The Cytotoxicity and Apoptosis Induced by 4-Tertiary Butylphenol in Human Melanocytes are Independent of Tyrosinase Activity  Fan Yang, Rangaprasad Sarangarajan, I. Caroline Le Poole, Raymond E. Boissy  Journal of Investigative Dermatology  Volume 114, Issue 1, Pages 157-164 (January 2000) DOI: 10.1046/j.1523-1747.2000.00836.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Viability of melanocytes from donors with different pigmentary skin types after treatment of 4-TBP. NHM were cultured from three Caucasian (NHM 143, 617, and 630) and three African-American (NHM 637, 707, and 718) donors. (A) Tyrosine hydroxylase and melanin content were determined as described in Materials and Methods. Tyrosine hydroxylase activity ranged from 1.84 to 5.57 DPM per mg protein per h ×  106 and melanin content ranged from 80.60 to 1133.33 μg per mg protein. Values are the mean ± SD of three determinations. (B) After treatment with 4-TBP (100, 500, and 750 μM) for 3 d, viable cells were counted by a Coulter Counter. Viability of all six melanocyte lines demonstrated comparable concentration-dependent response. Controls without treatment of 4-TBP represent 100% viability. Values are the mean ± SD of four determinations. The results were similar to two additional independent experiments. Journal of Investigative Dermatology 2000 114, 157-164DOI: (10.1046/j.1523-1747.2000.00836.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Viability of OCA 1 melanocytes after treatment of 4-TBP. Normal melanocytes (NHM 69) derived from an African-American donor and OCA 1 melanocytes (OHM 43) were treated with 4-TBP (100, 500, and 750 μM) for 3 d. Viable cells were counted by a Coulter Counter. Viability of OCA 1 and normal melanocytes demonstrated comparable concentration-dependent responses. Controls without treatment of 4-TBP represent 100% viability. Values are the mean ± SD of four determinations. The results were similar to independent experiment. Each set per treatment group were compared statistically by the Student’s t test; *p <  0.05. Journal of Investigative Dermatology 2000 114, 157-164DOI: (10.1046/j.1523-1747.2000.00836.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Viability of autologous cutaneous fibroblasts, keratinocytes, and melanocytes after treatment of 4-TBP. Fibroblasts, keratinocytes, and melanocytes were established from one African-American (A) and one Caucasian (B) donor, respectively. After treatment of 4-TBP (100, 500, and 750 μM) for 3 d, viable cells were counted by a Coulter Counter. Viability of autologous fibroblasts and melanocytes demonstrated comparable concentration-dependent responses. Keratinocytes demonstrated more viability than autologous melanocytes at 4-TBP dosages of 500 and 750 μM. Controls without treatment of 4-TBP represent 100% viability. Values are the mean ± SD of six determinations. Journal of Investigative Dermatology 2000 114, 157-164DOI: (10.1046/j.1523-1747.2000.00836.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Analysis of 4-TBP cytotoxicity in UCD and IIB-MEL-J cells expressing exogenous human tyrosinase cDNA. (A) Presents tyrosinase activity and melanin content in sense transfected and untransfected pigmented IIB-MEL-J melanoma cells and transfected (vector alone, anti-sense, or sense) and untransfected UCD cells and normal human fibroblasts. Sense transfected cells demonstrate induction of tyrosinase activity in both transfected cell lines and a concomitant increase in melanin content in the IIB-MEL-J line only. Values are the mean ± SD of three determinations. Large asterisk indicates that respective values for tyrosinase activity were not detected. Small asterisk indicates that value is significantly increased over the value for the respective untransfected counterpart. (B) Presents immunoblotting analysis of tyrosinase. Cell lysates were prepared from transfected UCD cells (sense, anti-sense, or vector alone), untransfected UCD cells, transfected (sense and vector alone) IIB-MEL-J cells, untransfected IIB-MEL-J cells and normal human fibroblasts. Detection of tyrosinase protein by immunoreaction was performed as described in Materials and Methods. A 75 kDa tyrosinase positive band was detected only in the sense transfected UCD and in all the IIB-MEL-J cells where the relative amount of tyrosinase was increased after transfection of tyrosinase sense cDNA. (C) Presents viability in transfected (sense, anti-sense, or vector alone) and untransfected UCD cells, and (D) presents viability in transfected (sense or vector alone) and untransfected IIB-MEL-J cells after treatment with 4-TBP (100, 250, 500, and 750 μM) for 3 d. Viable cells were counted by a Coulter Counter. Controls (without 4-TBP) represent 100% viability. Viability of all cell lines demonstrated comparable concentration-dependent response. Values are the mean ± SD of six determinations. Journal of Investigative Dermatology 2000 114, 157-164DOI: (10.1046/j.1523-1747.2000.00836.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Electron microscopy of changes in the plasma membrane of cultured melanocytes with or without 4-TBP treatment. (A) Melanocytes, treated with 400 μM 4-TBP for 16 h, demonstrated blebbing of the plasma membrane (arrow). (B) Melanocytes, treated with vehicle alone, did not exhibit any changes in the plasma membrane. Scale bars: 0.77 μm (A) and 0.7 μm (B). Journal of Investigative Dermatology 2000 114, 157-164DOI: (10.1046/j.1523-1747.2000.00836.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Detection of DNA fragments in apoptotic cells by the in situ apoptosis assay. Cultured human melanocytes were treated with 4-TBP (750 μm) or vehicle alone for 3 d. DNA fragments were detected using the Klenow enzyme conjugated to streptavidin–fluorescein. DNA fragmentation was not apparent in (A) control melanocytes treated with vehicle alone, but apparent in (B) detached melanocytes treated with 750 μM 4-TBP. Scale bar: 11 μm. Journal of Investigative Dermatology 2000 114, 157-164DOI: (10.1046/j.1523-1747.2000.00836.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Detection of phosphatidylserine by annexin V during the early stage of apoptosis. Cultured human melanocytes were treated with (a, b) 4-TBP (400 μM) or (c, d) vehicle alone for 3 d. Attached cells were collected and labeled with annexin V-FITC. Twenty-five percent of melanocytes treated with 400 μM 4-TBP (arrow) exhibited positive staining. In contrast, melanocytes treated with vehicle alone exhibited no staining (a, c: without; b, d: with fluorescent filter). Scale bar: 10 μm. Journal of Investigative Dermatology 2000 114, 157-164DOI: (10.1046/j.1523-1747.2000.00836.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Detection of annexin V-FITC and PI staining in detached cells after treatment of 750 μM 4-TBP. Both annexin V (a: with fluorescent filter) and PI (b: with rhodamine filter) staining was exhibited in all of these cells. Scale bar: 11 μM. Journal of Investigative Dermatology 2000 114, 157-164DOI: (10.1046/j.1523-1747.2000.00836.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions