Mutagenic response to benzene and tris(2,3-dibromopropyl)-phosphate in the lambda lacI transgenic mouse mutation assay: a standardized approach to in vivo mutation analysis.

The genotoxic response of benzene and tris(2,3-dibromopropyl)-phosphate (TDBP) have been evaluated in several tissues using the standardized lambda/lacI (Big Blue) transgenic mouse mutation assay. Separate groups of four to five male B6C3F1 transgenic lambda/lacI mice were given oral administrations of benzene or TDBP at varying concentrations. Tissues evaluated include lung, bone marrow, and spleen in benzene-treated animals, and liver, kidney, and stomach in TDBP-treated animals. Significant increases in lacI mutations were observed in the spleen and bone marrow of benzene treated mice, and the kidneys of TDBP-treated mice. Where applicable, mutagenesis patterns of tissue sensitivity were consistent with what has been observed previously in other assays. In addition, mutagenicity in tissues not traditionally evaluated for mutations correlated to sites of carcinogenicity for the chemicals tested.

Spectrum of mutations in kidney, stomach, and liver from lacI transgenic mice recovered after treatment with tris(2,3-dibromopropyl)phosphate.

The flame retardant tris(2,3-dibromopropyl)phosphate (TDBP), once used in cotton sleep wear for children, is presently banned from commerce. It produces tumors in rodents in both a sex- and tissue-specific manner. The kidney is the main target for tumor formation in male and female rats, as well as in male mice. In contrast, tumors are formed in the liver of female animals. We have used lacI transgenic male B6C3F1 mice (Big Blue) to examine the induction of mutation in kidney, liver, and stomach after exposure to 150 mg/kg (2 days), 300 mg/kg (4 days), and 600 mg/kg (4 days) of TDBP. At the highest dose, the mutant frequency was approximately 50% above control values in the kidney (P < 0.01). A smaller increase was observed in the liver (P = 0.07), while no increase was seen in the stomach (P = 0.28). Sequence analysis of the recovered mutants showed a TDBP-specific change in mutation spectrum in kidney, which was not observed in liver and stomach. In kidney, a dose-dependent decrease in G:C-->A:T transitions, including at 5'-CpG-3' sites, was observed. This was accompanied by an increase in the loss of single G:C base pairs from approximately 3% to 15%. These results illustrate both the sensitivity and specificity of the lacI transgenic system in the analysis of tissue-specific mutation. This study also reinforces the importance of examining mutational spectra when mutant induction levels are low.

Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP/lacI shuttle vector.

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74-fold) over background levels for a variety of compounds, including N-ethyl-N-nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well-characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.

Evaluation of the transgenic Lambda/LacI mouse model as a short-term predictor of heritable risk.

Transgenic male C57BL/6 lambda/lacI mice were used to assess the mutagenic response in seminiferous tubules and epididymal spermatozoa 3 days after exposure to ethylnitrosourea (ENU), iso-propyl methanesulfonate (iPMS) and methyl methanesulfonate (MMS). No significant mutagenic response was observed in epididymal spermatozoa for all three compounds, as expected 3 days after treatment. However, ENU and iPMS treated samples demonstrated significant mutagenic inductions relative to controls in seminiferous tubules while MMS treated samples did not. The failure of MMS to induce a mutagenic response in lambda/lacI transgenic mice is likely due to a combination of the low dose used, the short expression time after exposure and the reduced sensitivity to large deletion events in transgenic lambda/lacI shuttle vectors. In addition, ex vivo mutations were measured in control samples and iPMS treated samples, where 33% of mutants from control samples and 35% of mutants from iPMS treated samples were mosaic.

The use of selection in recovery of transgenic targets for mutation analysis.

Transgenic animal mutagenesis assays using lambda shuttle vectors have recently been described for isolation and characterization of spontaneous and chemical induced DNA mutations. Extensive information on lambda and E. coli genetics provides a wealth of techniques to allow selection of mutant target genes. Here we describe the modification of an E. coli host which permits two methods for the direct selection of mutant genes. These methods reduce the number of plates needed to be screened for a comparable amount of frequency data by 20-100-fold and thus provide a significant savings of the materials and time required for the screening of mutations. In addition, mutants selected by these approaches described here may alter or broaden the spectrum of mutations that are recoverable. Ultimately, a combination of selective and nonselective techniques may prove valuable for the analysis of mutations produced in vivo in transgenic animals.

Interlaboratory comparison: liver spontaneous mutant frequency from lambda/lacI transgenic mice (Big Blue) (II).

Spontaneous mutant frequency in livers of two transgenic mouse strains, each carrying identical lambda shuttle vectors with a lacI target gene, was evaluated by two laboratories. These studies investigated variability in spontaneous mutant frequency between animals and as a function of the number of phage screened. Liver DNA was independently isolated from 7-11 week old C57BL/6 and B6C3F1 Big Blue transgenic mice. At least 500,000 phage were screened for mutation at lacI for each animal using standardized assay procedures. In the two labs, the C57BL/6 liver spontaneous mutant frequency was 45 +/- 9 x 10(-6) and 41 +/- 7 x 10(-6). The B6C3F1 liver spontaneous mutant frequency was 42 +/- 10 x 10(-6) at one lab and 43 +/- 12 x 10(-6) and 41 +/- 8 x 10(-6) in two trials at the second lab. Mean mutant frequency data from both labs, calculated in increments of 100,000 plaque forming units (pfu) scored for each mouse strain, show stabilized mean mutant frequency and standard deviation after approximately 200,000-300,000 pfu screened. The frequency of spontaneous lacI mutants was reproducible both within and between labs and was comparable between the two transgenic mouse strains.

Intralaboratory optimization and standardization of mutant screening conditions used for a lambda/lacI transgenic mouse mutagenesis assay (I).

A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has been optimized and standardized for reproducible mutant detection. The mutagenic endpoints are blue lacI- phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal. Non-mutant lacI phage plaques remain colorless. Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant plaque visibility. In vivo mutant frequencies for liver in untreated animals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10(-5) and 4.1 +/- 0.8 x 10(-5)). These studies analyze the use of internal controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant frequencies obtained when using standardized procedures.

The use of shuttle vectors for mutation analysis in transgenic mice and rats.

The establishment in recent years of transgenic shuttle vector-based mutagenicity assays has provided improved systems for analysis of mutagenic and carcinogenic processes. Results in the mouse have stimulated the development of an alternate species suitable for mutation analysis and have increased our understanding of the existing models. A previously described shuttle vector (lambda LIZ), based on a lacI target gene, was constructed in this laboratory for the study of mutagenesis in transgenic mice and in cultured cell lines. The shuttle vector allows for several options in its recovery from the host genome and in mutant identification. Of the 9 transgenic lineages that were generated with the lambda LIZ vector, one was chosen for use in a standardized mutagenicity assay (Big Blue, mouse lineage A1). Characterization of this lineage included copy-number determination, chromosomal localization of transgene integration and analysis of copy-number stability. As part of the validation process, the standardized color-screening assay has been tested in the mouse, both for spontaneous mutant frequencies and with a variety of model mutagenic compounds, and has been shown to identify most major classes of mutations as evidenced by mutant spectra data. A discussion of the relative sensitivity of the shuttle vector to each of these classes of mutations is included. These studies have now been extended to the generation of transgenic rats containing the same shuttle vector for cross-species analysis. Spontaneous mutant frequencies in two transgenic rat lineages were measured in liver and in germ cells. Preliminary data suggest that spontaneous mutant frequencies in somatic tissue are lower in rats than in mice, a result consistent with historical observations of DNA damage and repair in these two species. Also under evaluation are alternative selectable systems for mutant identification, and hybrid animals obtained from mating lambda LIZ transgenics with genetically engineered mice possessing an inactivated tumor suppressor gene. It is expected that each of these widely varying endeavors will contribute, not only in furthering our understanding of the role transgenic systems should play in human risk assessment, but in illuminating the mechanisms of mutation in general.

Transgenic systems for in vivo mutation analysis.

Transgenic mice carrying shuttle vectors containing the lacI gene as the target permit the in vivo measurement of mutations in multiple tissues and have been used to test the mutagenic effects of several compounds. Tissue-specific and time-dependent responses have been observed, and the spectrum of mutations determined by sequencing allows analysis of the role of expression time in mutagenesis. The results obtained from sequencing analysis have demonstrated spectra paralleling those observed in alternative in vivo assays. In addition to color screening, modifications to this system have permitted direct selection for mutations in the lacI target by a variety of methods. Transgenic rats containing the same lambda/lacI shuttle vector have been developed for inter-species comparison of mutagenesis testing results, which may offer a better understanding of the specific mechanisms involved in mutagenesis at the molecular level in vivo.

Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F1 mice.

Transgenic B6C3F1 mice carrying a lacI target gene were exposed to acute and multiple doses of ethylnitrosourea (ENU), and germ cells from the seminiferous tubules were assayed for mutation 3 and 90 days after treatment. Relative to untreated controls, the mutation frequency increased 3.2- and 19.9-fold at 3 and 90 days after treatment, respectively. Mutant lacI genes recovered from untreated and treated groups were sequenced, and the spectra of mutations were determined. Eighty-five percent (11/13) of the spontaneous mutations resulted in G.C-->A.T transitions, all of which occurred at CpG dinucleotides. Fifteen of 22 sites (68%) found mutated 3 days after ENU treatment occurred at G.C base pairs, although some of these are expected to be spontaneous mutations. Ninety days after treatment, 13 of 19 sites (68%) found mutated occurred at A.T base pairs. The mutation spectra seen are consistent with proposed mechanisms of ENU mutagenesis and correlate with the in vivo spectra seen in ENU studies by using transmissibility assays and the hprt gene. These findings represent significant progress toward defining the in vivo spectra of ENU mutagenesis in mammalian germ cells.

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.

Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.

Databases and software for the analysis of mutations in the human p53 gene, the 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.

The use of transgenic mice for short-term, in vivo mutagenicity testing.

In order to develop a short-term, in vivo assay to study the mutagenic effects of chemical exposure, transgenic mice were generated using a lambda shuttle vector containing a lacZ target gene. Following exposure to mutagens, this target can be rescued efficiently from genomic DNA prepared from tissues of the treated mice using restriction minus, in vitro lambda phage packaging extract and restriction minus Escherichia coli plating cultures. Mutations in the target gene appear as colorless plaques on a background of blue plaques when plated on indicator agar. Spontaneous background levels were approximately 1 x 10(-5) in each of three mouse lineages analyzed. Exposure of lambda transgenic mice to N-ethyl-N-nitrosourea resulted in as much as a 14-fold induction in detected mutations over background levels. The assay is currently being modified to incorporate lacI as the target for ease of mutation detection as well as in vivo excision properties of the Lambda ZAP vector, facilitating sequence analysis of mutant plaques.

Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice.

Transgenic mice with a lambda shuttle vector containing a lacI target gene were generated for use as a short-term, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal). Phage with mutations in the lacI gene form blue plaques, whereas phage with a nonmutated lacI form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10(-5) to 1.7 x 10(-5), depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the mice with N-ethyl-N-nitrosourea (EtNU), benzo[a]pyrene (B[a]P), or cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the lacI gene, when replicated in Escherichia coli, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the lacI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance of mutations (81%) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.

Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/lacI transgenic rats and mice following exposure to aflatoxin B1.

In vivo mutations were studied in lambda/lacI (Big Blue) transgenic C57BL/6 mice and F344 rats following exposure to either AFB1 (aflatoxin B1) or DMSO vehicle. Fourteen days after exposure, livers were removed for DNA extraction and subsequent mutational analysis of the lacI gene. Mice injected with a single i.p. dose of AFB1 at 2.5 mg/kg did not show a significant increase in liver mutant frequency relative to vehicle-treated controls. DNA sequence analysis of lacI mutations collected from the AFB1-treated mice showed a pattern of mutation similar to that of the previously observed spontaneous mouse liver mutational spectrum. In contrast, rats subjected to one-tenth the mouse AFB1 dosage responded with an approximate 20-fold induction in liver mutant frequency over background. Sequencing of lacI mutations also revealed spectral differences between vehicle- and AFB1-treated rats. A large increase in G:C-->T:A transversions was observed among lacI mutations isolated from the AFB1-treated rats. This work is among the first multi-species in vivo mutagenicity studies using transgenic rodents harboring the same shuttle vector. Such multi-species in vivo assays may prove to be valuable in the areas of mechanistic analysis and risk assessment.

Induction of hepatic mutations in lacI transgenic mice.

Transgenic B6C3F1 and C57BL/6 mice containing a lambda shuttle vector that carries a lacI target and an alpha lacZ reporter gene have been constructed for use in in vivo mutagenesis assays. After chemical treatment of mice carrying the lacI target gene, genomic DNA is isolated and the shuttle vector is recovered by exposing the DNA to lambda phage packaging extracts in vitro. Mutations in the lacI target gene that inactivate the repressor gene allow expression of the alpha lacZ reporter gene, resulting in blue mutant plaques. We have examined the ability of two genotoxic agents, dimethylnitrosamine (DMN) and methylmethane sulfonate (MMS), to induce mutations in these transgenic mice. Both compounds induce a variety of DNA adducts in mouse liver; DMN is a hepatocarcinogen that induces significant hepatic cell proliferation, but MMS is not hepatocarcinogenic and does not induce hepatic cell proliferation. The effects of animal age, differences in strain and dosing regimen, and length of expression time were evaluated. Mice were treated for 5, 14 or 21 days and were sacrificed 1, 8 or 22 days after the final dose to evaluate the effects of increased expression time on mutant frequency in liver. In 3 week old mice, DMN (2 mg/kg/day) produced 10- to 20-fold elevations in mutant frequency that increased with expression time and the number of treatments. In contrast, MMS (20 mg/kg/day) failed to increase the mutant frequency. DMN failed to induce mutations in 6 week old mice at 2 mg/kg/day, but 4 mg/kg/day yielded significant elevations in hepatic mutations. Sequencing results indicate that treatment of mice with DMN produced predominantly C:G-->T:A transitions.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenicity of o-anisidine to the bladder of lacI- transgenic B6C3F1 mice: absence of 14C or 32P bladder DNA adduction.

Earlier studies have established that the rodent bladder carcinogen o-anisidine (OA) gives negative results in all of the standard rodent genetic toxicity assays. In the present study, a single oral administration of the maximum tolerated dose level (750 mg/kg) of OA to B6C3F1 mice yielded negative results in 32P-post-labelling assays of bladder and liver DNA (24 h after dosing). Likewise, 14C-ring-labelled OA administered orally to B6C3F1 mice gave no evidence of DNA binding 6, 12 or 24 h later. Administration of OA (750 mg/kg) to transgenic lacI- mice (Big Blue) led to a small increase in mutation frequency (MF) in the bladder, but not in the liver. Increased MFs were observed in the bladder following 1, 3 or 10 daily doses with sampling times of 1 or 2 weeks after the final dose. However, statistical significance (P < 0.01) was only reached 2 weeks after either 3 or 10 daily administrations of OA. The positive control chemical (dimethylnitrosamine) gave a positive result (P < 0.01) in the liver, but not the bladder, 7 days after a single administration of 10 mg/kg. The possibility that OA is mutagenic and carcinogenic to the rodent bladder via formation of radical species is suggested.