David P. Gibson

The pH 6.7 Syrian hamster embryo cell transformation assay for assessing the carcinogenic potential of chemicals

Cell transformation models have been established for studying the cellular and molecular basis of the neoplastic process. Transformation models have also been utilized extensively for studying mechanisms of chemical carcinogenesis and, to a lesser degree, screening chemicals for their carcinogenic potential. Complexities associated with the conduct of cell transformation assays have been a significant factor in discouraging broad use of this approach despite their reported good predictivity for carcinogenicity. We previously reported that many of the experimental difficulties with the Syrian hamster embryo (SHE) cell transformation assay could be reduced or eliminated by culturing these cells at pH 6.7 culture conditions compared to the historically used pH 7.1-7.3. We and others have shown that morphological transformation (MT), the earliest recognizable phenotype in the multi-step transformation process and the endpoint used in the standard assay to indicate a chemicals transforming activity, represents a pre-neoplastic stage in this model system. In the collaborative study reported here, in which approx. 50% of the chemicals were tested under code in one laboratory (Hazelton) and the other 50% evaluated by several investigators in the second laboratory (P & G), we have evaluated 56 chemicals (30 carcinogens, 18 non-carcinogens, 8 of inconclusive carcinogenic activity) in the SHE cell transformation assay conducted at pH 6.7 culture conditions with a standardized, Good Laboratory Practices-quality protocol. An overall concordance of 85% (41/48) between SHE cell transformation and rodent bioassay results was observed with assay sensitivity of 87% (26/30) and specificity of 83% (15/18), respectively. The assay exhibited a sensitivity of 78% (14/18) for Salmonella assay negative carcinogens, supporting its value for detecting non-mutagenic carcinogens. For maximum assay sensitivity, two exposure durations were required, namely a 24-h exposure and a 7-day exposure assay. Depending on the duration of chemical treatment required to induce transformation, insight into the mechanism of transformation induction may also be gained. Based on the data reported here, as well as the larger historical dataset reviewed by Isfort et al. (1996), we conclude that the SHE cell transformation assay provides an improved method for screening chemicals for carcinogenicity relative to current standard genotoxicity assays.

SFTG international collaborative study on in vitro micronucleus test: III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using mouse lymphoma L5178Y cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, 5-fluorouracil, colchicine and griseofulvin. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in mouse lymphoma L5178Y cells. Mannitol was the only exception, being tested in only one laboratory. Mannitol was negative, while bleomycin induced a concentration-dependent increase in micronucleated cells. Equivocal results were obtained for 5-fluorouracil, colchicine and griseofulvin. High levels of cytotoxicity interfered with the assessment of aneuploidy for colchicine and griseofulvin, preventing the ability to obtain clear results in all the treatment schedules. Experiments with 5-fluorouracil, colchicine and griseofulvin showed that both short and long treatment times are required as each compound was detected using one or more treatment protocol. No clear differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. It was also found that a recovery period may help to detect compounds which induce a genotoxicity associated to a reduction in cell number or cell proliferation. Overall, the results of the present study show that mouse lymphoma L5178Y cells are suitable for the in vitro micronucleus assay.

Sodium fluoride-induced chromosome aberrations in different stages of the cell cycle : a proposed mechanism

In an attempt to clarify the controversy about sodium fluoride (NaF) clastogenicity, the induction of chromosome aberrations in Chinese hamster ovary cells (CHO) by NaF was investigated. Following a protocol used for screening chemicals for clastogenic activity, significant increases of aberrant cells were observed when cells were exposed to NaF for 4 h and harvested 8 h later. Cell-cycle kinetic studies demonstrated most cells were exposed in G2 of the cell cycle. Smaller increases in aberrant cells were observed when cells were harvested 20 h later (most cells were exposed in G1/S). The sensitivity of G2 cells to NaF was investigated further, along with the induction of aberrations at low doses. The results indicated that G2 cells are sensitive to NaF and the percent of aberrant cells increased with dose and length of exposure. With a 3-h exposure until harvest, no statistically significant increase in aberrant cells was observed at doses below 10 micrograms/ml NaF. These data are consistent with a threshold for NaF-induced clastogenicity around 10 micrograms/ml, as has been proposed previously (Scott and Roberts, 1987). It thus may be predicted that clastogenic effects would not occur in humans exposed to the levels of fluoride that are present in drinking water or dentifrices. An understanding of the mechanism of NaF-induced clastogenicity would help to clarify this point. It has previously been reported that NaF inhibits DNA synthesis/repair. The types of aberrations, mostly deletions and gaps, the induction of endoreduplicated cells, the cell-cycle delay and the sensitivity of G2 cells to NaF observed are similar to that reported in the literature for DNA synthesis/repair inhibitors like aphidicolin (APC). Similarities in the induction of aberrations by NaF and APC were confirmed in experiments with G2 cells. Based on these results and those previously reported for NaF and APC, it is proposed that NaF-induced aberrations may occur by an indirect mechanism involving the inhibition of DNA synthesis/repair.

Induction of micronuclei in Syrian hamster embryo cells: comparison to results in the SHE cell transformation assay for National Toxicology Program test chemicals.

Sixteen chemicals currently being tested in National Toxicology Program (NTP) carcinogenicity studies were evaluated in the Syrian hamster embryo (SHE) cell in vitro micronucleus assay. Results from these studies were compared to the results from the SHE cell transformation assay for the same chemicals The overall concordance between induction of micronuclei and transformation of SHE cells was 56%, which is far lower that the 93% concordance between these two tests reported previously by Fritzenschaf et al. (1993; Mutation Res. 319, 47-53). The difference between our results appears to be due to differences in the types of chemicals in the two studies. Overall, there is good agreement between the SHE cell micronucleus and transformation assays for mutagenic chemicals, but, as our study highlights, the SHE cell transformation assay has the added utility of detecting nonmutagenic carcinogens. The utility of a multi-endpoint assessment in SHE cells for carcinogen screening is discussed.

Detection of aneuploidy-inducing carcinogens in the Syrian hamster embryo (SHE) cell transformation assay

As evidenced by the recent report of the Commission of the European Communities (CEEC) project (Detection of Aneugenic Chemicals-CEEC project, 1993), there currently is a great deal of effort towards developing and validating assays to detect aneuploidy-inducing chemicals. In this report, we describe the utility of the Syrian hamster embryo (SHE) cell transformation assay for detecting carcinogens with known or suspected aneuploidy-inducing activity. The following carcinogens were tested: asbestos, benomyl, cadmium chloride, chloral hydrate, diethylstilbestrol dipropionate, and griseofulvin. Thiabendazole, a noncarcinogen, was also tested. Chemicals of unknown or inconclusive carcinogenicity data, colcemid, diazepam, econazole nitrate, and pyrimethamine were also evaluated. All of the above chemicals except thiabendazole induced a significant increase in morphological transformation (MT) in SHE cells. Based on these results as well as those published in the literature previously, the SHE cell transformation assay appears to have utility for detecting carcinogens with known or suspected aneuploidy-inducing ability.

Tissue-specific mutant frequencies and mutational spectra in cyclophosphamide-treated lacI transgenic mice

The induction and nature of mutations in the lacI transgene were evaluated in multiple tissues after exposure of adult male B6C3F1 lacI transgenic mice to cyclophosphamide (CP). Mice were given a single i.p. injection of 25 mg CP/kg, 100 mg CP/kg, or vehicle (PBS) and then necropsied 6 weeks after treatment to allow DNA extraction and lacI mutant recovery. Tissues evaluated included target tissues for tumorigenesis (lung, urinary bladder) and sites not susceptible to tumor formation in B6C3F1 mice (kidney, bone marrow, splenic T‐lymphocytes). After exposure to the high dose of CP, a significant increase in the mutant frequency (Mf) was detected in the lungs and urinary bladders, compared to the respective tissues from vehicle‐treated controls. In contrast, the Mfs in kidney, bone marrow, and splenic T cells from CP‐treated mice were not significantly different from controls. The spectra of mutations in lacI from lung and urinary bladder were significantly changed after high‐dose CP treatment, with a significant increase in the frequency of A · T → T · A transversions found in both tissues and a significantly elevated frequency of deletions in the lungs. Conversely, in vehicle‐treated mice, the two predominant classes of lacI mutations recovered in lung and urinary bladder were G · C → A · T transitions at CpG sites and G · C → T · A transversions. These CP exposures were also genotoxic as measured by the significant induction of micronuclei in peripheral blood 48 hr after exposure. These data indicate that under these study conditions, CP‐induced mutations are detectable in the lacI transgene in the target tissues, but not in nontarget tissues for CP‐induced cancer. With the lacI assay it is possible to study mutagenicity in a variety of critical tissues to provide mechanistic information related to genotoxicity and carcinogenicity in vivo. Environ. Mol. Mutagen. 34:154–166, 1999

A refined protocol for conducting the low pH 6.7 Syrian hamster embryo (SHE) cell transformation assay

The Syrian hamster embryo (SHE) cell transformation assay evaluates the potential of chemicals to induce morphological transformation in karyotypically normal primary cells. Induction of transformation has been shown to correlate well with the carcinogenicity of many compounds in the rodent bioassay. Historically the assay has not received wide-spread use due to technical difficulty. An improved protocol for a low pH 6.7 assay was developed by LeBoeuf et al. [R.A. LeBoeuf, G.A. Kerckaert, M.J. Aardema, D.P. Gibson, R. Brauninger, R.J. Isfort, Mutat. Res., 356 (1996) 85-127], that greatly reduced many of the technical difficulties associated with the SHE assay. The purpose of this paper is to describe the most current execution of the pH 6.70 protocol including protocol refinements made since the publication of a comprehensive protocol for this assay in Kerckaert et al. [G.A. Kerckaert, R.J. Isfort, G.J. Carr, M.J. Aardema, Mutat. Res., 356 (1996) 65-84].

Comparative genotoxicity of quinolone and quinolonyl‐lactam antibacterials in the in vitro micronucleus assay in Chinese hamster ovary cells

The in vitro micronucleus assay is gaining increased attention as a potential alternative to the standard in vitro metaphase analysis assay. In particular, the in vitro micronucleus assay has been proposed as a useful method for chemicals that induce both structural and numerical chromosome alterations, such as DNA gyrase/topoisomerase inhibitors. In this study, we compared the micronucleus‐inducing activity of quinolonyl‐lactam antibacterials that inhibit DNA‐gyrase and bind to penicillin‐binding proteins relative to the activity of structurally related quinolone antibacterials that also inhibit DNA‐gyrase. All of the quinolones that were structurally related to the quinolonyl‐lactams were cytotoxic and induced large increases in the frequency of micronucleated binucleated cells (MNBC) at concentrations between 0.02 and 0.16 mM. These changes were larger than those seen with the commercial quinolones, ciprofloxacin (cytotoxic at ≥ 0.57 mM and MNBC at ≥ 0.3 mM) and nalidixic acid (cytotoxic at 1.8 mM and no MNBC up to this dose). In contrast, the quinolonyl‐lactams were not cytotoxic up to 1.0 mM concentrations and induced either no MNBC or a low frequency of MNBC at higher concentrations compared to the quinolones. Quinolonyl‐lactams appear to be less cytotoxic and genotoxic than structurally related quinolones. These results add to the growing database on the in vitro micronucleus assay in general, and more specifically to the relatively small database for the in vitro micronucleus assay in Chinese hamster ovarycells. Environ. Mol. Mutagen. 31:345–351, 1998.

Evaluation of lacI mutation in germ cells and micronuclei in peripheral blood after treatment of male lacI transgenic mice with ethylnitrosourea, isopropylmethane sulfonate or methylmethane sulfonate.

Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study. Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle. Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations. The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213). Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries. As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice. The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%). In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995). These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment. Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells.

Aneuploidy and Consistent Structural Chromosome Changes Associated with Transformation of Syrian Hamster Embryo Cells

To gain a better understanding of the role of specific numerical and structural chromosome changes in the multistage process of transformation of Syrian hamster embryo (SHE) cells, we analyzed seven benzo(a)pyrene (BP)-induced immortal SHE cell lines, and one spontaneously immortalized cell line. In addition, we analyzed chromosome changes in early passage tumor-derived cell lines induced by injection of four immortalized cell lines into neonate hamsters. Of particular interest was the observation of a deletion in the short arm of chromosome 2 in four of the seven BP-immortalized cell lines. Other types of alterations in chromosome 2 were observed in two other cell lines. Loss of one copy of chromosome 16 was also observed in more than 90 to 100% of the cells in three of seven BP-immortalized cell lines. In contrast, the only chromosome alteration seen in the spontaneously immortalized cell line was a deletion in the short arm of chromosome 20. Genetic instability, as indicated by increased numerical or structural chromosome changes, was observed in all tumor-derived cell lines compared to the immortal cell line from which they originated. These results, along with previous reports in the literature, suggest that alterations in specific chromosomes, like chromosome 2, may be involved in transformation of SHE cells.