In vivo and in vitro exposures for the evaluation of the genotoxic effects of lead on the Neotropical freshwater fish Prochilodus lineatus.

In the present study, in vivo and in vitro exposures were used to assess the genotoxicity of lead (Pb) to the freshwater fish Prochilodus lineatus. The comet assay using blood, liver and gill cells, and the occurrence of micronuclei (MN) and other erythrocytic nuclear abnormalities (ENA) were used to assess the genotoxic potential of lead in vivo. Metallothionein content (MT) was measured in fish liver in order to evaluate the protection of fish against Pb toxicity. Fish erythrocytes were exposed to Pb in vitro (1, 3 and 6 h) and the number of viable cells, DNA integrity, using the comet assay, and lysosomal membrane stability, measured by the neutral red retention assay (NRRA) were analyzed. The results of the comet assay after in vivo toxicity tests (6, 24 and 96 h) showed that Pb was genotoxic for all the three tissues analyzed after 96 h exposure. A significant increase in liver MT content was observed after 6 and 24 h of Pb exposure. MN frequency did not increase after Pb exposures, but the frequency of the other ENA, such as kidney-shaped nuclei, segmented nuclei and lobed nuclei, showed a significant increase after 24 and 96 h, indicating that ENA is a better biomarker for Pb exposure than MN alone after short-term exposures. The results of the comet assay performed with erythrocytes in vitro exposed to lead confirmed its genotoxic effect and showed that DNA damage increased with increasing exposure time. Moreover, the NRRA clearly indicated that Pb induces a destabilization of the lysosomal membrane. These results demonstrate the potential genotoxicity and cytotoxicity of lead after acute exposures.

Genetic damage in the bivalve mollusk Corbicula fluminea induced by the water-soluble fraction of gasoline.

Although gasoline is an important contaminant of aquatic ecosystems, information concerning the potential effects of this petroleum derivative on the DNA of aquatic biota is lacking. The present study aimed to evaluate the genotoxic and mutagenic effects of acute exposures (6, 24, and 96h) to gasoline water-soluble fraction (GWSF), diluted to 5%, on the Asian clam Corbicula fluminea. The comet assay and the micronucleus (MN) test were performed on hemocytes and gill cells of C. fluminea. For the three different times tested, the comet assay indicated DNA damage in hemocytes and gill cells of C. fluminea exposed to GWSF. The MN test detected significant damage in the genetic material of the hemocytes only after 96h of exposure to GWSF. The recovery capacity of organisms previously exposed for 6h to GWSF was also evaluated with the comet assay. The results revealed a great capacity of this species to repair DNA damage; following 6h of recovery, the comet score returned to that of the control groups. Overall, our findings indicate that GWSF has genotoxic and mutagenic effects on C. fluminea. In addition, the present results confirm the sensitivity of C. fluminea to damage caused by exposure to environmental contaminants, and, therefore, its suitability for use in environmental monitoring studies.

Genotoxic effects of Roundup on the fish Prochilodus lineatus.

Glyphosate-based herbicides, such as Roundup, represent the most extensively used herbicides worldwide, including Brazil. Despite its extensive use, the genotoxic effects of this herbicide are not completely understood and studies with Roundup show conflicting results with regard to the effects of this product on the genetic material. Thus, the aim of this study was to evaluate the genotoxic effects of acute exposures (6, 24 and 96 h) to 10 mg L(-1) of Roundup on the neotropical fish Prochilodus lineatus. Accordingly, fish erythrocytes were used in the comet assay, micronucleus test and for the analysis of the occurrence of nuclear abnormalities and the comet assay was adjusted for branchial cells. The results showed that Roundup produces genotoxic damage in erythrocytes and gill cells of P. lineatus. The comet scores obtained for P. lineatus erythrocytes after 6 and 96 h of exposure to Roundup were significantly higher than respective negative controls. For branchial cells comet scores were significantly higher than negative controls after 6 and 24 h exposures. The frequencies of micronucleus and other erythrocyte nuclear abnormalities (ENAs) were not significantly different between Roundup exposed fish and their respective negative controls, for all exposure periods. In conclusion, the results of this work showed that Roundup produced genotoxic effects on the fish species P. lineatus. The comet assay with gill cells showed to be an important complementary tool for detecting genotoxicity, given that it revealed DNA damage in periods of exposure that erythrocytes did not. ENAs frequency was not a good indicator of genotoxicity, but further studies are needed to better understand the origin of these abnormalities.

Genotoxic and mutagenic effects of diesel oil water soluble fraction on a neotropical fish species.

Numerous spills and leakages involving petroleum and its derivatives have recently occurred in Brazilian rivers. Considering the lack of information regarding the genotoxic response of neotropical fish to these events and the predominance of information regarding saltwater fish, which offers no genuine comparisons, the present work aimed to evaluate the genotoxicity and mutagenicity of the diesel water soluble fraction (DWSF) on the neotropical fish Prochilodus lineatus under acute (6, 24 and 96h) and subchronic (15 days) exposures, using the comet (SCGE) and micronucleus assays. The results indicated genotoxic and mutagenic damage in erythrocytes of P. lineatus exposed to DWSF. Comet scores for fish exposed to DWSF in all experimental periods were significantly higher than the respective negative control groups (fish exposed to clean water for the same period). The relative frequencies of micronucleated erythrocytes for P. lineatus exposed to DWSF under acute and subchronic treatment were also significantly higher than their respective negative controls. Taken together these results showed that acute and subchronic exposures to DWSF produce mutagenic and genotoxic effects on the blood cells of P. lineatus and that the combination of comet and micronucleus assays proved to be both suitable and useful in the evaluation of the genotoxicity of diesel oil due to their complementary action.

Acute morphological and physiological effects of lead in the neotropical fish Prochilodus lineatus

O presente estudo investigou os efeitos do chumbo na morfologia branquial, nos hematócritos e nas concentrações plasmáticas de sódio, glicose, lipídeos, proteínas e colesterol de Prochilodus lineatus exposto a duas concentrações subletais de chumbo durante 96 h. Inicialmente, testes agudos (96 h) e estáticos determinaram a CL50 (96 h) de chumbo para P. lineatus em 95 mg Pb.L-1. As concentrações de chumbo utilizadas nos testes subletais foram 24 e 71 mg Pb.L-1, que correspondem a 25% e 75%, respectivamente, da CL50 (96 h). As brânquias de P. lineatus expostos a ambas as concentrações de chumbo apresentaram maior incidência de lesões histopatológicas, como elevação epitelial, hiperplasia e aneurisma lamelar. P. lineatus não apresentou alterações significativas no hematócrito durante a exposição a ambas as concentrações de chumbo. Peixes expostos a 71 mg Pb.L-1 apresentaram decréscimo significativo do Na+ plasmático após 48 h, o que pode estar se refletindo na redução das taxas de influxo desse íon. P. lineatus expostos a ambas as concentrações de chumbo apresentaram resposta clássica ao estresse, como verificado pela hiperglicemia associada ao decréscimo dos lipídeos e proteínas plasmáticas. A magnitude da resposta ao estresse foi dose-dependente. A resposta apresentada na concentração mais baixa representa um processo adaptativo, enquanto na maior concentração caracteriza a exaustão.

Acute morphological and physiological effects of lead in the neotropical fish Prochilodus lineatus.

The present study investigated lead effects on gill morphology, hematocrit, blood sodium, glucose, lipids, protein, and cholesterol of Prochilodus lineatus exposed to two sublethal lead concentrations for 96 h. Preliminary series of short-term static toxicity tests were run to determine LC50 (96 h) of lead in P. lineatus, which was 95 mg Pb.L-1. Therefore, lead concentrations tested in the sublethal experiments were 24 and 71 mg Pb.L-1, which correspond to 25% and 75% of the LC50 (96 h), respectively. Gills of P. lineatus exposed to both lead concentrations during 96 h presented a higher occurrence of histopathological lesions such as epithelial lifting, hyperplasia, and lamellar aneurism. P. lineatus did not show significant alterations in hematocrit during exposure to both lead concentrations. Fish exposed to the highest lead concentration showed a significant decrease in Na+ plasma concentration after 48 h, possibly reflecting a sodium influx rate decrease. P. lineatus exposed to both lead concentrations presented a "classical general adaptation syndrome to stress", as hyperglycemia associated with lowered lipids and proteins was reported. Stress-response magnitude was dose-dependent. While the response to the lowest lead concentration might represent adaptation, the highest concentration seems to characterize exhaustion.

Effect of benzene on the swimming activity of Mysidopsis juniae (Crustacea, Mysidacea)

The effects of benzene on the swimming activity of the crustacean Mysidopsis juniae were investigated. The swimming activity of M. juniae was observed after 1.3 and 6 h of exposure to 5, 10 15 and 20 ppm benzene in sea water (35 ñ 1 grade/00 S, 25 ñ 1 grade C). The mysids were observed with a pair of coupled to a camara lucida, and the swimming activity was measured in a Petri dish, corresponding to the distance (cm) covered by the animals in 1 min (N=150 animals). The swimming activity of mysids exposed to 20 ppm benzene decreased significantly after all three periods of exposure to values which were 80-90% smaller than the control value. On the other hand, after 6 h exposure to 5 ppm benzene, the swimming activity of the mysids was significantly higher, increasing by 87% in relation to the control (29.8 ñ 8.9 cm?min). Furthermore, a slight nonsignificant increase in swimming activity was also observed in mysids submitted to 5, 10, 15 and even 20 ppm benzene, as the period of exposure to the pollutant increased from 1 to 6 h. These results indicate that short-term exposure to sublethal benzene concentrations can affect the swimming activity of mysids. in these situations, mysid populations can be seriously damaged since alterations in swimming activity could lead to a reduction in food intake and to a marked increase in their susceptibility to predation by other organisms