Nancy J. Gorelick

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage‐based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of ∼3 × 10−5 mutants/locus and 125,000–300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two‐thirds the MTD, and one‐third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose‐response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control. Environ. Mol. Mutagen. 35:253–259, 2000

Databases and software for the analysis of mutations in the human p53 gene, human hprt gene and both the lacI and lacZ gene in transgenic rodents

We have created databases and software applications for the analysis of DNA mutations at the humanp53gene, the humanhprtgene and both the rodent transgeniclacIandlacZlocus. The databases themselves are stand-alone dBASE files and the software for analysis of the databases runs on IBM-compatible computers. Each database has a separate software analysis program. The software created for these databases permit the filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web (WWW). Open the following home page with a Web Browser: http://sunsite.unc.edu/dnam/mainpage.ht ml . Alternatively, the databases and programs are available via public FTP from: anonymous@sunsite.unc.edu . There is no password required to enter the system. The databases and software are found beneath the subdirectory: pub/academic/biology/dna-mutations. Two other programs are available at the site-a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

Detection of cyclophosphamide-induced mutations at the Hprt but not the lacI locus in splenic lymphocytes of exposed mice.

The relative sensitivities and specificities of the endogenous Hprt gene and the lacI transgene as mutational targets were evaluated in splenic lymphocytes from male standard B6C3F1 mice (only Hprt assayed) and from lacI transgenic B6C3F1 mice treated at 6–7 weeks‐ of‐age with the indirect‐acting agent, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were given single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then necropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mfs were found at 6 weeks postexposure, with mean Mf values ranging from 2.27 to 3.27 × 10–5 using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 × 10–5 in various experiments). To determine the dose response for Hprt Mfs, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. These treatments produced a supralinear dose response curve for CP‐induced Hprt Mfs. Based on these experiments, CP mutagenicities at Hprt and lacI were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using another lot of CP in ISOPAC® bottles; Sigma) and necropsied 6 weeks later. There was a significant increase in Hprt Mfs in treated transgenic mice (100 mg CP/kg: 0.75 ± 0.09 × 10–5; 25 mg CP/kg: 0.39 ± 0.05 × 10–5) versus controls (0.10 ± 0.01 × 10–5); however, the Mfs in lacI of lymphocytes from the same CP‐treated animals were not significantly different from controls (100 mg CP/kg: 9.4 ± 1.1 × 10–5; 25 mg CP/kg: 6.7 ± 0.8 × 10–5; control: 7.7 ± 0.7 × 10–5). Hprt mutational spectra data in CP‐treated transgenic and nontransgenic mice were different from those of control mice, whereas the spectra of mutations in lacI of lymphocytes from Big Blue® transgenic mice were not significantly changed after CP treatment. These data indicate that, under these treatment conditions, CP‐induced mutations in splenic lymphocytes were detectable in the Hprt gene but not the lacI transgene of this nontarget tissue for CP‐induced cancer. Environ. Mol. Mutagen. 34:167–181, 1999

Mutation assays in male germ cells from transgenic mice: overview of study and conclusions

Three confirmed mouse germ cell mutagens, ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS), have been evaluated for their activity as mutagens to the germ cell DNA of two strains of transgenic mice (lac I, Big Blue and LacZ, Muta Mouse). Both testicular DNA and epididymal sperm DNA were evaluated. A range of sampling times was studied, from 3 days post-dosing to 100 days post-dosing. ENU and iPMS were mutagenic to both testicular DNA and epididymal sperm DNA. Mutant frequencies were higher for both chemicals in DNA recovered from testicular tissue than in epididymal sperm DNA. Likewise, mutant frequencies were higher for both DNA samples at the later sampling times. MMS was not mutagenic under any condition of test. A good level of qualitative agreement in test results was seen for the two assays and for the same assays conducted in different laboratories. The level of quantitative agreement was not as high, but was, nonetheless, generally good. Recommendations for the future conduct of transgenic rodent germ cell mutation assays are made. The test data are discussed within the context of the larger question of how such assays should be integrated into the chemical hazard assessment process.

Mutational specificity: Mutational spectra in transgenic animal research: Data analysis and study design based upon the mutant or mutation frequency

Understanding chemically induced changes in mutational spectra can aid in deciphering mechanisms of mutagenesis. In this paper, we propose the use of statistical methods that are based upon the mutation frequency, rather than simple mutant counts which have no relationship to the mutation frequency. These methods have a number of advantages over the current standard analysis: an improved means of identifying those classes/sites of mutation which have treatment‐related induction, greater sensitivity to localized differences in spectra (e.g., limited to a single base pair), one‐sided tests for induction of mutations, tests of dose‐response, and a framework for sample‐size estimation in terms of the number of mutants to sequence. As examples, the methods are applied to data from transgenic mutation assays.

Sources of variability in data from a positive selection lacZ transgenic mouse mutation assay: An interlaboratory study

Experimental features of a positive selection transgenic mouse mutation assay based on a lambda lacZ transgene are considered in detail, with emphasis on results using germ cells as the target tissue. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined, with the goal of identifying sources of excess variation in the observed mutant frequencies. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal variability. Data from five laboratories are evaluated in detail. Results suggest only scattered patterns of excess variability below the animal-to-animal level, but, generally, significant excess variability at the animal-to-animal level. Using source of variability analyses to guide the choice of statistical methods, control-vs-treatment comparisons are performed for assessing the male germ cell mutagenicity of ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), and methyl methanesulfonate (MMS). Results on male germ cell mutagenesis of ethyl methanesulfonate (EMS) and methylnitrosourea (MNU) are also reported.

Prospects for safety testing: A strategy for the application of transgenic rodent mutagenesis assays

The past several years have seen an enormous increase in the development and use of transgenic animal models to measure mutations in specific inserted reporter genes. These systems provide gene mutation data in vivo in a wide range of relevant tissues. Numerous laboratories are now using these systems with consistent results. This paper describes the unique niche that transgenic mutagenesis systems can fill in product development and registration strategies. In addition to tissue‐specific mechanistic studies, transgenic assays are available to follow up mutagenic effects demonstrated in Salmonella, Escherichia coli, mouse lymphoma (L5178Y) cells, or other in vitro systems.

Tissue-specific mutant frequencies and mutational spectra in cyclophosphamide-treated lacI transgenic mice

The induction and nature of mutations in the lacI transgene were evaluated in multiple tissues after exposure of adult male B6C3F1 lacI transgenic mice to cyclophosphamide (CP). Mice were given a single i.p. injection of 25 mg CP/kg, 100 mg CP/kg, or vehicle (PBS) and then necropsied 6 weeks after treatment to allow DNA extraction and lacI mutant recovery. Tissues evaluated included target tissues for tumorigenesis (lung, urinary bladder) and sites not susceptible to tumor formation in B6C3F1 mice (kidney, bone marrow, splenic T‐lymphocytes). After exposure to the high dose of CP, a significant increase in the mutant frequency (Mf) was detected in the lungs and urinary bladders, compared to the respective tissues from vehicle‐treated controls. In contrast, the Mfs in kidney, bone marrow, and splenic T cells from CP‐treated mice were not significantly different from controls. The spectra of mutations in lacI from lung and urinary bladder were significantly changed after high‐dose CP treatment, with a significant increase in the frequency of A · T → T · A transversions found in both tissues and a significantly elevated frequency of deletions in the lungs. Conversely, in vehicle‐treated mice, the two predominant classes of lacI mutations recovered in lung and urinary bladder were G · C → A · T transitions at CpG sites and G · C → T · A transversions. These CP exposures were also genotoxic as measured by the significant induction of micronuclei in peripheral blood 48 hr after exposure. These data indicate that under these study conditions, CP‐induced mutations are detectable in the lacI transgene in the target tissues, but not in nontarget tissues for CP‐induced cancer. With the lacI assay it is possible to study mutagenicity in a variety of critical tissues to provide mechanistic information related to genotoxicity and carcinogenicity in vivo. Environ. Mol. Mutagen. 34:154–166, 1999

The genetic analysis of lacI mutations in sectored plaques from Big Blue® transgenic mice

The Big Blue® lacl transgenic rodent assay, which uses the λLIZ/lacl gene as the target for mutation, provides a convenient short‐term assay for the study of mutation in viva [Kohler et al. [1991]: Proc Natl Acad Sci USA 88:7958–7962; Provost et al. (1993): Mutat Res 288:133–149]. However, the interpretation of data from transgenic animal assays is sometimes complicated by mutants that appear as sectored mutant lambda plaques. These mutants can form a significant fraction of the mutant plaques [Hayward et al. (1995): Carcinogenesis 16:2429–2433]. Thus, in order to accurately determine in vivo mutant frequencies and mutational specificities, it is necessary to score sectored plaques and partition them from the rest of the data. In this study, the specificity of mutation in sectored plaques recovered from untreated and UVB‐treated Big Blue® mouse skin was analyzed and compared to mutations recovered from λLIZ/lacl grown on the Escherichia coli host. The mutational spectra of sectored plaques from untreated and UVB‐treated mice were remarkably similar to each other and resembled those recovered from the λLIZ/lacl phage plated directly on E. coli. Both the sectored mutants and those recovered in λLIZ/lacl phage differed from the spectra of spontaneous mutants in E. coli and in Big Blue® mouse skin. While sectored mutants from UVB‐treated mouse skin and λLIZ/lacl mutants were also different from spontaneous mutants recovered from Big Blue® liver, there was little difference between sectored mutants from untreated mouse skin and spontaneous liver mutants (P = 0.07). The mutational spectra of sectored plaques is thus largely consistent with their origin as spontaneous mutations arising in vitro during growth of the λLIZ/lacl shuttle vector DNA on the E. coli host, although the potential contribution from lesions in mouse DNA being expressed ex vivo in the E. coli host cannot be excluded.

Genotoxicity of trans-anethole in vitro

Trans-anethole genotoxicity has been evaluated previously both in vitro and in vivo. To ascertain the reproducibility and relevance of previously conducted gene mutation studies, the Salmonella/microsome test and the L5178Y mouse lymphoma TK+/- assay were repeated according to the protocols that previously produced positive results. For the mouse lymphoma TK+/- assay, standard conditions were employed. For the Salmonella/microsome tests, however, metabolic cofactors were supplemented relative to standard protocols. In addition, trans-anethole was evaluated for its ability to induce chromosome aberrations in vitro in Chinese hamster ovary cells. The results presented here indicate that trans-anethole does not increase the mutant frequency in the Salmonella/microsome test, whereas a dose-related response was confirmed in the L5178Y mouse lymphoma TK+/- assay with metabolic activation. The metabolic conditions used in each of the published gene mutation assays may explain the various responses to trans-anethole. Trans-anethole did not induce chromosome aberrations in Chinese hamster ovary cells. The molecular nature of the genetic change induced in mouse lymphoma cells by trans-anethole has not been identified but the available genotoxicity data are consistent with either a recombination event or a non-DNA reactive mechanism. Considering the trans-anethole genotoxicity data base as a whole, including the positive response observed only in the L5178Y mouse lymphoma TK+/- assay, the irreproducible response in the Salmonella/microsome test, the negative result in the chromosome aberration test in vitro and the results from 32P-postlabeling studies in vivo, as well as the occurrence of liver tumors in the rat bioassay only at doses which exceeded the MTD and caused significant liver toxicity, repeated toxic insult followed by compensatory cell proliferation is favored as an underlying mechanism for the observed rat tumorigenic response.