Literature Review

Induction of cytochrome P450 by toluene

Article· Literature ReviewinInternational Journal of Biochemistry 26(12):1333-40 · January 1995with 42 Reads
Abstract
At least six cytochrome P450 (P450) isoenzymes, including CYP1A1/2, CYP2A1, CYP2B1/2, CYP2C6, CYP2C11 and CYP2E1, are involved in the metabolism of toluene in rat liver. Toluene exposure induces CYP1A1/2, CYP2B1/2, CYP2E1 and CYP3A1, but decreases CYP2C11/6 and CYP2A1 in adult males. Both sex and age influence the induction of P450s by toluene: in general, the inductive effect is more prominent in younger than in older animals; in males than in females. Neonatal exposure to toluene causes significant changes in liver microsomal P450 dependent monooxygenase activities during the early stage of life, whereas the effects on the rats of more than 3 weeks of age are small. Although structurally related chemicals of toluene also influence similar hepatic P450 isoenzymes, the degree of CYP2B1/2 induction increases, whilst that of CYP2E1 decreases with increasing molecular weight and aliphatic moieties. Unlike liver, exposure to toluene does not influence the distribution of pulmonary or renal microsomal P450-related enzyme activity in rats. In humans, occupational exposure to toluene is so low that it could not lead to the induction of P450. However, the induction may be seen in toluene sniffers who are exposed to high concentrations.
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    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5 -flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10-760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0-9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30-3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons.
  • Article
    Cytochrome (CYP) P450 2E1 is clinically and toxicologically important and it is constitutively expressed in the liver and many other tissues. In contrast to many other CYP isoenzymes, indisputable evidence for a functionally important polymorphism of CYP2E1 in the human population is lacking. CYP2E1 metabolizes a wide variety of chemicals with different structures, in particular small and hydrophobic compounds, including potential cytotoxic and carcinogenic agents. In addition, chlorzoxazone and trimethadione metabolism are good CYP2E1 probes for liver disease in vivo and in vitro. In the future, methods for fully analysing the function of CYP2E1 using knockout mice will be established. This article reviews recent advances in our understanding of the role of human CYP2E1 in drug metabolism.
  • Article
    'Sick house syndrome' (SHS) is a health issue that closely resembles sick building syndrome (SBS) that had occurred in European countries. The aim of this review is to clarify the characteristics of SHS by reviewing previous reports rigorously. We propose the definition of SHS as "health impairments caused by indoor air pollution, regardless of the place, causative substance, or pathogenesis". Cases of SBS are reported to occur predominantly in offices and sometimes schools, whereas those of SHS are usually found in general dwellings. In many cases, SHS is caused by biologically and/or chemically polluted indoor air. Physical factors might affect the impairments of SHS in some cases. It is considered that symptoms of SHS develop through toxic, allergic and/or some unknown mechanisms. Psychological mechanisms might also affect the development of SHS. It is still unclear whether SBS and SHS are very close or identical clinical entities, mostly because a general agreement on a diagnostic standard for SHS has not been established. Previous research gradually clarified the etiology of SHS. Further advances in research, diagnosis, and treatment of SHS are warranted with the following measures. Firstly, a clinical diagnostic standard including both subjective and objective findings must be established. Secondly, a standard procedure for assessing indoor air contamination should be established. Lastly, as previous research indicated multiple causative factors for SHS, an interdisciplinary approach is needed to obtain the grand picture of the syndrome.
  • Article
    Full-text available
    Polymorphism and the induction/inhibition of drug-metabolizing enzymes, such as cytochrome P450, aldehyde dehydrogenase (ALDH), glutathione S-transferase (GST), N-acetyltransferase (NAT), and NAD(P)H-quinone oxidoreductase (NQO1), were reviewed in relation to susceptibility to disease and to inter-individual difference in biological monitorings. A number of genetic and acquired factors can influence the susceptibility of an individual to chemicals, creating a so-called predisposition. Most cases in which genetic factors were present resulted from polymorphism of drug-metabolizing enzymes. However, conflicting reports have appeared on the relationship between polymorphism and risk of disease; in some cases, biologically plausible mechanisms linking genotypes and disease are not yet in evidence. Current findings based on biological monitoring of chemicals are insufficient to evaluate the relationship between genetic polymorphism and acquired risk when exposure has occurred in an occupational area. Investigation of such situations has generated data implicating GSTT1, GSTM1, NAT2, and NQO1 polymorphisms in biological monitoring of some chemicals; the ALDH2 polymorphism is the likely link between the genotype and the metabolism of low molecular aliphatic aldehydes. Although this polymorphism is limited in the case of Japanese as well as other Asian subjects, the inhibitors of ALDH2 activity such as trichloroethylene may produce a false polymorphism of this gene. As to the effect of factors influencing acquired predisposition, such as ethanol intake, intake of low carbohydrate diet or diabetes, corroborative epidemiological studies may be further required.
  • Article
    Full-text available
    We investigated the expressional regulation of cytochrome P450 (CYP) 2D isoforms in rat brain by toluene. Toluene (10 mmol/kg) was injected intraperitoneally into male rats daily for 3 days. CYP2D4 level in the brain was determined by Western blotting. The level of CYP2D4 in the brain and the catalytic activity specific for this isoform were significantly increased by toluene administration. The expressions of CYP2D mRNAs in the brain were quantified by real time PCR. The mRNA level of CYP2D4 increased similar to the expression of the protein and enzymatic activity. These results showed that CYP2D4 was transcriptionally induced by toluene administration for 3 days. This is the first report that subacute exposure to toluene activated the expression of CYP2D4 mRNA in the brain. The response of CYP2D4 expression to toluene is thought to relates to the regulation of the physiological and pharmacological functions of CYP2D isoforms in the brain.
  • Article
    Short-term exposure to a high concentration (TWA > 100 ppm) of toluene can cause hepatotocixity and neurotoxicity in humans. Data on the effects of exposure to low levels of toluene, however, are controversial. In addition, few studies on the effects of toluene exposure on the autonomic nervous system have been conducted. Urine samples from 34 male factory workers in Taiwan who were exposed to low levels of toluene either intermittently (n = 13) or continuously (n = 21) were taken on a Monday morning after a 2-day hiatus and at the end of the workweek on Friday evening. Urinary hippuric acid levels were measured using high-performance liquid chromatography (HPLC). A complete blood work-up was also performed for each subject. The prevalence and severity of neurotoxic symptoms were investigated by a self-reported questionnaire, a neuropsychiatric battery, and sympathetic and peripheral nerve function tests. The mean value of urinary hippuric acid corrected for creatinine (Cr) was 0.34 ± 0.18 g/g Cr on Monday morning and 0.43 ± 0.26 g/g Cr on Friday evening. The difference in the mean value of urinary hippuric acid between the two periods (p < 0.01) and the odds ratio of impairment of sympathetic (OR = 4.13, p = 0.11) and peripheral nerves (OR = 6.94, p = 0.074) were higher in workers continuously exposed to toluene. In addition, workers who were continuously exposed to toluene had a lower mean platelet count (216 ± 41 × 10(6) /µL) than workers who were intermittently exposed (252 ± 40 × 10(6)/µL), (p = 0.018). Furthermore, there was a positive relationship between neurological abnormalities and a self-reported neuropsychiatric measurement (r = 0.35-0.66, p < 0.05) in all workers. These data suggest that continuous exposure to low levels of toluene may be associated with sympathetic and peripheral nerve dysfunction and sub-clinical hematological damage. Further research needs to be carried out regarding how chronic exposure to low-levels of toluene affects workers.
  • Article
    A panel of 17 hybridomas producing (MAbs) against human cytochrome P450 2E1 (h2E1) was generated by immunizing mice with baculovirus-expressed h2E1. All 17 hybridoma clones gave positive ELISA or immunoblots with either baculovirus-or vaccinia virus-expressed h2E1. Two of the latter were further developed due to their desirable characteristics. MAb 1-73-18 was found to be a powerful inhibitor of P450 h2E1; however, it did not yield a positive immunoblot. MAb 2-106-12 was found to be noninhibitory but formed a strong positive immunoblot with P450 h2E1. These MAbs to h2E1 were highly specific and did not recognize six other human P450s as tested with ELISA or immunoblot analyses. The MAbs to baculovirus-expressed h2E1 also reacted with h2E1 expressed from a vaccinia virus vector system as well as with microsomal fractions of human and acetone-treated rat liver. MAb 1-73-18 inhibited h2E1 enzyme activity catalyzing the metabolism of phenanthrene by 85%, p-nitroanisole by 90%, 4-methylanisole by 60-80%, toluene by 90%, and chlorzoxazone by 90%. The inhibitory MAb 1-73-18 is uniquely useful for determining the contribution of h2E1 to the metabolism of h2E1 substrates in human liver containing multiple P450s. The quantitatively determined contribution of h2E1 to the metabolism of the above substrates ranged from 25% to 75%. Thus, h2E1 was responsible for the following percentages of the total metabolism in human liver: p-nitroanisole (35%), phenanthrene (23%), methylanisole to cresol (25%), methylanisole to methoxybenzyl alcohol (12%), toluene (40%), and chlorzoxazone (72%). The MAb 2-106-12 forming a strong immunoblot is useful for determining the amount of h2E1 protein in a tissue. Thus the utility of the inhibitory and immunoblot positive MAbs is complementary and can determine both the contribution of h2E1 to the metabolism of specific substrates and the amount of h2E1 protein in human tissue. The analyses of metabolism with the inhibitory MAb 1-73-18 can be generalized and applicable to all h2E1 substrates.
  • Article
    Full-text available
    Among various human CYPs, CYP2E1 is of particular interest because of its involvement in the metabolic activation of many low molecular mass procarcinogens. CYP2E1 induction, which may be a consequence of genetic polymorphism or/and gene induction by xenobiotics, is the first step leading to the development of certain chemically-mediated cancers. The aim of this review is to outline the current knowledge on chemically-induced cancers through activation by CYP2E1, with emphasis on the association between polymorphisms of the CYP2E1 gene and incidence of different neoplasias. Literature searches of MEDLINE (1966 to July 2009) for English articles in CYP2E1-induced carcinogenesis were conducted. CYP2E1 genetic polymorphisms leading to enhanced CYP2E1 gene transcription have been associated with increased risk of development of malignant tumours, through increased biotransformation of procarcinogens. Likewise, long-term intake of CYP2E1 inducers, such as ethanol, isoniazid, various solvents and chemicals, also increase the probability of developing malignancy, especially for carriers of certain CYP2E1 alleles. Genetic screening for CYP2E1 'carcinogenic' polymorphisms and CYP2E1 phenotype determination of susceptible subjects, as well as the development of effective CYP2E1 inhibitors, could be a future perspective towards prevention of CYP2E1-mediated cancers.
  • Article
    Full-text available
    We conducted an inhalation toxicity test on the alternative animal model, Drosophila melanogaster, to investigate potential hazards of indoor air pollution. The inhalation toxicity of toluene and formaldehyde was investigated using comprehensive transcriptomics and computational behavior analyses. The ingenuity pathway analysis (IPA) based on microarray data suggests the involvement of pathways related to immune response, stress response, and metabolism in formaldehyde and toluene exposure based on hub molecules. We conducted a toxicity test using mutants of the representative genes in these pathways to explore the toxicological consequences of alterations of these pathways. Furthermore, extensive computational behavior analysis showed that exposure to either toluene or formaldehyde reduced most of the behavioral parameters of both wild-type and mutants. Interestingly, behavioral alteration caused by toluene or formaldehyde exposure was most severe in the p38b mutant, suggesting that the defects in the p38 pathway underlie behavioral alteration. Overall, the results indicate that exposure to toluene and formaldehyde via inhalation causes severe toxicity in Drosophila, by inducing significant alterations in gene expression and behavior, suggesting that Drosophila can be used as a potential alternative model in inhalation toxicity screening.
  • Article
    Freshwater clams Corbicula fluminea were exposed in aquariums to four doses of trichloroethylene-TCE-(1.56 up to 100 mg/1) or toluene-TOL-(7.5 up to 60 mg/1) for 5 days. At the end of exposure, components of (de)toxification metabolism of phases I and II, parameters related to oxidative stress and propionylcholinesterase activity were assayed. Determination of TCE and TOL concentrations in water revealed an important evaporative loss during the experiment, characteristic of acute and occasional contaminations by such products occurring in the environment. Appropriate statistical methods such as ANOVA, Tukey test and discriminant analysis underlined the relevance of cytochromes P450 and P418, NADH-cytochrome c reductase, catalase, peroxided and peroxidizable lipids and net peroxidation as biomarkers of exposure to these solvents in C. fluminea. This experiment emphasised the importance of a multi-biomarker approach in environmental surveys and will be completed further by mesocosm studies.
  • Article
    Background Herb-drug interactions may be of particular concern in the elderly. Single time-point, phenotypic metabolic ratios were used to determine whether supplementation of Ginkgo biloba, Panax ginseng, garlic oil, or St. John's wort extracts affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity in elderly subjects.Methods Twelve healthy volunteers between the ages of 60 and 76 (mean= 66 years) were randomly assigned to receive each supplement for 28 days followed by a 30-day washout period. Probe drug cocktails of midazolam, caffeine, chlorzoxazone, and debrisoquine were administered before and at the end of supplementation. Pre- and post-supplementation phenotypic ratios were determined for CYP3A4, CYP1A2, CYP2E1, and CYP2D6 using 1-hydroxymidazolam/midazolam serum ratios (1-hr), paraxanthine/caffeine serum ratios (6-hr), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hr), and debrisoquine urinary recovery ratios (8-hr), respectively.ResultsComparisons of pre- and post-St. John's wort phenotypic ratios revealed significant induction of CYP3A4 (~140%) and CYP2E1 activity (~28%). Garlic oil inhibited CYP2E1 (~22%). Ginseng inhibited CYP2D6, but the magnitude (~7%) did not appear clinically relevant. CYP1A2 activity in the elderly was unaffected by herb supplementation.Conclusions Elderly subjects, like their younger counterparts, are susceptible to herb-mediated changes in CYP activity, especially those involving St. John's wort.Clinical Pharmacology & Therapeutics (2005) 77, P16-P16; doi: 10.1016/j.clpt.2004.11.063
  • Article
    The mechanisms of action involved in the neurotoxicity of solvents are poorly understood. In vitro studies have suggested that the effects of some solvents might be due to the formation of reactive oxygen species (ROS). This study assesses hydroxyl radical ((.)OH) generation and measures malondialdehyde (MDA) levels in the cerebral tissue of rats exposed to six solvents (n-hexane, n-octane, toluene, n-butylbenzene, cyclohexane and 1,2,4-trimethylcyclohexane). Three of these solvents have been shown to generate ROS in studies carried out in vitro on granular cell cultures from rat cerebellum. We assessed (.)OH production by quantifying the rate of formation of 3,4-dihydroxybenzoic acid using a trapping agent, 4-hydroxybenzoic acid, infused via the microdialysis probe, into the prefrontal cortex of rats exposed intraperitoneally to the solvents. Extracellular MDA was quantified in microdialysates collected from the prefrontal cortex of rats exposed, 6hours/day for ten days, to 1000ppm of the solvents (except for n-butylbenzene, generated at 830ppm) in inhalation chambers. Tissue levels of free and total MDA were measured in different brain structures for rats acutely (intraperitoneal route) and sub-acutely (inhalation) exposed to solvents. None of the six solvents studied increased the production of hydroxyl radicals in the prefrontal cortex after acute administration. Nor did they increase extracellular or tissue levels of MDA after 10 days' inhalation exposure. On the other hand, a decrease in the concentrations of free MDA in brain structures was observed after acute administration of n-hexane, 1,2,4-trimethylcyclohexane, toluene and n-butylbenzene. Therefore, data of this study carried out in vivo did not confirm observations made in vitro on cell cultures.
  • Article
    Niclosamide produces genotoxic effects, such as point mutations in Salmonella sp., sperm-head abnormalities in mice and clastogenic effects in human lymphocytes in vitro and in vivo. As cytochrome P450 could be involved in the bioactivation of niclosamide, we investigated which subfamily was involved. We used liver microsomal fractions from rats treated with phenobarbital/β-naphthoflavone (PB/β-NF), benzo[a]pyrene (BaP) or cyclohexanol, which are known to induce different cytochrome P450 subfamilies, such as CYP2B, CYP1A1, CYP1A2 and CYP2E1. We also inhibited CYP1A and CYP2E using α-NF and diethyldithiocarbamate to identify the cytochrome P450 involved. Liver-S9 fractions obtained from PB/β-NF- and BaP-treated rats significantly increased the number of revertants induced by niclosamide, while the CYP1A1 inhibitor α-NF decreased the number of revertants. The incubation of niclosamide with CYP1A1 Supersomes™ increased the number of revertants, suggesting that CYP1A1 is responsible for the bioactivation of niclosamide. Nitroreduction is also involved in niclosamide bioactivation, as the nitroreductase-deficient strain YG7132 did not respond to the niclosamide treatment. Our findings indicated that a metabolite, derived from the action of CYP1A1 and a nitroreduction-reaction process, has a key role in the bioactivation of niclosamide.
  • Article
    In the oil sands of Alberta, Canada, toxicology research has largely neglected the effects of air contaminants on biota. Captive Japanese quail (Coturnix c. japonica) and American kestrels (Falco sparverius) were exposed to mixtures of volatile organic compounds and oxidizing agents (benzene, toluene, NO2 and SO2) in a whole-body inhalation chamber, to test for toxicological responses. Hepatic biotransformation measured through 7-ethoxyresorufin-O-dealkylase (EROD) tended to be increased in exposed kestrels (p=0.06) but not in quail (p=0.15). Plasma corticosterone was increased in the low dose group for quail on the final day of exposure (p=0.0001), and midway through the exposure period in exposed kestrels (p=0.04). For both species, there was no alteration of T and B-cell responses, immune organ mass, or histology of immune organs (p>0.05). This study provides baseline information valuable to complement toxicology studies and provides a better understanding of potential health effects on wild avifauna. Copyright © 2014 Elsevier Inc. All rights reserved.
  • Article
    Full-text available
    Some animal experiments have shown that mutual metabolic inhibition takes place between n-hexane and toluene, but we have found only one report dealing with their metabolic interaction at occupationally relevant exposure levels (Baelum et al. 1998). In order to evaluate the effect of dose- dependent metabolic interaction between toluene and n-hexane, especially in occupationally relevant exposure conditions such as relevant exposure levels, physical activities and exposure patterns, a physiologically based pharmacokinetic (PBPK) model for co-exposure to n-hexane and toluene was developed. The PBPK model for the binary co-exposure was established by initially validating or refining the existing PBPK models for n-hexane and toluene and then linking the individual solvent models via the hepatic metabolism terms. In reporting previous findings, noncompetitive inhibition was assumed and the inhibition constant of toluene on n-hexane biotransformation and that of n-hexane on toluene biotransformation used in simulation were 7.5, 30 μm, respectively, in previous data. According to the model, 8 h of constant exposure to 50 ppm n-hexane and 25, 50, 100 and 500 ppm toluene will cause about 7%, 18%, 62% and 96% decreases in the urinary excretion of 2,5-hexanedione (2,5-HD) and 4%, 10%, 25% and 30% increases in the n-hexane concentration in blood at the end of the fifth day of exposure simulated in a standard man at a 25 W work load. Simulations of co-exposure to 50 ppm n-hexane and 50 ppm toluene in a standard man who inhaled 50 ppm n- hexane with 0 or 50 ppm toluene for 8 h at different work loads suggest that toluene causes a slight decrease in urinary 2,5-HD in the resting condition, a 17% decrease at 25 W, and a 41% decrease at 50 W work load. The simulations of co-exposure in different exposure patterns with the same time-weighted concentration (TWA) of 50 ppm, i.e. 50 ppm for 8 h, 100 ppm of 4 times for 1 h and 200 ppm of twice for 1 h, showed reductions in urinary 2,5-HD of 17%, 40% and 67%, respectively. These simulations suggest that coexposure to n- hexane and toluene around 50 ppm (TWA) could affect urinary n-hexane metabolites to various degrees depending on the fluctuations in exposure concentrations and variety of work activities in the workplace.
  • Article
    Full-text available
    The cytochrome P450 enzymes are of great importance and interest because they catalyze reactions which have profound effects on the biological activities of drugs, environmental chemistry, and endogenous compounds. As a consequence, the literature on these enzymes is too voluminous to read, to scan, or even to cite. And while the pace accelerates and the scope broadens to include more biochemical aspects such as amino acid sequencing and three-dimensional structural models of isoenzymes, the bench scientists struggles tp perceive where the new information fits and what applicability it has, if any, zo his/her work.Pharmacologists and toxicologists also feel swamped by the exponential increase in cytochrome P450 publications. The present paper is designed to assist by providing framework for prediction and interpreting new data. It is an effort to summarize the functions of known human cytochrome P450s in terms of their specific catalytic activities, substrates, inducers, and inhibitors. Summaries are presented in tables for the ready access of information. (Tables 5-21) The literature through 1996 is covered in the reference section. In some instances references are to reviews which should be consulted for citations of the original literature. (Full text of the paper available through Informa Healthcare Publisher only, or download updated article: Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metabolism Reviews 01/2002; 34(1-2):83-448.)
  • Article
    Toluene is one of well-known neuro-toxicants. It can readily cross the blood-brain-barrier and mainly affect the central nervous system (CNS). An inhalation is the typical route of its human exposure and show a variety of symptoms involving cerebral and cerebellar dysfunction, bronchial asthma, chemical pneumonitis, toxic hepatitis, renal tubular acidosis, intestinal obstruction, and myelo-dysplastic syndrome. In this case, toluene-inhalated showed numerous CNS and systemic symptoms. Authors performed such gait analysis, gross and fine motor evaluations, computerized neurocognitive test that we noticed the impaired results. After comprehensive rehabilitation for four weeks, patient improved as much as she could carry out the independent life, and finally discharged home. Authors report the case of toluene-induced encephalopathy in Republic of Korea for the first time.
  • Article
    Sudden sniffing death is rare but serious during volatile substance sniffing or in the subsequent hours, especially in patients with toluene (super-glues) sniffing. We present the case of a 38-year-old female who sustained out-of-hospital cardiac arrest temporally related to toluene sniffing. Resuscitation of sudden sniffing death is seldom successful in the previous published reports. Our patient initially survived after aggressive resuscitation. Toluene abuse is associated with major toxicities including severe metabolic acidosis with lactate accumulation, CNS depression, ventricular arrhythmias, rhabdomyolysis, and liver toxicity. A possible mechanism of sudden sniffing death related to toluene abuse is ventricular arrhythmias, yet the true mechanism is still unknown. Severe metabolic acidosis may be another possible and important cause of sudden death in our patient. Sudden sniffing death should be considered in evaluating out-of-hospital cardiac arrest related to toluene sniffing, especially in the severe metabolic acidosis condition.
  • Chapter
    History Identifying Characteristics Exposure Dose Effect Toxicokinetics Histopathology and Pathophysiology Clinical Response Diagnostic Testing Treatment References
  • Article
    Toluene and verapamil are subject to extensive oxidative metabolism mediated by CYP enzymes, and their interaction can be stereoselective. In the present study we investigated the influence of toluene inhalation on the enantioselective kinetic disposition of verapamil and its metabolite, norverapamil, in rats. Male Wistar rats (n = 6 per group) received a single dose of racemic verapamil (10 mg/kg) orally at the fifth day of nose-only toluene or air (control group) inhalation for 6 h/day (25, 50, and 100 ppm). Serial blood samples were collected from the tail up to 6 h after verapamil administration. The plasma concentrations of verapamil and norverapamil enantiomers were analyzed by LC-MS/MS by using a Chiralpak AD column. Toluene inhalation did not influence the kinetic disposition of verapamil or norverapamil enantiomers (p > 0.05, Kruskal-Wallis test) in rats. The pharmacokinetics of verapamil was enantioselective in the control group, with a higher plasma proportion of the S-verapamil (AUC 250.8 versus 120.4 ng x h x mL(-1); p < or = 0.05, Wilcoxon test) and S-norverapamil (AUC 72.3 versus 52.3 ng x h x mL(-1); p < or = 0.05, Wilcoxon test). Nose-only exposure to toluene at 25, 50, or 100 ppm resulted in a lack of enantioselectivity for both verapamil and norverapamil. The study demonstrates the importance of the application of enantioselective methods in studies on the interaction between solvents and chiral drugs.
Literature Review
  • Article
    Lactating Sprague-Dawley rats and their pups were exposed on postnatal days 1-7, 6 h/day, to 80, 500 and 1000 ppm toluene, respectively, by inhalation. Exposure to 80 ppm toluene decreased the liver microsomal AHH activity and the rate of 7 alpha- and 6 beta-hydroxylation of androstenedione in 8-day-old-pups. On the other hand, neonatal exposure to 500 or 1000 ppm toluene resulted in a significant increase in AHH and 7-ethoxyresorufin O-deethylase activities and in the formation of 16-oxygenated metabolites of androstenedione in 8-day-old animals. Exposure to toluene increased the cytochrome P-450 content at all 3 dose levels in male but not in female pups. Twenty-one days after neonatal exposure no such effects were seen in young animals of either sex. In 56-day-old male rats, however, neonatal exposure to 80 ppm toluene resulted in a decreased rate of 6 beta-hydroxylation of androstenedione and a reduced AHH activity. No such effects were seen in female rats of the same age. Neonatal exposure to toluene affected the body and liver weights in 8-day-old pups of both sexes but had no effect on these parameters in 21-day-old animals of either sex. Exposure to 80 ppm toluene during the neonatal period gave a significantly increased body weight of 56-day-old male but not of female rats of the same age although this treatment increased liver weight in both sexes at this age. Serum testosterone levels were decreased in 21-day-old male rats following neonatal exposure to 80 or 500 ppm toluene and in 56-day-old male rats exposed neonatally to 1000 ppm toluene. In conclusion, exposure to toluene during the first week of life caused significant changes in various liver microsomal cytochrome P-450 dependent enzyme activities in 8-day-old pups, whereas the long-term effects on liver metabolism of the adult animal were small.
  • Article
    Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.
  • Article
    Monoclonal antibodies (MAbs) were used to study the contribution of cytochromes P450IA1/IA2, P450IIB1/IIB2, P450IIC11/IIC6 and P450IIE1 to toluene side-chain (benzyl alcohol, BA, formation) and ring (o- and p-cresol formation) oxidation in liver microsomes from fed, one-day fasted, and phenobarbital (PB)-, 3-methylcholanthrene (MC)- and ethanol-treated rats. All rats were fed synthetic liquid diets. MAb 1-7-1 against P450IA1/IA2 inhibited markedly o-cresol formation and slightly p-cresol formation but not BA formation only in microsomes from MC-treated rats. MAbs 2-66-3, 4-7-1 and 4-29-5 against P450IIB1/IIB2 strongly inhibited BA, o-cresol and p-cresol formation only in PB-induced microsomes. MAb 1-68-11 against P450IIC11/IIC6 inhibited BA formation at high toluene concentration in the following order: fed > fasted > ethanol = MC > PB, and ethanol ⩾ fed = fasted > MC > PB on the basis of the percentage and net amount inhibition, respectively. MAb 1-91-3 against P450IIE1 inhibited BA formation at low toluene concentration, but not at high concentration, in the following order: ethanol > fasted = fed > MC, and ethanol > fasted > fed > MC on the basis of percentage and net inhibition, respectively. MAbs 1-68-11 and 1-91-3 also inhibited p-cresol formation at high and low toluene concentrations, respectively. These results indicate that (i) both P450IIE1 and P450IIC11/IIC6 are constitutive isozymes mainly responsible for the formation of BA and p-cresol from toluene as low-and high-Km isozymes, respectively; (ii) P450IIE1, but not P450IIC11/IIC6, is induced by one-day fasting and ethanol treatment; (iii) both P450IIE1 and P450IIC11/IIC6 are decreased by PB and MC treatments; (iv) P450IIE1 is inhibited by high concentration of toluene; (v) P450IIB1/IIB2 can contribute to the formation of BA, o- and p-cresol from toluene, while P450IAI/IA2 preferentially contributes to the formation of o-cresol.
  • Article
    The enzymatic components of the rabbit pulmonary monooxygenase system, cytochromes P-450I and P-450II and NADPH-cytochrome P-450 reductase, are immunochemically distinct proteins. In pulmonary microsomes, the N-demethylation of benzphetamine, amino-pyrine, and ethylmorphine, and the O-deethylation of 7-ethoxycoumarin are dependent only on cytochrome P-450I, and the hydroxylation of coumarin is apparently catalyzed by both cytochromes. Cytochrome P-450II is immunochemically distinct from the major forms of hepatic cytochrome P-450 induced by phenobarbital or 3-methylcholanthrene, whereas cytochrome P-450I is indistinguishable from the former on the basis of physical and catalytic as well as immunochemical characteristics. Pulmonary and hepatic NADPH-cytochrome P-450 reductases also have identical physical, catalytic, and immunochemical properties. The lack of response of the lung monooxygenase system to phenobarbital, therefore, is apparently not due to an inability of the lung to synthesize the enzymes induced by phenobarbital in the liver. The relatively high proportion of cytochrome P-450I in the lung appears to be responsible for the higher rates (per nmol of P-450) of N-demethylation that have been observed in rabbit pulmonary as compared to hepatic microsomal fractions.
  • Article
    The induction of hepatic P-450 hemoprotein-dependent mono-oxygenase systems was studied in fetal and neonatal rats. The fetal liver was refractive to phenobarbital induction of aminopyrine and ethylmorphine N-demethylase. which are cytochrome P-450-dependent mono-oxygenases, but was not refractive to the 3-methylcholanthrene induction of benzo[a]pyrene hydroxylase. a cytochrome P1-450-dependent mono-oxygenase. After parturition, all three enzyme activities were inducible. These and other observations suggest that a control mechanism operates in the fetal rat which selectively suppresses the induction of cytochrome P-450. but allows induction of cytochrome P1-450. This selective suppression of phenobarbital induction in the fetus was reversed in part by the simultaneous administration of 3-methylcholanthrene. Several other inducing agents also partially reversed the suppression of phonobarbital induction in Fetal livers: dibenz-[a,c]anthracene, 2-diethylaminoethyl-2.2-diphenyl-valerate (SKF 525-A), and 3β-hydroxy-20-oxopregn-5-ene-16α-carbonitrile (PCN). Other inducing agents were inactive: α-naphthoflavone, β-naphthoflavone. 1,1-bis[p-chlorophenyl]2,2,2-trichloroethane (DDT). 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). dieldrin. chlordane and chlorpromazine.
  • Article
    Full-text available
    The concentrations of trichloroethylene in breath and blood and the urinary excretion of its metabolites following 30 minutes' direct immersion of one hand in the liquid, were compared with those obtained after four hours' inhalation exposure to the vapour of 100 ppm, described in a previous paper. The comparison shows that the end-tidal air concentrations during the first two hours of the post-exposure period were about twice as high in the case of skin exposure as in that of inhalation exposure, although the uptake of the solvent through the skin was only about one-third of the inhaled uptake. A kinetic approach suggested that differences in trichloroethylene movement in the body would be a principal cause of this discrepancy. The results of a similar series of experiments using toluene suggested that it is less readily taken up than trichloroethylene through the skin. It was concluded from the present investigation that analyses of not only breath but also of blood or urine are necessary and toluene would rarely be absorbed through the skin in toxic quantities during normal industrial use.
  • Article
    Developmental curves of liver microsomal cytochrome P-450 total content and five monooxygenase activities from control and 3-methylcholanthrene-treated New Zealand White rabbits were determined from 5 days prior to birth to adulthood (110 days of age). Microsomes were concomitantly analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Acetylarylamine (2-acetylaminofluorene) N-hydroxylase induction by 3-methylcholanthrene parallels quite closely increases in an electrophoretic band of 54,000 daltons and the approximately 2 nm spectral shift to the blue in the Soret peak of the reduced hemoprotein CO complex, and the induced N-hydroxylase activity is markedly inhibited by ..cap alpha..-naphthoflavone in vitro; these parameters develop in rabbits of age 10 days and older. Biphenyl 4-hydroxylase induction by 3-methylcholanthrene is at least 2-fold in the fetus and neonate for the first 10 days of age, is less than 2-fold between 16 and 40 days of age, and is absent in rabbits 50 days and older. Cytochrome P-450-mediated monooxygenases are implicated in the metabolic potentiation of certain carcinogens and other environmental pollutants. These data provide evidence for at least three different tissue-specific forms of regulation of inducible monooxygenase activities and their associated P-450 subunits by tissue-specific genes and the modification of these by temporal genes. These two types of genetic control may play a role in the susceptibility to chemical carcinogenesis or toxicity as a function of either age or tissue localization.
  • Article
    Full-text available
    Rats were exposed to toluene at a wide range of concentrations from 50 to 4000 ppm for six hours, and the effects of ethanol and phenobarbital (PB) treatments on the pharmacokinetics of toluene metabolism were investigated. Ethanol treatment influenced toluene metabolism mainly at low exposure concentrations. Thus ethanol accelerated the clearance of toluene from blood only when the blood concentration of toluene was not high (less than 360 microM), and ethanol increased hippuric acid (HA) excretion in urine more significantly at low (less than 250 ppm) than at high atmospheric toluene concentrations. Ethanol also expressed a similar effect on p-cresol excretion as on HA, but had little effect on o-cresol. Phenobarbital treatment promoted the urinary excretion of all of the metabolites of toluene, especially after exposure to high toluene concentration. As well as HA, benzoylglucuronide (BG) and free benzoic acid were found in urine. These are the products of the side chain metabolism of toluene. Amounts of BG could be detected when the urinary excretion of free benzoic acid exceeded 5 mumol/kg/6 h, indicating that a great deal of benzoic acid is required for the formation of BG. The Michaelis constant (Km) and the maximum rate of metabolic excretion in urine during six hours exposure (Vmax) of isozymes involved in the excretion of toluene metabolites were calculated, and correlated with the subtypes of cytochrome P-450. The significance of the result was suggested in the biological monitoring of exposure to toluene.
  • Article
    Sex-, age- and pregnancy-induced changes in the metabolism of toluene and trichloroethylene in rat liver were investigated in relation to the regulation of cytochrome P450IIE1 and P450IIC11 content using monoclonal antibodies. Immature male rats had a higher level of microsomal protein than females, and this increased with development; however, no difference by sex was found at puberty. No difference in cytochrome P450 content was seen between immature male and female rats; the content increased with development only in males, so that a sex difference in cytochrome P450 content occurred at puberty. Pregnancy decreased the cytochrome P450 content but not that of the microsomal protein. The rate of formation of benzyl alcohol from toluene was 4 times higher in mature than in immature male rats at a high concentration of toluene, but no difference was seen at a low toluene concentration. In contrast, the rate was lower in mature female rats than in immature ones at a low toluene level and no difference was seen at the high concentration. A sex difference was thus found in benzyl alcohol formation at puberty at both concentrations of toluene. The levels of o- and p-cresol formation in liver were similar in males and females but the rate decreased during development of females. The rate of metabolism of trichloroethylene was higher in immature than in mature male and female rats, especially at a low substrate level, and no sex difference in metabolism was seen with either age or concentration of trichloroethylene. Pregnancy decreased the metabolism of both toluene and trichloroethylene.(ABSTRACT TRUNCATED AT 250 WORDS)
  • Article
    The contribution of cytochrome P-450 isozymes to benzene metabolism in liver microsomes from fed, fasted, pyrazole-, phenobarbital (PB)- and ethanol-treated rats and in respective isocaloric controls was investigated using monoclonal antibodies (mAbs). Clone 1-7-1 mAb did not inhibit benzene metabolism, whereas clone 2-66-3 inhibited only in PB-induced microsomes at a high concentration of benzene (6.26 mM), and clone 1-91-3 mAb inhibited benzene metabolism in all cases. The degree of inhibition was as follows: fed congruent to isocaloric control congruent to PB less than fasted less than pyrazole congruent to ethanol. The pattern of inhibition was similar with clone 1-91-3 for low (0.23 mM) and high concentrations of benzene, except in PB-induced microsomes. Western blot analysis showed that clone 1-7-1 mAb did not bind any liver microsomal protein in the region of cytochrome P-450s, whereas with clone 2-66-3 a clear-cut band was seen only in liver microsomes from PB-treated rats, with clone 1-98-1, a band was detected in microsomes from all treated groups, in the following order: PB = isocaloric control less than fed less than fasted less than pyrazole less than ethanol. These results indicate that (i) cytochromes P-450b,e and P-450j contribute to benzene metabolism in rat liver; (ii) the former has a low affinity to benzene and is induced by PB; and (iii) P-450j has a high affinity to benzene and is induced by 1-day fasting, pyrazole and ethanol, but decreased by PB treatment.
  • Article
    The N-nitrosodimethylamine demethylase (P450I-IE1) is induced severalfold in liver by giving rats ethanol, acetone, pyrazole, and other related small molecular weight compounds. This induction is not the result of an increase in IIE1 mRNA, but could be due to either an increase in translation rate or a decrease in protein degradation. To determine the mechanism of induction, we measured IIE1 synthesis and degradation rates in untreated and acetone-treated rats. This was accomplished by immunopurification of radiolabeled IIE1 protein using a specific monoclonal antibody subsequent to in vivo labeling of total cellular protein with either NaH14CO3 or [3H]leucine. We found that in rats fed acetone, the rate of IIE1 synthesis was not changed; however, IIE1 degradation was markedly altered. In untreated rats, IIE1 protein was degraded via a biphasic pathway consisting of both a rapid and slow component with approximate half-lives of 7 and 37 h, respectively. However, in acetone-treated rats, only a monophasic curve with a half-life of 37 h was observed. The abolition of the rapid degradation component of the IIE1 turnover cycle indicates that induction of IIE1 by acetone is primarily due to specific stabilization of IIE1 protein. Since acetone is also metabolized by IIE1, we believe that this may be a substrate-induced enzyme stabilization.
  • Article
    Male Sprague-Dawley rats were injected ip with benzene, toluene, or a mixture of xylene isomers at 20 mmol hydrocarbon/kg daily for 3 days. The effects of administration of these hydrocarbons upon their own in vitro metabolism, as well as upon cytochrome P-450, NADPH-cytochrome c reductase, aminopyrine N-demethylase, aniline hydroxylase, glutathione, glutathione S-transferase, and UDPglucuronyltransferase in liver were studied. Each hydrocarbon studied increased its own in vitro metabolism. Benzene had no effect on the metabolism of toluene or xylenes. Toluene and xylenes increased the metabolism of benzene, toluene, and xylenes. Cytochrome P-450 was elevated by toluene and xylenes, but was not affected by benzene. NADPH-cytochrome c reductase was induced by all three hydrocarbons. Aminopyrine N-demethylase and aniline hydroxylase were induced by toluene and xylenes and were not affected by benzene. Glutathione was elevated by benzene, decreased by xylenes, and not affected by toluene. Glutathione S-transferase was induced differentially by these hydrocarbons toward various substrates: toward 1-chloro-2,4-dinitrobenzene by benzene and toluene, toward 1,2-dichloro-4-nitrobenzene by benzene and xylenes, and no effect toward 1,2-epoxy-3-(p-nitrophenoxy)propane by any hydrocarbons. UDPglucuronyltransferase was induced by benzene and toluene when o-aminophenol and phenol were used as the substrate. Xylenes had no effect. Benzene was more effective at inducing conjugation enzymes. Xylenes were more effective at inducing cytochrome P-450 dependent enzymes. Toluene was equipotent at inducing both types of enzymes. The results indicate that the addition of methyl groups to the aromatic ring affects the inductive pattern of these monocyclic aromatic hydrocarbons.
  • Article
    The instability of the solubilized/purified form, the lack of catalytic activity of the stabilized, macrolide-complexed form, and the compromised catalytic activity of the decomplexed form of steroid-inducible cytochrome P450IIIA1 motivated further investigations of the substrate specificity of this isozyme. A major complementary goal was to identify reactions utilizable as sensitive, specific diagnostic probes for the detection and partial characterization of this isozyme in tissues for which isolation and purification are not practical (e.g., extrahepatic, embryonic tissues, etc.). The approach utilized a combination of a specific, purified inducer, specific inhibitors including triacetyloleandomycin and inhibitory antibodies, and diagnostic probe substrates including the phenoxazone ethers, testosterone, warfarin, 2-acetylaminofluorene, estradiol-17 beta and benzo[a]pyrene. The results obtained indicated that steroid-inducible, rat hepatic P450IIIA1 would catalyze minimal or no O-dealkylation of methoxy-, ethoxy- or pentoxyphenoxazone but catalyzed rapid O-debenzylation of benzyloxyphenoxazone. Hydroxylation of testosterone was specific for the beta face of the molecule at the 2-, 6-, 15- and 16-positions with no detectable conversion to androstenedione and minimal hydroxylation on the alpha face. Both the R- and S-enantiomers of warfarin were attacked at positions 9 and 10, and these reactions appeared to be specific to isozymes of the IIIA family. Aromatic hydroxylation of estradiol-17 beta was efficiently catalyzed, particularly at the 2-position. Hydroxylations of 2-acetylaminofluorene at positions 5 and 7 were catalyzed at relatively rapid rates, but N-hydroxylation of the same substrate was not catalyzed effectively. Hydroxylation of benzo[a]pyrene occurred preferentially at carbon 3 with much lesser activity at carbon 9 and little or no detectable attack at positions 7 or 1. The results indicated that the 2 beta- and 15 beta-hydroxylation of testosterone and the 10-hydroxylation of warfarin would serve as the most useful probes thus far available for detection of the presence of functional P450IIIA1 isozymes in tissues for which isolation and purification are impractical. The results also indicated a very broad, yet selective substrate specificity for the steroid-inducible P450IIIA1.
  • Article
    I feel very honored to have been selected as the first Bernard B. Brodie Lecturer at Pennsylvania State University. Dr. Brodie, with his fertile and questioning mind, has made so very many seminal contributions to our understanding of the function and regulation of neurotransmitters and to our understanding of drug metabolism and the multiple factors that influence drug metabolism that he is considered by many of us to be the father of modern biochemical pharmacology. In addition to his research contributions, Dr. Brodie has trained large numbers of scientists from all over the world, and many of these scientists have become today's leaders in pharmacology and in the related biomedical sciences. I am particularly pleased to be here today because of my very great esteem for Dr. Brodie.
  • Article
    The effects of pretreatment with benzene and various methylbenzenes, ethyl- and propylbenzene, cumene and styrene on hepatic and pulmonary microsomal enzymes were studied in male rats. In the lungs, all the substituted benzenes, but not benzene itself, decreased cytochrome P-450 concentration, and most of them also decreased 7-ethoxycoumarin O-deethylase activity, whereas 7-ethoxyresorufin O-deethylase activity was increased by the same treatment. The change in aryl hydrocarbon hydroxylase activity was negligible. Neither NADPH-cytochrome c reductase activity, nor cytochrome b5 concentration were changed after hydrocarbon treatment. In the liver, all the compounds studied, except for benzene, increased 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase, and most of them also cytochrome P-450, aryl hydrocarbon hydroxylate and NADPH-cytochrome c reductase. The effect on cytochrome b5 in the liver was less marked. In the liver, all the monooxygenases studied seemed to be inducible by alkylbenzenes and styrene, whereas the effect was selective in the lung; depending on the monooxygenase, the activity can increase, decrease or remain unchanged.
  • Article
    The tissue-specific expression of cytochrome P-450b and P-450e mRNAs was examined with synthetic 18-mer oligomer probes in the liver, lung, kidney, and testis of control and inducer pretreated adult rats. RNAs homologous to the P-450e probe were detected in trace amounts in control and 3-methylcholanthrene (MC) induced livers and at high levels in livers from phenobarbital (PB) induced animals. P-450e mRNA levels were below detection limits in the other tissues examined, regardless of pretreatment. In contrast, mRNAs homologous to the P-450b oligomer were detected at low levels in control and inducer pretreated lung and testis, and at high levels in PB induced liver. No P-450b mRNAs were detected in these assays in RNA isolates from the kidney or from control or MC pretreated liver. Solution hybridization data indicated that the rat lung contained 9–12%, and the testis, 6–9%, respectively, of the levels of P-450b mRNA measured in the PB induced liver. Results from oligo(dT)-cellulose and poly(U)-affinity experiments indicated that the hepatic mRNAs for P-450b and P-450e were present predominantly in the bound, polyadenylated fraction, whereas the homologous lung and testes P-450b mRNAs predominated in the flow-thru fractions.
  • Article
    The influence on the kinetics of toluene from long-term occupational exposure, cigarette smoking, and ethanol consumption was studied in 26 male spray painters. A group of spray painters with reported subjective symptoms such as concentration deficits, fatigue, and dizziness due to the solvent exposure did not differ in the uptake and disposition of toluene from a group of spray painters with no symptoms. In occupationally exposed workers, a tendency for an enhanced clearance of toluene from the blood was observed in relation to personal habits such as smoking and/or moderate chronic ethanol intake. Long-term occupational exposure to a mixture of organic solvents does not exert any effect on the metabolic rate of toluene as compared with that of an unexposed group.
  • Article
    The influence of age, sex, and hormonal status on the expression of eight rat hepatic cytochrome P-450 (P-450) isoenzymes was evaluated by both catalytic and immunochemical methods. The male specificity of P-450 2c(male)/UT-A, the major microsomal steroid 16 alpha-hydroxylase of uninduced rat liver [Waxman, D.J. (1984) J. Biol. Chem. 259, 15481-15490], was shown to reflect its greater than or equal to 30-fold induction at puberty in male but not in female rats. The female specificity of P-450 2d(female)/UT-I was shown to reflect its developmental induction in females. P-450 PB-2a/PCN-E was shown to mediate greater than or equal to 85% of microsomal steroid 6 beta-hydroxylase activity; the male specificity of this P-450 largely reflects its developmental suppression in female rats. Neonatal gonadectomy and hormonal replacement experiments established that neonatal androgen "imprints" or programs the male rat for developmental induction of P-450 2c(male)/UT-A, for maintenance of P-450 PB-2a/PCN-E, and for suppression of P-450 2d(female)/UT-I, all of which occur in male rats at puberty. By contrast, the expressed levels of P-450 isoenzymes PB-1/PB-C, 3/UT-F, PB-4/PB-B, ISF-G, and beta NF-B were mostly unaffected by the rats' age, sex, and hormonal status. Studies on the sex specificity of P-450 induction established that the response of these latter five isoenzymes to the P-450 inducers phenobarbital, beta-naphthoflavone, pregnenolone-16 alpha-carbonitrile, and isosafrole is qualitatively and quantitatively equivalent in females as in males.
  • Article
    In the liver microsomes of toluene-treated and control Sprague-Dawley rats (n=148, males and females aged 13–93 days), the contents of cytochrome P-450 and b5 and the activities of NADPH – cytochrome c reductase and four monooxygenases were studied. In male control rats, cytochrome contents and NADPH – cytochrome c reductase, aminopyrine N-demethylase and aniline hydroxylase activities increased to the age of one month, and after a slight decrease in cytochrome concentrations, the average adult level was reached by the age of two months. Aniline hydroxylase and 7-ethoxycoumarine O-deethylase activities decreased to about half at the same age period. In control female rats, the activities of aminopyrine N-demethylase and aniline hydroxylase decreased after the age of one month, and they remained at a considerably lower level in adult females than in males. The sex-dependence of other enzymes was negligible. Toluene induction was already well developed in the youngest age group of both sexes; in most cases the induced enzyme levels in young rats were as high or higher than in adults. In adult female rats, toluene induction of all enzymes was weaker than in males. In male rats, the toluene-induced level of aniline hydroxylase and 7-ethoxycoumarine O-deethylase showed deep minima at the age of 43–53 days, at the puberty of rats.
  • Article
    Full-text available
    Sex differences exist in steroid and xenobiotic metabolism in the liver of a number of species. In the rat, the differences are regulated through the hypothalamo-pituitary axis. The previously postulated "feminizing factor" responsible for a female-type liver metabolism appears to be identical to growth hormone. The different effects of this peptide on hepatic metabolism in male and female rats may be related to the sexual dimorphism of the growth hormone secretory pattern; serum levels of growth hormone do not fluctuate as markedly in female as in male rats and may be simulated by administration of the hormone via osmotic minipumps, a procedure resulting in "feminization" of liver metabolism of male or hypophysectomized rats. This newly discovered system, the hypothalamo-pituitary-liver axis, represents a novel concept in endocrinology.
  • Article
    In the companion report we used primary cultures of adult rat hepatocytes to demonstrate that glucocorticoids comprise a "class" of compounds that stimulate de novo synthesis of a form of cytochrome P-450 (P450PCN) indistinguishable from that induced by the nonhormonal steroid pregnenolone 16 alpha-carbonitrile (PCN). Because induction of P450PCN is stereospecific for glucocorticoids and is dependent on the concentration of and the length of exposure to steroids it seemed possible that P450PCN represented another of the many genes whose expression is coordinately regulated by glucocorticoids bound to their specific cytoplasmic receptor and translocated into the nucleus. However, in cultured hepatocytes treated with glucocorticoids, synthesis of P450PCN failed to parallel synthesis of a typical glucocorticoid-responsive liver function, tyrosine aminotransferase, in the time course of induction, in the concentrations of glucocorticoids required for half-maximal induction, and in the order of effective steroids ranked by potency. Indeed, two moderately potent inducers of P450PCN either failed to induce tyrosine aminotransferase (spironolactone) or actually antagonized induction of tyrosine aminotransferase synthesis by glucocorticoids (PCN). Moreover, in the same cultures in which glucocorticoid induction of tyrosine aminotransferase was blocked by the presence of PCN or other previously identified antiglucocorticoids, synthesis of P450PCN was actually enhanced. We conclude that synthesis of P450PCN is a specific glucocorticoid-responsive liver function evoked by a novel mechanism readily distinguishable from the classic glucocorticoid receptor pathway.
  • Article
    Full-text available
    We administered a series of steroid hormones to primary nonproliferating cultures of adult rat hepatocytes and found that dexamethasone and other glucocorticoids but not sex steroid hormones, mineralocorticoids, or derivatives of pregnenolone other than pregnenolone 16 alpha-carbonitrile (PCN) stimulated de novo synthesis of an immunoreactive protein, indistinguishable from the form of cytochrome P-450 (P450PCN) induced by PCN in rat liver. No difference were discerned among purified liver cytochromes from rats treated with dexamethasone, PCN or dexamethasone plus PCN, among proteolytic digests of these proteins, or among the immunoprecipitated cytochromes prepared from cultured hepatocytes treated with these steroids as judged by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate followed by immunoblot analysis. Of the steroids tested, dexamethasone proved to be the most efficacious inducer increasing the rate of synthesis of P450PCN from 0.05% of total cellular protein synthesis in incubated control cultures (measured as incorporation of [3H]leucine into immunoprecipitable P450PCN) to as much as 9.4% in cultures incubated for 5 days in medium containing dexamethasone (10(-5) M). As with traditional glucocorticoid-responsive liver functions, induction of immunoreactive P450PCN was dependent on the concentration of dexamethasone (10(-8) to 10(-5) M) and was promptly reversed upon withdrawal of the steroid. However, during the 24-h interval between 24 to 48 h of culture age the hepatocytes were refractory to either induction or de-induction of immunoreactive P450PCN even though continuous exposure of the cells to dexamethasone (including this interval) was mandatory for maximal induction of P450PCN at 120 h in culture. Unlike cultured rat hepatocytes, HTC hepatoma cultures failed to exhibit dexamethasone-responsive expression of immunoreactive P450PCN. We conclude that glucocorticoids and PCN constitute a specific "class" of synthetic and endogenous inducers of a single form of cytochrome P-450.
  • Article
    Sprague-Dawley rats were exposed, by inhalation, to toluene and dichloromethane (500, 1,500 or 3,000 p.p.m.) and to benzene (1,500 p.p.m.) for three days. Toluene and benzene increased the concentration of liver microsomal cytochrome P-450. A dose dependent increase in the in vitro liver microsomal formation of several metabolites of biphenyl and benzo(a)pyrene was observed for both dichloromethane and toluene. At the highest dose-level the increase in the vitro formation of benzo(a)pyrene-7,8-dihydrodiol was more than three-fold for both dichloromethane and toluene whereas the formation of benzo(a)pyrene-4,5-dihydrodiol increased more than five-fold following exposure to toluene but less than two-fold after exposure to dichloromethane. Our results suggest that dichloromethane and toluene can modify the metabolism and thereby the toxicity of other environmental contaminants.
  • Article
    Full-text available
    Specific immunochemical techniques were used to quantitate the levels of eight isozymes of cytochrome P-450 (P-450) and epoxide hydrolase in liver microsomes of untreated rats and rats treated with phenobarbital, 3-methylcholanthrene, a mixture of these two compounds, nine individual polybrominated biphenyl (PBB) congeners, and a commercial mixture of PBBs. Levels of two 3-methylcholanthrene-inducible P-450s (designated P-450 beta NF-B and P-450 beta NF/ISF-G) varied over two orders of magnitude and were highly correlated. The levels of four phenobarbital-inducible P-450s (designated P-450PB-B, P-450PB-C, P-450PB-D, and P-450PB/PCN-E) were all correlated to each other. The level of one form, P-450UT-A, which was present at substantial levels in untreated rats, was inversely correlated to the levels of P-450 beta NF-B and P-450 beta NF/ISF-G. Among the PBB congeners which were examined, the presence of bromine at carbons o to the biphenyl bridge favored the induction of P-450PB-B, P-450PB-C, P-450PG-D, and P-450PB/PCN-E but did not necessarily eliminate the ability to induce P-450 beta NF-B and P-450 beta NF-ISF-G. PBB congeners with 2,2'-dibromo substitution induced P-450 beta NF-B and P-450 beta NF/ISF-G if one of the biphenyl rings contained bromines at positions 2,3, and 4. The induction of P-450UT-F was found to occur to a small extent with three of the compounds and is not readily explained in terms of structure-activity relationships. Although correlations were found among levels of some of the forms of P-450, several important exceptions were noted in relative levels of the individual enzymes. While the correlative data are useful in predicting induction patterns, all eight forms of P-450 appear to be independently regulated to some extent.
  • Article
    Current evidence in the rat suggests that the activity of various adult hepatic microsomal polysubstrate monooxygenases (PSMO) is programmed by neonatal exposure to hormones or hormonally active compounds. Since most chemical carcinogens are either metabolically activated or detoxified via the PSMO system, it is possible that neonatal exposure to pharmacologically active compounds may subsequently alter carcinogen metabolism in the adult animal. To investigate this question, two studies were conducted to examine the effect of phenobarbital (PB) exposure during the neonatal period on (a) the age-dependent activation of the hepatocarcinogen, aflatoxin B1 (AFB1) and (b) hepatic PSMO activities. Lactating rat dams were gavaged with either PB or an equal volume of 0.9% NaCl solution for 7 successive days after parturition. The dose of PB administered to the lactating rats was either 40 or 60 mg/kg for Studies 1 and 2, respectively. Thus, progeny were exposed to PB and its metabolites in the milk via suckling. Prior to sacrifice, progeny were given i.p. injections with [3H]AFB1 (1.0 mg/kg) in order to examine the effect of early PB exposure on the level of aflatoxin (AF):DNA adducts. In study 1 (i.e., PB, 40 mg/kg), early PB exposure did not alter the level of AF-DNA adducts at 28 or 84 days; however, at 168 and 259 days, the level of AF:DNA adducts were increased 19 and 39%, respectively, in males but not in females. Moreover, at 259 days, ethylmorphine N-demethylase activity was increased 160% in males exposed to PB during the neonatal period but not in females. In study 2, we also found that neonatal exposure to PB did not alter the levels of AF-DNA adducts or hepatic microsomal PSMO enzyme activities (i.e., ethylmorphine N-demethylase, cytochrome P-450, reduced nicotinamide adenine dinucleotide-phosphate-cytochrome c reductase) in 23-day-old weanling animals. However, at 252 days, early PB exposure significantly decreased the level of AF:DNA adducts by approximately 30% without affecting microsomal PSMO enzyme activities. These data are in contrast to our earlier study where a lower dose of neonatal PB (i.e., 40 mg/kg) increased the level of AF:DNA adducts (40%) and ethylmorphine N-demethylase (160%) in 259-day-old males. Presently, the mechanism(s) responsible for these differences is not known. Thus, the neonatal period appears to be very sensitive to compounds which alter the ontogenetic expression of carcinogen-metabolizing enzymes, and this sensitivity appears to be dose dependent. Interestingly, a similar finding has been reported for neonatal androgen imprinting of steroid metabolizing enzymes. These findings suggest that neonatal exposure to PSMO inducers may be an important determinant of cancer risk, insofar as carcinogen metabolism is involved in the neoplastic process.
  • Article
    Inhalation of toluene vapour of 2000 ppm increased the activities of aniline hydroxylase, aminopyrine N-demethylase, aryl hydrocarbon hydroxylase and NADPH-cytochrome c reductase and the concentrations of cytochromes P-450 and b5 in liver microsomes of adult male rats after an exposure period of 1 day or less. Repeated treatments, 8 h daily for 1-16 days, had only a slight further effect. In lung microsomes, the activities of monooxygenases and the concentration of cytochrome P-450 decreased after 6-24 h toluene exposure, but those of cytochrome b5 and NADPH-cytochrome c reductase did not change. In kidney microsomes the changes were mostly insignificant. After discontinuation of exposure the activities of enzymes and the concentrations of cytochromes returned to the control level in 1-4 days. The results obtained resemble the time-courses for the induction of monooxygenases by other inducers. The tissue differences suggest the unequal distribution of various cytochrome P-450 forms and their individual responsiveness to induction in liver, kidneys and lungs.
  • Article
    Cytochrome P-450 isozyme 1 (PB-1) (Mr congruent to 53 000) was purified to apparent homogeneity from phenobarbital (PB)-induced rat liver microsomes, and its spectral, structural, immunochemical, and catalytic properties were determined. PB-1, present in significant amounts in uninduced rat liver microsomes, is induced approximately 2-4-fold by phenobarbital, as compared to the greater than 30-fold induction typical of the major PB isozymes characterized previously. PB-1 was distinguished from the major PB-induced isozymes PB-4 and PB-5 [Waxman, D. J., & Walsh, C. (1982) J. Biol. Chem. 257, 10446-10457] by the absence of a Fe2+-metyrapone P446 complex, by its unique NH2-terminal sequence and distinct peptide maps, by the lack of immuno-cross-reactivity to PB-4, and by its characteristic substrate-specificity profile. Metyrapone effected a saturable enhancement of several PB-1-catalyzed reactions in the reconstituted system [Km(metyrapone) congruent to 200 microM], which varied in magnitude with the substrate, with a maximal stimulation of 5-8-fold in the case of acetanilide 4-hydroxylation. That metyrapone enhanced the corresponding microsomal activities only in cases where the metyrapone-sensitive PB-4 did not catalyze the same reaction at significant rates suggested that PB-1 is probably responsible for the substrate-dependent stimulatory effects of metyrapone on microsomal monooxygenations. In contrast to PB-4 and PB-5, PB-1 was characterized by a marked, but not absolute, dependence on cytochrome b5 (b5) for catalytic activity, with 4-7-fold stimulations typically effected by inclusion of stoichiometric b5 in the reconstituted system. That these b5-stimulations were lipid dependent and were abolished with specific proteolytic fragments lacking b5's COOH-terminal membranous segment evidenced the importance of this segment for efficient, b5-mediated electron transfer to P-450 PB-1 in the reconstituted monooxygenase system.
  • Article
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    Poly(A)+-RNA obtained from the livers of 3-methylcholanthrene (3MC)-treated rats was translated into cytochrome P-450c in a cell-free reticulocyte system. In this translational system, no precursor cytochrome P-450c was observed. The mRNA responsible for the synthesis of this cytochrome was isolated by immunoprecipitation of liver polyribosomes obtained at 15 hr after 3MC treatment, and a cDNA was constructed by the reverse transcriptase reaction. The cDNA was further purified by hybridizing at a high R0t (product of RNA concentration and incubation time) to poly(A)+-RNA isolated from control rat liver, and the nonhybridized, single-stranded cDNA was isolated by hydroxylapatite chromatography. This cDNAp-450c was employed in hybridization reactions with poly(A)+-RNA isolated from the livers of rats treated with 3MC for various times. These studies indicated a maximal induction of mRNAp-450c at about 15 hr after 3MC injection, although levels of this mRNA were significantly increased by 7 hr. The mRNAp-450c concentration had diminished by 24 hr but remained higher than control levels for at least 48 hr. These studies establish an effect of 3MC upon the accumulation of mRNAp-450c in rat liver.
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    Microsomal proteins were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and transferred to sheets of nitrocellulose. Specific forms of cytochrome P-450 (P-450) were identified on the sheets with the use of an immunoperoxidase staining technique and rabbit antibodies raised to electrophoretically homogeneous forms of rat and human liver P-450. The amounts of each form of P-450 present in microsomal preparations could be detected and quantitated by densitometry at the picomole level. A form of P-450 denoted P-450 PB-B2 accounted for the majority of the P-450 present in liver microsomes of rats treated with phenobarbital, trans-stilbene oxide, or dimethylnitrosamine. A similar protein was detected in rat lung microsomes regardless of treatment. Another form of P-450 denoted P-450 BNF-B2 accounted for the majority of the P-450 present in liver microsomes of rats treated with 5,6-benzoflavone, 3-methylcholanthrene, or 2,3,7,8-tetrachlorodibenzo-p-dioxin. A similar protein was induced by 5,6-benzoflavone in rat lung and kidney. Both forms of P-450 were induced in rat liver by the polychlorinated biphenyl mixture Aroclor 1254. Detectable levels of P-450s resembling these two forms were very low in livers of untreated rats, mice, and rabbits, in livers of rats treated with 2-(acetylamino)-fluorene or pregnenolone-16α-carbonitrile, and in rat hearts and brains. Antibodies raised to liver P-450s reacted with the inducible rabbit liver P-450s denoted P-450 LM-2 and P-450 LM-4 and were used to quantitate induction of those proteins. Antibodies raised to human liver P-450 purified from a single patient recognized a protein with identical electrophoretic mobility in liver microsomes prepared from ten different patients and also recognized a protein with a higher apparent monomeric molecular weight in the lung microsomes of two of these patients examined. The portion of total human liver microsomal P-450 that reacted with the antibody varied from 6% to 56% among ten different patients. The sensitivity and specificity of these techniques may be of further use in the study of P-450 multiplicity.
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    Administration of phenobarbital to mother rats during early lactation causes long-term, perhaps permanent, alteration of hepatic microsomal mixed-function oxidase activity and aflatoxin B1 adduct formation in the adult male offspring. These findings suggest that perinatal exposure to pharmacologically active compounds may be a determinant of cancer risk.
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    We provide here a list of 221 P450 genes and 12 putative pseudogenes that have been characterized as of December 14, 1992. These genes have been described in 31 eukaryotes (including 11 mammalian and 3 plant species) and 11 prokaryotes. Of 36 gene families so far described, 12 families exist in all mammals examined to date. These 12 families comprise 22 mammalian subfamilies, of which 17 and 15 have been mapped in the human and mouse genome, respectively. To date, each subfamily appears to represent a cluster of tightly linked genes. This revision supersedes the previous updates [Nebert et al., DNA 6, 1-11, 1987; Nebert et al., DNA 8, 1-13, 1989; Nebert et al., DNA Cell Biol. 10, 1-14 (1991)] in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene and cDNA, we recommend that the italicized root symbol "CYP" for human ("Cyp" for mouse), representing "cytochrome P450," be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen should precede the final number in mouse genes. "P" ("p" in mouse) after the gene number denotes a pseudogene. If a gene is the sole member of a family, the subfamily letter and gene number need not be included. We suggest that the human nomenclature system be used for all species other than mouse. The mRNA and enzyme in all species (including mouse) should include all capital letters, without italics or hyphens. This nomenclature system is identical to that proposed in our 1991 update. Also included in this update is a listing of available data base accession numbers for P450 DNA and protein sequences. We also discuss the likelihood that this ancient gene superfamily has existed for more than 3.5 billion years, and that the rate of P450 gene evolution appears to be quite nonlinear. Finally, we describe P450 genes that have been detected by expressed sequence tags (ESTs), as well as the relationship between the P450 and the nitric oxide synthase gene superfamilies, as a likely example of convergent evolution.
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    Cytochrome P450 isozymes induced in rat liver by a range of concentrations of toluene were studied with monoclonal antibodies (MAbs) to specific P450 isozymes and by enzyme assays. Nitrosodimethylamine demethylase activity was significantly increased in microsomes from rats exposed to more than 1000 ppm of toluene, an increase that was dose-dependent. Anti-CYP2E1 significantly inhibited the metabolism of toluene to benzyl alcohol (BA) by about 50%, in microsomes from 1000 to 4000 ppm toluene-exposed rats, at low substrate concentration (0.2 mM). With anti-CYP2B1/2, the rate of BA formation was decreased by 15-17% in microsomes from rats of 2000 and 4000 ppm toluene exposures at high substrate concentration (5.0 mM). On the other hand, anti-CYP2C11/6 inhibited the rate of formation of BA in all of the microsomes, but the extent of inhibition was progressively decreased from 55% in control to 33% in 4000 ppm exposure. Immunoblot analysis with anti-CYP2E1 and anti-CYP2B1/2 revealed stronger immunoreactive bands in microsomes from rats exposed to more than 1000 and 2000 ppm of toluene, respectively. Stronger bands were also observed in microsomes from rats of 2000-4000 ppm toluene exposures with anti-CYP3A1/2, but no immunoreactivity appeared with anti-CYP1A1/2. These results suggest that toluene induces CYP2E1, CYP2B1/2 and CYP3A1/2, but reduces CYP2C11/6, and has no effect on CYP1A1/2.
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    Factors influencing the metabolism of toluene were investigated in rats using monoclonal antibody (MAb) to cytochrome P450 (P450). At low toluene concentrations, P450 IIE1 was primarily involved in the metabolism of toluene, whereas P450 IIC11/6 was involved at high concentrations. A low-carbohydrate diet induced P450 IIE1 and resulted in an increase in toluene metabolism. The intake of fat did not influence the metabolism. A lowered protein intake decreased not only the total content of P450 but also the P450 IIC11/6. Fasting and ethanol consumption also enhanced toluene metabolism via the induction of P450 IIE1. The metabolic rate of toluene in adult male rats was 4-fold higher than in immature males and adult females at a high substrate concentration because of the high level of P450 IIC11/6 in adult males, whereas no difference was noted between adult and immature females. Although development did not influence toluene metabolism in males at a low substrate concentration, the metabolic rate in adult female rats was significantly lower than that of immature females and males; this may be due to the decrease in P450 IE1 with development. Diabetic status influenced toluene metabolism in rats by affecting several kinds of P450 isozymes. Toluene exposure also affected its own metabolism by increasing P450 IIE1 and P450 IIB 1/2, and decreasing P450 IIC11/6. A significant difference in toluene metabolism was observed among rat, mouse and human liver microsomes. Thus, when considering the factors affecting toluene metabolism, it is important to elucidate the change in specific P450 isozyme composition related to the modifications, and their affinities to toluene.
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    Neonatal exposure to monosodium glutamate (MSG) permanently blocks growth hormone (GH) secretion, which results in the development of a well-defined syndrome characterized by stunted body growth, obesity and impaired drug metabolism. We have found that restoration of the normal masculine circulating profile of GH (i.e., six daily pulses) by use of an external pumping apparatus is ineffective in restoring the normal expression of hepatic cytochrome P450 2C11, a major GH-dependent drug and steroid metabolizing enzyme that is eliminated by MSG treatment. Moreover, administering GH at two, four or seven plasma pulses per day with amplitudes ranging from physiologic to 7 times normal were similarly ineffective in restoring the expression (at both an activity and mRNA level) of the cytochrome. Additionally, multicytochrome P450-dependent hexobarbital hydroxylase was also unresponsive to GH administration in the MSG-treated rats. Because GH replacement was unable to correct the enzyme defects, our results suggest that the developmental abnormalities produced by neonatal MSG are not simply a result of a GH deficiency per se, but are due to an irreversible insensitivity of the target cell to GH.
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    Benzene (B), toluene (T), ethylbenzene (EB), styrene (S) and xylene isomers (oX, mX, pX) are important environmental pollutants and B is a proved human carcinogen. Their inhalation by male Wistar rats (4 mg/l, 20 h/day, 4 days) caused cytochrome P450 (P450) induction. The degree of P450 2B1 induction increased and that of 2E1 decreased in the series B, T, EB, S, oX, mX and pX, as estimated by Western blots, while neither solvent was as effective for 2B1 induction as phenobarbital and B was more effective for 2E1 than ethanol. The levels of several other P450s decreased after exposure to these solvents, B being most effective. Exposure to these solvents increased in vitro hepatic microsomal oxidation of B and aniline (AN) (2E1 substrates) 3 to 6-fold, indicating induction of this P450. T oxidation was increased 2 to 4-fold and chlorobenzene (ClB) oxidation 3-fold. Sodium phenobarbital (PB, 80 mg/kg/day, 4 days, i.p.) did not increase ethylmorphine (EM) and benzphetamine (BZP) demethylation (2B1 substrates), neither of the B derivatives did so, and oX decreased it; however, pentoxyresorufin O-dealkylation was well related to the immunochemically detected 2B1 levels in control, PB and B microsomes. PB did not increase B, but increased T and ClB oxidation 2-4 and 3-fold, respectively, indicating possible 2B1 role in their oxidation. B oxidation after various inducers was related to immunochemical 2E1 levels, T and ClB oxidation to both 2B1 and 2E1 and AN oxidation to 2E1 and 1A2 levels.(ABSTRACT TRUNCATED AT 250 WORDS)