Syrian Hamster Embryo Assay Research Articles

In Vitro-In Vivo Carcinogenicity.

The evaluation of the carcinogenic potential of chemicals constitutes an essential step in assessing the risk that the chemicals pose to human health. The "gold standard" method to evaluate the carcinogenic potential of chemicals is the carcinogenicity test in laboratory animals. However, because carcinogenicity studies in vivo are extremely time-consuming, expensive, make use of a high number of animals, and cannot be used to screen a high number of compounds at the same time, various different in vitro cell transformation assays have been developed. In this report, procedures to test the carcinogenicity in vivo and in vitro are described, whereby in the latter case three extensively evaluated test systems (the BALB/c 3T3 cell transformation assay, the Bhas 42 cell transformation assay, and the Syrian hamster embryo assay) are presented. Their performance shows that they are a useful complement to in vitro genotoxicity test batteries, can be used to identify non-genotoxic carcinogens, and as screening assays may significantly limit the number of chemicals to undergo an in vivo carcinogenicity testing, thereby strongly reducing the number of laboratory animals to be used. In the future, the development of human cell line-based transformation assays may contribute to increase further their relevance and the willingness to incorporate them into existing in vitro toxicity test batteries.

Classification of agents using Syrian hamster embryo (SHE) cell transformation assay (CTA) with ATR-FTIR spectroscopy and multivariate analysis.

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzo[e]pyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzo[a]pyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fisher's linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents.

Miniaturization of cytotoxicity tests for concentration range-finding studies prior to conducting the pH 6.7 Syrian hamster embryo cell-transformation assay

The Syrian hamster embryo (SHE) cell-transformation assay (SHE assay) is a promising alternative method to animal testing for the identification of potential carcinogens in vitro. Prior to conducting the SHE assay the appropriate concentration range for each test chemical must be established, with a maximum concentration causing approximately 50% cytotoxicity. Concentration range-finding is done in separate experiments, which are similar to the final SHE assay but with less replicates and more concentrations. Here we present an alternative for the cytotoxicity testing by miniaturization of the test procedure by use of 24-well plates and surpluses from feeder-cell preparations as target cells. In addition, we integrated the photometry-based neutral red (NR) assay. For validation of the assay, incubations with dimethyl sulf-oxide, p-phenylenediamine–2HCl, aniline, o-toluidine–HCl, 2,4-diaminotoluene, and 2-naphthylamine were carried out in the miniaturized approach and compared with the standard procedure in terms of calculating the relative plating efficiencies (RPEs). To directly compare both methods, concentrations that produced 50% cytotoxicity (IC50) were calculated. Excellent associations were observed between the number of colonies and NR uptake. For all test substances a concentration-dependent, concomitant decrease of NR uptake in the miniaturized approach and RPEs in the standard test was observed after a 7-day incubation. The results from both test setups showed a comparable order of magnitude and the IC50 values differed by a factor <2 (1.4–1.9), depending on the substance in question. Overall, the miniaturized approach should be considered an improved alternative for cytotoxicity testing in the SHE assay, as it saves valuable SHE cells and speeds-up the time, to obtain test results more rapidly.

Classification of test agent-specific effects in the Syrian hamster embryo assay (pH 6.7) using infrared spectroscopy with computational analysis

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has utility in the assessment of potential chemical carcinogenicity (both genotoxic and non-genotoxic mechanisms of action). The assay uses morphological transformation as an end point and has a reported sensitivity of 87%, specificity of 83% and overall concordance of 85% with in vivo rodent bioassay data. However, the scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with benzo[a]pyrene, 3-methylcholanthrene, anthracene, N-nitroso-N-methylnitroguanidine, ortho-toluidine HCl, 2,4-diaminotoluene or D-mannitol for 7 days before fixation with methanol. Identified colonies were interrogated by acquiring a minimum of five infrared (IR) spectra per colony using attenuated total reflection Fourier-transform IR spectroscopy. Individual IR spectra were acquired over a spatial area of approximately 250 × 250 μm. Resultant data were analysed using Fisher's linear discriminant analysis and feature histogram algorithms to extract classifying biomarkers of test agent-specific effects or transformation in SHE cells. Clustering of spectral points suggested co-segregation or discrimination of test agent categories based on mechanism of action. Towards transformation, unifying alterations were associated with alterations in the Amide I and Amide II peaks; these were consistently major classifying biomarkers for transformed versus non-transformed SHE cells. Our approach highlights a novel method towards objectively screening and classifying SHE cells, be it to ascertain test agent treatment based on mechanism of action or transformation.

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7 for assessment of carcinogenic potential of chemicals

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is an important in vitro method that is highly predictive of rodent carcinogenicity. It is a key method for reducing animal usage for carcinogenicity prediction. The SHE assay has been used for many years primarily to investigate and identify potential rodent carcinogens thereby reducing the number of 2-year bioassays performed in rodents. As for other assays with a long history of use, the SHE CTA has not undergone formal validation. To address this, the European Centre for the Validation of Alternative Methods (ECVAM) coordinated a prevalidation study. The aim of this study was to evaluate the within-laboratory reproducibility, test method transferability, and between-laboratory reproducibility and to develop a standardised state-of-the-art protocol for the SHE CTA at pH 6.7. Formal ECVAM principles for criteria on reproducibility (including the within-laboratory reproducibility, the transferability and the between-laboratories reproducibility) were applied. In addition to the assessment of reproducibility, this study helped define a standard protocol for use in developing an Organisation for Economic Co-operation and Development (OECD) test guideline for the SHE CTA. Six compounds were evaluated in this study: benzo(a)pyrene, 3-methylcholanthrene, o-toluidine HCl, 2,4-diaminotoluene, phthalic anhydride and anthracene. Results of this study demonstrate that a protocol is available that is transferable between laboratories, and that the SHE CTA at pH 6.7 is reproducible within- and between-laboratories.

The Syrian hamster embryo (SHE) assay (pH 6.7): mechanisms of cell transformation and application of vibrational spectroscopy to objectively score endpoint alterations

Using morphological transformation as an endpoint, the Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) is an in vitro system with a high sensitivity and specificity for testing the carcinogenic potential of test agents. Advantages of the assay are that SHE cells are metabolically competent, genetically stable and acquire spontaneous transformation with a low frequency; additionally, it detects both genotoxic and non-genotoxic carcinogens. However, in comparison with other short-term mammalian cell assays, it is time consuming, laborious and, most importantly, the visual scoring of morphological transformation might be subjective. In this review, we examine the background to the test and why it has the potential for use in safety risk assessment. Additionally, we propose a novel approach to objectively interrogate and classify SHE colonies using vibrational spectroscopy coupled to a mathematical framework for high-throughput screening. It is our view that this alternative approach has the potential to improve the sensitivity and specificity of the in vitro SHE assay.

Syrian hamster embryo (SHE) assay (pH 6.7) coupled with infrared spectroscopy and chemometrics towards toxicological assessment

The Syrian hamster embryo (SHE) assay (pH 6.7) is an in vitro candidate to replace in vivo carcinogenicity tests. However, the conventional method of visual scoring of foci (non-transformed vs. transformed colonies) can be time-consuming and is open to subjectivity. Infrared (IR) spectroscopy has the potential to provide objective assessment of such SHE colonies with the added advantage of potentially providing mechanistic information. In this study, SHE cells were treated with one of eight different chemical regimens, allowed in culture to attach and form foci on IR-reflective glass slides; these were subsequently interrogated by attenuated total reflection (ATR) Fourier-transform IR (FTIR) spectroscopy. Derived mid-IR spectra (n = 13,406) were subjected to chemometric analysis focusing primarily on the extraction of biochemical information related to test agent treatment and/or morphological transformation. The use of ATR-FTIR spectroscopy with chemometrics to analyze the SHE assay is a novel approach to toxicological assessment.

Phenolphthalein and Bisacodyl: Assessment of Genotoxic and Carcinogenic Responses in Heterozygous p53 (+/−) Mice and Syrian Hamster Embryo (SHE) Assay

Phenolphthalein (800 and 2400 mg/kg/day by gavage and 2400 mg/kg/day by diet) and bisacodyl (800-500, 4000-2000, and 8000 mg/kg/day by gavage) were administered to 15 male and 15 female and 20 male and 20 female p53(+/-) mice respectively for 26 weeks to investigate the potential carcinogenicity of each compound. Toxicokinetic analyses confirmed systemic exposure. p-Cresidine was administered by gavage (400 mg/kg/day) and served as the positive control agent in each study. Dietary phenolphthalein reduced survival in both sexes and early deaths were attributed to thymic lymphoma. No bisacodyl-related neoplasms were observed. Regardless of route of administration to p53(+/-) mice, phenolphthalein but not bisacodyl was unequivocally genotoxic, causing increased micronuclei in polychromatic erythrocytes. In the Syrian hamster embryo (SHE) cell transformation assay, phenolphthalein caused increases in morphologically transformed colonies, thereby corroborating NTP's earlier reports, showing phenolophthalein has potential carcinogenic activity. Bisacodyl was negative in the SHE assay. Results of these experiments confirm an earlier demonstration that dietary phenolphthalein causes thymic lymphoma in p53(+/-) mice and show that (1) phenolphthalein causes qualitatively identical results in this transgenic model regardless of route of oral administration, (2) phenolphthalein shows evidence of micronucleus induction in p53(+/-) mice for up to 26 weeks, (3) phenolphthalein induced transformations in the in vitro SHE assay, and (4) bisacodyl in p53(+/-) mice induces neither drug-related neoplasm, nor micronuclei in polychromatic erythrocytes, and did not induce transformations in the in vitro SHE assay.

The results of five coded compounds: genistein, metaproterenol, rotenone, p-anisidine and resorcinol tested in the pH 6.7 Syrian hamster embryo cell morphological transformation assay

The pH 6.7 Syrian hamster embryo (SHE) cell morphological transformation assay is a short-term in vitro test that has been used to predict rodent carcinogenicity. Previous reports have indicated that the SHE assay has an overall concordance of approximately 80% with the 2 year rodent bioassay. We selected five compounds, genistein, metaproterenol, rotenone, p-anisidine and resorcinol, that had extensive genotoxicity and carcinogenicity data and tested them in the standard 7 day exposure SHE assay. Somewhat surprisingly, the SHE assay misclassified the actual rodent carcinogenicity of four out of the five test compounds. It is difficult to explain these findings as the actual mechanisms of SHE cell morphological transformation are currently unknown. However, it is obvious that in these studies there was no simple correlation between in vitro genotoxicity, morphological transformation in SHE cells and rodent carcinogenicity. Clearly, further research is required to accurately assess the role of the SHE assay in the carcinogenic risk assessment of new chemical entities.

The testing of chemicals in the Syrian hamster embryo (SHE) cell transformation assay for assessment of carcinogenic potential

The Syrian hamster embryo (SHE) cell transformation assay was used to test 28 chemical substances for their ability to induce morphologically transformed colonies. The purpose was to determine how well the assay method could be transferred from an experienced laboratory by including 18 chemicals previously evaluated and 10 new chemicals. Technical training was obtained in the experienced lab prior to testing. The assay was conducted at pH 6.7, a treatment period of 7 days was used, and single experiments were performed for each chemical. With this limited testing, 78% concordance with rodent bioassay results was obtained, and this high concordance would have increased if small, but statistically negative responses from single trials were overturned by positive data from repeat trials. Similarly, the results were highly concordant (90%) with the experienced lab results; only 2 chemical evaluations were discordant, and the use of repeat experiments would likely have eliminated these apparent disagreements. Thus, with appropriate training, the pH 6.7 SHE assay was successfully and reliably transferred.

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].

Oxymetholone: I. Evaluation in a comprehensive battery of genetic toxicology and in vitro transformation assays.

Oxymetholone is generally assumed to be a nongenotoxic carcinogen. This assumption is based primarily on the results of an Ames test, existing data in repeat-dose toxicology studies, and the predicted results of a 2-yr National Toxicology Program (NTP) rat carcinogenicity bioassay. To provide a comprehensive assessment of its genotoxicity in a standard battery of mutagenicity assays, oxymetholone was tested in microbial and mammalian cell gene mutation assays, in an in vitro cytogenetics assay (human lymphocytes), and in an in vivo micronucleus assay. Oxymetholone was also tested in an in vitro morphologic transformation model using Syrian hamster embryo (SHE) cells. These studies were initiated and completed prior to the disclosure of the results of the NTP bioassay. Oxymetholone was tested at doses up to 5,000 microg/plate in the bacterial plate incorporation assay using 4 Salmonella strains and the WP2 uvrA (pKM101) strain of Escherichia coil. There was no induction of revertants up to the highest dose levels, which were insoluble as well as toxic. In the L5178Y tk+/- mouse lymphoma assay, doses up to 30 microg/ml reduced relative survival to approximately 30% with no increase in mutants. Male or female human lymphocytes were exposed in vitro to oxymetholone for 24 hr without S9 or 3 hr with S9 and evaluated for the induction of chromosomal aberrations. There was no increase in aberration frequency over control levels and no difference between male and female cells. Peripheral blood from Tg.AC transgenic mice treated dermally for 20 wk with 0, 1.2, 6.0, or 12.0 mg/day of oxymetholone and from p53 transgenic mice treated orally by gavage for 26 wk with 125, 625, or 1,250 mg/kg/day of oxymetholone was evaluated for micronuclei in polychromatic and normochromatic erythrocytes. There was no difference in micronuclei frequency between control and treated animals. These results confirm that oxymetholone is not genotoxic in a comprehensive battery of mutagenicity assays. In the SHE assay, oxymetholone produced a significant increase in morphologically transformed colonies at dose levels of 13-18 microg/ml. The lack of genotoxicity of oxymetholone, the positive response in the in vitro transformation assay, and the results of transgenic mouse carcinogenicity assays will provide an interesting perspective on the results of an on-going NTP rat carcinogenicity bioassay.

Use of the Syrian hamster embryo cell transformation assay for determining the carcinogenic potential of heavy metal compounds.

Cobalt sulfate hydrate, gallium arsenide, molybdenum trioxide, vanadium pentoxide, and nickel sulfate heptahydrate were tested in the Syrian hamster embryo (SHE) assay in order to increase the SHE assay database for heavy metals. All five compounds produced significant morphological transformation at one or more doses in a dose-responsive manner. Cobalt sulfate hydrate, gallium arsenide, molybdenum trioxide, and nickel (II) sulfate heptahydrate were all positive with a 24-hr exposure, suggesting direct DNA perturbation. Vanadium pentoxide was negative with a 24-hr exposure, but positive with a 7-day exposure. This pattern of response (24-hr SHE negative/7-day SHE positive) has been seen with other chemicals which have tumor promotion-like characteristics. Since the inception of the use of the SHE cell transformation assay for detecting the neoplastic transformation potential of chemicals, over 42 heavy metal compounds have been tested in this assay. Based on the 24 metal compounds which have been tested in the SHE, Salmonella, and some type of rodent bioassay, the SHE assay is 92% concordant with rodent bioassay carcinogenicity results, including a sensitivity of 95% (21/22) and a specificity of 50% (1/2). At this time, the measure of SHE assay specificity for rodent carcinogenicity of metals is limited by the paucity of metal compounds which are rodent noncarcinogens. The Salmonella assay results are only 33% concordant with the rodent bioassay for these same chemicals. This relatively high concordance between the SHE assay and the rodent bioassay carcinogenicity results demonstrates the utility of the SHE assay for determining the carcinogenic potential of heavy metal compounds in rodent cancer bioassays.

SHE Cell Transformation Assay as a Predictive System for Carcinogenicity of PAH and Non-Genotoxic Carcinogens

Abstract The Syrian hamster embryo (SHE) cell transformation assay is an in vitro method for the detection of genotoxic and non-genotoxic carcinogens. Its reliability as a multistage carcinogen screening method was demonstrated by several authors who showed that cells derived from transformed colonies were capable of tumour formation when inoculated in a suitable host animal. The SHE assay offers the advantage of using normal diploïd cells with metabolizing capacities and low spontaneous transformation frequency. Epithelial cultures can be selected from the mixed cellular suspension obtained from the Syrian hamster embryos in order to develop an in vitro model for the detection of carcinogens inducing carcinomas. Polycyclic aromatic hydrocarbons, such as BaP and DMBA were studied in depth for their cell transforming potency. The synergistic effects of PAH and tumor promoters can be demonstrated by using a sequential treatment consisting in an initiation-promotion scheme1. This protocol also revealed interacting effects between non-genotoxic carcinogens2.

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.

Use of the Syrian Hamster Embryo Cell Transformation Assay for Determining the Carcinogenic Potential of Heavy Metal Compounds

Cobalt sulfate hydrate, gallium arsenide, molybdenum trioxide, vanadium pentoxide, and nickel sulfate heptahydrate were tested in the Syrian hamster embryo (SHE) assay in order to increase the SHE assay database for heavy metals. All five compounds produced significant morphological transformation at one or more doses in a dose-responsive manner. Cobalt sulfate hydrate, gallium arsenide, molybdenum trioxide, and nickel (II) sulfate heptahydrate were all positive with a 24-hr exposure, suggesting direct DNA perturbation. Vanadium pentoxide was negative with a 24-hr exposure, but positive with a 7-day exposure. This pattern of response (24-hr SHE negative/7-day SHE positive) has been seen with other chemicals which have tumor promotion-like characteristics. Since the inception of the use of the SHE cell transformation assay for detecting the neoplastic transformation potential of chemicals, over 42 heavy metal compounds have been tested in this assay. Based on the 24 metal compounds which have been tested in the SHE, Salmonella, and some type of rodent bioassay, the SHE assay is 92% concordant with rodent bioassay carcinogenicity results, including a sensitivity of 95% (21/22) and a specificity of 50% (1/2). At this time, the measure of SHE assay specificity for rodent carcinogenicity of metals is limited by the paucity of metal compounds which are rodent noncarcinogens. The Salmonella assay results are only 33% concordant with the rodent bioassay for these same chemicals. This relatively high concordance between the SHE assay and the rodent bioassay carcinogenicity results demonstrates the utility of the SHE assay for determining the carcinogenic potential of heavy metal compounds in rodent Cancer bioassays.

The Syrian hamster embryo (SHE) cell transformation system: a biologically relevant in vitro model--with carcinogen predicting capabilities--of in vivo multistage neoplastic transformation.

Neoplastic transformation is a multistep process that can be modeled in vitro using Syrian hamster embryo (SHE) cells. SHE cells multistage transformation involves several intermediate stages, including morphological transformation, immortality, acquisition of tumorigenicity, and malignant progression. Analysis of the molecular alterations that occur at each stage indicated that morphological transformation results from both carcinogen-induced irreversible chromosomal/genetic mutations and reversible genetic events, including altered DNA methylation. Morphological transformation results from a block in the cellular differentiation of progenitor and determined stem-like cells in the SHE cell population via alternation in the expression of the H19 tumor suppressor gene and other genes. Immortality results from genetic mutations in growth factor responsiveness, including loss of growth suppression by TGF beta and autocrine growth factor production, and genomic stability, resulting in genomic instability and an increased mutation rate. Acquisition of tumorigenicity involves loss of tumor suppressor gene function, altered mitogenic signal transduction, mutation of oncogenes, acquisition of anchorage independent growth, and chromosomal aberrations. Malignant progression is associated with alterations in extracellular matrix growth characteristics, alterations in cytoskeleton structure, elevated fibrinolytic activity, secretion of proteases, and changes in extracellular matrix protein secretion. Together, these changes model the alterations observed during in vivo neoplastic transformation and possibly explain why the SHE assay, as a carcinogen screening tool, is able to identify carcinogens with a 80 to 85% accuracy.

Morphological transformation of Syrian hamster embryo cells by mezerein

Mezerein (MEZ) has been described as a weak complete tumor promoter but an effective stage II promoter in the mouse skin initiation-promotion tumor model. In this study MEZ produced a strong transformation response when tested under code in the Syrian hamster embryo (SHE) clonal morphological transformation assay. Using a standard 7-day exposure protocol designed to detect complete carcinogens, MEZ was active at non-toxic concentrations, producing a linear response between 0.3 – 10 ng/ml in a log-log plot of transformation activity versus concentration. These concentrations were in the same range as those which had been shown to elicit promotion activity in several in vitro cell culture systems. Our data suggest that the SHE assay has the ability to detect some tumor promoters under the same conditions used to test for complete carcinogens. The possibility that MEZ may possess in vivo carcinogenic activity cannot be excluded.

An interlaboratory evaluation of the Syrian hamster embryo cell transformation assay using eighteen coded chemicals

Eighteen coded chemicals were evaluated in the Syrian hamster embryo (SHE) cell transformation assay in three different laboratories using the same basic experimental protocol with minor modifications. In addition, individual cell and serum sources were selected. Major factors influencing intra-and interlaboratory reproducibility were the source of cells and serum, the toxicity of the chemicals, and the dose-range selected for transformation evaluation. Two or three assays from each laboratory were required to determine the transformation-inducing potential of a chemical because of the low number of transformants scored in any single assay and the difficulty of interpreting morphological variations. Rodent carcinogenicity data were available for 16 of the 18 chemicals tested and the transformation response of 14 of those chemicals was in agreement with the rodent carcinogenicity data (if the positive results are adopted for the four chemicals that produced contradictory results). Four rodent carcinogens, di-(2-ethylhexyl) phthalate, diphenylhydantoin, methapyrilene hydrochloride and o-toluidine hydrochloride, that were negative in the Salmonella/microsome assay, induced morphological transformation in the SHE assay. Although the labour, cost and lack of reproducibility might preclude application of this transformation assay for routine screening, it might, nevertheless, prove valuable for distinguishing between non-mutagenic carcinogens and non-carcinogens.

Transformation of Syrian hamster embryo cells by synfuel mixtures

Existing Syrian hamster embryo (SHE) cell-transformation assay methods were modified to allow the assay of complex synfuel mixtures. A shale oil, high-molecular-weight, high-boiling distillates from two different solvent-refined coal (SRC) processes, and two crude petroleum samples caused transformation of SHE cells. Chemical fractionation of SRC-II heavy distillate and assay of resultant fractions allowed detection of significant transforming activity in chemical fractions, where such activity had not previously been observed. The polyaromatic-hydrocarbon-enriched fraction, neutral aliphatic hydrocarbon fraction, and neutral tar fraction all showed a positive response in the SHE assay. In addition, we confirmed earlier data showing that both the tar fractions (basic and neutral) and the soluble basic fractions from both SRC processes and shale oil contained genetically active materials.