Perfluorooctanoic acid (PFOA) acts as a tumor promoter on Syrian hamster embryo (SHE) cells.

Perfluorooctane sulfonate (PFOS) (C(8)F(17)SO(3)) and perfluorooctanoic acid (PFOA) (C(8)HF(15)O(2)) are synthetic chemicals widely used in industrial applications for their hydrophobic and oleophobic properties. They are persistent, bioaccumulative, and toxic to mammalian species. Their widespread distribution on earth and contamination of human serum raised concerns about long-term side effects. They are suspected to be carcinogenic through a nongenotoxic mode of action, a mechanism supported by recent findings that PFOS induced cell transformation but no genotoxicity in Syrian hamster embryo (SHE) cells. In the present study, we evaluated carcinogenic potential of PFOA using the cell transformation assay on SHE cells. The chemical was applied alone or in combination with a nontransformant concentration of benzo[a]pyrene (BaP, 0.4 µM) in order to detect PFOA ability to act as tumor initiator or tumor promoter. The results showed that PFOA tested alone in the range 3.7 × 10(-5) to 300 µM did not induce SHE cell transformation frequency in a 7-day treatment. On the other side, the combination BaP/PFOA induced cell transformation at all PFOA concentrations tested, which revealed synergistic effects. No genotoxicity of PFOA on SHE cells was detected using the comet assay after 5 and 24 h of exposure. No significant increase in DNA breakage was found in BaP-initiated cells exposed to PFOA in a 7-day treatment. The whole results showed that PFOA acts as a tumor promoter and a nongenotoxic carcinogen. Cell transformation in initiated cells was observed at concentrations equivalent to the ones found in human serum of nonoccupationally and occupationally exposed populations. An involvement of PFOA in increased incidence of cancer recorded in occupationally exposed population cannot be ruled out.

Morphological cell transformation of Syrian hamster embryo (SHE) cells by the cyanotoxin, cylindrospermopsin.

Cylindrospermopsin (CYN) is a cyanotoxin which has been implicated in human intoxication and animal mortality. Genotoxic activity of this hepatotoxin is known but its carcinogenic activity remains to be elucidated. In this work, CYN was assessed for its cell-transforming activity using the Syrian hamster embryo (SHE) cell transformation assay. This in vitro assay is used to evaluate the carcinogenic potential of chemical, physical and biological agents in SHE cells, which are primary, normal, diploid, genetically stable and capable of metabolic activation. We demonstrated that CYN induced a significant increase in morphological cell transformation in SHE cells following a 7-day continuous treatment in the range of non-cytotoxic concentrations 1 x 10(-7)-1 x 10(-2) ng/mL.

2,4-Dichlorophenoxyacetic acid: effects on Syrian hamster embryo (SHE) cell transformation, c-Myc expression, DNA damage and apoptosis.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a selective, systemic auxin-type herbicide extensively used throughout the world. The present research was aimed at studying effects of low and non-cytotoxic concentrations of 2,4-D on SHE cells in relation with carcinogenicity. Effects were studied on Syrian hamster morphological cell transformation, c-Myc expression - both at the gene and protein level - DNA damage and apoptosis. 2,4-D significantly induced cell transformation at 11.5 microM and 23 microM (i.e. 2.5 microg/mL and 5 microg/mL). An increase in the expression of the transcription factor c-Myc, measured by use of RT-PCR with respect to mRNA level and by Western blotting for protein level was registered at these concentrations, as well as genotoxic effects evaluated with the single-cell gel electrophoresis (Comet) assay. Consequences for apoptosis of 2,4-D treatment were also investigated. The fluorochrome acridine orange was used to study DNA fragmentation as a marker of apoptosis. No effect on apoptosis was found at 2,4-D concentrations that induced cell transformation. This was confirmed by the unchanged expression of Bcl-2 and Bax, two regulator genes of the mitochondrial pathway of apoptosis. Our results demonstrate the transforming and genotoxic effects of low concentrations of 2,4-D in mammalian cells. This information contributes to a better understanding of the mechanism of 2,4-D toxicity in mammalian cells and demonstrates that 2,4-D should be considered as potentially hazardous to humans.

Ecotoxicity of a polycyclic aromatic hydrocarbon (PAH)-contaminated soil.

Soil samples from a former cokery site polluted with polycyclic aromatic hydrocarbons (PAHs) were assessed for their toxicity to terrestrial and aquatic organisms and for their mutagenicity. The total concentration of the 16 PAHs listed as priority pollutants by the US Environmental Protection Agency (US-EPA) was 2634+/-241 mg/kgdw in soil samples. The toxicity of water-extractable pollutants from the contaminated soil samples was evaluated using acute (Vibrio fischeri; Microtox test, Daphnia magna) and chronic (Pseudokirchneriella subcapitata, Ceriodaphnia dubia) bioassays and the EC values were expressed as percentage water extract in the test media (v/v). Algal growth (EC50-3d=2.4+/-0.2% of the water extracts) and reproduction of C. dubia (EC50-7d=4.3+/-0.6%) were the most severely affected, compared to bacterial luminescence (EC50-30 min=12+/-3%) and daphnid viability (EC50-48 h=30+/-3%). The Ames and Mutatox tests indicated mutagenicity of water extracts, while no response was found with the umu test. The toxicity of the soil samples was assessed on the survival and reproduction of earthworms (Eisenia fetida) and collembolae (Folsomia candida), and on the germination and growth of higher plants (Lactuca sativa L.: lettuce and Brassica chinensis J.: Chinese cabbage). The EC50 values were expressed as percentage contaminated soil in ISO soil test medium (weight per weight-w/w) and indicated severe effects on reproduction of the collembola F. candida (EC50-28 d=5.7%) and the earthworm E. fetida (EC50-28 d=18% and EC50-56 d=8%, based on cocoon and juvenile production, respectively). Survival of collembolae was already affected at a low concentration of the contaminated soil (EC50-28 d=11%). The viability of juvenile earthworms was inhibited at much lower concentrations of the cokery soil (EC50-14 d=28%) than the viability of adults (EC50-14 d=74%). Only plant growth was inhibited (EC50-17d=26%) while germination was not. Chemical analyses of water extracts allowed us to identify inorganic water-extractable pollutants as responsible for toxicity on aquatic species, especially copper for effects on D. magna and C. dubia. The soil toxicity on collembolae and earthworms could be explained by 4 PAH congeners-fluorene, phenanthrene, pyrene, and fluoranthene. Yet, toxicity of the cokery soil as a whole was much lower than toxicity that could be deduced from the concentration of each congener in spiked soils, indicating that pollutants in the soil became less bioavailable with ageing.

Di-(2-ethylhexyl)phthalate (DEHP) increases Bcl-2/Bax ratio and modifies c-myc expression in Syrian hamster embryo (SHE) cells.

The objective of this work was to study the anti-apoptotic properties of the non-genotoxic rodent carcinogen, di(2-ethylhexyl)phthalate (DEHP) in Syrian hamster embryo (SHE) cells. We demonstrated that a 24 h pre-treatment of SHE cells with 50 microM DEHP inhibited apoptosis triggered by growth factors deprivation. The RNA expression levels of the regulator genes involved in the apoptotic pathway, bcl-2, bax and of c-myc were measured using Western blotting and RT-PCR. We showed that a 24 h treatment of SHE cells with 50 microM DEHP increased (P < 0.05) the bcl-2 expression, while c-myc expression was decreased. No effect on bax expression was observed in the range of 10-50 microM. The defective regulation of apoptosis caused by DEHP treatment could contribute to its carcinogenicity.

Changes in expression of bcl-2 and bax in Syrian hamster embryo (SHE) cells exposed to ZnCl2.

Zinc is involved in many physiological processes and plays a critical role in functional and structural cells. Zinc at concentrations ranging from 100 to 150 micromol L(-1) has been shown to induce morphological transformation of Syrian hamster embryo (SHE) cells. At these concentrations, zinc inhibited apoptosis in SHE cells. The objective of this study was to elucidate the mechanisms of action of zinc on the apoptotic pathway. Effects of 100 and 150 micromol L(-1) ZnCl(2) on the expression of two members of the Bcl-2 family of proteins and on the transcription factor c-Myc in SHE cells was investigated using RT-PCR. No effect on the proto-oncogene c-myc was observed. Up-regulation of bcl-2 expression was found and bax expression was reduced. These changes have been corroborated by immunoblotting. Effects of Zn(2+) on bcl-2/bax ratio were confirmed in apoptotic camptothecin-treated SHE cells. Cloned and sequenced cDNAs obtained from RT-PCR amplifications allowed us to check the RT-PCR products encoded the expected proteins. This study demonstrated that zinc acts in the early phases of the apoptotic process by modification of the bcl-2/bax ratio in normal and apoptotic SHE cells.

ZnCl2 induces Syrian hamster embryo (SHE) cell transformation.

In order to test the hypothesis of a relationship between apoptosis and neoplastic transformation, we studied the transforming potency of zinc, known for its antiapoptotic effects. In this study, zinc chloride (100 microM) was shown to induce morphological transformation (MT) in Syrian hamster embryo (SHE) cells. It was also tested in combination with benzo(a)pyrene (BaP), a positive control for carcinogenicity, or fomesafen, a carcinogenic pesticide with hepatic peroxisomal proliferation properties. A co-exposure of the two carcinogens with 100 microM zinc increased cell transformation in SHE cells. These results were in agreement with the theory of a relationship between the inhibition of apoptosis and induction of cell transformation. The cloning efficiency (CE) of SHE cells seeded at clonal density was raised by zinc, fomesafen and furthermore by the mixture of the two chemicals, which could be explained by the antiapoptotic action of zinc and fomesafen on SHE cells. No change in myc and bax expressions was observed in zinc-treated SHE cells.

ZnCl(2) prevents c-myc repression and apoptosis in serum-deprived Syrian hamster embryo cells.

In order to understand the c-myc implication in the apoptotic process better, we investigated the influence of ZnCl(2) on its expression in normal and transformed Syrian hamster embryo (SHE) cells in relation to apoptosis induced by serum withdrawal. Normal primary SHE cells exposed to a serum-free medium undergo rapid apoptosis characterised by a dramatic down-regulation of c-myc transcription. In these normal cells treated with ZnCl(2), c-myc expression is maintained in serum-starved conditions while apoptosis is inhibited. The results shed light on the involvement of c-myc expression in the survival of normal cells in the absence of growth factors. The regulation of c-myc expression appears to be influenced by zinc treatment as an inhibitor of apoptosis, but mechanisms sustaining the level of c-myc transcription remain to be demonstrated. The hypothesis that maintenance of c-myc expression allows cells to escape apoptosis is in accordance with results in transformed SHE cells that underwent low apoptosis and poor down-regulation of c-myc in serum-deprived conditions.

PARP degradation in apoptotic Syrian hamster embryo (SHE) cells compared to HL60 cell line.

In this study, we attempted to identify apoptotic Syrian hamster embryo (SHE) cells by detecting the specific cleavage of poly(ADP-ribose)polymerase (PARP). Apoptosis was unequivocally identified in serum-deprived SHE cells. After protein electrophoresis and transfer, the anti-PARP antibody (C-2-10) was applied in order to visualize PARP degradation and the anti-polymer antibody (LP96-10) was used to identify PARP and its expected 89-kDa fragment on the membrane after renaturation and NAD+ addition. Results showed that PARP rapidly disappeared during apoptosis in SHE cells, but the resulting fragment remained undetectable with the anti-PARP antibody and no stable polymerase activity of this fragment was measured using anti-polymer antibody. Serum-starved SHE cells were compared to the etoposide-treated HL60 cell line as a control for typical apoptosis-related PARP cleavage. These results underline the fact that while PARP degradation is a criterion for apoptosis, the diagnosis of apoptosis can not rely exclusively on the appearance of its 89-kDa fragment as this signal may fail to appear in some cell systems.

Detection of apoptosis induced by topoisomerase inhibitors and serum deprivation in syrian hamster embryo cells.

The sensitivity of normal diploid Syrian hamster embryo (SHE) cells to apoptosis was tested after treatment with the topoisomerase inhibitors camptothecin and etoposide and after serum withdrawal. Programmed cell death (PCD) was identified through morphological, biochemical, and molecular changes and compared with that of HL60 cell line. The results showed that topoisomerase inhibitors, which were shown to be potent PCD inducers in the HL60 cell line, induced a weaker apoptotic response in SHE cells than after growth factor deprivation. In addition, serum-free medium, which rapidly induced apoptosis in SHE cells, did not affect the HL60 cell line. In both cell types, PCD was expressed by condensed chromatin, fragmented nuclei, and DNA laddering on electrophoretic gels, an indisputable sign of apoptosis. In apoptotic HL60 cells, the cleavage of 113-kDa poly(ADP-ribose)polymerase (PARP) resulted in the so-called apoptotic 89-kDa fragment and was associated with increased caspase-3 activity. In apoptotic SHE cells, PARP degraded early but the degradation profile was not characterized by the appearance of an 89-kDa fragment. Moreover, no activation of caspase-3 was noted. ZnCl(2), which is known to prevent protease activity responsible for apoptosis features, inhibited PARP cleavage and nuclear modifications induced by apoptotic stimuli in both cell types, but with a higher sensitivity in SHE cells. Apoptosis induced by serum deprivation was linked with c-myc negative regulation in SHE cells, but not with p53 protein accumulation, while topoisomerase inhibitors led to p53 stabilization without any change in c-myc expression. Serum-free medium and topoisomerase inhibitors did not modify c-myc expression in the HL60 cell line. The overall results demonstrated that apoptosis, which is a carefully regulated process of cell death, may proceed through mechanisms varying according to cell type or apoptosis inducer. In addition, markers which are generally considered hallmarks of apoptosis may fail to appear in some cell types.

Peroxisome Proliferator-induced Transformation of Syrian Hamster Embryo Cells: Influence of Experimental Procedures.

We compared the ability of four hepatic peroxisome proliferators (HPPs) to induce morphological transformation (MT) in Syrian hamster embryo (SHE) cells under different experimental conditions including the composition of the test medium (DMEM at pH7.35 and 7.0, and in LeBoeuf's modified DMEM at pH6.7) and the modalities of exposure. The HPPs studied were two structurally-related hypolipidaemic agents, clofibrate and methyl clofenapate (MCP), an industrial plasticizer, di(2-ethylhexyl)phthalate (DEHP) and one of its primary active metabolite in vivo, mono(2-ethylhexyl)phthalate (MEHP). SHE cells were exposed to the HPP tested either alone, or in sequential treatments with other carcinogens such as benzo[a]pyrene (BaP) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in order to study possible interactions. A two-stage exposure assay was applied with DMEM at pH7.35 and 7.0. Structural analogues did not give similar results using the same experimental conditions. Indeed, while MCP was more potent at acidic pH, the transforming potency of clofibrate was higher at pH7.0 and 7.35. DEHP and MEHP also behaved differently: in contrast to DEHP, MEHP was more active at pH6.7 than at pH7.0. The MT induction, resulting from the interaction between MCP and BaP or TPA, appeared pH-dependent and higher at pH7.0 than at pH7.35. This study showed that: (i) pH actually influences SHE cell response to HPPs, (ii) the use of acidic medium (pH6.7) does not guarantee a better detection of HPPs' transforming effects and (iii) repeated applications of the test-medium within the 7 days of the assay are more efficient in detecting a transforming potency.

Two-stage exposure of Syrian-hamster-embryo cells to environmental carcinogens: superinduction of ornithine decarboxylase correlates with increase of morphological-transformation frequency.

As part of environmental toxicology, it is important to assess both the carcinogenic potential of xenobiotics and their mode of action on target cells. Since dysregulation of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, is considered as an early and essential component in the process of multistage carcinogenesis, we have studied the mode of ODC induction in Syrian-hamster-embryo(SHE) cells stage-exposed to carcinogens and to non-carcinogens. One-stage (5 hr) treatment of SHE cells with 50 microM clofibrate (CLF), a non-genotoxic carcinogen, or with 0.4 microM benzo(a)pyrene (BaP), a genotoxic carcinogen, slightly decreased basal ODC activity. Using the 2-stage exposure, 1 hr to carcinogen, then replacement by TPA for 5 hr, the ODC activity was higher than that obtained with TPA alone. This ODC superinduction was not observed when SHE cells were similarly pre-treated with non-carcinogenic compounds. Several environmental chemicals, pesticides, solvents, oxidizers and drugs were investigated with this SHE cell model. With one-stage exposure, some xenobiotics decreased basal ODC activity, while for others ODC changes were not noticeable. With 2-stage exposure (chemical followed by TPA), all carcinogens amplified the TPA-inducing effect, resulting in ODC superinduction. Comparative studies of the action of carcinogens and of non-carcinogens, using 2-stage exposure protocols, clearly show a close relationship between ODC induction rate and morphological transformation frequency.

Comparative effects of clofibrate and methyl clofenapate on morphological transformation and intercellular communication of Syrian hamster embryo cells.

The Syrian hamster embryo (SHE) cell system was used to evaluate the ability of two hepatocarcinogenic structurally related peroxisome proliferators (PPs) to induce morphological transformation (MT) of SHE colonies and to inhibit gap junctional intercellular communication (GJIC). Clofibrate and methyl clofenapate (MCP), which was shown to be a more active PP and a more potent carcinogen in vivo than clofibrate, were compared. MCP appeared slightly more active in vitro than clofibrate in affecting MT and GJIC of SHE cells. The morphological transformation of SHE colonies was induced by 50 microM MCP, against 100 microM clofibrate. Moreover, 50 microM MCP potentiated the transforming effects of both benzo[a]pyrene and 12-O-tetradecanoylphorbol-13-acetate. The inhibition of GJIC, measured by transfer of lucifer yellow, was transient and occurred at concentrations inducing morphological transformation. MCP inhibited dye transfer at 50 microM and the inhibition lasted up to 24 h at 100 microM. Inhibition of communication lasted only 4 h with clofibrate and occurred at a higher concentration (175 microM). This study showed that both the SHE cell transformation and dye transfer assays were able to display the different activities of the two PPs, even though the difference in potency observed was smaller than in vivo. It also revealed interactions between non-genotoxic carcinogens and the ability of the SHE cell transformation assay to detect these combined effects.

Synergistic effects of chlordane and TPA in multistage morphological transformation of SHE cells.

The cyclodiene pesticide chlordane has been reported to be a non-genotoxic carcinogen in rodents. The effects of chlordane on SHE cell transformation were investigated in this study. It appeared that chlordane exhibited a weak transforming activity when applied repeatedly at 8 micrograms/ml. No effect resulted from the combination of benzo[a]pyrene-chlordane. In contrast, chlordane in the range 5-20 micrograms/ml and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1 micrograms/ml) highly potentiated each other when applied sequentially. The synergistic effects could be inhibited by dexamethasone. These results led us to study the genotoxicity of chlordane on SHE cells: no DNA adduct formation could be detected in SHE cells treated with chlordane at a concentration potentiating the transforming effect of TPA. We also confirmed that this pesticide markedly inhibited intercellular communication between SHE as well as V79 cells. These results support literature data on the non-genotoxicity of chlordane. Overall, this study highlights the fact that interaction between-non genotoxic carcinogens may enhance the transformation frequency of SHE cells. Thus, combined effects must be taken into account in the evaluation of carcinogenic risk.

Interest of dynamic tests in acute ecotoxicity assessment in algae.

Sorption of toxics by algae may be important and occurs very early. Thus, a decrease of the experimental toxic concentrations in the medium results in understating toxicity when tests are conducted under static conditions. In this work, two different methods of exposure of algae (Chlorella vulgaris) are studied, the static test and the pseudodynamic test. Acute effects (biological and analytical effects) of inorganic compounds (Cu2+, Cd2+, Pb2+, Cr6+) have been evaluated for 96 hr of exposure; in each case, IC50 is much lower in the dynamic condition than in the static one. The percentage of reduction varies from 55 to 75% after 96 hr. Accumulation of metal by chlorellae is greater when testing by the pseudodynamic way, with Cu2+ and Pb2+. But in the case of Cd2+ and Cr6+, the concentration factors are similar in the two kinds of exposure. These results point out the advantage of the pseudodynamic test, of which the methodology is very easy, for a more realistic assessment of acute ecotoxicity in these organisms.