Effect of methyl methanesulfonate on hsp70 expression and tissue damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg

Abstract

Methyl methanesulfonate (MMS) is an anti-carcinogenic drug and its toxicity has been reported in various experimental models. The hsp70s are a family of ubiquitously expressed heat shock proteins. In the recent years, hsp70 has been considered to be one of the candidate genes for predicting cytotoxicity against environmental chemicals. Nowadays emphasis is given to the use of alternatives to mammals in testing, research and education. The European Centre for the Validation of Alternative Methods (EVCAM) has recommended the use of Drosophila as an alternative model for scientific studies. Almost all living organisms possess proteins with a similar structure to that of hsp70s. In the present study, the toxicity of MMS was evaluated by quantifying hsp70 expression and tissue damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg(9), at different doses and hours of exposure. We studied the effect of 0.25, 0.50, 0.75 and 1.0 µl/ml of MMS at 2, 4, 24 and 48 hours of exposure on hsp70 expression by using the soluble O-nitrophenyl-β-D-galactopyranoside (ONPG) assay and on establishing the tissue damage by the Trypan blue exclusion assay in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg(9). A dose-dependent increase in the expression of hsp70 was observed at 0.25, 0.50, and 0.75 µl/ml of MMS compared to the control. At the highest dose, i.e. 1.0 µl/ml of MMS, the activity of hsp70 was decreased due to tissue damage.

Keywords: Drosophila melanogaster (hsp70-lacZ) Bg9; Methyl methanesulfonate; hsp70.

Figure 1

Structure of methyl methanesulfonate.

Figure 2

Mean absorbance value after exposure to various doses of MMS.

Figure 3

Regression analysis for β-galactosidase activity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg ⁹ exposed to 0.25, 0.50, 0.75 and 1.0 µl/ml of MMS for 2 hrs.

Figure 4

Regression analysis for β-galactosidase activity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg ⁹ exposed to 0.25, 0.50, 0.75 and 1.0 µl/ml of MMS for 4 hrs.

Figure 5

Regression analysis for β-galactosidase activity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg ⁹ exposed to 0.25, 0.50, 0.75 and 1.0 µl/ml of MMS for 24 hrs.

Figure 6

Regression analysis for β-galactosidase activity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg ⁹ exposed to 0.25, 0.50, 0.75 and 1.0 µl/ml of MMS for 48 hrs.

Figure 7

Regression analysis for β-galactosidase activity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg ⁹ exposed to 0.25 µl/ml of MMS for 2, 4, 24 and 48 hrs.

Figure 8

Regression analysis for β-galactosidase activity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg ⁹ exposed to 0.50 µl/ml of MMS for 2, 4, 24 and 48 hrs.

Figure 9

Regression analysis for β-galactosidase activity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg ⁹ exposed to 0.75 µl/ml of MMS for 2, 4, 24 and 48 hrs.

Figure 10

Regression analysis for β-galactosidase activity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg ⁹ exposed to 1.0 µl/ml of MMS for 2, 4, 24 and 48 hrs.

Figure 11

Trypan blue staining pattern in the third instar larval tissues of D. melanogaster (hsp70-lacZ) Bg ⁹ for 48 hrs (control).

Figure 12

Trypan blue staining pattern in the third instar larval tissues of D. melanogaster (hsp70-lacZ) Bg ⁹ after exposure to 0.50 µl/ml of MMS for 48 hrs.

Figure 13

Trypan blue staining pattern in the third instar larval tissues of D. melanogaster (hsp70-lacZ) Bg ⁹ after exposure to 0.75 µl/ml of MMS for 48 hrs.

Figure 14

Trypan blue staining pattern in the third instar larval tissues of D. melanogaster (hsp70-lacZ) Bg ⁹ after exposure to 1.0 µl/ml of MMS for 48 hrs.