Constitutive androstane receptor transactivates the hepatic expression of mouse Dhcr24 and human DHCR24 encoding a cholesterogenic enzyme 24-dehydrocholesterol reductase

Phenobarbital treatment has long been known to influence serum and hepatic cholesterol levels in rodents and humans. Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, mediates various biological actions of phenobarbital. We have thus investigated whether CAR transactivates cholesterogenic genes in livers. Activation of CAR in mouse livers and cultured human hepatocytes increased mRNA levels of mouse Dhcr24 and human DHCR24, both of which encode 24-dehydrocholesterol reductase (DHCR24) catalyzing the last step of cholesterol biosynthesis. CAR transactivated the expression of these genes in reporter assays with cultured hepatoma cells. Furthermore, we have identified a DR4 (direct repeat separated by 4 nucleotides) motif in the human DHCR24 distal promoter as a binding site of CAR/retinoid X receptor α (RXRα) heterodimer. We have also demonstrated that the heterodimer of pregnane X receptor (PXR)/ RXRα binds to the DR4 motif and that human DHCR24 reporter gene is transactivated by the ligand-activated PXR. These results suggest a role of xenobiotic-responsive nuclear receptor CAR, and also possibly PXR, in cholesterol biosynthesis in the liver of mice and humans.     

Omeprazole alleviates benzo[a]pyrene cytotoxicity by inhibition of CYP1A1 activity in human and mouse hepatoma cells

Omeprazole is a drug used for treating gastro-oesophageal reflux disease and duodenal ulcers. Omeprazole induces a xenobiotic-metabolizing enzyme, cytochrome P450 1A1 (CYP1A1), as its ligand by aryl hydrocarbon receptor (AhR) activation without binding. CYP1A1-inducible chemicals, such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin, are known to have adverse effects (i.e. carcinogenesis, mutagenesis and malformation). Unlike these typical AhR activators, omeprazole has shown no experimental evidence of carcinogenic activity. The possibility, however, remains that omeprazole may aggravate the effect of environmental carcinogens through CYP1A1 induction. We exposed benzo[a]pyrene and omeprazole simultaneously to human and mouse hepatoma cells to investigate the synergistic effect of these chemicals. Contrary to our prediction, cytotoxicity of benzo[a]pyrene was inhibited by the omeprazole exposure in a dose-dependent manner. Omeprazole did not alter CYP1A1 mRNA and protein levels induced by benzo[a]pyrene. The 7-ethoxy-resorufin-O-deethylase assay revealed that omeprazole inhibited CYP1A1 enzyme activity. Kinetic analysis also demonstrated that it is a competitive inhibitor for CYP1A1. The K(m) value of omeprazole against CYP1A1 activity was 50.1 microM. We conclude that the effects of omeprazole on CYP1A1 involve not only induction through AhR activation but also inhibition of its enzyme activity, and that the protective effect of omeprazole against benzo[a]pyrene cytotoxicity depends on the latter.     

Genetic variations and haplotypes of ABCC2 encoding MRP2 in a Japanese population

The multidrug resistance-associated protein 2 (MRP2) encoded by the ABCC2 gene is expressed in the liver, intestine and kidneys and preferentially exports organic anions or conjugates with glucuronide or glutathione. In this study, all 32 exons and the 5'-flanking region of ABCC2 in 236 Japanese were resequenced, and 61 genetic variations including 5 novel nonsynonymous ones were detected. A total of 64 haplotypes were determined/inferred and classified into five *1 haplotype groups (*1A, *1B, *1C, *1G, and *1H) without nonsynonymous substitutions and *2 to *9 groups with nonsynonymous variations. Frequencies of the major 4 haplotype groups *1A (-1774delG), *1B (no common SNP), *1C (-24C>T and 3972C>T), and *2 [1249G>A (Val417Ile)] were 0.331, 0.292, 0.172, and 0.093, respectively. This study revealed that haplotype *1A, which has lowered activity, is quite common in Japanese, and that the frequency of *1C, another functional haplotype, was comparable to frequencies in Asians and Caucasians. In contrast, the haplotypes harboring 3972C>T but not -24C>T (*1G group), which are reportedly common in Caucasians, were minor in Japanese. Moreover, the allele 1446C>T (Thr482Thr), which has increased activity, was not detected in our Japanese population. These findings imply possible differences in MRP2-mediated drug responses between Asians and Caucasians.     

Effect of SMP-500, a novel acyl-CoA:cholesterol acyltransferase inhibitor,on serum cholesterol level and LDL cholesterol clearance in hamsters with induced hyperlipidemia

The effect of SMP-500, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on serum cholesterol levels was investigated in hyperlipidemic hamsters whose condition had been preestablished by diet. SMP-500 reduced the total serum cholesterol level in a dose-dependent manner. SMP-500 also reduced the hepatic free cholesterol content and markedly reduced the esterified cholesterol content compared with the control group. Interestingly, SMP-500 at a dose of 30 mg/kg increased LDL clearance in vivo. As SMP-500 at this dose potently lowered the total serum cholesterol level, the increased LDL clearance was identified as another mechanism for the cholesterol-lowering effect of SMP-500. However, unlike HMG CoA reductase inhibitors, SMP-500 did not affect cholesterol biosynthesis in HepG2 cells. Therefore the etiology of the increased LDL clearance is not yet clear, but the reduced hepatic free cholesterol may play an important role in this process. These results suggest that the cholesterol-lowering effect of SMP-500 is due, not only to the inhibition of ACAT, but also to the increase in cholesterol clearance from the blood. This finding supports the therapeutic potential of SMP-500 for the treatment of human hypercholesterolemia.     

Establishment of Healthy Japan 21 Regional plans according to local community type

OBJECTIVE: To examine the establishment of Health Japan 21 regional plans according to the local community type. METHODS: We sent all the Japanese local governments a questionnaire regarding the establishment of health Japan 21 regional plans with a request to provide a hardcopy of established regional plans. RESULTS: Of the respondent 1954 local communities, the proportions regarding establishment of plans (including "Completed" and "In the course of") according to the community type were: major, middle sized cities and Tokyo metropolitan wards, 100%; other cities, 64.9%; towns, 40.7%; and villages, 38.8%. Of the 500 local communities which answered "Completed establishment", the proportions with release onto the internet (including "Completed" and "In the course of") were: major cities, 100%; middle sized cities, 67.7%; Tokyo metropolitan wards, 85.7%; other cities, 38.8%; towns, 13.5%; and villages, 14.3%. We examined whether each of the 462 collected regional plans included each of the national Health Japan 21's target themes and items, and found a tendency for smaller community' regional plans to be less likely to give comprehensive coverage. CONCLUSION: More support for smaller local governments is necessary to achieve establishment and effective implementation of Health Japan 21 regional plans.     

Expression of transforming growth factor-beta receptors in normal rat retina and experimental choroidal neovascularization

PURPOSE: Transforming growth factor-beta (TGF-beta) plays an important role in the development of choroidal neovascularization. TGF-beta transduces signals through the mediation of type I and type II receptors. We investigated the expression of TGF-beta receptors in a normal rat retina and a model of experimentally induced choroidal neovascularization. METHODS: Choroidal neovascularization was induced by laser photocoagulation in rat eyes. The expression of TGF-beta receptors was determined using immunohistochemical and in situ hybridization methods. RESULTS: In normal adult rat retinas, immunoreactivity and mRNA expression of TGF-beta receptor type I (TbetaRI) and TGF-beta receptor type II (TbetaRII) were found in the ganglion cells. During the process of neovascularization, immunoreactivity and mRNA expression of TbetaRI and TbetaRII were widely distributed in laser lesions soon after photocoagulation; thereafter, these receptors were specifically detected in the endothelial cells of choroidal neovascularization. CONCLUSIONS: The expression of TGF-beta receptors in normal rat retinas suggests that TGF-beta plays an important role in the homeostasis of normal retina. The upregulation of TGF-beta receptors in choroidal neovascularization strongly suggests that TGF-beta is most likely transduced through specific receptors and plays an important role in the development of choroidal neovascularization.     

Aldehyde dehydrogenase 2 (ALDH2) genotype affects rectal cancer susceptibility due to alcohol consumption

BACKGROUND: Epidemiologic studies have shown the association between alcohol consumption and colorectal cancer, especially for rectal cancer. The alcohol related enzyme encoding gene ALDH2 has polymorphism Glu487Lys, and 487Lys allele is closely linked with phenotypic loss of enzyme activity. MATERIALS AND METHODS: A hospital-based case-control study was conducted with 72 colon and 70 rectal cancer cases and 241 non-cancer controls to evaluate the alcohol consumption and ALDH2 Glu487Lys polymorphism. The logistic regression model was applied to estimate the odds ratios (ORs). RESULT: The crude ORs for Glu/Lys and Lys/Lys genotype relative to Glu/Glu for colon and rectal cancer were not statistically significant. However, with the rectal cancer analysis, the ORs for high alcohol consumption were greater with 487Glu/Lys genotype compared with Glu/Glu, albeit not. CONCLUSIONS: These observations suggested rectal cancer risk might be influenced by ALDH2 gene polymorphism. The prevention effect by alcohol reduction might differ by ALDH2 genotype.     

Establishment of an animal model for radiation-induced vomiting in rats using pica

We investigated whether radiation-induced pica, a behavior characterized by the eating of a non-food substance, such as kaolin, can be used as an index of radiation-induced vomiting in rats. Since there was an individual difference in the susceptibility to pica, we selected rats that actually ate kaolin following X-ray irradiation, and used them for the experiment. The total-body irradiation (TBI) increased kaolin consumption in a dose-dependent manner (sham, 0.05 +/- 0.03 (SEM) g; 2 Gy, 0.38 +/- 0.11 g; 4 Gy, 1.54 +/- 0.28 g; 8 Gy, 3.55 +/- 0.67 g), and the increased kaolin consumption after 4 Gy of TBI was inhibited by a pretreatment with the serotonin 5-HT3 receptor antagonist ondansetron (2 mg/kg, i.p.) (saline, 1.49 +/- 0.33 g; ondansetron, 0.75 +/- 0.11 g). Furthermore, 4 Gy of abdominal irradiation was more effective to induce pica than that of head irradiation (abdomen: 0.37 +/- 0.05 g, head: 0.06 +/- 0.01 g). These findings suggested that peripheral serotonergic pathway is predominantly involved in the development of radiation-induced pica in rats and that the radiation-induced pica could be useful as a behavioral index for the severity of radiation-induced vomiting in rats.     

Oxidative DNA damage induced by carcinogenic dinitropyrenes in the presence of P450 reductase

Nitropyrenes are widespread in the environment due to mainly diesel engine emissions. Dinitropyrenes (DNPs), especially 1,8-dinitropyrene (1,8-DNP) and 1,6-dinitropyrene (1,6-DNP), are much more potent mutagens than other nitropyrenes. The carcinogenicity of 1,8-DNP and 1,6-DNP is stronger than 1,3-dinitropyrene (1,3-DNP). It is considered that adduct formation after metabolic activation plays an important role in the expression of carcinogenicity of nitropyrenes. However, Djuric et al. [(1993) Cancer Lett.] reported that oxidative DNA damage was also found as well as adduct formation in rats treated with 1,6-DNP. We investigated oxidative DNA damage by DNPs in the presence of NAD(P)H-cytochrome P450 reductase using 32P-5'-end-labeled DNA. After P450 reductase treatment, DNPs induced Cu(II)-mediated DNA damage in the presence of NAD(P)H. The intensity of DNA damage by 1,8-DNP or 1,6-DNP was stronger than 1,3-DNP. We also examined synthetic 1-nitro-8-nitrosopyrene (1,8-NNOP) and 1-nitro-6-nitrosopyrene (1,6-NNOP) as one of the metabolites of 1,8-DNP and 1,6-DNP, respectively, to find that 1,8-NNOP and 1,6-NNOP induced Cu(II)-mediated DNA damage in the presence of NAD(P)H but untreated DNPs did not. In both cases of P450 reductase-treated DNPs and NNOPs, catalase and a Cu(I) specific chelator attenuated DNA damage, indicating the involvement of H2O2 and Cu(I). Using a Clarke oxygen electrode, oxygen consumption by the reaction of NNOPs with NAD(P)H and Cu(II) was measured to find that NNOP was nonenzymatically reduced by NAD(P)H and that the addition of Cu(II) promoted the redox cycle. Therefore, these results suggest that DNPs are enzymatically reduced to NNOPs via nitro radical anion and that NNOPs are further reduced nonenzymatically by NAD(P)H. Subsequently, autoxidation of nitro radical anion and the reduced form of NNOP occurs, resulting in O2- generation and DNA damage. We conclude that oxidative DNA damage in addition to DNA adduct formation may play important roles in the carcinogenesis of DNPs via their metabolites.