Dicamba elevates concentrations of S-adenosyl methionine but does not induce oxidative stress or alter DNA methylation in rainbow trout (Oncorhynchus mykiss) hepatocytes.

Dicamba is a benzoic acid herbicide used to target woody and broadleaf weeds in industrial, domestic, and municipal spheres. Because of its widespread use, dicamba is frequently detected in surface waters near sites of application. However, little is known regarding the effects of dicamba on freshwater fishes. In the present study, primary cultures of hepatocytes from rainbow trout (Oncorhynchus mykiss) were exposed to either an environmentally relevant (0.22 or 2.2 µg L-1) or supra-environmental (22 µg L-1) concentration of dicamba for 48 h to investigate if oxidative stress is a mechanism of toxicity. mRNA abundances of genes involved in the response to oxidative stress, levels of lipid peroxidation, and concentrations of glutathione and s-adenosyl methionine (SAM) were quantified. Results indicate that dicamba does not induce oxidative stress. However, exposure to 2.2 µg L-1 of dicamba did cause a 5.24-fold increase in concentrations of SAM. To investigate the mechanisms of increased SAM, effects of dicamba on global and genome-wide DNA methylation were quantified. Dicamba did not cause changes to DNA methylation. Overall, dicamba was not acutely toxic to hepatocytes and did not cause oxidative stress or changes in DNA methylation at environmentally relevant concentrations.

Triclosan: environmental exposure, toxicity and mechanisms of action.

Triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol; TCS] is a broad spectrum antibacterial agent used in personal care, veterinary, industrial and household products. TCS is commonly detected in aquatic ecosystems, as it is only partially removed during the wastewater treatment process. Sorption, biodegradation and photolytic degradation mitigate the availability of TCS to aquatic biota; however the by-products such as methyltriclosan and other chlorinated phenols may be more resistant to degradation and have higher toxicity than the parent compound. The continuous exposure of aquatic organisms to TCS, coupled with its bioaccumulation potential, have led to detectable levels of the antimicrobial in a number of aquatic species. TCS has been also detected in breast milk, urine and plasma, with levels of TCS in the blood correlating with consumer use patterns of the antimicrobial. Mammalian systemic toxicity studies indicate that TCS is neither acutely toxic, mutagenic, carcinogenic, nor a developmental toxicant. Recently, however, concern has been raised over TCS's potential for endocrine disruption, as the antimicrobial has been shown to disrupt thyroid hormone homeostasis and possibly the reproductive axis. Moreover, there is strong evidence that aquatic species such as algae, invertebrates and certain types of fish are much more sensitive to TCS than mammals. TCS is highly toxic to algae and exerts reproductive and developmental effects in some fish. The potential for endocrine disruption and antibiotic cross-resistance highlights the importance of the judicious use of TCS, whereby the use of TCS should be limited to applications where it has been shown to be effective.

Purification and isolation of corticosteroidogenic cells from head kidney of rainbow trout (Oncorhynchus mykiss) for testing cell-specific effects of a pesticide.

The teleost head kidneys contain corticosteroidogenic cells, chromaffin cells, lymphoid cells, and melanomacrophages. We have developed and validated a method using a Percoll density gradient and differential staining for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) to prepare fractions enriched with specific head kidney cell types. The proportion of steroidogenic cells to other cells in the head kidney was 1:8000 in rainbow trout, Oncorhynchus mykiss. To test the hypothesis that steroidogenic cells are more vulnerable to a pesticide than other cell types in the head kidney, head kidney cells were separated by a Percoll gradient and the steroidogenic cell-enriched fractions and lymphoid cell-enriched fractions were exposed to the pesticide endosulfan in vitro, and their functional integrity and viability were assessed. The effective concentration of the pesticide (EC50, concentration that inhibits 50% of the secretory response to ACTH) in the mixed head kidney cell preparation was similar to the EC50 in the fraction enriched with steroidogenic cells, but differences in viability were detected. The Percoll method for isolation of different cell types from the head kidney facilitated a study of cell-specific effects of a pesticide.

Role of calcium channels in cadmium-induced disruption of cortisol synthesis in rainbow trout (Oncorhynchus mykiss).

The mechanisms of toxicity of cadmium (Cd(2+)) in adrenal steroidogenesis were investigated in vitro in adrenocortical cells of rainbow trout (Oncorhynchus mykiss). Toxicity of Cd(2+) was increased in absence of extracellular Ca(2+), but was prevented in Ca(2+)-supplemented medium. Pretreatment of cells with BAY K8644 (BAY), an agonist of voltage-dependent calcium channels, increased the Cd(2+)-mediated inhibition of ACTH-stimulated secretion but not pregnenolone (PREG)-stimulated secretion. Nicardipine, an antagonist of voltage-dependent calcium channels, also increased the inhibition of adrenocorticotropic hormone (ACTH)-stimulated secretion by Cd(2+). These results suggest that opening of voltage-dependent calcium channels with BAY may allow Cd(2+) entry at the same time as calcium, thus increasing toxicity of Cd(2+), however voltage-dependent calcium channels may not be the only way of entry into adrenocortical cells. The influx of Cd(2+), measured as intracellular Cd(2+) using Fluo-3 in PREG-stimulated adrenocortical cells, was significantly enhanced by the stimulation. These results suggest that the deleterious effect of Cd(2+) on cortisol steroidogenesis may be enhanced when the endocrine stress response is triggered.

Effects of Cu on plasma cortisol and cortisol secretion by adrenocortical cells of rainbow trout (Oncorhynchus mykiss).

Fish are exposed to multiple stressors, often acting concurrently, in their environment. To evaluate the potential of Cu to act as a chemical stressor, rainbow trout (Oncorhynchus mykiss) were exposed to Cu (30 or 80 microg/l) for 30 days in the laboratory and they were subjected to a physical stressor (1 min air exposure) before sampling. Physiological stress indicators in the whole fish as well as cortisol secretion by adrenocortical cells in vitro were measured. Fish exposed to Cu had a lower condition factor, hepatosomatic index, plasma glucose, hepatic glycogen and gill Na(+)/K(+)-ATPase activity compared to controls. Exposure to Cu did not have an effect on basal plasma cortisol (fish sampled without air exposure stress) however, the air exposure-induced increase in plasma cortisol was lower in fish exposed to Cu. Cortisol secretion stimulated by ACTH in vitro was greater in adrenocortical cells isolated from fish exposed to Cu in vivo but in vitro exposure to Cu consistently impaired cortisol secretion. Our results indicate that Cu at high concentrations disrupts cortisol secretion through a direct toxic effect on adrenocortical cells while low concentrations resulting from a 30-day exposure to environmentally relevant Cu concentrations enhances cortisol secretion in response to ACTH in vitro.

Oxidative stress and endocrine endpoints in white sucker (Catostomus commersoni) from a river impacted by agricultural chemicals.

The effects of agricultural chemicals on cortisol secretion, antioxidants, and lipid peroxidation were investigated in hepatic and adrenal tissue of white sucker (Catostomus commersoni) from a river (Yamaska) that drains an agricultural region in Québec (Canada). Plasma cholinesterase (ChE) activity, used as a biomarker of exposure to pesticides, was elevated in fish from the reference site compared to fish from the contaminated sites. Plasma concentrations of cortisol and thyroid hormones (T3 and T4) were higher in fish from the reference site compared to contaminated sites; reduced glutathione (GSH) levels, catalase (CAT), and glutathione peroxidase (GPx) activities were higher and lipid peroxidation (LPO) was lower. Levels of antioxidants (CAT, Gpx, and GSH) were higher (10-90%) and LPO levels were lower (50%) in the liver than in the adrenal tissue. The present in situ study provided evidence that antioxidants, lipid peroxidation, and plasma hormones were altered in fish sampled in areas impacted by agricultural chemicals. Endocrine-disrupting effects were associated with oxidative stress. The results suggest that antioxidants and lipid peroxidation could be used as markers of contaminant exposure in fish.

A comparative assessment of the adrenotoxic effects of cadmium in two teleost species, rainbow trout, Oncorhynchus mykiss, and yellow perch, Perca flavescens.

Rainbow trout (Oncorhynchus mykiss) and yellow perch (Perca flavescens) have a different sensitivity to cadmium (Cd) in vivo (trout < LC50 < perch). Metals and particularly Cd impair cortisol secretion by adrenocortical cells in both species. The purpose of the present study was to assess in vitro the effect of Cd on cortisol secretion by adrenocortical cells of trout and perch, to compare the sensitivity of adrenal steroidogenesis in these two teleosts. Adrenocortical cells were exposed to Cd for 60 min, then stimulated with ACTH, dbcAMP or with pregnenolone, a cortisol precursor. Cd inhibited ACTH-stimulated cortisol secretion in a dose-dependent manner in both fish species, however, the EC50s (concentration resulting in 50% inhibition of cortisol secretion) was significantly lower in trout (EC50 = 0.09 mM) than perch (EC50 = 0.26 mM). To test the specificity of Cd to act as an endocrine disrupter, the LC50 (concentration that kills 50% of the cells) was also evaluated to determine the LC50/EC50 ratio ( LC50/EC50(O.mykiss) = 175.6 > LC50/EC50(P.flavescens) = 37.7). Adrenocortical cells of trout were more sensitive than those of perch and Cd had a higher endocrine-disrupting potential and specificity in trout than in perch. However, in both species, Cd had the same effect on ACTH, dbcAMP and pregnenolone-stimulated cortisol secretion, with pregnenolone maintaining cortisol secretion until cell viability was impaired. These results confirm that for both species, Cd interferes in the signalling pathway of cortisol synthesis in a step prior to the pregnenolone formation. Data provided by the present study revealed important differences in vulnerability of adrenal steroidogenesis between rainbow trout and yellow perch.

Toxicity of cadmium, endosulfan, and atrazine in adrenal steroidogenic cells of two amphibian species, Xenopus laevis and Rana catesbeiana.

The effects of cadmium, endosulfan, and atrazine on corticosterone secretion and viability of adrenal cells of African clawed frog (Xenopus laevis) and bullfrog (Rana catesbeiana) were assessed in vitro using a new bioassay. The bioassay relies on stimulation with adrenocorticotropic hormone (ACTH), the endogenous secretagogue for corticosterone secretion, and with dibutyryl cyclic adenosine monophosphate (dbcAMP), an analogue of cAMP, to pinpoint the site of action of the xenobiotics within the steroidogenic cell. To compare the test toxicants according to their endocrine-disrupting potential, the lethal concentration needed to kill 50% of the cells:effective concentration of 50% (LC50:EC50) ratio was calculated, with LC50 as the concentration that kills 50% of the steroidogenic cells and the EC50 as the concentration that impairs corticosterone secretion by 50%. The higher the ratio, the higher the potential for endocrine disruption. Atrazine had no affect on cell viability and on corticosterone secretion in X. laevis, but its endocrine-disrupting potential was high in R. catesbeiana. The LC50:EC50 ratio for cadmium and endosulfan in X. laevis was 26.07 and 1.23, respectively, and for atrazine, cadmium, and endosulfan in R. catesbeiana it was 909, 41, and 3, respectively. The dbcAMP did not restore corticosterone secretion in the cells exposed to the test toxicants in both species. Our study suggests that the secretory capacity of adrenal cells of amphibians can be impaired by environmental chemicals, especially atrazine in the bullfrog, and that these adrenotoxicants disrupt the enzymatic pathways leading to corticosterone secretion downstream from the step-generating cAMP.

The organochlorine o,p'-DDD disrupts the adrenal steroidogenic signaling pathway in rainbow trout (Oncorhynchus mykiss).

The mechanisms of action of o,p'-DDD on adrenal steroidogenesis were investigated in vitro in rainbow trout (Oncorhynchus mykiss). Acute exposures to o,p'-DDD inhibited ACTH-stimulated cortisol secretion while cell viability decreased significantly only at the highest concentration tested (200 microM o,p'-DDD). Stimulation of cortisol secretion with a cAMP analogue (dibutyryl-cAMP) was inhibited at a higher concentration than that needed to inhibit ACTH-stimulated cortisol synthesis in cells exposed to o,p'-DDD. Forskolin-stimulated cortisol secretion and cAMP production, and NaF-stimulated cAMP production were inhibited in a concentration-dependent manner by o,p'-DDD. In contrast, basal cortisol secretion was stimulated while basal cAMP production was unaffected by o,p'-DDD. Pregnenolone-stimulated cortisol secretion was enhanced by o,p'-DDD at a physiologically relevant pregnenolone concentration, while o,p'-DDD inhibited cortisol secretion when a pharmacological concentration of pregnenolone was used. Our results suggest that the cAMP generation step is a target in o,p'-DDD-mediated disruption of ACTH-stimulated adrenal steroidogenesis in rainbow trout but that other downstream targets such as steroidogenic enzymes responsible for cortisol synthesis might also be affected.

Hormonal, morphological, and physiological responses of yellow perch (Perca flavescens) to chronic environmental metal exposures.

The effects of a chronic environmental exposure to metals on the hormonal, physiological, and reproductive status were assessed in yellow perch (Perca flavescens) sampled in six lakes situated along a contamination gradient of Cd, Zn, Cu, Pb, and Ni in the mining region of Rouyn-Noranda, Québec. Fish were captured in the summer and fall, and sampled before or after a confinement of one hour. Metal concentrations in the kidneys and the interrenal tissues (homologous to mammalian adrenals) were measured to compare tissue-specific metal accumulation. An exposure-related decrease of condition factor, gonadosomatic index (GSI), branchial Na+/K(+)-ATPase activity, plasma thyroxine (T4), triiodothyronine (T3), and 17 beta-estradiol and an impaired capacity to enhance cortisol levels after confinement were observed. Fish from the metal-contaminated lakes possessed gonads at less mature stages and exhibited structural alterations of their gills, interrenal cells, and thyroid follicle epithelium. A comparison of the morphological, biochemical, and physiological endpoints measured in the present study revealed that plasma concentrations of hormones and parameters of gill function were the most affected by metal contamination. The results of this study indicate that lifelong exposures to sublethal concentrations of metals alter the physiological functions of fish and delay reproduction.

Loss of capacity to elevate plasma cortisol in rainbow trout (Oncorhynchus mykiss) treated with a single injection of o,p'-dichlorodiphenyldichloroethane.

The organochlorine o,p'- dichlorodiphenyldichloroethane (o,p'-DDD) is a metabolite of dichlorodiphenyltrichloroethane (DDT), known for its adrenolytic actions in birds and mammals. The effects of o,p'-DDD on the cortisol stress response were investigated in rainbow trout, Oncorhynchus mykiss, in a dose-response study in vivo. A dose-dependent decrease in plasma cortisol levels was observed on days 7 and 14 after a single i.p. injection of o,p'-DDD. Treatment with o,p'-DDD had no effect on weight gain, hematocrit, and gonado- or hepatosomatic index but decreased liver glycogen reserves. The results indicate that o,p'-DDD is an adrenotoxic compound in rainbow trout and that its effects can be detected even 14 d postinjection.

Cytotoxic and endocrine-disrupting potential of atrazine, diazinon, endosulfan, and mancozeb in adrenocortical steroidogenic cells of rainbow trout exposed in vitro.

An in vitro bioassay for detection and quantitative assessment of chemicals with the capacity to disrupt adrenal steroidogenesis has been developed and used to compare the cytotoxic and endocrine-disrupting potential of four pesticides. Enzymatically dispersed adrenocortical cells of rainbow trout (Oncorhynchus mykiss) were exposed in vitro to atrazine, diazinon, endosulfan, and mancozeb, and cortisol secretion in response to ACTH or dibutyryl-cAMP (dbcAMP) and cell viability were determined. The effective concentration, EC50 (concentration that inhibits cortisol secretion by 50%), the median lethal concentration, LC50 (concentration that kills 50% of the cells), and the LC50/EC50 ratio were established for the test pesticides. The pesticides were ranked as follows: EC50, endosulfan < diazinon < mancozeb < atrazine; LC50, diazinon < endosulfan < mancozeb < atrazine, with diazinon as the most cytotoxic. Endosulfan and mancozeb disrupted sites downstream of the cAMP-generating step of the cortisol synthetic pathway while atrazine seemed to act upstream from the cAMP step. The in vitro adrenal bioassay can be used for screening of adrenotoxicants and for mechanistic studies of adrenotoxicity.