Grantee Research Project Results
2000 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, 1999 through September 30, 2000
Project Amount: $505,497
RFA: Issues in Human Health Risk Assessment (1997) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
Nucleophilic attack by DNA on an electrophilic chemical genotoxin in formation of a DNA adduct is regarded by most as an early and critical step in the multistage processes of reproductive and developmental toxicity as well as carcinogenesis. Much is known regarding metabolic activation of genotoxins to reactive electrophiles, and the chemical nature of various adducts have been elucidated. Little is known, however, about the mechanisms by which mixtures of genotoxins form adducts with DNA. Hypothesis: the presence of a chemical adduct in DNA can alter the site of formation of a second adduct upon exposure to a second genotoxin, and that the first adduct can block or shift the binding site for the second adduct, such that formation of multiple adducts can be other than additive; the modulation of adduct formation results from alterations in the nucleophilicity of the N7 atom in guanine, steric influences at neighboring binding sites and conformational changes in the helix at more distant binding sites.Progress Summary:
The currently accepted practice for assessing the risk associated with multiple exposures to carcinogens is the summation of potencies associated with each compound individually. There is then the assumption that changes resulting from the binding of a first genotoxin do not have an influential effect on the reception to subsequent compounds. Work described here challenges the validity of this assumption and the efficacy of this standard. Sequential exposure studies performed with N-acetoxy-2-acetylaminofluorene (N-AcOAAF), aflatoxin B1-8,9-epoxide (AFB1-epoxide), (?)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and ethidium bromide (EB) using the Ames mutagenicity assay as a testing system, provided support to in vitro indications that preexisting DNA modifications can potentially alter the mutagenic potential associated with a subsequent genotoxin exposure. DNA conformational changes that result from intercalation of the initial mutagen may be the primary factor involved in the modifications in binding reactivity associated with the second genotoxin, as indicated by the similarity in response generated by EB preexposure. Differential responses demonstrated by sequential exposures to N-AcOAAF and AFB1-epoxide as well as AFB1-epoxide and BPDE, dependent on the order of exposure, demonstrate the apparent involvement of the individual chemical reactivities of the compounds. The nature of the second compound with respect to binding preferences and requirements may be a determining factor in the response that is generated by the preexisting adduct, possibly more significant than the exact identity of the initial adduct. Conflicting findings associated with BPDE reactivity after N-AcO-AAF preexposure between previous in vitro findings and the results obtained with this in vivo system may be indicative of the importance of sequence context, emphasizing the necessity of acknowledging the possibility of sequence specific responses when conducting future studies. Though the studies were initially established to determine the influence that chemically distinct mutagens have on their relative reactivities with a specific binding sequence, it was also revealed that multiple exposures to the same compound displayed the same effect found as when an alternate genotoxin was utilized. These results are substantial when considering occupational situations, where exposures occur throughout the day.The in vivo bacterial system results substantiate the significance of previous in vitro findings performed using oligonucleotides, plasmid DNA fragments, and calf thymus DNA, doing so in a system that portrays the secondary structural influences conferred by DNA binding proteins and further packaging of the genome into a condensed state, analogous to the arrangement found in eukaryotic cells. The Salmonella strains initially used to investigate the influential effects of the initial mutagen exposure expressed proteins involved in a repair response nonexistent in the mammalian system. It was then necessary to examine the independence of the nonadditive relationship using strains that lack sensitivity to both the plasmidexpressed and genomeexpressed proteins involved in the SOS error-prone repair response. Further studies were therefore established for the pretreatment effect of AFB1-epoxide, BPDE, and EB on N-AcO-AAF. The mutagenic response of N-AcOAAF was previously shown to be independent from the UmuDC genomeexpressed proteins, thereby ensuring the complete separation of an SOSrelated influence from the mutagenic response to pretreatment. The enhancing effect of BPDE and EB were shown to exist regardless of the SOS status, suggesting that such influential responses to prior mutagen exposure are also foreseeable in a mammalian system. The effect of excision repair was also investigated, and was shown to efficiently alleviate the altered response demonstrated by pretreatment. The active excision repair system appeared to remove the preexisting lesions such that the influence on the reactivity of the subsequent genotoxin was alleviated. The bacterial excision repair system, though expressing activity analogous to the mammalian system, is greatly simplified in comparison to the components involved in the eukaryotic cell. The highly complex system in humans may then be susceptible to variability in expression or the complete inactivation of key proteins involved. Not only does variability in the excision repair response exist in humans, but also other genetic predispositions not directly related such as susceptibility due to the lack of p53 mediated G1 cell cycle arrest during periods of DNA insult. In individuals with such direct and indirect repair deficiencies, the excision repair system may become saturated and therefore show the inability to efficiently remove lesions, and ultimately affect the potential for nonadditivity in responses to multiple genotoxin exposures.
The enhanced reactivity of N-AcOAAF towards the hisD3052 sequence after pretreatment with AFB1-epoxide, BPDE, and EB was also further characterized by examining the mutation spectra portrayed by the increased frequency of revertants. Though the initial mutagen exposures influenced the binding of N-AcOAAF, their binding was apparently upstream from the sequence probed, therefore the 2 deletion characteristic of AAF alone was predominantly displayed. Further studies that investigate the span of nucleotides influenced by a preexisting adduct and whether the specificity of sequence is also involved will present necessary and interesting information.
The results from the sequential exposure studies using the in vivo Salmonella mutagenicity system indicate the potential for a mutagen to amplify the reactivity of hotspot regions of genes beyond the frequency that they are normally targeted when exposed individually. Though the addition of a functional excision repair system seemed to alleviate the presence of these nonadditive mutagenic relationships, it cannot be assumed that the response demonstrated in the bacterial system will be commensurate with that in the mammalian. Differences in repair activity may present substantial variability across the population, and therefore may establish susceptibility in multiple exposure situations. Influences on processes with indirect involvement in the repair process may also impact the response. Moreover, the significance of chronic exposure in the overall effect has yet to be considered. Such considerations substantiate the need to further examine the influential effects that exist with multiple genotoxin exposures and the many factors that may be involved in the response, such that risks associated with these situations can be more accurately represented.
Future Activities:
The next year of this study will focus on interactions of bulky genotoxins with oligonucleotides containing mutational hot spot sequences of the human tumor suppressor gene, p53. The investigation will include: (1) the effect of a single modified guanine on the formation of a genotoxin adduct in mutational hot spots in the p53 gene; (2) the effect of sequential adduct formation on the double helix conformation in mutational hot spots in the p53 gene; and (3) the effect of 5-methylcytosine and 8-hydroxyguanine on the formation of a genotoxin adduct in mutational hot spots in the p53 gene.Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 7 publications | 3 publications in selected types | All 3 journal articles |
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Ross MK, Mathison BH, Said B, Shank RC. 5-Methylcytosine in CpG sites and the reactivity of nearest neighboring guanines toward the carcinogen aflatoxin B1-8,9-epoxide. Biochemical and Biophysical Research Communications 1999;254(1):114-119. |
R825809 (1999) R825809 (2000) R825809 (Final) |
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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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Said B, Ross MK, Hamade AK, Matsumoto DC, Shank RC. DNA-damaging effects of genotoxins in mixture: nonadditive effects of aflatoxin B1 and N-acetylaminofluorene on their mutagenicity in Salmonella typhimurium. Toxicological Sciences 1999;52(2):226-231. |
R825809 (1999) R825809 (2000) R825809 (Final) |
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Supplemental Keywords:
genotoxic mixtures, teratogens, carcinogens, DNA damage by mixtures., RFA, Health, Scientific Discipline, Waste, Toxicology, Genetics, Environmental Chemistry, Chemistry, chemical mixtures, Risk Assessments, 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.