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Arylamines and nitroarenes are suspected of playing a key role in chemical carcinogenesis. Therefore, the study of DNA adduct formation is an important step to determine the genotoxic potential of these compounds. Calf thymus DNA was modified in vitro by reaction with activated N-hydroxyarylamines:  2-chloroaniline (2CA), 4-chloroaniline (4CA), 2-methylaniline (2MA), 4-methylaniline (4MA), 2,4-dimethylaniline (24DMA), 2,6-dimethylaniline (26DMA), 2-aminobiphenyl (2ABP), 3-aminobiphenyl (3ABP), and 4-aminobiphenyl (4ABP). Female Wistar rats (n = 2) were given a single dose of the above arylamines and their analogous nitro derivatives by oral gavage and sacrificed after 24 h. Hepatic DNA and in vitro modified DNA were hydrolyzed enzymatically to individual 2‘-deoxyribonucleosides. Adducts were determined using HPLC/MS/MS by comparison to synthesized standards. The hydrolysis efficiency was monitored by HPLC with UV detection. Each arylamine described above formed adducts to 2‘-deoxyguanosine and 2‘-deoxyadenosine after in vitro reaction with DNA. DNA adducts were found in rats dosed with 4ABP or with 4-nitrobiphenyl. DNA adducts were not detected in rats dosed with 2CA, 4CA, 2MA, 4MA, 24DMA, 26DMA, 2ABP, 3ABP, 2-chloronitrobenzene, 4-chloronitrobenzene, 2-nitrotoluene, and 4-nitrotoluene. All compounds formed hydrolyzable hemoglobin adducts. Therefore, biologically available N-hydroxyarylamines yielded hemoglobin adducts but not hepatic DNA adducts, except for 4ABP.

Toluenediisocyanates (TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. Protein adducts of TDI might be involved in the etiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. The functional groups of cysteine, tyrosine, serine, lysine, tryptophan, histidine, and N-terminal amino acids are potential reaction sites for isocyanates. Especially the N-terminal amino acids, valine, and aspartic acid of hemoglobin and albumin, respectively, are reactive toward electrophilic xenobiotics. To develop methods for the quantitation of protein adducts of 2,4- and 2,6-TDI, we reacted 3-nitro-4-methylphenyl isocyanate (1a) with single amino acids and reduced the nitro group using catalytic hydrogenation or ammonium formate with palladium on carbon yielding N-[(3-amino-4-methylphenyl)carbamoyl]valine (2a), N-[(3-amino-4-methylphenyl)carbamoyl]aspartic acid (8a), N^α-acetyl-N^ε-[(3-amino-4-methylphenyl)carbamoyl]lysine (12a), and N^α-acetyl-O-[(3-amino-4-methylphenyl)carbamoyl]serine (15a). The same reactions were performed with 5-nitro-2-methylphenyl isocyanate (1b) and 3-nitro-2-methylphenyl isocyanate (1c). The valine adducts were boiled in acid to obtain the corresponding hydantoins:  3-(3-amino-4-methylphenyl)-5-isopropylimidazoline-2,4-dione (5a), 3-(5-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5b), and 3-(3-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5c). A method for the detection of N-terminal adducts with valine in biological samples was developed. The tripeptide adduct N-[(3-amino-4-methylphenyl)carbamoyl]valyl-glycyl-glycine (19a) was hydrolyzed with acid in the presence of globin and the internal standard N-[(3-amino-4-methylphenyl-d6)carbamoyl]valyl-glycyl-glycine (19d). The released hydantoins were determined by LC/MS/MS and after derivatization with pentafluoropropionic anhydride by GC/MS. The determination limit was 0.16 pmol/sample. The same N-terminal adduct with valine was found in globin of a TDI-worker and in two women with polyurethane covered breast implants.

4,4‘-Methylenediphenyl diisocyanate (MDI) is the most important of the isocyanates used as intermediates in the chemical industry. Among the main types of damage after exposure to low levels of MDI are lung sensitization and asthma. Protein adducts of MDI might be involved in the etiology of sensitization reactions. It is therefore necessary to have sensitive and specific methods for monitoring the isocyanate exposure of workers. To date, urine metabolites or protein adducts have been used as biomarkers in workers exposed to MDI. However, with these methods it is not possible to determine if the biomarkers result from exposure to MDI or to the parent aromatic amine 4,4‘-methylenedianiline (MDA). This work presents a procedure for quantitating isocyanate-specific hemoglobin adducts. Blood proteins are used as markers of exposure and possibly as markers of dose size for the modifications of macromolecules in the target organs where the disease develops. For the quantitation of hemoglobin adducts, N¹-[4-(4-isocyanatobenzyl)phenyl]acetamide (AcMDI) was reacted with the tripeptide valyl-glycyl-glycine and with valine yielding N-[4-(4-acetylaminobenzyl)phenyl]carbamoyl]valyl-glycyl-glycine and N-[4-[4-(acetylaminobenzyl)phenyl]carbamoyl]valine, respectively. N-[4-[4-(Acetylamino-3,5-dideuteriobenzyl)-2,6-dideuteriophenyl]carbamoyl]valine was synthesized from valine, as was N¹-[4-(4-isocyanato-3,5-dideuteriobenzyl)-2,6-dideuteriophenyl]acetamide, for use as an internal standard. These adducts were cleaved in 2 M HCl to yield the corresponding hydantoins, 3-[4-(4-aminobenzyl)phenyl]-5-isopropyl-1,3-imidazoline-2,4-dione (MDA-Val-Hyd) and 3-[4-(4-amino-3,5-dideuteriobenzyl)-2,6-dideuteriophenyl]-5-isopropyl-1,3-imidazoline-2,4-dione, respectively. In globin of rats exposed to MDI, MDA-Val-Hyd could be found in a dose-dependent manner. The adduct was identified by HPLC/MS/MS and quantified by GC/MS after derivatization with heptafluorobutyric anhydride. The amount of MDA-Val-Hyd found after acid hydrolysis of globin at 100 °C is about 12 times larger than the sum of N-acetyl-4,4‘-methylenedianiline (AcMDA) and MDA obtained from mild base hydrolysis of hemoglobin. The MDA-Val-Hyd is an isocyanate-specific adduct. MDA and AcMDA released after mild base hydrolyses result most likely from a sulfinamide adduct which is a typical adduct of arylamines. According to these results, higher amounts of isocyanate adducts than arylamine adducts should be expected in workers exposed to isocyanates.

Aromatic diamino compounds, e.g., 4,4‘-methylenebis(2-chloroaniline) (MOCA) and 4,4‘-methylenedianiline (MDA), are used as curing agents in the production of elastomers. Since MOCA and MDA are mutagenic and carcinogenic, substitutes are of great commercial interest. For benzidine it has been shown that ortho substitution with methyl groups yields the nonmutagenic 3,3‘,5,5‘-tetramethylbenzidine. Therefore, MDA analogues with large substituents in the ortho position have been synthesized. The substituents are supposed to inhibit the formation of the N-hydroxyarylamines which are the putative genotoxic intermediates. We investigated the biological availability of the N-hydroxylamines of ortho-substituted diamines and of known carcinogenic diamines in female Wistar rats, by determining hemoglobin (Hb) adducts. Hb from rats dosed with 0.5 mmol/kg diamine and from controls was isolated and hydrolyzed in base. The released diamine and monoacetyldiamine were quantified by HPLC with electrochemical detection and/or GC/MS. MDA, 4,4‘-oxydianiline (ODA), 4,4‘-ethylenedianiline, and 4,4‘-thiodianiline (TDA) bound to hemoglobin as diamine and as monoacetyl-diamine. 4,4‘-Methylenebis(2,6-dimethylaniline), 4,4‘-methylenebis(2,6-diethylaniline), MOCA, and 4,4‘-sulfonyldianiline (dapsone) bound only as diamine to Hb. 4,4‘-Methylenebis(2,6-dichloroaniline) did not bind to Hb. Thus, the presence of two substituents in the ortho position and the presence of electron-withdrawing groups in the para position to the amino group drastically reduced the formation of Hb adducts. The amount of hemoglobin adducts was compared to their carcinogenic potency. The extent of hemoglobin binding of the bicyclic diamines (dapsone, 3,3‘-dichlorobenzidine, MDA, MOCA, TDA, ODA, and benzidine) increases with their carcinogenic potency.

4,4‘-Methylenedianiline (MDA) is used as a hardener in the manufacture of plastics and polyurethanes. MDA has been classified as a carcinogen in animals and is a suspected human carcinogen. Assuming that MDA would yield similar DNA adducts to other arylamines, we synthesized the following C-8 guanine adducts:  N‘-acetyl-N-(deoxyguanosin-8-yl)-MDA, N-(deoxyguanosin-8-yl)-MDA, N-(deoxyguanosin-8-yl)-4MA, and their corresponding 3‘-monophosphate derivatives. We developed methods to identify these adducts of MDA in liver DNA using ³²P-postlabeling, HPLC, and GC-MS techniques. Liver DNA was obtained from rats treated with radiolabeled MDA (1.11 and 116.5 μmol/kg body weight). The total radioactivity bound to the DNA corresponded to 0.06 and 2.7 adducts per 10⁷ nucleotides [covalent binding index (CBI = (μmol of adduct per mol of nucleotide)/(mmol of compound per kg body weight)) of 1.05 and 2.3]. This DNA-binding potency is in the range of weakly genotoxic compounds. The liver DNA was analyzed for the presence of the synthesized adducts by the following methods:  (I) HPLC analysis of nucleotides and purines after enzymatic and acid hydrolysis, and (II) ³²P-postlabeling after enzymatic hydrolysis. The major adducts found in vivo did not correspond to the synthesized standards. Further work was carried out to determine the structure of the unidentified adducts. It was possible to release MDA and MDA-d4 from DNA of rats dosed with MDA and/or MDA-d4 and from the synthesized adducts using strong base hydrolysis. Liver of two female Wistar rats given 500 μmol/kg MDA·2HCl was hydrolyzed in 0.1 M NaOH overnight at 110 °C. GC-MS analysis of the heptafluorobutyric anhydride derivatized dichloromethane extracts detected 428 ± 40 fmol of MDA/mg of DNA. In the control animals no MDA was found. The experiment was repeated with livers from animals dosed 500 μmol/kg MDA-d4·2DCl. In these rats 488 ± 19 fmol MDA-d4 was found to be bound at liver DNA. Taking into account a 68% yield of the method, the CBI found in these cases was 0.82 and 1.0, respectively.