The gradient plate assay: a modified Ames assay used as a prescreen for the identification of bacterial mutagens.

Bacterial test systems have been used extensively to identify the mutagenic potential of new compounds. In particular, the Ames test has gained worldwide acceptance and is required by many regulatory agencies to support product registration. The gradient plate assay (GPA) is a modification of the Ames test. It is used as a high capacity prescreen to detect the mutagenic potential of synthetic intermediates, impurities, and research compounds over a concentration gradient. Since the development of the GPA, over 4000 compounds have been tested in the assay. Selection and use of the GPA in our laboratory is due to many factors: reliability; sensitivity; capacity; timeliness of reporting results; and establishment of safety standard in the laboratory. In this manuscript, results of the GPA method are compared with results from the traditional Ames assay. To date, 113 compounds of identical lots have been evaluated in both tests, and in all but 3 instances the results are the same. Thus, the GPA is an ideal assay for use as a prescreen in determining the ability of a compound to induce bacterial mutation.

A comparison of the CHO/HGPRT+ and the L5178Y/TK+/- mutation assays using suspension treatment and soft agar cloning: results for 10 chemicals.

The mouse lymphoma assay (MLA) and Chinese hamster ovary (CHO) cell assay are sensitive indicators of mutagenicity. The CHO assay has been modified technically to permit treatment in suspension and soft agar cloning comparable to the MLA. This methodology eliminates the risk of metabolic cooperation and the trauma of trypsinization. In addition, a larger population of cells can be treated and cloned for mutant selection. In order to compare the effectiveness of the test systems, 10 chemicals were evaluated for the induction of forward mutations in the CHO and MLA. Several of these chemicals have been reported as clastogenic; therefore, abbreviated colony sizing was performed to gauge the extent of genetic damage to the MLA cells. Both test systems detected benzo[a]pyrene, mitomycin C, acridine orange, and proflavin, and, with the exception of proflavin, more large colonies were present than small colonies. The suspect clastogen, phenytoin, was not mutagenic in the MLA and produced inconclusive results in the CHO. Ethidium bromide, a clastogen and a bacterial mutagen, was not mutagenic in either the MLA or CHO. Four compounds (p-aminophenol, benzoin, methoxychlor, and pyrene) were positive in the MLA, generally inducing a large number of small colonies, while demonstrating no mutagenic activity in the CHO assay. They have also been shown to be generally nongenotoxic in other test systems. Overall, the modified CHO assay did not appear to be better than the MLA for the detection of mutagenic agents. However, the MLA does appear to have lower specificity.

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2. III. Effects in the Salmonella typhimurium/Escherichia coli reversion assay and the mouse lymphoma L5178Y TK+/- forward mutation assay.

The hair-dye ingredients, HC Blue No. 1 (HCB1) and HC Blue No. 2 (HCB2), were tested for the induction of bacterial mutation using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100; and Escherichia coli strains WP2uvrA-. In addition, both dyes were evaluated in the mouse lymphoma L5178Y TK+/- assay (MLA) for the potential to induce forward mutation. A liver homogenate (S9) prepared from Aroclor 1254-induced male Fischer 344 rats was used to provide a means for metabolic activation. HCB1 was not mutagenic in the Ames assay, but was weakly mutagenic in the MLA only in the presence of metabolic activation. In contrast, HCB2 was a strong mutagen in the Ames assay in tester strain TA98 both in the presence and absence of metabolic activation. A positive response was also noted with HCB2 in the MLA, both in the presence and absence of metabolic activation. Negative findings from the Ames assay of this study agree with other published results where an identical lot of HCB1 was used. Using the same lot, a weak positive result was observed in the MLA, however, the activation requirements and magnitude of the response were different from that of a lot evaluated by the NTP. In contrast, HCB2 appears to be both a bacterial and mammalian cell mutagen independent of lot variability.

An evaluation of the CHO/HGPRT mutation assay involving suspension cultures and soft agar cloning: results for 33 chemicals.

The Chinese hamster ovary cell assay (CHO), which measures forward mutation of the HGPRT locus, is used in several laboratories for the detection of mutagens. A procedure involving treatment of CHO cells in suspension culture and mutant selection in soft agar cloning has been developed (Oberly TJ, Bewsey BJ, Probst GS (1987): Mutat Res 182:99-111). In order to evaluate the effectiveness of these modifications, 33 chemicals representing six chemical classes were tested, and the results were compared to findings obtained in other tests for genotoxicity at Lilly Research Laboratories (LRL). A positive response was obtained with 21 chemicals, all of which are recognized mutagens. Of the 12 compounds that produced negative results, 4 were considered to be mutagens and/or carcinogens. Twelve of the compounds mentioned in this report have been previously tested in the CHO/HGPRT assay by other laboratories, and the results showed strong agreement between laboratories. These findings support the conclusion that the use of suspension cultures and soft agar cloning in the CHO assay provides a sensitive test for the identification of mutagens and is a viable alternative to the traditional monolayer procedure of O'Neill et al. (O'Neill JP, Couch DB, Machanoff R, San Sebastian JR, Brimer PA, Hsie AW (1977): Mutat Res 45:103-109).

A procedure for the CHO/HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft-agar.

A procedure involving treatment of cells in suspension culture and soft-agar cloning was developed for measuring mutation of Chinese hamster ovary (CHO) cells to 6-thioguanine (6TG) resistance. The use of suspension cultures precluded the need for trypsinization and also permitted a 5-fold increase in cell population for compound exposure and mutant selection as compared to former monolayer techniques. Soft-agar cloning reduced the opportunity for metabolic cooperation and permitted the use of non-dialyzed fetal calf serum which resulted in spontaneous mutant frequencies of 6.6 +/- 3.2 X 10(-6) and cloning efficiencies of 91 +/- 18%. Relative total growth values were calculated based on suspension growth and cloning efficiencies such that an assessment of toxicity could be estimated from treatment through cloning. Dose-dependent mutagenic responses were observed in CHO cells following treatment with ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline-1-oxide, methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Clones of 6TG-resistant cells harvested from soft agar maintained 6TG resistance and methotrexate sensitivity and did not incorporate [3H]hypoxanthine into DNA. These preliminary findings indicate that the use of suspension cultures and soft-agar cloning has improved the efficiency and cost effectiveness of the CHO/HGPRT mutation assay.

Thymidine kinase activity and trifluorothymidine resistance of spontaneous and mutagen-induced L5178Y cells in RPMI 1640 medium.

L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants.

Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays.

Ochratoxin A (OA), a nephrotoxic mycotoxin, was evaluated for genotoxic potential in a battery of in vitro and in vivo assays. OA was not mutagenic to Salmonella typhimurium, either with or without metabolic activation, in the plate incorporation (Ames) test at concentrations of 50-600 micrograms OA/plate or in the gradient plate assay at concentrations of 0.1-1000 micrograms OA/ml. No induction of unscheduled DNA synthesis was evident in primary cultures of rat hepatocytes exposed to concentrations of OA ranging from 0.000025 to 500 micrograms/ml. In the mouse lymphoma forward mutation assay, exposure of L5178Y TK+/- mouse lymphoma cells to OA did not increase the numbers of L5178Y TK-/- mutants. There was no significant difference between the numbers of sister-chromatid exchanges in cells from OA-treated Chinese hamsters and those in cells from the negative-control animals.

An evaluation of the L5178Y TK+/- mouse lymphoma forward mutation assay using 42 chemicals.

The L5178YTK+/- mouse lymphoma assay (MLA) has been utilized in several laboratories as a short-term test for chemical-induced forward mutation in cultured mammalian cells. In order to evaluate several technical modifications to the MLA, 42 chemicals representing 9 chemical classes were tested and the results were compared with those published elsewhere as well as with findings in a genetic toxicology test battery currently used in this laboratory. A positive response for the induction of TK-/- mutants was obtained for 26 chemicals. With the exception of p-aminophenol, all of these compounds were recognized mutagens or carcinogens and were representative of direct-acting and activation-dependent genotoxins. 16 compounds did not induce TK-/- mutants and among these were 5 compounds that were considered to be mutagens or carcinogens. A comparison of the results of this study with those published elsewhere revealed a strong agreement among findings for this test irrespective of minor technical variations. It was concluded that the MLA is a useful system for identifying chemical mutagens in mammalian cells and can serve as a valuable component in a genetic toxicology test battery.

Comparison of three in vitro assays for carcinogen-induced DNA damage.

DNA damage induced by five ultimate carcinogens and five procarcinogens was evaluated by three methods: (1) DNA strand breaks in Crandall feline kidney (CRFK) cells measured by alkaline sucrose gradient centrifugation; (2) unscheduled DNA synthesis (UDS) in CRFK, Syrian hamster embryo (SHE), and rat hepatocyte cultures measured by liquid scintillation counting; and (3) UDS in CRFK, SHE, and rat hepatocyte cultures measured by autoradiography. DNA strand breaks were observed with three procarcinogens and four ultimate carcinogens. Only two procarcinogens and two ultimate carcinogens could be identified by liquid scintillation counting. A positive autoradiographic response for UDS was observed in CRFK cells with four of five ultimate carcinogens and one procarcinogen. SHE cells showed a positive autoradiographic response for UDS with all ultimate carcinogens and three procarcinogens. The autoradiographic system with primary rat hepatocyte cultures was the only one in which a positive response for DNA damage was elicited for all 10 carcinogens.