Carlos Eduardo da Rosa

Effects of glyphosate on cholinesterase activity of the mussel Perna perna and the fish Danio rerio and Jenynsia multidentata: in vitro studies.

Although the herbicide glyphosate [N-(phosphonomethyl)glycine] is not classified as an acethylcholinesterase inhibitor, some studies have reported reduction in the acethylcolinesterase activity after in vivo exposure to both its pure form and its commercial formulations. Considering this controversy, the objective of the present study was to investigate, in vitro, the effects of glyphosate exposure on cholinesterase activity of the brown mussel Perna perna and of two fish species: zebrafish Danio rerio and onesided livebearer Jenynsia multidentata. For this purpose, samples of different tissues (brain and muscle for fish; gills and muscle for mussel) were homogenized and pre-incubated with different glyphosate concentrations before cholinesterase activity determination. Results demonstrated that cholinesterase from different fractions of all species tested was inhibited by glyphosate. The concentrations of glyphosate that inhibits 50% of cholinesterase activity (IC50) ranged from 0.62 mM for P. perna muscle to 8.43 mM for J. multidentata brain. According to this, cholinesterase from mussel seems to be more sensitive to glyphosate exposure than those from the fish D. rerio and J. multidentata.

Effect of glyphosate on the sperm quality of zebrafish Danio rerio.

Glyphosate is a systemic, non-selective herbicide widely used in agriculture worldwide. It acts as an inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase by interrupting the synthesis of essential aromatic amino acids. This pathway is not present in animals, although some studies have shown that the herbicide glyphosate can affect fish reproduction. In this study, the effect of glyphosate on sperm quality of the fish Danio rerio was investigated after 24 and 96 h of exposure at concentrations of 5mg/L and 10mg/L. The spermatic cell concentration, sperm motility and motility period were measured employing conventional microscopy. The mitochondrial functionality, membrane integrity and DNA integrity were measured by fluorescence microscopy using specific probes. No significant differences in sperm concentration were observed; however, sperm motility and the motility period were reduced after exposure to both glyphosate concentrations during both exposure periods. The mitochondrial functionality and membrane and DNA integrity were also reduced at the highest concentration during both exposure periods. The results showed that glyphosate can induce harmful effects on reproductive parameters in D. rerio and that this change would reduce the fertility rate of these animals.

Antioxidant mechanisms of the Nereidid Laeonereis acuta (Anelida: Polychaeta) to cope with environmental hydrogen peroxide.

Hydrogen peroxide (H2O2) is a naturally occurring prooxidant molecule, and its effects in the macroinvertebrate infauna were previously observed. The existence of a gradient of antioxidant enzymes activity (catalase [CAT], glutathione peroxidase [GPx], superoxide dismutase [SOD], and glutathione‐S‐transferase [GST]) and/or oxidative damage along the body of the estuarine polychaeta Laeonereis acuta (Polychaeta, Nereididae) was analyzed after exposure to H2O2. Because this species secretes conspicuous amounts of mucus, its capability in degrading H2O2 was studied. The results suggest that L. acuta deal with the generation of oxidative stress with different strategies along the body. In the posterior region, higher CAT and SOD activities ensure the degradation of inductors of lipid peroxidation such as H2O2 and superoxide anion (O2•−). The higher GST activity in anterior region aids to conjugate lipid peroxides products. In the middle region, the lack of high CAT, SOD, or GST activities correlates with the higher lipid hydroperoxide levels found after H2O2 exposure. Ten days of exposure to H2O2 also induced oxidative stress (lipid peroxidation and DNA damage) in the whole animal paralleled by a lack of CAT induction. The mucus production contributes substantially to H2O2 degradation, suggesting that bacteria that grow in this secretion provide this capability.

GH overexpression modifies muscle expression of anti-oxidant enzymes and increases spinal curvature of old zebrafish

Growth hormone (GH) excess causes an increment in the metabolic rate and in reactive oxygen species generation, which accelerate the ageing process in mammals. Considering that there is no information on this subject in fish, the aim of the present study was to evaluate the excess GH effect on senescence in a zebrafish (Danio rerio) transgenic model. In order to reach this objective, we analyzed the phenotype of spinal curvature and expression of genes related to the anti-oxidant defense system and myogenesis in muscle of 8 and 30 months old GH-transgenic males. Gene expression analyses revealed that both superoxide dismutase isoforms were down-regulated only in 30 months old animals, while glutamate cysteine ligase was down-regulated in GH-transgenic zebrafish. Acceleration of the spinal curvature and a reduction in the expression of miogenin at both ages and MyoD in the old fish were also observed. Although neurolipofuscin accumulation was not significant in GH-transgenic zebrafish, the estimation of maximum longevity based on the von Bertalanffy growth function was significantly lower in this group. The results obtained here indicate that GH overexpression reduces the transcription of anti-oxidant defense system and myogenesis-related genes, which probably accelerates senescence in the zebrafish transgenic model used.

GH overexpression decreases spermatic parameters and reproductive success in two-years-old transgenic zebrafish males

Growth hormone (GH) transgenesis has been postulated as a biotechnological tool for improving growth performance in fish aquaculture. However, GH is implied in several other physiological processes, and transgenesis-induced GH excess could lead to unpredictable collateral effects, especially on reproductive traits. Here, we have used two-years-old transgenic zebrafish males to evaluate the effects of GH-transgenesis on spermatic parameters and reproductive success. Transgenic spermatozoa were analyzed in terms of motility, motility period, membrane integrity, mitochondrial functionality, DNA integrity, fertility and hatching rate. We have also performed histological analyses in gonad, in order to verify the presence of characteristic cell types from mature testes. The results obtained have shown that, even in transgenic testes present in all cells in normal mature gonads, a significant general decrease was observed in all spermatic and reproductive parameters analyzed. These outcomes raise concerns about the viability of GH-transgenesis appliance to aquaculture and the environmental risks at the light of Trojan gene hypothesis.

GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.

Effects of increasing temperature on antioxidant defense system and oxidative stress parameters in the Antarctic fish Notothenia coriiceps and Notothenia rossii

Antioxidant defense system (ADS) and oxidative stress parameters were evaluated in the Antarctic fish Notothenia rossii and N. coriiceps exposed to increasing temperature. Acclimated fish were kept at 0°C or exposed to 4°C for 1day (N. rossii) or to 2 and 4°C for 1 and 6 days (N. coriiceps). Measurements were assessed in brain, gills, liver, white muscle and erythrocytes. Parameters analyzed included antioxidant capacity against peroxyl radicals (ACAP); reduced glutathione (GSH) and metallothionein-like proteins (MTLP) concentration; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutamate-cysteine ligase (GCL) activity; lipid peroxidation (LPO) level and protein carbonyl (PC) concentration. Increased liver GST activity was observed in N. rossii exposed to 4°C for 1day. Increased muscle GPx activity was observed in N. coriiceps after exposure to 2°C for 1day. Reduced gill GPx activity and increased liver SOD activity were observed after exposure to 4°C for 1day. In N. coriiceps, increased gill GCL activity and reduced gill GPx activity, as well as reduced liver MTLP were observed after exposure to 2°C for 6 days. Reduced brain SOD activity and increased brain LPO; reduced gill ACAP, GSH concentration and GPx activity, as well as increased gill GCL activity; reduced liver ACAP, MTLP, SOD activity, GST activity and increased liver and erythrocytes LPO were observed after exposure to 4°C for 6 days. These findings indicate that ADS is more responsive to short-term increasing temperature in the sluggish N. coriiceps than in the active N. rossii. However, responses of N. coriiceps to long-term increasing temperature were transient and did not prevent tissue oxidative damage. Considering the predicted increase in temperature in the Southern Ocean over the next decades, our findings suggest that Antarctic fishes are sensitive to ocean warming, displaying tissue oxidative damage associated with the thermal stress.

Genotype-dependent gene expression profile of the antioxidant defense system (ADS) in the liver of a GH-transgenic zebrafish model.

The aim of this study was to evaluate the effects of growth hormone (GH) overexpression on the gene expression profile of multiple components of the antioxidant defense system (ADS) of different genotypes of a GH-transgenic zebrafish (Danio rerio) model. Several ADS-related genes were analyzed by semiquantitative reverse transcription–PCR in the liver of hemizygous (HE) and homozygous (HO) transgenic zebrafish. The results showed a significant reduction in the glutamate cysteine ligase catalytic subunit (GCLC) and the gene expression of two glutathione S-transferase (GST) isoforms and an increase in the glutathione reductase gene in the HO group compared to non-transgenic controls. The expression of the Cu, Zn-superoxide dismutase (SOD1) and catalase (CAT) genes was reduced in HO and HE groups, respectively. Among the ten genes analyzed, two were altered in HE transgenic zebrafish and five were altered in HO transgenic zebrafish. These findings indicate a genotype-dependent gene expression profile of the ADS-related genes in the liver of our GH-transgenic zebrafish model and are in agreement with the general effects of GH hypersecretion in the fish and mouse, which involves a reduction in the capability of the tissues to deal with oxidative stress situations. The GH-transgenic zebrafish model used here seems to be an interesting tool for analyzing the effect of different GH expression levels on physiological processes.

Antioxidant defense system and oxidative status in Antarctic fishes: The sluggish rockcod Notothenia coriiceps versus the active marbled notothen Notothenia rossii

Adaptive responses of antioxidant defense systems (ADS) to changes in increased levels of activity are critical, especially in Antarctic fishes. The benthopelagic marbled notothen (Notothenia rossii) shows higher spontaneous activity than the benthonic and sluggish rockcod (N. coriiceps). Therefore, we hypothesize that species-related responses of ADS would occur to counteract different rates of reactive oxygen species formation in these two Antarctic fish. Here we evaluated ADS and oxidative damage in tissues (brain, gills, liver and white muscle) of the two Antarctic fish. Despite no significant differences in lipid and protein oxidative damage were observed, we actually found species- and tissue-specific differences in ADS. Gill metallothionein-like proteins (MTLP) and liver reduced glutathione (GSH) concentrations were higher in N. coriiceps than in N. rossii. Brain and gill antioxidant capacity against peroxyl radicals (ACAP); gill enzyme [glutamate-cysteine ligase (GSL), superoxide dismutase (SOD) and catalase (CAT)] activity; liver GCL and SOD activity; and white muscle CAT activity were higher in N. rossii than in N. coriiceps. Therefore, the more active fish (N. rossii) maintains higher activities of enzymes involved in superoxide ions (O2.-) detoxification and GSH production in peripheral tissues (gills, liver and white muscle). This allows the more active fish (N. rossii) to keep levels of lipid and protein oxidative damage similar to those observed in the sluggish fish (N. coriiceps). It is worth noting that the more active fish also shows a higher brain antioxidant capacity, which could involve other non-enzymatic antioxidants like vitamins C and E. In contrast, N. coriiceps shows lower consumption of non-enzymatic antioxidants in peripheral tissues than N. coriiceps. As hypothesized, our results indicate that differences in ADS profiles between fish species are likely related to their habits and metabolic rates. This would imply in different fish abilities to deal with oxidative stress associated with increasing seawater temperature.

Embryo-larval exposure to atrazine reduces viability and alters oxidative stress parameters in Drosophila melanogaster

The herbicide atrazine has been used worldwide with subsequent residual contamination of water and food, which may cause adverse effects on non-target organisms. Animal exposure to this herbicide may affect development, reproduction and energy metabolism. Here, the effects of atrazine regarding survival and redox metabolism were assessed in the fruit fly D. melanogaster exposed during embryonic and larval development. The embryos (newly fertilized eggs) were exposed to different atrazine concentrations (10μM and 100μM) in the diet until the adult fly emerged. Pupation and emergence rates, developmental time and sex ratio were determined as well as oxidative stress parameters and gene expression of the antioxidant defence system were evaluated in newly emerged male and female flies. Atrazine exposure reduced pupation and emergence rates in fruit flies without alterations to developmental time and sex ratio. Different redox imbalance patterns were observed between males and females exposed to atrazine. Atrazine caused an increase in oxidative damage, reactive oxygen species generation and antioxidant capacity and decreased thiol-containing molecules. Further, atrazine exposure altered the mRNA expression of antioxidant genes (keap1, sod, sod2, cat, irc, gss, gclm, gclc, trxt, trxr-1 and trxr-2). Reductions in fruit fly larval and pupal viability observed here are likely consequences of the oxidative stress induced by atrazine exposure.