Effectiveness of antioxidant treatments on cytochrome P450 2E1 (CYP2E1) activity after alcohol exposure in humans and in vitro models: A systematic review

ABSTRACT This systematic review aimed to describe the effects of antioxidant treatments after alcohol exposure in humans and in vitro models. A systematic review of quantitative studies to identify the effectiveness of antioxidant treatments on CYP2E1 activity after alcohol consumption. Multiple databases were searched from 2010 to May 2020 for studies in English, using MeSH terms and text words relating to antioxidant treatments, CYP2E1 and, alcohol consumption. The protocol is registered on PROSPERO CRD42021226123. Study quality was assessed using the NICE methodology. The review is reported according to PRISMA. A total of eight original articles were analyzed. All papers collected and reported quantitative data. Antioxidant treatments could be beneficial after alcohol exposure to improve oxidative stress by decreasing CYP2E1 activity and increasing GSH levels. Our results suggest that antioxidant treatments could be beneficial and effective during ethanol exposure in human or in vitro models.


Introduction
It has been described that excessive consumption of alcohol promotes the pathogenesis of many fatal diseases, including cancer, liver cirrhosis cardiovascular diseases, diabetes, and neuropsychiatric disorders. [1][2][3] In effect, causing liver injury in humans due to increased oxidative stress has been shown. [4,5] Ethanol metabolism produces alcoholic fatty liver, alcoholic hepatitis, or cirrhosis. [6,7] The major pathway of oxidative metabolism of ethanol in the liver involves alcohol dehydrogenase (ADH) present in the cytosol. [8] Both ADH, CYP450 2E1 (CYP2E1), and catalase (CAT) are responsible for the oxidative metabolism of ethanol, while via non-oxidative metabolism, fatty acid ethyl esters (FAEE) synthase produces FAEEs. [9] During alcohol intoxication, the CYP2E1-dependent microsomal monooxygenase system and the microsomal respiratory chain are the main sources of reactive oxygen species (ROS) within the hepatocytes. Cytochrome P450 2E1 is of special interest because of its ability to metabolize and activate numerous hepatotoxic substrates in the liver such as ethanol, carbon tetrachloride, acetaminophen, and N-nitroso dimethylamine, to more toxic products. [10] In this sense, induction of cytochrome CYP2E1 by ethanol appears to be one of the central pathways by which ethanol generates

Population/setting
People aged between 15 and 80 years old, living in the community; including healthy participants with no pre-conditions for later ill health such as diabetes, insulin resistance, high blood pressure, or high cholesterol. People on medication or studies that primarily focused on populations with ill health e.g. stroke, coronary heart disease, and mental health conditions were excluded. For in vitro studies, HepG2 or SK-Hep-1 cells were exposed to ethanol and protective molecules to evaluate their protective capacity on alcohol-induced liver disease.

Quality assessment/risk of bias
Methodological quality was assessed using the NICE methodology for quantitative studies by one reviewer and checked for accuracy by a second reviewer. [25] Differences between reviewers were resolved by discussion. No studies were excluded based on quality.

Data extraction and synthesis
Data relating to the population and study characteristics of the included studies were extracted by one reviewer (CC) and checked by another reviewer (DC) ( Table 1). To identify information relevant to variables involved in the effectiveness of antioxidant treatments on CYP2E1 activity after alcohol exposure, one researcher (CS) examined the results and discussion sections of each text, line by line, to identify data relating to antioxidant treatments, CYP2E1 activity, and alcohol exposure. Interpretation and concepts of the study authors were also included if they were developed from the original data. The text was then further examined and reorganized into themes ( Table 2). Further interpretation and analysis about possible treatments on CYP2E1 activity after alcohol exposure in identified from the texts. Figure 1 illustrates the flow chart for the study selection process from eight papers were identified. [26][27][28][29][30][31][32][33] A summary of included studies, and the populations/setting and context in which they were conducted is shown in Table 1.

Description of included studies
From the primary studies, four papers were conducted in Korea, two in China, and two in Indonesia. A total of eight original articles were analyzed. All papers collected and reported quantitative data through randomized controlled trials (Table 1). Six studies were realized in cell culture (in vitro models); [26][27][28][29]31,32] and two in human models. [30,33] Five studies were conducted in HepG2 cells, [26,27,29,31,32] one in SK-Hep-1 cells [28] and two in healthy adults. [30,33] Details of antioxidant treatment of each study where available are shown in Table 1. Two studies included S. quelpaertensis Nakai leaf extract, [31,32] one fermented sea tangle, [27] one baicalin, [29] one corn-peptides, [30] one use phytochemicals, [28] one in reduced-glutathione (GSH) [26 ; ] while one including a standardized polyphenolic extract of clove buds (Clovinol). [33] Quality assessment Quality assessment results and assessment criteria of individual studies are shown in Supplemental Table 1. Overall, the quality of studies was generally high or moderate for internal and external validity. No studies were excluded based on quality. Clovinol administration was found to inhibit the increase in the CRP and IL-6 levels significantly, and; survey of hangover severity: he mean overall hangover severity score for the Clovinol group was significantly lower than that for the placebo group with a reduction of 55.  Clovinol administration was found to maintain the baseline level of SOD with significant inhibition (92.5%; p < .001) in SOD depletion as compared with placebo.
Mammen et al. [33]a,c Clovinol administration inhibits the decrease in GSH levels with an average improvement of 34.5% compared to placebo.
Mammen et al. [33]a,c The level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation of the oxidative stress.
Chandrasekaran et al. [26]a,b The SQEA, rich in phenolic acids such as p-coumaric acid and flavonoids, particularly myristin, showed a hepatoprotective effect against EtOH (400 mM) in HepG2 cells.
Madushani Herath et al. [31]a,b Baicalin inhibits oxidative stress induced by the combination of alcohol and iron, mainly by chelation of iron.
Xu et al. [29]a,b Co-treatment of cells with ethanol and Qu, Ct, Cf and Py significantly inhibited oxidative ethanol metabolism-induced cytotoxicity by blocking ROS production.
Lee et al. [28]a,b Ct was very effective at recovering levels of intracellular ROS and MMP altered by ethanol treatment.
Lee et al. [28]a,b Cell viability Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells.
Chandrasekaran et al. [26]a,b Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells.
Madushani Herath et al. [32]a,b Co-treatment of cells with 100 mM ethanol and Qu, Ct, Cf or Py (1 or 5 μM) significantly increased proliferation of SK-Hep-1 cells to a range of 93-106%.
Madushani Herath et al. [31]a,b Pretreatment with Ba markedly decreased apoptosis (p < .05) in the EtOH-Fc-Ba group (25 µM) and the EtOH-Fc-Ba group (50 µM) compared to the EtOH-Fc group. The effect of Ba was concentration dependent.
Xu et al. [29]a,b SQEE80 treatments (250 and 500 μg/mL) decreased the apoptotic DNA in sub-G1 groups in ethanol-stimulated cells by 62.2% and 55.5% compared to those without SQEE80 treatment.
Madushani Herath et al. [32]a,b Cellular damage Clovinol administration reduced the extent of lipid peroxidation by 25% and helped it to remain in the normal range at all times after alcohol ingestion.
Mammen et al. [33]a,c Clovinol administration was found to maintain the baseline 8-isoprostane levels.
Mammen et al. [33]a,c Pretreatment with Ba reduced the level of ROS induced by EtOH-Fc in the EtOH-Fc-Ba group (25 µM) and the EtOH-Fc-Ba group (50 µM) after 1 h of EtOH treatment.
Xu et al. [29]a,b ROS production was decreased in ethanol stimulated HepG2 cells treated with SQEE80 compared to those without SQEE80 treatment. The reduction of ROS production was 22.9% (p < .05) and 37.7% (p < .05) with 250 μg/mL and 500 μg/mL of SQEE80 treatments, respectively.
Madushani Herath et al. [32]a,b Treatment with FSTJ at a concentration of 25 μg/mL increased GSH levels to 67.08%. Kang et al. [27]a,b In ethanol-treated cells, GSH content decreased to 44.35% of control (ethanol-untreated cell) value.
Lee et al. [28]a,b Lipid hydroperoxide levels were decreased by 14-18% with the addition of Qu, Ct, Cf and Py.
Lee et al. [28]a,b The level of reduced glutathione increased to a range of 6.61-6.71 nmol/μg protein with the addition of Qu, Ct, Cf and Py.
Lee et al. [28]a,b Protein expression Incubating VL-17A cells with 100 mM ethanol for 72 h resulted in a 2.6-fold increase in the level of CYP2E1 when compared to untreated VL-17A cells.
Chandrasekaran et al. [26]a,b The expression of CYP2E1 was completely inhibited in cells treated with FSTJ at a concentration of 25 µg/mL.
Kang et al. [27]a,b Biochemical parameters SQEE80 treatment with 250 µg/ml (p < .05) significantly increased catalase activity by 1.7-fold in ethanol-stimulated HepG2 cells compared to those without SQEE80 treatment. In addition, treatment with both doses of SQEE80 in ethanol stimulated cells significantly increased liver GPX-1 activity (p < .05) compared to cells without SQEE80 treatment.

Relation between antioxidants and alcohol exposure
Processes that protect against alcohol-induced deleterious effects include antioxidant enzymes, [19] such as superoxide dismutase, catalase, glutathione, and glutathione reductase [18,19 ; ] the antioxidant GSH, metal-binding proteins, vitamins C and E; the carotenoids that deactivate the free radicals [34,35 ; ]

Measurement Cell or metabolic changes References
Supplementation with CPs decreased ALT and AST, whereas participants who received WP or placebo showed no significant changes.
Wu et al. [30]a,b Decrease in the blood acetaldehyde concentrations upon Clovinol treatment, as compared with control.
Mammen et al. [33]  and other antioxidants such cinnamic acid and syringic acid that improving the inflammatory and oxidative damage produced by ethanol. [36] Few studies in humans have described the role of antioxidants on alcohol intake. [30,33] In effect, some variables involved during alcohol exposure, such as time of exposure, age, dose, ethnicity, or type of alcohol consumed could hinder the process. Meanwhile, in vitro assays allow controlling the studied substance, without subjecting humans or animals to the possible side effects or toxicity of a new drug. The possible antioxidant strategies to use on CYP2E1 activity after alcohol exposure are shown in Figure 2.

Antioxidant activity
The antioxidant effects were indicated in almost all articles (Table 2). However, if the antioxidant activity was not measured; others measurements such as cell viability, cellular damage, protein expression, or enzymatic analysis were analyzed to evaluate it. [27,30,32] The changes on GSH enzyme activity to evaluate antioxidant effects were often used. [26,31,32] Although one study used to measure more than one enzyme involved in the antioxidant activity to evaluate antioxidant effects such as catalase and glutathione peroxidase, [28] just one study used the ABTS radical cation to measure antioxidant activity. [29] Cell viability Viability levels and/or proliferation rates of cells are good indicators of cell health. In effect, physical and chemical agents can affect cell health and metabolism. Thus, some in vitro studies have shown that cells treated with Ethyl acetate fraction of 80% ethanol extract of Sasa quelpaertensis Nakai leaves extract (SQEA) or 80% ethanol extract of Sasa quelpaertensis (SQEE80) have enhanced cell viability due to attenuated oxidative stress by decreased expression of CYP2E1. [31,32] In the same way, the cotreatment of cells with ethanol and phytochemicals shown an increased proliferation of SK-Hep-1 cells due to suppression ROS and lipid hydroperoxide (LPO) production, protein oxidation, [28] and loss of mitochondrial membrane potential (MMP) and increased CAT and glutathione peroxidase-1 (GPX-1) activities during 4-methylpyrazole (4-MP) induced non-oxidative metabolism of ethanol.

Apoptosis
Flow cytometry is one of the most popular and versatile applications for studying apoptosis. In effect, antioxidants' effects on ethanol-induced free radicals and hepatic DNA strand breaks have been studied. In this sense, the treatment with SQEA or SQEE80 decreased the apoptotic DNA fraction confirming their hepatoprotective effect against ethanol-induced cell death. [31,32] Likewise, pretreatment with Baicalin has shown decreased apoptosis levels on groups treated with ethanol and 25 µM and 50 µM of Baicalin, due to a decreased accumulation of ROS and prevention on HepG2 cells from protein oxidation damage and apoptosis. [29]

Cellular damage
Reactive oxygen species plays an important role in alcohol-induced cell injury and disease; and it can cause various cellular injuries, such as DNA damage, lipid peroxidation, and protein modification. However, antioxidant exposure could decrease levels of lipid peroxidation and ROS. In this sense, Clovinol -standardized polyphenolic extract of clove buds-administration reduced lipid peroxidation and 8-isoprostane, a marker of oxidative stress, after alcohol ingestion. [33] While Baicalin and SQEE80 reduced the levels of ROS after ethanol treatment. [29,32] Likewise, there are non-enzymatic defense mechanisms that include: GSH, which is almost exclusively in its reduced form and detoxifies the ROS produced on the mitochondrial electron transport chain. Thus, compounds as Fermented Sea Tangle Juice (FSTJ) and phytochemicals, such as quercetin, catechin, caffeic acid, and phytic acid; have shown increased GSH levels after ethanol exposure, [27,28] promoting increased protection of this organelle to oxidative damage.

Protein expression
Induction of CYP2E1 is one of the central pathways by which ethanol generates oxidative stress, where incubating VL-17A cells with ethanol results in an increase in CYP2E1 levels. [26] Likewise, studies have described the expression of CYP2E1 was completely inhibited when cells were treated with FSTJ. [27]

Biochemical parameters
Antioxidants promote hepatoprotective effects and improve the activities of the enzymes that are associated with alcoholic liver disease. In this sense, SQEE80 has been shown to increase CAT and GPX-1 activity in ethanol-stimulated HepG2 cells compared to those without SQEE80 treatment. [32] Clove buds (Syzygium aromaticum L.), a popular kitchen spice, have been identified as one of the richest sources of antioxidants polyphenols among the various fruits, vegetables, and edible herbs. Thus, Clovinol has been shown to decrease acetaldehyde levels and inhibited the alcohol-induced elevation in IL-6 through its antioxidant and anti-inflammatory potential. [33] Likewise, Corn Peptides (CPs) -a novel food prepared from corn gluten meal-have shown beneficial effects on early alcoholic liver disease and acute alcoholic injury in mice. In effect, CPs decreased serum activities of ALT and AST. [30]

Discussion
The systematic review collates and synthesizes evidence from eight quantitative studies relating different kinds of antioxidant treatments and CYP2E1 activity after alcohol exposure in humans and in vitro models.

Summary of key findings and interpretation
It is important to note that the effects produced by alcohol can be counteracted by antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase as well as non-enzymatic mechanisms. [18,19] These non-enzymatic defense mechanisms include: GSH; metal-binding proteins; vitamins C and E; the carotenoids that deactivate the free radicals [34,35 ; ] and other antioxidants such cinnamic acid and syringic acid that suppress liver activity and/or protein expression of CYP2E1, ADH, NADPH oxidase, nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-kB), improving the inflammatory and oxidative damage produced by ethanol. [36] In addition, baicalin can alleviate ethanol-induced liver damage in rats, probably due to its antioxidant, anti-inflammatory properties, and the activation of Shh, which plays an important role in the morphogenesis of tissues and liver repair. [15,37] In this sense, in vitro studies on HepG2 cells have shown that SQEA and SQEE80 enhance cellular growth rates. [31,32] While, phytochemicals treatment has shown an increased proliferation on SK-Hep-1 cells. [28] Thus, cell viability has been enhanced through a decreased oxidative stress and CYP2E1 expression, suppression of ROS and LPO production, protein oxidation, loss of MMP, and increased CAT and GPX-1 activities; suggesting that SQEA, SQEE80, and phytochemicals, such as quercetin, catechin, caffeic acid, and phytic acid; have a direct action on oxidative and non-oxidative metabolism of ethanol.
It has been reported that ethanol promotes the formation of ROS within the mitochondria and reduces the concentration of GSH, promoting increased susceptibility of this organelle to oxidative damage. [12,38] Also, ROS can interact with lipids, proteins, and DNA in a process called peroxidation, leading to the production of malondialdehyde (MDA) and 4-hydroxynonenal (HNE). [39] However, previous studies have described that exposure to antioxidants as Clovinol, Baicalin, SQEE80, FSTJ, and phytochemicals as quercetin, catechin, caffeic acid, and phytic acid; could decrease levels of lipid peroxidation and ROS. [27][28][29]32,33] In effect, Clovinol reduces lipid peroxidation and 8-isoprostane in adult males after binge drinking, [33] whereas Baicalin and SQEE80 reduced the levels of ROS after ethanol treatment on HepG2 cells. [29,32] Likewise, compounds as FSTJ and phytochemicals increase GSH levels after ethanol exposure on HepG2 and SK-Hep-1 cells, respectively. [27,28] These findings support previous hypotheses, suggesting a decreased oxidative stress and increased protection to oxidative damage after antioxidant exposure.
Peroxidation increases the release of cytochrome c, as a result of the increase in mitochondrial permeability, promoting apoptosis. [40][41][42] However, the treatment with SQEA or SQEE80 decreased the apoptotic DNA fraction. [31,32] Likewise, Baicalin has shown decreased apoptosis levels. [29] These results confirming the protective effects of SQEA, SQEE80, and Baicalin against ethanol-induced cell death, probably due to a decreased accumulation of ROS and prevention from protein oxidation damage and apoptosis.
Biochemical tests can be useful in determining the toxic effects and health wellness. [43] It has been described that hepatic damage could be observed after 4 and 12 weeks of ethanol treatment, which is evidenced by higher levels of hepatic enzyme markers and malondialdehyde. [44] For alcoholics, abnormal values for two or more of the five parameters Alanine Aminotransferase (ALAT), Aspartate Aminotransferase (ASAT), gamma-glutamyl transferase (GGT), and creatinine gave a diagnostics sensitivity of 85% and a diagnostic specificity of 64%. Likewise, elevated ALAT and ASAT levels have been described as a general indicator of tissue and organ damage caused by alcohol, viruses, infections, drugs, or toxins. [45] In this sense, CPs have decreased serum activities of ALAT and ASAT promoting beneficial effects on early alcoholic liver disease and acute alcoholic injury. [30] In addition, antioxidant enzymes such CAT and GPX-1 increase their activities after SQEE80 exposure, enhanced alcohol oxidation. [32] Ethanol-induced oxidative stress is the result of the combined impairment of antioxidant defenses and the production of reactive oxygen species by the mitochondrial electron transport chain, the alcohol-inducible CYP2E1, and activated phagocytes. Currently, it is known that antioxidants can be used as a therapeutic strategy in various pathologies, including ethanol consumption. It has been found the activation of stellate pancreatic cells is inhibited when they are incubated with antioxidants. [46] Therefore, antioxidants described previously may represent a potential therapeutic strategy for damage caused by alcohol.

Scope and limitations
The goal of this study was to seek an explanation that relates antioxidant treatments and CYP2E1 activity after alcohol exposure. However, the lack of data linking CYP2E1 and alcohol dehydrogenase activities/expressions with antioxidants therapies remains a limitation of this study and needs to be addressed in future studies. Unfortunately, the results provide little support for this notion. At best, we could only discern a trend toward enhanced enzymatic activities/expressions in most of studies with significant improvement in those who had antioxidants exposure. Whereas in light of these data, it appears that antioxidants exposure, did significantly improve oxidative stress, cell viability, DNA damage, and biochemical parameters after alcohol exposure in humans, HepG2, and SK-Hep-1 cells.
Our systematic review excluded animal studies, due to different variables involves such as nutritional status; type of food ingested, genetic factors, environmental factors such as animal facility; and different amounts of daily ethanol intake or antioxidants, which could influence or interfere with the interpretation of the data related to the physiological response of ethanol consumption and antioxidant treatment.

Conclusion
Our results suggest that antioxidant treatments could be beneficial and effective during ethanol exposure in human or in vitro models due to decreasing CYP2E1 activity and apoptosis levels and; an increase in GSH levels and cell proliferation. However, despite all the advances that have been made in clarifying effects caused by antioxidants supplementation, future studies should clarify more efficiently the relationship between ethanol consumption, antioxidant treatments, and defense mechanisms against oxidative stress; including enzymatic and non-enzymatic mechanisms using specific cell lines or clinical trials.

Disclosure statement
No potential competing interest was reported by the authors.

Funding
This work was supported by the Universidad de La Frontera under Grant DI20-0003.

Author contributions
CS and VS designed this study; DC and CC supervised the study; CS, DC, CC, and VS conducted the literature searches, data extraction, and independent search and reviewing; CS and VS prepared a first draft of the manuscript; and CS, DC, CC, and VS finalized it.