Bruno Nunes

Behaviour and biomarkers of oxidative stress in Gambusia holbrooki after acute exposure to widely used pharmaceuticals and a detergent

Pharmaceuticals are continuously dispersed into the environment, as a result of human and veterinary use, and have become a relevant environmental concern. In the present study, the acute toxicity of three therapeutic agents (diazepam, clofibrate, and clofibric acid) and a detergent, sodium dodecylsulphate (SDS), to the euryhaline fish Gambusia holbrooki was evaluated. Special attention was devoted to oxidative stress parameters. G. holbrooki males, captured in the estuary of the Minho River (NW Portugal), were exposed for 96 h to the selected compounds. The following oxidative stress biomarkers were evaluated in gills and liver tissues: reduced and oxidised glutathione, lipid peroxidation, and several antioxidant enzymes, namely (1) total and selenium-dependent glutathione peroxidase (GPx), (2) glutathione reductase (GRed), (3) copper-zinc superoxide dismutase (Cu-ZnSOD) and manganese superoxide dismutase (MnSOD), and (4) glutathione-S-transferases (GSTs). In the particular case of diazepam, swimming behaviour was also evaluated. The obtained results indicate an overall diminished oxidative stress response caused by SDS and diazepam. Oxidative-based alterations were observed after exposure to clofibrate and clofibric acid, with modifications of several enzymatic activities. Diazepam caused evident behavioural changes: animals showed dark pigmentation and also abnormal postures, namely lethargy and anomalous movement.

The Use of Cholinesterases in Ecotoxicology

Cholinesterase (ChE) is one of the most employed biomakers in environmental analysis. Among ChEs, potentially the most significant in environmental terms is acetylcholinesterase (AChE), an enzymatic form that terminates the nerve impulse . Because of its physiological role, Ache has long been considered a highly specific biomarker for organisms exposed to anticholinesterasic agents, primarily agro-chemicals (organophosphate and carbamate pesticides). The effects of these pesticides depends upon their selective inhibition of AChE. Because large amounts of such pesticides are employed, it is plausible that they exert neurotoxic effects on some non-target species. Therefore, AChE is among the most valuable of diagnostic tools that can be used to verify exposure to such chemical agents. It is well known that assays are available for use quantifying AChE in multiple tissues of several test organisms. Enzymes other than AChE (e.g., butyrylcholinesterase and carboxylesterases) have also been used as putative markers for detecting the environmental presence of contaminating compounds. Researchers must use a step-by-step approach to identify the most prominent cholinesterasic form present in a given species, so that this form can be distinguished from others that may interfere with its use. Such fundamental work must be completed prior to using ChEs for any monitoring to assess for anticholinesterasic effects. Despite massive employment in environmental analysis, using ChE inhibition as an endpoint or effect criterion has been unsettled by the discovery the ChEs may interact in the environmental in previously unknown ways. Several chemicals, in addition to anticholinesterasic pesticides, are now known to inhibit ChE activity. Such chemical include detergents, metals, and certain organic compounds such as hydrocarbons. The situation is made worse, because the literature is contradictory as to the ability of such chemicals and elements to interact with ChEs. Some results indicate that ChE inhibition by metals, detergents , and complex mixtures do not or are unlikely to occur. These problems and contradictions are addressed in this review. It is purpose in this review to address the following practical issues related to the ChEs: 1. The situation and organisms in which ChEs have been employed as biomarkers in laboratory trials, and the need to fully characterize these enzymatic forms before they are used for environmental assessment purposes. 2. The ways in which ChEs have been used in field monitoring, and the potential for use of others complimentary markers to diagnose organophosphate exposure, and how drawbacks (such as the absence of reference values) can be overcome. 3. What requirements must be satisfied prior implementing the use of ChEs as biomarkers in species not yet studied. 4. How direct linkages have been established between ChE inhibition and effects from inhibition observed at higher levels of integration (e.g., behavioral changes and population effects, or others indices of ecological relevance). 5. The potential for ChE inhibition to be applied as an effective parameter of toxicity to detect for the environmental presence of compounds other than the organo-phosphate and carbamate pesticides, and the limitations associated therewith.

Biochemical effects of acetaminophen in aquatic species: edible clams Venerupis decussata and Venerupis philippinarum

Acetaminophen (paracetamol) is one of the most used pharmaceutical drugs, due to its antipyretic and analgesic properties that turn it into a primary choice in varied pathologies and conditions. However, and despite its massive use, acetaminophen is not exempt of adverse effects, especially when administered in over dosage, which are related to the formation of toxic metabolites by oxidative pathways. It is thus possible to observe that toxicity caused by acetaminophen is usually mediated by reactive oxygen species and can result in multiple effects, ranging from protein denaturation to lipid peroxidation and DNA damage. The occurrence of acetaminophen has been reported in the aquatic environment, being important to address the potential exertion of toxic effects on nontarget environmentally exposed organisms. The present study intended to characterize the effects of acute acetaminophen exposure on physiological traits (antioxidant defense, oxidative damage) of two species of bivalves, namely, the edible clams Venerupis decussata and Venerupis philippinarum. Results showed a significant increase in all oxidative stress biomarkers, evidencing the bioactivation of acetaminophen into a deleterious prooxidant, triggering the onset of deleterious effects. Furthermore, strong interspecific differences were observed among responses of the two tested species, which was a major issue due to intrinsic ecological implications when one considers that both species share the same habitat.

Short-term effects of neuroactive pharmaceutical drugs on a fish species: Biochemical and behavioural effects

The presence of pharmaceutical residues in the aquatic environment is receiving great attention since significant levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. In our study, the toxicity of three anticonvulsant drugs commonly found in the environment (diazepam, carbamazepine, and phenytoin) was evaluated in Lepomis gibbosus (pumpkinseed sunfish). This study focused on oxidative stress parameters, namely: glutathione reductase (GRed), glutathione S-transferases (GSTs), catalase (CAT), and lipid peroxidation (thiobarbituric acid reactive substances, TBARS) in the hepatic, digestive, and gill tissues of exposed animals. Simultaneously, we assessed the effects of these drugs in terms of behavioural parameters, such as scototaxis and activity. Exposure to diazepam caused an increase in GST activities in the gills and an inhibition of GRed in the digestive tract, relative to control, suggesting an antioxidant response. It also caused fish to spend more time swimming and less time in a refuge area (black compartment of an aquarium). Exposure to carbamazepine caused an increase in GSTs and GRed activity in the digestive tract, which is not always consistent with the literature. A significant positive correlation was found between carbamazepine concentration and time spent in motion and a negative correlation with time spent in black compartment. Exposure to phenytoin was responsible for adaptive responses in the activities of CAT and GSTs (in the liver), but it did not elicit any behavioural alterations. Although all three drugs seemed to induce oxidative stress in some organs, peroxidative damage (measured as TBARS concentrations) was not found at the selected range of concentrations. Our results enlighten the need for more research on the ecological consequences of pharmaceuticals in the aquatic environment, especially drugs that interfere with the CNS and behaviour, because the net outcome of these effects may be difficult to predict.

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.

The impact of paracetamol on selected biomarkers of the mollusc species Corbicula fluminea.

The Asian clam Corbicula fluminea is an invasive bivalve that has recently spread in Europe and currently represents a large portion of the aquatic biomass in specific areas. Because of the impacts that the species may have in invaded ecosystems, increased knowledge on the physiologic features of the species life‐cycle under different environmental scenarios (e.g., contamination events) is critical to understand the dynamics of the invasion and resulting ecosystem imbalance. The presence of pharmaceutical residues in the aquatic environment has recently received great attention since high levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. The present article reports toxicological biochemical effects of paracetamol to Corbicula fluminea following short‐ and long‐term exposures. Oxidative stress parameters were specially focused namely catalase (CAT), glutathione S‐transferases (GSTs), and glutathione reductase (GRed). The effect of tested substances on lipid peroxidation was also investigated. Paracetamol did not induce alterations on CAT activity, caused a significant decrease of GSTs activity following short‐ and long‐term exposure (LOEC values of 532.78 mg L−1 and 30.98 μg L−1, respectively), and was responsible for a significant and dose‐dependent decrease of GRed activity in short‐ and long‐term exposures. These results indicate that exposure to paracetamol can provoke significant alterations on the cellular redox status of C. fluminea. 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 74–83, 2014.

Toxic potential of paracetamol to freshwater organisms: a headache to environmental regulators?

Paracetamol is one of the most prescribed drugs globally, due to its antipyretic and analgesic properties. However, it is highly toxic at elevated doses, with involvement of an already described oxidative stress pathway. Despite this, the number of ecotoxicological studies on potential effects of paracetamol in wild organisms is still scarce. The present article presents a comprehensive series of standardized assays for the assessment of paracetamol effects in freshwater organisms. The results show that paracetamol toxicity is widely variable among species, even when these species are phylogenetically related. Furthermore, comparisons between data from the literature and our results reinforce this conclusion, providing evidence of the inadequacy of standardized toxicity testing guidelines for pharmaceutical compounds in wild organisms. Paracetamol toxicity can be modulated by unpredictable physiological conditions that might compromise extrapolations and comparisons of responsiveness among species. The ecological relevance of data obtained from classical tests for this compound is further discussed.

In situ bioassay with Eisenia andrei to assess soil toxicity in an abandoned uranium mine

The goal of this study was to develop an in situ bioassay with Eisenia andrei, deploying it in several locations of an abandoned mining area. Our objectives were two-fold: (i) we intended to validate the in situ soil bioassay procedures, while (ii) providing ecologically relevant data to complement the ongoing risk evaluation based on laboratorial assays. To promote cost- and time-effectiveness, the in situ exposure was short (48 h) and the endpoints analysed included oxidative stress biomarkers and metal content in soil and organisms. The bioassay was carried out under different experimental conditions, simulating local (natural soil) vs. control conditions (LUFA soil), and irrigation with artificial rainwater vs. irrigation with diluted acidic effluent. Variation in the data was mostly due to soil type, rather than irrigation water, and substantial spatial heterogeneity was observed. Oxidative stress biomarkers did not fully work as sensitive parameters to environmental contamination. Earthworm metal burdens suggested a potential concern in terms of bioaccumulation of some metallic elements.

Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.

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.