Determination of 2-Acetylaminofluorene Adducts by Immunoassay

Abstract

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.

Simultaneous measurement of benzo[a]pyrene‐induced Pig‐a and lacZ mutations, micronuclei and dna adducts in mutaTMmouse

“…We are not aware of previously published information on the kinetics of formation and elimination of BPDE DNA adducts following a 28-day subchronic repeat dose study in the mouse, however, studies by Poirier et al conducted in rats may help shed some light on murine DNA adduct kinetics. In Poirier et al [ 1982 ], Wistar-Furth rats were fed 2-acetylaminofluorene for 28-days and the kinetics of hepatic DNA adduct formation and elimination were measured. Adduct levels reached a plateau at approximately two weeks, and once treatment stopped, adduct levels began to decrease.…”

“…Adduct levels reached a plateau at approximately two weeks, and once treatment stopped, adduct levels began to decrease. However, at three-days post-treatment greater than 90% of adducts still remained [Poirier et al, 1982 ]. Based on this information, and the fact that the measured levels of BaP-DNA adducts showed dose-dependency and strong correlation with lacZ mutant frequency, it is safe to say that the BaP-DNA adduct levels at 28 days were at steady state, and the adduct levels measured at 72-hr post-exposure were a sufficiently accurate representation of BaP internal dose.…”

Structural Characterization of Two Interchangeable Conformations of an N-2-Aminofluorene-Modified DNA Oligomer by NMR and Energy Minimization

“…0006-2960/94/0433-13611 $04.50/0 vivo to a reactive intermediate which then covalently binds to DNA, primarily at the C8 position of guanine (Beland & Kadlubar, 1990;Miller & Miller, 1967;Singer & Grunberger, 1983). A-(Deoxyguanosin-8-yl)-2-aminofluorene DNA adducts (Figure 1) are persistent in mammalian systems (Gupta & Dighe, 1984;Poirier et al, 1982) and have been shown to induce base substitution or frame-shift mutations during DNA replication (Bichara & Fuchs, 1985;Carothers et al, 1993;Mah et al, 1989;Reid et al, 1990), although translesion bypass with correct incorporation of cytosine opposite the modification site is the predominant outcome (Lutgerink et al, 1985;Michaels et al, 1991; Shibutani & Grollman, 1993; van de Poll et al, 1992).…”

NMR and computational characterization of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] opposite adenosine in DNA: (AF)G[syn].cntdot.A[anti] pair formation and its pH dependence

“…oxyguanosin-8-yl)aminofluorene adduct [(AF)G], which is the major and most persistent adduct in mammalian systems (Irving, 1966;Kriek, 1969;Irving & Veazey, 1969;Beland et al, 1982;Poirier et al, 1982).…”