Mutagenesis Advance Access originally published online on January 25, 2005
Mutagenesis 2005 20(1):51-56; doi:10.1093/mutage/gei009
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Mutagenesis vol. 20 no. 1 © UK Environmental Mutagen Society 2005; all rights reserved.
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
Genetic Toxicology, GlaxoSmithKline, Park Road, Ware, Hertfordshire SG12 0DP, UK
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Abstract |
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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
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. |
Introduction |
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The pH 6.7 Syrian hamster embryo (SHE) cell morphological transformation assay is a short-term in vitro test which is claimed to predict rodent carcinogenicity (LeBoeuf et al., 1996
). The SHE assay measures morphological cell transformation, an endpoint that has been reported to correlate well with carcinogenicity in the 2 year rodent bioassay (LeBoeuf et al., 1990, 1996). The SHE assay is unique among in vitro assays in that it reportedly detects both genotoxic and non-genotoxic rodent carcinogens (LeBoeuf et al., 1996). Recent reviews of the SHE assay have demonstrated that it possesses a high level of concordance (80%) with carcinogenicity in the rodent bioassay (Myhr and Zhang, 2000). This high level of concordance has recently led to some regulatory agencies requesting a SHE assay to assess the carcinogenic potential of new chemical entities that are positive in standard in vitro genotoxicity tests (Bigger, 2003). However, as the actual mechanisms of SHE cell morphological transformation are not understood, it can be difficult to assess the relevance of the SHE assay data in the carcinogenic risk assessment process (Farmer, 2002). The re-emergence of the SHE assay in the field of carcinogenic risk assessment led us to conduct a series of studies to evaluate its ability to predict a compound's carcinogenicity in rodents. We selected five compounds—genistein, metaproterenol, resorcinol, rotenone and p-anisidine—for evaluation in the SHE assay. Although the compounds had extensive published genotoxicity, short term transgenic carcinogenicity and standard 2 year rodent carcinogenicity data (Table I), none of the compounds had any published 7 day SHE assay data. All five compounds were therefore tested using the standard 7 day continuous treatment version of the SHE assay that would satisfy the current regulatory expectations. Here, we present the results of the SHE assay for these five compounds and discuss the results in the context of the use of the SHE assay in carcinogenic risk assessment.
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Materials and methods |
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Chemicals
Genistein (100% pure), metaproterenol hemisulfate (99.9% pure), p-anisidine (98.8% pure), resorcinol (99.9% pure), rotenone (96% pure) and benzo[a]pyrene were purchased from Sigma Chemical Company. All chemicals were of the highest purity available.
SHE cell morphological transformation assay
The SHE assays were conducted at Covance Laboratories (Vienna, VA) in compliance with OECD, FDA and MHLW GLP regulations. The SHE assays, performed at pH 6.7, were based upon procedures described previously (Kerckaert et al., 1996). All compounds were coded and tested blind using the standard 7 day continuous treatment version of the assay.
Briefly, SHE cells were cultured in Le Boeuf's modification of DMEM supplemented with 20% fetal bovine serum and 4 mM L-glutamine. The sodium bicarbonate content of this medium was 0.75 g/l in order to maintain a pH of 6.65–6.75. In all tests, the cells were cultured at 37 ± 1°C in a humidified atmosphere of 10 ± 0.5% CO2.
Initial dose range finding experiments were conducted to determine the cytotoxicity of the five compounds in SHE cells. Target SHE cells expected to generate 25–45 colonies per culture dish were added to irradiated feeder SHE cells that had been seeded 24 h earlier. These target and feeder SHE cell cultures were incubated for a further 24 h prior to treatment with each test article. Ten cultures were prepared for each concentration of each test article and each concurrent vehicle control. After the incubation period, the cultures were washed with Hanks' balanced salt solution and the colonies were fixed with methanol and stained with 10% aqueous Giemsa. The SHE cell colonies were counted to determine the average number of colonies per culture dish for the vehicle control and each treatment group. This information was used to calculate the relative plating efficiency (i.e. the average number of colonies per treatment compared to the vehicle) to provide a measure of the cytotoxicity.
The results of these experiments were used to select an appropriate range of test concentrations for each compound in the SHE assay. The SHE assay was carried out as described previously, with the exception that 45 culture dishes were prepared for each treatment group and the target SHE cell population was adjusted to maintain an average of 35 ± 10 colonies per dish for each concentration. The SHE assay was considered acceptable if at least one concentration of the test article caused an 50% decrease in relative plating efficiency or relative colony density. If the test article was non-cytotoxic, then the compound was tested up to a maximum concentration of 10 mM. At least five treatment groups were selected for SHE cell morphological transformation evaluation. Morphologically transformed colonies were identified by light microscopy using the criteria outlined in previous studies (Kerckaert et al., 1996). Dimethyl sulfoxide or culture media was used as the vehicle control and benzo[a]pyrene was used as the positive control in all of the SHE assays.
Statistics and assay evaluation criteria
Data were analysed using standard procedures outlined in previous studies (Kerckaert et al., 1996). A Fisher's exact test was conducted to determine whether treatment groups were considered significantly different (P 
0.05) from the vehicle control. In addition, if one dose group was positive, a stratified Cochran–Armitage trend test was performed to determine if there was a statistically significant (P 0.05) positive dose-related response.
A compound was defined as positive if the test article induced a statistically significant increase in the morphological transformation frequency at two or more concentrations compared with the concurrent vehicle control. In addition, the test article was considered positive if one concentration showed a statistically significant increase and there was a statistically significant positive dose-related response. A compound was considered negative if it failed to match either of the criteria defined for a positive result.
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Results |
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Genistein was negative in the SHE assay (Table II). The compound did not induce an increase in the frequency of morphological transformation at any of the concentrations tested (2–4 µg/ml). The maximum concentration tested was limited by cytotoxicity. Metaproterenol was negative in the SHE assay (Table III). The compound did not induce an increase in the frequency of morphological transformation at any of the concentrations tested (132–2112 µg/ml). The maximum concentration tested was limited to 10 mM (in accordance with standard in vitro genetic toxicology regulatory guidelines). Rotenone was positive in the SHE assay (Table IV). The compound was tested at a range of concentrations (0.0025–0.02 µg/ml) and a statistically significant increase (P < 0.05) in the frequency of morphological transformation was observed at 0.005 and 0.01 µg/ml. The maximum concentration tested was limited by cytotoxicity. p-Anisidine was positive in the SHE assay (Table V). The compound was tested at a range of concentrations (6.1–18.5 µg/ml) and a statistically significant increase (P < 0.05) in the frequency of morphological transformation was observed at 18.5 µg/ml. Furthermore, the compound induced a dose-dependent increase in morphological transformation as defined by a statistically significant trend test. The maximum concentration tested was limited by cytotoxicity. Resorcinol was negative in the SHE assay (Table VI). The compound did not induce an increase in the frequency of morphological transformation at any of the concentrations tested (28–138 µg/ml). The maximum concentration tested was limited by cytotoxicity.
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Discussion |
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In these studies, the SHE assay predicted the rodent carcinogenicity of only one of the five test compounds correctly. Furthermore, an examination of the available data for these compounds indicates that there is no straightforward correlation between genotoxicity, SHE cell morphological transformation and rodent carcinogenicity.
Genistein
The phytoestrogen genistein is a naturally occurring isoflavone that possesses estrogenic and anti-estrogenic properties. The compound can also inhibit topoisomerases, tyrosine kinases and growth factor signalling pathways. Genistein was negative in bacterial mutation tests (Misra et al., 2002), positive in both the in vitro mouse lymphoma (Misra et al., 2002) and human lymphocyte chromosome aberration assays (Kulling et al., 1999), and negative in the in vivo bone marrow micronucleus assay (Misra et al., 2002). Previous studies have indicated that genistein induces morphological transformation in SHE cells (Tsutsui et al., 2003). However, these studies were not conducted using the standard 7 day protocol (Kerckaert et al., 1996) and, in our experience, would not satisfy the current regulatory requirements. Genistein was negative in a short-term in vivo p53-deficient transgenic mouse carcinogenicity assay (Misra et al., 2002). Therefore, it would be reasonable to assume that while genistein is genotoxic in vitro, the compound is not genotoxic in vivo and it is therefore unlikely to be a potential genotoxic rodent carcinogen.
Genistein has been shown to induce benign and malignant lesions of the rodent reproductive tract including uterine adenocarcinomas in mice at 18 months (Newbold et al., 2001). While the precise mechanism of the action of genistein is unclear, there is compelling evidence that its carcinogenicity is related to its estrogenic rather than its genotoxic properties (Ashby, 2001).
Despite being a rodent carcinogen, genistein was negative in the SHE assay using the standard 7 day protocol. Surprisingly, neither the genotoxic nor estrogenic properties of the compound appeared to increase the frequency of morphological transformation in SHE cells. Based on these data, it is apparent that simple correlations between in vitro genotoxicity assay data and SHE assay data can be misleading when assessing a compound's putative carcinogenicity in rodents.
Metaproterenol
Metaproterenol is a ß-adrenergic stimulator that is used therapeutically as a bronchodilator in patients with asthma (Nelson, 1995). The compound was negative in bacterial mutation tests, the in vitro mouse lymphoma and CHO chromosome aberration assays and also in the in vivo bone marrow micronucleus assay (ILSI HESI, 1998). Metaproterenol was also negative in a short-term in vivo p53 heterozygous transgenic mouse carcinogenicity assay (Storer et al., 2001), and therefore it may be concluded that it is not a potential genotoxic carcinogen.
Metaproterenol induced a non-significant increase in leiomyomas in female rats in a 2 year rat carcinogenicity study (Thomson PDR, 2004). The compound also induced a significant increase in benign hepatic adenomas in male rats and benign ovarian tumours in female rats in a 78 week carcinogenicity study in NMRI mice (Thomson PDR, 2004). Metaproterenol has been classified as a non-genotoxic rodent carcinogen in previous evaluations of short-term carcinogenicity models (Storer et al., 2001).
Despite being a non-genotoxic rodent carcinogen, metaproterenol was negative in the SHE assay using the standard 7 day protocol. As a chemical class, beta II agonists have been associated with the development of leiomyomas in female rodents. This carcinogenic activity is thought to be caused by excessive stimulation of tissues with beta II receptors and is linked to the pharmacological potency of the compound. One could argue that the SHE assay cannot be expected to detect a compound with such an unusual and specific carcinogenic mechanism in rodents. However, if this is the case and if a compound's carcinogenic mechanism of action does effect SHE cell morphological transformation, then simple correlations between the SHE assay and rodent carcinogenicity clearly become redundant.
Rotenone
Rotenone is a registered insecticide isolated from the plant genera Derris and Lonchorcarpus. The compound acts as an inhibitor of oxidative phosphorylation and has been used extensively to study mitochondrial respiration (Lummen, 1998). Rotenone was negative in bacterial mutation tests (NTP, 2004), positive in both the in vitro mouse lymphoma (Guadano et al., 1998) and human lymphocyte micronucleus assays (Amer and Aboul-ela, 1985), and negative in the in vivo bone marrow micronucleus assay (Eastin et al., 1998). The results of the in vitro mammalian genotoxicity studies provided evidence that the compound did not interact with DNA directly, but it was in fact aneugenic (Amer and Aboul-ela, 1985). The compound was negative in a short-term in vivo p53 heterozygous transgenic mouse carcinogenicity assay (McGregor et al., 1988). Therefore, it would be reasonable to assume that while rotenone is genotoxic in vitro, the compound is not genotoxic in vivo and it is unlikely to be a potential genotoxic rodent carcinogen.
Rotenone did not demonstrate any evidence of carcinogenic activity when tested in 2 year carcinogenicity studies in female F344/N rats and male and female B6C3F1 mice (NTP, 2004), although the compound did produce equivocal evidence of carcinogenic activity in male F344/N rats as indicated by an increased incidence of uncommon parathyroid gland adenomas. Despite the in vitro genotoxic activity and the equivocal carcinogenicity findings, rotenone has been classified as non-genotoxic and non-carcinogenic in rodents in previous evaluations of data from short-term carcinogenicity models (Storer et al., 2001).
While rotenone is generally considered to be non-carcinogenic in rodents, in the current study, the compound was positive in the SHE assay using the standard 7 day protocol. Again, these results highlight the fact that drawing correlations between in vitro genotoxicity assay data and SHE assay data could lead to an incorrect assessment of a compound's potential genotoxic rodent carcinogenicity. One could argue that the SHE assay correctly predicted rotenone's equivocal carcinogenic activity in male rats, although such a claim would be inappropriate without knowing the mechanism of rotenone-induced morphological transformation in SHE cells.
p-Anisidine
p-Anisidine is an aromatic chemical intermediate that has been used in the synthesis of azo dyes. p-Anisidine was positive in bacterial mutation tests (NTP, 2004), and the in vitro mouse lymphoma and CHO chromosome aberration assays (NTP, 2004) but negative in the in vivo bone marrow micronucleus assay (Pritchard et al., 2003). The compound was also negative in a short-term in vivo p53 heterozygous transgenic mouse carcinogenicity assay (Pritchard et al., 2003). Again, it is reasonable to assume that while p-anisidine is genotoxic in vitro, the compound is not genotoxic in vivo and it is unlikely to be a genotoxic rodent carcinogen.
p-Anisidine (HCl) did not demonstrate any evidence of carcinogenic activity when tested in 2 year carcinogenicity studies in female F344/N rats and male or female B6C3F1 mice (NTP, 2004). The compound did demonstrate equivocal evidence of carcinogenic activity in male F344/N rats indicated by a non-statistically significant increase in the incidence of squamous cell carcinomas and alveolar/bronchiolar adenomas (NTP, 2004). p-Anisidine has been classified as genotoxic but non-carcinogenic in rodents in previous evaluations of data from short-term carcinogenicity models (Storer et al., 2001). In fact, p-anisidine has been used as a negative control compound (i.e. a genotoxic rodent non-carcinogen) in previous short-term carcinogenicity studies (Tennant et al., 1995).
Despite being non-carcinogenic in rodents, somewhat surprisingly, p-anisidine was positive in the SHE assay using the standard 7 day protocol. Once again, the study highlights the fact that simple correlations between in vitro genotoxicity assay data, SHE assay data and rodent carcinogenicity can be inappropriate. Again, one could argue that the SHE assay correctly predicted p-anisidine's equivocal carcinogenic activity in male rats. However, such a claim would be inappropriate without knowing the mechanism by which p-anisidine induces SHE cell morphological transformation. Interestingly, the structurally related rodent carcinogen o-anisidine was also positive in the SHE assay (LeBoeuf et al., 1996). This would indicate that the assay may not be able to distinguish between structurally related non-carcinogenic and carcinogenic compounds.
Resorcinol
Resorcinol is a component of various adhesives, dyes and non-prescription pharmaceutical preparations used in the topical treatment of skin. Resorcinol was negative in bacterial mutation tests, but positive in the in vitro mouse lymphoma and CHO chromosome aberration assays and the in vivo bone marrow micronucleus assay (NTP, 2004). The compound was negative in a short-term in vivo p53 heterozygous transgenic mouse carcinogenicity assay (Pritchard et al., 2003). Therefore, it would be reasonable to assume that while resorcinol is genotoxic in vitro and in vivo, it is unlikely to be a genotoxic rodent carcinogen.
Resorcinol did not demonstrate any evidence of carcinogenic activity when tested in 2 year carcinogenicity studies in male and female F344/N rats or male and female B6C3F1 mice (NTP, 2004). No treatment-related increases in tumours or non-neoplastic lesions were seen in rats or mice administered resorcinol for 2 years (NTP, 2004). Despite the genotoxic activity, resorcinol has been classified as non-genotoxic and non-carcinogenic in rodents in previous evaluations of short-term carcinogenicity models (Storer et al., 2001).
Resorcinol was negative in the SHE assay using the standard 7 day protocol. The genotoxic properties of the compound did not appear to influence the frequency of morphological transformation in SHE cells. In the current study, resorcinol provided the only example in which the SHE assay demonstrated good concordance with the existing rodent carcinogenicity data.
Empirical correlations of in vitro genotoxicity assays, short-term carcinogenicity models and the 2 year rodent carcinogenicity bioassay
While these five compounds are not new chemical entities, the in vitro and in vivo genotoxicity data are quite typical of such compounds being developed within the pharmaceutical industry. If one views the data for each compound sequentially, it is apparent that simple correlations between in vitro genotoxicity assays and SHE cell transformation assays can be misleading when it comes to predicting a compound's rodent carcinogenicity (Table I).
In the current studies, only the in vitro and in vivo genotoxin resorcinol, which was negative in the SHE assay, would have been correctly predicted to be non-carcinogenic in the rodent 2 year bioassay. In contrast, the in vitro genotoxin genistein, which was negative in the SHE assay, would have been incorrectly predicted to be non-carcinogenic in rodents. Furthermore, the non-genotoxic metaproterenol, which was also negative in the SHE assay, would also have been incorrectly predicted to be non-carcinogenic in rodents. These last two results are particularly surprising given that the SHE assay is reported to be able to detect non-genotoxic rodent carcinogens and that both of these compounds are considered to be carcinogenic in rodents via a non-genotoxic mechanism. Finally, the in vitro genotoxins, rotenone and p-anisidine, which were positive in the SHE assay, would also have been incorrectly predicted to be potential genotoxic rodent carcinogens based upon the in vitro genotoxicity and SHE assay data.
Interestingly, for the current set of compounds, a combination of the standard battery of in vitro and in vivo genotoxicity assay data alongside the p53 heterozygous mouse assay data provided the best indication of whether or not a test compound was carcinogenic in rodents via a genotoxic mechanism. However, for both rotenone and p-anisidine, it is unlikely that even negative p53 heterozygous mouse assay data would have negated the potential non-genotoxic carcinogenic activity inferred by the positive SHE assay data. Ultimately, for these compounds, the positive SHE assay data would have been negated only by the absence of findings in subsequent 2 year rodent carcinogenicity bioassays.
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Conclusions |
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In these studies, the standard 7 day SHE cell morphological transformation assay did not predict the rodent carcinogenicity of four out of the five test compounds. It is difficult to explain these findings given that the actual mechanisms of SHE cell morphological transformation are currently unknown. While these studies suggest that the SHE assay cannot predict the carcinogenic activity of certain compounds in rodents, we accept that the results alone, do little to undermine the overall concordance of the SHE assay with the rodent bioassay. However, the results do demonstrate that for certain compounds there is no simple correlation between in vitro genotoxicity, morphological transformation in SHE cells and rodent carcinogenicity. In fact, the use of the SHE assay to clarify the carcinogenic potential of in vitro mammalian genotoxins could well result in compounds being incorrectly classified as potential genotoxic rodent carcinogens. Clearly, more research is required to accurately assess the role of the SHE cell morphological transformation assay in the carcinogenic risk assessment of new chemical entities.
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* To whom correspondence should be addressed. Tel: +44 1920 883049; Fax: +44 1920 882679; Email: James.S.Harvey{at}gsk.com
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References |
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Received on October 8, 2004; revised on December 21, 2004; accepted on December 22, 2004.
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