Nobuo Nemoto

Regulation of the expression of two female-predominant CYP3A mRNAs (CYP3A41 and CYP3A44) in mouse liver by sex and growth hormones

A second female-predominant murine CYP3A, CYP3A44, was isolated from liver and its mRNA expression was compared with that of the previously described CYP3A41. The expression of CYP3A44 was relatively constant after birth in females, whereas it gradually declined in males after 5 weeks of age. The expression of CYP3A41 increased with age in females after 3 weeks of age, whereas it gradually declined in males after 5 weeks of age. Hypophysectomy and growth hormone replacement indicated that expression of both CYP3A mRNAs in females was dependent on the feminine plasma growth hormone profile. Estradiol induced the expression of both mRNAs and the effect was dependent on the presence of the pituitary gland. These observations suggest that endocrine control of expression might be similar, but not identical, for two female-predominant CYP3A mRNAs.

Different expression of hepatic and renal cytochrome P450s between the streptozotocin-induced diabetic mouse and rat.

1. Since limited information is available about alterations of cytochrome P450 levels in diabetic animals other than rat, expression of P450s in the liver and kidney of the streptozotocin (STZ)-induced diabetic mouse was investigated. 2. The mRNA levels of CYP2B10, 3A11, 4A10 and 4A14 in the liver were increased in the STZ-induced diabetic mouse of both sexes. The CYP2B9 mRNA level was increased in the liver of the male diabetic mouse. These alterations were observed even at 2 weeks after administration. Insulin treatment restored these changes. The findings were consistent with changes reported in rat. 3. The levels of hepatic CYP1A2 and 2E1 and renal 2E1 and 4A did not change in the diabetic mouse at any time-point examined. No changes were seen in CYP2A- or 2Crelated proteins in the diabetic mouse. These findings were in contrast to those in rat. 4. The results indicate that mouse P450s respond to insulin-dependent diabetes mellitus differently from those of the rat, and suggest that the expression of P450s in diabetes is not generally the same across animal species.

Assay and properties of glutathione-S-benzo(a)pyrene-4,5-oxide transferase

Abstract Rat liver soluble fraction catalyzes the formation of a conjugate of glutathione and [3H]benzo(a)-4,5-oxide. The conjugate was isolated by thin-layer chromatography and identified by its radioactivity and its ninhydrin reactivity. When both [3H]benzo(a)-pyrene-4,5-oxide and [14C]glutathione were used, the radioactivity from each precursor was in the conjugate. The reaction was linear with protein concentration from 10 to 500 μg protein. Transferase activity was pH dependent, and sensitive to heat and pronase digestion. The amount of nonenzymatic conjugation was negligible under all the conditions described. This procedure measures a key enzyme in polycylic hydrocarbon metabolism and its activity may relate to the efficiency of detoxification of active carcinogenic intermediates.

Targets of Transcriptional Regulation by Transforming Growth Factor-β: Expression Profile Analysis Using Oligonucleotide Arrays

Transforming growth factor‐βs (TGF‐βs) are potent inhibitors of cell proliferation, and disruption of components of the TGF‐β signaling pathway leads to tumorigenesis. Mutations of transmem‐brane receptors and Smads mediating intracellular signaling have been reported in various cancers. To identify transcriptional targets of TGF‐β, we conducted an expression profile analysis. HaCaT cells derived from human keratinocytes and highly sensitive to TGF‐β were treated with TGF‐β in the absence or presence of cycloheximide (CHX). mRNAs extracted from the HaCaT cells were used for hybridization of oligonucleotide arrays representing approximately 5600 human genes. TGF‐β increased the expression of PAI‐1, junB, p21 cdk inhibitor, Smad7, βIG‐H3, and involucrin that have been reported to be up‐regulated by TGF‐β, validating the usefulness of this approach. The induction of βIG‐H3 by TGF‐β was completely abolished by CHX, suggesting that the transcription of βIG‐H3 is not directly regulated by TGF‐β. Unexpectedly, we identified more genes down‐regulated by TGF‐β than up‐regulated ones. TGF‐β repressed the expression of epithelial specific Ets that may be involved in breast and lung tumorigenesis, which could contribute to tumor suppression by TGF‐β. Among a panel of cell cycle regulators, TGF‐β induced the expression of p21 cdk inhibitor; however, the induction of other cdk inhibitors was not significant in the present study. Taken together, the results suggest that TGF‐β may suppress tumorigenesis through positive and negative regulation of transcription.

Mechanism of Growth Promoting Activity of Epiregulin in Primary Cultures of Rat Hepatocytes

In spite of lower receptor affinity, epiregulin exhibits a stronger stimulation of DNA synthesis than epidermal growth factor (EGF) in rat hepatocytes. To determine the mechanism of stimulation, we examined the activities of epiregulin on growth stimulation, signal transduction, and mRNA induction of hepatotrophic factors in primary cultures of rat hepatocytes. Epiregulin stimulated hepatocyte proliferation as efficiently as hepatotrophic factors, including heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor- f (TGF- f ). Epiregulin induced a more prolonged activation of EGF receptor (EGFR) and p42/44 mitogen-activated protein kinase (MAPK) than EGF. Furthermore, epiregulin up-regulated the mRNAs of TGF- f and HB-EGF, and in turn, these growth factors enhanced the expression of epiregulin mRNA. In vivo, increased production of epiregulin was noted in extracts of the remnant liver obtained 24 h after partial hepatectomy, and EGFR phosphorylation by these extracts was partially inhibited by anti-epiregulin antibody. Our results showed a more potent hepatocyte proliferative activity for epiregulin compared with EGF in vitro, which depends on prolonged activation of EGFR and p42/44 MAPK. Our findings suggest that epiregulin may play significant roles in liver regeneration following partial hepatectomy in cooperation with other growth factors.

Sex-associated expression of mouse hepatic and renal CYP2B enzymes by glucocorticoid hormones

The expression of Cyp2b9 and Cyp2b10 genes was investigated in kidney, liver, and cultured hepatocytes of adult C57BL/6NCrj mice. The constitutive expression level of CYP2B mRNA in kidney was higher in female than in male mice, as it was in the liver where more CYP2B9 than CYP2B10 was expressed in the females, and more CYP2B10 was expressed in the males. After treatment with dexamethasone (Dex), induction of CYP2B10 mRNA and protein in the kidneys was far greater in male than in female mice. In contrast to Dex, phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) did not induce the expression of the Cyp2b gene in the kidneys of either sex. In the liver, PB, PCN, and DDT induced both CYP2B9 and CYP2B10 in both sexes to the same extent, whereas Dex induced only CYP2B10 and simultaneously suppressed CYP2B9. Dex-inducible expression of CYP2B mRNA was decreased by 11 beta-[4-dimethylamino]phenyl-17 beta-hydroxy-17-[1-propynyl]estra-4,9-dien-3-one (RU-486), in both the kidneys and liver from male mice, and in cultured hepatocytes. However, RU-486 itself induced the expression of CYP2B mRNA in female liver and cultured hepatocytes. Interestingly, RU-486 increased PB-inducible expression of these species in cultured hepatocytes. Gonadectomy increased the expression of CYP2B mRNA in untreated male liver, but suppressed Dex-induced expression in the kidneys of both sexes. These observations suggest that (a) there are multiple regulatory pathways in the expression of Cyp2b genes, one of which used by Dex is mediated via the glucocorticoid receptor, which is different from that used by PB, and (b) sex hormones play a role in the regulation of the sex-dependent expression of Cyp2b genes in the mouse.

Preferential excision repair and non-preferential photoreactivation of pyrimidine dimers in the c-ras sequence of cultured goldfish cells

The time courses of excision repair and photoreactivation of pyrimidine dimers induced by 254-nm UV were examined in the genome overall and in the c-ras sequence of RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Excision repair was more efficient in the ras sequence than in the genome overall, whereas no differences in efficiency of photoreactivation were detected. These results suggest that excision repair is affected by the accessibility of chromatin, while photoreactivation is not.

Sexual dimorphic expression of mouse hepatic CYP2B: alterations during development or after hypophysectomy

The constitutive expression of CYP2B mRNA in the livers of mice in the prepubertal stage was sex-independent, with CYP2B9 as the principal isoform. During the maturation stage, CYP2B10 was expressed in both sexes, whereas CYP2B9 was diminished markedly in the males, resulting in a sexually dimorphic expression in adult mice. Hypophysectomy eliminated the sexual dimorphism in the mouse CYP2B subfamily by markedly increasing the expression of both CYP2B9 and CYP2B10 in males to levels similar to those in females.

Rapid loss of 7-methylguanine from liver nucleic acids in mice during the initial stage of liver carcinogenesis induced by dimethylnitrosamine.

Abstract Dimethylnitrosamine was administered to mice in their drinking water. The amount of 7-methylguanine in liver nucleic acids was found to increase initially and then to decrease. This decrease in 7-methylguanine was found to be due to decrease in the rate of its formation and increase in its excision from nucleic acids.

Enhancer elements in the mouse Cypla2 gene for constitutive expression

CYP1A2 is one of the major hepatic cytochrome P450s that is involved in the metabolism of many drugs, as well as in the activation of chemical carcinogens. To elucidate the transcriptional regulation of the constitutive expression of the mouse Cypla2 gene, the 4.8-kbp 5()-flanking region of the gene was analyzed for transcriptional activity using a primary cultured mouse hepatocyte system. With 5()- and 3()-deletion analysis, two enhancer elements, i.e., a 20-bp DNA fragment (E1) from -4401 to -4382 and a 9-bp (E2) from -4300 to -4292, were identified. E1 and E2 contain a phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE) and TRE-like element, respectively. Electrophoretic mobility shift assay confirmed specific binding between these two enhancer elements and nuclear proteins. Site-directed mutagenesis assay suggested that the TRE element in E1 is essential for constitutive expression of the mouse Cypla2 gene.