Effects of continuous bisphenol A exposure from early gestation on 90 day old rat testes function and sperm molecular profiles: A CLARITY-BPA consortium study.

Bisphenol A (BPA) is a ubiquitous industrial chemical that has been identified as an endocrine disrupting compound (EDC). There is growing concern that early life exposures to EDCs, such as BPA, can adversely affect the male reproductive tract and function. This study was conducted as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA) to further delineate the toxicities associated with continuous exposure to BPA from early gestation, and to comprehensively examine the elicited effects on testes and sperm. NCTR Sprague Dawley rat dams were gavaged from gestational day (GD) 6 until parturition, and their pups were directly gavaged daily from postnatal day (PND) 1 to 90 with BPA (2.5, 25, 250, 2500, 25,000, 250,000 µg/kg/d) or vehicle control. At PND 90, the testes and sperm were collected for evaluation. The testes were histologically evaluated for altered germ cell apoptosis, sperm production, and altered spermiation. RNA and DNA isolated from sperm were assessed for elicited changes in global mRNA transcript abundance and altered DNA methylation. Effects of BPA were observed in changes in body, testis and epididymis weights only at the highest administered dose of BPA of 250,000 µg/kg/d. Genome-wide transcriptomic and epigenomic analyses failed to detect robust alterations in sperm mRNA and DNA methylation levels. These data indicate that prolonged exposure starting in utero to BPA over a wide range of levels has little, if any, impact on the testes and sperm molecular profiles of 90 day old rats as assessed by the histopathologic, morphometric, and molecular endpoints evaluated.

Multicomponent folate-targeted magnetoliposomes: design, characterization, and cellular uptake.

Folate-targeted cationic magnetoliposomes (FTMLs) have been prepared with coencapsulated doxorubicin (DOX) and anionic superparamagnetic iron oxide (SPIO) nanoparticles (NPs) with 5 nm γ-Fe(2)O(3) cores and 16 nm hydrodynamic diameters. NP encapsulation (89%) was confirmed by cryogenic transmission electron microscopy (TEM), and the presence of the oppositely charged NPs did not cause liposome aggregation. The FTMLs had an average diameter of 174 ± 53 nm and existed as unilamellar and cup-shaped liposomes, which was attributed to dissimilar lipid packing parameters and the presence of PEG-lipids. A 3-fold increase in DOX release was achieved over 2 hours when the encapsulated SPIO NPs were heated by an alternating current electromagnetic field operating at radio frequencies (RF). Results with human cervical cancer cells (HeLa), which have been shown to exhibit high folate receptor (FR) expression, confirmed FTML surface binding and cellular uptake. In contrast, no uptake was observed for lower FR-expressing human breast carcinoma cells (ZR-75-1). FROM THE CLINICAL EDITOR: This study discusses the design and cellular uptake of multifunctional folate-targeted cationic magnetoliposomes enabling doxorubicin delivery and SPIO labeling.

Subfertility caused by altered follicular development and oocyte growth in female mice lacking PKB alpha/Akt1.

Mammalian females are endowed with a finite number of primordial follicles at birth. Immediately following formation of the primordial follicle pool, cohorts of follicles are either culled from the ovary or are recruited to grow until the primordial follicle population is depleted. The majority of ovarian follicles, including the oocytes, undergo atresia through apoptotic cell death. As PKB alpha/Akt1 is known to regulate apoptosis, we asked whether Akt1 functioned in the regulation of folliculogenesis in the ovary. Akt1(-/-) females display reduced fertility and abnormal estrous cyclicity. At Postnatal Day (PND) 25, Akt1(-/-) ovaries possessed a reduced number of growing antral follicles, significantly larger primary and secondary oocytes, and an increase in the number of degenerate oocytes. By PND90, there was a significant decrease in the number of primordial follicles in Akt1(-/-) ovaries relative to Akt1(+/+). In vivo granulosa cell proliferation was reduced, as were expression levels of Kitl and Bcl2l1, two factors associated with granulosa cell proliferation/survival. No compensation was observed by Akt2 or Akt3 at the mRNA/protein level. Significantly higher serum LH and trends for lower FSH and higher inhibin A and lower inhibin B relative to Akt1(+/+) females were observed in Akt1(-/-) females. Exposure to exogenous gonadotropins resulted in an increase in the number of secondary follicles in Akt1(-/-) ovaries, but few mature follicles. Collectively, our results suggest that PKB alpha/Akt1 plays an instrumental role in the regulation of the growth and maturation of the ovary, and that the loss of PKB alpha/Akt1 results in premature ovarian failure.

From the Cover: Sperm Molecular Biomarkers Are Sensitive Indicators of Testicular Injury following Subchronic Model Toxicant Exposure.

Traditional testis histopathology endpoints remain the gold standard for evaluating testicular insult and injury in a non-clinical setting, but are invasive and unfeasible for monitoring these effects clinically in humans. Assessing testicular injury in humans relies on semen and serum hormone analyses, both of which are insensitive and poor indicators of effect. Therefore, we hypothesized that sperm messenger RNA (mRNA) transcripts and DNA methylation marks can be used as translatable and sensitive indicators or testicular injury. Dose-response studies using adult male Fischer 344 rats subchronically exposed to model Sertoli cell toxicants (0.14, 0.21, and 0.33% 2,5-hexanedione, and 30, 50, and 70 mg/kg/day carbendazim), and a model germ cell toxicant (1.4, 3.4, and 5.1 mg/kg/day cyclophosphamide) for 3 months were evaluated for testicular injury by traditional histopathological endpoints, changes in sperm mRNA transcript levels using custom PCR arrays, and alterations in sperm DNA methylation via reduced representation bisulfite sequencing. Testis histopathological evaluation and PCR array analysis of the sperm transcriptome identified dose-dependent changes elicited by toxicant exposure (P < 0.05). Global sperm DNA methylation analysis of subchronic 0.33% 2,5-hexandione and 5.1 mg/kg/day cyclophosphamide exposure using a Monte Carlo approach did not identify differentially methylated regions (methylation difference > 10% and q < 0.05) with robust signatures. Overall, these results suggest that sperm mRNA transcripts are sensitive indicators of low dose toxicant-induced testicular injury in the rat, while sperm DNA methylation changes are not. Additionally, the Monte Carlo analysis is a powerful approach that can be used to assess the robustness of signals resulting from -omic studies.

Biomarkers of chemotherapy-induced testicular damage.

Increasing numbers of men are having or wanting children after chemotherapy treatment. This can be attributed to improvements in cancer therapies that increase survival. However, a side effect of most chemotherapy drugs is disruption of spermatogenesis and a drastic reduction in sperm count and quality. Although many men eventually recover reproductive function, as indicated by normal semen analyses, there is no clinical test that can assess sperm quality at a high level of sensitivity. Sperm fluorescent in situ hybridization (i.e., FISH) and several different tests for deoxyribonucleic acid (DNA) fragmentation have been used infrequently in clinical assessment. Animal models of chemotherapy-induced testicular damage are currently being used to identify potential molecular biomarkers that may be translatable to humans-these include sperm messenger RNAs, microRNAs, histone modifications, and DNA methylation patterns. Changes in these molecular measurements are quantitative and sensitive, potentially making them important clinical biomarkers of testicular function after chemotherapy treatment.

SOT Symposium Highlight: Translatable Indicators of Testicular Toxicity: Inhibin B, MicroRNAs, and Sperm Signatures.

Testicular toxicity is an important safety endpoint in drug development and risk assessment, but reliable and translatable biomarkers for predicting injury have eluded researchers. However, this area shows great potential for improvement, with several avenues currently being pursued. This was the topic of a symposium session during the 2013 Society of Toxicology Annual Meeting in San Antonio, TX, entitled "Translatable Indicators of Testicular Toxicity: Inhibin B, MicroRNAs, and Sperm Signatures." This symposium brought together stakeholders from academia, government, and industry to present the limitations and drawbacks of currently used indicators of injury and discussed the ongoing efforts in developing more predictive biomarkers of injury. The presentations highlighted the early challenges of using circulating inhibin B and microRNA levels, and sperm messenger RNA transcript abundance and DNA methylation profiles, as novel biomarkers of testicular toxicity.

The current status of biomarkers for predicting toxicity.

INTRODUCTION: There are significant rates of attrition in drug development. A number of compounds fail to progress past preclinical development due to limited tools that accurately monitor toxicity in preclinical studies and in the clinic. Research has focused on improving tools for the detection of organ-specific toxicity through the identification and characterization of biomarkers of toxicity. AREAS COVERED: This article reviews what we know about emerging biomarkers in toxicology, with a focus on the 2012 Northeast Society of Toxicology meeting titled 'Translational Biomarkers in Toxicology.' The areas covered in this meeting are summarized and include biomarkers of testicular injury and dysfunction, emerging biomarkers of kidney injury and translation of emerging biomarkers from preclinical species to human populations. The authors also provide a discussion about the biomarker qualification process and possible improvements to this process. EXPERT OPINION: There is currently a gap between the scientific work in the development and qualification of novel biomarkers for nonclinical drug safety assessment and how these biomarkers are actually used in drug safety assessment. A clear and efficient path to regulatory acceptance is needed so that breakthroughs in the biomarker toolkit for nonclinical drug safety assessment can be utilized to aid in the drug development process.

Optimization of a filter-lysis protocol to purify rat testicular homogenates for automated spermatid counting.

Quantifying testicular homogenization-resistant spermatid heads (HRSH) is a powerful indicator of spermatogenesis. These counts have traditionally been performed manually using a hemocytometer, but this method can be time consuming and biased. We aimed to develop a protocol to reduce debris for the application of automated counting, which would allow for efficient and unbiased quantification of rat HRSH. We developed a filter-lysis protocol that effectively removes debris from rat testicular homogenates. After filtering and lysing the homogenates, we found no statistical differences between manual (classic and filter-lysis) and automated (filter-lysis) counts using 1-way analysis of variance with Bonferroni's multiple comparison test. In addition, Pearson's correlation coefficients were calculated to compare the counting methods, and there was a strong correlation between the classic manual counts and the filter-lysis manual (r = 0.85, P = .002) and the filter-lysis automated (r = 0.89, P = .0005) counts. We also tested the utility of the automated method in a low-dose exposure model known to decrease HRSH. Adult Fischer 344 rats exposed to 0.33% 2,5-hexanedione in the drinking water for 12 weeks demonstrated decreased body (P = .02) and testes (P = .002) weights. In addition, there was a significant reduction in the number of HRSH per testis (P = .002) when compared to controls. A filterlysis protocol was optimized to purify rat testicular homogenates for automated HRSH counts. Automated counting systems yield unbiased data and can be applied to detect changes in the testis after low-dose toxicant exposure.

Sperm mRNA transcripts are indicators of sub-chronic low dose testicular injury in the Fischer 344 rat.

Current human reproductive risk assessment methods rely on semen and serum hormone analyses, which are not easily comparable to the histopathological endpoints and mating studies used in animal testing. Because of these limitations, there is a need to develop universal evaluations that reliably reflect male reproductive function. We hypothesized that toxicant-induced testicular injury can be detected in sperm using mRNA transcripts as indicators of insult. To test this, we exposed adult male Fischer 344 rats to low doses of model testicular toxicants and classically characterized the testicular injury while simultaneously evaluating sperm mRNA transcripts from the same animals. Overall, this study aimed to: 1) identify sperm transcripts altered after exposure to the model testicular toxicant, 2,5-hexanedione (HD) using microarrays; 2) expand on the HD-induced transcript changes in a comprehensive time course experiment using qRT-PCR arrays; and 3) test these injury indicators after exposure to another model testicular toxicant, carbendazim (CBZ). Microarray analysis of HD-treated adult Fischer 344 rats identified 128 altered sperm mRNA transcripts when compared to control using linear models of microarray analysis (q<0.05). All transcript alterations disappeared after 3 months of post-exposure recovery. In the time course experiment, time-dependent alterations were observed for 12 candidate transcripts selected from the microarray data based upon fold change and biological relevance, and 8 of these transcripts remained significantly altered after the 3-month recovery period (p<0.05). In the last experiment, 8 candidate transcripts changed after exposure to CBZ (p<0.05). The two testicular toxicants produced distinct molecular signatures with only 4 overlapping transcripts between them, each occurring in opposite directions. Overall, these results suggest that sperm mRNA transcripts are indicators of low dose toxicant-induced testicular injury in the rat.

Modulation of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) by 6-arylpyrrolo[2,1-d][1,5]benzothiazepine derivatives, ligands of peripheral benzodiazepine receptor (PBR).

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate xenobiotic sensing and metabolism through interactions with multiple exogenous and endogenous chemicals. Compounds that activate CAR are often ligands of PXR; attention is therefore given to discovery of new, receptor-specific chemical entities that may be exploited for therapeutic and basic research purposes. Recently, ligands of the peripheral benzodiazepine receptor (PBR), PK11195 and FGIN-1-27, were shown to modulate both CAR and PXR. PBR is a mitochondrial transport protein responsible for multiple regulatory functions, including heme biosynthesis, a major component in cytochrome P450 (CYP) enzymes. To investigate possible new roles for PBR involvement in metabolic regulation, expression of the CAR and PXR target genes, CYP2B6 and CYP3A4, was measured in human hepatocytes following treatment with a targeted PBR ligand set. Luciferase reporter assays with transiently expressed wild-type CAR (CAR1), splice variant CAR3, or PXR in HuH-7 cells were used to further study activation of these receptors. Four structurally related PBR ligands (benzothiazepines) differentially modulate CAR1, CAR3 and PXR activity. Benzothiazepine NF49 is an agonist ligand of CAR3, a partial agonist of PXR, exhibits greater inverse agonist activity on CAR1 than does PK11195, and is a new tool for studying these closely related nuclear receptors.

Rational quantitative structure-activity relationship (RQSAR) screen for PXR and CAR isoform-specific nuclear receptor ligands.

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related orphan nuclear receptor proteins that share several ligands and target overlapping sets of genes involved in homeostasis and all phases of drug metabolism. CAR and PXR are involved in the development of certain diseases, including diabetes, metabolic syndrome and obesity. Ligand screens for these receptors so far have typically focused on steroid hormone analogs with pharmacophore-based approaches, only to find relatively few new hits. Multiple CAR isoforms have been detected in human liver, with the most abundant being the constitutively active reference, CAR1, and the ligand-dependent isoform CAR3. It has been assumed that any compound that binds CAR1 should also activate CAR3, and so CAR3 can be used as a ligand-activated surrogate for CAR1 studies. The possibility of CAR3-specific ligands has not, so far, been addressed. To investigate the differences between CAR1, CAR3 and PXR, and to look for more CAR ligands that may be of use in quantitative structure-activity relationship (QSAR) studies, we performed a luciferase transactivation assay screen of 60 mostly non-steroid compounds. Known active compounds with different core chemistries were chosen as starting points and structural variants were rationally selected for screening. Distinct differences in agonist versus inverse agonist/antagonist effects were seen in 49 compounds that had some ligand effect on at least one receptor and 18 that had effects on all three receptors; eight were CAR1 ligands only, three were CAR3 only ligands and four affected PXR only. This work provides evidence for new CAR ligands, some of which have CAR3-specific effects, and provides observational data on CAR and PXR ligands with which to inform in silico strategies. Compounds that demonstrated unique activity on any one receptor are potentially valuable diagnostic tools for the investigation of in vivo molecular targets.