Mycopathologia (2011) 171:267–271 DOI 10.1007/s11046-010-9370-x
In Vitro Antifungal Activity of Dihydroxyacetone Against Causative Agents of Dermatomycosis Cheila Denise Ottonelli Stopiglia • Fabiane Jamono Vieira Andressa Grazziotin Mondadori • Te´rcio Paschke Oppe • Maria Lu´cia Scroferneker
•
Received: 29 May 2010 / Accepted: 22 September 2010 / Published online: 9 October 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Dihydroxyacetone (DHA), a three-carbon sugar, is the browning ingredient in commercial sunless tanning formulations. DHA preparations have been used for more than 50 years and are currently highly popular for producing temporary pigmentation resembling an ultraviolet-induced tan. In this work, the in vitro antifungal activity of dihydroxyacetone was tested against causative agents of dermatomycosis, more specifically against dermatophytes and Candida spp. The antifungal activity was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute C. D. O. Stopiglia M. L. Scroferneker Graduate Program in Medicine, Medical Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil C. D. O. Stopiglia F. J. Vieira A. G. Mondadori M. L. Scroferneker Laboratory of Pathogenic Fungi, Department of Microbiology, Institute of Basic Health Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil T. P. Oppe School of Pharmacy, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil C. D. O. Stopiglia (&) Instituto de Cieˆncias Ba´sicas da Sau´de, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 763/307, Porto Alegre, RS CEP 90050-170, Brazil e-mail:
[email protected];
[email protected]
guidelines for yeasts and filamentous fungi. The data obtained show that the fungicidal activity varied from 1.6 to 50 mg ml-1. DHA seems to be a promising substance for the treatment of dermatomycosis because it has antifungal properties at the same concentration used in artificial suntan lotions. Therefore, it is a potential low-toxicity antifungal agent that may be used topically because of its penetration into the corneal layers of the skin. Keywords Candida spp. Dermatophytes Antifungal activity Dihydroxyacetone Fungicidal activity
Introduction Dermatomycoses are fungal infections that involve the horny layer of the skin and the appendages, such as nails, hair, and the surface of mucous membranes [1]. They cause high morbidity, and according to a recent report, their world incidence has increased by 50% in patients older than 25 years and particularly in immunocompromised patients [2]. Although many antifungal drugs have been developed during the last two decades, they are confined to a relatively few chemical classes, and some of them exhibit problems such as poor efficacy, side effects, toxicity, drug interactions, and development of resistance [3–7]. Moreover, the activity spectrum of these drugs is
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variable, leading to therapeutic failure in 25–40% of treated patients [8]. With all these factors considered, there is an urgent need for novel drugs that combine safety with antifungal efficacy against the most common pathogens and against some fluconazole-resistant and amphotericin B-resistant agents [3]. The development of resistance to azole antifungal drugs, especially to fluconazole and itraconazole, is an emerging trend that may threaten the clinical effectiveness of these drugs. Moreover, resistance to one azole is usually associated with cross-resistance to other azoles, and consequently, the treatment of dermatomycoses has been problematic [4, 9–11]. Dihydroxyacetone (DHA), a three-carbon sugar, is the browning ingredient in commercial sunless tanning formulations. DHA preparations have been used for more than 50 years and are currently highly popular for producing temporary pigmentation resembling an ultraviolet-induced tan [12]. Besides its cosmetic use, there are reports of application of DHA for camouflaging segmental psoriasis [13], vitiligo and piebald lesions [14, 15]. The aim of this work was to evaluate the activity of dihydroxyacetone against dermatophytes and Candida species, determining the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC).
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Procedures to Evaluate Antifungal Activity The minimum inhibitory concentrations (MIC) of itraconazole, nystatin, and dihydroxyacetone (DHA) were determined by using broth microdilution techniques as described by the Clinical and Laboratory Standards Institute for yeasts (M27-A2) [16] and for filamentous fungi (M38-A) [17]. Culture Medium The culture medium used during the experiments was RPMI 1640 (Gibco) with l-glutamine and without sodium bicarbonate, pH 7.0, with morpholinepropanesulfonic acid (MOPS; 0.165 mol l-1; Sigma–Aldrich). The medium was filter-sterilized in 0.22-lm membrane (Millipore) [17]. Antifungal The commercial drugs used were itraconazole (Jansen-Cilag) and nystatin (Genix) prepared in dimethyl sulfoxide (DMSO; Vetec). The solutions were diluted in RPMI 1640 and the final drug concentrations ranged from 0.03 to 16 lg ml-1 [17]. The commercial DHA (Merck) was prepared in sterile distilled water. Ten different concentrations were used, ranging from 0.195 to 100 mg ml-1. Inoculum Preparation
Materials and Methods Fungal Strains A total of 48 strains were used. Thirty-four belonged to Candida species—Candida albicans (American Type Culture Collection—ATCC 10231, ATCC 18804, ATCC 28367, 0050-L, 0051-L, MG), C. dubliniensis (22, 23, 25, 27, 28, 29, ATCC 7987), C. glabrata (0030-L, 993, ATCC 2001, MG), C. guilliermondii (0031-L, 168, 992), C. krusei (ATCC 6258, ATCC 20298, 0037-L, 219, 990, MG), C. parapsilosis (ATCC 22019, 0052-L, 0053L, 0054-L, MG), C. tropicalis (0056-L, ATCC 750, 0055-L), and 14 dermatophyte strains - Microsporum gypseum (35, 44000, 44704), M. canis (22, 48, 43979), Trichophyton mentagrophytes (23, 59, 64, 85), T. rubrum (43316, 43553), and T. interdigitale (72, 87).
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Candida strains were subcultured onto Sabouraud dextrose agar (Difco) at 35°C for 24 h. The inoculum was suspended in saline solution. The dermatophyte strains were subcultured onto potato dextrose agar (PDA/Difco) at 28°C for 7 days. The surface was gently scraped with a sterile bent glass rod after flooding with sterile saline solution. The standard suspensions were adjusted and diluted in RPMI 1640 and MOPS to obtain a final inoculum of 103 and 104 CFU ml-1 for Candida and dermatophyte strains, respectively. Testing Procedures Sterile flat-bottomed 96-well microtiter plates (Cral Plast) were used, with addition of 100 ll of each drug to columns 1 to 10; 100 ll of RPMI 1640 medium was added in columns 11 and 12, which were used as
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the drug-free growth control well. The MIC was defined as the lowest concentration of the antifungal agent preventing visible fungal growth. In order to determine the minimum fungicidal concentration (MFC), 100 ll of the well with 100% of growth inhibition was seeded into culture tubes with 2 ml of Sabouraud dextrose broth medium. The tubes with Candida species and dermatophytes were
growth positive and medium sterile controls, respectively. Aliquots of 100 ll of the aforementioned standardized inoculum were added to the wells, and the microtiter plates with Candida species were incubated at 35°C for 24 h while those with dermatophytes were incubated at 28°C for 7 days. The determination of the minimum inhibitory concentration (MIC) was performed by visual observation with
Table 1 Range of antifungal activities of dihydroxyacetone, nystatin, and itraconazole against clinical isolates of Candida spp. and dermatophytes Dihydroxyacetone (mg ml-1)
Itraconazole (lg ml-1)
Nystatin (lg ml-1)
MICa
MFCb
MICa
MFCb
MICa
MFCb
Range GMd
12.5–25 19.8
25–50 35.3
0.5–1 0.9
[16 [16
1–2 1.8
2–4 3.6
Range
12.5–25
25–50
0.03–0.25
0.25–[16
1–2
2–8
GM
22.6
33.6
0.1
1.6
1.6
3.2
Range
12.5–25
25–50
0.06–1
0.25–[16
1–2
2–8
GMd
17.7
29.7
0.3
2.4
1.4
4.8
Strain
C. albicans (6)
c
C. dubliniensis (7)c
d
C. glabrata (4)c C. guilliermondii (3)c C. krusei (6)c C. parapsilosis (5)c C. tropicalis (3)c
Range
12.5–25
25
0.12–0.25
0.25–8
1–2
2
GMd
19.8
25
0.2
1
1.2
2
Range
12.5–50
25–50
0.12–0.5
0.5–8
2–4
4–8
GMd
22.3
35.4
0.3
1.8
2.2
4.5
Range
6.25–25
25–50
0.12–0.25
0.5–[16
2
4–8
GMd
16.5
28.7
0.2
1.3
2
5.3
Range
12.5–25
25
0.12–2
[16
2
2–4 3.2
d
GM
15.7
25
0.4
[16
2
All Candida strains (34)c
Range
6.2–50
25–50
0.03–2
0.25–[16
1–4
2–8
M. gypseum (3)c
GMd Range
19.6 1.6–3.1
31.3 3.1
0.3 0.12–0.25
2.9 0.5
1.8 0.5–2
4 0.5–4
GMd
2
3.1
0.2
0.5
1
1.6
Range
1.6–3.1
1.6–3.1
0.12
0.12–[16
1–4
2–8
GM
2
2.5
0.12
0.99
2
4
Range
1.6–3.1
1.6–3.1
0.5
1–[16
1–4
1–4
GMd
2.2
2.2
0.5
4
2
2
M. canis (3)c
d
T. rubrum (2)c T. mentagrophytes (4)c T. interdigitale (2)c All dermatophyte strains (14)c All strains (48)c
Range
3.1
3.1–6.2
0.12–0.25
0.12–16
1–4
1–4
GMd
3.1
4.4
0.17
0.99
2.4
2.4
Range
3.1–6.2
3.1–6.2
0.25
0.5–16
2–4
2–4
GMd
4.4
4.4
0.25
2.8
2.8
2.8
Range
1.6–6.2
1.6–6.2
0.12–0.25
0.12–[16
0.5–4
0.5–8
GMd
2.4
3.3
0.2
14.7
1.9
2.4
Range
1.6–50
1.6–50
0.03–2
0.12–[16
0.5–4
0.5–8
10.8
16.2
0.2
2.3
1.8
3.3
d
GM a
Minimum inhibitory concentration
b
Minimum fungicidal concentration
c
Number of isolates
d
Geometric mean
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incubated at 35°C for 3 days and at 28°C for 10 days, respectively, in order to determine fungal growth. The MFC was the minimum fungistatic concentration that prevented fungal growth [18].
Results DHA showed fungicidal activity against Candida strains and dermatophytes at the same concentrations used in artificial suntan lotions (Table 1). Considering the accepted clinical standard of resistance limit to itraconazole [16, 17], our results show different levels of resistance of Candida strains to itraconazole (66, 25, and 33% for C. albicans, C. glabrata, and C. tropicalis, respectively) and different fungicidal activity (all strains of C. albicans and C. tropicalis, 29% of C. dubliniensis, 50% of C. glabrata, and 20% of C. parapsilosis were resistant to itraconazole). As to the MIC of itraconazole against dermatophytes, 100% of M. canis, 30% of M. gypseum, and 50% of T. mentagrophytes strains were sensitive to this antifungal agent, whereas the remaining strains exhibited dose-dependent sensitivity. However, the MFC shows that only 25% of T. mentagrophytes and 50% of M. canis strains were sensitive to itraconazole, while 25, 33, 50, and 50% of T. mentagrophytes, M. canis, T. rubrum, and T. interdigitale strains, respectively, were resistant to this agent.
Discussion Considering the development of resistance and the adverse effects of the available antifungal drugs, there is a need for new pharmacological agents. Dihydroxyacetone (DHA) has been used for over half a century in sunless tanning products to darken the skin. The color emerges by non-enzymatic glycosylation of skin proteins in the stratum corneum, a process known as the Maillard reaction [19, 20]. Since DHA is covalently bound to free amino groups, it is not easily washed off and the color lasts for several days. The concentration range of DHA in self-tanning products can range from 25 to 100 mg ml-1 [21], being used as lotions, oils, creams, or gels. Although the use of sunless tanning products has been widespread, the antifungal activity of DHA
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against pathogenic fungi had not yet been described. This work shows the antifungal activity of DHA, which inhibits fungal growth, demonstrating fungicidal activity at concentrations close to the ones used with tanning purposes. Our results show that all strains of C. albicans and dermatophytes tested were sensitive to DHA in the concentration range analyzed and had fungicidal activity. Moreover, given the chemical characteristics of DHA and its toxicological profile, this agent can be considered nontoxic. It reacts quickly in the stratum corneum, thus minimizing systemic absorption. The acute toxicity of DHA was investigated in diabetics in the 1920s, proving to be well tolerated when given orally. The DHA phosphate is found naturally as one of the intermediates in the Kreb’s cycle [21, 22]. No adverse effects have been observed with the use of DHA creams in animal and human studies, which indicate that DHA is nontoxic and noncarcinogenic [14, 22–24]. Therefore, we acknowledge the potential value of DHA for the treatment of dermatomycoses because of its fungicidal activity, lower cost, and lower toxicity. Acknowledgments The authors are grateful to CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior), CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico), and FAPERGS (Fundac¸a˜o de Amparo a` Pesquisa do Rio Grande do Sul) for their financial support.
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