Ciro Alberto de Oliveira Ribeiro

Genetic damage induced by trophic doses of lead in the neotropical fish Hoplias malabaricus (Characiformes, Erythrinidae) as revealed by the comet assay and chromosomal aberrations

The effects of clastogenic or mutagenic agents have rarely been studied in neotropical fish species exposed to contaminated water. In this study, the genetic damage caused by lead in the widely distributed South American fish, Hoplias malabaricus, was assessed using the comet (SCGE) assay and by testing for chromosomal aberrations. Eighteen specimens were acclimatized to laboratory conditions and then chronically exposed to contaminated food by feeding prey (Cyprinus sp.) injected intraperitoneally with doses of inorganic lead adjusted to give a contamination level of 21 mg of Pb2+.g-1 net weight of H. malabaricus. Three fish were sampled for chromosomal analysis after four doses (18 days) and another three after eight doses (41 days) of lead and the results then compared with three untreated controls kept under lead-free conditions. An additional six treated fish and three controls were sampled for the comet assay after 13 doses (64 days). Exposure to lead significantly increased the frequency of chromosomal aberrations and the frequency of tailed cell nuclei, the latter indicating DNA damage. These results show that H. malabaricus is a useful biological model for screening the clastogenic effects of lead and possibly other xenobiotics. The genetic damage seen here illustrates the need to investigate the potential effects of heavy metals on fish species in South America.

Metals in liver and kidneys and the effects of chronic exposure to pyrite mine pollution in the shrew Crocidura russula inhabiting the protected wetland of Doñana.

Historically impacted by anthropogenic activities, the nature reserve of Doñana (SW Spain) was affected by an unprecedented spillage of mud and acidic water from the Aznalcóllar pyrite mine in April 1998. Although several studies have addressed the influence of this spill on soils, water, and biota, there is little information on mammals, especially carnivorous species. We measured the concentrations of Fe, Mg, Pb, Hg, Cd, Zn, Cu, Mn, Mo, Co, and Cr in specimens of the greater white-toothed shrew, Crocidura russula, inhabiting the protected area affected by the mine spillage. We also examined other parameters to approach at the physiological effects of pollution. We found an increase in non-essential metals (Pb, Cd, and Hg), and morphometric, histological and genotoxic alterations. Age and gender were two significant factors explaining metal bioaccumulation: adults had higher Hg and Cd levels than juveniles, whereas males bioaccumulated more Pb and Co and less Mo than females. The micronucleus frequencies in blood erythrocytes were significantly higher in specimens from the polluted site than animals from the control site. Shrews from the impacted area also had hepatic alterations, namely increased liver-body ratio, focal necrosis, and signs of apoptosis in hepatocytes. Due to the relevance of small mammals in the diet of endangered species such as carnivorous birds and mammals, the findings of our study are of practical use for the management of the Doñana wildlife reserve and other protected Mediterranean wetlands.

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.

Mercury and DDT exposure risk to fish-eating human populations in Amazon

In order to investigate the potential risk of mercury and DDTs exposure to fish-eating human populations in Samuel Reservoir, not affected directly by gold-mining activities, the axial muscle of Cichla monoculus was analyzed. Twenty-nine and thirty adults individuals were collected respectively on February (rainy season) and August (dry season) 2007. The specimens were sacrificed by spinal section before sex identification, body weight and total length determination. For total mercury, DDT and DDE quantifications and cholinesterase activity samples of the axial muscle were frozen at -20°C, and for histopathological studies gill and liver were fixed in ALFAC solution for 16 h. A value of 48.2% and 33% of the individuals, respectively from rainy and dry seasons, presented mercury concentrations higher than the maximum established for safe human consumption (0.5 μg g⁻¹) by World Health Organization. A positive correlation between body weight and Hg concentration was observed only in individuals from the rainy season, but no correlation was observed to DDT and DDE from both seasons. Differently from that observed to mercury, DDT levels presented a significant difference between both studied seasons, but no correlation was observed for both mercury and DDTs and sex. The levels of DDTs in muscle of C. monoculus are under the maximum established by FAO-Alimentarius CODEX and Swedish Food Regulation for human consumption. The histopathological and neurotoxic findings showed that the wild population of fish is affected by chronic exposure to mercury, meaning risk also to fish-eating populations. Finally, the results showed that C. monoculus is an important vehicle for human exposure to mercury and DDTs in Samuel Reservoir and that it is necessary a continuous biomonitoring of the levels of both pollutants in order to manage the risk of exposure to human populations.

Metabolic interactions between low doses of benzo[a]pyrene and tributyltin in arctic charr (Salvelinus alpinus): a long-term in vivo study.

We have previously reported that short-term, single exposure to a high dose of tributyltin (TBT), a widely used antifouling biocide, inhibited both the in vivo metabolism and metabolic activation of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) in fish; (BaP), in turn, stimulated TBT metabolism. Here, we provide further mechanistic evidence of mutual metabolic interactions between BaP and TBT in response to long-term, repeated exposures to low doses. Juvenile Arctic charr (Salvelinus alpinus) received 10 separate i.p. injections (a single injection every 6 days) of BaP (3 mg/kg), TBT (0.3 mg/kg), or both in combination; control fish received corn oil vehicle only. Two days after the 2nd (Day 8), 6th (Day 32), and 10th dose (Day 56), blood, bile, and liver samples were collected and analyzed for a suite of biomarkers. HPLC/fluorescence analysis indicated that TBT cotreatment inhibited the formation of (+)-anti-BaP diol-epoxide adducts with plasma albumin (53%, Day 8), hepatic DNA (27%, Day 32), or both albumin and globin (50 and 58%, Day 56) compared to BaP alone. This antagonistic interaction was associated with a time-dependent modulation (inhibition at Day 8, enhancement at Day 32) of both cytochrome P450 (P450)1A-mediated ethoxyresorufin O-deethylase (EROD) activity and biliary BaP metabolite formation. TBT cotreatment also inhibited (39%) the BaP-mediated induction of hepatic glutathione S-transferase (GST) activity observed at Day 8. Treatment with TBT alone increased EROD activity (60%) at Day 32, but decreased both GST activity (70 and 37%) and glutathione content (24% and 16%) at Days 32 and 56, respectively. GC/MS analysis revealed that, at Day 56, BaP cotreatment increased (200%) the levels of biliary butyltin compounds, including mono- and dibutyltin metabolites. This potentiative interaction was associated with a protective effect of BaP cotreatment against the TBT-mediated decreases in GST activity and glutathione content. The current results demonstrate that, whereas TBT inhibited BaP bioactivation via a time-dependent modulation of P4501A induction, BaP stimulated the Phase II metabolism of TBT and/or its biliary excretion. The mutual metabolic interactions between these two widespread aquatic pollutants reinforce the need for long-term in vivo interactive studies at low doses.

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.

Methylmercury localization in Danio rerio retina after trophic and subchronic exposure: A basis for neurotoxicology

Methylmercury is a known neurotoxic organometal which affects visual functions and few studies concerns to wild fish are available. The autometallography mercury distribution in the retina of Danio rerio was mapped using light and electron microscopy. Abundant mercury deposits were found in the photoreceptor layer (outer and inner segments of the photoreceptors) and in the inner and outer nuclear layers. Occasionally, the presence of mercury deposits in plexiform layers was observed and very rarely in the ganglion cell layer. Also the occurrence of mercury deposits in cells from the disc region was observed, but not in the nerve fiber layer. An interesting difference was found between mercury accumulation in the central and peripheral regions of the retina. These results demonstrate that mercury after trophic exposure to Danio rerio is able to cross the blood-retina barrier and accumulate in the cells of the retina even under subchronic exposure.

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.

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.

Effects of dietary methylmercury on zebrafish skeletal muscle fibres.

Mercury and more specifically methylmercury have been reported as hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. Adult male zebrafish (Danio rerio) were distributed in two groups: a control group (fed with uncontaminated food) and a MeHg-contaminated group (fed with food containing 13.5μgHgg(-1) (dry wt)). Five fish per condition were removed after 7, 21 and 63 days. Bioaccumulation of mercury was determined and muscle samples from control and exposed groups were fixed for histological and ultrastructural studies. In contaminated muscles were observed a decrease of the inter-bundle surface, mitochondria with variable shapes, sizes and cristae disorganization, also decreasing the surface area and inter-bundle surfaces. Indeed, damage in the endoplasmic reticulum cisternae was observed. For statistical evaluation the damages in mitochondria was quantified by image. According to the current results, methylmercury affects the structure of fibre cells of D. rerio after trophic and low dose exposure.