Grantee Research Project Results
1998 Progress Report: Improved Risk Assessment with an Intragenic Mutation Assay
EPA Grant Number: R825810Title: Improved Risk Assessment with an Intragenic Mutation Assay
Investigators: Wilson, Vincent L. , Lee, William R.
Institution: Louisiana State University - Baton Rouge
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1997 through September 30, 2000
Project Period Covered by this Report: October 1, 1997 through September 30, 1998
Project Amount: $428,305
RFA: Issues in Human Health Risk Assessment (1997) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The ability to detect intragenic mutations at sensitivities of better than one mutant allele in 106 or more cells provides the unique opportunity to directly evaluate the genotoxic activities of hazardous agents in model systems and ultimately in exposed human populations. Such a sensitive molecular test would enable more accurate assessment of the toxic potential of chemicals at very low doses and multiple exposures, similar to those commonly found in the human living and work environments. Some complex mixtures may also be decipherable by the determination of intragenic base substitution mutation spectra analysis, since mutation spectra are well known to be genotoxic agent specific if analyzed at the DNA nucleotide sequence level.This laboratory has established methods for the detection of single base substitution mutations at a sensitivity of one mutant in the presence of 106, 107, or more wild type cells. These methods are based on the combined sensitivity and specificity of the polymerase chain reaction (PCR), restriction endonuclease digestion (RE), and the ligase chain reaction (LCR) which enables the detection of these rare mutant cell(s) in minute tissue specimens. The sensitivity of this PCR/RE/LCR test can be adjusted to range from one mutant cell in 102 to 107 normal cells by varying the number of genomes in the sample. These PCR/RE/LCR molecular techniques, will be used to establish a universal intragenic molecular test for the in vivo induction of germline (or somatic) mutations by environmentally important genotoxic agents.
It is hypothesized that this molecular test for intragenic mutations will enable more accurate determinations of the genotoxic potential of very low dose exposures of hazardous agents in model systems, of multiple and complex exposure patterns, and the identification of the genetic effects of individual components of at least simple mixtures. This hypothesis will be experimentally approached with the use of two well characterized animal model systems: Drosophila melanogaster and Mus musculus. The quantitative identification of intragenic mutations in the germline of D. melanogaster and mice offers the unique opportunity to validate these methods with published data from the well established standard genetic mutation tests. Since PCR/RE/LCR procedures are dependent only on the knowledge of the DNA sequence of the gene under study, the present molecular test for the induction and frequency of base substitution mutations will ultimately be universally applicable to any species.
These studies will use a well tested standard reference mutagen, ethyl nitrosourea (ENU), and the environmentally important methyl bromide (MeBr). The genotoxic effects of single and multiple (5 daily doses per week for two weeks) dosing regimens will be determined in the germ cell populations of the D. melanogaster and mouse testis. The genes under study include the well characterized alcohol dehydrogenase (Adh) gene locus in D. melanogaster and the Harvey-ras and p53 genes in mice. Similarly, mixtures of these two agents and a mixture of 2-chloro-ethyl methanesulfonate and ENU will be subjected to the same singleEand multiple dose genotoxic analyses in mice. In addition, site specific mutagen frequency studies will be performed in the Adh gene of D. melanogaster and in the p53 gene of the mouse model.
The PCR/RE/LCR molecular test will complement the standard heritable mutation assays and will enable germ cell genotoxic testing to be performed relatively cheaply and routinely in the mammalian model and perhaps ultimately in the accidentally exposed human.
Objectives of Research:
The principle objective of this project is to develop a more accurate means of assessing the biological risk of human exposure to genotoxic agents. The ability to detect and identify fixed biological damage at relevant sites in genomic sequences of DNA at a sensitivity of one mutation in a million or more cells provides the necessary testing procedures to directly evaluate genotoxic effects of single and multiple dosing regimens, complex mixtures, complex environmental exposures, and perhaps ultimately identifying the family of agents and/or guilty compound(s) in questionable exposures. Determining the frequency of occurrence of fixed mutations at specific loci within the genomic sequences of an exposed individual(s) identifies the ultimate biological damage responsible for genetic disease(s). By using direct acting agents (methyl bromide and ethylnitrosourea) coupled with an agent that requires metabolic activation (2-chloroethyl methanesulfonate), the genetic effects of these agents can be determined with and without the interference of interindividual variation in metabolic activation. Thus, the following objectives are designed to determine the capabilities of these sensitive testing procedures in the germ and somatic cells of model species and develop a widely applicable model molecular test for evaluating the human risk of genotoxic effects from environmental pollutants, hazardous materials, and other potentially toxic compounds.
Objective 1) To Characterize and Validate the quantitative capabilities PCR/RE/LCR techniques for a molecular test.
This will be performed by using PCR/RE/LCR mutation detection and identification procedures on treated and control Drosophilia melanogaster. Natural clonal expansion that occurs during spermatogenesis following stem cell mutagenesis, can be used to both verify and demonstrate the quantitative capabilities of these PCR/RE/LCR techniques. The probability of a sample containing a mutation can be adjusted by changing the sample size, which for single copy genes is the number of haploid genomes, and the number of replications between mutation fixation and sampling can be determined by reference to the biology of the test organism. In a series of samples containing sperm DNA isolated from consecutively fewer treated D. melanogaster , there will be a successively lower number of mutant stem cell genomes represented by cloned DNA in each sample and an increasing number of samples that do not contain the mutation. By choosing the time following treatment, analyses can allow for clonal expansion that distinguishes between treatment effects and spontaneous mutations, thereby enhancing the sensitivity of detection of mutations at lower levels of exposure.
Objective 2) To extend the molecular assay to the mammalian mouse model.
Male mice will be injected with methyl bromide (MeBr) or ENU (reference standard mutagen) and after allowing sufficient time for spermatozoa to mature, the spermatozoa will be assayed by the same procedures as above in Objective 1. Individual tubules of the mouse testis will be studied for clonal expansion of mutant cells. Previous studies with molecular-dosimetry have produced a dose-response curve for ENU in mouse spermatogonia [17,50,53] that will be used as a reference. Base substitution mutation frequencies will also be determined at selected loci in somatic tissues, including the liver and brain of these treated mice.
Objective 3) To determine the cumulative effect of multiple doses of genotoxic agents on mutation frequencies.
Male mice will receive multiple low dose exposures to MeBr (or ENU, for reference studies) of 5 separate daily exposures per week for two weeks. Two separate dosages will be used, both of which will be below the TD10: one that provides for statistically significant mutation frequencies with only a single dose (i.e. 30-40 mg/kg for ENU), and one that is below the statistically significant level, as determined by Objective 2 (above).
Objective 4) To determine the combined effect of a mixture of genotoxic agents on mutation frequencies.
The present molecular test provides a unique opportunity of deciphering the individual and combined (synergistic, additive, or opposing) effects of two or more genotoxic agents. Thus, the procedures outlined in Objectives 2 and 3, above, will be repeated with a mixture of these genotoxic compounds.
Male mice will receive a single dose of a mixture of equal parts of MeBr and ENU or 2-chloro-ethyl methanesulfonate and ENU, that is equivalent to a genotoxic effect from a single low dose of the less potent mutagen.
Progress Summary:
In order to validate the molecular PCR/RE/LCR mutation test, we have used the well characterized D. melanogaster to develop this system. The standard protocol for testing germline mutations in D. melanogastor is to treat adults and sample post meiotic germ cells. For each new mutation induced this protocol will produce only one mutant gamete which is advantageous for statistical analysis of mutant F-1 progeny. The difficulty with the traditional approach is the very large number of F-1 individuals that must be produced and tested. To overcome this difficulty we are developing a molecular test that can quantitatively determine mutation frequency in germ cells of D. melanogastor. The standard protocol of treating adult males is not feasible because each DNA lesion will either remain a lesion until fertilization or if converted into a mutation will be represented by only one mutant molecule which will not permit verification of a sample. In vivo cloning of induced mutants is required to permit verification by repeat sampling and to provide a satisfactory margin between spontaneous and induced mutations. This is accomplished by treating a cell stage that will by replication of mutant cells produce a "jack pot" of mutations in each sample in which a mutation has been induced. The sample size, determined as the number of treated individuals, will be adjusted to prevent multiple "jack pots" in any one sample.In order to test its usefulness, we have developed and tested (with the established sex-linked recessive lethal, SLRL, test) a protocol of treating second instar D. melanogastor larvae containing only spermatogonial cells. We used treated and control flies that were crossed to females of the appropriate genotype for a SLRL test (Byrne and Lee, 1989; and Lee, et al. 1990). Our procedure is to collect an 8 hour sample of eggs from a cage with flies of the genotype that will produce only males for the SLRL test. Development of this 8 hour sample of eggs is timed so that a 12 hour period of treatment occurs entirely within the second instar stage of larval development, thereby treating only spermatogonial cells. Following the 12 hour treatment with either 0.5 mM or 1.0 mM ethylnitrososurea (ENU) in a 4.3 pH acetic acid, sodium acetate buffer, the treated larvae are transferred to standard Drosophilia media and allowed to complete their development. Upon emergence the adult males are aged 5 days so their seminal vesicles are engorged with sperm and then divided into two samples. In one sample the seminal vesicles are pulled out, homogenized, and digested with DNase. Sperm DNA is not digested with DNase while the somatic DNA is digested. We then treat the sperm cells with mercaptoethanol and extract germ line DNA from the sperm cells for a PCR/RE/LCR molecular analysis that is currently being used in human and mammalian studies.
Initially, the concentration for the 12 hour exposure was varied from control (zero) to 5 mM. Concentrations higher than 1 mM ENU were found to significantly inhibit reproduction and feeding behavior. Therefore, we are performing all our work at the 1 mM ENU and below. We have sufficient data for our 1 mM and 0.5 mM to report in the following table with the historical control. a concurrent control, 0.25 mM ENU concentration, and additional data at the 1 mM and 0.5 mM are currently being conducted.
Sex-Linked Recessive Lethal test for 0.5 mM and 1.0 mM ENU
ENU (mM) | Mutations | NonMutants | %Induced Mutations* |
0.5 | 11 | 938 | 1.1 |
1.0 | 22 | 894 | 2.3 |
*corrected for spontaneous mutations; historical spontaneous mutation frequency is 0.12%
Molecular Mutation Analyses:
Drosophilia melanogaster is being used as a model for the development of a new universally applicable test for the determination of the mutation frequency in the germ cells by a direct molecular, PCR/RE/LCR analysis of sperm cells. We have developed a procedure for the selective isolation of sperm DNA. Treatment of sperm with DNase does not lead to DNA degradation due to the special packaging and protective membranes of the sperm heads, as opposed to somatic cells which cannot prevent DNA digestion. The standard protocol of treating adult males is not feasible because each DNA lesion will either remain a lesion until fertilization or, if converted into a mutation, will be represented by only one mutant molecule which will not permit verification of a sample. We have developed the protocol noted above for treating second instar larvae which contain only spermatogonial cells. Each induced mutation will be replicated to produce a "jack pot" of mutant sperm cells which can then be detected by the PCR/RE/LCR assay. The sample size, or number of treated larvae, is adjusted to prevent multiple "jack pots" in any one sample. The established SLRL test has been used to develop this treatment protocol, as noted above, and will provide a validation of the PCR/RE/LCR molecular test. We are presently adjusting the sample size for the PCR/RE/LCR molecular mutation detection and identification test.
The PCR/RE/LCR assay has been established for the detection and identification of base substitution mutations at base sites 41 and 400 on the D. melanogaster Adh gene. These deoxyguanosine nucleotide sites have been previously demonstrated to produce null mutations in the Adh gene (Fossett et al., Mutation Res. 331: 213-224, (1995), with a erratum due to printer error: Mutation Res. 354: 143-144 (1996)). The molecular assay is also being developed for a deoxyadenosine nucleotide base site in order to contrast and compare the frequencies of clonally expanded ENU induced transition mutations.
Future Activities:
These procedures will be modified for use in the mammalian mouse model. The PCR/RE/LCR test has already been established for a single base site in codon 61 of the Harvey-ras gene and p53 base sites are being chosen for molecular test development. The increased complexity of the increased number of stem cells in the mouse testes over D. melanogaster, will require the selective dissection of the mouse testes prior to sperm isolation and PCR/RE/LCR molecular analyses. This increased complexity also offers opportunities for deciphering basic biological processes and the risk or probability of producing mutant offspring.Once the PCR/RE/LCR molecular test is characterized and validated in the D. melanogaster model, these mouse studies should enable the more accurate determination of the human germ cell risk to low levels of mutagens.
Journal Articles:
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Progress 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.