Search

Antibodies raised in rabbits against the bovine serum albumin conjugates of O6-ethylguanosine (O6-EtGuo) and N-(guanosin-8-yl)-N-acetyl-2-aminofluorene (Guo-8-AAF) have been used in a double peroxidase-antiperoxidase staining assay to visualize the localization of DNA-O6-ethyldeoxyguanosine (O6-EtdGuo) and some of the interaction products of N-acetyl-2-aminofluorene (AAF) with DNA guanine in liver sections of rats treated with diethylnitrosamine (DEN), ethylnitrosourea (ENU), or AAF respectively. O6-EtdGuo could be detected in nuclei of parenchymal cells after injection of DEN (12-50 mg/kg) or ENU (140 mg/kg). Clear time and dose dependencies were observed. The lowest dose of DEN which, at 5 h after injection, resulted in immunohistochemically detectable levels of O6-EtdGuo, was 12 mg/kg; 5 h after 6 mg/kg no consistent difference between treated and untreated rats could be observed. A striking heterogeneity in staining pattern was observed after DEN: centrilobular regions were stained much more than peripheral zones. At 7 days after a single DEN injection of 50 mg/kg small rims of significantly stained hepatocytes could still be observed around the central veins. No heterogeneity of staining pattern was observed 2 h after ENU. ENU, in contrast with DEN, also resulted in a significant staining of nonparenchymal cells. At 24 h after ENU no significant staining of hepatocytes could be detected, but positive staining was still present in bile duct cells, vascular endothelial cells and sinusoidal cells. The results indicate a detection level of approximately 5 mumol O6-EtdGuo/mol DNA-P, i.e., 5 X 10(4) O6-EtdGuo residues per diploid genome. Using the anti-Guo-8-AAF antiserum, positive results were obtained 6 days after a single AAF dose of 0.5-10 mg/kg, corresponding to a detection limit of less than or equal to 0.4 mumol dGuo-8-(A)AF/mol DNA-P. Staining was rather homogenously distributed over the liver lobules. Persistency of the AAF-DNA interaction products was investigated both after 10 and 2 mg/kg AAF. Increased nuclear staining could be observed up to 8 weeks after 10 mg/kg and 4 weeks after 2 mg/kg.

Reaction of deoxyguanosine in glacial acetic acid with chloroethylene oxide, a proposed reactive metabolite of vinyl chloride, led to a single, strongly fluorescent product in nearly quantitative yield. The u.v. spectra indicated alkylation of N-7 of guanine, which was confirmed following reduction of the reaction product by sodium borohydride to 7-(2-hydroxyethyl)guanine, and the synthesis of the same modified guanine via a stereoselective 7-N hydroxy alkylation using 2,3-epoxy-1-propanol. In agreement with the expected structure 7-(2-oxoethyl)guanine reacted with the carbonyl specific reagent 2,4-dinitrophenylhydrazine (2,4-DNPH). However, its i.r. and proton n.m.r. spectra did not support the existence of a simple aldehyde group. Moreover, the 2,4-dinitrophenylhydrazone was labile, 7-(2-oxoethyl)guanine being produced when excess 2,4-DNPH was removed. This instability was interpreted as being due to the reversible formation of a hemiacetal ring between O6 of the guanine residue and the aldehyde carbon of the 2-oxoalkyl group resulting in O6,7-(1'-hydroxyethano)guanine. This conformation was supported by the occurrence in field desorption mass spectra of the ions of m/e = 175 and 292 which are interpreted as O6,7-ethenoguanine and O6,7-ethenodeoxyguanosine resulting from the elimination of H2O of the hydroxyethano residue. O6,7-(1'-hydroxyethano)guanine might be expected to cause faulty base pairing during replication of DNA, which may be the molecular basis of the carcinogenicity of vinyl chloride.

Antibodies raised in rabbits against the bovine serum albumin conjugates of O6-ethylguanosine and N-(guanosin-8-yl)-N-acetyl-2-aminofluorene have been used to develop a high-sensitive enzyme-linked immunosorbent assay (HS-ELISA) for the quantification of adducts in DNA modified by ethylating agents and N-acetyl-2-aminofluorene. Linear dose-response relations were obtained in the non-competitive HS-ELISA between 0.5 fmol and 50 fmol O6-ethyldeoxy-guanosine per 2.8 microgram DNA, and between 0.1 fmol and 20 fmol N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene per 0.8 microgram DNA. The sensitivity of an ultrasensitive radioimmunoassay was in the same order of magnitude. Modification levels as low as 0.1 mumol of adduct/mol DNA-nucleotides (1: 10(7)) can be detected by each assay.