Mol Cell Toxicol (2015) 11:349-355 DOI 10.1007/s13273-015-0035-1
original Paper
Benzo(a)pyrene represses melanogenesis in B16F10 mouse melanoma cells Da Hye Joo1,*, Hwa Jun Cha1,*, Karam Kim1, Minhee Jung1, Jung Min Ko1, In Sook An1, Sung Nae Lee2, Hyun Hee Jang3, Seunghee Bae1, Nam Kyung Roh4, Kyu Joong Ahn4 & Sungkwan An1 Received: 9 March 2015 / Accepted: 15 July 2015 Ⓒ The Korean Society of Toxicogenomics and Toxicoproteomics and Springer 2015
Abstract Benzo(a)pyrene (BaP) is a chemically based
polycyclic aromatic hydrocarbon (PAH) that is readily absorbed by the skin. BaP is metabolized to BaP-diolepoxide by cytochromes P-450 1A1/2 (CYP1A1/2) and cytochromes P-450 1B1 (CYP1B1) in the cytosol. BaP and its metabolites induce genotoxicity and cancer. Although BaP easily accumulates in melanin-containing tissues as well as other tissue types, the effects of BaP on melanocytes are not fully understood. Here, we show that 40-100 μM BaP represses melanin synthesis in B16F10 cells. The decrease of melanin contents is induced by tyrosinase activity in BaP-exposed B16F10. However, this repression of melanin synthesis is not induced by direct inhibition of tyrosinase in in vitro assay. Therefore, we show whether BaP regulated melanin synthesis-related enzyme. BaP regulates melanin synthesis by Tyr and Tyrp1 expression. In addition, these genes expression is down-regulated by Mitf repressed by BaP. Importantly, the repression was provoked in the absence and presence of α-melanocyte stimulating hormone (α-MSH). Therefore, we hypothesize BaP interrupts the UV protection mechanism by repressing melanin synthesis in the skin. Taken together our results have revealed new side effects that ex1Korea
Institute for Skin and Clinical Sciences, Konkuk University, Seoul, Korea 2Department of Cosmetology, Kyung-In Women’s College, Incheon, Korea 3School of Art, Kyungbok University, Namyangju, Gyeonggi-do, Korea 4Department of Dermatology, Konkuk University School of Medicine, Seoul, Korea *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S. An (
[email protected])
posure of BaP abolished melanin synthesis in melanocytes. Keywords Benzo(a)pyrene, B16F10 cells, Melanogenesis, MITF, Tyrosinase, TYRP1 Benzo(a)pyrene (BaP) is chemically based on a polycyclic aromatic hydrocarbon (PAH) and is one of the various toxic substances in polluted air1. In particular, BaP is produced by incomplete combustion of organic matter, and humans are primarily exposed to BaP thro ugh tobacco smoke and diet2-4. BaP is metabolized to BaP-7,8-epoxide and BaP-7,8-dihydrodiol-9,10-epoxide by cytochromes P-450 1A1/2 (CYP1A1/2) and cytochromes P-450 1B1 (CYP1B1) in the cytosol5-7. BaP and these metabolites induce cancer by inactivating tumor suppression and inducing DNA adducts that cause genotoxicity7,8. Because BaP is readily absorbed by the skin, it has been reported that BaP exposure to skin is sufficient to induce skin cancer9. In addition, high BaP exposure to skin causes inflammation of the skin, hyperplasia, hyperkeratosis, pneumonitis, lymph node modifications, ulcerations, decreased growth and fertility rates, and the induction of immunosuppressive effects10,11. Melanogenesis is implicated to protect skin from UV irradiation, because melanin blocks the penetration of UV radiation12. Melanogenesis is regulated in melanocytes by the expression of melanin synthesis enzymes, such as tyrosinase and tyrosinase-related proteins 1 and 2 (TYRP1, 2)13,14. Melanin synthesis enzyme transcription is primarily regulated by microphthalmiaassociated transcription factor (MITF)14,15. In previous studies, Roberto and colleagues reported that BaP ac-
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cumulates in melanin-containing tissues due to an affinity between melanin and BaP16,17. Therefore, BaP exposure may have a significant function in the physiological roles of melanocytes. However, it remains unclear whether BaP regulates melanogenesis in melanocytes. In the present study, we examine the effects of BaP on the melanin synthesis pathway, as well as its effects on the repression of melanogenesis in B16F10 mouse melanoma cells. Mechanistically, the repression of melanogenesis occurred via BaP-induced repression of MITF expression and sequential repression of tyrosinase and Tyrp1 expression. BaP represses melanin synthesis in B16F10 cells
To determine the effect of BaP on melanogenesis, we first examined whether BaP influenced cell viability in B16F10 cells. As shown in Figure 1A, we found that treatment with 0-100 μM BaP is not cytotoxic for B16F10 cells. Interestingly, under the same conditions, we found that BaP repressed melanin content in a dosedependent manner (Figure 1B). At concentrations grea ter than 40 μM BaP, the melanin content significantly decreased. In addition, to evaluate whether BaP repressed α-MSH-induced melanin synthesis in B16F10 cells, we measured the melanin content in B16F10 cells treated with BaP (40 μM) or α-MSH (100 nM). As shown in Figure 1C, the cell pellets were black in α-MSH-treated B16F10 cells, but returned to white upon the addition of BaP. Additionally, the melanin content was increased by α-MSH and decreased by BaP (in both the absence and presence of α-MSH) (Figure 1C).
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(A)
(B)
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BaP inhibits α-MSH-induced cellular tyrosinase activity in B16F10 cells
To determine whether BaP directly or indirectly regul ates cellular tyrosinase activity, we performed L-DOPA oxidase activity assays (mushroom tyrosinase activity assays and cellular tyrosinase assays). As shown by our mushroom tyrosinase activity assays, BaP did not directly affect on tyrosinase activity (Figure 2A). However, as shown in Figure 2B, in B16F10 cells, BaP sig nificantly decreased cellular tyrosinase activity. These results indicate that BaP might decrease melanin synthesis by regulating the expression of melanogenesisrelated enzymes. BaP decreases the expression of melanogenesisrelated enzymes in B16F10 cells
Next, we examined whether BaP regulates expression of melanogenesis-related enzymes. Because Tyr,
Figure 1. BaP represses melanin synthesis in B16F10 cells. B16F10 cells were treated for 48 h with the indicated concentrations of BaP. (A) Cell viability was determined using MTT assay. (B) Melanin levels were determined separately by measuring the absorbance at 415 nm. Cells were co-treated with α-MSH (100 nM) and BaP (40 μM) for 48 h. (C) (Top panel) Melanin content was determined by measuring the absorbance at 415 nm. All results are expressed as a percentage compared with the untreated control samples. (Bottom panel) Color change of the cell pellet in BaP- and α-MSH-treated B16F10 cells. Values are the mean±SE of three independent experiments. *P<0.05 compared to vehicle-treated cells; #P<0.05 compared to α-MSH-treated cells.
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(A)
BaP inhibits MITF expression in B16F10 cells
(B)
MITF has a critical role as a nuclear transcription factor that initiates Tyr and Tyrp1 expression in melanogenesis15. Thus, we examined whether Mitf expression is regulated in BaP-treated B16F10 cells. As shown in Figure 4A, we found that Mitf protein expression was significantly downregulated by BaP. Moreover, BaP-mediated downregulation of Mitf was provoked in α-MSH-treated B16F10 cells. We also showed that BaP treatment inhibited Mitf mRNA expression as well as Mitf protein expression B16F10 cells (Figure 4B). In addition, BaP repressed Mitf mRNA and protein expression in α-MSH-treated B16F10 cells.
Discussion
Figure 2. BaP inhibits tyrosinase activity in B16F10 cells. (A) Direct inhibition of tyrosinase was evaluated using mushroom tyrosinase L-DOPA oxidase activity assays. L-DOPA was added with 0-100 μM BaP and incubated for 20 min with mushroom tyrosinase. The amount of dopachrome produced was determined using a microplate reader at 475 nm. (B) B16F10 cells were co-treated with α-MSH (100 nM) and BaP (40 μM) for 48 h, followed by detection of cellular tyrosinase activity using L-DOPA oxidase activity assays. The amount of dopachrome produced was determined using a microplate reader at 475 nm. All results are expressed as a percentage compared with the untreated control samples. Values are the mean±SE of three independent experiments. *P<0.05 compared with vehicle-treated cells; #P<0.05 compared with α-MSH-treated cells.
Tyrp1, and Tyrp2 are key melanogenesis enzymes, we examined their levels and observed that BaP treatment for 48 h in the presence of α-MSH decreased Typ and Tyrp1 levels (Figure 3A). In addition, to identify whe ther BaP-mediated inhibition of melanogenesis-related protein expression is due to decreased mRNA expression, we performed real-time RT-PCR analyses (Figure 3B). The mRNA expression of Typ and Tyrp1 were significantly inhibited in the presence of BaP for 48 h compared to control α-MSH-treated B16F10 cells (Fig ure 3). Because these genes have Mitf-binding sites in their promoter regions, we next examined Mitf expression in B16F10 cells.
BaP, a component of cigarette smoke, is a well-known carcinogen that promotes cancer through DNA adduct formation7. However, although BaP is absorbed by the skin and accumulates in melanin-containing tissues and cells15, how BaP affects the physiological role of melanocytes has not been studied. Here, we examined the effects of BaP on melanogenesis in B16F10 cells. In the present study, we first evaluated whether BaP influences the viability of B16F10 cells. Because B16F10 cell viability was not reduced by 100 μM BaP, we determined the melanin content in B16F10 cells treated with up to 100 μM BaP (Figure 1). At concen trations greater than 40 μM, BaP significantly decreas ed melanin synthesis (Figure 1B). Therefore, subsequent experiments were performed using 40 μM BaP. As shown in Figure 1C, BaP-mediated repression of melanogenesis also occurs in the presence of α-MSH. α-MSH stimulates melanin production and release in melanocytes by sequential pathways13-15. First, α-MSH binds the melanocortin receptor, which initiates an increase in intercellular cyclic AMP by adenylyl cyclase20. Second, cyclic AMP activates PKA, which elevates CREB transcriptional activity21. Next, activated CREB transcribes MITF, a transcription factor that controls melanin synthesis enzymes, including tyrosinase and Tyrp113-14. Finally, the melanin synthesis enzymes metabolize L-tyrosine to melanin15. Therefore, we determined whether BaP decreased production of the melanin synthesis enzymes. As shown in Figure 3, BaP significantly decreased the mRNA and protein levels of tyrosinase and Tyrp1 in B16F10 cells, in both the presence and absence of α-MSH. Studies have shown that MITF can regulate tyrosinase and Tyrp1 in melanocytes13-15. Therefore, we also examined whether MITF regulates the BaP-mediated decrease in tyrosinase and Tyrp1 expression (Figure 4). Overall, BaP
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(A) (B)
(C) (D)
Figure 3. BaP negatively regulates the expression of melanogenesis-related genes in B16F10 cells. (A) B16F10 cells were co-treated with α-MSH (100 nM) and BaP (40 μM) for 48 h. The expression of Tyr, Tyrp1, Tyrp2 proteins was examined by western blot analyses. (B-D) B16F10 cells were co-treated with α-MSH (100 nM) and BaP (40 μM) for 48 h. The mRNA expression of (B) Tyr, (C) Tyrp1, and (D) Tyrp2 were examined by qRT-PCR analyses. All results are expressed as a percentage compared with the untreated control samples. Values are the mean±SE of three independent experiments. *P<0.05 compared with vehicle-treated cells; #P<0.05 compared with α-MSH-treated cells.
significantly repressed melanin synthesis by inactivating the MITF-tyrosinase-Tyrp1 pathway in B16F10 cells. These results are different to previous studies. In generally, BaP induced expression of CYP1A1 using activation of aryl hydrocarbon receptor (AHR)22,23. BaP is also rapidly metabolized by CYP1A1 in feedback mechanism, which maintains homeostasis thro ugh balancing between BaP and CYP1A122. Additionally, in 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed melanocyte, the AHR is identified as novel regulator of melanogenesis24. Thus, BaP may have a potential as inducer of melanogenesis. However, our results suggested the opposite. To our knowledge, our findings that MITF-mediated alteration of tyrosinase and Tyrp1 expression underlies BaP-mediated repression of melanogenesis are novel. In addition, BaP-mediated repression of melanogenesis occurred equally in α-MSHtreated B16F10 cells. Because α-MSH-induced melanogenesis, the major protection mechanism in UVexposed skin25, is significantly decreased by BaP in B16F10 cells, we hypothesize that BaP interrupts the
UV protection mechanism by repressing melanin synthesis in the skin. However, although our current data demonstrate the importance of BaP in B16F10 cell depigmentation, the mechanism by which BaP regulates Mitf gene expression in these cells remains to be identified.
Materials & Methods Cell culture, antibodies, and chemicals
B16F10 mouse melanoma cells were obtained from ATCC and grown in DMEM (Life Technologies Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Life Technologies Gibco) and 1% penicillin-strepto mycin (Life Technologies Gibco) in a humidified atmosphere containing 5% CO 2 at 37℃. Antibodies against MITF, tyrosinase, Tyrp1, Tyrp2, as well as the bovine anti-goat antibody, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antimouse and anti-rabbit antibodies were purchased from
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BaP and α-MSH, cells were harvested and lysed with 1 N NaOH at 94℃ for 20 min. The melanin content was determined by optical density at 415 nm and normalized to the amount of total protein. Results are presented as the relative percentage of melanin content. Mushroom tyrosinase activity assays
(B)
In vitro tyrosinase activity was determined using mush room tyrosinase as described previously19. 3,4 Dihydroxy-L-phehylalanine (L-DOPA; Sigma-Aldrich) and mushroom tyrosinase (Sigma-Aldrich) were incubated with 0-100 μM BaP at 37℃ for 20 min. We measured dopachrome metabolized from L-DOPA by mushroom tyrosinase at 475 nm using a microplate reader. Cellular tyrosinase activity assays
Figure 4. BaP negatively regulates MITF expression in B16F10 cells. (A) B16F10 cells were co-treated with α-MSH (100 nM) and BaP (40 μM) for 48 h. MITF protein expression was examined by western blot analyses. (B) B16F10 cells were co-treated with α-MSH (100 nM) and BaP (40 μM) for 48 h. The mRNA expression level of Mitf was examined by qRT-PCR analyses. All results are expressed as a percentage compared with the untreated control samples. Values are the mean±SE of three independent experiments. *P<0.05 compared with vehicle-treated cells; #P< 0.05 compared with α-MSH-treated cells.
Cell Signaling Technology (Danvers, MA, USA). The β-actin antibody was supplied by Sigma-Aldrich (St. Louis, MO, USA). BaP and α-melanocyte stimulating hormone (α-MSH) were purchased from Sigma-Aldrich. Cell viability assays
B16F10 cells (2 × 103 cells/well) were seeded in 96 well plates and incubated for 24 h. Cells were treated with 0-100 μM BaP and incubated for 48 h. Cell viability was determined using MTT assays (Sigma-Aldrich) as described previously18. The absorbance of each well was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Melanin content assays
B16F10 cells (2 × 105) were cultured in 60 mm plates for 24 h, and exposed to BaP for 48 h in the presence or absence of α-MSH (600 nM). After treatment with
B16F10 cells (2 × 105) were cultured in 60 mm plates for 24 h, and then exposed to BaP for 48 h in the presence or absence of α-MSH (600 nM). Cells were harvested and lysed in cell lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100). Lysates were incubated for 20 min at 37℃ with L-DOPA, the tyrosinase substrate, in 0.1 M sodium phosphate buffer (pH 7.0). The absorbance of each sample was measured at 475 nm using a microplate reader. Western blotting analyses
B16F10 cells (2 × 105) were cultured in 60 mm plates for 24 h and exposed to BaP for 48 h in the presence or absence of α-MSH (600 nM). Cells were harvested and lysed in 1% SDS lysis buffer (1% SDS, 20 mM Tris-Cl (pH 7.4), 2 mM EDTA). Cell lysates were electropho resed in 12% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and detected with the following primary antibodies: anti-β-actin, anti-MITF, anti-tyrosinase, anti-TYRP1, and anti-TYRP2. Horseradish peroxidase-conjugated horse anti-mouse, goat anti-rabbit, and bovine anti-goat were used as secondary antibodies. Each protein was visualized using an enhanced chemiluminescence HRP substrate (Thermo Fisher Scientific, Waltham, MA, USA) and x-ray film (Agfa, Hiroshima, Japan). Western blot data are representative of at least three independent experiments. Quantitative real-time PCR
B16F10 cells (2 × 105) were cultured in 60 mm plates for 24 h and exposed to BaP for 48 h in the presence or absence of α-MSH (600 nM). Total RNA was isolated from cells using the TRIzol reagent (Life Technologies Invitrogen, Grand Island, NY, USA) and cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen). PCR was performed using Line-gene K (Bio-
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er Technology, Hangzhou, China) and the EvaGreen qPCR master mix (Solis BioDyne, Tartu, Estonia). Rel ative target mRNA expression was determined using an established comparative threshold (Ct) of target genes, which were normalized β-actin Ct values. Values represent the mean±standard error (SE) of triplicate experiments. The following primer sequences were used for PCR: Mouse β-actin forward primer : 5′-CGACAGGATGCAGAAGGAG-3′, Mouse β-actin reverse primer: 5′-ACATCTGCTGGAAGGTGGA-3′, Mouse Mitf forward primer: 5′-GGAACAGCAACGA GCTAAGG-3′, Mouse Mitf reverse primer: 5′-TGAT GATCCGATTCACCAGA-3′, Mouse Tyr forward primer: 5′-GGCAGATTGTCTGTAGCCGA-3′, Mouse Tyr reverse primer: 5′-CCTTGGGGTTCTGGATTT GT-3′, Mouse tyrp1 forward primer: 5′-GATGTCTG CACTGATGACTTG-3′, Mouse tyrp1 reverse primer: 5′-CCTGATTGGTCCACCCTCAG-3′, Mouse tyrp2 forward primer: 5′-CGTGCTGAACAAGGAATGCT3′, Mouse tyrp2 reverse primer: 5′-GCATGTCCGGTT GAAGAAT-3′. Statistical analysis
Statistical significance was determined by Student’s t-test. P<0.05 was considered to indicate a statistically significant difference. Authors contributions
D. H. Joo, H. J. Cha, K. Kim, M. Jung, J. M. Ko, I. S. An, and N. K. Roh performed experiments and analyz ed the data. S. N. Lee, H. H. Jang, S. Bae, K. J. Ahn, and S. An conceived and designed the study. D. H. Joo, H. J. Cha, and S. An prepared the manuscript. S. An was responsible for the overall project. All authors discussed the results and approved the final version of manuscript. Acknowledgements This study was supported by the Konkuk University in 2012. Conflict of Interest The authors state no conflict of interest.
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