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.

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/cm(2) 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-ß-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.

Cytotoxic and transforming effects of some iron-containing minerals in Syrian hamster embryo cells.

Four physicochemically characterized iron-containing minerals, one fibrous (a nemalite [brucite]) and three nonfibrous (a biotite [phyllosilicate], a magnetite (Fe3O4), and a goethite [FeOOH alpha]), were studied for cytotoxicity and morphological transformation of Syrian hamster embryo (SHE) cells. When colony-forming efficiency was used as a measure of cytotoxicity, it appeared that the nemalite was about 1.7-fold more cytotoxic than the biotite and magnetite. However, if the inhibitory effect on the cell growth was considered the nemalite appeared to be 8-fold more effective. The analysis of the cell cycle kinetics by flow cytometry revealed a time- and dose-dependent delay in the progression of cells through the cell cycle, with the accumulation of cells in S and G2-M phases, more particularly in the cultures treated with nemalite. While the goethite was neither cytotoxic nor transforming, the other three dusts were, in a dose-dependent manner, efficient in inducing morphological transformation of SHE cells. According to their transforming potency they ranged as follows: nemalite > biotite > magnetite. A 18-fold higher treatment concentration of magnetite than that of nemalite was necessary to induce the same transformation frequency. The iron chelator desferrioxamine abolished the transforming effect of nemalite. The results suggest that (i) the cytotoxicity and the transformation are induced by some divalent iron-containing minerals and that they are two distinct processes; (ii) there is a varying ability among these dusts to induce cell transformation; and (iii) the bioavailability of divalent iron leading to formation of reactive iron-oxygen species could mediate the transforming potency of a mineral. Physicochemical studies correlated to biological effects of many metallic mine dusts are the only approach for understanding their mechanisms of action and their role in occupational pathology.

Role of solubilized chromium in the induction of morphological transformation of Syrian hamster embryo (SHE) cells by particulate chromium(VI) compounds.

Chromium(VI) compounds--Ca, Sr, Zn and Pb chromates--were studied for cytotoxicity and morphological transformation in Syrian hamster embryo (SHE) cells in relation to their solubilization in cell culture conditions and intracellular Cr concentration. Ca, Sr and Zn chromates were completely solubilized after 1 day of incubation in cell cultures; for Pb chromate, 20-36% Cr was solubilized only after 7 days. In two parallel transformation assays, the SHE cells were treated with suspensions or with corresponding supernatants (containing only solubilized Cr) of these compounds. A statistically significant relationship was observed between the Cr treatment concentration and the amount of Cr per cell, irrespective of the compound (except suspensions of Pb chromate). The cytotoxicity was due to extracellular solubilized chromium because treatments with either supernatants or suspensions of Ca, Sr and Zn chromates gave the same LC50 of 0.31 +/- 0.01 microgram Cr/ml. A clear dose-response relationship was observed for the induction of morphological transformation for each compound, either previously solubilized or in suspension. The expression of the transformation frequencies as a function of the Cr concentration/cell revealed that (i) the transformation frequency is dependent on the Cr concentration/cell irrespective of the Cr compound Ca, Sr or Zn chromate, (ii) the transformation frequency induced by solubilized Pb chromate is higher than that induced by the other compounds at the same concentration of Cr/cell. A double treatment with solutions of Cr and Pb at corresponding concentrations induced the same transformation frequency as the solubilized Pb chromate. The results show that the solubilization of particulate Cr(VI) compounds is a critical step for their cytotoxic and transforming activities; the intracellular soluble Cr is the sole causative agent of the transforming activity of Ca, Sr and Zn chromates, while Pb appears to act synergistically with Cr in inducing the transformation by Pb chromate.

Cytotoxic and neoplastic transforming effects of industrial hexavalent chromium pigments in Syrian hamster embryo cells.

Twenty eight moderately water-soluble to insoluble chromium (VI) compounds, such as zinc and lead chromate, industrial and laboratory synthesized pigments, and the analytical reagents strontium, barium and calcium chromate, were physicochemically characterized and studied for cytotoxicity and morphological transformation in cultured Syrian hamster embryo (SHE) cells. In vivo validation of malignancy of transformed SHE cells was performed. A high physicochemical diversity among the complex chromium pigments was revealed. The solubility of the compounds was greatly increased after incubation in a complete medium and even higher under cell culture conditions. The cytotoxic effects appeared to be due principally to extracellular solubilized chromium because the most solubilized compounds. Zn, Ca and Sr chromates, were equitoxic at about the same Cr concentration treatment and 8-fold more cytotoxic than less soluble compounds such as some Pb chromates and Ba chromate. However, certain physicochemical properties of lead chromate pigments could also influence their cytotoxic activity. All test compounds were, in a dose-dependent manner, efficient in inducing morphological transformation of SHE cells. Many of the Cr pigments, although physicochemically different, were similarly effective in transformation induction. Nevertheless, compounds among Zn and Pb chromates had various transforming potencies. Ba chromate was the least active in inducing transformation. Certain physicochemical properties could mediate the transforming activity but no particular relationship could be established between any one of the physicochemical parameters and the transforming potency. Cloned morphologically-transformed colonies of SHE cells were grown in soft agar medium and showed true neoplastic behaviour by tumour formation in syngeneic animals. These results show that various chromate pigments containing either Zn or Pb, of medium to very low aqueous solubility, induced neoplastic transformation of SHE cells.

Chromosome aberrations in peripheral blood lymphocytes of welders and characterization of their exposure by biological samples analysis.

Chromosomal aberrations in cultured lymphocytes obtained from 55 welders and 55 matched controls were analyzed. Depending on the welding techniques and the nature of the consumables and metals welded, three separate groups of welders were examined. Chromium, nickel, and manganese levels in serum and urine were measured to assess the exposure to welding fumes. A statistically significant increase of chromosomal aberrations was found in one of the three analyzed groups of welders. This group used the semi-automatic metal active gas welding process with cored wire containing nickel for welding mild steel. These welders had significantly higher concentrations of serum and urine manganese and, unlike the other welders, significantly elevated concentrations of nickel, both in serum and urine. However, no significant correlations between nickel or manganese levels and the frequency of chromosomal aberrations were found. There was a significant correlation between the length of welding employment of these welders and the frequency of chromosomal breaks, although there was no significant correlation between age and the frequency of chromosomal aberrations. The other two groups of welders, for which the analyses of biologic fluids proved chromium and manganese exposure, had no statistically significant higher frequency of chromosomal aberrations. One of these groups used the manual metal arc welding process with coated electrodes for welding mainly mild steel and the other group used the tungsten inert gas welding process for welding stainless steel. A significant correlation between the daily amount of cigarettes smoked and the frequency of chromosomal breakages, in controls as in welders, was observed. The present data indicate that certain welding processes may generate fumes that seem to have a clastogenic activity.

Cellular uptake, cytotoxic and mutagenic effects of insoluble chromic oxide in V79 Chinese hamster cells.

The cellular uptake, the cytotoxicity and the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells exposed to insoluble crystalline trivalent chromium [Cr(III)], Cr2O3, were investigated. Intracytoplasmic Cr2O3 crystalline particle-containing vacuoles were observed by electron microscopy. Concentrations of 50-200 micrograms/ml did not have a marked killing effect but did show a predominantly concentration-dependent inhibitory effect on cell cycle progression with accumulation of cells in G2 phase. Exposure for 18 h to Cr2O3 induced a statistically significant (p less than 0.001) increase in the mutation frequency of up to 10-fold over the controls. Expression time was 6 days for the lowest concentration and 9 days for the highest. Culture of 6-TGr clones in selective media indicated that they were mutants at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. Examination of growth patterns of Cr2O3-induced mutants showed that, after a delay in reinitiating cell growth, they had varying growth kinetics. The results indicate the ability of a particulate (Cr(III) compound to induce mutation in a mammalian cell system and the usefulness of such systems for detecting genotoxic insoluble metal compounds.

Sister chromatid exchanges in Chinese hamster V79 cells treated with the trivalent chromium compounds chromic chloride and chromic oxide.

The induction of sister chromatid exchanges (SCEs) in Chinese hamster V79 cells exposed to soluble CrCl3 and insoluble Cr2O3, compounds of trivalent chromium (Cr3+), was determined. Their ability to induce SCEs was compared with those of three hexavalent chromium (Cr6+) compounds: K2CrO4, Na2CrO4 and Na2Cr2O7. Both the delay in progression through the cell cycle induced by Cr3+ compounds and the SCE frequencies in the delayed cells were also evaluated. The exposure for 28 h to CrCl3 and Cr2O3 at concentrations of 9.7-39 micrograms and of 34-136 micrograms of Cr3+ per ml, respectively, induced a statistically significant (p less than 0.001) dose-dependent increase in SCEs up to 1.9-fold (CrCl3) and 4-fold (Cr2O3) over control levels. Compared with the effective concentrations of Cr6+ compounds, which produced up to 4-fold increase of SCEs, inducing concentrations of CrCl3 and Cr2O3 were 300- and 1000-fold higher in terms of chromium. By prolongation of treatment time up to 48 h, a progressive dose- and time-related enhancement in SCE frequencies induced by Cr3+ compounds in delayed cells was observed. Lower concentrations of Cr2O3, without effect after 28 h of treatment, induced an increase of SCEs by prolongation of exposure time.