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DNA-damaging effects of genotoxins in mixture: modulation of covalent binding to DNA
1Department of Community and Environmental Medicine, University of California at Irvine, 92697-1820, USA.
Abstract:
Modulation of DNA adduct formation by pre-existing adducts was examined in synthetic oligonucleotides and genomic DNA (calf thymus); genotoxins studied were N-acetoxy-acetylaminofluorene (N-AcO-AAF), aminofluorene (AF), aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide), and dimethylsulfate (DMS). Oligodeoxynucleotides containing either guanine-C8-AAF (Gua-C8-AAF) or Gua-C8-AF adducts and a neighboring unadducted guanine (G) (target G), located 1, 2, or 4 nucleotides from the adduct, were reacted, as single- (ss) or double-stranded (ds) substrates, with dimethylsulfate (DMS) or AFB1-8,9-epoxide. A modified Maxam-Gilbert technique showed that the presence of the AAF adduct lowered the extent to which AFB1-8,9-epoxide, but not DMS, reacted with target G. Binding of AFB1-8,9-epoxide to the target G was attenuated (> or =5-fold) when the target was located immediately adjacent to an AAF, but not AF, adduct in ds-DNA. Reaction with AFB1-8,9-epoxide increased when the target G was located 2 or 4 nucleotides from the AAF adduct. Pretreatment of calf thymus DNA with AAF (0-1.8% nucleotides modified) reduced levels of Gua-N7-AFB1 adducts formed after subsequent treatment with AFB1-8,9-epoxide. Pretreatment of calf thymus DNA with AFB1 did not alter levels of adducts formed after subsequent treatment with N-AcO-AAF. The supposition that aflatoxin B1-binding to DNA may be altered by conformational changes in the helix, due to the presence of a pre-existing AAF adduct, is supported by the absence of an effect by AF and confirmation of local denaturation of the oligomer helix by use of chemical probes hydroxylamine and diethylpyrocarbonate. Nonetheless, the importance of changes in the nucleophilicity of neighboring nucleotides and local steric effects cannot be ruled out.
Insights
Pre-existing DNA adducts, like acetylaminofluorene (AAF), can alter how other genotoxins, such as aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide), bind to DNA. This modulation depends on the adduct type and proximity, influencing DNA damage and repair mechanisms.
Area of Science:
- Chemical Biology
- Molecular Toxicology
- Genetics
Background:
- DNA adducts are chemical modifications to DNA that can lead to mutations and cancer.
- Understanding how existing DNA adducts influence the formation of new adducts is crucial for assessing genotoxic risk.
Purpose of the Study:
- To investigate the modulatory effects of pre-existing DNA adducts on the formation of new adducts by various genotoxins.
- To determine if the sequence context and DNA structure (single- vs. double-stranded) influence these modulatory effects.
Main Methods:
- Utilized synthetic oligonucleotides and calf thymus genomic DNA.
- Studied genotoxins including N-acetoxy-acetylaminofluorene (N-AcO-AAF), aminofluorene (AF), aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide), and dimethylsulfate (DMS).
- Employed a modified Maxam-Gilbert technique and chemical probes (hydroxylamine, diethylpyrocarbonate) to analyze adduct formation and DNA structure.
Main Results:
- The presence of an acetylaminofluorene (AAF) adduct attenuated the binding of aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide) to adjacent guanines in double-stranded DNA.
- This attenuation was adduct-specific, as aminofluorene (AF) adducts did not show the same effect.
- AFB1-8,9-epoxide binding increased when the target guanine was located 2 or 4 nucleotides away from the AAF adduct.
- Pre-treatment with AAF reduced subsequent AFB1-8,9-epoxide adduct formation in genomic DNA, while AFB1 pre-treatment did not affect N-AcO-AAF adduct formation.
- Evidence suggested local denaturation of the DNA helix by the AAF adduct.
Conclusions:
- Pre-existing AAF adducts can significantly modulate the formation of subsequent AFB1-8,9-epoxide adducts, potentially through conformational changes in the DNA helix.
- The observed effects highlight the complexity of DNA damage and repair, where the order and type of genotoxic exposure matter.
- While conformational changes appear important, nucleophilicity and steric effects may also contribute to the observed modulation.