Evidence for magmatic evolution and diversity on Mars from infrared observations.

Compositional mapping of Mars at the 100-metre scale with the Mars Odyssey Thermal Emission Imaging System (THEMIS) has revealed a wide diversity of igneous materials. Volcanic evolution produced compositions from low-silica basalts to high-silica dacite in the Syrtis Major caldera. The existence of dacite demonstrates that highly evolved lavas have been produced, at least locally, by magma evolution through fractional crystallization. Olivine basalts are observed on crater floors and in layers exposed in canyon walls up to 4.5 km beneath the surface. This vertical distribution suggests that olivine-rich lavas were emplaced at various times throughout the formation of the upper crust, with their growing inventory suggesting that such ultramafic (picritic) basalts may be relatively common. Quartz-bearing granitoid rocks have also been discovered, demonstrating that extreme differentiation has occurred. These observations show that the martian crust, while dominated by basalt, contains a diversity of igneous materials whose range in composition from picritic basalts to granitoids rivals that found on the Earth.

Use of an anti-viral drug, Ribavirin, as an anti-glioblastoma therapeutic.

The median survival for glioblastoma patients is ~15 months despite aggressive surgery and radio-chemotherapy approaches. Thus, developing new therapeutics is necessary to improve the treatment of these invasive brain tumors, which are known to show high levels of the eukaryotic initiation factor, eIF4E, a potent oncogene. Ribavirin, the only clinically approved drug known to target eIF4E, is an anti-viral molecule currently used in hepatitis C treatment. Here, we report the effect of ribavirin on proliferation, cell cycle, cell death and migration of several human and murine glioma cell lines, as well as human glioblastoma stem-like cells, in vitro. In addition, we tested ribavirin efficacy in vivo, alone and in combination with temozolomide and radiation. Our work showed that ribavirin inhibits glioma cell growth and migration, and increases cell cycle arrest and cell death, potentially through modulation of the eIF4E, EZH2 and ERK pathways. We also demonstrate that ribavirin treatment in combination with temozolomide or irradiation increases cell death in glioma cells. Finally and most importantly, ribavirin treatment in vivo significantly enhances chemo-radiotherapy efficacy and improves survival of rats and mice orthotopically implanted with gliosarcoma tumors or glioma stem-like cells, respectively. On the basis of these results, we propose that ribavirin represents a new therapeutic option for glioblastoma patients as an enhancer of the cytotoxic effects of temozolomide and radiotherapy.

Frequency and spectrum of ethylnitrosourea-induced mutation at the hprt and lacI loci in splenic lymphocytes of exposed lacI transgenic mice.

The development of mouse models with the endogenous hypoxanthine-guanine phosphoribosyl transferase (hprt) gene and lacI transgene as mutational targets provides an excellent opportunity to compare the mutant frequency (Mf) and types of mutations induced in vivo in different sequence contexts. To this end, a study was conducted to determine the Mfs and spectrum of mutations induced at these loci in splenic T cells from male B6C3F1 Big Blue mice (6 weeks old) exposed to N-ethyl-N-nitrosourea (ENU). Six weeks after i.p. injection of 40 mg ENU/kg, T cells were isolated from control (n = 7) and treated (n = 8) mice for the culture of hprt mutants and for the extraction of DNA and recovery of lacI mutants. Mutations in hprt exon 3 and in lacI were quantified and analyzed using published procedures (S. W. Kohler et al., Proc. Natl. Acad. Sci. USA, 88: 7958-7962, 1991; T. R. Skopek et al., Proc. Natl. Acad. Sci. USA, 89: 7866-7870, 1992). In treated mice, the Mfs (average +/- SE) in hprt (6.0 +/- 0.2 x 10(-5)) and lacI (11.4 +/- 1.8 x 10(-5)) were approximately 16.2-fold (P = 0.006) and 3.4-fold (P = 0.009), respectively, above controls. However, the average induced Mfs (i.e., induced Mf = treatment Mf - background Mf) in hprt and lacI were similar, with the respective increases in Mf being 5.6 +/- 0.2 x 10(-5) and 8.0 +/- 2.3 x 10(-5) over background. Eleven of the 107 hprt mutants from treated Big Blue mice had mutations in exon 3, with 73% being substitutions at AxT bp. These data are similar to those observed in ENU-exposed nontransgenic B6C3F1 mice, in which 62 of 69 exon 3 mutations were substitutions at AxT bp (T. R. Skopek et al., Proc. Natl. Acad. Sci. USA, 89: 7866-7870, 1992). For comparison, the sequences of the lacI genes in two to five mutants from each mouse were determined, and a total of 75 mutations (70 different mutations) was detected. In exposed mice, 55% (24 of 44) of the mutations in lacI were substitutions at AxT bp. In controls, substitutions at AT bp comprised only 20% of the recovered mutations in either hprt exon 3 (1 of 5) or lacI (5 of 26). These data indicate that the lacI mutation assay is less sensitive than the hprt assay for detecting increases in Mf induced by ENU exposure of mice as indicated by the lower relative increase in Mf in the lacI gene, but, given a 6-week expression time, the types of mutations induced by ENU in the transgene reflect those observed in the native transcribed gene.

Detection of multiple polycyclic aromatic hydrocarbon-DNA adducts by a high-performance liquid chromatography-32P-postlabeling method.

A 32P-postlabeling procedure for identifying and quantifying hydrophobic DNA adducts was developed (by modifying the method of Randerath and co-workers) in which labeled adducts are separated by high-performance liquid chromatography (HPLC) and quantified by liquid scintillation counting. This method was first developed for fluoranthene-DNA adducts, and methods for optimal detection and quantification of DNA adducts with diol epoxide metabolites of benzo[a]pyrene (BPDE), chrysene (CHDE), and benz[a]anthracene (BADE) have now been established. Analytical conditions slightly different from those adopted for fluoranthene-DNA adducts are required for accurate quantification of BPDE-, CHDE-, and BADE-DNA adducts. In particular, HPLC analysis requires generation of nucleotide 5'-[32P]monophosphate adducts by treatment with nuclease P1, and polycyclic aromatic hydrocarbon adducts demonstrate variable sensitivity to nuclease P1, mediated dephosphorylation. Thus, multiple adducts can be detected in one sample as long as the recovery of adducts under the applied conditions has been determined and chromatographic separation of labeled adducts is achieved. A battery of postlabeling assays can thus make it possible to detect optimally multiple adducts in one DNA sample. Results from these studies indicate that the HPLC- 32P-postlabeling assay is complementary to immunoassays in which related polycyclic aromatic hydrocarbon diol epoxide adducts cross-react for the quantification of adducts.

Chemical and biochemical dosimetry of exposure to genotoxic chemicals.

Epidemiologic studies designed to evaluate the health significance of environmental chemicals are compromised by the lack of quantitative exposure data for individuals in exposed populations. Monitoring data on levels of compounds in environmental media often represent the only information available, and average population exposure is therefore the only quantitative parameter that can be calculated. Biological monitoring, i.e., measurements on cells, tissues or body fluids of exposed persons, has the objective of defining the so-called "internal dose" or "effective dose" on an individual basis. Such measurements can be used to ensure that current or past exposure does not entail unacceptable health risks, or can detect potentially excessive exposure before the appearance of adverse health effects. Results obtained through this approach can be interpreted on an individual basis and also used to estimate for that individual the amount of chemical absorbed during a specific time interval or the amount bound to critical sites. They may also be useful for characterization of community exposure by analyzing results obtained in groups of individuals within the general population. In this respect, biological monitoring data complement environmental measurements but have certain advantages in estimating health risks. Most importantly, the data obtained are more directly related to adverse effects and thus provide a better estimate of risk than ambient monitoring. Biological monitoring also takes into account absorption by all routes, integrates exposure from all sources, and therefore can be used as a basis for estimate of total risk from multiple chemicals.

Overview of mutation assays in transgenic mice for routine testing.

There is scientific and regulatory interest in using mutation assays in transgenic mice in safety assessments for new chemicals and drugs. Currently these assays are in the process of being validated, and protocols for routine testing are being defined. Some of the issues and results to date with regard to assay validation include reproducibility of the assay results (they are qualitatively reproducible), relevance of the test system (the transgene closely approximates an endogenous mammalian gene as a mutational target for the limited number of compounds tested), and the predictivity of the assay for heritable effects (unknown at this time) or carcinogenicity (the assays show good positive predictivity for carcinogenicity; the negative predictivity of the assay requires further investigation). Definition of appropriate study protocols for routine testing requires that applicable statistical methods are available and that the experimental parameters that affect the detection of mutations are known. Progress made in identifying these parameters is discussed. A proposal is made for the custom design of routine safety studies, which is based on the anticipated use of each individual test agent. A working group has been formed to conduct some of the studies still required for validation of these assays.

Statistical tests of significance in transgenic mutation assays: considerations on the experimental unit.

When significant animal-to-animal variability is present in binary response data, the usual statistical tests applied to such data do not always operate correctly. In transgenic mouse mutation data, some evidence of significant animal-to-animal variability already exists, suggesting that conventional statistical methods may not be appropriate. Here, we describe an alternative statistical method that treats the animal as the experimental (or statistically independent) unit, and contrast results of its application with those from methods that take the transgene as the experimental unit. Using data from two publications that report experimental results for individual animals, the transgene-based and animal-based analyses can yield very different interpretations of the experimental data. The performance of animal-based statistical methods should be improved by conducting future experiments with enough animals to adequately address animal-to-animal variability.

Statistical design and analysis of mutation studies in transgenic mice.

We have been working on identifying sources of variability in data from transgenic mouse mutation assays in order to develop appropriate statistical methods and designs for routine studies. Data from our lab and elsewhere point to the presence of significant animal-to-animal variability, which must be taken into account in statistical hypothesis tests. Here, the usual Cochran-Armitage (CA) test for trend in mutant frequencies, which takes the transgene as the experimental unit, and a generalized Cochran-Armitage test (GCA), which takes the animal as the experimental unit, are contrasted in computer simulations that help to quantify the differences between these statistical tests. The simulations report the statistical power of each test to detect treatment group differences, and their type I error rates. We find in general that the GCA test performs poorly compared to the CA test when it is appropriate to take the transgene as the experimental unit, and the study also uses a small number of animals. However, the CA test performs poorly in small group-size studies when the animal is the appropriate experimental unit. Extensions of the computer simulations allow for identification of cost-effective experimental designs. The results emphasize that the benefits of using additional animals in these mutation studies can be realized without substantial increases in costs. Here we illustrate the methods for liver studies in our lab. These methods can be used to derive optimal experimental designs for any combination of spontaneous mutant frequency and animal-to-animal variability.

Database and software for the analysis of mutations at the lacI gene in both transgenic rodents and bacteria.

The use of transgenic rodents for the study of genetic toxicology has increased dramatically in the past several years. A great deal of the recent work has employed the lacI locus in transgenic mice. In addition to the transgenic data, a substantial amount of information exists regarding mutation of the lacI gene in bacteria. In an effort to centralize the information regarding mutations in the lacI gene in both rodents and bacteria, we have created a computerized database that contains information about DNA sequence alterations on about 500 mutations in transgenic rodents and 8,000 mutations in bacteria. We have also produced a software package for the analysis of the lacI database. Routines have been developed for the analysis of single base substitutions, including programs to (i) determine if two mutational spectra are different; (ii) determine if mutations show a DNA strand bias; (iii) determine the frequency of transitions and transversions; (iv) display the number and kind of mutations observed at each base in the coding region; (v) perform nearest neighbor analysis; and (vi) display mutable amino acids in the lacI protein. The software runs only on IBM-compatible machines running Microsoft Windows. The software and lacI database are freely available via the internet (http:/(/)sunsite.unc.edu/dnam/mainpage.++ +html).

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.

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.

Overview of the workshops on statistical analysis of mutation data from transgenic mice.

Early results from two transgenic mouse mutation assays, Big Blue [Kohler SW et al. (1991): Proc Natl Acad Sci USA 88:7958-7962] and Muta Mouse [Myhr BC (1991): Environ Mol Mutagen 18:308-315], raised questions about appropriate study design and methods for statistical analysis. First, there were a number of potential sources of variability in the technical aspects of the assay. These are "how we do it in our laboratory" differences, which, if not controlled, ultimately make inter-laboratory comparison of data difficult. Second, separate from the technical sources of variability were a number of study design issues, e.g., how many animals are needed in each treatment group, how many times should the animal be dosed, what is the appropriate expression period for a particular tissue, how many plaques need to be collected from each animal, and so on. To address these questions and to identify and understand the sources of variability in mutation data from these systems, a workshop was held in June, 1992 in Cincinnati, Ohio, USA. A core group of biologists and statisticians discussed possible sources of bias (tissue sampling, phage recovery and transgene recovery), possible sources of variability in mutant frequency (between animals, protocol-based sources, and design-based sources) and assay sensitivity. Following two days of discussion on protocol design and assay procedures, three action steps were recommended: (1) compile a data base of existing mutation data in transgenic mice to study its statistical features, (2) develop standard protocols for the mutation assays; and (3) use the standard protocol to generate a large data base of mutant frequencies in liver DNA from untreated mice for statistical study and analysis. This report summarizes the proceedings and recommendations of the workshop. The progress made toward these recommendations was reviewed in a second workshop, held in April, 1993, in Norfolk, Virginia, part of which is the subject of the accompanying paper by Piegorsch et al. To date, a standard protocol has been developed for the Big Blue mutagenesis assay and a data base of over 90 million plaques from seven labs using either the Big Blue or Muta Mouse system has been assembled, including a large data set of spontaneous liver mutant frequencies in the Big Blue system.

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

The Big Blue lacI transgenic rodent assay, which uses the lambda LIZ/lacI gene as the target for mutation, provides a convenient short-term assay for the study of mutation in vivo [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 lambda LIZ/lacI 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 lambda LIZ/lacI phage plated directly on E. coli. Both the sectored mutants and those recovered in lambda LIZ/lacI 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 lambda LIZ/lacI mutants were also different from spontaneous mutants recovered from Big Blue liver, these 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 lambda LIZ/lacI 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.

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.

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 x 10(-5) using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 x 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((R)) 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 x 10(-5); 25 mg CP/kg: 0.39 +/- 0.05 x 10(-5)) versus controls (0.10 +/- 0.01 x 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 x 10(-5); 25 mg CP/kg: 6.7 +/- 0. 8 x 10(-5); control: 7.7 +/- 0.7 x 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((R)) 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.

Sources of variability in data from a lacI transgenic mouse mutation assay.

Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail. 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 fractions. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability. Data from two laboratories are evaluated, using various statistical methods to identify excess variability. Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues. Further study is encouraged to examine the validity and implications of this time/storage-related effect.

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 approximately 3 x 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.

Application of HPLC in the 32P-postlabeling assay.

The postlabeling procedure for the detection of DNA modifications entails enzyme-catalyzed incorporation of 32P into nucleotides and chromatographic separation of radiolabeled products for quantification. Alternate versions of this procedure have been developed which vary in sensitivity and in applicability for the detection of different DNA adducts. Methods that utilize HPLC in either of two steps in the procedure (i.e., the separation of modified and unmodified nucleotides before the labeling reaction or the resolution of 32P-labeled adducts) are applicable for the detection of alkyl adducts as well as bulky, hydrophobic adducts and are discussed in this review. In some cases, postlabeling assays have been tailored for the quantitative detection of specific adducts. Use of multiple optimized postlabeling methods to analyze one DNA sample may enable identification of multiple specific adducts in human DNA. The widest and most promising applications for adduct detection with the postlabeling assay are for previously characterized adducts, where adduct standards are available for optimization and characterization of recovery in the assay. 32P-Postlabeling is a powerful way to measure DNA adducts as it is very sensitive. However, caution should be applied in drawing conclusions from postlabeling studies without appropriate corroborative data using another adduct detection method or without appropriate method development preceding the study. Examples of applications in human, laboratory animal, and environmental studies are available.

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.

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.