Benzene Uptake and Glutathione S-transferase T1 Status as Determinants of S-Phenylmercapturic Acid in Cigarette Smokers in the Multiethnic Cohort

Research from the Multiethnic Cohort (MEC) demonstrated that, for the same quantity of cigarette smoking, African Americans and Native Hawaiians have a higher lung cancer risk than Whites, while Latinos and Japanese Americans are less susceptible. We collected urine samples from 2,239 cigarette smokers from five different ethnic groups in the MEC and analyzed each sample for S-phenylmercapturic acid (SPMA), a specific biomarker of benzene uptake. African Americans had significantly higher (geometric mean [SE] 3.69 [0.2], p<0.005) SPMA/ml urine than Whites (2.67 [0.13]) while Japanese Americans had significantly lower levels than Whites (1.65 [0.07], p<0.005). SPMA levels in Native Hawaiians and Latinos were not significantly different from those of Whites. We also conducted a genome-wide association study in search of genetic risk factors related to benzene exposure. The glutathione S-transferase T1 (GSTT1) deletion explained between 14.2–31.6% (p = 5.4x10^-157) and the GSTM1 deletion explained between 0.2%-2.4% of the variance (p = 1.1x10^-9) of SPMA levels in these populations. Ethnic differences in levels of SPMA remained strong even after controlling for the effects of these two deletions. These results demonstrate the powerful effect of GSTT1 status on SPMA levels in urine and show that uptake of benzene in African American, White, and Japanese American cigarette smokers is consistent with their lung cancer risk in the MEC. While benzene is not generally considered a cause of lung cancer, its metabolite SPMA could be a biomarker for other volatile lung carcinogens in cigarette smoke.     

Mercapturic Acids Derived from the Toxicants Acrolein and Crotonaldehyde in the Urine of Cigarette Smokers from Five Ethnic Groups with Differing Risks for Lung Cancer

The Multiethnic Cohort epidemiology study has clearly demonstrated that, compared to Whites and for the same number of cigarettes smoked, African Americans and Native Hawaiians have a higher risk for lung cancer whereas Latinos and Japanese Americans have a lower risk. Acrolein and crotonaldehyde are two important constituents of cigarette smoke which have well documented toxic effects and could play a role in lung cancer etiology. Their urinary metabolites 3-hydroxypropylmercapturic acid (3-HPMA) and 3-hydroxy-1-methylpropylmercapturic acid (HMPMA), respectively, are validated biomarkers of acrolein and crotonaldehyde exposure. We quantified levels of 3-HPMA and HMPMA in the urine of more than 2200 smokers from these five ethnic groups, and also carried out a genome wide association study using blood samples from these subjects. After adjusting for age, sex, creatinine, and total nicotine equivalents, geometric mean levels of 3-HPMA and HMPMA were significantly different in the five groups (P<0.0001). Native Hawaiians had the highest and Latinos the lowest geometric mean levels of both 3-HPMA and HMPMA. Levels of 3-HPMA and HMPMA were 3787 and 2759 pmol/ml urine, respectively, in Native Hawaiians and 1720 and 2210 pmol/ml urine in Latinos. These results suggest that acrolein and crotonaldehyde may be involved in lung cancer etiology, and that their divergent levels may partially explain the differing risks of Native Hawaiian and Latino smokers. No strong signals were associated with 3-HPMA in the genome wide association study, suggesting that formation of the glutathione conjugate of acrolein is mainly non-enzymatic, while the top significant association with HMPMA was located on chromosome 12 near the TBX3 gene, but its relationship to HMPMA excretion is not clear.     

Angiotensin II Triggers Expression of the Adrenal Gland Zona Glomerulosa-Specific 3β-Hydroxysteroid Dehydrogenase Isoenzyme through De Novo Protein Synthesis of the Orphan Nuclear Receptors NGFIB and NURR1

The 3β-hydroxysteroid dehydrogenase (3β-HSD) is an enzyme crucial for steroid synthesis. Two different 3β-HSD isoforms exist in humans. Classically, HSD3B2 was considered the principal isoform present in the adrenal. However, we recently showed that the alternative isoform, HSD3B1, is expressed specifically within the adrenal zona glomerulosa (ZG), where aldosterone is produced, raising the question of why this isozyme needs to be expressed in this cell type. Here we show that in both human and mouse, expression of the ZG isoform 3β-HSD is rapidly induced upon angiotensin II (AngII) stimulation. AngII is the key peptide hormone regulating the capacity of aldosterone synthesis. Using the human adrenocortical H295R cells as a model system, we show that the ZG isoform HSD3B1 differs from HSD3B2 in the ability to respond to AngII. Mechanistically, the induction of HSD3B1 involves de novo protein synthesis of the nuclear orphan receptors NGFIB and NURR1. The HSD3B1 promoter contains a functional NGFIB/NURR1-responsive element to which these proteins bind in response to AngII. Knockdown of these proteins and overexpression of a dominant negative NGFIB both reduce the AngII responsiveness of HSD3B1. Thus, the AngII-NGFIB/NURR1 pathway controls HSD3B1. Our work reveals HSD3B1 as a new regulatory target of AngII.     

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells

Background

Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established.

Methodology

Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3–30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity.

Results and Conclusions

In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC₅₀) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.     

Gender-dependent association of HSD11B1 single nucleotide polymorphisms with glucose and HDL-C levels

In this study, we investigated the influence of two SNPs (rs846910 and rs12086634) of the HSD11B1 gene that encodes 11β-hydroxysteroid dehydrogenase type 1(11β-HSD1), the enzyme that catalyzes the conversion of cortisol to cortisone, on variables associated with obesity and metabolic syndrome in 215 individuals of both sexes from southern Brazil. The HSD11B1 gene variants were genotyped using the TaqMan SNP genotyping assay. Glucose, triglycerides, total cholesterol, HDL-cholesterol and LDL-cholesterol were measured by standard automated methods. Significant results were found in women, with carriers of the G allele of SNP rs12086634 having higher glucose levels than non-carriers. Carriers of the A allele of SNP rs846910 had higher levels of HDL-cholesterol. The involvement of both polymorphisms as independent factors in determining the levels of glucose and HDL-cholesterol was confirmed by multiple regression analysis (β = 0.19 ±0.09, p = 0.03 and β= 0.22 ± 0.10, p = 0.03, respectively). Our findings suggest that the HSD11B1SNPs studied may indirectly influence glucose and HDL-cholesterol metabolism in women, possibly through down-regulation of the HSD11B1 gene by estrogen.     

Antitumor activity of IL-32β through the activation of lymphocytes,and the inactivation of NF-κB and STAT3 signals

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32β overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32β inhibited B16 melanoma growth in IL-32β-overexpressing transgenic mice (IL-32β mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32β also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32β-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32β also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32β mice, and athymic nude mice. The number of cytotoxic T (CD8⁺) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32β mice and athymic nude mice inoculated with IL-32β-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32β mice and athymic nude mice, as well as in IL-32β-transfected cultured cancer cells. These findings suggest that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.     

Prostatitis,Sexually Transmitted Diseases,and Prostate Cancer: The California Men's Health Study

Background

Prostatitis and sexually transmitted diseases (STDs) have been positively associated with prostate cancer in previous case-control studies. However, results from recent prospective studies have been inconclusive.

Methodogy/Principal Findings

We investigated the association between prostatitis, STDs, and prostate cancer among African American, Asian American, Latino, and White participants of the California Men''s Health Study. Our analysis included 68,675 men, who completed a detailed baseline questionnaire in 2002–2003. We identified 1,658 incident prostate cancer cases during the follow-up period to June 30, 2006. Cox proportional hazards models were used to estimate relative risks and 95% confidence intervals. Overall, men having a history of prostatitis had an increased risk of prostate cancer than men with no history (RR = 1.30; 95% CI: 1.10–1.54). Longer duration of prostatitis symptoms was also associated with an increased risk of prostate cancer (P trend = 0.003). In addition, among men screened for prostate cancer (1 or 2 PSA tests), a non-significant positive association was observed between prostatitis and prostate cancer (RR = 1.10; 95% CI: 0.75–1.63). STDs were not associated with overall prostate cancer risk. In racial/ethnic stratified analysis, Latinos reporting any STDs had an increased risk of disease than those with no STDs (RR = 1.43; 95% CI: 1.07–1.91). Interestingly, foreign-born Latinos displayed a larger risk associated with STDs (RR = 1.87; 95% CI: 1.16–3.02) than U.S. born Latinos (RR = 1.15; 95% CI: 0.76–3.02).

Conclusion

In summary, results from this prospective study suggest that prostatitis and STDs may be involved in prostate cancer susceptibility. While we cannot rule out the possible influence of incidental detection, future studies are warranted to further investigate the role of infectious agents related to prostatitis and STDs in prostate cancer development.     

Mapping of the HSD17B2 gene encoding type II 17β-hydroxysteroid dehydrogenase close to D16S422 on chromosome 16q24.1–q24.2

The enzymes of the 17β-hydroxysteroid dehydrogenase (17β-HSD) gene family are responsible for a key step in the formation and degradation of androgens and estrogens: catalyzing the interconversion of 17-ketosteroids and their active 17β-hydroxysteroid counterparts. The structure of human type II 17β-HSD cDNA was recently reported. This enzyme catalyzes the interconversion of Δ⁴-androstenedione and testosterone, androstanedione and dihydrotestosterone, and estrone and 17β-estradiol, whereas type I 17β-HSD catalyzes exclusively the interconversion of estrogens. To locate the HSD17B2 gene, the novel dinucleotide CA repeat sequence found 571 bp downstream from the end of exon 1 was genotyped into eight CEPH reference families by PCR. Two-point linkage analysis was performed between the latter polymorphism and the 2066 microsatellite markers of Généthon. The maximal pairwise lod score (Z_max = 33.3) with a maximal recombination fraction (θ_max) of 0.008 was obtained with the marker D16S422 located on 16q24.1–q24.2. To define further the localization of the HSD17B2 gene, we constructed a high-resolution genetic map of the region flanking the polymorphic HSD17B2 gene including eight Généthon markers. The order of the HSD17B2 gene and markers is qter-D16S516 — D16S504 — D16S507 — D16S505 — D16S511 — [HSD17B2—D16S422]—D16S520—D16S413—tel.     

Tissue-Specific Increases in 11β-Hydroxysteroid Dehydrogenase Type 1 in Normal Weight Postmenopausal Women

With age and menopause there is a shift in adipose distribution from gluteo-femoral to abdominal depots in women. Associated with this redistribution of fat are increased risks of type 2 diabetes and cardiovascular disease. Glucocorticoids influence body composition, and 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) which converts inert cortisone to active cortisol is a putative key mediator of metabolic complications in obesity. Increased 11βHSD1 in adipose tissue may contribute to postmenopausal central obesity. We hypothesized that tissue-specific 11βHSD1 gene expression and activity are up-regulated in the older, postmenopausal women compared to young, premenopausal women. Twenty-three pre- and 23 postmenopausal, healthy, normal weight women were recruited. The participants underwent a urine collection, a subcutaneous adipose tissue biopsy and the hepatic 11βHSD1 activity was estimated by the serum cortisol response after an oral dose of cortisone. Urinary (5α-tetrahydrocortisol+5β-tetrahydrocortisol)/tetrahydrocortisone ratios were higher in postmenopausal women versus premenopausal women in luteal phase (P<0.05), indicating an increased whole-body 11βHSD1 activity. Postmenopausal women had higher 11βHSD1 gene expression in subcutaneous fat (P<0.05). Hepatic first pass conversion of oral cortisone to cortisol was also increased in postmenopausal women versus premenopausal women in follicular phase of the menstrual cycle (P<0.01, at 30 min post cortisone ingestion), suggesting higher hepatic 11βHSD1 activity. In conclusion, our results indicate that postmenopausal normal weight women have increased 11βHSD1 activity in adipose tissue and liver. This may contribute to metabolic dysfunctions with menopause and ageing in women.     

Consistent Association of Type 2 Diabetes Risk Variants Found in Europeans in Diverse Racial and Ethnic Groups

It has been recently hypothesized that many of the signals detected in genome-wide association studies (GWAS) to T2D and other diseases, despite being observed to common variants, might in fact result from causal mutations that are rare. One prediction of this hypothesis is that the allelic associations should be population-specific, as the causal mutations arose after the migrations that established different populations around the world. We selected 19 common variants found to be reproducibly associated to T2D risk in European populations and studied them in a large multiethnic case-control study (6,142 cases and 7,403 controls) among men and women from 5 racial/ethnic groups (European Americans, African Americans, Latinos, Japanese Americans, and Native Hawaiians). In analysis pooled across ethnic groups, the allelic associations were in the same direction as the original report for all 19 variants, and 14 of the 19 were significantly associated with risk. In summing the number of risk alleles for each individual, the per-allele associations were highly statistically significant (P<10⁻⁴) and similar in all populations (odds ratios 1.09–1.12) except in Japanese Americans the estimated effect per allele was larger than in the other populations (1.20; P_het = 3.8×10⁻⁴). We did not observe ethnic differences in the distribution of risk that would explain the increased prevalence of type 2 diabetes in these groups as compared to European Americans. The consistency of allelic associations in diverse racial/ethnic groups is not predicted under the hypothesis of Goldstein regarding “synthetic associations” of rare mutations in T2D.     

17β-Hydroxysteroid dehydrogenase type 14 is a predictive marker for tamoxifen response in oestrogen receptor positive breast cancer

Introduction

17β-hydroxysteroid dehydrogenases (17βHSDs) are important enzymes regulating the pool of bioactive steroids in the breast. The current study was undertaken in order to evaluate implications of 17βHSD14 in breast cancer, measuring 17βHSD14 protein expression in breast tumours.

Methods

An antibody targeting the 17βHSD14 antigen was generated and validated using HSD17B14-transfected cells and a peptide-neutralising assay. Tissue microarrays with tumours from 912 post-menopausal women diagnosed with lymph node-negative breast cancer, and randomised to adjuvant tamoxifen or no endocrine treatment, were analysed for 17βHSD14 protein expression with immunohistochemistry.

Results

Results were obtained from 847 tumours. Patients with oestrogen positive tumours with high 17βHSD14 expression had fewer local recurrences when treated with tamoxifen (HR 0.38; 95% C.I. 0.19–0.77, p = 0.007) compared to patients with lower tumoural 17βHSD14 expression, for whom tamoxifen did not reduce the number of local recurrences (HR 1.19; 95% C.I. 0.54–2.59; p = 0.66). No prognostic importance of 17βHSD14 was seen for systemically untreated patients.

Conclusions

Using a highly specific validated antibody for immunohistochemical analysis of a large number of breast tumours, we have shown that tumoural expression levels of 17βHSD14 can predict the outcome of adjuvant tamoxifen treatment in terms of local recurrence-free survival in patients with lymph node-negative ER+ breast cancer. The results need be verified to confirm any clinical relevance.     

Genetic Susceptibility Factors on Genes Involved in the Steroid Hormone Biosynthesis Pathway and Progesterone Receptor for Gastric Cancer Risk

Background

The objective of the study was to investigate the role of genes (HSD3B1, CYP17A1, CYP19A1, HSD17B2, HSD17B1) involved in the steroid hormone biosynthesis pathway and progesterone receptor (PGR) in the etiology of gastric cancer in a population-based two-phase genetic association study.

Methods

In the discovery phase, 108 candidate SNPs in the steroid hormone biosynthesis pathway related genes and PGR were analyzed in 76 gastric cancer cases and 322 controls in the Korean Multi-Center Cancer Cohort. Statistically significant SNPs identified in the discovery phase were re-evaluated in an extended set of 386 cases and 348 controls. Pooled- and meta-analyses were conducted to summarize the results.

Results

Of the 108 SNPs in steroid hormone biosynthesis pathway related genes and PGR analyzed in the discovery phase, 23 SNPs in PGR in the recessive model and 10 SNPs in CYP19A1 in the recessive or additive models were significantly associated with increased gastric cancer risk (p<0.05). The minor allele frequencies of the SNPs in both the discovery and extension phases were not statistically different. Pooled- and meta-analyses showed CYP19A1 rs1004982, rs16964228, and rs1902580 had an increased risk for gastric cancer (pooled OR [95% CI] = 1.22 [1.01–1.48], 1.31 [1.03–1.66], 3.03 [1.12–8.18], respectively). In contrast, all PGR SNPs were not statistically significantly associated with gastric cancer risk.

Conclusions

Our findings suggest CYP19A1 that codes aromatase may play an important role in the association of gastric cancer risk and be a genetic marker for gastric cancer susceptibility.     

Analysis of 17β-hydroxysteroid dehydrogenase types 5, 7, and 12 genetic sequence variants in breast cancer cases from French Canadian Families with high risk of breast and ovarian cancer

A family history and estrogen exposure are well-known risk factors for breast cancer. Members of the 17β-hydroxysteroid dehydrogenase family are responsible for important steps in the metabolism of androgens and estrogens in peripheral tissues, including the mammary gland. The crucial biological function of 17β-HSDs renders these genes good candidates for being involved in breast cancer etiology. This study screened for mutations in HSD17B7 and HSD17B12 genes, which encode enzymes involved in estradiol biosynthesis and in AKR1C3, which codes for 17β-HSD type 5 enzyme involved in androgen and progesterone metabolism, to assess whether high penetrance allelic variants in these genes could be involved in breast cancer susceptibility. Mutation screening of 50 breast cancer cases from non-BRCA1/2 high-risk French Canadian families failed to identify germline likely high-risk mutations in HSD17B7, HSD17B12 and AKR1C3 genes. However, 107 sequence variants were identified, including seven missense variants. Assessment of the impact of missense variants on enzymatic activity of the corresponding enzymes revealed no difference in catalytic properties between variants of 17β-HSD types 7 and 12 and wild-type enzymes, while variants p.Glu77Gly and p.Lys183Arg in 17β-HSD type 5 showed a slightly decreased activity. Finally, a haplotype-based approach was used to determine tagging SNPs providing valuable information for studies investigating associations of common variants in these genes with breast cancer risk.     

Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris) Affected with Hypoadrenocorticism (Addison’s Disease)

Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison’s disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as ³⁵S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.     

Atenolol Induced HDL-C Change in the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study

We sought to identify novel pharmacogenomic markers for HDL-C response to atenolol in participants with mild to moderate hypertension. We genotyped 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) study on the Illumina HumanCVD Beadchip. During PEAR, participants were randomized to receive atenolol or hydrochlorothiazide. Blood pressure and cholesterol levels were evaluated at baseline and after treatment. This study focused on participants treated with atenolol monotherapy. Association with atenolol induced HDL-C change was evaluated in 232 whites and 152 African Americans using linear regression. No SNPs achieved a Bonferroni corrected P-value. However, we identified 13 regions with consistent association across whites and African Americans. The most interesting of these regions were seven with prior associations with HDL-C, other metabolic traits, or functional implications in the lipid pathway: GALNT2, FTO, ABCB1, LRP5, STARD3NL, ESR1, and LIPC. Examples are rs2144300 in GALNT2 in whites (P=2.29x10^-4, β=-1.85 mg/dL) and rs12595985 in FTO in African Americans (P=2.90x10^-4, β=4.52 mg/dL), both with consistent regional association (P<0.05) in the other race group. Additionally, baseline GALNT2 expression differed by rs2144300 genotype in whites (P=0.0279). In conclusion, we identified multiple gene regions associated with atenolol induced HDL-C change that were consistent across race groups, several with functional implications or prior associations with HDL-C.     

Folic Acid Supplementation during Pregnancy Induces Sex-Specific Changes in Methylation and Expression of Placental 11β-Hydroxysteroid Dehydrogenase 2 in Rats

In the placenta, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA) during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg) or normal low (1mg/kg, control) levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (−378 to −275) was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11β-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%). This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.