Sharon Miksys

Smoking, alcoholism and genetic polymorphisms alter CYP2B6 levels in human brain.

CYP2B6 metabolizes drugs such as nicotine and bupropion, and many toxins and carcinogens. Nicotine induces CYP2B1 in rat brain and in humans polymorphic variation in CYP2B6 affects smoking cessation rates. The aim of this study was to compare CYP2B6 expression in brains of human smokers and non-smokers and alcoholics and non-alcoholics (n=26). CYP2B6 expression was brain region-specific, and was observed in both neurons and astrocytes. CYP2B6 levels were higher in brains of smokers and alcoholics, particularly in cerebellar Purkinje cells and hippocampal pyramidal neurons, cells known to be damaged in alcoholics. Significantly more (p<0.05) CYP2B6 protein was seen in four brain regions of smoking alcoholics compared to non-smoking non-alcoholics: hippocampus (5.8-fold), caudate nucleus (3.3-fold), putamen (3.0-fold) and cerebellar hemisphere (1.6-fold). The genetic variant C1459T (R487C) has been associated with reduced hepatic enzyme levels, stability and activity. Preliminary genotyping of this small sample (n=24) suggested that individuals with the CC genotype had higher brain CYP2B6 than those with the CT or TT genotype. Higher brain CYP2B6 activity in smokers and alcoholics may cause altered sensitivity to centrally acting drugs, increased susceptibility to neurotoxins and carcinogenic xenobiotics and contribute to central tolerance to nicotine.

Regional and cellular expression of CYP2D6 in human brain: higher levels in alcoholics

Cytochrome P450 (CYP) 2D6 is expressed in liver, brain and other extrahepatic tissues where it metabolizes a range of centrally acting drugs and toxins. As ethanol can induce CYP2D in rat brain, we hypothesized that CYP2D6 expression is higher in brains of human alcoholics. We examined regional and cellular expression of CYP2D6 mRNA and protein by RT‐PCR, Southern blotting, slot blotting, immunoblotting and immunocytochemistry. A significant correlation was found between mean mRNA and CYP2D6 protein levels across 13 brain regions. Higher expression was detected in 13 brain regions of alcoholics (n = 8) compared to nonalcoholics (n = 5) (anovap < 0.0001). In hippocampus this was localized in CA1–3 pyramidal cells and dentate gyrus granular neurons. In cerebellum this was localized in Purkinje cells and their dendrites. Both of these brain regions, and these same cell‐types, are known to be susceptible to alcohol damage. For one case, a poor metabolizer (CYP2D6*4/*4), there was no detectable CYP2D6 protein, confirming the specificity of the antibody used. These data suggest that in alcoholics elevated brain CYP2D6 expression may contribute to altered sensitivity to centrally acting drugs and to the mediation of neurotoxic and behavioral effects of alcohol.

Brain CYP2E1 is induced by nicotine and ethanol in rat and is higher in smokers and alcoholics

Ethanol and nicotine are commonly coabused drugs. Cytochrome P450 2E1 (CYP2E1) metabolizes ethanol and bioactivates tobacco‐derived procarcinogens. Ethanol and nicotine can induce hepatic CYP2E1 and we hypothesized that both centrally active drugs could also induce CYP2E1 within the brain. Male rats were treated with saline, ethanol (3.0 g kg−1 by gavage) or nicotine (1.0 mg kg−1 s.c.) for 7 days. Ethanol treatment significantly increased CYP2E1 in olfactory bulbs (1.7‐fold), frontal cortex (2.0‐fold), hippocampus (1.9‐fold) and cerebellum (1.8‐fold), while nicotine induced CYP2E1 in olfactory bulbs (2.3‐fold), frontal cortex (3.0‐fold), olfactory tubercle (3.1‐fold), cerebellum (2.5‐fold) and brainstem (2.0‐fold). Immunocytochemical analysis revealed that the induction was cell‐type specific. Consistent with the increased CYP2E1 found in rat brain following drug treatments, brains from alcoholics and alcoholic smokers showed greater staining of granular cells of the dentate gyrus and the pyramidal cells of CA2 and CA3 hippocampal regions as well as of cerebellar Purkinje cells compared to nonalcoholic nonsmokers. Moreover, greater CYP2E1 immunoreactivity was observed in the frontal cortices in the alcoholic smokers in comparison to nonalcoholic nonsmokers and alcoholic nonsmokers. To investigate if nicotine could contribute to the increased CYP2E1 observed in alcoholic smokers, we treated human neuroblastoma IMR‐32 cells in culture and found significantly higher CYP2E1 immunostaining in nicotine‐treated cells (0.1–10 nM). CYP2E1 induction in the brain, by ethanol or nicotine, may influence the central effects of ethanol and the development of nervous tissue pathologies observed in alcoholics and smokers.

Regional and cellular induction of nicotine-metabolizing CYP2B1 in rat brain by chronic nicotine treatment

In the rat, nicotine is metabolized to cotinine primarily by hepatic cytochrome P450 (CYP) 2B1. This enzyme is also found in other organs such as the lung and the brain. Hepatic nicotine metabolism is unaltered after nicotine exposure; however, nicotine may regulate CYP2B1 in other tissues. We hypothesized that nicotine induces its own metabolism in brain by increasing CYP2B1. Male rats were treated with nicotine (0.0, 0.1, 0.3, or 1.0 mg base/kg in saline) s.c. daily for 7 days. CYP2B1 mRNA and protein were assayed in the brain and liver by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting, and immunocytochemistry. In control rats, CYP2B1 mRNA and protein expression were brain region- and cell-specific. CYP2B1 was not induced in the liver, but CYP2B1 mRNA and protein showed dose-dependent, region- and cell-specific patterns of induction across brain regions. At 1.0 mg nicotine/kg, the largest increase in protein was in the brain stem (5.8-fold, P < 0.05) with a corresponding increase in CYP2B1 mRNA (7.6-fold, P < 0.05). Induction of CYP2B1 was also observed in the frontal cortex, striatum, and olfactory tubercle. Immunocytochemistry showed that induction was restricted principally to neurons. These data indicate that nicotine may alter its own metabolism in the brain through transcriptional regulation, perhaps contributing to central tolerance to the effects of nicotine. CYP2B1 and its human homologue CYP2B6 also activate tobacco smoke procarcinogens such as NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Highly localized increases in CYP2B could result in increased mutagenesis. These data suggest roles for nicotine-induced CYP2B in central metabolic tolerance, nicotine-induced neurotoxicity, neuroplasticity, and carcinogenesis.

The Unique Regulation of Brain Cytochrome P450 2 (CYP2) Family Enzymes by Drugs and Genetics

Cytochrome P450 (CYP) enzymes in the brain may have a role in the activation or inactivation of centrally acting drugs, in the metabolism of endogenous compounds, and in the generation of damaging toxic metabolites and/or oxygen stress. CYPs are distributed unevenly among brain regions, and are found in neurons, glial cells and at the blood–brain interface. They have been observed in mitochondrial membranes, in neuronal processes and in the plasma membrane, as well as in endoplastic reticulum. Brain CYPs are inducible by many common hepatic inducers, however many compounds affect liver and brain CYP expression differently, and some CYPs which are constitutively expressed in liver are inducible in brain. CYP induction is isozyme‐, brain region‐, cell type‐ and inducer‐specific. While it is unlikely that brain CYPs contribute to overall clearance of xenobiotics, their punctate, region‐ and cell‐specific expression suggests that CNS CYPs may create micro‐environments in the brain with differing drug and metabolite levels (not detected or predicted by plasma drug monitoring). Coupled with the sensitivity of CNS CYPs to induction, this may in part account for inter‐individual variation in response to centrally acting drugs and neurotoxins, and may have implications for individual variation in receptor adaptation and cross‐tolerance to different drugs. In addition, genetic variation in brain CYPs, depending on the type of polymorphism (structural versus regulatory), will alter enzyme activity. These aspects of brain CYP expression regulation and genetic influences are illustrated in this review using mRNA, protein, and enzyme activity data for CYP2D1/6, CYP2E1 and CYP2B1/6 in rat and human brain. The role of CYP‐mediated metabolism in the brain, a highly heterogeneous and complex organ, is a new and relatively unexplored field of scientific enquiry. It holds promise for furthering our undestanding of inter‐individual variability in response to centrally acting drugs as well as risk for neurological diseases and pathogies.

Cytochrome P450-mediated drug metabolism in the brain.

Cytochrome P450 enzymes (CYPs) metabolize many drugs that act on the central nervous system (CNS), such as antidepressants and antipsychotics; drugs of abuse; endogenous neurochemicals, such as serotonin and dopamine; neurotoxins; and carcinogens. This takes place primarily in the liver, but metabolism can also occur in extrahepatic organs, including the brain. This is important for CNS-acting drugs, as variation in brain CYP-mediated metabolism may be a contributing factor when plasma levels do not predict drug response. This review summarizes the characterization of CYPs in the brain, using examples from the CYP2 subfamily, and discusses sources of variation in brain CYP levels and metabolism. Some recent experiments are described that demonstrate how changes in brain CYP metabolism can influence drug response, toxicity and drug-induced behaviours. Advancing knowledge of brain CYP-mediated metabolism may help us understand why patients respond differently to drugs used in psychiatry and predict risk for psychiatric disorders, including neurodegenerative diseases and substance abuse.

Regional and cellular distribution of CYP2D subfamily members in rat brain

1. Human CYP2D6 is present in brain, metabolizes many drugs and has been implicated in Parkinsons and Alzheimers diseases and some cancers. It is still unclear which of the six known rat CYP2D subfamily members is are homologous to human CYP2D6. 2. In this study, RT-PCR, Southern and Western blotting and immunohistochemical techniques were used to study the distribution of CYP2D subfamily member mRNA and proteins across 10 rat brain regions. CYP2D subfamily mRNA and protein levels were correlated with brain dextromethorphan O-demethylation (DOD), a measure of human CYP2D6 and rat CYP2D1 activities. 3. The data showed a strong relationship between CYP2D1 and CYP2D1-18 with brain DOD activity. In addition, it was shown that CYP2D proteins are present in brain mitochondrial as well as microsomal membranes. CYP2D subfamily member mRNA and proteins varied across brain regions and were highly concentrated in specific cell types. 4. These data strongly suggest that CYP2D1 and not CYP2D5 mediates DOD activity in rat brain, and may be the rat homologue of human CYP2D6. The highly localized nature of CYP2D indicates that in specific neurones enzyme levels may approach hepatic levels and, hence, contribute to local alterations in brain drug metabolism.

Brain Drug-Metabolizing Cytochrome P450 Enzymes are Active In Vivo, Demonstrated by Mechanism-Based Enzyme Inhibition

Individuals vary in their response to centrally acting drugs, and this is not always predicted by drug plasma levels. Central metabolism by brain cytochromes P450 (CYPs) may contribute to interindividual variation in response to drugs. Brain CYPs have unique regional and cell-type expression and induction patterns, and they are regulated independently of their hepatic isoforms. In vitro, these enzymes can metabolize endogenous and xenobiotic substrates including centrally acting drugs, but there is no evidence to date of their in vivo function. This has been difficult to demonstrate in the presence of hepatically derived metabolites that may cross the blood–brain barrier. In addition, because of the membrane location of brain CYPs and the rate limiting effect of endogenous heme levels on the activity and appropriate membrane insertion of some induced CYPs, it has been unclear whether sufficient cofactors and coenzymes are present for constitutive and induced CYP forms to be enzymatically active. We have developed a method using a radiolabeled mechanism-based inhibitor of CYP2B1, 3H-8-methoxypsoralen, to demonstrate for the first time that both the constitutive and induced forms of this enzyme are active in situ in the living rat brain. This methodology provides a novel approach to assess the function of enzymes in extrahepatic tissues, where expression levels are often low. Selective induction of metabolically active drug metabolizing enzymes in the brain may also provide ways to control prodrug activation in specific brain regions as a novel therapeutic avenue.

Induction of the drug metabolizing enzyme CYP2D in monkey brain by chronic nicotine treatment

Cytochrome P450 (CYP) 2D6, an enzyme found in the liver and the brain, is involved in the metabolism of numerous centrally acting drugs (e.g. antidepressants, neuroleptics, opiates), endogenous neurochemicals (e.g. catecholamines) and in the inactivation of neurotoxins (e.g. pesticides, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)). Although CYP2D6 is essentially an uninducible enzyme in the liver, we show that smokers have higher CYP2D6 in the brain, especially in the basal ganglia. In order to determine whether nicotine, a component of cigarette smoke, could increase brain CYP2D, African Green monkeys were treated chronically with nicotine (0.05 mg/kg for 2 days, then 0.15 mg/kg for 2 days followed by 0.3 mg/kg for 18 days s.c., b.i.d.). Monkeys treated with nicotine showed significant induction of CYP2D in brain when compared to saline-treated animals as detected by western blotting and immunocytochemistry. No changes in liver CYP2D were observed in nicotine-treated monkeys. Induction was observed in various brain regions including those affected in Parkinsons disease (PD) such as substantia nigra (3-fold, p = 0.01), putamen (2.1-fold, p = 0.001) and brainstem (2.4-fold, p = 0.001), with the caudate nucleus approaching significance (1.6-fold, p = 0.07). Immunocytochemistry revealed that the expression of CYP2D in both saline- and nicotine-treated monkeys is cell-specific particularly in the cerebellum, frontal cortex and hippocampus. These results suggest that monkey brain expresses CYP2D, which is induced in specific cells and brain regions upon chronic nicotine treatment. Smokers, or those using nicotine treatment, may have higher levels of brain CYP2D6 that may result in altered localized CNS drug metabolism and inactivation of neurotoxins.

The neuroprotective enzyme CYP2D6 increases in the brain with age and is lower in Parkinson's disease patients.

Cytochrome P450 2D6 (CYP2D6) is a drug-metabolizing enzyme expressed in the brain that also metabolizes endogenous neural compounds (e.g., catecholamines) and inactivates neurotoxins (e.g., 1-methyl-4-thenyl-1,2,3,6-tetrahydropyridine; MPTP). Genetically poor CYP2D6 metabolizers are at higher risk for developing Parkinsons disease (PD), a risk that increases with exposure to pesticides. As age is a risk factor for PD we measured the ontogenic expression of CYP2D6 in human brain, and compared brain CYP2D6 levels in PD cases with age-matched controls. CYP2D6 increased from fetal to 80 years of age (n = 76), exhibiting 3 distinct phases of change. Compared with PD controls, PD cases had approximately 40% lower CYP2D6 levels in the frontal cortex, cerebellum, and the hippocampus, even when controlling for CYP2D6 genotype. In contrast, CYP2D6 levels in cases were similar to controls in PD-affected brain areas, the substantia nigra, and caudate, consistent with higher astrocytic and cellular CYP2D6 staining observed in PD cases. In summary, the lower CYP2D6 levels in PD cases may have reduced their ability to inactivate PD-causing neurotoxins contributing to their disease risk.