The ultimate goal of toxicologic investigation of synthetic vitreous fibers (SVFs) is to provide essential input for the assessment of human risk to their exposure. Toxicity of mineral fibers is usually evaluated by testing biopersistence in rodent model. However, a cellular model would be much appreciated in order to reduce, refine, and replace animal models. Pulmonary disorders triggered by inhalation of occupational or environmental mineral particulates can be the endpoints of a chronic inflammatory process in which alveolar macrophages (AMs) play a crucial role. Depending on the type of SVF involved, phagocytosis of fiber leads to activation of macrophages, resulting in release of fiber components and potent mediators, such as reactive oxygen or nitrogen species and cytokines. As a matter of fact, macrophages should be the cells of choice since SVF toxicity is the consequence of fibers and alveolar macrophages interaction. Today, monocytes and macrophages culture are firmly established as a paradigm in toxicology when several endpoints are assayed in macrophages: (1) fiber durability, (2) fiber surface changes, (3) oxidative stress and genotoxicity in macrophage, and (4) macrophage cell viability and apoptosis. This article is a review of up-to-date knowledge of in vitro studies involving macrophages, and assesses endpoints of macrophage toxicity with an emphasis on (1) dissolution, (2) scanning electron microscopy analysis, (3) cytotoxicity, and (4) gene expression.

Evaluating the pathogenic potentials of man-made mineral fibers (MMMF) is an important task performed by the European Community. Noting that it has been proposed that the use of laboratory animals for scientific tests should be reduced or phased out, macrophages then become the cells of choice for conducting in vitro studies. We have evaluated the in vitro toxicity of six commercial stonewool fibers (A, B1, B2, C, D, and E) on U-937 cells. The physical interaction between U-937 cells and MMMF was observed using scanning electron microscopy, and the cytotoxicity was evaluated by studying cell viability using MTT assay and cell apoptosis with an ELISA detection kit. Scanning electron microscopy (SEM) analysis has shown that long fibers can be covered by several macrophages, and that a small fiber can be completely engulfed by one cell. With 50 microg/mL of MMMF, a decrease in cell viability appeared after seven days of incubation, whereas 200 microg/mL induced loss of viability and apoptosis after one day. Fiber D, comprising a high proportion of fibers >20 microm in length and a high concentration of MgO, induced the highest loss in viability and the highest rate of apoptosis compared to the other five fibers. Whether this toxic effect is related to either the physical characteristics of the fibers (such as length), or to the high concentration of magnesium is still to be determined. Because the results can be rapidly obtained, the proposed model is suitable for studying the toxicities of mineral components, even if the tested concentrations are far from the ones reached in the lung.

The toxicity of mineral fibers, whether they are natural or man made (MMMF), is usually evaluated in vivo using biopersistence tests in rodents. Development of an in vitro cellular model would be worthwhile in order to reduce, refine and finally replace animal models. For this purpose, we developed an in vitro assay using human monocytic cell line (U-937) to evaluate a new manufactured rock wool fiber (HDN) biodegradation. Experiments on earlier known mineral fibers asbestos (crocidolite) and glass wool fibers (CM44) were also performed. U-937 responded to HDN and CM44 only if they were activated. Among the different activators we used, Escherichia coli living cells as well as FS were the most efficient as evidenced by alterations of HDN and CM44 surface, detected by scanning electron microscopy, and by the measure of silicon released from the rock wool fibers. Asbestos fibers were not degraded when incubated in the presence of living bacteria. The MMMF modifications were function of the fiber composition, the time of exposure to activated cells and the concentration of activators. The pattern of MMMF degradation by our in vitro system was in accordance with those observed in an in vivo study, thus indicating that the fiber degradation by macrophage cells activated by E. coli living cells as well as FS is a valuable system to assess mineral fibers' biopersistence.

A human monocytes cell line, U-937, incubated in the presence of filtered medium from Escherichia coli culture (FS) has been previously reported to degrade man made mineral fiber and it has been indicated as a good paradigm of in vivo fiber biopersistence evaluation (manuscript accepted for publication). In the present paper, a study is reported aimed to define the molecular modification occurring in the U-937 monocytes during in vitro fiber degradation. The induction of gene expression was investigated in U-937 exposed to rock wool fibers (HDN) in the presence of FS by transcriptome analysis using 20 K DNA microarrays and quantitative RT-PCR. The over-expression of genes related to mobility and cellular adhesion, oxidative stress, immune system stimulation, enzymes, and ions transport protein systems were identified. Among them NCF1 gene, the gene encoding a subunit of NADPH oxidase, over-expression was detected. As the product of this gene allows the formation of superoxide anion that could lead to oxidative stress, HDN fibers were exposed to hydrogen peroxide. Fiber degradation similar to those observed upon incubation with U-937 in the presence of FS was obtained thus suggesting that reactive oxygen species production may be responsible for fiber degradation by U-937 monocytes.

The carcinogenic potential in the intraperitoneal cavity of three newly developed biosoluble insulation glass wool fibers (M, P, and V) and one newly developed biosoluble insulation stone wool fiber (O) was investigated and compared to that of a previously developed soluble glass fiber (B). The in vitro dissolution coefficient of the three glass wool fibers ranged from 450 to 1037 ng/cm(2) x h and was 523 ng/cm(2) x h for the stone wool fiber. The in vitro dissolution coefficient of the B fiber was 580 ng/cm(2) x h. Groups of female Wistar rats (strain Crl: Wi BR) were exposed by repeated injections to doses of 0.5, 2, and 5 x 10(9) WHO fibers, which corresponds to between 41 mg to 724 mg fiber injected. In addition, 2 groups of crocidolite were used as positive controls at doses of 0.1 x 10(9) and 1 x 10(9) WHO fibers (0.5 and 5 mg). The in vitro dissolution coefficient of crocidolite is estimated to be approximately 1 ng/cm(2) x h. The protocol of the study and the size distribution of the test samples conformed to the European Commission Protocol EUR 18748 EN, and the study was executed under Good Laboratory Practice conditions. Two of the new insulation wools, fibers M and 0, showed no statistically significant tumorigenic response even at the very high dose of 5 x 10(9) WHO fibers injected. Fibers P and V showed a small tumorigenic response in the ip cavity similar in magnitude to the B fiber, which has been declared in the German fiber regulations as a noncarcinogenic fiber. The response to the soluble insulation fibers was notably different from that of the known carcinogen crocidolite, which produced 53% tumors at a comparatively low dose of 0.1 x 10(9) WHO fibers. The incidence of mesothelioma was found to be highly correlated to the incidence of intra-abdominal nodules and masses at different sites. The incidence of abdominal nodules and masses was highly correlated to the number of animals with ascites. The incidence of chronic peritonitis with fibrotic nodules at different organs also correlated with the incidence of mesotheliomas. Differences in etiology were observed between the massive doses of the highly soluble insulation wools when injected directly into the ip cavity and the lower doses of the extremely insoluble fiber crocidolite. The variability in this reaction and the impairment of animal health put into question the value of these massive doses in evaluating the carcinogenic response of soluble insulation wools. All of the fibers tested fulfilled the exoneration criteria with respect to carcinogenicity according to the European Directive 97/69/EC ("an appropriate intra-peritoneal test has not expressed signs of excessive carcinogenicity"). The dose as defined in the EC-Protocol EUR 18748 EN was 1 x 10(9) WHO fibers with a defined geometric spectrum. The influence of fiber dimensions on the ip tumor response and the difficulty in assessing the influence of the difference in background levels between this and previous studies make direct application of the German TRGS 905 criteria difficult;...