Emerging and legacy contaminants on the Brazilian southern coast (Santa Catarina): A multi-biomarker approach in oysters Crassostrea gasar (Adanson, 1757).

Coastal environments, such as those in the Santa Catarina State (SC, Brazil), are considered the primary receptors of anthropogenic pollutants. In this study, our objective was to evaluate the levels of emerging contaminants (ECs) and persistent organic pollutants (POPs) in indigenous Crassostrea gasar oysters from different regions of SC coast in the summer season (March 2022). Field collections were conducted in the São Francisco do Sul, Itajaí, Florianópolis and Laguna coastal zones. We analyzed the bioaccumulation levels of 75 compounds, including antibiotics (AB), endocrine disruptors (ED), non-steroidal anti-inflammatory drugs (NSAIDs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Furthermore, we assessed biomarker responses related to biotransformation, antioxidant defense, heat shock protection and oxidative damage in oysters' gills. Prevalence of ECs was observed in the central and southern regions, while the highest concentrations of POPs were detected in the central-northern regions of SC. Oysters exhibited an induction in biotransformation systems (cyp2au1 and cyp356a1, sult and GST activity) and antioxidant enzymes activities (SOD, CAT and GPx). Higher susceptibility to lipid peroxidation was observed in the animals from Florianópolis compared to other regions. Correlation analyses indicated possible associations between contaminants and environmental variables in the biomarker responses, serving as a warning related to climate change. Our results highlight the influence of anthropogenic activities on SC, serving as baseline of ECs and POPs levels in the coastal areas of Santa Catarina, indicating more critical zones for extensive monitoring, aiming to conserve coastal regions.

Biochemical responses of the mussel Perna perna after exposure to environmental concentrations of the UV filter Benzophenone-3.

Personal care products, such as UV filters, are frequently present in aquatic ecosystems, but studies on their impact on marine organisms are still scarce. Here we addressed the effects of benzophenone-3 (BP-3) on the antioxidant status of Perna perna mussels exposed to concentrations of 0.1 and 3 µg.L-1 for 72 h and 7 days. Glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH) activity and lipoperoxidation (MDA) were evaluated in the gills. A significant reduction (p < 0.05) in the activity of G6PDH and GPx was observed after exposure for 7 days to 0.1 µg.L-1. However, no significant differences were observed in GST activity and MDA levels, independently of the exposure time. Principal component analysis (PCA) showed an association of BP-3 highest concentration with GR and MDA at 72 h and only with GR at 7 days of exposure. Similarly, the integrated biomarker response (IBR) demonstrated GR and MDA alterations. In conclusion, environmentally relevant concentrations of BP-3 altered antioxidant and auxiliary enzymes, which could cause long-term damage to P.perna mussels. The need to implement more efficient techniques in wastewater treatment systems is pointed out, especially in summer, when UV filters are used more frequently and abundantly.

Thiol oxidation of hemolymph proteins in oysters Crassostrea brasiliana as markers of oxidative damage induced by urban sewage exposure.

Urban sewage is a concerning issue worldwide, threatening both wildlife and human health. The present study investigated protein oxidation in mangrove oysters (Crassostrea brasiliana) exposed to seawater from Balneário Camboriú, an important tourist destination in Brazil that is affected by urban sewage. Oysters were exposed for 24 h to seawater collected close to the Camboriú River (CAM1) or 1 km away (CAM2). Seawater from an aquaculture laboratory was used as a reference. Local sewage input was marked by higher levels of coliforms, nitrogen, and phosphorus in seawater, as well as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), linear alkylbenzenes (LABs), and fecal steroid in sediments at CAM1. Exposure of oysters to CAM1 caused marked bioaccumulation of LABs and decreased PAH and PCB concentrations after exposure to both CAM1 and CAM2. Protein thiol oxidation in gills, digestive gland, and hemolymph was evaluated. Lower levels of reduced protein thiols were detected in hemolymph from CAM1, and actin, segon, and dominin were identified as targets of protein thiol oxidation. Dominin susceptibility to oxidation was confirmed in vitro by exposure to peroxides and hypochlorous acid, and 2 cysteine residues were identified as potential sites of oxidation. Overall, these data indicate that urban sewage contamination in local waters has a toxic potential and that protein thiol oxidation in hemolymph could be a useful biomarker of oxidative stress in bivalves exposed to contaminants. Environ Toxicol Chem 2017;36:1833-1845. © 2016 SETAC.

Hsp70 expression in shrimp Litopenaeus vannamei in response to IHHNV and WSSV infection.

The effects of environmental changes, on non-target organisms may impact and bring consequences at the molecular level in marine organisms. There is a lack of data supporting the hypothesis according to which environmental stress modulates the immune system, leading to an increased susceptibility to infectious agents in shrimps. The present study was focused on changes occurring in cellular defense proteins in Litopenaeus vannamei infected by virus infection hypodermal and hematopoietic necrosis (IHHNV) and white spot syndrome (WSSV). Western blot analysis was used to evaluate expression of protein Hsp70, in gill tissue. Up-regulation levels were supported by immune detection analysis, suggesting that IHHNV and WSSV-shrimp infection promotes changes in the expression of these proteins.

Assessment of the sublethal toxicity of organochlorine pesticide endosulfan in juvenile common carp (Cyprinus carpio).

This study is aimed at evaluating the sublethal effects of endosulfan (EDS) in juvenile common carp (Cyprinus carpio). For this purpose, fish were exposed for 15 days to the technical EDS (95% pure) diluted in dimethyl sulfoxide (DMSO) 0.1% of the total volume in water solution in a semi-static system at sublethal concentration (1 µg/L). Subsequently, the liver somatic index (LSI) and factor condition (K) were determined. The total cytocrome P450 (CYP), CYP1A isoform, and the ethoxyresorufin-O-deethylase (EROD) activity were determined from the hepatic microsomal fraction as well as the activity of the oxidative stress enzyme system such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GP(X)), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH). Among the parameters assessed, EDS at the sublethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (Cyprinus carpio). Thus, it is seen that the use of biochemical biomarkers of environmental contamination in this study proved to be an extremely important tool for detecting the adverse effects of xenobiotics in the aquatic environment, even at low concentration.

Genotyping of white spot syndrome virus (WSSV) geographical isolates from Brazil and comparison to other isolates from the Americas.

White spot syndrome virus (WSSV) is a viral pathogen that has caused significant economic losses in shrimp farming. Variable-number tandem repeats (VNTRs) (open reading frame [ORF] 94, 125 and 75), a large deletion (ORF 23/24) and a transposase were proposed as molecular markers for genotyping. WSSV-infected shrimp Litopenaeus vannamei were collected in 2 Brazilian regions (Santa Catarina and Bahia) from 2005 to 2008. DNA was extracted and PCR of the variable regions was performed, followed by sequencing. All Santa Catarina samples showed the same number of repeats for the minisatellites analyzed. Bahia samples showed a different pattern for the regions, indicating that there are at least 2 different WSSV genotypes in Brazil. Both Brazilian isolates have an 11453 bp deletion in ORF 23/24 when compared with WSSV-TW (Taiwan), which has the full sequence for this locus. The Brazilian WSSV isolates were compared with WSSV isolates from other countries in the Americas (USA, Panama, Honduras, Mexico and Nicaragua); the repeat number patterns for the 3 VNTR regions analyzed were different between the Brazilian isolates and the other western-hemisphere isolates. This may be due to mutations in WSSV after its introduction into the different countries. Our results also show that WSSV found in Bahia and Santa Catarina very likely originated from different sources of contamination.

Zinc reverses malathion-induced impairment in antioxidant defenses.

Malathion toxicity has been related to the inhibition of acetylcholinesterase and induction of oxidative stress, while zinc has been shown to possess neuroprotective effects in experimental and clinical studies. In the present study the effect of zinc chloride (zinc) was addressed in adult male Wistar rats following a long-term treatment (30 days, 300mg/L in tap water ad libitum) against an acute insult caused by a single malathion exposure (250mg/kg, i.p.). Malathion produced a significant decrease in hippocampal acetylcholinesterase, as well as a decrease in the activity of several hippocampal antioxidant enzymes: glutathione reductase, glutathione S-transferase, catalase and superoxide dismutase. The pretreatment with zinc did not completely prevent acetylcholinesterase activity impairment; however, antioxidant activity was completely restored. Zinc administration significantly increased HSP60, but not HSP70, expression. The HSP60 increase suggests a novel zinc-dependent pathway, which may be related to a counteracting mechanism against malathion effects. Based on these results the following hypothesis can be presented: the published "pro-oxidative" effect of malathion may be related, among others, to compromised antioxidant defenses, while the zinc "antioxidant" action may be related to the preservation of antioxidant defenses. In conclusion, our data points to the inhibition of antioxidant enzymes as an important non-cholinergic effect of malathion, which can be rescued by oral zinc treatment.

Biochemical alterations in juvenile carp (Cyprinus carpio) exposed to zinc: glutathione reductase as a target.

The aim of this study was to investigate biochemical changes in juvenile carp (Cyprinus carpio) exposed to zinc chloride (10, 30 and 100 microM) for a period of 48 h. Zinc exposure caused a concentration-dependent reduction in glutathione reductase (GR) activity in gills, liver and brain. Gill glutathione S-transferase (GST) was reduced when animals were exposed to the highest concentration of 100 microM zinc. The phosphorylation of p38(MAPK) increased in the brain of fish exposed to zinc 100 microM, while phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and c-Jun N-terminal protein kinase 1/2 (JNK1/2) remained unchanged. Expression of proteins HSP60 and HSP70 were not affected by zinc exposure. Considering the significant concentration-dependent inhibition of GR in all tissues analyzed, this enzyme could be a potential biomarker of exposure to zinc, which has to be confirmed.

Induced gene expression in oyster Crassostrea gigas exposed to sewage.

Pacific oysters, Crassostrea gigas, were exposed to untreated sewage diluted in seawater. After 48h of exposure, the expression of genes associated to biotransformation pathways (CYP356A1, GSTO, MDR, FABP and ALAS) were analyzed in gills through semi-quantitative RT-PCR. A significant induction in all genes analyzed in the sewage-exposed oysters was observed. These genes are related to phase I (CYP356A1), phase II (GSTO) and phase III (MDR) biotransformation systems, to the uptake and transport of hydrophobic ligands (FABP) and to the synthesis of prosthetic group heme (ALAS). The organisms were able to survive in contaminated conditions since protective mechanisms have been properly stimulated.

Glutathione S-transferase cytosolic isoform in the pink-shrimp, Farfantepenaeus brasiliensis, from Conceição Lagoon, Santa Catarina Island, SC, Brazil.

Contaminant input into the environment can affect the biochemical responses of exposed organisms. Activity of conjugation enzymes, such as glutathione S-transferase (GST) has been proposed as a biomarker of susceptibility to the presence of potentially damaging xenobiotics. The aim of the present study was to evaluate the activity of total and Pi-class GST in the hepatopancreas of pink-shrimp Farfantepenaeus brasiliensis. The shrimp were caught at a single site at Conceição Lagoon and transplanted to another site (Canto da Lagoa - CA) in the same lagoon, and to the laboratory (LAB), where they were kept for 15 days. This experiment was carried out in winter 2003 and in summer 2004. Shrimp were sampled before the exposure, at a zero time (t0), and after 15-days at CA site and LAB. The activity of total GST and Pi-isoform were analyzed using CDNB and ethacrynic acid (EA) as substrates, respectively. Shrimp caught in winter showed higher levels of Pi-class GST than those caught in summer, for both t0 and CA groups. Moreover, differences in the activity of this GST isoform between groups were observed only in winter, where the animals from t0 and CA groups presented higher activity when compared to those kept in the laboratory. This difference could be associated either to changes in the contaminant input or to other water quality parameters in this ecosystem. The elevated GST Pi activity observed in the shrimp of both t0 and CA groups sampled in winter could be related to salinity, since it was higher in winter than in summer. We could also suggest a possible association of this finding to a seasonal metabolic variation in this penaeid. If that is the case, one should take into account these alterations when considering the use of this GST isoform as biomarker in F. brasiliensis in environmental monitoring programs.

Antioxidant status and stress proteins in the gills of the brown mussel Perna perna exposed to zinc.

Zinc, at low levels, has several basic housekeeping functions in metalloenzymes, transcription factors, immunoregulation, growth, and cytoprotection, displaying antioxidant, anti-apoptotic, and anti-inflammatory roles. At high levels, however, the metal can be highly toxic. The aim of this work is to investigate the toxic effect of zinc on antioxidant status and stress proteins in the gills of the brown mussel Perna perna exposed for 48 h to zinc chloride (zinc) at 10, 30 and 100 microM. Glutathione reductase (GR) activity was drastically reduced at 30 and 100 microM zinc. At the lower levels, i.e. 10 microM zinc, antioxidant defenses were up-regulated, as were glutathione levels and the activities of glutathione peroxidase and catalase, in spite of the absence of effect on glutathione S-transferase and glucose 6-phosphate dehydrogenase activity. At the higher tested concentration of 100 microM zinc, oxidative stress was apparent as reflected by the increased lipid peroxidation end products and decreased protein thiol and glutathione levels, associated with an inability to up regulate antioxidant defenses. Using 30 microM zinc, higher gill rhodamine B efflux was observed, indicating an activation of multixenobiotic resistance (MXR) activity, which is reinforced by increased immunoreactive P-glycoprotein detection. Zinc also increased the HSP60-immunoreactive protein, whereas the HSP70-immunoreactive protein remained unchanged. Overall, the results indicate that zinc toxicity -- at higher levels -- may be connected to a strong inhibition of GR activity, and related to the pro-oxidative state found. Mussels showed an adaptive-like response to 10 microM zinc by increasing antioxidant defenses. Increased P-glycoprotein and HSP60 expression, and rhodamine B efflux were also remarkable features in the gill response to zinc.