Noriko Nakamura

Background: Perfluorooctane sulfonic acid (PFOS) belongs to a group of synthetic chemicals referred to as per‐ and polyfluoroalkyl substances (PFAS) that are known to degrade slowly. Exposure to PFOS has been reported to cause birth defects and disrupt female sex hormone levels in mice. However, to date, the direct effects of PFOS exposure on folliculogenesis in young animals have not been evaluated. Therefore, this study was conducted to examine the potential effects of in utero and prepubertal exposure to PFOS on follicle development in juvenile, female mouse pups. Methods: ICR dams and young, female mouse pups were dosed with 0.1 mg/kg PFOS or saline (vehicle) via gavage beginning on gestation day (GD) 6 (plug positive = GD 0) through GD 17 ( n = 11–17 per group) [in utero exposure] or postnatal day (PND) 7 (PND 0 = day of birth) through PND 21 ( n = 22–29 per group) [prepubertal exposure]. Ovaries and blood were then collected from a pup/litter on PND 28 for analysis. Results: There were no adverse effects of PFOS exposure observed in the measured endpoints upon in utero exposure; however, statistically significant differences following prepubertal exposure were noted for serum estradiol levels, the total number of primordial follicles, and the transcript levels of Foxo1 and Gdf9 . Conclusion: The preliminary findings suggest that prepubertal exposure to PFOS may affect follicle development by disrupting estradiol production and altering Foxo1 and Gdf9 gene expression. Further studies will be important to examine the mechanisms underlying prepubertal exposure to PFOS on folliculogenesis.

Background: Dexamethasone (DEX) is used during pregnancies at risk of early delivery or congenital adrenal hyperplasia. DEX exposure is also known to cause placental damage. Although placental cytokines/chemokines protect the fetus and regulate placental development, few studies have examined placental cytokine/chemokine transcript levels in DEX‐dosed pregnant mice. Methods: To examine this, quantitative PCR and histological analysis in humanized mice were performed. Mice were injected once daily for five consecutive days with DEX (5 mg/kg) or saline (0.9%) via the tail vein on gestation days (GDs) 10–14, respectively ( n = 3–5). All mice were intravenously injected with human immunoglobulin G (2 mg/kg) on GD14. Results: No statistically significant changes in maternal body weights by GD 12, absolute or relative placental weights in the dosed group were observed compared to concurrent controls. Fetal weights in the DEX‐dosed group were lower than in concurrent controls, and statistically significant changes were observed on GD 18. Necrosis/apoptosis of cytotrophoblasts in the placenta's labyrinth zone was observed in the DEX‐dosed dams. The placental transcript levels of interferon lambda receptor 1, interleukin 6, and C‐X‐C motif chemokine ligand 10 ( Cxcl10 ) were higher in the DEX‐dosed than the control group on GDs 15 and 16; the difference of Cxcl10 transcript level was statistically significant ( p = 0.016) on GD 16. Conclusions: Cxcl10 is overexpressed during DEX‐induced placental damage in the mouse models, suggesting it as a potential biomarker of placental damage. Further studies are needed to confirm Cxcl10 changes during placental damage induced by other placental toxicants.

Background/Aim: Prostate cancer (PCa) is one of the most common types of cancer in men. Prostate-specific antigen (PSA) is currently the only biomarker used to screen for the risk of developing PCa. Because PSA tests may show false positives, identifying novel PCa-specific biomarkers would improve prediction and diagnosis at an early stage. Previously, we identified a number of genes/microRNAs (miRNAs) in prostate tissue as potential biomarkers of chronic prostatitis in a rat model of chemical-induced prostatitis. The current study aimed to evaluate their potential for use as translational, diagnostic markers in humans. Materials and Methods: We performed quantitative polymerase chain reaction analysis using pathologically clear (normal) or confirmed PCa tissue samples from the same patients (N=18 per group). Results: Levels (relative fold changes) of bone morphogenetic protein 7 (BMP7) transcripts were significantly lower in PCa tissues, compared with clear tissues, in a paired t-test (p=0.0075). Although neural cell adhesion molecule 1 (NCAM1) transcripts tended to be altered in PCa tissues, statistically insignificant differences were observed (p=0.0521). No statistically significant differences were observed for the other genes/miRNAs analyzed in PCa tissues due to a high degree of individual variance in expression. Conclusion: Similar to the results previously observed in rats, changes in the levels of BMP7 and NCAM1 transcripts were evident in human PCa tissues, suggesting that these genes may serve as potential diagnostic biomarkers during the early stages of PCa. Further studies are needed to determine the potential use of these molecules as biomarkers.

Rats are the standard model for male reproductive toxicity testing. Rat prostates are physiologically and anatomically different from those of humans. Drug and chemical toxicity testing would benefit from an in vitro model of human prostate cells. Recently, spheroids derived by three-dimensional culture of human cell lines have been used for assessing drug and chemical toxicity in vitro as they mimic in vivo environments more closely than two-dimensional culture. However, forming consistently sized, uniform spheroids is technically challenging for toxicity testing. The purpose of this study was to identify potential genetic markers for assessing prostatic toxicity in spheroids. We formed prostate spheroids using agarose-coated plates seeded with human primary prostate epithelial cells. Prostate spheroids were treated with either 17β-estradiol (E2) or testosterone (T) on days 2–7 of culture. Samples were harvested on culture day 7. qPCR was used to examine gene expression levels previously identified in rats with chronic inflammation exposed to estradiol benzoate, E2 and/or T. Changes in some gene expression levels were observed in the spheroids treated with E2 or T. We found that treatment with 1 nM E2 and/or 10 μM T significantly altered spheroid proliferation and viability, as well as the expression levels of genes including Nanog homeobox (NANOG), C-C motif chemokine ligand 2 (CCL2) and bone morphogenetic protein receptor type 2 (BMPR2). Further studies using biologically active molecules with prostatic toxicity are needed to verify the results and to determine whether gene expression changes in the spheroid are specific to E2 or T treatment.

Background: Spermatogenesis complexity makes reliable in vitro testis model development challenging. Previously, we evaluated an in vitro mouse testis organ culture system for assessing testicular toxicity. However, rat models are commonly used for drug/chemical toxicity testing; therefore, we assessed the effects of media on germ cell differentiation in cultured rat testis fragments.Methods: Testes from postnatal day 5 Sprague–Dawley (Hsd:SD) rats were cultured in knockout serum replacement (KSR) or Albumax™ I (Albumax) medium. For testis morphology and germ cell differentiation, rat testis fragments were collected on days 20, 27, 35, 42, 49, and 63 of culture for histology/immunohistochemistry using antibodies to spermatogenesis‐specific markers. The fragments collected on days 20, 27, 42, 49, and 63 were used for qPCR.Results: Pachytene spermatocyte (PS) differentiation was observed in rat testis fragments cultured in KSR and Albumax. However, there were more seminiferous tubules (STs) with PS in rat testis fragments cultured in Albumax than in KSR. Over 60% of STs with germ cell differentiation were observed in rat testis fragments when cultured in Albumax on days 20, 27, and 35, whereas this figure showed only on day 20 when cultured in KSR.Conclusions: This study found only PS differentiation in rat testis fragments. Compared to KSR, Albumax appears to contribute to increased PS production. This in vitro rat testis organ culture system may be useful for assessing testicular toxicity. However, PS differentiation per ST is lower in rat testis fragments; further studies are required to improve this rat testis organ culture system.

Introduction Prostatitis is likely to occur in younger or middle-aged men, while prostate cancer is likely to occur in older men. Although amino acids and lipids as biomarkers of prostate cancer have been examined using prostate cancer cell lines/tissues, no previous studies have evaluated amino acids or lipids as potential chronic prostatitis biomarkers. Objectives The study’s aim was to identify amino acids and lipids that could serve as potential biomarkers of chronic prostatitis. Methods We profiled the amino acids and lipids found in plasma from rats collected in a previous study. In brief, a total of 148 Sprague–Dawley rats (offspring) were dosed with estradiol benzoate (EB) on postnatal days (PNDs) 1, 3 and 5, and subsequently dosed with testosterone (T)/estradiol (E) tubes via subcutaneous implants from PND 90 to 200. Plasma was collected on PNDs 30, 90, 100, 145 and 200. Analysis was conducted with a Xevo TQ-S triple-quadrupole mass spectrometer using a Biocrates AbsoluteIDQ p180 kit. Results Plasma acylcarnitines [(C2, C16:1, C18, C18:1, C18:1-OH, and C18:2)], glycerophospholipids (lysophosphatidylcholine-acyl, -di-acyl, and -di-acyl acyl-alkyl) and sphingomyelins [SM (OH) C16:1, SM C18:0, SM C18:1, and SM C20:2] significantly increased on PND 145, when chronic inflammation was observed in the dorsolateral prostate of rats dosed with EB, T, and E. No statistical significances of amino acid levels were observed in the EB + T + E group on PND 145. Conclusion Exposure to EB, T, and E altered lipid levels in rat plasma with chronic prostate inflammation. These findings suggest that the identified lipids may be predictive chronic prostatitis biomarkers. The results require confirmation through additional nonclinical and human studies.

Background: Elevated estrogen (E) levels caused by aging or exposure to endocrine disrupting chemicals are related to prostate disease development. Sixty to seventy percent of prostate cancer or benign prostatic hyperplasia patients are over the age of 65, while prostatitis is likely to occur in men under 45 years. MicroRNAs currently represent a class of distinctive biological indicators to be used for clinical disease diagnosis and treatment monitoring. This study aims to identify microRNAs that could serve as potential biomarkers for prostate disorders induced by elevated E levels according to their altered expression in prostate or plasma.Materials and methods: Groups of Sprague Dawley rats (offspring) were dosed with estradiol benzoate (EB) on postnatal days 1, 3, and 5, and subcutaneously implanted with tubes containing testosterone (T)/E on postnatal day 90. Expression levels of prostate and plasma microRNAs were evaluated using microRNA microarray and validated via qRT‐PCR. The expression levels of the potential targeted genes of a set of identified microRNAs were also examined by qRT‐PCR.Results: Postnatal administration of EB, T, and E elevated serum E levels with decreased serum T levels in rats. Chronic inflammation was observed in the dorsolateral prostate. Significant changes in expression levels of several microRNAs (rno‐miR‐146‐5p, rno‐miR‐329‐3p, and rno‐miR‐126a‐3p) in the dorsolateral prostate and of a microRNA (rno‐miR‐329‐3p) in the plasma were found in the dosed rats. The target gene expression levels of the altered microRNAs also changed accordingly.Conclusion: Chronic inflammation in the dorsolateral prostate of rats dosed with EB, T, and E resulted in deregulated expression in a set of microRNAs whose target genes were related to tumor growth or abnormal proliferation. Our findings suggest the identified microRNAs and their target genes the potential use as biomarkers to predict prostate cancer development. Validation using human samples is warranted.