CYP2E1 Levels Research Articles

Modulation of Paracetamol-Induced Hepatotoxicity by Acute and Chronic Ethanol Consumption in Mice: A Study Pilot

Paracetamol (APAP) overdose is the leading cause of drug-induced liver injury, leading to acute liver failure. However, the role of concurrent acute or chronic ethanol ingestion in this context requires further clarification. In this study, we investigated the effects of acute and chronic ethanol ingestion on APAP-induced hepatotoxicity. Male C57BL/6 mice were randomly allocated into four groups: control (C; water 2×/day for 7 days); APAP (single dose of APAP, 500 mg/kg); acute ethanol (AE; a single ethanol dose—10 mL/kg, and one hour later an overdose of APAP—500 mg/kg); chronic ethanol (CE; ethanol—10 mL/kg, 2×/day for 7 days; and on the last day, an overdose of APAP—500 mg/kg). The results showed that AE induced heightened liver damage, increased necrotic area, and elevated levels of ALT, AST, TBARS, and oxidized glutathione compared to the control group. The AE group exhibited diminished glutathione availability and elevated CYP2E1 levels compared to the other groups. CE maintained a hepatic profile similar to that of the control group in terms of necrosis index, ALT and AST levels, GSH/GSSG ratio, and CYP2E1 activity, along with the upregulation of gene expression of the glucuronidation enzyme compared to the APAP group. Proteomic analysis revealed that the AE protein profile closely resembled that of the APAP group, whereas the C and CE groups were clustered together. In conclusion, ethanol consumption differentially modulated APAP overdose-induced liver damage. Acute consumption exacerbated hepatotoxicity, similar to an APAP overdose alone, whereas chronic consumption appeared to mitigate this injury, at least within the parameters assessed in this study.

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.

Ameliorative effects of Monascus-fermented hawthorn extract on a high-fat diet-induced rat model of non-alcoholic fatty liver disease

ObjectiveThe aim of this study was to elucidate the effects of fermented hawthorn extract on high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats, and explore the possible underlying mechanisms. MethodsA total of 42 male adult Sprague-Dawley rats were randomly divided into five groups: normal control group (given a normal feed diet and distilled water by gavage), NAFLD model (given HFD and distilled water by gavage), low-, medium-, and high-dose fermented hawthorn extract treatment groups (given HFD and different doses of fermented hawthorn extract by gavage). After 12 weeks of gavage administration, changes in body weight, liver/body weight ratio, serum liver enzymes, as well as triglyceride (TG) content and oxidative stress levels in rat liver tissueswere detected. Histological evaluation was performed to observe the degree of fat accumulation (steatosis). qRT-PCR and western blotting were performed to detect the mRNA and protein expression of cytochrome P4502E1 (CYP2E1, a key enzyme associated with lipid peroxidation), and lipogenic factors (sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS)) in rat liver tissues. ResultsFermented hawthorn extract significantly reduced the body weight, decreased the levels of liver enzymes, improved hepatic steatosis, and exhibited obvious antioxidant effects. Fermented hawthorn extract also significantly down-regulated the mRNA and protein expression levels of CYP2E1, SREBP-1c and FAS. ConclusionOur findings suggested that fermented hawthorn extract can markedly reduce body weight, ameliorate HFD-induced NAFLD in rats, and exhibits significant antioxidant effects. Its underlying mechanism may depend on the inhibition of CYP2E1, SREBP-1c, and FAS expression.

Pre-pubertal obesity compromises ovarian oxidative stress, DNA repair and chemical biotransformation

Obesity in adult females impairs fertility by altering oxidative stress, DNA repair and chemical biotransformation. Whether prepubertal obesity results in similar ovarian impacts is under-explored. The objective of this study was to induce obesity in prepubertal female mice and assess puberty onset, follicle number, and abundance of oxidative stress, DNA repair and chemical biotransformation proteins basally and in response to 7,12-dimethylbenz(a)anthracene (DMBA) exposure. DMBA is a polycyclic aromatic hydrocarbon that has been shown to be ovotoxic. Lactating dams (C57BL6J) were fed either a normal rodent containing 3.5% kCal from fat (lean), or a high fat diet comprised of 60% kCal from fat, and 9% kCal from sucrose. The offspring were weaned onto the diet of their dam and exposed at postnatal day 35 to either corn oil or DMBA (1 mg/kg) for 7 d via intraperitoneal injection. Mice on the HFD had reduced (P < 0.05) age at puberty onset as measured by vaginal opening but DMBA did not impact puberty onset. Heart, spleen, kidney, uterus and ovary weight were increased (P < 0.05) by obesity and liver weight was increased (P < 0.05) by DMBA exposure in obese mice. Follicle number was largely unaffected by obesity or DMBA exposure, with the exception of primary follicle number, which were higher (P < 0.05) in lean DMBA exposed and obese control relative to lean control mice. There were also greater numbers (P < 0.05) of corpora lutea in obese relative to lean mice. In lean mice, DMBA exposure reduced (P < 0.05) the level of CYP2E1, EPHX1, GSTP1, BRCA1, and CAT but this DMBA-induced reduction was absent in obese mice. Basally, obesity reduced (P < 0.05) the abundance of CYP2E1, EPHX1, GSTP1, BRCA1, SOD1 and CAT. There was greater (P < 0.05) fibrotic staining in obese DMBA-exposed ovaries and PPP2CA was decreased (P < 0.05) in growing follicles by both obesity and DMBA exposure. Thus, prepubertal obesity alters the capacity of the ovary to respond to DNA damage, ovotoxicant exposure and oxidative stress.

Folic acid protects against isoniazid-induced liver injury via the m6A RNA methylation of cytochrome P450 2E1 in mice.

Cytochrome P450 2E1 (CYP2E1) converts isoniazid (INH) to toxic metabolites and is critical in INH-induced liver injury. The aim is to investigate the effect of folic acid (FA) on CYP2E1 and INH-induced liver injury. Male Balb/c mice were used. The mice in the control group only received an AIN-93M diet. The AIN-93M diet was supplemented with 0.66 g INH/kg diet for the mice in the INH and FA groups. The mice in the FA group were treated with additional 0.01 g FA/kg diet. The one-carbon cycle metabolites, the expressions of CYP2E1 and the DNA and RNA methylation levels were detected to reveal the potential mechanism. FA treatment significantly reduced the alanine aminotransferase level and alleviated the liver necrosis. The mRNA and protein expressions of CYP2E1 were significantly lower in the FA group than those in the INH group. The N6-methyladenosine RNA methylation level of Cyp2e1 significantly increased in the FA group compared with the INH group, while the DNA methylation levels of Cyp2e1 were similar between groups. Additionally, the liver S-adenosyl methionine (SAM)/S-adenosyl homocysteine (SAH) was elevated in the FA group and tended to be positively correlated with the RNA methylation level of Cyp2e1. FA alleviated INH-induced liver injury which was potentially attributed to its inhibitory effect on CYP2E1 expressions through enhancing liver SAM/SAH and RNA methylation.

Pharmacogene expression during progression of metabolic dysfunction-associated steatotic liver disease: Studies on mRNA and protein levels and their relevance to drug treatment

Metabolic dysfunction-associated steatotic liver disease (MASLD) is common worldwide. Genes and proteins contributing to drug disposition may show altered expression as MASLD progresses. To assess this further, we undertook transcriptomic and proteomic analysis of 137 pharmacogenes in liver biopsies from a large MASLD cohort.We performed sequencing on RNA from 216 liver biopsies (206 MASLD and 10 controls). Untargeted mass spectrometry proteomics was performed on a 103 biopsy subgroup. Selected RNA sequencing signals were replicated with an additional 187 biopsies.Comparison of advanced MASLD (fibrosis score 3/4) with milder disease (fibrosis score 0–2) by RNA sequencing showed significant alterations in expression of certain phase I, phase II and ABC transporters. For cytochromes P450, CYP2C19 showed the most significant decreased expression (30 % of that in mild disease) but significant decreased expression of other CYPs (including CYP2C8 and CYP2E1) also occurred. CYP2C19 also showed a significant decrease comparing the inflammatory form of MASLD (MASH) with non-MASH biopsies. Findings for CYP2C19 were confirmed in the replication cohort. Proteomics on the original discovery cohort confirmed decreased levels of several CYPs as MASLD advanced but this decrease was greatest for CYP2C19 where levels fell to 40 % control. This decrease may result in decreased CYP2C19 activity that could be problematic for prescription of drugs activated or metabolized by CYP2C19 as MASLD advances. More limited decreases for other P450s suggest fewer issues with non-CYP2C19 drug substrates. Negative correlations at RNA level between CYP2C19 and several cytokine genes provided initial insights into the mechanism underlying decreased expression.

Kaempferol and nicotiflorin ameliorated alcohol-induced liver injury in mice by miR-138-5p/SIRT1/FXR and gut microbiota

AimsExcessive alcohol consumption can lead to alcoholic liver diseases (ALDs). Tetrastigma hemsleyanum Diels et Gilg is a rare Chinese medicinal herb. Tetrastigma hemsleyanum Diels et Gilg has been validated to be highly effective for treating hepatitis. Kaempferol and nicotiflorin are two highly representative flavonoids, which have exhibit therapeutic effects on liver disease. Therefore, the protective mechanism of kaempferol and nicotiflorin on alcohol-induced liver injury were investigated. Main methodsForty mice were used in this study. After treatment of Kaempferol and nicotiflorin, serum and liver were collected and used for determination of biochemical indicators, H&E staining, and molecular detection. The interaction of miRNAs from serum extracellular vehicles (EVs) with mRNAs and 16S rRNA sequencing of gut microbiota were also investigated. Key findingsThe results showed that kaempferol and nicotiflorins significantly ameliorated alcohol-induced liver damage and observably regulated gut microbiota. Specifically, the levels of malondialdehyde (MDA) and CYP2E1 in the liver significantly reduced, and the activity of superoxide dismutase (SOD) and glutathione (GSH) in the liver evidently increased. They also significantly relieved liver oxidative stress and lipid accumulation by suppressing miR-138-5p expression, inversely enhancing deacetylase silencing information regulator 2 related enzyme-1 (SIRT1) levels and then decreasing farnesoid X receptor (FXR) acetylation, which then modulated Nrf2 and SREBP-1c signaling pathways to regulate oxidative stress and lipid metabolism induced by alcohol. SignificanceKaempferol and nicotiflorin reduced alcohol-induced liver damage by enhancing alcohol metabolism and reducing oxidative stress and lipid metabolism. The intestinal microorganism disorder was also ameliorated after oral kaempferol and nicotiflorin.

The therapeutic effect of wine-processed Corni Fructus on chronic renal failure in rats through the interference with the LPS/IL-1-mediated inhibition of RXR function

Ethnopharmacological relevanceCorni Fructus, derived from the fruit of Cornus officinalis Sieb. et Zucc, is a widely utilized traditional Chinese medicine (TCM) with established efficacy in the treatment of diverse chronic kidney diseases. Crude Corni Fructus (CCF) and wine-processed Corni Fructus (WCF) are the main processed forms of Corni Fructus. Generally, TCM is often used after processing (paozhi). Despite the extensive use of processed TCM, the underlying mechanisms of processing for most TCMs have been unclear so far. Aim of the studyIn this study, an integrated strategy combined renal metabolomics with proteomics was established and investigated the potential processing mechanisms of CCF or WCF on chronic renal failure (CRF) models. Materials and methodsFirstly, the differences in biochemical parameters and pathological histology were compared to evaluate the effects of CCF and WCF on CRF model rats. Then, the tissue differential metabolites and proteins between CCF and WCF on CRF model rats were screened based on metabolomics and proteomics technology. Concurrently, a combined approach of metabolomics and proteomics was employed to investigate the underlying mechanisms associated with these marker metabolic products and proteins. ResultsCompared to the MG group, there were 27 distinct metabolites and 143 different proteins observed in the CCF-treatment group, while the WCF-treatment group exhibited 24 distinct metabolites and 379 different proteins. Further, the integration interactions analysis of the protein and lipid metabolite revealed that both WCF and CCF improved tryptophan degradation and LPS/IL-1-mediated inhibition of RXR function. WCF inhibited RXR function more than CCF via the modulation of LPS/IL-1 in the CRF model. Experimental results were validated by qRT-PCR and western blotting. Notably, the gene expression amount and protein levels of FMO3 and CYP2E1 among 8 genes influenced by WCF were higher compared to CCF. ConclusionThe results of this study provide a theoretical basis for further study of Corni Fructus with different processing techniques in CRF. The findings also offer guidance for investigating the mechanism of action of herbal medicines in diseases employing diverse processing techniques.

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.

Association between acetaminophen metabolites and CYP2E1 DNA methylation level in neonate cord blood in the Boston Birth Cohort

BackgroundAcetaminophen is a commonly used medication by pregnant women and is known to cross the placenta. However, little is known about the biological mechanisms that regulate acetaminophen in the developing offspring. Cytochrome 2E1 (CYP2E1) is the primary enzyme responsible for the conversion of acetaminophen to its toxic metabolite. Ex vivo studies have shown that the CYP2E1 gene expression in human fetal liver and placenta is largely controlled by DNA methylation (DNAm) at CpG sites located in the gene body of CYP2E1 at the 5’ end. To date, no population studies have examined the association between acetaminophen metabolite and fetal DNAm of CYP2E1 at birth.MethodsWe utilized data from the Boston Birth Cohort (BBC) which represents an urban, low-income, racially and ethnically diverse population in Boston, Massachusetts. Acetaminophen metabolites were measured in the cord plasma of newborns enrolled in BBC between 2003 and 2013 using liquid chromatography-tandem mass spectrometry. DNAm at 28 CpG sites of CYP2E1 was measured by Illumina Infinium MethylationEPIC BeadChip. We used linear regression to identify differentially methylated CpG sites and the “DiffVar” method to identify differences in methylation variation associated with the detection of acetaminophen, adjusting for cell heterogeneity and batch effects. The false discovery rate (FDR) was calculated to account for multiple comparisons.ResultsAmong the 570 newborns included in this study, 96 (17%) had detectable acetaminophen in cord plasma. We identified 7 differentially methylated CpGs (FDR < 0.05) associated with the detection of acetaminophen and additional 4 CpGs showing a difference in the variation of methylation (FDR < 0.05). These CpGs were all located in the gene body of CYP2E1 at the 5’ end and had a 3–6% lower average methylation level among participants with detectable acetaminophen compared to participants without. The CpG sites we identified overlap with previously identified DNase hypersensitivity and open chromatin regions in the ENCODE project, suggesting potential regulatory functions.ConclusionsIn a US birth cohort, we found detection of cord biomarkers of acetaminophen was associated with DNAm level of CYP2E1 in cord blood. Our findings suggest that DNA methylation of CYP2E1 may be an important regulator of acetaminophen levels in newborns.

Chronic-binge ethanol feeding aggravates systemic dyslipidemia in Ldlr-/- mice, thereby accelerating hepatic fibrosis.

Chronic ethanol consumption is known to cause alcohol-associated liver disease, which poses a global health concern as almost a quarter of heavy drinkers develop severe liver damage. Alcohol-induced liver disease ranges from a mild, reversible steatotic liver to alcoholic steatohepatitis and irreversible liver fibrosis and cirrhosis, ultimately requiring liver transplantation. While ethanol consumption is associated with dysregulated lipid metabolism and altered cholesterol homeostasis, the impact of dyslipidemia and pre-existing hypercholesterolemia on the development of alcohol-associated liver disease remains to be elucidated. To address the influence of systemic dyslipidemia on ethanol-induced liver disease, chronic-binge ethanol feeding was applied to female C57BL/6J (wild type) mice and mice deficient for the low-density lipoprotein receptor (Ldlr-/-), which display a human-like lipoprotein profile with elevated cholesterol and triglyceride levels in circulation. Respective control groups were pair-fed an isocaloric diet. Chronic-binge ethanol feeding did not alter systemic lipid levels in wild type mice. While increased systemic cholesterol levels in Ldlr-/- mice were not affected by ethanol feeding, chronic-binge ethanol diet aggravated elevated plasma triglyceride levels in Ldlr-/- mice. Despite higher circulatory triglyceride levels in Ldlr-/- mice, hepatic lipid levels and the development of hepatic steatosis were not different from wild type mice after ethanol diet, while hepatic expression of genes related to lipid metabolism (Lpl) and transport (Cd36) showed minor changes. Immunohistochemical assessment indicated a lower induction of infiltrating neutrophils in the livers of ethanol-fed Ldlr-/- mice compared to wild type mice. In line, hepatic mRNA levels of the pro-inflammatory genes Ly6g, Cd11b, Ccr2, Cxcl1 and F4/80 were reduced, indicating less inflammation in the livers of Ldlr-/- mice which was associated with reduced Tlr9 induction. While systemic ALT and hepatic MDA levels were not different, Ldlr-deficient mice showed accelerated liver fibrosis development after chronic-binge ethanol diet than wild type mice, as indicated by increased levels of Sirius Red staining and higher expression of pro-fibrotic genes Tgfb, Col1a1 and Col3a1. Ldlr-/- and wild type mice had similar plasma ethanol levels and did not show differences in the hepatic mRNA levels of Adh1 and Cyp2e1, important for ethanol metabolism. Our results highlight that chronic-binge ethanol feeding enhances systemic dyslipidemia in Ldlr-/- mice which might accelerate the development of hepatic fibrosis, independent of hepatic lipid levels.

Phenylpropionic acid produced by gut microbiota alleviates acetaminophen-induced hepatotoxicity

ABSTRACT The gut microbiota affects hepatic drug metabolism. However, gut microbial factors modulating hepatic drug metabolism are largely unknown. In this study, using a mouse model of acetaminophen (APAP)-induced hepatotoxicity, we identified a gut bacterial metabolite that controls the hepatic expression of CYP2E1 that catalyzes the conversion of APAP to a reactive, toxic metabolite. By comparing C57BL/6 substrain mice from two different vendors, Jackson (6J) and Taconic (6N), which are genetically similar but harbor different gut microbiotas, we established that the differences in the gut microbiotas result in differential susceptibility to APAP-induced hepatotoxicity. 6J mice exhibited lower susceptibility to APAP-induced hepatotoxicity than 6N mice, and such phenotypic difference was recapitulated in germ-free mice by microbiota transplantation. Comparative untargeted metabolomic analysis of portal vein sera and liver tissues between conventional and conventionalized 6J and 6N mice led to the identification of phenylpropionic acid (PPA), the levels of which were higher in 6J mice. PPA supplementation alleviated APAP-induced hepatotoxicity in 6N mice by lowering hepatic CYP2E1 levels. Moreover, PPA supplementation also reduced carbon tetrachloride-induced liver injury mediated by CYP2E1. Our data showed that previously known PPA biosynthetic pathway is responsible for PPA production. Surprisingly, while PPA in 6N mouse cecum contents is almost undetectable, 6N cecal microbiota produces PPA as well as 6J cecal microbiota in vitro, suggesting that PPA production in the 6N gut microbiota is suppressed in vivo. However, previously known gut bacteria harboring the PPA biosynthetic pathway were not detected in either 6J or 6N microbiota, suggesting the presence of as-yet-unidentified PPA-producing gut microbes. Collectively, our study reveals a novel biological function of the gut bacterial metabolite PPA in the gut-liver axis and presents a critical basis for investigating PPA as a modulator of CYP2E1-mediated liver injury and metabolic diseases.

Pharmacokinetic evaluation of Sinisan containing vinegar-processed products in depressive rats, a comprehensive perspective of ‘individual herb, herb-pair, and herbal formula’

Ethnopharmacological relevanceAs a classical formula for the treatment of depression, the clinical application of vinegar-processed products of Bupleuri Radix (Bupleurum chinense DC., BR) and Paeoniae Radix Alba (Paeonia lactiflora Pall., PRA) contained in Sinisan (SNS) is still controversial. Aim of the studyThree levels of ‘individual herb, herb-pair, and herbal formula’ were employed to investigate whether and how the processing of main drugs affected the active constituents of pharmacokinetics in SNS, as well as their impacts on the hepatic CYP450 enzyme. Materials and methodsRats were subjected to construct a chronic unpredictable mild stimulation (CUMS) model. A rapid and sensitive ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analytical method was developed and validated for simultaneously quantitative evaluation of thirteen potential active compounds of SNS in depressive rat plasma, and successfully applied to a holistic comparison of pharmacokinetics. The differences in pharmacokinetic parameters based on three different forms of drug composition from BR and PRA before and after vinegar-processing were compared. Meanwhile, qRT-PCR and Western Blot were utilized to explore the metabolic activity of three isoforms of CYP450 enzyme scattered in the livers of depressive rats. ResultsThe characteristic pharmacokinetics profiles of thirteen representative constituents in CUMS rats were influenced by vinegar-processing of BR and PRA and/or the compatibility. In detail, there were significant differences in the Cmax, AUC0-24, AUC0-∞, t1/2, and MRT0-24 of most constituents among the three different forms of drug composition from BR and PRA before and after vinegar-processing, with the most obvious changes in six constituents from the adjuvant and mediating guide drugs. And also, the pharmacokinetic parameters of seven constituents from BR and PRA in SNS containing vinegar-processed products obviously changed after compatibility. Additionally, the mRNA and protein levels of CYP1A2, CYP2E1, and CYP3A1 were observed to increase significantly with the processing of BR and PRA and the combination/formulation. ConclusionsIn conclusion, SNS containing vinegar-processed products was more conducive to the absorption of most activated constituents compared to the original formula in vivo. The vinegar-processing of BR and PRA and the compatibility co-contribute to the pharmacokinetic variability of active compounds of SNS in CUMS rats, and the extent of contribution varies among drugs, which might be related to the regulation of the hepatic drug metabolizing enzymes. The finding of the investigation could help to better understand how active compounds metabolized in vivo, which might be helpful for guiding the clinical application of SNS containing vinegar-processed products.

Synergistic effects of combined treatment of 1,2-dichloroethane and high-dose ethanol on liver damage in mice and the related mechanisms.

Our previous studies have shown that the metabolism of 1,2-dichloroethane (1,2-DCE) mediated by CYP2E1 could result in oxidative damage in the liver of mice. In the current study, we further investigated the effects of combined treatment with 1,2-DCE and high dose ethanol on liver and the mechanisms since both of them can be metabolized by CYP2E1 in the liver. There are several novel findings in the current study. First, combined treatment of mice with 1,2-DCE and high-dose ethanol could synergistically upregulate both protein and mRNA levels of CYP2E1, which might aggravate liver damage through CYP2E1-mediated oxidative stress. Second, the combined treatment could also synergistically trigger NLRP3 inflammasome activation and inflammatory responses in the liver. Third, the combined treatment synergistically upregulated the antioxidant defence systems in response to oxidative stress, however the compensatory mechanisms of antioxidant defence systems appeared to be insufficient to protect liver damage in the mice. Finally, the upregulated CYP2E1 expression was confirmed by using its specific inhibitor to play the crucial roles in liver damage in the mice during the combined treatment.

Sex- and lifestyle-related factors are associated with altered hepatic CYP protein levels in people diagnosed with mental disorders.

In this study, we used human postmortem tissue to investigate hepatic protein expression levels of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 by LC‒MS/MS in a population of people suffering from mental disorders (n=171). We report hepatic protein levels of these six CYP isoforms in 171 individuals in total, and define a focused population dataset of 116 individuals after excluding 55 samples due to low Microsomal Protein Per Gram of Liver (MPPGL) yield. Postmortem decay was most likely the reason for the low MPPGL yield in the 55 samples. In the focused population, we found women to have significantly higher protein levels of CYP3A4 than men in addition to decreased CYP3A4 protein levels among obese individuals. Furthermore, MPPGL was negatively correlated with body mass index (BMI). An increase in CYP1A2 protein levels was observed among smokers and increased CYP2E1 protein levels were observed among individuals with a history of alcohol abuse. Finally, individuals who received phenobarbital (CYP3A4 inducer) had significantly higher CYP3A4 levels. In conclusion, lifestyle-related factors prevalent among people suffering from mental disorders are associated with altered CYP protein levels which may alter drug metabolism and affect the efficacy of commonly prescribed drugs. Furthermore, this investigation demonstrates that postmortem hepatic tissue can be used to study how lifestyle and effectors affect hepatic CYP-levels in a large cohort of patients. Significance Statement Using a large number of postmortem hepatic tissue specimens (n=116) originating from the autopsy of individuals diagnosed with mental disorders, we were able to show that hepatic CYP-levels were affected by alcohol, smoking, BMI, and sex and that MPPGL was affected by BMI. Theses lifestyle-related changes may alter drug metabolism and affect the efficacy of commonly prescribed drugs. It is a novel approach to use a large postmortem cohort to investigate how lifestyle and effectors affect hepatic CYP-levels.

Cyp2e1 knockdown attenuates high glucose-induced apoptosis and oxidative stress of cardiomyocytes by activating PI3K/Akt signaling.

Cyp2e1 is a crucial CYP450 enzyme participating in diabetes and cardiovascular disorder. However, the role of Cyp2e1 in diabetic cardiomyopathy (DCM) has never been reported. Thus, we intended to identify the effects of Cyp2e1 on cardiomyocytes under high glucose (HG) conditions. Identification of differentially expressed genes in DCM and control rats was performed using bioinformatics analysis based on GEO database. The Cyp2e1-knockdown H9c2 and HL-1 cells were established through transfection with si-Cyp2e1. Western blot analysis was performed to determine the expression levels of Cyp2e1, apoptosis-related proteins and PI3K/Akt signaling-associated proteins. TUNEL assay was performed to assess apoptotic rate. Reactive oxygen species (ROS) generation was examined by DCFH2-DA staining assay. From the bioinformatics analysis, Cyp2e1 was confirmed as an upregulated gene in DCM tissues. In vitro assays proved that Cyp2e1 expression was markedly increased in HG-induced H9c2 and HL-1 cells. Cyp2e1 knockdown attenuated HG-induced apoptosis in both H9c2 and HL-1 cells, as proved by deceased apoptotic rate, relative cleaved caspase-3/caspase-3 level, and caspase-3 activity. Cyp2e1 knockdown reduced ROS generation and elevated the expression level of nuclear Nrf2 in HG-induced H9c2 and HL-1 cells. Increased relative levels of p-PI3K/PI3K and p-Akt/Akt were found in Cyp2e1-knockdown H9c2 and HL-1 cells. Inhibition of PI3K/Akt using LY294002 reversed the inhibitory effects of Cyp2e1 knockdown on cell apoptosis and ROS generation on cardiomyocytes. Cyp2e1 knockdown attenuated HG-induced apoptosis and oxidative stress by activating PI3K/Akt signaling in cardiomyocytes. These findings suggested that Cyp2e1 might be potentially used as an effective therapeutic strategy for DCM.

Acetaminophen for the patent ductus arteriosus: has safety been adequately demonstrated?

Patent ductus arteriosus (PDA) is the most common cardiovascular condition diagnosed in premature infants. Acetaminophen was first proposed as a potential treatment for PDA in 2011. Since that time acetaminophen use among extremely preterm neonates has increased substantially. The limited available data demonstrate that acetaminophen reduces PDA without evident hepatotoxicity. These findings have led some to suggest that acetaminophen is a safe and effective therapy for PDA closure. However, the lack of apparent hepatoxicity is predictable. Acetaminophen induced cellular injury is due to CYP2E1 derived metabolites; and hepatocyte CYP2E1 expression is low in the fetal and neonatal period. Here, we review preclinical and clinical data that support the hypothesis that the lung, which expresses high levels of CYP2E1 during fetal and early postnatal development, may be particularly susceptible to acetaminophen induced toxicity. Despite these emerging data, the true potential pulmonary risks and benefits of acetaminophen for PDA closure are largely unknown. The available clinical studies in are marked by significant weakness including low sample sizes and minimal evaluation of extremely preterm infants who are typically at highest risk of pulmonary morbidity. We propose that studies interrogating mechanisms linking developmentally regulated, cell-specific CYP2E1 expression and acetaminophen-induced toxicity as well as robust assessment of pulmonary outcomes in large trials that evaluate the safety and efficacy of acetaminophen in extremely preterm infants are needed.

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.

Generation and Characterization of CYP2E1-Overexpressing HepG2 Cells to Study the Role of CYP2E1 in Hepatic Hypoxia-Reoxygenation Injury.

The mechanisms of hepatic ischemia/reperfusion (I/R) injury, which occurs during liver transplantation or surgery, are poorly understood. The purpose of the current study was to generate and characterize a HepG2 cell line with a stable overexpression of CYP2E1 to investigate the role of the enzyme in hypoxia/reperfusion (H/R) injury in an ex vivo setting. GFP-tagged CYP2E1 and control clones were developed, and their gene expression and protein levels of GFP and CYP2E1 were determined using RT-PCR and ELISA/Western blot analysis, respectively. Additionally, the CYP2E1 catalytic activity was determined by UPLC-MS/MS analysis of 6-hydroxychlorzoxazone formed from the chlorzoxazone substrate. The CYP2E1 and control clones were subjected to hypoxia (10 h) and reoxygenation (0.5 h), and cell death and reactive oxygen species (ROS) generation were quantitated using LDH and flow cytometry, respectively. Compared with the control clone, the selected CYP2E1 clone showed a 720-fold increase in CYP2E1 expression and a prominent band in the western blot analysis, which was associated with a 150-fold increase in CYP2E1 catalytic activity. The CYP2E1 clone produced 2.3-fold more ROS and 1.9-fold more cell death in the H/R model. It is concluded that the constitutive CYP2E1 in the liver may play a detrimental role in hepatic I/R injury.