(4-Nitrobenzyl)pyridine - American Chemical Society

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Chemoassay Screening of DNA-Reactive Mutagenicity with 4‑(4-Nitrobenzyl)pyridine − Application to Epoxides, Oxetanes, and Sulfur Heterocycles Diana Thaens,†,‡ Daniel Heinzelmann,†,‡ Alexander Böhme,† Albrecht Paschke,† and Gerrit Schüürmann*,†,‡ †

UFZ Department of Ecological Chemistry, Helmholtz Centre for Environmental Research, Permoserstraße 15, 04318 Leipzig, Germany ‡ Institute for Organic Chemistry, Technical University Bergakademie Freiberg, Leipziger Straße 29, 09596 Freiberg, Germany S Supporting Information *

ABSTRACT: Organic electrophiles have the potential to covalently attack DNA bases, and thus initiate mutagenic and carcinogenic processes. In this context, aromatic nitrogen sites of the DNA bases are often particularly nucleophilic, with guanine N7 being one of the most favored sites of adduct formation with electrophilic xenobiotics. Employing 4-(4-nitrobenzyl)pyridine (NBP) as model nucleophile with a respective aromatic N− unit, a new kinetic variant of a photometric chemoassay for sensing the DNA reactivity of organic compounds is introduced and applied to 21 three- and four-membered oxygen and sulfur heterocycles (15 epoxides, two thiiranes, three oxetanes, and one thietane). Besides six unreactive compounds (oxetanes, thietane, and aliphatic epoxides with six or more side-chain carbons), second-order rate constants of the electrophile-NBP reaction, kNBP, were obtained for 15 compounds, ranging from (1.16 ± 0.05)·10−3 to (36.5 ± 0.6)·10−3 L mol−1 min−1 in a methanol/tris-HCl buffer (16/84 v/v) reaction medium. Solvolysis as confounding factor was addressed by determining respective first-order rate constants ksolv. Analysis of the kNBP values resulted in structure−reactivity relationships, and comparison with literature data from the Ames test bacterial strains TA100, TA1535, and TA97 (Salmonella typhimurium) as well as from WP2 uvrA (Escherichia coli) revealed significant log−log relationships between the mutagenic potency of the heterocycles and their reactivity toward NBP. The latter demonstrates the potential of the NBP chemoassay as a nonanimal component of integrated testing strategies for REACH, enabling an efficient screening of organic electrophiles with respect to their DNA reactivity and associated mutagenicity and carcinogenicity.

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INTRODUCTION Most DNA-reactive agents are either electrophiles or radical intermediates.1 An example of the latter is the OH radical that may be generated through oxidative stress, and attacks the DNA through homolytic H abstraction or addition to double bonds. By contrast, electrophiles have the potential to alkylate DNA-base nitrogen (endocyclic: −NH− and N−; exocyclic: −NH2) and oxygen (exocyclic: O and −OH) as well as phosphodiester residue oxygen (−O−P(O)(OR)2). Guanine N7 appears to be particularly nucleophilic and is a favored site of attack by small and thus sufficiently diffusible electrophiles; nevertheless, alkylation can take place at virtually all DNA heteroatoms with respective lone-pair electrons.1 DNA damage may result in metabolic dysfunctions through modified gene expression, in the inhibition of cell replication or cell death, or in gene mutation, the latter of which may (but need not) yield cancer. There is evidence that the regioselectivity of electrophilic attack is related to the Pearson HSAB (hard and soft acid and base) concept.1−3 While DNA alkylation of exocyclic N and © 2012 American Chemical Society

monoamidic O as well as of endocyclic amidic N typically results in stable products, electrophilic attack at endocyclic sp2 N and at exocyclic carbamide O leads to labile lesions, facilitating deglycosylation and ring-opening.1 Given that N is generally more nucleophilic than O and that the most nucleophilic DNA-base sites are of the N− type that is also softer than both amino and amidic N, a simple strategy for screening the alkylation potential of electrophiles is to focus on their reactivity toward a reference nucleophile carrying an aromatic N− unit. More than 50 years ago, a respective approach had been undertaken, employing 4-(4-nitrobenzyl)pyridine (NBP) with an aromatic nitrogen as the nucleophilic site.4 Since then, NBP has been applied to sense the alkylating potency of a number of organic electrophiles.5−16 Indeed, the alkylation of NBP is easy to observe because of the blue colored product that is formed through the reaction with the electrophile (see Scheme 1). Received: March 30, 2012 Published: August 13, 2012 2092

dx.doi.org/10.1021/tx3001412 | Chem. Res. Toxicol. 2012, 25, 2092−2102

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previously analyzed data sets and taking into account solvolysis as confounding factor. The chemicals tested were selected to investigate the effects of structural features such as length and branching of aliphatic side chains, ring size, nonaliphatic side chains, and ring oxygen vs sulfur on the reactivity toward NBP. The resultant second-order rate constants of the electrophileNBP reaction, kNBP, lead to structure−reactivity relationships that can be used for screening-level estimates of the NBP reactivity. Moreover, comparison with literature data on Ames test results reveals a significant log−log relationship between kNBP and the mutagenic potency, demonstrating the potential of the NBP chemoassay as a nonanimal tool for the DNA-reactive mutagenicity and carcinogenicity screening of organic electrophiles.

Scheme 1. Reaction of 4-(4-Nitrobenzyl)pyridine (NBP) (A) with an Epoxide (B)a

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a

Deprotonation of the resulting colorless intermediate product (C) by a base yields the final blue product (D) that can be determined photometrically.

MATERIALS AND METHODS

Chemicals. 4-(4-Nitrobenzyl)pyridine (NBP), methanol, tris(hydroxymethyl)aminomethane (tris), hydrochloric acid (HCl), potassium carbonate, and acetone were obtained from Merck (Darmstadt, Germany). The test compounds (see Scheme 2) were from Merck, Alfa (Karlsruhe, Germany), and Sigma-Aldrich (Steinheim, Germany). All chemicals were at least of p.a. grade. Kinetic NBP Chemoassay. The solutions of the reagents were prepared freshly for every experiment. NBP was dissolved in methanol and 10 mM tris-HCl buffer (pH 7.4) (1:4 (v/v)). The concentrations of the NBP stock solutions ranged from 0.541 to 5.14 mmol L−1. The stock solution of the test compound consisted of methanol and trisHCl buffer (2:3 (v/v)) with test compound concentrations ranging from 16.5 to 151 mmol L−1. The amounts of NBP and test compound were selected such that the concentration of the test compound’s stock solution is about 30 times higher than the one of NBP. The reactions were performed in glass-coated deep well plates (LabHut, Maisemore, Gloucestershire, UK). Every reaction mixture consisted of 720 μL of tris-HCl buffer, 720 μL of NBP stock solution, and 360 μL of test compound stock solution. Immediately after filling the 96 wells by using an electronic 8-channel pipet (Proline 50 − 1200 μL from Biohit, Helsinki, Finland), the plates were capped with a sealing film (Platemax Cyclerseal from Axygen, Union City, USA) to avoid evaporation and contamination from surrounding air. The reactions were performed under plate agitation using a microplate shaker (PMS-1000 from Grant-bio, Shepreth, Cambridgeshire, UK) at 25 °C in the climate chamber (Binder, Tuttlingen, Germany). The progress of the reaction was observed by determining the absorbance of the reaction product using UV−vis spectrometry. Therefore, 160 μL of the reaction mixture from each well of a column

Given the scope of efficient and mechanistically sound screening tools in the context of integrated testing strategies for REACH,17 a correspondingly adapted NBP test procedure could serve as a chemoassay primarily sensing the reactivity of electrophiles toward DNA bases. As compared to the Ames test that requires the cultivation and testing of five bacterial strains, the NBP chemoassay requires less laboratory time, is more cost-effective, and provides more specific information about the contaminant reactivity toward DNA-type aromatic nitrogen. Moreover, NBP and glutathione (GSH) could be used as complementary components of a respective chemoassay battery, informing about the potential of electrophiles to attack DNA or proteins18,19 or both. Epoxides (oxiranes) are alkylating agents that can react in vivo with nucleophilic sites of proteins and the DNA,20 with a preference for guanine N7 among the DNA bases.21 Moreover, monosubstituted epoxides are more potent mutagens toward the bacterial strains Salmonella typhimurium TA100 and TA1535 than higher substituted derivatives.22 Regarding aquatic toxicity, recent investigations with bacteria and ciliates revealed systematic relationships between the substitution pattern of oxiranes and their toxic potency.23,24 In the present study, a modified variant of the NBP chemoassay is introduced and applied to 15 epoxides, two thiiranes, three oxetanes, and one thietane, going beyond

Scheme 2. Chemical Structures of the 21 Oxygen and Sulfur Heterocycles Listed in Table 1a

a

Epoxides: 1,2-propylene oxide (A1), 1,2-epoxybutane (A2), 1,2-epoxyhexane (A3), 1,2-epoxyoctane (A4), 1,2-epoxy-9-decene (A5), 1,2epoxydodecane (A6), isobutylene oxide (A7), 1,2-epoxy-3-methylbutane (B1), 3,3-dimethyl-1,2-epoxybutane (B2), styrene oxide (B3), allyl glycidyl ether (B4), n-butyl glycidyl ether (B5), iso-propyl glycidyl ether (B6), glycidyl phenyl ether (B7), and glycidyl methacrylate (C1); oxetanes: trimethylene oxide (C2), 3-methyl-3-oxetanemethanol (C3), and 3,3-dimethyloxetane (C4); thiiranes: ethylene sulfide (C5) and propylene sulfide (C6); thietane: trimethylene sulfide (C7). 2093


2094

C1 B7 B3 B5 B4 B6 A7 A1 A2 A3 B1 C5 C6 A4 B2 A5 A6 C2 C3 C4 C7

glycidyl methacrylate glycidyl phenyl ether styrene oxide n-butyl glycidyl ether allyl glycidyl ether iso-propyl glycidyl ether isobutylene oxide 1,2-propylene oxide 1,2-epoxybutane 1,2-epoxyhexane 1,2-epoxy-3-methylbutane ethylene sulfide propylene sulfide 1,2-epoxyoctane 3,3-dimethyl-1,2-epoxybutane 1,2-epoxy-9-decene 1,2-epoxydodecane trimethylene oxide 3-methyl-3-oxetanemethanol 3,3-dimethyloxetane trimethylene sulfide

106-91-2 122-60-1 96-09-3 2426-08-6 106-92-3 4016-14-2 558-30-5 75-56-9 106-88-7 1436-34-6 1438-14-8 420-12-2 1072-43-1 2984-50-1 2245-30-9 85721-25-1 2855-19-8 503-30-0 3143-02-0 6921-35-3 287-27-4

CAS 588−596 576−592 587−592 584−592 584−592 584−592 588−596 589−595 590−597 588−597 590−596 574−592 572−592 587−594 592−604

λmax (nm) (5.41 (5.61 (5.65 (5.72 (5.50 (5.63 (5.83 (5.53 (5.71 (5.64 (5.37 (5.73 (5.73 (5.63 (5.67

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.09)·104 0.07)·104 0.11)·104 0.12)·104 0.09)·104 0.01)·104 0.27)·104 0.05)·104 0.04)·104 0.15)·104 0.04)·104 0.04)·104 0.03)·104 0.05)·104 0.08)·104

ε592 nm (L mol−1 cm−1) (36.5 ± 0.6)·10−3b (32.4 ± 2.5)·10−3b (22.2 ± 0.4)·10−3 (12.8 ± 0.4)·10−3b (11.5 ± 0.3)·10−3 (10.4 ± 0.4)·10−3b (9.01 ± 0.07)·10−3 (6.51 ± 0.33)·10−3 (4.71 ± 0.33)·10−3b (4.48 ± 0.36)·10−3b (2.81 ± 0.03)·10−3b (2.43 ± 0.38)·10−3 (2.28 ± 0.03)·10−3 (1.54 ± 0.11)·10−3 (1.16 ± 0.05)·10−3b not reactivec not reactivec not reactive not reactive not reactive not reactive

kNBP (L mol−1 min−1) (35.3 (32.1 (26.6 (13.8 (13.1 (10.1 (11.1 (7.23 (4.98 (5.34 (2.82 (3.86 (3.55 (2.84 (1.19

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.2)·10−3 3.2)·10−3 1.2)·10−3b 1.2)·10−3 0.6)·10−3b 0.8)·10−3 0.1)·10−3b 0.27)·10−3b 0.20)·10−3 0.71)·10−3 0.21)·10−3 0.87)·10−3b 0.39)·10−3b 0.21)·10−3b 0.06)·10−3

corr. kNBP (L mol−1 min−1) (7.44 (3.59 (1.13 (3.12 (5.45 (4.48 (1.44 (4.05 (5.37 (1.04 (3.96 (2.15 (1.91 (5.48 (5.75

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.26)·10−5 1.00)·10−5 0.13)·10−4 2.26)·10−5 1.35)·10−5 6.72)·10−5 0.89)·10−3 2.73)·10−4 3.19)·10−4 0.91)·10−4 3.56)·10−5 0.21)·10−3 0.58)·10−3 1.51)·10−5 3.50)·10−6

ksolv (model) (min−1) ± ± ± ± ± ±

0.04)·10−4 0.32)·10−5 0.06)·10−4 0.17)·10−5 0.42)·10−5 0.37)·10−5

(7.72 ± 0.70)·10−5

(4.45 ± 0.14)·10−5 (1.89 ± 0.31)·10−5 (1.04 ± 0.06)·10−3

(1.34 (6.87 (2.83 (2.96 (5.11 (3.00

ksolv (measured) (min−1)d

The compound numbering refers to Scheme 2. corr. kNBP: The kNBP values from simple linear data evaluation through eq 3 were corrected for solvolysis using eq 5. The ksolv (measured) values were determined separately by GC-MS, GC-FID, or LC-DAD analysis of compound concentration and calculated using eq 1. The ksolv (model) values were calculated from the kinetic NBP reaction setups using eq 5. bkNBP values used for discussion. cAt saturation limit. dThe respective solvolysis half-lives (t1/2 = ln 2/ksolv) range from 11.1 h to 16.2 days.

a

no.

compound

Table 1. Second-Order Rate Constants kNBP for the Reaction of 21 Heterocyclic Compounds with NBP Together with Associated Solvolysis Rate Constants ksolv, Wavelengths of the Maximum Absorbance λmax, and Extinction Coefficients ε592 nma

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were transferred to a microplate. Then, 10 μL of 2 M potassium carbonate solution as base and 130 μL acetone were added. The intensity of the developed blue color was measured directly with a UV−vis spectrometer with microplate reader (SpectraMax Plus 384 from Molecular Devices, Sunnyvale, USA) at 592 nm. Solvolysis of the Test Compounds. The stability of the test compounds in the reaction medium can be limited due to solvolysis effects. Therefore, the solvolysis of 10 of the test compounds was investigated in addition to the kinetic chemoassay experiments. The amount of the test compound was determined by GC-MS, GC-FID, or LC-DAD in triplicate (for details see the Supporting Information). The concentration−time relationships were used to determine the first-order solvolysis rate constants, ksolv (measured), according to the following equation, with cE denoting the electrophile concentration:

ln

c E(0) = ksolv ·t cE

values, an approximate equation (Taylor expansion confined to second order) covering both the reaction with NBP and the solvolysis was derived (for details see the Supporting Information). This equation was applied to the kinetic measurement results for determining ksolv (model) and kNBP values, yielding a correction of the latter for the additional electrophile loss through solvolysis: ln

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(1)

dc p dc NBP = kNBP· c E· c NBP = dt dt

RESULTS AND DISCUSSION Chemoassay Optimization. The goal of the chemoassay optimization was to extend its application range to less soluble compounds, to adapt the procedure to physiologically relevant conditions regarding pH, temperature and the test medium (aqueous solution), and to make the approach thus fit as a mechanistic ITS component for the predictive DNA reactivity assessment of organic electrophiles. While previous reaction test procedures with NBP as nucleophile focused on ethylene glycol and acetone as solvents,4−12,15,26,27 we employed methanol28 that is significantly less volatile than acetone and a much better solvent for NBP than ethylene glycol. Test solutions with 16% methanol turned out as a good compromise between aqueous conditions and a sufficient NBP solubilization to obtain proper reaction times. Moreover, a buffer of 10 mM tris-HCl was found to keep the pH value sufficiently constant at 7.4 (physiological milieu) for all test compounds, contrasting with much higher buffer concentrations (0.1 to 0.2 M) or the lack of buffer in previous procedures.5,6,8−13,16,27 Instead of using high temperatures (100−180 °C)4,8,11−13,29 for shortening the reaction time, we preferred employing 25 °C to avoid monitoring reactions not feasible at physiological conditions, and to reduce the volatilization tendency and allow for an overall convenient handling. So far, the base for deprotonating the reaction intermediate (see Scheme 1) has often been triethylamine in pure form or as a mixture with 50% acetone.4−7,9,10,15,16,26,27 However, triethylamine is quite caustic, and indeed destroyed the microplates used for the absorbance measurements. Among the alternatives NaOH, KOH, Na2CO3, and K2CO3, absorbances comparable to triethylamine were obtained with K2CO3, which is thus recommended for the presently used microplate variant of the NBP chemoassay. The design of the latter followed the procedure introduced previously for the kinetic glutathione (GSH) chemoassay,18 employing deep-well plates with a small headspace (sealed after filling to hinder further evaporation) and an electronic 8-channel pipet device that provide a capacity for eight blanks and 88 reaction mixtures. Hence, UV−vis absorbance measurements could be made at 11 different reaction times, with eight replicates per reaction time. Finally, instead of confining the data evaluation to first-order rate constants, which are always linked to the concentration of the reactant being in excess, we report concentrationindependent second-order rate constants kNBP, thus enabling direct comparisons with reactivities toward other model nucleophiles such as GSH18,19 as well as for respective computational toxicology investigations.30−34 In this context,

(2)

where cNBP denotes the NBP concentration, cE the concentration of the test compound, and cp the concentration of the product. Pseudo-firstorder conditions with cE ≫ cNBP yield a respective rate constant k′ = kNBP·cE. Under these conditions, the maximum product concentration is given by the initial NBP concentration, cpmax = cNBP(0). According to Lambert−Beer’s law, the absorbance A and its maximum value Amax correspond to cp and cpmax, respectively. The integrated rate law can be written as c pmax − c p A −A ln = − k′·t = ln max c pmax A max (3) For all test compounds, Amax was determined separately (see below), and the relationship between ln[(Amax − A)/Amax] and t was monitored through 5−11 measurements (and eight replicates per measurement) over the relevant concentration range (see Supporting Information, Figure S5), yielding r2 values of 0.877 to 0.999 and compound-specific slopes k′. The associated second-order rate constants kNBP were then obtained through dividing k′ by cE(0). For each compound, at least three kinetic experiments were performed using different initial values for cNBP and cE, and the correspondingly averaged kNBP values and their standard errors are listed in Table 1. Determination of Maximum Absorbances and Extinction Coefficients. Using the same stock solutions (7.92 mL tris-HCl buffer, 7.92 mL NBP stock solution, and 3.96 mL test compound stock solution) as for the above-described kinetics, the absorbance A at 592 nm was measured over a much longer period of time (weeks to months) until the maximum absorbance Amax was reached (see Supporting Information, Figures S1−S4). For these measurements, aliquots of the reaction mixture were diluted with methanol/tris-HCl buffer (16/84 v/v) before adding potassium carbonate and acetone to ensure absorbances below 6. The measured absorbances were multiplied by the dilution factor to obtain the absorbance of the reaction mixture. Application of Lambert−Beer’s law with a path length d of 1 cm yielded the compound-specific extinction coefficients ε592,

ε592 (L mol−1 cm−1) =

A max c NBP(0)(mol L−1)·d(cm)

(5)

Statistical Parameters. For the quality evaluation of the performed regressions, the following parameters have been applied: squared correlation coefficient r2, root-mean-square error of calibration rms, F-test value F1,n‑2 (with n = number of compounds), leave-1-out cross-validated predictive squared correlation coefficient qcv2,25 and cross-validated root-mean-square error of prediction rmscv.

Determination of Second-Order Reaction Rate Constants. The reactivity of the test compounds toward NBP was quantified through second-order reaction rate constants kNBP as follows:

−

c NBP 1 ≈ − kNBP·c E(0)·t + ·kNBP·ksolv ·c E(0)·t 2 c NBP(0) 2

(4)

which are also listed in Table 1. These extinction coefficients were used for quantifying Amax of eq 3 through multiplication with the cNBP(0) values employed for the respective kinetic experiments with the individual test compounds, thus saving the need for experimentally determining Amax for every single cNBP(0) value. Correction of kNBP for Solvolysis. To consider solvolysis of the electrophile (= hydrolysis and alcoholysis) and its impact on the kNBP 2095



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compounds with detectable reactivities, some of the resultant ksolv (model) values show unusually large standard errors. Possible reasons include the current limitation to 5−11 measurement points per compound and the restriction of the Taylor expansion underlying eq 5 (see Supporting Information) to second order, keeping in mind that inclusion of higher orders would still increase the miminum number of measurement points. Nevertheless, ksolv (measured) agrees with ksolv (model) within a factor of 2 for the 10 compounds where both experimental and calibration-derived solvolysis rate constants are available, indicating that eq 5 as an approximate model for two concurrent reactions enables a reasonable estimation of the impact of electrophile solvolysis on kNBP. Reactivity toward NBP. The reactivity toward NBP was determined for a set of 21 test compounds (see Scheme 2) containing 15 oxiranes (epoxides), two thiiranes, three oxetanes, and one thietane. The respective kNBP values according to eq 3 (without considering the additional electrophile loss through concurrent solvolysis) are listed in the sixth column of Table 1. They span a factor of 30, ranging from (1.16 ± 0.05)·10−3 L mol−1 min−1 (log kNBP = −2.94: 3,3dimethyl-1,2-epoxybutane, B2) to (36.5 ± 0.6)·10−3 L mol−1 min−1 (log kNBP = −1.44: glycidyl methacrylate, C1). Inclusion of the concurrent electrophile solvolysis through calibration with eq 5 yields the corrected kNBP values in the seventh column of Table 1. These range from (1.19 ± 0.06)·10−3 L mol−1 min−1 (B2) to (35.3 ± 2.2)·10−3 L mol−1 min−1 (C1). Comparison between both kNBP data sets shows the largest deviations for ethylene sulfide (2.43·10−3 vs 3.86·10−3), propylene sulfide (2.28·10−3 vs 3.55·10−3), and 1,2-epoxyoctane (1.54·10−3 vs 2.84·10−3). Based on the present data set and our experience with the NBP chemoassay gained so far, we recommend correcting the reactivity toward NBP when the electrophile concentration changes by more than 10% through solvolysis within the observed reaction time. In terms of pseudo-first-order kinetics this condition reads

a particular advantage has been achieved through deriving the concentration-independent extinction coefficients ε592. To this end, experiments were carried out over a time period of several weeks. To check the possible loss of electrophile through solvolysis, the latter was analyzed separately employing the chemoassay reaction medium but without NBP. In Figures S1−S4 of the Supporting Information, the respective results are shown for glycidyl methacrylate (C1) and glycidyl phenyl ether (B7) as the two compounds with largest reactivities toward NBP, styrene oxide (B3) as the compound with the shortest hydrolysis half-life according to an earlier estimation,24 and 1,2-epoxyoctane (A4) as a representative of epoxides at the low end of NBP reactivity. As can be seen from Figures S2 and S4, Supporting Information, after 29 days of solvolysis the concentrations of glycidyl phenyl ether and 1,2-epoxyoctane are still large enough to deplete the whole initial NBP concentration as applied for the NBP chemoassay. By contrast, 29-day solvolysis reduces the concentrations of glycidyl methacrylate (Figure S1, Supporting Information) and styrene oxide (Figure S3, Supporting Information) to values below the initial NBP concentration. During regular NBP chemoassay experiments, however, the electrophile-NBP reaction is faster than the electrophile-solvent reaction (see below), making sure that there is always enough electrophile to react with NBP until all NBP is depleted. As can be seen from Table 1, ε592 turned out to be essentially constant for all electrophile-NBP reaction products (Scheme 1, right) with an average value of (5.62 ± 0.12)·104 L mol−1 cm−1. These results confirm the expectation that because none of the presently analyzed electrophiles participates in the chromophore system of the NBP-electrophile adduct, the electrophile moiety has no significant impact on the absorbance of the colored product. It demonstrates further that this product is relatively stable in the methanol/tris-HCl solution, and that in all experiments for determining ε592 all of the initial NBP was converted by the epoxide. The essentially constant ε592 value implies further that for future investigations with test compounds where the chromophore remains confined to the deprotonated NBP moiety (or where additional chromophoric moieties would neither be conjugated with this moiety nor absorb in the relevant wavelength range), the time-consuming determination of maximum absorbances is no longer needed. Moreover, typical second-order reaction setups with similar concentrations for both NBP and the electrophile can be applied, extending the range of suitable test compounds to a lower minimum solubility as well as to a larger maximum reactivity. Electrophile Solvolysis Concurrent to the Reaction with NBP. The solvolysis of the electrophile (= reaction with water and methanol) forms a confounding factor for the determination of kNBP according to pseudo-first-order kinetics (see eq 3) because cE may change significantly. Therefore, pseudo-first-order rate constants of the solvolysis were measured (ksolv (measured)) for compounds B3−B7, C1, A3, B1, C5, and A4, and calculated for all electrophiles applying eq 5 (ksolv (model)). The results are summarized in the last two columns of Table 1. As can be seen from the table, ksolv (model) ranges from (5.75 ± 3.50)·10−6 min−1 to (2.15 ± 0.21)·10−3 min−1 (B2 and C5, respectively), while the minimum and maximum experimental ksolv values are (1.89 ± 0.31)·10−5 min−1 (B1) and (1.04 ± 0.06)·10−3 min−1 (C5), respectively. Note that despite r2 values of 0.87 to 0.96 when calibrating eq 5 for the 15

c E(t ) = e−ksolv·t < 0.9 c E(0)

(6)

as an empirical criterion whether solvolysis should be considered as a confounding factor. In case eq 6 indicates that electrophile solvolysis may play a role, a comparison of kNBP (eq 3) and corrected kNBP (eq 5) taking into account their standard deviations will show whether this is indeed the case, provided the recorded time course of cNBP(t) enables a reasonable calibration of eq 5. Following this approach, the NBP reactivity of seven electrophiles (A1, A4, A7, B3, B4, C5, and C6) was corrected for concurrent solvolysis when referred to in subsequent discussions. Because ksolv is derived as a first-order rate constant, a direct comparison with kNBP is not possible. Considering the composition of the reaction medium with 84% water and 16% methanol, division of ksolv by the water molarity (55.56 mol/L) yields approximate second-order solvolysis rate constants, ranging for the 10 experimental values from 1.87·10−5 L mol−1 min−1 (ethylene sulfide, C5) to 3.40·10−7 L mol−1 min−1 (1,2-epoxy-3-methylbutane, B1), and for the 15 data points calibrated through the concurrent-reaction model from 3.87·10−5 L mol−1 min−1 (ethylene sulfide) to 1.03·10−7 L mol−1 min−1 (3,3-dimethyl-1,2-epoxybutane, B2). In all cases, the accordingly estimated second-order solvolysis rate constants are smaller than kNBP by at least two and up to four 2096



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orders of magnitude, indicating a substantially larger electrophile reactivity for NBP than for water/methanol. Structure−Reactivity Trends. The data listed in Table 1 reveal the following structure−reactivity trends: First, the heterocyclic reactivity toward NBP decreases with increasing size of the alkyl side chain. Examples are the kNBP values of the compounds A1−A4 as well as of the series A2, B1, and B2. Second, kNBP decreases with increasing alkyl group branching, as can be seen from comparing the two isomers A3 and B2. Note further that the n-butane derivative (A3) is more reactive than the iso-propyl counterpart (B1), indicating the sensitivity of kNBP toward α-C branching. Third, 1,2-propylene oxide (A1) is more reactive by factors of 2.04 and 1.87 than the corresponding thiirane (C6) and ethylene sulfide (C5), indicating that kNBP is larger for epoxides than for corresponding thiiranes. This trend contrasts with recent findings on their reactivity-driven toxicity enhancements Te = EC50(narcosis)/EC50(exp.) (48-h EC50, effective concentration yielding 50% growth inhibition) toward the ciliates Tetrahymena pyriformis that implied essentially identical reactivity contributions to toxicity for C6 and A1, and a somewhat larger electrophilic reactivity for C5.24 A possible explanation for this discrepancy would be that NBP, although it could also mimic the heterocyclic N of tryptophan and histidine, may be less suited as model for those nucleophilic sites of proteins that are predominantly attacked by electrophiles. Fourth, side chains with heteroatoms increase kNBP as compared to alkyl side chains, probably because the electrophilic reactivity of the heterocyclic ring carbon is enhanced through the respective negative inductive effect. A particularly striking example is n-butyl glycidyl ether (B5) vs 1,2epoxyhexane (A4) that are isomeric except for one O vs C atom and differ in kNBP by a factor of 8. In fact, all epoxides with heteroatom-containing side chains (B4−B7, C1) are in the upper kNBP range of the presently analyzed data set, keeping in mind that some of them also contain unsaturated moieties. The latter leads to rule five, signifying an increase in kNBP through the presence of unsaturated groups. Again, the cause is likely to be an increase in ring-carbon electrophilicity through side-chain electron attraction, caused by either negative inductive or mesomeric effects (or both). Indeed, styrene oxide (B3) is significantly more reactive than its saturated nalkyl counterpart 1,2-epoxyoctane (A4), with a difference in kNBP by a factor of 9.4 due to a tautomeric stabilization of the intermediate resulting from SN2-type ring-opening as the initial step of the electrophile−nucleophile reaction.23,24 The reactivity enhancement through mesomeric stabilization can also be illustrated by comparing glycidyl phenyl ether (B7) with the glycidyl alkyl ethers B5 and B6. The former is more reactive toward NBP than the latter by factors of 2.5 and 3, probably reflecting the fact that the phenoxy moiety offers an additional side-chain SN2 attack at the methylene carbon with the phenolate ion as leaving group (a route not possible for the corresponding alcoholate group because of its much larger basicity and nucleophilicity; see Scheme 3). In this context, the similar reactivities of allyl glycidyl ether (B4, unsaturated C3 side chain) and n-butyl glycidyl ether (B5, saturated C4 side chain) are somewhat surprising, considering the fact that the allyl moiety would offer nucleophilic addition at its terminal sp2 carbon as a further reaction pathway.23 However, additional investigation of the reactivity of allyl methyl ether (H2C CH−CH2−O−CH3) toward NBP yielded a very low kNBP value

Scheme 3. Reaction of a Nucleophile (NuH) with Phenyl Glycidyl Ether via an Alternative SN2 Reaction Pathway

of 1.77·10 −4 L mol −1 min −1 , demonstrating that the corresponding side-chain reaction of allyl glycidyl ether cannot compete with the SN2-type ring-opening. Sixth, kNBP comparison of glycidyl ethers (B4−B7, C1) with alkyl epoxides (A1−A6) indicates a generally enhanced electrophilic reactivity of the former. It suggests an involvement of the lone pair electrons of the ether oxygen. In Scheme 4, two Scheme 4. Reaction Pathways of Glycidyl Ethers with a Nucleophile (NuH and Nu−)a

a

Left and bottom: nucleophilic attack at the unsubstituted epoxide carbon leads to ring-opening, followed by tautomeric carbanion formation stabilized through partial intramolecular H bonding, and protonation to the ultimate product. Right: initial intramolecular SN2 of the glycidyl oxygen at an epoxide carbon (neighbor group effect), followed by a second SN2 reaction with NuH at the one or other epoxide carbon.

possible respective explanations are depicted. On the one hand, one ether oxygen lone pair could invoke an initial intramolecular SN2 attack at the epoxide ring carbon attached to the methylene group (neighbor group effect), followed by a second SN2 attack of the nucleophilic reaction partner (here: NBP) at one of the two epoxide carbons with accordingly different products (top and right in Scheme 4). On the other hand, initial SN2 attack of the nucleophilic reaction partner could be supported by tautomerism coupled with a mesomeric stabilization through H-bonding to the ether oxygen lone pair (left and bottom in Scheme 4), yielding a still different product. Further support for a respective neighbor group effect of the glycidyl ether oxygen is provided by the finding that neither methyl methacrylate (H3C−C(CH2)−C(O)−OCH3) nor ethyl methacrylate showed any reactivity toward NBP in our chemoassay, thus making a corresponding side-chain reaction of glycidyl methacrylate (C1) unlikely. Note also that in previous studies of the GSH reactivity and bacterial toxicity of acrylates and methacrylates, the latter turned out to have a significantly lower Michael-acceptor reactivity due to the α-C substitution.19,35 2097



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Table 2. Literature Data for the Alkylation of NBP in Terms of the Change of the Absorbance at 560 nm per Hour, ΔA560/h, Pseudo-First-Order Reaction Rate Constants, log k(1) NBP, Absorbances per mmol (A/mmol), and Literature Data for the Alkylation of Deoxyguanosine Expressed as the Difference in Fluorescence Intensity per Hour, ΔI/h, by Some of the Compounds Tested alkylation of NBP name

CAS

no.

ΔA560/h

1,2-propylene oxide 1,2-epoxybutane 1,2-epoxyhexane 1,2-epoxyoctane 1,2-epoxydodecane styrene oxide allyl glycidyl ether n-butyl glycidyl ether iso-propyl glycidyl ether glycidyl phenyl ether

75-56-9 106-88-7 1436-34-6 2984-50-1 2855-19-8 96-09-3 106-92-3 2426-08-6 4016-14-2 122-60-1

A1 A2 A3 A4 A6 B3 B4 B5 B6 B7

90 80

10

−1 7 log k(1) NBP (day )

A/mmol11

alkylation rate of deoxyguanosine (ΔI/h)10

−0.11 −0.19 −0.26 −0.25 −0.16 −0.03

0.625 0.360 0.209 0.159

95 75

130 145 160 240

0.085 130 135 90 290

Figure 1. Bacterial mutagenicity in terms of (a) Ames test results with different Salmonella typhimurium strains, (b) Ames test results with Escherichia coli WP2 uvrA (expressed as revertant fraction ΔRF (number of revertants divided by number of viable cells of control sample)), (c) results from the SOS-Chromotest using Escherichia coli PQ37 (SOS-inducing potency expressed as induction factor I related to the compound concentration), and (d) results from the fluctuation test with Klebsiella pneumoniae (expressed as mutation rate fraction (ratio of observed over average spontaneous mutation rate) per compound concentration) vs reactivity of some of the test compounds toward NBP in terms of the second-order reaction rate constants log kNBP (L mol−1 min−1) (for details see Supporting Information).

associated (predicted)36 Sw was 2.8·10−3 mol L−1, we estimate that the NBP chemoassay application range in the pseudo-firstorder kinetics mode is limited to compounds with Sw > 1.0·10−3 mol L−1 (log Sw > −3.0). In terms of the logarithmic octanol/ water partition coefficient log Kow, the respective threshold is about 3.5. Comparison to Literature Data. For 10 of the presently analyzed 21 test compounds, literature data on their reactivity toward NBP are available.7,10,11 In one study, the change of the absorbance at 560 nm per hour, ΔA560/h, was determined for the alkyl epoxides A1 and A2 and for four alkyl glycidyl ethers

The three oxetanes (C2−C4), the thietane (C7), and the oxiranes 1,2-epoxy-9-decene (A5) and 1,2-epoxydodecane (A6) showed no reactivity toward NBP. Concerning the fourmembered heterocycles, the ring strain and ring-carbon electrophilicity appear to be too low for a corresponding SN2type reaction under the presently used chemoassay conditions. With regard to the C10 and C12 epoxides, the low water solubilities Sw (predicted as 9.5·10−6 mol L−1 and 2.5·10−5 mol L−1)36 did not enable setting up a reactive concentration. Taking into account that among all test compounds with an experimental kNBP determined so far in our lab, the lowest 2098



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(B4−B7).10 As can be seen from Table 2, the respective trend correlates well with the one for kNBP from the present work, yielding an r2 value of 0.88. For the same subset, rates of alkylation of deoxyguanosine expressed as difference in fluorescence intensity per hour, ΔI/h, were reported,10 resulting in r2 = 0.98 for the correlation with kNBP. This latter finding supports the use of NBP as a model nucleophile for the aromatic ring N of DNA bases such as N1, N3, and N7 of guanine. For the subset of five alkyl epoxides (A1−A4 and A6) and styrene oxide (B3), logarithmic pseudo-first-order rate −1 constants of their reaction with NBP, log k(1) NBP (day ), are 7 (1) available from the literature. Inspection of the kNBP vs kNBP (our second-order data) data distribution reveals styrene oxide (B3) as a significant outlier (with k(1) NBP as the overall largest value still being much lower than expected from the trend observed for the other compounds). A further discrepancy concerns 1,2-epoxydodecane (A6) for which the reactivity toward NBP was not detectable under our chemoassay conditions but reported to be more reactive than the smaller butane, hexane, and octane counterparts (A2−A4),7 the latter of which is unlikely in view of the otherwise opposite trend of decreasing NBP reactivity with increasing alkyl group size. In a further study, absorbances (A/mmol) were determined for A1−A4 and for styrene oxide (B3).11 The latter yielded the overall lowest reactivity, in contrast with the other literature data7 and with our present results. For the remaining four compounds, the correlation between A and kNBP has an r2 value of 0.91, which decreases to 0.16 when including styrene oxide. Note further that according to a recent investigation,37 oxetanes do not react with NBP at neutral pH, which is in agreement with our present findings. Relationship of Chemical Reactivity to Mutagenicity. Twelve oxiranes (A1−A4, A6, A7, B3−B7, and C1) and the three oxetanes (C2−C4) of the present compound set had been tested earlier with respect to their mutagenic potential in bacteria10,37−42 and in Chinese hamster V79 cells.43 Accordingly, to check the applicability of the NBP chemoassay to screen chemicals for their mutagenic potential, the results of common mutagenicity screening assays are compared with our presently determined log kNBP values in Figures 1 and 2; the respective mutagenicity data are provided in the Supporting

Information (Table S2). In this context, it should be noted that the Ames test results are from different literature sources (because no single study covered all compounds subject to the present analysis), which may lead to some additional data uncertainty. Figure 1a shows the data distributions of mutagenic potency in three Ames test strains (bacteria Salmonella typhimurium) vs log kNBP for 12 oxiranes (TA100: without A7; TA1535: without A7, B3; TA97: without A2−A4, A7, B3, B4, and B6) and the three oxetanes (C3 and C4: only TA100). For each bacterial strain, the mutagenic potency increases with increasing reactivity toward NBP, demonstrating the suitability of the NBP chemoassay for screening the DNA reactivity of chemical compounds. With TA100, two epoxides with large alkyl groups (A4, A6) and the three oxetanes (C2−C4) were not mutagenic; for the remaining nine epoxides, linear regression of the mutagenic potency (decadic logarithm, [revertants/μmol]) on log kNBP yields log muta TA100[rev./μmol] = (1.94 ± 0.37) ·log kNBP(L mol−1min−1) + (5.24 ± 0.70)

(7)

where n = 9, r2 = 0.80, rms = 0.31, F1,7 = 28.0, q2cv = 0.71, and rmscv = 0.40. In the strains TA1535 and TA97, the numbers of revertants that occur by treatment with the test compounds were 1.5 to 46 times lower than in strain TA100. With strain TA98, negative results were always obtained. This may be caused by the fact that TA98 is reverted back to the original state only by frameshift mutations, which are apparently not induced by the test compounds. In Figure 1b (top right), the experimental mutagenicity refers to E. coli WP2 uvrA bacteria as a further Ames test strain, and is expressed as the revertant fraction ΔRF (number of revertants divided by number of viable cells of control sample). The respective ΔRF potency increases linearly with increasing log kNBP (A1, A2, and B4−B7). A linear correlation with log kNBP is also obtained for the SOS chromotest induction factor I related to the compound concentration as available40 for B3−B7 and C1 (Figure 1c). In this assay, however, no significant mutagenic response had been obtained for the alkyl epoxides (A1−A4 and A6; not included in Figure 1c), contrasting with both the above-mentioned TA100 and TA1535 results (where only A4 and A6 were not mutagenic) and with our kNBP values for A1−A3 of 4.5·10−3 to 7.2·10−3 L mol−1 min−1 (see Table 1). A further literature data set was available for A1−A4, A7, B4, C1, and C2 from the fluctuation test with Klebsiella pneumoniae,42 expressed as the mutation rate fraction (ratio of observed over average spontaneous mutation) per compound concentration. In accordance with our kNBP results, 1,2-epoxyoctane (A4) and trimethylene oxide (C2) were not mutagenic, and except for isobutylene oxide (A7), the remaining compounds show an increase in mutagenic potency with increasing log kNBP (Figure 1d). In Figure 2, results from the SCE (sister chromatid exchange) test with Chinese hamster V79 cells (SCE induction potency per cell and compound concentration)43 are plotted against log kNBP for the 10 epoxides A1−A4 (A6 was inactive), B3−B7, and C1. It is seen that for these mammalian cells, mutagenicity is observed only beyond a certain reactivity

Figure 2. Mutagenic action in mammalian cells in terms of the results of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells (expressed as SCE-inducing potency per cell and compound concentration) vs the reactivity toward NBP in terms of the secondorder reaction rate constants log kNBP (L mol−1 min−1) for some of the test compounds (for details, see Supporting Information). 2099



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Scheme 5. Structural Alerts for the NBP Reactivity (Expressed as log kNBP (L mol−1 min−1)) and Associated Mutagenic Potency in Salmonella typhimurim TA100 (Expressed in Revertants per μmol) of Epoxides and Oxetanes

threshold (kNBP > 0.02 L mol−1 min−1), and that in this latter range glycidyl phenyl ether exerts a significantly lower mutagenic potency than would be expected from the other (however only) two compounds. Clearly, the respective compound set is too small to draw general conclusions, but the observed data distribution suggests the SCE test to be less sensitive to DNA damage than the NBP chemoassay, which may contribute to an overall lower correlation between log kNBP and SCE induction potency. The overall trends for both NBP reactivity (see above) and the Ames test mutagenicity as related to the substitution pattern of the epoxides and oxetanes are summarized in Scheme 5 in terms of corresponding structural alerts. Epoxides with the glycidyl ether unit as well as with unsaturated moieties are likely to be mutagenic in the Ames test, with log kNBP (L mol−1 min−1) typically ranging between −1 and −2. Small aliphatic epoxides with up to four side-chain carbons are weakly mutagenic. Their log kNBP values range from about −2 to −2.35, the latter of which is the currently determined threshold above which their mutagenicity begins to become relevant. Oxetanes are neither mutagenic nor reactive toward NBP, and aliphatic epoxides with six or more side-chain carbons are also not mutagenic, with NBP reactivities being below −2.5 log kNBP units. At present, the NBP chemoassay does not include a system for metabolic activation and thus, in contrast to the Ames test S9 mix variant, is not applicable to pro-electrophilic mutagens. Although a first step in this direction was reported using a chemical activation system,11 addressing this issue in a full kinetic approach would require separate generation and analysis of electrophilic metabolites, obtaining guidance from established knowledge44 and computerized models45 as well as from detailed studies of rate-determining biotransformation steps.46 Future investigations may address this issue; for the time being, however, the NBP chemoassay appears useful to inform about the potential of organic compounds for exerting the type and extent of reactivity related to DNA N− attack and associated mutagenicity.

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aromatic nitrogen sites of DNA bases. As a general trend, kNBP decreases with increasing side-chain alkyl size and branching. Moreover, glycidyl ethers are expected to be systematically more NBP-reactive and thus also more mutagenic than epoxides without this particular ether functionality, and a correspondingly enhanced NBP reactivity and mutagenicity is likely to hold for epoxides with aromatic residues as compared to aliphatic epoxides. The results suggest further that neither oxetanes nor thietanes are likely to be mutagenic (except for separate side-chain sites with a corresponding potential). From the perspective of a predictive toxicity profile such as through integrated testing strategies for REACH, application of both the NBP chemoassay and the previously introduced GSH chemoassay18 could be employed as complementary tools, informing about the potential of organic electrophiles to form adducts with DNA or proteins or both.

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ASSOCIATED CONTENT

S Supporting Information *

Description of the methods for the solvolysis measurements including four figures, one figure showing the dependence of absorbance on reaction time, the derivation of the equation for the calculation of kNBP with competing solvolysis of the electrophile, one table containing log kNBP values, log Kow values, and log Sw values of the test compounds, and one table containing the mutagenicity data from the literature for some of the test compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-341-235-1262. Fax: +49-341-235-1785. E-mail: [email protected]. Funding

This study was financially supported by the European Union through the Integrated Project OSIRIS (Optimized Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-Test and Test Information, contract no. GOCE-CT-2007-037017), which is gratefully acknowledged.

CONCLUSIONS

Notes

The newly introduced kinetic variant of the 4-(4-nitrobenzyl)pyridine (NBP) chemoassay provides a convenient way to screen the DNA reactivity and the associated mutagenic potency of organic electrophiles. For three- and four-membered oxygen and sulfur heterocycles, analysis of the second-order rate constants of their reaction with NBP, kNBP, unravelled distinct relationships between the substitution pattern of the compounds and their reactivity toward NBP as surrogate for

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to our colleagues Ralf-Uwe Ebert for calculating the log Sw values with the OSIRIS Edition of the ChemProp software,47 and Torsten Thalheim for the calculation of statistical parameters. Furthermore, we thank Nadin Ulrich 2100



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for performing some HPLC measurements, and Uwe Schröter for his assistance on the GC measurements.

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ABBREVIATIONS NBP, 4-(4-nitrobenzyl)pyridine; kNBP, second-order rate constant of the reaction with NBP; GSH, glutathione; HSAB, hard and soft acid and base; tris, tris(hydroxymethyl)aminomethane; Amax, maximum value of absorbance; ε592, extinction coefficient for the absorbance of the NBP-electrophile reaction product at 592 nm; Te, toxicity enhancement; EC50, effective concentration 50%; Sw, water solubility; Kow, octanol/water partition coefficient; SCE, sister chromatid exchange; ksolv, first-order rate constant of the solvolysis of the test compounds; GC, gas chromatography; LC, liquid chromatography; MS, mass spectrometry; FID, flame ionization detector; DAD, diode array detector

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(4-Nitrobenzyl)pyridine - American Chemical Society

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