Sequential enhanced tyrosine phosphorylation during progressive malachite green induced malignant transformation of Syrian hamster embryo cells in culture is associated with no change in the activity levels of tyrosine phosphatases.

Malachite green (MG) consisting green crystals with a metallic lustre is extremely soluble in water and is highly cytotoxic to mammalian cells and also acts as liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported that MG induces malignant transformation in Syrian hamster embryo (SHE) cells. Since tyrosine phosphorylation and dephosphorylation reactions are known to play critical roles during normal and abnormal cellular proliferation, in this study we have studied the tyrosine phosphorylation, tyrosine phosphorylated proteins and protein tyrosine phosphatases in malignantly transformed cells and during sequential development of cellular transformation by MG compared to control cells. The present investigation shows that enhanced tyrosine phosphorylation and tyrosine phosphorylated proteins associated with the static levels of tyrosine protein phosphatases may probably contribute to the abnormal cellular proliferation during malignant transformation of SHE cells by MG.     

Malignant transformation of Syrian hamster embryo (SHE) cells in culture by malachite green: an agent of environmental importance

Malachite green (MG), consisting of green crystals with a metallic lustre, is very soluble in water and is highly cytotoxic to mammalian cells in culture and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. Accordingly, we have studied the effect of MG on the formation of free radicals using Electron Spin Resonance (ESR) analysis with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. ESR analysis showed formation of reactive free radicals during exposure of MG to Syrian hamster embryo (SHE) cells. As per mechanism-based toxicology in cancer risk assessment, the chemicals that have the potential to be metabolized to active free radical species could be human cancer hazards. So, we have investigated the effect of MG on the formation of Type II and Type III morphologically transformed foci using SHE cell transformation assay. MG induced dose related transformed foci. Some of these transformed foci were taken out using selective trypsinisation and established immortal cell lines. One of these immortal cell lines was characterized extensively. This immortal cell line showed enhanced DNA synthesis in the form of BrdU incorporation, increased presence of proliferating cell nuclear antigen (PCNA), bcl-2 and p53 proteins by immunohistochemistry. When these immortal cells were injected subcutaneously into nude mice, they developed tumors which were transplantable and histopathologically sarcomas. The present studies indicate that MG could be a potential candidate for two year chemical carcinogenesis rodent bioassays.     

Hyperphosphorylation of extracellular regulated kinase 2 (ERK2) and inhibition of JNK2 phosphorylation are associated with increased S-phase during transformation of Syrian hamster embryo cells by Malachite Green

Malachite Green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the mitogen activated protein (MAP) kinase signal transduction pathway in preneoplastic cells induced by MG. Western blots of MG induced preneoplastic cells showed no phosphorylation of ERK1, an increased phosphoactive ERK2 associated with a decreased expression of phosphoactive JNK2. However, total forms of ERKs, JNKs and p38 Kinases showed similar levels of expression in control and preneoplastic SHE cells. Indirect immunofluorescence studies have shown a distinct nuclear localisation of phosphoactive ERKs in MG induced preneoplastic cells. Flow cytometric analysis showed an increase of S-phase cells in preneoplastic cells compared to control SHE cells. The present study indicates that hyperphosphorylation of ERK2, decreased JNK2 phosphorylation and an increase in S-phase cells seems to be the early changes associated with the MG induced malignant transformation of SHE cells in primary culture.     

Differential role of MAP kinase isoforms in malachite green transformed Syrian hamster embryo fibroblasts in culture

Malachite green (MG) induces DNA damage and malignant transformation of Syrian hamster embryo (SHE) cells in primary culture. In the present study, we have studied the role of all the three isoforms of mitogen activated protein (MAP) kinases i.e. ERK (extracellular regulated kinase), JNK (JUN- N- terminal kinase) and p38 kinase during transformation of SHE cells by MG. The results showed that transformed cells were associated with a decreased expression of phosphoactive ERK and JNK and increased expression of p38 kinase as evident from the Western blot, immunofluorescence and flow cytometry studies. Also, a persistent nuclear localization of p38 kinase was observed in the transformed cells. The present study indicated that p38 kinase was present at higher levels and seemed to be associated with transformation, which suggested that inhibitors of p38 kinase could serve in general as potential agents for selective cancer therapy.     

Insect virus: assays for toxic effects and transformation potential in mammalian cells

The nuclear polyhedrosis virus of Autographa californica (AcNPV) was evaluated by using in vitro test systems for toxicity and transforming potential in mammalian cells. Mass cell cultures of CV-1 and WI38 cells appeared unaffected by AcNPV at a multiplicity of infection of 5. Human foreskin cells grew more slowly after inoculation but eventually produced healthy monolayers. The sensitivities of the inhibition of reproductive survivability assays were greater and demonstrated slight AcNPV toxicity to CV-1, WI38, and human foreskin cells. Toxicity was not ameliorated when gradient-purified or psoralen-inactivated virus was used, suggesting that the toxic component of the preparation is part of the virion or copurifies with it. AcNPV was not toxic to and did not transform BALB/c 3T3 cells or primary cell cultures derived from Syrian hamster embryo cells (SHE). Unlike the BALB/c 3T3 transformation assay, the SHE assay detected no spontaneous transformants. The SHE transformation assay can employ simian adenovirus 7 as a positive control. SHE are transformed by numerous viruses and so are useful in assessment protocols. This study suggests that in vitro assessment of viral pesticide toxicity should employ the inhibition of reproductive survivability assay and that transformation assessment is best done with the SHE-simian adenovirus 7 procedure.     

Insect virus: assays for toxic effects and transformation potential in mammalian cells.

The nuclear polyhedrosis virus of Autographa californica (AcNPV) was evaluated by using in vitro test systems for toxicity and transforming potential in mammalian cells. Mass cell cultures of CV-1 and WI38 cells appeared unaffected by AcNPV at a multiplicity of infection of 5. Human foreskin cells grew more slowly after inoculation but eventually produced healthy monolayers. The sensitivities of the inhibition of reproductive survivability assays were greater and demonstrated slight AcNPV toxicity to CV-1, WI38, and human foreskin cells. Toxicity was not ameliorated when gradient-purified or psoralen-inactivated virus was used, suggesting that the toxic component of the preparation is part of the virion or copurifies with it. AcNPV was not toxic to and did not transform BALB/c 3T3 cells or primary cell cultures derived from Syrian hamster embryo cells (SHE). Unlike the BALB/c 3T3 transformation assay, the SHE assay detected no spontaneous transformants. The SHE transformation assay can employ simian adenovirus 7 as a positive control. SHE are transformed by numerous viruses and so are useful in assessment protocols. This study suggests that in vitro assessment of viral pesticide toxicity should employ the inhibition of reproductive survivability assay and that transformation assessment is best done with the SHE-simian adenovirus 7 procedure.     

Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.     

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].     

Application of a two-stage Syrian hamster embryo cell transformation assay to cigarette smoke particulate matter

The induction of transformation in Syrian hamster embryo (SHE) cells is a multifactorial process, in comparison to endpoints induced in in vitro genotoxicity assays such as Ames, mouse lymphoma and cytogenetics [Y. Berwald, L. Sachs, In vitro cell transformation with chemical carcinogens, Nature (London) 200 (1963) 1182-1184]. Furthermore, a number of non-genotoxic carcinogens and promoters such as clofibrate and diethylhexylphthalate, have been positively identified in this assay, while giving false negative results in traditional genotoxicity assays [H. Yamasaki, J. Ashby, M. Bignami, W. Jongen, K. Linnainmaa, R.F. Newbold, G. Nguyen-Ba, S. Parodi, E. Rivedal, D. Schiffmann, J.W.I.M. Simons, P. Vasseur, Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project, Mutat. Res. 353 (1996) 47-63]. A high concordance between results obtained in this assay when compared with rodent carcinogenesis bioassays has also been noted [R.J. Isfort, G.A. Kerckaert, R.A. LeBoeuf, Comparison of the standard and reduced pH Syrian hamster embryo (SHE) in vitro cell transformation assays to predict the carcinogenic potential of chemicals, Mutat. Res. 356 (1996) 11-63]. Carcinogenesis is known to be a multistage process, with agents potentially acting at each stage. Specifically, mouse skin painting experiments established that tumour induction could be mechanistically divided into two distinct phases, termed initiation and promotion. Initiation, is defined as the stage at which a normal cell is converted to a latent tumour cell, followed by promotion where the latent tumour cell progresses to a tumour [W.F. Friedwald, P. Rous, The initiating and promoting elements in tumour production: analysis of the effects of tar, benzpyrene and methylcholanthrene on rabbit skin, J. Exp. Med. 80 (1944) 101-125]. A protocol for the pH 6.7 SHE transformation assay has been developed which allows separation of cell transformation process into two phases, potentially analogous to initiation and promotion in vivo. This allows chemicals found to be positive in the traditional SHE cell transformation assay to be further classified as initiators or promoters. Following validation with known initiators, benzo(a)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine and promoters, 12-O-tetradecanoyl-phorbol-13-acetate and phenobarbitone, the two-stage model was applied to cigarette smoke particulates which was found to act both at the initiation and promotion stage of cell transformation.     

Comparison between mutagenesis in normal and transformed syrian hamster fibroblasts: Difference in the temporal order of HPRT gene replication

A highly tumorigenic subdiploid cell line, BP6T, derived in our laboratory from Syrian hamster embryo (SHE) cells, is amenable to studies of somatic mutation in vitro. Cellular and biochemical characterization of clonally derived BP6T cells resistant to 6-thioguanine (TG^r) or ouabain (Oua^r) demonstrated these mutants to be similar qualitatively to mutants of SHE cells characterized previously (Barrett et al., 1978). BP6T TG^r mutants resistant to 6-thioguanine are cross-resistant to 8-azaguanine, lack HPRT activity, exhibit a low frequency of reversion and arise spontaneously at a rate of 5 × 10⁻⁷ mutants per cell per generation. BP6T Oua^r mutants were shown to be highly resistant to ouabain-mediated inhibition of ⁸⁶Rb influx, indicating an alteration in the Na⁺/K⁺ ATPase. These studies on the BP6T cell line provide the experimental basis for a comparative study of the mutagenic responses of normal, diploid SHE cells versus those of related, but transformed aneuploid cells. Highly synchronized cultures of these 2 cells were mutagenized by pulse treatment with BrdU during different periods of S phase, followed immediately by near-UV irradiation. The induced mutation frequencies so obtained provided information about the temporal order of replication of genes encoding HPRT and Na⁺/K⁺ ATPase in both SHE and BP6T cells. The temporal pattern of replication of Na⁺/K⁺ ATPase gene loci is similar in both cell types, but the temporal order of replication of the HPRT gene is significantly different between SHE and BP6T cells (mid-late S phase, versus early S phase, resp.). This observed difference emphasizes the caution required in the study of mutagenesis and DNA replication using transformed, aneuploid cells under the assumption that the underlying mechanisms are the same for normal, diploid cells.     

An Imperfect G2M Checkpoint Contributes to Chromosome Instability Following Irradiation of S and G2 Phase Cells

DNA double strand break (DSB) repair and checkpoint control represent two major mechanisms that function to reduce chromosomal instability following ionising irradiation (IR). Ataxia telangiectasia (A-T) cells have long been known to have defective checkpoint responses. Recent studies have shown that they also have a DSB repair defect following IR raising the issue of how ATM’s repair and checkpoint functions interplay to maintain chromosomal stability. A-T and Artemis cells manifest an identical and epistatic repair defect throughout the cell cycle demonstrating that ATM’s major repair defect following IR represents Artemis-dependent end-processing. Artemis cells show efficient G2/M checkpoint induction and a prolonged arrest relative to normal cells. Following irradiation of G2 cells, this checkpoint is dependent on ATM and A-T cells fail to show checkpoint arrest. In contrast, cells irradiated during S phase initiate a G2/M checkpoint which is independent of ATM and, significantly, both Artemis and A-T cells show a prolonged arrest at the G2/M checkpoint likely reflecting their repair defect. Strikingly, the G2/M checkpoint is released before the completion of repair when approximately 10-20 DSBs remain both for S phase and G2 phase irradiated cells. This defined sensitivity level of the G2/M checkpoint explains the prolonged arrest in repair-deficient relative to normal cells and provides a conceptual framework for the co-operative phenotype between checkpoint and repair functions in maintaining chromosomal stability.     

Induction of selective DNA amplification and morphological cell transformation in Syrian hamster embryo cells

DNA amplification is a frequently observed event in continuous cell lines and in tumors. It is likely that a common mechanism underlies the amplification of specific DNA sequences which confer drug resistance and genes which give a growth advantage to the tumor. To find a correlation between the induction of DNA amplification by chemicals and morphological cell transformation we treated Syrian hamster embryo (SHE) cells with diverse antineoplastic agents of different classes. Analysis of these agents seems to be important since they are potentially carcinogenic and resistance inducing. For the measurement of DNA amplification we established a new system using adeno-associated virus type 2 (AAV)-infected primary SHE cells as target cells and amplification of viral DNA as marker of DNA amplification. Simultaneously we determined morphological cell transformation in SHE cells. Our findings demonstrate that there is only a limited correlation between the induction of AAV DNA amplification and the morphological cell transformation in SHE cells. The newly established system of AAV DNA amplification appears to be a useful tool for the investigation of drug resistance in target cells of choice.     

Recommended protocol for the Syrian hamster embryo (SHE) cell transformation assay

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is a short-term in vitro assay recommended as an alternative method for testing the carcinogenic potential of chemicals. SHE cells are "normal" cells since they are diploid, genetically stable, non-tumourigenic, and have metabolic capabilities for the activation of some classes of carcinogens. The CTA, first developed in the 1960s by Berwald and Sachs (1963,1964) [3,4], is based on the change of the phenotypic feature of cell colonies expressing the first steps of the conversion of normal to neoplastic-like cells with oncogenic properties. Pienta et al. (1977) [22] developed a protocol using cryopreserved cells to enhance practicality of the assay and limit sources of variability. Several variants of the assay are currently in use, which mainly differ by the pH at which the assay is performed. We present here the common version of the SHE pH 6.7 CTA and SHE pH 7.0 CTA protocols used in the ECVAM (European Centre for the Validation of Alternative Methods) prevalidation study on CTA reported in this issue. It is recommended that this protocol, in combination with the photo catalogues presented in this issue, should be used in the future and serve as a basis for the development of the OECD test guideline.     

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: III. Cell transformation in C3H/10T1/2 mouse embryo cell, BALB/c 3T3 mouse fibroblast and Syrian hamster embryo cell cultures

A standardized protocol and guidelines for the performance of cell transformation testing in mouse embryo (C3H/10T1/2), mouse fibroblast (BALB/c 3T3) and Syrian hamster embryo (SHE) cells have been developed. The protocol is based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with cell transformation experimentation. This report identifies those modifications to previously described methodologies which are being used on a regular basis and also serves to clarify confusing or inconsistent practices.     

Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2.

Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s).     

Focus formation and neoplastic transformation by herpes simplex virus type 2 inactivated intracellularly by 5-bromo-2''-deoxyuridine and near UV light

The induction of focus formation in low serum and of neoplastic transformation of Syrian hamster embryo cells was examined after the expression of herpes simplex virus type 2 functions. Syrian hamster embryo cells infected at a high multiplicity (5 PFU/cell) with 5-bromo-2'-deoxyuridine-labeled herpes simplex virus type 2 (11% substitution of thymidine residues) were exposed to near UV light irradiation at various times postinfection. This procedure specifically inactivated the viral genome, while having little, if any, effect on the unlabeled cellular DNA. Focus formation in 1% serum and neoplastic transformation were observed in cells exposed to virus inactivated before infection, but the frequency was enhanced (15- to 27-fold) in cells in which the virus was inactivated at 4 to 8 h postinfection. Only 2 to 45 independently isolated foci were capable of establishing tumorigenic lines. The established lines exhibited phenotypic alterations characteristic of a transformed state, including reduced serum requirement, anchorage-independent growth, and tumorigenicity. They retained viral DNA sequences and, even at relatively late passage, expressed viral antigens, including ICP 10.     

Syrian hamster embryo (SHE) cell transformation assay with conditioned media (without X-ray irradiated feeder layer) using 2,4-diaminotoluene, 2,6-diaminotoluene and chloral hydrate

The Syrian hamster embryo (SHE) cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The feeder layer of cells consists of X-ray irradiated cells which are still viable but unable to replicate. We have tried seeding the target cells in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer. Three SHE cell isolates were tested to investigate the feasibility of this approach. With freshly prepared conditioned medium (LeBoeuf's Dulbecco's Modified Eagle's Medium with 2 mM L-glutamine and 20% fetal bovine serum), used within 2 weeks of preparation, there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In each experiment, the positive control benzo(a)pyrene [B(a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without feeder cells. Three compounds, 2,4-diaminotoluene (2,4-DAT), 2,6-diaminotoluene (2,6-DAT), and chloral hydrate were tested in the SHE cell transformation assay without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable to those reported in the published literature using the standard methodology with feeder cells, with 2,4-DAT and chloral hydrate eliciting a significant increase in MTF, and 2,6-DAT not eliciting any increase in MTF. The results of this study demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and facilitating the scoring of morphologically transformed colonies.     

Rodent cell transformation assays-a brief historical perspective

In vitro cell transformation is a process characterized by a series of progressive distinctive events that often emulate manifestations occurring in vivo and which are associated with neoplasia. Attendant cellular and sub-cellular alterations include, among others: cellular immortality, phenotypic changes, aneuploidy, genetic variability, cellular disarray, anchorage-independent growth, and tumorigenicity in vivo. Early chemically induced neoplastic transformation studies involved the use of normal diploid (Syrian) hamster embryo (SHE) cells and monitored the formation of morphologically altered colonies. Later investigations employed primarily two established mouse cell lines, i.e. the BALB/c 3T3 A31 cell line and the C3H 10T 1/2 cell line, and monitored the induction of morphologically aberrant foci. In either case, such transformed cellular clusters (colonies and foci) could induce tumors upon inoculation in vivo. Some subsequent noteworthy advancements using these systems included pH adjustments, metabolic supplementation, amplification of expression of formerly latent transformed foci, concurrent detection of mutagenesis and transformation, and use of a Bhas 42 cell line (v-Ha-ras transfected BALB/c 3T3 cells) to detect both tumor initiators and promoters. Over time, such transformation assay systems have been found useful in academic, industry and regulatory laboratories, generally for research purposes, but also occasionally as screening tools for potential chemical carcinogens. Nevertheless, to date, use of these assays for decision-making purposes in the regulatory arena remains elusive and will require comprehensive validation to gain universal acceptance.     

Morphological transformation and catalase activity of syrian hamster embryo cells treated with hepatic peroxisome proliferators,TPA and nickel sulphate

The abilities of the hepatic peroxisome proliferators (HPPs) clofibrate, di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)- phthalate (MEHP), 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) and tiadenol to induce morphological transformation and to increase the catalase activity of Syrian hamster embryo (SHE) cells were studied. DEHP, MEHP, clofibrate and tiadenol induced morphological transformation of SHE cells and increased the catalase activity. DEHP was more potent than clofibrate and tiadenol in both inducing catalase and morphological transformation, while MEHP seemed more potent than DEHP in inducing catalase, but not morphological transformation, 2,4,5-T and 2,4-D did not induce morphological transformation, but 2,4,5-T was more potent than clofibrate in increasing the catalase activity. These results show that several HPPs induce morphological transformation of SHE cells and an increase in the catalase activity. There is, however, no direct connection between these two parameters, as seen from the results of 2,4,5-T. The tumor promoter TPA, and the metal salt nickel sulphate, induced morphological transformation of SHE cells without any appreciable increase in the catalase activity. These results further corroborate the dissociation between induction of morphological transformation and the increase in catalase activity.Abbreviations Clofibrate ethyl-2-(p-chlorophenox) isobutyrate - 2,4-D 2,4-dichlorophenoxy acetic acid - DEHP di(2-ethylhexyl)phthalate - HPP hepatic peroxisome proliferator - MEHP mono(2-ethylhexyl)phthalate - SHE Syrian hamster embryo - 2,4,5-T 2,4,5-trichlorophenoxy acetic acid - tiadenol di(hydroxyethylthio)-1,10-decane