Free radical-induced carbonyl content in protein of estrogen-treated hamsters assayed by sodium boro[3H]hydride reduction

M.L Winter,J.G Liehr

doi:10.1016/s0021-9258(18)98706-5

Abstract

Oxidative damage to proteins is known to occur via conversion of side chain amino groups to corresponding carbonyl derivatives. Such damage to enzymes and purified proteins has been quantified previously by reduction with sodium boro[3H]hydride and subsequent measurement of the incorporation of 3H into amino acid fractions. In this study, the NaB3H4 reduction assay was modified to permit the quantitation of free radical-mediated oxidative damage to proteins obtained from animals. Modifications included additional extractions of protein isolates with organic solvents to remove lipids and with nitric acid to remove metal ions. The modified assay has first been validated in vitro by measuring changes in levels of oxidative damage to bovine serum albumin exposed to xanthine plus xanthine oxidase (2-fold increase), to hydrogen peroxide and iron(II) sulfate (5-fold increase), or to gamma radiation (30-fold increase over controls, respectively). gamma radiation of isolated hamster kidney protein also raised the carbonyl content in a dose-dependent manner. The modified assay has then been validated in vivo by measuring the changes in oxidative damage to lung tissue in animals exposed to approximately 85% oxygen (2-fold increase) or to different doses of paraquat (5-fold increase with the high dose over controls, respectively). The assay was then used to examine free radical-mediated oxidation introduced by acute or chronic treatment of hamsters with estrogens, since both synthetic and natural estrogens induce kidney tumors in this species. Priming of hamsters for 3 days with 20 mg/kg/day diethylstilbestrol and treatment with 100 mg/kg of this drug on the 4th day resulted in a 160% increase in free radical modification of renal proteins. Oxidative damage to kidney proteins was also assayed in hamsters treated with estradiol implants for up to 7 months, a regimen known to induce kidney tumors. Significant increases in covalent oxidative modification to renal proteins over values in age-matched controls were detected after 1, 2, and 7 months of continuous estradiol exposure. It is concluded that the modification of the NaB3H4 reduction assay is a useful postlabeling method for monitoring free radical action in vivo. Furthermore, it is postulated that free radical damage in estrogen-treated hamster kidney plays a role in estrogen-induced carcinogenesis.

Highlights

Oxidative damage to proteinsis known to occur via Estrogens,such as diethylstilbestrol (DES)’ or catechol conversion of side chain amino groups to correspond- estrogens, undergo redox cycling i n vitro [1]between quinone ing carbonyl derivatives
In this study,we quantified the free radical-induced cluded that the modification of the NaB3H4reduction damage to proteins by both short term and chronicestrogen assay is a useful postlabeling method for monitoring treatment preceding E,induced carcinogenesis
The carbonyl content in protein was lowered to control levels by 50 pmol/liter of ascorbic acid ( p < 0.01, t test for grouped data)

Summary

Introduction

Oxidative damage to proteinsis known to occur via Estrogens,such as diethylstilbestrol (DES)’ or catechol conversion of side chain amino groups to correspond- estrogens, undergo redox cycling i n vitro [1]between quinone ing carbonyl derivatives. The levels of tritium were increased over2-fold from 0.15 in controls to 0.38 pmol of 3H/mg of protein in animals exposed to 85%oxygen for 3 days ( p < 0.05, t test for grouped data).

Results

Conclusion