Reaction products in hemoglobin and DNA after in vitro treatment with ethylene oxide and N-(2-hydroxyethyl)-N-nitrosourea

Abstract

Reaction products with DNA and blood proteins have been used for monitoring human exposure to electrophilic compounds and metabolites. The formation of products with nucleophilic sites in DNA after treatment with such reagents has been well characterized (especially for alkylating agents). It is therefore of great importance to collect corresponding data for nucleophilic groups in proteins. The formation of reaction products in hemoglobin (Hb) and DNA was studied after in vitro treatment with ethylene oxide (EtO) and N-(2-hydroxyethyl)-N-nitrosourea (HOEtNU). In DNA N-7-(2-hydroxyethyl)guanine was the main product of EtO, whereas O6-(2-hydroxyethyl)guanine was much lower (0.5% of the alkylation of guanine-N-7). For HOEtNU O6-(2-hydroxyethyl)guanine was found to be 63% of N-7-(2-hydroxyethyl)guanine. These relative reactivities are in agreement with what has been reported for related alkylating agents. The main reaction products in Hb were 2-hydroxyethylations of cysteine, N-terminal valine, the two imidazole nitrogens in histidine and carboxylic groups. The reactivities of human, mouse and rat Hb towards EtO were compared. The main difference between species was the 12 and 170 times higher reactivity of cysteine in mouse and rat Hb, respectively, than in human Hb. As expected from relative reactivities of alkylating agents with model nucleophiles, products with cysteine dominated for EtO (except in human Hb) whereas for HOEtNU products with carboxylic groups were far larger than any other. Relative amounts of cysteine and carboxylic group alkylation in mouse Hb were compared with corresponding data for other alkylating agents. The comparison showed that these amounts were close to what could be expected from known reactivities of these compounds with model nucleophiles, but that sterical and other modifying factors have to be taken into account. Each compound gives therefore a species-specific pattern that might be used for tracing human exposure of unknown origin.