Pregnane X receptor (PXR) deficiency promotes hepatocarcinogenesis via induction of Akr1c18 expression and prostaglandin F2α (PGF2α) levels.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, plays a critical role in the metabolism of endogenous and exogenous substances in the liver. Here, we investigate whether PXR plays a role in pathogenesis of HCC. We show that liver tumors were developed in diethylnitrosamine (DEN)-treated in PXR knockout (KO) mice. Hepatic levels of prostaglandin F2α (PGF2α) and aldo-keto reductase family 1 member C18 (Akr1c18), a prostaglandin synthase of catalyzing reduction of PGH2 to PGF2α, were significantly elevated in DEN-treated PXR KO mice. Hepatic mRNA levels of alpha fetoprotein (AFP), cyclin D1 (Ccnd1), fibroblast growth factor 21 (FGF21), and inflammatory cytokine interleukin 6 (IL-6) were significantly increased in DEN-treated PXR KO mice. Other members of Akr1c family, liver metabolizing enzymes including Cyp1a2, Cyp2b10 and Cyp3a11, and bile acid synthesis enzyme Cyp7a1 mRNA levels were significantly decreased in DEN-treated PXR KO mice. Our findings revealed that PXR deficiency promoted DEN-induced HCC in mice via induction of Akr1c18 expression and PGF2α levels and the increased PGF2α levels synthetized by Akr1c18 enhanced hepatocytes proliferation and induced inflammatory cytokine production, which accelerated liver tumor development after DEN treatment, suggesting that PXR deficiency may create a microenvironment that is more prone to DEN-induced liver tumors and targeting PXR and Akr1c18 to reduce PGF2α biosynthesis may be a potential and novel therapeutic strategy for HCC.

Mechanism of the Visible-Light-Promoted C(sp3)-H Oxidation via Uranyl Photocatalysis.

Uranyl cation, as an emerging photocatalyst, has been successfully applied to synthetic chemistry in recent years and displayed remarkable catalytic ability under visible light. However, the molecular-level reaction mechanisms of uranyl photocatalysis are unclear. Here, we explore the mechanism of the stepwise benzylic C-H oxygenation of typical alkyl-substituted aromatics (i.e., toluene, ethylbenzene, and cumene) via uranyl photocatalysis using theoretical and experimental methods. Theoretical calculation results show that the most favorable reaction path for uranyl photocatalytic oxidation is as follows: first, hydrogen atom transfer (HAT) from the benzyl position to form a carbon radical ([R•]), then oxygen addition ([R•] + O2 → [ROO•]), then radical-radical combination ([ROO•] + [R•] → [ROOR] → 2[RO•]), and eventually [RO•] reduction to produce alcohols, of which 2° alcohol would further be oxidized to ketones and 1° would be stepwise-oxygenated to acids. The results of the designed verification experiments and the capture of reactive intermediates were consistent with those of theoretical calculations and the previously reported research that the active benzylic C-H would be stepwise-oxygenated in the presence of uranyl. This work deepens our understanding of the HAT mechanism of uranyl photocatalysis and provides important theoretical support for the relevant application of uranyl photocatalysts in organic transformation.

Case report: Recurrent pontine stroke and leukoencephalopathy in a patient with de novo mutation in COL4A1.

This report presents a case of pontine autosomal dominant microangiopathy with leukoencephalopathy (PADMAL) in a 35 year-old male patient. The patient exhibited a consistent history of recurrent ischemic strokes, concentrated primarily in the pons region, accompanied by concurrent manifestations of leukoencephalopathy and microbleeds. Genetic evaluation revealed a heterozygous missense mutation consistent with c.3431C>G, p. Thr1144Arg substitution within exon 40 of the COL4A1 gene. This mutation was also identified in the patient's mother, affirming an autosomal dominant inheritance model. Our findings serve as testament to the potential role of mutation in the exon 40 of COL4A1 in the pathogenesis and progression of PADMAL, contributing to ongoing efforts aimed at better understanding the genetic basis of this debilitating disorder.

Stiffness changes in internal rotation muscles of the shoulder and its influence on hemiplegic shoulder pain.

Background: Hemiplegic shoulder pain (HSP) is a common complication in patients with stroke. The pathogenesis of HSP is complex, and muscle hypertonia, especially the hypertonia of internal rotation muscles of the shoulder, may be one of the important causes of shoulder pain. However, the relationship between muscle stiffness and HSP has not been well studied. The purpose of this study is to explore the correlations between the stiffness of internal rotation muscles and clinical symptoms in patients with HSP. Methods: A total of 20 HSP patients and 20 healthy controls were recruited for this study. The stiffness of internal rotation muscles was quantified using shear wave elastography, and Young's modulus (YM) of the pectoralis major (PM), anterior deltoid (AD), teres major ™, and latissimus dorsi (LD) were measured. Muscle hypertonia and pain intensity were evaluated using the Modified Ashworth Scale (MAS) and Visual Analog Scale (VAS), respectively. The mobility of the shoulder was evaluated using the Neer score. The correlations between muscle stiffness and the clinical scales were analyzed. Results: YM of internal rotation muscles on the paretic side was higher than that of the control group in the resting and passive stretching positions (P < 0.05). YM of internal rotation muscles on the paretic side during passive stretching was significantly higher than that at rest (P < 0.05). YM of PM, TM, and LD during passive stretching were correlated with MAS (P < 0.05). In addition, the YM of TM during passive stretching was positively correlated with VAS and negatively correlated with the Neer score (P < 0.05). Conclusion: Increased stiffness of PM, TM, and LD was observed in patients with HSP. The stiffness of TM was associated with pain intensity of the shoulder and shoulder mobility.

6PPD and its metabolite 6PPDQ induce different developmental toxicities and phenotypes in embryonic zebrafish.

The automobile tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone metabolite 6PPDQ have recently received much attention for their acute aquatic toxicity. The present study investigated the mechanistic developmental toxicity of 6PPD and 6PPDQ in embryonic zebrafish. Neither compound induced significant mortality but significantly decreased spontaneous embryo movement and heart rate. Both compounds induced malformations with different phenotypes; the 6PPD-exposed larvae manifested a myopia-like phenotype with a convex eyeball and fusion vessels, while the 6PPDQ-exposed embryonic zebrafish manifested enlarged intestine and blood-coagulated gut, activated neutrophils, and overexpressed enteric neurons. mRNA-Seq and quantitative real-time PCR assays showed that 6PPD- and 6PPDQ-induced distinct differential gene expression aligned with their toxic phenotype. 6PPD activated the retinoic acid metabolic gene cyp26a, but 6PPDQ activated adaptive cellular response to xenobiotics gene cyp1a. 6PPD suppressed the gene expression of the eye involved in retinoic acid metabolism, phototransduction, photoreceptor function and visual perception. In contrast, 6PPDQ perturbed genes involved in inward rectifier K+ and voltage-gated ion channels activities, K+ import across the plasma membrane, iron ion binding, and intestinal immune network for IgA production. The current study advances the present understanding the reason of why many fish species are so adversely impacted by 6PPD and 6PPDQ.

Cold stress during the perinatal period leads to developmental and neurobehavioral toxicity in zebrafish larvae.

In nature, cold stress is a core threat to aquatic organisms. But the neurodevelopmental effects of cold stress during the perinatal period on the offspring development were unknown. In the present study, adult zebrafish were cold-stressed at 18 °C for five days before spawning, and then the fertilized eggs were raised at 18, 24, or 28 °C from 0 to 120 h post fertilization (hpf). The resulting embryos and larvae were assessed for developmental and neurobehavioral responses. Our findings showed that embryos raised at 18 °C (Cold+++) suffered hatching failure and death, at 24 °C (Cold++) had decreased hatching, while those raised at 28 °C (Cold+) exhibited no developmental adversity. The neurobehavioral assessment showed that embryos from Cold+ and Cold++ groups displayed decreased motor behaviors, including spontaneous movement at 20-24 hpf, touch response at 48 hpf, and swimming speed at 120 hpf. In addition, cold stress during perinatal stage irreversibly affected larval social behaviors examined during 10-13 days post fertilization (dpf), such as unconsolidated shoaling, increased mirror attacks, and decreased social contacts. Notably, behavioral adversity was more pronounced in larvae from the Cold ++ group than those from the Cold+ group. Mechanistically, cold stress increased cell apoptosis, evidenced by increased acridine orange positive cells at 24 hpf and upregulation of casp8 at 120 hpf, increased oxidative stress (upregulation of cat and nos1) at 120 hpf, delayed motor neuron extension at 72 hpf, and upregulated nrxn2 and rab33a at 120 hpf. Our data indicate that cold stress during the perinatal period impaired neural development in zebrafish larvae, showing high mental health risk. These findings highlight cold stress should be avoided during the perinatal period for both aquatic fish or even humans.

An Update on Nondopaminergic Treatments for Motor and Non-motor Symptoms of Parkinson's Disease.

Nondopaminergic neurotransmitters such as adenosine, norepinephrine, serotonin, glutamate, and acetylcholine are all involved in Parkinson's disease (PD) and promote its symptoms. Therefore, nondopaminergic receptors are key targets for developing novel preparations for the management of motor and non-motor symptoms in PD, without the potential adverse events of dopamine replacement therapy. We reviewed English-written articles and ongoing clinical trials of nondopaminergic treatments for PD patients till 2014 to summarize the recent findings on nondopaminergic preparations for the treatment of PD patients. The most promising research area of nondopaminergic targets is to reduce motor complications caused by traditional dopamine replacement therapy, including motor fluctuations and levodopa-induced dyskinesia. Istradefylline, Safinamide, and Zonisamide were licensed for the management of motor fluctuations in PD patients, while novel serotonergic and glutamatergic agents to improve motor fluctuations are still under research. Sustained- release agents of Amantadine were approved for treating levodopa induced dyskinesia (LID), and serotonin 5HT1B receptor agonist also showed clinical benefits to LID. Nondopaminergic targets were also being explored for the treatment of non-motor symptoms of PD. Pimavanserin was approved globally for the management of hallucinations and delusions related to PD psychosis. Istradefylline revealed beneficial effect on daytime sleepiness, apathy, depression, and lower urinary tract symptoms in PD subjects. Droxidopa may benefit orthostatic hypotension in PD patients. Safinamide and Zonisamide also showed clinical efficacy on certain non-motor symptoms of PD patients. Nondopaminergic drugs are not expected to replace dopaminergic strategies, but further development of these drugs may lead to new approaches with positive clinical implications.

Constitutive androstane receptor (CAR) mediates pyrene-induced inflammatory responses in mouse liver, with increased serum amyloid A proteins and Th17 cells.

BACKGROUND AND PURPOSE: The constitutive androstane receptor (CAR), a known xenobiotic sensor, plays an important role in drug metabolism by regulating numerous genes. The polycyclic aromatic hydrocarbon pyrene, an environmental pollutant, is a CAR activator and induces mouse hepatotoxicity via CAR. Here, we investigate the molecular mechanisms of the inflammatory response in pyrene-caused mice liver injury. EXPERIMENTAL APPROACH: Effects of pyrene on the liver were investigated in wild-type and CAR knockout (KO) mice. Levels of pyrene and its urinary metabolite were analysed by high performance liquid chromatography (HPLC). Inflammatory responses were measured by qRT-PCR, western blotting, and ELISA for cytokines. KEY RESULTS: Serum amyloid A proteins (SAAs) were markedly increased in the liver and serum of pyrene-exposed wild-type mice. IL-17-producing helper T cells (Th17 cells) and IL-17 levels were increased in the liver of pyrene-exposed wild-type mice. Hepatic mRNA levels of inflammatory cytokines including IL-1ß, IL-6 and TNFα, and serum IL-6 levels were significantly elevated in pyrene-treated wild-type mice. However, these changes were not observed in CAR KO mice. CONCLUSION AND IMPLICATIONS: CAR plays a crucial role in pyrene-caused mice liver inflammatory response with increased SAAs and Th17 cells. Our results suggest that serum SAAs may be a convenient biomarker for early diagnosis of liver inflammatory response caused by polycyclic aromatic hydrocarbons, including pyrene. CAR and Th17 cells may be potential targets for novel therapeutic strategies for xenobiotic-induced liver inflammation.

Increased Stiffness of the Superficial Cervical Extensor Muscles in Patients With Cervicogenic Headache: A Study Using Shear Wave Elastography.

Background: Cervicogenic headache (CEH) is a secondary headache caused by lesions of the cervical spine and surrounding soft tissues. Cervical muscle dysfunction may be related to the onset of CEH. However, whether cervical muscle stiffness changes in patients with CEH has not been well studied. The purpose of this study was to explore changes in superficial cervical extensor muscle stiffness in patients with CEH using shear wave elastography (SWE). Methods: In this study, 19 patients with CEH and 20 healthy controls were recruited. Superficial cervical extensor muscle stiffness was obtained from SWE, and the SuperLinear SL10-2 MHz linear array probe in the musculoskeletal muscle mode was chosen as the transducer. Regions of interest in the trapezius (TRAP), splenius capitis (SPL), semispinalis capitis (SCap), and semispinalis cervicis (SCer) were manually segmented. Correlations between superficial cervical extensor muscle stiffness and visual analog scale (VAS) scores, age, and body mass index (BMI) were analyzed using Pearson's correlation. Receiver operating characteristic (ROC) curve was used to investigate the diagnostic ability of superficial cervical extensor stiffness for CEH. Results: Superficial cervical extensor muscle stiffness on the headache side of patients with CEH was higher than that on the non-headache side and in healthy controls (p < 0.05). Increased stiffness was also observed in SCer on the non-headache side of patients with CEH compared to healthy controls (p < 0.01). In patients with CEH, SCer stiffness was positively correlated with VAS scores (r = 0.481, p = 0.037), but no correlation was found between other muscles and VAS scores (p > 0.05). The areas under the curve of TRAP, SPL, SCap, and SCer in diagnosing CEH were 0.766, 0.759, 0.964, and 1.000, respectively. Conclusions: Increased stiffness was observed in the superficial cervical extensor muscles on the headache side of patients with CEH. SCer stiffness was correlated with headache intensity in patients with CEH and may provide clues for the diagnosis of CEH.

Increased Magnetic Susceptibility in the Deep Gray Matter Nuclei of Wilson's Disease: Have We Been Ignoring Atrophy?

Background: Histopathological studies in Wilson's disease (WD) have revealed increased copper and iron concentrations in the deep gray matter nuclei. However, the commonly used mean bulk susceptibility only reflects the regional metal concentration rather than the total metal content, and regional atrophy may affect the assessment of mean bulk susceptibility. Our study aimed to quantitatively assess the changes of metal concentration and total metal content in deep gray matter nuclei by quantitative susceptibility mapping to distinguish patients with neurological and hepatic WD from healthy controls. Methods: Quantitative susceptibility maps were obtained from 20 patients with neurological WD, 10 patients with hepatic WD, and 25 healthy controls on a 3T magnetic resonance imaging system. Mean bulk susceptibility, volumes, and total susceptibility of deep gray matter nuclei in different groups were compared using a linear regression model. The area under the curve (AUC) was calculated by receiver characteristic curve to analyze the diagnostic capability of mean bulk susceptibility and total susceptibility. Results: Mean bulk susceptibility and total susceptibility of multiple deep gray matter nuclei in patients with WD were higher than those in healthy controls. Compared with patients with hepatic WD, patients with neurological WD had higher mean bulk susceptibility but similar total susceptibility in the head of the caudate nuclei, globus pallidus, and putamen. Mean bulk susceptibility of putamen demonstrated the best diagnostic capability for patients with neurological WD, the AUC was 1, and the sensitivity and specificity were all equal to 1. Total susceptibility of pontine tegmentum was most significant for the diagnosis of patients with hepatic WD, the AUC was 0.848, and the sensitivity and specificity were 0.7 and 0.96, respectively. Conclusion: Brain atrophy may affect the assessment of mean bulk susceptibility in the deep gray matter nuclei of patients with WD, and total susceptibility should be an additional metric for total metal content assessment. Mean bulk susceptibility and total susceptibility of deep gray matter nuclei may be helpful for the early diagnosis of WD.

Chrysene, a four-ring polycyclic aromatic hydrocarbon, induces hepatotoxicity in mice by activation of the aryl hydrocarbon receptor (AhR).

Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic global environmental pollutants and cause harmful effects on human health. Here, we evaluated adverse effects of chrysene, which is a four-ring PAH and an important member of 16 priority PAHs, on the liver. Chrysene was detected in some common raw and cooked Chinese food samples. Hepatotoxicity including increased relative liver weight, hepatocyte swelling and degeneration, and elevated serum alanine aminotransferase (ALT) levels were observed in chrysene-exposed C57BL/6 mice. Glutamine treatment effectively ameliorated chrysene-induced mice liver injury by decreasing serum ALT levels. Chrysene induced mice hepatic glutathione depletion and oxidative DNA damage with increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels. Hepatic expression levels of the aryl hydrocarbon receptor (AhR), AhR-related target genes including CYP1A1, CYP1A2 and CYP1B1, and AhR nuclear translocator (ARNT) were significantly increased in chrysene-exposed C57BL/6 mice. Chrysene induced mice hepatic mRNA levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-mediated phase II detoxifying and antioxidant enzymes including NQO1, UGT1A1, UGT1A6, SULT1A1, GSTm1, GSTm3, Catalase (CAT), GPx1, and SOD2. We found that chrysene had toxic effects including increased relative liver weight and elevated serum ALT levels on AhR+/+ mice but not AhR-/- mice. Chrysene significantly induced hepatic mRNA levels of CYP1A1 and CYP1A2 in AhR+/+ mice but not AhR-/- mice. To our knowledge, this study is the first to demonstrate that hepatotoxicity causes by chrysene is dependent on AhR, and Nrf2 plays an important regulation role in protection against oxidative liver injury induced by chrysene.

Phenanthrene, but not its isomer anthracene, effectively activates both human and mouse nuclear receptor constitutive androstane receptor (CAR) and induces hepatotoxicity in mice.

Polycyclic aromatic hydrocarbons (PAHs) are a class of pervasive global environmental pollutants and adversely affect human health. Among PAHs, phenanthrene and anthracene are isomers consisting of three benzene rings. In the present study, we have made comparisons of constitutive androstane receptor (CAR) activation and toxic effects on the liver between these two isomers. Phenanthrene, but not anthracene, significantly induced promoter activity and gene expression of human drug metabolizing enzyme CYP2B6 in HepG2 cells and human primary hepatocytes, respectively. Phenanthrene, but not anthracene, significantly increased CYP2B10 expression levels and caused hepatotoxicity in mice. Phenanthrene induced the nuclear accumulation of CAR in the liver of wild-type mice, but not CAR-/- mice. Hepatocellular necrosis, elevated expression levels of some CAR-related genes such as CYP2B10, CYP3A11, UGT1A1, SULT2A1 and GSTM3, and lower hepatic glutathione levels were found in phenanthrene-exposed wild-type mice but not CAR-/- mice. Additionally, phenanthrene and anthracene were detected in both raw and grilled lamb samples. The average concentrations of phenanthrene were much higher than those of anthracene in these samples. This study is the first to demonstrate that phenanthrene, but not its isomer anthracene, effectively activates both human and mouse nuclear receptor CAR, and CAR plays a crucial role in phenanthrene-induced mouse hepatotoxicity. Compared with anthracene, K region may be an important electronic structure of phenanthrene for activation of CAR. Dietary consumption of PAHs-contaminated food is an important exposure route for humans. Exposure to phenanthrene may affect human health especially associated with liver.

Author Correction: Design of amidobenzimidazole STING receptor agonists with systemic activity.

Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

Identification of hepatotoxicity and renal dysfunction of pyrene in adult male rats.

Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic pollutants primarily formed from the incomplete combustion of carbonaceous materials, and have adverse effects on human health. In this study, we investigated whether pyrene, a PAH consisting of 4 fused benzene rings, has adverse effects on rat. Adult male Sprague-Dawly rats were treated daily by oral gavage with vehicle (corn oil) or pyrene at doses of 375, 750, 1500, or 2200 mg/kg/day for 4 days. The results showed that pyrene caused hepatotoxicity in rats. When compared with the control group, relative liver weights, plasma alanine aminotransferase, and direct bilirubin levels significantly increased after pyrene exposure. Hepatocyte swelling and degeneration and decreased hepatic total glutathione (GSH) levels were also found in pyrene-exposed rats. We further observed that mRNA levels of several hepatic metabolizing enzymes regulated by constitutive androstane receptor (CAR) such as CYP2B1 and CYP2B2 significantly increased in pyrene-exposed rats. These results suggest that decreased GSH levels, elevated hepatic metabolizing enzyme gene expression, and CAR activation are important contributors for pyrene-induced hepatotoxicity in rats. Additionally, we found pyrene significantly induced plasma inflammatory indices including white blood cell and lymphocyte counts. We also observed that pyrene exposure increased relative weight of kidneys and disrupted kidney function with elevated urea and creatinine levels in rats.

Pregnane X receptor (PXR) deficiency improves high fat diet-induced obesity via induction of fibroblast growth factor 15 (FGF15) expression.

Obesity has become a significant global health problem, and is a high risk factor for a variety of metabolic diseases. Fibroblast growth factor (FGF) 15 plays an important role in the regulation of metabolism. Xenobiotic-sensing nuclear receptors pregnane X receptor (PXR/NR1I2) and constitutive androstane receptor (CAR/NR1I3) play important roles in xenobiotic detoxification and metabolism, and also are involved in the regulation of energy metabolism. However, the effects that PXR and CAR have on the regulation of FGF15 are unknown. Here, we found that body weight, hepatic triglyceride levels, liver steatosis, and hepatic mRNA expression levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1), the key enzymes in the bile acid classical synthesis pathway, were significantly decreased in high fat diet (HFD)-fed PXR knockout (KO) mice compared to HFD-fed wild-type mice. Interestingly, intestinal FGF15 expression levels were significantly elevated in HFD-fed PXR KO mice compared with HFD-fed wild-type mice. Additionally, serum total bile acid levels were significantly decreased in PXR KO mice than those in wild-type mice when fed a control diet or HFD. Total lipids in feces were significantly increased in HFD-fed PXR KO mice compared to HFD-fed wild-type mice. However, these alterations were not found in HFD-fed CAR KO mice. These results indicate that PXR deficiency improves HFD-induced obesity via induction of FGF15 expression, resulting in suppression of bile acid synthesis and reduction of lipid absorption, hepatic lipid accumulation and liver triglyceride levels. Our findings suggest that PXR may negatively regulate FGF15 expression and represent a potential therapeutic target for the treatment for metabolic disorders such as obesity.

Upregulation of human CYP2C9 expression by Bisphenol A via estrogen receptor alpha (ERα) and Med25.

Bisphenol A (BPA) is an important industrial chemical, mainly used in the manufacture of polycarbonate plastic and epoxy resins. Due to its widespread use, humans have a high risk of exposure to BPA. BPA has been found to have adverse health effects such as interfering with hormone-related pathways and is well-known to act as an endocrine disruptor. The present study is the first to show the induction effect of BPA on gene expression and enzyme activity of CYP2C9, an important hepatic drug metabolizing enzyme in human. We further identify the mechanism of BPA upregulation of CYP2C9 expression. We show that BPA is able to transcriptionally activate CYP2C9 promoter through ERα and ERE site within the CYP2C9 promoter region in HepG2 cells, and can induce CYP2C9 gene expression and enzyme activity in human primary hepatocytes. Moreover, we demonstrate that Med25, a variable member of the Mediator complex, is a coactivator of ligand-activated ERα that interacts with ERα through its C-terminal LXXLL motif after BPA exposure, and is functionally involved in BPA-induced transcriptional regulation of CYP2C9 expression and enzyme activity. Our findings suggest that BPA exposure has a potential risk for adverse health effects in human liver metabolism by upregulation of CYP2C9 expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 970-978, 2017.

Pyrene is a Novel Constitutive Androstane Receptor (CAR) Activator and Causes Hepatotoxicity by CAR.

Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous persistent environmental pollutants which are primarily formed from the incomplete combustion of organic materials. Many potential sources of human exposure to PAHs exist, including daily exposures from the ambient environment or occupational settings. PAHs have been found to cause harmful effects on human health. Here, we evaluated the adverse effects of pyrene, a common PAH, on the liver. The present study demonstrates that pyrene is able to activate mouse constitutive androstane receptor (CAR). CAR protein, as measured by Western blot analysis, was observed to translocate into the nucleus from the cytoplasm in mouse liver after exposure to pyrene. Utilizing CAR null mice, we identified that CAR mediates pyrene-induced hepatotoxicity. Increased relative liver weight, hepatocellular hypertrophy, and elevated serum alanine aminotransferase levels were found in wild-type but not CAR null mice after orally administered pyrene. We further show that pyrene induced the expression of mouse liver metabolism enzymes including CYP2B10, CYP3A11, GSTm1, GSTm3, and SULT1A1, and caused hepatic glutathione depletion in wild-type but not CAR null mice. Moreover, by luciferase reporter assay and quantitative real-time PCR analysis, pyrene was found to be a potential inducer of CYP2B6 expression via activation of human CAR in HepG2 cells and human primary hepatocytes. Our observations suggest that pyrene is a novel CAR activator and that CAR is essential for mediating pyrene-induced liver injury.

The BRAF and MEK Inhibitors Dabrafenib and Trametinib: Effects on Immune Function and in Combination with Immunomodulatory Antibodies Targeting PD-1, PD-L1, and CTLA-4.

PURPOSE: To assess the immunologic effects of dabrafenib and trametinib in vitro and to test whether trametinib potentiates or antagonizes the activity of immunomodulatory antibodies in vivo. EXPERIMENTAL DESIGN: Immune effects of dabrafenib and trametinib were evaluated in human CD4(+) and CD8(+) T cells from healthy volunteers, a panel of human tumor cell lines, and in vivo using a CT26 mouse model. RESULTS: Dabrafenib enhanced pERK expression levels and did not suppress human CD4(+) or CD8(+) T-cell function. Trametinib reduced pERK levels, and resulted in partial/transient inhibition of T-cell proliferation/expression of a cytokine and immunomodulatory gene subset, which is context dependent. Trametinib effects were partially offset by adding dabrafenib. Dabrafenib and trametinib in BRAF V600E/K, and trametinib in BRAF wild-type tumor cells induced apoptosis markers, upregulated HLA molecule expression, and downregulated certain immunosuppressive factors such as PD-L1, IL1, IL8, NT5E, and VEGFA. PD-L1 expression in tumor cells was upregulated after acquiring resistance to BRAF inhibition in vitro. Combinations of trametinib with immunomodulators targeting PD-1, PD-L1, or CTLA-4 in a CT26 model were more efficacious than any single agent. The combination of trametinib with anti-PD-1 increased tumor-infiltrating CD8(+) T cells in CT26 tumors. Concurrent or phased sequential treatment, defined as trametinib lead-in followed by trametinib plus anti-PD-1 antibody, demonstrated superior efficacy compared with anti-PD-1 antibody followed by anti-PD-1 plus trametinib. CONCLUSION: These findings support the potential for synergy between targeted therapies dabrafenib and trametinib and immunomodulatory antibodies. Clinical exploration of such combination regimens is under way.

Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK inhibitor.

Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss) while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.