Letter pubs.acs.org/journal/estlcu
Comparative Toxicity of High-Molecular Weight Iopamidol Disinfection Byproducts Friedrich M. Wendel,† Thomas A. Ternes,† Susan D. Richardson,‡ Stephen E. Duirk,§ Justin A. Pals,∥ Elizabeth D. Wagner,∥ and Michael J. Plewa*,∥ †
Department of Water Chemistry, Federal Institute of Hydrology (BfG), Am Mainzer Tor 1, D-56068 Koblenz, Germany Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States § Department of Civil Engineering, University of Akron, Akron, Ohio 44325, United States ∥ Department of Crop Sciences and The Safe Global Water Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States ‡
S Supporting Information *
ABSTRACT: When the X-ray contrast pharmaceutical iopamidol was chlorinated in natural source water, a toxic mixture of many disinfection byproducts (DBPs) was formed. We isolated and identified five high-molecular weight iopamidol chlorination DBPs and analyzed their cytotoxicity and genotoxicity using mammalian cells. Of the five DBPs, four exhibited low levels of cytotoxicity, while none of the iopamidol DBPs was genotoxic. These data support the hypothesis that these high-molecular weight DBPs were not the forcing agents that induced the cytotoxicity and genotoxicity observed after iopamidol was disinfected in natural source waters.
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INTRODUCTION Iopamidol is an iodinated X-ray contrast medium (ICM) commonly used to allow the medical imaging of soft tissues (e.g., organs and blood vessels). Up to 200 g of this pharmaceutical is used per application with approximately 75 million patient applications annually.1 ICMs were designed to be inert in the human body and are excreted within 24 h. Conventional wastewater treatment plants (WWTPs) cannot degrade or adequately process ICMs.2,3 In Germany, iopamidol was found at a level of 15 μg/L in WWTP effluent and 98 ng/L in finished drinking water, while 2.7 μg/L was measured in a U.S. drinking water source.4,5 We demonstrated that iopamidol in the presence of natural organic matter (NOM) with chlorination generated disinfection byproducts (DBPs) and iodinated DBPs,5,6 which was recently confirmed by others.7,8 Chlorination of an iopamidol solution induced cytotoxicity and genotoxicity in bacterial and mammalian cells.5,9 During chlorination of iopamidol, two classes of products were formed: high-molecular weight DBPs that are structurally similar to the ICM parent compound (HMWDBPs) and low-molecular weight DBPs that incorporated iodine cleaved from iopamidol (LMWDBPs). While iopamidol is not toxic itself, many LMWDBPs are cytotoxic and genotoxic, especially iodinated DBPs.10−13 This has important implications for drinking water safety. Of the agents generated with the chlorination of iopamidol, it is unknown whether the © XXXX American Chemical Society
primary HMWDBPs are involved in generating the enhanced toxicity of the finished drinking water. The hypothesis tested was whether the HMWDBPs or the LMWDBPs were the forcing agents that induced the increased toxicity of chlorinated iopamidol solutions.
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MATERIALS AND METHODS Standards and Reagents and Biologicals. Purified water (18 MΩ cm, DOC < 0.1 mg/L) was obtained from a Milli-Q system (Merck Millipore). Other solvents (acetonitrile and methanol) were purchased from Merck (Darmstadt, Germany) at high-performance liquid chromatography (HPLC) grade purity, hypochlorite solutions (10−15%) from Sigma-Aldrich (Seelze, Germany). Iopamidol was supplied by Bayer Schering Pharma (Berlin, Germany) at a purity of >95%. General biological reagents, cell culture medium (F12), and fetal bovine serum (FBS) were purchased from Fisher Scientific (Itasca, IL). Chinese hamster ovary (CHO) cell line AS52 (clone 11-4-8) was used for the biological assays.14 CHO cells were maintained on glass culture plates in Ham’s F12 medium containing 5% FBS, 1% antibiotics (100 units/mL sodium penicillin G, 100 μg/mL streptomycin sulfate, and 0.25 μg/mL amphotericin B Received: January 31, 2016 Accepted: February 3, 2016
A
DOI: 10.1021/acs.estlett.6b00037 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Letters in 0.85% saline), and 1% glutamine at 37 °C in a humidified atmosphere of 5% CO2. Isolation of DBPs. A hypochlorite solution [10−15%, in an excess of 4:1 to 20:1 (v/v)] was added to stirred solutions of iopamidol (up to 1 g/L) in water with borate buffer at pH 8.5 in 1 L glass bottles. The reaction was quenched by the addition of solid sodium thiosulfate either after 1 h or after 24 h to obtain relatively large amounts of the chlorinated iopamidol DBPs. The HMWDBPs were separated from the reaction mixture using a semipreparative Agilent 1260 Infinity HPLC system with an automatic fraction collector. The chromatographic methods are described in Tables S1 and S2. The isolated fractions were frozen, and the solvent was evaporated by lyophilization. Five HMWDBPs were isolated and identified and are mentioned by their MW (Table 1).
microplates with replicates per dilution as well as the negative controls. The microplate wells were covered with AlumnaSeal to prevent volatilization during the 72 h exposure period. After exposure, the cell density per microplate well was determined by staining using crystal violet and absorbency at 595 nm using a Spectramax Paradigm plate reader (Molecular Devices). A cytotoxicity concentration−response curve for each HMWDBP was generated from the summary data from combined replicate experiments. The concentration associated with a 50% reduction in cell density as compared to the concurrent negative control (LC50) was calculated for each HMWDBP using nonlinear regression analysis of the concentration− response curve. The LC50 values were used to quantify the relative cytotoxicity of different water samples or specific DBPs. The detailed procedures and calibration for the CHO cell cytotoxicity assay and their use with water samples were published.13,16−18 A detailed description of the assay and results of the cytotoxicity experiments are presented in the Supporting Information. CHO Single-Cell Gel Electrophoresis (SCGE) Assay. SCGE (Comet) is a molecular genetic assay that quantitatively measures the level of genomic DNA damage induced by a test agent in individual nuclei of cells.19−22 Induced DNA lesions that lead to single- and double-strand DNA breaks allow the DNA to migrate out of the nucleus within a microgel subjected to electrophoresis. The SCGE metric for genomic DNA damage induced by the DBPs is the %tail DNA value, which is the amount of DNA that migrated from the nucleus into the microgel.23 We employed a CHO cell microplate SCGE assay; the exposure time to the HMWDBPs was 4 h, which maximizes the ability to detect genomic damage while limiting the effect of DNA repair. For each SCGE experiment, acute cytotoxicity was measured to ensure that there was no excessive cell killing within the concentration range of the HMWDBPs. Within each concentration range with >70% cell viability, a concentration− response curve was generated for each HMWDBP from repeated experiments. Each experiment contained concurrent negative and positive controls. Detailed procedures for this assay and the results of the HMWDBP genotoxicity experiments are presented in the Supporting Information.13,22
Table 1. Identification, Structure, and Toxicity of Iopamidol High-Molecular Weight DBPs
a
The molecular weight of the iopamidol disinfection byproduct as a designator for each HMWDBP. bLowest cytotoxic concentration of the HMWDBP in the cytotoxicity concentration−response curve that induced a statistically significant reduction in cell density as compared with the concurrent negative controls. cThe LC50 value is the concentration of the HMWDBP, determined from a regression analysis of the data, that induced a CHO cell density of 50% as compared to the concurrent negative controls. dGenotoxicity as measured by single-cell gel electrophoresis (SCGE) using CHO cells. Abbreviations: NS, not statistically significant; NA, not applicable.
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RESULTS AND DISCUSSION In our previous work, 19 high-molecular weight DBPs were identified after chlorination of water containing iopamidol.6 An overview of these HMWDBPs and their reaction pathway is provided in Figure S1. To assess the cytotoxicity and genotoxicity of selected HMWDBPs, they were isolated in milligram quantities. We chlorinated iopamidol solutions at concentrations higher than what would be expected under water treatment conditions to determine whether the same products are still formed. Solutions containing up to 1 g/L iopamidol were brought to reaction with a 20-fold molar excess of hypochlorite in purified water buffered at pH 8.5, and samples were withdrawn at regular intervals. Iopamidol chlorination products formed at low concentrations were formed at these high concentrations. However, the intensity of signals caused by dimeric compounds increased, which indicated that they are likely not relevant for reactions occurring in drinking water treatment where concentrations of iopamidol are much lower. The isolated HMWDBPs were DBP705, DBP778, DBP735, DBP643, and DBP551. These could be isolated in sufficiently large quantities because they were formed in amounts larger than those of the other
Chinese Hamster Ovary Cell Chronic Cytotoxicity Assay. Chinese hamster ovary (CHO) cell line AS52 (clone 11-4-8) served as a mammalian cell model for in vitro experiments.14 CHO cells are widely used in analyzing the in vitro toxicology of DBPs. 13 The cells exhibit normal morphology, express cell contact inhibition, and grow as a monolayer without expression of neoplastic foci.14,15 Via measurement of the reduction in cell viability compared to untreated controls, cytotoxicity captures a wide array of toxic insults.13 The assay measured cytotoxicity as the reduction in cell density after exposure of the cells to individual HMWDBPs for 72 h compared to untreated controls. The HMWDBPs were stored in dimethyl sulfoxide (DMSO) at −20 °C. For each experiment, a dilution series (generally 10 concentrations for each HMWDBP) was prepared by diluting the HMWDBPs into culture medium just prior to the experiment. The DMSO concentrations were not toxic to CHO cells (Figure S7). These dilutions were rapidly transferred to the CHO cells in 96-well B
DOI: 10.1021/acs.estlett.6b00037 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Letters
DBP551 > DBP735 > DBP643 ≈ DBP705; DBP778 was not toxic (Tables S3 and S4). The individual concentration− response curves for the genotoxicity of the HMWDBPs are presented in Figures S14−S18. None of the HMWDBPs induced genomic DNA damage in CHO cells (Table S5). The HMWDBP concentrations were higher than those detected in drinking water to generate a response, which is standard practice in comparative in vitro toxicology. To the best of our knowledge, these data represent the first mammalian cell cytotoxicity and genotoxicity of iopamidolmediated high-molecular weight DBPs. The results indicate that these HMWDBPs are not the forcing agents in the enhanced cytotoxicity and genotoxicity observed after iopamidol was chlorinated in natural source water.5 Because of the high cytotoxicity and genotoxicity of low-molecular weight iodinated DBPs,13 we contend that these LMWDBPs are driving the toxicity with possible additional LMWDBPs not yet identified.
HMWDBPs. The total amounts of the isolated high-molecular weight DBPs are listed in Table 1. LC−UV chromatograms of the compounds are presented in Figures S2−S6. These DBPs represent most of the degradation pathway of iopamidol (Figure S1). DBP778 was formed from iopamidol by the inversion of a side chain with subsequent hydrolysis of an amide group. DBP705 is the key intermediate product formed directly from iopamidol by cleavage of a side chain; most of the HMWDBPs were formed by subsequent reactions from DBP705. DBP735 is one of these products formed from DBP705 by oxidation of an amino function to a nitro group. The other two products, DBP643 and DBP551, differ from DBP735 only in that one and two atoms of iodine have been replaced with chlorine, respectively. Most of the selected highmolecular weight DBPs were relatively stable and hence could be isolated from the reaction mixture after 24 h. However, DBP705 was formed at the beginning of the reaction pathway and within
