Norethindrone alters mating behaviors, ovary histology, hormone production and transcriptional expression of steroidogenic genes in zebrafish (Danio rerio).

The impact of progestins (i.e. synthetic forms of progesterone) on aquatic organisms has drawn increasing attention due to their widespread occurrence in the aquatic environments and potential effects on the endocrine system of fish. In this study, the effects of norethindrone (NET, a progestin) on the reproductive behavior, sex hormone production and transcriptional expressions were evaluated by exposing female zebrafish to NET at 0, 3.1, 36.2 and 398.6 ng L-1 for 60 days. Results showed that NET impaired the mating behaviors of female at 36.2 and 398.6 ng L-1 exhibited by males and increased the frequency of atretic follicular cells in the ovary exposed to NET at 398.6 ng L-1. As for sex hormones, plasma testosterone concentration in zebrafish increased, while estradiol concentration decreased. Up-regulation of genes (Npr, Mpra, Mprß, Fshß, Lß, Tshb, Nis and Dio2) was detected in the brain of fish exposed to NET at 398.6 ng L-1. The transcriptional levels of genes (Esr1, Vtg1, Ar, Cyp19a, Cyp11b and Ptgs2) were generally inhibited in the ovary of zebrafish by NET at 398.6 ng L-1. Moreover, the transcripts of genes (Vtg1, Esr1, Ar and Pgr) in the liver were reduced by NET at 36.2 and 398.6 ng L-1. Our findings suggest that NET can potentially diminish the of fish populations not only by damaging their reproductive organs, but also by altering their mating behavior through the changes in the expressions of genes responsible for the production of sex hormones.

Transcriptomic and physiological changes in western mosquitofish (Gambusia affinis) after exposure to norgestrel.

Norgestrel (NGT) is a synthetic progestin used in human and veterinary medicine. Adult female mosquitofish were exposed to NGT for 42 d at 377 ng L-1. The fin morphology and the liver transcriptome were assessed. NGT exposure increased ray 4:6 length ratio. As compared to the control, NGT treatment affected the expression of 11,772 annotated transcripts in female mosquitofish. Specifically, we found 5780 were repressed while 5992 were significantly induced. Gene ontology (GO) analysis showed that 53 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 158 GO terms were significantly over expressed. Genes showing the largest magnitude of expression changes were related to fin development, androgen biosynthesis, and lipid and fatty acid metabolisms, suggesting the involvement of these biological processes in response to NGT exposure in G. affinis. This first comprehensive study on the transcriptomic alterations by NGT in G. affinis not only provides valuable information on the development of molecular markers but also opens new avenues for studies on the molecular mechanisms of effects of NGT in particular and possibly other progestins in G. affinis.

The interactive effects of ethinylestradiol and progesterone on transcriptional expression of genes along the hypothalamus-pituitary-thyroid axis in embryonic zebrafish (Danio rerio).

Progestins and estrogens are widespread in various aquatic environments and their potential endocrine disruption effects to aquatic organisms have drawn growing concern. However, their combined effects in aquatic organisms remain elusive. The aim of the present study was to assess the effects of the binary mixtures of gestodene (GES) and 17α-ethinylestradiol (EE2) on the hypothalamic-pituitary-thyroid (HPT) axis of zebrafish (Danio rerio) using the eleuthero-embryos. Embryos were exposed to GES and EE2 alone or in combination at concentrations ranging from 41 to 5329 ng L-1 (nominal ones from 50 to 5000 ng L-1) for 48 h, 96 h and 144 h post fertilization (hpf). The results showed that the transcripts of the genes along the HPT axis displayed pronounced alterations. There was no clear pattern in the change of the transcripts of these genes over time and with concentrations. However, in general, the transcripts of the genes were inversely affected by EE2 (increase 0.5 to 4.2-folds) and GES (inhibition 0.4 to 4.9-folds), and their mixtures showed interactive effects in embryonic zebrafish. In addition, physiological data (mortality, malformation, body length and heart rate etc.) denoted higher toxicity of the two chemicals in combination than alone based on the developmental toxicity and neurotoxicity (locomotor behavior). These results indicated that the interactive effects of these two chemicals might be different between at the transcriptional level and at the whole organismal level. In summary, GES and EE2 affect the HPT axis (related genes expression and thyroid hormones (THs) levels) and exhibit developmental toxicity and neurotoxicity.

Norethindrone causes cellular and hepatic injury in zebrafish by compromising the metabolic processes associated with antioxidant defence: Insights from metabolomics.

Progestins, such as norethindrone (NET), have been increasingly detected in aquatic environments due to their extensive use for medical applications. While NET is notorious for its endocrine disrupting effects, it has been recently shown to cause cellular damage, suggesting its potential impacts on the body defence of organisms. Hence, we examined the histological features and antioxidant defence of zebrafish (Danio rerio) after exposing to NET (50 ng/L and 500 ng/L) for 72 days, followed by analysing its metabolome to explore whether NET disturbs the metabolic processes responsible for antioxidant defence. While acute mortality was not triggered, we found that antioxidant defence was substantially weakened by NET at 500 ng/L (i.e. reduced SOD and GSH levels) and hence liver injury was inflicted (i.e. elevated ALT and MDA levels), as manifested by vacuolization of liver tissues and reduced number of normal cells in the liver. Metabolomic analysis showed that the metabolic processes responsible for antioxidant defence were disrupted by NET (e.g. upregulation of nervonyl carnitine and chenodeoxycholic acid 3-sulfate; downregulation of homolanthionine and acevaltrate) and these changes can undermine antioxidant defence by suppressing Nrf2-ARE and NF-κB pathways that contribute to the synthesis of SOD and GSH. This study demonstrates how NET can compromise the body defence of aquatic organisms via metabolic disruption, suggesting that the impacts of progestins on their fitness are more detrimental than previously thought.

Reproductive potential of mosquitofish is reduced by the masculinizing effect of a synthetic progesterone, gestodene: Evidence from morphology, courtship behaviour, ovary histology, sex hormones and gene expressions.

The ever-increasing use of synthetic hormones, especially progestins, for medical applications has drawn growing concerns due to their potential endocrine disrupting effects that may diminish the reproductive outputs of aquatic organisms. Using mosquitofish (Gambusia affinis) as a model species, we tested whether gestodene (GES), a commonly used progestin, can alter the expressions of genes associated with sex hormone synthesis and cause ensuing changes in morphological features, courtship behaviour and oocyte development. After exposing to GES at environmentally relevant concentrations (2.96, 32.9 and 354 ng L-1) for 40 days, we found that GES, especially at 354 ng L-1, induced masculinization of female fish, indicated by the reduced body weight to length ratio and development of gonopodia (i.e. anal fins of male fish). Thus, the males showed less intimacy and mating interest towards the GES-exposed females, indicated by the reduced time spent on attending, following and mating behaviours. While oocyte development was seemingly unaffected by GES, spermatogonia were developed in the ovary. All the aforementioned masculinizing effects of GES were associated with the increased testosterone level and decreased estradiol level, driven by upregulating androgen receptor genes (Arα and Arß). Overall, our findings suggest that progestins could undermine the reproductive potential of aquatic organisms and hence their persistence in the progestin-contaminated environment.