Assessment of mechanisms driving non-linear dose-response relationships in genotoxicity testing.

In genetic toxicology, risk assessment has traditionally adopted linear dose-responses for any compound that causes genotoxic effects. Increasing evidence of non-linear dose-responses, however, suggests potential cellular tolerance to low levels of many genotoxicants with diverse modes of action. Such putative non-linear dose-responses need to be substantiated by strong mechanistic data that identifies the mechanisms responsible for the tolerance to low doses. This can be achieved by experimental demonstration of cytoprotective mechanisms and by providing experimental support for the existence of tolerance mechanisms against low dose effects. By highlighting key experiments into low dose mechanisms, this review aims to clarify which mechanistic data are required to support the use of non-linear dose-response models in risk assessment. Such key experiments are presented and discussed for alkylating agents, oxidants, particulate matter, nucleoside analogues, topoisomerase inhibitors and aneugens and exemplify the use of gene knockout models or transgenic models as well as chemical modulators of key effectors of relevant pathways and their impact on dose-response relationships. In vitro studies are particularly valuable to elucidate mechanisms of low-dose protection or lack thereof, while in vivo experiments are most appropriate for deriving a safe dose. In order to evaluate the existence of non-linear dose-response relationships for genotoxicants, we suggest that careful attention should be given to the mode of genotoxic action, relevant biomarkers of exposure, as well as to the existence and impact of potential cytoprotective mechanisms like detoxifying metabolism and DNA repair.

Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis.

The in vitro micronucleus test (MNT) is a well-established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and a specific parameter. Various automated systems to replace the tedious and time-consuming visual slide analysis procedure as well as flow cytometric approaches have been discussed. The ROBIAS (Robotic Image Analysis System) for both automatic cytotoxicity assessment and micronucleus detection in human lymphocytes was developed at Novartis where the assay has been used to validate positive results obtained in the MNT in TK6 cells, which serves as the primary screening system for genotoxicity profiling in early drug development. In addition, the in vitro MNT has become an accepted alternative to support clinical studies and will be used for regulatory purposes as well. The comparison of visual with automatic analysis results showed a high degree of concordance for 25 independent experiments conducted for the profiling of 12 compounds. For concentration series of cyclophosphamide and carbendazim, a very good correlation between automatic and visual analysis by two examiners could be established, both for the relative division index used as cytotoxicity parameter, as well as for micronuclei scoring in mono- and binucleated cells. Generally, false-positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyse 24 slides within 65h by automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS which is supporting various assays at Novartis.

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.

Quantitative correlation between radiation-induced mutagenesis in endogenous genes and transgenes of mouse spermatogonial stem cells.

In order to evaluate the pUR288-plasmid transgenic mouse model, utilizing the bacterial lacZ gene as the mutational target, radiation-induced mutagenesis was primarily analyzed in spermatogonial stem cells. A combined hydroxyurea (HU)-X-ray treatment protocol was used, known to sensitize dramatically the induction of mutations in endogenous genes. In the testes of untreated animals, a mutant frequency of 6.7 +/- 4.4 x 10(-5) was found. In animals treated with HU or X ray alone, moderate elevations were seen (factors of about 4 and 2 over untreated animal values). In testes of mice having received the HU + X-ray combination treatment, a mutant frequency of 63.0 +/- 36.1 x 10(-5) was found. The results obtained showed a good quantitative correlation between endogenous genes and the transgene, indicating the suitability of pUR288 transgenic mice for also efficiently recording radiation-induced genetic damage. Radiosensitization, seen in spermatogonial stem cells, was not observed in other studied organs such as spleen, brain, or lung.

Characterization of color mutants in lacZ plasmid-based transgenic mice, as detected by positive selection.

The plasmid-based transgenic mouse model, which uses the lacZ gene as the target for mutation, is sensitive to a wide range of in vivo mutations, ranging from point mutations to insertions and deletions extending far into the mouse genome. In this study, the nature of subtle lacZ mutations, which do not completely abolish beta-galactosidase activity, as detected by positive selection, was investigated. These subtle mutants are called 'color mutants' due to their light blue staining on X-gal medium. Replating of color mutants and retransformation of plasmid DNA, purified from individual color mutants, resulted in the same phenotype as the original color mutant. The p-gal positive selection system tolerates approximately 10% of wild-type activity as indicated by spectrophotometric determination of beta-galactosidase activity of individual color mutants. Restriction digestion and size separation of plasmid DNA revealed no visible change in the size of the plasmid in color mutants. Sequence analysis confirmed the presence of a point mutation in each lacZ gene of nine different color mutants. The results indicate that color mutants are caused neither by the presence of a mixture of wild-type and mutated lacZ plasmids within the same host cell nor by a mixture of cells within the original mutant colony which carry either wild-type or mutated lacZ plasmids. In addition, it was discovered that the mouse line studied harbors four polymorphic base changes among the integrated plasmid copies.

Rapid accumulation of genome rearrangements in liver but not in brain of old mice.

Somatic mutations have long been considered a possible cause of ageing. To directly study mutational events in organs and tissues of ageing mammals, a transgenic mouse model has been generated that harbours lacZ reporter genes as part of chromosomally integrated plasmids. Using this model, we determined spontaneous mutant frequencies and spectra in mouse liver and brain as a function of age. In the liver, mutant frequencies increased with age from birth to 34 months; in the brain, an increase was observed only between birth and 4-6 months. Molecular characterization of the mutations showed that a substantial portion involved genome rearrangement events, with one breakpoint in a reporter gene and the other in the mouse flanking sequence. In the liver, these genome rearrangements did not increase with age until after 27 months, when they increased rapidly. In brain, the frequency of genome rearrangements was lower than in liver and did not increase with age.

Transgenic mouse models for studying mutations in vivo: applications in aging research.

To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed. The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations. Procedures have been worked out to efficiently recover the plasmids into E. coli host cells. A positive selection system, permitting only E. coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered. Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain. Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Transgenic mouse models for studying mutations in vivo: applications in aging research.

To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed. The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations. Procedures have been worked out to efficiently recovery the plasmids into E. coli host cells. A positive selection system, permitting only E. coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered. Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain. Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations.

To study in vivo somatic mutations a C57BL/6 transgenic mouse model was constructed harboring multiple chromosomally integrated copies of the plasmid pUR288, which carried the lacZ reporter gene as the mutational target. We previously demonstrated that lacZ-containing plasmids could be rescued from their integrated state efficient enough to detect mutations in lacZ by positive selection. The smaller size of the plasmid vector, as compared with our earlier transgenic mouse model based on bacteriophage lambda vectors, should offer considerable advantages in terms of rescue efficiency and sensitivity to large size alterations in the lacZ gene. To evaluate the plasmid-based mouse model for its suitability to detect in vivo mutations, we determined mutant frequencies in different organs of untreated and ethyl nitrosourea (ENU)-treated animals using a new, improved protocol. The rescue efficiencies obtained were as high as 200,000/micrograms genomic DNA; millions of transformants could be obtained in one single experiment. The average spontaneous mutant frequency in four different organs of 4- to 8-week-old mice ranged from 4.41 to 6.82 x 10(-5), compared with a mutant frequency of the same plasmid grown in Escherichia coli of approximately 1 x 10(-5) or less. Single treatments with 100 and 250 mg ENU/kg body wt resulted in a 7- and 14-fold increase, respectively, in spleen mutant frequency at 14 days after i.p. administration of the alkylating agent. Restriction enzyme analysis showed that a considerable portion of spontaneous mutants were size changes varying from approximately 100 to 3000 bp. Some mutant plasmids contained mouse genomic sequences, which is indicative of large genetic rearrangement events involving the 3' flanking regions of the transgene cluster. Among the ENU-induced mutants, size changes comprised only a minor fraction of the total, which is in keeping with the known ENU mutation spectra in vitro and in vivo. The high rescue efficiency of this plasmid-based model, in combination with its sensitivity to a broad spectrum of mutations, including large deletions, makes it very suitable as a general in vivo mutagenicity test system.

Use of transgenic mouse models for studying somatic mutations in aging.

Theories on the causes of aging, based on the accumulation of somatic mutations in tissues of an organism, were formulated decades ago, but remain insufficiently tested. Transgenic animals, equipped with integrated bacterial reporter genes that can be efficiently rescued from total genomic DNA of all tissues and organs, represent ideal tools for investigating the types and frequencies of spontaneous mutants accumulating during aging. The first of such systems, based on the transgenic integration of bacteriophage lambda shuttle vectors that contain the bacterial lacZ gene as mutational target, was constructed in our laboratory and is now routinely used. Results obtained with this and the related LacI system that are relevant for the somatic mutation theory of aging will be discussed. One conclusion is that, due to the nature of the transgene, lambda-based systems have the disadvantage that deletion type mutations are underrepresented in comparison to point mutations. To overcome those limitations, we constructed a new transgenic mouse model carrying a pUR288 plasmid shuttle vector with the lacZ reporter gene. Some preliminary data obtained with this model serve to illustrate its potential use to extensively test the somatic mutation theory of aging.

Plasmid-based transgenic mouse model for studying in vivo mutations.

A new transgenic mouse model for studying in vivo somatic mutations is based on the efficient recovery of chromosomally integrated lacZ-containing plasmids, using magnetic beads.

Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model.

Transgenic mouse mutation models carrying bacterial marker genes in bacteriophage lambda shuttle vectors have been applied to study spontaneous or induced mutations in vivo. However, due to the nature of the shuttle vector these models are insensitive to large deletions. Clastogenic agents, which predominantly induce large deletions, were therefore found to yield very low responses in these assays. Here we report the use of LacZ plasmid-based transgenic mice, allowing the detection of a broad spectrum of mutations. Treatment of mice with X-rays (5 x 50 rads) resulted in induction of up to about 5-fold higher mutation frequencies in lung, spleen and liver. Analysis of spontaneous and induced mutant LacZ genes indicated that at least 40-50% of all mutations were caused by deletions. The possibility of detecting a broad spectrum of mutations with this system suggests that the LacZ plasmid-based transgenic mouse may be the mammalian model of choice for studying spontaneous and induced mutations in vivo.

Malignantly transformed non-parenchymal liver epithelial cells and transformed oval cells suppress the homotypical gap junctional intercellular communication of co-cultured rat liver parenchymal cells.

Isolated rat liver parenchymal cells (PC) were co-cultured with a non-parenchymal rat liver epithelial cell line (NEC) or with an oval cell line. The homotypical gap junctional intercellular communication (GJIC) between the liver PC was measured after microinjection of Lucifer Yellow by dye transfer. The rat liver PC were dye coupled between 87% and 100% for at least 1 week in both co-cultures, in contrast to PC In monoculture between which no dye coupling was left after 1 week. When liver PC were co-cultured with a transformed and tumorigenic NEC or with a transformed and tumorigenic oval cell line the homotypical GJIC between the liver PC was drastically decreased with culture time, and the PC were then compressed and displaced by the expansive growth of the transformed cell lines. The disturbance of the GJIC between normal cells by adjacent tumorigenic cells might be a new mechanism to explain the expansive growth of tumors.