Delayed effects of embryonic exposure of zebrafish (Danio rerio) to methylmercury (MeHg)

Since previous short-term bioassays of methylmercury (MeHg) indicated no morphological effects in zebrafish (Danio rerio) after embryonic exposures below 20 microg/l MeHg, studies were done to determine whether embryonic exposure to MeHg at lower concentrations would induce behavioral effects. Newly fertilized embryos were exposed to 0, 5, 10 or 15 microg MeHg/l for selected exposure durations: single day, multiple day or continuous exposure from fertilization through hatching. Larvae were maintained in an essential salt solution after hatching. Spontaneous swimming performance and prey capture experiments were conducted. Continuous embryonic exposure to 15 microg/l caused delayed mortality syndrome (DMS). These larvae hatched normally and appeared normal, but beginning at Day 3 post-hatch (ph), general activity was severely reduced and by Day 5 ph, larvae were completely moribund; many had faint heartbeats, severely enlarged body cavities and upward flexures of the spinal cord. Most of these larvae were dead by Day 6 ph. Multi- and single-day embryonic exposures to 15 microg/l caused reduced swimming activity and prey capture ability, and by Day 4 ph, these larvae also began to show signs of DMS. Continuous embryonic exposure to 10 microg/l significantly reduced spontaneous swimming activity, which did not improve after 5 days in clean water. Similar results were seen in larvae exposed during the last 24 h of embryonic development. Prey capture ability was also impaired in larvae exposed continuously to 10 microg/l, even after 4 days in clean water. Single-day exposures to 10 microg/l did not affect prey capture ability. Larvae from the 5-microg/l exposures were not significantly different from controls for either parameter. This study reinforces the idea that functional impairment is a more subtle response to developmental toxicants than mortality or the production of morphological defects.     

Estrogenicity of butylparaben in rainbow trout Oncorhynchus mykiss exposed via food and water

The estrogenic effect of butylparaben was investigated in a rainbow trout Oncorhynchus mykiss test system. Butylparaben was administered orally to sexually immature rainbow trout every second day for up to 10 days in doses between 4 and 74 mgkg(-1)2d(-1) and in the water at 35 and 201 microgl(-1) for 12 days. Plasma vitellogenin was measured before and during the exposures and the concentrations of butylparaben in liver and muscle were determined at the end of experiments. Increases in average plasma vitellogenin levels were seen at oral exposure to 9 mg butylparaben kg(-1)2d(-1). The ED50 values for increase in vitellogenin synthesis were 46, 29 and 10.5 mg butylparaben kg(-1)2d(-1), respectively, at day 3, 6 and 12. Exposure to 201 microg butylparaben l(-1) increased vitellogenin synthesis, but exposure to 35 microgl(-1) did not. Butylparaben showed little tendency to bioaccumulation in rainbow trout; less than 1 per thousand of the total amount of butylparaben administered orally at 51 mgkg(-1)2d(-1) over the 12 days experimental period was retained in liver at the end of the experiment. After 12 days exposure to 35 and 201 microg butylparaben l(-1), plasma concentrations were 9 and 183 microgl(-1), respectively, and for the fish exposed to 201 microgl(-1) there was a positive correlation between concentrations of vitellogenin and butylparaben in the plasma. On the assumption that butylparaben removed from the water phase during water exposure were taken up into the fish, butylparaben uptake rates in the fish exposed to 35 and 201 microg butylparaben l(-1) were 13 and 78 mgkg(-1)day(-1), respectively.     

The effect of intraperitoneally administered microcystin-LR on the gastrointestinal tract of Balb/c mice.

Microcystin-LR (MCLR) has been associated with the development of gastrointestinal complaints in people ingesting cyanobacterial bloom contaminated water. The symptoms usually present a day or two after exposure raising questions as to the toxic effects of MCLR on the gastrointestinal tract. This study investigated the apoptotic effect of ip administered MCLR over time on the duodenum, jejenum and ileum of mice receiving a single 75% LD(50) dose. The apoptotic index was significantly raised in all sections at 8 h post exposure (pe) and continued to rise for the 16, 24 and 32 h pe groups, while the glycogen levels were normal at 24 h pe. The duodenum exhibited the most significant increase in apoptotic index overall, followed by the jejenum and ileum. Immunohistochemistry indicated the presence of MCLR in the lamina propria suggesting a role for MCLR in the induction of apoptosis in the GIT of mice exposed to a single sublethal dose of MCLR.     

A combination of budesonide and the SH-metabolite I of erdosteine acts synergistically in reducing chemiluminescence during human neutrophil respiratory burst

Activated neutrophils can release superoxide anion and nitric oxide (NO), which subsequently combine with each other to yield peroxynitrite anions, powerful and harmful oxidants that preferentially mediate the oxidation of the thiol groups in proteins and non-protein molecules. These oxidants play a direct role in the inflammatory process in chronic obstructive pulmonary disease and asthma by increasing the number of neutrophils and macrophages that induce a self-sustaining phlogogenic loop. Budesonide (BUD) and erdosteine (a muco-active drug which, after metabolization, produces an active metabolite (Met I) with a sulfhydryl group) are both active in reducing the release of superoxide anion, NO and peroxynitrite, and can be administered to patients with respiratory diseases. The aim of this study was to investigate the possible synergistic in vitro effect of BUD and Met I on chemiluminescence generation during fMLP-stimulated respiratory bursts of human neutrophils with the NO donor L-arginine, added to the incubating medium. The investigated BUD concentrations ranged from 6 x 10(-8) to 1 x 10(-6) mol/l in logarithmic scale and a significant and progressive reduction in luminol-amplified chemiluminescence (LACL) was observed at concentrations ranging from 2.5 x 10(-7) to 1 x 10(-6) mol/l. The investigated concentrations of Met I varied from 0.62 to 10 microg/ml. No significant changes were observed at 0.62, 1.25, and 2.5 microg/ml, but a significant decrease in LACL was observed at 5 and 10 microg/ml. When the two drugs were combined, there was a greater significant decrease in LACL versus the single drugs with the combinations of BUD 1 x 10(-6) mol/l plus Met I 10 microg/ml, BUD 5 x 10(-7) mol/l plus Met I 5 microg/ml, BUD 2.5 x 10(-7) mol/l plus Met I 2.5 microg/ml, and BUD 1.25 x 10(-7) mol/l plus Met I 1.25 microg/ml. A further interesting finding was that the combination of BUD 2.5 x 10(-7) mol/l plus Met I 2.5 microg/ml and BUD 1.25 x 10(-7) mol/l plus Met I 1.25 microg/ml significantly decreased LACL, whereas the single concentrations had no significant effect, thus indicating the possibility of extending the duration of the effect. Our findings indicate a synergistic antioxidant effect when BUD and Met I are given together, which is of interest for counteracting the airway phlogosis involved in many respiratory diseases.     

Decreased hepatic ALT synthesis is an outcome of subchronic microcystin-LR toxicity

Alanine aminotransferase (ALT; EC 2.6.1.2) is important for the transamination of amino acids and is also an important serum marker of hepatic damage. However, we had previously shown that hepatic ALT activity decreases with subchronic exposure to the hepatotoxin, microcystin-LR (MCLR), a potent inhibitor of serine/threonine protein phosphatases types 1 and 2A. These previous findings suggest that one outcome of subchronic MCLR toxicosis is decreased ALT synthesis by hepatocytes. This could affect the diagnostic sensitivity of serum ALT activity and metabolic processes within the cell. This study was done to investigate the mechanism by which ALT activity decreases following prolonged MCLR exposure. Immunoblots were first performed on liver tissue from 12 Harlan-Sprague-Dawley rats that had been treated with 0, 16, 32, or 48 microg/kg of microcystin-LR per day by continuous intraperitoneal infusion for 28 days. These revealed a dose-dependent decrease in ALT protein concentrations that correlated directly with hepatic ALT activity (r = 0.8132; P = 0.0013). Sixteen additional rats, treated with the same doses of MCLR showed a dose-dependent decrease in hepatic ALT activity to approximately 19% of values in saline-treated controls. Northern blot analysis revealed a decrease in hepatic ALT mRNA that correlated directly to hepatic ALT activity (r = 0.7909; P = 0.0004). It was concluded that subchronic MCLR exposure causes decreased hepatic ALT protein and mRNA concentrations. These findings suggest that one sequela of MCLR toxicosis is decreased hepatic ALT synthesis.     

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters retinoid homeostasis in maternal and perinatal tissues of the Holtzman rat

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most widely studied environmental contaminants, causes a variety of adverse health effects including teratogenesis and altered development which may be related to disruptions in retinoid homeostasis. The purpose of this study was to determine the effect that gestational administration of TCDD has on retinoid homeostasis in both pregnant Holtzman rats and developing fetuses and neonates. A single oral dose of TCDD (0, 1.5, 3, or 6 microg/kg) was administered to pregnant rats on gestation day 10, with fetuses analyzed on gestation days 17 and 20, and neonates analyzed on post natal day 7. Exposure to TCDD generally produced decreases in the concentrations of retinyl esters, such as retinyl palmitate, and retinol in maternal and perinatal liver and lung, while increasing levels in the maternal kidney. Additionally, perinatal hepatic retinol binding protein 1-dependent retinyl ester hydrolysis was also decrease by TCDD. Sensitivity of the developing perinates to TCDD appeared to have an age-related component demonstrated by an increased rate of mortality and significant alterations to body weight and length on post natal day 7 relative to that observed at gestation day 20. A unique observation made in this study was a significant decrease in lung weight observed in the perinates exposed to TCDD. Taken together, these data demonstrate that TCDD significantly alters retinoid homeostasis in tissues of the developing fetus and neonate, suggesting that their unique sensitivity to TCDD may at least be in part the result of altered retinoid homeostasis.     

Inhaled particle-bound sulfate: effects on pulmonary inflammatory responses and alveolar macrophage function

Acid sulfate-coated solid particles are a significant environmental hazard produced primarily by the combustion of fossil fuels. We have previously described a system for the nascent generation of carbonaceous particles surface coated with approximately 140 microg/m(3) acid sulfate [cpSO(4)(2-); 10 mg/m(3) carbon black (CB) and 10 ppm sulfur dioxide (SO(2)) at 85% relative humidity (RH)]. The effects of inhaled cpSO(4)(2-) on pulmonary host defenses are assessed in the present work. Mice were acutely exposed (4 h) to either 10 mg/m(3) CB, 10 ppm SO(2), or their combination at 10% or 85% RH in a nose-only inhalation chamber. No evidence of an inflammatory response was found following any of the exposures as assessed by total cell counts and differential cell counts from bronchoalveolar lavage fluid. However, alveolar macrophage Fc receptor-mediated phagocytosis decreased only following exposure to 140 microg cpSO(4)(2-), significant suppression occurred after 24 h, maximal suppression occurred at 3 days postexposure, and recovery to preexposure levels required 7-14 days. Intrapulmonary bactericidal activity (IBA) was also suppressed only after exposure to 140 microg cpSO(4)(2-); suppression was maximal at 1 day postexposure and recovered by day 7. To assess the effects of lower cpSO(4)(2-) concentrations, mice were repeatedly exposed to 1 mg/m(3) CB and 1 ppm SO(2) at 85% RH ( approximately 20 microg/m(3) cpSO(4)(2-) for 4 h/day) for up to 6 days. A significant decrement in IBA was observed following 5 and 6 days of exposure. These studies indicated that acute or repeated exposure to cpSO(4)(2-) could alter pulmonary host defense mechanisms.     

Pulmonary responses of acute exposure to ultrafine iron particles in healthy adult rats

As critical constituents of ambient particulate matter, transition metals such as iron may play an important role in health outcomes associated with air pollution. The purpose of this study was to determine the respiratory effects of inhaled ultrafine iron particles in rats. Sprague Dawley rats 10-12 weeks of age were exposed by inhalation to iron particles (57 and 90 microg/m(3), respectively) or filtered air (FA) for 6 h/day for 3 days. The median diameter of particles generated was 72 nm. Exposure to iron particles at a concentration of 90 microg/m(3) resulted in a significant decrease in total antioxidant power along with a significant induction in ferritin expression, GST activity, and IL-1beta levels in lungs compared with lungs of the FA control or of animals exposed to iron particles at 57 microg/m(3). NFkappaB-DNA binding activity was elevated 1.3-fold compared with that of control animals following exposure to 90 microg/m(3) of iron, but this change was not statistically significant. We concluded that inhalation of iron particles leads to oxidative stress associated with a proinflammatory response in a dose-dependent manner. The activation of NFkappaB may be involved in iron-induced respiratory responses, but further studies are merited.     

Fate and toxic effects of inhaled ultrafine cadmium oxide particles in the rat lung

Female Fischer 344 rats were exposed to ultrafine cadmium oxide particles, generated by spark discharging, for 6 h at a concentration of 70 microg Cd/m(3) (1 x 10(6)/cm(3)) (40 nm modal diameter). Lung morphology and quantification of Cd content/concentration by inductively coupled plasma (ICP)-mass spectrometry were performed on days 0, 1, 4, and 7 after exposure. Cd content in the lung on day 0 was 0.53 +/- 0.12 microg/lung, corresponding to 19% of the estimated total inhaled cumulative dose, and the amount remained constant throughout the study. In the liver no significant increase of Cd content was found up to 4 days. A slight but statistically significant increase was observed in the liver on day 7. We found neither exposure-related morphological changes of lungs nor inflammatory responses in lavaged cells. Another group of rats were exposed to a higher concentration of ultrafine CdO particles (550 microg Cd/m(3) for 6 h, 51 nm modal diameter). The rats were sacrificed immediately and 1 day after exposure. The lavage study performed on day 0 showed an increase in the percentage of neutrophils. Multifocal alveolar inflammation was seen histologically on day 0 and day 1. Although the Cd content in the lung was comparable between day 0 and day 1 (3.9 microg/lung), significant elevation of Cd levels in the liver and kidneys was observed on both days. Two of 4 rats examined on day 0 showed elevation of blood cadmium, indicating systemic translocation of a fraction of deposited Cd from the lung in this group. These results and comparison with reported data using fine CdO particles indicate that inhalation of ultrafine CdO particles results in efficient deposition in the rat lung. With regard to the deposition dose, adverse health effects of ultrafine CdO and fine CdO appear to be comparable. Apparent systemic translocation of Cd took place only in animals exposed to a high concentration that induced lung injury.     

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.     

Inhibition of nuclear protein phosphatase activity in mouse hepatocytes by the cyanobacterial toxin microcystin-LR.

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin and protein phosphatase inhibitor that contaminates water reservoirs worldwide. MCLR localizes to the cytosol of hepatocytes, however, immunohistochemical studies indicate that it accumulates in the nucleus. MCLR toxicosis is associated with decreased hepatic protein phosphatase activity, but effects in nuclear protein phosphatase activity have not been investigated. Balb/c mice were given lethal (100 microg/kg) or sublethal (12, 23 and 45 microg/kg) i.p. doses of MCLR and hepatic nuclear extracts were analyzed for protein phosphatase 1 and 2A activity. There was profound inhibition of nuclear protein phosphatase activity within 50 min of lethal dosing, however an inhibition was not detected with sublethal doses. MCLR immunohistochemistry revealed widespread lobular staining in the lethal group and centrilobular staining in the sublethal groups. At the cellular level there was nuclear and cytoplasmic staining of equal intensity. As an indicator of nuclear protein phosphatase activity, the phosphorylation of p53, a nuclear phosphoprotein and known substrate for protein phosphatases 1 and 2A, was evaluated. Balb/c mice were treated with sublethal doses of MCLR or saline vehicle after induction of hepatic p53 by the DNA damaging agent diethylnitrosamine (DEN). P53 was immunoprecipitated and probed with phosphoserine specific antibodies by Western blotting. There was greater phosphoserine reactivity of p53 protein in animals treated with MCLR relative to saline treated controls, consistent with increased phosphorylation of serine sites. It is concluded that an interaction of this toxin with nuclear protein phosphatases occurs within 50 min of lethal dosing, which leads to a profound inhibition of enzymatic activity. Even sublethal doses of MCLR that do not result in significant inhibition of activity in bulk nuclei, result in detectable changes in phosphorylation of p53.     

The ultraviolet filter 3-benzylidene camphor adversely affects reproduction in fathead minnow (Pimephales promelas).

The ultraviolet (UV) filter 3-benzylidene camphor (3BC) is used in personal care products and in a number of materials for UV protection. 3BC has been shown in vitro and in vivo in fish to be estrogenic, but possible effects on fertility and reproduction are unknown. In this study we evaluate whether 3BC affects reproduction of fish Pimephales promelas. After a preexposure period of 21 days, reproductively mature fathead minnows were exposed to increasing concentrations of 3BC for 21 days in a static-renewal procedure. Actual 3BC concentrations decreased to 23% of initial levels and median concentrations were 0.5, 3, 33, 74, and 285 microg/l. 3BC affected reproduction in a dose-dependent manner with weak effects on fecundity at 3 microg/l, a significant decrease at 74 microg/l, and a cessation of reproduction at 285 microg/l. 3BC was accumulated in fish with an average bioconcentration factor of 313 +/- 151. Dose-dependent demasculinization in secondary sex characteristics of male fish and dose-dependent induction of plasma vitellogenin occurred, which was significant at 74 microg/l and higher. 3BC had a profound and dose-dependent effect on the histology of gonads of male and female fish at 3 microg/l and higher. At 74 and 285 microg/l, oocyte and spermatocyte development was inhibited in male and female gonads. Testes of exposed males had much fewer spermatogenic cysts, and ovaries of exposed females had much fewer mature but more atretic, follicles. This study shows significant effects of the UV filter 3BC on fertility, gonadal development, and reproduction of fish after short-term exposure that may have negative consequences on the population level.     

Effects of pre-acclimation to aluminium on the physiology and swimming behaviour of juvenile rainbow trout (Oncorhynchus mykiss) during a pulsed exposure

Anthropogenic acidification of the freshwater environment causes aluminium to be mobilised into the aquatic environment. When pH falls below 5.5, exposure to aluminium concentrations as low as 12.5 microg.l(-1) can cause serious physiological disturbances in freshwater fish. However, under constant laboratory exposures fish can acclimate and recover physiological status within 5-30 days. In reality, fish in the wild are likely to experience chronic sub-lethal exposure, with occasional elevations (pulses) to much higher levels. The experiment described here investigated the effects of an environmentally realistic, 4-day pulse exposure to a high level of aluminium (36 microg.l(-1)) in two groups of juvenile rainbow trout. One group was exposed to a lower level of aluminium (24 microg.l(-1)) for 16 days before and 10 days after the pulse ('aluminium-acclimated' fish). A second group was exposed to pH 5.2 alone for 16 days before and 10 days after the pulse ('aluminium-na?ve' fish). A third group exposed to pH 5.2 alone for 30 days (no aluminium added) acted as controls. Triplicate groups of 24 juvenile rainbow trout (2.3-16.7 g) were randomly allocated to one of these three treatments. Swimming behaviour was monitored throughout and samples were taken on days 14, 20, 22, 26 and 30 for assessment of physiological status. No treatment effects were recorded in the control group (pH 5.2 alone). Fish in the 'aluminium-acclimated' treatment became hypo-active upon initiation of the exposure to 24 microg.l(-1) aluminium, but recovered after just 4 days of this exposure. Subsequent challenge on day 16 with the 36 microg.l(-1) aluminium 'pulse' caused these fish to became hypo-active again, but they recovered normal swimming behaviour whilst still subject to the 4-day pulse. The 'aluminium-na?ve' fish also became hypo-active during the pulse exposure (36 microg.l(-1) aluminium). However, they did not exhibit any recovery of swimming behaviour, either during the pulse, or even 6 days after the cessation of the pulse, despite a rapid depuration of gill aluminium load (within 2 days of the pulse finishing). Mortality was low in the aluminium-acclimated fish (4%) and significantly higher in the aluminium-na?ve fish (26%). Haematological disturbances were most extreme in the aluminium-na?ve fish and had not recovered to control levels 6 days after the end of the pulse. This study provides new evidence, using behavioural responses, that previous exposure to low levels of aluminium may be an important factor abating the impact of aluminium on fish in the natural environment.     

Interactions between cytochromes P450, glutathione S-transferases and Ghanaian medicinal plants

Inhibition of cytochrome P450s (CYPs) is a major cause of adverse drug-drug interactions. Alternatively, inhibition of glutathione S-transferases (GSTs) may increase harmful effects of electrophilic compounds or metabolites. In the present study, aqueous extracts of seven Ghanaian medicinal plants were investigated for their inhibitory potential towards recombinant human CYP1A2, CYP2C9, CYP2D6 and CYP3A4, heterologously expressed in Escherichia coli. Effects of these extracts on recombinant human GSTA1-1, GSTM1-1, GSTP1-1, human and rat cytosolic GSTs were also investigated. Seven extracts, including Phyllanthus amarus whole plant, leaf, stem and root, Cassia siamea and Momordica charantia, inhibited CYP1A2 and CYP2C9 with IC50 values ranging between 28.3-134.3microg/ml and between 63.4-425.9microg/ml, respectively. Similarly, both CYP2D6 and CYP3A4 were inhibited by five extracts including Phyllanthus amarus whole plant, leaf, stem and root and Cassia alata, with IC50 values ranging between 45.8-182.0microg/ml and between 79.2-158.8microg/ml respectively. Human and rat liver cytosolic GSTs were inhibited with IC50 values ranging between 25.2-95.5microg/ml and between 8.5-139.4microg/ml, respectively. GSTM1-1 was most susceptible to the inhibition by the extracts, with IC50 values ranging between 3.6-50.0microg/ml, whilst IC50 values of 8.9-159.0microg/ml and 68.6-157.0microg/ml were obtained for GSTA1-1 and GSTP1-1, respectively. These findings show a significant potential both for CYP- and GST-mediated herb-drug interactions of the Ghanaian medicinal plants investigated.     

Route of exposure affects the oestrogenic response of fish to 4-tert-nonylphenol

When toxicants cause effects to aquatic organisms, it is often unclear by what route, or routes, the toxicant entered the affected organism. The toxicity of a compound depends on its degree of uptake, distribution and metabolism, as well as its molecular interactions at the site of action. It was hypothesised, that a hydrophobic chemical such as 4-tert-nonylphenol (4-NP), entering via the gills/skin, may be more oestrogenic than one entering through the diet, because in the latter case it will undergo metabolism in the small intestine and liver before entering the bloodstream. In this way, metabolism may reduce or eliminate the oestrogenic potential of 4-NP before it reaches target organs such as the gonads or liver. To compare the potency of 4-tert-nonylphenol when administered via different routes, male fathead minnows (Pimephales promelas) were exposed to 4-NP either through waterborne exposure (experiment 1), or via the diet (experiment 2). Fish were exposed to 4-NP for 2 weeks either via the water at one of three nominal concentrations: 1, 10 or 50 microg/l (experiment 1) or 100, 500 or 1000 microg/day via the diet (experiment 2). Liver and blood samples were taken for vitellogenin mRNA and plasma vitellogenin quantification, respectively.Exposure of male fathead minnows to 50 microg/l of 4-NP in the water (experiment 1) and 500 and 1000 microg/day of 4-NP via the diet (experiment 2) induced vitellogenin mRNA. A similar pattern occurred for plasma vitellogenin induction, however, there was also a significant increase in plasma vitellogenin concentration in the fish exposed via the water to 10 microg/l of 4-NP.Using data from pharmacokinetics studies, an estimate for the total amount of 4-NP that entered the fish during each exposure was compared with the concentrations of plasma vitellogenin in each group of fish.The result showed a 10-fold greater sensitivity for 4-NP in fish exposed via the water compared with exposure via the oral route.Results obtained from this study indicate that a chemical such as 4-NP has a higher oestrogenic potential when it enters the bloodstream via the gills/skin of a fish compared with exposure through the diet.     

Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants

Epidemiological studies on arsenic contamination in drinking water indicated presence of arsenic in fetal tissues. Experiments on human fetal brain explants on exposure to arsenic in culture showed disturbance in lipid peroxidation, generation of nitric oxide (NO), reactive oxygen species (ROS) and apoptosis. The oxidative stress challenged by antioxidant vitamins C, E or chelator dimercaptosuccinic acid (DMSA) may reverse arsenic toxicity on neuronal development. The concept was tested with the models: (A) human fetal brain explants exposed to arsenic, 0.3 mg/l in culture for 24 h; (B) rat neonatal brain explants from 1-day-old litters exposed to 0.3 mg/l arsenic in drinking water during gestation. Rats (n=10) were given oral administration of vitamin C, 2.5 mg/kg/day, vitamin E, 148 microg/kg/day during gestation and DMSA, 50 mg/kg for 2 days at the end of gestation. (A) The arsenic induced in human fetal brain explants increase in production of NO, 20% and ROS, 25%, and decrease in DNA, 62% and protein, 54% synthesis. The morphological analyses showed growth of viable cells, neural networking vis-à-vis apoptosis on exposure to arsenic for 24 h and necrosis and loss of ground matrix on arsenic exposure for 18 days. The occurrence of two processes of apoptosis and necrosis in different neurons of same culture indicated existence of a selective cellular defense against arsenic toxicity. (B) The rats exposed to arsenic showed increased generation of NO, 25% and ROS, 22%, loss of glutathione content from 42 to 35 microg/mg protein, 40% increase in lipid peroxidation and decreased superoxide dismutase at 32%. The administration of vitamins C, E and DMSA showed partial reversal of the effects indicating possible protection from arsenic toxicity.     

Impairment of mysid (Neomysis integer) swimming ability: an environmentally realistic assessment of the impact of cadmium exposure

Neomysis integer (Crustacea: Mysidacea), a euryhaline member of the hyperbenthos of the upper reaches of European estuaries, has been identified as a suitable animal for assessing the impacts of chemical pollutants on these estuarine regions. In this study, the effect of a 7 day pre-exposure to environmentally relevant concentrations of cadmium (0.5 and 1.0 microg l(-1)) on the swimming behaviour of N. integer was examined using an annular flume. Cadmium speciation at two salinities (1 and 10 per thousand) that dominate these upper estuarine regions was modelled to ensure mysids were exposed to the same concentration of the toxic free-ion at each salinity. There was no significant difference in the swimming behaviour of mysids exposed to the same free-ion cadmium concentration at the two different salinities. At each salinity, exposure to 0.5 microg Cd2+ (aq) l(-1)resulted in fewer mysids moving forward into the current (normal behaviour) at free stream velocities typical of their natural habitat (e.g. 3-9 cm s(-1)) than non cadmium-exposed mysids. At these low current speeds, cadmium-exposed mysids were either able to maintain position or were swept by the current. The same general responses were recorded for mysids exposed to 1.0 microg Cd2+ (aq) l(-1)except that more mysids showed disrupted swimming ability compared with 0.5 microg Cd2+ (aq) l(-1). At higher current speeds (>12 cm s(-1)), current velocity was the dominant factor affecting mysid swimming behaviour and there was no effect of cadmium on mysids maintaining position. Exposure to cadmium also caused significant disruption of the hyperbenthic behaviour of N. integer and more cadmium-exposed individuals were in the water column than control mysids; this result was more variable at 10 per thousand than 1 per thousand. Results indicate that exposure to cadmium concentrations of 0.5 microg Cd2+(aq) l(-1)would result in displacement of N. integer from its optimum region within the estuarine environment. This conclusion would not be achieved from standard LC(50) tests (e.g. 7 day LC50 = 2.95 microg Cd2+ (aq) l(-1)), highlighting the value of behavioural disruption as a sensitive indicator of environmental chemical contamination.     

Cadmium affects the social behaviour of rainbow trout,Oncorhynchus mykiss

The present study investigated both the effects of cadmium on the social interactions of rainbow trout and the differential accumulation of waterborne cadmium among social ranks of fish. Fish exposed to waterborne cadmium concentrations of 2 microg l(-1) for 24 h, followed by a 1, 2 or 3 day depuration period in clean water, had a decreased ability to compete with non-exposed fish. However, the competitive ability of exposed fish given a 5 day depuration period was not significantly impaired. Cadmium accumulated in the olfactory apparatus of fish exposed to waterborne cadmium for 24 h and decreased significantly only after 5 days depuration in clean water. Among groups of ten fish held in stream tanks, where all fish were exposed to cadmium, there were significant effects on social behaviour and growth rate. Dominance hierarchies formed faster among fish exposed to cadmium than among control fish, and overall growth rates were higher in the cadmium treatment. In groups of ten fish, social status also affected tissue accumulation of cadmium during waterborne exposure, with dominant fish accumulating more cadmium at the gill. In conclusion, exposure to low levels of cadmium, affects the social behaviour of fish, in part due to accumulation in the olfactory apparatus, and dominant fish accumulate more gill cadmium than subordinates during chronic waterborne exposure.     

Tissue differences,dose-response relationship and persistence of DNA adducts in blue mussels (Mytilus edulis L) exposed to benzo[a]pyrene

Baltic Sea blue mussels (Mytilus edulis) were experimentally exposed to the genotoxic model substance benzo[a]pyrene (B[a]P) to study DNA adduct formation. The specific aims were (a). to examine where in the mussels the DNA adducts were formed, in gills or digestive gland; (b). to study the dose-response relationship between B[a]P exposure and DNA adduct formation; and (c). to examine the persistence of the formed adducts. A Scope for growth (SFG) study was also run to compare physiological responses of the mussels with the degree of DNA adduct formation. In an initial dose-response experiment, the mussels were exposed to 0, 5, 50, and 100 microg/l of tritium labelled B[a]P under semi-static conditions for 4 days, and thereafter the bioaccumulation of B[a]P and DNA adduct formation in different tissues was determined using liquid scintillation counting and 32P-postlabelling analysis, respectively. In a following exposure-depuration experiment, mussels were exposed to 17 microg/l of radiolabelled B[a]P under semi-static conditions for 6 days. B[a]P accumulation and DNA adduct formation were determined during the exposure, and B[a]P elimination and persistence of DNA adducts were studied during 28 days of depuration in uncontaminated water. The results revealed large tissue differences in DNA adduct formation. DNA adduct levels were not elevated in the digestive gland of the mussels at any exposure concentration (0-100 microg/l), even though the highest B[a]P tissue concentrations were found in the digestive gland (1.0+/-0.1 mg B[a]P/g tissue dry wt at 100 microg/l, mean+/-SE, n=12). DNA adducts were on the other hand formed in the gills, with the highest levels found in mussels exposed to 50 and 100 microg B[a]P/l, and a dose dependent increase in adduct levels (from 1.6 to 5.9 nmol adducts/mol nucleotides) from 0 to 50 microg B[a]P/l. In gills, DNA adduct levels increased with time during the 6-day exposure period in the exposure-depuration experiment, and then persisted for at least 2 weeks after exposure cessation while B[a]P tissue levels exhibited a rapid decrease (half-life of 8 days). No significant differences were observed in SFG between the control and exposed groups. Since DNA adducts exhibited a relatively high persistence in gills compared to B[a]P tissue concentrations, they seem to be a more integrated measure of genotoxic exposure than only chemical analysis of the contaminant bioaccumulation. The results also suggest that if using analysis of DNA adducts in M. edulis for monitoring purposes, analysis of gills in addition to the more commonly used digestive gland should be taken into consideration.     

Antioxidant enzyme activity and lipid peroxidation in liver and kidney of rats exposed to microcystin-LR administered intraperitoneally.

The effect of acute exposure of intraperitoneal injection of microcystin-LR (MCLR) on antioxidant enzymes and lipid peroxidation has been studied in liver and kidney of rats. Rats were treated with two doses, i.e. 100 and 150 microg of pure MCLR/kg body weight or saline solution. The enzyme activities of glutathione peroxidase (GSH-Px), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) in the liver were significantly decreased in MCLR-treated rats. The decrease of GR activity in the liver was 60%, followed by GSH-Px, SOD and CAT. Similarly, a decrease in the antioxidant enzymes was found in the kidney of MCLR-treated rats, such as GSH-Px (27-31%), GR (22%), SOD (42%) and CAT (25-28%). Concomitantly, significant increases in lipid peroxidation levels were recorded in liver (121 and 196% for 100 and 150 microg/kg, respectively) and kidney (48 and 58% for 100 and 150 microg/kg, respectively) from MCLR-treated rats. In conclusion, acute exposure to MCLR results in a decrease in the antioxidant enzymes and an increase in lipid peroxidation in liver and kidney rats, suggesting the oxidative stress as an important role in the pathogenesis of MCLR-induced toxicity. Antioxidant enzymes were significantly consumed in the liver and a minor decrease was found in kidney, confirming the organ-specific effects of MCLR.