Hazardous effects of effluent from the chrome plating industry: 70 kDa heat shock protein expression as a marker of cellular damage in transgenic Drosophila melanogaster - hsp70-lacZ - Research

Hazardous effects of an effluent from the chrome plating industry were examined by exposing transgenic Drosophila melanogaster (hsp70-lacZ) to various concentrations (0.05, 0.1, 1.0, 10.0, and 100.0 [micro]L/mL) of the effluent through diet. The emergence pattern of adult flies was affected, along with impaired reproductive performance at the higher dietary concentrations of the effluent. Interestingly, the effect of the effluent was more pronounced in male than in female flies. The effect of the effluent on development of adult flies was concurrent with the expression pattern of the heat shock protein 70 gene (hsp70), both in larval tissues and in the reproductive organs of adult flies. We observed a dose- and time-dependent expression of hsp70 in third instar larvae exposed for different time intervals. Absence of hsp70 expression in larvae exposed to 0.1 [micro]L/mL of the effluent indicated that this is the highest nontoxic concentration for Drosophila. The stress gene assay in the reproductive organs of adult flies revealed hsp70 expression in the testis of male flies only. However, trypan blue dye exclusion tests in these tissues indicate tissue damage in the male accessory gland of adult flies, which was further confirmed by ultrastructural observations. In the present study we demonstrate the utility of transgenic Drosophila as an alternative animal model for evaluating hazardous effects of the effluent from the chrome plating industry and further reveal the cytoprotective role of hsp70 and its expression as an early marker in environmental risk assessment. Key words: alternative animal model, biomarker, chrome plating industry, cytotoxicity, effluent, hsp70, reproduction, tissue damage, transgenic Drosophila melanogaster.

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With rapid industrialization, many metallic wastes have become widely distributed over the earth's surface and are excessively concentrated in localized areas, particularly in regions of high population density. Chrome plating is one of the most actively operating industries worldwide. The problem becomes more pronounced with small-scale and unorganized growth of such industries in the vicinity of densely populated areas. These industries are more prevalent in underdeveloped and developing countries such as India. Effluent discharged from the chrome plating industry contains a large number of metals, including chromium, copper, nickel, zinc, manganese, and lead. The specific difficulty with industrial effluents is that they are very persistent and may be transported considerable distances by air, water, or the food chain, where they tend to accumulate, reaching concentrations in the upper trophic levels that are several orders of magnitude higher than those that originally existed, thus representing a continuous threat to the biota (Edmund et al. 1976). In complex effluents, toxic interactions among various toxicants can be synergistic (more than additive), antagonistic (less than additive), or simply additive. Only whole-effluent toxicity tests allow practical and effective assessment of combined effects of toxic substances (Gomez et al). 2001).

Over the last decade, biomarkers have been used in considerable research and have been valuable to the field of molecular epidemiology (Bennett and Waters 2000). In recent years, heat shock proteins have evolved as a very successful first-tier diagnostic tool to screen for adverse biologic effects induced by environmental chemicals. Because heat shock proteins are part of the machinery for defense, repair, and detoxification of cells, they become direct and specific markers of exposure and effect (Bierkens 2000). Homologues of heat shock proteins occur in every species of every kingdom of living things (Feder and Hofman 1999). Thus, the heat shock proteins represent a remarkable example of molecular "descent with modification" (De Maio 1999) at the levels of gene sequence, genomic organization, regulation of gene expression, and protein structure and function. Among the different heat shock proteins, the stress-inducible heat shock protein 70 (Hsp70) family is the most conserved and is also the most highly induced by cellular stresses (Feige and Polla 1995; Macario et al. 1999). Because of its responsiveness to diverse forms of stress, the heat shock response has undergone widespread application in biomonitoring and environmental toxicology (Ait-Aissa et al. 2003; de Pomerai 1996; Mukhopadhyay et al. 2002a, 2002b; Radlowska et al. 2002; Ryan and Hightower 1996).

In the present study we explored the toxic potential of an effluent from the chrome plating industry, using hsp70 expression as a marker of cytotoxicity in transgenic Drosophila melanogaster (hsp70-lacZ)[Bg.sup.9] flies. We examined the emergence pattern of adult flies and their reproductive performance to determine whether the effluent affects their development. Finally, we attempted to determine the highest nontoxic concentration of the effluent in D. melanogaster by stress gene assay. Drosophila was used as a model organism because of its well-understood genetics and development. Moreover, the use of Drosophila falls within the recommendations of the European Centre for the Validation of Alternative Methods (Festing et al. 1998), whose goal is to promote the scientific and regulatory acceptance of alternative methods that are of importance in the field of bioscience and that reduce, refine, or replace the use of laboratory animals (Benford et al. 2000).