| Objective To study the cytotoxicity, oxidative stress and genotoxicity induced by three kinds of bentonite particles (BPs, including native BPs, active BPs and organic BPs) on human B lymphoblast cells in vitro and investigate DNA damage, micronucleus formation and lipid peroxidation in workers occupationally exposed to organic BPs. Methods Human B lymphoblast cells were served as target cells in vitro. Vincristine and adriamycin were used as positive controls. Normal solution (NS) and gypsum were used as negative controls. DQ-12 was used as a reference compound. The cytotoxic effects and oxidative stress induced by three kinds of BPs on human B lymphoblast cells were detected using CCK-8 assay, neutral red uptake (NRU) assay, lactate dehydrogenase (LDH) leakage assay, apoptosis assay, reactive oxygen species (ROS) assay, superoxide dismutase (SOD) assay and malondialdehyde (MDA) assay in vitro. In the CCK-8 and NRU assays, the concentrations of three kinds of BPs were 10,20,30, 60,120,240,480 and 960μg/ml. In the other five assays, the concentrations of three kinds of BPs were 30,60,120 and 240μg/ml. The cells were exposed for 4h and 24h in seven assays. The genotoxicity induced by three kinds of BPs were detected using comet assay and cytokinesis-block micronucleus (CBMN) assay in vitro. The concentrations of native and active BPs were 30,60,120 and 240μg/ml, and the concentrations of organic BPs were 1.88,3.75,7.5 and 15μg/ml. The exposure times of three kinds of BPs were 24,48 and 72 h. Genetic damage and lipid peroxidation in 60 subjects (exposure group I included 20 workers exposed to organic BPs for 3.95±0.55 years; exposure groupⅡincluded 20 workers exposed to organic BPs for 2.20±0.17 years); 20 controls were matched with workers on the basis of age, gender and smoking) were investigated using comet assay, CBMN assay and MDA assay, respectively. Results The order of cytotoxicity was:alkyl quaternary ammonium salt (AQAS)> organic BPs> active BPs> native BPs> quartz particles (DQ-12)> gypsum, according to the IC50 values in CCK-8 assay and neutral red uptake (NRU) assay. The lactate dehydrogenase (LDH) leakage, the proportions of early apoptotic cells, the reactive oxygen species (ROS) generation, the superoxide dismutase (SOD) inhibition and the malondialdehyde (MDA) release in the native and active BPs groups were significantly higher than those in the gypsum and DQ-12 groups (P< 0.05 or P< 0.01). Moreover, the cytotoxicity of BPs was enhanced with adsorption capacity of phenol. The oxidative stress induced by active BPs was significantly higher than that induced by native BPs (P < 0.05 or P< 0.01).%tail DNA and micronucleus frequency (MNF) in three BPs groups were significantly higher than those induced by gypsum control (P< 0.05 or P< 0.01). When the cells were exposed to BPs for 72 h,% tail DNA in active BPs group at the doses of 120和240μg/ml was significantly higher than that in native BPs group (P<0.01),% tail DNA in 240μg/ml native BPs and ative BPs groups was significantly higher than that in 240μg/ml DQ-12 group (P＜0.01), and% tail DNA in 15μg/ml orgnic BPs group was significantly higher than that in 30μg/ml DQ-12, native and active groups (P<0.05 or P＜0.01). When the cells were exposed to BPs for 24 h and 48 h, MNF in 15μg/ml orgnic BPs group was significantly higher than that in 30μg/ml DQ-12, native BPs and active BPs groups (P＜0.01). The water-soluble fractions of three kinds of BPs did not induce the cytotoxicity, ROS generation, DNA damage and micronuclei formation. The exposure concentrations of organic BPs in exposure group I were similar to those in exposure group II. The% tail DNA, MNF, and MDA levels of two exposure groups were significantly higher than those of control group (P< 0.01). The% tail DNA, MNF and MDA levels of exposure group I were significantly higher than those of exposure group II (P< 0.01). There were positive correlations between exposure years and% tail DNA and MNF (P< 0.05). Conclusion Native BPs, active BPs and organic BPs can induce significantly cytotoxicity, oxidative stress and genotoxicity on human B lymphoblast cells in vitro. The cytotoxicity and genotoxicity of BPs may be associated with the adsorption capacity of BPs, oxidative stress induced by BPs and pariticle contents to a certain extent. The insoluble particle fractions may play a main role in the cytotoxic effects, oxidative stress and genotoxicity induced by BPs. The cyto-genotoxicity induced by organic BPs was significantly higher than that induced by native BPs and active BPs, there were high detectable genetic damage and lipid peroxidation in workers occupationally exposed to organic BPs.
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