Enhancement of the morphological transformation of Syrian hamster embryo (SHE) cells by reducing incubation time of the target cells.

Syrian hamster embryo (SHE) cell transformation has been used for many years to study chemical carcinogenesis in vitro. It has been shown that this assay is probably the most predictive short-term test system for identifying rodent carcinogens. Although most of the operational difficulties encountered in the early stage of application of this assay have been overcome by culturing the SHE cells under slightly acidic conditions (pH 6.7), a relatively low level of induction of morphological transformation (MT) by known carcinogens still occurs for many cell isolates. In order to improve the response of this assay system to known carcinogens, the effect of incubation time of target SHE cells on the frequency of morphological transformation induced by benzo(a)pyrene (BaP) was investigated. It was shown that the morphological transformation frequency induced by BaP increased significantly (1.4-2.5-fold) when the incubation time of target cells was reduced from the usual 24h to less than 6h prior to seeding onto feeder layers. This improvement in sensitivity was consistent for different cell isolates. In addition, the enhanced response appeared to be a property of carcinogens because treatment with two non-carcinogens, l-ascorbic acid and 4-nitro-o-phenylenediamine, did not induce significant increases in the transformation frequency under the shortened incubation period for target cells. These results suggest that the response of the SHE cell transformation assay may be improved by optimizing the incubation time of the target SHE cells. In addition, the results of the present study provide further evidence to support the idea that morphological transformation of SHE cells results from a block of cellular differentiation of stem or stem-like cells.

Mouse lymphoma thymidine kinase locus gene mutation assay: International Workshop on Genotoxicity Test Procedures Workgroup Report.

The Mouse Lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24-hr treatment; (4) microwell colony counting and sizing; and (5) data acceptability/statistical analysis. Although the International Conference on Harmonisation (ICH) indicated a preference for the microwell over the soft agar method, all of the workgroup members agreed that both versions of the MLA are equally acceptable. The workgroup agreed that it is desirable for both assay versions to use the same measure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. Laboratories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the RSG, the RS, and the RTG and to develop further guidance. In the interim, the workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24-hr treatment in the absence of S9 when negative results are obtained with short (3-4 hr) treatments. The workgroup agreed to retain this requirement but acknowledged that more data are needed prior to making final recommendations concerning the need for and the specific protocol for the 24-hr treatment. Environ. Mol. Mutagen. 35:185-190, 2000 Published 2000 Wiley-Liss, Inc.

Transgenic mouse mutation assay systems can play an important role in regulatory mutagenicity testing in vivo for the detection of site-of-contact mutagens.

Transgenic mouse mutation assays, such as MutaMouse (lacZ, CD2F1) and Big Blue (lacI, B6C3F1), afford the opportunity to evaluate the mutagenic potential of chemicals in any target organ in vivo. This paper discusses published data collected from the analysis of the skin, stomach and lung DNA after topical, oral and inhalation exposure, respectively. These data indicate that both MutaMouse and Big Blue should play an important part in the evaluation of genotoxicity in vivo, particularly where the endpoint or target tissue available in the more conventional tests is inappropriate. It is concluded that there is a distinct role for this type of assay in genetic toxicology testing. For substances applied to the skin or dosed orally or by inhalation and which are unlikely to reach either the bone marrow or the liver, then data derived from these assays may be more relevant to an assessment of possible risk to man than the currently used unscheduled DNA synthesis in liver and cytogenetics assays in bone marrow or peripheral blood.

Lack of genotoxic effects of sucrose acetate isobutyrate (SAIB).

Sucrose acetate isobutyrate (SAIB) was tested for potential genotoxic activity in four different in vitro assay systems. Two independent trials of a Salmonella reverse mutation assay (using strains TA98, TA100, TA1535, TA1537 and TA1538) showed no increases in revertant frequencies at doses up to 10,000 microg/plate which was non-toxic but exceeded the solubility limit. Similarly, no mutagenic response was observed at doses up to 1000 microg/ml at the HGPRT locus in cultured CHO cells; SAIB was toxic and its solubility limit was exceeded at 50 microg/ml. No clastogenic activity was detected in cultured CHO cells at concentrations up to 2000 microg/ml. All three preceding in vitro tests were conducted both in the presence and absence of Aroclor 1254-induced rat liver S-9 metabolic activation systems. An unscheduled DNA synthesis assay also was performed using rat primary hepatocyte cultures with doses up to 1000 microg/ml, and no DNA repair was detectable. Thus, SAIB was stringently tested at doses exceeding the solubility limit in culture medium and causing toxicity to CHO cells without obtaining any evidence for genotoxic activity as a mutagen, clastogen, or DNA-damaging agent.

A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta Mouse).

We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50-200 mg/kg) and EMS (100-400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0-4.6 x 10(-6). MF in bone marrow was increased by ENU to 3.4 x 10(-5) at 200 mg/kg and induced by EMS to 1.8 x 10(-5) at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O6-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O6-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis.

The L5178Y/tk+/- mouse lymphoma specific gene and chromosomal mutation assay a phase III report of the U.S. Environmental Protection Agency Gene-Tox Program.

The L5178Y/tk+/- (-)3.7.2C mouse lymphoma assay (MLA) which detects mutations affecting the heterozygous thymidine kinase (tk) locus is capable of responding to chemicals acting as clastogens as well as point mutagens. Improvements in the assay to enhance detection of this spectrum of genetic events are summarized, and criteria for evaluating the data are defined. Using these criteria, the Phase III Work Group reviewed and evaluated literature containing MLA results published from 1976 through 1993. The data base included 602 chemicals of which 343 were evaluated as positive, 44 negative, 18 equivocal, 54 apparently inappropriate for evaluation in this test system with the published protocols, and 142 that were inadequately tested, and thus a definitive call could not be made. The overall performance of the assay is summarized by chemical class, and the outcome of testing 260 chemicals in the MLA is compared with Gene-Tox and National Toxicology Program evaluations of rodent carcinogenesis bioassay results for the same chemicals. Based on the Work Group's evaluation of published MLA data for chemicals that were considered adequately tested, it is concluded that for most chemicals the L5178Y/tk+/- mouse lymphoma assay is eminently well suited for genotoxicity testing and for predicting the potential for carcinogenicity.

Mammalian genotoxicity assessment of methylene blue in plasma: implications for virus inactivation.

BACKGROUND: The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems. STUDY DESIGN AND METHODS: Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent. RESULTS: Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x 10(-6). CONCLUSION: Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.

A preliminary assessment of the toxic and mutagenic potential of steroidal alkaloids in transgenic mice.

Impregnated CD2 transgenic mice, which contain multiple copies of a lambda gt10lacZ construct integrated into the genome of each cell, were given a predetermined estimated maximum tolerated dose of several steroidal alkaloids: Solanum glycoalkaloids from potato, alpha-chaconine and alpha-solanine; aglycones, solanidine and solasodine, and a Veratrum alkaloid, jervine. Observations were made of dams and foetuses for indications of toxicity and/or terata; some dam livers and foetuses were assayed for mutagenicity using the lacZ gene. Other dams were gavaged with a single dose of 75 mg all-trans-retinol/kg to serve as a reference teratogen. Unexpectedly, this level of retinol was not clearly teratogenic. The results of both positive and non-positive selection systems showed that the mutation frequencies in the livers of the dams dosed with alpha-chaconine, alpha-solanine and solanidine were three to four times higher than historically normal in the livers of this transgenic mouse strain.

The LacZ transgene in MutaMouse maps to chromosome 3.

Transgenic mouse models are being used with increasing frequency for mutational and toxicological studies. One such system. MutaMouse, contains a stably integrated lambda-gt10LacZ shuttle vector in the mouse genome. We describe the use of dual color fluorescence in situ hybridization (FISH) with Mus musculus whole chromosome paints and lambda DNA to map the integration site of the lambda transgene to band C on mouse chromosome 3.

Measurement of gene mutation in vivo using Muta Mouse and positive selection for lacZ- phage.

The use of transgenic animals for in vivo mutagenicity studies following rescue of the bacterial lacZ transgene has been hindered by the sheer scale of the experimental work involved. We describe here a new positive selection protocol which is based upon a modified E. coli bacterial host. This new system is potentially capable of generating mutation data much faster and more cheaply than previous methods.

Correlation of carcinogenic potency with mouse-skin 32P-postlabeling and muta-Rmouse lac Z- mutation data for DMBA and its K-region sulphur isostere: comparison with activities observed in standard genotoxicity assays.

The genotoxicities in vitro and in vivo of the mouse-skin carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) have been compared with those of its weakly carcinogenic 4,5-sulphur analogue, 6,11-dimethylbenzo[b]naphtho-[2,3-d]thiophene (S-DMBA). The only datasets that correlated with the relative carcinogenicity of these agents to the skin were those conducted using topically exposed mouse skin. Thus, both chemicals induced lacZ- mutations in the skin of lacZ+ transgenic mice, and both produced DNA adducts on mouse-skin DNA as assessed using the 32P-postlabeling technique. In each case, DMBA gave a stronger response than did S-DMBA. In contrast to these responses, only DMBA was active in the mouse bone-marrow micronucleus assay and in the C3H10T1/2 in vitro cell transformation assay. Both chemicals were mutagenic to Salmonella and of approximately equal potency. The molecular geometry of DMBA and S-DMBA are compared, and divergent CASE predictions of activity in the Salmonella assay and skin-painting bioassay are discussed. The importance of conducting predictive genotoxicity assays in systems close to those in which carcinogenicity is to be assessed is emphasized by these data.

The concomitant detection of gene mutation and micronucleus induction by mitomycin C in vivo using lacZ transgenic mice.

A new assay system that can simultaneously provide gene mutagenicity and clastogenicity data in vivo is described. Transgenic mice (Muta Mouse) harboring the lacZ gene as a target for mutation analysis were injected intraperitoneally with mitomycin C (MMC), either once or on 5 successive days. Micronucleus assays were performed with small amounts of peripheral blood collected from a tail vessel. The spontaneous frequency of micronucleated reticulocytes was 0.42%. For the mutation analysis, DNA was extracted from bone marrow and liver cells at several harvest times. The lacZ gene was rescued by lambda packaging and infection of E. coli C (lac-), followed by plating on agarose plates containing X-gal. The spontaneous lacZ mutant frequencies were 37 and 29 x 10(-6) in bone marrow and liver, respectively. In the micronucleus assay, single treatments with 1.0 and 2.0 mg/kg of MMC induced micronuclei in 3.6 and 5.8% of reticulocytes, respectively, peaking 48 h after treatment. Muta Mouse sensitivity to micronucleus induction was similar to nontransgenic strains used routinely for the micronucleus test. On the other hand, single treatments with MMC at 1.0 and 2.0 mg/kg did not induce any significant increases in the frequency of lacZ- mutants in bone marrow or liver. N-Ethyl-N-nitrosourea, used at 100 mg/kg as a positive control, yielded a 5-fold increase in mutant frequency above untreated animals in bone marrow only. After 5-day treatments, MMC induced approximately a 2-fold increase in mutant frequency in bone marrow only for the sublethal dose of 2 mg/kg. Therefore, this study indicated that the strong clastogenic activity of MMC in bone marrow was not accompanied by significant gene mutagenic activity.

Detection of chemical mutagens using Muta Mouse: a transgenic mouse model.

A transgenic mouse strain with a high copy number of rescuable lacZ sequences was evaluated for its effectiveness in detecting lacZ- mutations in selected tissues. Procarbazine, cyclophosphamide, ethylnitrosourea, 7,12-dimethylbenz[a]anthracene (DMBA), acrylamide and chlorambucil were tested following either single or repeated dosing regimens. Bone marrow, liver, skin and testis tissues were selected to assess as target sites for mutation. Bone marrow, liver and testis tissues were examined for mutation following exposures to ethylnitrosourea and chlorambucil. Increased mutant frequencies were found for both chemicals in all three tissues. Bone marrow tissue was examined for mutation following procarbazine, cyclophosphamide and acrylamide exposures, and skin was examined for mutation following dermal application of DMBA. Mutation induction was observed in all cases. The results obtained from this investigation demonstrate the applicability of this transgenic mouse as an effective model to detect and analyze gene mutation in selected organs including germinal tissues. Studies of organotrophic chemical mutagens and carcinogens are possible with this model as are studies of the susceptibility of germinal tissues to mutagen exposures.

Determination of mutagenicity in tissues of transgenic mice following exposure to 1,3-butadiene and N-ethyl-N-nitrosourea.

1,3-Butadiene (BD) is carcinogenic in the B6C3F1 mouse in multiple organs, including lung and liver. We conducted a study to measure the frequency of BD mutations in mouse tissues using a transgenic mouse (Muta mouse; MM). MM is a BALB/c x DBA/2 (CD2F1) mouse that has a bacteriophage lambda shuttle vector with the target gene lacZ integrated into the mouse genome. Mice were exposed by inhalation to 625 ppm BD (6 hr/day) for 5 days and the lacZ- mutant frequency (mf) was determined in lung, bone marrow, and liver. The lacZ- mf in lung increased twofold above air-exposed control animals, but the bone marrow and liver samples did not exhibit an increase above background. N-ethyl-N-nitrosourea (250 mg/kg ip) was mutagenic in all three tissues examined. Studies on the biotransformation of BD using MM liver microsomes showed that the ratio between the rates of BD bioactivation to BD monoepoxide (BMO) and hydrolysis of BMO by epoxide hydrolases was approximately 40% less than this ratio using B6C3F1 mouse liver microsomes. Quantitation of adducts of BMO to N-terminal valine in hemoglobin (Hb) in the MM revealed an adduct level of 3.7 pmol/mg globin. Using this value, the predicted Hb adduct level in MM would be approximately one-half of that measured in the B6C3F1 mouse following similar exposures. These results indicate that BD induces mutations in vivo in a known murine target tissue, but strain differences in the biotransformation of BD should be considered in comparing the susceptibility of transgenic mouse strains to mutation.

Assessment of the potential genotoxicity of perfluorodecanoic acid and chlorotrifluoroethylene trimer and tetramer acids.

Perfluoro-n-decanoic acid (PFDA) is a perfluorinated fatty acid that produces hepatomegaly and increased peroxisomal beta-oxidation when administered to rodents. Chlorotrifluoroethylene (CTFE) trimer acid and CTFE tetramer acid are metabolites of the six- and eight-carbon oligomers of CTFE, respectively. They are structurally related to PFDA, and CTFE tetramer acid has caused toxic effects in rodents that are similar to those observed following PFDA administration. Because of the correlation between peroxisome proliferation and hepatocarcinogenesis, CTFE trimer acid, CTFE tetramer acid, and PFDA were evaluated in in vitro and in vivo/in vitro bioassays to assess their potential genotoxic activity. The assays conducted were the Ames Salmonella/microsomal mutagenicity assay, the hypoxanthineguanine phosphoribosyltransferase (HGPRT) locus Chinese hamster ovary gene mutation assay, the sister chromatid exchange (SCE) assay, chromosomal aberration assay, and an in vivo/in vitro unscheduled DNA synthesis (UDS) and S-phase DNA synthesis assay. All test articles were negative in the Ames assay, the HGPRT assay, and the SCE assay. In the chromosomal aberration assay CTFE trimer acid and CTFE tetramer acid were negative in cultures with and without S9 metabolic activation. PFDA was also negative in the absence of metabolic activation, but chromosomal aberrations were observed when PFDA was incubated in the presence of S9 fraction. All test articles were negative for inducing UDS but all induced S-phase replicative DNA synthesis 16 hr after administration of the test article to the test animals; only CTFE tetramer acid and PFDA induced S-phase synthesis 48 hr after dosing: the usual timepoint examined for this response.

Chemical mutagenesis at the thymidine kinase locus in L5178Y mouse lymphoma cells: results for 31 coded compounds in the National Toxicology Program.

Experimental data from the testing of 31 chemicals for mutagenicity at the TK locus in L5178Y mouse lymphoma cells are presented and evaluated. If mutagenic activity was not obtained for the chemical added to suspension cultures for 4 hr, then the testing was repeated in the presence of hepatic S9 mix prepared from Aroclor 1254-induced male Fischer 344 rats. Multiple trials were performed for each chemical, and mutagenic treatments were analyzed for the induction of small and large mutant colony populations. Twelve chemicals were not detected as mutagenic, one (ascorbic acid) was questionable, and 18 were evaluated as mutagenic. These results were used in the evaluations presented by Tennant et al. [Science 236:933-941, 1987] in a critical comparison of four in vitro genotoxicity assays with rodent carcinogenicity results. The mouse lymphoma assay results were in general agreement with the carcinogenicity studies. Discordant evaluations with respect to carcinogenicity (four false negatives and six false positives) were discussed from the standpoint of how the predictive performance of the in vitro mutation assay might be improved.

Validation studies with Muta Mouse: a transgenic mouse model for detecting mutations in vivo.

MutaMouse is a transgenic mouse engineered to detect mutations in vivo in any tissue of choice by using simple laboratory methods. The target is a bacterial lacZ gene incorporated via lambda phage into the genome of each mouse cell such that a concatamer of approximately 40 copies exists at a single site on both chromosomes of a homologous pair. In order to assess the potential usefulness of MutaMouse in detecting in vivo mutagenesis, several known mutagens/carcinogens were applied to male animals of 8-10 weeks in age. Intraperitoneal injections (single or 5 daily doses) of N-ethyl-N-nitrosourea (ENU), chlorambucil, procarbazine, cyclophosphamide, and acrylamide were investigated for mutagenic effects in bone marrow, liver, and testes. In addition, skin painting studies (single application) were performed with dimethylbenzanthracene (DMBA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and acetic acid. Increases in mutant frequency were clearly induced by all eight chemicals, the magnitudes of which were dependent on the chemical, dose, method of dosing, tissue analyzed, and the time lapse between treatment and isolation of DNA. Data on variability in mutant frequency was presented relative to the analyzed population of lacZ genes and number of animals per treatment group. Application of the MutaMouse model to the detection of heritable mutations was discussed.

L5178Y mouse lymphoma cell mutation assay results with 41 compounds.

Forty-one chemicals were tested for their abilities to induce trifluorothymidine resistance in L5178Y mouse lymphoma (MOLY) cells. These chemicals were included in the National Toxicology Program's evaluation of four in vitro short-term toxicity assays for predicting carcinogenicity in the rodent bioassay. Of the 41 chemicals examined for this report, 8 were equivocal in the rodent bioassay, and 7 were questionable in- the MOLY assay. If these chemicals are eliminated from an analysis of concordance, the remaining 26 chemicals lead to a concordance of 69% with a sensitivity of 71%. The specificity could not be determined because only two non-carcinogens were detected.