Enhanced In Vitro Recapitulation of In Vivo Liver Regeneration by Co-Culturing Hepatocyte Organoids with Adipose-Derived Mesenchymal Stem Cells, Alleviating Steatosis and Apoptosis in Acute Alcoholic Liver Injury.

Hepatocyte organoids (HOs) have superior hepatic functions to cholangiocyte-derived organoids but suffer from shorter lifespans. To counteract this, we co-cultured pig HOs with adipose-derived mesenchymal stem cells (A-MSCs) and performed transcriptome analysis. The results revealed that A-MSCs enhanced the collagen synthesis pathways, which are crucial for maintaining the three-dimensional structure and extracellular matrix synthesis of the organoids. A-MSCs also increased the expression of liver progenitor cell markers (KRT7, SPP1, LGR5+, and TERT). To explore HOs as a liver disease model, we exposed them to alcohol to create an alcoholic liver injury (ALI) model. The co-culture of HOs with A-MSCs inhibited the apoptosis of hepatocytes and reduced lipid accumulation of HOs. Furthermore, varying ethanol concentrations (0-400 mM) and single-versus-daily exposure to HOs showed that daily exposure significantly increased the level of PLIN2, a lipid storage marker, while decreasing CYP2E1 and increasing CYP1A2 levels, suggesting that CYP1A2 may play a critical role in alcohol detoxification during short-term exposure. Moreover, daily alcohol exposure led to excessive lipid accumulation and nuclear fragmentation in HOs cultured alone. These findings indicate that HOs mimic in vivo liver regeneration, establishing them as a valuable model for studying liver diseases, such as ALI.

The anti-tumor effects of cosmosiin through regulating AhR/CYP1A1-PPARγ in breast cancer.

Breast cancer is one of the threatening malignant tumors with the highest mortality and incidence rate over the world. There are a lot of breast cancer patients dying every year due to the lack of effective and safe therapeutic drugs. Therefore, it is highly necessary to develop more effective drugs to overcome breast cancer. As a glycoside derivative of apigenin, cosmosiin is characterized by low toxicity, high water solubility, and wide distribution in nature. Additionally, cosmosiin has been shown to perform anti-tumor effects in cervical cancer, hepatocellular carcinoma and melanoma. However, its pharmacological effects on breast cancer and its mechanisms are still unknown. In our study, the anti-breast cancer effect and mechanism of cosmosiin were investigated by using breast cancer models in vivo and in vitro. The results showed that cosmosiin inhibited the proliferation, migration, and adhesion of breast cancer cells in vitro and suppressed the growth of tumor in vivo through binding with AhR and inhibiting it, thus regulating the downstream CYP1A1/AMPK/mTOR and PPARγ/Wnt/ß-catenin signaling pathways. Collectively, our findings have made contribution to the development of novel drugs against breast cancer by targeting AhR and provided a new direction for the research in the field of anti-breast cancer therapy.

Unraveling the molecular significance of CYP1A2 (rs762551; c.-9-154 C>A) genetic variant on breast carcinoma susceptibility: Insights from case-control study and meta-analysis.

BACKGROUND: The human cytochrome P450 (CYP) superfamily encompasses different categories of isoenzymes that contribute to multiple metabolic processes involving drug detoxification, cellular signaling, and the proliferation of malignant tissues. Using genetic technology, customized bioinformatic analysis, and meta-analysis design, the main goal of this study was to identify the association between the CYP1A2*rs762551 variant and the susceptibility to breast carcinoma (BRCA). METHODS: The case-control study was conducted based on 104 BRCA women and 102 healthy controls. Using the TaqMan allelic discrimination assay, the CYP1A2 (rs762551; c.-9-154 C>A) variant was genotyped. Bioinformatic frameworks and logistic regression analysis were used to assess the involvement of this genetic variant in BRCA development. A meta-analysis design was accomplished based on our case-control study and other previously published records. Publication bias, heterogeneity between studies, and trial sequential analysis (TSA) were analyzed. RESULTS: The CYP1A2*rs762551 variant conferred protection against BRCA development under allelic (OR = 0.48, p-value < 0.001), dominant (OR = 0.34, p-value < 0.001), and recessive (OR = 0.44, p-value = 0.011) models. However, this intronic variant was correlated with a decreased risk of BRCA among late-onset menopause women compared to other cases. Bioinformatic analysis confirmed that this genetic variant has a functional impact on the progression of tumorgenesis. Moreover, this meta-analysis design included 12922 BRCA women and 15603 healthy controls. Our findings disclosed the contribution of the CYP1A2*rs762551 variant with protection against cancer development among Caucasian females under allelic (OR = 0.75, p-value = 0.025), and dominant (OR = 0.58, p-value = 0.015) models. CONCLUSIONS: This case-control study confirmed the contribution of the CYP1A2*rs762551 variant with decreased risk of BRCA development among Egyptian subjects. Moreover, BRCA women with late-onset menopause conferred protection against cancer progression compared to other subjects. Our findings identified that this meta-analysis design achieved protection against BRCA development among Caucasian women compared to other ethnicities.

Polycyclic aromatic hydrocarbon and its adducts in peripheral blood: Gene and environment interaction among Chinese population.

BACKGROUND: Benzo(a)pyrene (B[a]P) is the most widely concerned polycyclic aromatic hydrocarbons (PAHs), which metabolizes benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) in vivo to produce carcinogenic effect on the body. Currently, there is limited research on the role of the variation of metabolic enzymes in this process. METHODS: We carried out a study including 752 participants, measured the concentrations of 16 kinds PAHs in both particle and gaseous phases, urinary PAHs metabolites, leukocyte BPDE-DNA adduct and serum BPDE- Albumin (BPDE-Alb) adduct, and calculated daily intake dose (DID) to assess the cumulative exposure of PAHs. We conducted single nucleotide polymorphism sites (SNPs) of metabolic enzymes, explored the exposure-response relationship between the levels of exposure and BPDE adducts using multiple linear regression models. RESULT: Our results indicated that an interquartile range (IQR) increase in B[a]P, PAHs, BaPeq, 1-hydroxypyrene (1-OHP), 1-hydroxynaphthalene (1-OHNap) and 2-hydroxynaphthalene (2-OHNap) were associated with 26.53 %, 24.24 %, 28.15 %, 39.15 %, 12.85 % and 14.09 % increase in leukocyte BPDE-DNA adduct (all P < 0.05). However, there was no significant correlation between exposure with serum BPDE-Alb adduct (P > 0.05). Besides, we also found the polymorphism of CYP1A1(Gly45Asp), CYP2C9 (Ile359Leu), and UGT1A1(downstream) may affect BPDE adducts level. CONCLUSION: Our results indicated that leukocyte BPDE-DNA adduct could better reflect the exposure to PAHs. Furthermore, the polymorphism of CYP1A1, CYP2C9 and UGT1A1affected the content of BPDE adducts.

Rutaecarpine Aggravates Acetaminophen-Induced Acute Liver Injury by Inducing CYP1A2.

In this study, we investigated whether rutaecarpine could aggravate acetaminophen-induced acute liver damage in vivo and in vitro. CCK-8 and apoptosis assays were performed to verify the cytotoxicity of acetaminophen to L02 cells with or without rutaecarpine. The expression levels of the target proteins and genes were determined using Western blotting and qRT-PCR. The liver pathological changes were evaluated with hematoxylin and eosin staining, while the aspartate aminotransferase (AST) and alanine aminotransferase (AST) levels in plasma were measured to assess the liver damage. Our results revealed that pretreatment of the cell and mice with rutaecarpine significantly aggravated the acetaminophen-induced liver damage. Mechanistically, rutaecarpine induces the CYP1A2 protein, which accelerates the metabolism of acetaminophen to produce a toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI), leading to severe liver inflammation. Rutaecarpine exacerbated the liver damage by upregulating CYP1A2 and proinflammatory factors. These findings highlight the importance of carefully considering the dosage of rutaecarpine when combined with acetaminophen in drug design and preclinical trials.

Inhibition of CYP1A1 expression enhances diabetic wound healing by modulating inflammation and oxidative stress in a rat model.

OBJECTIVE: Wound therapies utilizing gene delivery to the skin offer considerable promise owing to their localized treatment benefits and straightforward application. This study investigated the impact of skin electroporation of CYP1A1 shRNA lentiviral particles on diabetic wound healing in a streptozotocin (STZ)-induced rat model. METHODS: Male Sprague Dawley (SD) rats were made diabetic by injecting STZ and subsequently creating foot skin wounds. The rats were randomly divided into four groups: normal, diabetic foot ulcers (DFU), DFU + control shRNA (electroporation of control shRNA lentiviral particles), and DFU + CYP1A1 shRNA (electroporation of CYP1A1 shRNA lentiviral particles). Wound healing progress was monitored at multiple time points (0, 1, 3, 5, 7, 10, 14 days). On day 14, wound tissue specimens were collected for histological examination. Wound samples collected at days 7 and 14 were used for gene expression analysis via qRT-PCR, assessment of CYP1A1 protein levels using western blotting, and evaluation of oxidative stress markers. RESULTS: Treatment with CYP1A1 shRNA significantly enhanced diabetic wound healing rates compared to untreated controls over the observation period. Histological analysis revealed improved wound characteristics in the CYP1A1 shRNA-treated group, including enhanced epithelial regeneration, reduced inflammation, and increased collagen deposition, indicative of improved tissue repair. Furthermore, suppression of CYP1A1 corresponded with decreased expression levels of pro-inflammatory cytokines (interleukin-1ß, tumor necrosis factor-α, and interleukin-6) and diminished oxidative stress markers (malondialdehyde, superoxide dismutase) within wound tissues. CONCLUSION: Targeted suppression of CYP1A1 represents a promising therapeutic strategy to enhance diabetic wound healing by modulating inflammation and oxidative stress.

Coffee Consumption and CYP1A2 Polymorphism Involvement in Type 2 Diabetes in a Romanian Population.

Cytochrome P450 1A2 (CYP1A2) is known to be the main enzyme directly responsible for caffeine metabolism. Rs762551 (NC_000015.10:g.74749576C>A) is a single nucleotide polymorphism of the CYP1A2 gene, and it is known mainly for metabolizing caffeine. A significant worldwide health issue, type 2 diabetes (T2DM), has been reported to be negatively associated with coffee consumption. Yet, some studies have proven that high intakes of coffee can lead to a late onset of T2DM. OBJECTIVES: This study aims to find any significant correlations among CYP1A2 polymorphism, coffee consumption, and T2DM. METHODS: A total of 358 people were enrolled in this study-218 diagnosed with T2DM, and 140 representing the control sample. The qPCR technique was performed, analyzing rs762551 (assay C_8881221) on the LightCycler 480 (Roche, Basel, Switzerland) with Gene Scanning software version 1.5.1 (Roche). RESULTS: Our first observation was that the diabetic patients were likely to consume more coffee than the non-diabetic subjects. People with the AA genotype, or the fast metabolizers, are the least common, yet they are the highest coffee consumers and present the highest glucose and cholesterol levels. Another important finding is the correlation between coffee intake and glucose level, which showed statistically significant differences between the diabetic group (p = 0.0002) and the control group (p = 0.029). CONCLUSIONS: The main conclusion of this study is that according to genotype, caffeine levels, glucose, and cholesterol are interconnected and proportionally related, regardless of type 2 diabetes.

The cytochrome P4501A1 (CYP1A1) inhibitor bergamottin enhances host tolerance to multidrug-resistant Vibrio vulnificus infection.

PURPOSE: Vibrio vulnificus (V. Vulnificus) infection is characterized by rapid onset, aggressive progression, and challenging treatment. Bacterial resistance poses a significant challenge for clinical anti-infection treatment and is thus the subject of research. Enhancing host infection tolerance represents a novel infection prevention strategy to improve patient survival. Our team initially identified cytochrome P4501A1 (CYP1A1) as an important target owing to its negative modulation of the body's infection tolerance. This study explored the superior effects of the CYP1A1 inhibitor bergamottin compared to antibiotic combination therapy on the survival of mice infected with multidrug-resistant V. Vulnificus and the protection of their vital organs. METHODS: An increasing concentration gradient method was used to induce multidrug-resistant V. Vulnificus development. We established a lethal infection model in C57BL/6J male mice and evaluated the effect of bergamottin on mouse survival. A mild infection model was established in C57BL/6J male mice, and the serum levels of creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase were determined using enzyme-linked immunosorbent assay to evaluate the effect of bergamottin on liver and kidney function. The morphological changes induced in the presence of bergamottin in mouse organs were evaluated by hematoxylin and eosin staining of liver and kidney tissues. The bacterial growth curve and organ load determination were used to evaluate whether bergamottin has a direct antibacterial effect on multidrug-resistant V. Vulnificus. Quantification of inflammatory factors in serum by enzyme-linked immunosorbent assay and the expression levels of inflammatory factors in liver and kidney tissues by real-time quantitative polymerase chain reaction were performed to evaluate the effect of bergamottin on inflammatory factor levels. Western blot analysis of IκBα, phosphorylated IκBα, p65, and phosphorylated p65 protein expression in liver and kidney tissues and in human hepatocellular carcinomas-2 and human kidney-2 cell lines was used to evaluate the effect of bergamottin on the nuclear factor kappa-B signaling pathway. One-way ANOVA and Kaplan-Meier analysis were used for statistical analysis. RESULTS: In mice infected with multidrug-resistant V. Vulnificus, bergamottin prolonged survival (p = 0.014), reduced the serum creatinine (p = 0.002), urea nitrogen (p = 0.030), aspartate aminotransferase (p = 0.029), and alanine aminotransferase (p = 0.003) levels, and protected the cellular morphology of liver and kidney tissues. Bergamottin inhibited interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α expression in serum (IL-1ß: p = 0.010, IL-6: p = 0.029, TNF-α: p = 0.025) and inhibited the protein expression of the inflammatory factors IL-1ß, IL-6, TNF-α in liver (IL-1ß: p = 0.010, IL-6: p = 0.011, TNF-α: p = 0.037) and kidney (IL-1ß: p = 0.016, IL-6: p = 0.011, TNF-α: p = 0.008) tissues. Bergamottin did not affect the proliferation of multidrug-resistant V. Vulnificus or the bacterial load in the mouse peritoneal lavage fluid (p = 0.225), liver (p = 0.186), or kidney (p = 0.637). CONCLUSION: Bergamottin enhances the tolerance of mice to multidrug-resistant V. Vulnificus infection. This study can serve as a reference and guide the development of novel clinical treatment strategies for V. Vulnificus.

Caffeine Consumption and Interaction with ADORA2A, CYP1A2 and NOS1 Variants Do Not Influence Age at Onset of Machado-Joseph Disease.

BACKGROUND: The age at onset (AO) of Machado-Joseph disease (SCA3/MJD), a disorder due to an expanded CAG repeat (CAGexp) in ATXN3, is quite variable and the role of environmental factors is still unknown. Caffeine was associated with protective effects against other neurodegenerative diseases, and against SCA3/MJD in transgenic mouse models. We aimed to evaluate whether caffeine consumption and its interaction with variants of caffeine signaling/metabolization genes impact the AO of this disease. METHODS: a questionnaire on caffeine consumption was applied to adult patients and unrelated controls living in Rio Grande do Sul, Brazil. AO and CAGexp were previously determined. SNPs rs5751876 (ADORA2A), rs2298383 (ADORA2A), rs762551 (CYP1A2) and rs478597 (NOS1) were genotyped. AO of subgroups were compared, adjusting the CAGexp to 75 repeats (p < 0.05). RESULTS: 171/179 cases and 98/100 controls consumed caffeine. Cases with high and low caffeine consumption (more or less than 314.5 mg of caffeine/day) had mean (SD) AO of 35.05 (11.44) and 35.43 (10.08) years (p = 0.40). The mean (SD) AO of the subgroups produced by the presence or absence of caffeine-enhancing alleles in ADORA2A (T allele at rs5751876 and rs2298383), CYP1A2 (C allele) and NOS1 (C allele) were all similar (p between 0.069 and 0.516). DISCUSSION: Caffeine consumption was not related to changes in the AO of SCA3/MJD, either alone or in interaction with protective genotypes at ADORA2A, CYP1A2 and NOS1.

Influence of the CYP1A2 c.-163 A > C polymorphism in the effect of caffeine on fat oxidation during exercise: a pilot randomized, double-blind, crossover, placebo-controlled trial.

PURPOSE: The aim of this study was to determine the influence of the CYP1A2 c.-163 A > C (rs762551) polymorphism on the effect of oral caffeine intake on fat oxidation during exercise. METHODS: Using a pilot randomized, double-blind, crossover, placebo-controlled trial, 32 young and healthy individuals (women = 14, men = 18) performed an incremental test on a cycle ergometer with 3-min stages at workloads from 30 to 70% of maximal oxygen uptake (VO2max). Participants performed this test after the ingestion of (a) placebo; (b) 3 mg/kg of caffeine; (c) 6 mg/kg of caffeine. Fat oxidation rate during exercise was measured by indirect calorimetry. The influence of the CYP1A2 c.-163 A > C polymorphism in the effect of caffeine on fat oxidation rates during exercise was established with a three-way ANOVA (substance × genotype × intensity). RESULTS: Eight participants were genotyped as AA, 18 participants were CA heterozygotes, and 6 participants were CC. There was a main effect of substance (F = 3.348, p = 0.050) on fat oxidation rates during exercise with no genotype effect (F = 0.158, p = 0.959). The post hoc analysis revealed that, in comparison to the placebo, 3 and 6 mg/kg of caffeine increased fat oxidation at 40-50% VO2max in AA (all p < 0.050) and 50-60% VO2max in CA and CC participants (all p < 0.050). CONCLUSION: Oral intake of 3 and 6 mg/kg of caffeine increased fat oxidation rate during aerobic exercise in individuals with AA, CA and CC genotypes. This suggests that the effect of caffeine to enhance fat oxidation during exercise is not influenced by the CYP1A2 c.-163 A > C polymorphism. TRIAL REGISTRATION: The study was registered on clinicaltrials.gov with ID: NCT05975489.

Metformin attenuates PM2.5-induced oxidative stress by inhibiting the AhR/CYP1A1 pathway in proximal renal tubular epithelial cells.

The harmful effects of PM2.5 on human health, including an increased risk of chronic kidney disease (CKD), have raised a lot of attention, but the underlying mechanisms are unclear. We used the Shanghai Meteorological and Environmental Animal Exposure System (Shanghai-METAS) to simulate the inhalation of PM2.5 in the real environment and established an animal model by exposing C57BL/6 mice to filtered air (FA) and Particulate Matter (PM2.5) for 8 weeks. PM2.5 impaired the renal function of the mice, and the renal tubules underwent destructive changes. Analysis of NHANES data showed a correlation between reduced kidney function and higher blood levels of PM2.5 components, polychlorinated biphenyls (PCBs) and dioxins, which are Aryl hydrocarbon Receptor (AhR) ligands. PM2.5 exposure induced higher levels of AhR and CYP1A1 and oxidative stress as evidenced by the higher levels of ROS, MDA, and GSSG in kidneys of mice. PM2.5 exposure led to AhR overexpression and nuclear translocation in proximal renal tubular epithelial cells. Inhibition of AhR reduced CYP1A1 expression and PM2.5-increased levels of ROS, MDA and GSSG. Our study suggested metformin can mitigate PM2.5-induced oxidative stress by inhibiting the AhR/CYP1A1 pathway. These findings illuminated the role of AhR/CYP1A1 pathway in PM2.5-induced kidney injury and the protective effect of metformin on PM2.5-induced cellular damage, offering new insights for air pollution-related renal diseases.

Association between CYP1A2 gene variants -163 C/A (rs762551) and -3860 G/A (rs2069514) and bladder cancer susceptibility.

BACKGROUND: Bladder cancer (BLCA) poses a significant global health challenge due to its high incidence, poor prognosis, and limited treatment options. AIMS AND OBJECTIVES: This study aims to investigate the association between two specific polymorphisms, CYP1A2-163 C/A and CYP1A2-3860G/A, within the Cytochrome P450 1A2 (CYP1A2) gene and susceptibility to BLCA. METHODS: The study employed a case-control design, genotyping 340 individuals using Polymerase Chain Reaction-High-Resolution Melting Curve (PCR-HRM). Various genetic models were applied to evaluate allele and genotype frequencies. Genetic linkage analysis was facilitated using R packages. RESULTS: The study reveals a significant association with the - 163 C/A allele, particularly in the additive model. Odds ratio (OR) analysis links CYP1A2-163 C/A (rs762551) and CYP1A2-3860G/A(rs2069514) polymorphisms to BLCA susceptibility. The rs762551 C/A genotype is prevalent in 55% of BLCA cases and exhibits an OR of 2.21. The A/A genotype has an OR of 1.54. Regarding CYP1A2-3860G/A, the G/A genotype has an OR of 1.54, and the A/A genotype has an OR of 2.08. Haplotype analysis shows a predominant C-C haplotype at 38.2%, followed by a C-A haplotype at 54.7%, and a less frequent A-A haplotype at 7.1%. This study underscores associations between CYP1A2 gene variants, particularly rs762551 (CYP1A2-163 C/A), and an increased susceptibility to BLCA. Haplotype analysis of 340 individuals reveals a predominant C-C haplotype at 38.2%, followed by a C-A haplotype at 54.7%, and a less frequent A-A haplotype at 7.1%. CONCLUSION: In conclusion, the - 163 C/A allele, C/A genotype of rs762551, and G/A genotype of rs2069514 emerge as potential genetic markers associated with elevated BLCA risk.

Development of FluoAHRL: A Novel Synthetic Fluorescent Compound That Activates AHR and Potentiates Anti-Inflammatory T Regulatory Cells.

Aryl Hydrocarbon Receptor (AHR) ligands, upon binding, induce distinct gene expression profiles orchestrated by the AHR, leading to a spectrum of pro- or anti-inflammatory effects. In this study, we designed, synthesized and evaluated three indole-containing potential AHR ligands (FluoAHRL: AGT-4, AGT-5 and AGT-6). All synthesized compounds were shown to emit fluorescence in the near-infrared. Their AHR agonist activity was first predicted using in silico docking studies, and then confirmed using AHR luciferase reporter cell lines. FluoAHRLs were tested in vitro using mouse peritoneal macrophages and T lymphocytes to assess their immunomodulatory properties. We then focused on AGT-5, as it illustrated the predominant anti-inflammatory effects. Notably, AGT-5 demonstrated the ability to foster anti-inflammatory regulatory T cells (Treg) while suppressing pro-inflammatory T helper (Th)17 cells in vitro. AGT-5 actively induced Treg differentiation from naïve CD4+ cells, and promoted Treg proliferation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression and interleukin-10 (IL-10) production. The increase in IL-10 correlated with an upregulation of Signal Transducer and Activator of Transcription 3 (STAT3) expression. Importantly, the Treg-inducing effect of AGT-5 was also observed in human tonsil cells in vitro. AGT-5 showed no toxicity when applied to zebrafish embryos and was therefore considered safe for animal studies. Following oral administration to C57BL/6 mice, AGT-5 significantly upregulated Treg while downregulating pro-inflammatory Th1 cells in the mesenteric lymph nodes. Due to its fluorescent properties, AGT-5 could be visualized both in vitro (during uptake by macrophages) and ex vivo (within the lamina propria of the small intestine). These findings make AGT-5 a promising candidate for further exploration in the treatment of inflammatory and autoimmune diseases.

Colistin Induces Oxidative Stress and Apoptotic Cell Death through the Activation of the AhR/CYP1A1 Pathway in PC12 Cells.

Colistin is commonly regarded as the "last-resort" antibiotic for combating life-threatening infections caused by multidrug-resistant (MDR) gram-negative bacteria. Neurotoxicity is a potential adverse event associated with colistin application in clinical settings, yet the exact molecular mechanisms remain unclear. This study examined the detrimental impact of colistin exposure on PC12 cells and the associated molecular mechanisms. Colistin treatment at concentrations of 0-400 µM decreased cell viability and induced apoptotic cell death in both time- and concentration-dependent manners. Exposure to colistin triggered the production of reactive oxygen species (ROS) and caused oxidative stress damage in PC12 cells. N-acetylcysteine (NAC) supplementation partially mitigated the cytotoxic and apoptotic outcomes of colistin. Evidence of mitochondrial dysfunction was observed through the dissipation of membrane potential. Additionally, colistin treatment upregulated the expression of AhR and CYP1A1 mRNAs in PC12 cells. Pharmacological inhibition of AhR (e.g., using α-naphthoflavone) or intervention with the CYP1A1 gene significantly decreased the production of ROS induced by colistin, subsequently lowering caspase activation and cell apoptosis. In conclusion, our findings demonstrate, for the first time, that the activation of the AhR/CYP1A1 pathway contributes partially to colistin-induced oxidative stress and apoptosis, offering insights into the cytotoxic effects of colistin.

Tizanidine-Induced Bradycardia Without Concomitant Medications: A Case Report.

A 37-year-old woman was admitted to our hospital due to a loss of consciousness. She had been taking 2 mg of tizanidine for two months to manage shoulder muscle pain at night. On admission, an electrocardiogram showed sinus bradycardia with a heart rate of 30 bpm and QT prolongation (QTc 495 msec). She had a temporary pacemaker inserted in the catheterization room, after which an improvement in her level of consciousness was observed. There were no apparent endocrine disorders or structural heart diseases. The administration was discontinued after admission, and 12 hours after admission, her heart rate normalized to a sinus rhythm of 70-100 bpm, and QTc improved to 431 msec. Therefore, she was diagnosed with tizanidine-induced bradycardia. Although reports of tizanidine-induced bradycardia are rare, tizanidine's central α2 agonistic effects can cause bradycardia, necessitating caution.

Development of a Normal Porcine Cell Line Growing in a Heme-Supplemented, Serum-Free Condition for Cultured Meat.

A key element for the cost-effective development of cultured meat is a cell line culturable in serum-free conditions to reduce production costs. Heme supplementation in cultured meat mimics the original meat flavor and color. This study introduced a bacterial extract generated from Corynebacterium that was selected for high-heme expression by directed evolution. A normal porcine cell line, PK15, was used to apply the bacterial heme extract as a supplement. Consistent with prior research, we observed the cytotoxicity of PK15 to the heme extract at 10 mM or higher. However, after long-term exposure, PK15 adapted to tolerate up to 40 mM of heme. An RNA-seq analysis of these heme-adapted PK15 cells (PK15H) revealed a set of altered genes, mainly involved in cell proliferation, metabolism, and inflammation. We found that cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), lactoperoxidase (LPO), and glutathione peroxidase 5 (GPX5) were upregulated in the PK15H heme dose dependently. When we reduced serum serially from 2% to serum free, we derived the PK15H subpopulation that was transiently maintained with 5-10 mM heme extract. Altogether, our study reports a porcine cell culturable in high-heme media that can be maintained in serum-free conditions and proposes a marker gene that plays a critical role in this adaptation process.

Circadian time-dependent effects of experimental colitis on theophylline disposition and toxicity.

BACKGROUND AND PURPOSE: Drug disposition undergoes significant alteration in patients with inflammatory bowel disease (IBD), yet circadian time-dependency of these changes remains largely unexplored. In this study, we aimed to determine the temporal effects of experimental colitis on drug disposition and toxicity. EXPERIMENTAL APPROACH: RNA-sequencing was used to screen genes relevant to colitis induced by dextran sodium sulfate in mice. Liver microsomes and pharmacokinetic analysis were used to analyze the activity of key enzymes. Dual luciferase assays and chromatin immunoprecipitation (ChIP) were employed to elucidate regulatory mechanisms. KEY RESULTS: RNA sequencing analysis revealed that colitis markedly influenced expression of cytochrome P450 (CYP) enzymes. Specifically, a substantial down-regulation of CYP1A2 and CYP2E1 was observed in livers of mice with colitis at Zeitgeber Time 8 (ZT8), with no significant changes detected at ZT20. At ZT8, the altered expression corresponded to diminished metabolism and enhanced incidence of hepato-cardiac toxicity of theophylline, a substrate specifically metabolized by these enzymes. A combination of assays, integrating liver-specific Bmal1 knockout and targeted activation of BMAL1 showed that dysregulation in CYP1A2 and CYP2E1 during colitis was attributable to perturbed BMAL1 functionality. Luciferase reporter and ChIP assays collectively substantiated the role of BMAL1 in regulating Cyp1a2 and Cyp2e1 transcription through its binding affinity to E-box-like sites. CONCLUSION AND IMPLICATION: Our findings establish a strong link between colitis and chronopharmacology, shedding light on how IBD affects drug disposition and toxicity over time. This research provides a theoretical foundation for optimizing drug dosage in patients with IBD.

METTL3-induced circ_0008345 contributes to the progression of colorectal cancer via the microRNA-182-5p/CYP1A2 pathway.

BACKGROUND: Circular RNA (circRNAs) have been found to play major roles in the progression of colorectal cancer (CRC). However, the functions of circ_0008345 (transcribed by PTK2) in regulating CRC development remain undefined. In this study, we aimed to explore the roles and underlying mechanisms of circ_0008345 in CRC. METHODS: RNase R-treated total cellular RNA was used to verify the circular structure of circ_0008345, and a subcellular fractionation assay was performed to detect the subcellular localization of circ_0008345. RNA pull-down and dual-luciferase assays were used to verify the binding relation between microRNA (miR)-182-5p and circ_0008345 and/or CYP1A2. Colony formation assay, EdU, and Transwell assays were performed to detect the biological behavior of CRC cells in vitro, and CRC cells were injected into mice to observe the tumor formation. m6A immunoprecipitation was used to detect the m6A modification of circ_0008345 in CRC cells. RESULTS: Circ_0008345, upregulated in CRC tissues and cells, was mainly present in the cytoplasm. Circ_0008345 bound to miR-182-5p, and miR-182-5p targeted CYP1A2, an oncogene in CRC. The colony formation, mobility, EdU-positive cell rate in vitro, and tumor growth in mice were inhibited after the knockdown of circ_0008345. However, the suppressing effects of sh-circ_0008345 on CRC and CYP1A2 expression were significantly reversed after further knockdown of miR-182-5p. METTL3 was the m6A modifier mediating circ_0008345 expression, and the suppression of METTL3 reduced the expression of circ_0008345. CONCLUSIONS: METTL3-dependent m6A methylation upregulated circ_0008345, which blocked the inhibitory effect of miR-182-5p on CYP1A2, thereby exacerbating the malignant phenotype of CRC cells.

Computer-aided discovery of novel aryl hydrocarbon receptor ligands to regulate CYP1A1 expression in inflammatory macrophages.

The environmental factor aryl hydrocarbon receptor (AhR), a key protein connecting the external environmental signals (e.g., environmental endocrine disruptor TCDD) to internal cellular processes, is involved in the activation of peripheral macrophages and inflammatory response in human body. Thus, there is widespread interest in finding compounds to anti-inflammatory response in macrophages by targeting human AhR. Here, ensemble docking based-virtual screening was first used to screen a library (~200,000 compounds) against human AhR ligand binding domain (LBD) and 25 compounds were identified as potential inhibitors. Then, 9 out of the 25 ligands were found to down-regulate the mRNA expression of CYP1A1 (a downstream gene of AhR signaling) in AhR overexpressing macrophages. The most potent compound AE-411/41415610 was selected for further study and found to reduce both mRNA and protein expressions level of CYP1A1 in mouse peritoneal macrophage. Moreover, protein chip signal pathway analysis indicated that AE-411/41415610 play a role in regulating JAK-STAT and AKT-mTOR pathways. In sum, the discovered hits with novel scaffolds provided a starting point for future design of more effective AhR-targeted lead compounds to regulate CYP1A1 expression of inflammatory peritoneal macrophages.

New insights into aflatoxin B1 mechanistic toxicology in cattle liver: an integrated approach using molecular docking and biological evaluation in CYP1A1 and CYP3A74 knockout BFH12 cell lines.

Aflatoxin B1 (AFB1) is a pro-carcinogenic compound bioactivated in the liver by cytochromes P450 (CYPs). In mammals, CYP1A and CYP3A are responsible for AFB1 metabolism, with the formation of the genotoxic carcinogens AFB1-8,9-epoxide and AFM1, and the detoxified metabolite AFQ1. Due to climate change, AFB1 cereals contamination arose in Europe. Thus, cattle, as other farm animals fed with grains (pig, sheep and broiler), are more likely exposed to AFB1 via feed with consequent release of AFM1 in milk, posing a great concern to human health. However, knowledge about bovine CYPs involved in AFB1 metabolism is still scanty. Therefore, CYP1A1- and CYP3A74-mediated molecular mechanisms of AFB1 hepatotoxicity were here dissected. Molecular docking of AFB1 into CYP1A1 model suggested AFB1 8,9-endo- and 8,9-exo-epoxide, and AFM1 formation, while docking of AFB1 into CYP3A74 pointed to AFB1 8,9-exo-epoxide and AFQ1 synthesis. To biologically confirm these predictions, CYP1A1 and CYP3A74 knockout (KO) BFH12 cell lines were exposed to AFB1. LC-MS/MS investigations showed the abolished production of AFM1 in CYP1A1 KO cells and the strong increase of parent AFB1 in CYP3A74 KO cells; the latter result, coupled to a decreased cytotoxicity, suggested the major role of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved lower AFB1-induced cytotoxic effects in engineered cells versus naïve ones. Overall, this study broadens the knowledge on AFB1 metabolism and hepatotoxicity in cattle, and it provides the weight of evidence that CYP1A1 and CYP3A74 inhibition might be exploited to reduce AFM1 and AFBO synthesis, AFB1 toxicity, and AFM1 milk excretion.