Juliano Zanette

Biochemical biomarkers and metals in Perna perna mussels from mariculture zones of Santa Catarina, Brazil

The activity of cholinesterase (ChE), glutathione-S transferase (GST), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH) and catalase (CAT) was evaluated in the gill and digestive glands of the Perna perna mussel transplanted to three non-contaminated mariculture zones under the influence of distinct physical-chemical characteristics. Differences among sites for ChE, GST and CAT activities in gill, as well as ChE, GST and G6PDH activity in digestive gland of mussels, were found and possibly related to differences in physicochemical characteristics of the sites and/or biological status of the mussels. Mussels that were transplanted to another, more urbanized site (Ponta do Lessa) with similar physicochemical characteristics to one of the farming sites (Sambaqui), was also chosen to evaluate biomarker responses to pollution. Activities of ChE, GST and GR in the digestive glands and CAT in the gills were higher in the polluted site. GR was the only biomarker to be unaltered in different farming sites, but induced in the pollution site. The trace metal concentrations in the mussels were low and unlikely to cause the changes observed in the biomarker levels. The present study strongly suggests that monitoring programs should compare sites with similar physicochemical characteristics when using a complementary biomarker approach. In addition, the baselines for the biomarkers and metal used in the present study can serve as a reference for the monitoring of these mariculture zones in future monitoring programs employing P. perna.

Salinity influences glutathione S-transferase activity and lipid peroxidation responses in the Crassostrea gigas oyster exposed to diesel oil

Biochemical responses in bivalve mollusks are commonly employed in environmental studies as biomarkers of aquatic contamination. The present study evaluated the possible influence of salinity (35, 25, 15 and 9ppt) in the biomarker responses of Crassostrea gigas oysters exposed to diesel at different nominal concentrations (0.01, 0.1 and 1mL.L(-1)) using a semi-static exposure system. Salinity alone did not resulted in major changes in the gills catalase activity (CAT), glutathione S-transferase activity (GST) and lipid peroxidation levels (measured as malondialdehyde, MDA), but influenced diesel related responses. At 25ppt salinity, but not at the other salinity levels, oysters exposed to diesel showed a strikingly positive concentration-dependent GST response. At 25ppt and 1mL.L(-1) diesel, the GST activity in the gills remained elevated, even after one week of depuration in clean water. The increased MDA levels in the oysters exposed to diesel comparing to control groups at 9, 15 and 35ppt salinities suggest the occurrence of lipid peroxidation in those salinities, but not at 25ppt salinity. The MDA quickly returned to basal levels after 24h of depuration. CAT activity was unaltered by the treatments employed. High toxicity for 1mL.L(-1) diesel was observed only at 35ppt salinity, but not in the other salinities. Results from this study strongly suggest that salinity influences the diesel related biomarker responses and toxicity in C. gigas, and that some of those responses remain altered even after depuration.

Expression and activity of glutathione S-transferases and catalase in the shrimp Litopenaeus vannamei inoculated with a toxic Microcystis aeruginosa strain

Microcystin (MC) produced during cyanobacteria blooms is notably toxic to human and wildlife. Conjugation with reduced glutathione (GSH) by glutathione S-transferase (GST) and the antioxidant enzymes defenses (e.g. catalase, CAT) are important biochemical defense mechanisms against MCs toxicity. We investigated the enzymatic activity of CAT and GST and the gene expression levels of CAT and eight GST isoforms in the hepatopancreas of the globally farmed shrimp Litopenaeus vannamei 48-h after injection with a sub-lethal dose of 100 μg kg⁻¹ of a toxic Microcystis aeruginosa extract. MCs caused up-regulation for GSTΩ, μ and a MAPEG isoform, by 12-, 2.8- and 1.8-fold, respectively, and increases in the total GST enzyme activity and CAT enzyme activity. The study points to the importance of further characterization for the L. vannamei GST isoforms and GST/CAT post-translational regulation processes to better understand the key mechanisms involved in the shrimps defense against MC exposure.

Biomarkers of Organic Contamination in the South American Fish Poecilia vivipara and Jenynsia multidentata

South American cyprinodontiform fish are potential candidates to be used as model biomarker species of exposure in environmental toxicology. The aim of this study was to identify molecular and biochemical biomarkers of pollution using Poecilia vivipara (Poecilidae) and Jenynsia multidentata (Anablepidae). Partial nucleotide sequences for cytochrome P-450 1A (CYP1A), a classical biomarker of exposure to organic contaminants in fish, were identified in P. vivipara and J. multidentata (approximately 650 nucleotides) using degenerated primers and polymerase chain reaction (PCR). These sequences shared approximately 90% identity in the predicted amino acid sequence with the corresponding CYP1A region of Fundulus heteroclitus. Real-time quantitative PCR (RT-qPCR) analysis confirmed that CYP1A transcription was markedly induced in the liver and gills of J. multidentata (approximately185-fold and 20-fold, respectively) and P. vivipara (122-fold and 739-fold, respectively) 24 h after exposure to 1 μM synthetic CYP1A inducer β-naphthoflavone (BNF). At 24 h after injection with 1 μg/g environmental carcinogenic contaminant benzo[a]pyrene (BaP), a decreased total antioxidant capacity against peroxyl radicals was observed both in liver of J. multidentata and gills of P. vivipara. BaP injection in both fish did not produce changes in lipid peroxide (thiobarbituric acid-reactive substances, TBARS) levels, suggesting an absence of an oxidative stress condition. The newly identified CYP1A may thus serve as general biomarker of exposure to organic contaminant in future studies using P. vivipara and J. multidentata. Data also indicate the importance of species-specific differences in biomarker responses in these South American cyprinodontiform fish, suggesting distinct resistance/susceptibility properties to polycyclic aromatic hydrocarbons.

Biochemical biomarkers in barnacles Balanus improvisus: pollution and seasonal effects.

Biochemical biomarkers were evaluated in the barnacle Balanus improvisus (Crustacea: Cirripedia) sampled from both polluted and reference sites in the Patos Lagoon Estuary, Southern Brazil. During winter, higher glutathione S-transferase (GST) activity was recorded in the barnacles from the polluted sites, indicating environmental exposure to contaminants. Relatively low lipid peroxide levels (LPO) were also observed in barnacles from polluted sites, indicating that oxidative stress by lipid peroxidation was not a major threat in barnacles from those sites. Seasonal differences in the GST and total oxyradical scavenging capacity (TOSC) could have contributed to the low LPO levels in the summer relative to the levels in the winter. Catalase activity and metallothionein levels were not affected by contamination or seasonality. The seasonal changes observed in biomarker responses were paralleled by the differences in temperature, which could have affected physiological responses, including the balance between pro-oxidants and antioxidants.

Basal and 3-methylcholanthrene-induced expression of cytochrome P450 1A, 1B and 1C genes in the Brazilian guppy, Poecilia vivipara

In fish there are four cytochrome P450 (CYP1) subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Here we cloned Poecilia vivipara CYP1A, with an inferred amino acid sequence 91% identical to CYP1A from the killifish Fundulus heteroclitus, another member of the Cypriniformes, and an important model in ecotoxicology. In addition, we examined the expression of CYP1A, CYP1B1, and CYP1C1 by qPCR in liver, gill, and intestine of adult P. vivipara injected with 3-methylcholanthrene (3-MC) or held in clean water (control group) for 24h. All three tissues examined showed basal expression of the three CYP1 genes. CYP1A was most strongly expressed in the liver, while CYP1B1, and CYP1C1 were most strongly expressed in the gill and intestine respectively. 3-MC induced CYP1A, CYP1B1, and CYP1C1 significantly (20-120-fold) in the three organs, consistent with the regulation of CYP1A, CYP1B1 and CYP1C1 via the aryl hydrocarbon receptor. Validation of CYP1 gene biomarkers in fish collected from a contaminated urban mangrove environment was confirmed with significant induction of CYP1A and CYP1C1 in gills (10-15-fold) and CYP1B1 in liver (23-fold), relative to fish from a control site. The responsiveness of these CYP1 genes indicates P. vivipara is suitable as a model for environmental toxicology studies and environmental assessment in Brazil.

Salinity-dependent copper accumulation in the guppy Poecilia vivipara is associated with CTR1 and ATP7B transcriptional regulation

Copper (Cu) accumulation and regulation of key-genes involved in Cu homeostasis were evaluated in freshwater- and saltwater-acclimated guppies Poecilia vivipara. Fish were exposed (96h) to environmentally relevant concentrations of dissolved Cu (0, 5.0, 9.0 and 20.0μg/L). In freshwater guppies, gill and liver Cu accumulation was dependent on Cu concentration in the exposure medium. In saltwater guppies, this dependence was observed only in the gut. These findings indicate that Cu accumulation was salinity- and tissue-dependent. Key genes involved in Cu metabolism were sequenced for the first time in P. vivipara. Transcripts coding for the high-affinity copper transporter (CTR1) and copper-transporting ATPase (ATP7B) were identified using polymerase chain reaction (PCR) and gene sequencing. The full-length CTR1 open reading frame (1560bp) and a partial ATP7B (690bp) were discovered. Predicted amino acid sequences shared high identities with the CTR1 of Fundulus heteroclitus (81%) and the ATP7B of Sparus aurata (87%). Basal transcriptional levels addressed by RT-qPCR in control fish indicate that CTR1 and ATP7B was highly transcribed in liver of freshwater guppies while CTR1 was highly transcribed in gut of saltwater guppies. This could explain the higher Cu accumulation observed in liver of freshwater guppies and in gut of saltwater guppies, because CTR1 is involved in Cu uptake. Reduced gill mRNA expression of CTR1 was observed in freshwater guppies exposed to 20.0μg/L Cu and in saltwater guppies exposed to 5.0μg/L Cu. In turn, reduced mRNA expression of gut ATP7B was observed in freshwater and salt water guppies exposed to 9.0 and 20.0μg/L Cu. Liver CTR1 and ATP7B transcription were not affected by Cu exposure. These findings suggest that gill CTR1 and gut ATP7B are down-regulated to limit Cu absorption after exposure to dissolved Cu, while liver CTR1 and ATP7B levels are maintained to allow Cu storage and detoxification. In conclusion, findings reported here indicate that Cu accumulation in the euryhaline guppy P. vivipara is tissue specific and dependent on water salinity. They also suggest that Cu homeostasis involves a differential transcriptional regulation of the newly identified Cu transporters, CTR1 and ATP7B.

Identification of differentially transcribed genes in shrimp Litopenaeus vannamei exposed to osmotic stress and challenged with WSSV virus

The effects of hyposmotic stress and white spot syndrome virus (WSSV) challenge in expression was studied in the marine shrimp Litopenaeus vannamei. Messenger RNA from gills of shrimp submitted to osmotic stress was isolated to identify genes differentially expressed through the suppressive subtractive hybridization (SSH) method. Two subtractive libraries forward and two reverse were constructed to identify up and down-regulated genes under these conditions. About 192 clones were sequenced, of which 46 genes were identified. These genes encode proteins corresponding to a wide range of biological roles, including defense, cell signaling, electron transfer, cell proliferation and differentiation, apoptosis, intermediary metabolism, cytoskeleton and digestion. Among the identified genes, 19 were up-regulated and 27 were down-regulated in the animals kept at a lower ion concentration. We evaluated the expression of eight genes by RT-qPCR in shrimp submitted to hyposmotic conditions with and without WSSV challenge. The SSH enabled the identification of genes that are influenced by hyposmotic stress. A significant up-regulation was observed in lectin-C, QM, TGF beta inducible nuclear protein 1, ciclophilin, malate dehydrogenase, mitochondrial ATP synthase F chain and ATP synthase subunit 9 precursor transcripts. However, the expression of these genes in L. vannamei was not affected by WSSV infection both at isosmotic and hyposmotic conditions.

Modulation of antioxidant and detoxification responses induced by lipoic acid in the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) subjected to hypoxia and re-oxygenation

The effect of lipoic acid supplementation and moderate hypoxia (3 mg/L), followed by re-oxygenation, was analyzed in terms of antioxidant and oxidative damage responses in juvenile shrimp Litopenaeus vannamei. Lipoic acid (LA)-enriched rations (D1: 76.4 ± 6.4; D2: 196.4 ± 70.2; and D3: 397.2 ± 79.97 mg LA/kg) were offered to shrimps. A control group without LA adding was also run. After 45 days, LA-enriched ration increased the activity of the detoxifying enzyme glutathione-S-transferase in gills. Total antioxidant capacity against peroxy radicals was augmented in gills and hepatopancreas at doses D2 and D3. Doses D1 and D2 of LA reduced the levels of oxidative damage (lipid peroxidation) in gills and hepatopancreas. The results showed that certain LA doses (particularly D2) improved not only antioxidant responses but also weight gain. It can be concluded that LA triggered antioxidant and detoxification protection in L. vannamei, allowing the shrimp to cope with environmental stressful factors.

Responses of the CYP1A biomarker in Jenynsia multidentata and Phalloceros caudimaculatus and evaluation of a CYP1A refractory phenotype.

The level of cytochrome P450 1A (CYP1A) in fish is used as a typical environmental biomarker for the presence of organic contaminants. We used RT-qPCR to investigate CYP1A mRNA levels in the liver, gill and gonopodium of guppies Jenynsia multidentata and Phalloceros caudimaculatus in wetlands within the Rio Grande city (RG) which is under the influence of the Patos Lagoon Estuary (RS, Brazil). The CYP1A mRNA levels evaluated in fish liver from two locations that receive non-treated wastewater effluents (S3 and S4) and another locations near an oil refinery (S6) and an industrial complex (S7), were higher than in locations remote from those sites (S1, S2 and S5). The sum of 16 priority PAHs in sediment confirmed high levels in S4 and S6 (3914.0 and 4414.0 ng g(-1) dw, respectively) comparing to S7>S2>S3>S5>S1 (119.3, 66.3, 62.8, 16.4 and 1.7 ng g(-1) dw). J. multidentata from sites S1 to S4 that were transferred to the laboratory exhibited CYP1A induction after 24 h waterborne exposure to 1 µM betanaphtoflavone (BNF) in all organs compared to controls, except in the liver of fish from site S4. This lack of CYP1A induction by BNF indicates a CYP1A refractory phenotype in guppy. Although this characteristic possibly involves the alteration in AHR signaling or control, the mechanism of resistance is unknown. The present study provides information about the use of the use of CYP1A in South American guppies as an useful biomarker tool for environmental contamination studies.