Environ Sci Pollut Res (2015) 22:4763–4770 DOI 10.1007/s11356-014-3939-8

SHORT RESEARCH AND DISCUSSION ARTICLE

Photoinitiators enhanced 1,2-dichloropropane-induced cytotoxicity in human normal embryonic lung fibroblasts cells in vitro Yoichi Kawasaki & Chiaki Tsuboi & Kenta Yagi & Miwa Morizane & Yasuyuki Masaoka & Satoru Esumi & Yoshihisa Kitamura & Toshiaki Sendo

Received: 22 September 2014 / Accepted: 1 December 2014 / Published online: 12 December 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Dichloromethane (DCM) and 1,2-dichloropsropane (DCP) have various uses, including being solvents for paint removers. Photoinitiators are also used in a wide range of commercial applications such as printing. These chemicals have been shown to induce cytotoxic effects. In the present study, we evaluated the combined effects of DCM or DCP from paint removers and photoinitiators used in printing on normal human embryonic lung fibroblasts with the aim of preventing occupational injuries. We showed that DCP, 2,2dimethoxy-2-phenylacetophenone (2,2-DMPAP), 2ethylhexyl-4-(dimethylamino) benzoate (2-EHDAB), 1hydroxycyclohexyl phenyl ketone (1-HCHPK), and methyl 2-benzoylbenzoate (MBB) induced cytotoxicity, whereas DCM and 2-isopropylthioxanthone (2-ITX) did not. In addition, 2-methyl-4′-(methylthio)-2-morpholinopropiophenone (MTMP) caused a slight increase in cytotoxicity. The combination of DCP and the four photoinitiators (2,2-DMPAP, 2EHDAB, MBB, and MTMP) significantly induced cytotoxicity and also led to apoptosis. In conclusion, the combination of DCP and photoinitiators may increase the risk of respiratory diseases. Keywords 1,2-Dichloropropane . Photoinitiator . Combination effect . Cytotoxicity . Apoptosis . Occupational injury Responsible editor: Philippe Garrigues Y. Kawasaki : Y. Masaoka : S. Esumi : Y. Kitamura (*) : T. Sendo Department of Pharmacy, Okayama University Hospital, 2-5-1, Shikata-cho, Kita-ku, Okayama 700-8558, Japan e-mail: [email protected] C. Tsuboi : K. Yagi : M. Morizane : Y. Kitamura : T. Sendo Department of Clinical Pharmacy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama 700-8558, Japan

Introduction Dichloromethane (DCM) is an important solvent with unique chemical properties that have led to its widespread use in industries such as printing. It has been classified as a possible human carcinogen by the International Agency for Research on Cancer (IARC) (ISRC 1999). DCM is used in the USA as follows: paint remover, 40 %; exports, 20 %; aerosol sprays, 17 %; chemical specialties, 10 %; plastics processing, 6 %; other uses, 7 % (Fishbein 1979). The results of a 2-year DCM inhalation study revealed that the incidence of benign mammary tumors in male and female Sprague–Dawley rats exposed to concentrations up to 3,500 ppm was increased (Burek et al. 1984). US National Toxicology Program studies reported that the incidences of hepatic and pulmonary tumors in B6C3F1 mice exposed to DCM by inhalation at concentrations of 2000 and 4000 ppm for 2 years were also increased (NTP 1986). Paint stripping is an activity that requires caution because of the increased risk of potential exposure to DCM, a solvent that has been identified as a potential human carcinogen and neurotoxicant (Van Veen et al. 2002). A previous study reported the significant risk of pancreatic cancer associated with occupational exposure to chlorinated hydrocarbon solvents (Ojajärvi et al. 2001). A previous in vitro study detected DNA single-strand breaks following DCM exposure in mice (Graves et al. 1994; Graves et al. 1995). Furthermore, chronically inhaled DCM was shown to cause lung adenoma or carcinoma (Kari et al. 1993). 1,2-Dichloropropane (DCP) is another widely used solvent for oil- and fat-based furniture finishes, dry cleaning fluids, and paint removers (National Toxicology Program 1986). DCP was previously shown to induce nephrotoxicity in rats (Trevisan et al. 1992), and exposure to DCP also led to inflammation in the respiratory epithelium (Umeda et al. 2010). The inhalation of DCP was reported to cause

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pulmonary edema (Buie et al. 1986) and also increased the combined incidence of bronchiolo-alveolar adenomas and carcinomas in mice (Matsumoto et al. 2013). In the 1970s, halogenated saturated hydrocarbons such as DCM were found to increase the number of genomic mutations (Jongen et al. 1978), and this finding was corroborated by other mutagen studies (Kundu et al. 2004; Nestmann et al. 1981). Photoinitiators are used in a broad range of commercial and biological applications including printing (Bohonowych et al. 2008), dentistry (Kostoryz et al. 1999), the encapsulation of pancreatic islet cells (Cruise et al. 1999; Hill et al. 1997), and as blood vessel adhesives (Dumanian et al. 1995). Thus, a high level of daily exposure to photoinitiators is possible. We recently reported that two photoinitiators, 2-methyl4′-(methylthio)-2-morpholinopropiophenone (MTMP) and 1-hydroxycyclohexyl phenyl ketone (1-HCHPK), induced cytotoxicity in normal human peripheral blood mononuclear cells (Kawasaki et al. 2012; Yamaji et al. 2012). The incorporation of benzophenone, a photoinitiator, in sunscreen has also been shown to cause allergic skin reactions (Cook and Freeman 2001) similar to other skin irritants that cause photoallergic reactions, allergic contact dermatitis (Alanko et al. 2001), and facial erythema (Nedorost 2003). Therefore, the cytotoxic effects induced by photoinitiators are similar to those of chemicals such as DCM or DCP. Previous studies showed that exposure to DCM or DCP caused cholangiocarcinoma among workers in a printing company in Japan (Kubo et al. 2014; Kumagai et al. 2013). In the present study, we evaluated the combined effects of DCM or DCP from paint removers and photoinitiators used in printing on normal human embryonic lung fibroblasts with the aim of preventing occupational injuries.

Materials and methods Reagents and cells MRC-5, normal human embryonic lung fibroblasts, were obtained from the RIKEN BioResource Center (Ibaraki, Japan). Minimum essential medium (MEM) alpha and fetal bovine serum were purchased from Life Technologies Japan Ltd., Japan. Dichloromethane, 1,2-dichloropropane, 1hydroxycyclohexyl phenyl ketone (1-HCHPK), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co., USA. 2,2-Dimethoxy-2-phenylacetophenone (2,2-DMPAP), 2-ethylhexyl 4-(dimethylamino) benzoate (2-EHDAB), 2isopropylthioxanthone (2-ITX), methyl-2-benzoylbenzoate (MBB), and 2-methyl-4′-(methylthio)-2morpholinopropiophenone (MTMP) were purchased from Tokyo Chemical Industry Co., Ltd., Japan. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Wako Pure Chemical Industries Ltd., Japan.

Environ Sci Pollut Res (2015) 22:4763–4770

Cell culture The MRC-5 cell line was cultured in MEM alpha supplemented with 10 % (v/v) heat-inactivated fetal bovine serum. Cells were maintained at 37 °C in an incubator with 5 % (v/v) CO2 in a water-saturated atmosphere. Cell viability assay We performed cell seeding for 24 h and then drug treatment for 24 h. MRC-5 cells were cultured in a 96-well culture plate (1.0×104 cells/200 μl/well) along with various concentrations of DCM, DCP, and the photoinitiators for 24 h. The final concentrations of DCM and DCP used 10–100 mM, while those of the six photoinitiators were 1, 2, 3, 4, and 5 mM. Cells cultured in complete medium without DCM, DCP, or photoinitiators served as the control. DCM (50 mM) and photoinitiators (1 mM), or DCP (50 mM) and photoinitiators (1 mM) were used in the combination study. Cells cultured in complete medium without photoinitiators served as the control. After 24 h of exposure, 10 μl (5 mg/ml) of the MTT reagent was added to each well, and the cells were incubated for an additional 3 h. Absorbance was monitored at 570 nm. The percentage viability of cells was calculated by comparing the untreated group to the DCM- or DCP-treated group. Annexin V/propidium iodide staining and flow cytometric analysis Cells were treated with DCP with/without photoinitiators for 24 h. Early apoptotic cells were then detected with annexin V– fluorescein isothiocyanate (FITC) (Invitrogen, Tokyo, Japan) combined with propidium iodide (PI) by flow cytometry. Briefly, cells were plated in six-well plates at a density of 1×103 cells/cm2. Treated and untreated cells were incubated with FITC-conjugated annexin V and PI for 15 min at 20 °C. They were immediately analyzed on a flow cytometer (Beckton Dickinson, FACSCalibur) in their staining solution. Data were collected when the number of apoptotic cells reached 100. Data analysis was performed using Flow Jo software on cells characterized by their forward/side scatter (FSC/SSC) parameters. Samples stained with annexin V– FITC and PI were represented by dot plots of PI versus the intensity of annexin V. Plots were divided into four regions as follows. The lower left region included cells that failed to stain with either annexin Vor PI and were considered to be undamaged. The lower right region included cells stained with annexin V that were still PI negative and were considered to be early apoptotic cells. The upper right region included cells stained with both annexin Vand PI; they were classified as late apoptotic or necrotic cells. The upper left region included cells that were annexin V negative, but PI positive and were dead cells.

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Results

1 mM 2-ITX for 24 h (44.27 %) than in those treated solely with 1 mM 2-ITX (106.11 %). Cell viability was significantly less in MRC-5 cells treated with the combined treatment of 50 mM DCP and 1 mM MBB for 24 h (30.17 %) than in those treated solely with 1 mM MBB (89.39 %) or 50 mM DCP (51.40 %). Cell viability was significantly less in MRC-5 cells treated with the combined treatment of 50 mM DCP and 1 mM MTMP for 24 h (26.81 %) than in those treated solely with 1 mM MTMP (85.55 %) or 50 mM DCP (48.48 %) (Fig. 4).

Cell viability assay

Flow cytometric assay

DCM and DCP

Based on the result that cell viability was less with the DCP (50 mM) and photoinitiator (1 mM) treatment than with the DCP treatment, we investigated whether this effect was due to an increase in apoptosis. MRC-5 cells were treated for 24 h with DCP with/without photoinitiators, stained for the expression of annexin V and permeability to PI, and analyzed by flow cytometry (Fig. 5). Figure 6 shows that the induction of apoptosis was significantly stronger with the combination of DCP and 2,2-DMPAP, 1-HCHPK, MBB, or MTMP than with DCP alone [F(6, 14)=9.561, P

Photoinitiators enhanced 1,2-dichloropropane-induced cytotoxicity in human normal embryonic lung fibroblasts cells in vitro.

Dichloromethane (DCM) and 1,2-dichloropsropane (DCP) have various uses, including being solvents for paint removers. Photoinitiators are also used in ...
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