4-chloro-1,2-phenylenediamine Induces Apoptosis in Mardin–Darby canine kidney cells via activation of caspases
Abstract
4-Chloro-1,2-phenylenediamine (4-Cl-o-PD) is a halogenated aromatic diamine that was used as a precursor for manufacturing permanent hair dyes. Despite its well-documented mutagenic and carcinogenic effects in a number of in vitro and in vivo models, its cytotoxicity and mode of action have not received similar attention. Here, we investigated the effect of 4-Cl-o-PD on Mardin–Darby canine kidney cells. It induced apoptosis and the evidence suggests its initiation by reactive oxygen species (ROS). The results of various assays used show a dose-dependent (i) decrease in cell viability, (ii) increase in cells at sub-G1 phase and the G0/G1 phase arrested in cell cycle, (iii) increase in intracellular ROS accompanied by depletion of glutathione, and (iv) that apoptotic cell death probably involves activation of both intrinsic and extrinsic pathways. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 655–664, 2014.
INTRODUCTION
The great demand for hair dyes can be seen by the proliferation of hair salons and their ability to impart temporary or permanent color change to the hair, satisfying the desire of consumers for beauty, fashion, and a look-younger image. However, their potential toxicity to humans and the environment has emerged from numerous studies, and thus there is the need to institute regulations for their safety assessment, use, disposal, and warning labels stating such risks (Van Duuren, 1980 ; Wang et al., 2008; FDA, 2009). One type of hair dye in widespread use is the so-called oxidative or permanent hair dye, which uses a combination of dye intermediate (such as an aromatic diamine) and an oxidizing agent (e.g., hydrogen peroxide). One such dye intermediate is 4-chloro-1,2-phenylenediamine (4-Cl-o-PD), a halogenated aromatic amino compound. The U.S. Environmental Protection Agency reported that about 15 million people are potentially exposed to hair dye ingredients as a result of personal use or through application of hair dyes to other people (Wang et al., 2008). In animal studies, 4-Cl-o-PD induced hepatocellular carcinomas, papillomas, and urinary bladder cancer in mice and rats (Weisburger et al., 1980). Epidemiological studies also demonstrated that hair dye users and barbers incurred a high risk of bladder cancer, non-Hodgkin's lymphoma, multiple myeloma, and hematopoietic cancers (Thun et al., 1994; Yu et al., 1998; Gago-Dominguez et al., 1999; Rauscher et al., 2004). Apart from its potential carcinogenicity, genotoxic effects of 4-Cl-o-PD have also been documented. We and others showed that 4-Cl-o-PD induces DNA single-strand breaks in human lymphocytes, and induced significant dose-related genotoxicity in mouse micronucleus assay (Soler-Niedziela et al., 1991; Chye et al., 2008). However, recent reviews stated that the results of genetic toxicity, carcinogenicity, and reproductive toxicity studies suggest that modern hair dyes and their ingredients pose no genotoxic, carcinogenic, or reproductive risk to humans (Nohynek et al., 2004, 2010). The toxicity of chemicals, including hair dyes and their ingredients, should encompass carcinogenicity, mutagenicity, and cytotoxicity (Zhu et al., 1991; Chen et al., 2006; Hu et al., 2009). This article deals with the less investigated cytotoxicity of 4-Cl-o-PD.
MATERIALS AND METHODS
Materials
Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), trypsin-EDTA, trypan blue, trichloroacetic acid, and penicillin–streptomycin were obtained from GIBCO Laboratories (Grand Island, NY). Phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), Hoechst 33258, and paraformaldehyde were purchased from Merck (Darmstadt, Germany). 4-Cl-o-PD, propidium iodide (PI), and dichlorodihydroflourescein diacetate (DCFH-DA) were purchased from Sigma Chemical (St. Louis, MO). Glutathione (GSH) apoptosis detection kit, caspase-8, −9, and −3/7 activities assay kits were purchased from Calbiochem (La Jolla, CA).
Cell Line and Cell Culture
Mardin–Darby canine kidney (MDCK) cell line was obtained from the American Type Culture Collection (Rockville, MD). MDCK cell line was cultured in DMEM containing 10% FBS in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. The stock solution of 4-Cl-o-PD (100 mg/mL) was dissolved in DMSO, and different concentrations were prepared in the culture medium with a final DMSO concentration not exceeding 0.1%. The presence of 0.1% DMSO had no marked effect on any of the parameters monitored.
Determination of Cell Viability by Trypan Blue Exclusion Method
MDCK cells were seeded in 12-well flat-bottom plate at a density of 1 × 105 cells per well and grown for 24 h. The culture medium was replaced upon 80% confluence. Different concentrations of 4-Cl-o-PD were added to the cells at a final concentration of 50, 100, 200, or 300 μg/mL and grown at 37°C for 24 h. Cell viability was determined by trypan blue exclusion assay as described previously (Chen et al., 2006). Briefly, about 10 μL of cell suspensions in PBS was mixed with 10 μL of trypan blue, and the numbers of stained (dead cells) and unstained cells (live cells) were examined by using phase contrast microscope (Clement and Stamenkovic, 1994). At least 500 cells were counted in each experiment.
Hoechst 33258 Staining
MDCK cells were incubated with 200 μg/mL of 4-Cl-o-PD at 37°C for 24 h. Subsequently, cells were fixed with 3.7% paraformaldehyde at room temperature for 30 min and stained with 10 μM Hoechst 33258 at 37°C for 30 min. The cells were then washed with PBS and subjected to morphological observation using a fluorescence microscope at 400× (Leica, Wetzlar, Germany).
Cell Cycle Analysis by Flow Cytometry
Using a previously published procedure for this purpose, MDCK cells were first treated with 50, 100, 200, or 300 μg/mL of 4-Cl-o-PD for 24 h and then harvested for flow cytometric analysis (Chen et al., 2006). Briefly, adherent and floating cells were collected, washed twice with 1% FBS/PBS, and fixed with 70% ice-cold ethanol at 4°C overnight. Before fluorescence activated cell sorting (FACS) analysis, fixed cells were washed twice with filtered PBS and were suspended in fluorochrome solution containing 20 μg/mL of PI, 50 μg/mL of RNase A, and 0.1% of Triton X-100 at 37°C for 30 min. Flow cytometric analysis was performed using a BD FACSCalibur flow cytometer (BD Bioscience, San Jose, CA), a four-color and dual-laser system. The air-cooled 15-mW argon-ion laser was tuned to 488 nm, and list-mode data files consisting of 10,000 events gated on FL2-A versus FL2-W were acquired. The data were then analyzed using CellQuest PRO software (BD Bioscience) (Nicoletti et al., 1991).
Annexin V Staining and Flow Cytometry
MDCK cells were cultured in 60-mm tissue-culture dishes. The culture medium was replaced with new medium when the cells were 80% confluent and then exposed to 50, 100, 200, or 300 μg/mL of 4-Cl-o-PD for 24 h. Apoptosis was determined by staining with Annexin-V-FLUOS staining kit (Roche Diagnosis GmbH, Mannheim, Germany) (Zhu et al., 2005). After the incubations, floating as well as adherent cells that were later trypsinized were pooled and centrifuged for 5 min at 1000 × g. Pelleted cells were washed with PBS. Thereafter, cells were centrifuged again for 5 min at 1000 × g and resuspended in 100 μL of Annexin-V-FLUOS and PI labeling solution for 10 min. The stained cells were analyzed by flow cytometry, where the fluorescence emission was measured at 530 nm (Alexa Fluor 488). The percentage of viable, apoptotic, and necrotic cells was calculated using the Cellquest software (BD Biosciences, Franklin Lakes, NJ).
Measurement of Intracellular Reactive Oxygen Species
Detection of intracellular reactive oxygen species (ROS) was carried out using a previously published procedure by Hsieh et al. (2009). Briefly, MDCK cells were incubated with 50, 100, 200, or 300 μg/mL of 4-Cl-o-PD for 3 h and subsequently incubated with 10 μM of DCFH-DA for 30 min in the dark. Cells were then resuspended in PBS and subjected to fluorescence measurement at excitation wavelength of 400 nm and emission wavelength of 505 nm using the Tecan infinite F200 multifunctional microplate reader.
Measurement of Intracellular GSH
Intracellular GSH was determined by using Calbiochem® glutathione apoptosis detection kit (Calbiochem, Darmstadt, Germany). MDCK cells were treated with 50, 100, 200, or 300 μg/mL of 4-Cl-o-PD for 3 h. Cells were harvested and centrifuged at 700 × g for 5 min. The pellets were washed with PBS and resuspended with 100 μL of cell lysis buffer and incubated for 10 min on ice. The cell lysates were centrifuged at 1000 × g for 10 min and the supernatants were collected. Two microliters of glutathione-S-transferase reagent was added to each supernatant followed by 2 μL of monochlorobimane (MCB) reagent. For negative control, one microliter of MCB reagent was added to 100 μL of cell lysis buffer. The samples were incubated in the dark at 37°C for 15–30 min. Absorbance was measured using a fluorescence multiwell plate reader with excitation wavelength of 380 nm and emission wavelength of 460 nm. Soluble protein concentration was measured using the Bradford method (1976), and the bovine serum albumin was used as standard reference material.
Measurement of Caspase-8, −9, and −3/7 Activities
MDCK cells were cultured in 35-mm dishes at a density of 5 × 104 cells and treated with 50, 100, 200, or 300 μg/mL of 4-Cl-o-PD for 24 h. Cells were harvested by scraping and centrifugation at 1000 × g for 5 min. Caspase-8, −9, and −3/7 activities were determined using caspase-8, −9, or-3/7 activity assay kits, respectively (Calbiochem, Darmstadt, Germany). Briefly, the cell pellets were resuspended in 55 μL of sample buffer and vortexed for 5 min. After centrifugation, 50 μL of clear lysates was transferred to a black multiwell plate. Ten microliters of the prepared caspase-8, −9, or -3/7 substrates was added into each well containing respective cell lysates. Fluorescence was measured at excitation wavelength of 400 nm and emission wavelength of 505 nm using the Tecan infinite F200 multifunctional microplate reader.
Statistical Analysis
The experiments were repeated three times, and the results were expressed as mean ± SE. Statistical analysis was done using two-tailed Student's t-test, and p-values at a level of 95% confidence limit were considered as statistical significance.
RESULTS
Morphology Assessment of Apoptosis by Light Microscopy
Apoptosis, cell death associated with autophagy, and necrosis are the three major types of cell death that can be distinguished by morphological studies (Krysko et al., 2001). MDCK cells treated with increasing concentration of 4-Cl-o-PD (50, 100, 200, or 300 μg/mL) for 24 h were observed under light microscope. Swelling and rounding of cells, condensed chromatin, convolution of cellular surface, nuclear disintegration, formation of apoptotic bodies, and vacuolation of cytoplasm can be observed. These observations suggest apoptosis of MDCK cells treated with 50–200 μg/mL of 4-Cl-o-PD (Fig. 1). No obvious changes were observed in MDCK cells treated with medium alone or with vehicle alone (0.1% DMSO, data not show). Thus, 4-Cl-o-PD triggers morphological changes suggestive of apoptotic cell death in MDCK cells in a dose-dependent manner.
The morphology of MDCK cells after exposure to various concentrations of 4-Cl-o-PD for 24 h was photographed by phase microscope (×200). The cell damage was apparently evident after treatment with different doses of 4-Cl-o-PD when compared with control plate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Cell Viability of MDCK Cells with Trypan Blue Exclusion Assay
MDCK cell viability following 4-Cl-o-PD treatment was evaluated using trypan blue dye exclusion assay. Cells were treated with 50, 100, 200, and 300 μg/mL of 4-Cl-o-PD. The plot of viability against concentration of 4-Cl-o-PD shows that 4-Cl-o-PD had inhibited cell growth in MDCK cells in a dose-dependent manner (Fig. 2: dark blue plot). Cell death caused by 4-Cl-o-PD could be slightly prevented by presence of added vitamin E, a very permeable membrane antioxidant. Both caspase-8 inhibitor (z-EITD-FMK) and caspase-9 inhibitor (z-LEHD-FMK) could greatly abrogate the cytotoxicity of 4-Cl-o-PD (Fig. 2: yellow and sea-blue plots).
The viability of MDCK cells treated with various concentrations of 4-Cl-o-PD in the absence or presence of vitamin E, z-IETD-fmk, or z-LEHD-fmk before 4-Cl-o-PD treatment was evaluated by trypan blue exclusion method. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Assessment of Nuclear Damage by Hoechst Staining
To observe for nuclear damage following 24 h 4-Cl-o-PD exposure, MDCK cells were stained with Hoechst 33258. Substantial nuclear morphological changes characterized by loss of nuclear membrane integrity, chromatin condensation, and nuclear fragmentation and disintegration were observed when MDCK cells were treated with 200 μg/mL of 4-Cl-o-PD, indicative of DNA damage (Fig. 3).
Effect of 4-Cl-o-PD in MDCK cells was stained by Hoechst 33258. MDCK cells treated with 200 μg/mL of 4-Cl-o-PD for 24 h and stained with Hoechst 33258 and observed under fluorescence microscope (×400). Arrows in B, C, and D indicate DNA fragmentation, chromatin condensation and cell shrinkage, and DNA fragmentation, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Cell Cycle Analysis by Flow Cytometry
The biological effects of 4-Cl-o-PD on cell cycle progression of MDCK cells were investigated by PI staining coupled with flow cytometry analysis. A dose-dependent increase of cells at the sub-G1 population (from 3 to 56%) suggested that 4-Cl-o-PD induced apoptosis in a dose-dependent manner (Fig. 4). Concurrently, the S phase increased from 5 to 14%, whereas the G2/M phase decreased from 29 to 6% with increasing concentration of 4-Cl-o-PD added to the MDCK cells. Cell cycle arrest at S phase is suggested. Although a large proportion of cells undergo apoptosis, there is a small proportion of cells that could be activated at the sub-G1 phase, and these cells could progress up to S phase but could not complete their mitosis.
Flow cytometry detection of cell cycle of 4-Cl-o-PD-treated cells. MDCK cells treated with various concentrations of 4-Cl-o-PD for 24 h. After staining with propidium iodide, DNA content was analyzed by flow cytometry. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Annexin V Analysis by Flow Cytometry
Using fluorescent probe Annexin V-FITC and PI, apoptotic and necrotic cells were determined by flow cytometry analysis. The scatter plot (Fig. 5) of Annexin V-FITC to PI showed that at low dose of 4-Cl-o-PD, viable cells (AV−/PI−) shifted to upper right quadrant (AV+/PI+, apoptosis), whereas at high dose, cells shifted to upper-left quadrant (AV−/PI+, necrosis). The result shows that both apoptosis and necrosis increased with increasing 4-Cl-o-PD doses.
Flow cytometry analysis of cells death induced with 4-Cl-o-PD. Cells were treated with indicated concentration of 4-Cl-o-PD for 24 h and then stained with Annexin V-FITC and PI. A total of 20,000 cells were analyzed by flow cytometry.
Investigation of Caspase-Dependent Apoptosis Pathway
To further elucidate the mechanism of the cytotoxicity of 4-Cl-o-PD, we investigated whether 4-Cl-o-PD induced apoptosis was through the activation of caspase cascades. Indeed, when MDCK cells were treated with 4-Cl-o-PD, significant increase of caspase-8, −9, and −3/7 activities was detected [Fig. 8(A–C)]. When cells were pretreated with caspase-8 and −9 caspase inhibitors, the cytotoxic effects of 4-Cl-o-PD were greatly abrogated (Fig. 1).
Oxidative Stress Assays
As many cytotoxic and genotoxic agents induce apoptosis through induction of intracellular ROS production, we investigated whether oxidative stress was involved in 4-Cl-o-PD-induced MDCK cells apoptosis. The intracellular ROS levels and GSH were in MDCK cells following 4-Cl-o-PD treatment for 3 h. Intracellular GSH is an important defense system against ROS damage, and its depletion has been shown to be required for apoptosis (Han and Park, 2004). The results demonstrated that cells treated with 4-Cl-o-PD induced intracellular ROS and GSH depletion in a dose-dependent manner (Figs. 6 and 7). When cells were cotreated with vitamin E as an antioxidant, the cytotoxic effects of 4-Cl-o-PD were only partially abrogated (Fig. 1).
4-Cl-o-PD affected ROS levels of MDCK cells. The MDCK cells were treated with different concentrations of 4-Cl-o-PD for 3 h, and the ROS levels were detected after staining with DCFH-DA. p < 0.05 compared with control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
DISCUSSION
We found that incubation of MDCK cells for 24 h with 4-Cl-o-PD carcinogen induced apoptosis in the cells as evidenced by results of morphological study (Fig. 1). Clearly, 4-Cl-o-PD is cytotoxic to MDCK cells with an IC50 value of 135 μg/mL (947 μM), which was determined for a 24 h incubation period (Fig. 2). This was confirmed by flow cytometry, which showed that the percent apoptotic cells increased with increasing concentration of 4-Cl-o-PD from 50 to 300 μM (Fig. 4).
Separate experiments show that the viability of MDCK cells decreased while the ROS levels of MDCK cells increased with increase in concentration of 4-Cl-o-PD for the same incubation time (Fig. 6). However, this same compound was reported to be not cytotoxic to keratinocytes up to 1000 μM in the absence of light (Mosley-Foreman et al., 2008). Even in the presence of light, it was found to be cytotoxic to this epidermal cell type only at high concentration (1000 μM). However, the authors reported that they failed to identify the relevant species responsible for the phototoxicity. As the ROS detected in MDCK cells increased with increasing concentration of 4-Cl-o-PD, it is reasonable to assume the involvement of ROS in apoptosis in cells treated with 4-Cl-o-PD. Such involvement of ROS generated by similar ROS-producing compounds in apoptosis has been reported (Sakurai et al., 2001; Calviello et al., 2006; McLean et al., 2008; Thayyullathil et al., 2008). Investigation into the mechanism of apoptosis has shown the connection between cell death and increasing oxidative stress due to ROS production (Chandra et al., 2000; Kannan and Jain, 2008; Han et al., 2004; Thayyullathil et al., 2008). For example, it was shown that curcumin-mediated rapid generation of ROS could lead to apoptosis by modulating different apoptotic pathways in mouse fibroblast L929 cells (Thayyullathil et al., 2008). In one of the apoptotic pathways, ROS generated by curcumin caused release of apoptosis-inducing factor (AIF) from the mitochondria and the activation of the caspase-independent apoptotic pathway. AIF can induce chromatin condensation and large-scale DNA fragmentation when it reaches the nucleus (Susin et al., 1999).
Many studies have demonstrated that carcinogens could induce apoptosis via different pathways. For instance, the lung carcinogen chromium (VI)-induced apoptosis resulted from cell cycle arrest and activation of p38 mitogen-activated protein kinase (Wakeman et al., 2005). This metal also causes depolarization of mitochondria, activation of caspase-3, −8, and −9 (Muranaka et al., 2004), and generation of ROS in lymphocytes (Vasant et al., 2001). Benzene induced oxidative stress and cell cycle alterations, which was followed by programmed cell death in lymphocytes (Martínez-Velázquez et al., 2006).
As mentioned in the preceding paragraph, there may be a correlation between increase in apoptotic death and amount of ROS generated. Increasing depletion of antioxidant GSH in MDCK cells incubated with increasing concentration of 4-Cl-o-PD suggests that the cells attempted to neutralize the damaging effect of ROS (Fig. 7). However, addition of antioxidant vitamin E did not appreciably reduce cell death by 4-Cl-o-PD (Fig. 2). Likewise, vitamin E and beta-carotene, unlike vitamin C, were not able to inhibit chromium-induced cytotoxicity, ROS generation, and apoptosis in murine macrophages (Vandana et al., 2006). Here, it was found that the lipophilic vitamin E was not a good inhibitor because the chromium (VII)-induced free radicals were hydrophilic. Similarly, apoptosis in synoviocytes induced by water-soluble superoxide anions could not be prevented by vitamin E (Galleron et al., 2010). Thus, the ineffectiveness of vitamin E in inhibiting the cytotoxicity of 4-Cl-o-PD may be because of aqueous soluble free radicals generated, similar to those produced by chromium (VII) mentioned above. Nevertheless, apoptosis in rat pancreatic β-cell line (INS-1) caused by mitochondrial damages as a result of ROS generated from alloxan could be prevented by vitamin E, antioxidant butylated hydroxyanisol, and catalase (Sakurai et al., 2001). Apoptosis in LLC-PK1 cells induced by cyclosporin A, which was caused by mitochondrial damage, could also be prevented by vitamin E (Arriba et al., 2009).
4-Cl-o-PD affected antioxidant GSH depletion of MDCK cells. The MDCK cells were treated with different concentrations of 4-Cl-o-PD for 3 h. The GSH depletion was detected by assay kit. p < 0.05 compared with control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Besides generating ROS and inducing apoptosis, 4-Cl-o-PD caused DNA damage (Fig. 3). In response to DNA damage, cyclin-dependent kinase inhibitors can arrest cell cycle progression. Thus, flow cytometry was used to investigate cell cycle arrest in MDCK cells incubated with increasing concentration of 4-Cl-o-PD (Fig. 4). The sub-G1 phase increases in a dose-dependent manner from 15 to 56%. This correlates with the increase in apoptotic cells with increase in concentration of 4-Cl-o-PD (Fig. 5; Yang et al., 2000; Ansah et al., 2009). The S population shows significant increase (from 5% to between 10 and 17%). Other compounds, such as guanosine-5′-triphosphate and toxic arsenic trioxide, could also induce cell death by cell cycle arrest at S phase (Moosavi et al., 2006; Yong et al., 2007).
Overall, it seems that 4-Cl-o-PD induced apoptosis as a major pathway of cell death with an unknown cause for cell death by cell cycle arrest as a minor pathway. In the viability assay by using trypan blue exclusion method, cell death by 4-Cl-o-PD could be substantially abrogated by adding caspase-8 and caspase-9 inhibitors. In fact, when MDCK cells were treated with increasing concentration of 4-Cl-o-PD, the levels of caspase-8, −9, and −3/7 activities were found to increase (Fig. 8). Clearly, 4-Cl-o-PD induces apoptosis by activating both the intrinsic (caspase-3 and −9) and extrinsic (caspase-8) pathways.
4-Cl-o-PD affected caspase-8, −9, and −3/7 activities of MDCK cells. The MDCK cells were treated with difference concentrations of 4-Cl-o-PD for 24 h. The caspase-8, −9, and −3/7 activities were detected by assay kits. p < 0.05 compared with control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
In conclusion, the present data suggest that the cytotoxicity of 4-Cl-o-PD in MDCK cells involved: (1) induction of intracellular ROS; (2) depletion of intracellular GSH; (3) activation of both intrinsic and extrinsic apoptotic pathways followed by apoptosis; and (4) a minor cell death pathway by cell cycle arrest at S phase. This report suggests that risk assessment should monitor environmental pollution by 4-Cl-o-PD waste water and people (workers and consumers) directly exposed to this chemical. Absorption of this cytotoxic compound into the bloodstream through the skin and its ingestion pose potential risk.
