRNA. However, in cells exposed to PBDEs in combination with TCDD, a concentration-dependent decrease in TCDD-induced EROD activity occurred. Co-exposure of BDE153 (10 muM) and a maximally inducing concentration of TCDD (1 nM) reduced EROD activity to 49% of the maximum induction by TCDD alone. All tested PBDEs showed similar effects in each cell line, though quantitative differences were observed. The observed decrease in CYP1A1 activity was not due to PBDE-dependent catalytic inhibition of EROD activity or cytotoxicity, nor were decreased CYP1A1 mRNA levels observed. However, inhibition of luciferase induction in mouse (Hepa) and rat (H4IIE) hepatoma cells containing a stably transfected AhR-responsive luciferase reporter gene, suggests that BDE77 is a weak AhR antagonist or partial agonist.     

In vitro toxicity of aflatoxin B1 and its photodegradation products in HepG2 cells

The effects of aflatoxin B₁ (AFB₁) were studied using the HepG2 cell line. Cytotoxicity, apoptosis and p53 expression were assessed after exposure to different concentrations of AFB₁ (0–100 μm ) and its two types of degradation products, namely the mixtures of photodegradation products in water (Pw) and the mixtures of photodegradation products in peanut oil (Po) for different time periods (0, 24, and 48 h). After exposure of the HepG2 cells to these compounds for different times and concentrations, the cytotoxicity of Pw and Po decreased approximately 40 and 100% compared with AFB₁, respectively. The expression of p53 protein decreased significantly in AFB₁‐exposed cells, decreased slightly in Pw‐treated cells and did not decrease compared to the untreated cells. The results of the in vitro cytotoxicity assay indicate that Pw is less toxic than AFB₁, and Po has almost no toxicity, which can be explained by the differences in the chemical nature of the various kinds of the test compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Protective effect of ebselen on aflatoxin B1-induced cytotoxicity in primary rat hepatocytes

Recent studies have shown that aflatoxin B1 enhances reactive oxygen species formation and causes oxidative damage, which may ultimately contribute to the cytotoxicity and carcinogenic effect of aflatoxin B1. Ebselen, 2-phenyl-1,2-benzoisoseleazol-3(H)-one, a synthetic seleno-organic compound has been shown to possess glutathione peroxidase-like activity and free radical scavenging ability. Thus present study was designed to investigate the protective effect of ebselen on aflatoxin B1-induced cytotoxicity in primary rat hepatocytes. Aflatoxin B1-induced cytotoxicity and lipid peroxidation were determined by lactate dehydrogenase leakage and malondialdehyde generation, respectively. Intracellular reactive oxygen species level was measured using the fluorescent probe 2',7'-dichlorofluorescin diacetate, and the intracellular reduced glutathione concentration was determined with a fluorometric method. Ebselen was found to display a dose-dependent protective effect on lactate dehydrogenase leakage and malondialdehyde generation caused by aflatoxin B1 exposure. The results also demonstrate that ebselen efficiently inhibits the intracellular reactive oxygen species formation in aflatoxin B1-treated hepatocytes in a dose and time-dependent manner. It was also noted that ebselen was able to increase the intracellular reduced glutathione concentration, both in the control and in aflatoxin B1-treated hepatocytes. The protection of ebselen against aflatoxin B1 cytotoxicity, however, was not affected by lowering the concentration of intracellular reduced glutathione. The overall data indicate that ebselen possesses a potent protective effect against aflatoxin B1-induced cytotoxicity, and the main mechanism involved in the protection may be its strong capability in inhibiting intracellular reactive oxygen species formation and preventing oxidative damage.     

Ruthenium red protects HepG2 cells overexpressing CYP2E1 against acetaminophen cytotoxicity

We studied the mechanisms of acetaminophen (APAP) cytotoxicity in HepG2 cells overexpressing cytochrome p4502E1, particularly the role of oxidative/nitrosative stress and ryanodine Ca²⁺ channel. Cells were grown for 24 h with APAP in the presence or absence of 4-methylpyrazole (4MP), l-arginine methyl ester (L-NAME), superoxide dismutase (SOD), or ruthenium red (RuR). Drug cytotoxicity was also tested in cells pretreated overnight with V-PYRRO/NO. APAP was without effect on empty vector-transfected cells, but damaged CYP2E1-transfected cells and this was abolished by RuR, reduced by 4MP, or V-PYRRO/NO but affected by L-NAME or SOD. APAP increased microsomal [³H]-ryanodine binding, while microsomal Ca²⁺ uptake was significantly lowered. RuR increased net microsomal Ca²⁺ uptake and normalized cytosolic Ca²⁺ levels. We can conclude that neither oxidative nor nitrosative stress is relevant to APAP cytotoxicity in cultured HepG2 cells, but our results point to ryanodine receptors as a potential crucial protein in the early stages of APAP cytotoxicity.     

Suicide inhibitors of cytochrome P450 1A1 and P450 2B1.

The inhibition of the P450 1A1 dependent de-ethylation of 7-ethoxyphenoxazone (7EPO) and the P450 2B1 dependent de-pentylation of 7-pentoxyphenoxazone (7PPO) by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 1-ethynylanthracene (1EA), 2-ethynylanthracene (2EA), 9-ethynylanthracene (9EA), 2-ethynylphenathrene (2EPh), 3-ethynylphenanthrene (3EPh), 9-ethynylphenanthrene (9EPh), 1-ethynylpyrene (1EP) and 2-ethynylpyrene (2EP) was studied in hepatic microsomal preparations from rats. Although all of the polycyclic aromatic acetylenes studied inhibited the dealkylation of 7EPO or 7PPO, only some of the acetylenes produced a mechanism-based irreversible inactivation (suicide inhibition) of the P450 dependent dealkylation of 7EPO or 7PPO. Of the molecules tested, only 1EP, 1EN, 2EN, 2EPh and 3EPh were effective suicide inhibitors of the P450 1A1 dependent de-ethylation of 7EPO and only 1EN, 2EN, 1EA and 9EPh were effective suicide inhibitors of the P450 2B1 dependent de-pentylation of 7PPO. In addition to the size and shape of the polycyclic aromatic ring system, placement of the carbon--carbon triple bond on the ring system was critical for suicide inhibition. In contrast to 1EP, 2EP was not a mechanism-based inhibitor of P450 1A1; 9EPh, but not 2EPh or 3EPh, was a suicide inhibitor of P450 2B1. None of the aryl acetylenes tested produced heme destruction under assay conditions that produced the suicide inhibition of the P450 dependent 7EPO or 7PPO dealkylation activities. Because a precise orientation of the terminal acetylene is required to produce suicide inhibition without heme destruction, acetylenic suicide inhibitors can potentially be used to differentiate between P450 isozymes and to establish some distinguishing geometric features of the active site of these isozymes.     

Tribuli fructus constituents protect against tacrine-induced cytotoxicity in HepG2 cells

A new phenolic amide, tribulusimide D (4-hydroxy-N-[3-(4-hydroxy-3-methoxyphenyl)-1-oxo-2-propen-1-yl]-3-methoxybenzamide) (1), together with a known phenolic amide, terrestriamide ((E)-3-(4-hydroxy-3-methoxyphenyl)-N-[2-(4-hydroxyphenyl)-2-oxoethyl]-prop-2-enamide) (2) and a flavonol glycoside, quercetin-3-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside (3) were isolated from the H₂O extract of Tribuli Fructus. Compounds 1 and 3 showed significant hepatoprotective activities, with EC₅₀ values of 13.46 ± 0.2 and 7.06 ± 0.7 μM, respectively, against tacrine-induced cytotoxicity in HepG2 cells.     

Metabolism and cytotoxicity of aflatoxin b1 in cytochrome p-450-expressing human lung cells

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.     

Cytochrome P450 2A13 is an efficient enzyme in metabolic activation of aflatoxin G1 in human bronchial epithelial cells

Cytochrome P450 2A13 (CYP2A13) is an extrahepatic enzyme that mainly expresses in human respiratory system, and it is reported to mediate the metabolic activation of aflatoxin B1. Due to the structural similarity, AFG1 is predicted to be metabolized by CYP2A13. However, the role of CYP2A13 in metabolic activation of AFG1 is unclear. In present study, human bronchial epithelial cells that stably express CYP2A13 (B-2A13) were used to conduct the effects of AFG1 on cytotoxicity, apoptosis, DNA damages, and their response protein expression. Low concentrations of AFG1 induced significant cytotoxicity and apoptosis, which was consistent with the increased expressions of pro-apoptotic proteins, such as C-PARP and C-caspase-3. In addition, AFG1 increased 8-OHdG and γH2AX in the nuclies and induced S phase arrest and DNA damage in B-2A13 cells, and the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and γH2AX, were activated. All the above effects were inhibited by nicotine (a substrate of CYP2A13) or 8-MOP (an inhibitor of CYP enzymes), confirming that CYP2A13 mediated the AFG1-induced cytotoxicity and DNA damages. Collectively, our findings first demonstrate that CYP2A13 might be an efficient enzyme in metabolic activation of AFG1 and helps provide a new insight into adverse effects of AFG1 in human respiratory system.     

Induction of rat brain and liver cytochrome P450 1A1/1A2 and 2B1/2B2 isoenzymes by deltamethrin

Deltamethrin, an α-cyano type II pyrethroid, administered orally (5, 10 and 15 mg/kg body weight for 7 consecutive days or at 5 mg/kg for further 15 and 21 days) to young albino Wistar rats (approximately 8 weeks old) produced a dose- and time-dependent increase in the activity of cytochrome P450-dependent 7-ethoxyresorufin-O-dealkylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) in rat liver and brain. However, significant induction in the enzyme activities was observed at higher doses or prolonged exposure. The magnitude of induction in rat liver microsomes was less at 15 mg/kg dose as compared to 10 mg/kg dose. Western blot analysis revealed a similar dose-related and time-dependent increase in the expression of P450 2B1/2B2 and 1A1 isoenzymes as indicated by the increased cross-reactivity of liver microsomes isolated from deltamethrin-treated animals with anti-P450 2B1/2B2 and 1A1. Inhibition of EROD and PROD observed after in vitro addition of anti-P450 2B1/2B2 and 1A1/1A2 or organic inhibitors, metyrapone and α-naphthoflavone, to the brain and liver microsomes of deltamethrin-pretreated animals (5 mg/kg×21 days), further provided support that the induction observed in the EROD and PROD activity in brain is due to the increased expression of P450 2B1/2B2 and 1A1/1A2, while, in the liver, isoenzymes other than these are also involved.     

Protective effects of coffee diterpenes against aflatoxin B1-induced genotoxicity: mechanisms in rat and human cells.

The coffee-specific diterpenes cafestol and kahweol (C + K) have been reported to be anticarcinogenic in several animal models. Proposed mechanisms involve a co-ordinated modulation of several enzymes responsible for carcinogen detoxification, thus preventing reactive agents interacting with critical target sites. To address the human relevance of the chemoprotective effects of C + K against aflatoxin B(1) (AFB1) genotoxicity observed in rat liver, and to compare the mechanisms of protection involved in both species, animal and human hepatic in vitro test systems were applied. In rat primary hepatocytes, C + K reduced the expression of cytochrome P450 CYP 2C11 and CYP 3A2, the key enzymes responsible for AFB1 activation to the genotoxic metabolite aflatoxin B1-8,9 epoxide (AFBO). In addition, these diterpenes induced significantly GST Yc2, the most efficient rat GST subunit involved in AFBO detoxification. These effects of C + K resulted in a marked dose-dependent inhibition of AFB1-DNA binding in this rat in vitro culture system. Their relevance in humans was addressed using liver epithelial cell lines (THLE) stably transfected to express AFB1 metabolising cytochrome P450s. In these cells, C + K also produced a significant inhibition of AFB1-DNA adducts formation linked with an induction of the human glutathione S-transferase GST-mu. Altogether, these results suggest that C + K may have chemoprotective activity against AFB1 genotoxicity in both rats and humans.     

Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by n-propylxanthate

n-Propylxanthate (nPX) inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified, reconstituted rat hepatic P450 2B1 or human P450 2B6 in a mechanism-based manner. The inactivation followed pseudo-first-order kinetics and was entirely dependent on both NADPH and nPX. The maximal rate constant for inactivation of P450 2B1 at 30 degrees C was 0.2 min-1. The apparent KI was 44 microM, and the half-time for inactivation was 4.1 min. Purified, reconstituted human P450 2B6 was also inactivated by nPX with a KI of 12 microM. The kinactivation for P450 2B6 was 0.06 min-1, and the t1/2 was 11 min. Incubations of P450 2B1 with nPX and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 25% loss of the enzyme's ability to form a reduced CO complex. Little loss in the absolute spectrum of nPX-inactivated P450 2B1 was observed. With P450 2B6, an 83% loss in enzymatic activity and a 12% loss in the CO-reduced spectra were observed. The extrapolated partition ratio for nPX with P450 2B1 was 32. P450 2B1 could be protected from inactivation by nPX by adding an alternate substrate to the reaction mixture. Removal of unbound nPX by dialysis did not reverse the inactivation. The alternate oxidant iodosobenzene was able to partially restore enzymatic activity to nPX-inactivated P450 2B1 samples. A stoichiometry for labeling of 1.2:1 for binding of radiolabeled nPX metabolite to P450 2B1 was seen. These results indicated that nPX inactivated P450 2B1 and P450 2B6 in a mechanism-based manner. P450 2B1 was inactivated primarily by a nPX reactive intermediate that bound to the apoprotein.