Gary A. Kerckaert

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.

Comparison of the standard and reduced pH Syrian Hamster Embryo (SHE) cell in vitro transformation assays in predicting the carcinogenic potential of chemicals

A comprehensive review of the Syrian Hamster Embryo (SHE) cell transformation literature was performed in order to catalogue the chemical/physical entities which have been evaluated for in vitro cell transformation potential. Both reduced pH (pH 6.7) and standard pH (pH 7.1-7.3) SHE cell testing protocols were considered. Based upon this analysis, over 472 individual chemical/physical agents and 182 combinations of chemical/physical agents have been tested under the standard pH conditions, while over 56 chemical/physical agents have been tested under reduced pH conditions. Of the 472 chemical/physical agents tested at the standard pH, 213 had in vivo carcinogenicity data available. Of these 213 chemical/physical agents, 177 were carcinogens while 36 were non-carcinogens. The results of testing the SHE transformability of these 213 chemical/physical agents indicates that the standard pH SHE cell transformation assay had a concordance of 80% (171/213), a sensitivity of 82% (146/177), and a specificity of 69% (25/36). Of these 213 chemical/physical agents, 53% (112/213) were tested more than once often in more than one laboratory, with a 82% (92/112) interlaboratory agreement rate, thus providing confirmatory results. Carcinogenicity data were available for 48 of the 56 chemical/physical agents tested for SHE cell transformation under the reduced pH conditions. The SHE cell transformation assay under reduced pH conditions had a concordance of 85% (41/48), a sensitivity of 87% (26/30), and a specificity of 83% (15/18). For Salmonella-negative carcinogens, the standard pH SHE assay correctly predicted carcinogenicity 75% (48/64) of the time while the reduced pH SHE assay correctly predicted carcinogenicity for Salmonella-negative carcinogens 78% (14/18) of the time. For chemical/physical agents tested under both the reduced pH and standard pH conditions, the standard pH and reduced pH SHE cell assays had a 69% (22/32) agreement rate. Under the reduced pH conditions, the SHE assay correctly predicted rodent carcinogenicity in 86% (25/29) of the chemicals tested under both reduced and standard pH conditions. Under standard pH conditions, the SHE assay correctly predicted rodent carcinogenicity in 69% (20/29) of the chemicals tested under both reduced and standard pH conditions. Collectively, these data indicate that the SHE cell transformation assay is predictive for rodent carcinogenicity under either reduced or standard pH conditions. Importantly, the assay displays better performance and appears to have improved carcinogen prediction capability under reduced pH conditions.

A comprehensive protocol for conducting the Syrian hamster embryo cell transformation assay at pH 6.70.

Studies from our laboratory have demonstrated several advantages of conducting the Syrian hamster embryo (SHE) cell transformation assay at pH 6.70 compared to that done historically at higher pH values (7.10-7.35). These include reduction of the influence of SHE cell isolates and fetal bovine serum lot variability on the assay, an increase in the frequency of chemically induced morphological transformation (MT) compared to controls, and an increased ease in scoring the MT phenotype. The purpose of this paper is to report a comprehensive protocol for conduct of the pH 6.70 SHE transformation assay including experimental procedures, a description of criteria for an acceptable assay and statistical procedures for establishing treatment-related effects. We have also identified several assay parameters in addition to pH which can affect transformation frequencies, particularly the critical role colony number per plate can have on transformation frequency. Control of this parameter, for which details are provided, can greatly increase the reproducibility and predictive value of the assay.

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.

ROLE OF THE H19 GENE IN SYRIAN HAMSTER EMBRYO CELL TUMORIGENICITY

Carcinogen‐induced transformation in Syrian hamster embryo (SHE) cells is a multistage process characterized by specific genetic alterations at each stage in the transformation process. Loss of H19 gene expression is one of the earliest events observed, occurring in approximately 75% of the morphologically transformed cells and the subsequently derived tumorigenic cells. To investigate the effect the loss of H19 expression has on SHE cell tumorigenicity, H19 expression was reestablished in a tumorigenic SHE cell lineage that lacked H19 expression. H19 reexpression had little effect on cellular growth in vitro but did retard tumor growth in nude mice. Analysis of the tumors that did develop from cells containing the H19 gene indicated that loss of exogenous H19 gene expression was probably due to changes in DNA methylation. These results demonstrate that alterations in H19 gene expression play an important role in SHE cell tumorigenicity. Mol. Carcinog. 20:189–193, 1997.

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.

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.