Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: Modulation by reactive oxygen species

Abstract

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015–15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg⁹ to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.

Introduction

Organisms across kingdoms are subjected to a large number of stresses and respond by altering their cellular metabolism and activating their defense mechanisms. The first response of an organism to any environmental alteration inducing stress is at the biochemical level, which underlies all effects at higher organizational levels. Stress response and anti-oxidant defense system consists of stress proteins (also termed as heat shock proteins; Hsps) and anti-oxidants (both enzymatic and non-enzymatic), which are the primary protective responses that are highly conserved components of cellular stress responses found in all phyla from bacteria to man [1].

Originally discovered as inducible proteins against heat shock, the Hsps counter proteotoxic effects and play various roles in a cell including chaperoning proteins during synthesis, folding, assembly and degradation. Among the stress genes family, hsp70 is one of the highly conserved genes and first to be induced against various stressors. During the last decade, application of hsp70 was found to offer potential to be one of the candidates for predicting cellular toxicity against environmental chemicals [2], [3], [4]. Molecular oxygen is the key to aerobic life and is also converted into cytotoxic byproducts referred to as reactive oxygen species (ROS). In addition to their involvement in the normal metabolic activities, ROS have been reported to play a major role in enhancing the toxicity of several xenobiotics including metals and pesticides. Aerobic organisms have therefore developed multiple defense mechanisms where either the cellular antioxidant molecules or the antioxidant enzymes remove ROS to rescue cells from oxidative stress [5].

Though cells are endowed with protective responses, however, an enhancement in the stress beyond the capacity of a cell to cope up may result in cellular damage leading to cell death. One of the major forms of cell death, apoptosis, has been shown to be genetically regulated. Recently, ROS produced during oxidative stress have been implicated in apoptosis as possible signaling molecules [6]. Hsp70 in addition to having chaperoning function may also interfere with the signaling events that triggers apoptotic process at multiple points in the apoptotic pathway [7].

Organophosphate (OP) compounds have diverse functions including insecticidal properties and are often used indiscriminately due to the fact that they have low environmental persistence. Dichlorvos (2,2-dichlorovinyl dimethyl phosphate; DV), an organophosphate, primarily acts by irreversibly inhibiting acetylcholinesterase (AchE) at cholinergic junctions of the nervous system [8], produces hepatotoxicity in rats and has been reported to induce oxidative stress [3]. One of the mechanisms through which DV could elicit toxicity is by inhibiting mitochondrial ATP production through the uncoupling of oxidative phosphorylation that could lead to the generation of ROS [9].

An earlier study showed that many cellular stresses leading to induction of Hsps, also induce intracellular ROS [10]. In this context, studies from our laboratory showed that organophosphates induce hsp70 and antioxidant enzymes in Drosophila [3], [11] and ROS has been speculated as one of the causative factors. The present study was thus conducted to examine the possibility of correlation, if any, between Hsp70 expression and that of antioxidant enzymes with possible implications of free radicals using chemical modulators in D. melanogaster after DV exposure. The study was further extended to examine the effect of this OP compound on cell death in the exposed organisms.