Minro Watanabe

Microsatellite polymorphism in the human heme oxygenase-1 gene promoter and its application in association studies with Alzheimer and Parkinson disease

Abstract Oxidative stress has been suggested to be involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer disease (AD) and Parkinson disease (PD). Heme oxygenase-1 (HO-1), a key enzyme in heme catabolism, also functions as an antioxidant enzyme. Here, we show that a (GT)n repeat in the human HO-1 gene promoter region is highly polymorphic, although no particular alleles are associated with AD or PD. This newly identified genetic marker should allow us to study the possible involvement of HO-1 in certain human diseases.

Restriction fragment length polymorphism of the human CYP2E1 (cytochrome P450IIE1) gene and susceptibility to lung cancer: possible relevance to low smoking exposure.

Polymorphic metabolism of certain chemical carcinogens may result in differences in susceptibility to cancers. Human CYP2E1 (cytochrome P450IIE1) is an enzyme involved in the metabolic activation of precarcinogens such as nitrosamines. We detected a restriction fragment length polymorphism (RFLP) of the human CYP2E1 gene for the restriction endonuclease Dra I. The distribution of this polymorphism was examined among lung cancer patients (n = 91), patients with cancer of the digestive tract (n = 45) and controls (n = 76). A significant difference in the distribution was observed between lung cancer patients and controls (chi 2 = 11.4 with 2 df; p < 0.005). On the other hand, there was no significant difference between patients between cancer of the digestive tract and controls (chi 2 = 4.87 with 2 df; NS). This finding suggests that the Dra I polymorphism of the CYP2E1 gene is associated with susceptibility to lung cancer. In addition, an association was found between the amount of lifelong smoking exposure and the distribution of the genotypes of the RFLP among lung cancer patients. The distribution pattern seemed deviated from that of controls especially in the population of low smoking exposure. Our Northern blot analysis data using RNA from human liver autopsy samples suggest that the Dra I polymorphism might be associated with the gene expression of CYP2E1 at mRNA level.

THE AH RECEPTOR IS NOT INVOLVED IN 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN-MEDIATED APOPTOSIS IN HUMAN LEUKEMIC T CELL LINES

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental pollutant causing public concern. Its toxic effects include disruption of the immune, endocrine, and reproductive systems, impairment of fetal development, carcinogenicity, and lethality in rodents. Here, we report that TCDD induces apoptosis in two cultured human leukemic lymphoblastic T cell lines. This cell death was found not to be dependent on an aryl hydrocarbon receptor and to be inhibited by the inhibitor of tyrosine kinases and caspases. Apoptosis-linked c-Jun N-terminal kinase is rapidly activated in these cells by the treatment with TCDD. A dominant-negative mutant of c-Jun N-terminal kinase prevented cell death in the treatment with TCDD. Furthermore, TCDD decreases the Bcl-2 protein level in these cell lines. These findings will help in the understanding of the molecular mechanism underlying TCDD-mediated immunotoxicity.

Detection of point mutation in the tyrosinase gene of a Japanese albino patient by a direct sequencing of amplified DNA

SummaryEnzymatic DNA amplification and direct DNA sequencing were used to detect a mutation in the tyrosinase gene of an albino patient. Single-base change could be detected by direct sequencing. This base change (G to A) is thought to result in an amino acid change (Arg to Gln) in tyrosinase of the patient.

Liver microsomal drug metabolism in ethanol-treated hamsters

Administration of ethanol in drinking water to Syrian golden hamsters for 1-3 weeks caused alterations of microsomal cytochrome P-450-dependent monooxygenase activities in the liver accompanied by a slight elevation in cytochrome P-450 content. Ethanol treatment resulted in an increase in the activities for ethanol oxidation, aniline p-hydroxylation and dimethylnitrosamine N-demethylation. In particular, when dimethylnitrosamine was used as a substrate, the rate of formaldehyde formation was enhanced by 2- to 2.7-fold, while ethanol oxidation and aniline p-hydroxylation were increased by 1.5- to 2- and 1.2- to 1.3-fold, respectively. On the other hand, the activities of 7-ethoxycoumarin O-deethylase, benzphetamine N-demethylase and benzo[a]pyrene 3-hydroxylase were apparently decreased after ethanol treatment. These results for hamsters were significantly different from those reported for rats.

Significance of use of amino acids and histamine for the elution of nonhistone proteins in copper-chelate chromatography.

Abstract A method utilizing coppor-chelate chromatography has been developed for the fractionation of nonhistone proteins under mild conditions. Specific proteins adsorbed on the column were eluted with the buffer containing glycine, histamine, and histidine. Using this method, RNA polymerase B stimulatory factor was partially purified from nonhistone proteins of rat liver nuclei.

Assessment of cancer susceptibility in humans by use of genetic polymorphisms in carcinogen metabolism.

Prevention is an important and effective measure for reducing death caused by cancer. Thus information on individual susceptibility to cancer is valuable in suggesting high risk individuals to avoid intake of carcinogenic substances and receive frequent physical screening. To this end, polymorphisms found within cytochrome P450 (CYP) genes implicated in the metabolism of procarcinogens are expected to be good genetic targets in assessing human cancer susceptibility. We have found polymorphisms in the CYP2E1 and CYP1A1 genes associated with lung cancer susceptibility, though there were some discrepancies from observations made by other investigators. Discrepancies among investigators from different regions, however, are very common in these pharmacogenetic studies. We present an explanation for these discrepancies, difficulties associated with prediction of relative risk of individuals, and future directions.

Induction by interleukin-1 (IL-1) of the mRNA of histidine decarboxylase, the histamine-forming enzyme, in the lung of mice in vivo and the effect of actinomycin D

It is known that the activity of histidine decarboxylase (HDC), the histamine-forming enzyme, is induced in response to various stimuli. However, it has repeatedly been reported that actinomycin D (Act D), a typical inhibitor of RNA synthesis, is either ineffective, or actually potentiates induction of this enzyme. Thus, it has been suggested that the induction of HDC may not require the formation of mRNA, i.e. that pre-formed, long-lived mRNA molecules may be responsible for the induction. In the present study, we examined the effects of interleukin-1alpha (IL-1alpha) on the amount of HDC mRNA present during the induction of HDC activity. In mice injected with IL-1alpha, HDC mRNA increased in the lung, spleen and stomach, but was hardly detectable in these tissues in control (saline-injected) mice. In the lung, the time course of the rise and fall in HDC mRNA was shorter than that of the rise and fall in HDC activity. In the present study, actinomycin D (Act D) did not inhibit the increase in HDC mRNA induced by IL-1alpha; in fact, it potentiated the elevation of both HDC mRNA and HDC activity. These results suggest that IL-1alpha induces HDC activity or its enzyme protein through the formation of short-lived HDC mRNA molecules. This is the first demonstration that Act D can enhance an increase in HDC mRNA: this potentiating, rather than inhibiting, effect is discussed.

Cytochrome P-450 system dependent depression of δ-aminolevulinic acid dehydratase activity by bromobenzene in rats

Male Wistar rats (200-230 g) were treated with bromobenzene in soybean oil intraperitoneally (i.p.) (4 mmol/kg) once a day for 1 or 2 days while control rats received soybean oil alone. delta-Aminolevulinic acid dehydratase (ALA-D) activity was depressed to 80% and 43% in bone marrow after 24 h and 48 h, respectively. ALA-D activity was also depressed significantly in the liver after the administration of bromobenzene while the activity in peripheral erythrocytes was not altered. After the administration of bromobenzene, the concentration of reduced non-protein sulfhydryls in liver was the lowest at 24 h and increased thereafter. No significant change was observed in the activity of delta-aminolevulinate synthase in liver. The decrease of ALA-D activity was also reproducible in vitro. The 105 000 g supernatant fractions of rat bone marrow lyzates as ALA-D source were incubated with liver microsomes prepared from rats treated with phenobarbital. ALA-D activity was decreased by bromobenzene but no decrease was observed when the microsomes were preincubated with CO to inhibit cytochrome P-450. The effect of bromobenzene on ALA-D purified from rat erythroid cells was studied in incubations containing a reconstituted cytochrome P-450 system prepared from rat liver. The decrease of ALA-D activity was proportional to both the incubation time and to the concentration of P-450 while no decrease was detected when P-450 was inhibited by CO before the incubation.

Genetic differences in the induction of aryl hydrocarbon hydroxylase and its components by 3-methylcholanthrene in liver and lung microsomes among four strains of guinea pigs

Four strains of guinea pigs (Hartley, No. 2, No. 13 and JY-1) were examined for the effects of intraperitoneal treatment with 3-methylcholanthrene on aryl hydrocarbon hydroxylase activity, total cytochrome P-450 content in liver and lung microsomes, and NADPH-cytochrome c reductase activity in liver microsomes. Following treatment with 3-methylcholanthrene at a dose of 50 mg/kg body weight, aryl hydrocarbon hydroxylase activity and cytochrome P-450 content in liver were both increased in all the strains used, and the activity of NADPH-cytochrome c reductase in liver was also increased in all strains except No. 13. While the cytochrome P-450 content in lung was increased in all the strains except No. 13, there was no increase in the aryl hydrocarbon hydroxylase activity in lung from any strain of guinea pig examined. When the dose of 3-methylcholanthrene was increased to 250 mg/kg body weight, an apparent induction of aryl hydrocarbon hydroxylase was detected in the lung from the Hartley strain of guinea pigs, but not in the other three strains. In summary, marked differences were seen in sensitivity to 3-methylcholanthrene between liver and lung, and apparent strain differences were observed among the guinea pigs used in this experiment.