P450 2E1 Research Articles

Cisplatin induces kidney damage through the down-regulation of Prx I by autophagic degradation

In this study, we investigated the potential role of PrxI in cis-diamminedichloroplatinum (cisplatin)-induced renal damage in mice. The anticancer drug cisplatin is a chemotherapeutic agent that is widely used to treat solid tumors. Cisplatin-induced nephrotoxicity is a serious dose-limiting side effect, primarily caused by oxidative stress. The oxidative stress further damages DNA, membranes, and mitochondria, and increases endoplasmic reticulum (ER) stress. Cisplatin produces reactive oxygen species (ROS) through Cytochrome P450 2E1 (CYP2E1) and localizes to the surface of the ER, where CYP2E1 is located.Among the six Prx isoforms, Prx I was selectively degraded in cisplatin-treated kidneys during severe renal function damage. Prx I degradation is blocked in mouse proximal tubular cells treated with 3-methyladenine, an autophagy inhibitor, and in MEF lacking ATG7. Moreover, increased ROS levels on the ER surface due to CYP2E1 overexpression further accelerated Prx I degradation. These results suggest that Prx I degradation is largely mediated through autophagy, which is promoted by cisplatin-induced ER stress. Ablation of Prx I exacerbated cisplatin-induced nephrotoxicity and significantly increased the abundance of oxidative stress, ER stress, and inflammatory markers in the kidney, indicating that Prx I plays a protective role against cisplatin-induced nephrotoxicity.

Transgenic expression of human cytochrome P450 2E1 in C. elegans and rat PC-12 cells sensitizes to ethanol-induced locomotor and mitochondrial effects

Chronic alcohol (ethanol) use is increasing in the United States and has been linked to numerous health issues in multiple organ systems including neurological dysfunction and diseases. Ethanol toxicity is mainly driven by the metabolite acetaldehyde, which is generated through three pathways: alcohol dehydrogenase (ADH2), catalase (CAT), and cytochrome P450 2E1 (CYP2E1). ADH2, while the main ethanol clearance pathway in the liver, is not expressed in the mammalian brain, resulting in CAT and CYP2E1 driving local metabolism of ethanol in the central nervous system. CYP2E1 is known to generate reactive metabolites and reactive oxygen species and localizes to the mitochondria (mtCYP2E1) and endoplasmic reticulum (erCYP2E1). We sought to understand the consequences of mtCYP2E1 and erCYP2E1 in the nervous system during acute ethanol exposure. To answer this question, we generated transgenic Caenorhabditis elegans roundworms expressing human CYP2E1 in the mitochondria, endoplasmic reticulum, or both and exposed them to ethanol. We found that at lower concentrations, wild-type and mtCYP2E1-expressing worms had a small but significant inhibition of locomotion, whereas the erCYP2E1-expressing worms showed protection from this inhibition. At higher doses, all strains had reduced locomotion, but the erCYP2E1-expressing worms recovered faster than wild-type controls. CYP2E1 expression, regardless of organellar targeting, reduced mitochondrial respiration in response to ethanol. Similarly, transgenic expression of CYP2E1 in either organelle in PC-12 rat neuronal cell lines sensitized them to ethanol-induced cell death. Together, these findings suggest that subcellular localization of CYP2E1 impacts behavioral effects of ethanol and should be further studied in the mammalian central nervous system.

Different ethanol exposure durations affect cytochrome P450 2E1-mediated sevoflurane metabolism in rat liver

BackgroundChronic alcohol users often exhibit an increased minimum alveolar concentration (MAC) of sevoflurane, yet the specific mechanism remains unclear. It has been reported that ethanol exposure can upregulate the protein expression and enzyme activity of cytochrome P450 2E1 (CYP2E1). CYP2E1 is a key enzyme that converts 2–5% of sevoflurane into equimolar amounts of hexafluoroisopropanol (HFIP) and F−. This study aims to explore whether ethanol exposure could alter sevoflurane metabolism through CYP2E1 modulation, potentially explaining the increased MAC observed in alcohol users.MethodsEighty adult male Sprague-Dawley (SD) rats were randomly divided into two groups and received either 50% ethanol (dose: 3 g/kg) or 0.9% saline twice daily by gavage. After 1, 2, 3, and 4 weeks of gavage, ten rats were randomly selected from each group to undergo 1-hour anesthesia with 2.3% sevoflurane. Blood samples were collected after anesthesia to measure the concentration of free HFIP using gas chromatography. Additionally, the left lobe tissue of the liver was collected for the analysis of CYP2E1 protein expression by Western blot and CYP2E1 enzyme activity by colorimetric assay. Correlations between these parameters were analyzed using Pearson’s correlation.ResultsIn the ethanol group, CYP2E1 expression, activity, and the concentration of free HFIP were significantly higher at all time points compared to the control group (P < 0.05), except for protein expression in the first week (P > 0.05). Within-group comparisons indicated no significant changes in any of the parameters for the control group (P > 0.05). In the ethanol group, there was no difference in free HFIP concentration between the first and second weeks (P > 0.05), but a significant increase was observed in the third and fourth weeks (P < 0.01); protein expression and enzyme activity significantly varied over time, especially showing a notable increase from the first to the third and fourth weeks (P < 0.05). Correlation analysis revealed strong positive correlations between free HFIP concentration and CYP2E1 activity (r = 0.7898), free HFIP concentration and CYP2E1 expression (r = 0.8418), and CYP2E1 activity and expression (r = 0.8740), all with P < 0.001.ConclusionsEthanol exposure increased both the expression and enzymatic activity of CYP2E1, consequently enhancing the metabolism of sevoflurane.

Genetic polymorphisms in FABP2, CYP2E1, and TP53 genes are potentially associated with colorectal cancer susceptibility

Colorectal cancer (CRC) is among the most prevalent cancers with a high mortality rate. Both genetic and environmental factors contribute to CRC development. This study aimed to assess the association of single nucleotide polymorphisms (SNPs) in the fatty acid binding protein-2 (rs1799883), Cytochrome P450 2E1 (rs3813865), TP53 (rs1042522), and Murine double minute 2 (rs1042522) genes with CRC. A cross-sectional case–control study was conducted at the Institute of Molecular Biology and Biotechnology from May 2020 to March 2021, involving CRC patients (N = 100) and controls (N = 100) recruited from the Multan district in Pakistan. Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) were employed to investigate the studied SNPs. The association of SNPs in all genes with CRC was examined either individually or in various combinations. Genotypes at three SNPs, rs1799883 in FABP2, rs3813865 in CYP2E1, and rs1042522 in TP53, were found to be associated with the development of CRC, while rs1042522 in MDM2 was not. Patients who were married, smoked, lacked exercise habits or had a family history of CRC were at a greater risk of acquiring the disease. FABP2 gene rs1799883, CYP2E1 gene rs3813865, and TP53 gene rs1042522 polymorphisms are significant in the development of CRC in Pakistani participants.

Spatial Transcriptomics Reveals the Transcriptomic Signatures in a Mouse Model of Pediatric Metabolic Dysfunction–Associated Steatohepatitis

Metabolic dysfunction-associated steatohepatitis (MASH) is considered the progressive form of metabolic dysfunction-associated steatotic liver disease (MASLD), which is the leading cause of chronic liver disease in children. However, the pathogenesis of pediatric MASH remains poorly understood due to the lack of animal models. In this study, we developed a mouse model of pediatric MASH and characterized the hepatic transcriptomic profile using spatial transcriptomics (ST) technology. C57BL/6J mice were fed a western diet (WD) along with weekly injections of carbon tetrachloride (CCl4) from 3 weeks old to 8 weeks old. After 5 weeks of feeding, WD+CCl4-treated mice showed significant liver injury without the development of insulin resistance. Histologically, WD+CCl4 induced key features of type 2 MASH, the most common type observed in children, characterized by liver steatosis, portal inflammation, and portal fibrosis. Through ST analysis of liver tissues, we identified that cluster 0 in the mouse from the WD+CCl4 group was enriched in pathways associated with lipid metabolism. Further investigation revealed that cytochrome p450 2E1 (CYP2E1) was the top marker gene of cluster 0, and its expression was increased in the periportal area of mice from the WD+CCl4 group. These findings suggest that our mouse model of pediatric MASH mirrors the histological features of human MASH, and the upregulation of CYP2E1 may be linked to the disease pathogenesis.

MitoTracker Red for isolation of zone-specific hepatocytes and characterization of hepatic sublobular metabolism

BackgroundThe liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics. AimOur goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones. MethodsMice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed. ResultsMultiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes. ConclusionsMTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease.

Jujuboside A through YY1/CYP2E1 signaling alleviated type 2 diabetes-associated fatty liver disease by ameliorating hepatic lipid accumulation, inflammation, and oxidative stress

Non-alcoholic fatty liver disease (NAFLD) was a chronic complication of type 2 diabetes mellitus (T2DM), and this comorbid disease lacked therapeutic drugs. Semen Ziziphi Spinosae (SZS) was the seed of Ziziphus jujuba var. Spinosa (Bunge) Hu ex H.F. Chow, and it could alleviate the symptoms of T2DM patients. As a triterpene saponin, Jujuboside A (Ju A) was the main active substance isolated from SZS and could improve hyperglycemia of diabetic mice. However, it was still unknown whether Ju A has protective effects on T2DM-associated NAFLD. Our study showed that Ju A attenuated T2DM-associated liver damage by alleviating hepatic lipid accumulation, inflammatory response, and oxidative stress in the liver of db/db mice, and high glucose (HG) and free fatty acid (FFA) co-stimulated human hepatocellular carcinomas (HepG2) cells. Along with the improved hyperglycemia and liver injury, Ju A restrained Yin Yang 1 (YY1)/cytochrome P450 2E1 (CYP2E1) signaling in vivo and in vitro. YY1 overexpression intercepted the protective effects of Ju A on T2DM-induced liver injury via promoting hepatic lipid accumulation, inflammatory response, and oxidative stress. While, the blocking effect of YY1 overexpression on Ju A's hepatoprotective effect was counteracted by further treatment of CYP2E1 specific inhibitor diethyldithiocarbamate (DDC) in vitro. In-depth mechanism research showed that Ju A through YY1/CYP2E1 signaling promoted hepatic fatty acid β-oxidation, and inhibited inflammatory response and oxidative stress by activating peroxisome proliferator-activated receptor alpha (PPARα), leading to the improvement of T2DM-associated NAFLD. Ju A might be a potential agent in the treatment and health care of T2DM-associated liver disease, especially NAFLD.

Astrocytes in Amyloid Generation and Alcohol Metabolism: Implications of Alcohol Use in Neurological Disorder(s).

As per the National Survey on Drug Use and Health, 10.5% of Americans aged 12 years and older are suffering from alcohol use disorder, with a wide range of neurological disorders. Alcohol-mediated neurological disorders can be linked to Alzheimer's-like pathology, which has not been well studied. We hypothesize that alcohol exposure can induce astrocytic amyloidosis, which can be corroborated by the neurological disorders observed in alcohol use disorder. In this study, we demonstrated that the exposure of astrocytes to ethanol resulted in an increase in Alzheimer's disease markers-the amyloid precursor protein, Aβ1-42, and the β-site-cleaving enzyme; an oxidative stress marker-4HNE; proinflammatory cytokines-TNF-α, IL1β, and IL6; lncRNA BACE1-AS; and alcohol-metabolizing enzymes-alcohol dehydrogenase, aldehyde dehydrogenase-2, and cytochrome P450 2E1. A gene-silencing approach confirmed the regulatory role of lncRNA BACE1-AS in amyloid generation, alcohol metabolism, and neuroinflammation. This report is the first to suggest the involvement of lncRNA BACE1-AS in alcohol-induced astrocytic amyloid generation and alcohol metabolism. These findings will aid in developing therapies targeting astrocyte-mediated neurological disorders and cognitive deficits in alcohol users.

Obesity Induces DNA Damage in Mammary Epithelial Cells Exacerbated by Acrylamide Treatment through CYP2E1-Mediated Oxidative Stress.

Obesity and environmental toxins are risk factors for breast cancer; however, there is limited knowledge on how these risk factors interact to promote breast cancer. Acrylamide, a probable carcinogen and obesogen, is a by-product in foods prevalent in the obesity-inducing Western diet. Acrylamide is metabolized by cytochrome P450 2E1 (CYP2E1) to the genotoxic epoxide, glycidamide, and is associated with an increased risk for breast cancer. To investigate how acrylamide and obesity interact to increase breast cancer risk, female mice were fed a low-fat (LFD) or high-fat diet (HFD) and control water or water supplemented with acrylamide at levels similar to the average daily exposure in humans. While HFD significantly enhanced weight gain in mice, the addition of acrylamide did not significantly alter body weights compared to respective controls. Mammary epithelial cells from obese, acrylamide-treated mice had increased DNA strand breaks and oxidative DNA damage compared to all other groups. In vitro, glycidamide-treated COMMA-D cells showed significantly increased DNA strand breaks, while acrylamide-treated cells demonstrated significantly higher levels of intracellular reactive oxygen species. The knockdown of CYP2E1 rescued the acrylamide-induced oxidative stress. These studies suggest that long-term acrylamide exposure through foods common in the Western diet may enhance DNA damage and the CYP2E1-induced generation of oxidative stress in mammary epithelial cells, potentially enhancing obesity-induced breast cancer risk.

A computational approach to evaluate caffeoylquinic acids and flavonoids in Pluchea indica Less. leaves as potential anti-HIV agents

Context: The attachment of human immunodeficiency virus type 1 glycoprotein 120 (HIV-1 gp120) to the CD4 receptor of human immune cells is the beginning of HIV-1 infection. Stimulation of reactive oxygen species (ROS) production through upregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2) and -4 (NOX-4), and cytochrome P450 2E1 (CYP2E1) of the virus can be a potential target for anti-HIV agents. Aims: To evaluate the inhibitory effects of caffeoylquinic acids (CQAs) and flavonoids of Pluchea indica leaves against the binding of HIV-1 gp120 with CD4 receptor and their antioxidant activities via interactions with NOX-2, NOX-4, and CYP2E1 through in silico study. Methods: Ten CQAs and nine flavonoids of P. indica were docked to the 3TGS (gp120 HIV-1), 2CDU (NOX-2), 3A1F (NOX-4), and 3T3Z (CYP2E1) receptors using the AutoDockTools 1.5.7. Physicochemical and pharmacokinetics properties were predicted using the pkCSM online tool, while toxicity was predicted using the ProTox-II webserver. Results: Mostly, all of the CQAs and flavonoids were able to bind to all receptors. 3,4-Di-O-caffeoylquinic acid has the lowest binding energy (-8.79 kcal/mol) against 3TGS (gp120). 5-O-Caffeoylquinic acid and apigenin have great potential as antioxidants due to their good binding with NOX-2 and CYP2E1. However, CQAs might have ADME problems. Most test compounds did not cause hepatotoxicity, carcinogenicity, or mutagenicity. All test compounds have no cytotoxic potential. However, all CQAs have the potential to be immunotoxins. Conclusions: The findings indicated that 3,4-di-O-caffeylquinic acid could be a potential inhibitor of HIV-1 gp120-CD4 binding, while 5-O-caffeoylquinic acid and apigenin demonstrated strong antioxidant activities via NOX-2 and CYP2E1 inhibition. However, in-depth studies, including experimental in vitro and in vivo studies, are required to validate the anti-HIV activity of the compounds further.

Valorization of broiler edible byproducts: a chicken-liver hydrolysate with hepatoprotection against binge drinking

Over 10,000 metric-ton broiler livers are produced annually in Taiwan. Concerning unpleasant odor and healthy issue, broiler livers are not attractive to consumers. Although the patented chicken-liver hydrolysates (CLHs) through pepsin digestion possess several biofunctionalities, there is no study on hepatoprotection of CLH-based formula capsule (GBHP01) against binge drinking (Whiskey, 50% Alc./Vol.). GBHP01 led to an accelerated blood-alcohol clearance in rats, as evidenced by lowering blood-alcohol increment within 0 to 4 h, increasing blood-alcohol decrement within 4 to 8 h, and smaller blood alcohol concentration areas under the curve (BAC AUC) in the 8-h period (p < 0.05). The ameliorative effects of GBHP01 against binge drinking in rats over 6 wk were attributed to accelerated alcohol metabolism by further increasing alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities while downregulating cytochrome P450 2E1 (CYP2E1) protein expression, elevating antioxidant capacity, decreasing zonula occludens-1 (ZO-1) protein decrement and serum endotoxin, and reducing inflammation related protein levels, that is, toll-like receptor 4 (TLR4) and mitogen-activated protein kinase (MAPK), and proinflammatory cytokines. The development of CLH supplements could not only enhance the added value of broiler livers through nutraceutical development but also offer a strategy to maximize the utilization of poultry processing residues, as shown in this study.

Hepatoprotective effects of Juglans regia on carbon tetrachloride‐induced hepatotoxicity: In silico/in vivo approach

AbstractJuglans regia L. is a well‐known therapeutic plant in Nepal, employed in traditional medicine for treating liver ailments. This study aimed to evaluate the in vivo and in silico liver‐protective effects of J. regia extract using a carbon tetrachloride (CCl4)‐induced hepatic damage rat model. Healthy male rats were randomly divided into six groups: normal control (distilled water 10 mL/kg), toxic control (distilled water 10 mL/kg), standard test (silymarin 100 mg/kg), and three groups receiving oral J. regia extracts (125, 250, and 500 mg/kg/day) for seven days. On the eighth day, carbon tetrachloride (CCl4) was administered intraperitoneally (i.p.) (1.5 mL/kg in 1:1 olive oil ratio for all groups, except the normal control). Rats were sacrificed on the ninth day, and blood was collected retro‐orbitally for liver blood injury tests and histopathological studies. Molecular docking was performed against cytochrome P450 2E1 (CYP450 2E1) enzyme for 16 selected phytoconstituents. J. regia, at doses of 125, 250, and 500 mg/kg, significantly reduced liver enzyme levels (alanine aminotransferase, alkaline phosphatase, direct bilirubin, and total bilirubin), while increasing serum albumin. Histological analysis revealed mitigation of carbon tetrachloride (CCl4)‐induced liver injury, reducing fatty degeneration and necrosis. Molecular docking supported the findings, with Beta‐sitosterol and Betulinic acid exhibiting the best binding affinity of −9.2 and −9.1 kcal/mol, respectively. In conclusion, result suggests that J. regia showed dose‐dependent hepatoprotective activity in CCl4‐induced hepatotoxicity and it could be utilized as a promising hepatoprotective agent. This study suggests the hepatoprotective potential of J. regia bark extracts, emphasizing the need for further clinical validation.

CYP2E1 mediated advanced oxidation protein products exacerbate acetaminophen induced drug-derived liver injury in vitro and in vivo

Drug-induced liver injury (DILI) is prevalent in the treatment of chronic kidney disease (CKD). Advanced oxidation protein products (AOPPs) are markers of CKD progression and participate in the occurrence and development of liver diseases. However, the mechanisms underlying the regulation of DILI in CKD have not been established. Herein, we demonstrate the involvement of Cytochrome p450 2E1 (CYP2E1) in DILI induced by AOPPs is exacerbated by exposure to acetaminophen (APAP). We used a adenine-induced CKD model, a model of DILI induced by APAP, and the AOPPs model was generated by intraperitoneal injection. The decline in renal function was associated with a significantly increased concentration of Scr, BUN and AOPPs, and renal tissue fibrosis. The ALT, AST, and AOPPs levels and liver tissue necrosis increased significantly in CKD model group compared with the sodium carboxymethyl cellulose (CMCNa) group. In the AOPPs model, compared to the PBS controls, ALT, AST, and AOPP levels, and liver tissue necrosis increased significantly. In HepG2 or L0–2 cell lines, cell survival was significantly reduced in the AOPP + APAP treatment and CYP2E1 protein expression was increased. FPS-ZM1 or NAC attenuated the hepatocyte toxicity induced by AOPP + APAP and suppression of CYP2E1 expression. AOPPs exacerbated APAP-induced DILI through CYP2E1 signaling pathways. Protein uremic toxins, such as AOPPs, can modify drug toxicity in patients with CKD. This study provides new a rationale to reduce the generation of DILIs in clinical treatment in patients with CKD. AOPPs targeting may present a novel approach to reduce the occurrence of DILI.

Uncovering the impact of alcohol on internal organs and reproductive health: Exploring TLR4/NF-kB and CYP2E1/ROS/Nrf2 pathways.

This review delves into the detrimental impact of alcohol consumption on internal organs and reproductive health, elucidating the underlying mechanisms involving the Toll-like receptor 4 (TLR4)/Nuclear factor kappa light chain enhancer of activated B cells (NF-kB) pathway and the Cytochrome P450 2E1 (CYP2E1)/reactive oxygen species (ROS)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. The TLR4/NF-kB pathway, crucial for inflammatory and immune responses, triggers the production of pro-inflammatory agents and type-1 interferon, disrupting the balance between inflammatory and antioxidant responses when tissues are chronically exposed to alcohol. Alcohol-induced dysbiosis in gut microbes heightens gut wall permeability to pathogen-associated molecular patterns (PAMPs), leading to liver cell infection and subsequent inflammation. Concurrently, CYP2E1-mediated alcohol metabolism generates ROS, causing oxidative stress and damaging cells, lipids, proteins, and deoxyribonucleic acid (DNA). To counteract this inflammatory imbalance, Nrf2 regulates gene expression, inhibiting inflammatory progression and promoting antioxidant responses. Excessive alcohol intake results in elevated liver enzymes (ADH, CYP2E1, and catalase), ROS, NADH, acetaldehyde, and acetate, leading to damage in vital organs such as the heart, brain, and lungs. Moreover, alcohol negatively affects reproductive health by inhibiting the hypothalamic-pituitary-gonadal axis, causing infertility in both men and women. These findings underscore the profound health concerns associated with alcohol-induced damage, emphasizing the need for public awareness regarding the intricate interplay between immune responses and the multi-organ impacts of alcohol consumption.

In vivo upstream factors of mouse hepatotoxic mechanism with sustained hepatic glutathione depletion: Acetaminophen metabolite-erythrocyte adducts and splenic macrophage-generated reactive oxygen species

Investigation of acetaminophen (APAP)-induced liver damage recently indicated the significance of phagocytic NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and ferroptosis in the liver. Here, we focused on phagocytosis by iron-containing erythrocyte-devouring splenic macrophages and explored upstream factors of known APAP hepatotoxic mechanisms in vivo. Splenectomy did not alter hepatic cytochrome P450 (CYP) 2E1 activity or hepatic glutathione (GSH) content. APAP injection into splenectomized mice almost completely suppressed increases in plasma alanine aminotransferase levels and centrilobular hepatic necrosis showing the spleen to be a critical tissue in APAP-induced liver damage. Hepatic GSH was recovered to approximately 50 % content at 8 h. In non-splenectomized mice, liver damage was dramatically suppressed by a sensitive redox probe (DCFH-DA), macrophage-depleting clodronate (CL), and a NOX2 inhibitor. APAP treatment resulted in markedly stronger fluorescence intensity from DCFH-DA due to excessive ROS around splenic macrophages, which was lost upon co-treatment with a CYP inhibitor and CL. Deformed erythrocytes disappeared in mice co-treated with DCFH-DA, CL, the NOX2 inhibitor, and the CYP inhibitor. Simultaneously, these four compounds significantly improved APAP-depleted GSH levels. The CYP inhibitor also prevented the formation of APAP-cell adducts in the blood and spleen. In the spleen, CL co-treatment markedly reduced the number of adducts. Splenic ferrous iron levels were significantly elevated by APAP. Therefore, we demonstrated that splenic macrophages devoured APAP metabolite-erythrocyte adducts and subsequently splenic macrophage-related ROS caused sustained hepatic GSH depletion and excessive erythrocyte deformation around 7 h. Our data indicate in vivo upstream factors of known APAP hepatotoxic mechanisms.

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.

Common anesthetic used in preclinical PET imaging inhibits metabolism of the PET tracer [18F]3F4AP.

Positron emission tomography (PET) imaging studies in laboratory animals are almost always performed under isoflurane anesthesia to ensure that the subject stays still during the image acquisition. Isoflurane is effective, safe, and easy to use, and it is generally assumed to not have an impact on the imaging results. Motivated by marked differences observed in the brain uptake and metabolism of the PET tracer 3-[18F]fluoro-4-aminopyridine [(18F]3F4AP) between human and nonhuman primate studies, this study investigates the possible effect of isoflurane on this process. Mice received [18F]3F4AP injection while awake or under anesthesia and the tracer brain uptake and metabolism was compared between groups. A separate group of mice received the known cytochrome P450 2E1 inhibitor disulfiram prior to tracer administration. Isoflurane was found to largely abolish tracer metabolism in mice (74.8 ± 1.6 vs. 17.7 ± 1.7% plasma parent fraction, % PF) resulting in a 4.0-fold higher brain uptake in anesthetized mice at 35 min post-radiotracer administration. Similar to anesthetized mice, animals that received disulfiram showed reduced metabolism (50.0 ± 6.9% PF) and a 2.2-fold higher brain signal than control mice. The higher brain uptake and lower metabolism of [18F]3F4AP observed in anesthetized mice compared to awake mice are attributed to isoflurane's interference in the CYP2E1-mediated breakdown of the tracer, which was confirmed by reproducing the effect upon treatment with the known CYP2E1 inhibitor disulfiram. These findings underscore the critical need to examine the effect of isoflurane in PET imaging studies before translating tracers to humans that will be scanned without anesthesia.

Pharmacogenetic variability of tuberculosis biomarkers in native and mestizo Peruvian populations.

In Peru, 29 292 people were diagnosed with tuberculosis in 2022. Although tuberculosis treatments are effective, 3.4%-13% are associated with significant adverse drug reactions, with drug-induced liver injury (DILI) considered the most predominant. Among the first-line antituberculosis drugs, isoniazid is the main drug responsible for the appearance of DILI. In liver, isoniazid (INH) is metabolized by N-acetyltransferase-2 (NAT2) and cytochrome P450 2E1 (CYP2E1). Limited information exists on genetic risk factors associated with the presence of DILI to antituberculosis drugs in Latin America, and even less is known about these factors in the native and mestizo Peruvian population. The aim of this study was to determine the prevalence of NAT2 and CYP2E1 genotypes in native and mestizo population. An analytical cross-sectional analysis was performed using genetic data from mestizo population in Lima and native participants from south of Peru. NAT2 metabolizer was determined as fast, intermediate and slow, and CYP2E1 genotypes were classified as c1/c1, c1/c2 and c2/c2, from molecular tests and bioinformatic analyses. Of the 472 participants, 36 and 6 NAT2 haplotypes were identified in the mestizo and native population, respectively. In mestizo population, the most frequent NAT2*5B and NAT2*7B haplotypes were associated with DILI risk; while in natives, NAT2*5G and NAT2*13A haplotypes were associated with decreased risk of DILI. For CYP2E1, c1/c1 and c1/c2 genotypes are the most frequent in natives and mestizos, respectively. The linkage disequilibrium of NAT2 single nucleotide polymorphisms (SNPs) was estimated, detecting a block between all SNPs natives. In addition, a block between rs1801280 and rs1799929 for NAT2 was detected in mestizos. Despite the limitations of a secondary study, it was possible to report associations between NAT2 and CYP2E alleles with Peruvian native and mestizo by prevalence ratios. The results of this study will help the development of new therapeutic strategies for a Tuberculosis efficient control between populations.

Effect of SNPs in the Promoter Region on the Expression of Cytochrome P450 2E1 (CYP2E1) in Pig Liver.

Boar taint, an unfavorable odor in the meat of intact male pigs, is caused primarily by the accumulation of two compounds: androstenone and skatole. This multifactorial trait is regulated by numerous dietary, management and genetic factors. At the mechanistic level, there are many genes known to be involved in boar taint metabolism. Cytochrome P450 2E1 (CYP2E1) impacts boar taint through the phase I metabolism of skatole. The aim of this study was to identify single-nucleotide polymorphisms (SNPs) within the CYP2E1 gene promoter and explore their relationship with the expression of CYP2E1 mRNA and protein. Sequencing of the promoter region using pools of genomic DNA identified seven promoter region SNPs at -159, -586, -1693, -1806, -2322, -2369 and -2514 bp upstream of the ATG start site. Genomic DNA was obtained from 65 boars from the three major swine breeds: Duroc, Landrace and Yorkshire, and individual animals were genotyped for the identified SNPs. RNA was isolated from liver tissue and quantitative PCR was performed to measure CYP2E1 gene expression, while levels of CYP2E1 protein in liver were measured by Western blotting. Significant within-breed variation in CYP2E1 protein and mRNA expression was observed, indicating significant differences in gene expression among individuals. However, levels of CYP2E1 mRNA and protein were not significantly correlated. Two SNPs within the promoter were significantly associated with CYP2E1 mRNA expression, but not with protein expression. This study provides evidence of additional mutations affecting the gene expression of CYP2E1 and suggests that factors that affect the differences in translation of CYP2E1 mRNA may also be important in affecting skatole metabolism.

Augmented reparative potential of adipose-derived stem cells with silver nanoparticles against CCl4-induced liver injury in rats

Cymbopogon citratus (lemongrass) is a widely used medicinal plant. The previous literature studies demonstrated its diverse pharmacological activities such as anti-inflammatory, anti-diabetic, antibacterial and antifungal properties. The present study involves the green synthesis of silver nanoparticles from lemongrass and their characterization by X-ray diffraction (XRD), scanning electron microscope (SEM) and Fourier transmission infrared (FTIR). The purpose of this study is to evaluate the hepatoprotective potential of silver nanoparticles derived from the aqueous extract of lemongrass (LG-NPs) in comparison with the rat adipose-derived mesenchymal stem cells (ASCs) treated with lemongrass-derived silver nanoparticles (LG-NPs + ASCs). To assess the hepatoprotective effect of LG-NPs + ASCs, CCl4-induced fibrosis model rats were employed and it was revealed that transplanting LG-NPs + ASCs decreased fibrosis development compared to transplanting naive ASCs and LG-NPs, as demonstrated by improved hepatic architecture and functions. Additionally, the liver parenchyma's expression of fibrosis-associated gene markers Interleukin 1 beta (IL-1β), Cytochrome P450 2E1 (CYP2E1), Transforming Growth Factor Beta 1 (TGF-β1), collagen type I alpha 1(Col1α1), α-smooth muscle actin (α-SMA) reduced after LG-NPs + ASCs transplantation. These results suggest that pretreating ASCs with LG-NPs increase their effectiveness.