Effects of Arsenic and UVB on Normal Human Cultured Keratinocytes

Jan 28, 2005 - Inorganic arsenic is an environmental toxin and a human carcinogen. Being a co-mutagen, arsenic enhances carcinogenesis of ultraviolet ...
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Chem. Res. Toxicol. 2005, 18, 139-144

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Effects of Arsenic and UVB on Normal Human Cultured Keratinocytes: Impact on Apoptosis and Implication on Photocarcinogenesis Po-Hung Chen, Cheng-Che E. Lan, Min-Hsi Chiou, Ming-Chu Hsieh, and Gwo-Shing Chen* Department of Dermatology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China Received June 28, 2004

Inorganic arsenic is an environmental toxin and a human carcinogen. Being a co-mutagen, arsenic enhances carcinogenesis of ultraviolet irradiation on the mouse skin. Apoptosis, a wellregulated cell death process, is essential for cell development and tissue homeostasis. Dysregulation of apoptosis will lead to various kinds of pathological conditions, such as cancers. The purpose of this study is to investigate the apoptotic effect induced by the interactions of arsenic and UVB on cultured human keratinocytes. Cultured keratinocytes were treated with sodium arsenite (1 µM) and/or UVB 50 mJ/cm2 irradiation in different combinations, including arsenic alone (As group), UVB alone (UVB group), arsenic followed by UVB (As/UVB group), and UVB followed by As (UVB/As group) treatments. Our results revealed that a low concentration of sodium arsenite did not induce keratinocytes apoptosis. The UVB group showed obvious elevation of caspase-8, -9, and -3 activities in addition to strong induction of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling (TUNEL) assay. Similar pro-apoptotic effects were observed in the UVB/As group. In contrast, only subtle changes of cell morphology and survival rate were noticed in the As/UVB group. In addition, the results of Western blot and activity assay of caspase-8, -9, and -3 revealed that neither the receptor nor the mitochondrial apoptotic signaling pathway was activated in the As/UVB group. Therefore, we conclude that the pretreatment of keratinocytes with sodium arsenite decreased the pro-apoptotic effects induced by UVB. This finding corroborated with the animal model studying the effects of arsenic and UVB on carcinogenesis. The molecular mechanisms by which arsenic decreased UVB-induced apoptosis remain to be elucidated.

Introduction On the southwestern coast of Taiwan, the Blackfoot disease endemic area, increased incidence of neoplasms, including skin cancers, have been documented (1-4). Epidemiological research showed that a high concentration of arsenic in the artesian well water is the candidate carcinogen (5). A dose-response relationship was established between the prevalence of arsenic-induced skin cancers and the arsenic concentration present in the drinking water (3, 4). Inorganic arsenic is a well-known environmental toxin distributed throughout the earth’s crust (6). There are four main forms of arsenic found in mammals: arsenite (As(III)), arsenate (As(V)), monomethylarsenic acid (MMA), and dimethylarsenic acid (DMA) (7). However, which form of the arsenic poses the greatest potential of carcinogenesis is still controversial (8). The carcinogenic effects of arsenic are believed to be related to its effects on cellular differentiation, proliferation, and apoptosis. No animal model has yet shown arsenic alone to be directly involved in the carcinogenic process. Instead of being a complete carcinogen, arsenic could enhance the mutagenicity of other carcinogens such as UV irradiation on the mouse skin (9). This co-mutagenic effect of arsenic with UV irradiation has also been demonstrated in Escherichia coli (10), Chinese hamster V79 cells (11, 12), and human lymphoblastoid TK6 cells (13). * To whom correspondence should be addressed. Tel.: +886-73121101 ext 6103. Fax: +886-7-3216580. E-mail: [email protected].

Apoptosis, a well-regulated cell death process, can eliminate cells that are redundant, damaged, or mutated. This genetic program is essential for maintenance of tissue homeostasis and for an effective immune system (14-16). Once apoptosis is activated, apoptotic cells characterized by membrane blebbing, cell shrinking, nuclear condensation, chromatin aggregation, and degradation of DNA are observed (17-19). Induction of apoptosis can occur by external or internal stimuli. There are two major pathways of apoptosis: the receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway (18, 20, 21). Both apoptotic pathways converge at the level of the aspartatespecific cysteine proteases, the caspases (22). Based on their function, caspases can be classified into (1) initiator caspases such as caspase-8, -9, and -10, which initiate and amplify a death signal, and (2) executioner caspases such as caspase-2, -3, -6, and -7, which degrade vital cellular components and lead cell to death (23). Because apoptosis is important for the proper functioning of living organisms, any cause that disturbs the regulation of apoptosis will lead to various kinds of pathological conditions, including degenerative disorders, autoimmune diseases, and cancers (24-28). The present study was designed to investigate the apoptotic effect induced by the interactions of arsenic and UVB on cultured human keratinocytes.

Materials and Methods Keratinocyte Culture and Treatments. The normal human keratinocytes were obtained from adult foreskin through routine circumcision. The method for keratinocyte cultivation

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was described in our previous report. Briefly, skin specimens were washed with phosphate-buffered saline (PBS; pH 7.2), cut into small pieces, and incubated in medium containing 0.25% trypsin (Gibco, Grand Island, NY) overnight at 4 °C. The epidermal sheet was lifted from the dermis using fine forceps. The epidermal cells were pelleted by centrifugation (500g, 10 min) and dispersed into individual cells by repeated aspiration with a 2-mL pipet. The keratinocytes were gently resuspended in approximately 5 mL of keratinocyte-SFM medium (Gibco), which contained 25 µg/mL bovine pituitary extract (BPE) and 5 ng/mL recombinant human epidermal growth factor (rhEGF). The medium was changed every 2 days. Keratinocytes at the third passage were then grown in keratinocyte-SFM medium without BPE and rhEGF for 24 h before experimentation. Cells were then treated with various regimens of sodium arsenite (1 µM) and/or UVB (50 mJ/cm2) as follows: (1) no treatment (control group); (2) only incubated with As for 48 h (As group1); (3) only irradiated with UVB after a 48 h incubation (UVB group); (4) incubated with As for 24 h then irradiated with UVB, followed by a 24 h incubation in culture medium (As/UVB group); (5) irradiated with UVB then incubated for 24 h in culture medium, followed by a 24 h As exposure (UVB/As group). The cellular morphology of all groups was observed under phase contrast microscopy. Trypan Blue Exclusion Assay. Both adherent and nonadherent cells in every group were harvested by trypsinization. The cells were pelleted by centrifugation at 1000g for 10 min and were resuspended in PBS in the presence of an equal volume of trypan blue solution. Cell viability was assessed microscopically by trypan blue dye exclusion using a hemocytometer. Detection of Apoptosis. Terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling (TUNEL) assay was conducted for detection of apoptosis in keratinocytes. The commercially available apoptosis detection system (Promega, Madison, WI) was used. Keratinocytes were harvested by trypsinization and centrifugation. After being washed with PBS, the cells were fixed in 4% phosphate-buffered paraformaldehyde on ice for 20 min. The cells were then washed with PBS, followed by permeabilization with 0.2% Triton X-100 solution for 5 min on ice. After equilibration with terminal transferase buffer (TT buffer, 25 mM Tris-HCl, pH 6.6, 0.2 mM DTT, 200 mM potassium cacodylate, 2.5 mM cobalt chloride, and 0.25 mg/mL BSA) at room temperature for 5 min, the cells were then exposed to 0.5 U/µL terminal transferase and 5 µM fluoresceinated dUTP in TT buffer for 60 min at 37 °C in a moist chamber. The reaction was terminated by adding 1 mL of 20 mM EDTA and washed with phosphate buffer. Ten thousand keratinocytes in total were assessed by FACScan (Beckman Coulter, Brea, CA), and the positive staining rate was analyzed with Windows Multiple Document Interface (WinMDI) software for flow cytometry. Specificity of staining was ascertained with negative control omitting terminal transferase enzyme. Apoptosis was also measured by determining the DNA content of cells by propidium iodide (PI) staining and flow cytometry. Briefly, keratinocytes were collected by trypsin and washed once with PBS. Cell pellets were resuspended in 50% cold ethanol and fixed at -20 °C. After fixation, cells were washed once with cold PBS and incubated in 0.5 mL of PBS containing 100 µg/mL RNase A for 20 min at 37 °C. Keratinocytes were then pelleted by centrifugation, and 250 µL of PBS containing 50 µg/mL PI was added to the pellet. Thirty minutes later, flow cytometric analysis was carried out. Cells with DNA content less than that in untreated cells in G0/G1 were considered apoptotic. Protein Extraction and Western Blotting. Total cell extracts from cultured human keratinocytes were obtained by lysing the cells in cold Lysis buffer (50 mM Tris-HCL, pH 7.4, 1 As group, arsenic group; As/UVB group, arsenic treatment followed by UVB irradiation group; UVB/As group, UVB irradiation then arsenic treatment group; TUNEL, terminal deoxynucleotidyl transferasemediated deoxyuridine nick-end labeling; PI, propidium iodide.

Chen et al. 5 mM EDTA, 1% Triton X-100, 250 mM β-mercaptoethanol) in the presence of protease inhibitors (Roche, Molecular Biochemicals, Mannheim, Germany). After centrifugation to remove cell debris, 30 µg/well supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 10% acrylamide gels under reducing conditions (in the presence of 250 mM β-mercaptoethanol). Proteins were subsequently electrotransferred onto a nitrocellulose membrane (Sartorius AG, Go¨ttingen, Germany) following conventional protocols. Blots were blocked in 5% skimmed milk/PBS with 0.1% Tween-20 (PBS-T) for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4 °C. The working concentrations of primary antibodies in PBS-T with 5% skimmed milk were 1 µg/mL for procaspase-8, -9 (PharMingen, USA). After five washes in PBS-T, the blots were incubated with secondary antibodies (horseradish peroxidase coupled with antimouse or anti-rabbit immunoglobulin G, Santa Cruz, USA) at 1:5000 dilution. The enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, NJ) detection system and BioMax film (Kodak, Rochester, NY) were used to visualize the presence of specific proteins on the blots according to the manufacturer’s instructions. The exposure times for each protein ranging from 1 min to 1 h were calibrated for documenting linearity and permitting semiquantitative analysis of density. The visualized films were recorded on a digital imaging system (Alpha Imager 2000, Alpha Innotech Corp., San Leandro, CA). Caspasaes Activity Assays of Caspase-3, -8, -9. ApoAlert Caspase Profiling plate assay was conducted for detection of apoptosis in keratinocytes. The commercially available apoptosis detection system (BD Biosciences, CA) was used. Keratinocytes were collected by trypsinization, washed once with PBS, and pellets of 1/2 × 105 cells were counted and resuspended in 50 µ ice-cold lysis buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM dithiothreitol, and 100 µM EDTA) and homogenized. Homogenates were centrifuged at 12 000 rpm for 10 min at 4 °C. Supernatants were used for measuring caspase activity using an ELISA-based assay, according to the manufacturer’s instructions. Statistical Analysis. All results were expressed as mean values ( standard deviation (S.D.) and analyzed by using the statistical analysis system (SPSS, SPSS Inc.). Differences among groups were analyzed by one-way analysis of variance (ANOVA) with Scheffe’s posttest. A difference of P < 0.05 is considered as statistically significant.

Results Effects of Arsenic and UVB on Induction of Apoptosis. The morphological changes of cultured keratinocytes treated with arsenic and UVB were recorded with phase contrast microscopy. Keratinocytes of the control group adhered to plate and grew into a confluent monolayer. Similar morphology was also seen in the As and the As/UVB groups. In the UVB and the UVB/As groups, keratinocytes showed typical morphology of apoptosis including cellular contraction, plasma membrane blebbing, and contracted nuclei (Figure 1). Using trypan blue exclusion assay, a significant decrease in cell viability was detected in the UVB (56.63 ( 7.59%) and the UVB/As (42.27 ( 3.42%) groups but not in the As (86.74 ( 17.17%) and the As/UVB (88.95 ( 7.89%) groups (Figure 2). By TUNEL assay (Table 1), arsenic treatment (3.52 ( 0.47%) showed no significant induction of apoptosis as compared to the control group (4.02 ( 0.61%). UVB exposure alone (9.59 ( 1.42%) or followed by arsenic treatment (10.82 ( 2.23%) exhibited a significantly inductive effect on keratinocytes apoptosis. Contrary to the UVB/As group, no significant increase of apoptotic keratinocytes was detected in the As/UVB (6.75 ( 1.19%)

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Figure 1. Cell morphology of cultured human keratinocytes treated with sodium arsenite and/or UVB irradiation. Keratinocytes showing intact cell morphology attaching to the plate: (a) control group, (b) As group, and (d) As/UVB group. Keratinocytes showing shrinkage and round morphology: (c) UVB group and (e) UVB/As group. Cell detachment from the plate was also observed. Table 1. Effects of Sodium Arsenite and UVB on Apoptosis Inductiona TUNEL positive rate (%) control As (1 µM) UVB (50 mJ/cm2) As/UVB UVB/As

Figure 2. Influence of 1 µM sodium arsenite and UVB 50 mJ/ cm2 on cell viability in keratinocytes. The number of viable cells was counted by trypan blue exclusion assay. As shown in the figure, a significant decrease in cell viability was detected in the UVB and the UVB/As groups but not in the As and the As/ UVB groups. “*” indicates that p < 0.01 as compared to either the control or between groups.

group. Because DNA fragmentation is an early and characteristic event of apoptosis, we next examined the effect of arsenic and/or UVB-induced DNA fragmentation using PI staining on cultured human keratinocytes. The result of DNA fragmentation was compatible with TUNEL assay (Figure 3). The sub-G0 DNA content was obviously increased in the UVB and the UVB/As groups (21.32% and 26.02%, respectively).

4.02 ( 0.61 3.52 ( 0.47 9.59 ( 1.42b 6.75 ( 1.19 10.82 ( 2.23b

a TUNEL reaction and flow cytometry were used for the detection of apoptosis. The percentage of the positive cells was analyzed. The data are expressed as mean ( SD. b p < 0.01, n ) 3.

Effects of Arsenic and UVB on Procaspase-8 and -9 Expressions. To determine the possible apoptotic pathway, we measured the relative levels of procaspase-8 and -9 proteins in cultured human keratinocytes using Western blotting of whole cell lysates after exposure to different treatment conditions (Figure 4). It is conceivable that if a certain caspase pathway were activated, then the proenzyme of the corresponding caspase would show a decrease in protein level. For the receptor-signaling pathway, no prominent change in protein level of procaspase-8 was measured in the As group. The protein expression of procaspase-8 in the UVB and the UVB/As groups displayed a marked decrease of procaspase-8 as

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Figure 3. Effects of sodium arsenite and UVB on apoptotic induction. PI stain was used for the detection of apoptosis. The percentage (%) of the positive cells was analyzed. The sub-G0 DNA content was obviously increased in the UVB and the UVB/As groups.

Figure 4. Effects of sodium arsenite and UVB on protein expressions of procaspase-8 and -9. The protein expression of procaspase-8 and -9 showed a marked decrease in the UVB group as compared to the normal control. The proenzyme of caspase-8 and -9 showed a marked decrease and a relative decrease in the UVB/As group, respectively, as compared to the normal control. The As/UVB group showed no obvious changes in proenzyme levels as compared to the control group.

compared to the control group. In contrast to the UVB and the UVB/As groups, keratinocytes in the As/UVB group showed no obvious change in procaspase-8 protein level. The protein level of procaspase-9, the initiator caspase of mitochondrial-signaling pathway, was also measured. Similar to the result from procaspase-8, the protein level of procaspase-9 was significantly decreased in the UVB and relatively decreased in the UVB/As group as compared to the control group. These results indicated that procaspase-8 and -9 were catalyzed in the UVB and the UVB/As groups. To confirm our result, activity assays of caspase-8 and -9 were also performed. Effects of Arsenic and UVB on Caspase-3, -8, and -9 Activities. To determine the relative extent of caspase activation in response to the arsenic and/or UVB irradiation, the activities of caspase-3, -8, and -9 were measured. The effects of arsenic, UVB, and their interactions on caspase-3, -8, and -9 activities on cultured keratinocytes were shown as Figures 5-7. The caspase-8 and -9 activities of the As and the As/UVB groups were not increased. Yet keratinocytes treated with UVB and UVB/

Figure 5. Effects of sodium arsenite and UVB on caspase-9 activity. The activity level of caspase-9 was expressed in relative ratios as compared to the normal control (NC). The activity level of caspase-9 was not increased in the As and the As/UVB groups. In both the UVB and the UVB/As groups, the caspase-9 activity increased significantly. “*” indicates that p < 0.01 as compared to either the control or between groups.

As displayed at least 2.1- and 1.6-fold increases in caspase-8 and -9 activities, respectively. These results confirmed our finding from the Western blot analyses. Caspase-3, the only effector caspase assayed, showed

Effects of Arsenic and UVB on Keratinocytes

Figure 6. Effects of sodium arsenite and UVB on caspase-8 activity. The activity level of caspase-8 was expressed as relative ratios as compared to the normal control (NC). The activity level of caspase-8 was not increased in the As and the As/UVB groups. In both the UVB and the UVB/As groups, the caspase-8 activity increased significantly. “*” indicates that p < 0.01 as compared to either the control or between groups.

Figure 7. Effects of sodium arsenite and UVB on caspase-3 activity. The activity level of caspase-3 was expressed as relative ratios as compared to the normal control (NC). In both the UVB and the UVB/As groups, the caspase-3 activity increased significantly. “*” indicates that p < 0.01 as compared to either the control or between groups.

increased activities (∼16-fold) in the UVB and the UVB/ As groups.

Discussion Arsenic-contaminated drinking water has created a serious health hazard in some parts of the world. Recently, arsenic was shown not only to induce apoptosis in certain tumor cells but also to promote cell differentiation, proliferation, or transformation under restricted situation (29-33). Unlike arsenic pollution in limited area, UV irradiation is among the most ubiquitous damaging environmental factors that human skin is continuously exposed to. In this study, we investigated the interactions of arsenic and UVB on cultured human keratinocytes in terms of apoptosis by various assays. In our results, a low concentration of arsenic (1 µM) showed no pro-apoptotic effect, while UVB irradiation alone promoted significant apoptosis via both receptor and mitochondrial pathways in cultured keratinocytes. A similar finding has been reported by Sitailo et al. (34). Because UVB at 50 mJ/cm2 resulted in a modest degree of apoptosis, this dosage was used to access the contribution of arsenic on keratinocyte apoptosis. Comparing the two different combinations of arsenic and UVB treatment, a significant increase of apoptosis was noted in the UVB/As group but not in the As/UVB group. Because significant apoptosis can be induced within 24 h after UVB irradiation (34), our result cannot be explained by the difference in duration after UVB irradiation (24 vs 48 h) alone. Therefore, we propose that pretreatment with arsenic may provide an unknown mechanism that

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prevents keratinocytes from undergoing apoptosis induced by UVB. Being a co-mutagen, arsenic enhanced carcinogenesis of UV radiation on the mouse skin (9). In fact, arsenic seemed to divert photodamaged cells from going out of the mitotic cycle. Vogt and Rossman have reported that combined exposure of human fibroblast to arsenic and ionizing radiations lacked characteristic induction of p53 expression that normally blocks cell cycle progression (35). In addition, arsenic can prevent the UVB-induced proliferative blockage (36). In our study, the pretreatment of cultured human keratinocytes with arsenic followed by UVB irradiation decreased the apoptotic rate induced by UVB irradiation alone. This result implies that pretreatment with arsenic allows UVB-transformed keratinocytes to avoid apoptosis and proliferate. This abnormal proliferation, if taking place in vivo, may lead to formation of skin cancer. In summary, our results showed that pretreating keratinoctyes with arsenic could prevent UVB transformed cells from undergoing apoptosis. This finding correlated well with the animal model studying the effects of arsenic and UVB on carcinogenesis. However, our result seems to conflict the common clinical observation: skin cancer often arises from sun-protected areas in patients with chronic arsenism. Further investigations are needed to elucidate this paradoxical phenomenon.

Acknowledgment. We acknowledge the excellent technical assistance of Ms. Hsuan-Yu Kuo in cell culture. This study was supported by the grant from the National Science Council, Taiwan, Republic of China (NSC-892314-B-037-137).

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