1 DOI: /2013PM0018 PERIODICO di MINERALOGIA established in 1930 An International Journal of MINERALOGY, CRYSTALLOGRAPHY, GEOCHEMISTRY, ORE DEPOSITS, PETROLOGY, VOLCANOLOGY and applied topics on Environment, Archaeometry and Cultural Heritage Preliminary results of the spectroscopic and structural characterization of mesothelioma inducing crocidolite fibers injected in mice Alessandro Francesco Gualtieri 1, Carlotta Giacobbe 2, Caterina Rinaudo 3,*, Alessandro Croce 3, Mario Allegrina 3, Giovanni Gaudino 4, Haining Yang 4 and Michele Carbone 4 1 Dipartimento di Scienze Chimiche e Geologiche, Università di Modena e Reggio Emilia, Italy 2 Dipartimento di Scienze e Alta Tecnologia, Università dell Insubria, Como, Italy 3 Department of Science and Technological Innovation, Università del Piemonte Orientale Amedeo Avogadro, Alessandria, Italy 4 University of Hawai i Cancer Center - University of Hawai i, USA and Department of Pathology, John A. Burns School of Medicine - University of Hawai i, HI 96813, USA * Corresponding author: Abstract To investigate the structure and microstructure changes of crocidolite asbestos incorporated in biological tissues, fibers of this mineral phase were injected in mice peritoneum. Histological sections of different organs of mice developing mesothelioma after crocidolite inoculation were prepared and analysed by optical microscopy. The tumours developed within the peritoneal cavity, wrapped around the surrounding organs. Many fibers were observed in the fibrotic areas of the peritoneum lining pancreas and spleen. The raw fibers before inoculation and those embedded in mice tissues were characterized using Micro-Raman spectroscopy and in situ synchrotron X-ray diffraction at ESRF- Grenoble. Preliminary results indicate shifts of some bands on the Raman spectra and enlargement of the X-Ray diffraction peaks of the fibers localized in the mice tissue sections. A preliminary structural picture of the fibers incorporated in mice tissues suggests inter-crystalline migration of the iron and sodium ions. Key words: Raman spectroscopy; Synchrotron diffraction; Rietveld method; crocidolite; asbestos; mesothelioma. Introduction As demonstrated by many scientific works, crocidolite is an asbestos phase responsible of the development of mesothelioma (MM), a deadly cancer characterized by the transformation of mesothelial cells (HM) lining the pleural, peritoneal or pericardial cavities (Aust et al.,
2 300 A.F. Gualtieri et al. 2011; Carbone et al., 2012; Case, 2011; De Vuyst et al., 1997; Fubini and Mollo, 1995; Goodglick and Kane, 1990; Huang et al., 2011; Koerten et al., 1990; Mossman et al., 1983; Tannapfel, 2011; Wagner et al., 1984; Werner et al., 1995; Yao et al., 2010; Zucali et al., 2011). Although many scientific papers were addressed to understand the mechanisms of interaction between the cells and the mineral fibers causing the pathogenicity of this mineral (see for example: Cole et al., 1991; Fubini and Mollo, 1995; Guthrie, 1992; Huang et al., 2011; Mossman et al., 1983; Rihn et al., 2000; Yang et al., 2010), the issue is still debated. Different approaches to the problem are possible: one may be to investigate the chemical and biochemical response of the cells and extracellular microenvironment to the mineral fibers (Bernstein et al., 2005; Brody, 1993; Guthrie, 1992; Mastrangelo et al., 2011; Toyokuni, 2009); another possible approach is the one used in this study: the analysis of the chemical-crystallographic changes of the fibers induced by the prolonged permanence inside the biological milieu. The characterization of fibers and inorganic phases in histological sections of lung and pleural plaque of patients affected by MM has been carried out by our research group using micro-raman spectroscopy and the results were described in previous works (Croce et al., 2013a; Musa et al., 2012; Rinaudo et al., 2010a, b). It has also been demonstrated that micro- Raman analysis may be performed directly on the sections prepared for routine diagnostic purpose with no additional modifications. Notwithstanding, the latency time for the development of mesothelioma in humans may be very long and medical treatments may be a confounding element influencing the persistence of the respired fibers. To better understand the structure-microstructure changes of fibers induced by the contact with the tissues in situ, in this work we have attempted a combined spectroscopic and diffraction study of the standard UICC crocidolite fibers, injected in mice. In these animals MM developed in the peritoneal cavity and histological sections of the different surrounding organs were prepared. With the aid of an optical microscope crocidolite fibers were identified. Such fibers were therefore characterized by micro-raman spectroscopy and Rietveld structure refinement, by use of data collected in situ at ESRF (Grenoble, France). We describe here preliminary results on crocidolite fibers observed at the peritoneal surface adjacent to pancreas and spleen, where they appear free or coated by ferruginous material, forming the well-known asbestos bodies or ferruginous bodies (Arul and Holt, 1980; Croce et al., 2013b; Dodson et al., 1993; Gellert et al., 1986; Ghio et al., 2004; Langer et al., 1972; Pascolo et al., 2011; Sakai et al., 2010). To highlight variation in structural-spectroscopic characteristics after permanence in the mice tissues, raw fibers of the powder used for injection are characterized using the two techniques under the same experimental conditions. Materials and methods Fifteen BALB/c mice were injected intraperitoneum (i.p.) using crocidolite purchased from UICC (Union for International Cancer Control) at the University of Hawai i Cancer Center. A total amount of 4 mg was subdivided in 10 doses and given every two weeks in 50 µl PBS. During the following 28 months, animals were euthanized when they developed ascites. Formalin-fixed, Paraffin Embedded (FFPE) tissue sections of the different organs were prepared to identify the presence of mineral fibers with optical microscopy. In this preliminary work, FFPE tissue sections from pancreas and spleen were analysed. In order to avoid Raman band interference with those produced by the inorganic fiber, unstained FFPE sections were analyzed. The spectroscopic study was carried out using a Jobin Yvon HR800
3 Preliminary results of the spectroscopic and LabRam μ-spectrometer, equipped with an Olympus BX41 microscope on which a rotating plate with vernier was mounted, a HeNe 20 mw laser working at nm and a charge-coupled device (CCD) air-cooled detector. Instrument calibration was carried out before each analysis, by checking the position and intensity of the Si band at ± 0.05 cm -1. To balance signal against noise, 100 cycles of 100 s (about 11 h) were performed. The spectral region recorded ranged from 1200 cm -1 to 200 cm -1, where vibrational lattice modes in the asbestos occur (Foresti et al., 2009; Fornero et al., 2008; Giacobbe et al., 2010; Rinaudo et al., 2003, 2004, 2005). A confocal hole fixed at 200 μm allowed to record spectra with well-defined Raman bands. To enhance the differences in the relative intensities of the Raman bands in fibers after a period in the mice biological system, all the fibers were placed with the same orientation and precisely with their elongation axis exactly at 45 with respect to the N-S direction of the cross-hair of the microscope, annexed to the spectrometer. All the Raman spectra were recorded keeping the experimental conditions fixed. The recorded spectra were processed using ORIGIN software v 6.0. The chemical characterization of the fibers was performed under VP-SEM/EDS, using an E- SEM Quanta 200 model of the FEI group, equipped with EDS detector of EDAX system. This allows sample characterization on unpolished coating-free samples. The analyses were carried out under a pressure value of 90 Pa and 20 kv as the accelerating voltage. The registered spectra were processed using the GENESIS software v 3.6. Tentative structural characterization of the raw crocidolite fibers and of those inoculated in the peritoneum and detected around the spleen and pancreas was attempted to verify the structure changes induced by the interaction of the crocidolite fibers with the organic matrix. The in situ experimental data were collected at BM01 beamline at ESRF facility, using an X-ray beam of 100x100 µm. The organic tissues inoculated with the fibers were prepared on plastic substrates, sometimes glued with paraffin, and fixed by a thin kapton foil. Data were collected with a 200 mm sample to detector distance, a wavelength of Å on the MARR CCD two-dimensional detector with a 360 s exposure time and a 2300x2300 pixel area. Data reduction of the two dimensional pictures to obtain powder patterns was done using the FIT2D (Hammersley et al., 1996) software. All the collected powder patterns show broad peaks from the components used for the sample preparation (kapton, plastic and paraffin), which severely overlap with crocidolite peaks. Notwithstanding, differences in the peak intensity of the raw sample with respect to the fibers inoculated in the organic media were detected. The Rietveld structure refinements were performed using GSAS (Larson and Von Dreele, 1994) and its graphical interface EXPGUI (Toby, 2001). For all the data sets, the pseudo-voigt profile function coefficients GW (angleindependent term of the Gaussian broadening) and Lx (crystal size term of the lorentzian broadening) were refined together with the unit cell, scale factor and background shifted Chebyshev coefficients. Several excluded regions were considered to remove the peaks generated by the sample holder. This causes the loss of some crocidolite Bragg peaks, significantly reducing the accuracy of the results. For this reason, all the structure parameters (coordinates, site populations and overall adp s) were refined only for the starting raw crocidolite sample whereas only the site populations of the M and Am sites were refined in the spleen and pancreas crocidolite samples. The riebeckite structure calculated by Hawthorne (1978) was taken as initial model for crocidolite. For all the samples, magnesium was refined in the M1 position with a population kept fixed to the value
4 302 A.F. Gualtieri et al. obtained from the ideal chemical composition (0.50 a.f.u.) taken from the literature (Kohyama et al., 1996). Results and discussion A morphological and chemical characterization of the raw crocidolite is shown in Figure 1: as expected EDS peaks from Si, Fe, Na and Mg are detected (Bowes and Farrow, 1997). At first, a thorough characterization of these raw crocidolite fibers was performed recording Raman spectra and placing, thanks to the rotating plate, the different fibers at the same orientation with respect to the laser beam direction. The Raman spectra were therefore summarized and the average for each Raman band calculated, obtaining the result shown in Figure 2. The Raman spectrum contains many bands, generated by the vibrations of the different coordination polyhedra forming the crystal structure (Burns and Prentice, 1968). The tetrahedral units (mainly Si-O bonds) are responsible for the intense bands in the spectrum. In particular the band at 1087 cm -1 may be ascribed to antisymmetric (ν as ) stretching vibrations of the Si-O b -Si linkages, the band at 970 cm -1 to ν s modes of O-Si-O bonds, the bands at 666, 579, 540 and 510 cm -1 to the symmetric stretching modes (ν s ) and to the deformation modes of Si 4 O 11 groups (Apopei and Buzgar, 2010; Bard et al., 1997; Lewis et al., 1996; Rinaudo et al., 2004). More complicated is the assignment of the bands in the spectral range cm -1, due to the vibrational modes of the cations in cubic or octahedral coordination, the bending modes of the Si-centred tetrahedra and in general all the lattice modes. In this spectral region, exact assignment of the different bands to the vibrational mode of a specific chemical group represents a challenge. The optical analysis of the tissue sections from mice organs showed many crocidolite fibers in the fibrotic areas of the peritoneal surface of spleen and pancreas, as seen on Figure 3. As reported in literature, crocidolite fibers in the biological medium often appear coated by ironprotein material forming the well-known ferruginous bodies or asbestos bodies (Arul and Holt, 1980; Croce et al., 2013b; Dodson et al., 1993; Gellert et al., 1986; Ghio et al., 2004; Langer et al., 1972; Musa et al., 2012; Pascolo et al., 2011; Rinaudo et al., 2010b; Sakai et al., 2010). In the histological sections of mice tissues studied in the present work, the mechanism of fiber-coating occurred at different stages. At the Figure 1. a) EDS spectrum of raw crocidolite sample used for injection in mice; b) SEM image of the bundle of crocidolite undergone to EDS spot analysis.
5 Gua 13/09/13 12:06 Pagina 303 Preliminary results of the spectroscopic and Figure 2. Raman spectrum of the raw crocidolite. The spectrum is obtained by merging the Raman spectra recorded on different ﬁbres and hence calculating the mean value for each band. Figure 3. a) Optical photograph showing crocidolite ﬁbres aggregated in the peritoneal surface of spleen; b) higher magniﬁcation of the area delimited in a).
6 304 A.F. Gualtieri et al. beginning, the coating appears as a black film on the fiber (Figure 4a). In a further step, the characteristic features of the asbestos bodies are formed (Figure 4b). During this study only fibers free from coating, as that represented in Figure 4c, were selected for the Raman characterization. In this way, interference of the bands produced by the mineral phase with those generated by the coating was avoided. On several fibers observed at the surface of spleen, Raman spectra were recorded, all the spectra summarized and the average for each band calculated, obtaining the spectrum shown in Figure 5. Comparison with the spectrum in Figure 2 allows highlighting some differences: the band at 578 cm -1, which appeared with intensity higher than that of the band at 970 cm -1 in raw crocidolite, exhibits now a lower intensity (Figure 2); the band near 375 cm -1 is asymmetric; the underlying components show different intensities, in particular the band at 363 cm -1 (Figure 5) appears more intense in the Raman spectra from fibers in mice; in the spectral region from 230 cm -1 to 250 cm -1, shift of 4-5 cm -1 of the Raman bands of the fibers concentrated in spleen peritoneum is detected. The fibers identified on the peritoneal surface of pancreas were analysed with the same experimental procedure. Often the Raman bands appeared with very low intensity. In other cases, good spectra could be recorded. Merging the spectra and calculating the average wavenumber for each band we obtained the result shown in Figure 6. In the cm -1 region of the spectra, reduction in band signal from these fibers is observed. In particular, the band at 375 cm -1 shows reduced intensity and a component at 384 cm -1 appears. The band near 335 cm -1 splits in two components at 328 and 334 cm -1. In the Raman spectra of the crocidolite fibers in mice tissues, the vibrational modes of the Si- O bonds basically occur at the same wavenumbers as in raw crocidolite (Figures 2, 5 and 6). On the contrary, in the spectral region which displays the vibrational modes of the cations in the cubic or octahedral coordination shifts, splitting of bands or reduction of band intensity are evident. All these observations support a degradation or modification of the crystalline structure mainly affecting the cubic or octahedral polyhedra. As far as the Rietveld structure refinements are concerned, the following agreement factors were obtained: (i) raw crocidolite fibers: background Figure 4. a) Optical photograph of a crocidolite fibre, showing the early step of the coating process (arrowed) in mice tissue; b) characteristic features of the asbestos bodies, in a later advanced stage of the coating, arrowed; c) crocidolite fibre, free from coating- arrowed- selected for the Raman characterization.
7 Preliminary results of the spectroscopic and Figure 5. Raman spectrum of crocidolite fibres in spleen thin section. The spectrum is obtained by merging the Raman spectra recorded on different fibres and hence calculating the mean value for each band. Circled bands are produced by the paraffin film covering the thin section. Figure 6. Raman spectrum of crocidolite fibres in pancreas thin section. The spectrum is obtained by merging the Raman spectra recorded on different fibres and hence calculating the mean value for each band. Circled bands are produced by the paraffin film covering the thin section.
8 306 A.F. Gualtieri et al. Table 1. The structure parameters calculated from the Rietveld refinement of raw crocidolite. site x/a y/b z/c Cation Occ. U iso (Ǻ 2 )** T (13) (7) (25) Si T (14) (7) (29) Si O (18) (16) 0.232(6) O O (19) (14) 0.731(6) O O-H (4) (9) O O (28) (10) 0.788(6) O O (27) (12) 0.079(4) O O (27) (11) 0.597(4) O O (31) (6) O M (10) 0.5 Mg 0.125* M (10) 0.5 Fe (2) M (6) 0.0 Fe M Fe M (15) 0.5 Na 0.83(4) Am (11) (23) Na+Ca 0.182(26) * = population fixed to the value from the chemical analysis; ** = an overall parameter for each chemical species was refined. subtracted R wp = 4.78% and R p = 2.77%, χ 2 = for 1573 observations, 255 reflections and 77 variables; (ii) peritoneum spleen crocidolite fibers: background subtracted R wp = 2.77% and R p = 1.97%, χ 2 = for 947 observations, 289 reflections and 47 variables; (iii) peritoneum pancreas crocidolite fibers: background subtracted R wp = 2.08% and R p = 1.66%, χ 2 = for 947 observations, 248 reflections and 48 variables. As an example, Figure 7 reports the graphical output of the refinement of raw crocidolite. The calculated structure parameters of raw crocidolite are reported in Table 1. The chemical composition of the sample calculated from the refinement is: (Na,Ca) 0.37 Na 1.66 (Mg 0.50 Fe Fe 3+ 2)Si 8 O 22 (OH) 2. With respect to the chemical composition reported by Kohyama et al. (1996), a slight overestimation of Na+Ca (2.03 against 1.77) is observed. The calculated interatomic distances are reported in Table 2 whereas Table 3 illustrates the cation content of the raw crocidolite fibers and of those identified in the peritoneum surfaces of spleen and pancreas. The distribution of the iron atoms in the M1, M2 and M3 sites is compatible with the findings of Martra et al. (1999), who suggest that Fe 2+ and Fe 3+ are located in the three cases at fixed position and are not interchangeable one with other. The Figure 7. Graphical output of the refinement of raw crocidolite. Regions with peaks from the sample holder/environment were excluded.
9 Preliminary results of the spectroscopic and Table 2. The interatomic distances calculated from the Rietveld refinement of raw crocidolite. T1-O1 = 1.591(14) T1-O5 = 1.652(13) T1-O6 = 1.677(14) T1-O7 = 1.681(12) T2-O2 = 1.594(13) T2-O4 = 1.595(13) T2-O5 = 1.648(14) T2-O6 = 1.689(13) M1-O1 = 2.090(27) M1-O1 = 2.090(27) M1-O2 = 2.062(27) M1-O2 = 2.062(27) M1-O-H3 = 2.107(24) M1-O-H3 = 2.107(24) M2-O1 = 2.186(27) M2-O1 = 2.186(27) M2-O2 = 1.941(27) M2-O2 = 1.941(27) M2-O4 = 2.074(23) M2-O4 = 2.074(23) M3-O1 = 2.284(26) M3-O1 = 2.284(26) M3-O1 = 2.284(26) M3-O1 = 2.284(26) M3-O-H3 = 2.09(4) M3-O-H3 = 2.09(4) M4-O2 = 2.341(29) M4-O2 = 2.341(29) M4-O4 = 2.466(34) M4-O4 = 2.466(34) M4-O5 = 2.840(30) M4-O5 = 2.840(30) M4-O6 = 2.343(31) M4-O6 = 2.343(31) Am-O5 = 2.75(5) Am-O5 = 2.75(5) Am-O6 = 2.92(9) Am-O6 = 2.92(9) Am-O7 = 2.958(23) Am-O7 = 3.06(14) Am-Am = 1.31(17) calculated interatomic distances are very similar to those reported by Hawthorne (1978) for fluorriebeckite, the only exception being the large M3-O mean distance (2.217 Å for our sample and Å for the fluor-riebeckite). The shorter M-O mean distance found for the M2 site (2.068 Å for our sample and Å for the fluorriebeckite) may confirm the ordering of Fe 3+ in that site. Although the quality of these results is too poor to depict an indisputable model for the structure modification of the crocidolite fibers in organic media, some differences between the raw fibers and those inoculated in the peritoneum medium are detected. It is known from the literature (Crawford and Miller, 1981) that Table 3. Cation site distribution in raw, pancreas and spleen crocidolites. site x/a y/b z/c Cation Occ. Occ. Occ. U iso (Ǻ 2 )** Raw In situ In situ crocidolite crocidolite crocidolite in pancreas in spleen M (10) 0.5 Mg 0.125* 0.125* 0.125* M (10) 0.5 Fe (1) 0.77(3) 0.84(6) M (6) 0.0 Fe (6) 0.82(11) M Fe M (15) 0.5 Na 0.83(4) 0.94(8) 0.92(8) Am (11) (23) Na+Ca 0.182(26) 0.09(5) 0.10(5) Legend: * = population fixed to the value from the chemical analysis; ** = an overall parameter for each chemical species was refined.
10 308 A.F. Gualtieri et al. crocidolite in lung tissues shows reduced crystallinity due to erosion associated with the (100) planar defects and surface dissolution. The latter is likely a result of the leaching of Mg atoms, leaving a silicate skeleton, in an analogous manner to the mechanism proposed for chrysotile (Morgan et al., 1977). Although it is not possible to confirm the microstructure models proposed in the literature, for which a dedicated TEM study is mandatory, an indirect evidence of reduced crystallinity comes from the calculated values of the profile broadening coefficients. A lower crystallinity is expected for the pancreas and spleen crocidolites as, on equal values of Lx (1.07, 1.3 and 1.22 for spleen, pancreas and raw crocidolite, respectively), their GW values are notably larger (29.0 and 28.4, respectively) with respect to that of the raw sample (22.9). The ph of the peritoneum chemical environment, hosting the crocidolite fibers in the pancreas and spleen tissues, is Such acidic environment prompts solubility of Fe 2+ (whereas Fe 3+ is not soluble) and may be the chemical gradient promoting the dissolution of the crocidolite fibers. The mobilization of iron from crocidolite phagocytised by cultured human lung carcinoma cells, even at higher ph values (6.8), has already been reported by Chao et al. (1994). In fibers identified both at spleen and at pancreas peritoneal surface, intra-structure diffusion is apparently observed, with iron migrating from site M2 to site M1 and sodium migrating from site Am to site M4 (Figure 8). Cation redistribution is not surprising in crocidolite in contact with acidic solutions such as water-base solution of ferric salts used for detoxification (see for example Hearne et al., 1993). On the other hand, it is not possible to assess whether iron migrated in site M1 is Fe 3+ or the reduced species Fe 2+. In fact, the limited quality of the collected data does not permit the refinement of the position of M sites for the pancreas and spleen crocidolite, preventing the opportunity to detect the distribution of Fe 2+ /Fe 3+ in the crystallographic sites by the analysis of interatomic distances. As a matter of fact, it is also possible that iron migration from site M2 to site M1 actually counter-balances a migration of Mg from site M1 to site M2. In any case, the redistribution of positive charges seems to call for a counter-balance migration of sodium (and eventually calcium). According to Pascolo et al. (2013), cation mobilization is the first step of interaction of asbestos fibers with tissues: Fe 2+ is oxidized to Fe 3+ and migrates outside the structure to form ferrihydrate-rich ferritin and hematite, calcium and magnesium migrate to prompt phagocytosis induced calcification. For the reasons explained above, this model needs further confirmation and the basic chemical factor that promotes this ion migration is still unclear. To accomplish a robust and sound general model of the crocidolite fiber modification and dissolution mode in organic media and unequivocally understand the mechanism of the fiber-cell interaction at a molecular level, it is necessary to further investigate their structures possibly using a X-ray microfocus (coupled, when available, with micro-raman) at a synchrotron source. Gunter et al. (2011) recently performed such an analysis using synchrotron radiation to show different Fe oxidation states of asbestiform and nonasbestiform amphiboles from the same deposit. Figure 8. Crocidolite structure calculated from the Rietveld refinements showing iron migration from site M2 to site M1 and sodium migration from site Am to site M4 observed in fibres found in both pancreas and spleen peritoneum.
11 Preliminary results of the spectroscopic and Conclusions Crocidolite fibers injected in mice peritoneum were studied by micro-raman and in situ synchrotron diffraction to contribute to the understanding of the MM developed by this mineral fibers. The identification of fibers in the different sections performed by means of an optical microscope allowed to reckon crocidolite fibers in the peritoneum lining spleen and pancreas. The fibers occurred free or coated by ferro-protein aggregates, which form around asbestos fibers to constitute the well-known asbestos bodies. Our attention was focused on changes in the chemical or structural characteristics of the crocidolite fibers in contact with the biological medium. The main result obtained from the micro-raman spectroscopy experiments was that on the spectra from crocidolite fibers in mice tissue sections, the Raman bands produced by the vibrations of the tetrahedral part of the crystalline structure -Si-O bonds- do not show sensible shifts or broadening, but only reduction in intensity. On the contrary in the spectral region containing the vibrational modes of the cubic and octahedral components of the crystalline structure, the bands appear less intense, eventually broadened and constituted by more components, or shifted with respect to the wavenumbers of the same bands in raw crocidolite. These results may be indicative of a partial degradation of the crystalline structure when the fibers reside in the biological matrix. This hypothesis is confirmed by X-ray diffraction study. In the analysed tissues, intra-structure diffusion is observed, with iron migrating from site M2 to site M1 although it is not possible to assess whether intrastructural iron oxidation occurs. The cation mobilization seems to regard sodium and possibly calcium too, in agreement with the picture drawn by Pascolo et al. (2013) who postulate cation mobilization as the first step of interaction of asbestos fibers with tissues. On the same experimental trace marked out by Pascolo et al. (2011, 2013), the use of a microfocus synchrotron X-ray beam, coupled with Raman spectroscopy, is required to characterize single crocidolite fiber incorporated in mice and confirm the preliminary picture of the structure model of crocidolite in contact with the biological medium. The structure/microstructure features of each fiber should be correlated with its spectroscopic behaviour obtaining more detailed information about the change of the crystal structure and relation with Raman bands ascribed to different polyhedra. Acknowledgements The authors wish to thank the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities (project HS- 4422, BM01A), we would like to thank Volodymir Svitlyk for assistance in using beamline and M.E. Gunter for the revision of the manuscript. The authors also wish to thank the second anonymous referee and the editors for their work. Animal experiments were supported by the Hawaii Community Foundation (to H.Y. and to G.G.), by NCI P01 CA and Butitta Mesothelioma Foundation (to M.C.). References Apopei A.I. and Buzgar N. (2010) - The Raman study of amphiboles. Analele Ştiinţifice Ale Universităţii Al. I. Cuza Iaşi, Seria Geologie, Tomul LVI, Arul K.J. and Holt P.F. (1980) - Clearance of asbestos bodies from the lung: a personal view. British Journal of Industrial Medicine, 37, Aust A.E., Cook P.M. and Dodson R.F. (2011) - Morphological and chemical mechanisms of elongated mineral particle toxicities. Journal of Toxicology and Environmental Health, Part B: Critical Reviews, 14, Bard D., Yarwood J. and Tylee B. (1997) - Asbestos fiber identification by Raman microspectroscopy.
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582 THE APPLICATION OF A MATHEMATICAL MODEL DESCRIBING THE TIMES OF OCCURRENCE OF MESOTHELIOMAS IN RATS FOLLOWING INOCULATION WITH ASBESTOS G. BERRY AND J. C. WAGNER From the Medical Research Council's