A systematic evaluation of microRNAs in regulating human hepatic CYP2E1.

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.

Functional single nucleotide polymorphism in IL-17A 3' untranslated region is targeted by miR-4480 in vitro and may be associated with age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. Genetic factors contributing to AMD include single nucleotide polymorphisms (SNPs) in immune-related genes including CFH, C2, CFI, C9, and C3, thus implicating these pathways in AMD pathogenesis. MicroRNAs (miRNAs) are powerful regulators of gene expression and execute this function by binding to the 3' untranslated region (3'UTR) of target mRNAs, leading to mRNA degradation. In this study, we searched for the possible association of SNPs in the 3'UTR region of IL-17A, a gene implicated in AMD pathogenesis without any previous SNP association with AMD. Using two independent sample cohorts of Caucasian subjects, six SNPs in the IL-17A 3'-UTR were selected for genotyping based on bioinformatic predictions of the SNP effect on microRNA binding. The SNP rs7747909 was found to be associated with AMD (P < 0.05) in the NEI cohort, using a dominant model logistic regression. Luciferase reporter gene assays and RNA electrophoretic mobility shift assays were performed using ARPE-19 cells to confirm the preferential binding of microRNAs to the major allele of the SNP. Our findings support the hypothesis that microRNA-mediated gene dysregulation may play a role in the pathogenesis of AMD.

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.

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.

Differential promoter activities of functional haplotypes in the 5'-flanking region of human sulfotransferase 1A1.

Previously, we reported five common single nucleotide polymorphisms (SNPs), -624G>C, -396G>A, -358A>C, -341C>G, and -294T>C, and six common haplotypes (CGACT, GAACT, GGAGC, GGACC, CAACT, and GAACC) in the 5'-flanking region of the SULT1A1 gene that were associated with altered enzymatic activity. In the present study, we performed in vitro assays to determine the functional impact of these genetic variations on the promoter activity. Dual luciferase reporter assays revealed that these SNPs are located in a negative regulatory fragment of the SULT1A1 gene. Further experiments demonstrated that these SNPs and haplotypes affected promoter activities of SULT1A1. Electrophoretic mobility shift assays showed distinctive binding patterns for the SNPs -396G>A and -294T>C, due to differential binding affinities of the G/A alleles and the T/C alleles to nuclear proteins extracted from the liver carcinoma cell lines, HepG2 and Huh7.

Technical reproducibility of genotyping SNP arrays used in genome-wide association studies.

During the last several years, high-density genotyping SNP arrays have facilitated genome-wide association studies (GWAS) that successfully identified common genetic variants associated with a variety of phenotypes. However, each of the identified genetic variants only explains a very small fraction of the underlying genetic contribution to the studied phenotypic trait. Moreover, discordance observed in results between independent GWAS indicates the potential for Type I and II errors. High reliability of genotyping technology is needed to have confidence in using SNP data and interpreting GWAS results. Therefore, reproducibility of two widely genotyping technology platforms from Affymetrix and Illumina was assessed by analyzing four technical replicates from each of the six individuals in five laboratories. Genotype concordance of 99.40% to 99.87% within a laboratory for the sample platform, 98.59% to 99.86% across laboratories for the same platform, and 98.80% across genotyping platforms was observed. Moreover, arrays with low quality data were detected when comparing genotyping data from technical replicates, but they could not be detected according to venders' quality control (QC) suggestions. Our results demonstrated the technical reliability of currently available genotyping platforms but also indicated the importance of incorporating some technical replicates for genotyping QC in order to improve the reliability of GWAS results. The impact of discordant genotypes on association analysis results was simulated and could explain, at least in part, the irreproducibility of some GWAS findings when the effect size (i.e. the odds ratio) and the minor allele frequencies are low.

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.

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.

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. CBPR's 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.

A variant of the Cockayne syndrome B gene ERCC6 confers risk of lung cancer.

Cockayne syndrome B protein (ERCC6) plays an essential role in DNA repair. However, the Cockayne syndrome caused by the ERCC6 defect has not been linked to cancer predisposition; likely due to the fact that cells with severe disruption of the ERCC6 function are sensitive to lesion-induced apoptosis, thus reducing the chance of tumorigenesis. The biological function and cancer susceptibility of a common variant rs3793784:C>G (c.-6530C>G) in the ERCC6 was examined. We show that the c.-6530C allele has lower binding affinity of Sp1 by EMSA and displays a lower transcriptional activity in vitro and in vivo. We then examined the contribution of this polymorphism to the risk of lung cancer in a case-control study with 1,000 cases and 1,000 controls. The case-control analysis revealed a 1.76-fold (P= x 10(-9)) excess risk of developing lung cancer for the c.-6530CC carriers compared with noncarriers. The c.-6530CC interacts with smoking to intensify lung cancer risk, with the odds ratio (OR)=9 for developing lung cancer among heavy smokers. Our data constituted strong evidence that ERCC6 rs3793784:C>G alters its transcriptional activity and may confer personalized susceptibility to lung cancer.

Examination of human tissue cytosols for expression of sulfotransferase isoform 1A2 (SULT1A2) using a SULT1A2-specific antibody.

Sulfotransferase isoform 1A2 (SULT1A2) is a member of the cytosolic sulfotransferase family of phase II detoxification enzymes. Studies with recombinant enzymes have shown that SULT1A2 can catalyze the bioactivation of several procarcinogens, indicating a potential role in chemical carcinogenesis. However, previous studies have suggested that the SULT1A2 transcript has a splicing defect that might prevent it from becoming translated into protein; therefore, we sought to determine the expression of SULT1A2 in tissues. An antibody directed against a region of human SULT1A2 that differs from other known sulfotransferase isoforms was developed and used to screen a large number of cytosolic fractions from various tissues. Although the SULT1A2 antibody recognized recombinant SULT1A2 and did not cross-react with other SULT isoforms, the expression of SULT1A2 was not detected in any tissue examined. These studies suggest that if SULT1A2 is expressed as protein, the levels are very low and that SULT1A2 probably does not play a physiological role in chemical 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.

Simultaneous generation of multiple mitochondrial DNA mutations in human prostate tumors suggests mitochondrial hyper-mutagenesis.

Multiple somatic mitochondrial DNA mutations are frequently reported in human tumors, but the process leading to homoplasmic transformation and accumulation of multiple mutations in the same tumor cell lineage remains a mystery. We address possible mechanisms responsible for the generation of multiple mitochondrial (mt)DNA mutations observed in a high frequency of prostate tumors using sensitive mutant-specific PCR coupled with laser capture microdissection. Analysis of prostate tumors with multiple mtDNA mutations in the control region indicates that the mutations are locally confined, that the multiple mutations exist on the same molecules and that more than one mtDNA mutant species co-exists in the same neoplastic lesion. These results suggest an unusually rapid process in mtDNA mutagenesis during tumor progression. On the basis of prostate tumor cell kinetics, we propose a unique process of mitochondrial hyper-mutagenesis, probably mediated by cellular oxidative stress, to account for a burst of multiple mtDNA mutations in human prostate tumors.

The CYP3A4*1B variant is related to the onset of puberty, a known risk factor for the development of breast cancer.

Breast development, one of the first signs of puberty, is closely associated with age at menarche; and early menarche is in turn a well-established risk factor for female breast cancer. We examined the relationships between the onset of puberty and gene variants for certain enzymes that regulate hormone metabolism among 137 healthy nine-year-old girls from two pediatric clinics. High-activity CYP17 alleles, involved in estrogen formation, and high-activity CYP1A2 and CYP1B1 alleles, whose gene products metabolize estradiol, were not associated with pubertal stage. High activity CYP3A4, but not CYP3A5, which primarily metabolizes testosterone, showed a striking association with the onset of puberty (adjusted odds ratio, 3.21; 95% confidence interval, 1.62-6.89 for the genotype 0-1-2 rapid alleles). Of the homozygous CYP3A4*1B/1B girls, 90% had reached puberty; whereas, for the low-activity homozygous CYP3A4*1A/1A individuals, only 40% had done so. In heterozygotes, 56% had reached puberty. CYP1B1, CYP3A4, and CYP3A5 rapid variants were more common in African-American than in Hispanic or Caucasian girls.

Evidence for the presence of mutagenic arylamines in human breast milk and DNA adducts in exfoliated breast ductal epithelial cells.

Aromatic and heterocyclic amines are ubiquitous environmental mutagens present in combustion emissions, fried meats, and tobacco smoke, and are suspect human mammary carcinogens. To determine the presence of arylamines in breast tissue and fluid, we examined exfoliated breast ductal epithelial cells for DNA adducts and matched human milk samples for mutagenicity. Breast milk was obtained from 50 women who were 4-6 weeks postpartum, and exfoliated epithelial-cell DNA was evaluated for bulky, nonpolar DNA adducts by (32)P-postlabeling and thin-layer chromatography. Milk was processed by acid hydrolysis, and the extracted organics were examined in the standard plate-incorporation Ames Salmonella assay using primarily strain YG1024, which detects frameshift mutations and overexpresses aryl amine N-acetyltransferase. DNA adducts were identified in 66% of the specimens, and bulky adducts migrated in a pattern similar to that of 4-aminobiphenyl standards. The distribution of adducts did not vary by NAT2 genotype status. Of whole milk samples, 88% (22/25) had mutagenic activity. Among the samples for which we had both DNA adduct and mutagenicity data, 58% (14/19) of the samples with adducts were also mutagenic, and 85% (11/13) of the mutagenic samples had adducts. Quantitatively, no correlation was observed between the levels of adducts and the levels of mutagenicity. Separation of the milk showed that mutagenic activity was found in 69% of skimmed milk samples but in only 29% of the corresponding milk fat samples, suggesting that the breast milk mutagens were moderately polar molecules. Chemical fractionation showed that mutagenic activity was found in 67% (4/6) of the basic fractions but in only 33% (2/6) of acidic samples, indicating that the mutagens were primarily basic compounds, such as arylamines. Although pilot in nature, this study corroborates previous findings of significant levels of DNA adducts in breast tissue and mutagenicity in human breast milk and indicates that breast milk mutagens may be moderately polar basic compounds, such as arylamines.