Comparative genotoxicity of nitrosamine drinking water disinfection byproducts in Salmonella and mammalian cells

https://doi.org/10.1016/j.mrgentox.2011.11.006Get rights and content

Abstract

Nitrosamine water disinfection byproducts (DBPs) are an emerging class of non-halogenated, nitrogen-containing water contaminants. Five nitrosamine DBPs were analyzed for genotoxicity (N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) and N-nitrosodiphenylamine (NDPhA). Using Salmonella typhimurium strain YG7108 the descending rank order of mutagenicity was NDMA > NPIP > NMOR > NPYR; NDPhA was not mutagenic. We developed and calibrated an exogenous S9 mix that was highly effective in activating NDMA in Chinese hamster ovary (CHO) cells using the SCGE (Comet) assay. The descending rank order for genotoxicity was NDMA > NPIP > NMOR. NDPhA was genotoxic only at one concentration and NPYR was not genotoxic. The genotoxic potencies in S. typhimurium and CHO cells were highly correlated. Based on their comparative genotoxicity attention should be focused on the generation and occurrence of NDMA, NPIP and NMOR. Current drinking water disinfection processes may need to be modified such that the generation of nitrosamine DBPs is effectively limited in order to protect the environment and the public health.

Highlights

► Five nitrosamine DBPs were analyzed for genotoxicity in Salmonella and CHO cells. ► An exogenous S9 mix was developed for nitrosamine activation in CHO cells. ► The nitrosamine genotoxic potencies in both cell assays were highly correlated.

Introduction

An outstanding public health achievement of the 20th century was the disinfection of drinking water [1]. Drinking water treatment facilities reduce risks due to acute infection by waterborne pathogens yet they unintentionally generate a diversity of disinfection byproducts (DBPs) of regulatory and chronic health concern [2], [3], [4]. The U.S. Environmental Protection Agency (U.S. EPA) limits the levels of 11 DBPs in drinking water while over 600 DBPs are known [5]. The World Health Organization guidelines include 14 DBPs (http://www.who.int/water_sanitation_health/dwq/gdwq3/en). The toxicity of most of the non-regulated emerging DBPs is poorly understood with systematic in vitro toxicity studies conducted on only a small percentage of these environmental agents [4], [6], [7]. Earlier studies on the toxicity of DBPs were primarily confined to the formation and adverse biological effects of carbonaceous DBPs (C-DBPs). Currently there is a shift to focus on the comparative toxicity of nitrogen-containing DBPs (N-DBPs) [8], [9], [10]. N-DBPs include DBP chemical classes such as halonitroalkanes, halonitriles, haloamides, and nitrosamines. Enhanced interest in N-DBPs is due to increasing use by utilities of source waters impaired by wastewater discharges and algal blooms; these waters feature higher organic nitrogen concentrations that serve as N-DBP precursors [8]. In addition, changes in regulatory policy to reduce trihalomethanes and haloacetic acids have fostered a switch from chlorination to alternative disinfectant combinations; some of these combinations may generate higher levels of unregulated N-DBPs [11]. Most of the halogenated N-DBPs are direct-acting mutagens or genotoxins [4]; they are significantly more toxic than C-DBPs [6], [9]. Our laboratory has conducted a systematic study on the chronic cytotoxicity and genomic DNA damaging activity of these direct-acting N-DBPs [6], [12], [13], [14], [15].

A major class of indirect-acting (promutagenic) N-DBPs is the nitrosamines. Nitrosamine formation is primarily associated with chloramine disinfection, although there are cases where nitrosamine formation may be significant when chlorination is conducted in the presence of nitrite, or when specific precursors (e.g., N,N-dimethylsulfamide) react with ozone [16]. Nitrosamine DBPs that were identified in drinking water include N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosopiperidine (NPIP), N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosodibutylamine, N-nitrosodipropylamine and N-nitrosodiphenylamine (NDPhA) [5], [17], [18], [19], [20], [21], [22]. Of the nitrosamine DBPs found in disinfected water, NDMA has the highest levels of occurrence and concentration [19], [23]. The U.S. EPA Integrated Risk Information System lists NDMA with a 10−6 lifetime cancer risk at a concentration in drinking water of 0.7 ng/L (http://www.epa.gov/IRIS/). NDMA is regulated in Ontario, Canada at 9 ng/L;a national guideline is under development in Canada. NDMA, N-nitrosodiethylamine and N-nitrosodipropylamine are regulated in California, U.S.A. at 10 ng/L in drinking water. Although these agents are not regulated by the U.S. EPA, nitrosamines are listed in the Unregulated Contaminants Monitoring Rule (http://permanent.access.gpo.gov/lps21800/www.epa.gov/safewater/ucmr.html) and in the final Contaminant Candidate List 3 (http://water.epa.gov/scitech/drinkingwater/dws/ccl/ccl3.cfm). Although NDMA has been the predominant nitrosamine detected in disinfected waters, only ∼10% of the total nitrosamine in disinfected recreational waters were accounted for by specific nitrosamines, including NDMA [24].

The objective of this research was to conduct comparative and systematic in vitro toxicity analyses of five nitrosamine DBPs (NDMA, NDPhA, NMOR, NPIP, and NPYR) in bacterial and mammalian cells. The in vitro assays included mutagenicity and cytotoxicity in Salmonella typhimurium strain YG7108, and acute cytotoxicity and genotoxicity in Chinese hamster ovary (CHO) cells. Since the nitrosamines require metabolic activation another objective was to develop an improved S9 methodology for treating mammalian cells using the single cell gel electrophoresis (SCGE, Comet) assay.

Section snippets

Chemicals and reagents

Five nitrosamine DBPs were analyzed in this study, N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodiphenylamine (NDPhA), and N-nitrosopiperidine (NPIP). A description of each nitrosamine, their CASN, and their source and purity is presented in Table 1; the chemical structures are included in Fig. 1. Concentrated stock solutions of each nitrosamine were made in dimethylsulfoxide (DMSO) and stored at −22 °C.

General reagents were purchased from

Mutagenicity of nitrosamine DBPs in S. typhimurium

Four of the five nitrosamines were mutagenic with S9 activation in S. typhimurium strain YG7108. None were directly mutagenic and no decrease in viability was observed in the concentration ranges analyzed. Concentration–response curves are presented in Fig. 1. NDMA was the most potent mutagen with a doubling of revertants at 10 μM and a statistically significant increase at 50 μM (Table 2). A regression analysis was conducted within the linear portion of each concentration response curve; the r2

Discussion

For the bacterial mutagenicity assay we chose S. typhimurium strain YG7108 because of its enhanced sensitivity to alkylating agents [25]. It is responsive to a series of nitrosoguanidines [37], as well as to other alkylating agents including NDMA [25]. NDMA was mutagenic under preincubation conditions with S9 activation in a concentration range from 0.1 to 2.5 mg/plate [25]. Using mutagenic potency as the comparative metric, each μmol of NDMA induced 924 YG7108 revertants compared to 0.16 TA1535

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This research was supported by grants from the National Science Foundation (CBET-0651732 and CBET-0651333). We appreciate the support by the Center of Advanced Materials for the Purification of Water with Systems, National Science Foundation Science and Technology Center, under Award CTS-0120978.

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