Transcriptomic profile in carbendazim-induced developmental defects in zebrafish (Danio rerio) embryos/larvae.

Carbendazim is a widely used fungicide to protect agricultural and horticultural crops against a wide array of fungal species. Published reports have shown that the wide usage of carbendazim resulted in reprotoxicity, carcinogenicity, immunotoxicity, and developmental toxicity in mammalian models. However, studies related to the developmental toxicity of carbendazim in aquatic organisms are not clear. To address this gap, an attempt was made by exposing zebrafish embryos to carbendazim (800 µg/L) and assessing the phenotypic and transcriptomic profile at different developmental stages [24 hour post fertilization (hpf), 48 hpf, 72 hpf and 96 hpf). At 48 hpf, phenotypic abnormalities such as delay in hatching rate, deformed spinal axial curvature, and pericardial edema were observed in zebrafish larvae over its respective controls. At 72 hpf, exposure of zebrafish embryos exposed to carbendazim resulted in scoliosis; however, unexposed larvae did not exhibit signs of scoliosis. Interestingly, the transcriptomic analysis revealed a total of 1253 DEGs were observed at selected time points, while unique genes at 24 hpf, 48 hpf, 72 hpf and 96 hpf was found to be 76.54 %, 61.14 %, 92.98 %, and 68.28 %, respectively. Functional profiling of downregulated genes revealed altered transcriptomic markers associated with phototransduction (24 hpf and 72 hpf), immune system (48 hpf), and SNARE interactions in the vesicular pathway (96 hpf). Whereas functional profiling of upregulated genes revealed altered transcriptomic markers associated with riboflavin metabolism (24 hpf), basal transcription factors (48 hpf), insulin signaling pathway (72 hpf), and primary bile acid biosynthesis (96 hpf). Taken together, carbendazim-induced developmental toxicity could be ascribed to pleiotropic responses at the molecular level, which in turn might reflect phenotypic abnormalities.

Antioxidant effects of α-lipoic acid against epididymal oxidative damage in adult offspring rats exposed to maternal hypothyroidism stress.

It is well known that the epididymis promotes post-testicular sperm maturation events. However, its malfunction during congenital hypothyroidism is relatively less understood as compared to the testis. The present study evaluated the probable effect of α-lipoic acid on epididymal oxidative stress parameters in rats exposed to antithyroid drug, carbimazole during fetal period. Time-mated pregnant rats in unexposed and carbimazole (1.35 mg/Kg body weight exposed were allowed to deliver pups and weaned. At postnatal day 100, the F1 male pups were assessed for epididymal endpoints. Among the epididymal regions, significant elevation of lipid peroxidation levels, superoxide anion, and hydrogen peroxide contents with a concomitant reduction in the activity levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and reduced glutathione levels were observed in cauda epididymis of carbimazole exposed rats over controls. Significant elevation in sperm DNA fragmentation (comet assay), accelerated cauda epididymal sperm transit time and reduction in epididymal sialic acid content was observed in carbimazole exposed rats. RT-qPCR studies revealed that embryonic exposure to carbimazole resulted in down regulation of androgen receptor, nuclear factor eryrthoid 2 like 2, 5α-reducatse 1 mRNA levels, while up regulation of caspase 3 mRNA was observed in epididymal regions of rats. In addition, fetal exposure to carbimazole resulted in disorganization of cauda epididymal architecture in rats. Conversely, supplementation of α-lipoic acid (70 mg/Kg bodyweight) during PND 3 to 14 restored epididymal functions in carbimazole exposed rats and the ameliorative effects of lipoic acid could be attributed to its antioxidant and steroidogenic effects.

Transcriptomic profiling identified altered expression of genes associated with testicular functions in adult F1 rats exposed to carbimazole during fetal period.

The central goal of this study was to investigate the alterations in transcriptome of testis in F1 generation adult rats exposed to carbimazole prenatally. At post-natal day 100, the testis of rats delivered to carbimazole exposed (time-mated pregnant rats orally administered with carbimazole from gestation day 9 to 21) and control (untreated pregnant rats) groups were subjected to transcriptomic analysis using NGS platform. A total of 187 differentially expressed (up regulated: 49 genes; down regulated: 138) genes were identified in carbimazole exposed rats over controls and the major processes associated with these altered testicular transcripts were examined. Functional clustering analysis suggest that the involvement of identified DEGs were linked to intrinsic and extrinsic apoptotic pathways, mitochondrial solute carriers slc25a members, nuclear receptors/zinc family members, steroidogenic pathway and cholesterol synthesis, and growth factors and protein kinases and thus represent potential mediators of the developmental toxic effects of carbimazole in F1 generation rats. Based on the findings, it can be concluded that prenatal exposure to carbimazole prominently affects expression of multiple transcripts implicating key regulatory events associated with testicular functions, spermatogenesis and steroidogenesis in rats at their adulthood. These results support our earlier findings and hypothesis. This background information obtained at the testicular transcriptome during gestational hypothyroidism might be helpful for future studies and experiments to gain additional in-depth analysis and to develop strategies to protect F1 generation male reproductive health. SIGNIFICANCE: The rationale for the paper described thyroid gland changes in the off springs. Antithyroid drugs are widely used to control thyroid disorders and used to control thyroid hormone levels during surgeries. Carbimazole is one of the antithyroid drugs and is a parent molecule of methimazole. Both the drugs can able to cross placenta. During fetal period, the development of thyroid gland is not completely formed and hence, the fetus entirely depends on the maternal thyroid hormones. Therefore, it is conceivable that the disturbances at the level of maternal thyroid hormones could interfere with the development of vital organs such as testis and glands including thyroid gland (Kala et al., 2012). To address this notion, the present study was designed and executed.

An ancestral nuclear receptor couple, PPAR-RXR, is exploited by organotins.

Environmental chemicals have been reported to greatly disturb the endocrine and metabolic systems of multiple animal species. A recent example involves the exploitation of the nuclear receptor (NR) heterodimeric pair composed by PPAR/RXR (peroxisome proliferator-activated receptor/retinoid X receptor), which shows lipid perturbation in mammalian species. While gene orthologues of both of these receptors have been described outside vertebrates, no functional characterization of PPAR has been carried in protostome lineages. We provide the first functional analysis of PPAR in Patella sp. (Mollusca), using model obesogens such as tributyltin (TBT), triphenyltin (TPT), and proposed natural ligands (fatty acid molecules). To gain further insights, we used site-directed mutagenesis to PPAR and replaced the tyrosine 277 by a cysteine (the human homologous amino acid and TBT anchor residue) and an alanine. Additionally, we explored the alterations in the fatty acid profiles after an exposure to the model obesogen TBT, in vivo. Our results show that TBT and TPT behave as an antagonist of Patella sp. PPAR/RXR and that the tyrosine 277 is important, but not essential in the response to TBT. Overall, these results suggest a relation between the response of the mollusc PPAR-RXR to TBT and the lipid profile alterations reported at environmentally relevant concentrations. Our findings highlight the importance of comparative analysis between protostome and deuterostome lineages to decipher the differential impact of environmental chemicals.

Phenotypic and transcriptomic changes in zebrafish (Danio rerio) embryos/larvae following cypermethrin exposure.

Cypermethrin is one of the widely used type-II pyrethroid and the indiscriminate use of this pesticide leads to life threatening effects and in particular showed developmental effects in sensitive populations such as children and pregnant woman. However, the molecular mechanisms underlying cypermethrin-induced development toxicity is not well defined. To address this gap, the present study was designed to investigate the phenotypic and transcriptomic (next generation RNA-Seq method) impact of cypermethrin in zebrafish embryos as a model system. Zebrafish embryos at two time points, 24 h postfertilization (hpf) and 48 hpf were exposed to cypermethrin at a concentration of 10 µg/L. Respective control groups were maintained. Cypermethrin induced both phenotypic and transcriptomic changes in zebrafish embryos at 48 hpf. The phenotypic anomalies such as delayed hatching rate, increased heartbeat rate and deformed axial spinal curvature in cypermethrin exposed zebrafish embryos at 48 hpf as compared to its respective controls. Transcriptomic analysis indicated that cypermethrin exposure altered genes associated with visual/eye development and gene functional profiling also revealed that cypermethrin stress over a period of 48 h disrupts phototransduction pathway in zebrafish embryos. Interestingly, cypermethrin exposure resulted in up regulation of only one gene, tnnt3b, fast muscle troponin isoform 3T in 24 hpf embryos as compared to its respective controls. The present model system, cypermethrin exposed zebrafish embryos elaborates the toxic consequences of cypermethrin exposure during developmental stages, especially in fishes. The present findings paves a way to understand the visual impairment in sensitive populations such as children exposed to cypermethrin during their embryonic period and further research is warranted.

The evolutionary road to invertebrate thyroid hormone signaling: Perspectives for endocrine disruption processes.

Thyroid hormones (THs) are the only iodine-containing hormones that play fundamental roles in chordates and non-chordates. The chemical nature, mode of action and the synthesis of THs are well established in mammals and other vertebrates. Although thyroid-like hormones have been detected in protostomes and non-chordate deuterostomes, TH signaling is poorly understood as compared to vertebrates, particularly in protostomes. Therefore, the central objective of this article is to review TH system components and TH-induced effects in non-vertebrate chordates, non-chordate deuterostomes and protostomes based on available genomes and functional information. To accomplish this task, we integrate here the available knowledge on the THs signaling across non-vertebrate chordates, non-chordate deuterostomes and protostomes by considering studies encompassing TH system components and physiological actions of THs. We also address the possible interactions of thyroid disrupting chemicals and their effects in protostomes and non-chordate deuterostomes. Finally, the perspectives on current and future challenges are discussed.

α-lipoic acid inhibits oxidative stress in testis and attenuates testicular toxicity in rats exposed to carbimazole during embryonic period.

The aim of this study was to evaluate the probable protective effect of α-lipoic acid against testicular toxicity in rats exposed to carbimazole during the embryonic period. Time-mated pregnant rats were exposed to carbimazole from the embryonic days 9-21. After completion of the gestation period, all the rats were allowed to deliver pups and weaned. At postnatal day 100, F1 male pups were assessed for the selected reproductive endpoints. Gestational exposure to carbimazole decreased the reproductive organ indices, testicular daily sperm count, epididymal sperm variables viz., sperm count, viable sperm, motile sperm and HOS-tail coiled sperms. Significant decrease in the activity levels of 3ß- and 17ß-hydroxysteroid dehydrogenases and expression of StAR mRNA levels with a significant increase in the total cholesterol levels were observed in the testis of experimental rats over the controls. These events were also accompanied by a significant reduction in the serum testosterone levels in CBZ exposed rats, indicating reduced steroidogenesis. In addition, the deterioration of the testicular architecture and reduced fertility ability were noticed in the carbimazole exposed rats. Significant reduction in the activity levels of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and reduced glutathione content with a significant increase in the levels of lipid peroxidation were observed in the testis of carbimazole exposed rats over the controls. Conversely, supplementation of α-lipoic acid (70 mg/Kg bodyweight) ameliorated the male reproductive health in rats exposed to carbimazole during the embryonic period as evidenced by enhanced reproductive organ weights, selected sperm variables, testicular steroidogenesis, and testicular enzymatic and non-enzymatic antioxidants. To conclude, diminished testicular antioxidant balance associated with reduced spermatogenesis and steroidogenesis might be responsible for the suppressed reproduction in rats exposed to the carbimazole transplacentally. On the other hand, α-lipoic acid through its antioxidant and steroidogenic properties mitigated testicular toxicity which eventually restored the male reproductive health of carbimazole-exposed rats.

Mode of action of dopamine in inducing hyperglycemia in the fresh water edible crab, Oziothelphusa senex senex.

The objective of this study was to investigate the mode of action of dopamine in regulating hemolymph sugar level in the fresh water edible crab, Oziothelphusa senex senex. Injection of dopamine produced hyperglycemia in a dose-dependent manner in intact crabs but not in eyestalkless crabs. Administration of dopamine resulted in a significant decrease in total carbohydrates and glycogen levels with a significant increase in glycogen phosphorylase activity levels in hepatopancreas and muscle of intact crabs, indicating dopamine-induced glycogenolysis resulting in hyperglycemia. Bilateral eyestalk ablation resulted in significant increase in the total carbohydrates and glycogen levels with a significant decrease in the activity levels of phosphorylase in the hepatopancreas and muscle of the crabs. Eyestalk ablation resulted in significant decrease in hemolymph hyperglycemic hormone levels. The levels of hyperglycemic hormone in the hemolymph of dopamine injected crabs were significantly higher than in control crabs. However, no significant changes in the levels of hemolymph hyperglycemic hormone and sugar and tissue carbohydrate and phosphorylase activity were observed in dopamine injected eyestalk ablated crabs when compared with eyestalk ablated crabs. These results support an earlier hypothesis in crustaceans that dopamine acts as a neurotransmitter and induces hyperglycemia by triggering the release of hyperglycemic hormone in the crab, O. senex senex.

What do we (need to) know about the melatonin in crustaceans?

Melatonin (N-acetyl-5-methoxy-tryptamine) was first discovered from the bovine pineal gland extract in 1958. Since then, its synthesis, metabolism, physiological, and patho-physiological functions are well studied in vertebrates; there is an increasing recognition of melatonin in invertebrates and especially in crustaceans. The presence of melatonin in crustaceans is now well documented and some functional aspects in the framework of crustacean biology have been demonstrated. This review aims at giving a comprehensive overview of the various physiological events regulated by this pleiotropic hormone. Topics include: glucose homeostasis, regulation of reproduction, molting, limb regeneration, and antioxidant properties. Finally, perspectives on current and possible research are offered.

Protective effects of N-acetylcysteine against arsenic-induced oxidative stress and reprotoxicity in male mice.

Arsenic is a well-known environmental toxic metalloid element and carcinogen that affects multiple organ systems including tissue lipid peroxidation and reproduction. The present study was aimed to investigate the protective role of N-acetylcysteine (NAC) on arsenic-induced testicular oxidative damage and antioxidant and steroidogeneic enzymes and sperm parameters in mice. Arsenic was administered through drinking water to mice at a concentration of 4.0 ppm sodium arsenite (actual concentration 2.3 ppm arsenic) for 35 days. The body weight of treated mice did not show significant change as compared with the control mice. In arsenic exposed mice there was a significant decrease in the weight of the testis, epididymis and prostate gland as compared with the control animals. Significant reduction was observed in epididymal sperm count, motile sperms and viable sperms in mice exposed to arsenic indicate decreased spermatogenesis and poor sperm quality. The activity levels of testicular 3ß- and 17ß-hydroxysteroid dehydrogenases and circulatory levels of testosterone were also decreased in arsenic treated mice indicating reduced steroidogenesis. A significant increase in the activities of lipid peroxidation and a significant decrease in the activities of antioxidant enzymes were observed in the testis of mice exposed to arsenic. In addition, significant increase in the testicular arsenic levels was observed during arsenic intoxication. No significant changes in the oxidation status and selected reproductive variables were observed in the N-acetylcysteine alone treated mice. Whereas, intra-peritoneal injection of NAC to arsenic exposed mice showed a significant increase in the weights of reproductive organs, reduction in arsenic-induced oxidative stress in the tissues and improvement in steroidogenesis over arsenic-exposed mice indicating the beneficial role of N-acetylcysteine to counteract arsenic-induced oxidative stress and to restore the suppressed reproduction in male mice.

Protective role of Centella asiatica on lead-induced oxidative stress and suppressed reproductive health in male rats.

Centella asiatica has been mentioned in ancient ayurvedic text of the Indian system of medicine for its properties to promote intelligence. The objective of the present study was to investigate the beneficial effects of C. asiatica on lead-induced oxidative stress and suppressed reproductive performance in male rats. Significant decrease in the weights of testes and epididymis were observed in lead treated animals. Exposure to lead acetate significantly increased malondialdehyde levels with a significant decrease in the superoxide dismutase and catalase activities in the liver, brain, kidneys and testes of rats. Epididymal sperm count, viable sperms, motile sperms and HOS-tail coiled sperms decreased significantly in lead-exposed rats. Testicular steroidogenic enzyme activities also decreased significantly in lead-exposed rats. No significant changes in the selected reproductive variables were observed in the plant extract alone treated rats. Whereas, co-administration of aqueous extracts of C. asiatica to lead exposed rats showed a significant increase in the weights of reproductive organs, reduction in lead-induced oxidative stress in the tissues and improvement in selected reproductive parameters over lead-exposed rats indicating the beneficial role of C. asiatica to counteract lead-induced oxidative stress and to restore the suppressed reproduction in male rats.

Embryonic exposure to octylphenol induces changes in testosterone levels and disrupts reproductive efficiency in rats at their adulthood.

The purpose of the present study was to investigate the effects of prenatal exposure to octylphenol (OP) at the dose of 50mg/kg body weight on days 1, 7 and 14 of pregnancy on reproductive health of male rats at adulthood. F1 male rats from control and OP exposed animals were weaned and maintained up to postnatal day (PND) 100. The indices of testis, epididymis and seminal vesicles were significantly decreased in male rats exposed to OP during embryonic development when compared with controls. Significant reduction in the epididymal sperm count, viable sperms and motile sperms and number of tail coiled sperms (HOS-test) were observed in experimental rats when compared to control rats. The levels of serum testosterone and also activity levels of testicular hydroxysteroid dehydrogenases were significantly decreased with a significant increase in the serum follicle stimulating and leutinizing hormones in experimental rats. Furthermore, embryonic exposure to OP caused significant down regulation of StAR, 3ß hydroxysteroid dehydrogenase and 17ß hydroxysteroid dehydrogenase mRNAs in testis of adult rats as compared to control rats. The results of fertility studies revealed that there was an increase in the mating index in experimental rats with an increase in the pre- and post-implantation losses in rats cohabited with treated animals indicating poor male reproductive performance.

Lead acetate induced reproductive and paternal mediated developmental toxicity in rats.

Lead was administered orally to adult male rats at exposure level of 273 or 819 mg/L (0.05% or 0.15% lead acetate, respectively) for 45 days via drinking water. At the end of the exposure period, control and experimental males were mated with untreated females. Of the females mated with treated males, 73.3% in the 0.05% group and 53.33% in the 0.15% group showed copulatory plugs. Significant decrease in number of implantations and pre- and post-implantation loss was also observed in females mated with treated males. Significant decrease in the weight of the reproductive organs, reduction in epididymal sperm count, motile sperm and viable sperm were observed in lead-exposed rats indicating decreased sperm production and deteriorated sperm quality. Significant decrease in serum testosterone levels were also observed in treated rats indicating decreased steroidogenesis. The decreased serum testosterone levels and deteriorated sperm quality might be responsible for the suppressed reproduction in rats after exposure to lead.

Cadmium and mercury-induced hyperglycemia in the fresh water crab, Oziotelphusa senex senex: involvement of neuroendocrine system.

The effect of exposure to sublethal concentrations of cadmium chloride and mercuric chloride on hemolymph glucose levels of the freshwater crab, Oziotelphusa senex senex, was studied. Intact crabs exposed to cadmium or mercury exhibited a significant hyperglycemia compared to controls, but no significant differences in hemolymph glucose level were detected among the eyestalkless crabs after exposure to metals, suggesting that the effect of metals could be on the sinus gland in the eyestalks, increasing secretion of the hyperglycemic hormone. To test this hypothesis, eyestalks were collected from control and metal exposed crabs, and tested for hyperglycemic effect and also for the hyperglycemic hormone levels. The levels of hyperglycemic hormone and the hyperglycemic effect were significantly low in the eyestalks collected from metal exposed crabs when compared with eyestalks from control crabs. These results strongly suggest that metals act, at least in part, by triggering the secretion of hyperglycemic hormone from the eyestalk.

Melatonergic regulation of hemolymph sugar levels in the freshwater edible crab, Oziotelphusa senex senex.

In this study, the hyperglycemic effect of melatonin in the freshwater edible crab, Oziotelphusa senex senex, is investigated. Injection of melatonin induced hyperglycemia in a dose-dependent manner. Administration of melatonin produced hyperglycemia in both intact and eyestalk-ablated crabs. Bilateral eyestalk ablation resulted in significant increase in the total carbohydrates and glycogen levels with a significant decrease in phosphorylase activity in the hepatopancreas and muscle of the crabs. Injection of melatonin resulted in significant decrease in the total carbohydrate and glycogen levels, with an increase in phosphorylase activity in hepatopancreas and muscle of both intact and eyestalk-ablated crabs. From the results, it is hypothesized that melatonin-induced hyperglycemia in the crab, O. senex senex, is not mediated by eyestalk hyperglycemic hormone.

Recovery of suppressed male reproduction in mice exposed to progesterone during embryonic development by testosterone.

The present study aimed to examine whether transplacental exposure to progesterone caused male reproductive abnormalities and whether the changes can be reversed after testosterone administration. Progesterone was injected to mice on day 1, 3, and 7 of pregnancy. The male pups (F1 generation) were allowed to grow for 50 days and assessed for reproductive performance. Gestational exposure to progesterone (7 mg/kg body weight) resulted in significant body weight gain with a decrease in reproductive tissue indices in mice. Total sperm count, viable sperm, and motile sperm decreased in experimental mice. Hypo-osmotic swelling test revealed that experimental mice sperm membrane integrity was severely altered. The activity levels of testicular steroidogenic marker enzymes (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B) and hydroxysteroid (17-beta) dehydrogenase 1 (HSD17B)) decreased significantly in mice exposed to progesterone during embryonic development when compared with the controls. The levels of serum testosterone decreased with an increase in serum FSH and LH in mice exposed to progesterone during embryonic development. Prenatal exposure to progesterone caused significant reduction in the number of spermatozoa and increase in the lumen of seminiferous tubule. The experimental mice that cohabited with normal females showed fertility reduction. Administration of testosterone (4.16 mg/kg body weight) on postnatal day 20, 30, and 40 to progesterone-exposed prenates resulted in recovery of progesterone-induced suppressed male reproduction. It is suggested that the impairment of male reproduction in mice exposed to progesterone during embryonic development could be mediated through the inhibition of testosterone production. These results also indicate that in utero exposure to progesterone affects male reproduction and that supplementation of testosterone restores the suppressed male reproduction.

Effect of retinoic acid on hemolymph glucose regulation in the fresh water edible crab Oziotelphusa senex senex.

9-cis-Retinoic acid (9CRA) and all-trans-retinoic acid (ATRA) are known to be involved in the regulation of glucose homeostasis in vertebrates by inducing insulin release and expression of glucose reporter proteins. In view of the fact that 9CRA and ATRA are endogenous in crustaceans and a retinoic acid X-receptor exists in crabs, we investigated whether 9CRA and ATRA also plays a role in glucose homeostasis in freshwater crab, Oziotelphusa senex senex. Injection of 9CRA into intact crabs significantly increased the hemolymph glucose level in a dose-dependent manner. Such 9CRA-induced hyperglycemia was apparently mediated by the CHH since injection of 9CRA into eyestalk-ablated crabs did not result in hyperglycemia. In support of this, administration of 9CRA in to crabs resulted in reduced hyperglycemic activity of eyestalks and elevated titers of CHH in hemolymph. ATRA injection did not cause any changes in hemolymph glucose and CHH levels. The results provide the first evidence that 9-cis-retinoic acid, but not all-trans-retinoic acid, is involved in the regulation of glucose homeostasis and apparently mediated by the eyestalk hormone CHH.