Beverly Word

Increased expression of Toll-like receptors (TLRs) 7 and 9 and other cytokines in systemic lupus erythematosus (SLE) patients: Ethnic differences and potential new targets for therapeutic drugs

Increased expression of pro-inflammatory cytokines such as interferon, tumor necrosis factors (TNFs) and specific interleukins (ILs) has been found in a number of autoimmune diseases, including systemic lupus erythematous (SLE). These cytokines are induced by toll-like receptors (TLRs). Toll-like receptors are activated in response to accumulation of apoptotic bodies. These receptors play critical roles in innate immune systems. Increased levels of interferon-alpha (INF-α) have also been found in many SLE patients and often correlate with disease severity. The objectives of this study were to examine the expression of selected TLRs and cytokines that have been identified in animal models and some limited human studies in a group of African Americans (AA) and European Americans (EA) women with lupus in comparison to age-matched non-lupus women. Blood samples were consecutively obtained by informed consent from 286 patients, 153 lupus and 136 non-lupus, seen in the rheumatology clinics at East Carolina University. Cytokines were analyzed from blood serum using enzyme linked immunoassay (ELISA) for IL-6 and INF-α. Total RNA was isolated, using a Paxgene kit, from peripheral blood mononuclear cells of African American and European American women blood samples. Quantitative real-time PCR using the CFX real-time system was conducted on all samples to determine TLRs 7 and 9, as well as INF-α expression. Toll-like receptor 7 (p<0.01) and 9 (p=0.001) expression levels were significantly increased in lupus patients compared to age-matched controls. African American women with lupus had a 2-fold increase in TLR-9 expression level when compared to their healthy controls or European American lupus patients. However, there was no ethnic difference in expression of TLR-7 in lupus patients. INF-α expression was significantly higher in lupus patients (p<0.0001) and also showed ethnic difference in expression. Serum levels revealed significant increases in expression of IL-6, IFN-γ and TNF-α in lupus patients compared to non-lupus patients. African American women with lupus had significantly higher serum levels of IL-6 and TNF-α. African American women with lupus demonstrated increased levels of specific pro-inflammatory cytokines and Toll-like receptors when compared to EA women. Increased expression in these lupus patients provides an opportunity for targeting with antagonist as a new therapy for systemic lupus erythematous.

Increased levels of NAD(P)H: quinone oxidoreductase 1 (NQO1) in pancreatic tissues from smokers and pancreatic adenocarcinomas: A potential biomarker of early damage in the pancreas

NAD(P)H:quinone oxidoreductase 1 (NQO1) is elevated in several human tumors. This study was conducted to determine whether increased levels of NQO1 expression also occur in human pancreatic tumor tissue, and to compare expression levels in nontumorous tissue from smokers with those in nonsmokers. The expression of NQO1 was examined in pancreatic tissue samples from 82 human donors. These samples included normal (n = 20), smokers (n = 25), pancreatitis (n = 7), and adenocarcinomas of the pancreas (n = 30). Genotyping for the C609T polymorphism in NQO1 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis was also performed. Polymorphic variants were confirmed by automatic sequencing. Higher levels of NQO1 expression were demonstrated in pancreatic adenocarcinomas (0.831 ± 0.021) compared to those in nontumorous tissues from nonsmokers (0.139 ± 0.024). These high levels were also found in smokers (0.729 ± 0.167) and in pancreatitis tissues (0.923 ± 0.184). NQO1 activity was also higher in smokers (2.43 ± 0.61 nmol/min per mg protein) compared to nonsmokers (0.44 ± 0.05 nmol/min per mg protein; p < 0.05). No differences were found in genotype distribution and frequencies of the variant alleles between normal and cancer tissues in this relatively small sample pool. Seventy-five percent of the normal pancreatic tissues showed 609(C/C) and 25% 609(C/T). In pancreatic adenocarcinomas the frequency distribution was 65% C/C, 30% C/T and 5% T/T. The increased expression in noncancer pancreatic tissue from smokers and the fact that smoking is a moderate risk factor for pancreatic cancer suggest that NQO1 expression may be a good candidate as a biomarker for pancreatic cancer, especially in risk groups such as smokers.

Ethnic Differences in DNA Methyltransferases Expression in Patients with Systemic Lupus Erythematosus

PurposeSystemic lupus erythematous (SLE) is a systemic autoimmune inflammatory disease with both genetic and epigenetic etiologies. Evidence suggests that deregulation of specific genes through epigenetic mechanisms may be a contributing factor to SLE pathology. There is increasing evidence that DNA methyltransferase activity may be involved. This study demonstrated modulation in expression of DNA methyltransferases (DNMTs) according to ethnicity in patients diagnosed with SLE. Furthermore, differential expression in one of the DNMTs was found in a subset of lupus patients on dehydroepiandrosterone (DHEA) therapy.MethodsReal-time PCR analyses of DNMT1, DNMT3A and DNMT3B in peripheral blood mononuclear cells from a cohort of African American and European American lupus and non-lupus women were conducted. Also, global DNA methylation was assessed using the MethylFlashTM methylated quantification colorimetric assay.ResultsSignificant increase in DNMT3A (p < 0.001) was shown in lupus patients when compared to age-matched healthy controls. This increase was associated with a higher SLEDI index. More striking was that expression levels for African American (AA) women were higher than European American women in the lupus populations. A subset of AA women on DHEA therapy showed a significant decrease (p < 0.05) in DNMT3A expression in comparison to lupus patients not on the therapy. DHEA is an androgenic steroid found in low levels in the serum of lupus patients. Supplementation of this hormone has been shown to be beneficial to some lupus patients. DHEA was not shown to effect DNMT1 or DNMT3B expression. Increased expression was also noted in DNMT3B (p < 0.05) in lupus patients compared to age-matched healthy controls. However, no significant difference was noted in DNMT1 (p = 0.2148) expression between lupus patients and healthy controls. Although increases were detected in de novo methyltransferases, a global decrease (p < 0.001) in 5-methycytosine was observed in lupus patients when compared to age-matched healthy controls.ConclusionThese findings suggest that epigenetic changes may play a critical role in the manifestations of the disease observed among ethnic groups, particularly African American women who often have a higher incidence of lupus. DHEA therapy effects on DNMT3A expression in AA women warrant further investigation in a larger population.

Cigarette Smoke Condensate Induces Differential Expression and Promoter Methylation Profiles of Critical Genes Involved in Lung Cancer in NL-20 Lung Cells In Vitro Short-Term and Chronic Exposure

Establishing early diagnostic markers of harm is critical for effective prevention programs and regulation of tobacco products. This study examined effects of cigarette smoke condensate (CSC) on expression and promoter methylation profile of critical genes (DAPK, ECAD, MGMT, and RASSF1A) involved in lung cancer development in different human lung cell lines. NL-20 cells were treated with 0.1-100 μg/ml of CSC for 24 to 72 hrs for short-term exposures. DAPK expression or methylation status was not significantly affected. However, CSC treatment resulted in changes in expression and promoter methylation profile of ECAD, MGMT, and RASSF1A. For chronic studies, cells were exposed to 1 or 10 μg/ml CSC up to 28 days. Cells showed morphological changes associated with transformation and changes in invasion capacities and global methylation status. This study provides critical data suggesting that epigenetic changes could serve as an early biomarker of harm due to exposure to cigarette smoke.

Expression of drug transporters in human kidney: impact of sex, age, and ethnicity.

BackgroundDifferences in expression of drug transporters in human kidney contribute to changes in pharmacokinetics and toxicokinetics of a variety of drug compounds. The basal expression levels of genes involved in drug transport processes in the kidney introduces differences in bioavailability, distribution, and clearance of drugs, possibly influencing drug efficacy and adverse reactions. Sex differences in gene expression of transporters are a key cause of differences in sex-dependent pharmacokinetics, which may characterize many drugs and contribute to individual differences in drug efficacy and toxicity. Therefore, evaluating the expression of drug transporters in normal human kidneys is important to better understand differences in drug bioavailability, distribution, and clearance of drugs in humans. Other factors such as age and ethnicity may also contribute to individual differences in gene expression of drug transporters in the human kidney.MethodsQuantitative real-time PCR (QRT-PCR) was performed to determine the gene expression of 30 drug transporters in 95 age-matched normal human kidney tissues. Multiple Student’s t-tests (Sidak-Bonferroni correction) and two-way ANOVA (Bonferroni correction) analyses were used to determine statistically significant differences.ResultsIn the 30 transporter genes examined, sex, ethnicity, and age differences in gene expression were exhibited in normal human kidney tissue. These changes in expression were not found to be differentially significant. However, sex-age and sex-ethnicity interactions were found to be statistically significant. For sex-age interactions, SCL22A12 was found to be significantly higher expressed in females <50 years compared to males <50 years. Expression levels of SLC22A2, SLC22A12, SLC6A16, and ABCB6 were significantly higher in females <50 years compared to females ≥50 years. In sex-ethnicity interactions, expression levels of ATP7B and KCNJ8 were found to be significantly higher in African American females compared to European American females. Also, the expression of SLC31A2 was significantly higher in European American males compared to European American females.ConclusionsSex, age, and ethnic differences impacted the expression of drug transporters in normal human kidneys, which suggests that the analysis of gene expression of drug transporters will aid in improving the usage/dosage of drug therapies influencing personalized medicine and susceptibility to adverse drug reactions.

Evaluation of Batch Variations in Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes from 2 Major Suppliers

Drug-induced proarrhythmia is a major safety issue in drug development. Developing sensitive in vitro assays that can predict drug-induced cardiotoxicity in humans has been a challenge of toxicology research for decades. Recently, induced pluripotent stem cell-derived human cardiomyocytes (iPSC-hCMs) have become a promising model because they largely replicate the electrophysiological behavior of human ventricular cardiomyocytes. Patient-specific iPSC-hCMs have been proposed for personalized cardiac drug selection and adverse drug response prediction; however, many procedures are involved in cardiomyocytes differentiation and purification process, which may result in large line-to-line and batch-to-batch variations. Here, we examined the purity, cardiac ion channel gene expression profile, and electrophysiological response of 3 batches of iPSC-hCMs from each of 2 major cell suppliers. We found that iPSC-hCMs from both vendors had similar purities. Most of the cardiac ion channel genes were expressed uniformly among different batches of iCells, while larger variations were found in Cor.4U cells, particularly in the expression of CACNA1C, KCND2, and KCNA5 genes, which could underlie the differences in baseline beating rate (BR) and field potential duration (FPD) measurements. Although, in general, the electrophysiological responses of different batches of cells to Na+, Ca2+, Ikr, and Iks channel blockers were similar, with Ikr blocker-induced proarrhythmia, the sensitivities were depended on baseline BR and FPD values: cells that beat slower had longer FPD and greater sensitivity to drug-induced proarrhythmia. Careful evaluation of the performance of iPSC-hCMs and methods of data analysis is warranted for shaping regulatory standards in qualifying iPSC-hCMs for drug safety testing.

Transcriptional activity of DNMT3B in pancreatic cancer cells: Effects of −149 (C → T) promoter polymorphism

Polymorphic C-to-T change in the promoter region of DNA-methyltransferase-3B (DNMT3B) gene is associated with risk of several cancers. The aim of this study was to investigate the effect of DNMT3B promoter genetic variant on its transcriptional activity and to compare activity in several pancreatic cell lines. DNMT3B promoter constructs carrying either -149C allele or -149T allele were transiently transfected into pancreatic cancer cells. In promoter assaying, carriage of -149T allele showed only a slight activity (1.1-fold) in Mia cells (p=0.462). In contrast, significant increase (3.8-fold) in activity of -149T allele was shown in SU86.86 pancreatic cancer cells (p=0.0001). These preliminary findings suggest that genetic variance may influence DNMT3B expression in pancreatic cancer. Further studies are needed.

Cytotoxicity of chronic exposure to 4 cigarette smoke condensates in 2 cell lines.

Tobacco use is the leading preventable cause of death. The cytotoxicity of cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke without the vapor phase, has mostly been tested in short-term in vitro studies lasting from a few hours to a few days. Here, we assessed the toxicity of CSCs from 2 reference cigarettes, 3R4F and CM6, using a primary human small airway epithelial (PSAE) cell line by quantifying adenosine 5′-triphosphate (ATP), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]), and lactate dehydrogenase (LDH) release over the course of 28 days. The CSCs, 0.3 to 10 μg/mL, promoted cell proliferation at 120 hours of exposure, but demonstrated cytotoxicity at days 14 and 28. Interestingly, CSCs, 0.3 to 3 μg/mL, showed a cell death effect at day 14 but induced cell proliferation at day 28. Consistently, transformation associated with morphological changes began by day 14 and the transformed cells grew dramatically at day 28. The LDH assay appeared to be sensitive for assessing early cell damage, whereas the ATP, MTS, and GSH assays were more suitable for determining later stage CSCs-induced cytotoxicity. The ATP assay showed greater sensitivity than the MTS and GSH assays. We also assessed the toxicity of CSCs in an human Telomerase Reverse Transcriptase (hTERT)-immortalized Barrett esophagus cell line (CP-C). The CP-C cells demonstrated dose- and time-dependent cytotoxicity over the course of 28 days but displayed higher resistance to CSCs than PSAE cells. This study demonstrates that CSCs cause cytotoxicity and induce transformation related to cell resistance and cell invasion properties.

Cigarette smoke condensate and individual constituents modulate DNA methyltransferase expression in human liver cells

Objectives: Previous studies found higher expression levels of DNA methyltransferase 1 in liver samples from smokers compared to those from non-smokers. In contrast, expression levels of DNA methyltransferase 3a and DNA methyltransferase 3b were similar in smokers and non-smokers. This study extends these studies to establish a causal linkage to cigarette smoke exposure by examining whether DNA methyltransferase expression is modulated by cigarette smoke condensate. Methods: These experiments were conducted in an in vitro system using HepG2 human liver cells. The dose range of cigarette smoke condensate was 0.1–120 µg/mL. The duration of exposure was up to 72 h. Results: In a 24-h exposure, DNA methyltransferase 1 expression was found to increase significantly in a dose-dependent manner (greater than threefold at 100 µg/mL cigarette smoke condensate). Expression levels of DNA methyltransferase 3a and DNA methyltransferase 3b were, however, not affected under these conditions. The effect of two cigarette constituents, nicotine and cotinine, on DNA methyltransferase 1 expression was also examined. Nicotine exposure significantly increased DNA methyltransferase 1 expression in a dose-dependent manner (greater than twofold at 50 µM). However, under these conditions, cotinine did not increase DNA methyltransferase 1 expression. Conclusion: These results clearly provide additional support of the modulating effect of cigarette smoke on DNA methyltransferase 1 expression. Given the potential of alterations in DNA methyltransferase expression to affect cellular function, this pathway may play a critical role in cigarette smoke-induced toxicity.

In vitro analysis of factors influencing CYP1A2 expression as potential determinants of interindividual variation

Individual differences in drug metabolism contribute to interindividual variation that characterizes responses to drugs and risk in exposure to foreign chemicals. Large individual differences are found in expression levels of CYP1A2, a major drug‐metabolizing enzyme. Underlying causes for this variation are not well understood. Several factors, including tobacco smoking, consumption of cruciferous vegetables, and sex, have been associated with modulating CYP1A2 expression. To understand factors regulating expression of CYP1A2 in establishing a causal relationship, this study examined effects of cigarette smoke condensate (CSC), indole‐3‐carbinol (I3C), and 17β‐estradiol (estradiol) on CYP1A2 expression in in vitro systems using human liver and lung cells. Treatment with CSC (2–25 μg/mL) significantly increased levels of CYP1A2 in six cell lines examined, in a concentration‐ and time‐dependent manner. Fold changes in expression levels relative to controls varied among cell lines. CYP1A2 enzymatic activity also increased with CSC exposure. Treatment of H1299 and HepB3 cells with dietary agent I3C (50 and 100 μmol/L) increased CYP1A2 expression. In human cell lines H1299 and H1395, treatment with estradiol (10 and 100 nmol/L) significantly reduced expression of CYP1A2. Using ChIP assays, effects of CSC on histone modifications were analyzed. Increases in H3K4me3 and H4K16ac were observed at several segments in the CYP1A2 gene, whereas H3K27me3 decreased, following CSC treatment. These results suggest that CYP1A2 expression is affected epigenetically by CSC. Additional studies will be needed to further establish regulatory mechanisms underlying effects of various environmental, dietary, and endogenous factors on CYP1A2 expression in better predicting individual variation.