APAP is a commonly used analgesic found in mono and combination formulations and thus can be easily overdosed, especially with improper use or duplicate therapy. This explains why it is a common cause of acute liver injury ^[1][2][3][37,38,39]. Further, the COVID-19 pandemic has amplified reports of APAP drug-induced liver injury, as it is used for managing COVID-19 infection-related symptoms ^[4][40]. Although the majority of APAP is quickly detoxified, a small amount of APAP metabolite, NAPQI, binds to GSH or proteins. While binding of NAPQI to GSH leads to phase II metabolic clearance, its binding to protein causes loss of protein functions leading to necrosis or apoptosis ^[5][6][41,42]. APAP-induced liver injury significantly increases reactive oxygen species (ROS), hepatic malondialdehyde (MDA), serum aspartate transaminase, alanine transaminase, and lactate dehydrogenase levels ^[7][8][43,44]. Approximately 90% of APAP is excreted by sulfation and glucuronidation pathways, and 5–10% is removed in the form of NAPQI ^[1][9][37,45]. In obese COVID-19 patients with metabolic dysfunction-associated fatty liver disease (MAFLD), Ferron et. al., reported increased activity of CYP2E1, thus creating another complexity in the treatment regimen of these patients leading to an increased risk of drug-induced liver injury ^[10][46].

A study conducted by Licong et al. found that fisetin (FST), a flavonoid, reverses APAP-induced toxicity and reduces ROS in human fetal hepatocytes (L-02 cells) and male C57 mice ^[11][47]. The Jiang group studied APAP-induced liver injury in murine models that were treated intragastrically with Artemisia (Eucalyptol), an essential oil. Eucalyptol, an inhibitor of Kelch-like ECH-associated protein 1 (Keap1), activates nuclear factor erythroid 2-related factor 2 (Nrf2) and inhibits CYP2E1 activity leading to the suppression of NAPQI formation ^[2][7][38,43]. Another natural compound, shikonin (nutraceutical from the roots of Lithospermum erythrorhizon plants) in the study by Guo et al., was injected intraperitoneally to investigate its effects on APAP-induced toxicity in alpha mouse liver 12 cells and Balb/c mice ^[8][44]. Moreover, the anti-inflammatory and antioxidant properties of shikonin decreased interleukins (IL-6 and IL-1β), tumor necrosis factor-α (TNF-α), and CYP2E1 mRNA expression ^[8][44]. When APAP-challenged human hepatocellular carcinoma cells (HepG2) were treated with red betel leaf extract (from Indonesia) at 25 and 100 μg/mL, Ginting et al., showed hepatoprotective effects with elevated CYP2E1 and glutathione peroxidase gene expression ^[12][48]. Choi et al. investigated the effects of the natural alkaloid, rutaecarpine, on APAP-induced liver injury in mice ^[13][49]. Toxicity in mice was induced via intraperitoneal injection of APAP followed by oral administration of rutaecarpine; it decreased CYP2E1 protein expression and inhibited the expression of inflammatory cytokines by reducing nuclear factor kappa B cell (NF-κB) expression. Furthermore, diallyl disulfide (DADS) was used to suppress APAP-induced chronic liver injury by inhibiting CYP2E1 protein expression, anti-inflammatory, and antiapoptotic effects by inhibition of NF-κB, thus demonstrates its protective effects ^[14][50].

The metabolism of alcohol by hepatic CYP2E1 leads to ROS production, consequently generates damaging toxic effects in the liver through mitochondrial dysfunction, DNA damage, and lipid peroxidation in a chronic-binge mice model ^[15][51]. Enhanced superoxide and H₂O₂ production are mainly due to poor coupling of CYP2E1 with NADPH-cytochrome P450 reductase (CPR) ^[16][52]. ROS influence posttranscriptional modifications through small ubiquitin-like modifications that covalently attach to epsilon-amino groups of lysine residues and modulate protein stability, activity, and localization ^[17][53]. To prevent inflammatory and oxidant responses seen in alcohol-induced liver injury, Han et al. administered astaxanthin, a xanthophyll carotenoid compound, to mice, which blocked STAT3, reduced ROS and decreased CYP2E1 protein expression ^[15][51]. HepG2 cells were pretreated with gastrodin for 4 h prior to ethanol treatment in a study by Zhang et al. The authors reported nullification of ethanol-induced toxicity and apoptosis with gastrodin treatment. Furthermore, in mice with ethanol-induced acute liver injury, gastrodin decreased the enhanced alcohol-mediated induction of both protein and enzyme activity of ADH and CYP2E1 ^[18][54]. In an intragastric ethanol-fed mouse model, ubiquitin-conjugation enzyme 9 was expressed in the liver; silencing this enzyme by siRNA lowered CYP2E1 mRNA and protein expression and prevented ROS production involved in alcoholic liver disease (ALD) ^[17][53]. In the Nagappan et al., study with chronic ethanol-fed mice, the use of cryptotanshinone from Salivamiltiorrhiza Bunge plants protected hepatocytes from ethanol-induced acute liver injury by inhibiting oxidative stress and lipogenesis ^[19][55]. Cryptotanshinone conferred its hepatoprotective effects through the activation of the AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1) as well as Nrf2 and the inhibition of CYP2E1 pathways. Recently, Avila and coworkers discussed the benefits of inhibiting overexpressed CYP2E1 protein levels for the treatment of ALD ^[20][56]. In mouse models, pharmacological inhibition of CYP2E1 with clomethiazole and genetic deletion of CYP2E1 partly prevented ALD ^[21][22][57,58]. In contrast, CYP2E1 overexpression enhanced the severity of ALD, suggesting the detrimental role of CYP2E1 activity in alcohol-induced liver damage ^[23][59].

The N-terminal signal of CYP2E1 protein regulates its targeting to mitochondria ^[24][60]. CYP2E1 N-terminal signal variant W23R/W30R preferentially targets CYP2E1 to mitochondria and poorly targets it to the endoplasmic reticulum ^[25][61]. Mitochondrial CYP2E1 stimulates significantly higher alcohol-induced ROS generation and induces cell injury in comparison to microsomal CYP2E1 ^[25][61]. Further, previous investigations have shown that mitochondrial CYP2E1 promotes direct damage to mitochondrial cytochrome c oxidase, which can be rescued with mitochondrial antioxidant treatment ^[25][26][61,62], suggesting the role of mitochondrial CYP2E1 in alcohol-induced mitochondrial ROS production and hepatotoxicity.

Multi-drug combinations are given to increase the therapeutic efficacy and success of drugs in treatment regimens of different diseases and comorbidities. However, these same drug combinations can also lead to unfavorable outcomes such as toxicity, treatment failure, or adverse drug interactions ^[27][28][63,64]. Drug-drug interactions (DDI) occur when a drug inhibits or induces the level of a cytochrome CYP enzyme, especially the major drug-metabolic CYP3A4, which causes an alteration in the metabolism of another drug ^[29][30][31][65,66,67]. Inhibition of CYP enzymes can cause decreased metabolism of other substrates leading to increased drug plasma concentration and eventually drug-induced toxicity ^[32][33][68,69]. Induction of CYP enzymes by a drug causes increased metabolism of other substrates leading to suboptimal plasma drug concentration or elevated levels of drug metabolites, which can lead to metabolite-induced toxicity and reduced drug efficacy ^[32][68].

A study conducted by Liu et al. on rats, using APAP and roxithromycin in combination, showed the induction of CYP2E1 mRNA and protein, enhanced levels of NAD(P)H, quinone oxidoreductase 1 (NQO1), TNFα, malondialdehyde, and lower mRNA and protein expression of glutathione peroxidase and superoxide dismutase (SOD) ^[27][63]. They also showed an increased mRNA and protein expression of CYP2E1 resulting in oxidative stress-mediated liver damage. In another study with 10-week-old C57BL/6J mice, the co-administration of valproic acid (VPA) and APAP showed an increase in severity of liver damage consistent with high expression of CYP2E1 mRNA, the presence of NAPQI-protein, and depletion of glutathione in the liver ^[34][70]. This study also revealed upregulated expression of steroidogenic acute regulatory protein 1 (STARD1) with endoplasmic reticulum (ER) stress-causing hepatotoxicity mediated by phosphorylation of c-Jun N-terminal kinase (JNK1/2) and SH3BP5 (SH3 domain-binding protein 5 also called SAB). Furthermore, administration of N-acetylcysteine (NAC; 2.5 mmol/kg IP) demonstrated protection against VPA- and APAP-induced liver toxicity by preventing ER stress and STARD1 induction. Cepharanthine, a biscoclaurine alkaloid, has been shown to prevent DDI via CYP2E1 in vitro human liver microsomes ^[35][71]. Anemarsaponin BII (ABII) is an active saponin and competitive inhibitor of CYP2E1 activity with half-maximal inhibitory concentration (IC50) and dissociation constant (Ki) values of 19.72 μM and 9.82 μM, respectively, suggesting a DDI between ABII and drugs metabolized by CYP2E1 ^[36][72]. A study conducted on rats and hepatic cells revealed that anti-tuberculosis drugs, such as rifampicin and isoniazid, induce liver injury by regulating the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome and enhancing CYP2E1 mRNA expression ^[37][73].

Heavy alcohol consumption with other drugs leads to liver damage stemming from the generation of superoxide anion radicals and hydrogen peroxide after CYP2E1 metabolism. ^[38][74]. In alcohol-drug interactions, CYP2E1 is regulated by NF-κB, which activates other factors involved in liver injuries, such as inducible nitric oxide synthase, TNF-α, and IL-1β ^[39][75]. Chlormethiazole, a sedative drug, and certain phytochemicals (phenethyl isothiocyanate and sulforaphane), are used as potent inhibitors of CYP2E1 for protection and treatment of liver damage in alcohol consumers ^[40][76]. A study by Jiang et al. in mice showed that compounds such as isoquercetin (50 mg/kg), hyperoxide (50 mg/kg), 3-hydroxyphenyl acetic (50 mg/kg), 4-hydroxyphenyl acetic acid (50 mg/kg), and 3,4-hydroxyphenyl acetic acid (50 mg/kg) inhibit the protein expression of CYP2E1 and protect the liver from alcohol-induced (50%, 15 mL/kg) and APAP-induced (300 mg/kg) liver damage ^[41][77]. In chronic alcohol-drinking rodent models, simulating liver injury, the administration of CYP2E1 inhibitors chlormethiazole, phenethyl isothiocyanate, and diallyl sulfide (DAS) reduced lipid peroxidation and alcohol-induced oxidative stress ^[38][42][74,78].