Effect of ampicillin on hepatic microsomal mixed-function oxidase system in male mice

The effect of ampicillin [(D)-alpha-aminobenzyl penicillin] administration on the hepatic mixed-function oxidase (MFO) system was studied in male mice. Ampicillin (100 mg/kg, i.p., 3 days) decreased the levels of cytochrome P-450, aminopyrine N-demethylase, acetanilide hydroxylase and cytochrome c-reductase activity significantly. In carbon tetrachloride (CCl4)-pretreated mice, ampicillin increased acetanilide hydroxylation compared with CCl4 treatment alone; however, all other parameters of the MFO system remained unchanged. Ampicillin exhibited type II binding with microsomes (trough at 388 nm, peak at 430 nm). Thus ampicillin acts as an inhibitor of the MFO system.     

Effect of Picroliv on impaired hepatic mixed-function oxidase system in carbon tetrachloride-intoxicated rats

A drastic impairment in hepatic mixed-function oxidase (MFO) system was observed in rats 24 h following ip injection of CCl₄ (1.0 ml/kg body weight), as indicated by a decrease in the activities of various microsomal enzymes related to drug metabolism (e.g., aniline hydroxylase, aminopyrine-N-demethylase, benzo(a)pyrene hydroxylase, NADH/NADPH cytochrome c reductase, glutathione-s-transferase, cytochrome P-450, and cytochrome b₅). In addition, total protein and reduced glutathione content in liver were decreased, while a significant elevation in the level of lipid peroxides was observed. Hepatoprotection afforded by oral pretreatment of CCl₄ intoxicated rats with multiple doses (6.0 mg/kg × 7 days) of Picroliv (standardized iridoid glycoside fraction, obtained from roots and rhizomes of Picrorhiza kurroa), was evidenced by a significant improvement in the activities of various enzymes related to drug metabolism. Alterations in levels of total proteins, reduced glutathione, and lipid peroxides in liver were also significantly prevented by Picroliv pretreatment. Drug Dev. Res. 41:44–47, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of intestinal cytochrome P-450 and heme by dietary nutrients. Critical role of selenium

The intestinal cytochrome P-450 (I-P-450)-dependent mixed function oxidase (MFO) system is regulated to a remarkable extent by various ingested xenobiotics, including drugs and carcinogens, as well as dietary nutrients. Accordingly, acute dietary iron deprivation is found to result in a marked decrease in I-P-450 content and activity. This decrease is most pronounced in the villous tip cells, the very cells committed to absorption of ingested materials. We investigated the mechanistic basis for such acute reduction and report that iron was not only required as a co-substrate for I-P-450 heme formation, but also as a regulator of two key heme-synthetic enzymes, delta-aminolevulinic acid synthetase and ferrochelatase. In addition, our studies revealed that dietary deprivation of selenium for a single day dramatically reduced I-P-450-dependent MFO activity. This prompt reduction apparently reflects impaired I-P-450 formation resulting from lowered ferrochelatase activity and consequently decreased intestinal heme availability, and was not a consequence of intracellular peroxidation presumably enhanced by concomitant lowering of the seleno-dependent glutathione peroxidase. Thus, we report the novel observation that dietary selenium also appears to be a critical modulator of intestinal cytochrome P-450-dependent metabolism of ingested drugs, carcinogens, and toxins that are absorbed by the intestinal mucosa.     

Alterations in hepatic mixed-function oxidase activity of rainbow trout after acute treatment with polybrominated biphenyl isomers and FireMaster BP-6

The effects of FireMaster BP-6 and several pure isomers on hepatic microsomal mixed-function oxidase (MFO) activity in rainbow trout have been investigated. After one parenteral injection of these agents at 150 mg/kg and sacrifice 5 d later, there was no elevation of hepatic cytochrome P-450. The noncoplanar isomers 2,4,5,2',4',5'-hexa and 2,3,4,5,2',4',5'-heptabromobiphenyl did not increase any MFO activity generally associated with an increase in cytochrome P-450 levels, including the dealkylations of ethoxycoumarin and benzphetamine. The coplanar isomer 3,4,5,3',4',5'-hexabromobiphenyl produced an increase in O-deethylation of both ethoxycoumarin and ethoxyresorufin, suggesting enhancement of cytochrome P-448 associated activity, but produced no increase in benzphetamine N-dealkylation. The "mixed inducer" 2,4,5,3',4',5'-hexabromobiphenyl, produced a small but insignificant elevation of cytochrome P-448 associated MFO activities and no increase in cytochrome P-450 associated activities. At both 150 and 500 mg/kg, the commercial mixture of polybrominated biphenyls FireMaster BP-6 produced marked elevations of both ethoxycoumarin and ethoxy-resorufin dealkylations but had no effect on demethylation of benzphetamine. When microsomes from rainbow trout treated with FireMaster BP-6 were examined electrophoretically, A coomassie Blue-staining band at 57,000 daltons was intensified, as seen after treatment of fish with the cytochrome P-448 inducer beta-naphthoflavone. It is concluded that coplanar polybrominated isomers produce an induction of hepatic cytochrome P-448 associated activity, while noncoplanar isomers are ineffective as inducers of cytochrome P-450 related MFO activities in rainbow trout.     

Depression of the hepatic cytochrome P-450 monooxygenase system by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The effects of the neurotoxic compound, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the hepatic cytochrome P-450 monooxygenase system were assessed using C57 BL/6J mice. Treatment with MPTP caused a marked depression of hepatic cytochrome P-450 content, ethoxyresorufin O-dealkylase and NADPH cytochrome C reductase activities. This effect was maximal 3 to 6 hours after treatment and was dependent on the dose of MPTP administered. Depression of spectrophotometrically measured cytochrome P-450 content was associated with increase in cytochrome P-420 content and lipid peroxidation. In vitro studies showed the formation of a metabolic-intermediate complex with cytochrome P-450 which may partially explain the depression of cytochrome P-450 content and activity by MPTP.     

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.     

Effect of phenobarbital treatment on carbon tetrachloride-mediated cytochrome P-450 loss and diene conjugate formation.

The effect of phenobarbital treatment on the linkage between carbon tetrachloride-mediated cytochrome P-450 loss and lipid peroxidation in rat liver microsomes was studied. Male Sprague-Dawley rats, pretreated with 3 daily i.p. doses of phenobarbital (50 mg/kg) or saline, were orally dosed with carbon tetrachloride (0.01-2.5 ml/kg), with liver microsomes prepared at 7.5-180 min after carbon tetrachloride treatment. In vivo cytochrome P-450 loss displayed pseudo-first-order kinetics, and the initial rates of diene conjugate formation were saturable with dose. Phenobarbital pretreatment decreased the in vivo t0.5,max from 27.0 to 15.6 min, and increased the Kd,app from 0.78 to 1.30 ml/kg for carbon tetrachloride mediated cytochrome P-450 loss. Phenobarbital had no effect on the in vivo Vmax (1.03 to 1.04 delta OD232 nm/min/mg phospholipid) for carbon tetrachloride mediated diene conjugate formation, but decreased the Km,app from 0.22 to 0.10 ml/kg. These results are consistent with destruction of cytochrome P-450 heme resulting from a metabolite which does not leave the site of generation, and with phenobarbital pretreatment enhancing the initiation of lipid peroxidation.     

Suppressive effect of bifemelane on lipid peroxidation in rat liver

The effects of Bifemelane (BF) on lipid peroxidation, the activities of hepatic drug metabolizing enzymes, and the function of cell membranes were examined in rats. In the liver ischemia-reperfusion model, BF suppressed the elevation of the lipid peroxidation level during the period of reperfusion. BF did not exhibit a radical-trapping action using a stable free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), which was estimated by electron spin resonance (ESR). BF remarkably inhibited NADPH-dependent lipid peroxidation in vitro. BF had no effect on the contents of cytochrome P-450 and b5 and the activities of NADPH cytochrome P-450 reductase and Cu,Zn-superoxide dismutase (SOD). BF suppressed phorbol myristate acetate (PMA)-induced superoxide formation of polymorphonuclear leukocytes (PMNs), protected hypotonic hemolysis of erythrocyte and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and serum phospholipase A activity. These results suggest that BF has neither radical-trapping activity nor any influence on the drug metabolizing enzymes, but BF has a membrane-stabilizing action and it attributes to the suppressive effect of lipid peroxidation.     

Lack of in vitro and in vitro effects of fenbendazole on phase I and phase II biotransformation enzymes in rats, mice and chickens.

Intraperitoneal administration of 10 mg fenbendazole/kg bw daily for 5 d caused no significant alterations in the activities of hepatic microsomal drug-metabolizing enzymes viz aminopyrine N-demethylase, aniline hydroxylase and cytosolic glutathione S-transferase in rats, mice and chickens. Similarly no significant difference in the amount of microsomal cytochrome P-450 and NADPH-cytochrome c reductase was found between control and treated animals. In vitro incubation of fenbendazole with rat, mouse and chicken microsomes suggests that the drug neither binds to microsomal protein cytochrome P-450 nor inhibits the activities of aminopyrine N-demethylase and aniline hydroxylase. Similarly in vitro addition of fenbendazole to cytosolic glutathione S-transferase from the above species did not alter the activity of this enzyme. The results indicate that fenbendazole does not alter the activity of hepatic microsomal monooxygenase system significantly in rats, mice and chickens at a dosage level of 10 mg/kg body weight. In vitro studies also indicate that fenbendazole does not interact with the hepatic microsomal monooxygenase system, indicating it is not a substrate for cytochrome P-450-dependent monooxygenase system.     

蕊木宁F对小鼠实验性肝损伤的保护作用

蕊木宁(kopsinine)F(K-F)系自云南蕊木提出的一种吲哚类生物碱。该成分对小鼠因注射CCl₄,硫代乙酰胺,TAA及扑热息痛引起的肝损伤有明显的保护作用,使血清谷丙转氨酶降低,肝脏病理损害减轻。该成分对CCl₄引起的肝微粒体脂质过氧化反应和¹⁴CCl₄与肝微粒体进行的共价结合亦有抑制作用。此外,K-F还能提高小鼠的肝微粒体细胞色素P-450活性。以上结果提示蕊木宁F对小鼠实验性肝损害有保护作用。     

Biochemical toxicology of argemone oil. I. Effect on hepatic cytochrome P-450 and xenobiotic metabolizing enzymes

The in vivo effect of argemone oil on hepatic xenobiotic metabolizing enzymes was investigated in albino rats following either a single (10 ml kg-1 body wt.) or multiple intraparenteral doses (5 ml kg-1 body wt.) for three days. Animals sacrificed 72 h after a single intraparenteral dose of argemone oil exhibited a significant loss of hepatic cytochrome P-450 (35%) and cytochrome b5 (34%) contents and inhibition of aminopyrine-N-demethylase (APD), aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin-O-deethylase (ECD) activities (21-39%). Three successive 24-hourly intraparenteral injections of argemone oil followed by sacrificing the animals after 24 h of the last injection, showed a greater degree of inhibition of the content of cytochrome P-450 (58%) and its dependent mixed-function oxidases (35-63%). Also, multiple treatment of argemone oil caused a depletion of endogenous hepatic glutathione (GSH) content (72%) with a concomitant increase in lipid peroxidation (177%) and decrease in glutathione-S-transferase (GST) activity (30%). A significant decrease in relative liver weight (39%) was observed in animals treated with multiple treatment of argemone oil. These results suggest that argemone oil can alter both membrane and cytosolic defences and destabilizes the hepatic cytochrome P-450 dependent mixed-function oxidase system, so that it tips in the direction of autooxidative peroxidation of lipids.     

Modulatory influence of noscapine on the ethanol-altered hepatic biotransformation system enzymes, glutathione content and lipid peroxidation in vivo in rats.

The modulatory potential of noscapine, an opium alkaloid was assessed on the ethanol-induced changes in hepatic drug metabolizing enzyme systems, glutathione content and microsomal lipid peroxidation. Noscapine was administered orally to male Wistar rats at a dose level of 200 mg/kg bw alone as well as in combination with 50% ethanol (v/v) for 5 days. Noscapine administration was associated with a approximately 91% decrease in hepatic microsomal cytochrome P-450 content. A decline of approximately 36% was observed in the NADPH-cytochrome c reductase activity on noscapine administration. The lowering of cytochrome P-450 levels on noscapine administration was accompanied by a concomitant increase in heme oxygenase activity as well as serum bilirubin levels. Our results indicate that the combination dosage of noscapine and ethanol antagonised the ethanol-induced elevation of cytochrome P-450 levels. Noscapine fed rats had decreased glutathione (GSH) content and enhanced lipid peroxidation compared to control rats as indexed by MDA method. Further, noscapine and ethanol coexposure produced a more pronounced elevation in lipid peroxidation and the glutathione levels also decreased significantly. We speculate on the basis of our results that the significant enhancement of lipid peroxidation on combination dosage of noscapine and ethanol is a consequence of depletion of glutathione to certain critical levels. The inhibition of glutathione-S-transferase (GST) as well as lowering of cytochrome P-450 suggests that the biotransformation of noscapine and ethanol is significantly altered following acute coexposures.     

Effects of arsenite on hepatic mixed-function oxidase activity in rats.

1. Injection of arsenite (As3+) to control rats results in losses of total hepatic cytochrome P-450 and significant decreases of ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD) activities. However, As3+ appears to decrease the activity of these enzymes differentially, with EROD showing greater sensitivity than ECOD. 2. Injection of As3+ to rats treated with phenobarbital and isosafrole significantly decreases the total content of hepatic cytochrome P-450 and various mixed function oxidase (MFO) activities, with the exception of ECOD which appears to be insensitive to As3+. 3. 3-Methylcholanthrene administration apparently protects against the effects of As3+ on the cytochrome P-450 system, since total content of the cytochrome P-450 and various MFO activities were all insensitive to this treatment.     

Species differences in stimulation of intestinal and hepatic microsomal mixed-function oxidase enzymes

The effect of intraperitoneal (i.p.) administration of phenobarbital (PB) or 3-methylchol-anthrene (3-MC) on some mixed-function oxidase (MFO) enzymes was studied in small intestine and liver of male rats, mice, guinea pigs and rabbits. PB treatment enhanced intestinal and 7-ethoxycoumarin deethylase activities in the mouse and rat, whereas benzo[a]pyrene hydroxylase (AHH) activity was increased only in the mouse. Ethylmorphine demethylase and aniline hydroxylase activities in small intestine were not stimulated by PB in any species. Administration of 3-MC increased the activity of intestinal AHH in rat, mouse and guinea pig, but intestinal 7-ethoxycoumarin deethylase activity was elevated only in the rat. The guinea pig and mouse intestinal ethoxycoumarin deethylase activity was inhibited by 3-MC treatment. None of the enzymes tested in rabbit intestine was induced by PB or 3-MC. The hepatic activities of ethylmorphine demethylase, aniline hydroxylase, 7-ethoxycoumarin deethylase and AHH, and the cytochrome P-450 content were increased by PB in all species. In contrast, 3-MC enhanced hepatic aniline hydroxylase and AHH activities in rats, mice and guinea pigs, and hepatic 7-ethoxycoumarin deethylase activity in mice and rats. In rabbits, these hepatic enzymes were inhibited by 3-MC pretreatment. The hepatic cytochrome P-450 absorption spectra was shifted to 448 nm in all species. These results suggest that there are differences in induction of intestinal and hepatic MFO enzymes which are influenced by the type of inducing agent, substrate and animal species used.     

Triphenyltin acetate-mediated in vitro inactivation of rat liver cytochrome P-450

The in vitro effects of the organotin (OT) compound triphenyltin acetate (TPTA) on cytochrome P-450 content and functions were investigated in liver microsomes from untreated, phenobarbital (PB)- or beta-naphthoflavone- (betaNAF) pretreated rats. At a concentration of 0.5 mM, TPTA caused a marked loss in the spectrally detectable content of cytochrome P-450 up to 27% of its original value, along with an increase in the inactive form cytochrome P-420. Both effects were most pronounced in betaNAF-treated microsomes, which showed a shift in the hemoprotein absorption maximum from 448 nm to 451 nm, but in all cases TPTA failed to affect either cytochrome b5 or total heme content, or to increase the production of malondialdehyde. These results suggest that lipid peroxidation of microsomal membranes or damage to the heme moiety should be excluded as contributing factors in the hemoprotein loss. TPTA also produced a concentration-related functional inactivation of cytochrome P-450 that was most pronounced in betaNAF-exposed microsomal preparations, as denoted by a striking reduction in the ethoxyresorufin O-deethylase (EROD) activity (IC50 = 0.088 mM). In contrast, the activities of cytochrome P-450-independent microsomal enzymes such as NADPH cytochrome c reductase and indophenyl acetate esterase (IPA-EST) were not markedly affected even by 0.5 mM TPTA (-30%). As assessed by Lineweaver-Burk plots, the mechanism of inhibition appeared to be noncompetitive for IPA-EST and of mixed type (competitive-noncompetitive) for EROD. Among sulfhydryl-containing compounds, dithiothreitol was considerably more effective than albumin and reduced glutathione in preventing cytochrome P-450 inactivation and even was able to partially reverse the hemoprotein damage when added after TPTA; glycerol, which is known to protect the hydrophobic environment of cytochrome P-450, was as effective as albumin. This study indicates that TPTA behaves as an almost specific and powerful in vitro inhibitor of cytochrome P-450-dependent monooxygenases, apparently through the interaction with critical sulfhydryl groups of the hemoprotein.     

利福定和利福平对小鼠肝微粒体酶的影响比较

利福定和利福平具有相似的化学结构,为了比较二者对肝微粒体酶的影响,我们考察了它们对小鼠体内安替比林血浆水平、戊巴比妥钠睡眠时间和肝脏重量的影响,测定了肝微粒体蛋白和细胞色素P-450含量及两种酶活性的改变,用凝胶电泳法比较了微粒体多肽组份相对含量的变化。结果表明,利福平在这些方面均表现出显著的肝微粒体酶诱导作用,而利福定则否。这提示利福定可能在临床很少导致严重的药物相互作用发生。     

Inhibition of hepatic microsomal drug-metabolizing enzymes by imperatorin

The effect of imperatorin on hepatic microsomal mixed function oxidases (MFO) was investigated. On acute treatment, imperatorin (30 mg/kg, i.p.) caused a significant reduction in activities of hepatic aminopyrine N-demethylase, hexobarbital hydroxylase and aniline hydroxylase as well as cytochrome p-450 content in rats and mice. Kinetic studies on rat liver enzymes revealed that imperatorin appeared to be a competitive inhibitor of aminopyrine N-demethylase (Ki, 0.007 mM), whereas a non-competitive inhibitor of hexobarbital hydroxylase (Ki, 0.0148mM). Imperatorin also inhibited non-competitively aniline metabolism (Ki, 0.2mM). Imperatorin binds to phenobarbital-induced cytochrome p-450 to give a typical type 1 binding spectrum (max. 388nm, min 422nm). Multiple administrations of imperatorin (30 mg/kg, i.p. daily for 7 days) to mice shortened markedly the duration of hexobarbital narcosis and increased activities of hepatic aminopyrine N-demethylase and hexobarbital hydroxylase and the level of cytochrome p-450 whereas aniline hydroxylase activity was unaffected.     

Inhibition of hepatic microsomal lipid peroxidation by endogenous glycogen in the rat

The effect of endogenous glycogen on lipid peroxidation was examined in hepatic microsomes from rats. Microsomes were prepared to retain endogenous hepatic glycogen (Pg+) or to minimize it (Pg-). The indices of lipid peroxidation examined included the rate of NADPH-dependent formation of malondialdehyde (MDA) and the concomitant destruction of cytochrome P-450 and decline in the linearity of benzphetamine N-demethylase activity in microsomes. Cytochrome P-450 was destroyed during benzphetamine N-demethylation in microsomes with the loss being more extensive in Pg- than in Pg+. The destruction of cytochrome P-450 and the concomitant loss in linearity of benzphetamine N-demethylation in Pg- were prevented by added EDTA. Added linoleic acid hydroperoxide (LAHP) also caused a time-dependent loss of cytochrome P-450 in microsomes with the rate being greater in Pg- than in Pg+. The results show that glycogen inhibits hepatic microsomal lipid peroxidation and suggest that variations in glycogen content may contribute to disparities in in vitro oxidative activities between different microsomal samples. Such disparities may be minimized by the removal of glycogen during the preparation of microsomes and then supplementing the incubation mixtures with EDTA. The in vivo relevance of the observed antioxidant effect of glycogen is discussed in terms of the possible modulation by the polysaccharide of hepatotoxicity by agents whose effects may be mediated by lipid peroxidation.     

Comparison of the effects of piperine administered intragastrically and intraperitoneally on the liver and liver mixed-function oxidases in rats

Piperine, a major pungent constituent of black and red peppers, was administered to rats intragastrically and intraperitoneally to study whether it alters the activities of hepatic mixed-function oxidases (MFO) and serum enzymes as specific markers of hepatotoxicity. An intragastric dose of 100 mg/kg of piperine to adult, male Sprague-Dawley rats caused an increase in hepatic microsomal cytochrome P-450 and cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h following treatment. On the other hand, a 10 mg/kg dose given i.p. exhibited no effect on the activities of the aforementioned parameters of the hepatic drug-metabolizing enzyme system. However, when the intragastric and intraperitoneal doses were increased to 800 mg/kg and 100 mg/kg, respectively, the black pepper alkaloid produced a significant decrease in the levels of cytochrome P-450, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h after treatment. None of the treatments significantly elevated the activities of serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and isocitrate dehydrogenase (ICD), suggesting that piperine is not a hepatotoxic agent.     

Potentiation of aflatoxin B1 induced hepatotoxicity in male Wistar rats with ethanol pretreatment

The interaction of ethanol and aflatoxin B1 (AFB1)-induced hepatotoxicity was studied in male Wistar rats using the activity of plasma GOT and GPT, liver triglyceride and histopathologic changes of liver necrosis as indices. Pretreatment of four oral doses of ethanol (4.0 g/kg BW each) at 48, 45, 24 and 21 hrs prior to AFB1 (0.5 to 2.0 mg/kg BW) single i.p. administration caused a significant increase in the activity of PGOT (6 folds) and PGPT (5 folds), liver triglycerides (2 folds) and severity of liver necrosis at 48 hrs after AFB1 administration. Ethanol pretreatment potentiated AFB1-induced hepatotoxicity by increasing MFO enzymes, aniline hydroxylase and p-nitroanisole-O-demethylase activity and lipid peroxidation, and decreasing in cytochrome b5, epoxide hydrolase activity and hepatic glutathione content. However, it did not cause any significant change in the activity of NADPH-cytochrome c reductase and glutathione-S-transferase and cytochrome P-450. These results suggest that potentiation of ethanol pretreatment on AFB1-induced hepatotoxicity may be due to an increase in the metabolic formation of AFB1-2, 3-oxide and subsequent binding to DNA.