Genotoxic potential evaluation of a cosmetic insoluble substance by the micronuclei assay

N. Dayan; V. Shah; T. Minko

TEST FOR GENOTOXIC POTENTIAL BY MICRONUCLEI ASSAY 39 (15) L. M. Green, J. L. Reade, and C. F. Ware. Rapid colometric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines, J. Immunol. Meth., 70, 257–268 (1984). (16) H. Ahu, I. Rusyn, A. Richard, and A. Tropsha, Use of cell viability assay data improves the prediction accuracy of conventional quantitative structure activity relationship models of animal carcinogenicity, Environ. Health Perspect., 116(4), 506–513 (2008).

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TEST FOR GENOTOXIC POTENTIAL BY MICRONUCLEI ASSAY 39 (15) L. M. Green, J. L. Reade, and C. F. Ware. Rapid colometric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines, J. Immunol. Meth., 70, 257–268 (1984). (16) H. Ahu, I. Rusyn, A. Richard, and A. Tropsha, Use of cell viability assay data improves the prediction accuracy of conventional quantitative structure activity relationship models of animal carcinogenicity, Environ. Health Perspect., 116(4), 506–513 (2008).

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TEST FOR GENOTOXIC POTENTIAL BY MICRONUCLEI ASSAY 37 CONCLUSIONS AND FUTURE ASPECTS Under the conditions of the study, the test substance, OB, did not show promotion of the generation of micronuclei with and without enzymatic activation. The measurements of cellular viability showed that cyclosphosphamide + S9 mix (10 μg/ml), ethyl methane- sulfonate (EMS, 400 μg/ml), or OB (0.2 mg/ml and 0.3 mg/ml) alone or in combination with S9 did not inhibit the growth of cells (Figure 4) at the concentrations used. Conse- quently, we conclude that all tested substances do not promote cell death at the concen- trations used and that the positive controls may have generated a shift in cellular function that led to enhanced proliferation. While this method indentifi es the genotoxic potential of the tested compounds, there are ad- ditional end points that can be studied to better assess the effect of the compounds tested on cell survival. Such is the characterization of cell death by either apoptosis or necrosis. In addition, this study demonstrates a way to test a compound that is not fully solublized in the media. Stages of introduction of the OB to the cell culture included dissolution in Figure 3. Number of micronuclei per 1000 cells. CHO-K1 cells were incubated for 24 hours with the fol- lowing substances: (1) Media (negative control) (2) DMSO (negative control) (3) Cyclosphosphamide + S9 mix (positive control) (4) Ethyl methanesulfonate (positive control) (5) OB, 0.2 mg/ml in DMSO (6) OB, 0.3 mg/ml in DMSO (7) OB, 0.2 mg/ml in DMSO + S9 mix and (8) OB, 0.3 mg/ml in DMSO + S9 mix. Means ± S.D. are shown. *P0.05 when compared with negative control. Figure 4. Cellular viability. CHO-K1 cells were incubated for 24 hours with the following substances: (1) DMSO (negative control) (2) Cyclophosphamide + S9 mix (positive control) (3) Ethyl methanesulfonate (positive control) (4) OB, 0.2 mg/ml in DMSO (5) OB, 0.3 mg/ml in DMSO (6) OB, 0.2 mg/ml in DMSO + S9 mix and (7) OB, 0.3 mg/ml in DMSO + S9 mix. Means ± S.D. are shown (1).

JOURNAL OF COSMETIC SCIENCE 38 DMSO (which had no effect on the cells when tested alone), sonication, centrifugation, and drawing the supernatant. This is to mimic real-life exposure wherein the particle does not penetrate healthy intact skin, but its impurities and residuals can dissolve in skin fl uids. REFERENCES (1) A. Vanhauwaert, P. Vanparys, and M. Kirsch-Volders, The in vivo gut micronucleus test detects clasto- gens and aneugens given by gavage, Mutagenesis, 16, 39–50 (2001). (2) B. Miller, F. Potter-Locher, A. Seelbach, H. Stopper, D. Utesch, and S. Madle, Evaluation of the in vitro micronucelus test as an alternative to the in vitro chromosomal aberration assay: Position of the GUM Working Group on the in vitro micronucleus test, Mutat. Res., 410, 81–116 (1998). (3) OECD Guideline for the Testing of Chemicals # 487: In Vitro Micronucleus Test. Proposed on December 21, 2006. (4) ECVAM statement: http://ecvam.jrc.it/publication/ESAC25_statement_MNT_20061128_C.pdf. Retrieved on October 4, 2010. (5) Guidelines for Mutagenicity Risk Assessment. EPA Publications. Federal Register, 51(185), 34006– 34012 (1986). (6) M. Kirsche-Vilders, M. Sofuni, T. Aardema, S. Albertini, D. Eastmond, M. Fenech, M. Ishidate Jr., S. Kirchner, E. Lorge, T. Morita, H. J. Norppa, A. Surralles Vanhauwaert, and A. Wakata, Report from the in vitro micronucleus assay working group, Mutat. Res., 540, 153–163 (2003). (7) The Micronucleus Assay: LPI/CCP protocol for in vitro micronucleus assay using CHO-K1 cells, http:// lpi.oregonstate.edu/CCP/micronucleus.html. Oregon State University Corvallis. Accessed on December 4, 2009. (8) M. Fenech, The cytokinesis-block micronucleus technique: A detailed description of the method and its application to genotoxicity studies in human populations, Mutat. Res., 285, 35–44 (1993). (9) P. I. Countryman and J. A. Heddle, The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes, Mutat. Res, 41, 321–332 (1976). (10) Commonly used tissue culture techniques, Sigma Catalog, 1989, p. 1492. (11) O. Biondi, S. Motta, and P. Mosesso, Low molecular weight polyethylene glycol induce chromosome aberration in Chinese hamster cells cultured in vitro, Mutagenesis 17(3), 261–164 (2002). (12) SCCNFP/0532/01, Final opinion of the scientifi c committee on cosmetic products and non-food prod- ucts intended for consumers concerning benzoic acid and sodium benzoate (June 2002). (13) R. I. Pakunlu, Y. Wang, W. Tsao, V. Pozharov, T. J. Cook, and T. Minko, Enhancement of the effi cacy of chemotherapy for lung cancer by simultaneous suppression of multidrug resistance and antiapoptotic cellular defense: Novel multicomponent delivery system, Cancer Res., 64, 6214–6224 (2004). (14) J. M. Parry and E. M. Parry, The use of the in vitro micronucleus assay to detect and assess the aneugenic activity of chemicals, Mutat. Res., 607, 5–8 (2006). Figure 5. Infl uence of test substances on cellular proliferation. The same number of cells (3 × 104) were seeded in the fl ask and incubated with media (1) and a test substance in two concentrations: 0.2 mg/ml (2) and 0.3 mg/ml (3). Cells were counted 48 h after the addition of the test substances. Means ± S.D. are shown from four independent measurements.

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J. Cosmet. Sci., 62, 41–48 (January/February 2011) 41 Inhibitory effect of a water extract from Pemphis acidula on melanogenesis in mouse B16 melanoma cells HSIOU-YU DING, TE-SHENG CHANG, CHIEN-MIN CHIANG, and SORGAN SHOU-KU TAI, Institute of Cosmetics Science, Chia Nan University of Pharmacy and Science (H.-Y.D.), Department of Biological Science and Technology, National University of Tainan (T.-S.C.), Department of Biotechnology, Chia Nan University of Pharmacy and Science (C.-M.C.), and Department of Biotechnology, National Formosa University (S.S.-K.T.), Taiwan, R.O.C. Accepted for publication November 3, 2010. Synopsis The inhibitory effect of a water extract from Pemphis acidula on melanogenesis in mouse B16 melanoma cells was investigated. The results showed that the P. acidula extract (PAE) inhibited melanogenesis in 3-isobutyl- 1-methylxanthin (IBMX)-stimulated B16 cells in a dose-dependent manner, with an IC50 value of 33.5 μg/ml. In addition, PAE also inhibited cellular tyrosinase activity. Moreover, western blot and real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analyses respectively confi rmed that PAE down-regulated levels of tyrosinase protein and its mRNA in IBMX-stimulated B16 cells. These results demonstrated that PAE inhibits melanogenesis of B16 cells by reducing tyrosinase gene expression. From the present study, PAE is proven to be a good candidate as a skin-whitening agent for treatment of skin hyperpigmentation. INTRODUCTION Skin pigmentation is produced by dermal melanocytes. Melanogenesis has been defi ned as the entire process leading to the formation of dark macromolecular pigments, i.e., melanin, which are formed by a combination of enzymatically catalyzed and chemical reactions (1). Melanogenesis is initiated in melanosomes, the special organelles of mela- nocytes, with the fi rst step of l-tyrosine oxidation to l-dopa (l-3,4-dihydroxyphenylala- nine) and then to dopaquinone, which is catalyzed by tyrosinase. This is a rate-limiting step in melanin synthesis because the remainder of the reaction sequence can proceed spontaneously at a physiological pH value. Although melanin mainly plays a photopro- tective role, the accumulation of abnormal amounts of melanin in different parts of the skin, which results in pigmented patches of skin, might become an esthetic problem. Address all correspondence to Sorgan Shou-Ku Tai at 64 Wun-Hua Road, Huwei, Yunlin, Taiwan, R.O.C. (sorgan@nfu.edu.tw).

JOURNAL OF COSMETIC SCIENCE 42 Therefore, several studies have focused on the inhibition of tyrosinase activity and the prevention of abnormal pigmentation (2,3). A previous study has reported that some cytokines and growth factors play important regulatory roles in melanogenesis (4). α-Melanocyte stimulating hormone (α-MSH) is the most well-studied hormone. This hormone binds to its receptor, melanocortin recep- tor 1 (MC1R), on the membrane of melanocytes and stimulates melanogenesis via the GPCR (G protein-coupled receptor)-cAMP-MITF (microphthalmia-associated transcrip- tion factor) pathway where the melanogenesis-related enzymes, including tyrosinase and tyrosinase-related proteins 1 and 2 (TRP1 and TRP2) are up-regulated. In addition to α-MSH, other intracellular cAMP elevation agents such as forskolin or 3-isobuty1-1- methylxanthin (IBMX) also stimulate melanogenesis through the same signal pathway as does α-MSH. Accordingly, agents blocking the signal pathway would exhibit depigmen- tation against melanocytes (5,6). In the present study, we screened more than 200 crude extracts of traditional Chinese medicinal herbs to identify their applicability as skin-lightening agents. The P. acidula extract (PAE) was found to have strong inhibitory activity on melanogenesis in mouse B16 melanoma cells. The inhibitory effect of the extract on melanogenesis of the cells was investigated in advance. MATERIALS AND METHODS PREPARATION OF PAE The dried powder of the bark (235.0 g) of P. acidula was extracted with one liter of water at room temperature overnight four times, followed by fi ltration at the end of each extrac- tion. The fl ow-through extracts were concentrated in a vacuum and combined to yield a black syrup (14.3 g). CHEMICALS AND ANTIBODIES Arbutin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, phenylmethylsulfonyl fl uoride (PMSF), l-dopa, dimethyl sulfoxide (DMSO), trypsin/EDTA, synthetic melanin, and IBMX were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-tyrosinase antibodies (#62914) and protease inhibitor cocktail were obtained from Abcam (Cambridge, MA). Anti-β-actin antibodies (#3662) were pur- chased from Bio Vision Inc. (Irvine, CA). All other chemicals were obtained from Tokyo Chemical Industry Co. (Tokyo, Japan) and were of analytic reagent grade. CELL CULTURES AND DRUG TREATMENTS Mouse B16 melanoma cells (4A5) were obtained from the Bioresources Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan, ROC). The cells were cultured in Dulbecco’s modifi ed Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum at 37°C in a humidifi ed, CO2-controlled

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