Alberto Teodorico Correia

Effect of acetaminophen exposure in Oncorhynchus mykiss gills and liver: Detoxification mechanisms, oxidative defence system and peroxidative damage

The increasing presence of pharmaceutical drugs in nature is cause of concern due to the occurrence of oxidative stress in non-target species. Acetaminophen is widely used in human medicine as an analgesic and antipyretic drug, and it is one of the most sold non-prescription drugs. The present study aimed to assess the toxic effects of acetaminophen (APAP) in Oncorhynchus mykiss following acute and chronic exposures in realistic levels. In order to evaluate the APAP effects in the rainbow trout, gills and liver were analyzed with biochemical biomarkers, such as catalase (CAT), total and selenium-dependent glutathione peroxidase (GPx), glutathione reductase (GRed) and glutathione-S-transferases (GSTs) activity and also lipid peroxidation levels (TBARS). The results obtained in all tests indicate that a significant response of oxidative stress was established, along with the increase of APAP concentrations. The establishment of an oxidative stress scenario occurred with the involvement of all tested biomarkers, sustaining a generalized set of pro-oxidative effects elicited by APAP. Additionally, the occurrence of oxidative damage strongly suggests the impairment of the antioxidant defense mechanism of O. mykiss. It is important to note that the occurrence of oxidative deleterious effects and peroxidative damages occurred for concentrations similar to those already reported for several freshwater ecosystems. The importance of these assumptions is further discussed under the scope of ecological relevance of the assessment of effects caused by pharmaceuticals in non-target organisms.

Acute Effects of Tetracycline Exposure in the Freshwater Fish Gambusia holbrooki: Antioxidant Effects, Neurotoxicity and Histological Alterations

AbstractA large body of evidence was compiled in the recent decades showing a noteworthy increase in the detection of pharmaceutical drugs in aquatic ecosystems. Due to its ubiquitous presence, chemical nature, and practical purpose, this type of contaminant can exert toxic effects in nontarget organisms. Exposure to pharmaceutical drugs can result in adaptive alterations, such as changes in tissues, or in key homeostatic mechanisms, such as antioxidant mechanisms, biochemical/physiological pathways, and cellular damage. These alterations can be monitored to determine the impact of these compounds on exposed aquatic organisms. Among pharmaceutical drugs in the environment, antibiotics are particularly important because they include a variety of substances widely used in medical and veterinary practice, livestock production, and aquaculture. This wide use constitutes a decisive factor contributing for their frequent detection in the aquatic environment. Tetracyclines are the individual antibiotic subclass with the second highest frequency of detection in environmental matrices. The characterization of the potential ecotoxicological effects of tetracycline is a much-required task; to attain this objective, the present study assessed the acute toxic effects of tetracycline in the freshwater fish species Gambusia holbrooki by the determination of histological changes in the gills and liver, changes in antioxidant defense [glutathione S-transferase (GST), catalase (CAT), and lipoperoxidative damage] as well as potential neurotoxicity (acetylcholinesterase activity). The obtained results suggest the existence of a cause-and-effect relationship between the exposure to tetracycline and histological alterations (more specifically in gills) and enzymatic activity (particularly the enzyme CAT in liver and GST in gills) indicating that this compound can exert a pro-oxidative activity.

Acute and chronic effects of erythromycin exposure on oxidative stress and genotoxicity parameters of Oncorhynchus mykiss

Erythromycin (ERY) is a macrolide antibiotic used in human and veterinary medicine, and has been detected in various aquatic compartments. Recent studies have indicated that this compound can exert biological activity on non-target organisms environmentally exposed. The present study aimed to assess the toxic effects of ERY in Oncorhynchus mykiss after acute and chronic exposures. The here adopted strategy involved exposure to three levels of ERY, the first being similar to concentrations reported to occur in the wild, thus ecologically relevant. Catalase (CAT), total glutathione peroxidase (GPx), glutathione reductase (GRed) activities and lipid peroxidation (TBARS levels) were quantified as oxidative stress biomarkers in gills and liver. Genotoxic endpoints, reflecting different types of genetic damage in blood cells, were also determined, by performing analysis of genetic damage (determination of the genetic damage index, GDI, measured by comet assay) and of erythrocytic nuclear abnormalities (ENAs). The results suggest the occurrence of a mild, but significant, oxidative stress scenario in gills. For acutely exposed organisms, significant alterations were observed in CAT and GRed activities, and also in TBARS levels, which however are modifications with uncertain biological interpretation, despite indicating involvement of an oxidative effect and response. After chronic exposure, a significant decrease of CAT activity, increase of GPx activity and TBARS levels in gills was noticed. In liver, significant decrease in TBARS levels were observed in both exposures. Comet and ENAs assays indicated significant increases on genotoxic damage of O. mykiss, after erythromycin exposures. This set of data (acute and chronic) suggests that erythromycin has the potential to induce DNA strand breaks in blood cells, and demonstrate the induction of chromosome breakage and/or segregational abnormalities. Overall results indicate that both DNA damaging effects induced by erythromycin may be related to the oxidative damage observed, shown to occur at environmentally relevant concentrations of erythromycin.

Evidence for genetic differentiation in the European conger eel Conger conger based on mitochondrial DNA analysis

The European conger eel Conger conger is an important marine benthic fish in the North-East Atlantic and represents a valuable fishery resource. However, little is known about its reproductive biology. In an attempt to gain a better understanding of the conger eel population structure, mitochondrial DNA (mtDNA) sequences were examined. A region with 432 bp of the control region of the mtDNA was sequenced from 40 individuals from six different locations around the central and eastern North Atlantic Ocean. Thirty variable positions defined 28 distinct haplotypes. The average sequence difference within samples (1.3–4.2%) was comparable to those between samples (1.4–3.6%). MtDNA sequence-based statistical tests showed significant geographic differentiation between some local population samples, suggesting that the conger eel does not comprise a single panmictic population. However, given our sample sizes, these preliminary results should be interpreted with caution and more individuals from more sites, including the Mediterranean Sea, should be analyzed in detail. The genetic variability detected in this study is an initial step to elucidate the genetic back-ground of the conger eel population structure.

Rainbow trout (Oncorhynchus mykiss) pro-oxidant and genotoxic responses following acute and chronic exposure to the antibiotic oxytetracycline

Oxytetracycline (OTC), an antibacterial agent, is extensively used in aquaculture practices all over the world, but also in human and veterinary medicines. Because of its intensive use, low rates of absorption by treated animals, inadequate disposal, and low efficiency of removal in wastewater treatment plants, the potential harmful effects on aquatic organisms are of great concern. This work aimed to assess the effects of this antibiotic in rainbow trout, following both acute and chronic exposures. Catalase (CAT), total glutathione peroxidase (GPx), glutathione reductase (GRed) activities and lipid peroxidation (TBARS levels) were quantified as oxidative stress biomarkers, in gills and liver. Genotoxic endpoints, reflecting different types of genetic damage in blood cells, were also determined, by analysis of genetic damage (determination of the genetic damage index, GDI, measured by comet assay) and erythrocytic nuclear abnormalities (ENAs). The obtained results showed a mild pattern of antioxidant response, with modifications in CAT and GPx activities in gills, and lipid peroxidation in liver. These results suggest that despite the occurrence of oxidative effects, a full scenario of oxidative stress is not likely. However, exposure to OTC resulted in the establishment of genotoxic alterations with the induction of DNA strand breaks in blood cells (increase of GDI), and of chromosome breakage and/or segregational abnormalities (increase of ENAs). Considering that the oxidative response was not totally devisable, other mechanisms may be involved in the genotoxic effects reported.

Effects of chronic exposure to benzalkonium chloride in Oncorhynchus mykiss: cholinergic neurotoxicity, oxidative stress, peroxidative damage and genotoxicity

Benzalkonium chloride (BAC) is one of the most used conservatives in pharmaceutical preparations. However, its use is limited to a small set of external use formulations, due to its high toxicity. Benzalkonium chloride effects are related to the potential exertion of deleterious effects, mediated via oxidative stress and through interaction with membrane enzymes, leading to cellular damage. To address the ecotoxicity of this specific compound rainbow trouts were chronically exposed to BAC at environmental relevant concentrations (ranging from 0.100 to 1.050mg/L), and the biological response of cholinergic neurotoxicity, modulation of the antioxidant defense, phase II metabolism, lipid peroxidation and genotoxicity was studied. The obtained results showed a dual pattern of antioxidant response, with significant alterations in catalase activity (starting at 0.180mg/L), and lipid peroxidation, for intermediate (0.180 and 0.324mg/L) concentrations. No significant alterations occurred for glutathione-S-transferases activity. An unexpected increased of the acetylcholinesterase activity was also recorded for the individuals exposed to higher concentrations of BAC (starting at 0.180mg/L). Furthermore, exposure to BAC resulted in the establishment of genotoxic alterations, observable (for the specific case of the comet assay results) for all tested BAC concentrations. However, and considering that the oxidative response was not devisable, other mechanisms may be involved in the genotoxic effects reported here.

Alterations in gills of Lepomis gibbosus, after acute exposure to several xenobiotics (pesticide, detergent and pharmaceuticals): morphometric and biochemical evaluation.

Abstract In recent decades, scientific research about the effects of anthropogenic xenobiotics on non-target organisms has increased. Among the likely effects, some studies reported the evaluation of biochemical and morphological changes in specific tissues or organs of fishes, such as gills, which are key organs for the direct action of pollutants in the aquatic environment. This work intended to assess biochemical [oxidative stress/phase II conjugation isoenzymes glutathione S-transferase (GSTs)] and morphological [secondary lamellar length (SLL), secondary lamellar width (SLW), interlamellar distance (ID), basal epithelial thickness (BET) and proportion of the secondary lamellae available for gas exchange (PAGE)] changes in gills, after acute exposure to the pesticide chlorfenvinphos, the detergent sodium dodecylsulphate (SDS) and to the anticholinesterasic pharmaceuticals (neostigmine and pyridostigmine). Our results point to a significant, eventually hormetic, effect in the activity of GSTs following exposure to chlorfenvinphos that significantly increased the activity of GSTs at concentration of 0.2 mg/L. The activity of GSTs increased significantly after exposure to 100 mg/L of neostigmine. Considering the morphometric analysis of the gills, the data obtained showed that chlorfenvinphos exerted mainly minor architectural alterations in gills, with the exception of the highest tested concentration of chlorfenvinphos that produced also a slight decrease of the PAGE. The overall conclusions point to a null or negligible toxicity of the selected toxicants towards L. gibbosus, which may be reverted if exposure is withdrawn.

Effects of environmentally relevant concentrations of metallic compounds on the flatfish Scophthalmus maximus: biomarkers of neurotoxicity, oxidative stress and metabolism

Flatfish species, such as the turbot (Scophthalmus maximus), are common targets for toxic effects, since they are exposed through the food chain (ingestion of contaminated preys) and are in direct contact with the waterborne contaminant and sediments. Furthermore, these fish species live in close proximity to interstitial water that frequently dissolves high amounts of contaminants, including metals. Despite this significant set of characteristics, the present knowledge concerning flatfish contamination and toxicity by metals is still scarce. To attain the objective of assessing the effects of metals on a flatfish species, S. maximus specimens were chronically exposed to lead, copper and zinc, at ecologically relevant concentrations, and biochemical (oxidative stress: catalase and glutathione S-transferases activities, and lipid peroxidation; neurotoxicity: cholinesterase activity) parameters were assessed on selected tissues (gills and liver). Copper had no significant effects on all tested parameters; lead was causative of significant increases in liver GSTs activities and also in lipoperoxidation of gill tissue; exposure to zinc caused a significant increase in catalase activity of gill tissue. None of the tested metals elicited noteworthy effects in terms of neurotoxicity. The obtained results showed that only the metal lead is of some environmental importance, since it was able to cause deleterious modifications of oxidative nature at relevant concentrations.

Effects of anthropogenic metallic contamination on cholinesterases of Gambusia holbrooki.

Metal contamination causes multiple biological dysfunctions, including impairment of key physiological functions by targeting enzymes. This feature is a matter of concern, since it may imply significant disturbances in energy allocation, behaviour, reproduction, and survival. Inhibition of the cholinesterase (ChE) activity of aquatic organisms by metals has been described, and systematically used in biomonitoring studies as effect criterion of environmental exposure to these compounds. The present paper addresses the feasibility of using ChE inhibition to quantify the adverse acute and chronic effects of metals (copper, zinc, lead, and cadmium) on nervous tissue of Gambusia holbrooki. With the exception of acute exposure to copper, ChE activity was not significantly impaired. The meanings of the reported findings are further discussed, aiming at a more comprehensive use of this biomarker in environmental assessment. Based on the obtained results, the role of ChE inhibition in environmental metal contamination scenarios should be questioned or even discarded.

Histological alterations in gills and liver of rainbow trout (Oncorhynchus mykiss) after exposure to the antibiotic oxytetracycline

The aim of this study was to investigate the histopathological effects of oxytetracycline (OTC) on the gill and liver tissues of rainbow trouts (Oncorhynchus mykiss) following acute (96h: 0.005-50mg/L) and chronic (28days: 0.3125-5μg/L) exposures. Results suggest the existence of a cause-and-effect relationship between the exposure to OTC and tissue damage. Most predominant disorders observed in gills were progressive (e.g. hypertrophy of mucous cells and hyperplasia of epithelial cells) in acute exposure and regressive (e.g. lamellar fusion, epithelial lifting of lamellae and some changes in tissue architecture) in chronic exposure. However, only the acute exposure was responsible for a significant increase of the total gill pathological index. PAGE index, reflecting the extent of gill tissue available for gas exchanges in fish, remained unchanged for both exposures. In liver, circulatory (e.g. hemorrhage and increase of sinusoidal space), regressive (e.g. pyknotic nucleus, vacuolization and hepatocellular degenerations) and progressive (e.g. hypertrophy of hepatocytes) changes were observed, but just after acute exposure. After chronic exposure, only inflammatory changes (e.g. leucocytes infiltration) were observed. Following both exposures, a significant increase of the total liver pathological index was recorded. Despite the increase of the histological damage in individuals exposed to OTC, lesions observed were of minimal or moderate pathological importance, non-specific and reversible. The data gathered following acute and chronic exposures also suggest the onset of adaptive mechanisms of fish, namely for longer exposure periods. Furthermore the observed histological alterations appear to be result of several physio-metabolic disorders consequence of the biochemical and molecular modes of action of OTC.