DNA methylome signatures of prenatal exposure to synthetic glucocorticoids in hippocampus and peripheral whole blood of female guinea pigs in early life.

Synthetic glucocorticoids (sGC) are administered to women at risk of preterm delivery, approximately 10% of all pregnancies. In animal models, offspring exposed to elevated glucocorticoids, either by administration of sGC or endogenous glucocorticoids as a result of maternal stress, show increased risk of developing behavioral, endocrine, and metabolic dysregulation. DNA methylation may play a critical role in long-lasting programming of gene regulation underlying these phenotypes. However, peripheral tissues such as blood are often the only accessible source of DNA for epigenetic analyses in humans. Here, we examined the hypothesis that prenatal sGC administration alters DNA methylation signatures in guinea pig offspring hippocampus and whole blood. We compared these signatures across the two tissue types to assess epigenetic biomarkers of common molecular pathways affected by sGC exposure. Guinea pigs were treated with sGC or saline in late gestation. Genome-wide modifications of DNA methylation were analyzed at single nucleotide resolution using reduced representation bisulfite sequencing in juvenile female offspring. Results indicate that there are tissue-specific as well as common methylation signatures of prenatal sGC exposure. Over 90% of the common methylation signatures associated with sGC exposure showed the same directionality of change in methylation. Among differentially methylated genes, 134 were modified in both hippocampus and blood, of which 61 showed methylation changes at identical CpG sites. Gene pathway analyses indicated that prenatal sGC exposure alters the methylation status of gene clusters involved in brain development. These data indicate concordance across tissues of epigenetic programming in response to alterations in glucocorticoid signaling.

Chronic exposure of Brown (Hydra oligactis) and green Hydra (Hydra viridissima) to environmentally relevant concentrations of pharmaceuticals.

Low concentrations of pharmaceuticals in the environment (ng/L to µg/L) are an environmental concern. We used the invertebrates, Hydra oligactis and Hydra viridissima, as freshwater models for primary toxicity testing to study effects of chronic low concentrations of pharmaceuticals in the environment. H. oligactis were exposed to three concentrations (0.1, 1.0 and 10 µg/L) of either fluoxetine, carbamazepine, or triclosan; H. viridissima were exposed to three concentrations (0.1, 1.0 and 10 µg/L) of triclosan. Ecologically relevant endpoints including morphology, budding rate, feeding behaviour, and regenerative capacity were examined during the 14 days exposure period. The interstitial:epithelial stem cell ratios was also examined in H. oligactis. There were no significant effects on the morphology, budding rate and feeding behaviour of the H. oligactis across all concentrations of fluoxetine, carbamazepine, and triclosan. However, regenerative capacity significantly decreased in comparison to the controls when H. oligactis was exposed to 10 µg/L of triclosan and fluoxetine, although there was no significant difference when exposed to carbamazepine. Neither fluoxetine nor carbamazepine treatment altered stem cell ratios. Exposure to triclosan at any concentration did not impact H. viridissima morphology, budding rate, regeneration or feeding behaviour. These results show there are limited effects in Hydra after exposure to chronic, low concentrations of fluoxetine, carbamazepine, and triclosan, except for regeneration in H. oligactis. These endpoints can be used effectively (and cost effectively) to study the effects of environmentally relevant concentrations of pharmaceuticals in Hydra species.

Paternal Exposure to Carbamazepine Impacts Zebrafish Offspring Reproduction Over Multiple Generations.

Chronic low-concentration chemical exposures may have both direct health outcomes on adults and indirect effects on their offspring. Using zebrafish, we examined the impacts of chronic, low-concentration carbamazepine (CBZ) exposure on a suite of male reproductive endpoints in the parents and four generations of offspring reared in clean water. CBZ is one of the most frequently detected pharmaceutical residues in water, is a histone deacetylase inhibitor in mammals, and is reported to lower androgens in mammals and fish. Exposure of adult zebrafish to 10 µg/L CBZ for 6 weeks decreased reproductive output, courtship and aggressive behaviors, 11-ketotestosterone (11KT), and sperm morphology but did not impact milt volume or sperm swimming speed. Pairwise breeding generated lineages of offspring with both parents exposed and two lineages where only one parent was exposed; the control lineage had unexposed parents. Reproductive output and male reproductive indices were assessed in F1-F4 offspring to determine whether parental CBZ exposure had transgenerational impacts. The offspring of CBZ-exposed males had lower 11KT, reproductive output, altered courtship, aggression, and sperm morphology compared to the lineage from unexposed parents. Our results indicate that parental carbamazepine exposure history impacts the unexposed progeny up to the F4 generations and that paternal, but not maternal, exposure is most important for the reproductive health of male offspring.

Parental gemfibrozil exposure impacts zebrafish F1 offspring, but not subsequent generations.

Gemfibrozil (GEM) is a fibrate lipid regulator and one of the most commonly occurring fresh water pharmaceuticals. The negative effects of fibrates including GEM on fish reproduction have been frequently reported including effects of F0 GEM exposure on reproduction of the unexposed F1 offspring. We predicted that chronic, direct exposure of zebrafish with low concentrations of GEM would adversely affect parental male reproduction and unexposed offspring for multiple generations. Adult zebrafish were exposed to 10 µg/L GEM for 6 weeks and a range of reproductive indices were analyzed. The F1-F4 offspring were reared in clean water from 3 distinct lineages where only a single or both parents were exposed and compared to a control lineage where parents were unexposed. Reproductive indices were examined in unexposed F1-F4 offspring to test the hypothesis of multi- or trans- generational impacts. Exposure to GEM caused a decline in breeding success and mean embryo production in F0 parents and a reduction in whole body 11-ketotestosterone (11-KT), altered male courtship, aggression and sperm morphology. Our results indicate that paternal exposure alone is sufficient to result in reproductive effects in unexposed male offspring but that effects are mostly limited to F1. We suggest that GEM may act as a reproductive endocrine disruptor in fish and that chronic exposure reduced male reproductive fitness but not over multiple generations.

Cardiovascular and growth outcomes of C57Bl/6J mice offspring exposed to maternal stress and ionizing radiation during pregnancy.

Purpose: Developmental programming involves an adverse intrauterine environment which can result in offspring phenotype changes following birth. The developmental programming of hypertension has been reported to possibly involve oxidative stress at the cellular level. Ionizing radiation produces oxidative stress, even at low doses, and irradiation of animals is often coupled with potential sources of maternal stress such as transportation of animals or repeated handling. Materials and methods: Pregnant C57Bl/6J mice were irradiated on gestational day 15 with 5-1000 mGy 137Cs gamma radiation. Post-natal weight, blood pressure (BP) and heart rate (HR) were measured. Radiation had minimal effects at doses ≤300 mGy, but 1000 mGy caused a significant reduction in HR in male pups and growth reduction at 16 weeks of age in both genders. The sham-irradiation protocol included repeated transportation in order to acclimate animals to transport. However, it may have resulted in programming, as sham-irradiation alone resulted in elevated BP measures compared to the offspring of animals that were never transported. Results and conclusions: Overall, there were minimal effects on cardiovascular measures or offspring weight due to irradiation except at 1000 mGy. The presence of maternal stress, a known trigger of developmental programming, may have confounded any potential irradiation effects.

Daily, repeating fluctuations in embryonic incubation temperature alter metabolism and growth of Lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis) utilize overwintering embryonic development (up to 180 days), and such stenothermic, cold-water embryos may be particularly susceptible to thermal shifts. We incubated whitefish embryos in temperature treatments that were constant temperature (2.0 ±â€¯0.1 °C, 5.0 ±â€¯0.1 °C, and 8.0 ±â€¯0.1 °C; mean ±â€¯SD) or variable temperature (VT, mean = 5.0 ±â€¯0.3 °C). In the VT, a daily 2 °C temperature change followed a continuous pattern throughout development: 2-4-6-8-6-4-2 °C. Hatchling survival proportion from fertilization to hatch was significantly impacted by incubation temperature (P < 0.001): 2 °C (0.88 ±â€¯0.01) and 5 °C (0.91 ±â€¯0.01) showed higher survival than both the VT (0.83 ±â€¯0.02) and 8 °C groups (0.15 ±â€¯0.06), which were statistically distinct from each other. Time to hatch (dpf) was significantly different across all treatments (P < 0.001): 8 °C (68 ±â€¯2 dpf), VT (111 ±â€¯4 dpf), 5 °C (116 ±â€¯4 dpf), 2 °C (170 ±â€¯3 dpf). Likewise, hatchling yolk-free dry mass (mg) and total body length (mm) were significantly different across all treatments (P < 0.001): 8 °C (0.66 ±â€¯0.08 mg; 11.1 ±â€¯0.08 mm), VT (0.97 ±â€¯0.06 mg; 11.7 ±â€¯0.05 mm), 5 °C (1.07 ±â€¯0.03 mg; 12.0 ±â€¯0.02 mm), 2 °C (1.36 ±â€¯0.04 mg; 12.8 ±â€¯0.05 mm). Oxygen consumption rate (V̇o2) was significantly affected by the interaction between treatment and measurement temperature (P < 0.001). Hatchling VT whitefish showed mean V̇o2 that was higher compared to the 2 °C group measured at 2 °C, and lower compared to the 2 °C and 5 °C group measured at 8 °C. This study demonstrates that the VT incubation treatment produced fewer (increased mortality), smaller embryos that hatched earlier than 2 °C and 5 °C embryos. The plasticity of V̇o2 for this stenothermic-incubating fish species under variable incubation conditions reveals a metabolic cost to cycling thermal incubation conditions.

Gemfibrozil and carbamazepine decrease steroid production in zebrafish testes (Danio rerio).

Gemfibrozil (GEM) and carbamazepine (CBZ) are two environmentally relevant pharmaceuticals and chronic exposure of fish to these compounds has decreased androgen levels and fish reproduction in laboratory studies. The main focus of this study was to examine the effects of GEM and CBZ on testicular steroid production, using zebrafish as a model species. Chronic water borne exposures of adult zebrafish to 10 µg/L of GEM and CBZ were conducted and the dosing was confirmed by chemical analysis of water as 17.5 ±â€¯1.78 and 11.2 ±â€¯1.08 µg/L respectively. A 67 day exposure led to reduced reproductive output and lowered whole body, plasma, and testicular 11-ketotestosterone (11-KT). Testicular production of 11-KT was examined post exposure (42 days) using ex vivo cultures to determine basal and stimulated steroid production. The goal was to ascertain the step impaired in the steroidogenic pathway by each compound. Ex vivo 11-KT production in testes from males chronically exposed to GEM and CBZ was lower than that from unexposed males. Although hCG, 25-OH cholesterol, and pregnenolone stimulation increased 11-KT production in all treatment groups over basal levels, hCG stimulated 11-KT production remained significantly less in testes from exposed males compared to controls. 25-OH cholesterol and pregnenolone stimulated 11-KT production was similar between GEM and control groups but the CBZ group had lower 11-KT production than controls with both stimulants. We therefore propose that chronic GEM and CBZ exposure can reduce production of 11-KT in testes through direct effects independent of mediation through HPG axis. The biochemical processes for steroid production appear un-impacted by GEM exposure; while CBZ exposure may influence steroidogenic enzyme expression or function.

The effects of parental carbamazepine and gemfibrozil exposure on sexual differentiation in zebrafish (Danio rerio).

The effects of parental exposure to pharmaceuticals on sexual differentiation in F1 offspring were examined in zebrafish (Danio rerio). Adult zebrafish were exposed to 0 or 10 µg/L of carbamazepine or gemfibrozil for 6 wk and bred in pairwise crosses to generate 7 distinct lineages. Lineages were formed with both parents from the same treatment group or with only one parent exposed, to delineate between maternal and paternal effects. The F1 offspring from each lineage were reared in clean water and sampled at 45 and 60 d post fertilization (dpf). Gonadal differentiation was assessed by histology. The morphological stages of the gonads were converted to a quantitative day-equivalent based on data from offspring of untreated parents sampled from 15 to 75 dpf, which enabled a quantitative statistical analysis on the timing of sexual differentiation. Paternal, but not maternal, exposure to carbamazepine resulted in significantly faster sexual differentiation and a male-biased sex ratio; these effects were not observed when both parents were exposed. Combined paternal and maternal exposure to gemfibrozil resulted in significantly faster sexual differentiation, and paternal, but not maternal, exposure to gemfibrozil led to male-biased sex ratios. The present study demonstrates the ability of parental exposure to pharmaceuticals to disrupt sexual differentiation in the F1 offspring and also shows that effects may be uniquely influenced by which parent was exposed. Environ Toxicol Chem 2018;37:1696-1706. © 2018 SETAC.

The effects of increased constant incubation temperature and cumulative acute heat shock exposures on morphology and survival of Lake Whitefish (Coregonus clupeaformis) embryos.

Increasing incubation temperatures, caused by global climate change or thermal effluent from industrial processes, may influence embryonic development of fish. This study investigates the cumulative effects of increased incubation temperature and repeated heat shocks on developing Lake Whitefish (Coregonus clupeaformis) embryos. We studied the effects of three constant incubation temperatures (2°C, 5°C or 8°C water) and weekly, 1-h heat shocks (+3°C) on hatching time, survival and morphology of embryos, as these endpoints may be particularly susceptible to temperature changes. The constant temperatures represent the predicted magnitude of elevated water temperatures from climate change and industrial thermal plumes. Time to the pre-hatch stage decreased as constant incubation temperature increased (148d at 2°C, 92d at 5°C, 50d at 8°C), but weekly heat shocks did not affect time to hatch. Mean survival rates and embryo morphometrics were compared at specific developmental time-points (blastopore, eyed, fin flutter and pre-hatch) across all treatments. Constant incubation temperatures or +3°C heat-shock exposures did not significantly alter cumulative survival percentage (~50% cumulative survival to pre-hatch stage). Constant warm incubation temperatures did result in differences in morphology in pre-hatch stage embryos. 8°C and 5°C embryos were significantly smaller and had larger yolks than 2°C embryos, but heat-shocked embryos did not differ from their respective constant temperature treatment groups. Elevated incubation temperatures may adversely alter Lake Whitefish embryo size at hatch, but weekly 1-h heat shocks did not affect size or survival at hatch. These results suggest that intermittent bouts of warm water effluent (e.g., variable industrial emissions) are less likely to negatively affect Lake Whitefish embryonic development than warmer constant incubation temperatures that may occur due to climate change.