Grantee Research Project Results
1998 Progress Report: Mechanisms in Toxicological Interactions of Genotoxic Teratogens in Mixture with DNA
EPA Grant Number: R825809Title: Mechanisms in Toxicological Interactions of Genotoxic Teratogens in Mixture with DNA
Investigators: Shank, Ronald C. , Said, Boctor
Institution: University of California - Irvine
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1997 through September 30, 1999 (Extended to September 30, 2001)
Project Period Covered by this Report: October 1, 1997 through September 30, 1998
Project Amount: $505,497
RFA: Issues in Human Health Risk Assessment (1997) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The study is conducting experiments to investigate the mechanisms by which the presence of 1) a single modified guanine in a polynucleotide alters the formation of a second guanine adduct at another site in the oligomer, and 2) modified guanines in genomic DNA alter reaction of a second genotoxin with that DNA. Small and bulky carcinogens are represented by alkylating and arylating agents, respectively.Progress Summary:
Most animal studies on genotoxins focus on expos-ures to single chemicals, however, humans are usually exposed to mixtures of genotoxins. Cancer and developmental toxicity risks of geno-toxins in mixture are generally estimated by assum-ing additivity of the components. Two or more genotoxins acting sequentially or simultaneously may present a greater or lesser risk than could be predicted by assuming additivity. Previously we stud-ied the effect of one genotoxin on the binding of a second genotoxin to DNA in an in vitro system; binding of the two toxins was not additive. In the present study the effect of a pre-existing adduct on reaction with DNA by a second genotoxin was examined at the level of genomic DNA (calf thymus DNA, ctDNA) and on a sequence-specific level (oligonucleotides). Pretreatment of ctDNA with N-acetoxy-acetylaminofluorene (N-Aco-AAF) (0-1.8% nucleotides modified) reduced levels of Gua-N7-aflatoxin B1 (AFB1) adducts formed after subsequent treatment with AFB1-8,9-epoxide (~2x reduction). No binding modula-tions following N-Aco-AAF treatment of ctDNA previously modified with AFB1-8,9-epoxide were observed. Oligo-deoxynucleotides containing either Gua-C8-AAF or Gua-C8-aminofluorene adducts and a neighboring unadducted guanine (G) (target G), located 1, 2 or 4 nucleotides from the adduct, were reacted, as single or double-strand-ed 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 the target G. Binding of AFB1-8,9-epoxide to the target G was attenuated (;5-fold) when located immediately adjacent to the AAF, but not an amino-fluorene 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. An optical titration technique was used to measure non-covalent binding of AFB1 and ethidium to DNA. An enhanced binding affinity of both compounds to DNA was observed in spite of the reduced covalent adduct yields suggesting that the ease by which AFB1-8,9-epoxide forms an intercalative intermediate within DNA does not dictate its overall reaction kinetics. The steady-state presence of specific adducts (e.g. Gua-AAF) in certain sequence contexts may mask favorable covalent binding sites of selected genotoxins (e.g. AFB1-8,9-epoxide) thereby reducing productive adduct yields. Such a mechanism may account for non-additive mutagenic responses observed in Salmonella typhimurium.The effect of one genotoxin on the mutagenicity of a second genotoxin was evaluated in a Salmonella assay for several pairs of compounds. Pretreatment of frame-shift strain TA98 with UV-activated afla-toxin B1 (AFB1) (0.4 mg/plate) or synthetic AFB1-8,9-epox-ide (0.17 mg/plate) enhanced the mutageni-city of N-acetoxy-acetyl-amino-fluorene (N-AcO-AAF) 2-3 times above theoretical additive effects. Pre-treatment of base-substitution strain TA100 with N-AcO-AAF (0.1 mg/plate) inhibited the mutagenicity of AFB1, methylnitrosourea and benzo(a)pyrene by 3,10 and 1.5 times below theoretical additive effect; resp. When strain TA102, containing an excision repair system, was pretreated with N-AcO-AAF for 2 hr, inhibition of MNU muta-genicity was partially released. Dose- response relationships for these enhancing or inhibitory effects were demonstrated. These results show nonadditive effects for mixtures of genotoxins in a bacterial mutagenicity assay.
Future Activities:
The next series of studies will expand the number of genotoxic teratogen and carcinogen interactions to included benzo[a]pyrene and alkylnitrosoureas; the DNA targets will be expanded to included oligonucleotides containing sequences identical to mutational hotspots in the human p53 gene. Bacterial mutagenicity studies will be expanded to include more strains to test proposed mechanisms of action by mixtures of genotoxins.Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 7 publications | 3 publications in selected types | All 3 journal articles |
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Type | Citation | ||
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Ross MK, Said B, Shank RC. DNA-damaging effects of genotoxins in mixture: modulation of covalent binding to DNA. Toxicological Sciences 2000;53(2):224-236. |
R825809 (1998) R825809 (1999) R825809 (2000) R825809 (Final) |
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Supplemental Keywords:
RFA, Health, Scientific Discipline, Waste, Toxicology, Environmental Chemistry, Genetics, Chemistry, Risk Assessments, chemical mixtures, chemical probes, synthetic oligonucleotides, genetic analysis, genotoxic teratogens, human exposure, metabolic activation, DNA, toxic environmental contaminants, toxicodynamics, reproductive health, teratogen mixtures, cancer riskProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.