Introduction

Asbestos is an abundant, inexpensive, and geographically ubiquitous mineral fiber. Due to its distinctive properties of strength and high temperature tolerance, it is widely used in the textile, building, chemical, machinery, and defense industries. Currently, about 125 million people in the world are exposed to asbestos in the workplace (Ezzati et al. 2004). There are two types of asbestos: hornblende (crocidolite, blue asbestos) and serpentine (chrysotile). Chrysotile accounts for approximately 95% of all asbestos used in the United States (Britton 2002) and 90% of asbestos used worldwide (Geoffrey 2002; Burki 2010). Controversy over the safety of chrysotile use has been substantial (Yarborough 2006; Finkelstein and Meisenkothen 2011; Kagan 2013; Bernstein and Hoskins 2006). As of 2013, more than 50 countries, including all member states of the European Union, had banned the use of all forms of asbestos, including chrysotile (Bernstein et al. 2013). Some non-asbestos producers, especially in western developed countries, advocate use of substitutes, such as rock wool fiber, glass fiber, or ceramic fiber. Despite the bans on chrysotile, however, it is still widely mined and exported in developing countries (Burki 2010), and, in recent years, some countries have maintained or even increased their production or use of chrysotile. China is the largest consumer and second largest manufacturer of chrysotile in the world, and more than one million people in China experience occupational exposure to chrysotile (Wang et al. 2013; Courtice et al. 2012).

Scientific evidence found that exposure to asbestos, including chrysotile, can cause cancers of the lung, larynx, and ovary, as well as mesothelioma and asbestosis. According to global estimates, at least 107,000 people die each year from asbestos-related lung cancer, mesothelioma, and asbestosis resulting from occupational exposure (Driscoll et al. 2005a, b). Artificial substitutes can also be cytotoxic, even to the same degree as chrysotile (Gazzano et al. 2007; Paustenbach et al. 2006; Pugnaloni et al. 2013; Brown and Harrison 2014; Donaldson et al. 2013). Since genotoxicity is generally a prerequisite for the development of malignancy, the relationship between asbestos exposure and chromosomal aberrations and mutations has been explored. Chrysotile fibers induce chromosome aberrations in human lymphocytes, amniotic fluid cells, and peritoneal fluid cells and in bone marrow cells of mice (Stanton et al. 1977; Durnev et al. 1993). In the present study, we compared the genotoxicity of chrysotile from Mangnai, Qinghai, China, and rock wool fiber. Our findings add to the scientific evidence that can be used to evaluate the safety of chrysotile and rock wool fibers.

Materials and methods

Chrysotile and rock wool fibers

Chrysotile fiber from Mangnai, Qinghai, China, and rock wool fiber were generously donated by the Southwest University of Science and Technology, Mianyang, Sichuan, P. R. China (He et al. 2014).

Preparation of suspensions of ultrafine fibers

Fibers were crushed in a ceramic mortar, then cut and ground to 2–3 mm. This material was suspended in ethyl alcohol and ground for 12 h in a horizontal ball mill, and then passed through a 400-mesh screen. Particles smaller than 10 μm were collected and oven-dried to a powder. Fiber powder (0.2 g) was sterilized by incubating at 150 °C for 2 h. RPMI 1640 medium (20 ml) was added, and the mixture was swirled for 30 min to produce a 10 mg/ml ultrafine powder suspension. At the end of this process, no aggregation of the granules was observed, and the average granule diameter was less than 10 μm. Prior to use, suspensions were diluted to 50, 100, or 200 μg/ml with RPMI 1640 medium supplemented with 2% fetal bovine serum (FBS, Life Technologies/Gibco, Grand Island, NY, USA).

Cell line and culture conditions

V79 Chinese hamster lung fibroblast cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China) and were maintained in 5% CO2 at 37 °C in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies/Gibco, Gaithersburg, MD, USA). Cells were maintained as monolayers in 25 cm2 culture flasks at 37 °C in a 5% carbon dioxide humidified atmosphere. At 80–90% confluence, cells were detached with 0.25% trypsin and were passaged at an initial density of 0.5–1.0 × 106 cells/ml.

Micronuclei (MN) assay

Cells in the logarithmic growth phase were suspended at a concentration of 105 cells/ml, seeded on culture dishes, and incubated at 37 °C in 5% CO2 for 24 h. After the cells adhered, the culture medium was replaced with medium containing suspensions of fiber powder at three different concentrations. Cell culture medium was used as a negative control, and culture medium containing 200 μg/ml cyclophosphamide was used as a positive control. The culture dishes were incubated for an additional 24 h, were washed with PBS, were fixed with methanol/acetic acid (3:1 [v/v]) for 30 min at 4 °C, and then were covered with coverslips. After staining with Wright-Giemsa stain for 5 min, cell morphology was examined by light microscopy[26]. In addition, 1 × 103 cells per slide were examined, totaling 4 × 103 cells on 3–4 slides per treatment group and the numbers of cells containing MN were counted. The slides were blinded to the scorer. The MN scoring criteria were as follows: diameter no larger than one-third of that of the main nuclei, non-refractile, staining intensity same as or brighter than that of the main nuclei, and located within the cytoplasm but not in contact with the main nuclei. Binucleated cells with overlapping main nuclei were not scored. The results were expressed as percentages of total cells according to the following formula: MN = n1/n0 × 100%, where n1 was the number of cells containing MN and n0 was the total number of cells observed.

Single cell gel electrophoresis

Single cell gel electrophoresis (SCGE), also known as the alkaline comet assay, was performed as described (Behm et al. 2009). Briefly, 0.5–1.0 × 106 V79 cells were seeded onto 12-well culture plates and were incubated with a fiber suspension at 50, 100, or 200 μg/ml in culture medium for 12, 24, or 48 h. Medium containing no fiber was used as a negative control, and medium containing 0.1 mM K2Cr2O7 was used as a positive control. For electrophoresis, microscope slides with frosted ends were covered with normal-melting-point agarose (1% in distilled water) before the experiment. For the second layer, 50 μl of cell suspension mixed with 100 μl of low-melting-point agarose (0.5% in PBS, Sigma) was pipetted onto the slides, was spread by use of a cover slip, and was maintained on an ice-cold flat tray for 5 min for solidification. After removal of the cover slips, the slides were immersed in cold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris (pH 10), and 1% sodium sarcosinate), with 1% Triton-X 100 and 10% DMSO added immediately before the reaction, for at least 2 h at 4 °C. The slides were removed from the lysing solution and were placed in a horizontal gel electrophoresis tank. The tank was filled with fresh electrophoresis solution (1 mM Na2EDTA and 300 mM NaOH, pH 13) to a level approximately 0.5 cm above the slides. The slides were allowed to remain in the cold (4 °C) buffer for 20 min to denature cellular DNA. Electrophoresis was performed at 4 °C for 30 min at 300 mA and 25 V (~0.74 V/cm). After electrophoresis, the slides were washed with Tris buffer (0.4 M Tris, pH 7.5) 3 times for 5 min each. To stain the DNA, ethidium bromide (65 μl of 20 μg/ml) was added to each slide. Slides were covered with a cover slip, were placed in a humidified, airtight container to prevent drying of the gel, and were analyzed within 3–4 h. Cells were visualized with a fluorescent microscope equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. For analysis, 100 cells were selected randomly using two slides. The slides were blinded to the scorer. CASP comet analysis software was used for quantification of DNA damage. Olive tail moments (OTM = [distance between the center of gravity of DNA in the tail and the center of gravity of DNA in the head]/[percent of DNA in the tail]) were measured and calculated automatically.

Statistical analyses

SPSS 20.0 was used for all data analyses. Results were expressed as means ± standard deviations. One-way analysis of variance (ANOVA) was used for comparisons of SCGE results and MN frequencies from experimental and control samples. Post hoc analysis of group differences was performed by the Fisher’s least significant difference (LSD) test. The limit for statistical significance was fixed as p < 0.05.

Results

Chrysotile and rock wool fibers induce chromosome aberrations in V79 cells

MN, cytoplasmic chromatin masses with the appearance of small nuclei, arise from chromosome fragments or an intact chromosome lagging behind in the anaphase stage of cell division (Dopp et al. 1997). Their presence in cells is a reflection of structural and/or numerical chromosomal aberrations during mitosis. To determine if chrysotile and rock wool fibers cause an increase in MN of V79 cells, the frequencies of MN were determined after 24 h of exposure to 50, 100, or 200 μg/ml fiber suspensions and were compared with those for untreated cells. Images of cells with and without MN are shown in Fig. 1a. Chrysotile and rock wool fibers caused increases in MN. For these fibers, there was no statistical difference in MN rates between untreated cells and cells treated with 50 μg/ml fiber suspension. There was, however, a significant increase in MN in cells treated with 100 or 200 μg/ml fiber suspension (Fig. 1b). Thus, for V79 cells, chrysotile, and rock wool fibers induce increases in MN in a dose-dependent manner.

Fig. 1
figure 1

In V79 cells, chrysotile and rock wool fibers induce chromosome aberrations in dose-dependent manner. NC negative control group, cell culture medium with no fiber added, PC positive control group, medium containing 200 μg/ml cyclophosphamide. MN micronuclei, CA exposed to chrysotile fibers, FR exposed to rock wool fibers. V79 cells were exposed to 50, 100, or 200 μg/ml suspensions of chrysotile or rock wool fibers for 24 h. a MN in cells exposed to chrysotile or rock wool fibers (100 μg/ml, 40×). b The frequency of MN in cells exposed to chrysotile or rock wool fibers for 24 h. *P < 0.05, significantly different from the negative control. a P < 0.05 significantly different from 50 μg/ml dose group and b P < 0.05 significantly different from 100 μg/ml dose group

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Chrysotile and rock wool fibers induce DNA damage in V79 cells

The SCGE assay is also known as the comet assay because, upon electrophoresis, damaged DNA trails behind intact DNA in the nucleus, resembling a comet. SCGE is a widely used procedure for detection of DNA fragmentation due to single- or double-strand breaks (Gagné et al. 1996). To compare DNA damage, SCGE was performed for V79 cells exposed to chrysotile or rock wool fibers. Cells were incubated for 24 h with 50, 100, or 200 μg/ml of each fiber suspension before quantifying DNA damage. As reflected by the extended migration of fragmented DNA in comet olive tails, the treated cells showed an increase in DNA damage that dose-dependently correlated with the concentrations of chrysotile and rock wool fibers (Fig. 2a). After 24 h of exposure, a significant increase in OTM was observed for all concentrations of chrysotile and rock wool fibers. In addition, there was DNA migration for the V79 cells treated with 200 μg/ml chrysotile or rock wool fibers for 12, 24, or 48 h (Fig. 3a), and the magnitude of OTMs increased in a time-dependent manner. Chrysotile fibers had a greater effect than rock wool fibers (Fig. 3b). These results indicate that chrysotile and rock wool fibers induce DNA damage of V79 cells in a dose- and time-dependent manner and that the degree of DNA damage induced by chrysotile fibers is greater than that induced by rock wool fibers.

Fig. 2
figure 2

In V79 cells, chrysotile and rock wool fibers induce DNA damage in a dose-dependent manner. NC Cell culture medium with no fiber added was used as a negative control, PC medium containing 0.1 mM K2Cr2O7 was used as a positive control. CA exposed to chrysotile fibers, FR exposed to rock wool fibers. V79 cells were exposed to 50, 100, or 200 μg/ml suspensions of chrysotile or rock wool fibers for 24 h. a Typical DNA comet images, 400×. b DNA fragmentation was quantified by determining OTMs using CASP comet analysis software. *P < 0.05, significantly different from the negative control. a P < 0.05 significantly different from 50 μg/ml dose group and bP < 0.05 significantly different from 100 μg/ml dose group

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Fig. 3
figure 3

In V79 cells, chrysotile and rock wool fibers induce DNA damage in a time-dependent manner. NC Cell culture medium with no fiber added was used as a negative control, PC medium containing 0.1 mM K2Cr2O7 was used as a positive control. CA exposed to chrysotile fibers, FR exposed to rock wool fibers. V79 cells were exposed to 200 μg/ml suspension of chrysotile or rock wool fibers for 12, 24, or 48 h. a Typical DNA comet images, 400×. b DNA fragmentation was quantified by determining OTMs using CASP comet analysis software *P < 0.05, significantly different from the negative control. a P < 0.05 significantly different from exposed to for 12 h and b P < 0.05 significantly different from exposed to for 24 h

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Discussion

Chrysotile accounts for more than 90% of the asbestos used worldwide (Geoffrey 2002; Burki 2010). Studies concerning the mutagenicity of chrysotile, mainly epidemiological investigations, focus mainly on the incidence of lung cancer and mesothelioma of populations exposed to chrysotile (Driscoll et al. 2005a, b; Goodman et al. 2014). As determined by investigations of the pathology of mesotheliomas, chrysotile fibers, along with various other types of asbestos fibers, are present (Yano et al. 2009; Straif et al. 2009; Huang et al. 2007; Ishihara 2001). Thus, evaluation of the mutagenicity of chrysotile fibers alone may not reflect the cause of mesotheliomas. Further evidence is needed to establish the cause of these cancers.

Mutation refers to “permanent changes in the structure and/or amount of the genetic material of an organism that leads to heritable changes in its function, and includes gene mutations as well as structural and numerical chromosome alterations” (Eastmond et al. 2009). Cancer is the result of accumulation of mutations that result in autonomy, unlimited growth, and metastasis of target cells. Therefore, an evaluation of mutagenicity is used as a test for carcinogenicity. Although the SCGE and MN are conventional methods to assess carcinogenicity, the genetic endpoints of these methods are different. DNA damage were detected by SCGE, and MN assay were used to evaluate the chromosome damage induced by DNA breaking agent and Aneuploidy mutagen. In the present study, the tests were used to evaluate the genetic effects of chrysotile from Mangnai, Qinghai, China and rock wool fiber.

The results show that the chrysotile and rock wool fibers cause chromosome aberrations, as indicated by an increase in MN frequency, in a dose-dependent manner. Further, in V79 cells, both chrysotile and rock wool cause DNA damage in a time- and dose-dependent manner. The SCGE results indicate that chrysotile exposure leads to single- and/or double-strand breaks in DNA. This is in agreement with a previous study showing that asbestos causes a variety of DNA damage, including DNA cross-linking, DNA single- or double-strand breaks, and damage to bases (Yano et al. 2009). Our results are also in agreement with results of a study showing that exposure of V79 cells to chrysotile leads to an increase in reactive oxygen species and MN and to decreases in cell viability; it also causes genetic toxicity in a time- and a dose-dependent manner (Dopp et al. 2005).

Assessment of the severity of DNA damage, as measured by the comet assay, showed that the effects of chrysotile were greater than those for rock wool. Further research is required to determine the structural features and surface properties and to clarify how they are related to genotoxicity. Glass fibers have cytotoxic and genotoxic effects, even at low concentrations (Rapisarda et al. 2015). Further studies, including epidemiological investigations and experiments with animals, should be accomplished in regard to the safety of chrysotile and rock wool fibers.

Conclusion

For V79 cells, chrysotile and rock wool fibers cause chromosome aberrations in a dose-dependent manner and DNA damage in a dose- and time-dependent manner. Measurements of DNA damage show that chrysotile has a greater effect than rock wool. Further investigation of the safety of chrysotile and other fibers is warranted.