17ß-Estradiol and the glucocorticoid clobetasol propionate affect the blood coagulation cascade in zebrafish.

Fish are exposed to estrogens, progestins and glucocorticoids in the aquatic environment but effects on the blood coagulation cascade are unknown. Here we analyzed effects of 17ß-estradiol (E2) alone and in combination with mixtures of new generation progestins and the glucocorticoid clobetasol propionate (CLO) in zebrafish embryos to assess their effects on the blood coagulation system. We assessed transcripts of 13 genes, such as coagulation factors, and genes involved in the anticoagulation and fibrinolytic system upon exposure to concentrations of 0.01-10 µg/L at 144 h post fertilization. Transcripts of genes encoding coagulation factors VII, X and fibrinogen, as well as protein C from the anticoagulation system, and serpine1 from the fibrinolytic system were upregulated by 10 µg/L, and factor II by 1 µg/L E2, as well as in mixtures containing E2 with progestins. CLO alone or in mixtures with progestins and E2 induced genes encoding factor VII, IX and fibrinogen. Progestins induced expression of genes encoding factor IX (f9b) only, but in mixtures with E2 and CLO, also factor VII (f7) and fibrinogen (fga) were induced. Our study demonstrates that exogenous exposure to E2 and CLO alone and in mixtures with new generation progestins used in contraception alter the expression of blood coagulation genes. This ultimately may lead to adverse effects on blood coagulation in fish.

Effects of the glucocorticoid clobetasol propionate and its mixture with cortisol and different class steroids in adult female zebrafish.

Ecotoxicological effects of glucocorticoids and steroid mixtures in the environment are not sufficiently known. Here we investigate effects of 11-14 days exposure of female zebrafish to the glucocorticoid clobetasol propionate (Clo), cortisol (Cs), their mixture and mixtures with five different class steroids (Clo + triamcinolone + estradiol + androstenedione + progesterone) in liver, brain and gonads. Cs showed little activity, while Clo reduced the condition factor at 0.57 and 6.35 µg/L. Clo induced differential expression of genes in the liver at 0.07-6.35 µg/L, which were related to circadian rhythm (per1, nr1d2), glucose metabolism (g6pca, pepck1), immune system response (fkbp 5, socs3, gilz), nuclear steroid receptors (pgr and pxr), steroidogeneses and steroid metabolism (hsd11b2, cyp2k22). Clo caused strong transcriptional down-regulation of vtg. Similar upregulations occurred in the brain for pepck1, fkbp5, socs3, gilz, hsd11b2, and nr1d2a, while cyp19b was down-regulated. Effects of Clo + Cs mixtures were similar to Clo alone. Transcriptional alterations were different in mixtures of five steroids with no alteration of vtg in the liver due to counteraction of Clo and estradiol. Induction of fkbp5 (brain) and sult2st3 (liver) and downregulation of cyp19a (gonads) occurred at 1 µg/L. Histological effects of the five steroids mixture in gonads were characterized by a decrease of mature oocytes. Our data indicate that effects of steroids of different classes sum up to an overall joint effect driven by the most potent steroid Clo.

Clobetasol propionate causes immunosuppression in zebrafish (Danio rerio) at environmentally relevant concentrations.

Synthetic glucocorticoids (GCs) are potential endocrine disrupting compounds that have been detected in the aquatic environment around the world in the low ng/L (nanomolar) range. GCs are used as immunosuppressants in medicine. It is of high interest whether clobetasol propionate (CP), a highly potent GC, suppresses the inflammatory response in fish after exposure to environmentally relevant concentrations. Bacterial lipopolysaccharide (LPS) challenge was used to induce inflammation and thus mimic pathogen infection. Zebrafish embryos were exposed to ≤1000nM CP from ~1h post fertilization (hpf) to 96 hpf, and CP uptake, survival after LPS challenge, and expression of inflammation-related genes were examined. Our initial experiments were carried out using 0.001% DMSO as a solvent vehicle, but we observed that DMSO interfered with the LPS challenge assay, and thus masked the effects of CP. Therefore, DMSO was not used in the subsequent experiments. The internal CP concentration was quantifiable after exposure to ≥10nM CP for 96h. The bioconcentration factor (BCF) of CP was determined to be between 16 and 33 in zebrafish embryos. CP-exposed embryos showed a significantly higher survival rate in the LPS challenge assay after exposure to ≥0.1nM in a dose dependent manner. This effect is an indication of immunosuppression. Furthermore, the regulation pattern of several genes related to LPS challenge in mammals supported our results, providing evidence that LPS-mediated inflammatory pathways are conserved from mammals to teleost fish. Anxa1b, a GC-action related anti-inflammatory gene, was significantly down-regulated after exposure to ≥0.05nM CP. Our results show for the first time that synthetic GCs can suppress the innate immune system of fish at environmentally relevant concentrations. This may reduce the chances of fish to survive in the environment, as their defense against pathogens is weakened.

Occurrence of glucocorticoids discharged from a sewage treatment plant in Japan and the effects of clobetasol propionate exposure on the immune responses of common carp (Cyprinus carpio) to bacterial infection.

The present study evaluated the environmental risks to common carp (Cyprinus carpio) posed by glucocorticoids present in sewage treatment plant (STP) effluent. To gather information on the seasonal variations in glucocorticoid concentration, the authors sampled the effluent of a Japanese STP every other week for 12 mo. Six of 9 selected glucocorticoids were detected in the effluent, with clobetasol propionate and betamethasone 17-valerate detected at the highest concentrations and frequencies. The present study's results indicated that effluent glucocorticoid concentration may depend on water temperature, which is closely related to the removal efficiency of the STP or to seasonal variations in the public's use of glucocorticoids. In a separate experiment, to clarify whether glucocorticoids in environmental water increase susceptibility to bacterial infection in fish, the authors examined the responses to bacterial infection (Aeromonas veronii) of common carp exposed to clobetasol propionate. Clobetasol propionate exposure did not affect bacterial infection-associated mortality. In fish infected with A. veronii but not exposed to clobetasol propionate, head kidney weight and number of leukocytes in the head kidney were significantly increased (p < 0.05), whereas these effects were not observed in infected fish exposed to clobetasol. This suggests that clobetasol propionate alleviated bacterial infection-associated inflammation. Together, these results indicate that susceptibility to bacterial infection in common carp is not affected by exposure to glucocorticoids at environmentally relevant concentrations.

Comparative toxicity study of hydrocortisone 17-butyrate 21-propionate (HBP) ointment and other topical corticosteroids in rats.

Comparative systemic and topical toxicity in male rats treated on the dorsal skin for 14 consecutive days with a volume of 0.15 g/100 g (body weight) of 0.1% hydrocortisone 17-butyrate 21-propionate (HBP) ointment, 0.05% clobetasol propionate (CP) ointment, 0.1% predonisolone 17-valerate 21-acetate (PVA) ointment and 0.1% diflucortolone valerate (DV) ointment was studied. In all the treated groups body weight gain was suppressed, serum concentration of total cholesterol and triglycerides increased and the lymphatic tissues and skin were atrophic. The DV and CP groups had adrenal atrophy and renal lesions, and the DV group also had gastric and hepatic lesions. The systemic effect of HBP ointment was weaker than that of the other drugs (DV greater than CP much greater than PVA greater than HBP). All the drugs significantly reduced the skin fold thickness in treated areas throughout the application period. The dermal atrophic effect of HBP ointment was also relatively weaker than that of the other drugs. From the above evidence, it was concluded that HBP ointment was less toxic than the other topical corticosteroids.

[Studies on toxicity of clobetasone-17-butyrate (II)--chronic toxicity in rats (author's transl)].

Chronic toxicity of clobetasone-17-butyrate, an anti-inflammatory corticosteroid, was investigated in rats. Subcutaneous administration with the drug at dose of 0.003, 0.01 and 0.03 mg/kg/day for three and six months induced no significant changes in the rats. At 0.1 and 0.3 mg/kg/day, however, some dose-dependent symptoms such as suppression of body weight gain, emaciation, regressive changes in adrenals, lymphatic and hematopoietic tissues, decrease in circulating white blood cell and lymphocyte counts, which have been known as toxic effects of synthetic corticosteroids, were induced. The results indicates that the maximum no-toxic dose of clobetasone-17 butyrate was 0.03 mg/kg/day on this experimental condition. In the recovery test for two months no significant differences in the treated rats from controls were found, suggesting that the toxic effects were reversible in the animals treated at 0.3 mg/kg/day and lower than that.

[Studies on toxicity of clobetasone-17-butyrate (I)--acute toxicity in mice and rats and subacute toxicity in rats (author's transl)].

Acute and subacute toxicities of clobetasone-17-butyrate, a new anti-inflammatory corticosteroid, were studied in mice and rats. In the acute toxicity tests intraperitoneal LD50 values of the drug were estimated to be around 5 g/kg for both sexes of mice, 1.51 g/kg for male and 1.66 g/kg for female rats. Subcutaneous and oral administration induced no fatal cases at dose of 3.6 (mice, s.c.), 2.6 (rats, s.c.) and 6.0 g/kg (mice and rats, p.o.). As for the toxic signs in both mice and rats after the i.p. and s.c. administrations, emaciation was marked, and atrophy of thymus, spleen and adrenals were observed. No marked symptoms, however, were induced in animals administered orally. In the subacute toxicity tests male and female rats were subcutaneously administered with the drug at daily doses of 0.01, 0.03, 0.1, 1.0, 10 and 100 mg/kg for one month. Dose dependent symptoms such as suppression in body weight gain, emaciation, regressive changes in adrenal, lymphatic and hematopoietic tissues, decrease in circulating white blood cell and lymphocyte counts, and increase in total cholesterol level of serum were induced in the rats administered at 0.1 mg/kg/day and more than that, indicating that the maximum nontoxic dose in this experimental condition was 0.03 mg/kg/day. In recovery tests it was observed that the rats, which had been administered with the drug at 1.0 mg/kg/day for one month, were almost normal two months after the final administration.