In Vitro Studies on the Depigmenting Activity of 4-(p-Hydroxyphenyl)-2-Butanone

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1 J Occup Health 1998; 40: Journal of Occupational Health In Vitro Studies on the Depigmenting Activity of 4-(p-Hydroxyphenyl)-2-Butanone Yoshiharu FUKUDA 1 *, Megumi NAGANO 1, Katsuhiko TSUKAMOTO 2 and Makoto FUTATSUKA 1 1 Department of Public Health, Kumamoto University School of Medicine and 2 Department of Dermatology, Yamanashi Medical College Abstract: In Vitro Studies on the Depigmenting Activity of 4-(p-Hydroxyphenyl)-2-Butanone: Yoshiharu FUKUDA, et al. Department of Public Health, Kumamoto University School of Medicine The aim of this study is to investigate the enzymatic properties and the depigmenting activity of 4-(p-hydroxyphenyl)- 2-butanone (HPB) in vitro. The activity of HPB as a substrate of tyrosinase, its effect on tyrosinase enzymatic reactions, and its inhibition of the growth and the melanogenesis of cultured melanoma cells were examined. The HPB-tyrosinase reaction and the effect of HPB on tyrosine-tyrosinase and dopa-tyrosinase reactions were followed spectrophotometrically. Fifty percent growth inhibition concentrations (IC 50 ) of several chemicals for melanoma cells and non-pigmented cells were measured. Melanogenic activities in HPB-treated melanoma cells were assayed. The results showed that HPB was oxidized by tyrosinase and stimulated tyrosinetyrosinase and dopa-tyrosinase reactions. The IC 50 of HPB for melanoma cells was higher than those of the established depigmenting agents but it was lower than that of HPB for non-melanotic cells. Tyrosine hydroxylase in HPB-treated melanoma cells was stimulated compared with the control, but melanin product in HPB-treated cells was almost similar to the control. The results showed that HPB acts as a good substrate for tyrosinase and it stimulates tyrosinase enzymatic reactions, but it inhibits pigmented cell growth selectively. This study suggests that HPB-induced depigmentation is due to a selective cytotoxic effect on pigmented cells rather than to the inhibition to melanogenesis. (J Occup Health 1998; 40: ) Key words: Occupational leukoderma, Depigmentation, 4-(p-hydroxyphenyl)-2-butanone, Raspberry Ketone, B16 melanoma cells, Tyrosinase Received Aug 8, 1997; Accepted Oct 13, 1997 Correspondence to: Y. Fukuda, Department of Public Health, Kumamoto University School of Medicine, Honjo, Kumamoto , Japan We reported three cases of leukoderma in a chemical factory 1). In one of the workshops, 4-(p-hydroxyphenyl)- 2-butanone (HPB), which is generally called Raspberry Ketone and commercially used as a flavoring agent, was produced. Our report was the first one describing its depigmenting activity, and suggesting that these cases of leukoderma were induced by occupational exposure to HPB. Moreover, our previous study on C57 black mice showed the depigmenting activity of HPB in vivo 2). The topical application of HPB to the dorsal surface induced depigmentation of the animal hairs. But the depigmenting activity was weaker than that of monomethyl ether of hydroquinone (MMH), a well-known depigmenting agent. Little is known about the biochemical properties of HPB, and its depigmenting activity in vitro has not been proved. Several chemicals have been clearly shown to exhibit depigmenting activity 3 13). Two main mechanisms of the chemically-induced depigmentation are considered: inhibition of melanogenesis and melanocyte cytotoxicity. Some of the depigmenting agents had been found to inhibit tyrosinase activity in in vitro studies 10, 14, 15). And others have a selective cytotoxic effect on melanocyte or melanoma cells in vitro 16). The aim of this study is therefore to clarify the biochemical properties and the depigmenting activity of HPB in vitro. Its activity as a substrate for tyrosinase, its effect on tyrosinase enzymatic reactions, the inhibitory effect on melanoma cell and non-pigmented cell growth, and the melanogenic activities of HBP-treated melanoma cells were investigated. Discussion was focused on the potential depigmenting activity of HPB and the mechanisms of the depigmentation. Materials and Methods Chemicals HPB was obtained from the factory where the

2 138 J Occup Health, Vol. 40, 1998 occupational leukoderma had occurred. Its purity was 99.97% as determined by gas chromatography-mass spectrometry assay. MMH and p-tertiary-butylphenol (ptbp) as depigmenting agents and p-hydroxybenzaldehyde as a non-depigmenting agent were purchased from Wako Chemical Ind (Osaka, Japan). Mushroom tyrosinase (EC ) was purchased from Sigma Chemical Co (St. Louis, USA). L-tyrosine and L-dopa (L-3,4-dihydroxyphenylalanine) were purchased from Wako Chemical Ind. L-[3,5-3 H]tyrosine and L-[ 14 C]tyrosine were purchased from Amersham Intl plc (Amersham, UK). Cell lines and tissue culture B16 and B16F10, mouse melanoma cell lines, were used as pigmented cell lines. HT1080, a transformed human fibrosarcoma cell line, was used as a nonpigmented cell line. B16 and HT1080 were purchased from Health Science Research Resources Bank (Osaka, Japan) and B16F10 was described in detail in a previous report 17). The cells were cultured in Dulbecco s modified Eagle s medium with 10% fetal-bovine serum, 4 mm L- glutamine, 100 unit/ml penicillin and 100 µg/ml streptomycin at 37 C in a 5% CO 2 /95% air atmosphere. Activity of HPB as a substrate for mushroom tyrosinase For the enzymatic reactions, optimum conditions of ph, incubation time and the enzyme and substrate concentration were determined. A typical enzymatic reaction was as follows: reaction mixtures contained 0.33 mm HPB in 3 ml of 0.1 M sodium phosphate buffer (PB), ph6.5, and it was oxygenated for 5 min and pre-incubated at 25 C. The enzymatic reaction was then started by adding 100 units of mushroom tyrosinase to the mixtures. The incubations were carried at 25 C, and absorption spectra were scanned with a spectrometer (Beckman DU640). For determination of kinetic constants of HPB, tyrosine, and dopa for tyrosinase, reaction mixtures were prepared by adding different concentrations of substrates ranging from 0.05 mm to 5 mm final concentration in 0.1 M PB (ph 6.5). After adding 100 units of mushroom tyrosinase, the incubations were carried at 25 C, and product formation was followed spectrophotometrically at 290 nm for the HPB-tyrosinase reaction, which was the maximum absorption band of the product in this reaction, at 280 nm for the tyrosine-tyrosinase reaction, and at 475 nm for the dopa-tyrosine reaction. The Km and the Vmax of each chemical was calculated by means of double-reciprocal plots. Effect of HPB on oxidation of tyrosine and dopa by mushroom tyrosinase The reaction mixtures containing various concentrations (range 0.05 mm to 1.0 mm) of tyrosine in 2 ml of 0.1 M sodium phosphate buffer (ph 6.5) and 1 ml of 0.1 M PB (ph 6.5) with HPB (0.5 mm and 1.0 mm) or without HPB were incubated at 25 C after the addition of tyrosinase, and followed spectrophotometrically at 280 nm. Dopatyrosinase was followed at 475 nm. The effect of HPB on oxidation of tyrosine and dopa was investigated by means of double-reciprocal plots. Inhibition of HPB for cell growth Confluence cultured B16 and HT1080 were removed from plastic culture plates with 0.25% trypsine/edta. Cell were placed into Corning 12-well plastic culture plates at a density of cells/well and incubated for 24 h in medium prior to treatment with the chemicals. After 24 h, the medium was replaced with 2 ml of fresh medium. To this was added 20 µl of filter sterilized vehicle (50% propylene glycol, 30% ethanol and 20% distilled water) containing the chemicals HPB, MMH, TBP and p-hydroxybenzaldehyde at various concentrations. Final concentrations ranged from 0.01 mm to 10 mm. This procedure was repeated daily for 3 days with no treatment on the 4th day. On day 5, the remaining adherent cells were assayed. Cell growth of B16 was assayed according to method of Dooley et al. 16) Cultured cells were removed from the medium and washed with PBS 2 times. After washing, the cells were lysed by the addition of 1.0 ml 1N NaOH and repeated manual pipeting. The crude cell extracts were assayed with a spectrophotometer at 400 nm. Cell growth of HT1080 was assayed with crystal violet 16, 18, 19). The medium was decanted from the plates and replaced with 1 ml 0.1% crystal violet in 10% ethanol per well. The plates were stained for 5 min at room temperature on a platform shaker at low speed and then the excess stain was decanted and the entire plates were rinsed briefly 4 times. After rinsing, the plates were inverted and tapped to remove excess water. The crystal violet retained by the adherent cells was then extracted into 2.0 ml of 95% ethanol/well. The plates were rotated on an electric shaker for 15 min at room temperature. The optical densities of the 1.0 ml samples were then determined in a spectrophotometer at 590 nm. Results from duplicate samples were analysed as a percentage of the control vehicle-treated B16 and HT1080. The 50% growth inhibition concentration (IC 50 ) of each chemical was calculated by Probit analysis with SPSS 7.5/PC. Melanogenic activities in HPB-treated melanoma cells B16F10 melanoma cells were cultured and exposed to HPB at concentrations of 0.1 mm and 1 mm, and to MMH at 0.01 mm and 0.1 mm for 5 days the same as in B16 melanoma cell culture. Cells grown in tissue culture were harvested by trypsine/edta treatment, and then

3 Yoshiharu FUKUDA, et al.: In Vitro Studies on 4-(p-Hydroxyphenyl)-2-Butanone Depigmentation 139 Fig. 1. Spectrophotometrical change in 4-(p-hydroxyphenyl)-2-butanone and mushroom tyrosinase reaction. The reaction mixture was 3 ml of 0.1 M sodium phosphate buffer (ph 6.5) containing 0.33 mm 4-(p-hydroxyphenyl)-2-butanone and 100 units of mushroom tyrosinase. Observation interval was 1 min. solubilized with 1% NP ml/ cells at 4 C for 60 h. These cell extracts were used for the [ 3 H]- tyrosine and the [ 14 C]-melanin assays. The [ 3 H]-tyrosine assay and the [ 14 C]-melanin assay for melanogenic activities were performed as previously detailed 20, 21). Briefly, the [ 3 H]-tyrosine assay specifically measures tyrosine hydroxylase activity. This is the initial reaction in the conversion of tyrosine to melanin, and involved the hydroxylation of tyrosine to dopa. The formation of [ 3 H]-water specifically and stoichiometrically released in this reaction was quantified with a liquid scintillation counter. The [ 14 C]-melanin assay measures the production of acid-insoluble melanin from the labeled tyrosine precursor and quantifies the entire reaction sequence, including tyrosinase and post tyrosinase catalytic activities. The production of the acidinsoluble [ 14 C]-melanin is measured with a liquid scintillation counter. Results Activity of HPB as substrate for mushroom tyrosinase Spectrometric change in the reaction mixture containing HPB and tyrosinase is shown in Fig. 1. HPB was rapidly changed by tyrosinase and it produced an increase in absorbency at 250 nm and 290 nm. In tyrosinase enzymatic reactions, the Km of HPB, tyrosine and dopa were 0.52 mm, 0.22 mm and 0.42 mm, respectively. The Vmaxs of the absorbent increase in HPB, tyrosine and dopa were 0.13, and 0.25 ( A/ min), respectively. Effect of HPB on oxidation of tyrosine and dopa by mushroom tyrosinase Double reciprocal plots of the effect of HPB on oxidation of tyrosine and dopa by tyrosinase are shown in Fig. 2, which shows that tyrosinase enzymatic reactions were not inhibited but stimulated. The absorbent increase of oxidation of tyrosine in the mixture with HPB was elevated compared with that without HPB. That of oxidation of dopa with HPB was also increased (Fig. 3). Effect of HPB on cell growth IC 50 s of test chemicals for B16 and HT080 are shown in Table 1. For B16, the IC 50 of HPB was 0.13 mm and it was higher than those of MMH and ptbp but not of p- hydroxybenzaldehyde. For HT1080, the IC 50 of HPB was 1.56 mm and it was higher than those of MMH and ptbp but similar to that of p-hydroxybenzaldehyde. The IC 50 s of HPB, MMH and ptbp for B16 were lower than those for HT1080, but the IC 50 of p- hydroxybenzaldehyde for B16 was higher than that for HT1080. Melanogenic activities in HPB-treated melanoma cells In this experiment, cell growth was only slightly inhibited in 0.1 mm HPB and 0.01 mm MMH exposure but distinctly inhibited in 1.0 mm HPB and 0.1 mm MMH exposure. Figure 4 shows the melanogenic activities in the HPB and MMH-treated melanoma cells. Though melanogenic activities in 0.1 mm HPB treated cells were very similar to the control, those in 1.0 mm HPB treated cells, particularly the activity of tyrosine hydroxylase, were stimulated compared with the control. In contrast, melanogenic activities in both 0.1 mm MMH and 0.01 mm MMH treated cells were inhibited.

4 140 J Occup Health, Vol. 40, 1998 Fig. 2. Effect of 4-(p-hydroxyphenyl)-2-butanone (HPB) on oxidation of tyrosine by mushroom tyrosinase. Fig. 3. Effect of 4-(p-hydroxyphenyl)-2-butanone (HPB) on oxdation of dopa by mushroom tyrosinase. Table 1. Inhibitiory potentiality of chemicals for B16 and HT1080 cell growth IC 50 (mm) a B16 (95% CI) HT1080 (95% IC) Monomethyl ether of hydroquinone (MMH) 0.04 ( ) 0.35 ( ) p-tertiary-butylphenol (ptbp) 0.10 ( ) 0.29 ( ) p-hydroxybenzaldehyde 4.10 ( ) 2.03 ( ) 4-(p-hydroxyphenyl)-2-butanone (HPB) 0.13 ( ) 1.56 ( ) a : 50% growth inhibition concentration. The IC 50 s of MMH, ptbp and HPB for B16 melanoma cell growth were lower than those for non-pigmented cells (HT1080). The results showed that MMH, TBP and HPB selectively inhibit pigmented cell growth. Discussion Naish-Byfield et al. 22) investigated the tyrosinedependent cytotoxicity of 30 compounds and showed that compounds which were good substrates for tyrosinase showed high potential cytotoxicity for melanoma and those which were not oxidized by tyrosinase were nontoxic for the melanoma. Moreover, Inoue et al. 23) investigated the biochemical properties and antimelanoma effect of 4-S-cysteaminylcatechol (4-S-CAC), an activated form of 4-S-cysteaminylphenol (4-S-CAP), and 4-S-CAC was found to be a better substrate for tyrosinase and had a more potent anti-melanoma effect than its isomers. These results suggested that depigmenting agents can be substrates for tyrosinase. This study demonstrated that HPB is a good substrate of tyrosinase as well as of tyrosine and dopa, and that HPB satisfies the conditions of a depigmenting agent. Dooley et al. 16) established the screening method for depigmenting agents and anti-melanoma agents. They tested the cytotoxic effect on melanocyte and a nonpigmented cell line of 50 compounds related to tyrosine, dopa and hydroquinone and showed that hydroquinone, 4-S-CAP and several compounds exhibited high melanocyte-specific cytotoxicity. They concluded that the agents that demonstrated the IC 50 at <100 µg/ml were melanocyte-active and their method was useful as an in vitro screen for skin depigmenting and anti-melanoma agents. In this study, as the IC 50 of HPB for melanoma cells was 0.13 mm (=21.3 µg/ml) and HPB qualifies as a depigmenting agent according to their criteria. The established depigmenting agents, MMH and TBP

5 Yoshiharu FUKUDA, et al.: In Vitro Studies on 4-(p-Hydroxyphenyl)-2-Butanone Depigmentation 141 Fig. 4. Melanogenic activities in B16F10 melanoma cells treated with 4-(p-hydroxyphenyl)-2-butanone (HPB) or monomethyl ether of hydroquinone (MMH). Melanin product activity was mesured by the [ 14 C]- melanin assay and tyrosine hydroxylase activity was measured by the [ 3 H]-tyrosine assay. The activities of melanin product and tyrosine hydroxylase in MMH-treated cells were inhibited compared with the control. In contrast, in HPB-treated cells, tyrosine hydroxylase activity, particulary in 1.0 mm HPBtreated cells, was stimulated, but melanin product activity was similar to the control. had a lower IC 50 for melanoma cells than for nonpigmented cells. The IC 50 of HPB for melanoma cells was also lower than that for non-pigmented cells. These results suggested that, like the established depigmenting agents, HPB had selective cytotoxicity for the melanoma cells. While the biochemical mechanisms of chemicallyinduced depigmentation have not been completely elucidated, two main mechanisms are under consideration: inhibition of melanogenesis and selective cytotoxicity to pigmented cells. Except for a few agents such as arbutin and methylgentisate, which inhibited melanogenesis without cytotoxicity for pigmented cells 14, 16), most depigmenting agents have cytotoxicity to pigmented cells 16). The fact that HPB stimulated tyrosinase enzymatic reactions and melanogenic activities in cultured cells and inhibited the growth of melanoma cells suggested that HPB-induced depigmentation is due not to inhibition of melanogenesis but to the cytotoxicity to pigmented cells. Most of the phenols which induced depigmentation are para-compounds 8), and Riley et al. 24), Ito et al. 25), and Naish-Byfield et al. 22) suggested that ortho-quinones generated from tyrosinase reaction were toxic. Considering the molecular structure of HPB, orthoquinones could be generated by the HPB-tyrosinase reaction. It is very interesting that HPB stimulated the tyrosinase enzymatic reactions in vitro and the melanogenic activities of cultured cells. Several depigmenting agents inhibit tyrosinase reactions as a competitive inhibitor in in vitro studies 10, 26) but p-octylphenol and p-tertiarybutylcathecol stimulate the tyrosinase reaction at low concentrations 10, 15). In recent studies, new enzymes related to melanogenic pathways other than tyrosinase were found, namely tyrosinase relative protein (TRP)-1 and TRP-2 27, 28). In our study, HPB stimulated tyrosine hydroxylase which is the first reaction of the melanogenic pathway for tyrosinase, but HBP did not stimulate total melanin product compared with tyrosine hydroxylase. It is possible that HPB stimulated the tyrosinase reactions but not the TRP-1 and TRP-2 reactions, with no resulting increase in melanin product. Dopa, 5,6-dihydroxyindole, and DHI-2-carboxylic acid, which are intermediate products through the melanogenic pathway, have inherent cytotoxicity for pigmented cells 17). The cytotoxitity of HPB for pigmented cells may be due to the increase in intermediates by melanogenic stimulation of HPB. In any case, this and our companion study 2) confirmed the depigmenting activity of HPB in vivo and in vitro. It was found that HPB acts as a substrate for tyrosinase and that the cytotoxicity is selective for the pigmented cells, but HPB stimulates tyrosinase enzymatic reactions. Considering that, unlike other depigmenting agents, HPB has a keto group in its molecule, it may have unique biochemical properties and a different mechanism of depigmentation from the other agents. Acknowledgments: The authors thank Dr. Shosuke Ito (Institute for Comprehensive Medical Science, Fujita- Gakuen Health University), Toshiro Kageshita and Shunji Hirai (Department of Dermatology, Kumamoto University School of Medicine) for their helpful advice. This study was funded by Higo Medical Foundation (Kumamoto, Japan), and Research grant No from the Japanese Ministry of Education, Science, and Culture. References 1) Fukuda Y, Nagano M, Futatsuka M. Occupational leukoderma of workers engaged in 4-(p-hydroxyphenyl)- 2-butanone manufacturing. J Occup Health 1998; 40: ) Fukuda Y, Nagano M, Arimatsu Y, Futatsuka M. An experimental study on depigmenting activity of 4-(phydroxyphenyl)-2-butanone in C57 black mice. J Occup Health 1998; 40: ) Oliver EA, Schwartz L, Warrex LH. Occupational

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