Biomonitoring studies and susceptibility markers for acrolein congeners and allylic and benzyl compounds.

The importance of genotoxic acrolein congeners and allylic and benzyl compounds as industrial compounds, ubiquitous environmental pollutants, and naturally occurring substances necessitates the availability of adequate biomonitoring techniques. Endogenously formed acrolein congeners are considered to play an important role in carcinogenesis. Our studies have demonstrated that acrolein congeners react with DNA components and form adducts with the guanine moiety. We have identified and characterized cyclic 1,N2-deoxyguanosine adducts, cyclic 7,8-guanine adducts, linear 7-guanine adducts, 1,N2,7,8-bis-cyclic adducts, and 1,N2-cyclic, 7-linear bis adducts. Both the reactivity of the acroleins toward nucleosides and their mutagenicity in S. typhimurium TA100 decrease with increasing degree of alkyl substitution. Adducts are now available as reference substances for developing sensitive detection methods. Of the biomonitoring methods investigated for allylic and benzyl compounds, the detection of cysteine and histidine adducts isolated from hemoglobin seems to be the most sensitive. Gas chromatography with electron capture detection of heptafluorobutyric acid derivatives allows a detection limit in the femtomole range, HPLC-fluorescence detection of O-phthalic dialdehyde derivatives allows a limit in the picomole range, and detection of 9-fluorenylmethyl-chlorofomiate derivatives allows a limit in the femtomole range.

Introduction c1,0-Unsaturated carbonyl compounds (acrolein congeners) and allylic and benzylic compounds are important industrial chemicals, ubiquitous environmental pollutants, and natural products and are found in foodstuffs (1,2). They form DNA adducts, are genotoxic, mutagenic, and carcinogenic (1)(2)(3)(4). The great importance of these compounds necessitates the availability of adequate biomonitoring techniques. a,O4-Unsaturated carbonyl compounds are also formed endogenously, e.g., during lipid peroxidation or after oxidative stress, and are considered to play an important role in human carcinogenesis (5). Although these endogenously formed acroleins are a constant source of DNA damage, no clear data are available for the severity of this damage and whether or to what extent these compounds lead to mutation and induce tumors in animals or humans (5). We have systematically studied the interaction of acrolein congeners with DNA components.

DNA Adducts with Acrolein Congeners
The detection of DNA adducts in animal or human tissue samples allows an estimation ofthe extent to which acrolein con-geners are involved in cytotoxic and genotoxic processes and the role of these substances in mutagenesis and carcinogenesis. A first prerequisite for a specific and sensitive detection of small amounts of such adducts in human tissue is their in vitro isolation, identification, and characterization. Therefore, we have isolated and characterized the nucleoside and nucleotide adducts of 12 ai,f-unsaturated carbonyl compounds by infrared, ultraviolet, 'H-nuclear magnetic resonance (NMR), '3C-NMR, and mass spectrometry as well as by melting point determination and elementary analysis. The deoxyguanosine moiety was the most reactive target in DNA. No adducts other than those with deoxyguanosine, guanine, or deoxyguanosine monophosphate were found except with acrolein, where we also found adenine adducts. All compounds (Table 1) formed cyclic 1, N2deoxyguanine adducts. Cyclic 1, N2-deoxyguanosine adducts were first described for acrolein and crotonaldehyde (6) and for x-bromoacrolein (7). We can confirm these results and have identified similar adducts with 2-methyl acrolein, pentenal, hexenal, hexadienal, 3,3-dimethylacrolein, 2,3-dimethylacrolein, methylvinylketone, ethylmethyl-ketone, and a-chloroacrolein. In principle, two types of regioisomers of the 1,N2-cyclic adducts were identified. First, one of the amino groups binds to the activated double bond (Michael addition), and then the other amino group reacts with the carbonyl carbon:  (Table 1), probably due to steric effects. In general, several enantiomers ofeach type of regioisomer were observed (Fig. 1).

Reactivity and Mutagenicity of Acroleins
In general, the reactivity of the acrolein congeners toward deoxyguanosine decreases with increasing alkyl substitution. This is consistent with the mutagenicity in S. typhimurium strain TAIOO, which also decreases with increasing degree of substitution.
Biomonitoring Assay for Detecting Low-level Exposure to Allylic and Benzyl Compounds In principle, biomonitoring can be performed by a) detection of typical metabolites in urine, b) detection of specific DNA adducts, or c) detection of modified amino acids from hemoglobin. Sixty to eighty percent ofreactive allyl compounds are excreted as mercapturic acids (10). Thus, it is practicable to use gas chromatography or gas chromatography-mass spectrometry (GC-MS) detection of mercapturic acids for effective bio-  (3), which can be used as distinctive markers for carcinogenic risk assessment.
In this paper we focus on the detecting modified amino acids from hemoglobin, which seems to be the most sensitive technique. Globin was isolated from exposed human hemoglobin and subjected to acid hydrolysis (11)

GLC and HPLC Analysis
After derivatization of the hydrolyzed hemoglobin mixture with heptafluorobutyric acid anhydride, aliquots were analyzed by GC with electron capture (ECD) detection under the conditions shown in Figure 3. The adducts were assigned by the retention times determined from the reference substances. The identity of the adducts was additionally confirmed by co-chromatography with the reference substances. The detection limit for these derivatives was in the femtomole range.
For HPLC analysis the respective adducts were derivatized either with OPA (Fig. 4) or with FMOC and assigned by retention times as well as by co-chromatography. The detection limit for the OPA derivatives was in the picomole range and that of FMOC derivatives was in the femtomole range.

Conclusions
Identication of acrolein congener adducts demonstrates that these acroleins can interact with DNA components. Well-characterized adducts are now available as reference substances for use in the development of highly sensitive techniques for the detection of trace amounts in human tissue. We are currently adapting the 32P-postlabeling method using HPLC and have shown that this technique is practicable (unpublished results).
Identification ofthese adducts in human tissue allows a clearer estimation ofthe role of formed a3,o-unsaturated carbonyl compounds in mutagenesis and carcinogenesis but also provides the basis for evaluation ofcancer risks associated with occupational and environmental exposure. Protective measures can be taken, e.g., by providing scavengers, once the genotoxic mechanism and the extent of endogenous and exogenous exposure to compounds are known. Our studies on techniques for sensitive biomonitoring of exposure to allyl and benzyl compounds provide a basis for practicable routine methods.