Elsevier

Journal of Environmental Sciences

Volume 58, August 2017, Pages 224-230
Journal of Environmental Sciences

Monohalogenated acetamide-induced cellular stress and genotoxicity are related to electrophilic softness and thiol/thiolate reactivity

https://doi.org/10.1016/j.jes.2017.04.027Get rights and content

Abstract

Haloacetamides (HAMs) are cytotoxic, genotoxic, and mutagenic byproducts of drinking water disinfection. They are soft electrophilic compounds that form covalent bonds with the free thiol/thiolate in cysteine residues through an SN2 reaction mechanism. Toxicity of the monohalogenated HAMs (iodoacetamide, IAM; bromoacetamide, BAM; or chloroacetamide, CAM) varied depending on the halogen substituent. The aim of this research was to investigate how the halogen atom affects the reactivity and toxicological properties of HAMs, measured as induction of oxidative/electrophilic stress response and genotoxicity. Additionally, we wanted to determine how well in silico estimates of electrophilic softness matched thiol/thiolate reactivity and in vitro toxicological endpoints. Each of the HAMs significantly induced nuclear Rad51 accumulation and ARE signaling activity compared to a negative control. The rank order of effect was IAM > BAM > CAM for Rad51, and BAM  IAM > CAM for ARE. In general, electrophilic softness and in chemico thiol/thiolate reactivity provided a qualitative indicator of toxicity, as the softer electrophiles IAM and BAM were more thiol/thiolate reactive and were more toxic than CAM.

Introduction

Disinfection of municipal drinking water is an essential safeguard of public health (Calderon, 2000). However, the addition of disinfectants to source waters containing organic and inorganic precursor molecules generates a mixture of toxic halogenated byproducts (Krasner et al., 1989). Many of these disinfection byproducts (DBPs) are cytotoxic, genotoxic, and mutagenic in vitro, and some are carcinogenic in rodents (Richardson et al., 2007). Moreover, epidemiologic studies suggested long-term exposures to DBPs increased risk of bladder cancer (Michaud et al., 2007, Villanueva et al., 2007). Still, the mechanisms of toxicity leading to the observed adverse effects are not fully understood, making it difficult to establish effective interventions. Additionally, since DBPs are formed at concentrations below their individual adverse effect levels, it is unlikely that any individual DBP can account for the aforementioned observed adverse effects highlighting the need for understanding the toxic effect of the whole mixture (Bull, 2006, Simmons et al., 2002). Identification of chemical groups that share a common mechanism within the DBP mixture would provide a better understanding of the mixture toxicity (U.S. EPA, 2002) and could provide valuable insight on how to improve chemical regulation.

Several DBPs can be classified as soft electrophilic compounds (highly polarizable chemicals deficient in electrons) that preferentially react with biological soft nucleophiles (e.g., thiol/thiolate groups) forming covalent bonds (Pearson, 1963). For example, many DBPs share an α-brominated carbonyl motif identified as a structural predictor of thiol (glutathione (GSH)) reactivity (Hughes et al., 2015). Additionally, cytotoxicity and genotoxicity correlated with predicted SN2 reactivity for several α-halogenated carbonyl (or nitrile) containing DBP chemical classes including haloacetonitriles (HANs), haloacetamides (HAMs), and haloacetic acids (HAAs) (Muellner et al., 2007, Plewa et al., 2008, Plewa et al., 2010). Genotoxicity induced by the α-halogenated carbonyl (or nitrile) containing DBPs, bromoacetamide (BAM), and bromoacetonitrile (BAN) were significantly reduced by co-treating with N-acetylcysteine (NAC) (Pals et al., 2016). Therefore, reactivity seems to play a significant role in the toxicity of DBPs, where directly perturbing the intracellular thiol pool through alkylation of cellular thiol/thiolates leads to a toxic response by disrupting various cellular processes (Schultz et al., 2006). Moreover, GSH depletion is observed in the progression of several diseases including cancer, cardiovascular, and neurodegenerative diseases (Ballatori et al., 2009).

Furthermore, reactive electrophiles with common cellular targets present an ideal opportunity for additive or synergistic toxicity (U.S. EPA, 2002). In fact, experiments with α,β-unsaturated carbonyl derivatives, showed additive toxicity in binary and ternary mixtures in vitro and in an in vivo model (Zhang et al., 2016). Work by Dawson and colleagues showed that HANs and halogenated ethyl acetates (α-halogenated carbonyl containing compounds) generated, in some cases, dose additive toxicity within and among these chemical classes (Dawson et al., 2010, Dawson et al., 2011, Dawson et al., 2014). Additive genotoxicity was observed in binary mixtures of BAM and BAN (Pals et al., 2016).

Soft electrophile reactivity can be predicted using in silico methods based on Hard Soft Acid Base (HSAB) theory (Karelson et al., 1996). HSAB theory makes use of frontier molecular orbital (FMO) energies to predict reactivity between electrophile–nucleophile pairs (Karelson et al., 1996). Several parameters for reactivity based on FMO energies were developed and utilized in Quantitative Structure Activity Relationship (QSAR) applications (Schwöbel et al., 2011); however, in its basic form, HSAB theory suggests that an electrophile is soft if it has a low energy lowest unoccupied molecular orbital (ELUMO) (Karelson et al., 1996). Estimations of ELUMO, therefore, provide a theoretical method for identifying soft electrophiles among identified DBPs. Our previous work showed that ELUMO was a useful predictor of thiol reactivity and genotoxicity in a set of mono-brominated DBPs (bromoacetic acid, BAM, and BAN) (Pals et al., 2016). However, because reactivity of alkyl halides is dependent on halogen leaving efficiency, the effect of variable halogen substitution requires additional investigation. The set of monoHAMs, iodoacetamide (IAM), BAM, and chloroacetamide (CAM), isolates the effect the halogen substituent asserts on thiol/thiolate reactivity and toxicity, and allows us to evaluate in silico softness parameters as predictors of these effects. In this study we measured the ability of each monoHAM to generate an Antioxidant Response Element (ARE) driven stress response, and to generate toxicity as DNA double strand breaks. We then compared toxic potencies with in chemico reactivity and in silico parameters derived from FMO energies to determine their ability to predict in vitro effects.

Section snippets

General reagents

General laboratory reagents were purchased from Fisher Scientific Co. (Itasca, IL) or Sigma Aldrich Co. (St. Louis, MO). IAM, BAM, CAM, DTNB (5,5′-dithiobis (2-nitrobenzoic acid)) and NAC were purchased from Sigma-Aldrich (St. Louis, MO).

In silico estimates of FMO energies

FMO energies, including energy of the highest occupied molecular orbital (EHOMO) and ELUMO were estimated using density function B3LYP 6-311 + G**, from Hartree Fock 6-311 + G** equilibrium geometries with Spartan 10 software (Wavefunction Inc., Irvine, CA).

Results and discussion

This work aimed to evaluate in silico parameters as predictors of thiol/thiolate reactivity, antioxidant/electrophilic stress response, and genotoxicity in a set of three monoHAMs. Compounds containing an α-halogenated carbonyl group are predicted to be thiol reactive (Hughes et al., 2015) to varying degrees based on the leaving efficiency of the halogen substituent (Plewa et al., 2008, Schultz et al., 2007). IAM, BAM, and CAM, which are genotoxic and mutagenic DBPs, contain this α-halogenated

Conclusion

The aim of this research was to investigate the relationship between thiol/thiolate reactivity, activation of ARE regulated stress response, and Rad51 foci formation. Within the selected group of HAMs, reactivity parameters derived from FMO energies as well as in chemico thiol/thiolate reactivity were able to qualitatively predict activation of antioxidant/electrophilic stress response and genotoxicity (measured as Rad51 foci formation). The softer electrophiles, IAM and BAM, were more reactive

Acknowledgments

We wish to thank the Chemistry Department at Eastern Illinois University for access to and assistance using Spartan 10 software. We acknowledge partial support from the U.S. Army Engineer Research and Development Center and the Army Environmental Quality Technology program, CESU W9132T-16-2-0005 (MJP). This work was partly supported by the interagency agreement IAG #NTR 12003 from the National Institute of Environmental Health Sciences/Division of the National Toxicology Program to the National

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