Taro E. Akiyama

Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver.

The occurrence of malignant tumors of the upper gastrointestinal tract and liver is, based largely on epidemiological evidence, causally related to the consumption of ethanol. It is widely recognized that oxidants play a key role in alcohol‐induced liver injury; however, it is unclear how oxidants may be involved in DNA damage. We asked whether nicotinamide adenine dinucleotide phosphate oxidase, cytochrome P450 CYP2E1, or both are responsible for the production of DNA damage. The rodent Tsukamoto‐French model of intragastric ethanol infusion was used. Wistar rats, Cyp2e1‐, p47phox‐null, and hCyp2e1 transgenic mice were used. The abundance of oxidative DNA adducts, mutagenic apurinic/apyrimidinic sites, and expression of base excision DNA repair genes was determined. In rats and wild‐type mice, ethanol treatment for 4 weeks led to an increase in oxidative DNA damage and induction of expression of the base excision DNA repair genes that are known to remove oxidative DNA lesions. No increase in either of the endpoints was observed in ethanol‐treated Cyp2e1‐null mice, whereas the magnitude of response in p47phox‐null mice and transgenic hCyp2e1 was identical to that in wild types. The increase in expression of DNA repair genes was completely abolished by treatment with the P450 inhibitor 1‐aminobenzotriazole. In conclusion, the data support the hypothesis that oxidative stress to DNA is induced in liver by ethanol. Furthermore, although it was shown that nicotinamide adenine dinucleotide phosphate oxidase‐derived oxidants are critical for the development of ethanol‐induced liver injury, CYP2E1 is required for the induction of oxidative stress to DNA, and thus may play a key role in ethanol‐associated hepatocarcinogenesis. (HEPATOLOGY 2005;41:336–344.)

Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors

Cytochrome P450s (P450s) constitute a superfamily of heme-proteins that play an important role in the activation of chemical carcinogens, detoxification of numerous xenobiotics as well as in the oxidative metabolism of endogenous compounds such as steroids, fatty acids, prostaglandins, and leukotrienes. In addition, some P450s have important roles in physiological processes, such as steroidogenesis and the maintenance of bile acid and cholesterol homeostasis. Given their importance, the molecular mechanisms of P450 gene regulation have been intensely studied. Direct interactions between transcription factors, including nuclear receptors, with the promoters of P450 genes represent one of the primary means by which the expression of these genes is controlled. In this review, several liver-enriched transcription factors that play a role in the tissue-specific, developmental, and temporal regulation of P450s are discussed. In addition, the nuclear receptors that play a role in the fine control of cholesterol and bile acid homeostasis, in part, through their modulation of specific P450s, are discussed.

Hepatic expression of cytochrome P450s in hepatocyte nuclear factor 1-alpha (HNF1α)-deficient mice

Hepatocyte nuclear factor 1 alpha (HNF1alpha) is a liver enriched homeodomain-containing transcription factor that has been shown to transactivate the promoters of several cytochrome P450 (CYP) genes, including CYP2E1, CYP1A2, CYP7A1, and CYP27, in vitro. In humans, mutations in HNF1alpha are linked to the occurrence of maturity onset diabetes of the young type 3, an autosomal dominant form of non-insulin-dependent diabetes mellitus in which afflicted subjects generally develop hyperglycemia before 25 years of age. Mice lacking HNF1alpha also develop similar phenotypes reminiscent of non-insulin-dependent diabetes mellitus. To investigate a potential role for HNF1alpha in the regulation of CYPs in vivo, the expression of major CYP genes from each family was examined in the livers of mice lacking HNF1alpha. Analysis of CYP gene expression revealed marked reductions in expression of Cyp1a2, Cyp2c29 and Cyp2e1, and a moderate reduction of Cyp3a11. In contrast Cyp2a5, Cyp2b10 and Cyp2d9 expression were elevated. There are also significant changes in the expression of genes encoding CYPs involved in fatty acid and bile acid metabolism characterized by a reduction in the expression of Cyp7b1, and Cyp27 as well as elevations in Cyp4a1/3, Cyp7a1, Cyp8b1, and Cyp39a1 expression. These results point to a critical role for HNF1alpha in the regulation of CYPs in vivo and suggest that this transcription factor may have an important influence on drug metabolism as well as lipid and bile acid homeostasis in maturity onset diabetes of the young type 3 diabetics.

A molecular link between the common phenotypes of type 1 glycogen storage disease and HNF1alpha-null mice.

The clinical manifestations of type 1 glycogen storage disease (GSD-1) in patients deficient in the glucose-6-phosphatase (G6Pase) system (e.g. growth retardation, hepatomegaly, hyperlipidemia, and renal dysfunction) are shared by Hnf1α−/− mice deficient of a transcriptional activator, hepatocyte nuclear factor 1α (HNF1α). However, the molecular mechanism is unknown. The G6Pase system, essential for the maintenance of glucose homeostasis, is comprised of glucose 6-phosphate transporter (G6PT) and G6Pase. G6PT translocates G6P from the cytoplasm to the lumen of the endoplasmic reticulum where it is metabolized by G6Pase to glucose and phosphate. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively.Hnf1α−/− mice also develop noninsulin-dependent diabetes mellitus caused by defective insulin secretion. In this study, we sought to determine whether there is a molecular link between HNF1α deficiency and function of the G6Pase system. Transactivation studies revealed that HNF1α is required for transcription of the G6PT gene. Hepatic G6PT mRNA levels and microsomal G6P transport activity are also markedly reduced in Hnf1α−/− mice as compared withHnf1α+/+ andHnf1α+/− littermates. On the other hand, hepatic G6Pase mRNA expression and activity are up-regulated inHnf1α−/− mice, consistent with observations that G6Pase expression is increased in diabetic animals. Taken together, the results strongly suggest that metabolic abnormalities in HNF1α-null mice are caused in part by G6PT deficiency and by perturbations of the G6Pase system.

Positive regulation of connexin32 transcription by hepatocyte nuclear factor-1α

Connexin32 (Cx32) encodes the predominant gap junction protein expressed by hepatocytes. We investigated the transcriptional control of Cx32 in expressing and nonexpressing rat liver cell lines and hypothesized that a putative hepatocyte nuclear factor-1 (HNF-1) binding site (centered at mp -187) in the liver-active, P1 promoter is essential for transcription of Cx32. HNF-1alpha was expressed by Cx32-expressing rat liver cell lines and bound the promoter at the -187 site, but was not expressed by non-Cx32-expressing hepatic lines. Stable transfection of non-Cx32-expressing WB-F344 rat liver epithelial cells with HNF-1alpha stimulated a transfected Cx32 promoter element (mp -244 to -33), binding of HNF-1alpha to the -187 site, and expression of endogenous Cx32. Site-directed mutagenesis of this HNF-1 binding site abolished HNF-1alpha binding and proximal promoter activity. Hepatic Cx32 expression was also significantly decreased in HNF-1alpha(-/-) mice. These data indicate that HNF-1alpha is a positive regulator of Cx32 expression in hepatic cells.