Helena C. Silva de Assis

Analyses of paralytic shellfish toxins and biomarkers in a southern Brazilian reservoir

The Alagados Reservoir (Brazil) is an important source for the supply of water, recreation and fishery. Since 2002, the occurrence of cyanobacterial blooms (paralytic shellfish toxins - PST producers) have been noted. This study was aimed at the monitoring of PST occurrence in the Reservoirs water and fish. Biomarkers such as ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), catalase (CAT), and acetylcholinesterase (AchE) activities, lipoperoxidation (LPO), histopathology, and comet assay were analyzed in fish. Water and fish were sampled in spring, summer and autumn. The PST concentrations in water were 5.15, 43.84, and 50.78 ng equiv Saxitoxin/L in the spring, summer and autumn, respectively. The PST muscle concentration was below the limit for shellfish. Gonyautoxins (GTX) were found in water samples and fish muscle, and GTX 5 was the major analogous found in muscle. In the summer samples, the LPO, genetic damage, and the GST and AchE activities increased while in the autumn an increase in EROD activity and genetic damage were observed. In all samplings, histopathological alterations in the fish gills and liver were found. The results showed a seasonal variation in the fishes health, which could be related also to farming activities and to the contaminants bioavailability during the year.

First report about saxitoxins in freshwater fish Hoplias malabaricus through trophic exposure.

Cyanobacterial waterblooms, such as the saxitoxin (STX) producer Cylindrospermopsis raciborskii, have been a worldwide concern in environmental health. However, the bioaccumulation of this neurotoxin in the trophic chain is not completely known. The aim of the present work was to evaluate STX bioaccumulation through chemical analyses and the toxic and trophic effects using biomarkers in the tropical freshwater fish Hoplias malabaricus. They were fed once every five days with Astyanax sp. before being subjected to intraperitoneal inoculation with STX extract (0.08 μg/100 g) obtained by lysis of toxic C. raciborskii strain (T3). After 20 days the brain was collected for acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione (GSH), lipoperoxidation (LPO), protein carbonylation (PCO), and comet assay analysis. The muscle was collected for STX chemical analysis. The activities of SOD and concentrations of PCO and LPO increased. The CAT, GST, and GPx activities decreased. Genotoxicity was observed in the experimental group. STX was not detected in muscle samples. Thus, an oxidative stress was observed in the brain, leading to the damage of lipids, proteins, and DNA. The mechanism of action of the neurotoxin in this subchronic exposure suggests an apoptotic cellular process.

Pre and postnatal exposure to endosulfan in Wistar rats.

The possible reproductive adverse effects of the pesticide endosulfan on male offspring rats exposed in utero and during lactation were investigated. Dams were treated orally with 0, 0.5 or 1.5 mg of endosulfan/kg 21 days prior to mating, during the mating, pregnancy and lactation. Maternal and reproductive outcome data and male sexual development landmarks (testis descent and preputial separation) were assessed. Reproductive endpoints of the male offspring were examined at adulthood: sex organ weights, daily sperm production, spermatid number, sperm transit, sperm morphology and testosterone level. No signs of maternal toxicity were detected at the dose levels tested. Sexual development landmarks were also unaffected. Moreover, with the exception of a significant increase in the relative epididymis weight seen in the group treated with the lowest dose, we have not found any statistically significant adverse effect in the reproductive endpoints investigated at adulthood. The results of the present study indicate that pre and post-natal exposure to low doses of endosulfan (0.5 and 1.5 mg/kg) do not induce significant adverse effects in the reproductive system of male offspring Wistar rats at adulthood.

Estrogen-like effects in male goldfish co-exposed to fluoxetine and 17 alpha-ethinylestradiol.

The antidepressant fluoxetine (FLX) and the synthetic estrogen, 17 alpha-ethinylestradiol (EE2), are present in municipal sewage discharges. To better understand possible interactions between them, male goldfish were exposed to an ethanol control or to nominal concentrations of FLX (0.54 μg/L) and EE2 (5 ng/L) alone and in combination for 14 days. Real-time reverse-transcription polymerase chain reaction was used to assess effects on hepatic gene expression and liquid chromatography tandem mass spectrometry to analyze the plasma proteome. The results showed an increase in estrogen receptor alpha (esr1) and vitellogenin (vtg) gene expression by 1.9-2.4-fold in the FLX and EE2 groups, but this did not reach statistical significance. In contrast, co-exposure up regulated esr1 and vtg gene expression by 5.5- and 5.3-fold, respectively. Fluoxetine and EE2 alone did not affect estrogen receptor beta (esr2), but the co-exposure down regulated esr2 expression by 50%. There was a significant increase in the number of plasma proteins that were related to endocrine system disorders in the FLX and FLX plus EE2 groups. The level of VTG protein was increased in the plasma from goldfish exposed to EE2, FLX, and FLX plus EE2. Our study demonstrates that low concentrations of FLX and EE2 in a simple mixture produce strong estrogen-like effects in the male goldfish.

Biochemical biomarkers of exposure to deltamethrin in freshwater fish, Ancistrus multispinis

ABSTRACT This study aimed to determine the effect of sublethal doses of deltamethrin, using biochemical biomarkers as activities of cholinesterase (ChE), ethoxyresorufin-O-deethylase (EROD) and the Na + K + - ATPase and levels of total cytochrome P450 (CYP450). Fishes received sublethal doses of deltamethrin and were sacrificed after 96 h of exposure. Samples of gills, heart, brain, liver and muscle were collected for enzymatic analyses. Deltamethrin inhibited the activity of the gills and heart Na + K + -ATPase, induced the liver total CYP450, as well as the liver EROD activity. The activity of the ChE was not inhibited by deltamethrin. Deltamethrin altered the hepatic metabolism and the normal ionic flux in Ancistrus multispinis . Key words: deltamethrin, cytochrome P450, fish, Ancistrus multispinis , biomarker * Author for correspondence: helassis@ufpr.br INTRODUCTION Pyrethroids are synthetic derivatives of pyrethrins, which are toxic components contained in the flowers of Chrysanthemum cinerariaefolium . Although synthetic pyrethroids are less persistent and less toxic to mammals and birds (Sayeed et al, 2003), they are highly toxic to a number of non-target organisms such as bees, freshwater fish and other aquatic organisms even at very low concentrations (Oudou et al, 2004). For this reason, these organisms are extremely sensitive to neurotoxic effects of pyrethroids when they reach surface water-courses (Bradbury and Coats 1989, Haya, 1989; Mittal et al, 1994). One of the pyrethroids that has found wide acceptability and is extremely used in agriculture and forestry because of its high activity against a broad spectrum of insect pests (Villarini et al, 1998) is deltamethrin ((S) α-cyano-3-phenoxybenzyl-(1R)-cis-3-(2.2-dibromovinyl)-2,2- dimethyl cyclopropane carboxylate). However, its effects on nervous, respiratory, and hematological systems in fishes have been reported (Ural and Sa glam, 2005, Pimpao et al, 2007). Balint et al (1995) observed 20% decrease in acetylcholinesterase activity of brain, heart, blood, liver and skeletal muscle of carp after the 3 days exposure to deltamethrin. In rats, the main reaction involved in the metabolism of deltamethrin is ester cleavage, by CYP450 and carboxyesterase action. Metabolism in fish is largely oxidative and deficient in esterases metabolization (Demoute, 1989). ATPase has been demonstrated to be one of the targets of pyrethroids. Some authors showed that ATPase including cell membrane-associated

Water quality assessment of the Tubarão River through chemical analysis and biomarkers in the Neotropical fish Geophagus brasiliensis

The Tubarão River rises in Santa Catarina, Brazil, and has been historically affected by coal mining activities around its springhead. To evaluate its water conditions, an investigation regarding a possible decontamination gradient associated with the increased river flow toward the estuary, as well as the influence of seasonality over this gradient was performed through a series of biomarkers (vitellogenin, comet assay, lipid peroxidation, protein carbonylation, gluthatione, gluthatione S-transferase, acetylcholinesterase, light microscopy in liver, and scanning electron microscopy in gills) and chemical analysis (polycyclic aromatic hydrocarbons (PAHs) in bile and metal analysis in sediment) in the cichlid Geophagus brasiliensis. Two collections (summer and winter) were made in four distinct sites along the river, while sediments were sampled between those seasons. As expected, the contamination linked exclusively to mining activities was not observed, possibly due to punctual inputs of contaminants. The decontamination gradient was not observed, although seasonality seemed to have a critical role in the responses of biomarkers and availability of contaminants. In the summer, the fish presented higher histopathological damages and lower concentrations of PAHs, while in the winter they showed both higher genetic damage and accumulation of PAHs. The Tubarão suffers impacts from diverse activities, representing health risks for wild and human populations.

Effects of trophic exposure to dexamethasone and diclofenac in freshwater fish

Steroidal and non-steroidalanti-inflammatories are pharmaceutical prescribed in human medicine and have the potential to contaminate water and sediments via inputs from sewage treatment plants. Their impacts on humans and ecosystems are emerging issues in environmental health. The aim of the present work was to evaluate the effects of diclofenac and dexamethasone in male fish Hoplias malabaricus after trophic exposure. Fish were fed twice every week with Astyanax sp. submitted to intraperitoneal inoculation with diclofenac (0; 0.2; 2.0 or 20.0 μg/kg) or dexamethasone (0; 0.03; 0.3 or 3.0 μg/kg). After 12 doses, blood was collected for testosterone dosage. The gonad and liver were collected to calculate gonadosomatic (GSI) and hepatosomatic index (HSI). Antioxidants enzymes activity and biotransformation were also evaluated in liver and gonads. In liver, diclofenac caused oxidative stress with increased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and lipoperoxidation (LPO). The GST activity was reduced by diclofenac in liver. Trophic exposure of H. malabaricus to dexamethasone caused an increase in antioxidant system (GPx, CAT, GST, and GSH) and LPO in liver. However, it reduced antioxidant system (GPX and GST activities and GSH) in gonads. Both diclofenac and dexamethasone reduced the levels of testosterone, causing impairment to reproduction. Diclofenac reduced HSI at the 0.2 μg/kg, but not GSI. Our results suggest that the anti-inflammatory drugs diclofenac and dexamethasone caused oxidative stress and reduced testosterone levels that can have a negative impact in aquatic organisms.

Sublethal Effects of Waterborne Herbicides in Tropical Freshwater Fish

The study evaluated the sublethal effects of the herbicides glyphosate (Roundup) and diuron (Hexaron) and the mixture of them, used extremely in agriculture, through biomarkers in fish. The glutathione S-transferase activity increased (74%) and catalase activity decreased (37%) at the higher exposure concentration of Hexaron in comparison to the control group, suggesting an activation of this metabolism route. Membrane damage was observed at the higher exposure of Roundup and in the mixture group compared to the control group, which can be related to the nuclear alterations observed in these exposed groups. The cholinesterase activity was also inhibited (37%) in mixture group compared to the control group and no gill morphology damage was found. The results suggested a potential synergic effect in some analysed parameters.

Chronic genetic damages in Geophagus brasiliensis exposed to anthropic impact in estuarine lakes at Santa Catarina coast--southern of Brazil.

Biological monitoring through animals exposed to pollutants using biomarkers provides a promising tool for the identification of pollutants that may cause damage to human health and/or to sustainability of ecosystems. The effects of pollutants in fish tissues are important tools to understand the impact of human activities in natural ecosystems. The aim of this work was to study the water quality of two estuarine lakes in Santa Catarina, Brazil (Camacho Lake and Santa Marta Lake). Geophagus brasiliensis is a species widely distributed in Brazil and was used in this work. Comet assays in peripheral red blood and kidney cells, micronucleus tests in peripheral red blood cells, measurements of acetylcholinesterase activity in axial muscle and histopathological analysis of liver were used as biomarkers. Three sampling campaigns were undertaken in November 2004, June 2005 and November 2005. Thirty adult animals were sampled from each of three different sites (P1—Santa Marta Lake, P2 and P3—Camacho Lake). A negative control was sampled in a non-polluted site at Costa Ecological Park, Paraná. The positive control for genotoxicity was obtained by treating animals with copper sulphate. The results showed that both studied lakes are impacted by potential genotoxic substances. Severe lesions in liver of G. brasiliensis were also observed. The inhibition of acetylcholinesterase activity suggests the presence of pesticides or metals in the studied sites. This work shows that the water quality of Santa Marta and Camacho Lakes have been compromised and further control source of pollutants into these ecosystems is required.

Hematologic and hepatic responses of the freshwater fish Hoplias malabaricus after saxitoxin exposure

The bioaccumulation of saxitoxins (STX) in the trophic chain, mainly in freshwater, are not completely known. This work aimed to elucidate the effects of STX on Hoplias malabaricus through trophic bioassay. The fish were fed once every five days with Astyanax sp. before being subjected to an intraperitoneal inoculation with the lysate of Cylindrospermopsis raciborskii culture containing 97% STX and 3% by neosaxitoxin and gonyautoxin during 20 days. The animals liver was assessed using biomarkers as activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx), and concentrations of reduced glutathione (GSH) and lipoperoxidation (LPO) and protein carbonylation (PCO). In the blood was analyzed the genotoxic and hematological parameters. The hepatosomatic index and the relative condition factor did not show a significant difference between the exposed and control groups. The values of mean corpuscular hemoglobin concentration and mean corpuscular hemoglobin increased in the STX group. The hepatic tissue from both groups exhibited a typical pattern that have been already described for most teleost fish. The results suggested the generation of reactive oxygen species, with increased activity of GPx and concentrations of LPO and GSH; whereas the specific activity of SOD decreased. However, no changes were observed in the CAT, PCO, and DNA damage. Although the STX effects are known as neurotoxic, this cyanotoxin caused liver biochemical alterations that can be considered ecologically relevant.