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CAF secreted HGF mediates HNSCC glycolysis.

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR.Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769–82. ©2018 AACR.

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Duloxetine (DLX) is a dual serotonin and norepinephrine reuptake inhibitor, widely used for the treatment of major depressive disorder. Although DLX has shown good efficacy and safety, serious adverse effects (e.g., liver injury) have been reported. The mechanisms associated with DLX-induced toxicity remain elusive. Drug metabolism plays critical roles in drug safety and efficacy. However, the metabolic profile of DLX in mice is not available although mice serve as commonly used animal models for mechanistic studies of drug-induced adverse effects. Our study revealed 39 DLX metabolites in human/mouse liver microsomes and mice. Of note, thirteen metabolites are novel, including five N-acetyl cysteine adducts and one GSH adduct associated with DLX. Additionally, the species differences of certain metabolites were observed between human and mouse liver microsomes. CYP1A2 and CYP2D6 are primary enzymes responsible for the formation of DLX metabolites in liver microsomes, including DLX-GSH adducts. In summary, a total of 39 DLX metabolites were identified and specie differences were noticed in vitro. The roles of CYP450s in DLX metabolite formation were also verified using human recombinant P450 enzymes and corresponding chemical inhibitors. Further studies are warranted to address the exact role of DLX metabolism in its adverse effects in vitro (e.g., human primary hepatocytes) and in vivo (e.g., Cyp1a2-null mice). Significance Statement Current study systematically investigated DLX metabolism and bioactivation in liver microsomes and mice. This study provided a global view of DLX metabolism and bioactivation in liver microsomes and mice, which are very valuable to further elucidate the mechanistic study of DLX-related adverse effects and drug-drug interaction from metabolic aspects.

Background Alternol is a natural compound isolated from fermentation products of a mutant fungus. Our previous studies demonstrated that Alternol specifically kills cancer cells but spares benign cells. Methods To investigate the mechanism underlying alternol-induced cancer cell-specific killing effect, we took a comprehensive strategy to identify Alternol's protein targets in prostate cancer cells, including PC-3, C4-2, and 22RV1, plus benign BPH1 cell lines. Major experimental techniques included biotin-streptavidin pulldown assay coupled with mass-spectrometry, in vitro enzyme activity assay for Krebs cycle enzymes and gas chromatography-mass spectrometry (GC-MS) for metabolomic analysis. Results Among 14 verified protein targets, four were Krebs cycle enzymes, fumarate hydratase (FH), malate dehydrogenase-2 (MDH2), dihydrolipoamide acetyltransferase (DLAT) in pyruvate dehydrogenase complex (PDHC) and dihydrolipoamide S-succinyltransferase (DLST) in a-ketoglutarate dehydrogenase complex (KGDHC). Functional assays revealed that PDHC and KGDHC activities at the basal level were significantly higher in prostate cancer cells compared to benign prostate BPH1 cells, while alternol treatment reduced their activities in cancer cells close to the levels in BPH1 cells. Although FH and MDH2 activities were comparable among prostate cancer and benign cell lines at the basal level, Alternol treatment largely increased their activities in cancer cells. Metabolomic analysis revealed that Alternol treatment remarkably reduced the levels of malic acid, fumaric acid, and isocitric acid and mitochondrial respiration in prostate cancer cells. Alternol also drastically reduced mitochondrial respiration and ATP production in PC-3 cells in vitro or in xenograft tissues but not in BPH1 cells or host liver tissues. Conclusions Alternol interacts with multiple Krebs cycle enzymes, resulting in reduced mitochondrial respiration and ATP production in prostate cancer cells and xenograft tissues, providing a novel therapeutic strategy for prostate cancer treatment.

Mitochondria damage plays a critical role in acetaminophen (APAP)-induced necrosis and liver injury. Cells can adapt and protect themselves by removing damaged mitochondria via mitophagy. PINK1-Parkin pathway is one of the major pathways that regulate mitophagy but its role in APAP-induced liver injury is still elusive. We investigated the role of PINK1-Parkin pathway in hepatocyte mitophagy in APAP-induced liver injury in mice. Wild-type (WT), PINK1 knockout (KO), Parkin KO, and PINK1 and Parkin double KO (DKO) mice were treated with APAP for different time points. Liver injury was determined by measuring serum alanine aminotransferase (ALT) activity, H&E staining as well as TUNEL staining of liver tissues. Tandem fluorescent-tagged inner mitochondrial membrane protein Cox8 (Cox8-GFP-mCherry) can be used to monitor mitophagy based on different pH stability of GFP and mCherry fluorescent proteins. We overexpressed Cox8-GFP-mCherry in mouse livers via tail vein injection of an adenovirus Cox8-GFP-mCherry. Mitophagy was assessed by confocal microscopy for Cox8-GFP-mCherry puncta, electron microscopy (EM) analysis for mitophagosomes and western blot analysis for mitochondrial proteins. Parkin KO and PINK1 KO mice improved the survival after treatment with APAP although the serum levels of ALT were not significantly different among PINK1 KO, Parkin KO and WT mice. We only found mild defects of mitophagy in PINK1 KO or Parkin KO mice after APAP, and improved survival in PINK1 KO and Parkin KO mice could be due to other functions of PINK1 and Parkin independent of mitophagy. In contrast, APAP-induced mitophagy was significantly impaired in PINK1-Parkin DKO mice. PINK1-Parkin DKO mice had further elevated serum levels of ALT and increased mortality after APAP administration. In conclusion, our results demonstrated that PINK1-Parkin signaling pathway plays a critical role in APAP-induced mitophagy and liver injury., Graphical abstract Image 1, Highlights • Cox8-GFP-mCherry is a novel molecular probe to monitor mitophagy in cultured hepatocytes and in mouse livers. . • Double deletion of PINK1 and Parking exacerbates acetaminophen-induced liver injury and mortality in mice.

Background & Aims Defects in lysosome function and autophagy contribute to the pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes by evaluating the functions of transcription factor EB (TFEB), which regulates lysosomal biogenesis. Methods We performed studies with GFP-LC3 mice, mice with liver-specific deletion of TFEB, mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB and TFE3 double-knockout mice), and Tfebflox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of regular ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice also were given injections of torin-1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time polymerase chain reaction to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. Results Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB compared with control mice, and hepatocytes had decreased lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting in increased mTOR activation. Administration of torin-1 increased liver levels of TFEB and decreased steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in the liver developed less severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared with mice carrying a control vector. Mice with knockdown of TFEB and TFEB-TFE3 double-knockout mice developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues. Conclusions We found that ethanol feeding plus an acute binge decreased hepatic expression of TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect the liver from ethanol-induced damage.

Early mammalian development is critically dependent on the establishment of oxidative energy metabolism within the trophectoderm (TE) lineage. Unlike inner cell mass (ICM), TE cells enhance ATP production via mitochondrial oxidative phosphorylation (OXPHOS) and this metabolic preference is essential for blastocyst maturation. However, molecular mechanisms that regulate establishment of oxidative energy metabolism in TE cells are incompletely understood. Here, we show that conserved transcription factor TEAD4, which is essential for pre-implantation mammalian development, regulates this process by promoting mitochondrial transcription. In the developing TE and TE-derived trophoblast stem cells (TSCs), TEAD4 localizes to mitochondria, binds to mitochondrial DNA (mtDNA) and facilitates mtDNA transcription by recruiting mitochondrial RNA Polymerase (POLRMT). Loss of TEAD4 impairs recruitment of POLRMT, resulting in reduced expression of mtDNA-encoded electron transport chain components, thereby inhibiting oxidative energy metabolism. Our studies identify a novel TEAD4-dependent molecular mechanism that regulates energy metabolism in the TE lineage to ensure mammalian development.

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR. Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769–82. ©2018 AACR.

Epidermal growth factor receptor (EGFR) plays a crucial role in hepatocyte proliferation. Its role in acetaminophen (APAP)-mediated hepatotoxicity and subsequent liver regeneration is completely unknown. Role of EGFR after APAP-overdose in mice was studied using pharmacological inhibition strategy. Rapid, sustained and dose-dependent activation of EGFR was noted after APAP-treatment in mice, which was triggered by glutathione depletion. EGFR-activation was also observed in primary human hepatocytes after APAP-treatment, preceding elevation of toxicity markers. Treatment of mice with an EGFR-inhibitor (EGFRi), Canertinib, 1h post-APAP resulted in robust inhibition of EGFR-activation and a striking reduction in APAP-induced liver injury. Metabolic activation of APAP, formation of APAP-protein adducts, APAP-mediated JNK-activation and its mitochondrial translocation were not altered by EGFRi. Interestingly, EGFR rapidly translocated to mitochondria after APAP-treatment. EGFRi-treatment abolished mitochondrial EGFR activity, prevented APAP-mediated mitochondrial dysfunction/oxidative-stress and release of endonucleases from mitochondria, which are responsible for DNA-damage/necrosis. Treatment with N-acetylcysteine (NAC), 4h post-APAP in mice did not show any protection but treatment of EGFRi in combination with NAC showed decrease in liver injury. Finally, delayed treatment with EGFRi, 12-h post-APAP, did not alter peak injury but caused impairment of liver regeneration resulting in sustained injury and decreased survival after APAP overdose in mice. Impairment of regeneration was due to inhibition of cyclinD1 induction and cell cycle arrest. Our study has revealed a new dual role of EGFR both in initiation of APAP-injury and in stimulation of subsequent compensatory regeneration after APAP-overdose.

Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis.

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 μM) and an ATPase activity with a Km of 0.99 mM. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.

The liver plays a key role in cholesterol metabolism. Impaired hepatic cholesterol homeostasis causes intracellular free cholesterol accumulation and hepatocyte injury. Sortilin 1 (SORT1) is a lysosomal trafficking receptor that was identified by genome-wide association studies (GWAS) as a novel regulator of cholesterol metabolism in humans. Here we report that SORT1 deficiency protected against cholesterol accumulation-induced liver injury and inflammation in mice. Using an LC-MS/MS-based proteomics approach, we identified liver carboxylesterase 1 (CES1) as a novel SORT1-interacting protein. Mechanistic studies further showed that SORT1 may regulate CES1 lysosomal targeting and degradation and that SORT1 deficiency resulted in higher liver CES1 protein abundance. Previous studies have established an important role of hepatic CES1 in promoting intracellular cholesterol mobilization, cholesterol efflux, and bile acid synthesis. Consistently, high cholesterol atherogenic diet-challenged Sort1 knock-out mice showed less hepatic free cholesterol accumulation, increased bile acid synthesis, decreased biliary cholesterol secretion, and the absence of gallstone formation. SORT1 deficiency did not alter hepatic ceramide and fatty acid metabolism in high cholesterol atherogenic diet-fed mice. Finally, knockdown of liver CES1 in mice markedly increased the susceptibility to high cholesterol diet-induced liver injury and abolished the protective effect against cholesterol lipotoxicity in Sort1 knock-out mice. In summary, this study identified a novel SORT1-CES1 axis that regulates cholesterol-induced liver injury, which provides novel insights that improve our current understanding of the molecular links between SORT1 and cholesterol metabolism. This study further suggests that therapeutic inhibition of SORT1 may be beneficial in improving hepatic cholesterol homeostasis in metabolic and inflammatory liver diseases.

Background & Aims Hepatic cholesterol accumulation and autophagy defects contribute to hepatocyte injury in fatty liver disease. Bile acid synthesis is a major pathway for cholesterol catabolism in the liver. This study aims to understand the molecular link between cholesterol and bile acid metabolism and hepatic autophagy activity. Methods The effects of cholesterol and cholesterol 7α-hydroxylase (CYP7A1) expression on autophagy and lysosome function were studied in cell models. The effects and mechanism of disrupting enterohepatic bile acid circulation on hepatic autophagy were studied in mice. Results The results first showed differential regulation of hepatic autophagy by free cholesterol and cholesterol ester, whereby a modest increase of cellular free cholesterol, but not cholesterol ester, impaired lysosome function and caused marked autolysosome accumulation. We found that CYP7A1 induction, either by cholestyramine feeding in mice or adenovirus-mediated CYP7A1 expression in hepatocytes, caused strong autophagy induction. Mechanistically, we showed that CYP7A1 expression markedly attenuated growth factor/AKT signaling activation of mechanistic target of rapamycin (mTOR), but not amino acid signaling to mTOR in vitro and in vivo. Metabolomics analysis further found that CYP7A1 induction not only decreased hepatic cholesterol but also altered phospholipid and sphingolipid compositions. Collectively, these results suggest that CYP7A1 induction interferes with growth factor activation of AKT/mTOR signaling possibly by altering membrane lipid composition. Finally, we showed that cholestyramine feeding restored impaired hepatic autophagy and improved metabolic homeostasis in Western diet–fed mice. Conclusions This study identified a novel CYP7A1–AKT–mTOR signaling axis that selectively induces hepatic autophagy, which helps improve hepatocellular integrity and metabolic homeostasis., Graphical Abstract

Overdose of acetaminophen (APAP) causes severe liver injury and even acute liver failure in both mice and human. A recent study by Kim et al. (2015, Metformin ameliorates acetaminophen hepatotoxicity via Gadd45β-dependent regulation of JNK signaling in mice. J. Hepatol. 63, 75-82) showed that metformin, a first-line drug to treat type 2 diabetes mellitus, protected against APAP hepatotoxicity in mice. However, its exact protective mechanism has not been well clarified. To investigate this, C57BL/6J mice were treated with 400 mg/kg APAP and 350 mg/kg metformin was given 0.5 h pre- or 2 h post-APAP. Our data showed that pretreatment with metformin protected against APAP hepatotoxicity, as indicated by the over 80% reduction in plasma alanine aminotransferase (ALT) activities and significant decrease in centrilobular necrosis. Metabolic activation of APAP, as indicated by glutathione depletion and APAP-protein adducts formation, was also slightly inhibited. However, 2 h post-treatment with metformin still reduced liver injury by 50%, without inhibition of adduct formation. Interestingly, neither pre- nor post-treatment of metformin inhibited c-jun N-terminal kinase (JNK) activation or its mitochondrial translocation. In contrast, APAP-induced mitochondrial oxidant stress and dysfunction were greatly attenuated in these mice. In addition, mice with 2 h post-treatment with metformin also showed significant inhibition of complex I activity, which may contribute to the decreased mitochondrial oxidant stress. Furthermore, the protection was reproduced in JNK activation-absent HepaRG cells treated with 20 mM APAP followed by 0.5 or 1 mM metformin 6 h later, confirming JNK-independent protection mechanisms. Thus, metformin protects against APAP hepatotoxicity by attenuating the mitochondrial oxidant stress and subsequent mitochondrial dysfunction, and may be a potential therapeutic option for APAP overdose patients.

ABCB6 is a mitochondrial transporter that regulates porphyrin biosynthesis. ABCB6 expression is upregulated in hepatocellular carcinoma (HCC) but the significance of this upregulation to HCC is not known. In the present study, we investigated: 1) ABCB6 expression in 18 resected human hepatocellular carcinoma (HCC) tissues and 3 human hepatoma cell lines; 2) pattern of ABCB6 expression during liver disease progression; and 3) functional significance of ABCB6 expression to HCC using the hepatoma cell line Huh7. ABCB6 expression was determined by real‐time quantitative reverse transcription‐polymerase chain reaction and western blotting. ABCB6 expression was upregulated in all the HCC specimens and the three‐hepatoma cell lines. Increased ABCB6 expression correlated with liver disease progression with the pattern of expression being HCC > cirrhosis > steatosis. Small hairpin RNA (shRNA)‐mediated knockdown of ABCB6 in Huh7 cells lead to decreased cellular proliferation and colony formation. Attenuation of ABCB6 expression did not affect Huh7 apoptosis but lead to a delay in G2/M phase of the cell cycle. In contrast, ABCB6 overexpression resulted in increased growth and proliferation of Huh7 cells. Since ABCB6 expression is induced in multiple tumor types we explored the role of ABCB6 in other cancer cells. ShRNA mediated knockdown of ABCB6 in HEK293 and K562 cells reduced cellular proliferation leading to a delay in G2/M phase, while ABCB6 overexpression promoted cell growth and proliferation. Collectively, these findings, obtained by loss of function and gain of function analysis, suggest that ABCB6 plays a role in cell growth and proliferation by targeting the cell cycle.

Leishmania donovani, causative organism for visceral leishmaniasis, is responsible for considerable mortality and morbidity worldwide. Generation of drug-resistant variants continue to challenge the chemotherapy, the mainstay to fight the disease. The aim of current study was proteomic profiling of wild type (Ld-Wt) and arsenite-resistant (Ld-As20) L. donovani. Significant differences in protein profiles were observed between Ld-As20 and its parent Ld-Wt strain. Proteomic analysis of 158 spots from Ld-Wt and 144 spots from, Ld-As20 identified 77 and 74 protein entries, respectively, through MALDI-TOF/TOF based mass spectrometry and database search. A shift in the isoelectric point of few proteins was observed both in Ld-Wt and Ld-As20, which raises the possibility of continuous arsenite stress, resulting in the differences in the protein profiles of drug-resistant strain from its parent wild type strain. The comparative proteomic data holds the key for elucidation of the multifactorial and complex drug resistance mechanism, like arsenite resistance, in the parasite.

The affinity of arsenic towards the cytoskeleton leading to disturbance of tubulin polymerization is well known. Tubulin undergoes extensive posttranslational modifications which effect stability and dynamics of microtubules but little is known about the effect of antimicrotubule drugs on their distribution and function in kinetoplastid parasites such as Leishmania. The current study was undertaken to investigate the effect of continuous sodium arsenite exposure on the tubulin distribution profile in wild type and sodium arsenite resistant Leishmania donovani together with effect of paclitaxel, a tubulin-polymerizing agent, on that distribution using confocal microscopy. Immunofluorescence studies using specific monoclonal antibodies against alpha-tubulin and posttranslationally modified tubulins (acetylated and tyrosinated) have revealed distinct differences in the organization of microtubule arrays in wild type and sodium arsenite resistant L. donovani that is further affected by paclitaxel treatment. Microtubules are arranged in spiral arrays in wild type as compared to the longitudinal arrays in arsenite resistant L. donovani. The difference in microtubular structure organization may explain the parasite response to continuous drug pressure and illustrate the fundamental impact of arsenite on microtubules in arsenite resistant L. donovani.

Gefitinib (GEF), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is widely used for the treatment of cancers, particularly non-small cell lung cancer. However, its clinical use is limited by multiple adverse effects associated with GEF, such as liver and lung injuries, severe nausea, and diarrhea. Although, the exact mechanism of GEF adverse effects are still unknown, xenobiotic-induced bioactivation is thought to play a significant role in GEF induced toxicity. Using a metabolomic approach, we investigated the metabolic pathways of GEF in human and mouse liver microsomes. Thirty four GEF metabolites and adducts were identified and half of them are novel. The potential reactive metabolites, two aldehydes and one iminium, were identified for the first time. The previously reported GSH adducts and primary amines were observed as well. The aldehyde and iminium pathways were further confirmed by using methoxylamine and potassium cyanide as trapping reagents. Using recombinant CYP450 isoforms, CYP3A4 inhibitor, and S9 from Cyp3a-null mice, we confirmed CYP3A is the major enzyme contributing to the formation of aldehydes, GSH adducts, and primary amines in liver. Multiple enzymes contribute to the formation of iminium. This study provided us more knowledge of GEF bioactivation and enzymes involved in metabolic pathways, which can be utilized for understanding the mechanism of adverse effects associated with GEF and predicting possible drug-drug interactions. Further studies are suggested to determine the roles of these bioactivation pathways in GEF toxicity.

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.

e18518Background: Insights into the biology of chemotherapy resistance may suggest novel strategies to treat Acute Myeloid Leukemia (AML). We examined the role of hyperbaric oxygen (HBO) in AML sen...

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Arsenic, an environmental carcinogen, remains a major public health problem. Arsenic damages biological systems through multiple mechanisms, including the generation of reactive oxygen species. ABCB6 is an ATP-binding cassette transporter that is highly expressed in cells resistant to arsenic. We have recently demonstrated that ABCB6 expression protects against cellular stressors. In the present study, we evaluated the significance of ABCB6 expression to arsenic toxicity both in mice and in cell culture. We show that sodium arsenite induces ABCB6 expression in a dose-dependent manner both in mice fed sodium arsenite in drinking water and in cells exposed to sodium arsenite in vitro. Arsenite-induced ABCB6 expression was transcriptionally regulated, but this induction was not mediated by the redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). We demonstrate that, in HepG2 and Hep3B cells, knockdown of ABCB6 expression using ABCB6-specific small interfering RNA sensitized the cells to arsenite toxicity. In contrast, stable overexpression of ABCB6 conferred a strong survival advantage toward arsenite-induced oxidative stress. Collectively, these results, obtained by both loss of function and gain of function analysis, suggest that ABCB6 expression in response to sodium arsenite might be an endogenous protective mechanism activated to protect cells against arsenite-induced oxidative stress.