Investigation of radical scavenging effects of acetaminophen, p-aminophenol and their O-sulfated conjugates

Abstract

Acetaminophen (APAP) and p-aminophenol (p-AP) are the analogous simple phenolic compounds that undergo sulfate conjugation (sulfation) by cytosolic sulfotransferases. Sulfation is generally thought to lead to the inactivation and disposal of endogenous as well as xenobiotic compounds. This study aimed to investigate the antioxidative effects of O-sulfated form of APAP and p-AP, i.e., APAPS and p-APS, in comparison with their unsulfated counterparts. Using a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay, the antioxidant capacity of APAPS was shown to be approximately 126-times lower than that of APAP. In contrast, p-APS displayed comparable activity as unsulfated p-AP. Similar trends concerning the suppressive effects of these chemicals on cellular O₂^- radical generation were found using an activated granulocytic neutrophil cell model. Collectively, these results indicated that, depending on the presence of an additional “active site”, sulfation may not always decrease the antioxidant activities of phenolic compounds.

INTRODUCTION

Sulfate conjugation (sulfation) has been generally thought to lead the inactivation and disposal of endogenous as well as xenobiotic compounds (Mulder and Jakoby, 1990; Falany and Roth, 1993; Weinshilboum and Otterness, 1994; Suiko et al., 2017). For instance, previous studies have demonstrated that sulfation of flavonoids can reduce their antiradical and biological activities in vitro (Rimbach et al., 2004; Roubalová et al., 2015). Moreover, sulfated metabolites of quercetin, a flavonoid, were found to display lower, but retain nonignorable, antioxidant activities (Dueñas et al., 2011). In regard to drug metabolism, sulfation is thought to be a major Phase-II detoxification pathway. In contrast to the above-mentioned examples, sulfation of certain chemicals such as hydroxyarylamines has been shown to result in their activation, thereby becoming reactive carcinogens (DeBaun et al., 1970). Therefore, it is essential to quantitatively investigate whether xenobiotic compounds, including drugs, can be inactivated or activated through the sulfation pathway on an individual basis.

Acetaminophen (N-acetyl-p-aminophenol or paracetamol; APAP) and p-aminophenol (p-AP) are simple phenolic compounds that may be subjected to sulfation by cytosolic sulfotransferases, generating APAP O-sulfate (APAPS) and p-AP O-sulfate (p-APS), respectively (Yamamoto et al., 2015; Allali-Hassani et al., 2007). APAP is a widely used antipyretic analgesic drug for relieving pain and reducing fever (Ohashi and Kohno, 2020; Jóźwiak-Bebenista and Nowak, 2014). APAP, administered orally, is absorbed from the gastrointestinal tract, predominantly metabolized in the liver, and excreted in the urine, mainly as glucuronidated and sulfated derivatives. Based on a recent analysis, APAPS constituted 20–46% of the APAP absorbed (Ohashi and Kohno, 2020). The overdose usage of APAP has been associated with adverse effects, including hepatotoxicity- and nephrotoxicity-related disorders (Jóźwiak-Bebenista and Nowak, 2014). It is noted that a small portion (< 5%) of APAP is metabolized through deacetylation to form p-AP or by oxidation under the action of cytochrome P450 enzymes to generate a reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) (Ohashi and Kohno, 2020). In industry, p-AP is used as a photographic developer, a precursor of hair-coloring dye, and a rubber antioxidant (Gomes and da Silva, 2005). In addition, p-AP is known as an intermediate for APAP synthesis or as a degradation byproduct, whereas significant nephrotoxic and hepatoxic effects of p-AP have been reported (Németh et al., 2008; SCCS, 2011). As noted above, p-AP is also a metabolic product of APAP, and may potentially be involved in the nephrotoxic and hepatoxic effects of APAP. A moderate radical scavenging capacity of APAP (Dinis et al., 1994; Alisi et al., 2012) and strong antioxidant activities of p-AP (Alisi et al., 2012; Bendary et al., 2013) have been demonstrated in vitro. It is an interesting question whether sulfation may inactivate the radical scavenging and antioxidant activities of APAP and p-AP. Along the same line, we have previously reported that sulfation of the naphthol isomers do not always lead to the decrease in their antioxidant activity (Sugahara et al., 2018). Indoxyl sulfate, a representative sulfated metabolite and a uremic toxin, has been shown to enhance intracellular oxidation level and decrease the phagocytic activity in macrophage model cells (Tsutsumi et al., 2020a, 2020b).

In addition to its antipyretic and analgesic effects, APAP can interact at different stages during the biosynthesis of prostaglandin by means of redox reactions, thereby exerting anti-inflammatory activity (Aronoff et al., 2006; Alisi et al., 2012). For example, it has been proposed that APAP plays a role as an antioxidant in reducing the active heme group or quenching the reactive tyrosyl radical in the active site of cyclooxygenase. A previous report has demonstrated that APAP is capable of protecting brain endothelial cells from menadione-induced oxidative stress (Tripathy and Grammas, 2009). Nevertheless, a systematic study using APAP and its possible metabolites generated upon phase-II sulfate conjugation has not been fully evaluated using antioxidant assays or other pharmacological experimental settings. As part of our overall goal to elucidate the functional relevance of sulfate conjugation of individual phenolic compounds, we attempted in the current study to specifically investigate the antioxidant capacity of APAP and its related metabolites, including APAPS, p-AP and p-APS.

The current study aimed to investigate the antioxidant capacity of APAPS and p-APS, in comparison with their unsulfated counterparts. To determine their antioxidant activities, 1,1-diphenyl-2-picrylhydrazyl (DPPH), a synthetic free radical, and a cellular O₂^- radical generation assay with an activated granulocytic cell model were employed. To gain additional insight into the structure-activity relationship, other derivatives of APAP and p-AP were concomitantly analyzed in DPPH radical assay.

MATERIALS AND METHODS

Materials

Acetaminophen (N-acetyl-p-aminophenol or paracetamol; APAP), p-aminophenol (p-AP), p-anisidine (p-aminophenol O-methylate; p-APM) and benzene were products of Nacalai Tesque Inc. (Kyoto, Japan). p-Aminophenol O-sulfate (4-aminophenyl hydrogen sulfate; p-APS) was from Ark Pharm, Inc. (Libertyville, IL, USA). Aniline, anisole, phorbol 12-myristate 13-acetate (PMA), and HBSS(-) without phenol red were purchased from Fujifilm-Wako Pure Chemical Co. (Osaka, Japan). Acetanilide was from Kishida Chemical Co., Ltd. (Osaka Japan). (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) was a product of Sigma-Aldrich, Inc. (St. Louis, MO, USA). p-Acetanisidide (N-(4-methoxyphenyl)acetamide or acetaminophen O-methylate; APAPM) was obtained from Oakwood Products, Inc. (Estill, SC, USA). Phenol was a product of Kanto Chemical Co., Inc. (Tokyo, Japan). Acetaminophen glucuronide and 4-aminophenyl β-glucuronide were purchased from Cayman Chemical Company (Ann Arbor, MI, USA) and Carbosynth Ltd. (Compton, UK), respectively. Aniline, anisole, and benzene were diluted with DMSO. A stock solution of Trolox was prepared in DMSO, while other test chemicals were dissolved in MilliQ water. Potassium phenyl sulfate and DPPH were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). A water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) was purchased from Dojindo Labs (Kumamoto, Japan). HL-60 human promyelocytic leukemia cells (JCRB0085) were obtained from Japanese Collection of Research Bioresources Cell Bank (Tokyo, Japan). All other chemicals were of the highest grade commercially available.

Acetaminophen O-sulfate (APAPS, as sodium salt with 97.0 ± 1.6% purity) was chemically synthesized from APAP and sulfuric acid, based on our recently established method (Morita et al., 2022), which was a modified procedure based on that used for the tyrosine O-sulfate synthesis (Jevons, 1963). Figure 1 shows chemical structures of APAP, p-AP and their derivatives used in this study.

Fig. 1

Chemical structures of APAP, p-AP and their derivatives used in this study. APAP; acetaminophen, APAPS; acetaminophen O-sulfate, APAPM; acetaminophen O-methylate, p-AP; p-aminophenol, p-APS; p-aminophenol O-sulfate, p-APM; p-aminophenol O-methylate, Phe; phenol, PheS; phenyl sulfate.

DPPH radical scavenging assay

DPPH radical scavenging activity was measured as described previously (Sugahara et al., 2018; Ueda et al., 2019). The reaction was started by adding of 0.5 mM DPPH dissolved in ethanol (50 μL) into the pre-assay mixture (200 μL) containing a test sample (10 μL), 70% EtOH (90 μL), and 0.1 M sodium acetate buffer (pH 5.5, 100 μL). The mixture, in a final volume of 250 μL, was incubated for 30 min at room temperature. The absorbance of the solution was measured at 517 nm using a grating microplate reader (SH-1000Lab, Corona Electric Co., Ltd., Ibaraki, Japan). Trolox was used as the control standard.

DPPH radical scavenging activity (%) = {1 − (A_Sample − A_Blank)/(A_Control − A_Blank)} x 100

where A_Sample is the Abs. measured in the presence of test sample. A_Blank is the Abs. measured in the absence of the test sample and the DPPH radical solution. A_Control is the Abs. measured in the absence of the test sample. The radical scavenging activity was calculated and expressed as the trolox equivalent antioxidant capacity (TEAC) value.

Cellular O₂^- radical generation assay

The cellular O₂^- radical generation assay was performed according to an established procedure (Ueda et al., 2019) with a minor modification. DMSO-differentiated HL-60 human granulocyte-like neutrophil cells (1 x 10⁶ cells/mL in HBSS, 250 μL) were prepared and preincubated with the sample (1.25 μL) in a test tube at 37°C for 15 min. A mixture (13.75 μL), containing 20 μM PMA (1.25 μL) in DMSO as an inducer for cellular O₂^- radical generation and 2.37 mM WST-1 solution (12.5 μL) in PBS as a colorimetric indicator for O₂^- radical, was added to the cell suspension and incubated at 37°C for 15 min. Thereafter, the microtube containing the assay mixture was immediately placed in ice-cold water for 5 min to terminate O₂^- radical generation. The activated cells were pelleted by centrifugation at 13,000 x g for 1–2 min and the supernatant was collected. The level of O₂^- radical generated was determined based on the colorimetric change of WST-1 at 450 nm.

Statistical analysis

Data, obtained from three or four experiments, were expressed as mean ± standard deviation. Data in part were analyzed using statistical add-on software program (Statcel, OMS Co., Saitama, Japan) for Excel 2004 (Microsoft Co., Redmond, WA, USA). In the Dunnett test, the significant difference was considered at *P < 0.05 or **P < 0.01. A post-hoc Tukey-Kramer test was conducted for the multiple comparison and differences at P < 0.05 were considered significant.

RESULTS AND DISCUSSION

The current study was designed to investigate the antioxidative capacity of APAPS and p-APS in comparison with their unsulfated counterparts. DPPH free radical scavenging assay and a cellular O₂^- radical generation assay with an activated granulocytic cell model were employed to determine their antioxidant activities.

Determination of free radical scavenging activities of sulfated vs. unsulfated APAP and p-AP

Previous studies have demonstrated differential radical scavenging activities of APAP and p-AP (Dinis et al., 1994; Alisi et al., 2012; Bendary et al., 2013). In this study, we first attempted to investigate the potential antioxidant activities of sulfated APAP and p-AP using a DPPH radical scavenging assay, which had previously been used to study naphthol isomers and their sulfated derivatives (Sugahara et al., 2018). As shown in Fig. 2, APAPS (EC₅₀; > 1,000 μM) demonstrated a much lower radical scavenging activity than unsulfated APAP (EC₅₀; 145 μM). In contrast, p-APS showed a radical scavenging activity (EC₅₀; 24.5 μM) comparable to that of p-AP (EC₅₀; 24.1 μM). Under the experimental settings adopted, the antioxidant activity of trolox (EC₅₀; 29.0 μM) was closer to those of p-AP and p-APS. A moderate free-radical scavenging activity of APAP and a stronger activity of p-AP as observed in this study were consistent with similar results reported previously (Dinis et al., 1994; Alisi et al., 2012; Bendary et al., 2013). It is noted that another previous study also demonstrated that p-AP displayed a stronger DPPH radical scavenging activity and suppressing effect on lipid peroxidation system than did APAP (Takahashi et al., 2002).

Fig. 2

Effects of APAP (gray circle), p-AP (gray triangle), and their sulfate conjugates, APAPS (black circle) and p-APS (black triangle), respectively, on the DPPH radical scavenging assay. Data shown represent mean ± S.D. from four experiments. Trolox (white circle) was used as a control standard. APAP; acetaminophen, APAPS; acetaminophen O-sulfate, p-AP; p-aminophenol, p-APS; p-aminophenol O-sulfate.

The EC₅₀ values and calculated TEAC values are compiled in Table 1. The ratio of EC₅₀ values (or 1/Ratio of EC₅₀) and that of TEAC values (or 1/Ratio of TEAC) for individual pairs of the tested compounds reflect quantitative differences in their activities. These ratio values indicated that the DPPH radical scavenging activity of APAPS was approximately 126-times lower than that of APAP. This result is in line with the general perspective that sulfation plays a deactivation. In contrast, the radical scavenging capability of p-APS was comparable to that of p-AP. For the sulfated compounds tested, the activity of p-APS was found to be almost 745-times higher than that of APAPS. Notably, p-AP showed approximately 6.0-times higher radical scavenging activity than that of APAP. This is the first time that the free radical scavenging activities of the O-sulfated metabolites of APAP and p-AP were quantitively determined in comparison with their unsulfated counterparts. According to a previous study concerning computational and theoretical chemistry, an initial hydrogen atom abstraction from the phenolic hydroxyl group, rather than that from the acetamide nitrogen atom, of APAP may occur preferentially (Diniz et al., 2004). In general, the reactivity of phenols towards DPPH radical depends on the capability of the aromatic system to stabilize the single electron produced (Alisi et al., 2012). Therefore, it is possible that the O-sulfation of APAP may lead to the masking of the active phenolic hydroxyl group, thereby resulting in a decrease of its antioxidant activity. The lower reactivity of APAP towards DPPH radical, compared to that of p-AP, could be due to the presence of the electron-withdrawing acetamido group, which may destabilize the phenoxyl radical (Alisi et al., 2012). Moreover, the amino group of p-AP, rather than its phenolic hydroxyl group, may also play an important role as a strong electron donor in the reaction with DPPH radical (Bendary et al., 2013; Ali et al., 2013; Ali and Ali, 2015). Based on the above-mentioned rationalization, it is likely that p-AP has a higher antioxidant capacity than APAP.

Table 1. EC₅₀ and TEAC values, and ratio of these values for APAP, p-AP, and their sulfate conjugates as determined in DPPH radical scavenging assay.*

	DPPH Radical Scavenging Assay
	EC50 (μM)**			TEAC (μmol TE/mmol)***
APAPS	> 1,000 (Max 14.7 ± 1.0%)			1.59	±	0.74a
APAP	145	±	12.8a	201	±	17.9b
p-APS	24.5	±	1.4b	1,185	±	63.0c
p-AP	24.1	±	0.5b	1,204	±	26.2c
Trolox	29.0	±	2.9b	-
	Ratio of EC50 values****			Ratio of TEAC values****
APAPS/APAP	-			0.00791 (126)
p-APS/p-AP	1.02 (0.980)			0.984 (1.02)
p-APS/APAPS	-			745 (0.00134)
p-AP/APAP	0.166 (6.02)			5.99 (0.167)

*Data shown represent mean ± S.D. from four experiments and values not sharing a common superscript letter are considered significantly different at P < 0.01. Trolox was used as a control standard.

**EC₅₀ values indicate effective concentration where 50% of the activity was shown in Fig. 2.

***TEAC values indicate trolox equivalent antioxidant capacity.

****Data shown in parentheses indicate calculated ‘1/Ratio of EC₅₀ values’ or ‘1/Ratio of TEAC values’.

APAP; acetaminophen, APAPS; acetaminophen O-sulfate, p-AP; p-aminophenol, p-APS; p-aminophenol O-sulfate.

Previous studies have demonstrated that APAP exerted significant suppressive effects on the H₂O₂-induced membrane lipid peroxidation in human erythrocytes (Orhan and Sahin, 2001) as well as the menadione-induced oxidative cell damage and the expression of inflammatory proteins in brain endothelial cells from rat brain microvessels (Tripathy and Grammas, 2009). It will be interesting to find out whether sulfate-conjugation of phenolic compounds such as APAP and p-AP is capable of interfering with the oxidative/antioxidative process-mediated pathogenesis and/or immune responses both in vitro and in vivo. As a well-established metabolic activation of APAP, a portion of APAP is subjected to N-hydroxylation in the liver by cytochrome P450 enzymes (particularly CYP2E1) to generate a toxic metabolite, namely NAPQI (Du et al., 2016; Ohashi and Kohno, 2020). NAPQI is normally detoxified via glutathione conjugation by glutathione S-transferase to form mercapturic acid, a harmless metabolite, which is subsequently excreted in the urine. Overusage of APAP and/or weakened hepatic functions may result in the depletion of glutathione. The resulting accumulation of NAPQI may promote interaction with mitochondrial proteins and induce oxidative stress through ROS generation. Incidentally, it has been demonstrated that the overuse of APAP resulted in hepatic cell death and cellular dysfunctions (Du et al., 2016). Further investigation is warranted in order to clarify whether APAP itself as well as its metabolites such as APAPS, p-AP, and p-APS may contribute to the attenuation of NAPQI-mediated oxidative stress and further suppression of liver injury.

To clarify further the biological relevance of the sulfate conjugation, APAPS, p-APS, and their unsulfated counterparts, APAP, p-AP, were analyzed for their effect on the cellular O₂^- radical generation using a PMA-stimulated granulocytic neutrophil cell model. As shown in Fig. 3, APAPS did not markedly attenuate the cellular O₂^- radical generation, while APAP showed a moderate effect. In contrast, the suppressive effect of p-APS on the O₂^- radical generation (IC₅₀; 4.65 μM) was comparable to that of p-AP (IC₅₀; 4.17 μM). Under the experimental settings adopted, no cytotoxic effects of these chemicals were observed during a 30-min incubation period, as indicated by trypan-blue dye-exclusion assay (data not shown). Neutrophils are known to play an important role in host defense against microorganisms and inflammatory responses. Once neutrophils are activated, excess amount of O₂^- radical is generated following the oxidative-burst process (Utsumi et al., 1992; Shintani, 2013). A previous study documented that PMA-stimulated leukocytes resulted in O₂^- radical generation through the enzymatic NAD(P)H oxidase reaction (Murakami et al., 1997). Our pilot study showed that both p-APS and p-AP exerted a comparable O₂^- radical suppressing effect (IC₅₀; 12.8 μM and 10.4 μM, respectively) on the NADH oxidase-based assay without enzyme inhibition (figure not shown). Therefore, it appeared that sulfation may not deactivate antioxidative potential of p-AP. Since APAP and APAPS both carry an amide bond, it will be important to investigate whether p-AP and p-APS, respectively, may be generated upon their spontaneous hydrolysis. Additional experiments using a previously established HPLC procedure (Morita et al., 2022) showed no decrease of the peaks corresponding to APAP and APAPS, and no appearance of the peaks corresponding to p-AP and p-APS, in two different antioxidant assay mixtures containing, respectively, APAP and APAPS (data not shown). Therefore, it appears unlikely that p-AP and p-APS could have been produced by spontaneous hydrolysis of, respectively, APAP and APAPS under these experimental settings.

Fig. 3

Effects of APAP (gray circle), p-AP (gray triangle), and their sulfate conjugates, APAPS (black circle) and p-APS (black square), respectively, on the cellular O₂^- radical generation in PMA-stimulated granulocytic neutrophil cell model. Data shown represent mean ± S.D. from three experiments. Trolox (white circle) was used as a control standard. Using the Dunnett test, significant difference from control was considered at *P < 0.05 or **P < 0.01, APAP; acetaminophen, APAPS; acetaminophen O-sulfate, p-AP; p-aminophenol, p-APS; p-aminophenol O-sulfate.

Examination of structure specificity of APAPS, p-APS and their derivatives using the DPPH radical assay

To gain insight into the structural specificity of APAPS and p-APS on their DPPH radical scavenging activity, related derivatives including O-methylated form (APAPM and p-APM, respectively), dehydroxylated form (acetanilide and aniline), phenol and phenyl sulfate were similarly tested at 100 μM and 1,000 μM concentrations. As shown in Fig. 4, APAPS showed lower radical scavenging activity than did APAP, while APAPM and acetanilide showed virtually no such activity. It is conceivable that the free radical scavenging activity of APAP, which is dependent on its hydroxyl group, may be diminished by O-sulfation and O-methylation. In addition, the activity of p-APS appeared higher than those of p-APM and aniline. Further experiments using varying concentrations of the test samples indicated that the activity of p-APS (EC₅₀; 24.5 μM, ref. Table 1) was higher than those of p-APM (EC₅₀; 40.3 μM) and aniline (EC₅₀; 101 μM) (figure not shown). The activity of p-AP, partly depending on a hydroxyl group, can be decreased by O-methylation but not by O-sulfation. In our previous report, O-sulfated metabolites of naphthol isomers showed relatively higher free radical scavenging activities than their methoxylated (O-methylated) derivatives (Sugahara et al., 2018). From the perspective of chemical structure, APAP, APAP-S, APAPM and acetanilide all contain an acetamido group, while their deacetylated form, p-AP, p-APS, p-APM and aniline, respectively, possess an amino group. It is possible that the higher antioxidant activity of p-AP may depend on the presence of an amino group as an active site, since phenol and phenyl sulfate as the tested chemicals showed much lower activities. Aromatic amines and aminophenols are known to be good antioxidants due to their electron donating substituents, which may produce a resonance stabilized radical (Iwatsuki et al., 1995; Gizdavic-Nikolaidis et al., 2004). The amino group present in these compounds is more effective than the hydroxyl group(s). Subsequently, anisole, a phenol derivative carrying a methyl group at the hydroxyl group, and benzene were tested at 100 μM and 1,000 μM in DPPH radical scavenging assay, and both exhibited virtually no antioxidant activity. These results further support the notion that the aromatic amino group plays an important role in the higher antioxidant capacity of p-APS. Thus, it is likely that O-sulfated chemicals with aromatic amines may retain their antioxidant activities. These results are in agreement with the proposal that O-sulfated metabolites possessing an additional active site may not always be inactivated upon Phase-II sulfate conjugation. Further studies under the physiological settings are warranted in order to clarify further the functional differences between a wide range of various sulfated metabolites and their unsulfated counterparts.

Fig. 4

DPPH radical scavenging activities of APAP, p-AP, and their derivatives. Activities were determined with each compound at 100 μM (gray) and 1,000 μM (black). Data shown represent mean ± S.D. from four experiments and values not sharing a common superscript letter (small at 100 μM, or capital at 1,000 μM) are considered significantly different at P < 0.05. Trolox was used as a control standard. APAP; acetaminophen, APAPS; acetaminophen O-sulfate, APAPM; acetaminophen O-methylate, p-AP; p-aminophenol, p-APS; p-aminophenol O-sulfate, p-APM; p-aminophenol O-methylate, Phe; phenol, PheS; phenyl sulfate.

In the Phase-II pathway, APAP can be conjugated not only by sulfation but also by glucuronidation (Ohashi and Kohno, 2020). Additional experiments using APAP glucuronide indicated that its DPPH radical scavenging activity, 2.85 ± 4.95% at 100 μM and 3.88 ± 1.52% at 1,000 μM, was comparable to that of negative control (0.00 ± 1.99%) (figure not shown). Therefore, it appears conceivable that APAP glucuronide possesses virtually no antioxidant effect compared with unconjugated APAP, possibly due to its more stable electronic state within the molecule. Interestingly, a pilot experiment demonstrated that p-AP glucuronide possessed a weaker but apparent DPPH radical scavenging activity (EC₅₀ 136 ± 16 μM) in comparison with p-AP (ref, EC₅₀ 24.1 μM) (figure not shown).

In conclusion, in this study, we demonstrated that the antioxidant capacity of APAPS was lower than that of APAP, while p-APS showed comparable activity as p-AP. These results support the notion that sulfate conjugation may not always result in inactivation of phenolic compounds due to the structural variations. It will be important to further investigate the physiological relevance of these sulfated metabolites, especially from the pharmacological and/or toxicological perspective. More work is warranted to achieve this goal.

ACKNOWLEDGMENTS

This work was partly supported by Grant-in-Aid for Scientific Research (C) (JSPS KAKENHI Grant Number JP20K05881), Research and Study Program/Project of Tokai University General Research Organization (Kanagawa, Japan).

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

•  Ali,  H.M.,  Abo-Shady,  A.,  Sharaf Eldeen,  H.A.,  Soror,  H.A.,  Shousha,  W.G.,  Abdel-Barry,  O.A. and  Saleh,  A.M. (2013): Structural features, kinetics and SAR study of radical scavenging and antioxidant activities of phenolic and anilinic compounds. Chem. Cent. J., 7, 53.
•  Ali,  H.M. and  Ali,  I.H. (2015): QSAR and mechanisms of radical scavenging activity of phenolic and anilinic compounds using structural, electronic, kinetic, and thermodynamic parameters. Med. Chem. Res., 24, 987-998.
•  Alisi,  M.A.,  Brufani,  M.,  Cazzolla,  N.,  Ceccacci,  F.,  Dragone,  P.,  Felici,  M.,  Furlotti,  G.,  Garofalo,  B.,  Bella,  A.L.,  Lanzalunga,  O.,  Leonelli,  F.,  Bettolo,  R.M.,  Maugeri,  C.,  Migneco,  L.M. and  Russo,  V. (2012): DPPH radical scavenging activity of paracetamol analogues. Tetrahedron, 68, 10180-10187.
•  Allali-Hassani,  A.,  Pan,  P.W.,  Dombrovski,  L.,  Najmanovich,  R.,  Tempel,  W.,  Dong,  A.,  Loppnau,  P.,  Martin,  F.,  Thornton,  J.,  Edwards,  A.M.,  Bochkarev,  A.,  Plotnikov,  A.N.,  Vedadi,  M. and  Arrowsmith,  C.H. (2007): Structural and chemical profiling of the human cytosolic sulfotransferases. PLoS Biol., 5, e97.
•  Aronoff,  D.M.,  Oates,  J.A. and  Boutaud,  O. (2006): New insights into the mechanism of action of acetaminophen: its clinical pharmacologic characteristics reflect its inhibition of the two prostaglandin H2 synthases. Clin. Pharmacol. Ther., 79, 9-19.
•  Bendary,  E.,  Francis,  R.R.,  Ali,  H.M.G.,  Sarwat,  M.I. and  EI  Hady, S. (2013): Antioxidant and structure–activity relationships (SARs) of some phenolic and anilines compounds. Ann. Agric. Sci., 58, 173-181.
•  DeBaun,  J.R.,  Smith,  J.Y.,  Miller,  E.C. and  Miller,  J.A. (1970): Reactivity in vivo of the carcinogen N-hydroxy-2-acetylaminofluorene: increase by sulfate ion. Science, 167, 184-186.
•  Dinis,  T.C.,  Maderia,  V.M. and  Almeida,  L.M. (1994): Action of phenolic derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys., 315, 161-169.
•  Diniz,  J.E.M.,  Borges,  R.S. and  Alves,  C.N. (2004): A DFT study for paracetamol and 3,5-disubstituted analogues. J. Mol. Struct. THEOCHEM, 673, 93-97.
•  Du,  K.,  Ramachandran,  A. and  Jaeschke,  H. (2016): Oxidative stress during acetaminophen hepatotoxicity: Sources, pathophysiological role and therapeutic potential. Redox Biol., 10, 148-156.
•  Dueñas,  M.,  Surco-Laos,  F.,  González-Manzano,  S.,  Gonzalez-Paramas,  A.M. and  Santos-Buelga,  C. (2011): Antioxidant properties of major metabolites of quercetin. Eur. Food Res. Technol., 232, 103-111.
• Falany, C. and Roth, J.A. (1993): Properties of human cytosolic sulfotransferases involved in drug metabolism. In: Human Drug Metabolism: From Molecular Biology to Man (Jeffery, E.H., ed.), pp.101-115, CRC Press, Boca Raton.
•  Gizdavic-Nikolaidis,  M.,  Travas-Sejdic,  J.,  Bowmaker,  G.A.,  Cooney,  R.P. and  Kilmartin,  P.A. (2004): Conducting polymers as free radical scavengers. Synth. Met., 140, 225-232.
•  Gomes,  J.R.B. and  da Silva,  M.A.V.R. (2005): Density functional theory study on the thermodynamic properties of aminophenols. Int. J. Quantum Chem., 101, 860-868.
•  Iwatsuki,  M.,  Komuro,  E. and  Niki,  E. (1995): Antioxidant activities of aminophenols against oxidation of methyl linoleate in solution. Bull. Chem. Soc. Jpn., 68, 620-624.
•  Jevons,  F.R. (1963): Tyrosine O-sulfate in fibrinogen and fibrin. Biochem. J., 89, 621-624.
•  Jóźwiak-Bebenista,  M. and  Nowak,  J.Z. (2014): Paracetamol: mechanism of action, applications and safety concern. Acta Pol. Pharm., 71, 11-23.
•  Morita,  C.,  Taniguchi,  R.,  Yoshida,  M.,  Tokunaga,  Y.,  Kinoshita,  H.,  Ono,  M.,  Kurogi,  K.,  Sakakibara,  Y.,  Suiko,  M. and  Yasuda,  S. (2022): Chemical synthesis of acetaminophen O-sulfate. Proc. Sch. Agric. Tokai Univ., 41, 39-46. (in Japanese)
• Mulder, G.J. and Jakoby, W.B. (1990): Sulfation. In: Conjugation Reactions in Drug Metabolism (Mulder, G.J., ed.), pp.107-161, Taylor and Francis, London.
•  Murakami,  A.,  Kuki,  W.,  Takahashi,  Y.,  Yonei,  H.,  Nakamura,  Y.,  Ohto,  Y.,  Ohigashi,  H. and  Koshimizu,  K. (1997): Auraptene, a citrus coumarin, inhibits 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in ICR mouse skin, possibly through suppression of superoxide generation in leukocytes. Jpn. J. Cancer Res., 88, 443-452.
•  Németh,  T.,  Jankovics,  P.,  Németh-Palotás,  J. and  Koszegi-Szalai,  H. (2008): Determination of paracetamol and its main impurity 4-aminophenol in analgesic preparations by micellar electrokinetic chromatography. J. Pharm. Biomed. Anal., 47, 746-749.
•  Ohashi,  N. and  Kohno,  T. (2020): Analgesic effect of acetaminophen: A review of known and novel mechanisms of action. Front. Pharmacol., 11, 580289.
•  Orhan,  H. and  Sahin,  G. (2001): In vitro effects of NSAIDS and paracetamol on oxidative stress-related parameters of human erythrocytes. Exp. Toxicol. Pathol., 53, 133-140.
•  Rimbach,  G.,  Weinberg,  P.D.,  de Pascual-Teresa,  S.,  Alonso,  M.G.,  Ewins,  B.A.,  Turner,  R.,  Minihane,  A.M.,  Botting,  N.,  Fairley,  B.,  Matsugo,  S.,  Uchida,  Y. and  Cassidy,  A. (2004): Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function. Biochim. Biophys. Acta, 1670, 229-237.
•  Roubalová,  L.,  Purchartová,  K.,  Papoušková,  B.,  Vacek,  J.,  Křen,  V.,  Ulrichová,  J. and  Vrba,  J. (2015): Sulfation modulates the cell uptake, antiradical activity and biological effects of flavonoids in vitro: an examination of quercetin, isoquercitrin and taxifolin. Bioorg. Med. Chem., 23, 5402-5409.
• SCCS (Scientific Committee on Consumer Safety). (2011): Opinion on p-Aminophenol. COLIPA n° A16 (https://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_078.pdf, 2022.3.31 Access)
•  Shintani,  H. (2013): Isolation of neutrophils/assay of O₂^- (superoxide anion radical) generation by cytochrome-c reduction. Pharm. Anal. Acta, 4, 243.
•  Sugahara,  S.,  Fukuhara,  K.,  Tokunaga,  Y.,  Tsutsumi,  S.,  Ueda,  Y.,  Ono,  M.,  Kurogi,  K.,  Sakakibara,  Y.,  Suiko,  M.,  Liu,  M.-C. and  Yasuda,  S. (2018): Radical scavenging effects of 1-naphthol, 2-naphthol, and their sulfate-conjugates. J. Toxicol. Sci., 43, 213-221.
•  Suiko,  M.,  Kurogi,  K.,  Hashiguchi,  T.,  Sakakibara,  Y. and  Liu,  M.-C. (2017): Updated perspectives on the cytosolic sulfotransferases (SULTs) and SULT-mediated sulfation. Biosci. Biotechnol. Biochem., 81, 63-72.
•  Takahashi,  N.,  Ohba,  T.,  Togashi,  S. and  Fukui,  T. (2002): Biological activity of p-methylaminophenol, an essential structural component of N-(4-hydroxyphenyl)retinamide, fenretinide. J. Biochem., 132, 767-774.
•  Tripathy,  D. and  Grammas,  P. (2009): Acetaminophen protects brain endothelial cells against oxidative stress. Microvasc. Res., 77, 289-296.
•  Tsutsumi,  S.,  Tokunaga,  Y.,  Shimizu,  S.,  Kinoshita,  H.,  Ono,  M.,  Kurogi,  K.,  Sakakibara,  Y.,  Suiko,  M.,  Liu,  M.-C. and  Yasuda,  S. (2020a): Investigation of the effects of indoxyl sulfate, a uremic toxin, on the intracellular oxidation level and phagocytic activity using an HL-60-differentiated human macrophage cell model. Biosci. Biotechnol. Biochem., 84, 1023-1029.
•  Tsutsumi,  S.,  Tokunaga,  Y.,  Shimizu,  S.,  Kinoshita,  H.,  Ono,  M.,  Kurogi,  K.,  Sakakibara,  Y.,  Suiko,  M.,  Liu,  M.-C. and  Yasuda,  S. (2020b): Effects of indole and indoxyl on the intracellular oxidation level and phagocytic activity of differentiated HL-60 human macrophage cells. J. Toxicol. Sci., 45, 569-579.
•  Ueda,  Y.,  Matsuda,  Y.,  Murata,  T.,  Hoshi,  Y.,  Kabata,  K.,  Ono,  M.,  Kinoshita,  H.,  Igoshi,  K. and  Yasuda,  S. (2019): Increased phenolic content and antioxidant capacity of the heated leaves of yacon (Smallanthus sonchifolius). Biosci. Biotechnol. Biochem., 83, 2288-2297.
•  Utsumi,  T.,  Klostergaard,  J.,  Akimaru,  K.,  Edashige,  K.,  Sato,  E.F. and  Utsumi,  K. (1992): Modulation of TNF-alpha-priming and stimulation-dependent superoxide generation in human neutrophils by protein kinase inhibitors. Arch. Biochem. Biophys., 294, 271-278.
• Weinshilboum, R. and Otterness, D. (1994): Sulfotransferase enzymes. In: Conjugation-Deconjugation Reactions in Drug Metabolism and Toxicity (Kaufmann, F.C., ed.), pp.45-78, Springer-Verlag, Berlin.
•  Yamamoto,  A.,  Liu,  M.-Y.,  Kurogi,  K.,  Sakakibara,  Y.,  Saeki,  Y.,  Suiko,  M. and  Liu,  M.-C. (2015): Sulphation of acetaminophen by the human cytosolic sulfotransferases: a systematic analysis. J. Biochem., 158, 497-504.