Development of a Biosensor for Methyl Parathion Detection and Genetic Risk Assessment of Exposed Population

Methyl parathion is one of the highly toxic organophosphate pesticides which contaminates \ud the environment and exerts toxic effects in the non-targeted living organisms. Its detection and removal from the environment is needed in order to protect both the public and the environment from its hazardous effects. We studied risk assessment to pesticide toxicity (based on case studies), detection of methyl parathion (by using purified green fluorescent protein as a biosensing molecule) and biodegradation of methyl parathion (by indigenous bacterial isolates immobilized in sol gel matrices).\ud Paraoxonase is a protein with enzymatic function which hydrolyzes toxic metabolites of methyl parathion and other organophosphate pesticides. We determined serum paraoxonase and serum arylesterase activities alongwith genotypes of respective groups recruited for the study. Various groups of the study population included healthy non-pesticide exposed male Punjabis, pesticide users, familial hypercholesterolaemia patients, glaucoma patients and appropriate controls. The results of the healthy population provided a baseline data (normal values). Study on pesticide users indicated significant differences of PON1 genotypes and phenotypes between the healthy pesticide users and the pesticide users who developed toxic effects of pesticides during spraying season. Serum paraoxonase and arylesterase activities were significantly low in hypercholesterolemia patients as compared to controls. There was no significant difference of PON1 genotype or phenotypes between glaucoma patients and age and gender matched controls. However age dependant decrease in serum paraoxonase and arylesterase activities explained the role of PON1 in glaucoma. The results of these studies will be helpful in understanding preventive measures to improve the status of public health.\ud Detection of methyl parathion is relatively costly with available methods and low cost and easy to use biosensor systems are needed for large scale screening of agricultural areas and other environmental segments. Thus we studied methyl parathion detection based on a simple method of fluorescence quenching. The linear decrease in the fluorescence of green fluorescent protein with increasing concentration of methyl parathion showed that fluorescence quenching based method can be utilized for low cost detection of methyl parathion. In addition, a novel fluorescent protein (HriGFP) was characterized for its physico-chemical properties to provide a baseline for future studies.\ud Bacteria play a major role in degrading pesticides in the environment. Bacterial enzymes (esterases) convert the pesticides into metabolites which can be used as nutrients by the bacteria for their growth and proliferation. We isolated bacterial strains from agricultural areas contaminated with methyl parathion and other pesticides and characterized them for their combined xipotential of antimicrobial and heavy metal resistance and utilizing methyl parathion as a source of nutrients. In addition, a short listed consortium of the bacterial strains was immobilized in sol-gel and their survival in sol gel was studied. The results indicate that these environmental bacteria are capable of utilizing methyl parathion as a source of nutrient and sol gel immobilization can be helpful in co-survival of different bacterial strains for their application in bioremediation.\ud On the whole, this thesis describes PON1 genotype and phenotype patterns in Pakistani populations, use of GFP as a biosensor molecule for detection of methyl parathion and isolation and characterization of bacterial strains for methyl parathion degrading activities. The study not only gives insight about the risk assessment and ultimately prevention of toxic effects of methyl parathion but also describes a proof of principle based on simple biotechnology approach for its detection. The results of this thesis will be a valuable source of information for public health managers, clinicians, policy makers, agricultural workers and bio-medical researchers for ultimate improvement of health status of general population as well as pesticide users