Transformation of the Ionic X-Ray Contrast Agent Diatrizoate and Related Triiodinated Benzoates by Trametes versicolor

Abstract

Iodinated X-ray contrast agents are considered to be nondegradable by microorganisms. The decomposition of the ionic X-ray contrast agents Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoic acid) and Iodipamide (3,3′-adipoyl-diimino-di(2,4,6-triiodobenzoic acid) and related triiodinated benzoates (Acetrizoate [3-acetylamino-2,4,6-triiodobenzoic acid] and Aminotrizoate [3-amino-2,4,6-triiodobenzoic acid]) by Trametes versicolor has been investigated. The fungus was able to transform all tested triiodinated benzoates cometabolically. During transformation of these compounds, iodide was released, but deiodination was not complete. T. versicolor liberated traces of ¹⁴CO₂ from uniformly ring-¹⁴C-labeled Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoate). Various extracellular metabolites were detected during transformation of the different substances. In the transformation of Diatrizoate, the three main metabolites were identified as 3,5-di(acetamido)-2,6-diiodobenzoic acid, 3,5-di(acetamido)-2,4-diiodobenzoic acid, and 3,5-di(acetamido)-2-iodobenzoic acid, suggesting reductive deiodinations in steps as initial transformation steps.

Triiodinated benzoates are used as X-ray contrast agents for intravenous injections. X-ray contrast agents permit visualization of the details of the internal structure of organs that would otherwise not be apparent. Strong growth in the number of medical imaging procedures performed has increased the use of contrast media dramatically, making this the fastest growing pharmaceutical use of iodine. From the world market of iodinated contrast media of 5.8 billion deutsche marks in 1993 (5.0 billion deutsche marks in 1992), a consumption of 3,500 tons per year of iodinated contrast agents in 1993 can be calculated (23).

As implied in their design, iodinated X-ray contrast agents are very stable and chemically inert (27). They are not degraded during use, and no degradation can be detected in sewage sludge. To date, there have been no reports on the biological degradation of ionic X-ray contrast agents. Attempts to isolate a microorganism which can degrade ionic X-ray contrast agents have failed (5, 13). Therefore, it is not surprising that these compounds were detected in rivers, especially near hospitals at fairly high concentrations (30).

White rot fungi are known to degrade a wide variety of polychlorinated aromatic compounds, such as polychlorinated biphenyls, lindane, 3,4-dichloroaniline, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol, and 2,4,5-trichlorophenoxyacetic acid (1, 3, 8, 12, 19, 20, 28, 32). This ability is due to their extracellular highly nonspecific free-radical-mediated oxidation system, consisting of oxidases, peroxidases, and peroxide-generating enzymes (9), which has been investigated with different white rot fungi (7, 11, 15, 29).

The fact that white rot fungi are capable of degrading polychlorinated aromatic compounds led us to propose that they are also able to degrade polyiodinated aromatic compounds, such as ionic X-ray contrast agents. In our previous studies, we found that the white rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus were not able to degrade these polyiodinated aromatic compounds (18). In this study, we report on the breakdown of the ionic X-ray contrast agents Diatrizoate and Iodipamide and of the related triiodinated benzoates Aminotrizoate and Acetrizoate by the white rot fungus Trametes versicolor (Fig. 1).

FIG. 1.

Structures of the triiodinated benzoates tested for transformation by T. versicolor. Ionic X-ray contrast agents (top) and their matrix compounds (bottom) are shown.

T. versicolor was grown for 7 days at 30°C on agar plates. The mycelium of one plate was then suspended in 7 ml of modified mycological broth (MB) (21). MB (100 ml) was inoculated with this mycelial suspension (2 to 5% [vol/vol]) and incubated in 250-ml Erlenmeyer flasks at 30°C. The triiodinated benzoates were added as 100-fold concentrate to give a final concentration of 1 mM. The following controls were always included: sterile controls consisting of MB with the addition of the triiodinated benzoates and background control cultures consisting of MB inoculated with the fungus with no triiodinated benzoates. The decrease in concentration of the X-ray contrast agents (Diatrizoate and Iodipamide) and the related triiodinated benzoates (Aminotrizoate and Acetrizoate) was monitored by high-pressure liquid chromatography (HPLC) (Hyperchrome Spherisorb octadecyl silane II column [250 by 4 mm; bead diameter, 5 μm]; water-methanol-phosphoric acid-triethylamine, 97:3:0.12:0.17 [by volume] [pH 4.0]; flow rate, 1 ml/min). All triiodinated benzoates tested were metabolized at similar rates (0.52 to 0.85 mM within 14 days). The transformation rate in the shaken flasks decreased with time and became negligible 14 days after inoculation.

The transformation of the various triiodinated benzoates was accompanied by a release of iodide ions. For all tested triiodobenzoates, the deiodination ratios (mole of iodide released per mole of substrate) were between 0.64 and 1.48 after 14 days of incubation. This iodide release was only a fraction of the amount calculated for the complete deiodination of the substrates. The iodide release rate of dimeric Iodipamide (6 mol of I/mol of substrate) was not significantly higher than those of the monomeric triiodobenzoates. The rates of transformation and deiodination seemed to be independent of the structures of the triiodinated benzoates. This is the first time that a biological transformation of iodinated ionic X-ray contrast agents was found.

The presence of triiodinated benzoates inhibited the growth of the fungus. When a defined amount of pregrown mycelium of T. versicolor was inoculated, the average dry weight of the cultures incubated with 0.1 mM triiodinated benzoates was 33% lower than that of cultures without triiodinated benzoates after 14 days.

To test whether the ring structure was attacked in the transformation of the triiodinated benzoates in addition to iodide release and to detect metabolites formed in the transformation, experiments with [U-ring-¹⁴C]Diatrizoate were performed. During 32 days of incubation of [U-ring-¹⁴C]Diatrizoate with T. versicolor, between 0.2 and 2.5% of the radioactivity was liberated as ¹⁴CO₂. This amount of ¹⁴CO₂ released from [U-ring-¹⁴C]Diatrizoate seems rather low, suggesting that no extensive mineralization had occurred. However, it should be noted that the absolute release of ¹⁴CO₂ from dichlorodiphenyltrichloroethane (DDT), pentachlorophenol, and 2,4,5-trichlorophenoxyacetic acid by the white rot fungus Phanerochaete chrysosporium, which was called extensive mineralization, was also in the range of 35 to 59 nM ¹⁴CO₂ during 30 days of incubation (2).

The liberation of ¹⁴CO₂ correlated with the transformation of Diatrizoate and the release of three radiolabeled metabolites (Fig. 2). These three metabolites were isolated from the culture liquid by preparative HPLC and by liquid chromatography.

FIG. 2.

Transformation of [U-ring-¹⁴C]Diatrizoate and formation of metabolites by T. versicolor. Symbols: □, Diatrizoate; ▴, metabolite 1; •, metabolite 2; ⧫, metabolite 3.

The molecular masses of the metabolites were determined by fast atom bombardment mass spectrometry. The substitution at the aromatic ring was determined by ¹H nuclear magnetic resonance (NMR) and ¹³C NMR. Metabolite 1 eluted at 11 min. It had a molecular mass of 488 Da and contained a single symmetric proton at the aromatic ring. Therefore, it was identified as 3,5-di(acetamido)-2,6-diiodobenzoate. Metabolite 2 eluted at 20 min. It had the same molecular mass and contained one asymmetric aromatic proton. It was identified as 3,5-di(acetamido)-4,6-diiodobenzoate. Metabolite 3 eluted at 36 min. It had a molecular mass of 362 Da and contained two nonidentical aromatic protons. Therefore, it was identified as 3,5-di(acetamido)-2-monoiodobenzoate. The spectral data leading to these identifications are given in Table 1. The structures of the metabolites and the proposed transformation scheme of Diatrizoate by T. versicolor are shown in Fig. 3.

TABLE 1.

Major spectroscopic data leading to the identification of the isolated metabolites

Metabolite	Spectroscopic data
	m/z (%) by FAB-MSa	1H NMR
1	489 (M + H+, 5.3), 361 (M − I, 4), 85 (100)	One aromatic proton at 7.38 ppm
2	489 (M + H+, 4), 85 (100)	One aromatic proton at 7.32 ppm
3	363 (M + H+, 3.8), 385 (M + Na+, 1.9), 85 (100)	Two nonidentical aromatic protons at 7.68 and 7.71 ppm

^aFAB-MS, fast atom bombardment mass spectroscopy. 

FIG. 3.

Proposed transformation of Diatrizoate by T. versicolor.

The isolated extracellular metabolites from Diatrizoate were partially deiodinated. The time course of the formation of the metabolites indicated that the metabolite deiodinated twice was formed by deiodination of the metabolites deiodinated once (Fig. 2). After 2 weeks, the concentration of the metabolites detected corresponded to about 50% of the amount of Diatrizoate that had disappeared. This indicates that in addition to reductive deiodination, other processes must have occurred. In addition to a further degradation of the metabolites, polymerization reactions, whose products would not have been detected under the HPLC conditions used, are the most likely candidates. However, initial reductive deiodination seems to be the main reaction.

To date, there have been only a few examinations into the biological degradation of iodinated hydrocarbons. It was shown that some of the microorganisms which degrade monochlorinated compounds can also degrade the corresponding iodinated compounds. In these cases, the isolated enzymes were also capable of dehalogenating the iodinated compounds (6, 10, 14, 16).

The degradation of polyiodinated aromatic compounds by microorganisms is documented in only one case. The degradation of triiodophenol with cell extracts from Flavobacterium sp. leads to a complete reductive deiodination. The inducible enzyme system was also able to dehalogenate the corresponding brominated or chlorinated compound as well as it dehalogenated pentachlorophenol (31).

Because X-ray contrast agents cannot pass through the cell membranes in humans, it seems likely that they also cannot enter the cells of T. versicolor. Therefore, the initial metabolization of the triiodinated benzoates by T. versicolor should be extracellular. White rot fungi are known to produce different extracellular enzymes, which are involved in the initial transformation of lignin in their natural environment. Extracellular peroxidases, lignin peroxidases, laccases, or manganese-dependent peroxidases from different white rot fungi have been found to be involved in the degradation of various haloaromatic compounds (1, 3, 8, 12, 17, 19, 20, 28, 32).

These peroxidases and laccases are able to perform one-electron oxidations of different aromatic substrates and redox mediators to yield substrate cation radicals. The substrate radicals then undergo a variety of reactions (22).

To check the dependence of the transformation of the triiodinated benzoates by T. versicolor on these extracellular ligninolytic enzymes, we have measured their activities during transformation of the contrast agents. No lignin peroxidase activity was detected at any time in any of the cultures. In contrast, nonspecific peroxidase, manganese-dependent peroxidase, and laccase activities appeared in all of the cultures at the same time and correlated with the transformation and deiodination of the substrates. The extracellular enzymes were produced constitutively during the logarithmic growth phase of the fungus, but their activities were influenced by the addition of the triiodinated benzoates. Because the extracellular enzymes appeared in parallel, we could not decide whether one or more enzymes were involved in the transformation of the triiodinated benzoates. The fact that the release of iodide from the tested triiodinated benzoates was independent of the other substituents of the triiodinated benzoates indicated that the reaction was not specific. Therefore, we propose that the initial transformation and deiodination of the triiodinated benzoates may be caused by the extracellular peroxidases and/or laccases of T. versicolor, which can metabolize the triiodinated benzoates indirectly.

The transformation of polyiodinated benzoates by T. versicolor is clearly different from the transformation of polychlorinated phenols by Phanerochaete chrysosporium. Here the di- and polychlorinated compounds were oxidatively dechlorinated by the extracellular lignin peroxidase or manganese-dependent peroxidase by formation of the corresponding quinones. The metabolites isolated in this study are not hydroxylated. The dehalogenation by T. versicolor is reductive. This is the first time that such a reductive deiodination by a white rot fungus is described. Chung et al. (4) and Shah and Aust (24, 25) described in several articles that the reactions of lignin peroxidase and manganese-dependent peroxidase can lead to reductions via a radical mechanism. For example, a reductive dechlorination of tetrachloromethane by lignin peroxidase was observed (26). Whether the deiodination of the triiodobenzoates follows a similar mechanism remains to be seen.