1 Toxicology Letters 114 (2000) Kerosene soot genotoxicity: enhanced effect upon co-exposure with chrysotile asbestos in Syrian hamster embryo fibroblasts Mohtashim Lohani a, Elke Dopp b, Dieter G. Weiss b, Dietmar Schiffmann b, Qamar Rahman a, * a Industrial Toxicology Research Centre, Di ision of Fibre Toxicology, Post Box 80, M. G. Marg, Lucknow , India b Institute of Cell Biology and Biosystems Technology, Department of Biological Sciences, Uni ersity of Rostock, Rostock, Germany Received 20 July 1999; received in revised form 3 November 1999; accepted 4 November 1999 Abstract Epidemiological and experimental studies have suggested an enhancement of asbestos-induced bronchogenic carcinoma by cigarette smoke. Further, our recent experimental and epidemiological studies have indicated that besides smoking, several other compounds including kerosene soot may accelerate disease processes in asbestos-exposed animals as well as in the humans. Incomplete combustion of kerosene oil generates large volumes of soot, which contains various polycyclic aromatic hydrocarbons and aliphatic compounds. As reported earlier, exposure to kerosene soot is known to cause biochemical and pathological changes in the pulmonary tissue, which may cause cardiopulmonary disorders. In this study we investigated genotoxic effects caused by kerosene soot and chrysotile asbestos as well as co-exposure of kerosene soot and chrysotile using Syrian hamster embryo fibroblasts (SHE). The micronucleus assay revealed a significant increase of induced micronuclei (MN), (P 0.05) in SHE cells after treatment with kerosene soot ( g/cm 2 ) for 66 h (36 MN/1000 cells). Combined treatment with chrysotile and soot induced up to 110 MN/1000 cells (chrysotile alone: 80 MN/1000 cells; concentrations: 1 g/cm 2, exposure times: 66 h). Kinetochore staining revealed mainly clastogenic effects in all cases (soot: 21.3% CRMN + ; chrysotile: 27%; soot+chrysotile: 27.6%; control: 20.8%). This is the first study showing that kerosene soot is not only genotoxic but it can also elevate the genotoxic potential of chrysotile asbestos. This information may be of importance for workers occupationally exposed to asbestos and domestically exposed to kerosene soot Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kerosene soot; Asbestos; Micronuclei; Kinetochores; Genotoxicity 1. Introduction * Corresponding author. Tel.: ; fax: address: q (Q. Rahman) The co-carcinogenic potential of asbestos in the development of bronchogenic carcinoma has been /00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S (99)
2 112 M. Lohani et al. / Toxicology Letters 114 (2000) reported (Selikoff et al., 1980). Studies implicate an enhancing effect of cigarette smoke on asbestos-induced toxicity (Eastman et al., 1983; McFadden et al., 1986a; Kamp et al., 1998). In addition to cigarette smoke other environmental pollutants have also been shown to influence asbestos-induced disease processes. According to earlier experimental as well as epidemiological observations from this laboratory, the influence of kerosene and its soot on the development of asbestos-induced disease processes has been reported (Arif et al., 1992, 1993, 1994, 1997). Coexposure of kerosene soot and chrysotile asbestos has been shown to enhance cytotoxicity and oxidative stress in comparison to the separate exposure of kerosene soot or chrysotile asbestos alone (Arif et al., 1994, 1997). Oxidative stress caused induction of lipid peroxidation, production of reactive oxygen species, alteration in the enzymes regulating the oxidative balance in the system as well as depletion of soluble antioxidants. Incomplete combustion of kerosene oil produces large amounts of kerosene soot, which contains naphthalene, pyrene and benzo(a)pyrene as major constituents along with acenaphthalene, fluorene, chrysene, anthracene and phenanthracene (Arif et al., 1992). Some of the hydrocarbons like benzene, 1,3-butadiene, styrene and xylene are of main concern because of their carcinogenic properties (Noa and Illnait, 1987). Exposure to soot may lead to cardiopulmonary disorders (Arif et al., 1991). Kerosene soot and some associated polycyclic aromatic hydrocarbons (PAHs) have already been shown to be mutagenic to Salmonella typhimurium (Kaden et al., 1979). In developing countries like India, a substantial portion of its population still uses kerosene oil as domestic fuel for cooking, heating and lighting purposes, because of its easy availability and low cost. Among these people, those working in asbestos-based factories are domestically exposed to kerosene soot and occupationally exposed to asbestos. According to our recent survey co-exposure causes increased pulmonary damage (unpublished). Kerosene oil is used commercially all over the world, and co-exposure with asbestos may cause problems in general. It seems possible that the co-exposure may increase the genotoxic potential in the exposed population. The Syrian hamster embryo (SHE) micronucleus assay was used to investigate the genotoxicity of fibers and particles by us and other authors as a shortterm test with a high predictive value (Fritzenschaf et al., 1993; Dopp et al., 1995). Therefore, the present study is designed to investigate the genotoxicity of kerosene soot alone and in combination with chrysotile asbestos. 2. Materials and methods 2.1. Cell culture and treatment conditions SHE fibroblasts were isolated from 13-day-old embryos as described by Pienta (1980). Cell cultures were grown in a humidified atmosphere containing 12% CO 2 at 37 C. The culture medium used was IBR-modified Dulbecco s Eagle s reinforced medium (Gibco Co.) supplemented with 15% fetal calf serum (Flow Laboratories, Meckenheim, FRG), 3.7 g/l NaHCO3 (7.5%), 1% glucose, IU penicillin, 10 mg/ml streptomycin and 0.01% tylosin. Kerosene soot was obtained by igniting kerosene oil (BP C) in a pressure stove under the conditions approximating those in a kitchen. Its composition was analyzed (Arif et al., 1997) and particles of a size less than 10 m were prepared (Zaidi, 1969). Information on particle size distribution was already published (Arif et al., 1997). Rhodesian chrysotile (UICC standard) was obtained from Dr Linnainmaa (University of Helsinki, Finland). Average length, diameter and number of fibers/ g were determined. Average dimensions of chrysotile were 0.10 m diameter, 2.24 m length and percentage of fibers greater than or equal to 5 m was approximately 5% (Dopp et al., 1997). Fibers and soot were sterilized by autoclaving (120 C for 2 h) and suspended in PBS (1 g/ l). Cells were treated with fibers according to the size of culture flasks (0.5, 1.0, 5.0 and 10.0 g/ cm 2 ).
3 M. Lohani et al. / Toxicology Letters 114 (2000) Micronucleus assay The micronucleus assay was performed as described by Schmuck et al. (1988). SHE cells were grown on coverslips and after treatment with asbestos, soot, or asbestos and soot, then cells were fixed and stored in cold methanol ( 20 C) for at least 30 min. After washing of cells with PBS, nuclei were stained with bisbenzimide (Hoechst 33258, concentration: 1 g/ml, 4 min). The slides were coded to avoid bias, mounted for microscopy and examined for the presence of micronuclei. Each data point represents the mean of treated cultures from at least three different independent experiments with 2000 nuclei evaluated in each case. The frequency of micronuclei was determined microscopically at 630 magnification; only micronuclei smaller than 1/3 of the nuclear diameter were taken into account Kinetochore staining For further analysis of the induced micronuclei, kinetochores were stained by incubating the fixed cell preparations with CREST serum (Chemicon, Temecula, USA) for 1 h in a humidified chamber at 37 C. After rinsing with phosphate-buffered saline, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-human antibodies (Sigma, Germany) before applying bisbenzimide (Hoechst). At least 100 micronuclei were examined for the presence of kinetochores in each case (CRMN + ) Statistical analysis The 2 -test was used to compare the results of the micronucleus assay of each treatment group with the control. 3. Results Fig. 1. Formation of MN in SHE fibroblasts after treatment (24, 48, 66 and 72 h) of cells with different concentrations of kerosene soot. Fig. 2. Formation of MN in SHE fibroblasts after treatment (48 and 66 h) of cells with different concentrations of chrysotile asbestos. Fig. 1 shows the formation of micronuclei in SHE cells after treatment with kerosene soot (exposure time: 24, 48, 66 and 72 h; concentration: 0.5, 1.0, 5.0 and 10.0 g/cm 2 ). The maximum number of micronuclei (MN) was observed at 5.0 g/cm 2 concentration and 66 h exposure (mean: 36 MN/1000 cells). At higher concentrations the number of micronuclei decreased probably due to increased cytotoxicity. Fig. 2 shows induction of micronuclei by chrysotile. The maximum number of micronuclei was induced at a concentration of 5.0 g/cm 2 and after 48 h of exposure (mean: 87 MN/1000) which is about four-fold higher compared to the control level (22 MN/1000 cells, 48 h; 22.5 MN/1000 cells, 66 h). After co-exposure of SHE cells to chrysotile and kerosene soot, additive genotoxic effects can be observed at a concentration of 0.5 g/cm 2 of each (mean: 90 MN/1000 cells, 48 h exposure; 95
4 114 M. Lohani et al. / Toxicology Letters 114 (2000) Discussion Fig. 3. Formation of MN in SHE cells after co-exposure of cells to chrysotile asbestos and kerosene soot (0, 0.5 and 1.0 g/cm 2 of each). The differences between treated and untreated cells are statistically significant (P 0.001) in all cases. MN/1000 cells, 66 h exposure) and 1.0 g/cm 2 concentration of each (mean: 104 MN/1000 cells, 48 h exposure; 111 MN/1000cells, 66 h exposure) which is up to five times higher compared to untreated controls (mean: 21 MN/1000 cells, 48 h; 21 MN/1000 cells, 66 h; Fig. 3). The kinetochore analysis revealed that the percentage of kinetochore positive micronuclei was not significantly increased after kerosene soot exposure (21.3%: 20.1% in control). Chrysotile-treated and co-exposed cells showed a slightly increased number of kinetochore-positive micronuclei (26.9 and 27.6%, respectively; Table 1). The induction of micronuclei by kerosene soot, chrysotile asbestos and co-exposure with kerosene soot and chrysotile asbestos was investigated in SHE fibroblasts. In addition, we investigated the presence of kinetochores in the induced micronuclei. Both kerosene soot and chrysotile asbestos were found to induce micronuclei in SHE cells in a dose-dependent manner. Cytotoxicity was observed after exposure of cells to higher concentrations of kerosene soot (10.0 g/cm 2 ) and chrysotile (10.0 g/cm 2 ). Chrysotile alone induced a significantly higher number of micronuclei than kerosene soot, when used in similar concentrations, revealing its higher genotoxic potential (Valerio et al., 1983). Co-exposure of kerosene soot and chrysotile induced higher numbers of micronuclei approximating the sum of the micronuclei induced separately by both these agents. It was shown in earlier investigations that amosite, chrysotile and crocidolite asbestos fibers induce micronuclei in SHE as well as in human amniotic fluid cells in a dose-dependent manner (Dopp et al., 1995, 1997). The micronucleus assay is a useful technique to assess genetic damage. The frequency of cells containing micronuclei reflects chromosomal damage and/or aneuploidy (Heddle et al., 1983). Further, the staining of kinetochores in micronuclei with anti-kinetochore (CREST) serum helps to discriminate between aneugenic and clastogenic effects. Kinetochore staining revealed an insignificant increase in kinetochore-positive micronuclei in kerosene soot treated cells indicating an involve- Table 1 Results of kinetochore staining of SHE cells after 48 and 66 h treatment with kerosene soot, chrysotile and chrysotile+soot Treatment Concentration No. of cells No. of CRMN + per CRMN per 1000 % CRMN +b ( g/cm 2 ) scored micronuclei a 1000 cells cells Kerosene soot (NS c ) Chrysotile (NS) Chrysotile+soot (NS) Control a Number of micronuclei scored for presence of kinetochores. b Percentage of detected micronuclei that reacted positively to anti-kinetochore serum. c NS, not significant.
5 M. Lohani et al. / Toxicology Letters 114 (2000) ment of mainly clastogenic events contributing to micronucleus induction, whereas chrysotile exposed cells showed slightly higher kinetochorepositive micronuclei. The co-exposed SHE cells revealed further increase in kinetochore-positive micronuclei compared to controls as well as compared to kerosene soot exposed cells. This study clearly indicates that loss of whole chromosomes is involved, but that mainly clastogenic events contribute to the formation of micronuclei. Our previous studies showed that both in vivo and in vitro co-exposure of kerosene soot and chrysotile asbestos resulted in significantly higher cytotoxicity, inflammatory reactions and oxidative stress (Arif et al., 1994, 1997). Asbestos fibers are phagocytosed by the cells within 24 h and accumulate in the perinuclear region after h (Hesterberg et al., 1986; Jensen et al., 1994). During mitosis, their physical presence results in missegregation of chromosomes. Therefore maximum numbers of micronuclei are produced at 66 h which is in agreement with the results of the present study. The possible cause of the aneugenic events may be a functional disturbance of the spindle apparatus, membrane components or centromere proteins (Mitchell et al., 1995) directly due to the presence of asbestos fibers or other particulates. Clastogenic events may be caused by reactive oxygen species (Kamp et al., 1992) generated as a result of the interaction between cell components and asbestos fibers or other particulates. Evidence has accumulated that reactive oxygen species produce stable clastogenic factors in response to the presence of fibres, causing genetic damage (Emerit et al., 1991; Kamp et al., 1992). As suggested by Kamp and Weitzman (1999), the production of free radicals may in this case also be due to polynuclear cation radicals. The most specific and representative marker for the damage caused by reactive oxygen species is 8-hydroxydeoxyguanosine (Dizdaroglu, 1991). It appears that according to the present findings the increased damage after co-exposure is attributed to an enhanced production of reactive oxygen species in addition to the physical presence of fibers/ particles in and around the mitotic apparatus. Asbestos fibres are found to facilitate the uptake and retention of benzo(a)pyrene and other polycyclic aromatic hydrocarbons into the hamster tracheal epithelial cells resulting in increased pathological effects (McFadden et al., 1986b). Presence of kerosene soot may also increase the penetration and retention of asbestos fibers from lung tissue. Thus, asbestos fibers stay longer in the lung, which results in enhanced pulmonary inflammatory reactions. Further, asbestos fibers may increase the absorption and retention of PAHs present in kerosene soot. Nevertheless, the whole mechanism still needs detailed studies. The occurence of increased pulmonary function abnormalities and respiratory impairments in asbestos-cement factory workers simultaneously exposed to cigarette smoke and kerosene soot together with asbestos, have clearly demonstrated the further deteriorating effects of the combined exposure (Rahman et al., 1998). Further studies in this direction are needed to prove a statistically relevant influence of kerosene soot on the development of bronchogenic carcinoma in the asbestos-exposed population. In conclusion, the present findings indicate that kerosene soot is a relatively weak, but effective genotoxic agent and induces micronuclei as a result of mainly clastogenic events. Co-exposure of kerosene soot and chrysotile asbestos results in additive genotoxic effects, which is quite alarming for the population simultaneously exposed to kerosene soot as well as to asbestos. Acknowledgements We would like to thank Ina Poser and Florian Schiffmann (University of Rostock, Germany) for technical support. The work was done under the Indian German collaborative program CSIR- DLR (project no. INI 086). References Arif, J.M., Khan, S.G., Aslam, M., Mahmood, N., Joshi, L.D., Rahman, Q., Early biochemical changes in kerosene exposed rat lungs. Chemosphere 22,
6 116 M. Lohani et al. / Toxicology Letters 114 (2000) Arif, J.M., Sikander, G.K., Aslam, M., Mahmood, N., Rahman, Q., Diminution in Kerosene-mediated induction of drug metabolizing enzymes by asbestos in rat lungs. Pharmacol. Toxicol. 71, Arif, J.M., Khan, S.G., Ashquin, M., Rahman, Q., Modulation of macrophage mediated cytotoxicity by kerosene soot: possible role of reactive oxygen species. Environ. Res. 61, Arif, J.M., Khan, S.G., Mahmood, N., Aslam, M., Rahman, Q., Effect of co-exposure of asbestos and kerosene soot on pulmonary drug-metabolizing enzyme system. Environ. Health Perspect. 102 (Suppl. 5), Arif, J.M., Khan, S.G., Ahmad, I., Joshi, L.D., Rahman, Q., Effect of kerosene and its soot on the chrysotile-mediated toxicity to the rat alveolar macrophages. Environ. Res. 72, Dizdaroglu, M., Chemical determination of free radicalinduced damage to DNA. Free Rad. Biol. Med. 10, Dopp, E., Saedler, J., Stopper, H., Weiss, D.G., Schiffmann, D., Mitotic disturbances and micronucleus induction in Syrian hamster embryo (SHE) fibroblast cells caused by different types of asbestos fibers. Environ. Health Perspect. 103, Dopp, E., Schuler, M., Schiffmann, D., Eastmond, D.A., Induction of micronuclei, hyperdiploidy and chromosomal breakage affecting the centric/pericentric regions of chromosomes 1 and 9 in human amniotic fluid cells after treatment with asbestos and ceramic fibres. Mut. Res. 377, Eastman, A., Mossman, B.T., Bresnick, E., Influence of asbestos on the uptake of benzo(a)pyrene and DNA alkylation in hamster trachial epithelial cells. Cancer Res. 43, Emerit, I., Jaurand, M.C., Saint-Etienne, L., Levy, F., Formation of a clastogenic factor by asbestos-treated rat pleural mesothelial cells. Agents Actions 34, Fritzenschaf, H., Kohlpoth, M., Rusche, B., Schiffmann, D., Testing of known carcinogens in the Syrian hamster embryo (SHE) micronucleus test in vitro; correlations with in vivo micronucleus formation and cell transformation. Mutat. Res. 319, Heddle, J.A., Hite, M., Krikhart, B., Mavournin, K., MacGregor, J.T., Newell, G.W., Salamone, M.F., The induction of micronuclei as a measure of genotoxicity. Mutat. Res. 123, Hesterberg, T.W., Butterick, C.J., Oshimura, M., Brody, A.R., Barrett, J.C., Role of phagocytosis in Syrian hamster cell transformation and cytogenetic effects induced by asbestos and short and long glass fibres. Cancer Res. 46, Jensen, C.G., Jensen, L.C.W., Ault, J.G., Osorio, G., Cole, R., Rieder, C.L., Time-lapse video light microscopic and electron microscopic observations of vertebrate epithelial cells exposed to crocidolite asbestos. In: Davis, I.M.G., Jaurand, M.C. (Eds.), Cellular and Molecular Effects of Mineral and Synthetic Dusts and Fibres. Heidelberg: NATO ASI series 85, pp Kaden, D.A., Hites, R.A., Thilly, W.J., Mutagenicity of soot and associated polycyclic hydrocarbons to Salmonella typhimurium. Cancer Res. 39 (10), Kamp, D.W., Weitzman, S.A., The molecular basis of asbestos-induced lung injury. Thorax 54, Kamp, D.W., Graceffa, P., Pryor, W.A., Weitzman, S.A., The role of free radicals in asbestos-induced diseases. Free Rad. Biol. Med. 12, Kamp, D.W., Greenberger, M.J., Sbalchierro, J.S., Cigarette smoke augments asbestos-induced alveolar epithelial cell injury. Free Rad. Biol. Med. 25, McFadden, D., Wright, J.L., Wiggs, B., Chrug, A., 1986a. Cigarette smoke increases the penetration of asbestos fibres into airway walls. Am. J. Pathol. 123, McFadden, D., Wright, J.L., Wiggs, B., Churg, A., 1986b. Smoking inhibits asbestos clearance. Am. Rev. Respir. Dis. 133, Mitchell, I., Lambert, T.R., Burden, M., Sunderland, J., Porter, R.L., Carlton, J.B., Is polyploidy an important genotoxic lesion? Mutagenesis 10, Noa, M., Illnait, J., Induction of Aortic plaques in guinea pigs by exposure to kerosene. Arch. Environ. Health 42, Pienta, R.J., Transformation of Syrian hamster embryo cells by diverse chemicals and correlation with their reported carcinogenic and mutagenic activities. In: de Serres, F.J., Hollaender, A. (Eds.), Chemical Mutagens, vol. 6. Plenum, New York, pp Rahman, Q., Prasad, R., Das, M., Pandey, U.S., Lohani, M., Ashquin, M., Follow up study in an asbestos cement factory with special emphasis on preventive and diagnostic measures: a project report submitted to Uttar Pradesh Council of Science and Technology Government of India. Schmuck, G., Lieb, G., Wild, D., Schiffmann, D., Henschler, D., Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts. Mutat. Res. 203, Selikoff, I.J., Seidman, H., Hammond, E.C., Mortality effects of cigarette smoking among amosite asbestos factory workers. J. Natl. Cancer Inst. 65, Valerio, F., de Ferrari, M., Ottaggio, L., Repetto, E., Santi, L., Chromosomal aberration induced by chrysotile and crocidolite in human lymphocytes in vitro. Mutat. Res. 122, Zaidi, S.H., Experimental Pneumoconiosis. Johns Hopkins Press, Baltimore, MD, pp