Olga Liliana Anguiano

Biomarkers of effect in toads and frogs.

Amphibians are good bioindicators of environmental pollution due to their susceptibility to chemicals during their freshwater cycles. The effects of environmental pollution, together with changes in human activity and climate, have contributed to the reduction in the amphibian population over recent decades. However, toxicological research on amphibians has been rather scarce compared with that on other vertebrates. In this article we review the biochemical alterations underlying xenobiotic action and/or the detoxifying responses described for anuran species, with the aim of establishing possible biomarkers of effect. During the embryonic development of anurans, morphological and behavioural alterations are the effects most frequently cited in connection with chemical exposures. However, such biomarkers have a low sensitivity and are unspecific compared with biochemical alterations. Some primary pesticide targets, in particular cholinesterases for organophosphates and carbamates, have been evaluated. Esterases change seasonally and with the stage of development, and their sensitivity to anticholinesterase agents varies between species. Thus their use as biomarkers in anurans must be carefully analysed. Enzymes and endogenous compounds related to oxidative metabolism may also be used as biomarkers of effect. Glutathione pool, glutathione-S-transferases and metallothioneins respond in different ways to pesticides and heavy metals in anuran embryos and tadpoles. Mixed-function oxidases, in turn, are less developed in amphibians, and show a reduced induction in response to pesticide exposures. Endogenous polyamine levels are also proposed as good age-related biomarkers of damage. Finally, molecular biomarkers related to receptor binding, signal transduction and genetic response have gained increasing relevance, as they have been implicated in the fertilisation process and the earliest events in anuran development. The identification of transcription factors associated with the exposure of amphibians to xenobiotics as well as other alterations in hormone signalling appears highly promising. However, these techniques are likely to complement other methods. In conclusion, the use of several biomarkers with multiple endpoints is needed to link exposure to response and to provide better predictive tools for the environmental protection of endangered anuran species.

Thiols and polyamines in the potentiation of malathion toxicity in larval stages of the toad Bufo arenarum.

Treatment with exogenous spermidine enhanced acute malathion toxicity during larval development of the toad Bufo arenarum Hensel. The polyamine was rapidly incorporated in the larvae with a subsequent metabolization to putrescine and spermine, which were excreted to the media. Endogenous polyamine levels were not changed by either spermidine or malathion treatments. However, 0.5-mM spermidine modified malathion uptake and bioavailability increasing the concentration of the xenobiotic in the larvae. The amount of reduced thiols was decreased by both compounds, but the depletion was insufficient to induce cytotoxicity. The oxidative degradation of polyamines competes for the pool of reduced glutathione used in the conjugation of malathion in the larvae, thus leading to the reported potentiation of toxicity. Our results suggest that exposure to thiols-depleting agents may induce alteration of organophosphate degradation in amphibian larvae.

The role of glutathion conjugation in the regulation of early toad embryos’ tolerance to pesticides

Reduced glutathion (GSH) content and glutathione S-transferase (GSH S-transferase) activity were investigated in developing toad embryos exposed to parathion, malathion, lindane and dieldrin. The embryonic GSH content was reduced after 96 h of incubation with 20.00 ppm malathion and 2.00 ppm lindane. Parathion and dieldrin did not produce any change. A similar effect was obtained in advanced stages of development (6-days larvae), but only with malathion. No correlation between the decrease in GSH level and mortality or morphologic abnormalities was observed. The four pesticides increased the activity of GSH S-transferase indicating that the enzyme is susceptible to induction during early development. The higher effect depicted by malathion may be related with an enhanced conjugation of the pesticide. Both GSH decrease and GSHS-transferase induction modifies the cell redox status and may indirectly influence transcription and translation. The early expression of GST genes provides the embryo with a useful mechanism for the regulation of tolerance against chemical stress.

Enhanced esterase activity and resistance to azinphosmethyl in target and nontarget organisms

The organophosphorous compound azinphosmethyl (AzMe) is applied extensively in northern Patagonia (southern Argentina) to manage codling moths (Cydia pomonella). This area is irrigated by fast-flowing channels that provide a favorable habitat for many species, including amphipods (Hyalella curvispina) and a field-mixed population of black flies (Simulium bonaerense, Simulium wolffhuegeli, and Simulium nigristrigatum). In the present study, AzMe susceptibility and carboxylesterase (CarbE) activity from both insecticide-exposed and nonexposed field populations were studied. The median lethal dose determined in codling moths from an insecticide-treated orchard was significantly higher (3.48 microg/insect) than that observed in those from an untreated orchard (0.69 microg/insect). Similarly, the median lethal concentration (LC50) determined in black flies collected from the treated area (0.021 mg/L) was significantly higher than that recorded in those from the untreated site (0.011 mg/L). For amphipods, both a subpopulation susceptible to AzMe (LC50, 1.83 microg/L) and a resistant one (LC50, 390 microg/L) were found in the treated area. Both subpopulations were more resistant to AzMe than the population from the untreated site (LC50, 0.43 microg/L). Significant differences (p < 0.001) in CarbE activities were observed between populations from pesticide-treated and untreated areas. Mean activities +/- standard deviation from treated and untreated sites were 0.21 +/- 0.16 and 0.016 +/- 0.008 micromol/min/mg protein, respectively, for codling moths; 2.17 +/- 1.71 and 0.81 +/- 0.35 micromol/min/mg protein, respectively, for black flies; and 0.27 +/- 0.10 and 0.14 +/- 0.07 micromol/min/mg protein, respectively, for amphipods. The results suggest that enhanced CarbE activity is one of the mechanisms that provide AzMe resistance in H. curvispina, Simulium spp., and C. pomonella populations from the insecticide-treated areas.

Comparative toxicity of parathion in early embryos and larvae of the toad, Bufo arenarum hensel

Received: 31 March 1993/Accepted: 24 October 1993 Parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate) is a widely used agricultural insecticide highly toxic to mammals; however, very little is known about the detoxication capacity in some natural enemies of insects such as amphibians (Harriet al 1979; Llamas et al. 1985; Caballero de Castro et al 1991; Gauna et al. 1991). The biota may be stressed by discharges of pesticides, and it is therefore essential to know its potential effect on non-target organisms before irreversible change occurs. Some monitoring techniques use amphibian larvae as an useful indicator for aquatic contamination (Dumpert and Zeitz 1984; Beiswenger 1988; Williams et al. 1989). An ideal insecticide should be efficacious against pest species, but relatively safe for non-target organisms. To develop selective insecticides it is important to understand the differences and similarities of the defense systems between insects and the beneficial fauna such as predators and parasitoids. Thus, this report deals with a comparative study of parathion susceptibility on early embryonic and larval stages of the Argentinian toad

Mechanisms of resistance to DDT and pyrethroids in Patagonian populations of Simulium blackflies.

Mixed populations of the pest blackflies Simulium bonaerense Coscarón & Wygodzinsky, S. wolffhuegeli (Enderlein) and S. nigristrigatum Wygodzinsky & Coscarón (Diptera: Simuliidae) are highly resistant to DDT and pyrethroids in the Neuquén Valley, a fruit‐growing area of northern Patagonia, Argentina. As these insecticides have not been used for blackfly control, resistance is attributed to exposure to agricultural insecticides. Pre‐treatment with the synergist piperonyl butoxide (PBO) reduced both DDT and fenvalerate resistance, indicating that resistance was partly due to monooxygenase inhibition. Pre‐treatment with the synergist tribufos to inhibit esterases slightly increased fenvalerate toxicity in the resistant population. Even so, biochemical studies indicated almost three‐fold higher esterase activity in the resistant population, compared to the susceptible. Starch gel electrophoresis confirmed higher frequency and staining intensity of esterase electromorphs in the resistant population. Incomplete synergism against metabolic resistance indicates additional involvement of a non‐metabolic resistance mechanism, such as target site insensitivity, assumed to be kdr‐like in this case. Glutathione S‐transferase activities were low and inconsistent, indicating no role in Simulium resistance. Knowing these spectra of insecticide activity and resistance mechanisms facilitates the choice of more effective products for Simulium control and permits better coordination with agrochemical operations.

Evolution of insecticide resistance in non-target black flies (Diptera: Simuliidae) from Argentina

Black flies, a non-target species of the insecticides used in fruit production, represent a severe medical and veterinary problem. Large increases in the level of resistance to the pyrethroids fenvalerate (more than 355-fold) and deltamethrin (162-fold) and a small increase in resistance to the organophosphate azinphos methyl (2-fold) were observed between 1996-2008 in black fly larvae under insecticide pressure. Eventually, no change or a slight variation in insecticide resistance was followed by a subsequent increase in resistance. The evolution of pesticide resistance in a field population is a complex and stepwise process that is influenced by several factors, the most significant of which is the insecticide selection pressure, such as the dose and frequency of application. The variation in insecticide susceptibility within a black fly population in the productive area may be related to changes in fruit-pest control. The frequency of individuals with esterase activities higher than the maximum value determined in the susceptible population increased consistently over the sampling period. However, the insecticide resistance was not attributed to glutathione S-transferase activity. In conclusion, esterase activity in black flies from the productive area is one mechanism underlying the high levels of resistance to pyrethroids, which have been recently used infrequently. These enzymes may be reselected by currently used pesticides and enhance the resistance to these insecticides.