Genotoxic xenobiotics bind covalently to hemoglobin in vivo. The major reaction product of aromatic amines is a sulfinic acid amide resulting from the reaction of arylnitroso derivatives with SH-groups. Alkylating compounds react with cysteine, histidine and the terminal valine. The adducts are formed proportional to dose down to extremely small doses, they are stable throughout the life-span of the erythrocytes and accumulate upon repeated exposure. Methods for their determination in blood samples from experimental animals and humans are becoming available. Moreover, it has been demonstrated that for a given agent, a constant ratio exists between the reaction with tissue DNA and hemoglobin over a wide range of doses, which indicates that the reactions follow apparent first order kinetics. The extent of hemoglobin binding is therefore considered to be a relative measure of tissue dose, and should correlate much better with risk than exposure levels calculated from concentrations in the environment. Not only can the actual uptake be monitored more reliably, but also the individual's capacity to metabolically activate the absorbed agent. Biomonitoring of hemoglobin-bound metabolites represents a novel approach to control exposure to potential carcinogens, to correlate environmental exposure with tissue dose and eventually also with human risk.

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Covalent binding of benzidine and some congeners to hemoglobin was studied in female Wistar rats after oral administration. Hemoglobin adducts were hydrolyzed under alkaline conditions, and the arylamines extracted and analysed by HPLC with electrochemical detector. With benzidine, three cleavage products were observed, the major component being monoacetylbenzidine. This indicates that 4-nitroso-4'-N-acetylaminobiphenyl is the major reactive metabolite in erythrocytes. In addition benzidine and 4-aminobiphenyl were identified. The latter indicates a hitherto unknown metabolic pathway of benzidine. With 3,3'-dichlorobenzidine-dihydrochloride, 3,3'-dimethoxybenzidine and 3,3'-dimethylbenzidine two cleavage products were observed, the parent diamines being present in excess to or in amounts comparable to the monoacetyl derivative. With 3,3',5,5'-tetramethylbenzidine a hemoglobin adduct could not be found. When the azo dye direct red 28 was administered to the animals, the three cleavage products typical for benzidine were found, indicating that benzidine became bioavailable after reductive cleavage of the azo compound. In this case the fraction of 4-aminobiphenyl was greater than after benzidine. It is proposed to use the analysis of hemoglobin adducts in human blood to control the exposure of individuals to these carcinogenic chemicals in the course of biochemical effect monitoring.

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1. The non-enzymic reactions of trans-4-nitrosostilbene, 4-nitrosobibenzyl, 2-nitrosofluorene and p-nitrosotoluene with glutathione were studied. 2. Three types of reaction products have been identified, namely, the corresponding hydroxylamine, amine, and a water-soluble adduct which hydrolyses under acidic or alkaline conditions to an amine and glutathione sulphinic acid. 3. Reduction to the amine is explained by formation of adducts which are reduced by glutathione with the production of oxidized glutathione. 4. The relevance of this reaction for metabolic activation and inactivation of aromatic amines in vivo is discussed.