Final Report: Improved Risk Assessment with an Intragenic Mutation Assay

EPA Grant Number: R825810
Title: Improved Risk Assessment with an Intragenic Mutation Assay
Investigators: Wilson, Vincent L. , Lee, William R.
Institution: Louisiana State University - Baton Rouge
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1997 through September 30, 2000
Project Amount: $428,305
RFA: Issues in Human Health Risk Assessment (1997) RFA Text |  Recipients Lists
Research Category: Human Health , Health Effects , Human Health Risk Assessment , Health


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). 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 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 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 ENU 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).

Summary/Accomplishments (Outputs/Outcomes):

This project's goals were based on the assumption that direct molecular analyses of intragenic mutations with a highly sensitive and adjustable molecular procedure would be comparable to the standard Drosophila phenotype germline mutagenesis test. However, our work has identified a new phenomenon that universally affects mutagenesis testing. Inherent in agent induced mutagenesis studies, this dose response curve altering issue causes an under representation of mutagenic activity in a direct molecular assay. In order to establish the intended direct molecular germline mutagenesis test, we have refocused our efforts on defining this new concept (Lee et al., 2001) and determining how to incorporate a solution to this problem within the molecular testing procedures. The discussions below detail our success in these two unexpected research excursions.

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.

D. melanogastor model and target cell lethality determination:

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, Rad. Res. 117: 469-479 (1989); and Lee, et al., Mut. Res. 231: 31-45 (1990)). Our procedure was to collect an 8 hour sample of eggs from a cage with flies of the genotype that produce only males for the SLRL test. Development of this 8 hour sample of eggs was timed so that the 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 were transferred to standard Drosophilia media and allowed to complete their development. Upon emergence the adult males were aged 5 days so their seminal vesicles were engorged with sperm and then divided into two samples. In one sample the seminal vesicles were pulled out, homogenized, and the sperm isolated (as noted below). We then treated 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 obtained sufficient data for 1 mM and 0.5 mM doses to compare with the historical control and report these findings (Lee et al., 2001).

These results amply demonstrated that the doubling of the dose of ENU doubled the induced mutation frequency. However, direct intragenic molecular assays will test samples consisting of sperm from one or more flies and not individual sperm as would be more comparable to the SLRL test. If this same SLRL data for ENU (noted above) is analyzed on a per male (Drosophila) basis instead of per F1 progeny, then there is no correction for "jackpot" mutation clusters and the higher dose (1.0 mM) of ENU presents an under representation of induced mutation frequency (Lee et al., 2001).

On a "per fly" basis, doubling of the dose did not double the induced mutation frequency due to the cell killing effect of the mutagen (ENU). The cell killing effiect reduces the number of target cells (spermatogonial cells) and increases the clonal expansion of mutant germ cells and thus increases the "jack pot" mutation frequency. These results are comparable to what will be found from direct molecular mutations assays such as PCR/RE/LCR.

As explained below, by adapting the PCR/RE selection procedures and adjusting the sensitivity of the PCR/RE/LCR assay, we have incorporated an internal correction for this universal cytotoxic phenomena of mutagens.

Molecular Mutation Analyses:

D. 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. However, unanticipated issues had to be addressed to accomplish this goal, including the successful removal of somatic cellular DNA from sperm preparations and the adaptation of the PCR/RE/LCR procedures for the appropriate detection sensitivities in multiple analyses per sample. Additional pitfalls also hampered the progress of this work.

Sperm Cell DNA Isolation:

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. Seminal vesicles are collected from one to ten flies and the sperm cells isolated. A combination of 0.02% periodic acid, hyluaronidase, collagenase, and dispase are used to disassociate the cells, followed by a DNase treatment. The sperm are then isolated by a ficoll gradient and the DNA subsequently isolated on a miniature scale with the help of mercaptoethanol and the use of 1 g of sheared salmon sperm DNA as a carrier. Using DAPI as a fluorescent marker, microscopic evaluation of sample preparations demonstrated the selective isolation of sperm cells prior to DNA isolation.

Development of PCR/RE/LCR testing procedures:

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. Based on these principles, we have adjusted the sample size as well as the PCR/RE selection protocol for the PCR/RE/LCR molecular mutation detection and identification test (as described below). The PCR/RE/LCR assay has been established for the detection and identification of base substitution mutations at the G:C basepair at site 41 on the D. melanogaster Adh gene. Procedures for establishing a PCR/RE/LCR assay for the A:T basepair at site 482 of the Adh gene, so that the germline selection of ENU induced transition (G to A) or transversion (A to T) mutations could be evaluated in our protocol, were unsuccessful. Work was initially delayed based on the interest of accommodating the simultaneous analyses of both sites 42 and 482 in the same set of specimens. Unfortunately, the procedures for site 482 were problematic at best and the delay was becoming too protracted.

Based on the assumption that ten seminal vesicle engorged (5 day old) flies contain approximately 10,000 sperm or less, the PCR/RE selection procedures have been altered to provide a sensitivity of one mutation in 104 wild type genomes. Only two rounds of PCR amplification restriction endonuclease digestion appear to be needed to obtain this sensitivity. This enables the analyses of samples of pooled sperm from ten flies, providing a positive for any sample containing one or more flies with mutations. However, the higher the dose of ENU the more target cell killing and the greater the chance for "jack pot" mutations. The above procedures do not enable a correction for the target cell killing effect of the mutagen. Fortunately, the PCR/RE selection procedures incorporate a dilution effect that enables the analyses of material at each step in the selection procedure. Thus, the same sample can be analyzed by LCR at two different levels in the same analyses providing two different sensitivities. With standard mutant mixtures, we have demonstrated that the LCR analysis of two different steps in the PCR/RE selection provide 10-2 and 10-4 detection sensitivities, respectively. The detection of mutant samples at both 10-4 and 10-2 enables the determination of "jack pot" mutations, since only "jack pot" mutations will be detectable at the 10-2 sensitivity. The mutation frequency bias of mutagen induced target cell killing can identified and corrected in the same data set with this direct intragenic molecular test.

Results from the Molecular Mutation Analyses:

The PCR/RE/LCR analyses of isolated DNA from control and treated flies has been problematic and hampered by technical difficulties, including contamination issues. These PCR/RE/LCR procedures require great care with meticulous bench work. This work has progressed only slowly and is still a focus in this laboratory. Interestingly, these same procedures designed for a number of base sites within the human genome have been much easier to consistently perform and accommodate in the laboratory (Wilson et al., 2000, 2001, and In Press). Thus, the data below represents the results of our efforts to date.

The procedures described above are being followed to determine the frequencies of both mutant flies and flies harboring mutation clusters (jack pot mutants). Sperm has been isolated from the pooled seminal vesicles dissected from ten flies, the DNA purified, and the mutations selected by two rounds of PCR with one restriction digestion in between. LCR analysis of the second PCR enables the detection and identification of mutants present in the sample at frequency of 10-4 or higher, while LCR analysis of the first PCR determines which samples contain mutations at the level of 10-2 or higher.

Table 1
PCR/RE/LCR Test for Mutations at site 41 of the Adh Gene
(Direct Molecular Test)

Samples* (10 flies each)

ENU (mM) 10-4 Positive 10-2 Positive Negative %Mutants (10-4) Total Flies
0 16 0/3 67 19.3 830
1.0 23 1/3 54 29.9 770

*Each sample pooled sperm from 10 flies;
Number of positive samples over the number of samples analyzed at this sensitivity;
ND, not done

None of the 0.5mM ENU treated flies have been analyzed at this time, while the analyses of the controls and 1.0mM ENU at the 10-2 sensitivity level has not provided sufficient numbers to evaluate jack pot mutation frequencies. The mutation (10-4) frequencies determined thus far are not inappropriate considering that a single positive mutant sperm in one of the flies will be detected in a sample (based on 10,000 sperm per sample and the detection sensitivity of one mutant in 10-4, as verified with standards). The molecular analysis of all positive samples at the 10-2 detection sensitivity will provide the number of samples containing mutant clusters (jack pot mutants).

Once the validation of the PCR/RE/LCR molecular germline mutagenesis assay is completed, this assay will offer a new molecular test that will be useful in mammalian models.

Benefits to the Environment and Human Health:

The present work has demonstrated that these techniques enable the quantitative evaluation of the target cell (spermatogonial) killing effects of mutagens, so that adaptation of the PCR/RE/LCR techniques should enable the study of induced cell death versus mutagenic and carcinogenic activities of toxic agents in mammalian models.

The PCR/RE/LCR molecular germline mutagenesis assay potentially offers a new molecular test that will be useful in mammalian models. Mammalian models for germline mutagenesis will provide a more accurate extrapolation of testing results to human risk assessment than the traditional D. melanogaster model. These PCR/RE/LCR procedures are already successfully in use in human studies, with potential for evaluation and monitoring of mutations in human semen specimens (Wilson et al., 2000, 2001, and In Press).

Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other project views: All 9 publications 5 publications in selected types All 5 journal articles
Type Citation Project Document Sources
Journal Article Lee WR, Perantie DC, Clark KB, Guillot DA, Wilson VL. Effect of mutagen induced cell lethality on the dose response of germline mutations. Environmental and Molecular Mutagenesis 2001;37(4):340-344. R825810 (Final)
not available
Journal Article Wilson VL, Tatford BC, Yin X, Rajki SC, Walsh MM, LaRock P. Species-specific detection of hydrocarbon utilizing bacteria. Journal of Microbiological Methods 1999;39(1):59-78. R825810 (Final)
not available
Journal Article Wilson VL, Yin X, Thompson B, Wade KR, Watkins JP, Wei Q, Lee WR. Oncogenic base substitution mutations in circulating leukocytes of normal individuals. Cancer Research 2000;60(7):1830-1834. R825810 (Final)
not available
Journal Article Wilson VL. Detecting rare mutations associated with cancer risk. American Journal of Pharmacogenomics 2001;1(4):283-293. R825810 (Final)
R825819 (Final)
  • Abstract from PubMed
  • Abstract: ADIS Online
  • Journal Article Wilson VL, Wade KR, Yin X, Albertini RJ. Temporal delineation of sequential HPRT mutations arising in vivo in a T-cell clone with a mutator phenotype. Mutation Research 2001;473(2):181-199. R825810 (Final)
    not available

    Supplemental Keywords:

    Jack pot mutations.,

    Progress and Final Reports:

    Original Abstract
  • 1998 Progress Report
  • 1999