Male-transmitted transgenerational effects of the herbicide linuron on DNA methylation profiles in Xenopus tropicalis brain and testis.

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic (igfbp4) and thyrotropic signaling (dio1 and tg) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the transgenerational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development (piwil1, spo11 and tdrd9) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation (dnmt3a and mbd2) and histone acetylation (hdac8, ep300, elp3, kat5 and kat14), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians.

Impaired spermatogenesis and associated endocrine effects of azole fungicides in peripubertal Xenopus tropicalis.

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3ß-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.

Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis.

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.

Combining elevated temperature with waterborne copper: Impacts on the energy metabolism of the killifish Poecilia vivipara.

We have previously demonstrated in a companion work that acclimation to 28 °C potentiated waterborne copper (Cu) toxic effects in Poecilia vivipara through oxidative stress-related processes. In the present study, we hypothesized that these results were related to kinetic metabolic adjustments in enzymes from aerobic and anaerobic pathways. To test this, P. vivipara was acclimated to two temperatures (22 °C or 28 °C) for three weeks and then exposed to Cu (control, 9 or 20 µg/L) for 96 h. The activity of enzymes from glycolysis (pyruvate kinase [PK] and lactate dehydrogenase [LDH]), Krebs cycle (citrate synthase [CS]) and the electron transport chain system (ETS) were assessed in gills, liver and muscle. Interactive effects were only seen for hepatic LDH activity, as both metal exposure and heat stress, combined or not, inhibited this enzyme, showing a suppression in anaerobic pathways. Conversely, a Cu main effect was present in the liver, expressed as an elevation in ETS activity, showing an enhancement in hepatic aerobic metabolism likely related with the very energy-demanding process of metal detoxification. Moreover, this study shows that P. vivipara has a remarkable ability to compensate heat stress in terms of energy metabolism, as we could not observe acclimation temperature effects for most of the cases. Nonetheless, a tissue-dependent effect of elevated temperature was observed, as we could observe an inhibition in muscular CS activity. Finally, it is concluded that kinetic adjustments in terms of the energy metabolism are not related with the temperature-dependent elevation of Cu toxicity in P. vivipara as we previously hypothesized.

Waterborne copper is more toxic to the killifish Poecilia vivipara in elevated temperatures: Linking oxidative stress in the liver with reduced organismal thermal performance.

In this study, we measured the interactive effect of temperature (22 °C and 28 °C) and waterborne copper (Cu) contamination (9 µg/L and 20 µg/L) on the killifish Poecilia vivipara. Endpoints analyzed included parameters involved in Cu-accumulation, antioxidant capacity (antioxidant capacity against peroxyl radicals [ACAP] and total antioxidant capacity [TAC]), oxidative damage (lipid peroxidation [LPO]) and upper thermal tolerance (critical thermal maximum [CTMax]). Results show that Cu hepatic accumulation was elevated in 28 °C in comparison to 22 °C in both exposure groups. For gills, this was true only in 20 µg/L. Moreover, hepatic and brachial accumulation were concentration-dependent in both acclimation temperatures. Additionally, Hepatic ACAP and TAC were elevated in animals acclimated to 28 °C and only the animals kept at this temperature had reduced ACAP and TAC levels facing metal exposure (9 and 20 µg/L). Similarly, the combination of elevated temperature and Cu exposure raised hepatic LPO levels. Finally, animals acclimated to 28 °C had higher CTMax levels in comparison to fish acclimated to 22 °C both in control and exposed animals, however, CTMax of contaminated fish were only reduced in comparison to control in animals kept at 28 °C. Concluding, we show that the physiological mechanism besides the potentiating effect of elevated temperature in Cu toxicity is related to higher hepatic and branchial metal accumulation and elevated oxidative stress in the liver, outlined by reduced antioxidant capacity and elevated oxidative damage. We also show that these outcomes lead to compromised organismal performance, characterized by reduced CTMax. Finally, it is concluded that Cu exposure in warmer periods of the year or within global warming predictions may be more hazardous to fish populations.

A glyphosate-based herbicide reduces fertility, embryonic upper thermal tolerance and alters embryonic diapause of the threatened annual fish Austrolebias nigrofasciatus.

Roundup is the most popular glyphosate-based herbicide (GBH) worldwide. These formulations kill a wide range of plants. Despite that, non-target species can be jeopardized by GBH, such as the annual fish Austrolebias nigrofasciatus. This species occurs in wetlands that dries annually. Key-adaptations permit them to live in such harsh habitats, e. i. Elevated fertility, drought-tolerant diapausing embryos and elevated thermal tolerance. We aimed to evaluate acute (96 h) effects of Roundup exposure (0.36 or 3.62 mg a. e./L) in reproduction, diapause pattern and embryonic upper thermal tolerance (EUTT) of A. nigrofasciatus. For such, we evaluated the number and diameter of embryos produced by exposed fish. Also, recently fertilized embryos were exposed and its diapause pattern was evaluated. Following 15 post exposure days (PED), we evaluated the number of somite pairs and following 30, 35 and 40 PED we evaluated the proportion of pigmented embryos (PPE). Finally, the critical thermal maximum (CTMax) of exposed embryos was assessed. Results demonstrated that couples exposed to 0.36 mg a. e./L Roundup produced less but larger embryos. Similarly, embryos exposed to 3.62 mg a. e./L Roundup had a reduced PPE following 30 PED. Finally, embryos exposed to 0.32 mg a. e./L Roundup had a CTMax reduction of 2.6 °C and were more sensitive to minor increases in heating rates. These results indicate that Roundup have negative outcomes in fish reproduction, embryonic development and EUTT. This information is of particular interest to the conservation of annual fish, considering that those are key-adaptations that permit these animals to survive the harsh impositions of ephemeral wetlands.