Smoking, alcoholism and genetic polymorphisms alter CYP2B6 levels in human brain
Introduction
Cytochrome P450 enzymes (CYPs) are expressed throughout the body and are involved in the activation or deactivation of endogenous and exogenous compounds such as hormones, drugs and dietary constituents. In the brain, CYPs are highly localized to specific regions and cell types (Miksys and Tyndale, 2002b) which may allow for microenvironments where drug and metabolite concentrations differ substantially from plasma concentrations (Britto and Wedlund, 1992). Under conditions of enzyme induction, brain CYPs may contribute to the observed interindividual differences in response to many centrally acting drugs and neurotoxins.
CYP2B6 has been identified in human brain (Gervot et al., 1999), but very little is known of its regional and cellular distribution or its regulation. In rat brain, CYP2B enzymes are inducible by several compounds such as nicotine (Miksys et al., 2000), phenobarbital (Schilter et al., 2000) and phenytoin (Kempermann et al., 1994, Rosenbrock et al., 1999). In humans and monkeys, hepatic CYP2B6 is inducible by a number of drugs (Gervot et al., 1999, Schoedel et al., 2003), but the inducibility of CYP2B6 in human brain is unknown.
The CYP2B6 enzyme metabolizes a wide variety of exogenous substrates (Ekins and Wrighton, 1999) and endogenous compounds such as testosterone (Rosenbrock et al., 1999). The induction of brain CYP2B6 by nicotine is of interest as human CYP2B6 can inactivate nicotine to its non-psychoactive metabolite, cotinine (Yamazaki et al., 1999) and it hydroxylates bupropion, a drug used in smoking cessation treatment, to a pharmacologically active metabolite (Hesse et al., 2000). In addition, CYP2B6 can metabolize several drugs of abuse, such as cocaine, phencyclidine and amphetamines to neurotoxic metabolites (Chun et al., 2000, Kreth et al., 2000, Pellinen et al., 2000). If, as in rats, nicotine induces CYP2B6 in human brain, then smokers may be at increased risk of neurotoxic effects of some xenobiotics, and may exhibit altered sensitivity to some centrally acting drugs such as bupropion.
A non-synonymous polymorphism has been identified within the coding region of the CYP2B6 gene, consisting of a base pair substitution C to T at position 1459 (C1459T) in exon 9, resulting in a predicted amino acid change from arginine to cysteine at position 487 (R487C; Lang et al., 2001). The amino acid change results in a decrease in hepatic CYP2B6 protein levels and activity (Lang et al., 2001). It is present in the Caucasian population at a frequency of 14% (Lang et al., 2001), and those with a T allele in their genotype had lower abstinence rates and relapsed more quickly than non-carriers in a smoking cessation trial (Lerman et al., 2002).
This paper reports for the first time the regional and cellular distribution of CYP2B6 in human brain, and also examines CYP2B6 enzyme levels in brains of human smokers compared to non-smokers. CYP2B6 brain levels in alcoholics were compared to non-alcoholics, as alcohol and tobacco are commonly co-abused, and more than 85% of alcoholics are heavy smokers (Patten et al., 1996), and also as ethanol can induce CYP2B1 in a rat model (Schoedel et al., 2001). To investigate the interaction of environmental and genetic effects, we also tested for the presence of the C1459T polymorphism in the CYP2B6 gene and assessed its impact on the levels of CYP2B6 in non-smokers, non-alcoholics, smokers and alcoholics. Parts of this study have been published in the form of an abstract (Miksys et al., 2001).
Section snippets
Human tissues and CYP2B6 genotyping
Human brain tissues were obtained from the University of Miami Brain Endowment Bank. The next of kin representing the deceased granted permission to retain brain tissue for research purposes. The protocols for obtaining postmortem specimens have received Institutional Review Board authorization from the University of Miami School of Medicine. Postmortem specimens were obtained during routine autopsy from 26 age-matched, drug-free control, smoking and alcoholic subjects (Table 1, Table 2). Three
Distribution of CYP2B6 among brain regions
A single immunoreactive protein band that co-migrated with cDNA-expressed human CYP2B6 was detected in each of the brain regions examined in all cases (n=24). No signal was detected when the primary antibody was omitted. CYP2B6 levels varied significantly among regions with a 2.5-fold range between the lowest (hippocampus) and highest (putamen) regions (Fig. 1, ANOVA p=0.02).
Cellular localization of CYP2B6 in non-smoking non-alcoholics
In NSNA, the cellular distribution (summarized in Table 3) was mainly neuronal, with some immuno-positive astrocytes
Discussion
Although CYP2B6 has been detected in human brain (Gervot et al., 1999), this is the first detailed quantitative and qualitative description of the distribution of this enzyme among different brain regions and cell types. CYP2B6 expression varied among brain regions, and was found primarily in neurons but also in astrocytes in specific brain regions. From studies in a rat model, we and others have observed that nicotine can induce CYP2B in rat brain (Anandatheerthavarada et al., 1993, Miksys et
Acknowledgements
This work was funded by CAMH, CIHR grant MT 14173, a Canadian Research Chair in Pharmacogenetics awarded to RFT and a Transdisciplinary Tobacco Use Research Center Grant P5084718 awarded to CL. The authors would like to thank Margaret Basile and Teresa Sanelli for expert technical assistance, and Dr. H. V. Gelboin for his generous gift of monoclonal anti-CYP2B1 antibody.
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