Luciana Fernandes de Oliveira

Multiple biomarker responses in Prochilodus lineatus subjected to short-term in situ exposure to streams from agricultural areas in Southern Brazil.

In order to assess the quality of streams susceptible to contamination by pesticides we apply biochemical and genotoxic biomarkers in the Neotropical fish Prochilodus lineatus submitted to in situ tests. Fish were caged, for 96 h, in two streams located in areas with intensive use of pesticides, the Apertados (AP) and the Jacutinga (JC), and in a small stream (Godoy stream — GD) found inside a forest fragment adjacent to a State Park. Biochemical parameters, such as biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), non-protein thiols (NPSH), lipoperoxidation (LPO), protein carbonylation (PCO) and acetylcholinesterase (AChE) were evaluated in various fish organs, as well as genotoxic biomarkers (damage to DNA and occurrence of micronuclei and erythrocyte nuclear abnormalities). Samples of water and sediment were collected for analysis of metals (Cu, Cr, Pb, Ni, Mn, Cd and Zn), organochloride pesticides, and triazine and glyphosate herbicides. We observed an increase in liver GST activity in fish at AP and gill GST activity in fish at JC. An increase in liver LPO was also observed in fish exposed to AP and JC. The same animals also exhibited increased DNA damage and erythrocyte nuclear abnormalities (ENAs) compared to the fish kept in GD. A number of compounds showed concentrations higher than the permitted levels, in particular, dichlorodiphenyltrichloroethane (DDT), its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), hexachlorocyclohexanes (HCH), heptachloride, diclofluanid and aldrins. These pesticides were detected at higher concentrations in water and sediment samples from AP, followed by JC and GD. The Integrated Biomarker Response Index (IBR) indicated that AP and JC (AP: 21.7 > JC: 18.5 > GD: 12.6) have the worst environmental quality. Integrated biomarker analysis revealed that the alterations observed related well with the levels of environmental contaminants, demonstrating the effectiveness of this biomonitoring approach.

Assessment of domestic landfill leachate toxicity to the Asian clam Corbicula fluminea via biomarkers

In order to evaluate the effects of domestic landfill leachate to bivalves Corbicula fluminea, clams were exposed to different leachate concentrations (v/v): 2, 3, 6 and 10 percent, corresponding to dilutions observed along a stream that receives this effluent, or only to clean water for comparisons. After 5 and 15 days of exposure the activity of the biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the multixenobiotic resistance mechanism (MXR) and lipid peroxidation (LPO) in gills and digestive gland and metallothionein (MT) content in gills were evaluated. Differences in biomarkers responses were observed between gills and digestive gland, except for MXR that decreased in both tissues of clams exposed to 6 percent for 5 days. EROD activity in gills was reduced in all leachate concentrations after 5 days and only in 2 percent after 15 days exposure, while an EROD increase was observed in digestive gland after 15 days exposure to 6 percent. GST activity increased only in the gills of clams exposed to 10 percent for 5 days. LPO varied between tissues and different conditions. A significant increase in LPO was observed in the gills, after 5 days exposure to 2 and 6 percent, and in digestive gland after 5 and 15 days exposure to 2 and 3 percent. MT content in the gills increased after 15 days exposure to 2 percent. In conclusion, different leachate concentrations tested here caused biochemical changes in C. fluminea, but due to the observed variability in biomarkers responses among leachate concentrations, it was difficult to determine patterns or thresholds concentrations.

Multi-biomarker investigation to assess toxicity induced by two antidepressants on Dreissena polymorpha

Antidepressants are one of the main pharmaceutical classes detected in the aquatic environment. Nevertheless, there is a dearth of information regarding their potential adverse effects on non-target organisms. Thus, the aim of this study was the evaluation of sub-lethal effects on the freshwater mussel Dreissena polymorpha of two antidepressants commonly found in the aquatic environment, namely Fluoxetine (FLX) and Citalopram (CT). D. polymorpha specimens were exposed to FLX and CT alone and to their mixture (MIX) at the environmental concentration of 500ng/L for 14days. We evaluated the sub-lethal effects in the mussel soft tissues by means of a biomarker suite: the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and the activity of the phase II detoxifying enzyme glutathione-S-transferase (GST). The oxidative damage was evaluated by lipid peroxidation (LPO) and protein carbonylation (PCC), while genetic damage was tested on D. polymorpha hemocytes by Single Cell Gel Electrophoresis (SCGE) assay, DNA diffusion assay and micronucleus test (MN test). Finally, the functionality of the ABC transporter P-glycoprotein (P-gp) was measured in D. polymorpha gills. Our results highlight that CT, MIX and to a lesser extent FLX, caused a significant alteration of the oxidative status of bivalves, accompanied by a significant reduction of P-gp efflux activity. However, only FLX induced a slight, but significant, increase in apoptotic and necrotic cell frequencies. Considering the variability in biomarker response and to perform a toxicity comparison of tested molecules, we integrated each endpoint into the Biomarker Response Index (BRI). The data integration showed that 500ng/L of FLX, CT and their MIX have the same toxicity on bivalves.

Metals bioaccumulation and biomarkers responses in the Neotropical freshwater clam Anodontites trapesialis: Implications for monitoring coal mining areas.

As one of the most impactful industries, coal mining can promote several alterations at surrounding environment. In surface water, elevated concentrations of metals like Mn, Zn, Fe and Al are often observed. Thus, the aim of this study was to investigate the bioaccumulation and the sub-lethal effects of these metals on various organs of the Neotropical bivalve Anodontites trapesialis confined along a stream located near a coal mine, in order to assess a set of biomarkers that could be used for effectively monitoring coal mining areas. Clams were caged, for 96h, at two sites located upstream (Up1 and Up2) and two sites downstream (Dw1 and Dw2) from the mine. Metals bioaccumulation was determined in gills, mantle, digestive gland, muscle and hemolymph and the following biomarkers were measured in A. trapesialis tissues: total antioxidant capacity against peroxyl radicals, metallothionein content, lipid peroxidation (LPO), proteins carbonylation, glutathione S-transferase activity, superoxide dismutase activity and acetylcholinesterase (AChE) activity. The results showed that Al and Fe bioaccumulation in the gills and hemolymph, Al bioaccumulation in the mantle and muscle, increased LPO in the gills (Dw1 and Dw2) and mantle (Dw1), as well as reduced AChE activity in the muscle (Dw1 and Dw2) should be considered effective biomarkers for monitoring coal mining areas. A. trapesialis proved to be an efficient biological model, considering that biomarkers responses were observed in the clams after only 96h of confinement at Dw sites, accordingly this species could be a good candidate for monitoring Neotropical freshwaters.

Effects of the water soluble fraction of gasoline on ZFL cell line: Cytotoxicity, genotoxicity and oxidative stress

This work aimed to evaluate the effects of different dilutions of gasoline water-soluble fraction (GSF) on Danio rerio hepatocyte cell line (ZFL). Two tests were used to assess cell viability, MTT reduction assay (MTT) and the Trypan blue (TB) exclusion test. Oxidative stress was evaluated through the quantification of reactive oxygen species (ROS) and the assessment of the total antioxidant capacity against peroxyl radicals (ACAP) and the comet assay was employed to assess DNA damage. ZFL cells were exposed to 5, 10, 25 and 50% GSF or only to saline for 1, 3 and 6h. The GSF exhibited concentration-dependent cytotoxicity, and longer exposure times resulted in lower cell viability as indicated by both MTT and TB assays. The establishment of oxidative stress in cells exposed to GSF was not observed at any exposure period and the lower ROS levels could be related to the increased antioxidant capacity after 6-hour exposure. DNA damage was significantly increased after exposure to GSF at the three experimental times. Taking together these results show that GSF has a genotoxic potential at the lower concentrations and becomes cytotoxic at higher concentrations and that ZFL can be considered a good biological model for in vitro toxicological studies.

Triple‐mixture of Zn, Mn, and Fe increases bioaccumulation and causes oxidative stress in freshwater neotropical fish

Metal bioaccumulation and oxidative stress biomarkers were determined in Prochilodus lineatus to understand the effects of short-term exposure to a triple-mixture of Zn, Mn, and Fe. Three independent tests were carried out, in which fish were exposed to 3 concentrations of Zn (0.18, 1.0, and 5.0 mg L-1 ), Mn (0.1, 0.5, and 5.0 mg L-1 ), and in the mix test to Fe (5.0 mg L-1 ) and a mixture of Zn (1.0 mg L-1 ) + Mn (0.5 mg L-1 ), with and without Fe. After exposure for 96 h, tissues were removed for metal bioaccumulation analysis and oxidative stress biomarkers were determined in liver, along with DNA damage in blood cells. Our results revealed that Zn and Mn were bioaccumulated in fish tissues after exposure to 5.0 mg L-1 , whereas Fe only bioaccumulated in muscle and gills after mixture exposure. Results indicated that 1 metal interfered with the others bioaccumulation. In P. lineatus, 5 mg L-1 of both Mn and Fe were toxic, because damage was observed (lipid peroxidation [LPO] in liver and DNA damage in blood cells), whereas Zn induced liver responses (metallothionein [MT] and reduced glutathione [GSH] increases) to prevent damage. In terms of bioaccumulation and alterations of oxidative stress biomarkers, we showed that Zn, Mn, and Fe triple-mixture enhances individual metal toxicity in Neotropical fish P. lineatus. Environ Toxicol Chem 2018;37:1749-1756.

Chromium accumulation and biomarker responses in the Neotropical fish Prochilodus lineatus caged in a river under the influence of tannery activities.

In order to assess the effects of tannery effluents (TE) in organism health, juveniles of Prochilodus lineatus were submitted to in situ tests at four different river locations: site A - upstream of the tannery; site B - next to the tannery; and sites C and D - downstream of the tannery. After 96 h exposure in the river, samples of fish tissue, river water and sediment were collected in order to quantify chromium (Cr) concentrations. Tissue samples were used to assess the activity of ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the content of glutathione (GSH) and metallothionein (MT) and the occurrence of lipid peroxidation (LPO) and DNA damage. Higher Cr concentrations were detected in the water and sediments from site B and in the liver of fish confined at site B, compared to the other sites. Fish caged at site B demonstrated higher levels of liver MT and hepatic EROD activity in relation to fish caged at the other sites. Moreover, fish from site B presented increased liver and branchial GST activities, as well as more GSH in the liver, than fish from site A. There were no significant variations in the occurrence of LPO and DNA damage among fish caged at the different sites. Thus, TE increased Cr levels in the water, sediments, and fish livers and stimulated the synthesis of MT and GSH and the activities of EROD and GST. In conclusion, TE affect the quality of the river and promote changes in biochemical biomarkers and Cr accumulation in P. lineatus.

Single and combined effects of Zn, Mn and Fe on the Neotropical freshwater bivalve Anodontites trapesialis: Bioaccumulation and biochemical biomarkers

Important concentrations of Zn, Mn and Fe were detected in a stream near a coal mining area and promoted, in field, biomarkers alterations in the bivalve Anodontites trapesialis. In order to understand the isolated and mixed effects of these metals on these Neotropical bivalves, we run short-term experiments under laboratory controlled conditions. After 96 h-exposure, tissues (gills, mantle, digestive gland, muscle, hemolymph) were removed for metal bioaccumulation analysis, oxidative stress biomarkers (reactive oxygen species (ROS), total antioxidant capacity, lipoperoxidation (LPO), proteins carbonylation (PC), metallothionein (MT), activity of superoxide dismutase and glutathione S-transferase and hemocytes DNA damage) and cholinesterase (ChE versus ASCh activity) activity evaluation. We run three independent tests. In Zn test, clams were exposed to three concentrations of Zn (0.18 mg L-1, 1.0 mg L-1, 5.0 mg L-1); in Mn test, clams were exposed to three concentrations of Mn (0.1 mg L-1, 0.5 mg L-1, 5.0 mg L-1) and in Mix test, clams were exposed to the mixture Zn (1 mg L-1) + Mn (0.5 mg L-1), with and without Fe (5.0 mg L-1). After single exposure to 5.0 mg L-1, Zn bioaccumulated in all tissues, but only in mantle and hemolymph after exposure to 1.0 mg L-1. The increased MT in gills of A. trapesialis exposed to Zn appears to be sufficient to avoid damage, since LPO occurred only in digestive glands from animals exposed to 5.0 mg L-1. We suggested that A. trapesialis had a metabolic suppression in consequence of Mn presence, based on the following results: the decrease of ROS in gills, the decrease of the Zn and Mn concentrations in tissues and the decrease of ChE versus ASCh activity in muscle. Despite this, animals exposed to Mn suffer oxidative damages (LPO and PC) in the mantle and digestive gland and MT increased in the mantle. These results showed A. trapesialis responded differently to each metal and Mn caused more damage. When exposed to Fe, gills level of ROS was increased, despite no changes in metal accumulation occurred. On the other hand, after exposure to the mixtures, tissues bioaccumulated Zn and previously observed damages caused by Mn and Fe disappeared. Consequently, biomarkers were less affected under mixture treatments, demonstrating mixtures effects or responses were not simply a combination of single exposures to Zn, Mn and Fe, but depend on metals toxicokinetics.