Processes involved in biochemical response to pesticides by lizard Podarcis siculus (Rafinesque-Schmaltz, 1810) - A field study.

Although reptiles are non-target organisms of pesticide applications, their ecological niche and trophic role suggest that the use of these compounds in agriculture can have toxicological effects on them. Our recent field study on Italian wall lizard Podarcis siculus in hazelnut orchards evidenced that the use of pesticides-mixtures, consisting of thiophanate-methyl (TM), tebuconazole (TEB), deltamethrin (DM), lambda-cyhalothrin (LCT), besides copper sulphate, induced an increase of the total antioxidant capacity toward hydroxyl radicals and caused DNA damage; however, it did not cause neurotoxicity, and did not induce the glutathione-S-transferases' activities. These results raised some questions which were answered in this study by carrying out analyses on 4 biomarkers and 5 chemical substances in the tissues of non-target organisms coming from treated fields: cytochrome P450, catalase, total glutathione, and malondialdehyde, TM, TEB, DM, LCT and Cu. Our results highlighted a partial accumulation of different chemicals, the involvement of two important mechanisms of defence, and some cellular damages after exposure to the considered pesticides. In details, 1) LCT and DM were not accumulated in lizard muscle, copper remained at basal levels, whereas TM and TEB were uptaken with a partial metabolization of TM; 2) the cytochrome P450 and the catalase were involved in lizard biochemical responses to pesticides-mixtures used for "conventional" farming treatment; 3) "conventional" treatment with pesticides caused damage to lipids, besides DNA, probably related to the excess of hydroxyl radicals.

Biological responses in pesticide exposed lizards (Podarcis siculus).

The release of contaminants as herbicides, fungicides and insecticides into the environment has been listed as one of the six major contributors to the global decline of reptiles. Although reptiles may face severe risk from contaminants due to their ecology and physiology, they are currently less studied than other vertebrate groups. In the present work, we investigated if and how different types of field treatment (conventional and organic) affected the health status of Italian wall lizard (Podarcis siculus) individuals in central Italy. We chose a multi-biomarker approach that evaluated the biological responses of lizards to the treatment by means of AChE activity in the nervous system, biotransformation enzymes activities and oxidative stress in the liver, micronuclei frequency measured in the erythrocytes, and rate of intestinal parasitic infection. Our findings showed evidence of effects of treatment in conventional areas and between sexes with significant oxidative stress due to hydroxyl radicals, that caused DNA damage. No difference of intestinal parasite infections was found among treatments. Podarcis siculus seems to be a good bioindicator in ecotoxicological studies and potentially in risk assessment of pesticides, although further analyses in laboratory and in the field are needed to achieve more accurate quantification of specific pesticide effects in relation to known exposure history and to understand if other mechanisms were involved in the toxicity and detoxification process of pesticides for this species.

Biological responses of the polychaete Hediste diversicolor (O.F.Müller, 1776) to inorganic mercury exposure: A multimarker approach.

Mercury (Hg) is a global priority pollutant given its relevance in terms of environmental damage and threat to human health. Its ecotoxicity was tested using the benthic keystone species Hediste diversicolor as target species. After 10 days of exposure to different levels of inorganic Hg (10 and 50 µg L-1), bioaccumulation and a wide range of biological responses were evaluated at different biological levels, including biomarkers of exposure, neurotoxicity, oxidative stress, genotoxicity and cytochemistry. In controlled laboratory conditions, Hg was taken up by H. diversicolor in a dose-response manner and caused a range of biological responses, including oxidative stress (GSTs, GPx, GSH-2GSSG, and TOSCA), neurotoxicity (AChE), and cellular damages at the membrane level (LFs, NLs, Ca2+-ATPase); however, it did not cause significant DNA damage or mortality. This study confirms the capability of H. diversicolor to tolerate high levels of metals and clarifies the mechanisms underlying the damage caused by waterborne Hg and the defense mechanisms, activated in this species. In particular, detoxification of the inorganic form of Hg in this species was found to be strongly related to glutathione expression and several antioxidant enzymes of the antioxidant system. This process also efficiently minimized negative effects on DNA and prevented death, but was not sufficient to avoid neurotoxicity and some cellular damages, mainly at the intestinal level.

Interactions between trace metals (Cu, Hg, Ni, Pb) and 2,3,7,8-tetrachlorodibenzo-p-dioxin in the Antarctic fish Trematomus bernacchii: oxidative effects on biotransformation pathway.

Biological effects of chemical mixtures are an emerging issue when using biomarkers in field conditions, because synergistic or inhibitory interactions, cascade, and indirect mechanisms can both enhance or suppress responses to specific classes of pollutants. The Antarctic rock cod (Trematomus bernacchii) was exposed to various trace metals (Cu, Hg, Ni, and Pb) and to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dosed alone or in combination, to generate hypotheses regarding the mechanisms of action at the cellular level. Concentrations of metals significantly increased in liver of T. bernacchii without significant differences between organisms exposed only to the elements or cotreated with TCDD. The marked induction of cytochrome P450 (CYP) by TCDD was greatly suppressed by cotreatment with Cu at both catalytic and protein expression levels, whereas no significant effects were caused by coexposures with Hg, Ni, and Pb. The oxidative status was measured by integrating individual antioxidants with the total oxyradical scavenging capacity toward peroxyl radicals (ROO*) and hydroxyl radicals (*OH). Synergistic oxidative effects appeared in fish coexposed to Cu and TCDD, suggesting that biotransformation efficiency is down-regulated by higher levels of hydrogen peroxide, hydroxyl radicals, and/or mechanisms limiting availability of heme groups. Major oxidative perturbations also were observed in organisms coexposed to TCDD and Hg; in these organisms, however, the marked increase of glutathione content could explain the absence of oxidative inhibition of the CYP system. More limited oxidative effects were caused by Ni and Pb, and the contemporary absence of inhibitory effects on CYP would further confirm the importance of pro-oxidant mechanisms in modulating the biotransformation pathway. The overall results indicate that a wide battery of biomarkers is necessary to assess the impact of chemical mixtures in field conditions.

Seasonal variations of exposure biomarkers, oxidative stress responses and cell damage in the clams, Tapes philippinarum, and mussels, Mytilus galloprovincialis, from Adriatic sea.

This work investigated the natural variability of several biomarkers in Tapes philippinarum and Mytilus galloprovincialis, sampled from Northern Adriatic where these organisms are important sentinel species for future environmental impact assessment. Levels of metallothioneins, peroxisomal enzymes and acetylcholinesterase, showed a significant seasonality and marked differences between clams and mussels. Among antioxidant enzymes, catalase and GST decreased during the warmer period, the latter enzyme activity resulting particularly high in clams. The total oxyradical scavenging capacity toward peroxyl radicals decreased in mussels from winter to summer, indicating a prooxidant challenge due to higher seawater temperature and intensity of light irradiance. Lysosomal membrane stability did not exhibit significant seasonal variations, while some variations were observed for DNA damages. Overall results indicated a significant influence of seasonal variability on several biomarkers and species-specific differences which should be considered to discriminate the appearance of anthropogenic disturbance.

Oxidative and modulatory effects of trace metals on metabolism of polycyclic aromatic hydrocarbons in the Antarctic fish Trematomus bernacchii.

Biological interactions between various classes of pollutants are of great relevance for the Antarctic marine environment, where the naturally elevated bioavailability of metals like cadmium might indirectly influence sensitivity of endemic organisms toward other environmental pollutants, e.g. polycyclic aromatic hydrocarbons (PAHs). To further investigate reciprocal effects of different chemicals, the fish Trematomus bernacchii was exposed to trace metals (Cd, Cu, Hg, Ni, Pb) and benzo[a]pyrene (BaP, as a model PAH), dosed alone and in combinations. Co-exposures revealed that BaP did not influence the accumulation of metals, while these elements caused significant changes on tissue levels of the PAH. The marked EROD induction caused by BaP was completely suppressed by co-exposure with Cd and Cu, but no effects were observed with Ni, Hg and Pb. Similar results were confirmed at the protein level by Western blot analyses while CYP1A1 mRNA levels were reduced only during Cd co-exposures. Clear evidence of oxidative perturbations was observed in fish co-treated with Cd and BaP and the reduced capability to absorb peroxyl and hydroxyl radicals suggested some oxidative pathways by which this element might indirectly modulate the biotransformation efficiency of Cytochrome P450. Partly different and post-transcriptional mechanisms of action could be hypothesized for Cu, while moderate oxidative effects of Hg, Ni and Pb during co-exposures would confirm their limited influence on metabolism of PAHs. In general, the overall results revealed a complex pathway of interactions between different chemicals during co-exposures and the importance of oxidative status in modulating induction and expression of CYP1A1.