ToxSci Advance Access originally published online on March 29, 2006
Toxicological Sciences 2006 91(2):510-520; doi:10.1093/toxsci/kfj177
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Surface Reactivity, Cytotoxic, and Morphological Transforming Effects of Diatomaceous Earth Products in Syrian Hamster Embryo Cells
,1
* Département Polluants et Santé, Institut National de Recherche et de Sécurité, 54501 Vandoeuvre Cedex, France; Dipartimento di Chimica IFM and Interdepartmental Center "G. Scansetti" for Studies on Asbestos and Other Toxic Particulates, University of Torino, 10125 Torino, Italy; and Dipartimento di Ingegneria Chimica e Scienza dei Materiali, Politecnico di Torino, 10129 Torino, Italy
1 To whom correspondence should be addressed. E-mail: christian.darne{at}inrs.fr and bice.fubini{at}unito.it.
Received January 10, 2006; accepted March 27, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTAL DATAREFERENCES |
|---|
In order to evaluate the effect of thermal treatments on the surface reactivity and carcinogenic potential of diatomaceous earth (DE) products, the physicochemical features of some specimens—derived by heating the same original material—were compared with their cytotoxic and transforming potency. The samples were an untreated DE (amorphous) progressively heated in the laboratory at 900°C (DE 900) and 1200°C (DE 1200) and a commercial product manufactured from the same DE (Chd) from which the finer fraction (< 10–µm diameter) was separated (Chd-F). Quartz (Min-U-Sil 5) and a vitreous silica (amorphous) smoothed up with hydrofluoric acid and were used as positive and negative controls, respectively. All samples were analyzed for their degree of crystallization, for their ability to release free radicals and reactive oxygen species, and for their cytotoxic and transforming potencies in Syrian hamster embryo (SHE) cells. X-ray diffractometry showed that DE 900, like DE, was still amorphous, whereas DE 1200 as well as the commercial product (Chd) were partially crystallized into cristobalite. The ability of the dust to release hydroxyl (•OH) radicals in the presence of hydrogen peroxide, as revealed by the spin-trapping technique, was as follows: Chd-F, DE 1200 > Chd > DE 900 > DE, suggesting that on heating, the surface acquires a higher potential for free radical release. Most of the silica samples generated COO• radicals from the formate ion, following homolytic rupture of the carbon-hydrogen bond, in the presence of ascorbic acid. A concentration-dependent decrease in cell proliferation and colony-forming efficiency was observed in SHE cultures treated with Chd-F, Chd, and DE. Heating abolished DE cytotoxicity but conferred a transforming ability to thermal treated particles. DE was the only sample that did not induce morphological transformation of cells. According to their transformation capacity, the samples were classified as follows: Chd-F > Chd, DE 1200 > DE 900 >> DE. Taken together, the reported results suggest that (1) the transforming potential of a biogenic amorphous silica is related to the thermal treatment that transforms the original structure in cristobalite and generates surface active sites; (2) the reactivity of samples in releasing •OH radicals correlates to their transforming ability; (3) the finer fraction of the commercial product is significantly more toxic and transforming than the coarse dust; and (4) opposite to silica dusts of mineral origin, which loose both cytotoxicity and transforming ability upon heating, heated diatomite acquires a cell-transforming potency. DE products should be thus considered a set apart of silica-based potentially toxic materials.
Key Words: diatomaceous earth; heated diatomaceous earth; cristobalite; hydroxyl radicals; surface reactivity; cytotoxicity; SHE cell transformation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTAL DATAREFERENCES |
|---|
The International Agency for Research on Cancer has classified quartz and cristobalite as carcinogenic to humans (group 1) on the basis of sufficient evidence for the carcinogenic effects in experimental animals and in humans (IARC, 1997
). Amorphous diatomaceous earths (DEs) were not classifiable for their carcinogenicity (group 3) considering the limited epidemiological and experimental data available. DE is the product of deposition over geological time of the siliceous skeleton of unicellular algae, diatoms. It is the largest source of biogenic amorphous silica. Occupational exposure to this dust may occur during either extraction of the native material or processing, and the calcined products are used in a variety of industries as a filtration agent, mineral charge, refractory, abrasive, carrier, or adsorbent material (IARC, 1997). The raw material is amorphous, but commercial dusts are partially crystallized into a cristobalite form, as a consequence of flux calcination.
The mechanisms of crystalline silica–induced carcinogenesis are only partially understood. Several studies have indicated that the pathogenesis of crystalline silica may be related to the generation of reactive oxygen species (ROS) both from particle and from inflammatory cells (Donaldson and Borm, 1998; Fubini, 1998; Fubini and Hubbard, 2003; Fubini and Otero-Aréan, 1999; IARC, 1997; Saffiotti et al., 1994; Shi et al., 1998). As most in vivo and in vitro studies on genotoxic effects have been focused on quartz, few data are available on the effects of DEs.
Previous studies by some of us (Elias et al., 2000, 2002a; Fubini et al., 2001) showed that, like crystalline silica, some commercial DEs were cytotoxic and induced morphological transformation of Syrian hamster embryo (SHE) cells, which further acquired tumorigenic properties (Elias et al., 2002b). Using various surface-modified dusts, we showed that the surface reactivity of the particle modulated the in vitro cellular response: silanol patches were involved in cytotoxicity effects, while ROS generated by surface active sites were involved in the transforming potency of silica. A direct relationship was found between the amount of hydroxyl radicals (•OH) released by the particles and the transformation frequency (TF, Fubini et al., 2001). The transforming activity was also related to previous thermal treatments of the diatomaceous dust (Elias et al., 2000).
We have therefore started up a systematic investigation on one original DE sample, as received, and heated at increasing temperatures in order to identify which physicochemical features, modified by heating, were related to the cellular effects elicited. The present paper reports data on the variation in free radical activity and on the cytotoxicity and morphological transformation of SHE cells induced by the original sample and its heated products. A commercial product, derived processing the same DE source, and its finer fraction were also included in the study in order to compare their activity with that of the products heated in the laboratory.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTAL DATAREFERENCES |
|---|
General Experimental Design
The effects of silica particles on cell growth and viability depend on cellular system, surface reactivity of particles, treatment concentrations, etc. and can be expressed by mitogenic, cytostatic, and/or cytotoxic responses (Elias et al., 2000
; Fubini and Otero-Aréan, 1999; Fubini et al., 1998, 1999). The three assays used in this study to measure alterations in SHE cell functions and cytotoxic potential of samples were the following.
Cell proliferation assay.
This assay provides information on the ability of proliferating culture cells to divide up to a stationary state. Particle treatments inducing inhibition of cell proliferation without a lethal effect (cytostatic effect) have been determined for each sample.
Cell division aberration assay.
This assay was used to assess the ability of cells to perform their normal mitotic division. Early effects of silica particle on the mitotic spindle and on the chromosome congression and segregation during mitosis can result in cell death or genomic changes of some surviving cells. Significant division abnormalities can lead to genomic imbalance in the following cell generations that appears to be one of the mechanisms of silica-induced cell transformation (Béna et al., 2000; Elias et al., 2002b).
Cloning efficiency.
This assay measures the ability of isolated cells to give rise to colonies of daughter cells at the end of several days. The cell death (cytotoxicity), as the final consequence of irreversible cell damage induced by the particles, was determined by the decrease of the number of cell colonies versus that of control cells. These data were obtained from the SHE cell transformation assay that has the advantage to assess both the cytotoxicity and the morphological transformation on the same treated cells and after the same incubation time.
The transforming potency of silica dusts was assessed using the following assay.
SHE cell transformation assay.
Among the current in vitro genotoxicity assays, the cell transformation is the most relevant for the process of carcinogenesis in vivo, involving many of the same genetic and epigenetic mechanisms. The SHE cell system, the most extensively used transformation assay, is applied also to understand the mechanisms of carcinogenesis as to detect carcinogens (Yamasaki, 1996). Taking into account the high predictivity of SHE cell assay for human and/or rodent solid carcinogens, the European Centre for Validation of Alternative Methods recommended it as an alternative method for investigating the carcinogenicity of solid materials (Fubini et al., 1998). We used SHE cell transformation assay in combination with the other end points because cellular proliferation, mitotic division, and viability are involved in the expression of transformation induced by silica particles. Moreover, data on the mechanisms of action of the samples can be obtained. This approach allows also to distinguish between a cytotoxic but not transforming sample and a transforming and/or not cytotoxic one.
To avoid misinterpretation of results, all the assays have been performed on the same cell type, SHE cells, using preselected batches (i.e., cells giving expected results with a positive and a negative control). The treatment concentrations of silica particles used in each assay were settled after preliminary experiments and are indicated subsequently.
The biological responses to silica samples were analyzed in relation with their degree of crystallization and surface reactivity.
Silica Samples
The set of samples investigated, described in Table 1, included (1) a biogenic DE, amorphous; (2) a commercial dust, industrially prepared by flux-calcining DE at high temperature and, consequently, partially crystallized into cristobalite (Chd); (3) a Chd sample depleted in particles with a diameter higher than 10 µm by sedimentation in isopropylic alcohol (finer particle fraction) (Chd-F); (4) a DE sample heated at 900°C (DE 900); and (5) a DE sample heated at 1200°C (DE 1200) in the laboratory. DE 900 and DE 1200 were prepared by heating DE particles for a period of 5 h in a platinum 98 bucket in order to avoid surface contamination during the thermal process and then analyzed by differential scanning calorimetry and thermal differential analysis; (6) Min-U-Sil 5 quartz, the most widely studied sample in in vitro and in vivo studies (IARC, 1997), was used as a positive control; (7) a vitreous amorphous silica (VHF), prepared by grinding a pure silica glass and treated with hydrofluoric acid (0.01M) in order to smooth it up and remove any surface reactivity, previously found biologically inert (Elias et al., 2002a), was used as a negative control.
|
Physicochemical Characterization of the Samples
The degree of crystallinity was determined by x-ray diffractometry (Philips Electronics, Eindhoven, The Netherlands). Micromorphology was explored by scanning electron microscopy (SEM) (Stereo Scan 420 Leica, Cambridge, UK). Impurities were analyzed by inductively coupled plasma spectrometry (Spectro Ciros CCD, Kleve, Germany) (Table 1). Particle size distribution was measured by SEM (JEOL 840A, Tokyo, Japan) (Table 2). The surface area of the dust specimens was evaluated by BET nitrogen adsorption method (Quantasorb, Quantachrome, Syosset, NY) (Table 2).
|
Free Radical Generation
Free radical release from aqueous suspensions of the particles was monitored by electron paramagnetic resonance (EPR) spectroscopy (PS 100x Adani EPR spectrometer, Adani, Minsk, Belarus) using the spin trap 5,5'-dimethyl-1-pirroline-N-oxide (Fluka, Buchs, Switzerland) (Fubini et al., 1995
). Hydrogen peroxide (78mM; Sigma, Milano, Italy) was used as target molecule to detect hydroxyl (•OH) radical production, and formate ions (1M sodium formate; Sigma) were used as target molecules to detect the formation of COO• radicals. In some experiments, COO• radical release was also tested in the presence of ascorbic acid (3mM; Sigma) used as reducing agent. The kinetics of free radical yield were followed up to 1 h. The amount of radicals released was evaluated by measuring the height of the EPR signal assuming that its width was identical for all signals. Each point represents the mean values ± SEM of at least three experiments. In order to preserve their physicochemical characteristics, the samples were not submitted to any preliminary sterilization.
Cell Cultures
SHE cell cultures were established from individual 13-day gestation fetuses (inbred colony, Institut National de Recherche et de Sécurité [INRS], Vandoeuvre, France). Culture medium was Dulbecco modified Eagles medium (DMEM; Invitrogen, Cergy Pontoise, France), pH 7, supplemented with 20% of preselected fetal calf serum (Dutscher, Brumath, France) and 2mM L-glutamine (Invitrogen) without antibiotics. Cells were incubated at 37°C, 10% CO2. Cryopreserved primary cultures were selected for cell growth, cloning efficiency (CE), and spontaneous and induced morphological transformation. Primary and secondary cultures from a batch that yielded results consistent with the historical range were used in the study. A unique embryo was used for cell transformation assay, and two different embryos were used for cell proliferation assay. The two embryos did not exhibit the same sensitivity in cell proliferation assay, but their transforming abilities were similar.
Cell Proliferation Assay
Cells (30,000 cells/ml) were cultured for 24 h at 37°C, 10% CO2, in culture medium. Cell cultures were then treated continuously with culture medium (control), or silica suspensions in final concentrations ranged between 1.82 and 43.7 µg/cm2 for up to 3 days. At the day of treatment and at 24, 48, and 72 h posttreatment, cells were removed by trypsination and counted (Coultronics, Beckman Coulter, Villepinte, France). Cell viability was determined by the trypan blue exclusion method. At least five concentrations of silica samples were analyzed, and triplicate cell cultures were used for each treatment concentration and time. The concentration that reduced cell proliferation to 50% of the control, inhibitory concentration 50, was calculated from cell proliferation curves obtained from four to six individual experiments. The mean number of cells ± SEM for each treatment concentration and time was calculated and compared to that of the control using Student t-test.
Cell Division Aberration Assay
SHE cells were seeded into chamber slides (2 x 104 cells in 1 ml DMEM/well) and exposed for 24 h later to particle suspension. For all silica samples, three concentrations per sample were tested in duplicate cell culture (4.5, 9, and 18 µg/cm2). The concentration of 36 µg/cm2 was only used for Chd and Chd-F treatment; the negative control received media alone, and the positive control was treated with colcemid (Invitrogen) at 0.02 µg/ml. After 24 h (
1 cell cycle), slides were fixed and stained differentially for chromosomes and mitotic spindle using the method of Wissinger et al. (1981). At least 100 mitoses per point were examined at x800 magnification (Axioplan, Zeiss, Jena, Germany). Mitotic division aberrations in each stage of mitosis were scored according to the criteria described by Parry et al. (1985). Between three and seven experiments per sample were performed. Statistical analysis of mitotic division aberrations was performed by Fisher exact test at the 95% confidence limit.
CE and Transformation Assay
The assay was performed as described previously (Elias et al., 1989, 2000). X-ray–irradiated SHE feeder cells were seeded at 3 x 104 cells/ml in 60-mm dishes. After 24 h of incubation (37°C, 10% CO2), 300 SHE target cells/dish were seeded onto the feeder cells. Cells were incubated for 24 h at 37°C, 10% CO2, and then exposed to silica sample of which at least three concentrations ranged between 1.9 and 30.4 µg/cm2 for most of the samples and up to 60.8 µg/cm2 for Chd and VHF. Control cells received culture medium alone.
After 7 days of incubation at 37°C, 10% CO2, dishes were washed (Hanks PBS, Invitrogen) and colonies were fixed (absolute methanol) and stained (10% Giemsa). Colonies were counted and examined for morphological transformation in a stereomicroscope (Wild, Leica Microsystems, Wetzlar, Germany). Ten cell cultures were used per treatment concentration and control. For each treatment concentration and control of an individual assay, were scored the following: (1) total colony number, (2) CE = (total colony number/total target cell number seeded) x 100, (3) relative CE = (CE of treated cells/CE of the control) x 100, (4) cytotoxicity = 100 – relative CE, (5) number of morphologically transformed colonies, (6) TF = (the number of transformed colonies/total number of colonies) x 100.
The treatment concentration which induced a 50% reduction in the relative CE, the lethal concentration 50 (LC50), was calculated from the curve for relative CE. The total number of analyzed colonies was between about 1000 and 6000 per treatment concentration. The mean CE ± SEM of the control cultures was of 23.55 ± 2.08% (n = 13). Only one spontaneous transformed colony was registered in one of a total of 13 experiments. For each treatment concentration, data reported the pooled results from three to eight individual assays. The TF (if the TF of control was not 0) and the CE of the treated cultures were compared to those of the concurrent control using Student t-test. The linear correlation coefficient (r) between the TF and the treatment concentration was calculated for each transforming sample.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTAL DATAREFERENCES |
|---|
Free Radical Release from the Particle Surface
In the presence of hydrogen peroxide as target molecule, all dusts generated •OH radicals. A sustained radical release was observed up to 60 min in all the samples examined, suggesting that free radicals were generated via a catalytic mechanism that occurred at the surface of the particles. The EPR signals obtained from this reaction are shown in Figure 1A. The signal intensity is proportional to the amount of radicals generated. The amorphous sample DE exhibited a low reactivity (spectrum a) but thermal treatments increased reactivity dramatically (spectra b and c), suggesting that during the heating process some surface sites on amorphous particles were transformed into active sites capable of generating •OH radicals. The abundance of the active sites at the surface has been evaluated by dividing the signal intensity by the specific surface area of each sample and reported as average of three or more separate experiments in Figure 1B. The large amount of •OH release observed for DE 1200, when compared to DE and DE 900 (approximately 10-fold more), indicates that a thermal treatment above 900°C is required to generate high density of active sites. Chd-F was approximately twice more reactive in releasing •OH radicals than Chd, suggesting that the finer and more homogenous size fraction of particles has an intrinsic higher surface reactivity than the coarse dust.
|
To evaluate the ability of the particle to initiate radical reactions with biomolecules, we measured the generation of COO• radicals from the formate ion, following homolytic rupture of a hydrogen-carbon bond. DE did not generate any COO• radical, opposite to DE 900 and DE 1200 which exhibited an increasing reactivity in COO• radical production (Fig. 2). These results suggest that a treatment at high temperature activates the surface not only in •OH radical release but also for the generation of COO•, except for the commercial product Chd, which was, in this case, unreactive. The difference between the reactivity of heated DE and Chd may depend on the different thermal treatments. Chd, in fact, undergoes a calcination in the presence of a fluxing agent (Na2CO3), whereas DE was only heated in air. COO• generation was also tested in the presence of an endogenous reducing agent (ascorbic acid) in order to simulate the physiological conditions when particles are inhaled and deposited in the lung-lining layer. Ascorbic acid keeps transition metals, typically iron, present at the surface of the particles as trace impurities, in a low redox state, which is required for the production of carbon-centered radicals (Fenoglio et al., 2001
, 2003; Fubini and Hubbard, 2003). All samples acquired or improved their activity in COO• radical generation in the presence of ascorbic acid (Fig. 3A). When the signal intensity was normalized per unit exposed surface (Fig. 3B), DE was the less active, DE 1200 was by far the most active and Chd was in this case more active than Chd-F.
|
|
Quartz, the positive control, released free radicals in both tests, whereas VHF, the negative control, did not present any surface reactivity.
Cell Proliferation
The inhibition of cell proliferation induced by DE, DE 900, DE 1200, Chd, and Chd-F is shown in Figures 4A and 4B. A concentration-dependent decrease in cell proliferation after exposure for 1–3 days to silica dusts was observed for all diatomite samples and quartz, while VHF (negative control) was nontoxic at all the concentrations tested (up to 43.7 µg/cm2).
|
- - -
- - -Compared to the other samples, on a per weight basis, Chd-F and DE exhibited more inhibiting effects, as early as at 24 h of treatment. However, on a per unit surface basis, Chd-F appeared significantly more active than DE. On this basis, the inhibitory potency of the samples ranks in the following order: Chd, Chd-F >DE 1200 > DE, DE 900 (Table 3). Note that DE 1200, Chd, and Chd-F exhibit an inhibitory effect even higher than the positive control (Min-U-Sil 5 quartz).
|
No cell death (trypan blue exclusion test) was observed after treatment with DE, Chd, and Chd-F up to 14.5 µg/cm2 and up to 29 µg/cm2 for DE 900 and DE 1200.
Cell Division
The increase of cell division aberrations induced by the various samples in SHE cells is shown in Figure 5. The most frequent types of aberrations observed were monopolar or tripolar spindles and chromosomes dislocating from mitotic spindle; some bridges in anaphase and scattered chromosomes were also observed.
|
A concentration-dependent increase in abnormal mitoses frequency was observed with all dusts tested, with the exception of DE at 4.5 and 9 µg/cm2. Chd-F sample induced more aberrations than Chd at treatment concentrations of 4.5–18 µg/cm2. Since the treatment with Chd and Chd-F particles at a concentration of 36 µg/cm2 for 1 day was highly inhibiting on cell proliferation and, for Chd-F, also highly cytotoxic, the effects on mitoses at this treatment concentration indicate broad cell damages rather than disturbance of particular cell division function. DE 900, as DE 1200, appeared less active than the DE. Min-U-Sil quartz (positive control) induced a significant concentration-dependent increase of cell division aberrations, as previously reported (Béna et al., 2000
), opposite to VHF which was negative in the assay. The comparison between the samples on a per unit surface basis revealed that a 3-fold increase in aberration mitoses over the control was induced in the order Chd-F > Chd > DE at 2, 2.4, and 9.1 x 10–5 m2/cm2 of culture surface dish, respectively (Table 4). Only a 2-fold increase in aberration mitoses was induced by DE 1200 (8.5 x 10–5 m2/cm2) followed by DE 900 (27.9 x 10–5 m2/cm2).
|
CE and TF of SHE Cells
Relative CE (upper part) and TF (lower part) induced by the different silica samples are reported in Figure 6. Three samples significantly decreased the CE, but their cytotoxic potency was different: Chd-F > DE > Chd. On a per unit surface basis (Table 3), the LC50 of Chd-F was 2.4- and 15-fold lower than that of Chd and DE, respectively. DE 900 and DE 1200 were not cytotoxic up to the highest concentrations tested (30.4 µg/cm2).
|
), DE 1200 (
), Chd-F (
) (negative control). Correlation coefficient (r) TF versus concentration: 
0.99. The solid bar marks 50% reduction in relative CE. Asterisks indicate statistically significant (p < 0.05) decrease of CE.Amorphous DE did not induce morphological transformation while a concentration-dependent increase of the TF was induced by all other samples. The heated samples exhibited a certain degree of transformation, DE 1200 > DE 900 (which was weakly active only above 15 µg/cm2). Chd-F was the most transforming sample and significantly more transforming than the coarse fraction Chd. The same order of activity was found when the transformation potency was calculated on per unit surface basis (Table 5).
|
As previously reported (Elias et al., 2000
, 2002a; Fubini et al., 2001), Min-U-Sil 5 did not express a cytotoxicity up to 30.4 µg/cm2 but induced morphological transformation in a dose-dependent manner at much lower concentrations (between 7.6 and 30.4 µg/cm2). The VHF sample was neither cytotoxic nor transforming (Fig. 6). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTAL DATAREFERENCES |
|---|
Effect of Heating
The main result of the present study is the parallel development of the potential to release free radicals and to cause morphological transformation in SHE cells on heating the original amorphous DE. DE itself did not induce any cell transformation but was cytotoxic. Thermal treatments on DE up to 900°C and above, while enhancing the transformation potency, blunt cytotoxicity, in contrast with that previously found in a cristobalite dust specimen (Fubini et al., 1999
).
On heating, a progressive crystallization of the amorphous particles takes place. At 1200°C (DE 1200) a crystallization of 48% of the original dust into cristobalite was attained, which is close to the value measured in the commercial dust Chd (47%) (Table 1). As expected, the thermal treatment also deeply modifies the state of the surface. Raw DEs are mainly composed by silica but contain a substantial amount of impurities, largely exceeding even what was found to modulate toxicity in commercial quartz dusts (Fubini et al., 2004). The iron content of both amorphous and commercial samples is thus much higher than that on the quartz dust considered as positive control. The presence of such impurities may cause an effect of calcination on hydrophilicity and surface reactivity, different from those observed on crystalline silica specimens of mineral origin, namely, quartz and cristobalite dusts. Thermal treatments on pure quartz were reported to decrease the amount of surface radicals (Fubini et al., 1989) which, together with isolated surface iron centers, are involved in the release of free radicals in aqueous suspensions (Fubini and Hubbard, 2003). In the present case, the thermal treatment increases the reactivity in •OH and COO• radical generation, suggesting that some modifications in the coordinative and oxidative state of iron ions at the surface occurred following the formation of a new crystalline phase (cristobalite).
ROS, Cell Transformation, and Cytotoxicity
The crucial role of ROS in the inflammatory and fibrogenic activity of crystalline silicas is well established, as recently reviewed (Fubini and Hubbard, 2003). Several studies have also shown the role of particle-derived ROS, such as the highly reactive •OH, in quartz-induced genotoxicity and carcinogenicity (Daniel et al., 1995a,b; Saffiotti et al., 1994; Schins et al., 2002; Shi et al., 1995, 1998; Zhang et al., 1999; Zhong et al., 1997). The role of ROS in the induction of cell transformation in vitro by quartz, cristobalite, and diatomite earth samples (Elias et al., 2000, 2002a; Fubini et al., 2001) was reported in previous studies by some of us, where a linear correlation was found between the amount of •OH released by the particles and the TF in SHE cells (Fubini et al., 2001).
Assuming that cytotoxic and transforming effects of silica particles are related to their surface properties, we have reported in Table 6 an overall comparison between the activity of the various dusts on a unit surface basis. In the present case, the •OH radicals released correlates well not only with the transforming potency but also with the inhibition of cell proliferation and, to some extent, with the amount of cell division aberrations. For amorphous and heated samples also the amount of active sites which cleave the carbon-hydrogen bonds, thus generating COO• radicals, follows the same order.
|
The cytotoxic and transforming potencies of the samples examined were unrelated, in agreement with that previously reported (Elias et al., 2000
), suggesting different mechanisms triggered by different surface properties in the two effects elicited. Cytotoxicity would not be related to the generation of free radicals but to other physicochemical features, such as micromorphology, surface charges, and, in particular, the degree of hydrophilicity (Fubini, 1998; Fubini et al., 1999).
As expected on the basis of previous studies (Elias et al., 2000, 2002a), Min-U-Sil quartz was found to release free radicals and to induce cell transformation, whereas VHF did not exhibit any surface reactivity and was fully inactive in cell transformation.
Mitotic Damages and Cell Transformation
Our previous results showed that Min-U-Sil quartz and, to a lesser degree, one transforming DE sample induced early abnormal mitoses, spindle disturbances, and micronuclei with whole chromosomes in SHE and human bronchial epithelial cells (Béna et al., 2000). These results suggested that some silica are potential aneugenic agents leading to a genomic imbalance which can be one of the mechanisms of silica-induced cell transformation. The study of karyotypic changes during the progression of silica-transformed cell to the neoplastic state has shown that aneuploidy is mechanistically important from initiation to tumorigenicity (Elias et al., 2002b). In the present study, all silica samples, except VHF, induced, at a different degree, cell division aberrations after 24 h treatment (Table 4). Only DE was nontransforming on SHE cells. This could indicate that only certain division aberrations induce aneuploid cells in the following cell divisions and that the mechanisms of morphological cell transformation involve several significant genetic and epigenetic changes.
A correlation between the surface reactivity of the particles in ROS generation and the induced cell division aberrations is suggested (Table 6), except for DE 1200, as reactive as Chd-F in •OH radical release but showing a weaker effect on cell division. A more evident relationship between the oxidative stress induced by some silicas and mitotic aberrations has been shown using cotreatment with antioxidants, which significantly decreased the frequency of abnormal mitoses (Elias et al., 2002a).
Effect of Particle Size
The parallel study of the commercial product, Chd, and of the finer fraction of the dust, Chd-F, reveals a higher activity of the latter with respect to the former (Table 6). This is not only due to the mere fact that the higher the surface area, the higher the biological effects caused by the same mass of the material, but also due to the fact that these effects are related to the dust surface reactivity. The finer fraction is in fact more cytotoxic and causes more cell division aberrations and morphological transformations than the coarse material, even when such effects are expressed on a per unit exposed surface basis (Table 6). This is due to a higher abundance of reactive surface sites on small versus larger particles. Interestingly, more •OH radicals—related to transforming potency—are released by the finer fraction than by the coarse per square meter of surface exposed (Table 6).
Variability of DE Hazard
The transforming inability of the DE sample tested in this study adds new information to our previous observations made on the transforming potency of four other DE samples, two of which reported to be fully amorphous but active (Elias et al., 2000; Fubini et al., 2001). As for crystalline silicas of mineral origin, not all DE products are in fact biologically active. The amorphous state has different degrees of structural disorders reflected in their surface reactivity. Inherent characteristics, determined by the origin of a given dust, state of surface, and external factors (contaminants, pretreatments), lead to a high diversity even among amorphous silica particles, which, consequently, affects their biological activity (IARC, 1997). Thus, in the absence of well-defined molecular mechanisms of silica toxicity, risk evaluation procedures require to perform physicochemical and cellular tests on each individual sample.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTAL DATAREFERENCES |
|---|
Heating an original amorphous DE material leads to a progressive crystallization into cristobalite, starting at about 900°C and proceeding up to about half of the material at 1200°C.
The level of impurities in this biogenic material, higher than in crystalline dusts of mineral origin, determines a different behavior on heating, reflected by the final state of the surface and the biological activity of the resulting particles. In contrast to that observed with cristobalite (Fubini et al., 1999) or quartz (Elias et al., 2000; Fubini et al., 2001), which lost both their cytotoxicity and transforming ability on heating, heated diatomite acquires a cell-transforming potency. The transformation potential appears to be correlated with the ability to generate radicals, mainly •OH, from the particle surface. Thus, DE products should be considered a set of silica-based potentially toxic materials, apart from the crystalline silica dusts of mineral origin.
The finding that the transforming potential of DE products largely depends on previous thermal treatments points out the control of the heating process as a key factor for reducing the toxic potential of manufactured DE products. Finally, in exposure conditions, a particular concern should be given to the finer fraction of the particles which have a higher surface reactivity and transforming potency, thus, when inhaled, would likely exhibit a higher pulmonary toxicity.
|
SUPPLEMENTAL DATA |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTAL DATAREFERENCES |
|---|
Supplementary data are available online at http://toxsci.oxfordjournals.org/.
|
ACKNOWLEDGMENTS |
|---|
The research carried out in the University of Turin was supported by INRS, Convention No. 5012587. The authors are grateful to Mr L. Gaté and Mr Y. Guichard for discussion and manuscript revision; to Mrs A. Peltier, INRS, for the chemical analysis of impurities; to Mrs C. Eypert-Blaison, INRS, for the SEM analysis; to Mr J. C. Moulut, INRS, for the x-ray analysis of the particle structure; to Dr M. Tomatis (Università di Torino) and Dr M. Ferraris (Politecnico di Torino) for discussion on the heating procedures; and to Mrs M. Roussel, INRS, for manuscript preparation.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTAL DATAREFERENCES |
|---|
Béna, F., Danière, M. C., Terzetti, F., Poirot, O., and Elias, Z. (2000). Study of cell division aberrations induced by some silica dusts in mammalian cells in vitro. Inhal. Toxicol. 12(Suppl. 3), 189–198.
Daniel, L. N., Mao, Y., and Saffiotti, U. (1995a). Direct interaction between crystalline silica and DNA: A proposed model for silica carcinogenesis. Scand. J. Work Environ. Health 21(Suppl. 2), 22–26.
Daniel, L. N., Mao, Y., Wang, T.-C. L., Markey, C. J., Markey, S. P., Shi, X., and Saffiotti, U. (1995b). DNA strand breakage, thymine glycol production, and hydroxyl radical generation induced by different samples of crystalline silica in vitro. Environ. Res. 71, 60–73.[Medline]
Donaldson, K., and Borm, P. J. A. (1998). The quartz hazard: A variable entity. Ann. Occup. Hyg. 42, 287–294.
Elias, Z., Poirot, O., Danière, M. C., Terzetti, F., Béna, F., Fenoglio, I., and Fubini, B. (2002a). Role of iron and surface free radical activity of silica in the induction of morphological transformation of Syrian hamster embryo cells. Ann. Occup. Hyg. 40(Suppl. 1), 53–57.
Elias, Z., Poirot, O., Danière, M. C., Terzetti, F., Marande, A. M., Dzwigaj, S., Pezerat, H., Fenoglio, J., and Fubini, B. (2000). Cytotoxic and transforming effects of silica particles with different surface properties in Syrian hamster embryo (SHE) cells. Toxicol. In Vitro 14, 409–422.[Medline]
Elias, Z., Poirot, O., Pezerat, H., Suquet, H., Schneider, O., Danière, M. C., Terzetti, F., Baruthio, F., Fournier, M., and Cavelier, C. (1989). Cytotoxic and neoplastic transforming effects of industrial hexavalent chromium pigments in Syrian hamster embryo cells. Carcinogenesis 10, 2043–2052.
Elias, Z., Poirot, O., Terzetti, F., and Danière, M. C. (2002b). Phenotypic and numerical chromosome changes during the neoplastic progression of Syrian hamster embryo cells transformed by silica. Med. Lavoro 93(Suppl.), 52 (Abstract).
Fenoglio, I., Fonsato, S., and Fubini, B. (2003). Reaction of cysteine and glutathione (GSH) at the freshly fractured quartz surface: A possible role in silica related diseases? Free Rad. Biol. Med. 35, 752–762.[CrossRef][ISI][Medline]
Fenoglio, I., Prandi, L., Tomatis, M., and Fubini, B. (2001). Free radical generation in the toxicity of inhaled mineral particles: The role of iron speciation at the surface of asbestos and silica. Redox Rep. 6, 235–241.[CrossRef][ISI][Medline]
Fubini, B. (1998). Surface chemistry and quartz hazard. Ann. Occup. Hyg. 42, 521–530.
Fubini, B., Aust, A. E., Bolton, R. E., Borm, P. J. A., Bruch, J., Ciapetti, G., Donaldson, K., Elias, Z., Gold, J., Jaurand, M. C., et al. (1998). Non-animal tests for evaluating the toxicity of solid xenobiotics. ECVAM Workshop 30, ATLA 26, 579–617.
Fubini, B., Fenoglio, I., Ceschino, R., Ghiazza, M., Martra, G., Tomatis, M., Borm, P., Schins, R., and Bruch, J. (2004). Relationships between the state of the surface of four commercial quartz flours and their biological activity in vitro and in vivo. Int. J. Hyg. Environ. Health 207, 89–104.[CrossRef][ISI][Medline]
Fubini, B., Fenoglio, I., Elias, Z., and Poirot, O. (2001). Variability of biological responses to silicas: Effect of origin, crystallinity, and state of surface on generation of reactive oxygen species and morphological transformation of mammalian cells. J. Environ. Pathol. Toxicol. Oncol. 20(Suppl. 1), 95–108.
Fubini, B., Giamello, E., Pugliese L., and Volante, M. (1989). Mechanically induced defects in quartz and their impact on pathogenicity. Solid State Ionics 32–33, 334–343.[CrossRef]
Fubini, B., and Hubbard, A. (2003). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Rad. Biol. Med. 34, 1507–1516.[CrossRef][ISI][Medline]
Fubini, B., Mollo, L., and Giamello, E. (1995). Free radical generation at the solid/liquid interface in iron containing mineral. Free Radic. Res. 23, 593–614.[ISI][Medline]
Fubini, B., and Otero-Aréan, C. (1999). Chemical aspects of the toxicity of inhaled mineral dusts. Chem. Soc. Rev. 28, 373–381.[CrossRef]
Fubini, B., Zanetti, G., Altilia, S., Tiozzo, R., Lison, D., and Saffiotti, U. (1999). Relationship between surface properties and cellular responses to crystalline silica: Studies on heat-treated cristobalite. Chem. Res. Toxicol. 12, 737–745.[CrossRef][ISI][Medline]
IARC (1997). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. 68. Silica, Some Silicates. Coal Dust and para-Aramid Fibrils. International Agency for Research on Cancer, Lyon, France.
Parry, E. M., Sharp, D. C., and Parry, J. M. (1985). The observation of mitotic division aberrations in mammalian cells exposed to chemical and radiation treatments. Mutat. Res. 150, 369–381.[Medline]
Saffiotti, U., Daniel, L. N., Mao, Y., Shi, X., Williams, A. O., and Kaighn, M. E. (1994). Mechanisms of carcinogenesis by crystalline silica in relation to oxygen radicals. Environ. Health Perspect. 102(Suppl. 10), 159–163.
Schins, R. P. F., Knaapen, A. M., Cakmak, G. D., Shi, T., Weishaupt, C., and Borm, P. J. A. (2002). Oxidant-induced DNA damage by quartz in alveolar epithelial cells. Mutat. Res. 517, 77–86.[ISI][Medline]
Shi, X., Mao, Y., Daniel, L. N., Saffiotti, U., Dalal, N. S., and Vallyathan, V. (1995). Generation of reactive oxygen species by quartz particles and its implication for cellular damage. Appl. Occup. Environ. Hyg. 10, 1138–1144.
Shi, X., Castranova, V., Halliwell, B., and Vallyathan, V. (1998). Reactive oxygen species and silica-induced carcinogenesis. J. Toxicol. Environ. Health B 1, 181–197.
Wissinger, W. L., Estervig, D. N., and Warg, R. J. (1981). A differential staining technique for simultaneous visualisation of mitotic spindle and chromosomes in mammalian cells. Stain Technol. 62, 221–226.
Yamasaki, H. (1996). Use of the Syrian hamster embryo (SHE) cell transformation assay for predicting the carcinogenic potential of chemicals. Mutat. Res. 356, 1–128.[Medline]
Zhang, Z., Shen, H. M., Zhang, Q. F., and Ong, C. N. (1999). Critical role of GSH in silica-induced oxidative stress, cytotoxicity and genotoxicity in alveolar macrophages. Am. J. Physiol. 277, L743–L748.
Zhong, B. Z., Whong, W. Z., and Ong, T. (1997). Detection of mineral dust-induced DNA damage in two mammalian cell lines using the alkaline single cell gel/comet assay. Mutat. Res. 393, 181–187.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||