Synthesis and Quantification of DNA Adducts of 4,4‘-Methylenedianiline

Abstract

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 mumol/kg body weight). The total radioactivity bound to the DNA corresponded to 0.06 and 2.7 adducts per 10(7) nucleotides [covalent binding index (CBI = (mumol 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 mumol/kg MDA-2HCl was hydrolyzed in 0.1 M NaOH overnight at 110 degrees 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 mumol/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.

An Unusual DNA Adduct Derived from the Powerfully Mutagenic Environmental Contaminant 3-Nitrobenzanthrone

“…Treatment of the adduct with carboxylesterase at 37°C was also found to be effective in the deacetylation of dG-N-Ac-ABA (19). HPLC analysis showed that dG-ABA was formed in 20% yield after incubation for 7 days.…”

Genotoxicity of Chemical Compounds Identification and Assessment by Yeast Cells Transformed With GFP Reporter Constructs Regulated by thePLM2orDIN7Promoter

“…Conventional and advanced physicochemical analyses were used to identify toxic compounds. 5,6 These methods mainly provide analytical information (ie, identity and quantification of known chemicals), rather than functional information regarding the possible biological effects of contaminants (cytotoxic or genotoxic) and their potential short- or long-term impact on the ecosystem, that is, on organisms, including humans. Additionally, when harmful substances are new, unknown, or not yet deposited in available databases, they cannot be identified by these approaches.…”

4,4′-Methylenedianiline-Induced Hepatotoxicity Is Modified by N-Acetyltransferase 2 (NAT2) Acetylator Polymorphism in the Rat

“…In addition, MDA is genotoxic (McQueen and Williams, 1990), forms DNA adducts in the liver (Schutze et al, 1996), and induces DNA damage in primary cultures of rat and human hepatocytes (Martelli et al, 2002). MDA is carcinogenic in both rats and mice with primary tumor sites in the liver, kidney, and thyroid (Lamb et al, 1986).…”