TEXT

Top Abstract Text References

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether; synonym, Irgasan DP 300) has been used as an antimicrobial compound in deodorants (6), soaps, and dentifrices (2, 8, 36) for many years. The mode of action of triclosan has, however, remained unclear. Recently, it was shown that triclosan blocks one step in bacterial fatty acid synthesis (20, 24, 25). Due to its widespread use, triclosan and some of its derivatives can be detected in the environment (22, 26, 27). The chemical structure of triclosan is related to many compounds which are well-known as xenobiotics, such as halogenated diphenyl ethers. Though some reports on the transformation of halogenated diphenyl ether compounds by bacteria are available (21, 30), triclosan itself was not metabolized by the strains tested (31) or by Rhodococcus chlorophenolicus, which can methylate several other chlorinated hydroxydiphenyl ethers (34). Several hydroxylated metabolites, as well as 2,4-dichlorophenol and 4-chlorocatechol, are formed from triclosan by rats (33), while guinea pigs mostly form glucuronide conjugates (3). No reports exist concerning the degradation of triclosan by fungi. The white rot fungi Trametes versicolor and Pycnoporus cinnabarinus are capable of transforming diphenyl ethers, like 4-chlorodiphenyl ether, up to ring cleavage (10, 11). Hydroxylated biarylic ethers are transformed to oligomerization products by laccases secreted by these strains (13). In this paper we describe some biotransformation reactions of triclosan by the white rot fungi T. versicolor and P. cinnabarinus.

The strains T. versicolor SBUG-M 1050, T. versicolor DSM 11269, T. versicolor DSM 11309, and P. cinnabarinus SBUG-M 1044 were cultivated in a nitrogen-rich (8 mM) medium. The cultures for transformation were prepared with 0.25 mM (wt/vol) triclosan (Mallinckrodt-Baker, Griesheim, Germany) and inoculated and incubated as described previously (10, 11).

Cell extracts were prepared from a 3-day culture of T. versicolor, harvested by centrifugation, and washed twice with 50 mM ice-cold Tris-HCl buffer (pH 7.5). Cells were broken at 1,000 lb/in² using a French press (SLM Amnico, Rochester, N.Y.) and were immediately resuspended in Tris-HCl buffer. The cell extracts (4 ml, approximately 5 mg of protein ml¹) were incubated at 30°C for 24 h with 2.88 mg of triclosan and 1 mg each of UDP-xylose, UDP-glucose, and UDP-glucuronic acid (Sigma, Deisenhofen, Germany).

Analysis of metabolites in culture supernatants and purification of intermediates were done by high-performance liquid chromatography (HPLC) (10).

Gas chromatography-mass spectrometry determinations were performed after extraction of whole cultures with ethyl acetate (11). Nonvolatile intermediates were silylated using a commercial kit (Silyl 991; Macherey & Nagel, Düren, Germany). The carbohydrate moieties of the metabolites were hydrolyzed by acid hydrolysis with 2 ml of concentrated HCl and analyzed by high-pH anion-exchange chromatography combined with a pulsed amperometric detector (Dionex, Idstein, Germany). A Dionex gradient pump (flow rate, 1 ml/min) was used. The carbohydrates were separated on a Dionex Carbo-Pac PA 100 column (250 by 4 mm) by gradient elution using 1.0 M NaOH, deionized water, and 0.5 M sodium acetate as solvents. Nuclear magnetic resonance (NMR) data were recorded on a multinuclear Fourier-transform-NMR ARX 300 spectrometer (Bruker, Rheinstetten, Germany) at 300 MHz (¹H) and 75.5 MHz (¹³C) in deuterated methanol with tetramethylsilane as an internal standard. The detoxification assay using the neutral red test with fibroblast-like cells was performed according to the method of Kusnick (18).

Triclosan (0.25 mM) markedly inhibited growth of T. versicolor SBUG-M 1050 over the first 3 days. However, after 10 days, there were no differences in dry weight recovered from cultures with and without triclosan.

After 48 to 72 h of incubation triclosan began to disappear from the supernatant and two products (products A and B) were detected by HPLC in increasing amounts. Due to their lower retention times (product A, 13.3 min; product B, 12.2 min), they were assumed to be more hydrophilic than triclosan (retention time, 14.9 min).

The UV spectra of these products were very similar to that of triclosan. Both metabolites were properly extractable with ethyl acetate. After acid hydrolysis of the products purified by HPLC and drying under nitrogen, the samples were analyzed by HPLC again, and triclosan was found in both samples. From this fact we inferred that metabolites A and B might be conjugates. Acid hydrolysis and silylation of product A yielded a peak with a mass spectrum characteristic of a silylated pentose. After acid hydrolysis and silylation, product B yielded a substance with a fragmentation pattern typical of hexoses. Furthermore, the initial substance, triclosan, was detected in both samples.

A third product (product C) was formed in small amounts from triclosan by T. versicolor and was identified as 2,4-dichlorophenol by comparison of its retention time and UV and mass spectra with those of a synthetic standard.

Identification of the carbohydrate moieties of products A and B was achieved by using high-pH anion-exchange chromatography and a pulsed amperometric detector. By comparing their retention times with those of known standards, we identified the carbohydrate moiety of product A as xylose and that of product B as glucose. All tested strains of T. versicolor formed these intermediates from triclosan during incubation in nitrogen-rich medium. The formation of the xylose conjugate with UDP-xylose was confirmed by experiments in a cell-free system. In contrast to these results, incubation of cell extracts in the presence of triclosan and UDP--D-glucose resulted in the formation of no metabolites. To provide more detailed information on the structures of metabolites A and B, NMR spectra were recorded. Their ¹H NMR data were as shown in Table 1. The spectra of both products contained six aromatic protons. From this fact it was concluded that the carbohydrates were connected to the hydroxyl group of triclosan. For product A the coupling constant of the proton H-C-1 (beta-D-xyloside) was determined to be 3J(H1,H2) 6.9 Hz, and the corresponding value of the glucose moiety of product B was determined to be 7.8 Hz. Due to the fact that the C-1 protons of -anomers show high coupling constants (approximately 7 to 11 Hz) in comparison to -anomers (3 to 4 Hz), it was concluded that both conjugates were -anomers (23).

The ¹³C NMR spectra of products A and B are shown in Fig. 1. The distortionless enhancement by polarization transfer 135 (DEPT-135) spectrum of metabolite A suggested that there was one secondary carbon atom (xylose, C-5) with two hydrogen substituents. The C-1 signal for xylose was at 102.9 ppm, and because of this characteristic chemical shift it was obvious that the xylose moiety was connected to the hydroxyl group of triclosan with the C-1 atom. The NMR data obtained also point to a pyranoside structure of xylose and glucose which predominates in aqueous solutions (>99.5% at 31°C).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   13C NMR spectra of products A and B of triclosan formed by T. versicolor SBUG-M 1050.

Under the same cultivation conditions, only product B (the glucoside conjugate) was formed by P. cinnabarinus. Furthermore, P. cinnabarinus formed one additional product. By comparing its UV and mass spectra with those of a standard compound synthesized by methylation of triclosan (7), it was determined to be 2,4,4'-trichloro-2'-methoxydiphenyl ether.

Using the neutral red test it was shown that both conjugates formed by T. versicolor were substantially less cytotoxic than triclosan. Both conjugates showed less than 5% inhibition when assayed at a concentration (40 µg per assay) at which triclosan fully inhibited the fibroblast-like cells. Furthermore, growth of Saccharomyces cerevisiae was inhibited by 0.1 mM triclosan but not by 0.25 mM concentrations of the conjugates. By days 3 and 5, most of the triclosan added to cultures of T. versicolor was converted to the conjugates and hence the inhibition of fungal growth was removed. Obviously, the formation of the conjugates represents a fungal mechanism of detoxification of xenobiotics.

White rot fungi are capable of degrading lignin efficiently. Though the degradation is performed mainly by the ligninolytic enzyme system, it has been suggested that glucosylating and xylosylating enzymes may also play a role (10). Probably they are of importance for the detoxification of some specific lignin degradation products (14, 15, 17). Furthermore, they seem to be involved in the detoxification of xenobiotics, like polycyclic aromatic hydrocarbons (4, 5, 32) and dibenzothiophen derivatives (12), or 2-chlorobenzylalcohol (1) and 2,4-dichlorophenol (28). From unhalogenated diphenyl ether and some halogenated derivatives, ring cleavage products, like 6-carboxy-4-phenoxy-2-pyrone, are formed by T. versicolor after several hydroxylations (10, 11). Some yeast strains are also able to degrade diphenyl ether (29), dibenzofuran (9), or biphenyl (19) up to ring cleavage. In contrast to these results, no ring cleavage products were formed from triclosan, and alternatively two carbohydrate conjugates accumulated in cultures of T. versicolor. This may result from the fact that triclosan has two chlorine substituents lying in para position. These substituents prevent the formation of an intermediate with three hydroxyl groups lying next to each other (29), which seems to be essential for the formation of a hydroxyphenoxymuconic acid as a ring cleavage product from diphenyl ether. As shown for monoaromatics (16), the xylosylation of these compounds was shown to be dependent on the presence of UDP-xylose. This suggests the involvement of a UDP-xylosyltransferase in this detoxification reaction. Furthermore, 2,4-dichlorophenol was formed from triclosan by T. versicolor SBUG-M 1050. Reddy et al. (28) showed that 2,4-dichlorophenol, a metabolite from 2,4-dichlorophenoxyacetic acid, was xylosylated by Dichomitus squalens. In our experiments, gas chromatography-mass spectrometry analysis of a silylated extract from T. versicolor incubated in the presence of triclosan yielded one peak with a spectrum of fragments typical for a dichlorophenol as well as for a silylated carbohydrate. Furthermore, HPLC analysis of concentrated extracts showed a peak with a lower retention time than 2,4-dichlorophenol but a very similar UV spectrum. Thus, it seems that 2,4-dichlorophenol was xylosylated in our experiments, too.

The fungus P. cinnabarinus SBUG-M 1044 methylated the hydroxyl group of triclosan during cultivation. Interestingly, methylated triclosan was reported to be present in environmental samples (26), although the origin remained unclear. In any case, several bacterial strains which were able to transform other chlorodiphenyl ether derivatives did not metabolize this substance (31, 34, 35).

To our knowledge, this is the first report about fungal biotransformation of triclosan. Besides the known hydroxylation and ring cleavage reactions of some diphenyl ethers (29), glycosyl conjugates and O-methylated intermediates can also be formed from these compounds by fungi.