Ravindranath R P Kommaddi and H V

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Division of Molecular and Cellular Neuroscience, National Brain Research Centre, Nainwal Mode, Manesar, Haryana, India

Summary. Cytochromes P450 is a family of heme proteins that metabolize xenobiotics including drugs. Unique human brain cyto-chrome P450 enzymes metabolize xenobiotics including drugs to active/inactive metabolites through biotransformation pathways that are different from the well-characterized ones in liver. We have identified an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 in human brain but not in liver or kidney from the same individual. Genotyping revealed the presence of the frame-shift mutation 138delT only in those subjects who expressed the brain variant CYP2D7, which metabolizes codeine exclusively to morphine unlike hepatic CYP2D6 that metabolizes codeine to nor codeine and morphine. CYP1A1 bioactivates polycyclic aromatic hydrocarbons to reactive DNA binding metabolites and initiates carcinogenesis. We have identified a unique splice variant of CYP1A1 having deletion of 87 bp of exon 6 which is present in human brain but not in liver of the same individual. We present evidence for the existence of biotransformation pathways in human brain that are dissimilar from known pathways in liver. Identification and characterization of novel CNS-specific P450 enzymes generated by alternate splicing of known genes or as yet unidentified genes may help predict consequences of exposure to xenobiotics including pesticides in the brain.

Metabolism of foreign compounds is an important prerequisite for detoxification of xe-nobiotics. A major enzyme involved in the metabolism of foreign compounds is cyto-chrome P-450 (P450). Generally, P450 mediated metabolism of xenobiotics leads to the formation of hydrophilic, non-toxic metabolites that are easily excreted from the body. However, there are instances wherein an inert, non-toxic compound is bioactivated to a reactive, toxic metabolite that can interact with cellular macromolecules leading to cell damage. Multiple forms of P450, which are selectively induced or inhibited by a variety of drugs are known to exist in liver, the major organ involved in P450 mediated metabolism (de Montellano, 1996). P450 enzymes, such as CYP2D6 exhibit genetic polymorphism and 7-10% of caucasians are poor metabolizers of debrisoquine (prototype substrates for P4502D6) while the remaining 90% are extensive metabolizers. The significance of P450 metabolism in extrahepatic organs (such as lung, kidney, skin, nasal epithelium) and pharmacological and toxi-cological consequences of in situ metabolism in target organs has been increasingly recognized (Gram et al., 1986; MeLemore et al., 1990). These studies have revealed the preferential localization of drug metabolizing enzymes within specific cell types in these organs rendering them vulnerable to damage by bioactivation, in situ, within these cells (Boyd, 1980).

Human brain is perhaps one of the most complex organs both functionally and anatomically. Brain exhibits a multitude of diversity both with respect to its distinct anatomical regions and cellular elements and is highly vulnerable to damage by toxic compounds due to the limited regenerative capability of the neurons, the major cell type involved in specialized functions of the brain. The distinctive features of the capillary endothelial cells surrounding the cerebral blood vessels render protection to the brain by preventing entry of circulating molecules. The blood-brain barrier, as this hypothetical barrier is commonly known, results from the presence of tight junctions and the paucity of pinocytic vesicles. However, xenobiotics that are lipophilic in character can diffuse through endothelial cells of brain capillaries and enter neuronal cells. Thus, bioactivation, in situ, in neuronal cell can have far-reaching consequences causing irreversible disruption of neuronal function. Further, metabolism of drugs in brain can lead to local pharmacological modulation at the site of action and result in variable drug response. Minor metabolic pathways, which are not of significance in liver could potentially produce significant pharmacological responses, if they were to occur at the site of action, within specific nuclei in the brain. This raises the question - can the brain metabolize xenobiotics? What is the P450 content in brain and where is the enzyme localized?

P450 content in rat brain is approximately 3-10% of the corresponding level in liver. The enzyme is not uniformly distributed amongst different regions of the brain and highest P450 levels have been detected in olfactory lobes, cerebellum and brain stem. Cytochrome P450 content and associated monooxygenase activities have been measured in microsomes from human brain tissue obtained at autopsy. Regional differences have been noted in the distribution of P450 in human brain and the hemeprotein levels are highest in the brain stem and cerebellum and lowest in striatum and hippocampus. Immu-nocytochemical localization of brain P450 has been performed using antisera to hepatic P450 enzymes and the preferential localization of P450 in neuronal cells has been documented (Ravindranath and Boyd, 1995).

The presence of P450 in neuronal cells, which have very limited regenerative capability brings forth the potential consequences of bioactivation of xenobiotics in situ in the CNS. In neurodegenerative diseases such as, Parkinson's, Alzheimer's and motor neuron disease specific cell populations within specialized regions of the brain are affected leading to selective loss of function. A role for environmental toxicants has been proposed in the pathogenesis of these disorders. The above hypothesis has gained ground following the incidence of Parkinson's disease (PD) in young adults who were exposed to MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The observations made with MPTP have demonstrated how the unique features of the CNS can potentially aid bioactivation of inert compounds to ultimate toxins, sequester them in target cells leading to irreversible damage to specific regions of the brain and thus bring about selective loss in function. The localization of P450 in specific cell types, such as the neurons of sub-stantia nigra, which are affected in Parkinson's disease points to the possible role of P450 mediated bioactivation or impaired detoxification of xenobiotics within these cells.

Following these discoveries several studies were carried out to ascertain the association between polymorphism in P450 enzymes, such as CYP2D6, 1A1, 1A2 and 2E1 and incidence of PD. These were based on the hypothesis that defective hepatic metabolism of environmental toxicants could potentially result in increased burden in the CNS leading to neurodegeneration. Several studies have been carried out in diverse population across the world with confounding

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Fig. 1. Expression of brain variant CYP2D7 and CYP2D6 in human samples. A RT-PCR analysis identifying brain variant CYP2D7 in 10 human brain autopsy samples (lanes 1-10). Human brain was obtained from Human Brain Tissue Repository, NIMHANS, India. The average age of the individuals was 30.7 ±4.1 years and postmortem delay between death and autopsy was 8.2 ± 1.7 hrs. Brain variant CYP2D7 could be detected in 4 samples wherein a band of 340 bp was detected. The PCR amplified product of 282 bp representing CYP2D6 was detected in all samples. B Membrane preparations from the 10 human autopsy brain samples (lanes 1-10) were subjected to immunoblotting and stained with antiserum to brain variant CYP2D7. Brain variant CYP2D7 protein could be detected only in 4 brain samples, which also showed the expression of the transcript by RT-PCR. 'M' represents molecular weight markers results and no clear association has emerged. This raises an important question: Are there brain-specific biotransformation pathways that differ from the well-characterized one in liver? While many P450 enzymes are expressed in brain, is there anything unique about their presence there?

One of the first evidence to emerge demonstrating the difference between brain and liver in the metabolism xenobiotics was the observation of differential metabolism of alpra-zolam. Alprazolam, an anti-anxiety agent, is metabolized in liver by P4503A to 4-hydroxy alprazolam (4-OHALP, pharmacologically less active) and a-hydroxy alprazolam (a-OHALP, pharmacologically more active). We observed that the relative amount of pharmacologically active metabolite, a-OHALP formed in brain was higher than liver. Since hydroxy metabolites of alprazolam are hydrophilic and not easily cleared through blood-CSF barrier, a-OHALP would potentially have a longer half-life in brain. While P450 levels in the brain are one-tenth to one-fifteenth of the corresponding hepatic levels and it is generally acknowledged that the capability of the brain to carry out P450 mediated metabolism of xenobiotics is substantially lower, the above considerations not withstanding bioactive metabolites can be formed in significantly high amounts in the brain (Pai et al., 2002).

Conclusive evidence for the presence of unique, brain-specific P450 enzymes was provided following the identification of a splice variant of CYP2D7 in human brain. A frame-shift mutation 138delT generates an open reading frame in the pseudogene, CYP2D7 and an alternate spliced functional transcript of CYP2D7containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 was detected in 4 out of 10 human autopsy brains (Fig. 1). Genotyp-ing revealed the presence of the frame-shift mutation 138delT only in those human subjects that expressed the brain variant

CYP2D7. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug) while nor-codeine, is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared to cells expressing CYP2D6. Morphine binds to m-opioid receptors in certain regions of the central nervous system, such as periaqueduc-tal gray and produces pain relief. The brain variant CYP2D7 and m-opioid receptor co-localize in neurons of periaqueductal gray area in human brain indicating that metabolism of codeine to morphine could occur at the site of opioid action (Pai et al., 2004).

CYP1A1, the cytochrome P450 enzyme, bioactivates polycyclic aromatic hydrocarbons to reactive metabolite(s) that bind to DNA and initiate carcinogenesis. We have identified in human brain a unique splice variant of cytochrome CYP1A1 cDNA having deletion of 87 bp of exon-6 (Chinta et al., 2005). This splice variant was present in human brain, but not in the liver from the same individual and was absent in rat brain and liver. Structural modelling of the putative protein indicated broadening of the substrate access channel indicating that it could potentially have broader substrate specificity. The presence of distinct cytochrome P450 enzymes in human brain generated by alternate splicing that are different from well-characterized hepatic forms indicates that metabolism of xenobiotics could occur in brain by pathways different from those known to occur in liver.

Current research on the pathogenesis of PD and its relationship to xenobiotic metabolizing enzymes, such as, P450 have been restricted to identifying association between genetic polymorphisms in P450 enzymes and incidence of sporadic PD (Riedl et al., 1998). Assessment of gene-environment interaction in PD, eg. exposure to pesticides and polymorphisms in P450 has provided evidence for increased incidence of PD in CYP2D6, PM (poor metabolizer) phenotype who have been exposed to pesticides (Elbaz et al., 2004). Although genetic polymorphism of CYP2D6 is one of the important determinants of inter-individual variation in drug response, functional polymorphism of P450 does not always correlate with outcome. We now present evidence for the existence of a pathway that can potentially mediate metabolism of xenobiotics at the site of action by mechanisms that are dissimilar from known pathways in liver.

Recent evidence indicating the presence of unique P450 enzymes in brain generated by alternate splicing indicates that potentially these unique P450 enzymes could play a role in determining the consequence of pesticide exposure in brain leading to neurodegeneration and potentially sporadic PD. Since the alternate spliced P450 enzymes are specifically generated in brain but not in other organs in the same individuals they are not related to differences in the genomic sequence. Nervous system has a propensity for generating alternate spliced genes and their expression is governed by the spliceo-somal complex and RNA binding proteins, which are poorly understood. This discovery adds a new dimension to the role of environmental toxins, such as, pesticides in the pathogenesis of PD, wherein the metabolism of the toxin, in situ, in the brain by brain-specific P450 enzymes would govern the outcome. Identification and characterization of novel histio-specific isoforms of P450 generated by alternate splicing of known genes or as yet unidentified genes may help predict outcome of exposure to xenobiotics including drugs that act on the CNS.

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