Induction Of CYP2E1 Research Articles

Characterization of cytochrome P450 2E1 activity by the [<sup>14</sup>C]nitrosodimethylamine breath test

The objective of this study was to measure the rate of demethylation of nitrosodimethylamine in vivo in the rat and determine its value to assess CYP2E1 activity in intact animals. Nitrosodimethylamine labeled with 14C on both methyl groups was administered to rats and exhaled 14CO2 was collected during 2-3 h. The nitrosodimethylamine breath test was increased by inducers of CYP2E1, such as ethanol (+139%) and 4-methylpyrazole (+115%), and decreased by the inhibitor diallyl sulfide (-53%). In addition, the nitrosodimethylamine breath test was not changed significantly by inducers specific for other cytochrome P450 such as beta-naphthoflavone, dexamethasone, and phenobarbital. The specificity of the induction by 4-methylpyrazole and of the inhibition by diallyl sulfide for CYP2E1 was determined using the [14C]caffeine (CYP1A2), [14C]aminopyrine (CYP2C11), and [14C]erythromycin (CYP3A2) breath tests. 4-Methylpyrazole treatment caused a small increase of the caffeine (+33%) and aminopyrine (+9%) breath tests and no change of the erythromycin breath test. Diallyl sulfide treatment led to a small decrease of the caffeine breath test (-33%) and of the aminopyrine breath test (-13%) but a 23% increase of the erythromycin breath test. It is concluded that the [14C]nitrosodimethylamine breath test is useful to assess CYP2E1 activity in vivo in the rat.

Minimal Effects of Acrylonitrile on Pulmonary and Hepatic Cell Injury Enzymes in Rats with Induced Cytochrome P450

ABSTRACTAcrylonitrile (AN) has many industrial applications but is a known carcinogen in animals and a suspect human carcinogen. Its toxicity is generally associated with its bioactivation, the initial step of which is epoxidation by cytochrome P450. While the hepatotoxicity and pneumotoxicity of AN in naive rats is generally low, the purpose of this study was to investigate the pneumotoxicity and hepatotoxicity of AN in adult male Sprague-Dawley rats and evaluate interactions with agents that may alter its metabolism. Five agents, phenobarbital, β -naphthoflavone, pyridine, ethanol, and acetone, were administered prior to AN as inducers of CYP2B, CYP1A, and CYP2E1. Pneumotoxicity was measured as increases in y-glutamyltranspeptidase (GGT) and lactate dehydrogenase (LDH) in bronchoalveolar lavage fluid (BALF). Hepatotoxicity was measured as increases in serum sorbitol dehydrogenase (SDH). AN (1 mmol/kg ip) had little effect on liver or lung, even when given following most of the inducing agents. AN (1.5 mmol/kg) caused an increase in GGT, but had little effect on SDH or LDH. Acetone plus AN caused an increase in mortality and some indication of pneumotoxicity, but lung and liver were histologically normal. Thus AN alone even at a high dose had no effect on the liver or lung and minimal effects following induction of cytochrome P450 by acetone.

Expression and Distribution of Cytochrome P450 Enzymes in Male Rat Kidney: Effects of Ethanol, Acetone and Dietary Conditions

Ethanol, acetone, diet and starvation, known modulators of the hepatic cytochrome P450 (CYP)-dependent microsomal monooxygenase system, were assessed for their effects on cytochrome P450 isozyme content and monooxygenase activities in the male rat kidney. In acute experiments, rats were either treated with acetone, fasted or given a combination of the two treatments. Acetone treatment alone induced CYP2E1-dependent p-nitrophenol hydroxylase activity in kidney microsomes by 8-fold. This was accompanied by a 6-fold increase in CYP2E1 apoprotein as determined by Western blot analysis. There was, however, no significant increase in steady-state levels of CYP2E1 mRNA as measured by Northern blot analysis. Starvation also induced CYP2E1 apoprotein in the kidney and, as has been reported previously in the liver, had a synergistic inductive effect with acetone. CYP2B and CYP3A apoproteins were also induced by acetone, starvation and starvation/acetone combinations in the kidney. Immunohistochemical analysis revealed localization of CYP2E1 and CYP2B principally in the cortex associated with tubular cells. This distribution was maintained upon starvation/acetone treatment. Two induction experiments were performed in which the ethanol was administered as part of a system of total enteral nutrition (TEN). A short-term study was conducted in which ethanol was administered for 8 days in two liquid diets of different composition, and a chronic experiment was performed in which ethanol was administered for 35 days. A diet-independent 6-fold increase in CYP2E1 apoprotein was observed in the short-term experiment. Expression of CYP3A and CYP2A cross-reactive apoproteins in kidney microsomes appeared to be affected by alterations in diet but, were unaffected by ethanol treatment. In the chronic 35-day ethanol exposure experiment, CYP2E1 apoprotein was also elevated 6-fold and this was found to be accompanied by a significant 3-fold increase in CYP2E1 mRNA. In the same study, no ethanol effects were apparent on expression of CYP2B and CYP3A apoproteins. Thus, acetone induced a variety of renal cytochrome P450 forms in addition to CYP2E1, while ethanol appeared to be a much more specific renal CYP2E1 inducer. Furthermore, as reported in the liver, acetone and ethanol appeared to induce CYP2E1 in the kidney by different mechanisms.

CYP2E1 inducers modulate valproic acid toxicity and induces 4-ene-valproic acid apoptosis in Hep G2 cells

CYP2E1 inducers modulate valproic acid toxicity and induces 4-ene-valproic acid apoptosis in Hep G2 cells

Impact of long-term ethanol consumption on CYP1A2 activity.

Ethanol is a well-known inducer of CYP2E1; whether or not it is an inducer of other cytochromes has not been investigated systematically. The aim of our study was to evaluate the impact of ethanol consumption on the activity of CYP1A2, which has been shown to be influenced by drugs (inhibited or induced). We evaluated CYP1A2 activity by the ratio of the molar urinary concentrations of the three end products of paraxanthine demethylation of caffeine to the molar concentration of a paraxanthine 8-hydroxylation product. This urinary metabolite ratio has previously been shown to correlate with caffeine clearance. The caffeine metabolites were measured in urine collected during the 3 hours after oral administration of 200 mg caffeine. The caffeine test was performed in 12 smokers (> 25 cigarettes/day) and 12 nonsmokers, all of whom were alcoholic inpatients (daily intake > 100 mg absolute ethanol), within the first 3 days of their hospital stay and after 14 days of abstinence from ethanol. In alcoholic patients who were smokers the molar urinary concentration ratio was 3.14 +/- 0.97 before withdrawal and 4.01 +/- 0.92 after 14 days of abstinence from ethanol. In contrast, in alcoholic patients who were nonsmokers it was 2.62 +/- 0.95 and 2.18 +/- 0.96 before and after withdrawal, respectively. In volunteers who were smokers the molar urinary concentration ratio was 5.02 +/- 1.51, whereas in volunteers who were nonsmokers it was 3.22 +/- 1.46. Our results confirm the well-known induction of CYP1A2 activity by tobacco smoking and show that this induction is masked by long-term ethanol consumption.

Noninvolvement of CYP 2E1 in the (ω-1)-hydroxylation of fatty acids in rat kidney microsomes

Pyrazole, acétone, and ethanol are known to induce cytochrome P450 2E1 (CYP 2E1) and fatty acid (co-l)-hydroxylation in rat liver microsomes. However, the nature of the P450 enzyme involved in this (co-l)-hydroxylation has not been clearly established in extrahepatic tissues such as kidney. Four enzymatic activities (hydroxylations of chlorzoxazone, 4-nitrophenol, and two fatty acids) were assayed in kidney microsomal preparations of rats treated with CYP 2E1 inducers. Per os treatment resulted in large increases (threefold to fivefold) in the chlorzoxazone and 4-nitrophenol hydroxylations, and up to a ninefold increase when ethanol was administered by inhalation. However, neither the ω-hydroxylation nor the (ω-1)-hydroxylation of fatty acids was modified. Immunoinhibition specific to CYP 2E1 did not significantly decrease the ω and (ω-1)-lauric acid hydroxylations, while the polyclonal anti-CYP4A1 antibody inhibited in part both the ω- and (ω-1)-hydroxylations. Chemical inhibitions using either CYP 2E1 competitive inhibitors (such as chlorzoxazone, DMSO, and ethanol) or P450 mechanism-based inhibitors (such as diethyldithiocarbamate and 17-octadecynoic acid) led to a partial inhibition of the hydroxylations. All these results suggest that fatty acid (ω-1)-hydroxylation, a highly specific probe for CYP2E1 in rat and human liver microsomes, is not mediated by CYP2E1 in rat kidney microsomes. In contrast to liver, where two different P450 enzymes are involved in fatty acid co- and (co-1)-hydroxylations, the same P450 enzyme, mainly a member of the CYP4A family, was involved in both hydroxylations in rat renal microsomes.

Gadolinium chloride blocks alcohol-dependent liver toxicity in rats treated chronically with intragastric alcohol despite the induction of CYP2E1.

Hepatic CYP2E1 is induced in several models of alcohol administration, but clinically relevant pathology is only observed in rats in a model involving the continuous intragastric administration of an ethanol-containing, corn oil-based, high-fat diet. The level of CYP2E1 correlates with the degree of liver pathology in the intragastric feeding model, which leads to the hypothesis that radical production by CYP2E1 is responsible for the pathology. Destruction of the Kupffer cells with gadolinium chloride (GdCl3) prevented the development of ethanol-dependent pathology and decreased the production of radicals that appeared in the bile of intragastrically alcohol-fed rats. If the induction of CYP2E1 and subsequent formation of oxidant species by the enzyme is causative in the ethanol-dependent hepatic pathology, then protection by GdCl3 could be due an inhibition of CYP2E1 induction. In the current study, ethanol-administration for 4 wk produced marked steatosis, necrosis, and inflammation not seen in control rats. Immunochemically, CYP2E1 was induced 5- to 6-fold in microsomes from the ethanol-treated animals. Rates of p-nitrophenol and chlorzoxazone hydroxylation were elevated approximately 3-fold, consistent with CYP2E1 induction. When GdCl3 was administered with ethanol, there was a decrease of approximately 80% in Kupffer cell receptor expression, and there was a significant decrease in hepatic pathology, which confirms previous studies. However, in the ethanol and GdCl3-treated animals, there was no significant decrease in the induction of CYP2E1. CYP2E1 was elevated approximately 5-fold, as estimated by immunoblot analysis, and rates of p-nitrophenol and chlorzoxazone hydroxylation were elevated 3- to 4-fold in ethanol + GdCl3-treated rats. Thus, these results clearly dissociate the induction of CYP2E1 by intragastric infusion of ethanol from the generation of early alcohol-induced liver disease. It is concluded that Kupffer cells rather than CYP2E1 play the major role in the initiation of hepatocyte damage caused by alcohol.

Evidence for a Tissue-Specific Induction of Cutaneous CYP2E1 by Dexamethasone

We studied in mouse the effect of topical application of dexamethasone or salicylic acid, on CYP2E1 and CYP3A expression (proteins and/or mRNA) in liver and skin. Dexamethasone was also administered by intraperitoneal injection. Topical application or intraperitoneal injection of dexamethasone increased cutaneous CYP2E1 (8 and 4-fold respectively) whereas the hepatic level of this isoform showed a slight decrease and hepatic CYP3A expression was increased (3-fold). Cutaneous CYP2E1 was increased (3-fold) after topical treatment by salicylic acid. This compound had no effect on hepatic CYP3A and CYP2E1 expression. Cutaneous CYP3A (protein and mRNA) was not detectable in all groups (control or treated animals). Dexamethasone and salicylic acid increased cutaneous CYP2E1 mRNA level (2.5 and 1.4-fold respectively). In conclusion, dexamethasone and salicylic acid induced cutaneous CYP2E1 protein and mRNA level. Cutaneous CYP2E1 induction by dexamethasone is a tissue-specific process.

Variation in Induction of Human Placental CYP2E1: Possible Role in Susceptibility to Fetal Alcohol Syndrome?

Fetal alcohol syndrome occurs in less than 10% of women who drink heavily during pregnancy. One potential mechanism for this intersubject variation is differences in placental alcohol metabolism. Alcohol dehydrogenase is present at low concentrations in the placenta and is not inducible. CYP2E1 has not been found in human placentas at early gestation time points or in random term placentas. Hepatic CYP2E1 is induced by alcohol and other environmental agents, but induction varies among heavy drinkers and may be genetically controlled. To test whether CYP2E1 is induced in placenta by heavy drinking during pregnancy, we performed a Western blot analysis on placental microsomes from women (n= 8) whose periconceptional average daily absolute alcohol intake was greater than 1 ounce. Using anti-human CYP2E1, bands consistent with CYP2E1 were identified in six samples, although considerable variation among individuals was observed. Among drinking mothers, offspring head size was smaller among those with placental CYP2E1 (p= 0.04). The association between the presence of the protein and smaller birth weight and birth length was equivocal (p= 0.09). Our data are consistent with placental CYP2E1 being inducible by drinking, but with induction being variable among heavy drinking women. We speculate intersubject variation in induction may have a genetic basis and may play a role in susceptibility to alcohol related defects.

Lipid Peroxidation, CYP2E1 and Arachidonic Acid Metabolism in Alcoholic Liver Disease in Rats

The role of cytochrome P450 metabolism of fatty acids and lipid peroxidation in the alterations of the fatty acid composition of the liver and liver pathology was investigated. The CYP2E1 inhibitors partially prevented CYP2E1 induction by ethanol and completely blocked lipid peroxidation. However, the liver pathology induced by ethanol was only partially prevented as was the decrease in arachidonic acid in total liver lipid, triglycerides and cholesterol esters. This means that liver peroxidation induced by ethanol can not completely account for the liver pathology or the decrease in arachidonic acid caused by ethanol. Lauric acid ω-1 hydroxidation by the liver microsomes in vitro was increased by ethanol and partially blocked by CYP2E1 inhibitors. However, although ethanol feeding increased the total hydroxidation and epoxidation of arachidonic acid, these were not inhibited by CYP2E1 inhibitors. Thus the ethanol-induced arachidonic acid depletion is not likely due to CYP2E1 metabolism of arachidonic acid, since the severity of liver pathology correlated negatively with the decrease in arachidonic acid in the ethanol-fed rats. The increase in its metabolism by microsomes and decrease in synthesis may be an important mechanism of ethanol-induced liver injury.

Pharmacokinetics and Metabolism of Vinyl Fluoridein Vivoandin Vitro

Vinyl fluoride (VF) is an inhalation carcinogen at concentrations of 25 ppm or greater in rats and mice. The main neoplastic lesion induced in rodents was hepatic hemangiosarcomas, and mice were more sensitive than rats. In a first set of experiments, groups of three rats or five mice were exposed to VF in a closed-chamber gas uptake system at starting concentrations ranging from 50 to 250 ppm. Chamber concentrations of VF were measured every 10–12 min by gas chromatography. Partition coefficients were determined by the vial equilibration technique and used as parameters for a physiologically based pharmacokinetic (PBPK) model. Mice showed a higher whole-body metabolic capacity compared to rats (Vmaxc= 0.3 vs 0.1 mg/hr-kg). Both species had an estimatedKmof ≤0.02 mg/liter. The specificity for the oxidation of VFin vivowas determined by selective inhibition or induction of CYP 2E1. Inhibition with 4-methylpyrazole completely impaired VF uptake in rats and mice, whereas induction with ethanol (rats only) increased the metabolic capacity by two- to threefold. The pharmacokinetics of VF were also investigatedin vitro.Microsomes from rat and mouse liver were incubated in a sealed vial with VF and an NADPH- regenerating system. Headspace concentrations (10–300 ppm) were monitored over time by gas chromatography. Consistent with thein vivodata, VF was metabolized faster by mouse microsomes than by rat microsomes (Vmax= 3.5 and 1.1 nmol/hr-mg protein, respectively). The rates of metabolism by human liver microsomes were generally in the same range as those found with rat liver microsomes (Vmax= 0.5–1.3 nmol/hr-mg protein), but one sample was similar to mice (Vmax= 3.3 nmol/hr-mg protein). Metabolic rates in human microsomes were found to correlate with the amount of CYP 2E1 as determined by Western blotting and by chlorzoxazone 6-hydroxylation. It is concluded that the greater metabolic capacity of mice for VF bothin vivoandin vitromay contribute to their greater susceptibility to tumor formation. CYP 2E1 is clearly the main isozyme involved in the oxidation of VF in all species tested. VF pharmacokinetics and metabolism in humans may depend upon the interindividual variability in the expression level of CYP 2E1. The excellent correspondence betweenin vivoandin vitrokinetics in rodents improves substantially the degree of confidence for humanin vivopredictions fromin vitrodata.

Kinetics and metabolism of 1,4-dichlorobenzene in male Wistar rats: no evidence for quinone metabolites

The biotransformation and kinetics of 1,4-dichlorobenzene (1,4-DCB) were studied in male Wistar rats at three oral dose levels (10, 50 and 250 mg/kg). The effect of induction of CYP2E1 by isoniazid on the kinetics and biotransformation was determined. Excretion was predominantly via the urine (78–85%) and to a small extent via the faeces (2–5%). The relative contributions of these routes were not dose dependent. Excretion via the bile ranged from less than 5% at the low dose level to 30% at the high dose level. The major biliary metabolite was the glucuronide of 2,5-dichlorophenol (2,5-DCP). The time point at which the plasma concentrations of the parent compound and the metabolites were maximal ( T Cmax) as well as the maximum concentrations ( C max) increased with higher dose level. Induction by isoniazid resulted in a faster urinary elimination, whereas T Cmax and C max were lower for induced rats. In addition, the area under the blood curve (AUC) was smaller and total clearance was higher for induced rats. 1,4-DCB was mainly metabolized to 2,5-DCP (ca. 90%), which was detected in the urine as its sulfate (50–60%), glucuronide (20–30%) and the free form (5–10%). Minor metabolites were the N-acetyl-cysteine- S-dihydro-hydroxy-1, 4-dichlorobenzene and the corresponding dehydrated N-acetyl-cysteine- S-1,4-dichlorobenzene, which comprised ca. 10% of total metabolites. No hydroquinones were observed for the male Wistar rat, not even under conditions of induced oxidative metabolism.

Characterisation of the toxic metabolite(s) of naphthalene

The toxicity of naphthalene and its metabolites has been investigated in vitro. Both naphthalene and its metabolite 1-naphthol were bioactivated by human hepatic microsomes to metabolite(s) which were toxic to mononuclear leucocytes (MNL). However, 1-naphthol was more cytotoxic than naphthalene (49.8 ± 13.9% vs. 19.0 ± 10.0% cell death; P < 0.01), indicating that the toxicity of naphthalene is dependent on the bioactivation of 1-naphthol. CYP2E1-induced rat liver microsomes increased metabolism of naphthalene by 13% compared to control microsomes with a concomitant increase in both 1-naphthol and dihydrodiol formation. The cytotoxicity of naphthalene but not of 1-naphthol was increased by CYP2E1 induction, indicating that separate enzymes are involved in the bioactivation of 1-naphthol. The metabolites of 1-naphthol, 1,2-naphthoquinone (51.4 ± 6.6% cell death) and 1,4-naphthoquinone (49.1 ± 3.4% cell death) were directly toxic to MNL and depleted glutathione to 1.0% of the control levels. Both quinones were also genotoxic to human lymphocytes. In contrast, the primary metabolite of naphthalene, the 1,2-epoxide (0–100 μM) was neither cytotoxic nor genotoxic, and did not deplete glutathione. In conclusion, our data suggests that the cytotoxicity and genotoxicity of naphthalene is associated with the formation of quinones from 1-naphthol rather than naphthalene-1,2-epoxide.

Dichloroacetic Acid Treatment Increases Hepatic CYP2E1 in Male and Female Rats

Previously, we showed that the pretreatment of dichloroacetic acid (DCA) increased CHCl3-induced hepatotoxicityin vivoand CHCl3metabolismin vitroin both male and female rats. The effects of DCA on hepatic cytochrome P450-dependent monooxygenases were studied in this experiment. Male and female Sprague–Dawley rats were treated with DCA (each 2.45 mmol/kg) three times (9AM, 4PM, and 9AM) and hepatic microsomes were prepared 3 hr after the last treatment (the same procedure as previous studies). After DCA treatment, the total content of cytochrome P450 (0.67 nmol/mg protein vs 0.79) and the activity of NADPH-dependent cytochrome P450 reductase (227 nmol/mg protein/min vs 332) were significantly increased in female rats, but they were unchanged in males (0.99 vs 0.98 for P450; 315 vs 311 for reductase). Induction of CYP2E1 was observed in both sexes, evidenced as increased activities of aniline andp-nitrophenol hydroxylases and increased CYP2E1 protein amount determined by immunoblot assay. In contrast, the CYP2B-related activity (dealkylation of pentoxyresorufin) and immunoreactive protein did not increase after DCA treatment in either males or females. The activity of ethoxyresorufin dealkylase was decreased in DCA-treated males compared to their controls (310 pmol/mg protein/min vs 229,p< 0.05), but it was not significantly affected in females. These data demonstrate that DCA treatment of both male and female rats altered the population of hepatic cytochrome P450. The results support the hypothesis that DCA increases CHCl3metabolism, and therefore hepatotoxicity, by inducing CYP2E1.

Acetaminophen metabolism in patients with different cytochrome P-4502E1 genotypes.

Cytochrome P-450 (CYP) 2E1 is the major ethanol-oxidizing enzyme of the nonalcohol dehydrogenase metabolic pathway in the liver. Recently, the presence of genetic polymorphisms of this enzyme was confirmed. In this study, to clarify the influence of CYP2E1 genotype on alcohol metabolism, we analyzed acetaminophen metabolism in subjects with different CYP2E1 genotypes. In normal subjects, a half-life of acetaminophen from blood was the longest in type A (c1/c1) and was the shortest in type C (c2/c2). The elimination rate in type C was more than twice that of type A and type B (c1/c2). In type A, both half-life and elimination rate of acetaminophen were not different between patients with noncirrhotic alcoholic liver disease within 1 week after abstinence and in normal subjects. In one patient with minimal change, there were no differences in both half-life and elimination rate within 1 and 6 weeks after abstinence. On the other hand, in type B, half-life was shorter and the elimination rate was greater in alcoholic noncirrhotic patients within 1 week after abstinence than in alcoholic patients with type A and in normal subjects with type B. In type B, half-lives were shorter, and the elimination rates were greater in patients with alcoholic liver disease within 1 week after abstinence than 4 to 6 weeks after abstinence. These results suggest the possibility that alcohol metabolism in individuals with the c2 gene may be greater than those with the c1 gene, and that the induction of CYP2E1 by ethanol in type B may occur more markedly than that in type A, although the sample number is too small to obtain final conclusions.

Transfer of PCBs via lactation simultaneously induces the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf in neonates

At the first day of lactation, maternal rats were injected with a single i.p. dose of 100 or 250 mg/kg body weight of a mixture of polychlorinated biphenyls (Aroclor 1254). This treatment caused significant increases in both material and neonatal hepatic cytochrome P-450, cytochrome b 5, and cytochrome-c-(P-450) reductase. Transfer of PCBs via lactation resulted in significant increases in hepatic enzyme activities catalysed by neonatal CYP1A1, CYP1A2, CYP2B1, CYPA1, and CYP2E1 using a variety of substrates. In contrast, the metabolism of dimethylnitrosamine and aminopyrine was only marginally (up to 2-fold) increased in maternal animals four days post treatment. Further measurements showed significant increases in maternal and neonatal epoxide hydrolase, glutathione-S-transferase, and UDP-glucuronyl transferase activities, thus suggesting a coordinated response in maternal and neonatal oxidative and post-oxidative drug metabolism. Western blot analysis provided evidence for an induction of CYP1Al, CYP1A2, CYP2A1, CYP2B1, CYP2E1, CYP 3A1, and CYP4A1 in both maternal and neonatal liver, albeit at varying intensities. However, PCBs did not modulate the expression of maternal and neonatal CYP2C6, and at the higher dose the expression of neonatal CYP2E1 was significantly reduced. Northern blot analysis provided further evidence for significant increases in maternal and neonatal hepatic CYP1A1, CYP1A2, CYP2B1, and CYP2E1 mRNA, but reduced amounts of CYP2C7 and CYP4A1 mRNA. Additional Northern blot hybridization experiments may suggest an increased expression of the protooncogenes c-Ha-ras and c-raf in the mother and the neonate upon treatment of maternal rats with Aroclor 1254. Lactation itself may result in an increased expression of the latter protooncogenes, but the mRNA of the protooncogenes c-erb A and c-erb B was not detected in any of the tissues examined.

Differences in Rates of Benzene Metabolism Correlate with Observed Genotoxicity

Benzene (BZ) requires oxidative metabolism via cytochrome P450 2E1 (CYP 2E1) to exert its hematotoxic and genotoxic effects. Male mice are two- to threefold more sensitive to the genotoxic effects of BZ as measured by micronuclei induction and sister chromatid exchanges. The purpose of our study was to investigate sex-related differences in the metabolism of BZ, phenol (PHE) and hydroquinone (HQ) in order to understand the metabolic basis for sex-dependent differences in BZ genotoxic susceptibility in mice. Rates of BZ oxidation were quantitated using closed chamber gas uptake studies with male and female B6C3F1 mice exposed to initial low (400–500 ppm), intermediate (1200–1300 ppm), and high (2600–2800 ppm) BZ concentrations. Acetone-pretreated and diethyldithiocarbamate-pretreated male mice were also studied to determine the extent to which induction and inhibition of CYP 2E1, respectively, would alterin vivoBZ oxidation rates. Elimination of PHE and HQ from blood was also compared in male and female mice to complement previously reported data on sex-related differences in urinary excretion of conjugated metabolites following iv administration of PHE. Based on PBPK model analysis, the optimized rate of metabolism (Vmax) of BZ was almost twofold higher in male mice (14.0 μmol/hr-kg) than in female mice (7.9 μmol/hr-kg); both male and female mice gas-uptake data were well fit with aKMof 3.0 μM. Pretreatment of male mice with 1% acetone in drinking water for 8 days to specifically induce CYP 2E1 enhanced the rate of BZ oxidation by approximately fivefold (Vmax= 75 μmol/hr-kg), while diethyldithiocarbamate pretreatment (320 mg/kg ip 30 min prior to uptake study) completely inhibited BZ oxidation (Vmax= 0 μmol/hr-kg). Thus, both pretreatment regimens are potentially useful investigative tools to study the metabolic basis for benzene toxicity. Elimination of PHE from blood was significantly faster in male mice, while elimination of HQ did not differ between male and female mice. Previous data indicated male mice produce more of the oxidized and conjugated metabolite, HQ glucuronide, after PHE administration, suggesting that HQ production from PHE is greater in male mice. Taken together, these data support the hypothesis that the greater sensitivity of male mice to the genotoxic effects of BZ compared to females is a function of greater oxidative metabolism toward both BZ and PHE in male mice. These data also suggest that differences in hepatic human CYP 2E1 activity may be an important factor to consider when evaluating human risk for benzene-induced hematotoxic and genotoxic effects.

Induction of hepatic cytochrome P4502E1 in rats by acetylsalicylic acid or sodium salicylate

Studies were conducted on the mechanism of the ethanol-inducible cytochrome P450 (cytochrome P4502E1, CYP 2E1) induction by acetylsalicylic acid (ASA) or its metabolite salicylate (SAL). Many exogenous inducers of CYP 2E1 seem to increase CYP 2E1 by post-transcriptional activation without elevation of its mRNA level. Administration of a single high dose of ASA or SAL produces a significant increase in the activity of the hepatic microsomal p-nitrophenol hydroxylase in rats. Pretreatment of ASA-treated rats with a blocker of mRNA transcription, actinomycin D, or a blocker of protein synthesis, cycloheximide, markedly suppressed this enhanced activity of microsomal p-nitrophenol hydroxylase. The CYP 2E1 mRNA levels in livers of control rats and rats treated with ASA or SAL were measured by Northern blot analysis. Significantly elevated CYP 2E1 mRNA levels were measured in livers of treated rats compared with mRNA amounts of the control group. These data suggest that mRNA elevation seems to be characteristic for ASA induction, while other inducing agents show different patterns and mechanisms of activation.

Comparison of rat hepatic and pulmonary microsomal metabolism of benzene and the lack of benzene-induced pneumotoxicity and hepatotoxicity

Since little is known about the toxicity of benzene in the lung or if the lung is capable of metabolizing benzene, the ability of the lung to bioactivate or detoxify benzene and the pneumotoxicity of benzene were determined. While overall metabolism was lower, pulmonary microsomes converted benzene (17.5 μM) to hydroquinone, considered to be a marker for benzene toxicity, in proportionately greater amounts than did hepatic microsomes. Treatment of rats with pyridine, an inducer of CYP2E1, enhanced hepatic microsomal metabolism of benzene, although benzene, which is also considered to be an inducer of CYP2E1, did not. Neither pyridine nor benzene treatment induced the pulmonary microsomal metabolism of benzene. When hepatic and pulmonary microsomes from control and pyridine-treated rats were incubated with benzene (17.5 μM) and the CYP2E1 inhibitor, diethyldithiocarbamate, benzene metabolism was significantly inhibited, indicating that CYP2E1 is the predominant cytochrome P-450 isozyme involved in hepatic and pulmonary metabolism in microsomes from control and pyridine-treated rats. Benzene (600 mg/kg body weight i.p.) did not cause significant lung cell damage as determined by measurement of γ-glutamyltransferase and lactate dehydrogenase in bronchoalveolar lavage fluid. Induction of CYP2E1 and CYP2B1/2 with pyridine and phenobarbital, respectively, did not alter this lack of effect. Thus, the primary concern about benzene and the lung should focus on benzene metabolism as opposed to acute toxicity.

Hematotoxic interactions: occurrence, mechanisms and predictability

The available data on the binary chemical interactions involving hematotoxicants, particularly organic chemicals causing a reduction in either the number of white/red blood cells or the capacity of hemoglobin to transport oxygen, are limited. These observations are limited to investigations in rodents of the enhancement or attenuation of the hematotoxicity of benzene, dichloromethane and dimethylanilines following prior administration of inducers of CYP 2E1 or co-administration of substrates for this isoenzyme. The relevance of these data on interactions for humans exposed at low concentrations can be assessed only when the mechanism of interaction is understood at a quantitative level, and incorporated within a physiological modeling framework. The present study exemplifies the predictability of the magnitude of binary chemical interactions in humans exposed to low concentrations, by developing a physiological model of the modulation by toluene of dichloromethane-induced carboxyhemoglobinemia. Consistent with the basic biochemical principles, this modeling exercise suggests that, with competitive metabolic inhibition mechanism, the threshold for binary chemical interactions will follow a downward trend with increasing number of substrates or structurally-similar substances in a mixture. The use of this kind of mechanistic models, along with data from descriptive chemical interaction studies, will form the very basis of mechanistic risk assessment methods for complex chemical mixtures.