How Does Acetaminophen Function? Metabolite, Electron Transfer

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Chapter 10

How Does Acetaminophen Function? Metabolite, Electron Transfer, Reactive Oxygen Species, Oxidative Stress and COX Peter Kovacic1,* and Ratnasamy Somanathan1,2 1Department

of Chemistry and Biochemistry, San Diego State University, San Diego, California 92182-1030, United States 2Centro de Graduados e Investigación del Instituto Tecnológico de Tijuana, Apdo postal 1166, Tijuana, B.C. Mexico *E-mail: [email protected]

A recent article presents mode of action by acetaminophen as an enigma involving various contributing factors. An alternative approach entails redox cycling by the N-acetyl benzoquinone imine metabolite. The resulting reaction oxygen species can play a role in both therapy and toxicity. The broader ramifications of the unifying mechanism are also addressed, including a multifaceted approach. Commonality with the widely accepted COX theory is treated. Keywords: acetaminophen; N-acetyl benzoquinone imine; electron transfer; reactive oxygen species; oxidative stress; COX; toxicity

Introduction A recent news item titled “The Enigma Pill” deals with action mode of acetaminophen (Fig 1) (Tylenol) (APAP), a widely used sedative and antipyretic (1). A 2013 article states that it is now generally accepted that cyclooxygenase (COX) enzymes play an important role (2). This chapter summarizes the numerous modes of action that have been proposed with contribution to the enigma. Also, focus is centered on an alternative approach involving metabolism, electron transfer (ET), reactive oxygen species (ROS), oxidative stress (OS) and

© 2015 American Chemical Society In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

COX. A leading actor is the N-acetyl benzoquinone imine (NAPQ1) (Fig 2) metabolite, which appears to function as an ET agent that generates ROS-OS.


Figure 1. Acetaminophen (APAP)

Figure 2. NAPQ1

The unifying mechanism applied here is an extension of prior work (3). “The preponderance of bioactive substances, usually as the metabolites, incorporate ET functionalities. We believe these play an important role in physiological responses. The main groups include quinones (or phenolic precursors), metal complexes (or complexors), aromatic nitro compounds (or reduced hydroxylamine and nitroso derivatives), and conjugated imines (or iminium species). The imine category is the main object of our review and is the least well known. Resultant redox cycling is illustrated in Scheme 1. Iminiums are usually better electron acceptors than imines, due partly to positive charge. In vivo redox cycling with oxygen can occur, giving rise to oxidative stress (OS) through generation of ROS, such as hydrogen peroxide, hydroperoxides, alkyl peroxides, and diverse radicals (hydroxyl, alkoxyl, hydroperoxyl, and superoxide) (Scheme 2).

Scheme 1. Redox cycling with superoxide formation 260 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Scheme 2. Other ROS from superoxide In some cases ET results in involvement with normal electrical effects (e.g., in respiration of neurochemistry). Generally, active entities possessing ET groups display reduction potentials in the physiologically responsive range, (i.e., more positive than about -0.5 V). Hence, ET in vivo can occur resulting in production of ROS which can be beneficial in cell signaling at low concentrations, but produce toxic results at high levels. Electron donors consist of phenols, N-heterocycles or disulfides in proteins which produce relatively stable radical cations. ET, ROS and OS have been increasingly implicated in the mode of action of drugs and toxins, (e.g., antiinfective agents (4), anticancer drugs (5), carcinogens (6), reproductive toxins (7), nephrotoxins (8) hepatotoxins (9), cardiovascular toxins (10), nerve toxins (11), mitochondrial toxins (12), abused drugs (13), pulmonary toxins (14), ototoxins (15), and various other categories (16). There is a plethora of experimental evidence supporting the ET-ROS theoretical framework. This evidence includes generation of the common ROS, lipid peroxidation, degradation products of oxidation, depletion of AOs, effect of exogenous AOs, and DNA oxidation and cleavage products, as well as electrochemical data. This comprehensive, unifying mechanism is consistent with the frequent observations that many ET substances display a variety of activities (e.g., multiple-drug properties), as well as toxic effects. It is important to recognize that mode of action in the biodomain is often multifaceted. In addition to the ET-ROS-OS approach, other aspects may pertain, such as, enzyme inhibition, allosteric effects, receptor binding, metabolism and physical factors. A specific example involves protein binding by quinones in which protein nucleophiles, such as amino or thiol, effect conjugate addition.”

Enigma Mode of Action When researchers are asked of knowledge about how APAP works, the responses ranged from “pretty clear” to “poorly” (1). The “mechanism seems messy enough to discourage even the most optimistic scientists”. Various action modes have been proposed with differing degrees of evidence, including chemical messengers associated with inflammation and pain, as well as neurotransmission in the brain and spinal cord (1). Another proposal invokes blockage of prostaglandin formation in the central nervous system (CNS) (17, 18). A focus was on COX inhibition with evidence that this could represent a primary central mechanism. The analgesic activity appears to be prevented by blockage of cannabinoid receptors (19, 20). Serotonin (5-HT) transmission in the CNS may also play a role (21). There is the suggestion that the drug indirectly employs communication systems similar to those of opioids (22). However, acetaminophen is neither an opioid nor an NSAID. 261 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

APAP, following deacetylation, undergoes acylation of the amine by the fatty acid arachidonic acid (23). The bioactive product, possessing enhanced lipid solubility, would be expected to undergo reactions similar to those of APAP. Another proposed mode of action consists of interaction at the brain or spinal cord in APAP-induced antinociception (22).


N-Acetyl p-Benzoquinone Imine (NAPQI) Metabolite There is appreciable literature linking the NAPQI metabolite to physiological activity. Some of the reports relate to the ROS unifying theory. APAP is known to play a prominent role in inducing liver toxicity (24). The toxic metabolite NAPQ1 Is believed to be importantly involved, including effector of apoptosis and depletion of GSH which is commonly an oxidant effect. Increase in GSH peroxides following APAP administration and the beneficial effect of a SOD mimic pointed to generation of ROS. Direct evidence for ROS generation was provided by the flow cytometers. ROS production was also associated with mitochondrial damage characterized by collapse of transmembrane potential. The various lines of evidence point to a role for ROS in APAP-induced liver insult. Another report links APAP hepatotoxicity to lipid peroxidation and covalent binding to macromolecules leading to mitochondrial damage (25). The result is attributed to the NAPQI metabolite via sulfhydryl oxidation or addition. The adverse influences were countered by bicyclol which is believed to elicit a variety of biological effects through its AO and antiinflammatory properties (25, 26). Another report associates NAPQ1 with the toxic side effects of the parent drug (27). Several other articles deal with reaction of NAPQ1 with thiol groups via conjugate addition which is likely involved in quinone binding to protein (25, 28–30). A related example entails reaction of NAPQ1 with proline and the resultant biological ramifications (31). In addition to GSH depletion by the quinone imine, there is a role for reactive nitrogen species in APAP induced toxicity (32). p-Benzoquinone, an electrophile, is also reported as a metabolite (29), capable of functioning in a manner similar to NAPQ1. In relation to basic chemistry, NAPQ1 is a close analog of p-benzoquinone which is well known ET agent (Scheme 3). Uptake of an electron to form a radical anion would be facilitated by the acetyl substituent of NAPQ1 in conjugation resulting in further, favorable delocalization as illustrated in Fig. 3. Important light is cast upon the mechanism by comparison with the α-carbonylimine structure which has been investigated by means of electrochemistry (33). Reduction potentials fell within the range favorable for in vivo ET protonation or hydrogen bonding which form iminium type species enhanced electron affinity. Relationship to physiological activity was discussed. The acetaminophen metabolite is electronically related as a vinylog of the parent α-carbonylimine model. The vinylogous character bestows enhanced favorable stabilization via conjugation in the generated radical anion. The captodative effect is discussed as a beneficial contributing factor.

262 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Scheme 3. p-Benzoquinone redox

Figure 3. Delocalized radical anion of NAPQ1

Toxicity This topic is also treated in other sections. Hepatotoxicity has been extensively investigated and presented as a serious threat in some cases. In addition, the Introduction presents numerous examples entailing application of ET-ROS-OS to toxicity, including that of the liver. There is substantial literature on the subject in more recent years. Hence, it should not be surprising that APAP fits the unifying theme via the ET iminoquinone metabolite leading to ROS-OS. The advese reactions have been associated with OS and NAPQ1 metabolite, including ROS (34). Various systems may be responsible for ROS generation. Studies indicate that APAP and NAPQ1 are the primary ototoxic agents (35). Data indicate involvement of ROS overproduction.



Commonality Involving COX and ET-ROS-OS Mechanisms There is wide acceptance that APAP operates by inhibition of COX enzymes which can function as peroxidases (13). In the process, the drug undergoes oxidation in which it functions as an AO, a common property of phenols (36), e.g., vitamin E. The phenols can also act as pro-oxidants (36), which applies to APAP via metabolism to a iminoquinone resulting in generation of ROS and OS. Oxidation of the drug leads to reduction or inhibition of the COX enzymes. Previously, the two mechanisms were regarded as separate and diverse pathways. This novel perspective reveals that the two are intimately associated with the COX enzyme acting as a peroxidase oxidant of the drug with conversion to the ET imnioquinone metabolite. This represents an expansion of the unifying feature. There has been extensive literature on COX enzymes. A book in 1998 addresses various aspects, including inhibition, mechanism of action, therapy, and drugs, e.g., aspirin (37).

Other Analgesics and Related Drugs Aspirin This related drug is treated in another Chapter in the book. Benzodiazepines (BDZs) The tranquilizers are widely involved in the CNS operating via electrical effects (13). The BDZs increase ionic conductance in connection with synapses and chloride ion channels. Electrical phenomena are observed in membrane neurons. Protonated BDZs display reduction potentials in the physiologically active range. There are correlations involving reduction potentials, structure and drug activity. A prominent member is Valium (Diazepam) (Fig. 4) which is a highly conjugated imine. Theoretical calculations support participation of ET and electronic effects. A report deals with activation of Clozapine to radical metabolites that produce OS and inhibit mitochondrial respiration, apparently by ET.

Figure 4. Valium 264 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Phenobarbital


Incorporation in the unifying theme has been previously addressed (13). Various modes of action are documented, including ion channel blockage, effect on membrane potentials and neurotransmission. An ET mechanism as anticonvulsant is reported, involving electrical phenomena. A key aspect entails aromatic hydroxylation leading to phenol and catechol metabolites (see Fig. 5) which can serve as precursors of ET quinone derivatives. o-Quinone are known to posses favorable reduction potentials making for facile participation in reductions in vivo.

Figure 5. Phenobarbital catechol metabolite Phenytoin The hydantoin is used as an epileptic (13). OS is suggested as being involved in the neurotoxicity. Similar to Phenobarbital, the drug is converted metabolically to phenol and catechol products with subsequent oxidation to an ET o-quinone (Fig. 6). Subsequent binding to protein can occur. The drug can induce oxidative damage to DNA and proteins, apparently from ROS generated by ET redox cycling. Also, an epoxide metabolite has been discussed, which may generate ROS via alkylation of DNA.

Figure 6. Phenytoin o-quinone metabolite 265 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Morphine and Heroin These are among the earliest analgetic abused drugs. Various reports incorporate them within the unifying mechanism (13). With morphine, apoptosis was linked to superoxide, with protection by the thiol AO N-acetylcysteine. Redox cycling with oxygen occurs with production of superoxide and hydrogen peroxide. Since heroin is metabolized to morphine, the above results would also pertain. Levels of lipid peroxides were enhanced in heroin abusers. Relation between toxicity and ROS was reported. Oxidation and peroxidation pointed to the presence of ROS. There are other drugs related to morphine which may operate in a similar manner mechanistically.

Broad Ramifications of the Unifying Theme The unifying theme of ET-ROS-OS emulates related aspects common in nature. For example, in living systems, one finds common structural threads in protein (amide), carbohydrate (acetal) and lipids (ester), as well as in reaction systems, such as enzymes (oxidases, esterases, hydrolases), electron transfer and electrochemistry. The ET-ROS-OS theme has found widespread application as exemplified in the Introduction. Many antibacterial agents appear to copy the immune systems in what is described as phagomimetic action (38). At high levels, ROS are commonly found as a unifying feature in toxicity, being associated with a wide variety of adverse reactions. It is notable that ROS at low concentrations can exert beneficial effects in a unifying aspect based on cell signaling (39). Another unifying aspect that lauds credence is the beneficial effect of AOs on deleterious processes supporting the common involvement of toxic ROS. Participation of ET agents (see Introduction) in ROS formation is a neglected aspect. Another case of unification applies to action mechanism of abused drugs, including banned ones and legal sedatives, such as APAP, aspirin and barbiturates (13).

Abbreviations APAP= Acetaminophen; NAPQ1= N-acetyl benzoquinone imine; ET= electron transfer; ROS= reactive oxygen species; OS= oxidative stress; AO= antioxidant; COX= cyclooxygenase; CNS= central nervous system

Acknowledgments Editorial assistance by Thelma Chavez and Bianca Aviña is acknowledged.

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How Does Acetaminophen Function? Metabolite, Electron Transfer

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