Exploring Chemical Routes Relevant to the Toxicity of Paracetamol

Article pubs.acs.org/crt

Exploring Chemical Routes Relevant to the Toxicity of Paracetamol and Its meta-Analogue at a Molecular Level Romina Castañeda-Arriaga and Annia Galano* Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, Col. Vicentina. Iztapalapa, C. P. 09340, México D. F., México S Supporting Information *

ABSTRACT: Several chemical routes related to the toxicity of paracetamol (APAP, also known as acetaminophen), its analogue Nacetyl-m-aminophenol (AMAP), and their deacetylated derivatives, were investigated using the density functional theory. It was found that AMAP is more resilient to chemical oxidation than APAP. The chemical degradation of AMAP into radical intermediates is predicted to be significant only when it is induced by strong oxidants. This might explain the apparent contradictions among experimental evidence regarding AMAP toxicity. All of the investigated species are incapable of oxidizing DNA, but they can damage lipids by H atom transfer (HAT) from the bis-allylic site, with the phenoxyl radical of AMAP being the most threatening to the lipids’ chemical integrity. Regarding protein damage, Cys residues were identified as the most likely targets. The damage in this case may involve two different routes: (i) HAT from the thiol site by phenoxyl radicals and (ii) protein arylation by the quinone imine (QI) derivatives. Both are not only thermochemically viable, but also are very fast reactions. According to the mechanism identified here as the most likely one for protein arylation, a rather large concentration of QI would be necessary for this damage to be significant. This might explain why APAP is nontoxic in therapeutic doses, while overdoses can result in hepatic toxicity. In addition, the QI derived from both APAP and AMAP were found to be capable of inflicting this kind of damage. In addition, it is proposed that they might increase •OH production via the Fenton reaction, which would contribute to their toxicity.

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INTRODUCTION Paracetamol, also known as acetaminophen, or N-acetyl-paminophenol (APAP, Scheme 1), is one of the most frequently used drugs as an analgesic and antipyretic. Albeit in therapeutic doses it is usually safe, overdoses can result in hepatic toxicity and are the most common cause of drug-induced liver injuries.1−3 Moreover, while APAP toxicity is mainly associated with liver damage, other organs can also be affected including the auditory4 and reproductive5 systems, the epidermis,6,7 and the brain.8 Albeit there is evidence that at low doses it protects brain cells against oxidative stress,9,10 APAP overdoses have been associated with increased oxidative and nitrosative stress in hepatic and renal tissues.11,12 Moreover, some studies show the beneficial effects of some antioxidants against APAP induced damage, which are assumed to arise from the deactivation of reactive oxygen and nitrogen species. Some examples of such antioxidant protectors are naringin,13 Nacetylcysteine amide,14 quercetin,12 and morin.15 The APAP hepatotoxicity is attributed to its metabolite Nacetyl-p-benzoquinone imine (NAPQI, Scheme 1),3,16 which is produced by the action of cytochrome P450 enzymes (CYP). It has been established that, at therapeutic doses, 5 to 10% of APAP is metabolized into NAPQI, which is then detoxified by glutathione.2 On the contrary, when administered at high doses, the glucuronidation and sulfation pathways are saturated and © 2017 American Chemical Society

NAPQI accumulates, exceeding the detoxification capacity of glutathione and yielding stable protein adducts.3,16,17 Such adducts arise from the reactions of NAPQI with the sulfhydryl group on cysteine residues and led to mitochondrial dysfunction, which in turn is involved in the formation of oxidative species.18 In fact, there is evidence that the concentration of glutathione disulfide, which is considered a marker for intracellular formation of reactive oxygen species, significantly increases when toxic doses of APAP are administered to mice.19 Additionally, it has been reported that NAPQI can, in principle, damage protein residues other than cysteine, such as methionine, tyrosine, and tryptophan.20 The characterization of the adducts revealed that they mainly involve the NAPQI site that is in ortho position with respect to the hydroxyl group (site 2, Scheme 1). In addition to NAPQI, there are other species arising from the metabolism of APAP that can be potentially damaging. It has been established that the overall two electron oxidation of APAP by CYP and also by other enzymes takes place in two steps (Scheme 1).21−24 Each corresponds to a one electron oxidation process, with the first step yielding the N-acetyl-paminophenoxyl radical (APAPR). In addition, when deacetyReceived: January 28, 2017 Published: May 5, 2017 1286

DOI: 10.1021/acs.chemrestox.7b00024 Chem. Res. Toxicol. 2017, 30, 1286−1301

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Chemical Research in Toxicology Scheme 1. Structures of Paracetamol and Related Species, and Site Numbering of Paracetamol

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lation is considered, the p-aminophenoxyl radical (PAPR) and p-benzoquinone imine (PBQI) arise as potential candidates to be involved in APAP toxicity.21 The viability of APAP deacetylation in living organisms has already been proven.25 Another interesting aspect, regarding APAP toxicity, is that while some studies indicate that its isomer N-acetyl-maminophenol (AMAP) does not lead to hepatotoxicity,26,27 other investigations indicate otherwise. It has been reported that AMAP is toxic in the rat and human liver and cannot be safely used as an alternative to APAP.28 AMAP has also been identified as hepatotoxic in human hepatocytes, which is assumed to be caused by the formation of mitochondrial protein adducts.29 Albeit the information gathered so far on the APAP toxicity is rather abundant, there are several aspects that have not been explored. They are mainly related to the chemical processes involved in the toxic effects of APAP. For example, there is no kinetic or thermochemical data on the possible reactions of NAPQI with biological targets. There is no information either on the possible role of other potential damaging species that can be produced after APAP consumption, such as APAPR, PAPR, and PBQI. Other oxidation processes that can also yield APAPR and PAPR radicals, for example, chemical reactions between APAP and oxygen reactive species, such as the hydroxyl radical, have not been explored yet. No data have been reported on the reactions of biological targets with the species that are expected to be formed from AMAP, i.e., N-acetyl-maminophenoxyl radical (AMAPR), m-aminophenoxyl radical (MAPR), N-acetyl-m-benzoquinone imines (NAMQIi), and mbenzoquinone imines (MBQIi). Such data and the comparison with the equivalent processes for the APAP derivatives might contribute to solve current controversies on the possible toxicity of AMAP. There is also a lack of information on the chemical interactions of all these species with metal ions. The main goal of the present study is to provide information on all these aspects and new physicochemical insights on the chemistry involved. Hopefully, the new data reported here might help to gain a better understanding on the molecular processes involved in APAP toxicity.

METHODS

All electronic calculations were performed with the Gaussian 09 package of programs.30 Geometry optimizations and frequency calculations were carried out using the M05-2X functional31 and the 6-31+G(d,p) basis set, in conjunction with the solvation model based on density (SMD)32 using pentylethanoate and water as solvents to mimic lipid and aqueous environments, respectively. The damage to unsaturated fatty acids was modeled in lipid solution, while the modeling of DNA and protein damage was carried out in aqueous solution. The M05-2X functional has been recommended and tested for kinetic calculations by their developers. It is among the best performing functionals for kinetic calculations in solution33 and for calculating reaction energies involving free radicals.34 SMD is considered to be a universal solvation model due to its applicability to any charged or uncharged solute in any solvent or liquid medium for which a few key descriptors are known.32 For the species including Cu and Fe and for all the related energies of reaction, the M05 functional31 has been used also with the 6-31+G(d,p) basis set and the SMD model. This functional has been chosen for this part of the study because it was parametrized including both metals and nonmetals, whereas M05-2X is a highly nonlocal functional with double the amount of nonlocal exchange (2X) that was parametrized for nonmetals. M05 has been recommended for studies involving both metallic and nonmetallic elements and has been reported to perform well not only for main-group thermochemistry and radical reaction barrier heights but also for interactions with transition metals.31 Unrestricted calculations were used for open shell systems. Local minima and saddle points were identified by the number of imaginary frequencies. Local minima have only real frequencies, while saddle points are identified by the presence of a single imaginary frequency that corresponds to the expected motion along the reaction coordinate. Relative energies are calculated with respect to the isolated reactants. Thermodynamic corrections at 298.15 K were included in the calculation of relative energies, which correspond to the 1 M standard state. The rate constants (k) were calculated using the 1 M standard state and the conventional transition state theory (TST)35−37 with harmonic vibrational frequencies and Eckart tunneling.38,39 The Eckart barrier was fit to reproduce the gas-phase zero-point-inclusive energies of reactants, saddle point, and product. It should be noted that since corner cutting effects are not included in the Eckart formalism, tunneling corrections obtained this way may differ to some extent from those obtained with more sophisticated approaches.40 1287


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Chemical Research in Toxicology Scheme 2. Models Used to Represent Biological Targets

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RESULTS AND DISCUSSION Models and Reaction Mechanisms. Different models (Scheme 2) have been used to represent three kinds of biological targets: lipids, DNA, and proteins. The lipid model (LM) was constructed to represent unsaturated fatty acids, and it can be considered as a reduced model of linoleic acid (LA) that retains its main chemical reactivity feature, i.e., two allylic H atoms. They only differ in the number of CH2 groups: LM, CH3−CH2−CHCH−CH2−CHCH−CH2−COOH, and LA, CH3-(CH2)4-CHCH−CH2−CHCH-(CH2)7-COOH, which is not expected to significantly modify the reactivity toward oxidants. However, to prove this point the rate constants of the reaction of both LM and LA with •OOH were calculated (Table 1). It was found that both values are very similar, which supports the hypothesis that LM is an adequate model for the task at hand.

For the electron transfer reactions, the Gibbs free energy of activation was calculated using the Marcus theory.41 In addition, since several of the calculated rate constants (k) are close to the diffusionlimit, the apparent rate constant (kapp) cannot be directly obtained from TST calculations. The Collins-Kimball theory was used for that purpose,42 in conjunction with the steady-state Smoluchowski43 rate constant for an irreversible bimolecular diffusion-controlled reaction, and the Stokes−Einstein44,45 approaches for the diffusion coefficient of the reactants. These computational details are in line with the quantum mechanics based test for overall free radical scavenging activity (QM-ORSA) protocol,46 which has been validated by comparison with experimental results; its uncertainties have been proven to be no larger than those arising from experiments.46 The kinetic calculations were performed only for the exergonic reaction pathways because endergonic pathways are expected to be reversible to a significant extent. Thus, albeit they might take place at a significant rate, the corresponding products will not be observed. The only exception was the reaction of MBQI3 (site 6), which actually corresponds to an isoergonic reaction. However, since all the other pathways for this particular species are even less thermochemically favored, this is the only channel (if any) that might cause protein arylation. The abundance of the anionic Cys species at physiological pH was considered in the calculation of the rate constants for route XI. For that purpose, the rate constant obtained by the TST for each quinone imine (QI) was multiplied by the molar fraction (mf) of the residue at the pH of interest:

Table 1. Rate Constants of the •OOH Reactions with Unsaturated Fatty Acids, at Room Temperature lipid model linoleic acid linoleic acid linolenic acid arachidonic acid

pH 7.4 pH 7.4 TST k QI,site k QI,site j j = mf Cys

method

ref

× × × × ×

calculated calculated experimental experimental experimental

this work this work 47 47 47

1.42 1.79 1.18 1.70 3.05

103 103 103 103 103

In addition, the calculated rate constant for the LA + •OOH reaction is in excellent agreement with the experimental value.47 This supports the reliability of the level of theory used in this work, as well as the performance of the QM-ORSA protocol to obtain kinetic data in solution. Another interesting fact is that the experimental rate constants for three unsaturated fatty acids (linoleic, linolenic, and arachidonic acids) are all similar, ranging from 1.18 × 103 to 3.05 × 103 M−1 s−1 (Table 1). This

In turn, the overall rate coefficients for each quinone imine was obtained as the sum of the rate coefficients corresponding to the reaction pathways identified as viable: n pH 7.4 k QI,overall =

k (M−1 s−1)

pH 7.4 ∑ k QI,site j j=1

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Chemical Research in Toxicology Table 2. Chemical Routes (CR) Investigated in This Work CR

target

(I) (II) (III) (IV) (V) (VI) (VII) (VIII) (IX) (X) (XI)

LM (bis-allylic site) 2dG G (site 8) NF-Leu (γ site) NF-Cys (SH site) NF-Tyr (OH site) NF-Tyr NF-Trp NF-Met (γ site) NF-His (β site) NF-Cys (SH site)

toxic species APAPR, APAPR, APAPR, APAPR, APAPR, APAPR, APAPR, APAPR, APAPR, APAPR, NAPQI,

reaction mechanism

PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PAPR, AMAPR, MAPR PBQI, NAMQI1, NAMQI2, NAMQI3, MBQI1, MBQI2, MBQI3

formal hydrogen atom transfer (HAT) single electron transfer (SET) radical adduct formation (RAF) HAT HAT HAT SET SET HAT HAT protein arylation

Scheme 3. Structures of AMAP and Related Species, and Their Site Numbering

indicates that any of them can be used to investigate oxidative damage to unsaturated fatty acids in general. 2′-Deoxyguanosine (2dG) (Scheme 2) was chosen to model oxidative damage to DNA since guanine (G) is the most easily oxidized of the nucleobases,48−51 which is why the one-electron oxidation of DNA mainly involves G sites.52 Although small differences in the ease of oxidation of guanine (G), guanosine (Gs), 2′-deoxyguanosine (2dG), and 2′-deoxyguanosine 5′monophosphate (2dGMP) are expected,53 these species all have the lowest one electron oxidation potential when compared with the equivalent species of the other nucleobases. Therefore, if a chemical oxidant is capable of oxidizing 2dG it can inflict oxidative damage on DNA. On the contrary, if an oxidant is not strong enough to oxidize 2dG it should be harmless to DNA, i.e., unable to oxidize any of the bases. Therefore, 2dG seems to be an appropriate model to investigate potential oxidative damage to DNA. In addition, radical adduct formation at site 8 in G was also investigated. Site 8 was chosen for this part of the investigation because it has been identified as the most likely addition site in the target molecule.54

A realistic model has been used to represent amino acid residues in proteins (Scheme 2). This model has been successfully used, and it is widely accepted as appropriate for investigating protein site reactions.55−66 Albeit it might seem simplistic, it is actually not, at least for this particular purpose. This is because typical oxidants are not protein targets. Thus, no particular protein conformation is required for the oxidation to take place, i.e., their secondary and tertiary structures are not relevant in this case, provided that the target residues are exposed. On the other hand, the lack of unsaturation in the protein backbone prevents electronic effects from propagating further than two sigma bonds. Therefore, as long as a model includes two amide groups next to the side chain of the investigated amino acid, it should be enough to model reactions with chemical oxidants. Therefore, at least for modeling the intrinsic reactivity of amino acid residues toward chemical oxidants, the used model is expected to be appropriate. However, it should be noted that protein environments may alter such reactivity, for example, by modifying the acidity of some residues such as cysteine.67,68 1289


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Chemical Research in Toxicology The models used in this work correspond to N-formylcysteinamide (NF-Cys), N-formyl-tyrosinamide (NF-Tyr), Nformyl-tryptophanamide (NF-Trp), N-formyl-leucinamide (NF-Leu), N-formyl-methioninamide (NF-Met), and Nformyl-histidinamide (NF-His). They were chosen based on previous data indicating that the residues cysteine (Cys), leucine (Leu), tyrosine (Tyr), tryptophan (Trp), methionine (Met), and histidine (His) are among the most susceptible to oxidative damage in proteins.57,69−74 In addition, they can react with oxidants through different reaction mechanisms including H transfer (Cys, Leu, Met, and His), electron transfer (Tyr, Trp), and adduct formation (Tyr, Trp, and His). On the basis of what is known about the most likely reaction mechanisms involved in the reactions of lipids, proteins, and DNA with oxidants, different chemical routes were investigated here for exploring the toxicity of APAP, AMAP, and related species to biological targets (Table 2). The considered APAPrelated toxic species are those shown in Scheme 1. Since the formation of similar species can be anticipated for AMAP (Scheme 3), their potential toxicity was also explored. The species labeled here as NAMQI1, NAMQI2, and NAMQI3 have been already identified using experimental techniques.75 In addition, APAP, PAP, AMAP, and MAP can be susceptible to radical attack, yielding the radical products shown in Schemes 1 and 3 or other radical species that in turn can also damage biological targets. Therefore, this point was also explored using two paradigmatic free radicals: (i) the hydroxyl radical (•OH); and (ii) the hydroperoxyl radical (HOO•). •OH is the most reactive of the oxygen-centered radicals in biological systems, while HOO• is the smallest of the peroxyl radicals, has moderate reactivity, and has been proposed to play an essential role in the toxic side effects associated with aerobic respiration.76 The reaction mechanisms investigated for the oxidation induced by these radicals are HAT, SET, RAF, and sequential proton loss electron transfer (SPLET). Oxidative Damage to APAP, PAP, AMAP, and MAP. The reaction mechanism explored in this work for the oxidative damaged induced by •OH and HOO• to APAP, PAP, AMAP, and MAP can be schematically represented as

Figure 1. Gibbs free energies (kcal/mol) for the reactions of APAP, PAP, AMAP, and MAP, reacting with •OH (A) and HOO• (B). Site numbers are shown in Schemes 1 and 3.

thermochemically viable except for the SET from AMAP. They were also found to be very fast reactions, with rate constants within or near the diffusion-limited regime (Table 3). These findings are in line with the known high reactivity of •OH. Table 3. Rate Constants (k, M−1 s−1) of the Thermochemically Viable Pathways of the Reactions of APAP, PAP, AMAP, and MAP with •OH and HOO•, in Aqueous Solution, at 298.15 Ka APAP

PAP

AMAP

MAP

× × × × × × × × × ×

9.25 × 109 7.85 × 109 7.85 × 109

•

HAT: HXAP + •R → XAP• + HR SET: HXAP + •R → HXAP+• + R−

SPLET: (a)HXAP ⇌ XAP− + H+ (b)XAP− + •R → XAP• + R− RAF: HXAP + •R → [HXAP − R]• a

In these chemical equations, HXAP represents APAP, PAP, AMAP, or MAP before any deprotonation takes place, and R stands for the free radicals (•OH or HOO•). It seems relevant to note that HAT, SET, and RAF are single-step reactions, while SPLET is a two-step process. It involves the deprotonation of the phenolic group followed by an electron transfer from the phenolate anion to the free radical. The first step is controlled by the pH and only depends on the acidity of the phenolic H in each molecule. On the contrary, the second step is expected to be the relevant one for kinetics. That is why the energy of the overall SPLET process and the energy of its second step (b) are both included in Figure 1 and Tables S1 and S2. It was found that for the reactions involving •OH (Figure 1A and Table S1), all of the investigated reaction pathways are

× × × × × × × ×

109 109 109 107 109 109 109 109

SPLET (b) HAT (OH) HAT (NH) HAT (CH3) RAF (site 1) RAF (site 2) RAF (site 3) RAF (site 4) RAF (site 5) RAF (site 6)

7.56 8.14 8.14 8.87 7.53 7.89 7.36 7.02

SPLET (b) HAT (OH)

2.02 × 108 1.55 × 104

OH 3.76 × 109 7.80 × 109 7.80 × 109 7.80 7.80 7.80 7.80

× × × ×

109 109 109 109

9.44 7.96 7.96 1.18 9.41 7.90 9.25 7.92 2.24 7.90

HOO• 8.98 × 109 6.07 × 106

109 109 109 108 108 109 107 109 108 109

6.96 7.84 7.67 7.84 2.06 7.84

× × × × × ×

109 109 109 109 109 109

3.82 × 108 8.65 × 103

Site numbers are shown in Schemes 1 and 3.

On the contrary, for the reactions involving HOO• (Figure 1B and Table S2), which is a much less reactive free radical, the only exergonic pathways are HAT from the phenolic OH and the electron transfer from the phenolate ions, i.e., SPLET (b). These two chemical routes are also the most exergonic ones for the reactions with •OH and yield the species labeled as APAPR, PAPR, AMAPR, and MAPR. Accordingly, they are expected to be the most likely free radicals formed from the oxidation of APAP, PAP, AMAP, and MAP, respectively, regardless of the intrinsic reactivity of the oxidant involved in their degradation. This is the reason why APAPR, PAPR, AMAPR, and MAPR are the species chosen to model chemical routes I to X. According 1290


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Table 4. Gibbs Free Energies of Reaction (ΔG, kcal/mol), Gibbs Free Energies of Activation (ΔG⧧, kcal/mol) and Rate Constants (k, M−1 s−1), at 298.15 K, for the Reactions of APAPR, PAPR, AMAP, and MAPR with Biological Targets target

CR

APAPR

AMAPR

PAPR

MAPR

ΔG LM 2dG G (site 8) NF-Leu NF-Cys NF-Tyr NF-Tyr NF-Trp NF-Met NF-His

I II III IV V VI VII VIII IX X

−11.03 26.67 12.86 9.91 0.19 3.40 32.55 20.14 8.32 6.48

−16.06 21.45 5.56 4.55 −5.17 −1.96 27.33 14.92 2.96 1.12

−4.25 39.07 19.85 19.23 9.50 12.72 68.26 55.85 17.63 15.80

−14.55 26.97 9.08 8.51 −1.21 2.01 32.85 20.44 6.92 5.09

14.29 15.70 9.92

22.23

16.89 9.39

4.19 × 103 3.74 × 103 7.40 × 105

7.46 × 10−1

3.10 × 102 1.76 × 106

ΔG⧧ LM NF-Cys NF-Tyr

I V VI

18.42 16.62

LM NF-Cys NF-Tyr

I V VI

2.45 × 101 2.34 × 102

k

found to be endergonic. On the contrary, they all can damage unsaturated fatty acids (LM). Their reactivity was found to follow the trend AMAPR > MAPR > APAPR > PAPR. The rate constants for the reactions of LM with APAPR, PAPR, and MAPR were found to be significantly lower than those corresponding to the reactions of unsaturated fatty acids with HOO• (1.18 × 103 to 3.05 × 103 M−1 s−1).47 Accordingly, the reactivity of these three radicals toward lipid targets can be considered rather low. On the contrary, the rate constant for the LM + AMAPR reaction is similar and slightly higher than that of the LM + HOO• reaction. This indicates that AMAPR represents a non-negligible hazard to the chemical integrity of lipids. These findings support the idea that AMAP can be toxic, even more than APAP, provided that it encounters a strong enough oxidant capable of converting it into AMAPR. The optimized structures of the corresponding transition structures (TS) are shown in Figure 2. Regarding the reactions with protein residues, PAPR is the only one of the four investigated radical species that cannot damage them. For this particular radical, chemical routes IV to

to the calculated kinetic data (Table 3), they are expected to be rapidly formed even when the oxidant is not very strong. On the other hand, it was found that the reactions involving APAP are systematically more thermochemically favored than those involving AMAP, regardless of the free radical they are reacting with. This indicates that AMAP is more resilient to oxidation than APAP. The same trend appears when comparing PAP with MAP. Moreover, in the particular case of the AMAP + HOO• reaction, all of the investigated chemical routes are predicted to be endergonic. This means that the degradation of AMAP into AMAPR would be determined by the strength of the oxidant and is expected to be only minor when the oxidant is HOO• or other radicals of similar reactivity. Accordingly, the potential toxicity of AMAP may be significantly influenced by the species present in its environment, which might explain the apparent contradictions among experimental evidence.26−28 The results discussed in this section also support other experimental evidence. The findings regarding the reactivity of APAP and AMAP toward •OH and HOO• indicate that they may act as free radical scavengers. This is in in line with previous reports showing that, at low doses, APAP protects brain cells against oxidative stress.9,10 In addition, such free radical scavenging activity would yield intermediate (phenoxyl) radicals, which are involved in the formation of the quinone imine. Accordingly, antioxidants capable of repairing such radicals, i.e., turning them back to their original chemical form, would inhibit further oxidation and the consequent damage inflicted by quinone imines. This might explain the observed protecting role of some antioxioxidants12−15 against APAP toxicity. Chemical Routes I to X. The gathered data for the reactions of APAPR, PAPR, AMAP, and MAPR with different biological targets are reported in Table 4. On the basis of both thermochemical and kinetic analyses, the acetylated species (AMAPR and APAPR) are more reactive toward biological targets than the desacetylated ones (MAPR and PAPR). According to the Gibbs free energies of reaction (ΔG), none of the investigated radicals is capable of directly damaging DNA, i.e., both the SET (II) and the RAF (III) reactions were

Figure 2. Optimized geometries of the transition structures corresponding to HAT from lipids, route (I). 1291


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Three possibilities can be envisioned: (a) The −SH group in the Cys residue is previously damaged by a free radical, so there is instead an −S• site that in turn reacts with the NAPQI-like species. (b) The adduct formation simultaneously involves the addition of the −SH group and a proton transfer from this group to the NAPQI-like framework, i.e., the formation of an S−C bond, the rupture of the S−H bond, and the formation of an X-H bond (where X is the N or the O atom in the NAPQIlike species). (c) The reaction involves the deprotonated fraction of the Cys residue (−S−) instead of the most abundant neutral species (−SH). Route XI-a was ruled out based on several considerations that arise from the fact that free radicals are short-lived species that, fortunately, are present only in very low concentrations in living systems. Therefore, it is highly unlikely that an −S• site previously formed from the reaction of a Cys residue with a free radical (nonrelated to APAP or AMAP) has a long enough life to encounter NAPQI-like species before reacting with any other of the many chemicals present in biological systems. It is even more unlikely that a free radical derived from APAP or AMAP damages a Cys residue and that later the same residue reacts with their quinone imine metabolites. In addition, the population of the −S• (route XI-a) sites in proteins is necessarily much lower than those of −SH (route XI-b) and −S− (route XI-c) sites. It might also be anticipated that route XI-b is less likely than route XI-c because of the larger number of forming and breaking bonds necessary for the former to take place. However, since this is not always the case, the necessary calculations were made to compare the thermochemical viability of both addition routes (Table 5). The reaction pathways leading to the formation of ipso adducts and thiohemiketal ipso adduct were not included in the calculations, based on previous experimental findings.77 Such findings suggest that the ipso adducts of NAPQI, if produced, are only unstable intermediates yielding the products of addition involving sites 2 and 3. In the same paper, it was proposed

X were all found to be endergonic with ΔG values ranging from 9.5 to 68.3 kcal/mol. Only four reaction pathways were found to be thermochemically viable in this case, and they all involve NF-Cys and NF-Tyr. This indicates that Cys and Tyr protein residues would be the most susceptible to be damaged by AMAPR, MAPR, and APAPR. The most exergonic reaction was found to be the HAT from the thiol group in NF-Cys (chemical route V) by AMAPR, which has a rate constant on the order of 103 M−1 s−1. On the other hand, the fastest reaction also corresponds to route V but with the damaging species being MAPR. The same reaction, when the oxidant is APAPR is isoergonic and has a lower rate constant (2.3 × 102 M−1 s−1). The optimized structures of the corresponding TSs are shown in Figure 3.

Figure 3. Optimized geometries of the transition structures corresponding to HAT from protein residues, routes (V) and (VI).

According to the gathered data, it seems to be a trend that AMAPR and MAPR are more reactive toward biological targets, in general, than their partners APAPR and PAPR. This indicates that albeit the investigated m-aminophenols are less oxidizable than the corresponding p-aminophenols, the meta species can be as dangerous as the para species or even more, once they are degraded into free radicals. This suggests that AMAP is not necessarily a safe alternative to APAP. Chemical Route XI (Protein Arylation). Since APAP hepatotoxicity has been mainly attributed to protein arylation by NAPQI, particular attention was paid to this chemical route XI. All of the NAPQI-like species, and reaction sites, shown in Schemes 1 and 3 were considered. The reactions were all investigated using the thiol group in the NF-Cys model as the binding site. Any attempt to obtain an adduct between NF-Cys and the NAPQI-like species invariably led to the separated fragments, regardless of the particular species and the reaction site. This indicates that the addition reaction does not involve the Cys residues in its original chemical form at physiological pH, i.e., with the thiol group intact. However, there is compelling experimental evidence on the formation of the adducts with the S atom in the Cys residues bonded to one of the C atoms in NAPQI. Therefore, some alternative routes to the direct addition must be involved, which yield these types of products.

Table 5. Gibbs Free Energies (ΔG, kcal/mol) for the Addition of NAPQI-Like Species to NF-Cys, in Aqueous Solution, at 298.15 K site 2 NAPQI PBQI NAMQI1 NAMQI2 NAMQI3 MBQI1 MBQI2 MBQI3 NAPQI PBQI NAMQI1 NAMQI2 NAMQI3 MBQI1 MBQI2 MBQI3 a

1292

site 3

site 4

NF-Cys (-S−), Route XI-c −9.50 0.79 13.24 2.62 −2.57 −8.76 −7.86 2.88 3.16 −9.44 4.18 2.91 13.42 NF-Cys (-SH), Route XI-b 20.53 17.55 43.80 8.29 33.05 13.80 15.94 11.90 35.51 6.17 19.07 9.60 nf a

site 5

−7.83 −8.24 −0.81 15.53

18.99 21.97 25.01 nf

site 6

−3.57 −10.51 −2.78 3.07 −6.19 −0.39

20.41 −8.60 8.60 16.19 −7.30 3.66

nf = not found. DOI: 10.1021/acs.chemrestox.7b00024 Chem. Res. Toxicol. 2017, 30, 1286−1301

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pKa of GSH (bound to the active site) to 6.1−6.6,92,93 which can increase the GSH reactivity toward NAPQI. In addition, the pKa of Cys-47 in human GST pi has a low pKa (∼4.2),94 which makes this enzyme itself a potential NAPQI detoxifier. The reaction pathway yielding the NAPQI adduct at site 2 is exergonic by 9.5 kcal/mol, while the pathway yielding the adduct at site 3 is isoergonic (Table 5). Therefore, according to the calculated data, the adduct formed at site 2 in NAPQI is predicted to be the most abundant one, which is in line with previously reported experimental evidence.20 On the other hand, both pathways for the reaction of PBQI with NF-Cys were found to be endergonic (Table 5). This suggests that deacetylation decreases the reactivity of NAPQI toward thiol groups. For the AMAP quinone imines, it was found that the most exergonic chemical pathways are those involving sites 4, 6, and 5 for NAMQI1, NAMQI2, and NAMQI3, respectively (Table 5). Therefore, adducts formed by the addition of Cys residues to those sites are proposed as the most abundant ones. A similar trend (NAMQIi vs MBQIi) was found for the quinone imines derived from AMAP, i.e., the reactions or Cys residues with any NAMQIi is thermochemically more viable than that involving the corresponding MBQIi species. Considering these results, together with the findings described for radical species in the previous section, deacetylated APAP derivatives, with the desired pharmacological effects, might constitute a safer alternative to APAP than AMAP seems to be. In addition, to check the reliability of the calculated Gibbs free energies, the equilibrium constant (K) for the reaction NFCys + NAPQI (site 2), has been estimated and compared with that experimentally measured for a similar reaction (N-acetyl-Lcysteine +4-methyl benzoquinone).95 Because the systems are not identical, the K values are not expected to be identical either. However, they should be similar in magnitude. The experimental values were reported to be 4.70 × 10−4 and 1.68 × 10−4 at pH 5 and pH 6, respectively. The calculated values, at the same pHs, are 4.53 × 10−4 and 4.52 × 10−3. The agreement is reasonable (considering the difference in reactants) and seems to support the reliability of the calculated data. The kinetic calculations were first performed without including any explicit solvent molecules. The optimized geometries of the corresponding TSs are provided as Supporting Information (Figures S1 to S3), as well as the reaction barriers and the rate constants (Tables S3 and S4). However, when comparing the calculated data for the NAPQI + NF-Cys with the available experimental data for similar systems (Table 7) it was found to be significantly overestimated (1.04 × 108, at pH 7.4). It should be noted that there is another work that reports kinetic data for reactions similar to those investigated here, i.e., cysteine or mercaptoacetic acid, with dopaminoquinone (DQ).96 However, the results from that work cannot be directly compared with those calculated in the present work.

that the latter can also be formed by direct addition reactions, i.e., without intermediates, which correspond to the chemical routes modeled here. In addition, for MBQI3 (route XI-b), reactions at sites 3 and 5 do not lead to the formation of proper adducts but to separate fragments. It was found that, in general, the ΔG values corresponding to route XI-c are lower than those of route XI-b, when comparing the same reactants and the same reaction sites. Therefore, we have chosen route XI-c as the most likely one. The results and analysis leading to such a choice are in line with experimental evidence for the reaction of NAPQI with glutathione (GSH), which indicates that the key species for the reaction is the deprotonated one (GS−).78 Route XI-c involves the deprotonated fraction of the Cys residue; thus its pKa becomes relevant since it rules the amount of −S− at each pH. However, albeit the pKa of Cys residues is generally around 8.5,68 this value can be significantly modified, depending on the protein (or peptide) environment. There are numerous estimations of the pKa of Cys (Table 6) with values ranging from 4.6 to 9.9. Table 6. Some of the pKa values reported for cysteine residues in peptides and proteins pKa

ref

pKa

ref

pKa

ref

4.60 4.67 5.0 5.2 5.23 5.3 5.4 5.54 5.57 5.6 5.7

79 68 82 82 79 86 80,82 79 89 88 82

5.84 5.94 5.99 6.0 6.1 6.4 6.53 6.7 6.86 7.2 7.35

80 80 68 80 82 82 85 88 90 84 68,81

8.11 8.16 8.2 8.31 8.43 8.64 8.79 8.84 9.0 9.08 9.9

81 81 83,84 81 85 87 81 81 83 81 83

Here, the pKa of Cys in glutathione has been used as an example (8.64).87 In this case, the population of species presenting the thiol site deprotonated, at physiological pH, is 5.4% of the total Cys concentration. Accordingly, provided that route XI-c is actually the one involved in protein arylation, only a rather small fraction of Cys residues would be susceptible to damage. Therefore, it would be necessary to have a rather large concentration of the other reactant (NAPQI-like species) for such damage to be significant. This might help explain why APAP is nontoxic in therapeutic doses, while overdoses can result in hepatic toxicity. Moreover, this also means that not all Cys residues would be equally susceptible to arylation by NAPQI-like species. The proteins’ vulnerability to such damage is expected to increase as the pKa of their Cys residues lowers. It should be noted, however, that the reaction of NAPQI with glutathione (GSH), in vivo, is not purely chemical but can be enzymatically influenced. For example, glutathione Stransferases (GST) can catalyze this reaction, altering the NAPQI hepatotoxicity.78,91 It has been demonstrated that the rate of the GSH + NAPQI can be increased from 3.2 × 104 M−1 s−1 in the absence of GST, up to 2.0 × 107 M−1 s−1 when GST is present.78 In addition, GST pi null mice were found to be highly resistant to the hepatotoxic effects of this APAP.91 This finding has led to the hypothesis that GST inhibitors may protect against the liver and renal damage induced by this painkiller. Moreover, it is well documented that GST lowers the

Table 7. Experimental Rate Constants (kexp, M−1 s−1) for Reactions Similar to Those Investigated in This Work reaction L-cysteine

+ 4-methyl benzoquinone N-acetyl-L-cysteine + 4-methyl benzoquinone glutathione + 4-methyl benzoquinone glutathione + NAPQI

1293

pH

kexp

6.0 6.5 6.0 7.0

× × × ×

7.0 5.2 5.4 3.2

ref 5

10 105 105 104

95 95 95 78


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found to be 7.03 × 104 M−1 s−1. This value is in between those experimentally measured for the reactions NAPQI + GSH (at pH 7) and N-acetyl-L-cysteine + 4-methyl benzoquinone (at pH 6.5). The calculated value is 2.2 times higher than the first one and 7.4 times lower than the second one. This good agreement strongly suggests that the model including two explicit water molecules is adequate to properly describe the target reactions. In addition, as mentioned before, the pKa of the Cys residue may significantly influence the protein arylation reactions by QI species. Therefore, rate constants calculations were also performed, in the 5.0 to 8.0 pH range, for the NAPQI + NFCys reaction considering several pKas selected from Table 6. The values of such rate constants are reported in Table S6, Supporting Information. Since the chemical route chosen as the most likely one involves thiolate sites (−S−) in the Cys residues, two clear trends arise (Figure 5). The first one is that

This is because in ref 96, it was assumed that the reaction mechanism involves the formation of cys-DQ intermediates. Therefore, the rate constants correspond to first-order processes, while the ones calculated here (as well as those reported in Table 7) are second-order rate constants. Although the rate constant for the NAPQI + NF-Cys reaction has not been experimentally measured, those reported in Table 7 correspond to systems that are chemically close enough. Therefore, all of them are expected to have similar, albeit not identical, rate constants. That is why the large discrepancies between calculated and experimental values were interpreted as a failure in the used model. To improve it, two explicit solvent molecules were included in the calculations. They were arranged in such a way that hydrogen bonding (HB)-like interactions between one H in each water molecule and the thiolate center were promoted. The transition structure corresponding to the addition of NAPQI to NF-Cys (site 2) is shown in Figure 4, while those corresponding to the other quinone imines are provided as Supporting Information (Figures S4 to S7).

Figure 4. Optimized geometry of the transition structure corresponding to the addition of NAPQI (site 2) to NF-Cys, route XI, including two explicit water molecules.

Figure 5. Dependence of the rate constants, for the NAPQI + NF-Cys reaction (at 298.15 K, in aqueous solution) with the pH and the pKa of the Cys residue.

The reaction barriers (Table S5, Supporting Information) were found to be significantly higher than those corresponding to the bare models. Furthermore, the rate constant of the NAPQI + NF-Cys reaction, calculated including explicit water molecules, is in good agreement with the experimental ones for similar systems (Table 8). However, it was calculated considering pH 7.4, which is to some extent higher than those in the experiments. The rate constant were also calculated at different pHs to facilitate comparisons with the experiments (Table S6, Supporting Information). At pH 6.5 and considering pKa = 8.64 for the Cys residue, the calculated rate constant was

(at each pH) the rate constant increases as the pKa of the Cys residue decreases. The second one is that (for each pKa) the rate constant increases with the pH. Both trends are consequences of the same effect, the increment in the deprotonated fraction of the Cys residue. This is in line with previous experimental evidence for similar reactions.78,95 The model including two explicit water molecules, already validated for the NAPQI + NF-Cys reaction, was then used for the reactions involving the other QI species investigated in this

Table 8. Rate Constants (M−1 s−1) for the Addition Pathways of NAPQI-Like Species to NF-Cys, in Aqueous Solution, at 298.15 K and pH 7.4, Considering pKa(Cys) = 8.64 site 2 NAPQI NAMQI1 NAMQI2 NAMQI3 MBQI1 MBQI2 MBQI3

site 3

site 4

5.99 × 104

2.10 × 105

site 5

site 6

1.02 × 107 5.44 × 106

1.07 × 103 2.11 × 107 7.50 × 104

5.32 × 10

5

7.01 × 105 9.22 × 104

8.01 × 105

1294

4.32 × 106 8.02 × 104

koverall 5.32 2.71 3.20 5.52 9.22 5.12 8.02

× × × × × × ×

105 105 107 106 104 106 104


Article

Chemical Research in Toxicology work. Figure 6 shows a comparison of the relative reactivity of the different QI species toward Cys residues in proteins. It was

Scheme 4. Reaction Mechanism Proposed for Protein Arylation Involving Quinone Imines and Cys Residuesa

Figure 6. Overall rate coefficients (M−1 s−1) for the addition of NAPQI-like species to NF-Cys, in aqueous solution, at 298.15 K and pH 7.4, including two explicit water molecules in modeling.

found that, while the reactivity of NAMQI1, MBQI1, and MBQI3 toward NF-Cys is slightly lower (∼2 to 7 times) than that of NAPQI, those of NAMQI2, NAMQI3, and MBQI2 are significantly higher (∼10 to 60 times). This data support the idea that if AMAP is metabolized (or chemically transformed) into its quinone imine derivatives, it can be as toxic as APAP. The data provided up to this point correspond to the first step of the protein arylation, i.e., the initial addition of the quinone imines to Cys residues. However, to reach the structures of the adducts that have been experimentally observed some additional chemical steps are necessary. The complete mechanism proposed here is shown in Scheme 4 where step 1 represents the initial addition (already analyzed), while steps 2 and 3 involve proton exchange. Step 2 is postulated to be an intramolecular process, while step 3 corresponds to protonation from the environment, i.e., from the solvent (water). Therefore, the viability of these two steps was also estimated. However, while steps 1 and 2 (Scheme 4) are independent of the pH, the equilibrium constant and Gibbs energy of step 3 explicitly depend on the pH. The conditional Gibbs energy of reaction (ΔG′) can be calculated in this case, at each particular buffered pH, using the following equation:

a

NAPQI is used as a representative example.

Table 9. Gibbs Free Energies (ΔG, kcal/mol) of the Proton Transfer and the Protonation Process (Steps 2 and 3, Respectively (Scheme 4)), in Aqueous Solution, at 298.15 K site 2

site 3

site 4

site 5

site 6

−20.97 −30.58

−14.41 −17.56 −32.51

Step 2

ΔG′ = ΔG 0 + 2.303RT (pH)

NAPQI NAMQI1 NAMQI2 NAMQI3 MBQI1 MBQI2 MBQI3

In this equation, ΔG represents the Gibbs energy calculated for standard conditions, i.e., 1 M concentrations, which means pH 0. The interested reader can find more details on how this equation is obtained elsewhere.97,98 The corresponding data are reported in Table 9, where the ΔG′ values for step 3 correspond to pH 7.4. It was found that these two steps are exergonic for all of the investigated species. Accordingly, after the initial addition (step 1) takes place the intramolecular proton transfer is a favorable process, which means that the equilibrium would evolve in its direction. Finally, at the pH of interest, the acid−base equilibria with the solvent would lead to the protonation of the intermediate, yielding the products that have been experimentally identified. Considering the gathered data altogether, it seems that the mechanism proposed in this work (Scheme 4) for the protein arylation (at Cys sites) by quinone imines derived from APAP and AMAP is viable. Moreover, it allows to explain some 0

−26.28 −21.02

−13.65

−22.44 −7.62

−18.74

−12.75 −23.51

Step 3 NAPQI NAMQI1 NAMQI2 NAMQI3 MBQI1 MBQI2 MBQI3

−16.22 −4.50

−5.69

−1.40

−4.17 −7.19

−7.94 −3.68 −7.37

−9.91 −4.53

−4.67 −11.13

experimental observations18,21 and some apparent contradictions26−28 among experimental results. 1295


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Chemical Research in Toxicology Reduction of Metal Ions (•OH Production). There are other possible reactions involving APAP and AMAP that might result in damage to biomolecules, i.e., that might contribute to their toxicity. In this work, one of them has been explored. It corresponds to the potential involvement of these amino phenols in the production of a potent oxidant, the hydroxyl radical (•OH), and involves the reduction of metal ions. Because Cu(II) and Fe(III) are the most abundant and stable oxidative states of copper and iron, respectively, they constitute the main sources of Cu(I) and Fe(II) in biological systems. At the same time, the latter can be involved in •OH production via the Fenton reaction. Thus, chemical species promoting the reduction of Cu(II) and Fe(III) would increase the amounts of • OH formation in living systems. This may also represent a chemical route related to the toxicity of chemicals since •OH is so reactive that it can damage almost any molecule in the vicinity of its site of formation.99 Accordingly, the potential role of APAP, AMAP, and related species as reductors of Cu(II) and Fe(III) has been investigated. The related species include PAP, MAP, the free radicals previously identified as the most likely ones (phenoxyl), and monoanions (phenolate). The latter were included in the investigation because phenolic compounds are involved in acid−base equilibria in aqueous solution, and their conjugated bases are expected to be stronger reductors than the neutral acids. However, to properly estimate the potential role of the anions in a particular chemical process it is important to know the corresponding pKa values. This is particularly important in this case because usually the pKa values of phenols are higher than 9, which means that only a small fraction of these species is deprotonated under physiological conditions. There are previous experimental determinations of the pKas of APAP, PAP, and MAP, but to our best knowledge, there are no previous reports on the pKa of AMAP. Therefore, it was estimated in this work using the parameter fitting (PF) method, which has been proven to produce excellent agreement with experimental data.100 The pKa values for APAP, PAP, and MAP were also estimated using the same strategy and compared to the experimental values to validate the calculated pKa of AMAP. It was found (Table 10) that the differences between

Table 11. Gibbs Free Energies of Reaction (ΔG, kcal/mol), Reorganization Energies (λ, kcal/mol), Gibbs Free Energies of Activation (ΔG⧧, kcal/mol), and Rate Coefficients (kTST and kpH 7.4, M−1 s−1) for the Reduction of Cu(II)a

exp

a

9.38a 10.46a 9.815a

10.30b 10.02c

calcd

mf (pH 7.4)

9.31d 9.39d 10.50d 9.99d

0.0122 0.0101 0.0008 0.0026

ΔG

APAPA PAPA AMAPA MAPA APAPR PAPR AMAPR MAPR O2•− Asc-

−18.35 −29.62 −13.43 −19.49 5.97 −4.96 24.29 21.92 −30.47 −6.42

ΔG⧧

kTST

34.15 35.60 32.12 33.58 35.78 32.27

1.83 0.25 2.72 1.48 12.18 5.78

7.35 7.44 6.74 7.40 7.33 3.44

× × × × × ×

38.82 36.90

0.45 6.29

7.52 × 1009 1.48 × 1008

λ

kpH 7.4 09

10 1009 1009 1009 1003 1008

8.97 5.95 6.81 1.92

× × × ×

1007b 1006b 1007b 1007b

O2•− and the ascorbate anion (Asc−) are included for comparison purposes. All of the values were calculated in aqueous solution at 298.15 K. bCalculated including the molar fraction of the anions. a

Table 12. Gibbs Free Energies of Reaction (ΔG, kcal/mol), Reorganization Energies (λ, kcal/mol), Gibbs Free Energies of Activation (ΔG⧧, kcal/mol), and Rate Coefficients (kTST, kpH 7.4, M−1 s−1) for the Reduction of Fe(III)a reductor

ΔG

APAPA PAPA AMAPA MAPA APAPR PAPR AMAPR MAPR O·− 2 Asc-

−24.00 −35.27 −19.08 −25.14 0.32 −10.61 18.64 16.27 −36.12 −7.87

ΔG⧧

kTST

19.36 20.81 17.32 18.79 20.99 17.48

0.28 2.51 0.04 0.54 5.41 0.68

7.44 6.85 7.45 7.41 6.18 7.39

× × × × × ×

24.03 17.91

1.52 1.41

7.57 × 1009 7.33 × 1009

λ

kpH 7.4 09

10 1009 1009 1009 1008 1009

9.08 5.48 7.52 1.93

× × × ×

1007b 1006b 1007b 1007b

O2•− and the ascorbate anion (Asc−) are included for comparison purposes. All of the values were calculated in aqueous solution at 298.15 K. bCalculated including the molar fraction of the anions. a

modeled as their aquo complexes. This model is more appropriate to represent “free” copper, under physiological conditions, than the bare ions. However, it should be noted that in biological systems these metal ions are frequently bound to other ligands with higher metal affinity. Since such ligands are widely diverse, it is not possible to model them all; that is why water was chosen for the models used here. It is expected that relative trends remain the same for other ligands, albeit the absolute chemical reactivity might vary to some extent, depending on the particular ligands bound to the metal ions. Cu(II) was modeled in an almost square-planar fourcoordinate geometry, which has been previously identified as the most likely configuration for Cu(II) in the aqueous phase.102,103 For consistency purposes, the hydrated Cu(I) ion was modeled with the same amount of water molecules, albeit in this case the linear two-coordinate configuration is preferred, i.e., Cu(I) is coordinated only to two water molecules, and the other two solvate the system. This linear, 2-coordinated, structure is consistent with previous experimental evidence.104−106 Both iron ions, Fe(III) and Fe(II), were modeled as octahedric hexa-aquo complexes, which correspond to their expected geometry and coordination number.107−109

Table 10. pKa Values for the Phenolic Species Investigated in This Work, and Molar Fractions (mf) of the Anions at Physiological pH APAP AMAP PAP MAP

reductor

PubChem. bRef 101. cRef 87. dThis work.

experimental and calculated values were lower than 0.1 pKa units. Moreover, the calculated value for MAP is between those experimentally obtained. This excellent agreement supports the reliability of the value estimated here for AMAP (pKa = 9.39). The molar fractions at pH 7.4 (Table 10) were obtained for all of the amino phenols using the calculated pKas for consistency purposes. The thermochemical and kinetic data estimated for the reduction of Cu(II) and Fe(III) are reported in Tables 11 and 12, respectively. To obtain this information, metal ions were 1296


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encounters a strong enough oxidant capable of turning it into AMAPR. Regarding protein damage, Cys residues were identified as the most likely targets. The damage induced by the investigated species to this residue can take place in two different ways: (i) HAT from the thiol site by the free radical intermediates arising from their oxidation and (ii) protein arylation by their quinone imine derivatives. Both are not only thermochemically viable but also are very fast reactions. Since route ii has been held responsible for the hepatotoxicity of AMAP, it was investigated in great detail. It is proposed that the most likely reaction mechanism involves thiolate sites, albeit the population of deprotonated species, at physiological pH, is only 5.4% of the total Cys concentration. Since this means that only a rather small fraction of Cys residues would be susceptible to damage, a rather large concentration of the other reactant (NAPQI-like species) would be necessary for such damage to be significant. This might explain why APAP is nontoxic in therapeutic doses, while overdoses can result in hepatic toxicity. The gathered data indicate that the quinone imines derived from APAP, AMAP, and MAP are all capable of protein arylation involving Cys residues. This supports the idea that if AMAP is metabolized (or chemically transformed) into its quinone imine derivatives, it can be as toxic as APAP when consumed in similar amounts. In addition, the potential role of APAP, AMAP, and their deacetylated partners in reducing metal ions, in particular Cu(II) and Fe(III), was also investigated. The obtained results suggest that their anionic fractions are capable of increasing • OH production via the Fenton reaction, which might also contribute to their toxicity. The results presented here for the diverse chemical routes are in line with diverse experimental evidence. Such agreement supports the reliability of the performed calculations and the conclusion derived from them.

The data on the reduction of Cu(II) and Fe (III) by the radical anion superoxide (O2•−) and the ascorbate anion (Asc−) are included in Tables 11 and 12 for comparison purposes. The reaction with O2•− corresponds to the first step of the HaberWeiss recombination, while the reaction with Asc− represents the possible role of other, less strong, reductants. This particular ion was selected because copper-ascorbate mixtures are frequently used in experiments to generate oxidative conditions. It was found that the reactions involving the anionic fractions are all thermochemically viable for both Cu(II) and Fe(III) reductions. The corresponding ΔG values are between those of the reactions involving O2•− and Asc− as reductants. As expected, based on charge considerations the radicals are worse reductants than the anions. In the particular case of AMAPR and MAPR, the reactions were found to be significantly endergonic, which indicates that they are not able to reduce Cu(II) and the Fe(III). Accordingly, their potential role in increasing •OH production via the Fenton reaction is expected to be negligible. The reaction APAPR + Cu(II) was also found to be endergonic, albeit moderately (∼6 kcal/mol), while the APAPR + Fe(III) reaction is isoergonic. Thus, they are less efficient as metal ion reductants than Asc−, but such a role is not necessarily negligible. Regarding kinetics, the reactions of O2•− and Asc− with both Cu(II) and Fe(III) are very fast, within or close to the diffusionlimited regime (Tables 11 and 12). That is also the case for the reactions of the anions and PAPR. Thus, they are all expected to significantly contribute to the production of the reduced metal ions, Cu(I) and Fe(II), and consequently to the •OH formation via the Fenton reaction. Logically, when the molar fraction of the anions is included in the calculation of the rate constants (kpH 7.4, Tables 11 and 12) their values substantially decrease. However, even when including this factor, all of the estimated rate constants remain larger than 106 M−1 s−1. This suggests that increasing the production of •OH might also contribute to the toxicity of APAP, AMAP, and their deacetylated partners. These findings are in line with experimental evidence indicating that the amounts of reactive oxygen species significantly increase when toxic doses of APAP are administered to mice.19

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00024. Gibbs free energies of the reactions of APAP, PAP, AMAP, and MAP with •OH and HOO•; Gibbs free energies of activation for the addition of NAPQI-like species to Cys residues; rate constants obtained from using models without explicit water molecules; overall rate constants in aqueous solution; optimized geometries of the transition structures (PDF)

■

CONCLUSIONS Several chemical routes related to the toxicity of paracetamol (APAP), its analogue N-acetyl-m-aminophenol (AMAP), and their deacetylated analogues (PAP and MAP) were investigated in this work. The formation of radical intermediates arising from the oxidation of these molecules by free radicals was investigated, and it was found that AMAP is more resilient to chemical oxidation than APAP (MAP and PAP follow the same trend). Thus, the chemical degradation of AMAP into AMAPR would be determined by the strength of the oxidant and is expected to be significant only when it is induced by strong oxidants. This finding might explain the apparent contradictions among experimental evidence regarding the AMAP toxicity. In addition, it was confirmed that phenoxyl radicals are the most abundant intermediates. According to the gathered data, none of these radical intermediates is able to oxidize DNA. On the contrary, they are all able to damage lipids by HAT from the bis-allylic site, albeit only AMAPR reacts fasts enough to represent a non-negligible hazard to the chemical integrity of lipids. These findings support the idea that AMAP can be toxic, provided that it

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AUTHOR INFORMATION

Corresponding Author

*Tel: 52 55 5804 4675. E-mail: [email protected]. ORCID

Annia Galano: 0000-0002-1470-3060 Funding

R.C.-A. acknowledges the financial support through a CONACyT postdoctoral fellowship. Notes

The authors declare no competing financial interest. 1297


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ACKNOWLEDGMENTS We gratefully acknowledge the Laboratorio de Supercómputo y Visualización en Paralelo at Universidad Autónoma Metropolitana-Iztapalapa for computing time.

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ABBREVIATIONS 2dG, 2′-deoxyguanosine; 2dGMP, 2′-deoxyguanosine 5′-monophosphate; AMAP, N-acetyl-m-aminophenol; AMAPR, Nacetyl-m-aminophenoxyl radical; APAP, N-acetyl-p-aminophenol (also known as paracetamol or acetaminophen); APAPR, N-acetyl-p-aminophenoxyl radical; CYP, cytochrome P450 enzymes; Cys, cysteine; DNA, deoxyribonucleic acid; DQ, dopaminoquinone; G, guanine; Gs, guanosine; GSH, glutathione; GST, glutathione S-transferase; HAT, formal hydrogen atom transfer; HB, hydrogen bonding; LA, linoleic acid; Leu, leucine; LM, lipid model; MAPR, m-aminophenoxyl radical; MBQIi, m-benzoquinone imines; NAMQIi, N-acetyl-m-benzoquinone imine; NAPQI, N-acetyl-p-benzoquinone imine; NFCys, N-formyl-cysteinamide; NF-Leu, N-formyl-leucinamide; NF-Trp, N-formyl-tryptophanamide; NF-Tyr, N-formyl-tyrosinamide; PAPR, p-aminophenoxyl radical; PBQI, p-benzoquinone imine; QM-ORSA, quantum mechanics based test for overall free radical scavenging activity; RAF, radical adduct formation; SET, single electron transfer; SMD, solvation model based on density; SPLET, sequential proton loss electron transfer; Trp, tryptophan; TST, transition state theory; Tyr, tyrosine

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