Toxicity of methoprene as assessed by the use of a model microorganism

Abstract

Methoprene is an insect juvenile growth hormone mimic, commonly used as a pesticide. Although widely used for the control of several pests, toxic effects on organisms of different phyla have been reported. These events triggered studies to clarify the mechanisms of toxicity of this insecticide putatively involved in ecological issues. Here we show the effect of methoprene on the normal cell growth and viability of a strain of the thermophilic eubacterium Bacillus stearothermophilus, previously used as a model for toxicological evaluation of other environment pollutants. Respiration studies were also carried out attempting to identify a putative target for the cytotoxic action of methoprene. Cell growth was affected and a decrease of the number of viable cells was observed as a result of the addition of methoprene to the growth medium, an effect reverted by the presence of Ca²⁺. Methoprene also inhibited the redox flow of B. stearothermophilus protoplasts before the cytochrome oxidase segment, an effect further studied by individually assessing the enzymatic activities of the respiratory complexes. This study suggests that methoprene membrane interaction and perturbation of cell bioenergetics may underlie the mechanism of toxicity of this compound in non-target organisms.

Introduction

Concerns about chemicals in the environment and their possible ecotoxic risks have emerged in the last decades. Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate), designed to mimic a juvenile growth hormone, was the first insect growth regulator approved by the Environmental Protection Agency (USA), in the 1970s, after extensive studies showing low toxicity to vertebrates (Zoecon Corporation, 1974) and rapid natural chemical degradation in the environment (Zoecon Corporation, 1973, Quistad et al., 1975) and through organism metabolism (Schooley et al., 1975, Quistad et al., 1975). However, deleterious effects induced by methoprene have been reported on non-target aquatic insects (Miura and Takahashi, 1973, Breaud et al., 1977), crustaceans (Breaud et al., 1977, Templeton and Laufer, 1983) and, at higher concentrations, some fish species (Quistad et al., 1976, McKague and Pridmore, 1978). These findings and an eventual relationship of methoprene with the increase of malformed amphibians in North America (LaClair et al., 1998, Stocum, 2000) motivated researches to reexamine the bioactivity of this compound and metabolites, in order to predict putative noxious effects on non-target organisms.

Alternative methods to animal testing for assessing chemical toxicity have been developed in the last decades. Mitochondria have provided data that have been correlated with cytotoxicity parameters derived from cell cultures and whole organisms (Blondin et al., 1987, Knobelock et al., 1990). Microorganisms, like yeasts (Haubenstricker et al., 1990) and bacteria (Sikkena et al., 1992, Carneiro de Melo et al., 1996), have also been used in toxicity tests that resulted in toxicity correlations concerning energy metabolism and cell viability (Sikkena et al., 1992, Donato et al., 1997b). A strain of the thermophilic eubacterium Bacillus stearothermophilus has been used as a model to study the cytotoxicity of several lipophilic drugs (Luxo et al., 2000, Rosa et al., 2000, Monteiro et al., 2003) and pollutants (Donato et al., 1997a, Donato et al., 1997b, Martins et al., 2003) and a remarkable parallelism of effects on this prokaryotic model and other model systems (Donato et al., 1997a) has been observed. These results prompted us to use the strain of B. stearothermophilus to evaluate the toxicity of methoprene resulting from unspecific interactions with the cytoplasmic membrane. As a lipophilic molecule, methoprene may be incorporated into biomembranes, common components to all living systems and potential targets of xenobiotics. It is attempted to correlate the effects of methoprene on growth and cell viability of B. stearothermophilus with effects in vitro at the molecular level, viz. the interaction of the compound with the enzymatic complexes of the redox system.