Reactivity-directed analysis – a novel approach for the identification of toxic organic electrophiles in drinking water

Abstract

Drinking water consumption results in exposure to complex mixtures of organic chemicals, including natural and anthropogenic chemicals and compounds formed during drinking water treatment such as disinfection by-products. The complexity of drinking water contaminant mixtures has hindered efforts to assess associated health impacts. Existing approaches focus primarily on individual chemicals and/or the evaluation of mixtures, without providing information about the chemicals causing the toxic effect. Thus, there is a need for the development of novel strategies to evaluate chemical mixtures and provide insights into the species responsible for the observed toxic effects. This critical review introduces the application of a novel approach called Reactivity-Directed Analysis (RDA) to assess and identify organic electrophiles, the largest group of known environmental toxicants. In contrast to existing in vivo and in vitro approaches, RDA utilizes in chemico methodologies that investigate the reaction of organic electrophiles with nucleophilic biomolecules, including proteins and DNA. This review summarizes the existing knowledge about the presence of electrophiles in drinking water, with a particular focus on their formation in oxidative treatment systems with ozone, advanced oxidation processes, and UV light, as well as disinfectants such as chlorine, chloramines and chlorine dioxide. This summary is followed by an overview of existing RDA approaches and their application for the assessment of aqueous environmental matrices, with an emphasis on drinking water. RDA can be applied beyond drinking water, however, to evaluate source waters and wastewater for human and environmental health risks. Finally, future research demands for the detection and identification of electrophiles in drinking water via RDA are outlined.

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Carsten Prasse

Dr Carsten Prasse is an Assistant Professor in the Department of Environmental Health & Engineering at Johns Hopkins University. His research is driven by a fascination with environmental chemistry and a deep concern about the public and environmental health impacts of chemicals. His research focuses on the fate and effects of organic contaminants in the environment. Prioritizing which compounds are most threatening to human and environmental health is a great challenge given that thousands of chemicals are present in our waters. To tackle this problem, he is implementing concepts and methods from toxicology and public health with the goal of developing new methodologies to assess environmental exposure to anthropogenic chemicals.

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Environmental significance

The increasing number of contaminants detected in drinking water presents a major challenge for (bio)analytical water quality assessment. Organic electrophiles are a pressing concern due to their toxicity and widespread formation during (oxidative) water treatment. This critical review assesses the state of scientific knowledge on organic electrophiles in drinking water and proposes the use of a new analytical approach called Reactivity-Directed Analysis (RDA). RDA simultaneously identifies and assesses the toxicity of electrophiles in drinking water, bridging chemical and toxicological methodologies to tackle complex chemical mixtures.

A Introduction

Drinking water consumption exposes humans to complex mixtures of organic chemicals, which are present as a result of natural processes, contamination from anthropogenic activities, and formation during drinking water treatment.^1,2 While modern analytical instruments allow us to detect an ever-increasing number of chemicals with increasing sensitivity, the complexity of these mixtures makes an evaluation of the resulting health impacts extremely challenging. Traditionally, the quality of drinking water has been assessed based on the occurrence of specific, regulated compounds with well-defined toxicities as determined by expensive, time-consuming animal studies.^3,4 However, the growing number of anthropogenic chemicals that are detected in drinking water renders this chemical-by-chemical assessment approach unfeasible. Similarly, there is also a rising number of treatment processes, in particular those that use chemical oxidants (e.g., O₃, O₃/H₂O₂, UV/H₂O₂, HOCl, HOCl/NH₃, UV/HOCl, and electrochemical oxidation) that result in the formation of a large spectrum of transformation products.^5–7 Decades of research have demonstrated the presence of more than 700 disinfection by-products (DBPs) in drinking water treated by chlorination or chloramination.⁸ Yet, the dominant fraction of halogenated compounds remains unknown. In addition, only a small subset of DBPs (trihalomethanes (THMs), haloacetic acids (HAAs), bromate, and chlorite) are regulated, despite evidence of unregulated DBPs that exhibit higher toxicities.⁹ Health effects associated with exposure to DBPs include cancer, reduced fertility and fetal loss, respiratory diseases such as asthma, and skin diseases like allergic contact dermatitis.^10–15 Based on epidemiological studies, clear evidence exists for the association between adverse health outcomes and exposure to regulated DBPs such as THMs.¹¹ However, due to the lack of monitoring data for most drinking water contaminants, it is impossible to determine whether these regulated and widely monitored DBPs are indeed responsible, or whether the adverse health effects are attributable to other co-occurring DBPs. This uncertainty emphasizes the need for approaches that identify and prioritize the most relevant toxicants for toxicity assessment and drinking water monitoring.

In toxicology, high-throughput toxicity screening approaches, in particular in vitro bioassays, were developed to enable the evaluation of an increasing number of anthropogenic chemicals that are present in the environment.^16–20 A large number of in vitro bioassays for most relevant toxicity endpoints are available today and have been utilized to assess thousands of chemicals. In addition, in vitro bioassays have also been increasingly applied for the toxicological assessment of environmental samples including wastewater, surface water, and drinking water.^21–23 While these methods provide useful insights into the overall toxicity of complex mixtures, they do not provide any information regarding the chemicals responsible for the toxicity. With regard to drinking water and the expanding use of oxidative treatment technologies, another key limitation of current in vitro assays is the use of a preconcentration step such as specific resins (e.g. XAD), solid-phase extraction, liquid–liquid extraction, and freeze-drying, which is often necessary to achieve a sufficient sensitivity. The preconcentration methods, however, bias the results toward compounds that survive the work-up procedure. As such, highly polar compounds as well as substances that are volatile or reactive are likely lost.^24–26

One approach gaining popularity to identify chemicals responsible for an observed toxic effect in complex environmental mixtures is the use of effect-directed analysis (EDA).^27,28 As a first step, EDA employs in vitro bioassays to assess the toxicity of the investigated environmental sample. If a toxic effect is observed, the mixture is fractionated (generally using a chromatographic system and separation based on retention time) followed by in vitro toxicity testing of the individual fractions. For those fractions showing toxic effects, the process is typically repeated to further isolate the chemicals present in each fraction, which are then identified using analytical chemistry approaches such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS).^27,29,30 However, similar to the aspects discussed for in vitro bioassays, the extensive manipulation of the samples, including sample extraction and fractionation, likely results in the loss of highly polar, volatile, and reactive compounds. This is particularly relevant for transformation products formed in oxidative drinking water, which typically show at least one of these characteristics.

Overcoming these shortcomings requires the development of approaches that enable a direct assessment of complex mixtures, i.e. that do not focus only on chemicals that survive the work-up procedure, but also allow for the identification of the responsible toxicants.^18,31 This review introduces the application of a novel approach called reactivity-directed analysis (RDA) to address these shortcomings. In contrast to existing in vitro and in vivo approaches, RDA represents an in chemico approach (i.e., in the absence of organisms or cells) for the identification and toxicological assessment of organic electrophiles, the largest class of known toxic chemicals.^32–36 This review is structured into four different sections that focus on (i) the relevance of electrophiles as environmental toxicants, (ii) occurrence of electrophiles in drinking water, (iii) application of RDA to identify the presence of electrophiles in complex environmental mixtures, and (iv) future research needs.

B Organic chemistry meets molecular toxicology: electrophiles as toxicants

Advances in molecular toxicology have fundamentally changed our understanding of how chemical exposure is linked to adverse health effects. This includes the development of the adverse outcome pathway conceptional framework that outlines the cascade of biochemical and cellular processes that lead to a toxic effect.³⁷ Of particular relevance is the molecular interaction of a chemical with a biomolecule as an initial step, also called the molecular initiating event (MIE). MIEs include non-covalent interactions of chemicals with biomolecules (e.g. receptor binding), the oxidation of biomolecules by reactive oxygen species and reactive nitrogen species (e.g. due to oxidative stress), and covalent binding of chemicals to biomolecules, thus leading to the formation of adducts.^38,39 For the latter, the reaction of electrophiles with nucleophilic sites in biomolecules such as proteins and DNA is of particular importance^36,40,41 and is the focus of this review. Reactive nucleophilic moieties in biomolecules that are common targets of electrophiles include S- (cysteine and glutathione) and N-centers (lysine, histidine, and amine groups in DNA bases). The covalent binding of electrophiles to nucleophiles is attributable to substitution or addition reactions that involve donation of an electron pair by the nucleophile and subsequent formation of a covalent bond. The toxicological consequences of these covalent reactions are well-established in the case of DNA adducts, which can result in chemical-induced carcinogenesis,^40,42–44 making electrophilicity one of the key characteristics of carcinogens.⁴⁵ Similarly, reactions of electrophiles with proteins can result in changes of the target protein function or provocation of an antigen response.^32,46,47 Increased exposure to electrophiles further results in the depletion of antioxidant capacities, e.g. by reaction with glutathione, and thus indirectly leads to a toxic response due to increased oxidative stress.^48,49 The abundance and diversity of potential effects makes electrophiles the largest class of known toxic chemicals.^32,33,50

Assessing the reaction mechanisms of electrophiles with nucleophiles requires an understanding of the physico-chemical characteristics of both species responsible for the reactivity.^51–53 According to the Hard and Soft Acid and Base (HSAB) theory,⁵⁴ electrophiles react preferentially with nucleophiles of similar hardness or softness, a characteristic that has been used to predict biological targets and molecular toxicological effects.⁵⁵ Hard electrophiles including alkyl carbonium ions, benzylic carbonium ions, iminium ions and aldehydes react primarily with hard nucleophiles such as endocyclic nitrogen atoms of purine bases in DNA.⁵⁶ Examples of soft electrophiles include epoxides, enones, quinone imines, quinone methides, and Michael acceptors such as α,β-unsaturated aldehydes and ketones, which react with soft nucleophiles such as protein thiol groups, sulfhydryl groups of glutathione, primary/secondary amino groups of proteins, and lysine and histidine residues.^42,56 In addition, some electrophiles such as α,β-unsaturated carbonyl compounds show a high reactivity with both soft and hard nucleophiles, as they contain both a hard (carbonyl carbon) and a soft (α-carbon) electrophile group.^51,55,57 The HSAB theory also provides useful insights into the potential toxicity endpoints resulting from exposure to different electrophiles based on the reaction with different nucleophile pools.⁵⁸ For example, weak electrophiles are more likely to form adducts with slow turnover proteins that contain activated cysteine and lysine residues and have been associated with neuro- and reproductive toxicities. In contrast, reaction of strong electrophiles with nucleic acids results in cytotoxic effects (hard–hard covalent interactions) or systemic toxicity and skin sensitization (soft–soft covalent interactions).⁵⁸

Evaluating the covalent interaction of electrophiles with nucleophiles and utilizing them to assess the presence of electrophiles in environmental matrices such as drinking water also requires an in-depth understanding of the underlying reaction mechanisms. The reaction of electrophiles with nucleophilic biomolecules can be categorized into different mechanistic or reactivity domains and include Michael addition, Schiff base formation, acylation, nucleophilic aromatic substitution (S_NAr) and nucleophilic substitution (S_N2) (Table 1).^36,59–62 Michael addition is the reaction of a nucleophile with an α,β-unsaturated carbonyl such as an α,β-unsaturated ester, ketone, or aldehyde.^63–66 The effectiveness of the reaction is dependent on the α-carbon substituent and its ability to stabilize a negative charge on the carbon it is bound to.⁶⁷ For example, a methyl group reduces the reactivity while electron withdrawing groups such as aldehydes, ketones, and nitro groups increase the reactivity. Schiff base formation is another important mechanistic domain of carbonyl compounds and is particularly relevant for the reaction of aliphatic aldehydes and ketones with primary amines.³⁵ Schiff base formation is reversible via hydrolysis of the amine adduct. To stabilize the adducts before chemical analysis, reducing agents like sodium cyanoborohydride are typically used to reduce the product to a stable secondary amine.⁶⁸ Aromatic electrophiles undergoing reaction with nucleophiles via S_NAr reactions require the presence of electron withdrawing groups in the ortho- and/or para-position to the leaving group.⁶⁹ S_NAr electrophiles are reactive with both soft and hard nucleophiles and thus react with a wide spectrum of biomolecules.⁷⁰ Nucleophilic substitution (S_N2) reactions are particularly important for compounds containing primary alkyl groups that are bound to leaving groups such as halides⁷¹ which include DBPs such as haloacetic acids, haloacetamides, and haloacetonitriles.^72–74 In contrast, the presence of a halogen substituent β to the leaving group is strongly deactivating due to adverse nonbonding interactions between the lone pairs of the halogen and the incoming nucleophile.⁵⁹ This explains the low reactivity of 1,2-dichloroethane with nucleophiles. Finally, acylation is the reaction of an electrophile via transfer of an acyl moiety to a nucleophile and is relevant for esters of acidic alcohols such as phenol esters, acyl halides, and cyclic anhydrides.⁶⁶

Table 1 Mechanisms of the reaction of electrophiles with nucleophilic biomolecules

Reaction mechanism		Examples of drinking water relevant electrophiles
Michael addition		Quinones, quinone imines, unsaturated aldehydes
Schiff base formation		Saturated and unsaturated aldehydes
Aromatic substitution (SNAr)		2,4-Dinitrochlorobenzene, tetrachloroisophthalonitrile (fungicide)
Nucleophilic substitution (SN2)		Haloacetic acids, haloaceto-nitriles, haloketones
Acylation		Phthalic anhydride, phenol esters

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C Occurrence of electrophiles in drinking water

Despite the importance of electrophiles as environmental toxicants, their presence in drinking water has not been systematically evaluated. However, there is a large body of literature that has described the presence and formation of specific organic electrophiles in drinking water. The literature indicates that electrophile formation in water treatment systems utilizing chemical oxidants including chlorine, chloramine, ozone, hydroxyl radicals, and ultraviolet (UV) light is of particular relevance (Table 2). For drinking water treated by chlorination and chloramination, halogenated furanones such as MX were among the first classes of electrophilic DBPs (eDBPs) that were identified in drinking water.^75,76 They contribute significantly to the mutagenicity in disinfected drinking water, especially in that containing high concentrations of humic substances.^77–79 Other eDBPs commonly detected in disinfected drinking water include haloaldehydes, haloacetic acids, and haloketones. Their formation is primarily attributable to the reaction of chlorine with aliphatic and aromatic compounds.^80–83 While their formation can be significantly reduced by the use of chloramination, this typically results in increased production of electrophilic nitrogenous DBPs (eN-DBPs) including haloacetamides and haloacetonitriles.⁸⁴ The formation of eN-DBPs has been associated with the reaction of amino acids and other dissolved organic nitrogen compounds.^84,85 More recently, halogenated quinones, in particular 2,6-dichloro-1,4-benzoquinone and 2,6-dibromo-1,4-benzoquinone, have been reported in drinking water treated with chlorine and chloramine.^86,87 Their formation can most likely be explained by the presence of natural and anthropogenic compounds that contain phenolic moieties. The formation of non-halogenated benzoquinones and benzoquinone imines has also been identified in the reaction of aminophenols with chlorine.⁸⁸ Another important class is aldehydes, for which elevated concentrations have been reported in chlorinated waters containing tertiary alkylamines and primary amines.^89–91 If ClO₂ is used as a disinfectant, aldehydes can also be formed by reaction with aromatic compounds.⁹² This includes the formation of α,β-unsaturated aldehydes, in particular 2-butene-1,4-dial and its chlorinated analogue chloro-2-butene-1,4-dial, which were recently identified as novel eDBPs formed when phenolic compounds react with chlorine.⁹³

Table 2 Electrophiles previously identified in drinking water, relevant precursors (pro-electrophiles), and treatment processes leading to their formation (see Table S1 for details on the chemical structures of the electrophiles)^a

Electrophile class	Examples	Relevant pro-electrophiles	Formation by natural or technical processes	Electrophile reactivity domain
a Abbreviations: AOP – advanced oxidation process; ClO2 – chlorine dioxide; DON – dissolved organic nitrogen; NOM – natural organic matter; MCA – 3,4-dichloro-5-hydroxy-2(5H)-furanone; MCF – 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone; MX – 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone; SN2 – nucleophilic substitution; UV – ultraviolet.
Aldehydes	Formaldehyde, acetaldehyde, glutaraldehyde, glyoxal, methylglyoxal	Aliphatic and aromatic compounds containing double bonds, amino acids^110	Ozonation,^96,111 AOPs,^112 UV photolysis,^113 chlorination,^89 chloramination,^89 ClO2 ^92	Schiff base
α,β-Unsaturated aldehydes	Acrolein, 2-butene-1,4-dial, crotonaldehyde, 3-methyl-crotonaldehyde fluorotelomer aldehydes	Phenols, aromatics, unsaturated fatty acids^93,108,114	Ozonation,^94 AOP,^93 chlorination^93	Michael addition
Haloaldehydes	Chloroacetaldehyde, bromoacetaldehyde, 2-chloropropenal	Amino acids, low molecular weight NOM, phenolics	Chlorination^8,115,116 chloramination^117	SN2
Chlorinated hydroxyfuranones	MX, MCF, MCA	Fulvic acids, humic acids^117–119	Chlorination,^4,120,121 chloramination^4,78	Michael addition
Haloketones	1,1-Dichloropropanone, 1,3-dichloropropanone and 1,1,1-trichloropropanone	Amino acids, humic acid^85,116	Chlorination,^8,116 chloramination^8,116	SN2
Haloacetonitriles	Chloroacetonitrile, bromoacetonitrile, dichloroacetonitrile	Phenols, DON, haloaldehydes^85,122–127	Chlorination,^8,128 chloramination^8,128	SN2
Haloacetic acids	Chloroacetic acid, bromoacetic acid, dichloroacetic acid	Hydrophilic, neutral NOM, hydrophobic NOM^83,129	Chlorination,^4 chloramination^4	SN2
Haloacetamides	Chloroacetamide, bromoacetamide, dichloroacetamide	Organic acids, phenols^122,127,130	Chlorination,^4,8,84 chloramination^4,8,84	SN2
Quinones	Benzoquinone, ortho-quinone, 2,6-dichlorobenzoquinone	Aromatic compounds, phenols, aromatic amines^88,109,131,132	Chlorination,^88 ozonation,^101 AOPs^109,133	Michael addition
Quinone imines	Benzoquinone imine, N-acetyl-p-benzoquinone imine	Aromatic amines^88,104	Ozonation,^104 chlorination^88	Michael addition
Epoxides	Carbamazepine epoxide, oxirane, methyloxirane	Unsaturated aliphatic and aromatic compounds^134–136	Ozonation,^134,137,138 chlorination,^134,139 chloramination^134	SN2
Cyclic anhydrides	Phthalic anhydride, maleic anhydride	Azo dyes, hydroxylated aromatics^140,141	Ozonation,^141 AOPs^140,142–144	Acylation
Organo-phosphorus esters	Chlorpyrifos oxon, diazinon oxon, malaoxon, parathion oxon	Thiophosphates such as chlorpyrifos, malathion, and parathion^145,146	Chlorination,^145 photolysis,^147 hydrolysis,^148 ozonation^146	SN2
Cyanogen halides	Cyanogen chloride, cyanogen bromide	Aliphatic amino acids, formaldehyde^149,150	Chloramination,^150,151 chlorination^152	SN2
Hydro-peroxides	Hydroxymethyl hydroperoxide, l-hydroxyethyl hydroperoxide	Unsaturated alkyl compounds, thymidine, vinyl chloride^107,137,153	Ozonation^107,137,153	Unstable, form radicals and epoxides

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In addition to chlorine-based disinfection, aldehydes are one of the most important eDBPs in drinking water treatment using ozone and hydroxyl radicals. Of particular relevance are low molecular weight saturated aldehydes, including acetaldehyde, formaldehyde, glyoxal, and methylglyoxal, which have been detected in concentrations in the low to medium μg L⁻¹-range in treated waters.^94–96 Relevant precursors of aldehydes formed in ozonation and advanced oxidation processes include aromatic compounds, amino acids, and unsaturated fatty acids.^97–99 Formation of quinones has been observed during ozonation and advanced oxidation processes with known precursors, including aromatic compounds, phenols, and azo dyes.^100,101 Azo dyes are also important precursors of quinone imines, as are anilines and other phenolic compounds containing nitrogen substituents in the para- or ortho-position.^102–104 Other classes of electrophiles likely to be formed during oxidative water treatment include peroxides, epoxides, and α,β-unsaturated aldehydes.³¹ Information about their presence in drinking water, however, is still widely lacking. In laboratory experiments, the formation of hydroperoxides has been observed for the reaction of ozone with unsaturated fatty acids, alkenes, and nucleic acids such as thymine and thymidine.^105–107 Prasse et al. recently demonstrated the formation of α,β-unsaturated aldehydes when phenols react with hydroxyl radicals and/or UV light.¹⁰⁸ The reaction of benzene and other monoaromatic compounds with hydroxyl radicals can further result in the formation of C₆-α,β-unsaturated aldehydes.¹⁰⁹ The results demonstrate the relevance of these electrophiles and highlight the importance of developing approaches to detect them in the environment and drinking water.

D Reactivity-directed analysis for the assessment of exposure to organic electrophiles

Despite the widespread occurrence of electrophiles in drinking water, their detection in complex environmental mixtures is challenging. This is particularly true for small, highly polar, and reactive electrophiles. Addressing this limitation requires the rethinking of the analytical approaches currently in use. This rethinking is necessary, too, if we want to move beyond the detection of individual, known electrophiles to investigate the formation and presence of unknown electrophiles.

In toxicology and biochemistry, novel analytical approaches have emerged to specifically identify individual electrophiles (Table 3).^34,154 Initially, these methods were predominantly used in combination with in vitro-based approaches to elucidate adverse drug reactions facilitated by electrophilic metabolites.^155–161 In addition, a large number of studies have focused on the identification of electrophiles used in cosmetics or associated with occupational exposure due to their importance as skin and respiratory sensitizers.^65,162 Originally, these assessments were performed using animal models. Recognition of electrophile and nucleophilic biomolecule covalent reactions as critical MIEs in the adverse outcome pathways of these toxicity endpoints led to the development of alternative strategies to simulate these interactions via in chemico assays.^163–168 In contrast to in vivo and in vitro assays, in chemico assays are used to investigate the interactions of chemicals with biomolecules in the absence of cells or organisms. In this review, the term Reactivity-Directed Analysis (RDA) is introduced, which encompasses existing in chemico assays as well as novel approaches to identify unknown electrophiles in complex environmental mixtures such as drinking water.

Table 3 Overview of existing RDA approaches used for the in chemico assessment of electrophiles, their main function, and their benefits and limitations^a

RDA approach	Analytical detection method	Benefits and limitations
a Abbreviations: ADRA – amino acid derivative reactivity assay; Cor1C420 – peptide derived from the sequence around Cys420 in the human Coronin1 protein; DCYA – dansylcysteamine; DPRA – direct peptide reactivity assay; EASA – electrophilic allergen screening assay; HPLC – high performance liquid chromatography; HTS – high-throughput screening; NMR – nuclear magnetic resonance; LC – liquid chromatography; MS – mass spectrometry; PPRA – peroxidase peptide reactivity assay; RDA – reactivity directed analysis; UV – ultraviolet.
ADRA	HPLC-UV	Naphthalene modified amino acids allow for higher sensitivity compared to DPRA; no information on adducts; requires high concentrations of electrophiles
ADRA-DM	LC-MS/MS	Use of LC-MS/MS enables the use of lower concentrations of electrophiles; provides information on both nucleophile depletion and adduct formation
Cor1C420	LC-MS	Contains multiple electrophiles; detection of both depletion and adduct formation
DPRA	HPLC-UV	Requires high concentrations of electrophiles; only addresses depletion of peptides
EASA	HPLC-UV and HPLC-fluorescence	Use of nitrobenzenethiol and pyridoxylamine nucleophile probes; only addresses depletion of the nucleophilic probe
HTS-DCYA	Fluorescence microplate reader	Allows for high-throughput processing of samples; no information on the chemical structure of adducts; potential interference by nucleophile dimerization (relevant for cysteine)
NMR-DCYA	NMR	NMR allows detailed structural insights into adducts; work intensive as DCYA adducts have to be purified or synthesized in high quantities prior to NMR analysis
Photo-DPRA	HPLC-UV	Requires high concentrations of electrophiles; can be used to study both direct and indirect photolytic formation of electrophiles; no information on the chemical structure of adducts
Photo-ADRA	HPLC-fluorescence	Higher sensitivity compared to photo-DPRA; no information on the chemical structure of adducts
PPRA	LC-MS/MS	Metabolic activation peroxidase has a low substrate specificity; limited to compounds that can be degraded by horseradish peroxidase

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Existing RDA approaches can be distinguished based on the type of molecule and the analytical techniques used to elucidate the reaction of electrophiles with nucleophilic targets. Most approaches focus on the identification of adducts with proteins, peptides, or amino acids.^41,169,170 In contrast, they are less commonly used to assess reactions with DNA. The formation of DNA adducts themselves is a poor predictor of toxicity, in particular carcinogenicity, because the adducts have to be converted into mutations, which is dependent on whether they are recognized by cellular repair mechanisms.¹⁷¹ Thus, this review only discusses RDA approaches focusing on proteins and protein subunits, i.e., amino acids and peptides. It should be noted, however, that most protein reactive chemicals are also likely to react with DNA.^172,173

The development of RDA approaches has been largely driven by efforts to develop new screening approaches to assess the skin and respiratory sensitization potential of chemicals. Up to now, more than 4,000 skin sensitizing compounds have been identified, and two-thirds of these are organic electrophiles.^67,174,175 The direct peptide reactivity assay (DPRA)¹⁷⁶ is the most widely used assay and has been approved by the Organization for Economic Co-operation and Development as an alternative to conventional animal testing methods.¹⁷⁷ The DPRA uses synthetic peptides containing either lysine or cysteine to evaluate the reaction of individual organic chemicals with nucleophilic biomolecules.^168,176 In this assay, the sensitizers are added in 10-fold excess to either a cysteine or lysine model peptide, and the sensitizer potency is evaluated based on the peptide depletion after 24 h of incubation at room temperature detected via high performance liquid chromatography-UV detection (HPLC-UV). To also determine the chemical kinetics of electrophile–nucleophile reactions, the high-throughput kinetic profiling (HTKP) assay¹⁷⁸ and the electrophilic allergen screening assay (EASA) were developed.^179,180 This EASA, which is currently undergoing evaluation by the National Toxicology Program, uses stopped-flow techniques and nitrobenzenethiol and pyridoxylamine as cysteine and lysine substitutes, respectively. While the previously mentioned RDA approaches primarily assessed the depletion of nucleophiles using HPLC-UV analysis, the low sensitivity of the methods requires the use of high concentrations of both test chemicals and nucleophiles, thus limiting the applicability of these methods for hydrophobic compounds. To address this issue, alternative approaches providing a higher sensitivity have been developed. The amino acid derivative reactivity assay (ADRA)¹⁸¹ uses naphthalene-modified lysine and cysteine amino acids which can be analyzed by HPLC-UV at 281 nm and significantly increases its sensitivity compared to the DPRA assay. The same is true when fluorescence-labelled amino acids are used.^182,183 To enable the testing of large numbers of suspected electrophiles, high-throughput approaches have been developed. The high-throughput screening dansylcysteamine (HTS-DCYA) assay¹⁸⁴ allows for the direct quantification of dansylcysteamine adducts by fluorescence detection in multi-well microplates. In this assay, the dosing of electrophiles is followed by addition of silica-supported maleimide, which reacts with the remaining, unreacted DCYA. Subsequently, the solutions are centrifuged, and the supernatants containing only the DCYA electrophile adducts are transferred into 96-well plates for fluorescence analysis via a microplate reader.

Although detection via UV or fluorescence allows for the quantification of nucleophile depletion, it does not provide any information on the identity of the formed adducts. To address this limitation, LC-MS and nuclear magnetic resonance (NMR) spectroscopy can be applied to simultaneously determine peptide depletion, peptide oxidation (e.g. dimerization of cysteine), and adduct formation.^185–187 The high sensitivity of LC-MS compared to HPLC-UV also allows the use of lower concentrations of both electrophiles and nucleophiles. The ADRA-dilutional method (ADRA-DM) assay developed by Yamamoto et al. uses 100-fold lower concentrations than those used by the DPRA or ADRA, which is particularly advantageous for compounds with low water solubility.¹⁸⁸ In addition to the RDA approaches that use individual nucleophilic targets, investigations have also focused on the use of peptides containing multiple nucleophilic sites. The Cor1C420 kinetic peptide reactivity assay^186,189,190 uses a peptide derived from the sequence around Cys420 in the human Coronin1 protein, which shows a high reactivity toward electrophiles, as it contains two lysine residues at the N-terminal side of the cysteine residue.¹⁹¹ Ahlfors et al. developed an assay that uses the synthetic model peptide containing essentially all nucleophilic amino acids (H-Pro-His-Cys-Lys-Arg-Met-OH) followed by identification of adduct structures via NMR and LC-MS.¹⁸⁷ This allows for the assessment of the relative affinity of electrophiles to different nucleophiles.

To also address the relevance of biotic and abiotic transformation processes leading to the formation of electrophiles, studies have also focused on the development of assays to identify precursors of electrophiles, the so-called pro-electrophiles. These assays include the peroxidase peptide reactivity assay (PPRA) that uses a horseradish peroxidase and hydrogen peroxide system.^164,192,193 The active site of HRP is accessible by structurally diverse pro-electrophiles, making it ideal for screening purposes. Examples of relevant pro-electrophiles include aniline, 2-amino-phenol, 3-methylcatechol, and 2-methoxy-4-methylphenol, which are activated to benzoquinone and quinone imine electrophiles.^65,194 The relevance of sensitization reactions caused by the degradation of skin care product ingredients has led to the development of the photo-DPRA and photo-ADRA to address the formation of electrophilic transformation products by exposure to sunlight.^195,196 To investigate the formation of electrophiles during the photodegradation of various commonly used organic UV-filters, Stiefel et al. developed a high performance thin-layer chromatography-based assay.¹⁹⁷ Exposure of UV-filters to both artificial and natural sunlight revealed the formation of electrophiles for ketone-containing UV filters such as benzophenone-3 and 4-t-butyl-4′-methoxydibenzoylmethane. Similar results were observed for the photodegradation of UV-filters and identification of electrophiles via reaction with lysine-containing model peptides and bovine serum albumin.¹⁹⁸ In addition, the photo-DPRA has also been used to investigate the photosensitization of glyphosate and glyphosate-containing agrochemical formulations.¹⁹⁹

E Application of reactivity-directed analysis in the analysis of electrophiles in drinking water

The existing studies on the application of RDA demonstrate the general applicability of this new methodology to the identification of electrophiles. For the assessment of drinking water relevant contaminants, Pals et al. used a thiol reactivity assay to determine the toxicity of brominated DBPs including bromoacetic acid, bromoacetonitrile, and bromoacetamide.^72,200 The authors observed a high correlation with an in vitro genotoxicity assay performed in parallel. Similar results were obtained by Wei et al., who investigated the relative toxicity of haloacetonitriles using mammalian cell cytotoxicity, genotoxicity, and a thiol reactivity assay.²⁰¹ Boehme et al. used a kinetic glutathione assay to determine the thiol reactivity of α,β-unsaturated ketones, acrylates, and propiolates.²⁰² The authors observed a wide range of reaction rates spanning over 5 orders of magnitude. In addition, a high correlation (r² = 0.91) was observed between the measured rate constants and the toxicity toward the ciliates Tetrahymena pyriformis, thus demonstrating that the toxicity of these compounds is driven by their electrophilic reactivity. A similar approach was used by Roberts et al. to assess the thiol reactivity of 60 haloaliphatic compounds.⁷¹ The highest correlation (r² = 0.889) with acute aquatic toxicity (IG₅₀) to Tetrahymena pyriformis was observed for compounds in which a primary halogen is α to a carbonyl or other electronegative unsaturated group (S_N2 electrophiles).

In addition to the assessment of individual (drinking) water contaminants, recent efforts have also focused on the assessment of the presence of electrophiles in different environmental matrices. Dong et al. used a plate reader-based assay with N-acetyl-cysteine as a nucleophilic probe to evaluate the thiol reactivity of different water samples, including tap water and wastewater.^203,204 Thiol reactivity differed significantly between the different samples and was highest in treated wastewater and lowest in tap water and water from a direct potable reuse system.²⁰⁴ Furthermore, the authors demonstrated a high correlation between thiol reactivity and mammalian genotoxicity as observed using Chinese hamster ovary cells, which indicated the suitability of this method to assess the toxicity of environmental mixtures. Using the same approach, Dong et al. also determined the toxicity of wastewater amended with Br⁻ and I⁻ during subsequent treatment with ozone, chlorine, or chloramine.²⁰⁵ Significantly higher thiol reactivity was observed in chlorinated and chloraminated wastewater, while the ozonated wastewater showed a similar reactivity to the untreated control. According to the authors, this can be explained by the formation of ozonation products that are less toxic than the DBPs formed in chlorination and chloramination. However, no attempts were made to elucidate the electrophiles responsible for the observed thiol reactivity. In addition, all previously mentioned studies used an extraction step based on XAD resins to concentrate the electrophiles prior to their toxicity assessment. As such, the lower thiol reactivity observed for some of the samples, in particular ozonated wastewater, could potentially be attributed to the loss of low molecular weight and/or highly polar compounds such as aldehydes during the extraction step. Preconcentration using XAD resins was also used by Cheh et al. for the detection of alkyl halides and epoxides in drinking water via their reaction with the nucleophilic probe compounds 4-nitrothiophenol and 4-nitrobenzylpyridine.²⁰⁶ The authors further investigated the impacts of the addition of different amounts of these nucleophiles on the mutagenicity of the extracted drinking water sample. They observed a decrease in toxicity with increasing amounts of nucleophiles, which again demonstrated the importance of electrophiles as contributors to the toxic effects of these environmental mixtures.

Another promising application of RDA is the identification of toxic electrophiles formed during the oxidative treatment of individual drinking water contaminants in simulated laboratory studies. Prasse et al. investigated the formation of electrophilic transformation products during the degradation of phenolic compounds by UV light, hydroxyl radicals, and HOCl.^93,108 Formation of electrophiles was investigated using N-α-acetyl lysine and N-acetyl cysteine as nucleophilic probes. The identity of electrophile adducts with both amino acids was elucidated using high-resolution mass spectrometry. The results of experiments with phenol revealed the formation of the highly toxic dialdehyde 2-butene-1,4-dial. The toxicity of this compound was further studied using chemoproteomics, which revealed the reaction of 2-butene-1,4-dial with several proteins using mouse liver proteome.¹⁰⁸ Experiments with other substituted phenolic compounds revealed that dicarbonyl compounds are common transformation products of hydroxyl radicals with this class of chemicals. Most importantly, this was the first time that these compounds were detected in aqueous environments, which can be attributed to the inadequacy of conventional approaches like GC-MS and LC-MS. As such, RDA offers an opportunity to identify electrophiles that are intrinsically challenging to detect. In this context, nucleophilic biomolecules or their analogues function as derivatization agents, thus enabling the indirect identification of electrophiles via analysis of nucleophile adducts.

F Future research needs

RDA represents a novel approach with the potential to substantially expand our knowledge about the presence and formation of toxic contaminants in drinking water and other aqueous matrices. As discussed in this review, electrophiles are commonly detected in drinking water, and their relevance will rise in the future due to the increased application of oxidative treatment technologies. This increase is contrasted by the limited availability of analytical methods to comprehensively assess the presence of electrophiles in drinking water. This is particularly true for electrophiles characterized by low molecular weight, high polarity, and low stability, as they are likely to be lost during current sample work-up procedures and difficult to directly detect by conventional analytical approaches such as LC- and GC-high-resolution mass spectrometry. Even though the environmental applications of RDA are still limited, the escalating importance of this approach for chemical risk assessment will likely contribute to the expansion of this technology toward the analysis of complex mixtures and assessment of environmental exposure.^207–209 In addition to electrophile detection in drinking water and reactive organic electrophile identification in (oxidative) drinking water treatment processes, RDA also offers new approaches to advance our understanding of the impacts of drinking water contaminants on human health. It provides a new tool to identify chemicals that are of particular health concern and that should be prioritized in future toxicity testing efforts that use in vitro and in vivo assays.

In addition, RDA can also support epidemiological studies that focus on the health impacts of drinking water exposure. Existing data strongly suggest an association between the presence of specific drinking water contaminants, in particular THMs, and health outcomes such as bladder cancer.^11,210 It is currently unknown, however, whether this association is indeed attributable to THMs or whether other co-occurring compounds are responsible, considering that the vast majority of drinking water contaminants (including DBPs) are not (or not widely) monitored. For skin cancer, exposure to drinking water contaminants has been associated with the presence of THMs, which are metabolized by skin enzymes to toxic intermediates.¹⁰ Thus, RDA could provide novel insights into the formation of electrophilic intermediates that lead to adverse health outcomes, e.g., in controlled laboratory experiments simulating these metabolic processes, and help to elucidate the toxic mode of action. RDA can also help identify the chemicals responsible for dermal and respiratory diseases associated with drinking water exposure.¹³ As discussed before, electrophiles represent the majority of (skin) sensitizers that are responsible for chemically induced allergic reactions. Known electrophilic sensitizers are characterized by molecular weights <500 Da,^211,212 which is also likely to be the case for electrophiles formed during oxidative drinking water treatment. As such, organic electrophiles are likely candidates responsible for these effects, and RDA represents an ideal platform to identify the relevant toxicants.

Despite the advantages of RDA as a novel tool to determine the presence and formation of organic electrophiles in drinking water, a number of challenges need to be addressed before the approach can be more widely applied. These include the detection of electrophiles at concentrations relevant for drinking water, the stability of nucleophilic probes—in particular thiol-containing compounds—and the stability of specific nucleophile adducts. Similar to other analytical approaches, the low concentrations of most drinking water contaminants represent a major challenge for their analysis. Existing strategies that use additional pre-concentration steps like SPE or resins like XAD are not suitable for the extraction of most electrophiles, as they are not sufficiently retained and/or reactive with the sorbents. For RDA, this issue could be addressed by direct addition of nucleophilic probe compounds to the (drinking) water samples (Fig. 1). This step would then be followed by extraction of the nucleophile adducts using conventional approaches such as SPE. Modified nucleophilic probes containing hydrophobic moieties, similar to those used to enhance their detection by UV or fluorescence, could be utilized to enhance the extraction efficacies. On the other hand, this approach would require the addition of large amounts of nucleophiles to reach sufficient nucleophile concentrations in the solutions. For the subsequent analysis of adducts, the low concentrations of electrophiles in drinking water might also be challenging due to the high concentrations of unmodified nucleophilic probes in the sample extracts. As such, an additional sample pre-treatment step might be required to separate the modified nucleophiles from the unmodified nucleophiles. Another challenge is the potential auto-oxidation of nucleophiles, in particular thiols, by residual chemical oxidants and trace metals in drinking water samples. Thiols present in cysteines are easily oxidized and form disulfides, which can significantly impact the availability of nucleophiles as well as their analysis.^213,214 To address this issue, additives such as ethylenediaminetetraacetic acid can be added to complex trace metals and reducing agents such as tris(2-carboxyethyl)phosphine can be added to reduce oxidized thiol species back to their reduced monomeric form.¹⁸² Similarly, sodium cyanoborohyride can be used to stabilize Schiff base adducts.⁶⁸

Fig. 1 Strategies for the identification of electrophiles in drinking water using RDA. The nucleophile depletion assay (a) allows for the general assessment of the presence of electrophiles in drinking water, while the nucleophile adduct identification assay (b) can be used to detect individual nucleophile adducts which can then be used to identify the relevant electrophiles responsible for the adduct formation.

Finally, the exposure time of the reaction between nucleophilic probe compounds and organic electrophiles needs to be considered. The reaction kinetics of electrophiles with nucleophiles vary substantially and span several orders of magnitude.^215,216 While nucleophiles are generally added in a large excess to prevent their complete depletion, shorter exposure times will result in bias toward highly reactive electrophiles, while longer exposure times would be required for slower reacting compounds. However, this could potentially be used to prioritize electrophiles likely to be of the highest concern as the electrophilic reactivity is highly correlated with toxicity.²¹⁷

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

I want to thank Daisy Grace, Veronica Wallace, Casey Smith and Jessica Goddard for valuable comments and edits on the draft and Daisy Grace for the design of the TOC art. I am grateful to all the incredible researchers who have inspired and influenced my research. I further acknowledge financial support by Johns Hopkins University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0em00471e

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