Effects of binary mixtures of xenoestrogens on gonadal development and reproduction in zebrafish

Previous studies exposing fish to xenoestrogens have demonstrated vitellogenin (VTG) induction, delayed gametogenesis, altered sex ratio, and decreased reproductive performance, with a majority of those studies focusing on exposure to single chemicals. The objective of this study was to determine the effects of binary mixtures of a weak estrogen receptor agonist, nonylphenol (NP) and a potent estrogen receptor agonist, 17-ethinylestradiol (EE) on sex ratios, gametogenesis, VTG induction, heat shock protein 70 (HSP70) expression and reproductive capacity in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to nominal concentrations of 10 or 100 μg/l NP (NP10 or NP100, respectively), 1 or 10 ng/l EE (EE1 or EE10, respectively), 1 ng/l EE + 10 or 100 μg/l NP (EE1 + NP10 or EE1 + NP100, respectively), 10 ng/l EE + 10 or 100 μg/l NP (EE10 + NP10 or EE10 + NP100, respectively) or solvent control (0.01% acetone, v/v) in a static-renewal system with replacement every 48 h. At 60 dph, fish from each treatment were euthanized for histological examination of gonads, and whole body VTG and HSP70 levels. Remaining fish were reared in clean water until adulthood (240 dph) for breeding studies. In all EE10 exposure groups (EE10, EE10 + NP10 and EE10 + NP100), increasing NP concentration acted antagonistically to the action of EE in terms of VTG induction at 60 dph. Similarly, non-additivity was observed with egg production, where EE1 + NP100 exposure resulted in significantly more eggs produced per breeding trial than EE1 alone. Histological staging of oogenesis revealed suppressed gametogenesis in an additive fashion in females at 60 dph. There were no differences among treatment groups in whole body HSP70 expression in 60 dph fish or in gonadal HSP70 expression in adult fish. Although there was no statistical evidence of non-additivity, breeding trials in adults revealed significant reductions in egg viability, egg hatchability and/or F₁ swim-up success, suggesting that developmental exposures to xenoestrogens may cause irreversible effects on egg quality and progeny even after periods of depuration. In conclusion, these results suggest that environmentally relevant mixtures of NP and EE can produce additive or non-additive effects that depend on the particular response being determined and the respective exposure concentrations of each chemical.     

Developmental estrogenic exposure in zebrafish (Danio rerio): I. Effects on sex ratio and breeding success

In vivo studies of fishes exposed to xenoestrogens have reported vitellogenin (Vtg) induction, ovatestes, altered sex ratios, and impaired reproductive capacity. The objective of this study was to determine concentration dependent effects of a weak estrogen receptor agonist, 4-nonylphenol (NP) and a potent estrogen receptor agonist, 17alpha-ethinylestradiol (EE) on sex ratios, gonad morphology, Vtg induction and breeding success in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to NP (10, 30, or 100 microg/l nominal), EE (1, 10, or 100 ng/l nominal), or solvent control (acetone; 0.2% v/v) in a static-renewal system with replacement every 48 h. At 60 dph, 20 fish from each treatment were euthanized for histological examination of gonads and Western blotting for Vtg using pooled heart homogenates. Remaining fish were reared in clean water until adulthood (120 dph) for breeding studies. Due to high mortality in the 100 ng/l EE group, insufficient fish were available for analyses. The percentage of males at 60 dph changed from 45% (9/20) in solvent controls to 0% at 10 ng/l EE and 10% at 100 microg/l NP. A concentration dependent increase in the number of fish with undeveloped gonads at 60 dph was observed in the EE exposure group. Two fish with ovatestes were observed at 100 microg/l NP, while one was observed at 30 microg/l NP. Western blotting showed induction of Vtg at 30 and 100 microg/l NP and 10 ng/l EE. The sex ratios of adults determined at 160 dph revealed no significant departure from 1 male:1 female, suggesting that exposure of zebrafish to estrogenic chemicals during sexual differentiation and early gametogenesis did not irreversibly alter phenotypic sex. The condition factor of adult fish and ovo-somatic index of adult female fish were also unaffected by developmental exposure to NP or EE. Despite this, breeding trials conducted in adult fish from 120 to 160 dph revealed significant reductions in the percent of viable eggs, hatchability, and swim-up success at 10 ng/l EE and 100 microg/l NP. Our results suggest that functional reproductive capacity (breeding success) may be more sensitive than gross morphological endpoints (condition, ovo-somatic index, sex ratio) in adult zebrafish exposed to xenoestrogens during sexual differentiation and early gametogenesis.     

Developmental estrogenic exposure in zebrafish (Danio rerio): II. Histological evaluation of gametogenesis and organ toxicity

Aquatic species can be exposed to endocrine disrupting chemicals (EDCs) in wastewater that often includes the weak estrogen, 4-nonylphenol (NP) and the potent estrogen, 17alpha-ethinylestradiol (EE). The goal of the present study was to determine concentration-dependent effects of developmental exposure to NP and EE on gametogenesis, as well as gonad, kidney and liver pathology using quantitative histological evaluation of hematoxylin/eosin-stained saggital sections of zebrafish (Danio rerio). The major finding of the present study was that exposure to NP (>/=100 microg/l nominal) and EE (>/=1 ng/l nominal) from 2 to 60 days post-hatch (dph) caused concentration-dependent suppression of gametogenesis in both male and female zebrafish. Severe kidney pathology was observed in 60 dph zebrafish, specifically glomerular dilation or degeneration, fibrosis, tubule enlargement and tubule necrosis, at a threshold of 10 ng/l EE. However, minor kidney histopathology indicated by increased pyknotic nuclei in kidney tubule and interstitial (hematopoietic) cells was detected at lower estrogenic exposures (>/=10 microg/l NP nominal) than delayed gametogenesis. Considering all histological parameters in the current study, the rank order of potency for pathological effects in 60 dph zebrafish was 10 ng/l EE>1 ng/l EE=100 microg/l NP>30 microg/l NP>10 microg/l NP10 (nominal concentrations). Zebrafish from the same cohort examined in the current study that had been placed in clean water from 60 to 300 dph had histologically normal testes and no kidney or liver histopathology. However, increased ovarian follicle atresia was detected at 300 dph in zebrafish exposed developmentally to 100 microg/l NP. Therefore, we conclude that functional rather than morphological changes may be more important for future evaluations of developmental exposure to estrogens in fish, and that negative effects in female rather than male gonads may contribute to prolonged breeding impairment.     

Steroid androgen 17α-methyltestosterone induces malformations and biochemical alterations in zebrafish embryos

The synthetic androgen 17α-methyltestosterone is widely used in fish aquaculture for sex reversion of female individuals. Little is known about the amount of MT residues reaching the aquatic environment and further impacts in non-target organisms, including fish early-life stages. Thus, in this work, zebrafish embryos were exposed to two forms of 17α-methyltestosterone: the pure compound (MT) and a formulation commonly used in Brazil (cMT). For MT, a 96 h-LC₅₀ of 10.09 mg/l was calculated. MT also affected embryo development inducing tail malformations, edemas, abnormal development of the head, and hatching delay. At biochemical level MT inhibited vitellogenin (VTG) and inhibited cholinesterase and lactate dehydrogenase. cMT elicited similar patterns of toxicity as the pure compound (MT). Effects reported in this study suggest a potential environmental risk of MT, especially since the VTG effects occurred at environmental relevant concentrations (0.004 mg/l).     

Effects of nonylphenol on motility and subcellular elements of epididymal rat sperm

Nonylphenol (NP) is an important environmental toxicant and potential endocrine disrupting chemical. The objective of these studies was to determine the effects of NP on epididymal rat sperm in vitro. Epididymal sperm samples from Sprague-Dawley rats were incubated in 1, 10, 100, 250, and 500 μg/ml NP for 1, 2, 3, or 4 h. Computer-assisted sperm analysis was used to determine motility. Epifluorescent microscopy was used to determine acrosomal status and flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity. Exposure of epididymal rat sperm to 250 or 500 μg/ml NP was highly detrimental to motility (P < 0.05), with complete loss of motility observed after exposure to 500 μg/ml NP (P < 0.05). The acrosomal integrity of sperm was significantly reduced with the lowest concentration (1 μg/ml) of NP, and higher concentrations resulted in a dose-dependent induction of the acrosomal reaction (P < 0.05). Similarly, the percentage of sperm with high MMP declined dramatically after exposure to 100, 250, and 500 μg/ml NP (P < 0.05). Duration of NP exposure did not have any effect on motility or MMP and NP did not appear to have detrimental effects on chromatin integrity (P > 0.05). These results indicate that major mechanism of action of NP on rat sperm is by adversely affecting their acrosomal integrity. However, NP-induced impaired sperm motility, decreased mitochondrial membrane potential also likely to play an important role in destruction of sperm function.     

Peroxisome proliferator-activated receptor gamma (PPARγ) in brown trout: Interference of estrogenic and androgenic inputs in primary hepatocytes

Peroxisome proliferator-activated receptor gamma (PPARγ) is a pivotal regulator of lipid and glucose metabolism in vertebrates. Here, we isolated and characterized for the first time the PPARγ gene from brown trout (Salmo trutta f. fario). Hormones have been reported to interfere with the regulatory function of PPARγ in various organisms, albeit with little focus on fish. Thus, primary hepatocytes isolated from juveniles of brown trout were exposed to 1, 10 and 50 μM of ethinylestradiol (EE2) or testosterone (T). A significant (3 fold) decrease was obtained in response to 50 μM of EE2 and to 10 and 50 μM of T (13 and 14 folds), while a 3 fold increase was observed at 1 μM of EE2. Therefore, trout PPARγ seems a target for natural/synthetic compounds with estrogenic or androgenic properties and so, we advocate considering PPARγ as another alert sensor gene when assessing the effects of sex-steroid endocrine disruptors.     

Sexual disruption in zebrafish (Danio rerio) exposed to mixtures of 17α-ethinylestradiol and 17β-trenbolone

Environmental estrogens and androgens can be present simultaneously in aquatic environments and thereby interact to disturb multiple physiological systems in organisms. Studies on interaction effects in fish of androgenic and estrogenic chemicals are limited. Therefore, the aim of the present study was to evaluate feminization and masculinization effects in zebrafish (Danio rerio) exposed to combinations of two synthetic steroid hormones detected in environmental waters: the androgen 17β-trenbolone (Tb) and the oestrogen 17α-ethinylestradiol (EE2). Juvenile zebrafish were exposed between days 20 and 60 post-hatch to different binary mixtures of Tb (1, 10, and 50 ng/L) and EE2 (2 and 5 ng/L). The endpoints studied were whole-body homogenate vitellogenin concentration at 40 days post-hatch, and sex ratio including gonad maturation at 60 days post-hatch. The feminizing potency of 5 ng/L of EE2, alone as well as in combination with Tb, was clear in the present study, with exposures resulting in almost all-female populations and females being sexually immature. Masculinization effects with male-biased sex ratios were observed when fish were exposed to 2 ng/L of EE2 in combination with Tb concentrations. Intersex fish were observed after exposure to mixtures of 2 ng/L EE2 with 50 ng/L Tb. Sexual maturity generally increased among males at increasing concentrations of Tb. The results of the present study show that exposure to environmentally relevant mixtures of an oestrogen and androgen affects the process of gonad differentiation in zebrafish and lead to sexual disruption.     

Estrogenicity of food-associated estrogenic compounds in the fetuses of female transgenic mice upon oral and IP maternal exposure

The present study investigated to what extent seven food-associated in vitro estrogenic compounds can induce estrogenic effects in the fetuses of pregnant female mice with an estrogen receptor (ER)-mediated luciferase (luc) reporter gene system. The luc-induction was determined either 8 h after maternal dosing with a single intraperitoneal (IP) dose or 24 h after the last of a series of 8 daily oral dosages. Three known estrogens, 17β-estradiol (E₂), 17α-ethynylestradiol (EE) and 17β-estradiol 3,17-dipropionate (EP) were used as positive controls at 1 mg/kg bw and DMSO as solvent control. The food-associated estrogenic compounds tested were: bisphenol A (BPA), nonylphenol (NP) both at 50 mg/kg bw, dichlorodiphenyldichloroethylene (p,p′-DDE) at 50 mg/kg bw, quercetin at 16.6 mg/kg bw, and di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 100 mg/kg bw. Exposure to E₂, EE and EP resulted in significant luc inductions upon both oral and/or IP dosing in a variety of tissues including liver, tibia and femurs, and upon IP dosing also in fetuses.BPA, NP, DEHA, DEHP, DIHP, DDE and quercetin were unable to significantly induce luc activity in fetuses. However, after maternal oral exposure during gestation to NP, BPA and DIHP placental luc activity was significantly lowered.The results indicate that at the current levels of exposure to food-associated estrogenic compounds, estrogenic effects to the fetus are not expected. The significant luc reduction in the placenta, should be further studied for its significance for fetal development and relevance for the human situation.     

Antidepressive behaviors induced by enriched environment might be modulated by glucocorticoid levels

Exposure to enriched environment (EE) can influence expression of depression symptoms, however, the underlying mechanism has not been established, although neurogenesis was probably involved. It has been reported that EE stimulates glucocorticoids release. However, the role of corticosterone (CORT) in effects of EE is still unknown. To address these issues, we examined depression-like behaviors of the animals exposed to EE with low dose CORT supplement following bilateral adrenalectomy (ADX + CORT). Two months after housing, tail suspension test and forced swim test were used to assess depression-related behavior of mice. Serum CORT levels were measured by radio-immunoassay. Signals of DNA synthesis marker bromodeoxyuridine and immature neuronal marker doublecortin were measured by immunohistochemistry. Results showed that EE significantly decreased immobility time of the mice in both the tail suspension test and forced swim test, showing distinctive antidepressive behaviors. Exposure to EE also increased serum CORT level, and prevention of this increase with ADX + CORT eliminated the decrease of immobility time of the animals. Both the mice exposed to EE and those receiving ADX + CORT treatment showed enhanced newly born cells and immature granule neurons in the hippocampus. Taken together, our data suggest that glucocorticoids elevation is required for antidepressive behaviors of EE.     

Fish oil decreases inflammation and reduces cardiac remodeling in rosiglitazone treated aging mice

Clinical studies suggest that rosiglitazone (RSG) treatment may increase the incidence of heart failure in diabetic patients. In this study, we examined whether a high corn oil diet with RSG treatment in insulin resistant aging mice exerted metabolic and pro-inflammatory effects that stimulate cardiac dysfunction. We also evaluated whether fish oil attenuated these effects. Female C57BL/6J mice (13 months old) were divided into 5 groups: (1) lean control (LC), (2) corn oil, (3) fish oil, (4) corn oil + RSG and (5) fish oil + RSG. Mice fed a corn oil enriched diet and RSG developed hypertrophy of the left ventricle (LV) and decreased fractional shortening, despite a significant increase in total body lean mass. In contrast, LV hypertrophy was prevented in RSG treated mice fed a fish oil enriched diet. Importantly, hyperglycemia was controlled in both RSG groups. Further, fish oil + RSG decreased LV expression of atrial and brain natriuretic peptides, fibronectin and the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α, concomitant with increased interleukin-10 and adiponectin levels compared to the corn oil + RSG group. Fish oil + RSG treatment suppressed inflammation, increased serum adiponectin, and improved fractional shortening, attenuating the cardiac remodeling seen in the corn oil + RSG diet fed C57BL/6J insulin resistant aging mice. Our results suggest that RSG treatment has context-dependent effects on cardiac remodeling and serves a negative cardiac role when given with a corn oil enriched diet.     

The effect of tramadol hydrochloride on early life stages of fish

The aim of this study was to perform the fish embryo acute toxicity test (FET) on zebrafish (Danio rerio) and the early-life stage toxicity test on common carp (Cyprinus carpio) with tramadol hydrochloride. The FET was performed using the method inspired by the OECD guideline 236. Newly fertilized zebrafish eggs were exposed to tramadol hydrochloride at concentrations of 10; 50; 100 and 200 μg/l for a period of 144 h. An embryo-larval toxicity test on C. carpio was performed according to OECD guideline 210 also with tramadol hydrochloride at concentrations 10; 50; 100 and 200 μg/l for a period of 32 days.Hatching was significantly influenced in both acute and subchronic toxicity assays. Subchronic exposure also influenced early ontogeny, both morphometric and condition characteristics and caused changes in antioxidant enzyme activity. The LOEC value was found to be 10 μg/l tramadol hydrochloride.     

High dietborne Cu and Cd induced genotoxicity of Nile tilapia (Oreochromis niloticus)

In this study, the effects of fish diet contaminated with Cu, Cd and Cu + Cd on Nile tilapia, was demonstrated by evaluating its bioaccumulation in the muscle and by testing the cytogenetic profile. Fish exposed to diet contaminated with Cu, Cd or their mixture had a significant increase in the number of chromosomal abnormalities and an inhibition of the mitotic index. Our study revealed high muscle Cu or Cd content in fish fed with diet contaminated with high dietborne Cu, Cd, Cu and Cd. It also revealed that the chromosomal abnormalities were higher for fish fed diet Cd contaminated and Cu + Cd contaminated diets than those fed diet Cu contaminated diet. Thus, maybe fish diets contaminated with Cu, Cd, Cu + Cd induced genotoxicity and mutation. Also, maybe high concentrations of Cu and Cd in fish tissue resulted from feeding on Cu and Cd contaminated diets, are dangerous for human consumption.     

Polymorphisms of endothelin 1 (G5665T and T-1370G) and endothelin receptor type A (C+70G and G-231A) in Graves' disease

PurposeEndothelin 1 (EDN1) is a strong angiogenic and mitogenic factor, playing a key role in hypervascularization, thyroid follicle cell hyperplasia, and lymphocyte infiltration in the thyroid gland of patients with Graves’ disease (GD). EDN1 induces angiogenesis and mitogenesis via endothelin receptor type A (EDNRA). This study examined the possible association of EDN1 (G5665T and T-1370G) and EDNRA (C + 70G and G-231A) single nucleotide polymorphisms (SNPs) with the occurrence of GD, and evaluates the relationship between genotypes and clinical/laboratory manifestations of GD.Materials and methodsWe analyzed genotype and allele distributions of EDN1 and EDNRA polymorphisms in 165 patients with GD and 181 healthy controls by real-time PCR combined with melting curve analysis.ResultsNo significant associations between GD and variant alleles of the studied polymorphisms were observed. However, the anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-TG) levels in EDN1 G5665T GG genotype were higher than those in T allele carriers (GT + TT) (p = 0.001 and p = 0.026, respectively). In addition, anti-TPO levels in EDN1 T-1370G wild-type homozygous patients were found to be higher than in mutant gene carrying patients (GT + GG) (p = 0.006). The presence of EDNRA + 70G allele was associated with 3.37-fold increased risk for development of ophthalmopathy in GD patients (p = 0.009).ConclusionAlthough there were no associations between EDN1 (G5665T and T-1370G) and EDNRA (C + 70G and G-231A) SNPs and susceptibility to GD, EDN1 G5665T and T-1370G polymorphisms were related to alterations of autoantibody production and EDNRA C + 70G polymorphism is related with increased risk for ophthalmopathy in GD patients.     

Hesperidin,a citrus bioflavonoid,alleviates trichloroethylene-induced oxidative stress in Drosophila melanogaster

Trichloroethylene (TCE) is a chlorinated organic pollutant of groundwater with diverse toxic effects in animals and humans. Here, we investigated the ameliorative role of hesperidin, a citrus bioflavonoid on TCE-induced toxicity in Drosophila melanogaster. Four groups of D. melanogaster (50 flies/vial, with 5 vials/group) were exposed to ethanol (2.5%, control), HSP (400 mg/10 g diet), TCE (10 μM/10 g diet) and TCE (10 μM/10 g diet) + HSP (400 mg/10 g diet) respectively in the diet for 5 days. Then, selected oxidative stress and antioxidant markers were evaluated. The results showed that TCE significantly increased the level of reactive oxygen species (ROS) and inhibited catalase, glutathione S-transferase and acetylcholinesterase (AChE) activities with concurrent depletion of total thiol level. However, co-administration of TCE and hesperidin mitigated TCE-induced depletion of antioxidants, and restored ROS level and AChE activity in the flies (p < 0.05). Overall, hesperidin offered protective potency on TCE-induced oxidative stress in the flies via anti-oxidative mechanism.     

Environmental estrogen(s) induced swimming behavioural alterations in adult zebrafish (Danio rerio)

The present study is an attempt to investigate the effects of long-term (75 days) exposure to environmental estrogens (EE) on the swimming behaviour of zebrafish (Danio rerio). Adult zebrafish were exposed semi-statically to media containing commonly detected estrogenic water contaminants (EE2, DES and BPA) at a concentration (5 ng/L) much lower than environmentally recorded levels. Time spent in swimming, surface preference, patterns and path of swimming were recorded (6 mins) for each fish using two video cameras on day 15, 30 60 and 75. Video clips were analysed using a software program. Results indicate that chronic exposure to EE leads to increased body weight and size of females, reduced (P < 0.05) swimming time, delay in latency, increased (P < 0.05) immobility, erratic movements and freezing episodes. We conclude that estrogenic contamination of natural aquatic systems induces alterations in locomotor behaviour and associated physiological disturbances in inhabitant fish fauna.     

Drinking of flavored solutions by high preferring (WHP) and low preferring (WLP) alcohol-drinking rats

BackgroundSelective breeding alcohol-preferring (P) and alcohol-nonpreferring (NP) rats showed a strong preference for the sucrose solutions, whereas P rats intake greater amounts than NP rats.The aim of this study was the estimation of selectively bred ethanol-preferring (WHP – Warsaw High Preferring) and ethanol-nonpreferring (WLP – Warsaw Low Preferring) rats for their preference for various tastes.MethodsThe oral drinking of the following substances was studied at a range of concentrations: sucrose (0.5–64.0 g/100 ml), NaCl (0.025–3.2 g/100 ml), citric acid (0.008–2.048 g/l), and sucrose octaacetate (0.002–0.512 g/l) solutions. Separate groups of 7–8 rats from each line were investigated of each of the four tastes. The investigated solutions were presented continuously keeping water and food always available. Concentrations of the various flavors were doubled every 48 h.ResultsRats from WHP and WLP lines clearly revealed the preference for the sucrose solution and the highest preference was at the 4.0 and 8.0 g/100 ml sucrose concentration. Similar to sucrose, both lines exposed strong preference for the NaCl solution and this preference enhanced together with the increase of the NaCl concentration. Nevertheless their preference for the NaCl solutions decreased when the concentration of NaCl reached 1.600 g/100 ml. Both lines of rats did not differ in citric acid or sucrose octaacetate intake at any of the concentrations studied.ConclusionSelective breeding of rats (WHP) for high and rats (WLP) for low ethanol drinking is favorably correlated with the drinking of sweet and salty solutions and negatively correlated with the consumption of sour and bitter tastes.     

Window of sensitivity for the estrogenic effects of ethinylestradiol in early life-stages of fathead minnow,Pimephales promelas

Sexual differentiation in fish occurs after hatching during early life-stages and is believed to be a time when the gonad has a heightened sensitivity to disruption by chemicals that mimic hormones. In this study fathead minnows (Pimephales promelas) were exposed to an environmentally relevant concentration of ethinylestradiol (EE2) for short intervals in fish early life-stages and vitellogenic and gonadal responses were measured at 30 and 100 dph (sexual maturity), respectively. All EE2 exposure regimes induced vitellogenin (VTG) synthesis and disruption in duct development (a feminization) in males, with a window of enhanced sensitivity between 10 and 15 dph (where 60% of the males had feminized ducts). There was an altered pattern in sex cell development in males (inhibition of spermatogenesis) in the solvent controls (ethanol 0.1 ml/l) and all EE2 treatments when compared with the dilution water controls. Furthermore, fewer spermatozoa were observed in the testis of males exposed to EE2 from 15 to 20 dph and fertilized eggs (<24 h post-fertilization)-20 dph, compared with both the solvent and dilution water controls. These data show that short exposures of embryos/very early life-stage fathead minnows to an environmentally relevant concentration of EE2 lead to alterations in gonadal development that potentially have reproductive consequences and thus population level effects.     

The potential effects of efavirenz on Oreochromis mossambicus after acute exposure

Antiretroviral drugs (ARVs) are hazardous therapeutic pharmaceuticals present in South African surface water. Efavirenz is an ARV commonly used in human immunodeficiency virus (HIV) treatment in South Africa. Although little is known about the toxic effects of efavirenz on fish health, threats of toxicity to the aquatic environment have been reported. Oreochromis mossambicus were exposed under controlled conditions to environmentally-relevant efavirenz concentrations (10.3 ng/l) as measured in rivers that flow into the Nandoni Dam in the Vhembe District, South Africa. Acute (96 h) exposures were conducted using efavirenz concentrations of 10.3 ng/l and 20.6 ng/l. The overall health of exposed fish was determined using a histology-based fish health assessment index. Necropsies and haematology were conducted and somatic indices calculated after which the liver, kidney, heart, gills and gonads were microscopically quantitatively assessed. Results indicated that fish exposed to 20.6 ng/l efavirenz had significantly (p < 0.02) higher liver indices than the control fish, indicating increased liver damage including steatosis and frank necrosis. Fish exposed to 20.6 ng/l efavirenz presented with significantly (p < 0.02) higher total fish indices, representative of declined overall health compared to control fish. It was concluded that the exposure of O. mossambicus to efavirenz resulted in liver damage and overall decline in fish health. These novel findings may indicate a health risk for O. mossambicus and other biota exposed to efavirenz in aquatic ecosystems. Thus, ARV’s in water sources of South Africa pose a definite threat to wildlife and ultimately human health.     

Transient effects of 2,4-dichlorophenoxyacetic acid (2,4-D) exposure on some metabolic and free radical processes in goldfish white muscle

This study aims to assess effects of 96 h goldfish exposure to 1, 10 and 100 mg/L of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), on metabolic indices and free radical process markers in white muscle of a commercial fish, the goldfish Carassius auratus L. Most oxidative stress markers and antioxidant enzymes were not affected at 2,4-D fish treatment. 2,4-D fish exposure induced the elevated levels of total (by 46% and 40%) and reduced (by 77% and 73%) glutathione in muscles of goldfish of 10 mg/L 2,4-D and recovery (after 100 mg/L of 2,4-D exposure) groups, respectively. However, in muscles of 100 mg/L 2,4-D exposed goldfish these parameters were depleted (by 47% and 64%). None of investigated parameters of protein and carbohydrate metabolisms changed in white muscles of 2,4-D exposed fish, with exception of lactate dehydrogenase activity, which was slightly (by 11–15%) elevated in muscles of goldfish exposed to 10–100 mg/L of 2,4-D, but also recovered. Thus, the short term exposure of goldfish to the selected concentrations of 2,4-D does not substantially affect their white muscle, suggesting the absence of any effect under the environmentally relevant concentrations.