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Applied and Environmental Microbiology, December 2007, p. 7759-7762, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01410-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Anaerobic Metabolism of 1-Amino-2-Naphthol-Based Azo Dyes (Sudan Dyes) by Human Intestinal Microflora{triangledown}

Haiyan Xu,1 Thomas M. Heinze,2 Siwei Chen,1 Carl E. Cerniglia,1 and Huizhong Chen1*

Division of Microbiology,1 Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079-95022

Received 25 June 2007/ Accepted 5 October 2007


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ABSTRACT
 
The rates of metabolism of Sudan I and II and Para Red by human intestinal microflora were high compared to those of Sudan III and IV under anaerobic conditions. Metabolites of the dyes were identified as aniline, 2,4-dimethylaniline, o-toluidine, and 4-nitroaniline through high-performance liquid chromatography and liquid chromatography electrospray ionization tandem mass spectrometry analyses. These data indicate that human intestinal bacteria are able to reduce Sudan dyes to form potentially carcinogenic aromatic amines.


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INTRODUCTION
 
Human exposure to azo dyes occurs through ingestion, inhalation, or skin contact. The human skin and gastrointestinal tract harbor a complex and diverse microflora composed of at least several thousand species (10, 12). This microflora also plays roles in the degradation of azo dyes, with azo reduction being the most important reaction related to toxicity and mutagenicity (9, 21). Ingested azo dyes are mainly metabolized by intestinal microflora to colorless aromatic amines by NAD(P)H-dependent azoreductases (5-9, 14, 17). There has been concern about contamination of hot chili, other spices, and baked foods with 1-amino-2-naphthol-based azo dyes (Sudan I, II, III, and IV and Para Red) (4, 16, 23). There is evidence that Sudan dyes have genotoxic effects (1, 18-20) and that ingestion of food products contaminated with Sudan I, II, III, and IV and Para Red could lead to exposure in the human gastrointestinal tract. Although azo dyes can be reduced by the mammalian liver to form aromatic amines, it has been suggested that intestinal microflora could be primarily responsible for the in vivo reduction of azo dyes (5, 9). Besides a few studies related to the metabolism of Sudan I in rats and rabbits, no reports regarding the metabolism of other Sudan dyes by the intestinal microflora have been published. In addition, attempts to isolate potential toxic metabolites, such as aromatic amines, of any of the Sudan dyes are limited (5, 9). Moreover, there are potential problems in translating the results obtained with animal models to humans. These problems include significant differences in the composition of the intestinal microflora and the difficulty in separating the metabolism of microbes from that of animals (15). The recent detection of Sudan dyes in various food commodities requires toxicological evaluation by regulatory agencies to determine the impact of these Sudan dyes on human health (4, 11, 16, 22, 23). Our investigation provides evidence for the importance of the human intestinal microflora in Sudan dye metabolism. In this study, we demonstrated that Sudan dyes were metabolized to potentially carcinogenic aromatic amines by intestinal microorganisms.


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Time course of Sudan dye degradation.
 
The potential for growth-linked decolorization of Sudan dyes by the intestinal microflora was investigated with a time course experiment. Stock solutions of Sudan I, II, III, and IV were made by dissolving each dye in 100% ethanol (1 mg/ml); that for Para Red was made by dissolving the dye in dimethyl sulfoxide (1 mg/ml). Fresh diluted human fecal suspensions (6 ml; 10%, wt/vol) were transferred under anaerobic conditions into flasks containing 300 ml brain heart infusion broth to observe the effect of the microflora on decolorization of the individual Sudan dyes (Fig. 1). Samples (supernatants, cell extracts, and debris) were extracted with ethyl acetate to ensure that dye bound to bacterial cells could be released from the cells as well. Each residue was dissolved in 1 ml acetonitrile, and 40 µl of each sample was analyzed by high-performance liquid chromatography with a Hewlett-Packard 1050 equipped with a variable-wavelength detector (the detection wavelengths used were 250 and 500 nm) and a reversed-phase Luna C18 (2) column (Fig. 1). The peak area was used to calculate the concentration of Sudan dye. Reduction of Sudan dyes was determined by monitoring the disappearance of the absorption peak for each dye at 500 nm and by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) when the cultures were extracted with ethyl acetate as well. After a lag of 4 h, the Sudan dyes began to be reduced. Sudan I was reduced more rapidly than the other dyes. Approximately 75% of the Sudan I present was metabolized in 16 h, and the dye completely disappeared after 20 h. The efficiencies of Para Red and Sudan II reduction in cultures were similar to that of Sudan I reduction. After 16 h, about 70% of the Para Red and 44% of the Sudan II present were reduced. Both dyes were completely metabolized in 24 to 30 h. The diazo Sudan dyes Sudan III and IV were reduced much more slowly than the monoazo Sudan dyes Sudan I and II and Para Red, probably because of the solubility and availability of the diazo dyes (3). When a higher concentration (10 µg/ml) of the diazo Sudan dyes was present in cultures, it was difficult to observe reduction of the dyes. However, at lower concentrations (0.3 to 0.5 µg/ml), dye reduction was observed. Both diazo Sudan dyes were reduced in 24 to 32 h (Fig. 1). The metabolites from Sudan dyes were detected by LC/ESI-MS/MS after 4 h incubation, and the amounts of the metabolites increased with the incubation time. No reduction of the tested Sudan dyes was observed in uninoculated controls.


Figure 1
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  		FIG. 1. Extent of metabolism of Sudan dyes by the human intestinal microflora. The concentrations used were as follows: Sudan I, 10 µg/ml; Sudan II, 10 µg/ml; Sudan III, 0.3 µg/ml; Sudan IV, 0.5 µg/ml; Para Red, 10 µg/ml. Error bars represent the standard deviations of triplicate cultures.


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Effects of Sudan dyes on the growth of the intestinal microflora.
 
Bacterial density in the cultures was determined by measuring the optical density of cultures at 600 nm (not shown). In the medium without Sudan dyes, maximal cell growth was obtained after 24 h. In the medium with Sudan I, II, III, and IV (10 µg/ml), a lag phase of 2 to 4 h was observed, with a final cell density of about 90% of that obtained without Sudan dyes. Para Red (10 µg/ml) had a minor inhibitory effect on the growth of the bacteria, with a lag phase of 6 h. The moderate inhibition of the growth of the intestinal microflora by the Sudan dyes indicated that the dyes were not toxic to the human intestinal microflora.


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Identification of metabolites of Sudan dyes degraded by the intestinal microflora.
 
To identify the Sudan dye metabolites, separate experiments were conducted in which the human intestinal microflora was incubated with Sudan I, II, III, or IV or Para Red for 30 h. Ethyl acetate extracts of incubation cultures were dried, and the residues were extracted with starting buffer (5% acetonitrile, 94.9% water, 0.1% formic acid). Much of the dried sample was insoluble, and soluble metabolites were analyzed by LC/ESI-MS/MS. For analyses of Sudan dye metabolites, the starting buffer composition was held for 20 min, ramped quickly (in 1 min) to 95% acetonitrile-4.9% water-0.1% formic acid at 21 min, and held to 40 min. For the Para Red metabolite, the method was modified to a linear 21-min gradient of 5% acetonitrile-94.9% water to 95% acetonitrile-4.9% water with a constant 0.1% formic acid.

Product ion spectra, retention times (Rts), and UV data for metabolites were compared to those for authentic compounds for identification. The protonated molecules typically lost 17 Da from aromatic amines and 15 Da from aromatic amines with a methyl substituent. The metabolites with an Rt of 4.1 min from Sudan I and III were identified as aniline on the basis of their Rts and product ion mass spectra (Fig. 2A and C). The protonated molecule at m/z 94 was fragmented to give ions at m/z 77 [MH+-17] and 51. The metabolite with an Rt of 18.7 min from Sudan II was identified as 2,4-dimethylaniline on the basis of its Rt and product ion mass spectrum (Fig. 2B). The protonated molecule at m/z 122 was fragmented to give ions at m/z 107 [MH+-15], 105 [MH+-17], 103, 79, and 77. The metabolite with an Rt of 7.5 min from Sudan IV was identified as o-toluidine on the basis of its Rt and product ion mass spectrum (Fig. 2D). The protonated molecule at m/z 108 was fragmented to give ions at m/z 93 [MH+-15], 91 [MH+-17], and 65. The metabolite with an Rt of 17.9 min from Para Red was identified as 4-nitroaniline on the basis of its Rt and product ion mass spectrum (Fig. 2E). The protonated molecule at m/z 139 was fragmented to give ions at m/z 122 [MH+-17], 93, 92, and 65. Traces of aniline were also found in the Sudan I and III controls but were only 4.9 and 1.8%, respectively, of those of the samples metabolized by the intestinal microflora. No 2,4-dimethylaniline or o-toluidine was found in the Sudan II or IV control. Because of impurity, some 4-nitroaniline was found in the Para Red control, which was 19.9% of that found in the samples metabolized by the intestinal microflora. Concentrations of 2.6 µg/ml aniline (71.4%), 2.3 µg/ml 2,4-dimethylaniline (52.8%), 0.054 µg/ml aniline (67.6%), 0.11 µg/ml o-toluidine (75.3%), and 2.5 µg/ml 4-nitroaniline (53.7%) were detected in the metabolites of Sudan I, II, III, and IV and Para Red, respectively. Interestingly, the expected 1-amino-2-naphthol from all of the Sudan dyes tested, 1,4-phenylenediamine from Sudan III, and 2,5-diaminotoluene from Sudan IV (Fig. 3) could not be detected in the extracted samples. Some brown substances in the metabolites of Sudan dyes did not dissolve in the starting buffer for LC/ESI-MS/MS analysis. Other workers have demonstrated that naphthylamines are unstable when they are exposed to oxygen and rapidly form brown polymerization products (13, 17). When the metabolites of the reduced Sudan dyes became aerobic, the hydroxyl and amino groups would be expected to be oxidized, like the bacterial degradation products of the azo dye Reactive Red 3.1 and the pigment GemSperse Orange EX5s (2, 17).


Figure 2
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  		FIG. 2. Product ion mass spectra of the aniline metabolite from Sudan I (A), the 2,4-dimethylaniline metabolite from Sudan II (B), the aniline metabolite from Sudan III (C), the o-toluidine metabolite from Sudan IV (D), and the p-nitroaniline metabolite from Para Red (E), which were obtained by the incubation of Sudan dyes with the human intestinal microflora.


Figure 3
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  		FIG. 3. Suggested possible pathway for the reduction of Sudan dyes by the human intestinal microflora. 1-Amino-2-naphthol, 1,4-phenylenediamine, and 2,5-diaminotoluene (in brackets) could not be detected in the extracted samples. Some brown substances in the metabolites of Sudan dyes did not dissolve in the starting buffer for LC/ESI-MS/MS analysis.

In conclusion, our results are the first to show that members of the human intestinal microflora are capable of degrading Sudan dyes to form toxic aromatic amines, which suggests a potential health risk in consuming foods contaminated with Sudan dyes.


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ACKNOWLEDGMENTS
 
We thank John B. Sutherland and Robin L. Stingley for their critical reviews of the manuscript.

This study was funded by the National Center for Toxicological Research, U.S. Food and Drug Administration, and supported in part by an appointment (H.X.) to the Postgraduate Research Fellowship Program and an appointment (S.C.) to the Summer Internship Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

The views presented in this article do not necessarily reflect those of the Food and Drug Administration.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Microbiology, National Center for Toxicological Research, U.S. FDA, 3900 NCTR Rd., Jefferson, AR 72079-9502. Phone: (870) 543-7410. Fax: (870) 543-7307. E-mail: huizhong.chen{at}fda.hhs.gov Back

{triangledown} Published ahead of print on 12 October 2007. Back


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Applied and Environmental Microbiology, December 2007, p. 7759-7762, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01410-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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