Bridgett Green

Effect of polymorphism in the human glutathione S-transferase A1 promoter on hepatic GSTA1 and GSTA2 expression

The patterns of expression of glutathione S-transferases A1 and A2 in human liver (hGSTA1 and hGSTA2, respectively) are highly variable, notably in the ratio of hGSTA1/hGSTA2. We investigated if this variation had a genetic basis by sequencing the proximal promoters (-721 to -1 nucleotides) of hGSTA1 and hGSTA2, using 55 samples of human liver that exemplified the variability of hGSTA1 and hGSTA2 expression. Variants were found in the hGSTA1 gene: -631T or G, -567T, -69C, -52G, designated as hGSTA1*A; and -631G, -567G, -69T, -52A, designated as hGSTA1*B. Genotyping for the substitution -69C > T by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), showed that the polymorphism was widespread in Caucasians, African-Americans and Hispanics, and that it appeared to conform to allelic variation. Constructs consisting of the proximal promoters of hGSTA1*A, hGSTA1*B or hGSTA2, with luciferase as a reporter gene, showed differential expression when transfected into HepG2 cells: hGSTA1*A approximately hGSTA2 > hGSTA1*B. Similarly, mean levels of hGSTA1 protein expression in liver cytosols decreased significantly according to genotype: hGSTA1*A > hGSTA1-heterozygous > hGSTA1*B. Conversely, mean hGSTA2 expression increased according to the same order of hGSTA1 genotype. Consequently, the ratio of GSTA1/GSTA2 was highly hGSTA1 allele-specific. Because the polymorphism in hGSTA1 correlates with hGSTA1 and hGSTA2 expression in liver, and hGSTA1-1 and hGSTA2-2 exhibit differential catalysis of the detoxification of carcinogen metabolites and chemotherapeutics, the polymorphism is expected to be of significance for individual risk of cancer or individual response to chemotherapeutic agents.

Similarities and Differences in the Expression of Drug-Metabolizing Enzymes between Human Hepatic Cell Lines and Primary Human Hepatocytes

In addition to primary human hepatocytes, hepatoma cell lines, and transfected nonhepatoma, hepatic cell lines have been used for pharmacological and toxicological studies. However, a systematic evaluation and a general report of the gene expression spectra of drug-metabolizing enzymes and transporters (DMETs) in these in vitro systems are not currently available. To fill this information gap and to provide references for future studies, we systematically characterized the basal gene expression profiles of 251 drug-metabolizing enzymes in untreated primary human hepatocytes from six donors, four commonly used hepatoma cell lines (HepG2, Huh7, SK-Hep-1, and Hep3B), and one transfected human liver epithelial cell line. A large variation in DMET expression spectra was observed between hepatic cell lines and primary hepatocytes, with the complete absence or much lower abundance of certain DMETs in hepatic cell lines. Furthermore, the basal DMET expression spectra of five hepatic cell lines are summarized, providing references for researchers to choose carefully appropriate in vitro models for their studies of drug metabolism and toxicity, especially for studies with drugs in which toxicities are mediated through the formation of reactive metabolites.

Human CYP1B1 Leu432Val gene polymorphism: ethnic distribution in African-Americans, Caucasians and Chinese; oestradiol hydroxylase activity; and distribution in prostate cancer cases and controls.

Cytochrome P4501B1 (CYP1B1) is involved in the activation of many carcinogens and in the metabolism of steroid hormones, including 17beta-oestradiol (E2) and testosterone. We report a significant difference in the allele frequencies of two point mutations in the coding region of the CYP1B1 gene among Caucasian (n = 189), African-American (n = 52) and Chinese (Linxian) (n = 109) populations. A (C to G) transversion at position 1666 in exon 3, which results in an amino acid substitution of Leu432 to Val, was present in African-Americans with an allele frequency for Va1432 of 0.75, in Caucasians of 0.43, and in Chinese of 0.17. A (C to T) transition at position 1719 in exon 3, with no amino acid change (Asp449), appeared to be closely linked with the Val432 variant. Results using human lung microsomal preparations from individuals with the CYP1B1Val/Val and CYP1B1Leu/Leu genotypes indicate that Val432 variant may be a high activity allele and thus may contribute to the interindividual differences in CYP1B1 activity. Because CYP1B1 is involved in hormone and carcinogen metabolism, and given the disparate rates of prostate cancer among ethnic groups, we also evaluated the association of the CYP1B1 Leu432Val polymorphism with prostate cancer risk in a pilot case-control study. Among Caucasians, 34% of men with cancer (n = 50) were homozygous for the Val432 polymorphism, while only 12% of matched control subjects (n = 50) had this genotype. These preliminary data indicate that genetic polymorphisms in CYP1B1 might play an important role in human prostate carcinogenesis.

Association of SULT1A1 phenotype and genotype with prostate cancer risk in African-Americans and Caucasians

Exposure to heterocyclic amines may increase prostate cancer risk. Human sulfotransferase 1A1 (SULT1A1) is involved in the bioactivation of some dietary procarcinogens, including the N-hydroxy metabolite of the food-borne heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo(4,5-b) pyridine. This study compares a polymorphism in the SULT1A1 gene, SULT1A1 enzyme activity, meat consumption, and the risk of prostate cancer in a population based case-control study. Prostate cancer patients (n = 464) and control individuals (n = 459), frequency matched on age and ethnicity, provided informed consent, answered a survey, and provided a blood sample. Platelets were isolated for phenotype analysis, and DNA was isolated from lymphocytes for genotype determination. Meat consumption was assessed using a dietary questionnaire. Caucasians homozygous for the SULT1A1*1 high activity allele were at increased risk for prostate cancer [odds ratio (OR), 1.68; 95% confidence interval (CI), 1.05–2.68] compared with individuals homozygous for the low-activity allele. The association between SULT1A1 genotype and prostate cancer risk in African-Americans did not reach significance (OR, 1.60; 95% CI, 0.46–5.62). When SULT1A1 activity was considered, there was a strong association between increased SULT1A1 activity and prostate cancer risk in Caucasians (OR, 3.04; 95% CI, 1.8–5.1 and OR, 4.96; 95% CI, 3.0–8.3, for the second and third tertiles of SULT1A1 activity, respectively) compared with individuals in the low enzyme activity tertile. A similar association was also found in African-American patients, with ORs of 6.7 and 9.6 for the second and third tertiles of SULT1A1 activity (95% CI, 2.1–21.3 and 2.9–31.3, respectively). When consumption of well-done meat was considered, there was increased risk of prostate cancer (OR, 1.42; 95% CI, 1.01–1.99 and OR, 1.68; 95% CI, 1.20–2.36 for the second and third tertiles, respectively). When SULT1A1 activity was stratified by tertiles of meat consumption, there was greater risk of prostate cancer in the highest tertile of meat consumption. These results indicate that variations in SULT1A1 activity contributes to prostate cancer risk and the magnitude of the association may differ by ethnicity and be modified by meat consumption.

Low frequency of CYP2A6 gene polymorphism as revealed by a one-step polymerase chain reaction method.

Human cytochrome P450 2A6 (CYP2A6) has been shown to metabolically activate carcinogens and mutagens. Genetic polymorphisms for CYP2A6 have been reported previously in different ethnic groups using a two-step polymerase chain reaction (PCR) method to identify CYP2A6*1, CYP2A6*2 and CYP2A6*3. Moreover, a new truncated allele has been recently identified in a Japanese population. We report here a one-step PCR amplification of the CYP2A6 gene from human genomic DNA and the detection of intact CYP2A6 alleles by restriction enzyme digestion. The diagnostic exon (exon 3) of the CYP2A6 gene was amplified from human genomic DNA with a primer pair. The forward primer is unique to the CYP2A6 gene, which eliminates previous problems in amplifying two highly homologous CYP2A genes, CYP2A7 and CYP2A13, in humans. The resulting PCR products (214 bp) were digested with XcmI or DdeI to detect the presence of CYP2A6*2 or CYP2A6*3 alleles, respectively. The allelic frequencies for CYP2A6*2 were 2.3% (n = 320) in the Caucasian and 0.7% (n = 71) in the Chinese populations, respectively. CYP2A6*3 allelic frequency in the Chinese population was 0.7%; while no CYP2A6*3 allele was detected in the Caucasian population. The allelic frequencies are relatively low and the reason for this discrepancy between different methods is discussed.

Personalizing nutrigenomics research through community based participatory research and omics technologies.

Personal and public health information are often obtained from studies of large population groups. Risk factors for nutrients, toxins, genetic variation, and more recently, nutrient-gene interactions are statistical estimates of the percentage reduction in disease in the population if the risk were to be avoided or the gene variant were not present. Because individuals differ in genetic makeup, lifestyle, and dietary patterns than those individuals in the study population, these risk factors are valuable guidelines, but may not apply to individuals. Intervention studies are likewise limited by small sample sizes, short time frames to assess physiological changes, and variable experimental designs that often preclude comparative or consensus analyses. A fundamental challenge for nutrigenomics will be to develop a means to sort individuals into metabolic groups, and eventually, develop risk factors for individuals. To reach the goal of personalizing medicine and nutrition, new experimental strategies are needed for human study designs. A promising approach for more complete analyses of the interaction of genetic makeups and environment relies on community-based participatory research (CBPR) methodologies. CBPRs central focus is developing a partnership among researchers and individuals in a community that allows for more in depth lifestyle analyses but also translational research that simultaneously helps improve the health of individuals and communities. The USDA-ARS Delta Nutrition Intervention Research program exemplifies CBPR providing a foundation for expanded personalized nutrition and medicine research for communities and individuals.

Suppression of CYP2C9 by MicroRNA hsa-miR-128-3p in Human Liver Cells and Association with Hepatocellular Carcinoma

Published studies have identified genetic variants, somatic mutations, and changes in gene expression profiles that are associated with hepatocellular carcinoma (HCC), particularly involving genes that encode drug metabolizing enzymes (DMEs). CYP2C9, one of the most abundant and important DMEs, is involved in the metabolism of many carcinogens and drugs and is down-regulated in HCC. To investigate the molecular mechanisms that control CYP2C9 expression, we applied integrative approaches including in silico, in vitro, and in vivo analyses to elucidate the role of microRNA hsa-miR-128-3p in the regulation of CYP2C9 expression and translation. RNA electrophoresis mobility shift assays demonstrated a direct interaction between hsa-miR-128-3p and its cognate target, the CYP2C9 transcript. Furthermore, the expression of a luciferase reporter gene containing the 3′-UTR of CYP2C9 and the endogenous expression of CYP2C9 were suppressed by transfection of hsa-miR-128-3p. Importantly, chemically-induced up- or down-regulation of hsa-miR-128-3p correlated inversely with the expression of CYP2C9. Finally, an association analysis revealed that the expression of hsa-miR-128-3p is inversely correlated with the expression of CYP2C9 in HCC tumor tissues. Altogether, the study helped to elucidate the mechanism of CYP2C9 regulation by hsa-miR-128-3p, and the inverse association in HCC.

Cytotoxicity and inhibitory effects of low-concentration triclosan on adipogenic differentiation of human mesenchymal stem cells.

Humans at all ages are continually exposed to triclosan (TCS), a widely used antimicrobial agent that can be found in many daily hygiene products, such as toothpastes and shampoos; however, the toxicological and biological effects of TCS in the human body after long-term and low-concentration exposure are far from being well understood. In the current study, we investigated the effects of TCS on the differentiation of human mesenchymal stem cells (hMSCs) by measuring the cytotoxicity, morphological changes, lipid accumulation, and the expression of adipocyte differentiation biomarkers during 21-day adipogenesis. Significant cytotoxicity was observed in un-induced hMSCs treated with high-concentration TCS (≥ 5.0 μM TCS), but not with low-concentration treatments (≤ 2.5 μM TCS). TCS inhibited adipocyte differentiation of hMSCs in a concentration-dependent manner in the 0.156 to 2.5 μM range as indicated by morphological changes with Oil Red O staining, which is an index of lipid accumulation. The inhibitory effect was confirmed by a decrease in gene expression of specific adipocyte differentiation biomarkers including adipocyte protein 2, lipoprotein lipase, and adiponectin. Our study demonstrates that TCS inhibits adipocyte differentiation of hMSCs under concentrations that are not cytotoxic and in the range observed in human blood.

MicroRNA hsa-miR-29a-3p modulates CYP2C19 in human liver cells

Cytochrome P450 2C19 (CYP2C19) is involved in the metabolism of many drugs. Extensive studies have demonstrated that genetic variants and endogenous and environmental factors play important roles in the expression of CYP2C19. However, the role of microRNAs (miRNAs) in controlling CYP2C19 expression has not been investigated completely. In the present study, we performed in silico analysis to rank putative miRNA/CYP2C19 hybrids with regards to the predicted stabilities of their duplexes and then we applied a series of biochemical and molecular assays to elucidate the underlying functional mechanisms for the regulation of CYP2C19 by miRNAs. In silico analysis indicated that hsa-miR-23a-3p and hsa-miR-29a-3p target the coding region of CYP2C19 with hybrid stabilities of -27.5kcal/mol and -23.3kcal/mol, respectively. RNA electrophoresis mobility shift assays showed that both hsa-miR-23a-3p and hsa-miR-29a-3p miRNAs were able to bind directly to their cognate targets in the CYP2C19 transcript. Further, a significant inverse correlation was found between chemically-induced up-regulation of hsa-miR-29a-3p and CYP2C19 expression in HepaRG cells. In addition, inverse correlations were also observed in human liver tissue samples between the level of CYP2C19 mRNA expression and both hsa-miR-23a-3p and hsa-miR-29a-3p levels. All these results demonstrated the suppressing role of hsa-miR-29a-3p on CYP2C19 expression.

A systematic evaluation of microRNAs in regulating human hepatic CYP2E1

ABSTRACT Cytochrome P450 2E1 (CYP2E1) is an important drug metabolizing enzyme for processing numerous xenobiotics in the liver, including acetaminophen and ethanol. Previous studies have shown that microRNAs (miRNAs) can suppress CYP2E1 expression by binding to the 3′‐untranslated region (3′‐UTR) of its transcript. However, a systematic analysis of CYP2E1 regulation by miRNAs has not been described. Here, we applied in silico, in vivo, and in vitro approaches to investigate miRNAs involved in the regulation of CYP2E1. Initially, potential miRNA binding sites in the CYP2E1 mRNA transcript were identified and screened using in silico methods. Next, inverse correlations were found in human liver samples between the expression of CYP2E1 mRNA and the levels of two miRNA species, hsa‐miR‐214–3p and hsa‐miR‐942–5p. In a HepG2‐derived CYP2E1 over‐expression cell model, hsa‐miR‐214–3p exhibited strong suppression of CYP2E1 expression by targeting the coding region of its mRNA transcript, but hsa‐miR‐942–5p did not inhibit CYP2E1 levels. Electrophoretic mobility shift assays confirmed that hsa‐miR‐214–3p recruited other cellular protein factors to form stable complexes with specific sequences present in the CYP2E1 mRNA open reading frame. Transfection of HepaRG cells with hsa‐miR‐214–3p mimics inhibited expression of the endogenous CYP2E1 gene. Further, hsa‐miR‐214–3p mimics partially blocked ethanol‐dependent increases in CYP2E1 mRNA and protein levels in HepG2 cells and they reduced the release of alanine aminotransferase from CYP2E1‐overexpressing HepG2 cells exposed to acetaminophen. These results substantiate the suppressing effect of hsa‐miR‐214–3p on CYP2E1 expression.