Philip A. Spear

Changes of thyroid hormone levels and related gene expression in Chinese rare minnow (Gobiocypris rarus) during 3-amino-1,2,4-triazole exposure and recovery

Thyroid hormones (THs) play an important role in the development and metabolism of fish through their influences on genetic transcription and are targets for endocrine disruptive agents in the aquatic environment. Amitrole is a pesticide potentially interfering with thyroid hormone regulation. In this study, the rare minnow (Gobiocypris rarus) was exposed to different levels of 3-amino-1,2,4-triazole (amitrole) and allowed to recover in clean water. Plasma TH levels and the expression of TH-related genes, including transthyretin (ttr), deiodinases (d1 and d2), and the thyroid hormone receptor (tralpha) from the livers and brains were evaluated. After exposure, the plasma TH levels did not change. Histopathological observations showed that livers were degenerated at 10,000 ng/l and these damages could be recovered by the withdrawal of amitrole. However, the ttr, d1, and d2 mRNA levels in the livers of males were significantly up-regulated in all exposure groups (p<0.05). The ttr and d2 mRNA levels were significantly up-regulated at 10,000 ng/l and 10, 100, and 1000 ng/l in the livers of females, respectively (p<0.05). In the brains of males, a twofold increase of d2 mRNA levels at > or = 100 ng/l and a fivefold decrease of tralpha mRNA levels at > or = 10 ng/l were observed (p<0.05), whereas no significant differences were observed in the expression of d2 and tralpha in the brains of females. After a recovery period, the ttr, d1, and d2 mRNA levels in the livers of males returned to control levels, but the tralpha mRNA levels were irreversibly decreased at all treatments (p<0.05). In addition, the d2 mRNA levels in the livers of females were significantly induced at > or = 100 ng/l. Moreover, the d2 mRNA levels in the brains of males and females were up-regulated at 10,000 ng/l. These results indicated that amitrole exposure could result in alternations of ttr, d1, d2, and tralpha gene expression in different tissues of the rare minnow. The expression of these TH-related genes in males was more sensitive to amitrole than those of females. Recovery in clean water was associated with the selective regulation of TH-related gene transcription in the rare minnow. Therefore, these TH-related genes can serve as biomarkers to screen the effects of thyroid disruption chemicals in rare minnow.

Combined effects of agricultural activity and parasites on biomarkers in the bullfrog, Rana catasbeiana.

Agricultural contaminants can have devastating impacts on amphibian survival and development, particularly considering their sensitivity to environmental perturbation. However, it is commonly overlooked that amphibians are infected with various parasites that can influence the overall health of the animal when exposed to a stressful environment. We investigated the interaction of agriculture and parasitism on the health of bullfrogs (Rana catesbeiana) in the field. Nine physiological and immunological biomarkers were related to naturally acquired parasite infections, along a gradient of agricultural activity. Most health biomarkers were affected by agriculture, parasitism, or both. Although bullfrogs residing in agricultural areas were infected with fewer parasite species, reflecting environmentally compromised ecosystems, certain persistent parasites interacted with agricultural disturbance to alter the physiology and immune competence of bullfrogs. The consequences of the combination for animal health highlight the importance of parasitism in ecotoxicological studies. Consideration of parasitism is warranted when evaluating the influence of anthropogenic disturbance on amphibian declines and environmental health.

Tamoxifen effects on the early life stages and reproduction of Japanese medaka (Oryzias latipes)

Tamoxifen is widely used in the treatment of breast cancer and can enter the aquatic environment in municipal wastewater. To evaluate potential effects on embryonic development of Japanese medaka, fertilized eggs were exposed to tamoxifen at 1-625μg/l for 14 days. Adverse effects on hatchability and time to hatching only occurred at 125 and 625μg/l. Reproductive effects were assessed by exposing adults for 21 days to the same dose range. At all concentrations tested, tamoxifen significantly increased plasma vitellogenin levels in males in a dose-dependent manner. Fecundity and fertility were detrimentally affected at 625μg/l. Additionally, F1 eggs were removed from tamoxifen-contaminated water to evaluate transgenerational effects. Hatchability was affected at 625μg/l but no morphological deformities were observed. A significant dose-dependent increase in the proportion of genotypic males occurred at all concentrations greater than 5μg/l.

Comparison of ethinylestradiol and nonylphenol effects on reproduction of Chinese rare minnows (Gobiocypris rarus)

Breeding pairs of rare minnows were exposed to 5, 10, 20 microg/L 4-nonylphenol (NP) or 4 ng/L 17alpha-ethinylestradiol (EE(2)) for 21 days. Among reproductive parameters investigated, fecundity, fertility, and laying interval were significantly affected by 4 ng/L EE(2), but were not affected at any exposure level of NP. These effects coincided with male feminization, ova-testis, increased plasma VTG in both males and females as well as the accumulation of eosinophilic material in kidneys and livers. Histopathologic evaluation revealed severe kidney lesions in both EE(2) and NP-exposed fish. The bioassay succeeded in demonstrating that EE(2) was several orders of magnitude more potent than the industrial chemical NP. The rare minnow was more sensitive to EE(2) than medaka, but comparable to zebrafish in terms of sensitivity. The results of the present investigation indicated that a short-term reproductive bioassay is feasible using the Chinese rare minnow as the test species.

Hepatic retinoids of bullfrogs in relation to agricultural pesticides

Agricultural pesticides often have been cited as a factor affecting indigenous amphibian populations, but possible effects of pesticides and other factors associated with agricultural practices are understood poorly. Adult bullfrogs (Rana catesbeiana) were collected within the Yamaska River basin (Quebec, Canada) in subwatersheds representing low, medium, and high agricultural activities and 53 pesticides were analyzed in surface water. More pesticides were detected in subwatersheds associated with high agricultural activities like Rivière Noire and Rivière à la Barbue and pesticide concentrations were higher compared to the other study sites. Female and male body weights differed between sites. In the case of males, body weight was significantly less at Rivière à la Barbue. Liver retinol stores were decreased significantly in male bullfrogs from Rivière Noire, although total retinyl esters concentrations varied between sites having the highest concentration at Yamaska-Nord where the agricultural activity was considered low. The ratio of hepatic retinyl palmitate to retinol tended to be higher for male bullfrogs from Rivière Noire and Rivière à la Barbue. These results suggest that factors associated with intensive agricultural practices may affect the body weight and retinoid stores in male bullfrogs living in these agroecosystems.

β-carotene and retinoids in eggs of Great Blue Herons (Ardea herodias) in relation to St Lawrence River contamination.

The potential use of retinoids and β-carotene as biomarkers in the eggs of the Great Blue Heron was investigated. In the spring of 1991, 65 eggs were collected from nine heronries (seven along the St Lawrence River and two reference sites). A method was specifically developed for the extraction and analysis of β-carotene and the retinoids in heron egg yolks by reversed-phase HPLC. When results were expressed either as the molar ratio of retinol: retinyl palmitate or as retinyl palmitate concentration, significant differences were found between colonies; however, retinyl palmitate concentration was deemed the better biomarker because it was not significantly influenced by embryonic stage of development. Retinyl palmitate concentrations in freshwater colonies were negatively related to PCB congeners Nos 105 and 118 as well as their TCDD-EQ values (p < 0.02, r2=0.78). Egg tetrachloro-mono-ortho biphenyl concentrations were also negatively related to retinyl palmitate (p < 0.005, r2=0.90). With the exception of the two mono-ortho co-planar congeners detected in the present study, the contamination levels found in heron eggs were well below those found for other bird species in the Great Lakes area and, so far, no detrimental effects have been reported in Great Blue Heron populations in Quebec. These results suggest that retinyl palmitate may be useful as a sensitive and non-invasive biomarker for monitoring organochlorine contaminant effects in the Great Blue Heron in freshwater sites.

Retinoids: From metabolism to environmental monitoring

Abstract Adult herring gulls, Larus argentatus , were captured on their nests at various colonies on the Great Lakes and retinoids (vitamin A) analyzed in the liver by reverse-phase HPLC. All- trans -retinol and all- trans -retinyl palmitate concentrations showed significant differences between colonies. Colonies having the highest concentrations of 2,3,7,8-tetrachlorodibenzo- p -dioxin had the lowest retinoid concentrations. Particularly low levels of hepatic retinoids occurred in Lake Ontario gulls in 1987; and all- trans -retinol was negatively correlated with liver aryl hydrocarbon hydroxylase activity. Liver retinoids were identified in the white sucker, Catostomus commersoni , using reverse-phase HPLC with diode array detection. All- trans -retinol, all- trans -retinyl palmitate, and 3,4-didehydroretinyl esters were detected. In the case of adult white suckers sampled from a contaminated river near Montreal and a control site, significantly lower levels of all- trans -retinol and all- trans -retinyl palmitate were found in the contaminated population.

Assessment of Contamination and Biomarker Responses in Two Species of Herons on the St. Lawrence River

This study was undertaken to validate potential biomarkers of exposure and effects due to chemical contaminants in breedingcolonies of the Great Blue Heron and the Black-crowned Night-Heron on the St. Lawrence River. Eggs and fledglings from both species were collected from many colonies along theRiver. The fledglings from colonies in freshwater and brackishwater were more contaminated by mercury and PCBs than those from estuarine and gulf colonies. With respect to fledglings ofthe two heron species, some morphometric and blood biochemicalmeasurements, including plasma thyroid hormones and retinol, were significantly different among colonies. Significant differences were also observed in liver retinoids, EROD and porphyrins among colonies. The results of this study suggestthat plasma retinoids and thyroid hormones are good biomarkersof exposure and effects, and are sufficiently sensitive to reflect local and regional variations in contamination. Along with the measure of contaminants in egg and plasma, they constitute non-invasive biomarkers which represent an importantcriteria for long term monitoring of wildlife species. It is concluded that the Great Blue Heron is an appropriate sentinelspecies in the surveillance network for the St. Lawrence River.

Retinoic acid hydroxylation in rainbow trout (Oncorhynchus mykiss) and the effect of a coplanar PCB, 3,3',4,4'-tetrachlorobiphenyl

When liver microsomes of rainbow trout (Oncorhynchus mykiss) were incubated with [14C]-labeled retinoic acid plus NADPH, high performance liquid chromatography revealed the presence of radioactive peaks having elution times identical to those of 4-hydroxy- and 4-oxoretinoic acid. In the absence of NADPH, the radioactive peaks were not detected which is consistent with cytochrome P450-dependent 4-hydroxylation. In trout injected intraperitoneally with 5 μg/g of 3,3′,4,4′-tetrachlorobiphenyl (TCB) and sacrificed 56 days later, the hydroxylation rate was higher (P < 0.0005) compared with control trout. No difference was observed between TCB-treated and control groups 7 days after injection. In contrast, cytochrome P4501-dependent ethoxyresorufin-O-deethylase activity was significantly higher in the TCB-treated group, both at 7 and 56 days. Liver retinoid stores were not affected by TCB treatment. These results clearly demonstrate that fish metabolize retinoic acid through a hydroxylation step which can be accelerated by a cytochrome P4501-inducing coplanar PCB. Given the pronounced biological activity of retinoic acid and closely related retinoids, increased retinoic acid metabolism could explain some of the effects of PCBs in fish.

Evidence for metabolic imbalance of vitamin A2 in wild fish chronically exposed to metals

In a recent study on indigenous yellow perch chronically exposed to metals, we reported a negative correlation between liver metal concentration and liver transcription levels of genes encoding for enzymes involved in the metabolism of retinoids. We therefore speculated that metals, and especially the non-essential metal Cd, could alter the metabolism of retinoids in wild fish. Thus the present field study investigates the impact of in situ metal exposure on retinoid storage. A total of 55 yellow perch (Perca flavescens) were sampled in six lakes representing a metal contamination gradient (8≤N≤10 per lake). Our results show that yellow perch from Cd-contaminated lakes had significantly higher concentrations of liver dehydroretinol and dehydroretinyl esters than did fish from reference lakes. However, the increase in retinyl ester stores with increasing Cd concentrations was quantitatively much more important than the increase in free dehydroretinol. As a result, a significant decrease in the percentage of hepatic free dehydroretinol with increasing renal Cd concentrations was observed. These results suggest that the enzymes and the binding proteins involved in vitamin A homeostasis are inhibited by the presence of Cd. Alternatively, the increase in tissue vitamin A (antioxidant) levels could serve to better counteract the oxidative stress engendered by Cd exposure. Overall our findings illustrate that vitamin A(2) homeostasis can be altered as a consequence of chronic exposure to low Cd concentrations. Thus, in the context of environmental risk assessment, the percentage of liver free dehydroretinol can be considered as a biomarker of for in situ Cd exposure.