Induction Of Morphological Transformation Research Articles

Abstract 1579: Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate

Abstract Hexavalent chromium [Cr(VI)]-containing compounds are human carcinogens. They cause cancers in the respiratory system when inhaled, and stomach/other internal cancers when ingested. Soluble and insoluble hexavalent chromium [Cr(VI)] compounds induced base substitution, deletion, addition, and frameshift mutations. Cr(VI) compounds also induce DNA-DNA cross links and DNA-protein cross-links in mammalian cells. We examined the ability of the soluble chromium compounds, sodium chromate (Na2CrO4), calcium chromate (CaCrO4) and potassium chromate (K2CrO4) to induce cytotoxicity and morphological transformation in C3H/10T½ Cl 8 (10T1/2) mouse embryo cells. We tested the hypothesis that the intracellular reductants, ascorbate and dehydroascorbate, can reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III) intracellularly, making it a strong cytotoxin in mammalian cells. Ascorbate is present in serum at concentrations in the mM range under physiological conditions in humans, but is only present in the μM range in mouse embryo cells grown in BME cell culture medium plus 10% fetal calf serum. We hypothesized the relatively weak responses for induction of morphological transformation of mammalian cells by Cr(VI) compounds in culture (dose-dependent but weak induction of foci by lead chromate, and no induction of foci by calcium chromate, potassium chromate, and sodium chromate) is due to the small amounts of ascorbate in cultures of mammalian cells that are insufficient to reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III). Therefore, we investigated the cytotoxic effects of ascorbate on 10T½ mouse embryo cells, and the effects of the highest non-cytotoxic concentrations of ascorbate on Cr(VI)-induced cytotoxicity in 10T½ cells, using reduction in plating efficiency as our cytotoxicity assay. Cell survival data showed that ascorbate itself exerted significant cytotoxic effects at concentrations of 0.00625 mM and higher on 10T½ mouse embryo cells. Furthermore, when 10T½ cells were treated with both Cr(VI) and ascorbate, ascorbate played dual roles. It served as a pro-oxidant (enhancer of the cytotoxicity of chromate) at concentrations up to 0.1 mM, and as an anti-oxidant (reducer of the cytotoxicity of chromate) at concentrations of 0.25 mM and higher. This information is important for our ongoing and future experiments, where we designed a protocol to incorporate ascorbate into our assays assessing the cytotoxicity and cell transforming activity of Cr(VI) compounds. We project that at concentrations of 0.1 mM and lower, ascorbate will enhance the cell transforming activity of Cr(VI) compounds. Supported by undergraduate fellowships from the Provost's Office at the University of Southern California (USC) (P. I., JRL), by funding from the M. S. Program in Molecular Microbiology and Immunology to JRL, by discretionary funding to JRL, and by prior grant ES03341 from NIEHS/NIH (P. I., JRL). Citation Format: Sophia A. Shahin, William Liao, Laureen Tran, Qasim A. Akinwumi, Farn-shuan Tseng, Alyssa Mathew-Joseph, Joseph R. Landolph. Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2014-1579

Effect of Thermal Treatment of Refractory Ceramic Fibres on the Induction of Cytotoxicity and Cell Transformation

Refractory ceramic fibres (RCFs) are amorphous fibres made up of various metal oxides (mainly Al2O3 and SiO2), used as high temperature insulation materials. We have examined the surface properties of three RCFs from the TIMA repository, RCF1, RCF3 and RCF4 (RCF1 heated at the expected working temperature), which showed different potentials for cytotoxicity and the induction of morphological transformation in Syrian hamster embryo cells, RCF4 being less active. The degree of surface hydrophilicity, evaluated by adsorption calorimetry, showed that RCF4 is less hydrophilic than RCF1, likely as a consequence of the prior thermal treatment. In order to investigate the effect of thermal treatment on the cellular response to RCFs we have heated (800°C for 24 h) the most active fibre (RCF3) in air until it was converted to a fully hydrophobic material. The cytotoxic (colony forming efficiency) and transforming potencies of heated and unheated RCF3, measured at concentrations between 1 and 16 µg/cm 2 of culture dish for 7 days, were then compared. Both cytotoxicity and potential to transform cells dramatically decreased when fibres were heated, indicating that hydrophobicity blunts the cellular response to RCF3. These results confirm that the state of the surface of RCFs modulates the biological response elicited and that a correlation exists between the degree of surface hydrophilicity and induction of cell damage and transformation. Thermal treatment, by lowering the surface affinity of RCFs for water, inhibits some cell–fibre interactions and decreases the extent of internalization of the fibres.

Role of Iron and Surface Free Radical Activity of Silica in the Induction of Morphological Transformation of Syrian Hamster Embryo Cells

Using various surface modified silica dusts we previously showed that the samples able to generate reactive oxygen species (ROS) induced transformation of Syrian hamster embryo (SHE) cells. The purpose of the present study was to determine in what measure trace iron on the surface of silica particles is involved in the cellular effects, through the formation of ROS. Min-U-Sil 5 quartz (Q) and an amorphous diatomite earth (DE), as well as the same samples pretreated with the Fe chelators ferrozine (fz, 3 mM) and desferrioxamine (dfx, 0.1 mM), were studied for: (i) morphological transformation; and (ii) aberrant mitotic division of SHE cells, which we reported as one of the possible mechanisms of silica-induced cell transformation. In order to determine the nature of the ROS involved in these effects, the antioxidants superoxide dismutase, catalase and mannitol were added to the treated cells. On a per weight basis, DE appeared more cytotoxic and slightly more transforming than Q. The suspensions of Q and DE preincubated in Fe chelators were significantly less transforming and, in particular Q particles, had reduced potency in inducing abnormal mitoses than the untreated samples. Although the amount of Fe mobilized from the silica particles by dfx was greater than that of Fe(II) mobilized by fz, a decrease in transforming potency was largely achieved after pretreatment of particles with fz. In the presence of antioxidants, both the transformation and mitotic aberration frequencies were significantly decreased. The results show that the iron associated with the surface of particles is implicated in the generation of ROS and is partly responsible for the transforming potency of silicas. This study confirms the role of free radical activity in the induction of cell transformation in vitro, suggesting that oxidative stress is one of the mechanisms of silica carcinogenesis.

Morphological transformation and oxidative stress induced by cyanide in Syrian hamster embryo (SHE) cells.

Cyanide is a well-established poison known for its rapid lethal action and toxicity. Although long-term mammalian studies examining the carcinogenic potential of cyanide have not been previously reported, cyanide was reported to be positive in Salmonella typhimurium mutagenesis assay and induced aneuploidy in Drosophila. To further evaluate the carcinogenic potential of cyanide, the ability of cyanide to induce morphological transformation in Syrian hamster embryo (SHE) cells was studied. Cyanide induced a dose-dependent increase in morphological transformation in SHE cells following a 7-day continuous treatment. A significant increase in transformation was observed at potassium cyanide doses of 200 microM and greater. Transformation induced by cyanide was inhibited in a dose-related manner by vitamin E, suggesting a role of oxidative stress in the induction of morphological transformation by cyanide. Further, it was shown that 500 microM cyanide induced oxidative DNA damage in SHE cells, evidenced by the formation of 8-hydroxy-2'-deoxyguanosine (50-66% increase over control). The induction of oxidative stress by cyanide involved an early and temporal inhibition of antioxidant enzymes (catalase and superoxide dismutase) as well as an increased production of reactive oxygen species (1.5- to 2.0-fold over control).

Morphological transformation by 8-hydroxy-2'-deoxyguanosine in Syrian hamster embryo (SHE) cells.

8-Hydroxy-2'-deoxyguanosine (OH8dG) is one of the most prevalent oxidative DNA modifications found in eukaryotic cells. Previous studies have suggested an association between OH8dG formation and carcinogenesis. However, it is unclear whether OH8dG formation results in the necessary genotoxic events for cancer development. In the present study, the formation of OH8dG and its ability to transform Syrian hamster embryo (SHE) cells was examined. Methylene blue, a photosensitizer that in the presence of light can generate singlet oxygen by a type II mechanism, was used to produce oxidative DNA damage (predominantly OH8dG) in SHE cells. Photoactivated methylene blue produced a dose-dependent increase in OH8dG as well as a dose-dependent increase in morphological transformation in SHE cells. SHE cells transfected with DNA that contained increasing concentrations of OH8dG displayed a dose-dependent increase in morphological transformation. Treatment with beta-carotene (a singlet oxygen quencher) inhibited both the formation of OH8dG and the induction of morphological transformation in photoactivated methylene blue-treated SHE cells. These results suggest that formation of OH8dG can induce morphological transformation and provide further support for a role of OH8dG formation in the carcinogenesis process.

Induction of morphological transformation in BALB/3T3 mouse embryo cells by okadaic acid

Okadaic acid is produced by several types of dinoflagellates (marine plankton) and has been implicated as a causative agent of diarrhoetic shellfish poisoning. Okadaic acid, a known tumour promoter in vivo, has been shown to promote morphological transformation of carcinogen-initiated BALB/3T3 cells. This study shows that okadaic acid is capable of inducing morphological transformation of BALB/3T3 cells in the absence of an initiator.

Cell transformation by c-fos requires an extended period of expression and is independent of the cell cycle.

The proto-oncogene transcription factors Fos and Jun form a heterodimeric complex that binds to DNA and regulates expression of specific target genes. Continuous expression of c-fos causes transformation of cultured fibroblasts and induces osteogenic sarcoma in mice. To investigate the molecular basis of fos-mediated oncogenesis, we developed a conditional cell transformation system in which Fos expression was regulated by isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis or repression of Fos in L1-3c-fos cells occurred rapidly, within 30 min, after the removal or addition of IPTG to the culture medium. However, there was a significant delay between the induction of Fos expression and the appearance of morphological transformation. No effect was observed after 12 h of Fos expression, partial transformation was detected after 24 h, and full transformation required approximately 3 days of continuous Fos expression. Similarly, the transformed cell morphology persisted for at least 2 days after repression of Fos, and a normal phenotype was observed only after 3 days. Fos-Jun complexes, capable of binding to AP-1 sequences, were present continuously during the delay in morphological transformation. Furthermore, increased expression of several candidate Fos target genes, including those encoding Fra-1, transin (stromelysin), collagenase, and ornithine decarboxylase, was detected shortly after Fos induction. The induction of morphological transformation was not dependent on the cell cycle, as it occurred in both cycling and noncycling cells. Thus, the Fos-Jun complexes present before L1-3c-fos cells become fully transformed are transcriptionally active. These complexes disappeared, and the Fos target genes were repressed at least 2 days prior to reversion. Our results suggest that cell transformation by Fos requires increased expression of a target gene(s) with a long-lived product(s) that must reach a critical level.

Cell Transformation by c-fos Requires an Extended Period of Expression and Is Independent of the Cell Cycle

The proto-oncogene transcription factors Fos and Jun form a heterodimeric complex that binds to DNA and regulates expression of specific target genes. Continuous expression of c-fos causes transformation of cultured fibroblasts and induces osteogenic sarcoma in mice. To investigate the molecular basis of fos-mediated oncogenesis, we developed a conditional cell transformation system in which Fos expression was regulated by isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis or repression of Fos in L1-3c-fos cells occurred rapidly, within 30 min, after the removal or addition of IPTG to the culture medium. However, there was a significant delay between the induction of Fos expression and the appearance of morphological transformation. No effect was observed after 12 h of Fos expression, partial transformation was detected after 24 h, and full transformation required approximately 3 days of continuous Fos expression. Similarly, the transformed cell morphology persisted for at least 2 days after repression of Fos, and a normal phenotype was observed only after 3 days. Fos-Jun complexes, capable of binding to AP-1 sequences, were present continuously during the delay in morphological transformation. Furthermore, increased expression of several candidate Fos target genes, including those encoding Fra-1, transin (stromelysin), collagenase, and ornithine decarboxylase, was detected shortly after Fos induction. The induction of morphological transformation was not dependent on the cell cycle, as it occurred in both cycling and noncycling cells. Thus, the Fos-Jun complexes present before L1-3c-fos cells become fully transformed are transcriptionally active. These complexes disappeared, and the Fos target genes were repressed at least 2 days prior to reversion. Our results suggest that cell transformation by Fos requires increased expression of a target gene(s) with a long-lived product(s) that must reach a critical level.

Effect of cAMP elevating compounds on inhibition of gap junctional communication and induction of morphological transformation in Syrian hamster embryo cells

An enhancement of the cellular cAMP level has been shown to protect against phorbol ester-induced inhibition of gap junctional intercellular communication (GJIC) and induction of morphological transformation in Syrian hamster embryo (SHE) cells. Cholera toxin, forskolin, 3-isobutyl-1-methylxanthine (IBMX) and theophylline counteracted the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inhibition of GJIC. The enhancement of communication by these compounds was independent of the TPA concentration, as well as whether the cells were treated with TPA prior to or after the cAMP elevating agents. The induced increase in the cAMP level by cholera toxin occurred in the same concentration range as the enhancement of GJIC. With forskolin the effect on GJIC was apparent at concentrations 10 times lower than needed to enhance the cAMP level. IBMX and theophylline were found to enhance GJIC with only a 20% elevation of cAMP. The cAMP elevating compounds also suppressed the response of TPA on induction of transformed morphology in SHE cells.

Induction of morphological transformation by coal-dust extract in BALB/3T3 A31-1-13 cell line

The transforming activity of coal dust extracts was studied using BALB/3T3 clone A31-1-13 cells. Coal-dust extracts, both nitrosated and nonnitrosated, induced cell transformation in a dose-response manner. However, the transformation frequency was higher with the nitrosated than with the nonnitrosated extract. All transformed cell lines derived from coal-dust extract-induced foci showed biological characteristics of neoplastic transformation such as loss of contact inhibition and anchorage-independent growth. These results appear to support a hypothesis of coal mine dust causation of gastric cancer in coal miners.

The non-phorbol ester tumor promoter okadaic acid does not promote morphological transformation or inhibit junctional communication in hamster embryo cells

Okadaic acid is a potent non-phorbol ester mouse skin tumor promoter. Unlike the phorbol ester tumor promoters, okadaic acid is unable to promote the induction of morphological transformation in Syrian hamster embryo cell colonies. On the contrary, okadaic acid seems to counteract the effect of phorbol esters on transformation. Also unlike phorbol ester tumor promoters, okadaic acid does not inhibit intercellular communication, neither in primary hamster embryo cells, nor in the phorbol ester sensitive cell line BPNi. Furthermore, okadaic acid has no effect on the reoccurrence of communication following removal of 12-O-tetradecanoylphorbol-13-acetate.

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.

Effects of dinitrotoluenes on morphological cell transformation and intercellular communication in Syrian hamster embryo cells

The effects of four isomers of dinitrotoluene (DNT) and technical DNT (a mixture of DNT isomers and other compounds, with 2,4-DNT as the major constituent) were studied in two short-term in vitro assays. None of the isomers or technical DNT induced an increase in morphological transformation of Syrian hamster embryo (SHE) cells. Four DNT metabolites (2,4-diaminotoluene, 2-amino-4-nitrotoluene, 2-amino-6-nitrotoluene, and 2,4-dinitobenzoic acid), representing different stages in reduction or oxidation of DNT isomers, were also negative for induction of morphological transformation. The DNT isomers were tested in an intercellular communication assay based on dye transfer. 2,4-DNT, 2,6-DNT, and technical DNT inhibited intercellular communication in the SHE cell line BPNi at toxic concentrations. This may be reminiscent of in vivo data showing promoting activity of these compound. 2,3-DNT and 3,4-DNT did not inhibit communication.

Enhancement of adenovirus transformation of cloned rat embryo fibroblast cells by gamma irradiation

We have analyzed the effect of gamma irradiation on the induction of morphological transformation of cloned rat embryo fibroblast (CREF) cells by the host-range cold-sensitive type 5 adenovirus mutant, H5hr1. Treatment of CREF cells with 1-6 Gy of gamma irradiation immediately prior to viral infection resulted in dose-dependent decrease in cell survival and concomitant increase in viral transformation frequency. Exposure of CREF cells to 1-6 Gy of gamma radiation alone resulted in a similar dose-dependent inhibition in cell survival but without any subsequent morphological transformation. The effect of gamma irradiation on viral transformation was greatest when cells were irradiated directly before viral infection. The reduction in the enhancement of transformation was both dose and time dependent. The ability of gamma irradiation to enhance viral transformation was substantially reduced if CREF cells were treated with inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Employing a single-cell colony transfer assay and in situ hybridization with a 32P-labeled Ad5 DNA probe, we found that gamma irradiation of CREF cells prior to infection with H5hr1 resulted, 10 and 17 d after infection and replating, in an increase in the percentage of surviving CREF colonies that contain Ad5 DNA. Analysis of viral DNA integration by DNA-filter hybridization (Southern blot analysis) in H5hr1-transformed CREF clones isolated from untreated and gamma-irradiated cultures indicates that gamma irradiation caused increases in both the number of copies of Ad5 E1A DNA sequences integrated into cellular DNA and the number of unique Ad5 E1A DNA integration sites in transformed cells. These results indicate that gamma irradiation enhancement of adenovirus transformation was a consequence of radiation-induced cellular factors with finite life spans that are mediators of enhanced viral transformation. Potentially important components of the radiation enhancement process appear to involve an alteration in both the retention of free Ad5 DNA in surviving cells and an alteration in the profile of viral-DNA integration in gamma-irradiated cells.

Transformation-Specific Tyrosine Phosphorylation of a Novel Cellular Protein in Chicken Cells Expressing Oncogenic Variants of the Avian Cellular src Gene

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.

Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene.

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.

O-057 - Induction of morphological transformation and chromosome aberrations of cultured Syrian hamster embryo (SHE) cells by estrogeni and their metabolites.

O-057 - Induction of morphological transformation and chromosome aberrations of cultured Syrian hamster embryo (SHE) cells by estrogeni and their metabolites.

Induction of morphological transformation and micronuclei in Syrian hamster embryo fibroblasts by 1,2-dioxetanes. Correlation with single-strand breaks in HL-60 cells.

The photochemical genotoxic and cell-transforming potential of 4-hydroxymethyl-3,3,4-trimethyl-1,2-dioxetane (HTMD) and 3-(N-[4-pyridino]carbamoyl)methyl-3,4,4-trimethyl-1,2-dioxetane , (APD), in mammalian cell was studied. Both dioxetanes, which are efficient sources of triplet-excited ketones on thermal decomposition, induced morphological transformation in Syrian hamster embryo (SHE) fibroblasts. Unscheduled DNA synthesis in SHE and in HeLa cells could not be detected with these dioxetanes, but the number of micronuclei scored after the first mitosis was dose-dependently increased. Single-strand breaks but not Micrococcus luteus u.v.-endonuclease sensitive sites were observed by alkaline elution in HL-60 cells when treated with sub-lethal doses of HTMD and APD. A possible mechanism for the transformation mediated by DNA and chromosomal damage as well as the intermediacy of triplet carbonyls in these events are discussed.

Persistence of sister chromatid exchanges and in vitro morphological transformation of Syrian hamster fetal cells by chemical and physical carcinogens.

The induction of neoplastic cell transformation is closely associated with DNA alterations which occur shortly after carcinogen exposure. Sister chromatid exchange (SCE) formation is a sensitive indicator of carcinogen-DNA interaction and correlates with the induction of morphological cell transformation. The persistence of lesions generating SCE produced by chemical and physical carcinogens and its relevance to the induction of morphologic transformation was evaluated in coordinated experiments with cultured Syrian hamster fetal cells (HFC). Exponentially growing HFC were exposed for 1 h to benzo[a]pyrene (BP), methyl-methanesulfonate (MMS), cis-platinum (II) diaminedichloride (cis Pt II), N-methyl-N'-nitrosourea (MNU), mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-acetoxy-2-fluorenyl-acetamide (AcAAF) or u.v. light irradiated. Cells were incubated for 24 h with 5-bromodeoxyuridine (BrdUrd) required for SCE visualization at 1, 24 and 48 h after carcinogen exposure. The induction of morphological transformation was determined on the quantitative colony assay at 6 days after carcinogen treatment. SCE analysis demonstrates that for a period of 48 h after carcinogen exposure, during which time the cells undergo at least four replicative cycles, DNA damage generating SCE induced by all chemical carcinogens either persisted or was partially removed, whereas u.v.-induced lesions were completely removed. An elevated SCE frequency persisted after two additional cell cycles after treatment with BP, AcAAF or MMC without increased cell lethality as compared to other carcinogens whose lesions were completely eliminated during the same period. Although a correlation between the persistence of SCE and the induction of transformation was not observed for all carcinogens, this study illustrates that DNA damage generating SCE can persist over several replicative cycles, thus raising the possibility that lasting DNA alterations are important for the induction of neoplastic cell transformation.

Induction of morphological transformation and unscheduled DNA synthesis in Syrian hamster embryo fibroblasts by hexachlorobutadiene and its putative metabolite pentachlorobutenoic acid

Hexachloro-1,3-butadiene (HCBD) is a well known environmental carcinogen. The genotoxic properties of HCBD and its monooxidation product pentachloro-3-butenoic acid (PCBA) were investigated by comparative induction of unscheduled DNA synthesis (UDS) and morphological transformation in the same cell system (Syrian hamster embryo fibroblasts). HCBD and PCBA induce unscheduled DNA synthesis both in the presence and absence of an exogenous metabolizing system. The lowest effective dose for UDS induction is smaller for PCBA (1 μg/ml) than for HCBD (2 μg/ml). The intensity of UDS induction is increased about 3-fold for both compounds after metabolic activation. HCBD and PCBA induce morphological transformation. The lowest effective dose for transformation differs considerably between PCBA (0.8 μg/ml) and HCBD (10 μg/ml). The results are indicative of a genotoxic mechanism for the carcinogenic actions of HCBD and PCBA.