Inactivation of Cytochromes P450 2B Protects Against Cocaine-Mediated Toxicity in Rat Liver Slices

doi:10.1006/taap.1994.1086

Toxicology and Applied Pharmacology

Volume 126, Issue 1, May 1994, Pages 26-32

https://doi.org/10.1006/taap.1994.1086 Get rights and content

Abstract

Mechanism-based inactivators of rat liver cytochrome P450 2B1 and 2B2 were used to evaluate the role of these enzymes in the hepatotoxicity of cocaine. Loss of liver microsomal androstenedione 16β-hydroxylation was monitored to determine the extent of P450 2B1/2 inactivation by chloramphenicol (CAP) or its 2B-selective analogue, N-(2-p-nitrophenethyl)chlorofluoroacetamide (pNO₂C1FA). The effect of P450 2B1/2 inactivation on cocaine-mediated hepatotoxicity was assessed in rat liver slices. Exposure of slices from phenobarbital-induced Lewis rats to CAP concentrations ranging from 100 to 500 μM resulted in a concentration-dependent decrease in P450 2B activity and a corresponding decrease in cytotoxicity as measured by K⁺ loss following exposure to 1 mM cocaine. Treating slices from PR-induced rats with 250 μM pNO₂C1FA protected slices against cocaine-mediated cytotoxicity after exposure to 500 μM cocaine. In vivo administration of 300 mg/kg CAP or 200 mg/kg pNO₂C1FA to phenobarbital-induced Lewis rats decreased androstenedione 16β-hydroxylation to 30 or 39% of control, respectively, and blocked cocaine-mediated K⁺ loss in rat liver slices. Rat liver microsomes from animals treated with either CAP or pNO₂C1FA displayed approximately 40% of the control rate of cocaine N-demethylation. Experiments with phenobarbital-treated Munich Wistar (WM) rats, which lack 2B2, revealed similar rates of microsomal N-demethylation and comparable in vitro hepatotoxicity to Lewis rats. The capacity of a specific P450 2B1/2 inactivator to protect against cocaine-mediated hepatotoxicity both in vivo and in vitro and the results with the WM rats support the identification of P450 2B1 as a major cocaine bioactivating form.

References (0)

Cited by (24)

In vitro measurements of metabolism for application in pharmacokinetic modeling
2008, Pharmacology and Therapeutics
Human risk and exposure assessments require dosimetry information. Species-specific tissue dose response will be driven by physiological and biochemical processes. While metabolism and pharmacokinetic data are often not available in humans, they are much more available in laboratory animals; metabolic rate constants can be readily derived in vitro. The physiological differences between laboratory animals and humans are known. Biochemical processes, especially metabolism, can be measured in vitro and extrapolated to account for in vivo metabolism through clearance models or when linked to a physiologically based pharmacological (PBPK) model to describe the physiological processes, such as drug delivery to the metabolic organ. This review focuses on the different organ, cellular, and subcellular systems that can be used to measure in vitro metabolic rate constants and how those data are extrapolated to be used in biologically based modeling.
The views expressed in this paper are those of the authors and do not necessarily reflect the views and policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
Xenobiotic metabolizing enzymes in the central nervous system: Contribution of cytochrome P450 enzymes in normal and pathological human brain
2008, Biochimie
Citation Excerpt :
This compound is also O-demethylated by CYP2D6 into a toxic substance (4-hydroxyamphetamine) [53,54]. In addition, CYP2B6 metabolizes also psychoactive substances such as cocaine [55,56], phencyclidine [57] and some amphetamines [58]. Both CYP2B6 and CYP2D6 are inducible by nicotine: CYP2B6 is described as the enzyme mainly responsible for the metabolism of nicotine in brain of smokers [11], but CYP2D6 may influence the disposition of nicotine in the small subset of the population who correspond with CYP2D6 ultrametabolizer phenotype [59].
The metabolism of xenobiotics in human brain constitutes a field of recent intensive research in relation to the potential implications in the pharmacological effect of drugs acting on the central nervous system. Cytochrome P450 enzymes (CYPs) play a crucial role in these metabolic pathways and the existence of functional CYP monooxygenases in brain is now well established. These enzymes are preferentially localized in the neuronal cells within the microsomal fraction and the inner membrane of mitochondria. Although low, the metabolism in situ could influence individual response to xenobiotics or produce reactive, toxic metabolites causing irreversible damage in the neuronal cells. The abundant presence of CYPs in selective cell populations within different regions of the brain has also suggested a role for these enzymes in brain physiology thus not restricted to xenobiotic-induced neurotoxicity. For instance, CYPs participate in the regulation of neurotransmitters and steroids and brain maintenance of cholesterol homeostasis. Recent advances support an additional role for these enzymes in the pathogenesis of psychiatric and neurodegenerative disorders such as depression, schizophrenia, and Alzheimer's and Parkinson's diseases. The characterization of brain CYP isoforms and their localization, the identification of their substrates and metabolic end-products will allow better understanding of the role of these enzymes in brain physiology, development and diseases.
CYP2B6 is expressed in African Green monkey brain and is induced by chronic nicotine treatment
2006, Neuropharmacology
Citation Excerpt :
The elevated brain CYP2B6 levels in smokers and in non-smokers exposed to second-hand smoke or in people treated with nicotine may contribute to the activation of nitrosamines to DNA adducts. In addition, CYP2B also metabolizes drugs of abuse such as cocaine (Poet et al., 1994), amphetamines, ecstasy (Kreth et al., 2000) and phencyclidine (Jushchyshyn et al., 2003). In smokers, some brain cells may be at greater risk for toxicity due to localized and induced drug metabolism depending on the relative toxicity and pharmacology of the parent drug and metabolism.
CYP2B6 is a drug-metabolizing enzyme expressed in human tissues that can activate bupropion (a smoking cessation drug) and tobacco smoke nitrosamines and can inactivate drugs such as nicotine. Smokers have higher brain CYP2B6 protein levels compared to non-smokers but the cause of this elevation is unknown. We investigated the basal expression and the effect of chronic nicotine treatment on CYP2B6 protein in African Green monkey (Cercopithecus aethiops) brain. Basal expression of brain CYP2B6 was strong in specific cells such as the frontal cortical pyramidal cells, the cerebellar Purkinje cells and the neurons in the substantia nigra. Basal CYP2B6 protein levels varied 2.7-fold (non-significant) among 12 brain regions. All monkeys were given a subcutaneous 0.1 mg/kg nicotine test dose prior to treatment and the maximum plasma concentration achieved was 87 ± 69 ng/ml and the half-life was 2.6 ± 1.5 h. Monkeys were treated subcutaneously twice daily with nicotine at 0.05 mg/kg for 2 days, 0.15 mg/kg for 2 days followed by 0.3 mg/kg for 18 days (n = 6) or saline (n = 6). Chronic nicotine treatment induced CYP2B6 expression in specific cells such as astrocytes and neurons in the frontal cortex, caudate, thalamus and hippocampus. CYP2B6 protein levels were induced 1.5-fold in the frontal cortex (p < 0.01). Hepatic CYP2B6 expression was not altered by nicotine. In conclusion, CYP2B6 protein is expressed in specific cells in monkey brain and is induced by chronic nicotine treatment which may impact central metabolism of CYP2B6 substrates such as bupropion and nicotine.
Induction of nicotine-metabolizing CYP2B1 by ethanol and ethanol-metabolizing CYP2E1 by nicotine: Summary and implications
2003, Biochimica et Biophysica Acta - General Subjects
Alcohol and tobacco are frequently co-abused. Increased alcohol use and alcoholism are associated with smoking, and vice versa. Functional and/or metabolic cross-tolerance may contribute to this occurrence. This review summarizes recent studies published from our laboratory focusing on metabolic aspects of tolerance, which demonstrate that in rat, subchronic, behaviourally relevant doses of ethanol induce hepatic nicotine-metabolizing cytochrome P450 (CYP) 2B1, and that subchronically administered nicotine, at behaviourally relevant doses, induces hepatic ethanol-metabolizing CYP2E1. Increased CYP2B1 protein, mRNA and CYP2B1-mediated nicotine metabolism was observed following ethanol treatments. CYP2E1 protein and activity were induced by nicotine, but no changes were seen in levels of CYP2E1 mRNA. These data indicate that metabolic cross-tolerance may occur between nicotine and ethanol, suggesting that nicotine use may increase the elimination of ethanol, and ethanol use may increase the elimination of nicotine. Other implications, such as altered pharmacology and toxicology of drugs metabolized by these enzymes, as well as changes in pro-carcinogen and pro-toxin activation are also discussed.
Induction of CYP2B1/2 and nicotine metabolism by ethanol in rat liver but not rat brain
2001, Biochemical Pharmacology
Citation Excerpt :
CYP2B1/2 also metabolize cocaine to a toxic metabolite [9], suggesting additive or synergistic increases in liver damage when ethanol and cocaine are used concurrently. In fact, inhibition of CYP2B enzymes protects against cocaine-mediated hepatotoxicity in rats [9]. The mechanism(s) by which CYP2B enzymes are induced by ethanol is not known.
A higher proportion of alcoholics than non-alcoholics smoke (>80 vs 30%). In animals, chronic administration of alcohol induces tolerance to some effects of nicotine. To investigate if chronic ethanol (EtOH) induces alterations in CYP2B1/2 and nicotine C-oxidation activity, male rats (N = 4–6/group) were treated once daily with saline or EtOH (0.3, 1.0, and 3.0 g/kg, p.o./by gavage) for 7 days. A quantitative immunoblotting assay was developed to detect CYP2B1/2 in the brain, where constitutive expression is low, and in the liver. Using this method, it was determined that EtOH did not alter CYP2B1/2 protein expression significantly in six brain regions (olfactory bulbs, olfactory tubercles, frontal cortex, hippocampus, cerebellum, and brainstem). However, a dose-dependent induction of CYP2B1/2 protein expression was detected in the liver. Significant induction of 2-, 3-, and 2.7-fold were observed for the 0.3, 1.0, and 3.0 g/kg doses, respectively. Increases were also observed in CYP2B1 mRNA, which was induced by 14, 38, and 43% at the same doses. Liver microsomal nicotine C-oxidation also was increased (1.3 to 4.5-fold). CYP2B selective inactivators demonstrated that approximately 70% of nicotine C-oxidation was mediated by CYP2B1/2 in both EtOH-induced and uninduced hepatic microsomes. In summary, chronic, behaviorally relevant doses of EtOH induce CYP2B1/2 protein, mRNA, and nicotine C-oxidation activity in rat liver but not in rat brain, and these increases could contribute to cross-tolerance and co-abuse of ethanol and nicotine.
The effects of phencyclidine pretreatment on cocaine-mediated hepatotoxicity in mice
2001, Toxicology and Applied Pharmacology
Cocaine-mediated hepatotoxicity (CMH) requires cocaine (CCN) bioactivation by microsomal monooxygenase enzymes that results in cell death. Proposed mechanisms of toxicity involve reactive metabolites that covalently bind to hepatocellular proteins, depletion of cellular reducing equivalents through redox cycling, and/or the generation of reactive oxygen and nitrogen species that alter lipids and proteins. We have previously shown that phencyclidine (PCP) pretreatment potentiated CMH in CF-1 mice without increasing in vitro N-demethylation or N-hydroxylation of CCN. We have now further characterized PCP-potentiated CMH and determined that it is a dose- and time-dependent process, with PCP doses as low as 2.5 mg/kg for 3 days significantly increasing CMH. Immunohistochemistry and histology of livers from mice pretreated with PCP before CCN administration revealed a marked correlation between the regions of CCN metabolite binding and that of necrosis, whereas there was little binding or necrosis in vehicle-pretreated mice. Although hepatic GSH levels were not altered after repetitive PCP treatment alone, a sustained decrease (at least 6 h) in these levels was observed following CCN administration. Inhibitors of inducible nitric oxide synthase (NOS) abrogated PCP-potentiated CMH, although repetitive PCP treatment alone did not increase nitric oxide synthesis systemically or locally in hepatic tissue nor did lipopolysaccharide induction of NOS (without PCP) directly potentiate CMH. The precise mechanisms of PCP potentiation of CMH and involvement of NOS in CMH remain unclear, however, sustained depletion of GSH levels and increased hepatocellular binding of reactive cocaine metabolites have been demonstrated.

View all citing articles on Scopus

View full text

Regular ArticleInactivation of Cytochromes P450 2B Protects Against Cocaine-Mediated Toxicity in Rat Liver Slices

Abstract

Regular Article
Inactivation of Cytochromes P450 2B Protects Against Cocaine-Mediated Toxicity in Rat Liver Slices