Transgenic Mutation Assay Research Articles

Tissue specificity of spontaneous point mutations in lambda supF transgenic mice.

Transgenic mice carrying multiple copies of a recoverable lambda phage shuttle vector carrying the supF mutation reporter gene (lambda supF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from mouse liver and skin were analyzed. The mutation frequency was similar in both tissues (in the range of 2 x 10(-5)), but the spectrum of point mutations was distinct, with transitions common in the skin and transversions more prominent in the liver (P = 0.01). These results may help to elucidate pathways of endogenous mutagenesis in vivo, and they illustrate potentially important tissue-specific differences in genetic instability.

System Issues: Organ variation in the mutagenicity of ethylnitrosourea in Muta™ Mouse: Results of the collaborative study on the transgenic mutation assay by JEMS/MMS

System Issues: Organ variation in the mutagenicity of ethylnitrosourea in Muta™ Mouse: Results of the collaborative study on the transgenic mutation assay by JEMS/MMS

Organ variation in the mutagenicity of ethylnitrosourea in Muta mouse: results of the collaborative study on the transgenic mutation assay by JEMS/MMS. The Collaborative Study Group for the Transgenic Mouse Mutation Assay Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan.

A collaborative study of the transgenic mouse mutation assay was performed by a subgroup of the Mammalian Mutagenesis Study Group (MMS), which is a suborganization of the Environmental Mutagen Society of Japan (JEMS). Twenty-six laboratories participated in this collaboration, and ethylnitrosourea (ENU) mutagenesis was studied in eight organs of lacZ transgenic mice (Muta Mouse)-liver, spleen, bone marrow, brain, lung, kidney, urinary bladder, and heart. Mice were treated by a single intraperitoneal (ip) injection of 150 mg/kg ENU, and the lacZ mutant frequency (MF) was analyzed by positive selection after 3- and 14-day expression times. The MF in the control group was similar for all organs, approximately 40-60 x 10(-6). ENU increased MF in all organs except the brain with the highest values (more than 10 times the control value) observed in bone marrow on days 3 and 14, and in spleen on day 14. The MF in urinary bladder increased over 400 x 10(-6) and MFs in liver and lung were more than 150 x 10(-6) on day 14, although no increases were apparent in these organs on day 3. Approximately a doubling of control values was observed in kidney and heart but these were less than 100 x 10(-6). These results demonstrated that ENU induces organ specific mutagenesis with specific expression periods.

Development of a novel mouse tk+/- embryonic stem cell line for use in mutagenicity studies.

A tk+/- mouse embryonic stem (ES) cell line, designated 1G2, has been created in which one allele of the thymidine kinase (tk) gene was inactivated by targeted homologous recombination. This line is an analog of the mouse lymphoma tk+/- L5178Y cell line, which is used widely to assess the mutagenicity of chemical agents. Treatment of 1G2 cells with the alkylating agent N-ethyl-N-nitrosourea (ENU) resulted in a dose-related increase in trifluorothymidine-resistant colonies. Mutant frequencies of 152 and 296 per 10(6) cells were determined for 0.1 and 0.3 mg/ml doses of ENU, compared with a spontaneous mutant frequency of 15 per 10(6) cells. The data indicate that tk+/- 1G2 ES cells may be useful for the creation of a transgenic mouse model for assessing in vivo mutation using an endogenous autosomal gene.

APRT: a versatile in vivo resident reporter of local mutation and loss of heterozygosity.

We describe an in vivo mutagenesis model that utilizes reverse mutation and forward mutation at the endogenous Aprt locus. Reverse mutation provides an in situ method for detecting environments or agents that cause point mutations. Forward mutation detects large chromosomal events, including mitotic recombination, chromosome loss, and large multilocus deletion, all of which can lead to loss of heterozygosity. Detection of reverse mutation in vivo is based on the differential capacity of Aprt and Aprt cells to sequester radiolabeled adenine by catalyzing its conversion to adenosine monophosphate with subsequent incorporation into nucleic acids. Cells lacking APRT activity cannot accumulate exogenously administered, tagged adenine, whereas Aprt+ cells can and will thereby become marked. Thus, genetically modified mice with mutant but revertible Aprt alleles should be a useful vehicle for in situ detection of mutagenic activity in the whole animal. the feasibility of this model has been illustrated, first, by showing that APRT-deficient mice are viable and, second, by demonstrating that the minority of Aprt+ cells within a chimeric tumor growing in an Aprt+ mouse can be selectively labeled following IP injection of [14C]-adenine and can be identified by autoradiography. Forward mutation, detected by growth in selective medium of primary cells derived from Aprt+/- heterozygous mice, provides on independent estimate of in vivo mutation frequency. The frequency with which Aprt colonies arise provides a measure of the frequency of Aprt(-)-negative cells in the tissue at that point in time. Culture of skin fibroblasts in 2,6-diaminopurine (DAP) produced Aprt+ colonies with a frequency of about 10(-4). This frequency is similar to that found for human T lymphocytes from individuals heterozygous at the Aprt locus. In both cases, the majority of mutagenic events involved allele loss. Polymerase chain reaction with linked polymorphic microsatellites on mouse chromosome 8 demonstrated that allele loss was mediated mostly by mitotic recombination, as was the case for human T lymphocytes. The high frequency of mitotic recombination and allele loss at a neutral locus has significant implications for the process of tumorigenesis and argues that spontaneous or induced mitotic recombination may play a causal role in the progression to cancer.

Initial consideration for use of transgenic mutation assays in a regulatory submission.

As an initial consideration for use of TM assays in a regulatory submission, we focus here especially on the following questions: (1) What conditions would trigger the performance of a TM assay? (2) What data set is sufficient to support a negative result? and (3) What increase in mutation frequency is considered to be a positive result? In the first question, we discuss outcomes from traditional tests such as RM, CA, GM, and MN assays. In the second question, we propose a provisional data set including several items, i.e., experimental size [number of animals per group, number of plaques per animal (tissue)], tissue selection, route of administration, top dose level, number of dosings (single vs. multiple), sampling time, number of samples, dose levels, and positive and negative controls. Some statistical and biological considerations are needed for the third question.

Mutation frequencies but not mutant frequencies in Big Blue mice fit a Poisson distribution.

Transgenic mutation assays generally use mutant frequencies to estimate mutation frequencies but the degree to which clonal expansion inflates mutant frequencies is largely unknown. Mutant frequency is defined as the fraction of cells carrying mutations in the gene of interest and, according to the standard Big Blue protocol, is determined by dividing the number of mutant plaques by the total number of plaques screened. Mutation frequency is determined as the fraction of cells carrying definitely independent mutations and therefore requires correction for clonal expansion. Mutant and mutation frequencies were determined for brain, thymus and male germ cells of four mice from two age groups (3-versus 10-month old). The mutant frequency in thymus differed significantly between 3- and 10-month old mice (P < 0.05). By sequencing all mutants, the mutation frequency (i.e., corrected for jackpot mutations) in thymus was determined and was not significantly different between 3- and 10-month old mice. Mutant frequency does not fit a Poisson distribution, but mutation frequency corrected for jackpot mutations is substantially less variable and does fit a Poisson distribution.

Transgenic animal models for detection of in vivo mutations.

Transgenic rodent models for measuring mutations provide a tool for assessing tissue-specific mutations following in vivo treatment. These systems are based on the insertion into the rodent genome Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations. Following in vivo treatment of animals, genomic DNA is isolated from tissues of interest, and the target gene is screened for mutations using either lambda-phage packaging or isolation of the target gene with magnetic affinity capture. In this paper we review the various experimental methods used in the conduct of transgenic mutation assays and discuss critical factors that affect the interpretations of results of these assays.

Transgenic germ cell mutation assays: A small collaborative study

Transgenic germ cell mutation assays: A small collaborative study

Treatment protocols for transgenic mutation assays in vivo.

Treatment protocols for transgenic mutation assays in vivo.

Evaluation of the Genetic Toxicity of the Peroxisome Proliferator and Carcinogen Methyl Clofenapate, Including Assays Usin Muta TM Mouse and Big Blue™ Transgenic Mice

The rodent liver carcinogen and hepatic peroxisome proliferator methylclofenapate (MCP) has been evaluated for genetic toxicity in a range of in vitro and rodent genotoxicity assays. It gave a negative response in each of the following assays: mutagenicity to S. typhimurium and E. coli (+/- S9 mix, plate and pre-incubation assays), clastogenicity to cultured human lymphocytes and CHO cells (+/- S9 mix), a mouse bone marrow micronucleus assay (24h and 48h sampling), a rat liver assay for UDS in vivo (12h sampling), assays for lac I (Big Blue) and lac Z (Muta Mouse) mutations in the liver of transgenic mice, and an assay of the ability of MCP to modify the mutagenicity to the liver of dimethylnitrosamine in both transgenic mutation assays. The micronucleus and UDS assays were conducted using a single administration of MCP at its maximum tolerated dose, while the transgenic assays were conducted using nine daily administrations of MCP at its cancer bioassay dose level. These nine daily administrations were shown to double the weight of the liver of non-transgenic, Big Blue and Muta Mice, as well as leading to a dramatic proliferation of peroxisomes (electron microscopy) in the livers of each strain. These changed parameters had returned to control levels when the mutation analyses were conducted (10 days after the final dose of MCP). Despite the liver enlargement observed following MCP administration, no evidence of mitotic activity was observed in treated livers, although an increased number of cells were undergoing replicative DNA synthesis during the final 3 days of the 9 days of administration (BUdR assessment of S-phase). Liver biochemistry parameters (ALT, AST, AP, CK, GGT and albumin) were unaffected by the chronic (9 day) administration of MCP indicating an absence of hepatic toxicity. These combined observations favour a non-genotoxic mechanism of action for the hepatic carcinogenicity of MCP. The clastogenicity in vitro of the perixisome proliferator Wyeth 14,643 has been confirmed in CHO cells, but it is noted that this chemical is more soluble than is MCP. In particular, at the highest dose level at which MCP could be tested, Wy 14,643 was also non-clastogenic.

Response of the Muta TMmouse lacZ/galE − transgenic mutation assay to DMN: comparisons with the corresponding Big Blue TM (lacI) responses

The lacZ MutaTMmouse transgenic mutation assay was recently adapted into a selective assay based on use of E. coli galE − bacteria and phenyl galactoside (p-gal). A preliminary assessment of this selective assay was undertaken using a single oral dose of 10 mg/kg of dimethyl nitrosamine (DMN). The livers of treated male mice were assessed for UDS 2 h after dosing, and for lacZ − mutations 7, 11 and 20 days after dosing. A strong UDS response was recorded and a clear mutagenic response was observed at each of the 3 timepoints. Comparison of these data with earlier data derived using the Big Blue (lacI) mutation assay reveals a marginally greater sensitivity to DMN of the selective MutaTMmouse assay, an effect probably related to a biochemical difference between the strains of animal, as evidenced by the larger UDS response seen in the MutaTMmouse system. The original MutaTMmouse assay protocol was impractical. The galE − adaption makes the assay emminently practical and cost-effective. We are continuing to assess the true role of both the Big Blue assay and the galE − MutaTMmouse assay in mutagenicity/ carcinogenicity prediction. The former assay enables access to B6C3F1 mice and F344 rats, the latter enables the rapid acquisition of data.

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.

The accumulation and persistence of somatic mutations in vivo.

Mutant frequencies at the native Dlb-1 and the lacI transgene reach a plateau within 1 week of mutagenesis in the small intestine of mice. This indicates that these mutations are selectively neutral and should accumulate additively. In a test with three mutagens, one potent (ethylnitrosourea) and two weak (1,2-dimethylhydrazine and methyl methanesulphonate), ten weekly treatments induced ten times the frequency of Dlb-1 mutations induced by a single treatment in each case, a result that is consistent with perfect additivity. These observations indicate that the mutation frequency at neutral loci will be an integrated measure of the effects of aging, endogenous mutagens, environmental mutagens and antimutagens. They suggest a simple protocol for transgenic mutation assays which would increase sensitivity, require fewer animals, and conform to standard toxicological practice.

The micronucleus assay with 1,3-propane sultone

The Pig-a assay is a useful in vivo mutation detecting test and is easier to perform than the in vivo transgenic mutation assay. This assay is now recognized to be able to detect a number of mutagenic chemicals administered to rats in sub-acute or sub-chronic dose studies. The present investigation was conducted to evaluate the usefulness of peripheral blood Pig-a assays with total red blood cells (RBC Pig-a assay) and with reticulocytes (PIGRET assay) using two genotoxic rodent carcinogens, 1,3-propane sultone (1,3-PS) and 2-acetylaminofluorene (2-AAF). Male rats were orally administered a single dose of each test compound, and both the RBC Pig-a and PIGRET assays were performed using flow cytometry to measure the Pig-a mutant frequency (MF) before and after dosing on Days 8, 15 and 29. In the experiment with 1,3-PS, significant increases in Pig-a MF were observed from Day 15 and Day 8 in the RBC Pig-a and PIGRET assays, respectively. The results of both assays demonstrated that the increases in Pig-a MF were detectable after a single treatment with 1,3-PS. Furthermore, the difference in the kinetics of the increase in Pig-a MF between the RBC Pig-a and PIGRET assays with 1,3-PS suggests that the PIGRET assay has an advantage in detecting the mutant erythrocytes earlier than the RBC Pig-a assay. In contrast, no significant increases were observed in the Pig-a assays using either RBC or reticulocytes with 2-AAF. The negative results in both assays with 2-AAF may indicate the limitation of the single dose method; however, further investigation at higher doses is necessary to determine limitation of the single dose method.

Can nongenotoxic carcinogens be detected with the lacI transgenic mouse mutation assay?

In a commentary in Vol. 20. No. 3, ofthis joumal, Ashby and Lcigibcl [ 19921 warned of possible confusion in the understanding of gcnotoxic and nongenotoxic carcinogens becausc thc ncw transgenic mutation assays might open thc possihility of dctecting mutagenic activitics of chemieals unrclated to their presence in the cell at the momcnt of mutation initiation. They reasoncd that carcinogcns ablc to stimulatc thc rate of ccll division I Buuerworth, 1990; Cohen and Ellwein, 19911 could aceeierate the accumulation of ··spontancous'' mutations in marker genes arising from endogenous DNA darnage IAmcs. 1989~ Loeb, 1989: Lutz. 1990]. Wehave just completed a study to investigate whcther the measurement of lad mutations in lac/ transgenic mice 1 Kohl er et al.. 1990 I could indccd be uscd to dctcct nongenotoxic carcinogens such as heptachlor and phenobarbital as indirect mutagens.'' following a 4-month feeding period at dosc Ievels positive in thc 2-yc:ar bioassays. Di(2-cthylhcxyl)phthalatc (DEHP) was includcd in the study as a carcinogen that could, in addition to having mitogenic activity, be indirectly genotoxk via oxygen radicals from peroxis~­ mal hydrogen pcroxide. 2-Acctylaminotluorenc (2-AAI·) served as positive control for thc induction of lacl mutations. Thc stimulation of Ii ver ccll divisionwas investigated in thc samc animals, using immunohistochemistry for bromodeoxyuridinc incorporatcd into DNA. The negative rcsults. hoth for lad mutations in Ii ver DNA and for the rate of hcpatocytc division. show that thc nongcnotoxic carcinogcns investigatcd do not givc risc tu a gencrally incrcascd Ievei or mutations or a sustained gencral increasc in thc rate of ccll division.