Immunohistochemical localization of O⁶-ethyldeoxyguanosine and deoxyguanosin-8-yl-(acetyl)aminofluorene in liver sections of rats treated with diethylnitrosamine, ethylnitrosourea or N-acetylaminofluorene

Abstract

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.

Formation and persistence of O6-(2-hydroxyethyl)-2′-deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2-hydroxyethyl)urea. An immuno-slot-blot study

“…Largely irrespective of the initial dose, the 72-h values were reduced to a basal level of 3-6 /imol C^-HEdG/mol deoxyguanosine. This low level probably represents persistence in non-parenchymal cells with a low repair capacity (25,26).…”

Bioactivation of N-nitrosomethylbenzylamine and N-nitrosomethyl-amylamine in oesophageal papillomas

“…Immunohistochernistry offers the advantage of identifying cells containing specific DNA adducts (19,20). In the present study, we used a rabbit antiserum to C^-methyldeoxyguanosine (12).…”

DNA methylation in rat stomach and duodenum following chronic exposure to N-methyl-N′-nitro-N-nitrosoguanidine and the effect of dietary taurocholate

“…The antiserum was used without prior absorption. The procedure of Heyting et al (12) and Menkveld et al (13) was used with several modifications, generously communicated by Dr E.Scherer and colleagues at The Netherlands Cancer Institute, Amsterdam. Cryostat sections (6-10 fim) were mounted on ovalbumin-coated slides.…”