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Apoptosis inhibition and ornithine decarboxylase superinduction as early epigenetic events in morphological transformation of Syrian hamster embryo cells exposed to 2-methoxyacetaldehyde, a metabolite of 2-methoxyethanol

We have conducted a study to determine the carcinogenic potential of ethylene glycol monomethyl ether (EGME), a member of the glycol ether family, as compared to its reactive metabolite 2-methoxy-acetaldehyde (MALD). Since disruption of equilibrium between cell proliferation and cell death is thought to play a key role in multistage carcinogenesis, we investigated, in Syrian hamster embryo (SHE) cells exposed to various doses of EGME and MALD, impairment in apoptosis rate and in ornithine decarboxylase (ODC) metabolism. The activity of this rate-limiting enzyme of polyamine biosynthesis is closely related to cell proliferation and cell transformation. At the end-point, comparative action of the two products on SHE cell morphological transformation frequency was evaluated. One-stage exposure of SHE cells to 2 mM EGME and 200 microM MALD for 5 h did not change basal apoptotic level, whereas 0.16 microM phorbol ester (TPA) decreased it. Using two-stage exposure protocol (1 h xenobiotic followed by 5 h TPA), MALD strongly inhibited apoptosis more than did TPA alone; the parent compound EGME did not have any effect on TPA inhibiting action. Western blotting analysis showed that sequential treatment (MALD/TPA) increased Bcl-2 oncoprotein expression, whereas Bcl-XL and Bax proteins were not changed. The same staged exposure of SHE cells to MALD/TPA strongly induced ODC activity, and the rate was higher than that obtained with TPA alone: this was accompanied by an increase of ODC protein level. This ODC superinduction was not observed with EGME/TPA treatment. In long-term SHE-cell morphological transformation assay, staged exposure to MALD (800 microM or 1 mM for 24 h) followed by TPA applications increased the number of transformed colonies at the seventh day. Such early cooperative events as apoptosis inhibition and ODC superinduction, followed by the increase of SHE-cell transformation frequency, are highly indicative of a carcinogenic potential for the metabolite, MALD.

Cytotoxicity and genotoxicity of panel of single- and multiwalled carbon nanotubes: in vitro effects on normal Syrian hamster embryo and immortalized v79 hamster lung cells.

Carbon nanotubes (CNTs) belong to a specific class of nanomaterials with unique properties. Because of their anticipated use in a wide range of industrial applications, their toxicity is of increasing concern. In order to determine whether specific physicochemical characteristics of CNTs are responsible for their toxicological effects, we investigated the cytotoxic and genotoxic effects of eight CNTs representative of each of the commonly encountered classes: single- SW-, double- DW-, and multiwalled (MW) CNTs, purified and raw. In addition, because most previous studies of CNT toxicity were conducted on immortalized cell lines, we decided to compare results obtained from V79 cells, an established cell line, with results from SHE (Syrian hamster embryo) cells, an easy-to-handle normal cell model. After 24 hours of treatment, MWCNTs were generally found to be more cytotoxic than SW- or DWCNTs. MWCNTs also provoked more genotoxic effects. No correlation could be found between CNT genotoxicity and metal impurities, length, surface area, or induction of cellular oxidative stress, but genotoxicity was seen to increase with CNT width. The toxicity observed for some CNTs leads us to suggest that they might also act by interfering with the cell cycle, but no significant differences were observed between normal and immortalized cells.

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 μM clofibrate (CLF), a non‐genotoxic carcinogen, or with 0.4 μM 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. Int. J. Cancer 75:744–749, 1998.© 1998 Wiley‐Liss, Inc.

Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: A 4-week inhalation study

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.

In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts

Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres.

Assessment of the Pulmonary Genotoxicity of Bitumen Fumes in Big Blue® Transgenic Rats

Occupational and environmental exposures to bitumen fumes during road paving or roofing activities represent a safety issue since this complex mixture of volatile compounds and particles contains carcinogenic polycyclic aromatic hydrocarbons (PAH). However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning bitumen fumes toxicity. In order to gain better insights on their genotoxic potential, we used an experimental device able to generate bitumen fumes at 170°C (upper range used during road paving operations) with a total particulate matter of 112 ± 13 mg/m3 and a mass median aerodynamic diameter of 4.6 μ m. The nose-only exposure of Big Blue® transgenic rats was performed 6 h/day for 5 consecutive days. Biological exposure to this aerosol was monitored by measuring the rat urinary levels of 1-hydroxypyrene and lungs were collected at various time-points after the end of treatment for further analysis. As determined by the 32P post-labeling method, no DNA bulky adduct was found in control animal lungs, while one was detected in exposed rats 3 and 30 days after the end of treatment, suggesting that this genetic alteration was rather stable. However, there was no correlation between the amount of DNA adduct and the level of urinary of 1-hydroxypyrene. The pulmonary mutagenic properties of bitumen fumes were determined by analyzing the mutation frequency and spectrum of the neutral reporter gene cII inserted into the transgenic rodent genome. Thirty days after exposure, the cII mutant frequency was similar in control and exposed rat lungs. However, a modification of the mutation spectrum was noticeable in treated animals. The most striking difference was an increase of G:C to T:A and A:T to C:G transversions in exposed rodents. In addition, the increased occurrence of G:C to T:A transversions at CpG dinucleotides in treated animals was consistent with DNA adduction by PAH. Even though these data failed to demonstrate a clear pulmonary mutagenic potential of bitumen fumes in our experimental conditions, the analysis of the mutational spectrum provides a sensitive measure of a quantitative increase of specific mutations which may be associated with DNA adduct formation. The weak mutagenic response might also be related to the small inhalable fraction of bitumen fume particles reaching the deep lung. In conclusion, these results could give insights on the mechanism of action of bitumen fumes and provide additional information concerning human risk assessment associated with bitumen exposure.