Research Article - (2012) Volume 2, Issue 5
1Kebbi State University of Science and Technology, PMB 1144, Aliero
2School of Health and Biosciences, University of East London (UEL), UK.
To assess the target organ toxicity of hydroquinone, liver (BRL3A) and dermal (A375p) derived cell lines were used. The cells viability was assessed by the use of MTT assay. There was significant reduction of cell viabilities of both the cells by hydroquinone standard concentrations. The reduction in cell viability was more pronounced in A375p (dermal) cell line with at least 50% reduction at all concentrations tested. While in BRL3A (liver) cell line, the reduction in cell viability by hydroquinone standard was slightly lower than in A375p cell line. This is a suggestive of hydroquinone specific toxicity to skin cells and to melanocytes, where it exerts its main function on tyrosinase and melanin synthesis. In addition, the possible involvement of reactive oxygen species (ROS) generation in the toxicity process was evaluated using the NBT reduction assay. This is based on the ability of treated cells to stimulate NBT reduction due to the generation of reactive oxygen species. There was significant NBT reduction on treated liver (BRL3A) cells, however the dermal (A375p) cells showed no significant levels of NBT reduction even with positive control suggesting adaptive response in those cells.
hydroquinone, NBT, MTT assay, cytotoxicity, cell viability.
Skin lightening creams and solutions are widely used in Africa and Asia to cure skin pigmentation disorders such as melasma, freckles, pregnancy marks as well as skin lightening for cosmetic purposes. This practice is widely seen among dark – skinned people in their quest for beautiful, attractive and good looking skin, especially the female for better appearance and acceptance [1]. Some common potent skin lightening agents, such as hydroquinone, mercury and corticosteroids are utilized in the manufacture of these creams [2].
Despite the established effectiveness of these agents, a number of dermatologic and systemic side effects have been associated with the use of these agents in skin lightening products. Some common complications such as exogenous ochronosis, impaired wound healing and deshiscence, fish odour syndrome, nephropathy, steroid addiction syndrome, predisposition to infections, cutaneous and endocrinology complications, irritation, acne, oedema, stretch marks, etc. were reported to be associated with the use of skin lightening creams containing hydroquinone, mercury and corticosteroids [3].
For example, dermatitis, exogenous ochronosis, cataract, pigmented colloid milia, sclera and nail pigmentation, patchy depigmentation, loss of skin elasticity, impaired wound healing and fish odour syndrome were reported due to chronic and prolonged use of hydroquinone – containing formulations [2], [3]. The use of hydroquinone in skin lightening practice was reported to be associated with the development of cutaneous and internal malignancies [3]. In addition, [4] reported the long term carcinogenicity of hydroquinone in test animals.
Several studies done in this area have to do with the complications and inherent health hazards associated with the skin lightening practice particularly in Africa and among Africans living in Europe [1], [2], [3], [4].
However, not much research has been carried out to shade more light on the nature of the potential cytotoxicities of individual skin bleaching agents such as hydroquinone on established human cell lines. This research used cell viability assays to assess the in vitro cytotoxic effect of hydroquinone on established liver and skin cells. The finding are intended to be of importance on both the sellers and the users in order to make an informed and sound decision regarding the use or other wise of hydroquinone containing skin lightening creams.
Chemicals and Reagents
Dulbecco’s modified eagle’s medium/nutrient mixture Ham’s 12 (DME/F-12) and foetal bovine serum (FBS) were purchased from Biosera, UK. Phosphate buffered saline (PBS), penicillin, streptomycin, amphotericin B, trypsin, EDTA (ethylene diamine tetra- acetic acid), trypan blue, DMSO (dimethyl sulfoxyde), KOH (potassium hydroxide), hydroquinone (HQ) and MTT were purchased from Sigma-Aldrich Company Ltd., UK. Ethanol and isopropanol were purchased from BDH, Poole, England. Bleach was purchased from Selden Research Ltd., Buxton, UK. NBT (nitroblue tetrazolium salts) was purchased from Melford Laboratories Ltd., Ipswich, UK. All the chemicals were of analytical grade.
Materials
Tissue culture plates and plastics were purchased from NUNC, Roskilde, Denmark. Inverted face contrast microscopes were purchased from HUND, Wetzlar, Germany. Hemocytometres were purchased from Neubauer Brightline, Germany. Multiscan plate reader was from Thermo Electron Corporation, Vantaa, Finland. Class II biological safety cabinet was from NUAIRE.
Cell Lines
Two cell lines were used in this research; the BRL 3A and A375P cell lines, obtained from the School of Bioscience, University of East London, Stratford Campus, Water Lane, London, United Kingdom. BRL 3A is a liver derived cell while A375P is a dermal derived cell line.
Cell Culture
The cells were grown and maintained in a phenol red free medium DMEM/F-12 (Dulbecco’s modified essential medium/nutrient mixture Ham’s 12 containing 10% foetal bovine serum (FBS) supplemented with glutamine (2mmol/l) Hepes (15mmol/l), penicillin, streptomycin and amphotericin B. All cultures were kept and maintained at 37°C in a humidified 5% carbon dioxide (CO2) incubator. On reaching confluence, the cells were removed and diluted again and again throughout the experiment duration to maintain continuous cultures.
The cell number per millilitre (ml) was then counted from the number of cells counted. The formula used was: cells counted × 104 × 2 = number of cell per millilitre (cell / ml). The cell number of 5 × 104 cells / ml (5 × 103 cells/well) was used in the initial experiments especially with the A375p (dermal) cells which were growing very slowly. But the need to increase the number of cells / ml resulted in incubating the cells for longer period of time and there was corresponding increase in cells density and cell number. 1 × 105 cells / ml (1 × 104 cells/well) for both the two cell lines were later used.
MTT Cytotoxic assay
MTT cytotoxic assay is an exceptionally fast colorimetric test that was described by [5] to measure cell growth and survival. The assay is based on the ability of active and viable cells to transform a soluble tetrazolium salt, 3-4,5- dimethyl-2-thiazolyl-2,5-diphenyl tetrazolium bromide (MTT), into an insoluble purple formazan precipitate.
The test procedure was performed as adopted by [6], [7], [8] with slight modification. The cells (BRL 3A and A375p) were cultured in 96-well plates at a density of 1 × 105 cell/ml (that is 1 × 104 cells / well containing 100μl) and incubated for 24 hours. The cells were then treated with test samples at different concentration for different treatment times. The negative control was not treated (i.e. containing only medium) while the positive control was treated with hydrogen peroxide, H2O2 (8.5mM). After appropriate incubation (24 or 48 hours), 10μl of 5mg/ml MTT dissolved in PBS was added to each well and incubated for 1 hour to allow for MTT metabolism. The plates were centrifuged at 2,000rpm for 5 minutes. The medium was aspirated, washed with PBS and 100μl of isopropanol added to each well. The plates were then incubated for 30 minutes to dissolve formazan crystals. Absorbance was then read with the multiskan plate reader at 570nm.
NBT (Nitro blue tetrazolium) Assay
The ROS generation as the possible mechanisms of cells death was evaluated using NBT reduction, a yellow watersoluble powder that becomes blue and insoluble upon reduction. The test is based on the ability of reactive oxygen species (such as superoxide anion, O2-) generated in the cytotoxic process within the cells to reduced the water soluble NBT dye to blue insoluble formazan [9]. The higher the ROS produced, the higher the optical density generated at 630nm.
The NBT reduction assay was described by [10]. The assay procedure was performed as adopted by [11], [12] with slight modification. 10μl of 0.05g / 5ml NBT solution was pipetted to each well of the 24 hours incubated cells (1×105 cells/ml) in 100μl DMEM/F12 medium. Test samples were then added onto the appropriate wells and incubated for approximately 1 hour after which the medium was carefully aspirated. 60μl of 2M potassium hydroxide (KOH) followed by 70μl of DMSO were added to each well and then read in a multiskan plate reader at 630nm.
The experiments were performed in duplicates. The results were expressed as the NBT reduction stimulation index (SI) which gives the levels of NBT reduction stimulation of different hydroquinone concentrations. Stimulation Index (SI) was calculated as the ratio of optical density (OD) of the treated and control cells. The SI for the control cells was taken as 1.
Data Presentation and Analysis
Data will be presented as mean ± S.E.M from at least two independent experiments, performed in duplicate. Data are expressed as means ± standard error (SE) and were analysed by one-way ANOVA followed by Duncan’s post hoc test using graph pad prism 5 software. Differences were considered significant at p≤0.05.
Cell Viability for Hydroquinone
The cells were treated with increasing concentration of hydroquinone standard (0 – 10mM) for 24 hours and the cell viability was assessed using the MTT cytotoxic assay. The choice of this concentration range was as a result of the absence of reduction in cell viability observed from the previous concentrations (500 - 5000μM) of hydroquinone standard used. The concentrations were therefore adjusted to 0 – 10mM. The cells were treated with 0mM (control), 5mM, 7mM and 10mM hydroquinone standard and incubated for 24 hours at 37°C in 5% humidified incubator. Hydrogen peroxide, H2O2 (8.5mM) was used as positive control.
The results obtained from two to three experiments run in duplicates were expressed as % viability calculated from the ratio of treated to that of control. % viability of the control was taken as 100%. The values plotted are mean±SEM. The cell viability (%) for hydroquinone standard is presented in figure 4.1; figure 4.1A represents the BRL 3A cell viability while ‘B’ represents that of A375p cell line. Treatment of the cells with hydroquinone standard resulted in a statistically significant (p<0.05) reduction in cell viability of the two cells. This effect is more pronounced on A375p cell line with up to 50% reduction in viability. However, the reduction in viability does not seem to be dose – dependent, in fact, it even increases with increasing dose of hydroquinone standard. This is in contrast to the visual inspection made under the inverted microscope in which it was found that the number and densities of viable cells decreases with increasing dose of hydroquinone standard (not shown).
Figure 4.1: effect of hydroquinone standard concentration (0 – 10mM) on the viability (%) of BRL 3A (figure 4.1A) and A375p (figure 4.1B). Values are means±SEM of values from two or three experiments. The control value (no addition of hydroquinone) was set at 100%. (*) indicates statistical significance (p<0.05) using one way analysis of variance (ANOVA).
Effect of Treatment Duration on Viability (%)
The effects of hydroquinone standard treatment duration on cell viability (%) of the two cell lines were compared. The cells were treated with 0mM (control), 5mM, 7mM and 10mM hydroquinone standard and incubated for 24 and 48 hours at 37°C in 5% humidified incubator to evaluate the effect of treatment duration on the viability (%) of the two cell. Hydrogen peroxide, H2O2 (8.5mM) was used as positive control.
The results were expressed as % viability are presented in figure 4.2, figure 4.2A represents the viability (%) of BRL 3A cell line while figure 4.2B represents that for A375p after 24 and 48 hours treatment. There was significant decrease (P<0.05) of cell viability (%) in BRL3A cell line after 48 hours treatment compared to 24 hours treatment. However, there was no significant decrease in viability (%) of A375p cell line.
Figure 4.2: effect of hydroquinone treatment (mM) duration on the viability (%) of BRL 3A (figure 4.2A) and A375p (figure 4.2B). Values are means±SEM of values from two or three experiments. The control value (no addition of hydroquinone) was set at 100%. (*) indicates statistical significance (p<0.05) using student t test.
IC50
The 50% inhibitory concentration, IC50 was deducted from the graphs and was found to be 4.10mM for BRL3A cell line while that of A375p cell line was found to be 4.55mM.
NBT Reduction Assay
To get an idea about the possible mechanism by which the hydroquinone standard resulted in the reduction of the viability of the two cell lines, NBT reduction assay was used. This method is usually used as an indication of possible involvement of reactive oxygen species (ROS) primarily superoxide anion radical, O2- in the process of toxicity and cell’s death.
The results were expressed as NBT reduction stimulation index (SI) which was calculated as the optical density (OD) ratio of the treated and control cells. The stimulation index (SI) for the control was taken to be 1.
The NBT reduction assay for hydroquinone standard is shown on figure 4.3. Treatment of BRL3A cells with hydroquinone standard showed significant (P<0.05) stimulation of NBT reduction at 7mM (SI1.42) and at 10mM (SI1.63) (figure 4.3A). However, there was no significant (P<0.05) stimulation of NBT reduction at 5mM (SI1.16). In addition, H2O2 (positive control) showed significant stimulation of NBT reduction (SI1.71)
On the other hand, treatment of A375p cells (figure 4.3B) with hydroquinone standard showed significant (P<0.05) stimulation of NBT reduction at 10mM (SI2.03) where as there was no significant stimulation of NBT reduction at 5mM (SI1.13) and at 10mM (SI1.06). In addition, H2O2 (positive control) did not give significant stimulation of NBT reduction (SI1.15).
Figure 4.3: effect of hydroquinone standard treatment on the NBT reduction on BRL3A (A) and A375p (B) cells. Values are means±SEM of values from two or three experiments. The control value (no addition of hydroquinone standard) was set at 1. (*) indicates statistical significance (p<0.05) using one way analysis of variance (ANOVA).
Cell viability
As already mentioned, the cell viability was assessed by the use of MTT assay. There was significant reduction of cell viabilities of both the cells by hydroquinone standard concentrations. The reduction in cell viability was more pronounced in A375p (dermal) cell line with at least 50% reduction at all concentrations tested. While in BRL3A (liver) cell line, the reduction in cell viability by hydroquinone standard was slightly lower than in A375p cell line. This is a suggestive of hydroquinone specific toxicity to skin cells and to melanocytes, where it exerts its main function on tyrosinase and melanin synthesis. This is as observed by [13], [3] that hydroquinone is skin and melanocytes – specific in action.
The IC50 was found to be 4.10mM and 4.55mM for BRL3A and A375 cells respectively. These are in contrast with that found by [14] who found an IC50 of 25μM on M1 cells and [15] that found an IC50 of 250μM on MRC-5 cells. This means that the inhibitory concentration of hydroquinone that will inhibit or kill 50% of the cells was 1000 × higher than what was obtained by the above previous researches. This may be due to lower sensitivity of MTT assay compared to other cell viability assays [8], [16], [17], [18].
As observed by the above researchers, MTT might not be very sensitive cytotoxic assay. This is supported by the fact that visual inspection of cells showed great reduction in cells’ densities in wells treated with hydroquinone in a dose related manner compared to control. It was further observed during the course of the experiment that wells treated with hydroquinone always changed to dark brown after incubation with MTT, which may suggest a reaction between hydroquinone and MTT and therefore interference in the assay. If time and materials were available, alternative tests such as neutral red uptake, ATP assay, alamar blue, etc would be employed to assess the viability of the cells treated with hydroquinone to evaluate the sensitivity of the different assays on the cells.
For example; [16] concluded that MTT assay yields a relatively lower result of growth inhibition than the alternative ATP assay in a study to assess the anti-growth effects of chemotherapeutics drugs on a lung cancer cell line (A549). Equally, [8] found out that alamar blue assay is more sensitive than MTT assay in assessing cell viability. In a related study to evaluate the sensitivity of eight different in vitro assays to assess the cytotoxicity of cigarette smoke condensate on Chinese hamster ovary (CHO) cells, [18] concluded that LDH (lactate dehydrogenase) release was the most sensitive detecting cytotoxicity at a very lower concentration of 100μm of the test compound.
NBT Reduction
The possible involvement of reactive oxygen species (ROS) generation in the toxicity process was evaluated using the NBT reduction assay. This is based on the ability of treated cells to stimulate NBT reduction due to the generation of reactive oxygen species.
The NBT reduction assay was adopted by many researchers as a fast, simple and relatively cheap approach in the in vitro detection of reactive oxygen species (ROS), particularly superoxide anion radical generation. However, the drawback is that NBT can also be reduced by other compounds such as diaphorase (without superoxide involvement). Interestingly, the involvement of superoxide can now be verified with the use of superoxide dismutase (SOD), which catalyses the dismutation of superoxide into hydrogen peroxide (H2O2) and thus inhibits the superoxide mediated NBT reduction [19], [20]. The involvement of superoxide has also been verified with the use of NEM which is known to inhibits some cytosolic cofactors of NADPH oxidase, the enzyme generating superoxide anion radical [19].
For BRL3A, there was significant stimulation of NBT reduction treated with hydroquinone standard at the concentration of 7mM and 10mM, whereas no significant stimulation of NBT reduction was seen with 5mM concentration. In addition, positive control (H2O2) has shown significant NBT reduction stimulation on BRL3A cell line. This suggest that the toxicity of hydroquinone is due to the established effects of its oxidation products, quinones and reactive oxygen species (ROS) which caused an oxidative damage to cellular proteins, nucleic acids and membranes [21], [22].
However, the A375p (skin) cell line treated with hydroquinone has shown relatively low levels of NBT reduction stimulation even with the positive control (H2O2). This might be due to adaptive response of these cells to hydroquinone as observed by [14], [15]. They found out that murine myeloblastic leukemia cell line and MRC – 5 cell lines are hydroquinone resistant, insensitive to lower levels of hydroquinone treatments.
Although hydroquinone has been very potent and effective in skin lightening practice, its use in skin lightening creams should be stopped as this is toxic to the cells and ultimately to the body and those who use these creams containing hydroquinone. Therefore, government should impose strict regulatory measures to curtail the practice and engage in post – marketing surveillance of these creams. The sellers and users of these creams should make an informed and sound decision regarding the use or otherwise of this cream.
The research recommends that more work be done in this area by making an attempt to adopt alternative cell viability assays such as neutral red uptake, lactate dehydrogenase (LDH) leakage, alamar blue, etc. to compare the specificity of the assay used in the research (MTT) and other assays.
