O. Poirot

Cytotoxic and transforming effects of silica particles with different surface properties in syrian hamster embryo (SHE) cells

Several crystalline and amorphous silica dusts (two quartz of natural origin, one cristobalite of natural and two of biogenic origin, three amorphous diatomite earths and one pyrogenic amorphous silica) were studied in the SHE cell transformation assay, in order to compare their cytotoxic and transforming potencies and examine the role of the structure and of the state of the surface on these effects. Some samples were modified by grinding, etching and heating with the aim of establishing relationships between single surface properties and biological responses. The results showed that some quartz and cristobalite dusts (crystalline) as well as the diatomaceous earths (amorphous), but not the pyrogenic amorphous silica, were cytotoxic and induced morphological transformation of SHE cells in a concentration-dependent manner. The ranking in cytotoxicity was different from that in transforming potency, suggesting two separate molecular mechanisms for the two effects. The cytotoxic and transforming potencies were different from one dust to another, even among the same structural silicas. The type of crystalline structure (quartz vs cristobalite) and the crystalline vs biogenic amorphous form did not correlate with cytotoxic or transforming potency of silica dusts. Comparison of cellular effects induced by original and surface modified samples revealed that several surface functionalities modulate cytotoxic and transforming potencies. The cytotoxic effects appeared to be related to the distribution and abundance of silanol groups and to the presence of trace amounts of iron on the silica surface. Silica particles with fractured surfaces and/or iron-active sites, able to generate reactive oxygen species, induced SHE cell transformation. The results show that the activity of silica at the cellular level is sensitive to the composition and structure of surface functionalities and confirm that the biological response to silica is a surface originated phenomenon.

Surface reactivity, cytotoxicity, and transforming potency of iron-covered compared to untreated refractory ceramic fibers.

Untreated and iron-coated refractory ceramic fibers (RCFs) 1, 3, and 4 were examined for their potential to generate free radicals and to catalyze hydrogen peroxide decomposition in cell-free assays and were compared for cytotoxic and transforming potencies in Syrian hamster embryo (SHE) cell system. Coating with a high quantity of iron increased the capability of RCFs to generate hydroxyl radicals and to catalyze the decomposition of hydrogen peroxide. In the SHE cells, the untreated RCFs had varying ability to induce inhibition of cell proliferation, cytotoxicity (as measured by the colony-forming efficiency, CE) and morphological transformation, in a concentration-dependent manner. According to cytotoxic and transforming potencies, they ranged as follows: RCF3 > RCF1 > RCF4. The lethal concentration 50 (LC50; decrease of CE to 50% of controls after 7 d of treatment) expressed per number of RCF3 and RCF1/cm 2 of culture dish was 2.5 2 10 4 and 3.7 2 10 4 , respectively, whereas RCF4 was not cytotoxic up to the highest concentration tested (23.7 2 10 4 fibers/cm 2 ). At LC50, RCF3 was 1.4-fold more transforming than RCF1, and the weakest, RCF4, induced less than 1% transformation. Iron coating of RCF1 and RCF3 markedly attenuated their cytostatic, cytotoxic, and transforming potencies without a linear concentration-transformation relationship. In contrast, iron coating of RCF4 affected slightly its low transforming potency, although the growth inhibitory effect was reduced. The observed decrease rather than increase in the cytotoxic and transforming potencies of the active samples RCF1 and RCF3 by their coating with large amounts of ferric iron suggests that it is not the quantity or any form of iron on the surface of fibers but the iron, even in trace, in a particular redox and coordinate state that might play a role in the fibers surface reactivity with regard to the biological material. Surface chemical functions involved in the interaction with the cell could be inactivated by the deposition of a high quantity of Fe(III) on the surface of fibers. Physicochemical studies correlated to biological effects is an approach for understanding the properties of solids related to a given biological response and for elucidating the cellular and molecular mechanisms.

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.

Study of Cell Division Aberrations Induced by Some Silica Dusts in Mammalian Cells in Vitro.

Previously we observed that some crystalline and amorphous (diatomaceous earths) silicas (but not pyrogenic amorphous silica) induced morphological transformation of Syrian hamster embryo (SHE) cells. In order to explore the mechanisms of the silica-induced cell transformation, in this study we have examined the possibility that silica may cause genomic changes by interfering with the normal events of mitotic division. The SHE cells were exposed to transforming samples of Min-U-Sil 5 quartz and amorphous diatomite earth (DE) as well as to inactive amorphous synthetic Aerosil 0X50 at concentrations between 9 and 36 μg/cm2 of culture slide. Effects on the mitotic spindle and on chromosome congression and segregation through the mitotic stages were concurrently examined by differential and indirect immunofluorescence stainings using anti-β-tubulin antibody. Min-U-Sil 5 and DE dusts induced a significant increase in the number of aberrant mitotic cells detected by differential staining. Increased frequencies of monopolar mitoses and scattered chromosomes as well as a small incidence of lagging chromosomes in DE-treated cells were observed. The immunostaining was more efficient in the detection of spindle disturbances. Min-U-Sil induced a significantly concentration-dependent increase of monopolar spindles. At the highest concentration, highly disorganized prophase spindles and prometaphase multipolars were observed. These damages caused a concentration-dependent decrease in metaphase to anaphase transition. DE-induced spindle aberrations did not reach significant levels over control, although increase in monopolar and multipolar spindles were recorded. Exposure to OX50 particles did not disrupt spindle integrity. To determine whether micronuclei (MN) arise from divisional abnormalities induced by the active samples, we performed in SHE and human bronchial epithelial cells kinetochore (K)-specific and centromere (C)-specific staining, respectively. A concentration-dependent increase in K+ and C+ MN with increase of K+/K− and C+/C− MN ratio were induced by Min-U-Sil in both cells systems. The DE sample was positive only in SHE cells. The results suggest that some silicas are potential aneugens by disturbance of cell division, leading to genomic imbalance that can be one of the mechanisms of silica-induced cell transformation.

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.