Heat shock protein 70 and cortisol as biomarkers for cadmium, chromium and nickel contamination in Oreochromis mossambicus

South Africa is one of the countries with the largest mining operations in the world. Most of these mines make use of natural dams and rivers in their water supply, which often are being pumped back into the natural environment. The reticulated water pumped back into the system may contain high concentrations of dissolved chemicals, which may lead to the reduction of the endemic organisms. Many of the heavy metals mined in South Africa are highly toxic at very low concentrations, and it is therefore very important to do frequent analysis on the aquatic environment. The value of chemical analysis per se has become limiting, as chemical analysis supplies information on the levels of chemicals at a certain time, and the new trend is to incorporate biological monitoring into existing monitoring strategies. Heat shock proteins are classified as stress proteins and are primarily expressed under stressful conditions, therefore having the potential to be used as possible biomarkers. Cortisol, also a known stress hormone, has been suspected of suppressing the expression of heat shock proteins by replacing the heat shock protein on the glucocorticoid receptor. This leads to reduced levels of heat shock proteins in the organism through a negative feedback mechanism. However, before information on heat shock proteins and cortisol can be successfully incorporated into ecological risk assessment, an understanding of how cortisol influences heat shock protein levels after heavy metal exposure is needed. This study aims to determine what effect cortisol had on the production of a specific member of the Hsp70 class of heat shock proteins at different time intervals after exposure of Oreochromis mossambicus to various metals. Proteins were determination using the Bradford method, while protein separation was done using Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis. Thereafter, separated proteins were subjected to Western blotting and immunoblotting in order to quantify the different Hsp70 family members in the hepatic tissue. Cortisol levels were determined using a commercially available Cortisol ELISA Test Kit. Hsp70 accumulation occurred in only two exposure groups, namely the cadmium and chromium exposure groups. Accumulation of Hsp70 demonstrated an increase in accumulation rates at the 24 hour time interval of the 10% cadmium exposure group, with accumulation remaining relatively constant in the 20% cadmium exposure group. Accumulation of Hsp70 occurred only at the 48 and 96 hour time intervals in the 10% chromium exposure group. The accumulation of Hsp76 and Hsp74 were observed to follow the same pattern throughout the 96 hour exposure. In the 10% exposure groups accumulation of both Hsp76 and Hsp74 indicated an increase in accumulation rates at the 72 hour of groups exposed to chromium and nickel, whereas the accumulation of Hsp76 and Hsp74 remains constant after exposure to cadmium. In the 20% cadmium exposure group, an increase in Hsp76 and Hsp74 accumulation was observed at the 24 hour time interval, whereby accumulation of Hsp76 and Hsp74 remains constant in both the chromium and nickel 20% exposure groups. Males accumulated higher levels of Hsp70 members than females in the cadmium and chromium exposure groups. In the 10% exposure groups the females accumulated higher levels of Hsp74, whereas the males accumulated higher levels of Hsp74 in the 20% exposure groups. Accumulation of Hsp76 proved to be higher in the females in all exposure groups, compared to the males. Cortisol concentrations remained constant throughout the 96 hour exposure period, with higher cortisol levels observed in the chromium exposure groups. Cortisol concentrations proved to increase at higher concentrations of metal exposure. Cortisol proved to have no significant effect on Hsp70 family member accumulation, except in the cadmium exposure group, where a negative regression was observed. Accumulation of the HSp70 member can be linked to possible metal specificity, due to the fact that Hsp70 accumulated in only two metal species (cadmium and chromium). However, the accumulation of Hsp76 and Hsp74 may possibly prove that the higher concentrations of specific metals leads to early accumulation of heat shock proteins. The higher accumulation levels of Hsp70 in males, compared to accumulation levels in females may be due to the greater need to discard damadged or denatured proteins, whereas higher Hsp74 and Hp76 accumulation levels in females, may be due to the higher levels of reproductive proteins present in females, compared to males. This study therefore concluded that cortisol may have no significant effect on the accumulation of the Hsp70 family members in the liver of fish.Prof. J.H.J. Van Vure