Cytochrome P4502E (CYP2E) in brain: constitutive expression, induction by ethanol and localization by fluorescence in situ hybridization.

Cytochrome P4502E (P4502E), the major ethanol-inducible P450 metabolizes ethanol to acetaldehyde and bioactivates procarcinogens to ultimate carcinogens. Metabolism of ethanol to acetaldehyde in the brain could be deleterious since it can react with cytoskeletal proteins, forming adducts. In the present study, rats were administered ethanol chronically to evaluate its effect on chlorzoxazone hydroxylation in rat brain regions. Chlorzoxazone hydroxylation in brains from the treated rats was induced in hippocampus and cortex, downregulated in brainstem, and unchanged in cerebellum, striatum, and thalamus. The presence of functionally active P4502E was also seen in human brain regions obtained at autopsy from traffic accident victims. Northern blot analysis of rat and human brain poly(A)(+) RNA hybridized with cDNA to rat CYP2E1 revealed the constitutive presence of a corresponding transcript in rat and human brain. Localization of CYP2E by fluorescence in situ hybridization demonstrated the constitutive expression of CYP2E preferentially in the neuronal cells in rat and human brain. CYP2E expression was seen in neurons within the cerebral cortex, Purkinje and granule cell layers of cerebellum, granule cell layer of dentate gyrus, and pyramidal neurons of CA1, CA2, and CA3 subfields of hippocampus in both rat and human brain. The present studies demonstrate constitutive expression of P4502E1 in brain, its differential induction in rat brain regions by chronic ethanol treatment, and its topographic distribution in rat and human brain.

Rat brain thioltransferase: regional distribution, immunological characterization, and localization by fluorescent in situ hybridization.

Thioltransferase (TTase) is a member of the family of thiol-disulfide oxidoreductases that are involved in the maintenance of sulfhydryl homeostasis in cells by catalyzing thiol-disulfide interchange reactions. One of the major consequences of oxidative stress in brain is the formation of protein-glutathione mixed disulfides (through oxidation of protein thiols), which can be reversed by TTase during the recovery of brain from oxidative stress. We therefore examined the presence of TTase in brain regions from rat. In the rat, TTase activity in the whole brain was comparable with the corresponding activity in liver, but significantly higher in hippocampus. The enzyme activity was significantly lower in striatum and cerebellum compared with activity in whole brain. Rat brain TTase shared immunological similarity with the human red blood cell enzyme, but not with the pig liver enzyme. The constitutive expression of the mRNA to TTase was demonstrable by northern blotting. Localization of the TTase mRNA in rat brain by fluorescent in situ hybridization showed the presence of high amounts of mRNA in the olfactory bulb, cortex, and hippocampus and its predominant localization in the neurons. TTase mRNA was also present in Purkinje cells in the cerebellum, in giant reticular neurons in the midbrain, and in the striatal and thalamic neurons. This study demonstrates the constitutive presence of a functional TTase system in brain and delineates the regional and cellular localization of the enzyme in rat brain.

Expression of multiple forms of cytochrome P450 and associated mono-oxygenase activities in rat brain regions.

Cytochrome P450 (P450) content and P450-mediated mono-oxygenase activities were measured in microsomes prepared from various regions of rat brain. The regional P450 content in brain varied between 0.1 and 0.15 nmol/mg of protein, with the brainstem and cerebellum showing the highest levels. NADPH cytochrome c reductase activity was highest in the cortex followed by cerebellum and brainstem as compared with the whole brain. Mono-oxygenase activities also varied among the various brain regions. Southern blot analysis of the cDNA synthesized from the poly(A)RNA isolated from rat brain regions and hybridized with cDNA to rat liver P4502B or P4502E1 revealed the presence of a transcript in untreated rat brain that had a molecular mass similar to that of the corresponding transcript from rat liver. Immunoblot analyses using antisera to purified rat liver P4502E1, P450(2B1/2B2), and a phenobarbital-inducible form of rat brain P450 revealed the presence of corresponding immunoreactive protein bands in all the brain regions examined. The present study demonstrated the diversity in the distribution of P450 and associated mono-oxygenase activities in brain and thus may reflect the differential capability of various regions of the brain to detoxify or bioactivate diverse xenobiotics.

Xenobiotic metabolism in brain.

Recent hypothesis suggesting a role for environmental toxins in the pathogenesis of neurodegenerative disorders has stimulated interest in research on xenobiotic metabolizing capability of the brain. In addition to possible irreversible loss of neurons through bioactivation in situ in the nervous tissue, the metabolism of psychoactive drugs in the target tissue can lead to local pharmacological modulation at the site of action. The major drug metabolizing enzymes, cytochromes P-450 (P450) and flavin-containing monooxygenase (FMO) have been detected in rodent brain and human brain tissue obtained at autopsy. The brain microsomal and mitochondrial P450 systems are capable of metabolizing a variety of xenobiotics, while the brain FMO efficiently metabolizes a variety of psychoactive drugs to their respective N-oxides. Immunocytochemical studies have revealed the regional heterogeneity in the distribution of multiple forms of P450 in the brain and the co-localization of P450 and FMO predominantly in the neuronal cells. Although the brain P450 and FMO share many common features with similar enzymes present in other tissues such as liver and lung, there are some distinctive differences. It is evident from the studies carried out so far that the brain can metabolize a variety of lipophilic xenobiotics that enter by way of the blood stream.

Regulation of calbindin-D28K gene expression in response to acute and chronic morphine administration.

The effect of acute and chronic morphine administration on calbindin-D28K (calbindin) gene expression has been studied. One group of adult male rats received a single injection of morphine (10 mg/kg, s.c.) or saline and were sacrificed 1 or 4 h later. Another group was injected with escalating doses of morphine sulfate twice daily for 15 days to induce tolerance and physical dependence. Rats were sacrificed 1 h after the last injection. In a third group, the effect of naloxone-precipitated withdrawal on gene expression in morphine-addicted rats was also analyzed 1 h after naloxone (1 mg/kg, i.p.). The cerebellum and remaining brain (minus the cerebellum) were removed, and total RNA was extracted and used for analysis. Calbindin mRNA levels in cerebellum were decreased to 30%-40% control at 1 and 4 h after a single morphine injection. Co-administration of the opiate antagonist, naloxone, reversed the effect of morphine. Tolerance developed to the acute effects in that levels were not altered significantly 1 h after morphine injection in chronically-treated rats. Unlike the cerebellum, calbindin mRNA in the remainder of the brain (minus the cerebellum) was unchanged 1 and 4 h following morphine administration to drug-naive rats, but was increased more than 2-fold compared to controls 1 h after morphine injection in chronically treated animals. Naloxone-precipitated withdrawal caused a small (20%) but significant decrease in calbindin mRNA in the cerebellum, with no change in the brain (minus the cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)

Human ribosomal protein L37a: cloning of the cDNA and analysis of differential gene expression in tissues and cell lines.

A cDNA corresponding to human ribosomal protein L37a (hL37a) was obtained by screening a SHSY5Y neuroblastoma library. The full-length cDNA contained 366 nucleotides (nt) in addition to a poly(A) tail. The pyrimidine-rich sequence, CTTTCT, that is common to many ribosomal protein-encoding cDNAs was present at the 5' terminus. The nt sequence displayed 85% identity with rat L37a (rL37a) cDNA. The predicted protein contains 92 amino acids with a M(r) of 10,277, is highly basic, and has 100% sequence identity with rL37a. A putative zinc-finger domain is present in the central region of the protein. Human lymphocytes and several human cell lines express hL37a mRNA at significantly higher levels than the rat cell lines and rat tissues tested. The hL37a gene does not contain introns.