CYP2E1 Activity Research Articles

Artemisia Argyi essential oil ameliorates acetaminophen-induced hepatotoxicity via CYP2E1 and γ-glutamyl cycle reprogramming

BackgroundThe hepatotoxicity induced by acetaminophen (APAP), a commonly used antipyretic, analgesic and anti-inflammatory drug in clinical practice, has received accumulated attention. Artemisia argyi essential oil (AAEO), a volatile oil component extracted from traditional Chinese medicine Artemisia argyi H.Lév. & Vaniot, has great hepatoprotective effects. However, the potential role of AAEO in APAP-induced hepatotoxicity has not been characterized. The present study aimed to investigate the effects of AAEO on hepatic metabolic changes in mice exposed to APAP. MethodsIn this study, 300.00 mg/kg acetaminophen was used to establish liver injury model in C57BL/6 J mice. Hepatoprotective effect of AAEO on APAP-induced hepatotoxicity in mice was investigated by detecting liver function enzymes and histopathological examination. Secondly, UPLC-MS/MS was used to analyze the to analyze the small molecule metabolites and metabolic pathways induced by AAEO treatment; In addition, the effect of AAEO on APAP-induced oxidative stress and inflammation were evaluated by detecting the levels of glutathione peroxidase 4, malondialdehyde, reactive oxygen species and inflammatory factors. Finally, the active components of AAEO were preliminarily screened by cellular assays. The hepatoprotective effect of AAEO against APAP-induced hepatotoxicity was examined through the Western blotting, after the CYP2E1 gene was knocked down in AML12 cells by siRNA transfection. ResultsCompared with the APAP group, AAEO could reduce the abnormal increase in the levels of liver function enzymes caused by APAP. AAEO could enhance the antioxidant capacity by down-regulating the biosynthesis pathway of unsaturated fatty acids and promoting the activity of antioxidant enzymes SOD and CAT in liver tissue induced by APAP. Our study revealed that AAEO promoted GSH synthesis and covalently combined to form APAP-GSH conjugates to reduce the accumulation of APAP in liver tissue. In addition, the chemical constituents in AAEO were analyzed by GC–MS/MS, and it was determined to identify that dihydro-beta-ionone and (-)-verbenone in AAEO might have a significant protective effect on hepatocyte survival after APAP exposure. Further studies on the hepatoprotective mechanism of AAEO indicated that it might reduce the production of toxic metabolites by regulating CYP2E1 levels. ConclusionAAEO exerted hepatoprotective effects on acetaminophen-induced hepatotoxicity in mice via regulating the activity of CYP2E1 and regulating the γ-glutamyl cycle pathway.

The impact of freeze-dried Baiyedancong-Oolong tea aqueous extract containing bioactive compounds on the activities of CYP450 enzymes, the transport capabilities of P-gp and OATs, and transcription levels in mice.

In this study, (-)-epigallocatechin gallate (EGCG) and caffeine extracted from freeze-dried autumn Baiyedancong Oolong tea (FBOT) were orally administered to mice for 7 consecutive days to explore the effects of BOT and its bioactive compounds on the activities and transcription levels of CYP450 enzymes, intestinal effluence transporter P-gp, and renal ingestion Organic Anion Transporters (OATs). Concurrently, EGCG and caffeine enhanced the activities of CYP3A, CYP2E1, and CYP2C37 in the liver of mice, while impairing the transport capabilities of P-gp and OATs. Reduced levels of MDR1 encoding P-gp transcription in the small intestine and renal OAT1 and OAT3 revealed that transcription was involved in the regulation of CYP450, P-gp, and OATs. The reduced transcription level of liver CYP2E1 suggested that CYP2E1 activity may have been elevated due to alternative mechanisms, but not through transcription. The absorption, metabolism, and excretion of drugs may be influenced by the daily consumption or high-dose administration of BOT and its related products, in which EGCG and caffeine may make great contributions.

Discovery of CYP2E1 as a novel target in rheumatoid arthritis and validation by a new specific CYP2E1 inhibitor

Considerable evidence indicates that CYP2E1 is associated with a variety of inflammatory diseases. Here we evaluated CYP2E1 as a potential therapeutic target for rheumatoid arthritis (RA) and established the protective effect of a new CYP2E1 inhibitor. Gene-expression datasets were used to analyze the change in expression of CYP2E1 in RA patients; CYP2E1 activity in collagen-induced arthritis (CIA) rats was determined by HPLC. We further evaluated the protective effects of Cyp2e1 knockout and a CYP2E1-specific inhibitor, Q11, synthesized by our group, in CIA and adjuvant-induced arthritis (AIA) rats. The expression of CYP2E1 in synovial tissue was elevated in RA patients and in CIA rats and the activity of CYP2E1 in vivo and in vitro in CIA rats was greater than that of controls. Cyp2e1 knockout significantly reduced the incidence of CIA and alleviated the severity of symptoms. Treatment with different doses of Q11 decreased paw thickness, volume and arthritis scores and reduced the serum levels of IL‐6, TNF‐α, IL‐1β and MDA, and increased the level of GSH in CIA rats. A similar inhibitory effect was exhibited for Q11 in the AIA rats. Moreover, Q11 significantly impeded proliferation, migration, and invasion of human rheumatoid arthritis synovial fibroblasts cells. Q11 decreased the release of ROS and enhanced Nrf2 nuclear translocation and HO-1 expression in the cell nucleus. Overall, our results indicated that CYP2E1 may be a new target for RA and Q11 has potential protective effects against RA by reducing oxidative stress and opposing the inflammatory response via the ROS/Nrf2/HO-1 signaling pathway.

Inflammation, oxidative stress and gut microbiome perturbation: A narrative review of mechanisms and treatment of the alcohol hangover.

Alcohol is the most widely abused substance in the world, the leading source of mortality in 15-49-year-olds, and a major risk factor for heart disease, liver disease, diabetes, and cancer. Despite this, alcohol is regularly misused in wider society. Consumers of excess alcohol often note a constellation of negative symptoms, known as the alcohol hangover. However, the alcohol hangover is not considered to have long-term clinical significance by clinicians or consumers. We undertook a critical review of the literature to demonstrate the pathophysiological mechanisms of the alcohol hangover. Hereafter, the alcohol hangover is re-defined as a manifestation of sickness behavior secondary to alcohol-induced inflammation, using the Bradford-Hill criteria to demonstrate causation above correlation. Alcohol causes inflammation through oxidative stress and endotoxemia. Alcohol metabolism is oxidative and increased intake causes relative tissue hypoxia and increased free radical generation. Tissue damage ensues through lipid peroxidation and the formation of DNA/protein adducts. Byproducts of alcohol metabolism such as acetaldehyde and congeners, sleep deprivation, and the activation of nonspecific inducible CYP2E1 in alcohol-exposed tissues exacerbate free radical generation. Tissue damage and cell death lead to inflammation, but in the intestine loss of epithelial cells leads to intestinal permeability, allowing the translocation of pathogenic bacteria to the systemic circulation (endotoxemia). This leads to a well-characterized cascade of systemic inflammation, additionally activating toll-like receptor 4 to induce sickness behavior. Considering the evidence, it is suggested that hangover frequency and severity may be predictors of the development of later alcohol-related diseases, meriting formal confirmation in prospective studies. In light of the mechanisms of alcohol-mediated inflammation, research into gut permeability and the gut microbiome may be an exciting future therapeutic avenue to prevent alcohol hangover and other alcohol-related diseases.

Abstract 1477: Acrylamide exposure in obesity enhances mammary epithelial DNA damage via the activity of its metabolite, glycidamide, and increased oxidative stress

Abstract Obesity is a world-wide epidemic, with 39% of adults considered overweight and 13% obese, and rates are rising. Obese women are at greater risk for developing breast cancer than lean women, however the mechanisms of how obesity increases breast cancer risk are not well understood. There is also limited knowledge on how environmental toxins may interact with obesity to promote breast cancer. Acrylamide, a probable carcinogen, is a biproduct in starchy foods cooked at high temperatures, which are prevalent in the obesity-inducing Western diet. Acrylamide is metabolized by the CYP2E1 enzyme into the epoxide, glycidamide, which is known to induce DNA damage. CYP2E1 activity is elevated in obesity, which could enhance DNA damage in obese individuals. Acrylamide exposure is associated with an increased risk of breast cancer in epidemiologic studies, but the evidence has been contradictory. To investigate how acrylamide impacts DNA damage in the obese mammary gland, three-week-old FVB female mice were randomized to receive a low-fat (LFD; 16% kcal from fat) or high-fat diet (HFD; 60% kcal from fat) and either 0.7 mM acrylamide water or control water. HFD-fed mice gained significantly more weight than LFD-fed mice, and exposure to acrylamide did not impact weight gain compared to respective controls. Mammary epithelial cells from acrylamide-treated mice had increased DNA damage compared to controls measured by COMET assays, with the highest DNA damage present in epithelial cells from obese acrylamide-treated mice. Mammary epithelial cells from acrylamide-treated mice also had enhanced oxidative DNA damage, measured by 8-OHdG adducts, and an additive effect was observed between acrylamide exposure and obesity. To investigate the impact of acrylamide in vitro, COMMA-D mammary epithelial cells were treated with vehicle, 9.8 µM acrylamide, or 0.5 mM glycidamide for 24 hours, then COMET assays and immunohistochemistry for markers of DNA damage were performed. COMMA-D cells treated with acrylamide-metabolite glycidamide showed significantly increased double strand DNA breaks compared to vehicle-treated cells, however acrylamide-treated cells did not. Interestingly, acrylamide-treated cells demonstrated significantly higher levels of intracellular reactive oxygen species compared to both glycidamide and vehicle-treated cells. These studies suggest acrylamide exposure enhances DNA damage in mammary epithelial cells via conversion to the genotoxic metabolite glycidamide and through generation of oxidative stress, which is exacerbated by obese conditions. These studies suggest that long-term acrylamide exposure through foods common in the Western diet may enhance DNA damage in mammary epithelial cells, potentially enhancing obesity-induced breast cancer risk. Citation Format: Brenna Walton, Lisa Arendt. Acrylamide exposure in obesity enhances mammary epithelial DNA damage via the activity of its metabolite, glycidamide, and increased oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1477.

Influence of Different Plant Extracts on CYP-Mediated Skatole and Indole Degradation in Pigs.

One of the primary substances responsible for the unpleasant odor in boar meat is skatole. Enzymes belonging to the cytochrome P450 (CYP) family play a pivotal role in the hepatic clearance of skatole. This study aimed to investigate the impact of oregano essential oil (OEO), Schisandra chinensis extract (SC), and garlic essential oil (GEO) on hepatic CYP2E1 and CYP2A activity in pigs. In three consecutive trials, cannulated castrated male pigs were provided with a diet containing 0.2-0.3% of one of these plant extracts. Following a 14-day feeding period, the animals were slaughtered, and liver and fat samples were collected. The findings indicate that the activities of CYP2E1 were unaffected by any treatment. However, GEO treatment demonstrated a significant reduction in CYP2A activity (p < 0.05). Pigs treated with GEO also exhibited a notable increase in skatole concentrations in both plasma and adipose tissue. In contrast, animals fed SC displayed elevated skatole concentrations in plasma but not in fat tissue. OEO did not influence skatole concentrations in either blood or fat. Furthermore, the study revealed that a supplementation of 6 g GEO per animal per day induced a significant increase in skatole concentrations in blood plasma within 24 h.

The Benzene-induced Hepatic Cytochrome P450 2E1 Expression and Activity are Reduced by Quercetin Administration in Mice.

Benzene as an environmental and industrial agent induces adverse effects that are mainly metabolism-dependent. Effects of Quercetin (QCN) on Benzene (BNZ)-induced changes in the hepatic Cytochrome P450 2E1 expression and activity were investigated. Thirty-six adult male mice were divided into 6 groups (n = 6) and nominated as control, BNZ (exposed to BNZ: 30 ppm), QCN (received QCN: 50 mg/kg, orally), and the fourth, fifth and sixth groups were exposed to 30 ppm BNZ and received 10, 50 and 100 mg/kg QCN respectively, for 28 days. The microsomal subcellular fraction was isolated from the liver samples and the activity of CYP 2E1 was measured based on the hydroxylation rate of 4-nitrophenol. The hepatic activity of myeloperoxidase also was assessed. Total antioxidant capacity and nitric oxide contents of the liver were determined. Expression changes of CYP 2E1 at the mRNA level were examined by qPCR technique. QCN lowered significantly (p < 0.05) the BNZ-increased hepatic nitric oxide levels and restored the BNZ-reduced antioxidant capacity. The BNZ-elevated activity of myeloperoxidase was declined in QCN-received mice. QCN downregulated the expression and activity of hepatic CYP 2E1 in BNZ-exposed animals. Our results suggest that QCN could be a novel hepatoprotective compound for BNZ-induced hepatotoxicities, which is attributed to its capability in the down-regulation of CYP 2E1 expression and activity.

Translocation of Adenosine A2B Receptor to Mitochondria Influences Cytochrome P450 2E1 Activity after Acetaminophen Overdose.

The adenosine A2B receptor (A2BAR) is a member of a family of G-protein coupled receptors (GPCRs), which has a low affinity for adenosine and is now implicated in several pathophysiological conditions. We have demonstrated the beneficial effects of A2BAR activation in enhancing recovery after acute liver injury induced by an acetaminophen (APAP) overdose. While receptor trafficking within the cell is recognized to play a role in GPCR signaling, its role in the mediation of A2BAR effects in the context of APAP-induced liver injury is not well understood. This was investigated here, where C57BL/6J mice were subjected to an APAP overdose (300 mg/kg), and the temporal course of A2BAR intracellular localization was examined. The impact of A2BAR activation or inhibition on trafficking was examined by utilizing the A2BAR agonist BAY 60-6583 or antagonist PSB 603. The modulation of A2BAR trafficking via APAP-induced cell signaling was explored by using 4-methylpyrazole (4MP), an inhibitor of Cyp2E1 and JNK activation. Our results indicate that APAP overdose induced the translocation of A2BAR to mitochondria, which was prevented via 4MP treatment. Furthermore, we demonstrated that A2BAR is localized on the mitochondrial outer membrane and interacts with progesterone receptor membrane component 1 (PGRMC1). While the activation of A2BAR enhanced mitochondrial localization, its inhibition decreased PGRMC1 mitochondria levels and blunted mitochondrial Cyp2E1 activity. Thus, our data reveal a hitherto unrecognized consequence of A2BAR trafficking to mitochondria and its interaction with PGRMC1, which regulates mitochondrial Cyp2E1 activity and modulates APAP-induced liver injury.

Allyl methyl disulfide (AMDS) prevents N,N-dimethyl formamide-induced liver damage by suppressing oxidative stress and NLRP3 inflammasome activation

N,N-dimethylformamide (DMF), a widely consumed industrial solvent with persistent characteristics, can induce occupational liver damage and pose threats to the general population due to the enormous DMF-containing industrial efflux and emission from indoor facilities. This study was performed to explore the roles of allyl methyl disulfide (AMDS) in liver damage induced by DMF and the underlying mechanisms. AMDS was found to effectively suppress the elevation in the liver weight/body weight ratio and serum aminotransferase activities, and reduce the mortality of mice induced by DMF. In addition, AMDS abrogated DMF-elicited increases in malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels and decreases in glutathione (GSH) levels in mouse livers. The increase in macrophage number, mRNA expression of M1 macrophage biomarkers, and protein expression of key components in the NF-κB pathway and NLRP3 inflammasome induced by DMF exposure were all suppressed by AMDS in mouse livers. Furthermore, AMDS inhibited DMF-induced cell damage and NF-κB activation in cocultured AML12 hepatocytes and J774A.1 macrophages. However, AMDS per se did not significantly affect the protein level and activity of CYP2E1. Collectively, these results demonstrate that AMDS effectively ameliorates DMF-induced acute liver damage possibly by suppressing oxidative stress and inactivating the NF-κB pathway and NLRP3 inflammasome.

Evaluation of Human Hepatocyte Drug Metabolism Carrying High-Risk or Protection-Associated Liver Disease Genetic Variants.

Metabolic-dysfunction-associated steatotic liver disease (MASLD), which affects 30 million people in the US and is anticipated to reach over 100 million by 2030, places a significant financial strain on the healthcare system. There is presently no FDA-approved treatment for MASLD despite its public health significance and financial burden. Understanding the connection between point mutations, liver enzymes, and MASLD is important for comprehending drug toxicity in healthy or diseased individuals. Multiple genetic variations have been linked to MASLD susceptibility through genome-wide association studies (GWAS), either increasing MASLD risk or protecting against it, such as PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438. As the impact of genetic variants on the levels of drug-metabolizing cytochrome P450 (CYP) enzymes in human hepatocytes has not been thoroughly investigated, this study aims to describe the analysis of metabolic functions for selected phase I and phase II liver enzymes in human hepatocytes. For this purpose, fresh isolated primary hepatocytes were obtained from healthy liver donors (n = 126), and liquid chromatography-mass spectrometry (LC-MS) was performed. For the cohorts, participants were classified into minor homozygotes and nonminor homozygotes (major homozygotes + heterozygotes) for five gene polymorphisms. For phase I liver enzymes, we found a significant difference in the activity of CYP1A2 in human hepatocytes carrying MBOAT7 (p = 0.011) and of CYP2C8 in human hepatocytes carrying PNPLA3 (p = 0.004). It was also observed that the activity of CYP2C9 was significantly lower in human hepatocytes carrying HSD17B13 (p = 0.001) minor homozygous compared to nonminor homozygous. No significant difference in activity of CYP2E1, CYP2C8, CYP2D6, CYP2E1, CYP3A4, ECOD, FMO, MAO, AO, and CES2 and in any of the phase II liver enzymes between human hepatocytes carrying genetic variants for PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438 were observed. These findings offer a preliminary assessment of the influence of genetic variations on drug-metabolizing cytochrome P450 (CYP) enzymes in healthy human hepatocytes, which may be useful for future drug discovery investigations.

Psoralen Ameliorates Acetaminophen-Induced Acute Liver Injury by Inhibiting the Enzymatic Activity of CYP2E1

Psoralen Ameliorates Acetaminophen-Induced Acute Liver Injury by Inhibiting the Enzymatic Activity of CYP2E1

Reducing Hepatotoxicity Mechanism of Radix Wikstroemia Indica by Processing with “Sweat Soaking Method” Using UPLC-MS/MS and a Cocktail Probe Substrate

Background: Radix Wikstroemia indica is a traditional Chinese medicine (TCM) used as anti-inflammatory and anti-tumor drug. However, it has serious hepatotoxicity, "Sweat soaking method" processed could effectively decrease its hepatotoxicity. Objective: The objective of this study is to study the effects of Radix Wikstroemia indica on six kinds of cytochrome P450(CYP450) isozymes of rat liver microsomes before and after processing, and to study the mechanism of Radix Wikstroemia indica processed by the "Sweat soaking method" to reduce liver toxicity in rats. Methods: In this study, the effects of Radix Wikstroemia indica and processed Radix Wikstroemia indica on the six main CYP450 isoforms (2E1, 1A2, 2C6, 2D1, 2C11, and 3A1) were investigated in vitro. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we carried out in vitro enzymatic studies in liver microsomal incubation systems via UPLC-MS/MS. Results: The results showed that the established UPLC-MS/MS method was precise and reliable. Compared with the blank group, the activities of six enzymes in the RWI and PRWI groups were higher than those in the blank group. At the same dose, the enzyme activities of CYP2E1, CYP1A2, CYP2C6, CYP2C11, and CYP3A1 increased with the increase in dose, and the enzyme activities of the RWI group were higher than those of the PRWI group. The enzyme activities of CYP2E1 and CYP1A2 in the Radix Wikstroemia indica group were significantly increased compared with the blank group, CYP3A1 in the RWI high-dose group was higher than that in the blank group and PRWI group with statistical differences (p&lt;0.05 or p&lt;0.01). Conclusion: The processed Radix Wikstroemia indica could reduce liver injury, and its detoxication mechanism might be related to the decrease in enzyme activity of CYP1A2, CYP2E1 and CYP3A1.

A Biomimetic Nanoparticle Exerting Protection against Acute Liver Failure by Suppressing CYP2E1 Activity and Scavenging Excessive ROS.

Acute liver failure (ALF) is a severe liver disease caused by many reasons. One of them is the overdosed acetaminophen (APAP), which is metabolized into N-acetyl-p-benzoquinone imine (NAPQI), an excessive toxic metabolite, by CYP2E1, resulting in excessive reactive oxygen species (ROS), exhausted glutathione (GSH), and thereafter hepatocyte necrosis. N-acetylcysteine is the Food and Drug Administration-approved drug for detoxification of APAP, but it has limited clinical application due to the short therapeutic time window and concentration-related adverse effects. In this study, a carrier-free and bilirubin dotted nanoparticle (B/BG@N) is developed, which is formed using bilirubin and 18β-Glycyrrhetinic acid, and bovine serum albumin (BSA) is then adsorbed to mimic the in vivo behavior of the conjugated bilirubin for hitchhiking. The results demonstrate that B/BG@N can effectively reduce the production of NAPQI as well asexhibit antioxidant effects against intracellular oxidative stress via regulating the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signal axis and reducing the production of inflammatory factors. In vivo study shows that B/BG@N can effectively improve the clinical symptom of the mice model. This study suggests that B/BG@N own increases circulation half-life, improves accumulation in the liver, and dual detoxification, providing a promising strategy for clinical ALF treatment.

Effects of aqueous extract from Baiyedancong-Oolong tea on cytochrome P450 enzymes activities, P-gp and OATs transport abilities and transcription levels in mice.

Recent studies have been conducted on its influence on drug metabolism and its potential mechanisms, among which the most studies have been focused on CYP3A enzymes. In this study, Baiyedancong Oolong tea (BOT) was processed by freeze- and hot air-drying techniques separately to obtain the aqueous extracts of freeze-and hot-dried BOT (FBOT and HBOT, respectively). High and low doses of FBOT (1463.7 and 292.74 mg/kg/d, respectively) and HBOT (1454.46 mg/kg/d, 290.89, respectively) were administered to mice for 7 days. Aqueous extracts from BOT simultaneously improved liver CYP3A, CYP2E1, and CYP2C37 activities and weakened the transport ability of P-gp and OATs in a dose-dependent manner, thus affecting multiple links of oral drug metabolism in liver, intestinal absorption and metabolism, and renal excretion. Moreover, aqueous extracts from BOT significantly increased the mRNA expressions of liver CYP3A11 and CYP2C37 as well as intestinal CYP3A11. Decreased transcription levels of MDR1 encoding P-gp in small intestine and renal OAT1 and OAT3, which was in the same direction as the regulation of the above enzyme activities and transport capacities. Besides, the transcription level of liver CYP2E1 was weakened, which was inconsistent with its corresponding enzyme activity, suggesting that the increased CYP2E1 activity may be caused by other mechanisms. Daily consumption or high dose administration of BOT and its related products may affect drug absorption, metabolisms, and excretion.

Hepatocyte-specific deletion of thromboxane-prostanoid receptor has protective effects on alcohol-associated liver disease

Alcohol-associated liver disease (ALD) is a major health issue worldwide with limited effective treatment options. Thromboxane-prostanoid receptor (TP-R), a G-protein-coupled receptor, is widely expressed in liver. Previous studies reported that an alcohol diet increased levels of TP-R ligands, thromboxane A2 and 8-isoprostane, in mouse liver. However, the role of TP-R signaling in ALD still remains unknown. Here, to test the hypothesis that deleting hepatocyte TP-R ameliorates liver injury caused by an alcohol diet, nine-week female hepatocyte-specific TP-R knock-out mice (KO) and their wild-type (WT) littermate control mice were fed the Lieber-DeCarli control (CONT) or 5% (v/v) ethanol (ET) diet for 4 weeks. Mice were divided into four groups: WT-CONT (n=8), KO-CONT (n=8), WT-ET (n=10), and KO-ET (n=8). Real-time PCR analyses showed that deleting hepatocyte TP-R attenuated alcohol-induced increase in mRNA levels of inflammatory markers including interleukin 6 ( P&lt;0.01) and lipocalin 2 ( P&lt;0.01). In addition, mRNA levels of other inflammatory markers including C-C motif chemokine ligand 2 ( P&lt;0.05) and interleukin 1β ( P&lt;0.01) were increased significantly only in WT-ET mice but not in KO-ET mice compared with controls. Of note, both real-time PCR and immunohistochemistry analyses showed that deleting TP-R in hepatocytes attenuates alcohol-induced increase in the expression of cluster of differentiation 36 (CD36, P&lt;0.05), a fatty acid transport protein. Previous studies documented that chronic alcohol consumption led to hepatic steatosis through promoting CD36-mediated fatty acid uptake. Interestingly, the gas chromatographic analysis of liver lipids revealed a remarkable increase in linoleic acid ( P&lt;0.05) level in liver triglyceride (TG) in WT-ET mice and this effect was blunted in KO-ET group. Additionally, the alcohol diet increased the levels of arachidonic acid ( P&lt;0.01), dihomo-gamma-linoleic acid ( P&lt;0.05), and docosatetraenoic acid ( P&lt;0.05) in liver TG of WT mice, but these fatty acids were not altered by alcohol diet in KO-ET group. Chronic alcohol feeding is known to elicit an oxidative stress in mouse livers though increasing the activity of cytochrome P450 family 2 subfamily E member 1 (CYP2E1). Accordingly, we noted that ablation of hepatocyte TP-R inhibited liver CYP2E1 activity in mice fed an alcohol diet ( P&lt;0.01). Moreover, the alcohol diet increased liver malondialdehyde level in WT mice ( P&lt;0.01) but not in KO mice. These findings suggest that hepatocyte TP-R plays an important role in regulating lipid metabolism and oxidative stress, thereby contributing to the development of AALD. This study was supported by R21 awards from NIH-National Institute on Alcohol Abuse and Alcoholism (R21- AA025445 and R21-AA027367) and a Merit Award from the Department of Veterans Affairs (I01CX002084). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protective effects of butylated hydroxytoluene on the initiation of N-nitrosodiethylamine-induced hepatocellular carcinoma in albino rats.

Diethylnitrosamine (DEN), a hepatocarcinogen, is found in a variety of smoked and fried foods and was reported to be hepatotoxic in mice. Butylated hydroxytoluene (BHT) is a potent antioxidant used in cosmetic formulations and as a food additive and preservative. As a result, BHT was studied as a potential inhibitor in the early stages of diethylnitrosamine (DEN)-induced HCC. Male Wistar albino rats (n = 24) were equally subdivided. Group 1 was the negative control; Group 2 and 3 administered BHT and DEN, respectively; Group 4 received BHT followed by DEN. Blood samples and rat livers were taken for biochemical and histological investigation. Hepatotoxicity was assessed by increased liver enzymes and HCC indicators, along with reduced antioxidant and pro-apoptotic factors. AFP, AFPL3, GPC3, GSH, SOD, MDA, CASP3 and BAX expression increased significantly after DEN treatment. DEN also reduced GPx, CAT, and CYP2E1 activity, and BCl-2 expression. Moreover, in the hepatic parenchyma, the DEN caused histological alterations. Pretreatment with BHT enhanced antioxidant status while preventing histopathological and most biochemical alterations. BHT pretreatment suppresses DEN-initiated HCC by decreasing oxidative stress, triggering intrinsic mitotic apoptosis, and preventing histopathological changes in liver tissue.

Inhibitory effect of quercetin-3-O-α-rhamnoside, p-coumaric acid, phloridzin and 4-O-β-glucopyranosyl-cis-coumaric acid on rats liver microsomes cytochrome P450 enzyme activities.

P-coumaric acid, phloridzin, quercetin-3-O-α-rhamnoside and 4-O-β-glucopyranosyl-cis-coumaric acid isolated in Malus micromalus Makino fruit were investigated the inhibitory activity of cytochrome CYP450 enzyme by the probe test method of rat liver microsomes in vitro, and determined the role in drug metabolism and/or toxicology. Enzymatic kinetics method was used to determine the inhibition type of these components and corresponding inhibition constants. The results demonstrated that all the 4 compounds had no significance to inhibit the activities of CYP2E1 and CYP2C11. P-coumaric acid, phloridzin and quercetin-3-O-α-rhamnoside had a weak inhibitory effect on CYP3A4, which belonged to the competitive inhibitory type with inhibitory constants of 10.56, 30.79 and 40.29μmolL-1, respectively. 4-O-β-glucopyranosyl-cis-coumaric acid had a moderate inhibitory effect on CYP3A4, which belonged to the anti-competitive inhibition type and the inhibition constant was 5.56μmolL-1. The CYP1A2 could be weakly inhibited by p-coumaric acid in the competitive type, and the inhibition constant is 25.20μmolL-1 4-O-β-glucopyranosyl-cis-coumaric acid exhibited anti-competitive inhibition of CYP1A2 with an inhibition constant of 19.91μmolL-1, and the inhibition effect was weak. The results will be useful to optimize the clinical dosage regimen and avoid drug-drug interactions when it is utilized comminating with other medicines in the clinic.

Antibiotics-Induced Depletion of Rat Microbiota Induces Changes in the Expression of Host Drug-Processing Genes and Pharmacokinetic Behaviors of CYPs Probe Drugs.

The metabolism of exogenous substances is affected by the gut microbiota, and the relationship between them has become a hot topic. However, the mechanisms by which the microbiota regulates drug metabolism have not been clearly defined. This study characterizes the expression profiles of host drug-processing genes (DPGs) in antibiotics-treated rats by using an unbias quantitative RNA-sequencing method and investigates the effects of antibiotics-induced depletion of rat microbiota on the pharmacokinetic behaviors of cytochrome P450s (CYPs) probe drugs, and bile acids metabolism by ultra-performance liquid chromatography-tandem mass spectrometry. Our results show that antibiotics treatments altered the mRNA expressions of 112 DPGs in the liver and jejunum of rats. The mRNA levels of CYP2A1, CYP2C11, CYP2C13, CYP2D, CYP2E1, and CYP3A of CYP family members were significantly downregulated in antibiotics-treated rats. Furthermore, antibiotics treatments also resulted in a significant decrease in the protein expressions and enzyme activities of CYP3A1 and CYP2E1 in rat liver. Pharmacokinetic results showed that, except for tolbutamide, antibiotics treatments significantly altered the pharmacokinetic behaviors of phenacetin, omeprazole, metoprolol, chlorzoxazone, and midazolam. In conclusion, the presence of stable, complex, and diverse gut microbiota plays a significant role in regulating the expression of host DPGs, which could contribute to some individual differences in pharmacokinetics. SIGNIFICANCE STATEMENT: This study investigated how the depletion of rat microbiota by antibiotics treatments influences the expression profiles of host DPGs and the pharmacokinetic behaviors of CYPs probe drugs. Combined with previous studies in germ-free mice, this study will improve the understanding of the role of gut microbiota in drug metabolism and contribute to the understanding of individual differences in the pharmacokinetics of some drugs.

Drug interaction potential of Ankaferd blood stopper® in human hepatocarcinoma cells.

Ankaferd blood stopper® (ABS) is an herbal extract consisting of mixtures of Alpinia officinarum, Gycyrrhiza glabra, Vitis vinifera, Thymus vulgaris, and Urtica dioica plants and has been used in recent years in Turkish medicine as a hemostatic agent. Despite its extensive usage, there is no information available about the drug interaction in HepG2 cells. The current work evaluated the effect of ABS on the expression of CYP1A1-1A2, CYP2E1, and CYP3A4 isozymes that are primarily involved in drug and carcinogen metabolism. We selected HepG2 cells as in vitro cellular models of the human liver. The cells were treated with different concentrations of ABS [0.25%-40% (v/v)]. A crystal violet staining assay was used to determine the cytotoxicity of ABS. We examined drug-metabolizing enzymes, including 7-ethoxyresorufin O-deethylase (CYP1A1), 7-methoxyresorufin O-demethylase (CYP1A2), aniline 4-hydroxylase (CYP2E1), and erythromycin N-demethylase (CYP3A4), in vitro in HepG2 cells. The expression (mRNA, protein) levels of drug-metabolizing enzymes were analyzed by qPCR and Western blotting, respectively. The EC05 and EC10 values for ABS were 0.37% and 0.52% (v/v), respectively. Therefore, 0.37% and 0.52% (v/v) doses were used for the remaining portion of this study. Investigation of the expression and activity levels revealed that CYP1A1-1A2, CYP2E1, and CYP3A4 activities were not affected by ABS significantly, with qPCR and Western blot results corroborating this result. Our study found that the activity, mRNA, and protein expression levels of CYP isozymes did not change with the application of ABS, suggesting that when humans are exposed to ABS, there may not be any risk associated with clinical drug toxicity, cancer formation, and drug metabolism disorders in humans.

Synbiotic Intervention Ameliorates Oxidative Stress and Gut Permeability in an In Vitro and In Vivo Model of Ethanol-Induced Intestinal Dysbiosis

Alcoholic liver disease (ALD) alters gut microbiota and tight junctions, causing bacterial components to enter the portal vein and induce oxidative stress-induced inflammation in the liver. Only corticosteroids and liver transplants are treatment options for severe alcoholic hepatitis. ALD's pathophysiology is unknown. However, acetaldehyde's toxic effects cause oxidative stress and intestinal permeability. This study investigates the influence of a synbiotic (a combination of aged garlic extract (AGE) and Lactobacillus rhamnosus MTCC1423) on colonic oxidative stress and inflammation in ALD male Wistar rats and Caco2 cells. MDA measurement by HPLC in CaCo2 cells, blood serum, and colon tissue demonstrated that synbiotic treatment in the ALD model reduces oxidative stress. Further, fecal high-throughput 16S rRNA gene sequencing revealed the microbiome's shift towards Firmicutes in the synbiotic group compared to ethanol. In addition, DCFDA labeling and H/E staining demonstrate that the synbiotic is beneficial in inhibiting the development of ALD. In the colon, the synbiotic reduces the activation of CYP2E1 and the inflammatory markers TNF-a and IL-6 while elevating the mRNA expression of ZO-1, occludin, and IL-10. Synbiotics colonize Lactobacillus to restore barrier function and microbiota and reduce colon oxidative stress. Thus, a synbiotic combination can be used in ALD treatment.