Grantee Research Project Results

Final Report: Mechanisms in Toxicological Interactions of Genotoxic Teratogens in Mixture with DNA

EPA Grant Number: R825809
Title: 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 Amount: $505,497
RFA: Issues in Human Health Risk Assessment (1997) RFA Text |  Recipients Lists
Research Category: Human Health

Objective:

Most animal studies on genotoxins focus on exposures to single chemicals; however, humans are usually exposed to mixtures of genotoxins. Developmental toxicity and cancer risks of genotoxins in mixture are generally estimated by assuming 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. Earlier, we studied 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 (Said and Shank, Nucleic Acids Research 1991;19(6):1311-1316; Said, et al., Carcinogenesis 1995;16:3057-3062). In this study, the effect of a preexisting adduct on reaction with DNA by a second genotoxin was examined at the level of genomic DNA (calf thymus DNA, ctDNA), on a sequence-specific level (oligonucleotides), and in vivo on bacterial systems. Small and bulky carcinogens were represented by alkylating and arylating agents, respectively. The following genotoxins were studied: N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), aflatoxin B1-8,9-epoxide (AFB1-epoxide), (?)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), N-methyl-N-nitrosourea (MNU), and ethidium bromide (EB).

Summary/Accomplishments (Outputs/Outcomes):

Pretreatment of ctDNA with N-AcO-AAF (0-1.8 percent nucleotides modified) reduced levels of Gua-N7-aflatoxin B1 adducts formed after subsequent treatment with AFB1-8,9-epoxide (~2x reduction). No binding modulations following N-AcO-AAF treatment of ctDNA previously modified with AFB1-8,9-epoxide were observed. Oligodeoxynucleotides 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-stranded 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 aminofluorene 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 noncovalent binding of AFB1 and ethidium bromide 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 (Ross, et al., 2000).

The determination that, in both in vitro and in vivo systems, the presence of an N-acetoxy-acetylaminofluorene (N-AcO-AAF)-guanine adduct, but not an N-aminofluorene adduct, in DNA inhibited the subsequent formation of aflatoxin B1-8,9-epoxide adducts but not methyl adducts from dimethylsulfate, generated the hypothesis that changes in the conformational state of the DNA helix was the basis for the nonadditive effect of these two genotoxins. The study was extended to investigate the changes in DNA conformation under the reaction conditions used in the above studies. Chemical probes were used to confirm that the AAF adduct caused local distortions in the helix. In studies using oligonucleotides, two probes, diethylpyrocarbonate and hydroxylamine, were used to show that there was significant hyperreactivity in the immediate area of Gua-C8-N-AcO-AAF adducts. Both probes sense the degree of denaturation or single strandedness of DNA, and the results support that there is local denaturation around AAF adducts in the test systems used (Ross, et al., 2000).

The reactivity of guanines in an oligonucleotide containing mutational hot-spots within the p53 gene (codons 248 and 249), 5 -CCG1G2AG3G4CCCA-3 , towards dimethylsulfate (DMS) and AFB1-8,9-epoxide was investigated by a modified Maxam-Gilbert technique. 5-Methylcytosine in the CpG site in codon 248 did not appear to modulate the reactivity of target guanines G1, G2, G3, and G4 towards either genotoxin when compared to the sequence containing a nonmethylated CpG site. An earlier study in which a 0.5 kb fragment of the human HPRT gene containing exon 1 and several CpG sites were treated with UV-activated aflatoxin B1 showed that guanine adduct formation was independent of the methylation status of the CpG site. These results were discussed by Ross, et al. (1999), in relation to other studies that have shown that cytosine methylation has an inhibiting effect, an enhancing effect, or no effect on adduct formation with nearby guanine nucleotides.

In an in vivo study, the effect of one genotoxin on the mutagenicity of a second genotoxin was evaluated in a Salmonella typhimurium assay for several pairs of compounds. Pretreatment of frameshift strain TA98 (AuvrB, +pKM101) or TA 1538 with AFB1-8,9-epoxide (17.3 ng/plate) enhanced the mutagenicity of N-acetoxy-acetylaminofluorene (N-AcO-AAF) approximately 3 times above theoretical additive effects. The same effect was seen in strain TA100 (AuvrB, -pKM101) in which a two-fold enhancement of N-AcO-AAF mutagenicity was seen. Pretreatment of TA 98 with trans-7,8-diol-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti) (BPDE) (75 ng/plate) enhanced N-AcO-AAF mutagenicity four to six times, and a nonadducting intercalator, ethidium bromide (20 ng/plate), enhanced N-AcO-AAF mutagenicity five times above the theoretical value assuming additivity. Neither of the initial genotoxins, AFB1-8,9-epoxide or BPDE, or the intercalator, ethidium bromide, was mutagenic by itself in TA 98. All of the initial genotoxin treatments followed clear dose-response relationships (Said, et al., 1999).

To study the effect of N-AcO-AAF on the mutagenicity of AFB1-8,9-epoxide, a base-substitution strain, TA100, was pretreated with N-AcO-AAF. In a dose-dependent manner, N-AcO-AAF (0.1-1000 ng/plate) inhibited the induction of revertants by AFB1-8,9-epoxide (43 ng/plate). A concentration of 1 ng/plate N-AcO-AAF reduced the total number of revertants to 44 percent of that induced by AFB1-8,9-epoxide alone. N-AcO-AAF itself induced mutations in this base-substitution strain. Pretreatment of TA100 with N-AcO-AAF (0.1 mg/plate) inhibited the mutagenicity of methylnitrosourea and benzo(a)pyrene by 10 and 1.5 times below theoretical additive effect, respectively. When strain TA102, containing an excision repair system, was pretreated with N-AcO-AAF for 2 hours, inhibition of MNU mutagenicity 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 (Said, et al., 1999).

The in vivo bacterial system results substantiated the significance of previous in vitro findings performed using oligonucleotides, plasmid DNA fragments, and calf thymus DNA 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 necessary to examine the independence of the nonadditive relationship using strains that lack sensitivity to both the plasmid-expressed and genome-expressed proteins involved in the SOS error-prone repair response. These studies investigated the pretreatment effect of AFB1-epoxide, BPDE, and EB on N-AcO-AAF. The mutagenic response of N-AcO-AAF was previously shown to be independent from the UmuDC genome-expressed proteins, thereby ensuring the complete separation of an SOS-related influence from the mutagenic response to pretreatment. The enhancing effects of BPDE and EB were shown to exist regardless of the SOS status, suggesting that such influential responses to prior mutagen exposure also are foreseeable in a mammalian system. The effect of excision repair also was investigated, and was shown to efficiently alleviate the altered response demonstrated by pretreatment. The active excision repair system presumably removed 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.

Another study investigated the mutational spectrum in the hisD3052 allele of Salmonella. Colonies treated with combinations of genotoxins were screened for the presence of a ?2 deletion of GC or CG within the hot spot target sequence CCGCGCGCGG, using a 32P end-labeled probe (5'-CTGCCGCGCGGACACCG-3'). Of the spontaneous mutations, 47-53 percent were ?2 deletions; in cells treated with N-AcO-AAF alone, 87-93 percent of the mutations were ?2 deletions. The frequency of ?2 deletions in bacteria pretreated with AFB1-8,9-epoxide, BPDE, or ethidium bromide was 92.5 percent, 91 percent, and 95 percent, respectively. Pretreatment of the Salmonella with the intercalating genotoxins increased the potency of N-AcO-AAF as a mutagen, but did not change the type of mutation. Further studies that investigate the span of nucleotides influenced by a preexisting adduct and whether the specificity of sequence also is involved will present necessary and interesting information (Shank, et al., 2001).

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 at which 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 also may 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.

These results of these studies demonstrate nonadditive effects of sequential exposures to two genotoxins on the formation of DNA adducts in model oligomers and the induction of mutations in a bacterial system, and suggest that changes in helix conformation induced by bulky agents are important in modulating the formation of subsequent adducts. The effects of individual genotoxins on the DNA damaging activity of other genotoxins in sequential exposure are summarized in Table 1.

Table 1. Summary of DNA damage induced by sequential exposure to genotoxins

Genotoxinsa	System	Effectb	Reference
DNA-adduct formation level:
BPDE/AAF	DNA fragment	>additive	Said, et al., 1995
AAF/BPDE	DNA fragment	<additive	"
BPDE/MNU	DNA fragment	additive	Said & Shank 1991
BPDE/CCNU	DNA fragment	<additive	"
AFB1/MNU	DNA fragment	additive	"
MNU/AFB1	DNA fragment	additive	"
AFB1/CCNU	DNA fragment	<additive	"
AAF/AFB1	Oligonucleotide	<additive	Ross, et al., 2000
MNU/AFB1	Oligonucleotide	additive	"
AAF/AFB1	Genomic DNA	<additive	"
AFB1/AAF	Genomic DNA	additive	"
DNA synthesis level:
MNU/AAF	Phage ssDNA	<additive	Said, et al., 1995
MNU/AFB1	Phage ssDNA	<additive	"
Mutagenic potencies level:
AFB1/AAF	TA98	>additive	Said, et al., 1999
AAF/AFB1	TA100	<additive	"
BPDE/AAF	TA98	>additive	Shank, et al., 2001
AAF/BPDE	TA100	additive	"
AAF/AAF	TA98	>additive	"
BPDE/BPDE	TA100	>additive	"
EtBr/AAF	TA98	>additive	"
AAF/MNU	TA100	<additive	"
BPDE/Safrol	TA100	additive	unpublished
Safrol/BPDE	TA100	additive	unpublished
Mutation spectrum level:
AFB1,BPDE or EtBr/AAF	TA98	>additive	unpublished
a sequential order, first exposure/second exposure; BPDE, benzo(a)pyrene diolepoxide; AAF, N-acetoxy-2-acetylaminofluorene; MNU, N-methyl-N-nitrosourea; CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; AFB1, aflatoxin-B1-8,9-epoxide; EtBr, ethidium bromide
b>additive, effect greater than that expected from theoretical additivity; <additive, effect less than that expected from theoretical additivity.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

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Other project views:	All 7 publications	3 publications in selected types	All 3 journal articles

Publications

Type	Citation	Project	Document Sources
Journal Article	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)	Abstract from PubMed Full-text: ScienceDirect-Full Text PDF Exit Abstract: ScienceDirect-Abstract HTML Exit
Journal Article	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)	Abstract from PubMed Full-text: Oxford - Full Texg Exit
Journal Article	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)	Abstract from PubMed Full-text: ToxSci-Full Text PDF Exit Abstract: ToxSci-Abstract Exit

Supplemental Keywords:

N-acetyoxy-2-acetylaminofluorene, aflatoxin B1-8,9-epoxide, bacteria, benzo(a)pyrenediolepoxide, carcinogen, chemicals, DNA adducts, DNA intercalators, dose-response, ethidium bromide, genotoxin mixtures, N-methyl-N-nitrosourea, mutagen, mutagenicity, teratogen, toxics., Health, RFA, Scientific Discipline, Waste, Risk Assessments, chemical mixtures, Chemistry, Environmental Chemistry, Toxicology, Genetics, chemical probes, toxic environmental contaminants, teratogen mixtures, genetic analysis, human exposure, DNA, synthetic oligonucleotides, genotoxins, reproductive health, cancer risk, genotoxic teratogens, metabolic activation

Progress and Final Reports:

Original Abstract

1998 Progress Report

1999 Progress Report

2000 Progress Report