The effects of para-phenylenediamine (PPD) on the skin fibroblast cells

Keywords: Para-phenylenediamine; fibroblast; reactive oxygen species; mitochondria; lysosome

The use of hair dye has roots in the past times and in Egypt. Furthermore, the hair dye has been used to enhance the attractiveness, health, femininity and beauty (Al-Shaikh et al., [2]). One of the most important components of the hair dye is para-phenylenediamine (PPD), which is a derivative of para-nitroaniline (Abd-ElZaher et al., [1]; Al-Shaikh et al., [2]; Blömeke et al., [5]; Chye et al., [11]; Hooff et al., [24]; Singla et al., [36]). Research has shown that about 30% of American women use PPD containing hair dyes (Abd-ElZaher et al., [1]). Documents show that the toxicological properties of aromatic amines such as PPD are very widespread. One of these important properties is the induction of apoptosis through increased reactive oxygen species (ROS) (Chye et al., [11]; Nohynek et al., [31]). The most important route of exposing humans to PPD is through the skin and inhalation during the dyeing process (Abd-ElZaher et al., [1]). PPD can cause skin irritation, contact dermatitis, rhabdomyolysis, renal impairment and severe edema. These complications indicate that this compound is very toxic (Al-Shaikh et al., [2]; Singla et al., [36]).

Previous studies have demonstrated that PPD induced apoptosis in the Mardin-Darby canine kidney (MDCK) cells and Chang liver cells through the mitochondrial pathway (Chen et al., [9]; Chye et al., [11]). Furthermore, reports indicate that the PPD induced apoptosis signaling through the increase of ROS level (Chen et al., [10]; Chye et al., [11]). Despite its widespread use, it has the ability to induce oxidative stress in different cells (Brans et al., [6]; Chen et al., [10]; PICAKDO et al., [32]; Rioux & Castonguay [35]). Observations show that changes in mitochondrial function lead to the generation of harmful ROS (Demers-Lamarche et al., [13]). Additionally, mitochondria are considered as an important source of ROS generation (often through complexes I and III) and start induction of apoptosis signaling (Dunn et al., [14]; Murphy [30]; Zorov et al., [44]). Also, it is proven that impairment of mitochondrial function leads to disruption of the lysosomal function and structure (Demers-Lamarche et al., [13]). Both mitochondria and lysosomes are sensitive to changes in the level of ROS, and these two intracellular organelles play a role in inducing apoptosis signaling (Ghosh et al., [21]; Zhang et al., [39]; Zhao et al., [42]).

In this study, we studied the mechanism of PPD toxicity on mitochondria and lysosomes of fibroblastic cells.

All chemicals (such as, PPD, dichloro-dihydro-fluorescein diacetate (DCFH-DA), rhodamine 123 (Rh 123) and Acridine Orange) were purchased from Sigma-Aldrich (St. Louis, MO) in the best pharmaceutical grade.

Male Wistar rats (200–250 g) were purchased from Pasteur Institute and placed in an air-conditioned room with the controlled temperature of 25 ± 2 °C and maintained on a 12:12 h light/dark cycle, humidity of 50–60% with free access to water and food. All experimental methods were conducted according to the ethical standards and protocols approved by the Animal Experimentation Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran.

At first, all the necessary equipment were sterilized via autoclaving (under conditions 121 °C and high pressure). Briefly, the small pieces (using sterile tools) of rat skin was removed and then moved to petri dishes including phosphate buffered saline (PBS) on ice. In the next step, animal skin pieces were fragmented into smaller pieces, and then transferred to Falcon tube together with the PBS. Samples were centrifuged twice (first, 168×g for 2 min and second, 203×g for 2 min). In the first step, the PBS was removed and the trypsin 0.1% was added, and in the second step, the added trypsin was removed. The trypsin was neutralized using culture medium including FBS. After centrifugation (203×g for 2 min), the medium containing trypsin was removed from the tubes and once again added to the cells and the cells were transferred to culture medium (Chashmpoosh et al., [8]).

Cell viability was assayed by MTT assay. Fibroblast cell was placed in 96-well plates. The cells were treated with 5–200 µM concentration of PPD for 24 h (cells were maintained in DMEM, supplemented with 10% FBS and antibiotics; 50 U/ml of penicillin and 50 µg/ml streptomycin). In the next step, after incubation for a specified time at 37 °C in a humidified incubator, MTT (5 mg/ml in DMEM) was added to each well and incubated for four hour after which the plate was centrifuged at 1800×g for 5 min at 4 °C. The buffer solution (containing PPD) was then removed from the wells by aspiration. After careful removal of the medium, 100 µl of buffered dimethyl sulfoxide (DMSO) (to dissolve the resulting formazan crystals) was added to each well, and plates were shaken. The absorbance was measured at 570 nm using ELISA reader (Tecan, Rainbow Thermo, Salzburg, Austria).

Fibroblast cells (1 × 106 cells) were treated with PPD (50, 100 and 150 µM) for 6, 12 and 18 hours. After treatment with PPD (50, 100 and 150 µM), fibroblast cells were washed with PBS and medium containing PPD (50, 100 and 150 µM) was removed. In the next step, cells were incubated with DCFH-DA at the concentration of 1.6 µM for 30 minutes. DCFH-DA penetrates fibroblast and becomes hydrolyzed to non-fluorescent dichlorofluorescein (DCFH). The latter then reacts with ROS to form the highly fluorescent dichlorofluorescein (DCF), which effluxes the cell. In the following, the fluorescence intensity of DCF at 6, 12 and 18 hours after the exposure was assayed using a Shimadzu RF5000U fluorescence spectrophotometer (Kyoto, Japan) (EXλ = 500 nm and EMλ = 520 nm) (Arast et al., [4]; Eskandari et al., [16]).

In this study, Rh 123 as a fluorescent dye was used for the evaluation of MMP via PPD (50, 100 and 150 µM) in the fibroblast cells (106 cells). Finally, the fluorescence intensity of Rh 123 at 6, 12 and 18 hours after the exposure was assayed using a Shimadzu RF5000U fluorescence spectrophotometer (Kyoto, Japan) (EXλ = 490 nm and EMλ = 520 nm). The capacity of mitochondria to take up Rh 123 was measured as the difference between treated and control fibroblast cells in Rh 123 fluorescence (at concentration of 1.5 µM). In fact, an increase in fluorescence intensity due to redistribution of Rh 123 represents a collapse in the MMP (Andersson et al., [3]; Fakharnia et al., [19]; Kwong & Molkentin [29]; Vaseva et al., [37]).

In this study, fibroblast lipid peroxidation (LPO) level at time interval 6, 12 and 18 hours was assayed via measuring the level of thiobarbituric acid-reactive substances (TBARS) formed during the decomposition of lipid hydroperoxides using a Beckman DU-7 spectrophotometer (Brea, CA) (at an absorbance of 532 nm) after treating 1.0 ml aliquots of fibroblast suspension (106 cells/ml) with trichloroacetic acid (70%, w/v) and oiling the suspension with thiobarbituric acid (0.8%, w/v). Malondialdehyde (MDA) content was expressed as µg/mg protein (Gasparovic et al., [20]; Zhang et al., [40]).

Briefly, the cytochrome c release was evaluated through the Quantikine Rat/Mouse cytochrome c Immunoassay kit provided by R&D Systems, Inc. (Minneapolis, MN). Cytochrome c specific antibodies have been pre-coated onto 96-well plates. Then, 50 μL of control and test samples are added to the wells along with 75 μL of biotinylated cytochrome c detection antibody and the microplate is then incubated at room temperature. Finally, the optical density of each well was determined through the aforementioned micro-plate spectrophotometer set to 450 nm (Faizi et al., [18]; Kamada et al., [26]).

The level of damage to lysosomal membrane in fibroblast cells exposed to PPD (50, 100 and 150 µM_B_)_b_ was evaluated through the redistribution of the acridine orange at concentration of 5 µM (as a fluorescent dye). Six, 12 and 18 after exposure, aliquots of the cell suspension (0.5 ml) stained with acridine orange (5 µM) were separated using centrifugation at 168×g for 1 min. The cell pellet was then re-suspended in 2 ml of fresh incubation medium. This washing process was carried out twice to remove the fluorescent dye from the media. Finally, acridine orange redistribution in the fibroblast cell suspension was assayed using a Shimadzu RF5000U fluorescence spectrophotometer (EXλ = 495 nm and EMλ = 530 nm) (Andersson et al., [3]; Brunk et al., [7]).

LDH release is used to evaluate the toxicity and survival of the cell. LDH activity was measured by specific commercial kit from Sigma-Aldrich Co. (St. Louis, MO). LDH released during the experiment was expressed as a percentage of total cellular LDH (Everse et al., [17]; Ho et al., [23]; Wallace et al., [38]).

Data are reported as mean ± SD. All statistical analyses were performed using GraphPad Prism, version 6 (La Jolla, CA). The assays were performed three times. Statistical significance was determined using the one-way ANOVA test, followed by the post hoc Tukey test, and the two-way ANOVA test, followed by the post hoc Bonferroni test. Statistical significance was set at p < 0.05. Furthermore, the one-way ANOVA test was used as a specific statistical analysis for the determinations of cell viability, LDH release and cytochrome c release. The two-way ANOVA test was used for the determinations of mitochondrial ROS level, collapse of MMP, LPO level and lysosomal membrane damage.

The effect of PPD at concentrations 0–200 µM on the viability of fibroblast cells isolated from the skin was examined for 24 h. As shown in Figure 1, PPD at all concentrations except 5 µM was able to significantly (p < 0.05) decrease cell viability in a dose-dependent pattern. The results of the cytotoxicity showed that the IC50 concentration of this compound is 100 µM. Subsequently, we used concentrations of 50 (1/2 IC50), 100 (IC50) and 150 (1/5 IC50) for subsequent experiments.

PHOTO (COLOR): Figure 1. Effect of PPD (0–200 μM) on fibroblast cells viability for 24 hr. All results were showed as the mean ± S.D (n=3). **p < 0.01 and ***p < 0.001: significantly different from the vehicle-only group (PPD concentration = "0").

In Figure 2, our results of showed that PPD at all applied concentrations (50, 100 and 150 µM) and at all times (6, 12 and 18 hours) has been able to significantly increase the level of ROS in the fibroblast cells. DCFH-DA penetrates fibroblast and becomes hydrolyzed to non-fluorescent dichlorofluorescein (DCFH). The latter then reacts with ROS to form the highly fluorescent dichlorofluorescein (DCF), which effluxes the cell. Finally, the measurement of fluorescence intensity of DCF indicates the level of ROS. Also, cyclosporine A (2 µM) decreases the generation of ROS from PPD (100 µM) (Figure 2).

PHOTO (COLOR): Figure 2. Effect of PPD (50, 100 and 150 µM) on fibroblast cells ROS generation for 6, 12 and 18 h. All results are shown as the mean ± SD (n = 3). *p < 0.05, ***p < 0.001 and ****p < 0.0001: significantly different from the vehicle-only group (PPD concentration="0"). ####p < 0.0001: significantly different between 100 µM and Cs.A (2 µM) + 100 µM.

The results shown in Figure 3 indicate that PPD in concentrations of 50, 100 and 150 µM significantly (p < 0.05) decreased the MMP in a time- and concentration-dependent manner in the rat fibroblast compared with control group. MMP collapse and fluorescence intensity (Rh 123) reported in our experiments are inversely proportional. Our results showed that cyclosporine A (2 µM) decreases the collapse of MMP from PPD (100 µM) (Figure 3).

PHOTO (COLOR): Figure 3. Effect of PPD (50, 100 and 150 µM) on fibroblast cells MMP collapse for 6, 12 and 18 h. All results are shown as the mean ± SD (n = 3). ****p < 0.0001: significantly different from the vehicle-only group (PPD concentration="0"). ####p < 0.0001: significantly different between 100 µM and Cs.A (2 µM) + 100 µM.

The effect of PPD at all applied concentrations (50, 100 and 150 µM) on the LPO content of fibroblast cells was examined for 6, 12 and 18 hours. As shown in Figure 4, PPD significantly (p < 0.05) increases the MDA content as an indicator of LPO (in a dose- and time-dependent manner) in fibroblast cells. Also, cyclosporine A at concentration of 2 µM decreases the content of LPO caused by PPD (100 µM) (Figure 4).

PHOTO (COLOR): Figure 4. Effect of PPD (50, 100 and 150 µM) on fibroblast cells lipid peroxidation for 6, 12 and 18 h. All results are shown as the mean ± SD (n = 3). *p < 0.05 and ****p < 0.0001: significantly different from the vehicle-only group (PPD concentration = "0"). ###p < 0.001 and ####p < 0.0001: significantly different between 100 µM and Cs.A (2 µM) + 100 µM. ns: not significantly different from the vehicle-only group (PPD concentration = "0").

As shown in Figure 5, PPD at the concentrations of 50, 100 and 150 µM raised cytochrome c release from rat fibroblast cells mitochondria. Furthermore, our results showed that Butylated hydroxytoluene (BHT; as an antioxidant) and cyclosporine A (as a pore sealing agent) inhibit the release of cytochrome c from PPD (100 µM) (Figure 5).

PHOTO (COLOR): Figure 5. Effect of PPD (50, 100 and 150 µM) on fibroblast cells cytochrome c release using Rat/Mouse cytochrome c ELISA kit. All results are shown as the mean ± SD (n = 3). *p < 0.05 and ***p < 0.001: significantly different from the vehicle-only group (PPD concentration = "0"). ###p < 0.001 significant difference in comparison with 100 µM PPD.

When fibroblast lysosomes were loaded with acridine orange (a lysosomotropic agent), a significant release of acridine orange ensued into the cytosolic fraction within 6, 12 and 18 hours of incubation with PPD (50, 100 and 150 µM) showing damage to the lysosomal membrane (Figure 6). The redistribution of acridine orange from lysosomes to cytosol indicates damage to lysosomes.

PHOTO (COLOR): Figure 6. Effect of PPD (50, 100 and 150 µM) on lysosomal membrane damage for 6, 12 and 18 h. All results are shown as the mean ± SD (n = 3). ***p < 0.001 and ****p < 0.0001: significantly different from the vehicle-only group (PPD concentration = "0").

The effect of PPD (0–200 µM) on the LDH release was examined for 24 h. LDH released into the medium was used as measures of cell death. As shown in Figure 7, PPD at all concentrations was able to significantly (p < 0.05) increase the release of LDH compared to the control group.

PHOTO (COLOR): Figure 7. Effect of PPD (0–200 µM) on LDH release for 24 h. All results are shown as the mean ± SD (n = 3). *p < 0.05 and ***p < 0.001: significantly different from the vehicle-only group (PPD concentration = "0").

In recent years, the use of PPD has increased. Therefore, assessing the effects and consequences of using it on human health is very necessary (Goyal et al., [22]). In this study, we examined PPD toxicity parameters, including oxidative stress, mitochondrial and lysosomal damage, and apoptosis in the fibroblast cells. Cytotoxicity in fibroblast cells was measured by MTT assay which shows a significant reduction in fibroblast cells viability treated with PPD (50, 100 and 150 µM) compared to control cells.

Furthermore, DCFH-DA assay displays the level of intracellular ROS in fibroblast cells. Our results showed that PPD in a dose- and time-dependent manner increased the generation of ROS in the fibroblastic cell. Our results are in agreement with the previous study that showed PPD induced ROS generation in a dose- and time-dependent manner in Mardin-Darby canine kidney cells and Murine myeloma cells (Chen et al., [10]; Elyoussoufi et al., [15]). It is well known that ROS are considered to cause damage to cells through different mechanisms. Furthermore, oxidants including ROS (As the initial signals) and lipid hydroperoxides play a role in the induction of apoptosis through the oxidative stress process (Elyoussoufi et al., [15]).

In our study, LPO was found to be raised through PPD. Our result is in agreement with the previous study (Elyoussoufi et al., [15]). The documentation shows that ROS increase the content of LPO. Furthermore, it has been shown that PPD raised LPO to support the significance that ROS are involved in PPD induced apoptosis signaling (Elyoussoufi et al., [15]). It has been shown that the mitochondrial pathway leads to the ROS attack of phospholipids on the membrane and decline in the MMP. Eventually, it leads to the activation of apoptotic factors and apoptosis signaling (Goyal et al., [22]; Zhou et al., [43]). Using an Rh 123 reagent, we showed that the PPD in a concentration- and time-dependent manner leads to a decline in the MMP of the fibroblast cells. This result is in agreement with previous studies (Goyal et al., [22]).

In our study, we found the release of cytochrome c into the cytosol in fibroblast cells treated to PPD. The level of cytochrome c was increased in fibroblast cells after the treatment with PPD. These results suggest that PPD (100 µM) affects mitochondrial permeability transition (MPT) pores through oxidative stress leading to raised cytochrome c release. The MPT is defined as the abrupt rise in the permeability of the inner mitochondrial membrane (IMM) to solutes material (1500 Da), mitochondrial swelling, and the collapse of MMP (Juhaszova et al., [25]). Furthermore, the opening of the MPT pores initiates the start of the MPT that is an element important in apoptosis signaling (Kim et al., [27]). Studies have shown that cytochrome c plays a vital role in beginning the process of apoptosis. The release of this protein from mitochondria to cytosol activates caspase cascade. Caspase 3 is one of the most important caspases that can induce apoptosis. Thus, cytochrome c release can induce apoptosis (Faizi et al., [18]; Ko et al., [28]).

In addition, our results showed that the PPD caused instability and damage to the lysosomal membrane in fibroblast cells. Studies have shown that free radicals (especially H2O2) have the potential to cross the lysosomal lipid membrane and can produce radical hydroxyl (OH•); according to the Haber–Weiss reaction. Furthermore, lysosomal is involved in the apoptosis process (Pourahmad et al., [33], [34]). In addition, reports show that lysosomes play an important role in the induction of apoptosis with the release of protease enzyme, such as cathepsins. The lysosomal pathway and mitochondrial pathway are connected together through Bcl2 family proteins (Cirman et al., [12]; Goyal et al., [22]; Zhang et al., [41]).

In conclusion, all the results of this study indicate that both mitochondria and lysosomes are targeted by PPD. Furthermore, PPD with changes in mitochondria and instability in lysosomes can induce apoptosis signaling.

The results presented in this paper were partly extracted from thesis of Dr. Mohsen Fatahi, Pharm.D. graduate of School of Pharmacy, Shahid Beheshti University of Medical Sciences, who performed his thesis under supervision of Professor Jalal Pourahmad. The investigation was carried out in Professor J. Pourahmad's laboratory in the School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

The authors declare that there is no conflict of interest.