Biochemical and cytological aspects of in vitro metabolism of parathion by rat liver cell fractions

Abstract

Signature was redacted for privacy.Signature was redacted for privacy.Signature was redacted for privacy. 1 Biochemical and cytological aspects of in vitro metabolism of parathion by rat liver cell fractions Ronald Lee Hybertson Under the supervision of P. A. Dahm This study presents a combined biochemical and cytological study of the in vitro metabolism of the organophosphate insecticide parathion by hepatic cell fractions of the laboratory rat. The cytological work in cludes an electron microscopical examination of liver homogenate and centrifugally prepared debris, mitochondrial, crude microsomal, rough micro somal, and smooth microsomal fractions. The biochemical work includes an analysis of the in vitro metabolism of parathion by gas-liquid chromato graphy, thin-layer chromatography, and liquid scintillation spectrometry. Protein determinations were made on each cell fraction, and RNA content of the microsomal fractions was determined and used in conjunction with electron microscopy to assess the purity of the rough and smooth micro somes. The effects of varying several incubation parameters on parathion metabolism by crude microsomes were examined. Regression analyses and analysis of variance tests were performed on these results. A comparison of the enzymatic activity by the six cell fractions showed the highest specific activity in the smooth microsomal fraction. The mean smooth microsometrough microsome response ratio for both paraoxon production and aqueous metabolite production was 1.4:1. After adjusting 2 the nitrogen level in the specific activity calculations to exclude the contribution by the ribosomal component, the ratio was 1.2:1. Specific activity differences as a function of the resuspension of crude microsomal pellets in several different aqueous solutions were not significant. The cesium chloride employed in the subfractionation of microsomes did not affect activity. An incubation mixture pH of 7.5 was optimal for parathion metabolism and maximal enzymatic activity occurred around the physiological temperatures of the rat (34.5-40.0 C). The specific activity of crude microsomes decreased with increasing microso mal concentration over a range of 0.3-3.1 mg protein/ml incubation mixture. The metabolism of parathion was not linear with time and showed a decreasing rate for both metabolic pathways. Crude microsomes retained their enzymatic activity without significant loss while stored at -20 C for six weeks. Microsomal activity towards parathion was destroyed by several methods of solubilization. ii