Transcript profiling in the testes and prostates of postnatal day 30 Sprague-Dawley rats exposed prenatally and lactationally to 2-hydroxy-4-methoxybenzophenone.

2-hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet light-absorbing compound that is used in sunscreens, cosmetics and plastics. HMB has been reported to have weak estrogenic activity by in vivo and in vitro studies, making it a chemical with potential reproductive concern. To explore if prenatal and lactational HMB exposure alters gene expression profiles of the developing reproductive organs, we performed microarray analysis using the prostate and testis of postnatal day (PND) 30 male Sprague-Dawley rats offspring exposed to 0, 3000, or 30,000 ppm of HMB from gestational day 6 through PND 21.

Testicular cells exhibit similar molecular responses to cigarette smoke condensate and .

Male exposure to cigarette smoke is associated with seminal defects and with congenital anomalies and childhood cancers in offspring. In mice, paternal exposure to cigarette smoke condensate (CSC) causes molecular defects in germ cells and phenotypic effects in their offspring. Here we used an testicular explant model and exposure to determine the concentration at which CSC impairs spermatogenesis and offspring development.

Evaluation of Culture Time and Media in an In Vitro Testis Organ Culture System.

Background: The complexity of spermatogenesis makes development of appropriate in vitro testis models challenging. A novel in vitro mouse testis culture system has been reported but not yet evaluated as an alternative model for male reproductive toxicity testing. We assessed the effects of media composition on sperm differentiation and testis morphology of cultured mouse testis fragments.

Evidence Favoring a Positive Feedback Loop for Physiologic Auto Upregulation of hnRNP-E1 during Prolonged Folate Deficiency in Human Placental Cells.

Previously, we determined that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) functions as an intracellular physiologic sensor of folate deficiency. In this model, l-homocysteine, which accumulates intracellularly in proportion to the extent of folate deficiency, covalently binds to and thereby activates homocysteinylated hnRNP-E1 to interact with folate receptor-α mRNA; this high-affinity interaction triggers the translational upregulation of cell surface folate receptors, which enables cells to optimize folate uptake from the external milieu. However, integral to this model is the need for ongoing generation of hnRNP-E1 to replenish homocysteinylated hnRNP-E1 that is degraded.

Transcriptomics analysis of early embryonic stem cell differentiation under osteoblast culture conditions: Applications for detection of developmental toxicity.

The mouse embryonic stem cell test (mEST) is a promising in vitro assay for predicting developmental toxicity. In the current study, early differentiation of D3 mouse embryonic stem cells (mESCs) under osteoblast culture conditions and embryotoxicity of cadmium sulfate were examined. D3 mESCs were exposed to cadmium sulfate for 24, 48 or 72h, and whole genome transcriptional profiles were determined.

Paternal exposure to cigarette smoke condensate leads to reproductive sequelae and developmental abnormalities in the offspring of mice.

Paternal smoking is associated with infertility, birth defects and childhood cancers. Our earlier studies using cigarette smoke condensate (CSC) demonstrated several deleterious changes in male germ cells. Here, we hypothesize that chronic paternal exposure to CSC causes molecular and phenotypic changes in the sire and the offspring, respectively.

Developing osteoblasts as an endpoint for the mouse embryonic stem cell test.

The mouse Embryonic Stem cell Test (EST) using cardiomyocyte differentiation is a promising in vitro assay for detecting potential embryotoxicity; however, the addition of another differentiation endpoint, such as osteoblasts, may improve the predictive value of the test. A number of variables such as culture conditions and starting cell number were investigated. A 14 day direct plating method of D3 mouse embryonic stem cells (mESCs) was used to test the predictivity of osteoblast differentiation as an endpoint in the EST.

Effects of silver nanoparticles on human and rat embryonic neural stem cells.

Silver nano-particles (Ag-NPs) are becoming increasingly prevalent in consumer products as antibacterial agents. The increased use of Ag NP-enhanced products will almost certainly increase environmental silver levels, resulting in increased exposures and the potential for increased adverse reactions including neurotoxic effects. In the present study, embryonic neural stem cells (NSCs) from human and rat fetuses (gestational day-16) were used to determine whether Ag-NPs are capable of causing developmental neurotoxicity.

Effects of maternal and lactational exposure to 2-hydroxy-4-methoxybenzone on development and reproductive organs in male and female rat offspring.

Background: 2-Hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet (UV) absorbing compound used in many cosmetic products as a UV-protecting agent and in plastics for preventing UV-induced photodecomposition. HMB has been detected in over 95% of randomly collected human urine samples from adults and from premature infants, and it may have estrogenic potential.

Methods: To determine the effects of maternal and lactational exposure to HMB on development and reproductive organs of offspring, time-mated female Harlan Sprague-Dawley rats were dosed with 0, 1000, 3000, 10,000, 25,000, or 50,000 ppm HMB (seven to eight per group) added to chow from gestation day 6 until weaning on postnatal day (PND) 23.

Cigarette smoke-induced cell cycle arrest in spermatocytes [GC-2spd(ts)] is mediated through crosstalk between Ahr-Nrf2 pathway and MAPK signaling.

Our earlier studies have demonstrated that the cigarette smoke in the form of cigarette smoke condensate (CSC) causes growth arrest of a mouse spermatocyte cell line [GC-2spd(ts)] through activation of the AHR-NRF2 pathway. The present study demonstrates the CSC-activated p38 and ERK MAPK signaling in GC-2spd(ts) via arylhydrocarbon receptor (AHR). Pharmacological inhibition by using AHR-antagonist, or p38 MAPK and ERK (MEK1) inhibitors significantly abrogates CSC-induced growth arrest by AHR and MAPK inactivation.

Modulation of cell cycle progression in the spermatocyte cell line [GC-2spd(ts) Cell-Line] by cigarette smoke condensate (CSC) via arylhydrocarbon receptor-nuclear factor erythroid 2-related factor 2 (Ahr-Nrf2) pathway.

Prior studies in our laboratory have demonstrated that cigarette smoke condensate (CSC) activates arylhydrocarbon receptor (Ahr) leading to upregulation of several antioxidant enzymes in murine spermatocytes. In this study, we show that exposure of the spermatocyte cell line GC-2spd(ts) to CSC induces an increase in Cyp1a1, demonstrating AHR activation, and simultaneous expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2), where it is believed to modulate Ahr expression by a feedback mechanism. Pharmacological inhibition by the AHR-antagonist CH223191 and interference by Ahr- and Nrf2-small interfering RNA followed by quantitative real-time PCR implicate the Ahr-Nrf2 pathway in the modulation of DNA damage and growth suppression genes such as Gadd45a and P21 and oxidative stress-related genes Cyp1a1, Nrf2, and Ahrr.

The aryl hydrocarbon receptor is important for proper seminiferous tubule architecture and sperm development in mice.

The aryl hydrocarbon receptor (AHR) is known for its roles in xenobiotic metabolism and essential physiologic processes such as cell growth, death, and differentiation. AHR is also an important regulator of male reproductive processes. However, no studies have characterized the consequences of loss of AHR in spermatogenesis.

Cardiovascular toxicity of Citrus aurantium in exercised rats.

When safety concerns forced the removal of ephedra from the market, other botanicals, including Citrus aurantium or bitter orange (BO) were used as replacements. A major component of the BO extract is synephrine, a chemical that is structurally similar to ephedrine. Because ephedrine has cardiovascular effects that may be exacerbated during physical exercise, the purpose of this study was to determine whether extracts containing synephrine produced adverse effects on the cardiovascular system in exercising rats.

Cigarette smoke condensate induces aryl hydrocarbon receptor-dependent changes in gene expression in spermatocytes.

Cigarette smoke contains numerous compounds that cause oxidative stress and alter gene expression in many tissues, and cigarette smoking is correlated with male infertility. To identify mechanisms by which this occurs, we evaluated expression of antioxidant genes in mouse spermatocytes in response to cigarette smoke condensate (CSC). CSC exposure led to oxidative stress and dose-dependent up-regulation of Hsp90aa1, Ahr, Arnt, Sod1, Sod2, and Cyp1a1 expression in a mouse spermatocyte cell line.

Molecular analysis of cell type-specific gene expression profile during mouse spermatogenesis by laser microdissection and qRT-PCR.

Laser microdissection (LMD) is a selective cell isolation technique that enables the separation of desired homogenous cell subpopulations from complex tissues such as the testes under direct microscopic visualization. The LMD accompanied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) represents an indispensable tool in quantifying messenger RNA (mRNA) expression among defined cell populations. Gene expression is temporally and spatially regulated at 3 sequential phases of mitotic, meiotic, and postmeiotic stages of spermatogenesis.

Physiological effects following administration of Citrus aurantium for 28 days in rats.

Background: Since ephedra-containing dietary supplements were banned from the US market, manufacturers changed their formulations by eliminating ephedra and replacing with other botanicals, including Citrus aurantium, or bitter orange. Bitter orange contains, among other compounds, synephrine, a chemical that is chemically similar to ephedrine. Since ephedrine may have cardiovascular effects, the goal of this study was to investigate the cardiovascular effects of various doses of bitter orange extract and pure synephrine in rats.

Alternative models in developmental toxicology.

In light of various pressures, toxicologists have been searching for alternative methods for safety testing of chemicals. According to a recent policy in the European Union (Regulation, Evaluation Authorisation and Restriction of Chemicals, REACH), it has been estimated that over the next twelve to fifteen years, approximately 30,000 chemicals may need to be tested for safety, and under current guidelines such testing would require the use of approximately 7.2 million laboratory animals [ Hofer et al.

Developmental toxicology: new directions workshop: refining testing strategies and study designs.

In April 2009, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute's (HESI) Developmental and Reproductive Toxicology Technical Committee held a two-day workshop entitled "Developmental Toxicology-New Directions." The third session of the workshop focused on ways to refine animal studies to improve relevance and predictivity for human risk. The session included five presentations on: (1) considerations for refining developmental toxicology testing and data interpretation; (2) comparative embryology and considerations in study design and interpretation; (3) pharmacokinetic considerations in study design; (4) utility of genetically modified models for understanding mode-of-action; and (5) special considerations in reproductive testing for biologics.

Incrimination of heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) as a candidate sensor of physiological folate deficiency.

The mechanism underlying the sensing of varying degrees of physiological folate deficiency, prior to adaptive optimization of cellular folate uptake through the translational up-regulation of folate receptors (FR) is unclear. Because homocysteine, which accumulates intracellularly during folate deficiency, stimulated interactions between heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) and an 18-base FR-α mRNA cis-element that led to increased FR biosynthesis and net up-regulation of FR at cell surfaces, hnRNP-E1 was a plausible candidate sensor of folate deficiency. Accordingly, using purified components, we evaluated the physiological basis whereby L-homocysteine triggered these RNA-protein interactions to stimulate FR biosynthesis.

Assessment of research models for testing gene-environment interactions.

Throughout the last century, possible effects of exposure to toxicants, nutrients or drugs were examined primarily by studies of groups or populations. Individual variation in responses was acknowledged but could not be analyzed due to lack of information or tools to analyze individual genetic make-ups and lifestyle factors such as diet and activity. The Human Genome, Haplotype Map, 1000Genomes, and Human Variome Projects are identifying and cataloging the variation found within humans.

Developmental toxicity of Citrus aurantium in rats.

Background: Ephedra was commonly used in herbal products marketed for weight loss until safety concerns forced its removal from products. Even before the ban, manufacturers had begun to replace ephedra with other compounds, including Citrus aurantium, or bitter orange. The major component in the bitter orange extract is synephrine which is chemically similar to ephedrine.

Study of valproic acid-induced endogenous and exogenous metabolite alterations using LC-MS-based metabolomics.

Background: Valproic acid (VPA; an anticonvulsant drug) therapy is associated with hepatotoxicity as well as renal toxicity. An LC-MS-based metabolomics approach was undertaken in order to detect urinary VPA metabolites and to discover early biomarkers of the adverse effects induced by VPA.

Results: CD-1 mice were either subcutaneously injected with 600-mg VPA/kg body weight or vehicle only, and urine samples were collected at 6, 12, 24 and 48 h postinjection.