Male Wistar-Furth rats were fed 0.02% 2-acetylaminofluorene (AAF) for 3 days or 0.02% AAF for 25 days followed by 0.02% [ring-3H]AAF for an additional 3 days. The concentration of hepatic DNA adducts was then monitored by both radioimmunoassay and radiolabeling during 28 days of control diet. This approach allowed comparisons to be made of adduct accumulation, removal and persistence at both the beginning and end of a four week carcinogen feeding period. DNA adduct formation remained constant during the month of AAF administration with an accumulation rate of 157 fmol adduct/micrograms DNA during days 1-3 and days 25-28 of the experiment. Furthermore, the rate of removal of adducts formed during these three day periods was similar when both groups were fed control diets for 28 additional days. Continued AAF administration resulted in a slow accumulation of persistent adducts; thus, 91 +/- 6% of the adducts detected after 3 days of AAF feeding were removed during a subsequent month of control diet, while only 65 +/- 11% of the adducts detected after 28 days of AAF diet were removed when rats were fed control diet for an additional 28 days. In a second experiment, the removal of adducts was compared in animals fed control or AAF diet after previously being fed 0.02% AAF for 17 days. Similar removal curves were observed in both groups; therefore, continued ingestion of AAF did not affect the rate of adduct removal. In both experiments, biphasic repair curves were observed. These data were used to develop a pharmacokinetic model. Two genomic regions were postulated, an area susceptible to fast repair and a region more resistant to the removal of AAF adducts. At equilibrium, which was reached after 2-3 weeks of AAF feeding, the concentration of adducts in each region was similar with approximately 150 fmol adduct/micrograms DNA. Although the total number of adducts formed in the fast repair region during one month of AAF administration was five times greater than in the resistant region, the model predicted that the adducts localized in regions resistant to repair were the persistent adducts detected after one month of control diet. Overall, the removal of adducts formed during chronic AAF feeding was very efficient since greater than 93% of the adducts were removed by the end of a subsequent month of control diet.
Antisera elicited in rabbits were used in radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) to determine femtomole quantities of deoxyguanosin-8-yl-acetylaminofluorene (dg-8-AAF) and deoxyguanosin-8-yl-aminofluorene (dg-8-AF). These adducts have been monitored in liver and kidney DNA of male Wistar-Furth rats fed 0.02% or 0.04% 2-acetylaminofluorene (2-AAF) either continuously or for a limited time followed by an interval on control diet. After 24 hr of 0.02% 2-AAF feeding, substantial levels of binding (80 fmole/4g DNA) were observed in liver DNA and increased with time, reaching a plateau of approximately 230 fmole/,Ag DNA at 30 days and thereafter. During the first week of continuous feeding about 80% of the total C-8 adducts in the liver DNA were deacetylated (dG-8-AF). By 25-60 days, dG-8-AF represented 97-100% of all C-8 adducts as measured by RIA and confirmed by HPLC. Values for C-8 adduct formation in kidney DNA were severalfold lower than in liver and dG-8-AF represented >90% of C-8 adducts at all times studied.In removal or repair experiments, rats were fed 2-AAF for 3, 7 or 28 days, the 2-AAF diet was discontinued and the liver adducts assayed after intervals on control diet. When dietary 2-AAF administration was for 3 or 7 days, removal of adducts was efficient and almost complete by 28 days on control diet, with preferential retention of dG-8-AF. However, when dietary 2-AAF administration was for 28 days, adduct levels were higher, the repair capacity was saturated and the removal of C-8 adducts was not complete after control diet for a 28-day interval. In a preliminary experiment when[3H]-2-AAF was fed for 3 days, after 25 days of 0.02% 2-AAF, the rates of newly formed adduct formation and removal were similar to those observed for the initial 3 days of 2-AAF feeding. These results demonstrate the predominance and persistence of dG-8-AF in liver and kidney DNA of 2-AAFfed rats and suggest that the repair capacity of the whole rat liver was not diminished after 1 month of 2-AAF feeding.
Antisera elicited in rabbits were used in radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) to determine femtomole quantities of deoxyguanosin-(8-yl)-acetylaminofluorene (dg-8-AAF) and deoxyguanosin-(8-yl)-aminofluorene (dg-8-AF). These adducts have been monitored in liver and kidney DNA of male Wistar-Furth rats fed 0.02% or 0.04% 2-acetylaminofluorene (2-AAF) either continuously or for a limited time followed by an interval on control diet. After 24 hr of 0.02% 2-AAF feeding, substantial levels of binding (80 fmole/μg DNA) were observed in liver DNA and increased with time, reaching a plateau of approximately 230 fmole/μg DNA at 30 days and thereafter. During the first week of continuous feeding about 80% of the total C-8 adducts in the liver DNA were deacetylated (dG-8-AF). By 25-60 days, dG-8-AF represented 97-100% of all C-8 adducts as measured by RIA and confirmed by HPLC. Values for C-8 adduct formation in kidney DNA were severalfold lower than in liver and dG-8-AF represented >90% of C-8 adducts at all times studied. In removal or repair experiments, rats were fed 2-AAF for 3, 7 or 28 days, the 2-AAF diet was discontinued and the liver adducts assayed after intervals on control diet. When dietary 2-AAF administration was for 3 or 7 days, removal of adducts was efficient and almost complete by 28 days on control diet, with preferential retention of dG-8-AF. However, when dietary 2-AAF administration was for 28 days, adduct levels were higher, the repair capacity was saturated and the removal of C-8 adducts was not complete after control diet for a 28-day interval. In a preliminary experiment when [3H]-2-AAF was fed for 3 days, after 25 days of 0.02% 2-AAF, the rates of newly formed adduct formation and removal were similar to those observed for the initial 3 days of 2-AAF feeding. These results demonstrate the predominance and persistence of dG-8-AF in liver and kidney DNA of 2-AAF-fed rats and suggest that the repair capacity of the whole rat liver was not diminished after 1 month of 2-AAF feeding.
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