Mol Cell Toxicol (2013) 9:51-56 DOI 10.1007/s13273-013-0008-1
ORIGINAL PAPER
Evaluation of comparative cytotoxicity of spray-type chemicals used in household products Jung-Taek Kwon1,*, Gyun-Baek Seo1,2,*, Hyun-Mi Kim1, Ilseob Shim1, Byoungcheun Lee1, Ji-Youn Jung2, Pilje Kim1 & Kyunghee Choi1 Received: 31 December 2012 / Accepted: 15 February 2013 �The Korean Society of Toxicogenomics and Toxicoproteomics and Springer 2013
Abstract Chemicals are widely used in our daily
lives for various purposes such as disinfectant, air fresher, paints and hair spray. However, their pulmonary toxicity has less studied compared with oral and dermal toxicity. Therefore, the purpose of this study was to examine comparative cytotoxicity of triclosan (TCS), benzisothiazolinone (BIT), dichlorophene (DCP) and citral (CTR) major using spray-type chemicals used in household products (CHPs) in Korea. TCS, DCP and BIT induced more severe mitochondria injury and cell membrane damage than CTR in lung epithelial cell during 24 hrs exposure. Furthermore, the result of clonogenic assay revealed that exposure of CHPs significantly decreased colony size and that BIT reduced cell growth at most compared with TCS, DCP and CTR. In summary, results of comparative cytotoxicity demonstrated that inhalation of TCS, DCP and BIT may cause pulmonary toxicity. Therefore, our results suggest that TCS, BIT and DCP are requiring inhalation toxicity assessment for maintaining a high quality of life. Keywords Cytotoxicity, Triclosan, Benzisothiazolinone, Dichlorophene, Citral
1
Environmental Health Research Department, National Institute of Environmental Research, Incheon 404-708, Korea 2 Department of Companion and Laboratory Animal Science, Kongju National University, Yesan 340-702, Korea *These authors contributed equally to this work Correspondence and requests for materials should be addressed to J. T. Kwon (
[email protected]) & H. M. Kim (
[email protected])
Various types of chemicals are widely used in our daily lives for different purposes including disinfectant, air fresher, paints, cosmetics and hair spray. Especially, spray type chemicals are commonly used in household products (CHPs) such as deodorant and air fresher generating respirable particles in the air. As a result, CHPs have been shown to be a major source of inhalation exposure to manufacturing factory workers1 and people during daily activities2,3. Nevertheless, their pulmonary toxicity has not been explored completely compared with oral and dermal toxicity study. Recently, in Korea, sterilizers in home humidifiers (SHHs) as CHPs caused lung fibrosis in baby and pregnant women4. The inhalation toxicity study of SHHs including polyhexamethyleneguanidine (PHMG), oligo (2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) and methylisothiazolinone (MIT)/chloromethylisothiazolinone (CMIT) in rats showed that the histopathological findings from exposure to products with polyhexamethyleneguanidine (PHMG) and oligo(2-(2ethoxy)ethoxyethyl guanidinium chloride (PGH) were identical to those of the human victims5. These accidents have drawn growing alarm about the potential adverse impact of CHPs on human health, especially on pregnant women and children6. In particular, young children are more susceptible to a contaminated indoor environment. Therefore, the purpose of this study was to determine comparative cytotoxicity of triclosan (TCS), benzisothiazolinone (BIT), dichlorophene (DCP) and citral (CTR) major using spray-type CHPs in Korea. Injury of mitochondrial
To determine the mitochondria injury of CHPs in A549 cells was treated with BIT, TCS, DCP and CTR for 24
52
Mol Cell Toxicol (2013) 9:51-56 Cl
OH
OH
OH
Cl
Cl
hours using the MTT assay. As shown in Figure 2A, exposure of CHPs significantly decreased the cell viability with dose dependant manner. The results were presented as a percentage relative to the control. The IC50 values (concentration at which 50% of mitochondrial dehydrogenase activity was inhibited) were 28.22 μM, 39.84 μM, 73.52 μM and 332.9 μM for the TCS, DCP, BIT and CTR, respectively. Also, cell morphology changes were observed at the TCS, BIT and DCP after 24 hours of treatment with 250 μM (Figure 2B).
O Cl
Cl
Triclosan
Dichlorophene
O
S
NH
O
Benzisothiazolinone
Membrane damage
Citral
To assess the damage of cell membranes, the concentration of LDH was quantified. Three different con-
Figure 1. The structure of chemicals used in household products (CHPs).
(A)
120
CTR DCP BIT TCS
110
Cell viability (% of control)
100 90 80 70 60 50 40 30 20 10 0
Con
31.3
62.5
125
250
Concentration (μM)
(B) Con
15.6
CTR
BIT
DCP
TCS
Figure 2. Cell viability and morphological changes after exposure of CHPs in A549 cells (A) A549 cells were treated with variable concentrations of CHPs for 24 hours. Mean±SEM. *P⁄0.05, **P⁄0.01 compared to control. (B) After incubation with CHPs for 24 hours, the cells were examined by inverted microscopy. ×200.
Mol Cell Toxicol (2013) 9:51-56
53
400 360
LDH release (relative to control)
320
CTR BIT DCP TCS
280 240 200 160 120 80 40 0 15.6
62.5
250.0
Concentration (μM)
Figure 3. LDH release from A549 cells following 24 hours exposure to CHPs. Mean±SEM. *P⁄0.05, **P⁄0.01 compared to control.
centrations (15.6, 62.5 and 250 μM) were tested for 24 hours exposure (Figure 3). Under these conditions CTR did not induce significant LDH release. However, TCS, DCP and BIT induced significant membrane damage at 250 μM. As shown in Figure 3, cell exposure to DCP and TCS induced the release of LDH to the culture medium which is 2-3 times more than control. Effect of colony-formation ability
We next investigated effect of cell growth by CHPs exposure of low-dose in long-term. The cells were treated with IC20 concentrations for 10 days. As shown in Figure 2A, the IC20 values of CHPs were approximately 15 μM. The results of clonogenic assay revealed that a significant difference was found in the number of colony forms of CHPs (Figure 4A). Also, exposure of CHPs significantly decreased colony size and BIT reduced cell growth in comparison with TCS, DCP and CTR (Figure 4B).
Discussion In this study, we investigated the cytotoxicity of BIT, TCS, DCP and CTR in human lung epithelial (A549) cells. We found that TCS, DCP and BIT have potential cytotoxicity and BIT inhibits cell growth in the long-term exposure of low concentration. Previous studies showed that while several studies
have been published on oral and dermal toxicity of the CHPs, quite limited studies have been conducted focusing on pulmonary toxicity7. To use CHPs in oral care product or dermal applied products are generally safe8,9, but inhalation of TCS induced death of rats and lung injury such as acute congestion and hemorrhagic foci10. Also, inhaled BIT leaded to asthma and rhinitis in workers1. These results suggest that inhalation exposure of CHPs can be dangerous for using personal care products, even if ingestion or dermal exposures have low toxicity. Damage of mitochondria and membrane in the cells is typical index of cytotoxicity11,12. Mitochondrial function is major indicator for living organism and it is important to affect metabolic activity as cell viability. In addition, the levels of LDH in culture medium indicate cell membrane damage as cytotoxicity marker. Previous in vitro studies showed that CHPs induced cytoxicity such as inhibits adipocyte differentiation in human mesenchymal stem cells13, inhibits cell proliferation and induces apoptosis in breast cells14 and impair the mitochondrial function in hepatocyte15. Our results of cytotoxicity showed that TCS, DCP and BIT affect mitochondria activity. Also, results of LDH analysis were consistent with those of MTT experiments at high concentrations. However, differences between MTT reduction and LDH release below treatment concentration of 62.5 μM were observed and it is suggested that exposure to low concentrations of CHPs in mitochondria appeared more sensitive. Therefore, the decrease in MTT reduction and the increase in LDH level demonstrated that TCS, DCP and BIT caused more severe mitochondria injury and cell membrane damage than CTR in the lung epithelial cells during 24 hours exposure. Personal care product contains low concentrations of CHPs ranging between 0.01 and 0.1 percent. Although these concentrations are low level, long term exposure to low doses of CHPs may cause lung damage16. Our clonogenic investigation is to assess cell growth effects during long term exposure of CHPs. In this study, cells were treated IC20 concentration of CHPs (15 μM) and did not show serious cytotoxicity for 24 hour treatment in MTT assay. Also, LDH results were no difference compared to the control group at 15.6 μM. However, our results of clonogenic assay demonstrated that long-term exposure of CHPs to low concentrations was more hazardous. In particular, exposure of BIT caused strong inhibition of colony formation ability. However, we do not know the exact mechanisms of this observation. These results suggested that cell proliferation and apoptosis reaction may be affected in long term exposure of CHPs. Thus, further studies are necessary to investigate the mechanism of cytotoxic
54
Mol Cell Toxicol (2013) 9:51-56
(A) 120
Colony number (% of control)
100 80 60 40 20 0
(B)
Con
TCS
CTR
DCP
BIT
Colony number (% of control)
120 100 80 60 40 20 0
Con
TCS
CTR
DCP
BIT
Figure 4. The inhibition effects of CHPs on colony formation in A549 cells after 10-day exposure to 15 μM of CHPs. (A) Effects on colony number (*P⁄0.05, **P⁄0.01 compared to control). (B) Effects on colony size. Mean±SEM (*P⁄0.05, **P⁄0.01 compared to control). Scale bar=100 μm.
Mol Cell Toxicol (2013) 9:51-56
effect of exposed CHPs. From the results of current study, we suggest that the long-term exposure to lowdose toxicity testing is important to complete toxicological evaluation of CHPs. Therefore, our study provides basic information of potential pulmonary toxicity for the risk assessment of CHPs. In summary, the results of cytotoxicity in the lung epithelial cells demonstrated that inhalation of TCS, DCP and BIT may cause pulmonary toxicity. Thus, inhalation exposure of TCS, DCP and BIT in indoor spaces should be considered potentially hazardous in the aspect of the pulmonary toxicity assessment. Based on the results above, it is suggested that TCS, BIT and DCP have the potential to cause pulmonary toxicity. Although more research is needed to confirm these results, careful regulation of CHPs would be necessary in maintaining a high quality of life.
Materials & Methods Materials
All chemicals used in this study were purchased from the Sigma-Aldrich (Sigma-Aldrich, St Louis, MO, USA). Cell culture
The A549 (human lung epithelial cell, Korea cell bank, Seoul, Korea) cells were maintained in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin, Gibco-BRL, Gaithersburg, MD, USA) under standard cell culture conditions (at 37� C, in 5% CO2 and 90% humidity). Cell viability assay
Cell viability of CHPs was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at a density of 1.0×104 cells per well, and then incubated for overnight. The cells were incubated at different concentrations (0, 15.6, 31.3, 62.5, 125 and 250 μM) of CHPs for 24 hours. After treatment, 10 μL of MTT solution (5 mg/mL in PBS) were added to each well and the plate was incubated for 4 hours at 37� C. To achieve solubilization of the formazan crystal formed in viable cells, 100 μL of dimethyl sulfoxide (DMSO) were added to each well. The plate was shaken for 15 min at room temperature and the absorbance was measured using a microplate reader (Tecan, Salzburg, Austria) at a wavelength of 540 nm.
55
LDH assay
The total lactate dehydrogenase (LDH) activity in cell lysates was measured according to the manufacturer’s instructions of the QuantiChrom Lactate Dehydrogenase Kit (BioAssay Systems, Hayward, CA, USA). Briefly, 1×105 cells were seeded in a 24-well plate one day before assaying. The cells were then exposed for 24 hours to concentrations of CHPs ranging from 0 μM to 250 μM. After exposure, we measured LDH level in the supernatants. Clonogenic assay
To assess cell growth effects of CHPs, the cells were investigated by clonogenic assay. The principle of the clonogenic assay, under which inhibition of cell growth and long-term effect by CHPs can be confirmed, is based on single cell growth to form colony17. Cells were seeded (250/well) in 6-well plate and allowed to adhere to the plate for overnight in a humidified incubator18. After incubation, cells were treated at IC20 concentrations of CHPs of 15 μM for 10 days and stained with crystal violet (0.1% crystal violet in 20% methanol). Statistical analysis
Results were analyzed by Student’s t-test, a multiple variance of analysis (ANOVA) test and calculation of IC50 (Graphpad Software, San Diego, CA, USA). All results were given as means±SE. A value of *P⁄ 0.05 was considered significant and **P⁄0.01 highly significant compared to the corresponding control. Acknowledgements The authors thank Mimi Lee, Environmental Health Research Department, National Institute of Environmental Research, for technical advice and assistance. This study was supported by Post Doctoral Course Program of National Institute of Environmental Research (NIER), Republic of Korea.
References 1. Moscato, G. et al. Occupational asthma and rhinitis caused by 1,2-benzisothiazolin-3-one in a chemical worker. Occup Med (Lond) 47:249-251 (1997). 2. Nazaroff, W. W. & Weschler, C. J. Cleaning products and air fresheners: Exposure to primary and secondary air pollutants. Atmos Environ 38:2841-2865 (2004). 3. Berger-Preiss, E. et al. Inhalational and dermal exposures during spray application of biocides. Int J Hyg Environ Health 208:357-372 (2005). 4. Korea Centers for Disease Control and Prevention. Interim report of epidemiological investigation on lung
56
injury with unknown cause in Korea. Public Health Weekly Report KCDC 4:817-832 (2011). 5. Ministry of Health and Welfare Press Release. Order for withdrawal of six humidifier disinfectants products from the market issued. Available via DIALOG (2011). http://english.mw.go.kr/front_eng/al/sal0201vw.jsp?P AR_MENU_ID=1002&MENU_ID=100203&page=1 &CONT_SEQ=260454&SEARCHKEY=&SEARCH VALUE. Accessed 5 Dec 2012. 6. Chang, M. H. et al. Characteristics of humidifier use in korean pregnant women: The mothers and children’s environmental health (moceh) study. Environ Health Toxicol 27:e20120003 (2012). 7. Henderson, J., Sherriff, A., Farrow, A. & Ayres, J. G. Household chemicals, persistent wheezing and lung function: Effect modification by atopy? Eur Respir J 31:547-554 (2008). 8. Rodricks, J. V., Swenberg, J. A., Borzelleca J. F., Maronpot, R. R. & Shipp, A. M. Triclosan: A critical review of the experimental data and development of margins of safety for consumer products. Crit Rev Toxicol 40:422-484 (2010). 9. Yamarik, T. & Andersen, F. Safety assessment of dichlorophene and chlorophene. Int J Toxicol 23:1-27 (2004). 10. Leutkemeier, H., Ullmann, L., Zak, F., Sachsse, K. & Shess, R. Irgasan DP 300 (FAT 80023/A) 21-day inhalation study on the rat. Ciba-Geigy Limited (1974). 11. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
Mol Cell Toxicol (2013) 9:51-56
cytotoxicity assays. J Immunol Methods 65:55-63 (1983). 12. Koh, J. Y. & Choi, D. W. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Method 20:83-90 (1987). 13. Guo, L. W. et al. Cytotoxicity and inhibitory effects of low-concentration triclosan on adipogenic differentiation of human mesenchymal stem cells. Toxicol Appl Pharmacol 262:117-123 (2012). 14. Chaouki, W., Leger, D. Y., Liagre, B., Beneytout, J. L. & Hmamouchi, M. Citral inhibits cell proliferation and induces apoptosis and cell cycle arrest in mcf-7 cells. Fundam Clin Pharmacol 23:549-556 (2009). 15. Newton, A. P., Cadena, S. M., Rocha, M. E., Carnieri, E. G. & Martinelli de Oliveira, M. B. Effect of triclosan (TRN) on energy-linked functions of rat liver mitochondria. Toxicol Lett 160:49-59 (2005). 16. Fujimaki, H. et al. Effect of long-term exposure to low-level toluene on airway inflammatory response in mice. Toxicol Lett 168:132-139 (2007). 17. Puck, T. T. & Marcus, P. I. A rapid method for viable cell titration and clone production with hela cells in tissue culture: The use of x-irradiated cells to supply conditioning factors. Proc Natl Acad Sci USA 41:432437(1955). 18. Herzog, E. et al. A new approach to the toxicity testing of carbon-based nanomaterials--the clonogenic assay. Toxicol Lett 174:49-60 (2007).
