Mutagenicity Assessment of Organophosphates using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay

Abstract

Objectives: In this study we have evaluated the mutagenicity of organophosphate pesticides acephate, chlorpyrifos, and profenofos using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the mosquito Culex quinquefasciatus taken as an experimental model.

Materials and methods: Second instar larvae were treated with LC20 of each pesticide for 24 h and mutations induced in the sequence of mitochondrial COII gene (690bp) were studied from restriction patterns generated with AluI, PacI, and PsiI restriction endonucleases.

Results: Variations in the number and size of digested fragments were recorded from treated individuals compared with controls showing that the restriction enzymes created a cut at different locations. In addition, sequences of COII gene from control and treated individuals were also used to confirm the RFLP patterns. From the sequence alignment data, it was found that mutations caused the destruction and generation of restriction sites in the gene sequence of treated individuals.

Conclusion: This study indicates that all the three pesticides had potential to induce mutations in the normal sequence of COII gene and also advocates the use of PCR-RFLP assay as an efficient, rapid, and sensitive technique to detect mutagenicity of pesticides.

Keywords: Acephate; Cx. quinquefasciatus; PCR-RFLP; chlorpyrifos; mutagenicity; profenofos.

Figure 1

RFLP patterns obtained after: AluI (a) and PsiI (b) digestion of the COII amplicon of control and acephate treated Culex. quinquefasciatus; AluI (c), PacI (d) and PsiI (e) digestion of the COII amplicon of control and chlorpyrifos treated Culex quinquefasciatus; AluI (f) and PsiI (g) digestion of the COII amplicon of control and profenofos treated Culex quinquefasciatus. Lane M: Gene ruler, Lane A: RFLP pattern from control individual, Lane B: RFLP pattern from treated individual, Lane N: Negative control

Figure 2

Restriction sites of AluI (AGCT) and PsiI (TTATAA) in COII gene sequences of control and acephate treated Culex quinquefasciatus

Figure 3

Restriction sites of AluI (AGCT), PacI (TTAATTAA) and PsiI (TTATAA) in COII gene sequences of control and chlorpyrifos treated Culex quinquefasciatus

Figure 4

Restriction sites of AluI (AGCT) and PsiI (TTATAA) in COII gene sequences of control and profenofos treated Culex quinquefasciatus