Mutagenesis Advance Access originally published online on May 20, 2008
Mutagenesis 2008 23(4):241-247; doi:10.1093/mutage/gen022
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New methods for assessing male germ line mutations in humans and genetic risks in their offspring
Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, The Netherlands 1Laboratory for Health Protection Research, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands 2Department of Genetics, University of Leicester, Leicester, UK
Germ line mutations resulting from chemical or radiation exposure are a particular problem in toxicology as they affect not only the exposed generation but also an infinite number of generations thereafter. Established methods to show that these mutations occur in an F1 or subsequent population require the use of a large number of progeny for statistical significance. Consequently, many thousands of animals have been used in the past. Such a use is no longer considered desirable and is also very expensive. Several new molecular techniques (including analysis of tandem repeats and randomly amplified polymorphic DNA) now provide alternative methods of assessment, which also allow the quantification of individual mutations in individual sperm cells. These can also be applied to human offspring, making extrapolation obsolete. The downside of these methods is that they effectively determine the mutation rate in certain regions of DNA and the relevance of these to diseases, particularly cancer, is not always apparent. Therefore, it must be assumed that an increase in mutation rates in these selected regions correlates with altered phenotype. However, disease types linked to changes in tandem repeat length indicate that these may act as relevant markers for the development of phenotypes. Further research and evaluation are required to more closely link changes in DNA with altered phenotype and validate the use of tandem repeats and randomly amplified polymorphic DNA in transgenerational genotoxicity testing. This paper introduces and compares recently developed methods to assess mutations in sperm due to stem cell damage.
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Introduction |
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New methods are needed to detect germ line mutations induced by exposures to genotoxins in humans. Too little is known about how genotoxic compounds induce mutations in human germ cells and the subsequent transmission of these to offspring. Transmittable genetic damage in humans has been studied in populations exposed to radiation (1
), whereas germ line mutations induced by environmental pollutants have only been studied in animals. Most of the mutations transmitted to offspring had a paternal origin (2–5). A similar result might be expected in humans although a direct relationship between mutations in human sperm and a genetic disease induced in the progeny has not been reported yet (6).
A parallelogram approach developed by Sobels (7) and extended by Adler (8) has been used until now to extrapolate measurable effects in rodent germ cells, their progeny and human germ cells in vitro to estimate the genetic risks in humans in vivo (Figure 1). This approach uses classical methods, such as the morphological specific locus (MSL) test and the dominant lethal test. These tests have low sensitivity, use large numbers of progeny to obtain the recommended sample size and are very time consuming and expensive. Recently developed germ cell mutagenicity tests seem to have rendered this parallelogram approach obsolete since these new tests can detect mutations in human germ cells and offspring and are suitable for studying genetic risks in human populations. A recent review by Singer et al. (9) compared classical approaches (e.g. MSL and heritable translocation assay) with new methodologies [e.g. expanded simple tandem repeat (ESTR) mutation assay and the gene mutation test in transgenic rodents] but too little data were available to compare the performances of these tests in detail.
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Of these new techniques, only the analysis of tandem repeat sequences seems suitable for testing in humans. We will therefore discuss this assay in greater detail together with randomly amplified polymorphic DNA PCR (RAPD-PCR), an assay that could be used to quickly screen exposed humans for the transmission of mutations to their offspring. Although these new methods seem to have a high sensitivity, they have yet to be validated and only a few laboratories are equipped to perform the analysis of tandem repeat sequences.
Germ cell tests play a decisive role in classifying and labelling chemicals for genotoxicity. To minimize animal use, tests that detect effects in germ cells should be performed first. However, if quantification of these effects is required, assays for mutations in the offspring are essential. In the guidance document for genotoxicity under Registration Evaluation and Authorisation of Chemical Substances, the new European Community (EC) policy for authorization of new and existing chemicals, a test that determines changes in hypervariable tandem repetitive regions is stated as an alternative to classical tests. As this test also allows the measurement of heritable damage in humans, it is a recommended alternative.
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Germ line mutagenicity tests |
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TopIntroductionGerm line mutagenicity testsDo differences between rodent...Analysis of germ line...Future perspectivesFundingReferences |
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RAPD-PCR
RAPD-PCR was first described by Williams et al. (10
) and is based on the amplification of random unknown DNA segments with a single primer of arbitrary nucleotide sequence. RAPD-PCR can be used to detect genomic alterations without prior information about the loci. Multiple fragments are generated in a single reaction that can be visualized after gel electrophoresis (Figure 2A). Any genomic alteration (point mutations, structural rearrangements, deletions, insertions, etc.) may change the place where the primer anneals resulting in different profiles (Figure 2B and C). For germ cell mutagenicity testing, the patterns obtained from the mother and father can be compared with those from the offspring. Differences between the patterns may indicate a genomic alteration if (i) the DNA fragment is only visible in the pattern of the child, (ii) the DNA fragment is present in the pattern of the father and/or the mother, but absent in the pattern of the child or (iii) bands are increased or decreased in intensity. However, PCR products that disappear or change in intensity should be interpreted with care because of the possibility of heterozygosity in the parents. Other PCR-based techniques have also been developed, like DNA amplification fingerprinting and arbitrarily primed PCR (11,12). However, these have yet to be used for germ line mutation detection.
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RAPD-PCR might detect single-base changes in genomic DNA (10
), although genomic rearrangements are probably detected the most (13). As RAPD-PCR only detects genomic alterations in or close to a priming site, the use of multiple primers may increase the sensitivity of this method. Moreover, the sensitivity of the test could be improved by using a more accurate method described by Valentini et al. (14) for the separation and detection of multiple amplified fragments. In toxicological exposure experiments to assess population genetic effects, this method successfully detected genomic alterations induced by benzo[a]pyrene (B[a]P), copper, mitomycin C, UV radiation and X-rays (15–18). Theoretically, all routes of exposure in which toxicants may affect the genetic structure can be analysed. Experimental studies usually require several hundred offspring for the powerful detection of germ line alterations using approximately 5–10 RAPD primers.
RAPD-PCR has several advantages: (i) it is relatively inexpensive and rapid, (ii) it yields information on a large number of loci, (iii) the same primers can be used for genomic analysis in all species, including humans, and (iv) it can be performed with relatively small sample sizes. However, there are also several drawbacks: (i) the results are considered to be qualitative and indicative and should be further corroborated by cloning, sequencing and probing techniques, (ii) the nature of the genomic change observed cannot be assessed from the sequence of the RAPD-PCR product, (iii) the technique may not screen the genome as randomly as expected and (iv) both nuclear and mitochondrial DNA may be amplified during PCR. Amplification of mitochondrial DNA may complicate the interpretation of RAPD fingerprints as it is not a Mendelian marker, but only inheritable from the mother (19). Reproducibility of RAPD profiles is another potential pitfall (20). However, this can be overcome by using appropriate PCR conditions and by cleaving DNA with restriction enzymes prior to the amplification (21).
So far, RAPD-PCR has mainly been used in ecotoxicological studies, for example, to detect genomic alterations in Daphnia magna exposed to B[a]P (13,22). Weinberg et al. (23) have used the method in humans and they detected a 7-fold increase in mutation frequencies in blood from children of families conceived after parental exposure to radiation from the Chernobyl accident compared to children conceived before parental exposure. They concluded that exposure to low-dose radiation induces genetic changes in the germ line. Although the finding of a 7-fold increase in mutation rate has been questioned by Jeffreys and Dubrova (24) and mutants have not been validated, the study nevertheless raises major concerns regarding radiation-induced heritable genetic effects.
In conclusion, RAPD-PCR seems to be a promising method for the analysis of germ line mutations in offspring. For human studies, however, PCR conditions and RAPD-PCR methods should be optimized to yield reproducible DNA fingerprints, and several verification methods should be used to validate and confirm results (13). Although the findings obtained by RAPD-PCR can only be regarded as indicative at present, the method can still be used as an effective screening tool in studying germ line mutagenesis.
DNA fingerprinting of tandem repetitive DNA sequences
Part of the genome consists of tandem repetitive sequences, which are known to be unstable and predominantly non-coding DNA. These sequences can be divided into microsatellites, minisatellites and ESTRs based on sequence size and number of repeats (Table I). Yauk (25) provides further information on the differences between minisatellites and ESTRs with respect to their structure and mutational mechanisms.
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Minisatellites and ESTRs can be sensitive tools for monitoring germ line mutations after mutagenic exposure since both exhibit high mutation rates. This facilitates the assessment of induced mutations in a relatively small number of samples following environmentally relevant exposure (26
). The DNA is digested with restriction enzymes and subsequently electrophoresed, before being hybridized with probes that are complementary to hypervariable loci. This gives a unique pattern of fragments (DNA fingerprint) (Figure 3). The use of multiple probes can increase the probability of detecting mutations, when comparing the patterns between parents and their offspring (each band should also be present in one of the parents). Therefore this is a statistically powerful technique that reduces the time needed and costs involved to quantify the number of germ line mutations.
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Recent experiments have shown that minisatellites and ESTRs are able to detect an increase in germ line mutations in humans and animals exposed to radioactive or chemical pollutants in their natural environment (2
–5). Multilocus DNA fingerprinting has also demonstrated significantly elevated mutation rates in the offspring of humans inhabiting the radioactive polluted areas of Belarus near Chernobyl, when compared to control families from the UK. Moreover, a significant radiation–dose effect relationship was observed (1,27).
These studies demonstrate that the analysis of tandem repetitive sequences could be a sensitive method for the detection of germ line mutations transmitted to the next generation and caused by long-term and/or low-dose environmental exposure. However, very little is currently known about germ line mutations induced at hypervariable tandem repeat sequences by specific chemicals. Two monofunctional alkylating agents, ethylnitrosourea (ENU) and isopropyl methanesulphonate, and the anti-cancer drug etoposide have already been investigated and led to elevated ESTR mutation rates in the germ line of male mice (28). Although these results demonstrate that tandem repeat sequences can be used to monitor germ line mutation induction, the effects of exposure to environmental or dietary mutagens other than radiation on germ line mutation induction at tandem repeat loci in rodents and humans clearly merits further evaluation.
Small-pool PCR
Small-pool PCR (SP-PCR), which is derived from DNA fingerprinting of minisatellites, can also be used as a germ cell mutagenicity test (29–31). However, in contrast to DNA fingerprinting, the tandem repeats are amplified by PCR using allele-specific PCR primers directed at the polymorphic sites in minisatellite regions. This allows the analysis of mutant alleles in individual gametes. Consequently, this method can be used to map minisatellite length polymorphisms directly in the germ cell DNA (29–31). SP-PCR uses amplification of small aliquots of sperm DNA to screen thousands of sperm cells from an individual in one PCR to detect changes in the repeat size of unstable minisatellite loci. Mutations detected by SP-PCR as products of altered length are counted and the rate of mutation expressed as a given number of events found in a given estimated number of sperm equivalents (32).
Quantitative SP-PCR methods were found to be appropriate for investigating the effects of chemotherapeutic mutagens and radiation on minisatellite mutation rates in human sperm (32–35). The advantage of this approach is that relatively small increases in mutation rate can be detected in individual samples, which makes it a sensitive method. However, this technique also has several limitations that are predominantly related to interpretation of the data. First, this technique may potentially underestimate the true number of expansion events since PCR is known to favour the amplification of the smaller allele. Second, the variants detected in this assay may be PCR artefacts rather than variants in repeat size. Third, SP-PCR is less precise in controlling for the number of cell equivalents analysed. Consequently, considerable care should be taken in accurately diluting and pipetting the DNA samples for SP-PCR in order to minimize variation, as the number of cells in each pool is the denominator in determining the rates of change. Furthermore, the efficiency of amplification of all the cell equivalents within a pool may be <100% (36).
In conclusion, SP-PCR seems less appropriate as a mutagenicity test than standard DNA fingerprinting of minisatellites and ESTRs. The disadvantages of this technique are mainly related to the use of PCR amplification, which causes misinterpretation of the results by PCR artefacts.
Single-molecule PCR
In addition to SP-PCR, single-molecule PCR (SM-PCR) has been developed as a method for detecting new mutations in both the germ line and somatic tissues (35). This method has been used to quantify the in vivo mutation frequency at the Ms6-hm locus in somatic and germ cells and to determine whether mutation induction by environmental exposure and ionizing radiation can be detected in pedigrees as well as directly in sperm DNA (35,37). This is an important addition to the pedigree approach for tandem repeat analysis because mutations in sperm are not necessarily transferred to the offspring and, vice versa, mutations in the offspring do not necessarily originate from the gametes, but can be induced during early embryogenesis. For this SM-PCR method, DNA samples are digested outside the ESTR array and distal to the PCR primer sites prior to amplification to render genomic DNA fully soluble prior to dilution. Digested DNA is serially diluted and amplified with flanking primers (38). PCR products are analysed by electrophoresis followed by hybridization with specific probes. The approximate number of amplifiable ESTR molecules in each dilution is estimated by Poisson analysis of the number of positive and negative reactions for ESTR-PCR products. Subsequently, a new set of PCRs is seeded with each containing a mean of approximately one amplifiable ESTR molecule. PCR products are subsequently separated on an agarose gel, followed by hybridization and scoring of the allele lengths. A band shift of at least 1 mm relative to the internal size marker indicates a mutation; smaller changes cannot be scored reliably (35).
SM-PCR has several advantages. First, experimental time might be reduced because mating and birth is unnecessary [it has been shown in laboratory mice that mutation rates in the offspring were indistinguishable from the rates in sperm DNA (35)]. Second, SM-PCR was found to be useful for studying mutation induction at low-dose exposures (35) and the majority of mutations were small changes in repeat copy number. These advantages allow a more thorough investigation of chemicals with different modes of action. Furthermore, using SM-PCR circumvents the requirement of sub-cloning. Yauk et al. (35) showed that SM-PCR can provide robust estimates of the frequency of ESTR mutations in somatic and sperm cells of irradiated and control male mice.
In addition to the limited number of in vivo results, in vitro studies have also shown that SM-PCR is a useful method for detecting mutations. Polyzos et al. (37,39) described the same method for the in vitro detection of de novo mutations in cultures of embryonic cells and noted that cells treated with ENU, B[a]P and etoposide showed a significant increase of the mutation frequency compared to untreated cells. They also indicated a similarity in mutation response between embryonic fibroblasts in culture and somatic and germ cells in vivo at the ESTR locus (39).
In conclusion, SM-PCR methods are appropriate for the detection of germ line mutations at the ESTR locus Ms6-hm. The reduction in time and costs make this method particularly suitable for screening large numbers of agents. A similar approach developed for minisatellites would be very useful for studying mutation frequencies in humans, but such methods are currently lacking.
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Do differences between rodent and human spermatogenesis affect germ line mutation induction? |
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TopIntroductionGerm line mutagenicity testsDo differences between rodent...Analysis of germ line...Future perspectivesFundingReferences |
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The advantage of the methods described above is that they can be used to detect germ line mutations in rodents and humans. This is essential because until recently extrapolation of animal data was necessary for assessing potential genetic risks in the human population. Nonetheless, extrapolation might still be very useful in certain situations. Human exposure to genotoxic chemicals usually involves chronic exposure to low doses, making it much more difficult to study mutagenicity during spermatogenesis. In theory, this could lead to a misinterpretation of the effects due to the differences in spermatogenesis between rodents and humans [reviewed by Ehmcke et al. (40
) and summarized in Figure 4]. There are differences in type of stem cell and in mitotic expansion. Finally, it must also be remembered that the vulnerability of germ cells towards genotoxins is known to alter during spermatogenesis due to cell turnover, differentiation and changes in DNA repair activity (41).
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Analysis of germ line mutations: where do they come from and where do they take us? |
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TopIntroductionGerm line mutagenicity testsDo differences between rodent...Analysis of germ line...Future perspectivesFundingReferences |
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Both RAPD-PCR and the analysis of repetitive sequences might increase our understanding of how best to extrapolate from rodents to humans. However, important concerns are (i) the mechanisms through which the mutations arise are unknown and (ii) their biological relevance for the health of offspring is not clear.
Mechanisms leading to mutations
Alterations in RAPD-PCR profiles may originate from genomic alterations, DNA lesions that block the polymerase and secondary structures within the PCR product (13). Therefore, studying the origin of RAPD-PCR detectable mutations from a mechanistic viewpoint will be difficult because multiple types of mutations can be involved. Gains or losses of tandem repeats are possibly easier to interpret, as the underlying mechanism might be the same. ESTR mutations have been observed after exposure to radiation but were also induced by chemical carcinogens that induce strand breaks or bulky DNA lesions (28,37,42). Although this non-specific detection of mutations induced by different types of DNA damage might be advantageous for the sensitivity of the assays, it complicates the mechanistic interpretation of the data. Therefore, more dedicated in vitro and in vivo experiments are needed to provide a definitive characterization of the mechanistic basis for this type of mutation analyses. Moreover, these mutations may also be the consequence of genomic destabilization during early embryogenesis, which is supported by the observation that an increased number of mutations can arise in the maternal allele of the offspring of irradiated males and unexposed mothers (43). Therefore, mutations in both sperm and offspring should be observed so that the two processes can be differentiated. There is evidence that indirect mechanisms are involved in the induction of tandem repeat mutations; for instance recent findings indicate a potential role for epigenetic alterations (44). Since the epigenome can be transferred to subsequent generations, this mechanism might also be relevant for exposed populations.
Transcriptomics could contribute to this mechanistic understanding of germ line mutagenesis by comparing gene expression in testes of exposed fathers. Despite the obvious difficulties in obtaining mRNA from human testes, gene expression can be studied in mRNA obtained from ejaculates. The presence of mRNA in an ejaculate was discovered by Pessot et al. (45) and is thought to reflect gene expression during spermatogenesis (46). Therefore, pathways involved in the induction of tandem repeat mutations may also be identifiable in humans. Studies in humans seem to be necessary to avoid problems with interspecies extrapolation. However, variability in the susceptibility towards genotoxic compounds may complicate the interpretation of the data and so controlled studies with laboratory animals are still required. Moreover, the use of genetically modified animals may allow further elucidation of mechanisms in germ line mutagenesis.
Biological significance of mutations detected
It is almost impossible to predict the biological impact of germ line mutations for offspring health. In principle, the RAPD-PCR assay can scan the entire genome including coding DNA regions, which can be identified by cloning and sequencing the altered PCR products (new PCR products or PCR products that disappeared due to exposure). However, most of the mutations will probably be found in non-coding DNA. No studies to date have linked an increased mutational load, as assessed by RAPD-PCR, to an actual phenotypic alteration. Furthermore, it is still unclear whether tandem repeat mutations reflect effects in coding regions or provide a measure of other heritable genomic outcomes. Although the tandem repeat loci do not confer any observed phenotypes, many tandem repeats are known to be related to transcribed genes and instability within these repetitive sequences has been shown to influence the function of these genes (47–50). For example, expansions within tandem repeat sequences are associated with 
20 well-characterized developmental and degenerative diseases (47). Nevertheless, mutations assessed by RAPD-PCR or tandem repeat analysis can be considered as biomarkers of adverse genetic events.
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Future perspectives |
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TopIntroductionGerm line mutagenicity testsDo differences between rodent...Analysis of germ line...Future perspectivesFundingReferences |
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The new approaches described are needed to test germ line mutagenicity in humans exposed to environmental and dietary mutagens (51
). Efforts need to be made to use new molecular techniques to overcome the limitations of classical tests with respect to time, costs and the number of animals needed for germ line mutagenicity testing. Further these tests should fit in a broad range of applications; genomic alterations should be measurable in humans, animals and their offspring, in multiple organs and at different stages during spermatogenesis. Table II provides an overview of the advantages and disadvantages of RAPD-PCR and DNA fingerprinting of tandem repetitive DNA sequences compared to classical germ cell assays. The two new methods fulfil most of the demands, including their applicability in humans, but still require further technical validation. Too few experiments have been performed to confirm the suitability of these methods for detecting germ line mutations. In addition, not enough is known about how tandem repeat mutations are formed and the type of mutations analysed by RAPD-PCR. Therefore, the biological consequences of these mutations for the offspring are unknown. However, the improved sensitivity, which results in the use of small sample sizes and consequently lower costs, means that these methods are promising for the testing of low doses of mutagens and long-term exposure to mutagens on germ line mutations and heritable effects.
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DNA fingerprinting for repetitive sequences is the most promising new method for mutagenicity testing, while RAPD-PCR can be considered as a semi-quantitative screening method. Furthermore, DNA fingerprinting is a sensitive method for detecting germ line mutations in sperm and for establishing whether these mutations can be transmitted to the progeny after long-term, low-dose exposure to pollutants in experimental animals, free-living animals and humans.
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Funding |
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TopIntroductionGerm line mutagenicity testsDo differences between rodent...Analysis of germ line...Future perspectivesFundingReferences |
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Cefic Long-Range Research Initiative Innovative Science award 2004 to R.W.L.G.; EU Integrated Project NEWGENERIS, 6th Framework Programme, Priority 5: Food Quality and Safety (FOOD-CT-2005 016320).
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Acknowledgments |
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Conflict of interest statement: None declared.
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Notes |
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* To whom correspondence should be addressed. Department of Health Risk Analysis and Toxicology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. Tel: +31 43 3881104; Fax: +31 43 3884146; Email: r.godschalk{at}grat.unimaas.nl
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References |
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1. Dubrova YE, Nesterov VN, Krouchinsky NG, Ostapenko VA, Neumann R, Neil DL, Jeffreys AJ. Human minisatellite mutation rate after the Chernobyl accident. Nature (1996) 380:683–686.[CrossRef][Medline]
2. Yauk CL, Fox GA, McCarry BE, Quinn JS. Induced minisatellite germline mutations in herring gulls (Larus argentatus) living near steel mills. Mutat. Res. (2000) 452:211–218.[Web of Science][Medline]
3. Somers CM, Yauk CL, White PA, Parfett CL, Quinn JS. Air pollution induces heritable DNA mutations. Proc. Natl Acad. Sci. USA (2002) 99:15904–15907.
4. Yauk CL, Quinn JS. Multilocus DNA fingerprinting reveals high rate of heritable genetic mutation in herring gulls nesting in an industrialized urban site. Proc. Natl Acad. Sci. USA (1996) 93:12137–12141.
5. Somers CM, McCarry BE, Malek F, Quinn JS. Reduction of particulate air pollution lowers the risk of heritable mutations in mice. Science (2004) 304:1008–1010.
6. Trasler JM, Doerksen T. Teratogen update: paternal exposures-reproductive risks. Teratology (1999) 60:161–172.[CrossRef][Web of Science][Medline]
7. Sobels FH. Models and assumptions underlying genetic risk assessment. Mutat. Res. (1989) 212:77–89.[Web of Science][Medline]
8. Adler ID. Future research directions to study genetic damage in germ cells and estimate genetic risk. Environ. Health Perspect. (1996) 104(Suppl. 3):619–624.[CrossRef][Web of Science][Medline]
9. Singer TM, Lambert IB, Williams A, Douglas GR, Yauk CL. Detection of induced male germline mutation: correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays. Mutat. Res. (2006) 598:164–193.[Web of Science][Medline]
10. Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. (1990) 18:6531–6535.
11. Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. (1990) 18:7213–7218.
12. Caetano-Anolles G. Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl. (1993) 3:85–94.[Web of Science][Medline]
13. Atienzar FA, Jha AN. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. Mutat. Res. (2006) 613:76–102.[CrossRef][Web of Science][Medline]
14. Valentini A, Timperio AM, Cappuccio I, Zolla L. Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA. Electrophoresis (1996) 17:1553–1554.[CrossRef][Web of Science][Medline]
15. Atienzar FA, Cheung VV, Jha AN, Depledge MH. Fitness parameters and DNA effects are sensitive indicators of copper-induced toxicity in Daphnia magna. Toxicol. Sci. (2001) 59:241–250.
16. Becerril C, Ferrero M, Sanz F, Castano A. Detection of mitomycin C-induced genetic damage in fish cells by use of RAPD. Mutagenesis (1999) 14:449–456.
17. Atienzar FA, Venier P, Jha AN, Depledge MH. Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations. Mutat. Res. (2002) 521:151–163.[Web of Science][Medline]
18. Jones C, Kortenkamp A. RAPD library fingerprinting of bacterial and human DNA: applications in mutation detection. Teratog. Carcinog. Mutagen. (2000) 20:49–63.[CrossRef][Web of Science][Medline]
19. Russell PJ. iGenetics (2002) Pearson Education, Inc: San Francisco, CA.
20. Benter T, Papadopoulos S, Pape M, Manns M, Poliwoda H. Optimization and reproducibility of random amplified polymorphic DNA in human. Anal. Biochem. (1995) 230:92–100.[CrossRef][Web of Science][Medline]
21. Savva D. Use of DNA fingerprinting to detect genotoxic effects. Ecotoxicol. Environ. Saf. (1998) 41:103–106.[CrossRef][Web of Science][Medline]
22. Atienzar FA, Jha AN. The random amplified polymorphic DNA (RAPD) assay to determine DNA alterations, repair and transgenerational effects in B(a)P exposed Daphnia magna. Mutat. Res. (2004) 552:125–140.[Web of Science][Medline]
23. Weinberg HS, Korol AB, Kirzhner VM, et al. Very high mutation rate in offspring of Chernobyl accident liquidators. Proc. Biol. Sci. (2001) 268:1001–1005.
24. Jeffreys AJ, Dubrova YE. Monitoring spontaneous and induced human mutation by RAPD-PCR: a response to Weinberg et al. (2001). Proc. Biol. Sci. (2001) 268:2493–2494.
25. Yauk CL. Advances in the application of germline tandem repeat instability for in situ monitoring. Mutat. Res. (2004) 566:169–182.[CrossRef][Web of Science][Medline]
26. Jeffreys AJ, Royle NJ, Wilson V, Wong Z. Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature (1988) 332:278–281.[CrossRef][Medline]
27. Dubrova YE, Nesterov VN, Krouchinsky NG, Ostapenko VA, Vergnaud G, Giraudeau F, Buard J, Jeffreys AJ. Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident. Mutat. Res. (1997) 381:267–278.[Web of Science][Medline]
28. Vilarino-Guell C, Smith AG, Dubrova YE. Germline mutation induction at mouse repeat DNA loci by chemical mutagens. Mutat. Res. (2003) 526:63–73.[Web of Science][Medline]
29. Buard J, Collick A, Brown J, Jeffreys AJ. Somatic versus germline mutation processes at minisatellite CEB1 (D2S90) in humans and transgenic mice. Genomics (2000) 65:95–103.[CrossRef][Web of Science][Medline]
30. May CA, Jeffreys AJ, Armour JA. Mutation rate heterogeneity and the generation of allele diversity at the human minisatellite MS205 (D16S309). Hum. Mol. Genet. (1996) 5:1823–1833.
31. Jeffreys AJ, Tamaki K, MacLeod A, Monckton DG, Neil DL, Armour JA. Complex gene conversion events in germline mutation at human minisatellites. Nat. Genet. (1994) 6:136–145.[CrossRef][Web of Science][Medline]
32. Armour JA, Brinkworth MH, Kamischke A. Direct analysis by small-pool PCR of MS205 minisatellite mutation rates in sperm after mutagenic therapies. Mutat. Res. (1999) 445:73–80.[Web of Science][Medline]
33. May CA, Tamaki K, Neumann R, Wilson G, Zagars G, Pollack A, Dubrova YE, Jeffreys AJ, Meistrich ML. Minisatellite mutation frequency in human sperm following radiotherapy. Mutat. Res. (2000) 453:67–75.[Web of Science][Medline]
34. Zheng N, Monckton DG, Wilson G, Hagemeister F, Chakraborty R, Connor TH, Siciliano MJ, Meistrich ML. Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy. Environ. Mol. Mutagen. (2000) 36:134–145.[CrossRef][Web of Science][Medline]
35. Yauk CL, Dubrova YE, Grant GR, Jeffreys AJ. A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus. Mutat. Res. (2002) 500:147–156.[Web of Science][Medline]
36. Crawford DC, Wilson B, Sherman SL. Factors involved in the initial mutation of the fragile X CGG repeat as determined by sperm small pool PCR. Hum. Mol. Genet. (2000) 9:2909–2918.
37. Polyzos A, Parfett C, Healy C, Douglas GR, Yauk CL. Instability of expanded simple tandem repeats is induced in cell culture by a variety of agents: N-Nitroso-N-ethylurea, benzo(a)pyrene, etoposide and okadaic acid. Mutat. Res. (2006) 598:73–84.[Web of Science][Medline]
38. Jeffreys AJ, Neumann R, Wilson V. Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis. Cell (1990) 60:473–485.[CrossRef][Web of Science][Medline]
39. Polyzos A, Parfett C, Healy C, Douglas G, Yauk C. A single-molecule PCR approach to the measurement of induced expanded simple tandem repeat instability in vitro. Mutat. Res. (2006) 594:93–100.[Web of Science][Medline]
40. Ehmcke J, Wistuba J, Schlatt S. Spermatogonial stem cells: questions, models and perspectives. Hum. Reprod. Update (2006) 12:275–282.
41. Adler ID. Comparison of the duration of spermatogenesis between male rodents and humans. Mutat. Res. (1996) 352:169–172.[CrossRef][Web of Science][Medline]
42. Dubrova YE. Radiation-induced mutation at tandem repeat DNA loci in the mouse germline: spectra and doubling doses. Radiat. Res. (2005) 163:200–207.[CrossRef][Web of Science][Medline]
43. Niwa O, Kominami R. Untargeted mutation of the maternally derived mouse hypervariable minisatellite allele in F1 mice born to irradiated spermatozoa. Proc. Natl Acad. Sci. USA (2001) 98:1705–1710.
44. Yauk C, Polyzos A, Rowan-Carroll A, et al. Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location. Proc. Natl Acad. Sci. USA (2008) 105:605–610.
45. Pessot CA, Brito M, Figueroa J, Concha II, Yanez A, Burzio LO. Presence of RNA in the sperm nucleus. Biochem. Biophys. Res. Commun. (1989) 158:272–278.[CrossRef][Web of Science][Medline]
46. Ostermeier GC, Goodrich RJ, Diamond MP, Dix DJ, Krawetz SA. Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility. Fertil. Steril. (2005) 83:1687–1694.[CrossRef][Web of Science][Medline]
47. Gatchel JR, Zoghbi HY. Diseases of unstable repeat expansion: mechanisms and common principles. Nat. Rev. Genet. (2005) 6:743–755.[Web of Science][Medline]
48. Dubrova YE, Jeffreys AJ, Malashenko AM. Mouse minisatellite mutations induced by ionizing radiation. Nat. Genet. (1993) 5:92–94.[CrossRef][Web of Science][Medline]
49. Dubrova YE, Plumb M, Brown J, Fennelly J, Bois P, Goodhead D, Jeffreys AJ. Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation. Proc. Natl Acad. Sci. USA (1998) 95:6251–6255.
50. Kelly R, Gibbs M, Collick A, Jeffreys AJ. Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events. Proc. Biol. Sci. (1991) 245:235–245.
51. Wyrobek AJ, Mulvihill JJ, Wassom JS, et al. Assessing human germ-cell mutagenesis in the Postgenome Era: a celebration of the legacy of William Lawson (Bill) Russell. Environ. Mol. Mutagen. (2007) 48:71–95.[CrossRef][Web of Science][Medline]
Received on May 22, 2007; revised on March 28, 2008; accepted on April 7, 2008.
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