Synergistic lignin degradation between Phanerochaete chrysosporium and Fenton chemistry is mediated through iron cycling and ligninolytic enzyme induction.

Removal of recalcitrant lignin from wastewater remains a critical bottleneck in multiple aspects relating to microbial carbon cycling ranging from incomplete treatment of biosolids during wastewater treatment to limited conversion of biomass feedstock to biofuels. Based on previous studies showing that the white rot fungus Phanerochaete chrysosporium and Fenton chemistry synergistically degrade lignin, we sought to determine optimum levels of Fenton addition and the mechanisms underlying this synergy. We tested the extent of degradation of lignin under different ratios of Fenton reagents and found that relatively low levels of H2O2 and Fe(II) enhanced fungal lignin degradation, achieving 80.4 ± 1.61 % lignin degradation at 1.5 mM H2O2 and 0.3 mM Fe(II). Using a combination of whole-transcriptome sequencing and iron speciation assays, we determined that at these concentrations, Fenton chemistry induced the upregulation of 80 differentially expressed genes in P. ch including several oxidative enzymes. This study underlines the importance of non-canonical, auxiliary lignin-degrading pathways in the synergy between white rot fungi and Fenton chemistry in lignin degradation. We also found that, relative to the abiotic control, P. ch. increases the availability of Fe(II) for the production of hydroxyl radicals in the Fenton reaction by recycling Fe(III) (p < 0.001), decreasing the Fe(II) inputs necessary for lignin degradation via the Fenton reaction.

Decreased serum voriconazole levels caused by hepatic enzyme induction after rifapentine discontinuation: a case report and literature review.

BACKGROUND: Rifapentine is a rifamycin with unique bactericidal activity against Mycobacterium tuberculosis. It is also a potent inducer of CYP3A activity. However, the duration of rifapentine-induced hepatic enzyme activity after withdrawal is unclear. CASE REPORT: We report a case of a patient with Aspergillus meningitis treated with voriconazole after discontinuing rifapentine. Within ten days of rifapentine discontinuation, serum levels of voriconazole failed to reach the effective treatment range. CONCLUSIONS: Rifapentine is a potent inducer of hepatic microsomal enzymes. The induction of hepatic enzymes may exceed ten days after rifapentine discontinuation. Clinicians should be reminded of residual enzyme induction by rifapentine, especially when treating critically ill patients.

Population Pharmacokinetic Analysis of Drug-Drug Interactions Between Perampanel and Carbamazepine Using Enzyme Induction Model in Epileptic Patients.

BACKGROUND: Perampanel (PER) is an oral antiepileptic drug and its concomitant use with carbamazepine (CBZ) leads to decreased PER concentrations. However, the magnitude of its influence may vary, depending on the dynamics of the enzyme induction properties of CBZ. This study aimed to develop a population pharmacokinetic (PPK) model considering the dynamics of enzyme induction and evaluate the effect of CBZ on PER pharmacokinetics. METHODS: We retrospectively collected data on patient background, laboratory tests, and prescribed drugs from electronic medical records. We developed 2 PPK models incorporating the effect of CBZ-mediated enzyme induction to describe time-concentration profiles of PER using the following different approaches: (1) treating the concomitant use of CBZ as a categorical covariate (empirical PPK model) and (2) incorporating the time-course of changes in the amount of enzyme by CBZ-mediated induction (semimechanistic PPK model). The bias and precision of the predictions were investigated by calculating the mean error, mean absolute error, and root mean squared error. RESULTS: A total of 133 PER concentrations from 64 patients were available for PPK modelling. PPK analyses showed that the co-administration of CBZ increased the clearance of PER. Goodness-of-fit plots indicated a favorable description of the observed data and low bias. The mean error, mean absolute error, and root mean square error values based on the semimechanistic model were smaller than those obtained using the empirical PPK model for predicting PER concentrations in patients with CBZ. CONCLUSIONS: We developed 2 PPK models to describe PER pharmacokinetics based on different approaches, using electronic medical record data. Our PPK models support the use of PER in clinical practice.

Boscalid shows increased thyroxin-glucuronidation in rat but not in human hepatocytes in vitro.

The fungicide boscalid induces thyroid histopathological and hormone effects in the rat, secondary to liver enzyme induction. To assess the human relevance of liver enzyme induction presumably leading to thyroid hormone disruption, a species comparative in vitro study on T4-glucuronidation was conducted. Currently, no guidelines how to evaluate Phase II induction are in place. Therefore, we investigated the optimal conditions to evaluate Phase I and Phase II induction potential of boscalid in primary rat (PRH) and human (PHH) hepatocytes. Endpoints included mRNA gene expression and enzyme activities (cytochrome P450 isozymes [CYPs] and uridine diphosphate-glucuronosyltransferases [UGTs]), measured after 3 (D3) and 7 (D7) days of exposure to reference compounds and to 5, 10, and 20 µM boscalid, focusing on T4-glucuronidation. Basal CYP activities and T4 glucuronidation were similar or higher on D7 than D3. The highest induction responses of CYPs were on D3, whereas UGT induction and T4-glucuronidation increases were highest on D7. Boscalid induced CYP1A, CYP2B, and CYP3A mRNA and/or increased related activities in PRH and PHH. Species differences in the induction pattern of UGT genes by reference inducers (ß-naphthoflavone [BNF], 5-pregnen-3ß-ol-20-one-16α-carbonitirile [PCN], rifampicin [RIF], and phenobarbital [PB]) and boscalid were seen: UGT1A1, UGT1A3, and UGT1A9 were predominantly induced in PHH, while UGT2B1 was predominantly induced in PRH. Basal activity levels for T4-glucuronidation were very low in humans and an order of magnitude higher in rat, for this reason increases in activities were assessed as delta activity to the control. Significant increases in T4-glucuronidation occurred with boscalid in rat but not in human hepatocytes.

Quantitative Cytochrome P450 3A4 Induction Risk Assessment Using Human Hepatocytes Complemented with Pregnane X Receptor-Activating Profiles.

Reliable in vitro to in vivo translation of cytochrome P450 (CYP) 3A4 induction potential is essential to support risk mitigation for compounds during pharmaceutical discovery and development. In this study, a linear correlation of CYP3A4 mRNA induction potential in human hepatocytes with the respective pregnane-X receptor (PXR) activation in a reporter gene assay using DPX2 cells was successfully demonstrated for 13 clinically used drugs. Based on this correlation, using rifampicin as a positive control, the magnitude of CYP3A4 mRNA induction for 71 internal compounds at several concentrations up to 10 µM (n = 90) was predicted within 2-fold error for 64% of cases with only a few false positives (19%). Furthermore, the in vivo area under the curve reduction of probe CYP substrates was reasonably predicted for eight marketed drugs (carbamazepine, dexamethasone, enzalutamide, nevirapine, phenobarbital, phenytoin, rifampicin, and rufinamide) using the static net effect model using both the PXR activation and CYP3A4 mRNA induction data. The liver exit concentrations were used for the model in place of the inlet concentrations to avoid false positive predictions and the concentration achieving twofold induction (F2) was used to compensate for the lack of full induction kinetics due to cytotoxicity and solubility limitations in vitro. These findings can complement the currently available induction risk mitigation strategy and potentially influence the drug interaction modeling work conducted at clinical stages. SIGNIFICANCE STATEMENT: The established correlation of CYP3A4 mRNA in human hepatocytes to PXR activation provides a clear cut-off to identify a compound showing an in vitro induction risk, complementing current regulatory guidance. Also, the demonstrated in vitro-in vivo translation of induction data strongly supports a clinical development program although limitations remain for drug candidates showing complex disposition pathways, such as involvement of auto-inhibition/induction, active transport and high protein binding.

Ensemble Docking Approach to Mitigate Pregnane X Receptor-Mediated CYP3A4 Induction Risk.

Three structurally closely related dopamine D1 receptor positive allosteric modulators (D1 PAMs) based on a tetrahydroisoquinoline (THIQ) scaffold were profiled for their CYP3A4 induction potentials. It was found that the length of the linker at the C5 position greatly affected the potentials of these D1 PAMs as CYP3A4 inducers, and the level of induction correlated well with the activation of the pregnane X receptor (PXR). Based on the published PXR X-ray crystal structures, we built a binding model specifically for these THIQ-scaffold-based D1 PAMs in the PXR ligand-binding pocket via an ensemble docking approach and found the model could explain the observed CYP induction disparity. Combined with our previously reported D1 receptor homology model, which identified the C5 position as pointing toward the solvent-exposed space, our PXR-binding model coincidentally suggested that structural modifications at the C5 position could productively modulate the CYP induction potential while maintaining the D1 PAM potency of these THIQ-based PAMs.

Effects of Enzyme Induction and Polymorphism on the Pharmacokinetics of Isoniazid and Rifampin in Tuberculosis/HIV Patients.

Tuberculosis is the most common cause of death in HIV-infected individuals. Rifampin and isoniazid are the backbones of the current first-line antitubercular therapy. The aim of the present study was to describe the time-dependent pharmacokinetics and pharmacogenetics of rifampin and isoniazid and to quantitatively evaluate the drug-drug interaction between rifampin and isoniazid in patients coinfected with HIV. Plasma concentrations of isoniazid, acetyl-isoniazid, isonicotinic acid, rifampin, and 25-desacetylrifampin from 40 HIV therapy-naive patients were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after the first dose and at steady state of antitubercular therapy. Patients were genotyped for determination of acetylator status and CYP2C19 phenotype. Nonlinear mixed-effects models were developed to describe the pharmacokinetic data. The model estimated an autoinduction of both rifampin bioavailability (0.5-fold) and clearance (2.3-fold). 25-Desacetylrifampin clearance was 2.1-fold higher at steady state than after the first dose. Additionally, ultrarapid CYP2C19 metabolizers had a 2-fold-higher rifampin clearance at steady state than intermediate or extensive metabolizers. An induction of isonicotinic acid formation from isoniazid dependent on total rifampin dose was estimated. Simulations indicated a 30% lower isoniazid exposure at steady state during administration of standard rifampin doses than isoniazid exposure in noninduced individuals. Rifampin exposure was correlated with CYP2C19 polymorphism, and rifampin administration may increase exposure to toxic metabolites by isoniazid in patients. Both findings may influence the risk of treatment failure, resistance development, and toxicity and require further investigation, especially with regard to ongoing high-dose rifampin trials.

Label-free chemical imaging of cytochrome P450 activity by Raman microscopy.

Although investigating drug modulation of cytochrome P450 (CYP) activity under physiological conditions is crucial in drug development to avoid severe adverse drug reactions, the current evaluation approaches that rely on the destructive and end-point analysis can be misleading due to invasive treatments and cellular heterogeneity. Here, we propose a non-destructive and high-content method for visualizing and quantifying intracellular CYP activity under drug administration by Raman microscopy. The redox-state and spin-state sensitive Raman measurement indicated that the induced CYPs in living hepatocytes were in oxidized and low-spin state, which is related to monooxygenase function of CYP. Moreover, glycogen depletion associated with CYP induction was simultaneously observed, indicating a relevant effect on glucose metabolism. By deciphering the overall changes in the biochemical fingerprints of hepatocytes, Raman microscopy offers a non-destructive and quantitative chemical imaging method to evaluate CYP activity at the single-cell level with the potential to facilitate future drug development schemes.

A novel approach to predict the comprehensive EROD potency: Mechanism-based curve fitting of CYP1A1 activity by PAHs.

Cytochrome P450 1A1 (CYP1A1) plays critical roles in polycyclic aromatic hydrocarbon (PAH) toxicity, including DNA adduction and ROS generation. Therefore, CYP1A1 activity quantified by the 7-ethoxyresorufin-O-deethylase (EROD) assay (named EROD potency) has been considered a typical biomarker of PAH exposure and toxicity. The EROD dose-response curve always presents a biphasic style, increasing at low concentrations and decreasing at high concentrations of PAHs, but relative effect potency (REP) commonly used in PAH risk assessment is only involved in the increasing phase. In this study, a full bell-shaped EROD curve fitting formula Eq. (1) was obtained by considering both CYP1A1 mRNA induction and enzyme inhibition to completely assess the EROD potency of PAHs. Correspondingly, in silico models of QSAR and docking methods successfully predicted the full EROD curves of PAHs, and the structure-activity relationship indicated that PAHs with heavy molecular weight and large diameter showed stronger EROD potency. Further EROD potency with predicted curve parameters (EC50,ind and area index) was confirmed by the reported REP (R2 = 0.697-0.977) and experimental data from human and mouse cells (R2 = 0.700-0.804). This study provides a novel curve fitting for the EROD dose-response relationship and a prediction model for PAH EROD potency.

Perspective of the Induction of Liver Microsomal Cytochrome P450s by Chemical Compounds.

The concept of hepatic induction of drug-metabolizing cytochrome P450s (P450s) by xenobiotics, including therapeutic drugs, was proposed in the early 1960s. A polycyclic aromatic hydrocarbon and phenobarbital have been the two major inducers used to investigate this induction mechanism. Currently, the mechanisms mediated by aryl hydrocarbon receptor and constitutive androstane receptor are well-established. In addition to mammals, insects and fungi also express P450s and induce them following exposure to insecticides. These inductions may have environmental consequences. Finding the molecular mechanism regulating these inductions will be of major interest in the future. SIGNIFICANCE STATEMENT: This paper summarizes present and future of investigations into induction of drug-metabolizing enzymes.

Mycobacterium tuberculosis Induces Irg1 in Murine Macrophages by a Pathway Involving Both TLR-2 and STING/IFNAR Signaling and Requiring Bacterial Phagocytosis.

Irg1 is an enzyme that generates itaconate, a metabolite that plays a key role in the regulation of inflammatory responses. Previous studies have implicated Irg1 as an important mediator in preventing excessive inflammation and tissue damage in Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the pattern recognition receptors and signaling pathways by which Mtb triggers Irg1 gene expression by comparing the responses of control and genetically deficient BMDMs. Using this approach, we demonstrated partial roles for TLR-2 (but not TLR-4 or -9), MyD88 and NFκB signaling in Irg1 induction by Mtb bacilli. In addition, drug inhibition studies revealed major requirements for phagocytosis and endosomal acidification in Irg1 expression triggered by Mtb but not LPS or PAM3CSK4. Importantly, the Mtb-induced Irg1 response was highly dependent on the presence of the bacterial ESX-1 secretion system, as well as host STING and Type I IFN receptor (IFNAR) signaling with Type II IFN (IFN-γ) signaling playing only a minimal role. Based on these findings we hypothesize that Mtb induces Irg1 expression in macrophages via the combination of two independent triggers both dependent on bacterial phagocytosis: 1) a major signal stimulated by phagocytized Mtb products released by an ESX-1-dependent mechanism into the cytosol where they activate the STING pathway leading to Type I-IFN production, and 2) a secondary TLR-2, MyD88 and NFκB dependent signal that enhances Irg1 production independently of Type I IFN induction.

Induction by Phenobarbital of Phase I and II Xenobiotic-Metabolizing Enzymes in Bovine Liver: An Overall Catalytic and Immunochemical Characterization.

In cattle, phenobarbital (PB) upregulates target drug-metabolizing enzyme (DME) mRNA levels. However, few data about PB's post-transcriptional effects are actually available. This work provides the first, and an almost complete, characterization of PB-dependent changes in DME catalytic activities in bovine liver using common probe substrates and confirmatory immunoblotting investigations. As expected, PB increased the total cytochrome P450 (CYP) content and the extent of metyrapone binding; moreover, an augmentation of protein amounts and related enzyme activities was observed for known PB targets such as CYP2B, 2C, and 3A, but also CYP2E1. However, contradictory results were obtained for CYP1A, while a decreased catalytic activity was observed for flavin-containing monooxygenases 1 and 3. The barbiturate had no effect on the chosen hydrolytic and conjugative DMEs. For the first time, we also measured the 26S proteasome activity, and the increase observed in PB-treated cattle would suggest this post-translational event might contribute to cattle DME regulation. Overall, this study increased the knowledge of cattle hepatic drug metabolism, and further confirmed the presence of species differences in DME expression and activity between cattle, humans, and rodents. This reinforced the need for an extensive characterization and understanding of comparative molecular mechanisms involved in expression, regulation, and function of DMEs.

Emerging Glycation-Based Therapeutics-Glyoxalase 1 Inducers and Glyoxalase 1 Inhibitors.

The abnormal accumulation of methylglyoxal (MG) leading to increased glycation of protein and DNA has emerged as an important metabolic stress, dicarbonyl stress, linked to aging, and disease. Increased MG glycation produces inactivation and misfolding of proteins, cell dysfunction, activation of the unfolded protein response, and related low-grade inflammation. Glycation of DNA and the spliceosome contribute to an antiproliferative and apoptotic response of high, cytotoxic levels of MG. Glyoxalase 1 (Glo1) of the glyoxalase system has a major role in the metabolism of MG. Small molecule inducers of Glo1, Glo1 inducers, have been developed to alleviate dicarbonyl stress as a prospective treatment for the prevention and early-stage reversal of type 2 diabetes and prevention of vascular complications of diabetes. The first clinical trial with the Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP)-a randomized, double-blind, placebo-controlled crossover phase 2A study for correction of insulin resistance in overweight and obese subjects, was completed successfully. tRES-HESP corrected insulin resistance, improved dysglycemia, and low-grade inflammation. Cell permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a prospective treatment for cancer-particularly for high Glo1 expressing-related multidrug-resistant tumors. The prototype Glo1 inhibitor is prodrug S-p-bromobenzylglutathione cyclopentyl diester (BBGD). It has antitumor activity in vitro and in tumor-bearing mice in vivo. In the National Cancer Institute human tumor cell line screen, BBGD was most active against the glioblastoma SNB-19 cell line. Recently, potent antitumor activity was found in glioblastoma multiforme tumor-bearing mice. High Glo1 expression is a negative survival factor in chemotherapy of breast cancer where adjunct therapy with a Glo1 inhibitor may improve treatment outcomes. BBGD has not yet been evaluated clinically. Glycation by MG now appears to be a pathogenic process that may be pharmacologically manipulated for therapeutic outcomes of potentially important clinical impact.

Characterization of Correction Factors to Enable Assessment of Clinical Risk from In Vitro CYP3A4 Induction Data and Basic Drug-Drug Interaction Models.

BACKGROUND AND OBJECTIVE: Induction of drug-metabolizing enzymes can lead to drug-drug interactions (DDIs); therefore, early assessment is often conducted. Previous reports focused on true positive cytochrome P450 3A (CYP3A) inducers leaving a gap in translation for in vitro inducers which do not manifest in clinical induction. The goal herein was to expand the in vitro induction dataset by including true negative clinical inducers to identify a correction factor to basic DDI models, which reduces false positives without impacting false negatives. METHODS: True negative clinical inducers were identified through a literature search, in vitro induction parameters were generated in three human hepatocyte donors, and the performance of basic induction models proposed by regulatory agencies, concentration producing twofold induction (F2), basic static model (R3) and relative induction score (RIS), was used to characterize clinical induction risk. RESULTS: The data demonstrated the importance of correcting for in vitro binding and metabolism to derive induction parameters. The aggregate analysis indicates that the RIS with a positive cut-off of < 0.7-fold area under the curve ratio (AUCR) provides the best quantitative prediction. Additionally, correction factors of ten and two times the unbound peak plasma concentration at steady state (Cmax,ss,u) can be confidently used to identify true positive inducers when referenced against the concentration resulting in twofold increase in messenger ribonucleic acid (mRNA) or using the R3 equation, respectively. CONCLUSIONS: These iterative improvements, which reduce the number of false positives, could aid regulatory recommendations and limit unnecessary clinical explorations into CYP3A induction.

Proteomic and Bioinformatics Analysis of Membrane Lipid Domains after Brij 98 Solubilization of Uninduced and Phenobarbital-Induced Rat Liver Microsomes: Defining the Membrane Localization of the P450 Enzyme System.

The proteomes of ordered and disordered lipid microdomains in rat liver microsomes from control and phenobarbital (PB)-treated rats were determined after solubilization with Brij 98 and analyzed by tandem mass tag (TMT)-liquid chromatography-mass spectrometry (LC-MS). This allowed characterization of the liver microsomal proteome and the effects of phenobarbital-mediated induction, focusing on quantification of the relative levels of the drug-metabolizing enzymes._The microsomal proteome from control rats was represented by 333 (23%) proteins from ordered lipid microdomains, 517 (36%) proteins from disordered lipid domains, and 587 (41%) proteins that uniformly distributed between lipid microdomains. Most enzymes related to drug metabolism were mainly localized in disordered lipid microdomains. However, cytochrome P450 (CYP) 1A2, multiple forms of CYP2D, and several forms of UDP glucuronosyltransferases (UGT) 1A1 and 1A6) localized to ordered lipid microdomains. Other drug-metabolizing enzymes, including several forms of cytochromes P450, were uniformly distributed between the ordered and disordered regions. The redox partners, NADPH-cytochrome P450 reductase and cytochrome b5, localized to disordered microdomains. PB induction resulted in only modest changes in protein localization. Less than five proteins were variably associated with the ordered and disordered membrane microdomains in PB and control microsomes. PB induction was associated with fewer proteins localizing in the disordered membranes and more being uniformly distributed or localized to ordered domains. Ingenuity Pathway Analysis (IPA) was used to ascertain the effect of PB on cellular pathways, resulting in attenuation of pathways related to energy storage/utilization and overall cellular signaling and an increase in those related to degradative pathways. SIGNIFICANCE STATEMENT: This work identifies the lipid microdomain localization of the proteome from control and phenobarbital-induced rat liver microsomes. Thus, it provides an initial framework to understand how lipid/protein segregation influences protein-protein interactions in a tissue extract commonly used for studies in drug metabolism and uses bioinformatics to elucidate the effects of phenobarbital induction on cellular pathways.

CYP450 drug inducibility in NAFLD via an in vitro hepatic model: Understanding drug-drug interactions in the fatty liver.

Drug-drug-interactions (DDIs) occur when a drug alters the metabolic rate, efficacy, and toxicity of concurrently used drugs. While almost 1 in 4 adults now use at least 3 concurrent prescription drugs in the United States, the Non-alcoholic fatty liver disease (NAFLD) prevalence has also risen over 25%. The effect of NALFD on DDIs is largely unknown. NAFLD is characterized by lipid vesicle accumulation in the liver, which can progress to severe steatohepatitis (NASH), fibrosis, cirrhosis, and hepatic carcinoma. The CYP450 enzyme family dysregulation in NAFLD, which might already alter the efficacy and toxicity of drugs, has been partially characterized. Nevertheless, the drug-induced dysregulation of CYP450 enzymes has not been studied in the fatty liver. These changes in enzymatic inducibility during NAFLD, when taking concurrent drugs, could cause unexpected fatalities through inadvertent DDIs. We have, thus, developed an in vitro model to investigate the CYP450 transcriptional regulation in NAFLD. Specifically, we cultured primary human hepatocytes in a medium containing free fatty acids, high glucose, and insulin for seven days. These cultures displayed intracellular macro-steatosis after 5 days and cytokine secretion resembling NAFLD patients. We further verified the model's dysregulation in the transcription of key CYP450 enzymes. We then exposed the NAFLD model to the drug inducers rifampicin, Omeprazole, and Phenytoin as activators of transcription factors pregnane X receptor (PXR), aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR), respectively. In the NAFLD model, Omeprazole maintained an expected induction of CYP1A1, however Phenytoin and Rifampicin showed elevated induction of CYP2B6 and CYP2C9 compared to healthy cultures. We, thus, conclude that the fatty liver could cause aggravated drug-drug interactions in NAFLD or NASH patients related to CYP2B6 and CYP2C9 enzymes.

Heme Oxygenase-1 Inhibits the Proliferation of Hepatic Stellate Cells by Activating PPARγ and Suppressing NF-κB.

BACKGROUND: Hepatic stellate cells (HSCs) are reported to play significant roles in the development of liver fibrosis. Heme oxygenase-1 (HO-1) is a key rate-limiting enzyme, which could decrease collagen synthesis and liver damage. Nevertheless, it was yet elusive towards the function and mechanism of HO-1. METHODS: An HO-1 inducer Hemin or an HO-1 inhibitor ZnPP-IX was used to treat the activated HSC-T6, respectively. MTT assay was adopted to detect cell proliferation. Immunocytochemical staining was employed to test the levels of alpha-smooth muscle actin (α-SMA), peroxisome proliferator-activated receptor-γ (PPARγ), and nuclear factor-kappa B (NF-kappa B) levels in HSC-T6. HO-1, PPARγ, and NF-κB expression levels were measured by qRT-PCR and Western blotting. ELISA was then used to detect the levels of transforming growth factor- (TGF-) beta 1 (TGF-ß1), interleukin-6 (IL-6), serum hyaluronic acid (HA), and serum type III procollagen aminopeptide (PIIIP). RESULTS: HSC-T6 proliferation was inhibited in Hemin-treated HSCs. The levels of α-SMA, HA, and PIIIP and the production of ECM were lower in Hemin-treated HSCs, whereas those could be rescued by ZnPP-IX. NF-κB activation was decreased, but PPARγ expression was increased after HO-1 upregulation. Furthermore, the levels of TGF-ß1 and IL-6, which were downstream of activated NF-κB in HSC-T6, were reduced. The PPAR-specific inhibitor GW9662 could block those mentioned effects. CONCLUSIONS: Our data demonstrated that HO-1 induction could inhibit HSC proliferation and activation by regulating PPARγ expression and NF-κB activation directly or indirectly, which makes it a promising therapeutic target for liver fibrosis.

Hypoxia Drives Centrosome Amplification in Cancer Cells via HIF1α-dependent Induction of Polo-Like Kinase 4.

Centrosome amplification (CA) has been implicated in the progression of various cancer types. Although studies have shown that overexpression of PLK4 promotes CA, the effect of tumor microenvironment on polo-like kinase 4 (PLK4) regulation is understudied. The aim of this study was to examine the role of hypoxia in promoting CA via PLK4. We found that hypoxia induced CA via hypoxia-inducible factor-1α (HIF1α). We quantified the prevalence of CA in tumor cell lines and tissue sections from breast cancer, pancreatic ductal adenocarcinoma (PDAC), colorectal cancer, and prostate cancer and found that CA was prevalent in cells with increased HIF1α levels under normoxic conditions. HIF1α levels were correlated with the extent of CA and PLK4 expression in clinical samples. We analyzed the correlation between PLK4 and HIF1A mRNA levels in The Cancer Genome Atlas (TCGA) datasets to evaluate the role of PLK4 and HIF1α in breast cancer and PDAC prognosis. High HIF1A and PLK4 levels in patients with breast cancer and PDAC were associated with poor overall survival. We confirmed PLK4 as a transcriptional target of HIF1α and demonstrated that in PLK4 knockdown cells, hypoxia-mimicking agents did not affect CA and expression of CA-associated proteins, underscoring the necessity of PLK4 in HIF1α-related CA. To further dissect the HIF1α-PLK4 interplay, we used HIF1α-deficient cells overexpressing PLK4 and showed a significant increase in CA compared with HIF1α-deficient cells harboring wild-type PLK4. These findings suggest that HIF1α induces CA by directly upregulating PLK4 and could help us risk-stratify patients and design new therapies for CA-rich cancers. IMPLICATIONS: Hypoxia drives CA in cancer cells by regulating expression of PLK4, uncovering a novel HIF1α/PLK4 axis.

Transcription factor paired related homeobox 1 (PRRX1) activates matrix metalloproteinases (MMP)13, which promotes the dextran sulfate sodium-induced inflammation and barrier dysfunction of NCM460 cells.

Paired related homeobox 1 (PRRX1) is a newly identified transcription factor that regulates the expression of various genes. We aimed to investigate the roles of PRRX1 and Matrix metalloproteinases (MMP)13 in dextran sulfate sodium (DSS)-induced inflammation and barrier dysfunction of NCM460 cells. PRRX1 expression in the mucosal tissues of patients with ulcerative colitis was analyzed using the GSE87466 microarray. PRRX1 and MMP13 expression was examined using Western blotting and RT-qPCR following the exposure of the NCM460 cells to DSS. The JASPAR database was used to predict the binding sites of PRRX1 to the MMP13 promoter, which was verified by luciferase reporter and chromatin immunoprecipitation assays. MMP13 expression was then detected following PRRX1 silencing or overexpression. The levels of inflammatory factors were determined using ELISA. Finally, the expression of intestinal barrier function-related proteins was evaluated using Western blotting and cellular permeability was detected by Transepithelial electrical resistance. PRRX1 was upregulated in the mucosal tissue samples of patients with UC. DSS induction upregulated PRRX1 and MMP13 expression. PRRX1 bound to the promoter of MMP13, which was further supported by the decreased expression of MMP13 observed following PRRX1 knockdown and its increased expression following PRRX1 overexpression. Furthermore, PRRX1 deletion decreased TNF-α, IL-1ß and IL-6 levels in the DSS-challenged NCM460 cells, which were subjected to MMP13 overexpression. Moreover, PRRX1 silencing upregulated ZO-1, occludin and claudin-1 expression and elevated the TEER value, whereas MMP13 overexpression attenuated these effects. Collectively, PRRX1 activates MMP13, which in turn promotes the DSS-induced inflammation and barrier dysfunction of NCM460 cells.

A Negative Feedback Loop and Transcription Factor Cooperation Regulate Zonal Gene Induction by 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin in the Mouse Liver.

The cytochrome P450 (Cyp) proteins Cyp1A1 and Cyp1A2 are strongly induced in the mouse liver by the potent environmental toxicant 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), acting through the aryl hydrocarbon receptor (AHR). The induction of Cyp1A1 is localized within the centrilobular regions of the mouse liver at low doses of TCDD, progressing to pan-lobular induction at higher doses. Even without chemical perturbation, metabolic functions and associated genes are basally zonated in the liver lobule along the central-to-portal axis. To investigate the mechanistic basis of spatially restricted gene induction by TCDD, we have developed a multiscale computational model of the mouse liver lobule with single-cell resolution. The spatial location of individual hepatocytes in the model was calibrated from previously published high-resolution images. A systems biology model of the network of biochemical signaling pathways underlying Cyp1A1 and Cyp1A2 induction was then incorporated into each hepatocyte in the model. Model simulations showed that a negative feedback loop formed by binding of the induced Cyp1A2 protein to TCDD, together with cooperative gene induction by the ß-catenin/AHR/TCDD transcription factor complex and ß-catenin, help produce the spatially localized induction pattern of Cyp1A1. Although endogenous WNT regulates the metabolic zonation of many genes, it was not a driver of zonal Cyp1A1 induction in our model. Conclusion: In this work, we used data-driven computational modeling to identify the mechanistic basis of zonally restricted gene expression induced by the potent and persistent environmental pollutant TCDD. The multiscale model and derived results clarify the mechanisms of dose-dependent hepatic gene induction responses to TCDD. Additionally, this work contributes to our broader understanding of spatial gene regulation along the liver lobule.