Yu Jui Yvonne Wan

Retinoid pathway and cancer therapeutics.

The retinoids are a class of compounds that are structurally related to vitamin A. Retinoic acid, which is the active metabolite of retinol, regulates a wide range of biological processes including development, differentiation, proliferation, and apoptosis. Retinoids exert their effects through a variety of binding proteins including cellular retinol-binding protein (CRBP), retinol-binding proteins (RBP), cellular retinoic acid-binding protein (CRABP), and nuclear receptors i.e. retinoic acid receptor (RAR) and retinoid x receptor (RXR). Because of the pleiotropic effects of retinoids, understanding the function of these binding proteins and nuclear receptors assists us in developing compounds that have specific effects. This review summarizes our current understanding of how retinoids are processed and act with an emphasis on the application of retinoids in cancer treatment and prevention.

Modulation of experimental alcohol‐induced liver disease by cytochrome P450 2E1 inhibitors

This study was done to determine if a relationship exists between CYP2E1 induction by ethanol, lipid peroxidation, and liver pathology in experimental alcohol-induced liver disease in the rat. Rats were fed ethanol with or without diallyl sulfide (DAS) or phenethyl isothiocyanate (PIC) intragastrically for 1 month. CYP2E1 induction by ethanol was correlated with lipid peroxidation, liver microsomal CYP2E1 hydroxylation of paranitrophenol, and the liver pathology score using the data from the PIC-fed rats. Some of the data from the ethanol and DAS-fed rats were not included here because they have been reported elsewhere. Microsomal CYP2E1 protein levels induction by ethanol was decreased by PIC ingestion. Similarly, PIC reduced the increase microsomal reduced form of nicotinamide-adenine dinucleotide (NADPH)-dependent lipid peroxidation and p-nitrophenol hydroxylase (PNPH) activity, induced by ethanol feeding. The lipid peroxidation was reduced to below control levels; however, the pathology score was partially but not significantly reduced by isothiocyanate feeding. CYP2E1 messenger RNA (mRNA) was decreased by both inhibitors of CYP2E1. Immunohistochemical staining of liver for CYP2E1 protein showed that the lobular distribution of the isozyme changed from the centrilobular to a diffuse pattern, with an increase in the periportal region when the CYP2E1 inhibitors were fed with ethanol, and that this change correlated with the change in the distribution of fat in the lobule. The data support the idea that there is a link between CYP2E1 induction by ethanol and the early phase of ethanol-induced liver injury in this rat model. This link may involve lipid peroxidation, but other factors related to CYP2E1 induction must also be involved.

Human carboxylesterases HCE1 and HCE2: Ontogenic expression, inter-individual variability and differential hydrolysis of oseltamivir, aspirin, deltamethrin and permethrin

Carboxylesterases hydrolyze chemicals containing such functional groups as a carboxylic acid ester, amide and thioester. The liver contains the highest carboxylesterase activity and expresses two major carboxylesterases: HCE1 and HCE2. In this study, we analyzed 104 individual liver samples for the expression patterns of both carboxylesterases. These samples were divided into three age groups: adults (>or= 18 years of age), children (0 days-10 years) and fetuses (82-224 gestation days). In general, the adult group expressed significantly higher HCE1 and HCE2 than the child group, which expressed significantly higher than the fetal group. The age-related expression was confirmed by RT-qPCR and Western immunoblotting. To determine whether the expression patterns reflected the hydrolytic activity, liver microsomes were pooled from each group and tested for the hydrolysis of drugs such as oseltamivir and insecticides such as deltamethrin. Consistent with the expression patterns, adult microsomes were approximately 4 times as active as child microsomes and 10 times as active as fetal microsomes in hydrolyzing these chemicals. Within the same age group, particularly in the fetal and child groups, a large inter-individual variability was detected in mRNA (430-fold), protein (100-fold) and hydrolytic activity (127-fold). Carboxylesterases are recognized to play critical roles in drug metabolism and insecticide detoxication. The findings on the large variability among different age groups or even within the same age group have important pharmacological and toxicological implications, particularly in relation to pharmacokinetic alterations of ester drugs in children and vulnerability of fetuses and children to pyrethroid insecticides.

Expression of Constitutive Androstane Receptor, Hepatic Nuclear Factor 4α, and P450 Oxidoreductase Genes Determines Interindividual Variability in Basal Expression and Activity of a Broad Scope of Xenobiotic Metabolism Genes in the Human Liver

Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4α (HNF4α) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4α expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4α, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

Pregnane X receptor is essential for normal progression of liver regeneration

Pregnane X receptor (PXR) mediates xenobiotic and endobiotic metabolism as well as hepatocyte proliferation. To determine the role of PXR in liver regeneration, 2/3 partial hepatectomy (PH) was performed on wild‐type and PXR‐null mice. Our results showed that hepatic steatosis was markedly suppressed in mice lacking PXR 36 hours after PH, concomitant with reduction of hepatocyte proliferation at the same time point. Gene expression analysis revealed the role of PXR in regulating the transcription of genes involved in lipid uptake, transport, biosynthesis, oxidation, and storage during liver regeneration. When PXR was absent, the second wave of hepatocyte proliferation was severely suppressed, which was accompanied by the inactivation of STAT3. Lack of PXR inhibited the second phase of liver growth, leading to 17% less liver mass at the anticipated end point of liver regeneration (day 10). Conclusion: PXR is required for normal progression of liver regeneration by modulating lipid homeostasis and regulating hepatocyte proliferation. (HEPATOLOGY 2008.)

EXPRESSION OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GENE IS DECREASED IN EXPERIMENTAL ALCOHOLIC LIVER DISEASE

Peroxisome proliferator-activated receptor (PPAR) and retinoid x receptor (RXR) play important roles in fatty acid metabolism. The present study examined the regulation of retinoic acid receptor (RAR alpha, beta, and gamma), RXR (alpha, beta, and gamma), PPAR, cytochrome P450 2E1 (CYP2E1), catalase, and beta-actin gene expression in chronic alcoholic liver disease in the rat. The results demonstrated that the expression of genes for RAR and RXR isoforms and catalase were not altered by ethanol in the fatty liver. In contrast, the levels of PPAR and CYP2E1 mRNAs were down- and up-regulated by ethanol in the liver, respectively. The levels of CYP2E1 mRNAs correlated positively with blood alcohol levels (BAL). In addition, ethanol induced expression of beta-actin mRNA was also proportional to the BAL. The level of PPAR mRNA and the content of polyunsaturated fatty acid decreased in ethanol-fed rat livers. Decreased PPAR gene expression in ethanol-fed rats might result from a decrease in the content of polyunsaturated fatty acid in the liver. However, the activities of enzymes involved in hepatic lipid metabolism, including acyl CoA synthetase, acyl CoA oxidase, catalase, and protein kinase C, were not changed by ethanol treatment. The significance of down-regulation of PPAR gene in alcohol liver disease is discussed.

Analysis of the CYP2D6 gene polymorphism and enzyme activity in African-Americans in southern California.

Despite its importance in drug metabolism and disease susceptibility, CYP2D6 activity and genetic polymorphism have rarely been investigated in African-American populations. In order to bridge this gap, we examined the genotype and phenotype of the enzyme in 154 African-American (AA) and 143 Caucasian (C) normal volunteers. AAs are significantly more likely to possess *17 and *5, but less likely to have *4. Overall, the two groups were similar in their CYP2D6 activity as measured with dextromethorphan as the probe (metabolic ratio 2.21 +/- 0.78 for AAs; 2.11 +/- 0.86 for Cs; t = 1.02, NS). Two of four AAs and six of seven Cs were classified as poor metabolizers and have two nonfunctioning alleles. CYP2D6 activity is determined by *17, *4, *5 and age in AAs (r2 = 0.33, f = 18.8, P < 0.001) and by *4 and *XN in Cs (r2 = 0.14, f = 10.8, P < 0.001). These results support previous findings demonstrating the importance of *17 in determining CYP2D6 activity in AAs.

ADH1B*1, ADH1C*2, DRD2 (−141C Ins), and 5-HTTLPR Are Associated With Alcoholism in Mexican American Men Living in Los Angeles

BACKGROUND The aim of the present study was to use a candidate gene approach to identify the genetic risk factors for alcoholism in Mexican Americans residing in the Los Angeles area. The genes selected include alcohol metabolizing genes and neurotransmitter genes, which have been shown in the literature to be associated with alcoholism in other ethnic groups. METHODS Thirteen allelic variants from seven genes were evaluated for their role in alcoholism using alcoholic (n = 200) and nonalcoholic (n = 251) Mexican Americans. Those polymorphic sites include alcohol dehydrogenase (ADH1B, ADH1C), aldehyde dehydrogenase (ALDH2), cytochrome P-450 2E1 (CYP2E1) TaqI, DraI, RsaI, dopamine D2 receptor (DRD2) TaqI A, B, intron 6, exon 7, -141C Ins/Del, serotonin transporter (5-HTTLPR), and GABAA receptor beta3 subunit (GABRbeta3). RESULTS The results demonstrate that Mexican Americans have extremely low allele frequency for both ALDH2*2 and ADH1B*2 and a relatively high frequency of ADH1C*2 and CYP2E1 c2 alleles. ADH1B*1, ADH1C*2, DRD2 (-141C Ins), and 5-HTTLPR were associated with alcoholism in Mexican Americans (p < 0.05). DRD2 Ins was associated with alcoholism in those alcoholics who carried the ADH1B*2 or ADH1C*1 protective alleles (p = 0.032 in genotype level and p = 0.015 in allele level). DRD2 TaqI A and B alleles were associated with early age of onset for drinking (p = 0.016 for TaqI A1 and p = 0.049 for TaqI B1 allele). CONCLUSIONS Together, the data reveal unique genetic patterns in Mexican Americans that may be in part responsible for the heightened risk for alcoholism and alcohol-associated health problems in this population.

Nuclear Receptors and Inflammatory Diseases

It is well known that the steroid hormone glucocorticoid and its nuclear receptor regulate the inflammatory process, a crucial component in the pathophysiological process related to human diseases that include atherosclerosis, obesity and type II diabetes, inflammatory bowel disease, Alzheimer’s disease, multiple sclerosis, and liver tumors. Growing evidence demonstrates that orphan and adopted orphan nuclear receptors, such as peroxisome proliferator-activated receptors, liver x receptors, the farnesoid x receptor, NR4As, retinoid x receptors, and the pregnane x receptor, regulate the inflammatory and metabolic profiles in a ligand-dependent or -independent manner in human and animal models. This review summarizes the regulatory roles of these nuclear receptors in the inflammatory process and the underlying mechanisms.

Retinoids Induce Cytochrome P450 3A4 through RXR/VDR-mediated Pathway

A panel of retinoids and carotenoids was screened as potential inducers of CYP3A4 through the RXR/VDR-mediated signaling pathway. Transient transfection assays revealed that 3 out of 12 retinoids screened transactivated RXRalpha/VDR and induced CYP3A4 reporter activity. These three retinoids are the active metabolites of retinoids, 9-cis-retinal, 9-cis-retinoic acid (9-cis-RA), and all-trans-retinoic acid (all-trans-RA). 9-cis-RA and all-trans-RA preferentially transactivated the RXR/VDR heterodimers and RXR homodimers. Retinoids and VDR agonist 1alpha, 25-dihydroxyvitamin D(3), but not PXR or CAR activator, could induce Cyp3a11 mRNA level in hepatocytes derived from PXR/CAR-double null mouse. Moreover, retinoids induced CYP3A4 enzyme activity in HepG2 human hepatoma and Caco-2 human colorectal adenocarcinoma cells. A direct role of retinoid-mediated CYP3A4 induction through RXRalpha/VDR was proved by the results that 9-cis-retinal, 9-cis-RA, and all-trans-RA recruited RXRalpha and VDR to CYP3A4 regulatory region pER6 (proximal everted repeat with a 6-nucleotide spacer) and dXREM (distal xenobiotic-responsive enhancer module). Thus, using various approaches, we have unequivocally demonstrated that retinoids transactivate RXR/VDR heterodimers and RXR homodimers and induce CYP3A expression at mRNA as well as enzyme activity levels in both liver and intestinal cells. It is possible that retinoids might alter endobiotic metabolism through CYP3A4 induction in vivo.