Biomarkers of DNA damage response improve in vitro micronucleus assays by revealing genotoxic mode of action and reducing the occurrence of irrelevant positive results.

We have previously described two flow cytometry-based in vitro genotoxicity tests: micronucleus (MN) scoring (MicroFlow®) and a multiplexed DNA damage response biomarker assay (MultiFlow®). Here, we describe a strategy for combining the assays in order to efficiently supplement MN analyses with a panel of biomarkers that comment on cytotoxicity (i.e. relative nuclei count, relative increased nuclei count, cleaved PARP-positive chromatin and ethidium monoazide-positive chromatin) and genotoxic mode of action (MoA; i.e. γH2AX, phospho-histone H3, p53 activation and polyploidy). For these experiments, human TK6 cells were exposed to each of 32 well-studied reference chemicals in 96-well plates for 24 continuous hours. The test chemicals were evaluated over a range of concentrations in the presence and absence of a rat liver S9-based metabolic activation system. MultiFlow assay data were acquired at 4 and 24 h, and micronuclei were scored at 24 h. Testing 32 chemicals in two metabolic activation arms translated into 64 a priori calls: 42 genotoxicants and 22 non-genotoxicants. The MN assay showed high sensitivity and moderate specificity (90% and 68%, respectively). When a genotoxic call required significant MN and MultiFlow responses, specificity increased to 95% without adversely affecting sensitivity. The dose-response data were analysed with PROAST Benchmark Dose (BMD) software in order to calculate potency metrics for each endpoint, and ToxPi software was used to synthesise the resulting lower and upper bound 90% confidence intervals into visual profiles. The BMD/ToxPi combination was found to represent a powerful strategy for synthesising multiple BMD confidence intervals, as the software output provided MoA information as well as insights into genotoxic potency.

In vitro evaluation of chromosomal damage and DNA strand breaks after treatment with the poppy seed alkaloid thebaine.

Thebaine is an alkaloid and can be found in poppy seeds in relatively high concentrations. Acute toxicity of thebaine is fairly high, but not much is known about chronic toxicity. To investigate the genotoxicity of thebaine, cytokinesis-block micronucleus test and comet assay were conducted in TK6 cells. In addition, effects of putative thebaine metabolites were analysed using metabolically active HepG2 cells and TK6 cells with S9 mix. FDA test and trypan blue test were used together with the frequency of mitotic and apoptotic cells to assess potential cytotoxicity of thebaine treatment. Micronucleus induction was observed after high doses (150 and 500 µM) of thebaine without metabolic activation in the presence of slight to moderate cytotoxicity. No effects were observed in the comet assay or after metabolic activation up to the highest dose of 500 µM. A potential protective effect on micronucleus induction after thebaine treatment was investigated via co-treatment with MMC and BaP in TK6 cells. Only after co-treatment with MMC, a reduction of micronucleus frequency was found. Overall, this study shows a potential of thebaine to induce genotoxic effects at high concentrations. The observation of cytotoxicity at these concentrations supports the hypothesis that genotoxicity may be caused by cytotoxic effects. Further studies will need to elucidate whether these effects are directly genotoxic or indeed result from cytotoxicity.

Human Newborn Monocytes Demonstrate Distinct BCG-Induced Primary and Trained Innate Cytokine Production and Metabolic Activation In Vitro.

Background: Newborns exhibit distinct immune responses and are at high risk of infection. Neonatal immunization with BCG, the live attenuated vaccine against tuberculosis (TB), is associated with broad protection against a range of unrelated pathogens, possibly reflecting vaccine-induced training of innate immune cells ("innate memory"). However, little is known regarding the impact of age on BCG-induced innate responses. Objective: Establish an age-specific human monocyte in vitro training platform to characterize and compare BCG-induced primary and memory cytokine responses and immunometabolic shifts. Design/Methods: Human neonatal and adult CD33-selected monocytes were stimulated for 24h with RPMI (control) or BCG (Danish strain) in 10% autologous serum, washed and cultured for 5 additional days, prior to re-stimulation with the TLR4 agonist LPS for another 24h. Supernatants were collected at Day 1 (D1) to measure primary innate responses and at Day 7 (D7) to assess memory innate responses by ELISA and multiplex cytokine and chemokine assays. Lactate, a signature metabolite increased during trained immunity, was measured by colorimetric assay. Results: Cytokine production by human monocytes differed significantly by age at D1 (primary, BCG 1:750 and 1:100 vol/vol, p<0.0001) and D7 (innate memory response, BCG 1:100 vol/vol, p<0.05). Compared to RPMI control, newborn monocytes demonstrated greater TNF (1:100, 1:10 vol/vol, p<0.01) and IL-12p40 (1:100 vol/vol, p<0.05) production than adult monocytes (1:100, p<0.05). At D7, while BCG-trained adult monocytes, as previously reported, demonstrated enhanced LPS-induced TNF production, BCG-trained newborn monocytes demonstrated tolerization, as evidenced by significantly diminished subsequent LPS-induced TNF (RPMI vs. BCG 1:10, p <0.01), IL-10 and CCL5 production (p<0.05). With the exception of IL-1RA production by newborn monocytes, BCG-induced monocyte production of D1 cytokines/chemokines was inversely correlated with D7 LPS-induced TNF in both age groups (p<0.0001). Compared to BCG-trained adult monocytes, newborn monocytes demonstrated markedly impaired BCG-induced production of lactate, a metabolite implicated in immune training in adults. Conclusions: BCG-induced human monocyte primary- and memory-innate cytokine responses were age-dependent and accompanied by distinct immunometabolic shifts that impact both glycolysis and training. Our results suggest that immune ontogeny may shape innate responses to live attenuated vaccines, suggesting age-specific approaches to leverage innate training for broad protection against infection.

Icotinib Induces Mechanism-Based Inactivation of Recombinant Human CYP3A4/5 Possibly via Heme Destruction by Ketene Intermediate.

Icotinib (ICT) is an antitumor drug approved by China National Medical Products Administration and is found to be effective against non-small cell lung cancer. The present study aimed at the interaction of ICT with CYP3A. ICT exhibited time-, concentration-, and NADPH-dependent inhibitory effect on recombinant human CYP3A4/5. About 60% of CYP3A activity was suppressed by ICT at 50 µM after 30 minutes. The observed enzyme inhibition could not be recovered by dialysis. Nifedipine protected CYP3A from the inactivation by ICT. The inhibitory effects of ICT on CYP3A were influenced neither by glutathione/N-acetyl lysine nor by superoxide dismutase/catalase. Incubation of ICT with human hepatic microsomes produced a ketene reactive intermediate trapped by 4-bromobenzylamine. CYP3A4 dominated the metabolic activation of ICT to the ketene intermediate. Ethyl and vinyl analogs of ICT did not induce inactivation of recombinant human CYP3A4/5, which indicates that acetylenic bioactivation of ICT contributed to the enzyme inactivation. Moreover, the metabolic activation of ICT resulted in heme destruction. In conclusion, this study demonstrated that ICT was a mechanism-based inactivator of recombinant human CYP3A4/5, and heme destruction by the ketene metabolite may be responsible for the observed CYP3A inactivation. SIGNIFICANCE STATEMENT: Cytochrome P450 enzymes play an important role in drug-drug interactions. The present study demonstrated that icotinib, an inhibitor of epidermal growth factor receptor used to treat non-small cell lung cancer, is a mechanism-based inactivator of recombinant human CYP3A4/5. The study provided solid evidence for the involvement of acetylene moiety in the metabolic activation as well as the inactivation of the enzyme. Furthermore, the resulting ketene intermediate was found to destroy heme, which is possibly responsible for the observed enzyme inactivation.

The aryl hydrocarbon receptor activates ceramide biosynthesis in mice contributing to hepatic lipogenesis.

Aryl hydrocarbon receptor (AHR) activation via 2,3,7,8-tetrachlorodibenzofuran (TCDF) induces the accumulation of hepatic lipids. Here we report that AHR activation by TCDF (24  µg/kg body weight given orally for five days) induced significant elevation of hepatic lipids including ceramides in mice, was associated with increased expression of key ceramide biosynthetic genes, and increased activity of their respective enzymes. Results from chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and cell-based reporter luciferase assays indicated that AHR directly activated the serine palmitoyltransferase long chain base subunit 2 (Sptlc2, encodes serine palmitoyltransferase 2 (SPT2)) gene whose product catalyzes the initial rate-limiting step in de novo sphingolipid biosynthesis. Hepatic ceramide accumulation was further confirmed by mass spectrometry-based lipidomics. Taken together, our results revealed that AHR activation results in the up-regulation of Sptlc2, leading to ceramide accumulation, thus promoting lipogenesis, which can induce hepatic lipid accumulation.

Effects of steric hindrance and electron density of ester prodrugs on controlling the metabolic activation by human carboxylesterase.

Carboxylesterase (CES) plays an important role in the hydrolysis metabolism of ester-type drugs and prodrugs. In this study, we investigated the change in the hydrolysis rate of hCE1 by focusing on the steric hindrance of the ester structure and the electron density. For 26 kinds of synthesized indomethacin prodrugs, the hydrolytic rate was measured in the presence of human liver microsomes (HLM), human small intestine microsomes (HIM), hCE1 and hCE2. The synthesized prodrugs were classified into three types: an alkyl ester type that is specifically metabolized by hCE1, a phenyl ester type that is more easily metabolized by hCE1 than by hCE2, and a carbonate ester type that is easily metabolized by both hCE1 and hCE2. The hydrolytic rate of 1-methylpentyl (hexan-2-yl) ester was 10-times lower than that of 4-methylpentyl ester in hCE1 solution. hCE2 was susceptible to electron density of the substrate, and there was a difference in the hydrolysis rate of up to 3.5-times between p-bromophenyl ester and p-acetylphenyl ester. By changing the steric hindrance and electron density of the alkoxy group, the factors that change the hydrolysis rate by CES were elucidated.

Effect of Microbial Short-Chain Fatty Acids on CYP3A4-Mediated Metabolic Activation of Human Pluripotent Stem Cell-Derived Liver Organoids.

The early and accurate prediction of the hepatotoxicity of new drug targets during nonclinical drug development is important to avoid postmarketing drug withdrawals and late-stage failures. We previously established long-term expandable and functional human-induced pluripotent stem cell (iPSC)-derived liver organoids as an alternative source for primary human hepatocytes. However, PSC-derived organoids are known to present immature fetal characteristics. Here, we treated these liver organoids with microbial short-chain fatty acids (SCFAs) to improve metabolic maturation based on microenvironmental changes in the liver during postnatal development. The effects of the three main SCFA components (acetate, propionate, and butyrate) and their mixture on liver organoids were determined. Propionate (1 µM) significantly promoted the CYP3A4/CYP3A7 expression ratio, and acetate (1 µM), propionate (1 µM), and butyrate (1 µM) combination treatment, compared to no treatment (control), substantially increased CYP3A4 activity and albumin secretion, as well as gene expression. More importantly, mixed SCFA treatment accurately revealed troglitazone-induced hepatotoxicity, which was redeemed on a potent CYP3A4 inhibitor ketoconazole treatment. Overall, we determined, for the first time, that SCFA mixture treatment might contribute to the accurate evaluation of the CYP3A4-dependent drug toxicity by improving metabolic activation, including CYP3A4 expression, of liver organoids.

Potent aneugenicity of 1-methylpyrene in human cells dependent on metabolic activation by endogenous enzymes.

1-Methylpyrene (1-MP) is a common environmental pollutant and animal carcinogen. After sequential activation by cytochromes P450 and sulfotransferases, it induced gene mutations and micronuclei in mammalian cells. The type of micronuclei formed, entire chromosomes or fragments, was not analysed. In this study, 1-MP and its primary metabolite, 1-hydroxymethylpyrene (1-HMP), were investigated for the induction of centromere-positive and -negative micronuclei in the human hepatoma cell line HepG2 and its derivative C3A, expressing relevant enzymes at higher levels. Under a short-exposure (9 h)/long-recovery regime (2 cell cycles in total), 1-MP and 1-HMP provided negative test results in HepG2 cells. However, they induced micronuclei in C3A cells, the effect being blocked by 1-aminobenzotriazole (inhibitor of cytochromes P450s) and reduced by pentachlorophenol (inhibitor of sulfotransferases). Immunofluorescence staining of centromere protein B in the micronuclei revealed purely clastogenic effects under this regime. Unexpectedly, 1-MP and 1-HMP at concentrations 1/5-1/4 of that required for micronuclei formation led to mitotic arrest and spindle aberrations, as detected by immunofluorescence staining of ß- and γ-tubulin. Following extended exposure (72 h, 2 cell cycles, no recovery), damage to the spindle apparatus and centrosomes was detected at even lower concentrations, with concurrent formation of micronuclei. At low concentrations (1-8 µM 1-MP, 0.25-0.5 µM 1-HMP), the micronuclei induced were unexceptionally centromere-positive. Thus, the chromosome-damaging mechanism of 1-MP was regime and concentration dependent: potently aneugenic under persistent exposure, while clastogenic at higher concentrations following a short-exposure/long-recovery regime. This is a convincing evidence for the existence of metabolic activation-dependent aneugens.

Metabolic activation and toxicological evaluation of polychlorinated biphenyls in Drosophila melanogaster.

Degradation of polychlorinated biphenyls (PCBs) is initiated by cytochrome P450 (CYP) enzymes and includes PCB oxidation to OH-metabolites, which often display a higher toxicity than their parental compounds. In search of an animal model reflecting PCB metabolism and toxicity, we tested Drosophila melanogaster, a well-known model system for genetics and human disease. Feeding Drosophila with lower chlorinated (LC) PCB congeners 28, 52 or 101 resulted in the detection of a human-like pattern of respective OH-metabolites in fly lysates. Feeding flies high PCB 28 concentrations caused lethality. Thus we silenced selected CYPs via RNA interference and analyzed the effect on PCB 28-derived metabolite formation by assaying 3-OH-2',4,4'-trichlorobiphenyl (3-OHCB 28) and 3'-OH-4',4,6'-trichlorobiphenyl (3'-OHCB 28) in fly lysates. We identified several drosophila CYPs (dCYPs) whose knockdown reduced PCB 28-derived OH-metabolites and suppressed PCB 28 induced lethality including dCYP1A2. Following in vitro analysis using a liver-like CYP-cocktail, containing human orthologues of dCYP1A2, we confirm human CYP1A2 as a PCB 28 metabolizing enzyme. PCB 28-induced mortality in flies was accompanied by locomotor impairment, a common phenotype of neurodegenerative disorders. Along this line, we show PCB 28-initiated caspase activation in differentiated fly neurons. This suggested the loss of neurons through apoptosis. Our findings in flies are congruent with observation in human exposed to high PCB levels. In plasma samples of PCB exposed humans, levels of the neurofilament light chain increase after LC-PCB exposure, indicating neuronal damage. In summary our findings demonstrate parallels between Drosophila and the human systems with respect to CYP mediated metabolism and PCB mediated neurotoxicity.

General Strategy for Integrated Bioorthogonal Prodrugs: Pt(II)-Triggered Depropargylation Enables Controllable Drug Activation In Vivo.

Bioorthogonal decaging reactions for controllable drug activation within complex biological systems are highly desirable yet extremely challenging. Herein, we find a new class of Pt(II)-triggered bioorthogonal cleavage reactions in which Pt(II) but not Pt(IV) complexes effectively trigger the cleavage of O/N-propargyl in a variety of ranges of caged molecules under biocompatible conditions. Based on these findings, we propose a general strategy for integrated bioorthogonal prodrugs and accordingly design a prodrug 16, in which a Pt(IV) moiety is covalently connected with an O2-propargyl diazeniumdiolate moiety. It is found that 16 can be specifically reduced by cytoplasmic reductants in human ovarian cancer cells to liberate cisplatin, which subsequently stimulates the cleavage of O2-propargyl to release large amounts of NO in situ, thus generating synergistic and potent tumor suppression activity in vivo. Therefore, Pt(II)-triggered depropargylation and the integration concept might provide a general strategy for broad applicability of bioorthogonal cleavage chemistry in vivo.

Metabolic Activation and Covalent Protein Binding of Berberrubine: Insight into the Underlying Mechanism Related to Its Hepatotoxicity.

INTRODUCTION: Berberrubine (BRB), an isoquinoline alkaloid, is a major constituent of medicinal plants Coptis chinensis Franch or Phellodendron chinense Schneid. BRB exhibits various pharmacological activities, whereas exposure to BRB may cause toxicity in experimental animals. METHODS: In this study, we thoroughly investigated the liver injury induced by BRB in mice and rats. To explore the underlying mechanism, a study of the metabolic activation of BRB was conducted. Furthermore, covalent modifications of cysteine residues of proteins were observed in liver homogenate samples of animals after exposure to BRB, by application of an exhaustive proteolytic digestion method. RESULTS: It was demonstrated that BRB-induced hepatotoxicities in a time- and dose-dependent manner, based on the biochemical parameters ALT and AST. H&E stained histopathological examination showed the occurrence of obvious edema in liver of mice after intraperitoneal (i.p.) administration of BRB at a single dose of 100 mg/kg. Slight hepatotoxicity was also observed in rats given the same doses of BRB after six weeks of gavage. As a result, four GSH adducts derived from reactive metabolites of BRB were detected in microsomal incubations with BRB fortified with GSH as a trapping agent. Moreover, four cys-based adducts derived from reaction of electrophilic metabolites of BBR with proteins were found in livers. CONCLUSION: These results suggested that the formation of protein adducts originating from metabolic activation of BRB could be a crucial factor of the mechanism of BRB-induced toxicities.

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/ß-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.

Molecular mechanisms of collateral sensitivity to the antibiotic nitrofurantoin.

Antibiotic resistance increasingly limits the success of antibiotic treatments, and physicians require new ways to achieve efficient treatment despite resistance. Resistance mechanisms against a specific antibiotic class frequently confer increased susceptibility to other antibiotic classes, a phenomenon designated collateral sensitivity (CS). An informed switch of antibiotic may thus enable the efficient treatment of resistant strains. CS occurs in many pathogens, but the mechanisms that generate hypersusceptibility are largely unknown. We identified several molecular mechanisms of CS against the antibiotic nitrofurantoin (NIT). Mutants that are resistant against tigecycline (tetracycline), mecillinam (ß-lactam), and protamine (antimicrobial peptide) all show CS against NIT. Their hypersusceptibility is explained by the overexpression of nitroreductase enzymes combined with increased drug uptake rates, or increased drug toxicity. Increased toxicity occurs through interference of the native drug-response system for NIT, the SOS response, with growth. A mechanistic understanding of CS will help to develop drug switches that combat resistance.

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression.

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8+ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.

Protective effects of epigallocatechin gallate against ischemia reperfusion injury in rat skeletal muscle via activating Nrf2/HO-1 signaling pathway.

AIMS: Previous studies have demonstrated that epigallocatechin gallate (EGCG) had certain protective effects on myocardial and renal ischemia reperfusion (I/R) injury. We aimed to research the special effects and underling mechanisms of EGCG on skeletal muscle I/R injury. MAIN METHOD: We established an experimental rat model of I/R skeletal muscle injury and treated with different doses of EGCG. Hematoxylin eosin staining, TUNEL assay, ELISA, qRT-PCR and Western blotting were used to evaluate the effects of EGCG. KEY FINDINDS: EGCG significantly improved skeletal muscle function of I/R injury rats. Moreover, EGCG had positive effects on decreasing apoptosis of skeletal muscle tissues, alleviating oxidative stress damage and suppressing the production of inflammatory cytokines. Further, EGCG had positive effects on activating Nrf2/HO-1 signaling pathway. SIGNIFICANCE: EGCG might be a powerful candidate compound for alleviating I/R injury in rat skeletal muscle.

Deletion of cytochrome P450 oxidoreductase enhances metabolism and DNA adduct formation of benzo[a]pyrene in Hepa1c1c7 cells.

The environmental carcinogen benzo[a]pyrene (BaP) is presumed to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. However, studies using the Hepatic Reductase Null (HRN) mouse model, in which cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes, is deleted specifically in hepatocytes, have shown that loss of hepatic POR-mediated CYP function leads to greater BaP-DNA adduct formation in livers of these mice than in wild-type (WT) mice. Here, we used CRISPR/Cas9 technology to knockout (KO) POR expression in mouse hepatoma Hepa1c1c7 cells to create an in vitro model that can mimic the HRN mouse model. Western blotting confirmed the deletion of POR in POR KO Hepa1c1c7 cells whereas expression of other components of the mixed-function oxidase system including cytochrome b5 (Cyb5) and NADH:cytochrome b5 reductase (which can also serve as electron donors to CYP enzymes), and CYP1A1 was similar in BaP-exposed WT and POR KO Hepa1c1c7 cells. BaP exposure caused cytotoxicity in WT Hepa1c1c7 cells but not in POR KO Hepa1c1c7 cells. In contrast, CYP-catalysed BaP-DNA adduct levels were ~10-fold higher in POR KO Hepa1c1c7 cells than in WT Hepa1c1c7 cells, in concordance with the presence of higher levels of BaP metabolite (e.g. BaP-7,8-dihydrodiol) in the medium of cultured BaP-exposed POR KO Hepa1c1c7 cells. As was seen in the HRN mouse model, these results suggest that Cyb5 contributes to the bioactivation of BaP in POR KO Hepa1c1c7 cells. These results indicate that CYP enzymes may play a more important role in the detoxication of BaP, as opposed to its bioactivation.

Oncogenic KRAS Sensitizes Lung Adenocarcinoma to GSK-J4-Induced Metabolic and Oxidative Stress.

Genetic and epigenetic changes (e.g., histone methylation) contribute to cancer development and progression, but our understanding of whether and how specific mutations affect a cancer's sensitivity to histone demethylase (KDM) inhibitors is limited. Here, we evaluated the effects of a panel of KDM inhibitors on lung adenocarcinomas (LuAC) with various mutations. Notably, LuAC lines harboring KRAS mutations showed hypersensitivity to the histone H3K27 demethylase inhibitor GSK-J4. Specifically, GSK-J4 treatment of KRAS mutant-containing LuAC downregulated cell-cycle progression genes with increased H3K27me3. In addition, GSK-J4 upregulated expression of genes involved in glutamine/glutamate transport and metabolism. In line with this, GSK-J4 reduced cellular levels of glutamate, a key source of the TCA cycle intermediate α-ketoglutarate (αKG) and of the antioxidant glutathione, leading to reduced cell viability. Supplementation with an αKG analogue or glutathione protected KRAS-mutant LuAC cells from GSK-J4-mediated reductions in viability, suggesting GSK-J4 exerts its anticancer effects by inducing metabolic and oxidative stress. Importantly, KRAS knockdown in mutant LuAC lines prevented GSK-J4-induced decrease in glutamate levels and reduced their susceptibility to GSK-J4, whereas overexpression of oncogenic KRAS in wild-type LuAC lines sensitized them to GSK-J4. Collectively, our study uncovers a novel association between a genetic mutation and KDM inhibitor sensitivity and identifies the underlying mechanisms. This suggests GSK-J4 as a potential treatment option for cancer patients with KRAS mutations. SIGNIFICANCE: This study not only provides a novel association between KRAS mutation and GSK-J4 sensitivity but also demonstrates the underlying mechanisms, suggesting a potential use of GSK-J4 in cancer patients with KRAS mutations.

Genome-Scale Characterization of Toxicity-Induced Metabolic Alterations in Primary Hepatocytes.

Context-specific GEnome-scale metabolic Network REconstructions (GENREs) provide a means to understand cellular metabolism at a deeper level of physiological detail. Here, we use transcriptomics data from chemically-exposed rat hepatocytes to constrain a GENRE of rat hepatocyte metabolism and predict biomarkers of liver toxicity using the Transcriptionally Inferred Metabolic Biomarker Response algorithm. We profiled alterations in cellular hepatocyte metabolism following in vitro exposure to four toxicants (acetaminophen, carbon tetrachloride, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six hour. TIMBR predictions were compared with paired fresh and spent media metabolomics data from the same exposure conditions. Agreement between computational model predictions and experimental data led to the identification of specific metabolites and thus metabolic pathways associated with toxicant exposure. Here, we identified changes in the TCA metabolites citrate and alpha-ketoglutarate along with changes in carbohydrate metabolism and interruptions in ATP production and the TCA Cycle. Where predictions and experimental data disagreed, we identified testable hypotheses to reconcile differences between the model predictions and experimental data. The presented pipeline for using paired transcriptomics and metabolomics data provides a framework for interrogating multiple omics datasets to generate mechanistic insight of metabolic changes associated with toxicological responses.

Similar Safety Profile of the Enantiomeric N-Aminoalkyl Derivatives of Trans-2-Aminocyclohexan-1-ol Demonstrating Anticonvulsant Activity.

Epilepsy is one of the most common neurological disorder in the world. Many antiepileptic drugs cause multiple adverse effects. Moreover, multidrug resistance is a serious problem in epilepsy treatment. In the present study we evaluated the safety profile of three (1-3) new chiral N-aminoalkyl derivatives of trans-2-aminocyclohexan-1-ol demonstrating anticonvulsant activity. Our aim was also to determine differences between the enantiomeric compounds with respect to their safety profile. The results of the study indicated that compounds 1-3 are non-cytotoxic for astrocytes, although they exhibit cytotoxic activity against human glioblastoma cells. Moreover, 1-3 did not affect the viability of HepG2 cells and did not produce adducts with glutathione. Compounds 1-3 demonstrated no mutagenic activity either in the Salmonella typhimurium or in Vibrio harveyi tests. Additionally, the compounds displayed a strong or moderate antimutagenic effect. Finally, the P-glycoprotein (P-gp) ATPase assay demonstrated that both enantiomers are potent P-gp inhibitors. To sum up, our results indicate that the newly synthesized derivatives may be considered promising candidates for further research on anticonvulsant drug discovery and development. Our study indicated the similar safety profile of the enantiomeric N-aminoalkyl derivatives of trans-2-aminocyclohexan-1-ol, although in the previous studies both enantiomers differ in their biotransformation pathways and pharmacological activity.

Arsenic-induced metabolic shift triggered by the loss of miR-199a-5p through Sp1-dependent DNA methylation.

Inorganic arsenic is an environmental carcinogen that poses a major global public health risk. A high percentage of drinking water from wells in the U.S. contains higher-than-normal levels of arsenic, suggesting an increased risk of arsenic-induced deleterious effects. In addition to primary preventive measures, therapeutic strategies need to effectively address and integrate multiple molecular mechanisms underlying arsenic-induced carcinogenesis. We previously showed that the loss of miR-199a-5p in arsenic-transformed cells is pivotal to promote arsenic-induced angiogenesis and tumor growth in lung epithelial cells. In this study, we further showed that subacute or chronic exposure to arsenic diminished miR-199a-5p levels largely due to DNA methylation, which was achieved by increased DNA methyltransferase-1 (DNMT1) activity, mediated by the formation of specific protein 1 (Sp1)/DNMT1 complex. In addition to the DNA hypermethylation, arsenic exposure also repressed miR-199a transcription through a transcriptional repressor Sp1. We further identified an association between miR-199a-5p repression and the arsenic-mediated energy metabolic shift, as reflected by mitochondria defects and a switch to glycolysis, in which a glycolytic enzyme pyruvate kinase 2 (PKM2) was a functional target of miR-199a-5p. Taken together, the repression of miR-199a-5p through both Sp1-dependent DNA methylation and Sp1 transcriptional repression promotes an arsenic-mediated metabolic shift from mitochondria respiration to aerobic glycolysis via PKM2.