Low mitochondrial DNA copy number in peripheral blood mononuclear cells is associated with future mortality risk: a long-term follow-up study from Japan.

OBJECTIVES: The mitochondrial DNA (mtDNA) is unique and circular with multiple copies of the genome. The lower mtDNA copy number (mtDNA-CN) in leukocytes is associated with the risk of all-cause mortality. However, its long-term association is unknown. Thus, the study examined the association between mtDNA-CN and the risk of all-cause mortality in a long-term follow-up study in the Japanese population. DESIGN: This longitudinal study included the study cohort from an annual, population-based health checkup in the town of Yakumo, Hokkaido, Japan. SETTING AND PARTICIPANTS: 814 participants (baseline age range: 38-80 years, mean: 56.3 years) were included in this study in 1990. They were followed-up regarding mortality for about 30 years (median: 28.1 years) till 2019. MEASURES: The genomic DNA was extracted from peripheral blood mononuclear cells and the mtDNA-CN was measured using real-time polymerase chain reaction. The level of the mtDNA-CN was divided into tertiles (low, middle, and high). The participants were categorized based on their age into middle-aged (<60 years old) or old-aged (≥60 years old). Survival analysis was performed for tertile of mtDNA-CN and compared using the log-rank test. Univariate and multivariable Cox proportional hazard regression analyses were performed to assess the association between mtDNA-CN and all-cause mortality. The model adjusted with age, sex, body mass index, systolic blood pressure, smoking habit, alcohol consumption, exercise habit, and education level. RESULTS: The low levels of mtDNA-CN resulted in a significant decrease in cumulative survival rate (P < 0.05). The risk of mortality was significantly higher in the middle-aged cohort when mtDNA-CN levels were low (hazard ratios [95% confidence intervals]: 1.98 [1.10-3.56]). CONCLUSION: This study demonstrated that leukocyte mtDNA-CN is associated with future mortality risk. Our study findings may lead to further research on the early prediction of mortality and its underlying mechanisms.

Serum carotenoid levels are positively associated with DNA methylation of thioredoxin-interacting protein.

Background: Carotenoids have been reported to exert protective effects against age-related diseases via changes in DNA methylation. Although lower thioredoxin-interacting protein (TXNIP) DNA methylation is associated with age-related diseases, only a few studies have investigated the factors influencing TXNIP DNA methylation. Carotenoids may be a factor linking TXNIP to specific pathophysiological functions. The aim of this study was to examine whether serum carotenoid levels are associated with TXNIP DNA methylation levels. Methods: We conducted a cross-sectional study using 376 health examination participants (169 men). DNA methylation levels were determined using a pyrosequencing assay. Serum carotenoid levels were determined by high-performance liquid chromatography. Multivariable regression analyses were performed to examine the associations between TXNIP DNA methylation levels and serum carotenoid levels with adjustment for age, BMI, HbA1c, CRP, smoking habits, alcohol consumption, exercise habits, and percentage of neutrophils. Results: Multiple linear regression analyses showed that TXNIP DNA methylation levels were positively associated with serum levels of zeaxanthin/lutein (ß [95%CI]: 1.935 [0.184, 3.685]), ß-cryptoxanthin (1.447 [0.324, 2.570]), α-carotene (1.061 [0.044, 2.077]), ß-carotene (1.272 [0.319, 2.226]), total carotenes (1.255 [0.040, 2.469]), total xanthophylls (2.133 [0.315, 3.951]), provitamin A (1.460 [0.402, 2.519]), and total carotenoids (1.972 [0.261, 3.683]) in men (all p<0.05). Of these, provitamin A showed the stronger association (standardized ß=0.216). No significant association of TXNIP DNA methylation and serum carotenoid was observed in women. Conclusions: The findings of this study suggest that carotenoid intake may protect against age-related diseases by altering TXNIP DNA methylation status in men.

Integration of methylation quantitative trait loci (mQTL) on dietary intake on DNA methylation levels: an example of n-3 PUFA and ABCA1 gene.

BACKGROUND: Epigenetic studies have reported relationships between dietary nutrient intake and methylation levels. However, genetic variants that may affect DNA methylation (DNAm) pattern, called methylation quantitative loci (mQTL), are usually overlooked in these analyses. We investigated whether mQTL change the relationship between dietary nutrient intake and leukocyte DNAm levels with an example of estimated fatty acid intake and ATP-binding cassette transporter A1 (ABCA1). METHODS: A cross-sectional study on 231 participants (108 men, mean age: 62.7 y) without clinical history of cancer and no prescriptions for dyslipidemia. We measured leukocyte DNAm levels of 8 CpG sites within ABCA1 gene by pyrosequencing method and used mean methylation levels for statistical analysis. TaqMan assay was used for genotyping a genetic variant of ABCA1 (rs1800976). Dietary fatty acid intake was estimated with a validated food frequency questionnaire and adjusted for total energy intake by using residual methods. RESULTS: Mean ABCA1 DNAm levels were 5% lower with the number of minor alleles in rs1800976 (CC, 40.6%; CG, 35.9%; GG, 30.6%). Higher dietary n-3 PUFA intake was associated with lower ABCA1 DNAm levels (1st (ref) vs. 4th, ß [95% CI]: -2.52 [-4.77, -0.28]). After controlling for rs180076, the association between dietary n-3 PUFA intake and ABCA1 DNAm levels was attenuated, but still showed an independent association (1st (ref) vs. 4th, ß [95% CI]: -2.00 [-3.84, -0.18]). The interaction of mQTL and dietary n-3 PUFA intake on DNAm levels was not significant. CONCLUSIONS: This result suggested that dietary n-3 PUFA intake would be an independent predictor of DNAm levels in ABCA1 gene after adjusting for individual genetic background. Considering mQTL need to broaden into other genes and nutrients for deeper understanding of DNA methylation, which can contribute to personalized nutritional intervention.

Intake of vegetables and fruits rich in provitamin A is positively associated with aryl hydrocarbon receptor repressor DNA methylation in a Japanese population.

DNA methylation can be affected by numerous lifestyle factors, including diet. Tobacco smoking induces aryl hydrocarbon receptor repressor (AHRR) DNA hypomethylation, which increases the risk of lung and other cancers. However, no lifestyle habits that might increase or restore percentage of AHRR DNA methylation have been identified. We hypothesized that dietary intakes of vegetables/fruits and serum carotenoid concentrations are related to AHRR DNA methylation. A total of 813 individuals participated in this cross-sectional study. A food frequency questionnaire was used to assess dietary intake of vegetables and fruits. AHRR DNA methylation in peripheral blood mononuclear cells were measured using pyrosequencing method. In men, dietary fruit intake was significantly and positively associated with AHRR DNA methylation among current smokers (P for trend = .034). A significant positive association of serum provitamin A with AHRR DNA methylation was observed among current smokers (men: standardized ß = 0.141 [0.045 to 0.237], women: standardized ß = 0.570 [0.153 to 0.990]). However, compared with never smokers with low provitamin A concentrations, percentages of AHRR DNA methylation were much lower among current smokers, even those with high provitamin A concentrations (men: ß = -19.1% [-33.8 to -19.8], women: ß = -6.0% [-10.2 to -1.7]). Dietary intake of vegetables and fruits rich in provitamin A may increase percentage of AHRR DNA methylation in current smokers. However, although we found a beneficial effect of provitamin A on AHRR DNA methylation, this beneficial effect could not completely remove the effect of smoking on AHRR DNA demethylation.

Increased risk of cancer mortality by smoking-induced aryl hydrocarbon receptor repressor DNA hypomethylation in Japanese population: A long-term cohort study.

BACKGROUND: Smoking is well known to be a major risk factor for cancer, and to decrease the levels of aryl hydrocarbon receptor repressor (AHRR) DNA methylation. AHRR is a key regulator for AHR signaling, which is involved in chemical metabolism and cancer development. Therefore, smoking-induced AHRR DNA hypomethylation may be associated with cancer development. However, it has not been reported that association between AHHR DNA methylation and cancer mortality in Asian population. Hence, we examined whether AHRR DNA methylation levels were associated with cancer mortality in a Japanese population. METHODS: This study was conducted with 812 participants (aged 38-80 years) who received a health check-up in 1990, and did not have a clinical histories. We followed up the participants until the end of 2019 (median: 27.8 years), and 100 participants died from cancer. The AHRR DNA methylation levels in peripheral blood mononuclear cells (PBMCs) were measured by the pyrosequencing method. We calculated the hazard ratio (HR) and 95% confidence interval (CI) for cancer mortality according to the baseline levels of AHRR DNA methylation. RESULTS: We found that AHRR DNA hypomethylation was associated with a higher risk of all cancer mortality, especially smoking related cancers and lung cancer. (all cancer: HR, 1.28, 95% CI, 1.09-1.51; smoking-related cancers: HR, 1.35, 95% CI, 1.12-1.62; lung cancer: HR, 1.68, 95% CI, 1.24-2.26). CONCLUSIONS: Smoking-induced AHRR DNA hypomethylation in PBMCs was associated with the risk of cancer mortality in Japanese population; therefore, hypomethylation of AHRR may be a useful biomarker of cancer mortality risk.

Adipsin-Dependent Secretion of Hepatocyte Growth Factor Regulates the Adipocyte-Cancer Stem Cell Interaction.

Adipose tissue is a component of the tumor microenvironment and is involved in tumor progression. We have previously shown that adipokine adipsin (CFD) functions as an enhancer of tumor proliferation and cancer stem cell (CSC) properties in breast cancers. We established the Cfd-knockout (KO) mice and the mammary adipose tissue-derived stem cells (mADSCs) from them. Cfd-KO in mADSCs significantly reduced their ability to enhance tumorsphere formation of breast cancer patient-derived xenograft (PDX) cells, which was restored by the addition of Cfd in the culture medium. Hepatocyte growth factor (HGF) was expressed and secreted from mADSCs in a Cfd-dependent manner. HGF rescued the reduced ability of Cfd-KO mADSCs to promote tumorsphere formation in vitro and tumor formation in vivo by breast cancer PDX cells. These results suggest that HGF is a downstream effector of Cfd in mADSCs that enhances the CSC properties in breast cancers.

Association between circulating microRNAs and changes in kidney function: A five-year prospective study among Japanese adults without CKD.

BACKGROUND: Although a number of microRNAs (miRNA) reflecting kidney function has been identified, prospective studies are now urgently needed to determine a clinical utility of these miRNAs among general populations. The purpose of this study was to examine the associations between serum miRNAs and kidney function in a population-based study. METHODS: We conducted a five-year prospective study (2012-2017) of 169 individuals without chronic kidney disease (CKD) at the baseline survey (mean age, 62.5; 96 women). The real-time qPCR was used to measure serum levels of five previously reported miRNAs. Participants with eGFR < 60 mL/min/1.73 m2 were defined as having CKD. Changes in eGFR were defined as eGFR2017 - eGFR2012. RESULTS: After adjusting for covariates including baseline eGFR, lower serum levels (1st tertile) of miR-126 were associated with a greater decline of eGFR (ß [SE] = -3.18 [1.50]) and a higher odds ratio (OR) of CKD onset over five years (OR [95% CI] = 3.85 [1.01-16.8]), compared with the 3rd tertile. CONCLUSIONS: We found baseline serum miR-126 levels were associated with changes in eGFR and new CKD cases in a five-year prospective study. This result suggests that miR-126 may be a potential biomarker of CKD even among general populations.

Global DNA hypermethylation in peripheral blood mononuclear cells and cardiovascular disease risk: a population-based propensity score-matched cohort study.

BACKGROUND: DNA methylation plays an important role in the pathogenesis and progression of cardiovascular disease (CVD) but the prospective association of DNA methylation with CVD has not been evaluated. Here, we conducted a prospective study to examine whether long interspersed nuclear element-1 (LINE-1) DNA methylation is associated with CVD mortality in a Japanese population. METHODS: We targeted 822 Japanese who participated in a health check-up in 1990 and had no clinical history of cancer, stroke or ischaemic heart disease. DNA was extracted from peripheral blood mononuclear cells and LINE-1 DNA methylation at three CpG sites was measured using a pyrosequencing method. We used propensity score (PS) matching to reduce the effect of potential confounding. RESULTS: During 18 118.7 persons-years of follow-up, there were 329 deaths from all-causes and 85 deaths from CVD. In PS-matched analysis, a significantly higher HR for CVD mortality was observed in the hypermethylation group than in the hypomethylation group for elderly participants (HR 2.77; 95% CI 1.55 to 4.93). No significant association between LINE-1 DNA methylation and CVD was observed for middle-aged participants. CONCLUSIONS: Based on this prospective study, we suggest that LINE-1 DNA hypermethylation is associated with increased CVD mortality risk in an elderly population.

Dietary fish and ω-3 polyunsaturated fatty acids are associated with leukocyte ABCA1 DNA methylation levels.

OBJECTIVES: A diet rich in fish and ω-3 polyunsaturated fatty acids (PUFAs) has been thought to reduce the risk for cardiovascular disease (CVD). The beneficial effects of fish oil and ω-3 PUFA on CVD can be mediated by epigenetic status of the genes associated with lipid metabolism and inflammation. The aim of this study was to investigate whether dietary fish and fatty acid (FA) intakes are associated with leukocyte ATP-binding cassette transporter A1 (ABCA1) DNA methylation levels in a Japanese population. METHODS: This cross-sectional study included 298 adults (137 men and 161 women) without clinical history of CVD or cancer. The pyrosequencing method was used to measure leukocyte ABCA1 DNA methylation levels. Dietary fish and FA intakes were assessed based on the validated food frequency questionnaire. RESULTS: Mean ABCA1 DNA methylation levels were significantly lower in the highest fish intake groups (≥5-6/wk) compared with the lowest intake group (≤1-2/wk; P = 0.004). In multivariable linear regression analyses, higher dietary intake of ω-3 PUFAs and ω-3 highly unsaturated fatty acids was significantly associated with decreased levels of ABCA1 DNA methylation (P = 0.001 and 0.005); whereas no significant associations were seen between intake of dietary saturated fatty acid, monounsaturated fatty acid, and ω-6 PUFAs and ABCA1 DNA methylation. CONCLUSION: Higher dietary fish and ω-3 PUFA intake were associated with lower ABCA1 DNA levels in a Japanese population. The present results may bring potential insights on biological mechanisms underlying the protective effects of dietary fish and ω-3 PUFA intakes on CVD.

Association of smoking habits with TXNIP DNA methylation levels in leukocytes among general Japanese population.

Thioredoxin-interacting protein (TXNIP) inhibits the activity of thioredoxin (TXN), leading to increased oxidative stress. Expression of the TXNIP gene is regulated by DNA methylation. However, no study has reported the influence of lifestyle factors on TXNIP DNA methylation. Our goal was to determine the association between smoking habits and TXNIP DNA methylation levels in a Japanese population. We conducted a cross-sectional study of 417 subjects (180 males and 237 females) participating in a health examination. We used a pyrosequencing assay to determine TXNIP DNA methylation levels in leukocytes. The mean TXNIP DNA methylation level in current smokers (75.3%) was significantly lower than that in never and ex-smokers (never: 78.1%, p < 0.001; ex: 76.9%, p = 0.013). Multivariable logistic regression analyses showed that the OR for TXNIP DNA hypomethylation was significantly higher in current smokers than that in never smokers, and significantly higher in current smokers with years of smoking ≥ 35 and Brinkman Index ≥ 600 compared to that in non-smokers. In conclusion, we found that current smokers had TXNIP DNA hypomethylation compared to never and ex-smokers. Moreover, long-term smoking and high smoking exposure also were associated with TXNIP DNA hypomethylation.

Associations between dietary vitamin intake, ABCA1 gene promoter DNA methylation, and lipid profiles in a Japanese population.

BACKGROUND: Higher intake of fruits and vegetables is associated with reduced risk of specific types of cancer and of cardiovascular disease (CVD), but the protective role of the vitamins contained in fruits and vegetables on CVD is controversial. This discrepancy can raise the question of the effects of antioxidants in vitamins on CVD. Recently, we reported that higher vegetable intake was significantly associated with the decreased DNA methylation level of ATP-binding cassette transporter A1 (ABCA1), a gene associated with HDL-cholesterol metabolism. OBJECTIVE: We investigated whether ABCA1 DNA methylation mediates an effect of dietary vitamin intake on lipid profiles, an important risk factor for CVD, in a Japanese population. METHODS: A total of 225 individuals (108 men and 117 women) with no clinical history and no drug use for dyslipidemia participated in this cross-sectional study. We used the pyrosequencing method to measure the ABCA1 DNA methylation levels at 8 CpG sites, and we used mean DNA methylation level in statistical analysis. Dietary vitamin intake was assessed with the FFQ and adjusted for the residual method. RESULTS: In women, higher dietary vitamin intake [vitamin A, ß-carotene, folic acid, vitamin C (VC), vitamin D, and vitamin E] was significantly associated with lower mean ABCA1 DNA methylation levels (P = 0.004, 0.03, 0.005, 0.001, 0.03, and 0.04, respectively). In addition, in women, we found a significant inverse association between mean ABCA1 DNA methylation and HDL cholesterol (P = 0.04) but not for other lipid indexes. Mediation analysis showed a significant indirect effect of VC intake on HDL cholesterol through ABCA1 DNA methylation level in women (P = 0.04). CONCLUSIONS: Although this study does not prove causality, the results suggest that ABCA1 DNA methylation mediates the protective effect of VC on HDL cholesterol in women, which could offer a novel biological mechanism in CVD prevention.

Maternal fructose-induced oxidative stress occurs viaTfam and Ucp5 epigenetic regulation in offspring hippocampi.

Fructose consumption is rising globally, but maternal high fructose intake might adversely affect offspring. Our previous report demonstrated that excess maternal fructose intake impairs hippocampal function in offspring, indicating that the hippocampi of offspring are highly sensitive to maternal fructose. Here, we examined the effect of maternal high fructose on mitochondrial physiology and uncoupling protein (UCP) expression. Rat dams received a 20% fructose solution during gestation and lactation. Immediately after weaning, offspring hippocampi were isolated. Maternal high fructose consumption attenuated the mitochondrial O2 consumption rate and stimulated lipid hydroperoxide production in the hippocampi of offspring. Reduced Ucp5 and mitochondrial transcription factor A (Tfam) mRNA levels were also observed after maternal exposure to fructose. We assessed the promoter regions of both genes and found that this treatment enhanced DNA methylation levels. In addition, luciferase assays showed that this DNA methylation could reduce the transcription of both genes. Chromatin immunoprecipitation analysis demonstrated that specificity protein 1 binding to the Ucp5 promoter regions was reduced by DNA methylation. In addition, Ucp5 knockdown induced the up-regulation of reactive oxygen species levels in a rat brain glioma cell line, whereas reduced O2 consumption was observed with Tfam knockdown. Maternal high fructose intake thus induces reduced O2 oxygen consumption and increases oxidative stress in offspring, at least partly through epigenetic mechanisms involving Ucp5 and Tfam.-Yamada, H., Munetsuna, E., Yamazaki, M., Mizuno, G., Sadamoto, N., Ando, Y., Fujii, R., Shiogama, K., Ishikawa, H., Suzuki, K., Shimono, Y., Ohashi, K., Hashimoto, S. Maternal fructose-induced oxidative stress occurs viaTfam and Ucp5 epigenetic regulation in offspring hippocampi.

Dietary vegetable intake is inversely associated with ATP-binding cassette protein A1 (ABCA1) DNA methylation levels among Japanese women.

OBJECTIVE: Dietary intake of vegetables is one of the key lifestyle factors associated with preventing cancer and cardiovascular disease (CVD). Although previous studies have provided evidence that dietary factors can alter global DNA methylation levels in humans, little work has been done on dietary factors influencing methylation levels of specific genes associated with CVD. The aim of this study was to examine whether dietary intake of vegetables was associated with adenosine triphosphate-binding membrane cassette transporter A1 (ABCA1) DNA methylation levels in leukocytes in a Japanese population. METHODS: This cross-sectional study included 279 Japanese adults (125 men, 154 women) without any clinical history of cancer, stroke, or ischemic heart disease. ABCA1 DNA methylation levels in leukocytes were measured using a pyrosequencing method. Information on dietary vegetable intake was obtained from the validated food frequency questionnaire. RESULTS: Mean ABCA1 DNA methylation levels in men and women were 35.6% ± 6.5% and 36.9% ± 6.7%, respectively. In women, multivariable linear regression analysis showed that the group with the highest dietary vegetable intake (carrot, broccoli, pumpkin, and all vegetables) showed significantly lower levels of ABCA1 DNA methylation than the lowest intake group (P = 0.04, <0.001, 0.001, and 0.02, respectively). No significant association was observed between dietary intake of vegetables and DNA methylation levels in men. CONCLUSIONS: High dietary intake of vegetables was associated with decreased ABCA1 DNA methylation levels in Japanese women. This may contribute to a better understanding of the protective effects of dietary vegetable intake on CVD.

Associations of serum microRNA-20a, -27a, and -103a with cognitive function in a Japanese population: The Yakumo study.

OBJECTIVES: MicroRNAs (miRNAs) dysregulate gene expression by binding to target messenger RNAs, and play an important role in the pathogenesis of various diseases, including cancers, cardiovascular diseases and diabetes. Circulating miRNAs have increasingly been recognized as biomarkers for detecting and diagnosing those diseases. Few studies have investigated the association of circulating miRNA with the early stages of cognitive impairment, such as mild cognitive impairment, in the general population. The purpose of this study was to examine the association between cognitive function and several serum miRNAs levels related to amyloid precursor protein (APP) proteolysis in a Japanese general population who had never been diagnosed with dementia. METHODS: We conducted a cross-sectional study of 337 Japanese subjects (144 men, 193 women) who attended a health examination. The short form of the Mini-Mental State Examination (SMMSE) was used to assess cognitive function. Serum levels of 6 miRNAs (let-7d, miR-17, miR-20a, miR-27a, miR-34a, miR-103a) were measured by quantitative real-time polymerase chain reaction. RESULTS: Multivariable-adjusted odds ratios (ORs) for lower SMMSE score (SMMSE score < 28) were significantly increased in the lowest tertile of serum miR-20a (OR, 2.08; 95% confidence interval (CI), 1.09-4.04) and miR-103a (OR, 1.91; 95%CI, 1.00-3.69) compared to the highest tertile. Moreover, serum levels of miR-20a, -27a, and -103a were linearly and positively associated with SMMSE scores after adjustment for confounding factors. CONCLUSION: Low serum levels of miR-20a, -27a, and -103a are independently associated with cognitive impairment.

Relationship between Long Interspersed Nuclear Element-1 DNA Methylation in Leukocytes and Dyslipidemia in the Japanese General Population.

AIM: Aberrant global DNA methylation is involved in the development of several diseases, including cardiovascular disease (CVD). We investigated whether the methylation of long interspersed nuclear element-1 (LINE-1) in leukocytes is associated with dyslipidemia, a major risk factor for CVD, in the Japanese general population. METHODS: We conducted a cross-sectional study consisting of 420 Japanese subjects (187 men and 233 women) without a clinical history of cancer, stroke, or ischemic heart disease. LINE-1 DNA methylation levels in leukocytes were measured using a pyrosequencing method. RESULTS: Significantly higher odds ratios (ORs) for hypermethylation were observed in the high LDL cholesterol and high LDL/HDL ratio groups than the corresponding normal group (high LDLC group: OR, 1.88; 95% confidence interval [CI], 1.20-2.96, high LDL/HDL ratio group: OR, 1.90; 95% CI, 1.20-3.01). Subjects with 2 or more lipid abnormalities had significantly higher ORs for hypermethylation than those with no lipid abnormality (OR, 2.31; 95% CI, 1.11-4.82). CONCLUSION: LINE-1 DNA hypermethylation in leukocytes was associated with CVD risk profiles: high LDLC, high LDL/HDL ratio, and the degree of abnormal lipid metabolism.

Maternal fructose intake disturbs ovarian estradiol synthesis in rats.

AIMS: Recent increases in fructose consumption have raised concerns regarding the potential adverse intergenerational effects, as maternal fructose intake may induce physiological dysfunction in offspring. However, no reports are available regarding the effect of excess maternal fructose on reproductive tissues such as the ovary. Notably, the maternal intrauterine environment has been demonstrated to affect ovarian development in the subsequent generation. Given the fructose is transferred to the fetus, excess fructose consumption may affect offspring ovarian development. As ovarian development and its function is maintained by 17ß-estradiol, we therefore investigated whether excess maternal fructose intake influences offspring ovarian estradiol synthesis. Rats received a 20% fructose solution during gestation and lactation. After weaning, offspring ovaries were isolated. KEY FINDINGS: Offspring from fructose-fed dams showed reduced StAR and P450(17α) mRNA levels, along with decreased protein expression levels. Conversely, attenuated P450arom protein level was found in the absence of mRNA expression alteration. Consistent with these phenomena, decreased circulating levels of estradiol were observed. Furthermore, estrogen receptor α (ERα) protein levels were also down-regulated. In accordance, the mRNA for progesterone receptor, a transcriptional target of ERα, was decreased. These results suggest that maternal fructose might alter ovarian physiology in the subsequent generation.

Metabolism and Action of 25-Hydroxy-19-nor-Vitamin D3 in Human Prostate Cells.

Since the discovery of 1α,25(OH)2D3 in the early 1970s, it has been widely accepted that this metabolite is responsible for the biological actions of vitamin D. Likewise, we have assumed that 25(OH)-19-nor-D3-dependent growth inhibition of human prostate PZ-HPV-7 cells was the result of its subsequent conversion to 1α,25(OH)2-19-nor-D3, catalyzed by CYP27B1 within the prostate cells. However, further in vitro studies in a reconstituted system using recombinant CYP27B1 revealed that 25(OH)-19-nor-D3 was hardly converted to 1α,25(OH)2-19-nor-D3 by the enzyme. The kinetic analysis of 1α-hydroxylation of 25(OH)D3 and 25(OH)-19-nor-D3 demonstrated that the k(cat)/K(m) for 25(OH)-19-nor-D3 is less than 0.1% of that for 25(OH)D3. When 25(OH)-19-nor-D3 was added to cultured PZ-HPV-7 cells, eight metabolites were detected, while no 1α,25(OH)2-19-nor-D3 was found. In addition, the time course of VDR translocation into the nucleus induced by 100 nM 25(OH)-19-nor-D3, and the subsequent transactivation of CYP24A1 gene were almost identical to those induced by 1 nM 1α,25(OH)2-19-nor-D3. These results strongly suggest that 25(OH)-19-nor-D3 binds directly to VDR as a ligand to transport VDR into the nucleus to induce CYP24A1 gene transactivation. Furthermore, knockdown of CYP27B1 gene did not affect the antiproliferative activity of 25(OH)-19-nor-D3, whereas VDR knockdown attenuated the effect, suggesting that the antiproliferative activity of 25(OH)-19-nor-D3 is VDR dependent but CYP27B1 independent. Finally, our recent studies using the same cell line demonstrate that 25(OH)D3 can act as a VDR agonist to induce gene transactivation. These findings suggest that vitamin D analogs without 1α-hydroxyl group could be developed as drugs for osteoporosis or cancer treatment.

Stimulation of estradiol biosynthesis by tributyltin in rat hippocampal slices.

Hippocampal functions are influenced by steroid hormones, such as testosterone and estradiol. It has been demonstrated that hippocampus-derived steroid hormones play important roles in neuronal protection and synapse formation. Our research groups have demonstrated that estradiol is de novo synthesized in the rat hippocampus. However, the mechanism(s) regulating this synthesis remains unclear. It has been reported that tributyltin, an environmental pollutant, binds to the retinoid X receptor (RXR) and modifies estrogen synthesis in human granulosa-like tumor cells. This compound can penetrate the blood brain barrier, and tends to accumulate in the brain. Based on these facts, we hypothesized that tributyltin could influence the hippocampal estradiol synthesis. A concentration of 0.1 µM tributyltin induced an increase in the mRNA content of P450(17α) and P450arom in hippocampal slices, as determined using real-time PCR. The transcript levels of other steroidogenic enzymes and a steroidogenic acute regulatory protein were not affected. The estradiol level in rat hippocampal slices was subsequently determined using a radioimmunoassay. We found that the estradiol synthesis was stimulated by ∼2-fold following a 48-h treatment with 0.1 µM tributyltin, and this was accompanied by transcriptional activation of P450(17α) and P450arom. Tributyltin stimulated de novo hippocampal estradiol synthesis by modifying the transcription of specific steroidogenic enzymes.

Anti-proliferative activity of 25-hydroxyvitamin D3 in human prostate cells.

1α-Hydroxylation of 25-hydroxyvitamin D3 is believed to be essential for its biological effects. In this study, we evaluated the biological activity of 25(OH)D3 itself comparing with the effect of cell-derived 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). First, we measured the cell-derived 1α,25(OH)2D3 level in immortalized human prostate cell (PZ-HPV-7) using [(3)H]-25(OH)D3. The effects of the cell-derived 1α,25(OH)2D3 on vitamin D3 24-hydroxylase (CYP24A1) mRNA level and the cell growth inhibition were significantly lower than the effects of 25(OH)D3 itself added to cell culture. 25-Hydroxyvitamin D3 1α-hydroxylase (CYP27B1) gene knockdown had no significant effects on the 25(OH)D3-dependent effects, whereas vitamin D receptor (VDR) gene knockdown resulted in a significant decrease in the 25(OH)D3-dependent effects. These results strongly suggest that 25(OH)D3 can directly bind to VDR and exerts its biological functions. DNA microarray and real-time RT-PCR analyses suggest that semaphorin 3B, cystatin E/M, and cystatin D may be involved in the antiproliferative effect of 25(OH)D3.

De novo synthesized estradiol protects against methylmercury-induced neurotoxicity in cultured rat hippocampal slices.

BACKGROUND: Estrogen, a class of female sex steroids, is neuroprotective. Estrogen is synthesized in specific areas of the brain. There is a possibility that the de novo synthesized estrogen exerts protective effect in brain, although direct evidence for the neuroprotective function of brain-synthesized estrogen has not been clearly demonstrated. Methylmercury (MeHg) is a neurotoxin that induces neuronal degeneration in the central nervous system. The neurotoxicity of MeHg is region-specific, and the molecular mechanisms for the selective neurotoxicity are not well defined. In this study, the protective effect of de novo synthesized 17ß-estradiol on MeHg-induced neurotoxicity in rat hippocampus was examined. METHODOLOGY/PRINCIPAL FINDINGS: Neurotoxic effect of MeHg on hippocampal organotypic slice culture was quantified by propidium iodide fluorescence imaging. Twenty-four-hour treatment of the slices with MeHg caused cell death in a dose-dependent manner. The toxicity of MeHg was attenuated by pre-treatment with exogenously added estradiol. The slices de novo synthesized estradiol. The estradiol synthesis was not affected by treatment with 1 µM MeHg. The toxicity of MeHg was enhanced by inhibition of de novo estradiol synthesis, and the enhancement of toxicity was recovered by the addition of exogenous estradiol. The neuroprotective effect of estradiol was inhibited by an estrogen receptor (ER) antagonist, and mimicked by pre-treatment of the slices with agonists for ERα and ERß, indicating the neuroprotective effect was mediated by ERs. CONCLUSIONS/SIGNIFICANCE: Hippocampus de novo synthesized estradiol protected hippocampal cells from MeHg-induced neurotoxicity via ERα- and ERß-mediated pathways. The self-protective function of de novo synthesized estradiol might be one of the possible mechanisms for the selective sensitivity of the brain to MeHg toxicity.