Mouse Hepatoma Hepa-1c1c7 Cells Research Articles

Molecular Networking, Docking, and Biological Evaluation of Licarin A from Myristica fragrans as a Potential Cancer Chemopreventive Agent.

Currently, clinically available cancer chemopreventive drug options are limited to mostly tamoxifen and its derivatives, such as raloxifene, and approved specifically for breast cancer. Thus, the availability of chemopreventive drug molecules for other types of malignant cancers would be desirable. In previous reports, the arils of Myristica fragrans (mace) have been found to exhibit cancer chemopreventive activity. Therefore, the purpose of the present study was to identify a natural product from this species with potential chemopreventive activity guided by chemoinformatic sample analysis via Global Natural Products Social (GNPS) molecular networking and molecular docking. The neolignan licarin A (1) was identified as a potential chemopreventive constituent, and subsequently submitted to several in vitro bioassays and a zebrafish toxicity evaluation. In this work, 1 afforded superior phosphoNF-κBp65 phosphorylation activity in DU-145 prostate cancer cells compared to isoliquiritigenin (2), which was used as a natural product chemopreventive control. Both 1 and 2 showed a longer-lasting reduction in cellular stress in a cell oxidative stress real-time dose-response assay than the positive control using Hepa1c1c7 mouse hepatoma cells. In addition, 1 displayed similar activities to 2, while also being less toxic to zebrafish (Danio rerio) than both this chalcone and the clinically used chemopreventive drug tamoxifen.

Quercetin Attenuates Acetaldehyde-Induced Cytotoxicity via the Heme Oxygenase-1-Dependent Antioxidant Mechanism in Hepatocytes.

It is still unclear whether or how quercetin influences the toxic events induced by acetaldehyde in hepatocytes, though quercetin has been reported to mitigate alcohol-induced mouse liver injury. In this study, we evaluated the modulating effect of quercetin on the cytotoxicity induced by acetaldehyde in mouse hepatoma Hepa1c1c7 cells, the frequently used cellular hepatocyte model. The pretreatment with quercetin significantly inhibited the cytotoxicity induced by acetaldehyde. The treatment with quercetin itself had an ability to enhance the total ALDH activity, as well as the ALDH1A1 and ALDH3A1 gene expressions. The acetaldehyde treatment significantly enhanced the intracellular reactive oxygen species (ROS) level, whereas the quercetin pretreatment dose-dependently inhibited it. Accordingly, the treatment with quercetin itself significantly up-regulated the representative intracellular antioxidant-related gene expressions, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase, catalytic subunit (GCLC), and cystine/glutamate exchanger (xCT), that coincided with the enhancement of the total intracellular glutathione (GSH) level. Tin protoporphyrin IX (SNPP), a typical HO-1 inhibitor, restored the quercetin-induced reduction in the intracellular ROS level, whereas buthionine sulphoximine, a representative GSH biosynthesis inhibitor, did not. SNPP also cancelled the quercetin-induced cytoprotection against acetaldehyde. These results suggest that the low-molecular-weight antioxidants produced by the HO-1 enzymatic reaction are mainly attributable to quercetin-induced cytoprotection.

The Metabolite of γ-Tocopherol, 2,7,8-Trimethyl-2-(2′-Carboxyethyl)-6-Hydroxychroman, Exerts Intracellular Antioxidant Activity via Up-Regulation of Heme Oxygenase-1 in Hepatocytes

γ-Tocopherol (γT) is the major form of vitamin E contained in plants and seed oils. Although it is readily metabolized in the liver, the function of the metabolites is not fully understood. This study investigated the antioxidant activities of the γT metabolite 2,7,8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman (γCEHC) in comparison to its parent compound. The pretreatment of mouse hepatoma Hepa1c1c7 cells with γCEHC showed a cytoprotective effect on the hydrogen peroxide-induced cytotoxicity to a lesser extent than that of γT. A mechanistic investigation revealed that both γ-CEHC and γT significantly up-regulated the gene and protein expressions of heme oxygenase-1 (HO-1) via the promotion of the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, the combination of γCEHC and γT significantly increased the gene and protein levels of HO-1 and the nuclear translocation of Nrf2, suggesting that it was an additive effect. Tin protoporphyrin IX (SnPP), a representative HO-1 inhibitor, significantly impaired the cytoprotection of γCEHC and γT against the hydrogen peroxide-induced cytotoxicity. These results suggested that not only γT but also its metabolite, γCEHC, are a promising cytoprotective factor against oxidative stress-induced cytotoxicity and that the cytoprotective effect is attributable to the cooperation of both compounds.

A Major Intestinal Catabolite of Quercetin Glycosides, 3-Hydroxyphenylacetic Acid, Protects the Hepatocytes from the Acetaldehyde-Induced Cytotoxicity through the Enhancement of the Total Aldehyde Dehydrogenase Activity.

Aldehyde dehydrogenases (ALDHs) are the major enzyme superfamily for the aldehyde metabolism. Since the ALDH polymorphism leads to the accumulation of acetaldehyde, we considered that the enhancement of the liver ALDH activity by certain food ingredients could help prevent alcohol-induced chronic diseases. Here, we evaluated the modulating effects of 3-hydroxyphenylacetic acid (OPAC), the major metabolite of quercetin glycosides, on the ALDH activity and acetaldehyde-induced cytotoxicity in the cultured cell models. OPAC significantly enhanced the total ALDH activity not only in mouse hepatoma Hepa1c1c7 cells, but also in human hepatoma HepG2 cells. OPAC significantly increased not only the nuclear level of aryl hydrocarbon receptor (AhR), but also the AhR-dependent reporter gene expression, though not the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent one. The pretreatment of OPAC at the concentration required for the ALDH upregulation completely inhibited the acetaldehyde-induced cytotoxicity. Silencing AhR impaired the resistant effect of OPAC against acetaldehyde. These results strongly suggested that OPAC protects the cells from the acetaldehyde-induced cytotoxicity, mainly through the AhR-dependent and Nrf2-independent enhancement of the total ALDH activity. Our findings suggest that OPAC has a protective potential in hepatocyte models and could offer a new preventive possibility of quercetin glycosides for targeting alcohol-induced chronic diseases.

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.

Effects of Maerua subcordata (Gilg) DeWolf on electrophile-responsive element (EpRE)-mediated gene expression in vitro.

Plant extracts and phytochemicals may prevent chronic diseases via activation of adaptive cellular stress response pathways including induction of antioxidant and phase II detoxifying enzymes. The regulatory regions of these inducible genes encode the electrophile-response element (EpRE). This study tested the EpRE induction ability of Maerua subcordata (fruit, leaf, root, seed) methanol extracts and selected candidate constituents thereof, identified by liquid chromatography coupled with multistage mass spectroscopy, employing an EpRE luciferase reporter gene assay using hepa-1c1c7 mouse hepatoma cells. A parallel Cytotox CALUX assay using human osteosarcoma U2OS cells was used to monitor any non-specific changes in luciferase activity or cytotoxicity. Results showed that fruit, root, and seed extracts were non-cytotoxic up to a concentration of 30 gram dry weight per litre but the leaf extract exhibited some cytotoxicity and that the leaf (despite some cytotoxicity), fruit, and seed extracts showed strong induction of EpRE mediated gene expression while induction by the root extract was minimal. Selected candidates included glucosinolates, isothiocyanates, and some biogenic amines. Subsequent studies showed that methyl-, ethyl-, isopropyl-, isobutyl- isothiocyanates, and sec-butyl thiocyanate as well as glucobrassicin induced concentration (1–100 μM) dependent EpRE-mediated gene expression while the biogenic amines stachydrine and trigonelline acted as inhibitors of EpRE-mediated gene expression at 100 μM. The identification of glucolepidiin, glucobrassicin, glucocapparin, stachydrine, and trigonelline in all extracts was confirmed using standards and based on multiple reaction monitoring; yet, glucobrassicin level in the root extract was negligible. In conclusion, this study provided a first report on EpRE mediated gene expression effects of M. subcordata; and despite detection of different glucosinolates in all extracts, those containing glucobrassicin particularly displayed high EpRE induction. Because EpRE inducers are cytoprotective and potential chemopreventive agents while inhibitors are suggested adjuvants of chemotherapy, results of this study imply that process manipulation of this plant may result in herbal preparations that may be used as chemopreventive agents or adjuvants of chemotherapies.

β-hydroxybutyrate protects hepatocytes against endoplasmic reticulum stress in a sirtuin 1-independent manner

β-hydroxybutyrate (BHB), a major ketone body in mammals, is produced from fatty acids through mitochondrial fatty acid oxidation in hepatocytes. To elucidate the role of BHB in the hepatic endoplasmic reticulum (ER), we examined the effects of BHB on hepatic ER stress induced by tunicamycin. In mouse hepatoma Hepa1c1c7 cells, BHB treatment suppressed the protein expression of ER stress responsive genes and increased cell viability, while reducing the protein expression of apoptosis inducible genes, without causing any alterations in the protein expression of sirtuin 1 (SIRT1) or the phosphorylation of AMP-activated protein kinase. The intraperitoneal administration of BHB also reduced the protein expression of ER stress responsive genes in mouse livers. In human hepatoma HepG2 cells, the protein expression levels of ER stress responsive genes were increased by the partial inhibition of BHB production with siRNA targeting endogenous 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase, whereas they were decreased by promoting BHB production with fenofibrate. These findings revealed that BHB helps to suppress hepatic ER stress via a SIRT1-independent pathway, and it might be possible to manipulate ER stress by regulating BHB production genetically or pharmacologically.

The nuclear receptor Rev-erbα participates in circadian regulation of Ugt2b enzymes in mice

Circadian clock is known to modulate phase I metabolism, however whether and how the phase II enzymes UDP-glucuronosyltransferases (UGTs) are regulated by circadian clock are largely unknown. In this study, we aimed to investigate a potential role of the clock gene Rev-erbα in regulation of Ugt2b enzymes. Ugt2b mRNA and protein expression in mouse livers were determined at a 4-h interval around the clock. Ugt2b activity was probed using morphine as a specific substrate. Regulation of Ugt2b by Rev-erbα was investigated using mouse hepatoma Hepa-1c1c7 cells and Rev-erbα knock-out (Rev-erbα−/−) mice. Luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays were performed to identify the Rev-erbα binding site in Ugt2b36 promoter. Circadian variations in hepatic mRNA expression were observed for six Ugt2b genes (Ugt2b1, Ugt2b5, Ugt2b35, Ugt2b36, Ugt2b37, and Ugt2b38) in mice. Likewise, the total Ugt2b protein showed a circadian fluctuation. Glucuronidation of morphine (an Ugt2b substrate) both in vitro and in vivo was dosing-time dependent. Morphine glucuronidation was more extensive at the dosing time of ZT2 than at ZT14 consistent with the Ugt2b protein levels. Furthermore, Rev-erbα knockdown significantly increased Ugt2b mRNA and protein in Hepa-1c1c7 cells, whereas Rev-erbα overexpression or activation down-regulated Ugt2b expression. Moreover, Rev-erbα ablation in mice up-regulated the mRNA and protein expression of Ugt2b and blunted Ugt2b rhythmicity in the liver. In addition, Rev-erbα repressed the transcription of Ugt2b36 through specific binding to the −30 to −18 bp of promoter region based on a combination of luciferase reporter, mobility shift and ChIP assays. In summary, the clock gene Rev-erbα negatively regulates the expressions of Ugt2b genes, contributing to their circadian variations.

Rev-erbα activation down-regulates hepatic Pck1 enzyme to lower plasma glucose in mice

REV-ERBα (NR1D1) is a nuclear heme receptor that controls many cellular processes including cell differentiation, lipid metabolism, and inflammatory responses. Although REV-ERBα has been also implicated in regulation of glucose homeostasis, the mechanism for this regulation remains unclear. Here we investigate a potential role of REV-ERBα in regulation of PCK1 (phosphoenolpyruvate carboxykinase 1), a rate-limiting enzyme in gluconeogenesis. Hepatoma cells (Hepa-1c1c7 and HepG2 cells), wild-type mice and streptozotocin-induced diabetic mice were treated with SR9009, a specific REV-ERBα agonist. The relative mRNA and protein levels of enzymes in the cells or mouse livers were determined by qPCR and Western blotting, respectively. The fasting plasma glucose test was performed to determine the effects of Rev-erbα on glucose homeostasis. Transcriptional regulation of Pck1 by Rev-erbα was investigated using a combination of luciferase reporter, mobility shift, and chromatin immunoprecipitation (ChIP) assays. SR9009 treatment significantly decreased the mRNA level of Pck1 in mouse hepatoma Hepa-1c1c7 cells, whereas other major enzymes involved in gluconeogenesis (pyruvate carboxylase, glucose-6-phosphatase, fructose bisphosphatase and Pck2) and in glycolysis (phosphofructokinase and hexokinase-1) were unaffected. Consistent with the mRNA change, the protein level of Pck1 was down-regulated. Similarly, a repressive action of REV-ERBα on PCK1 expression was observed in human HepG2 hepatoma cells. SR9009 administration to wild-type or diabetic mice significantly reduced the level of fasting plasma glucose. This coincided with decreased mRNA and protein levels of Pck1 in the liver. In addition, the diabetic mice showed an improvement in glucose tolerability after SR9009 treatment. Promoter analysis, mobility shift, and ChIP assays revealed that Rev-erbα trans-repressed Pck1 through direct binding to -325 to -320 bp region (a RevRE site) in the gene promoter. In conclusion, Rev-erbα activation down-regulates hepatic Pck1 to lower plasma glucose.

Lycii fructus extract ameliorates hydrogen peroxide-induced cytotoxicity through indirect antioxidant action.

LFE: Lycii Fructus extract; GSH: glutathione; NQO1: NAD(P)H:quinone oxidoreductase 1; HO-1: heme oxygenase-1; GCLC: glutamate-cysteine ligase, catalytic subunit; xCT: a component of cysteine/glutamate antiporter (cystine/glutamate exchanger); CYP1A1: cytochrome P450 1A1; GSH: glutathione; AhR: aryl hydrocarbon receptor; Nrf2: nuclear factor (erythroid-derived 2)-like 2; CGA: chlorogenic acid; RT-PCR: reverse transcription-polymerase chain reaction; DTT: dithiothreitol; PMSF: phenylmethylsulfonyl fluoride; ARE: antioxidant response element; XRE: xenobiotic responsive element.

E4bp4 regulates carboxylesterase 2 enzymes through repression of the nuclear receptor Rev-erbα in mice

Carboxylesterases (CES) are a family of phase I enzymes that play an important role in xenobiotic clearance and lipid metabolism. Here, we investigate a potential role of E4 promoter-binding protein 4 (E4bp4) in regulation of Ces and CPT-11 (irinotecan, a first-line drug for treating colorectal cancer) pharmacokinetics in mice. Mouse hepatoma Hepa-1c1c7 cells were transfected with Rev-erbα expression plasmid or siRNA targeting E4bp4. The relative mRNA and protein levels of Ces enzymes in the cells or the livers of wild-type and E4bp4-deficient (E4bp4−/−) mice were determined by qPCR and Western blotting, respectively. Transcriptional regulation of Ces by E4bp4/Rev-erbα were investigated using luciferase reporter, mobility shift, and co-immunoprecipitation (Co-IP) assays. Pharmacokinetic studies were performed with wild-type and E4bp4−/− mice after intraperitoneal injection of CPT-11. E4bp4 ablation down-regulated an array of hepatic Ces genes in mice. E4bp4−/− mice also showed reduced Ces-mediated metabolism and elevated systemic exposure of CPT-11, a well-known Ces substrate. Consistently, E4bp4 knockdown reduced the expression of Ces genes (Ces2b, Ces2e and Ces2f) in Hepa-1c1c7 cells. Furthermore, Rev-erbα repressed the transcription of Ces2b, whereas E4bp4 antagonized this repressive action. Co-IP experiment confirmed a direct interaction between E4bp4 and Rev-erbα. Through a combination of promoter analysis and mobility shift assays, we demonstrated that Rev-erbα trans-repressed Ces (Ces2b) through its specific binding to the -767 to-754 bp promoter region. In conclusion, E4bp4 regulates Ces enzymes through inhibition of the transrepression activity of Rev-erbα, thereby impacting the metabolism and pharmacokinetics of Ces substrates.

Potential Skin Anti‐Aging Effects of Korean Medicinal Plant Extracts

The development of a variety of human diseases is related to aging. During the aging process, a normal homeostatic condition of biological systems including free radicals, antioxidants and biomolecules, becomes disrupted, promoting the accumulation of oxidants that cause deterioration of vital cellular functions. Thus, alternative methods in delaying the signs of aging, such as functional food supplementation, have emerged in the past few decades. In previous research studies, antioxidants have been reported to stimulate the expression of the genes that regulate essential antioxidant signaling pathways. As part of the global continuous effort to inhibit the rate of skin aging, we screened various Korean traditional medicinal plant extracts by examining the effect on the cellular defense key transcription factors, particularly nuclear factor erythroid 2 (NF‐E2)‐related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase 1 (NQO1), and heme oxygenase 1 (HO‐1). Interestingly, two of the plant extracts screened, Inula japonica Thunberg (dogyeong) and Carpeslum abrotanoides Linne (hakseul), resulted in enhanced antioxidant gene expression in mouse hepatoma Hepa1c1c7 and human keratinocyte HaCaT cells as measured by Western blot and were found to provide strong beneficial effect against skin aging. Additionally, we evaluated the effect of the plant extracts on transcriptional activity of Nrf2 using a cell line stably transfected with antioxidant response element (ARE) and reporter luciferase gene. We also demonstrated that the non‐toxic administration of dogyeong and hakseul had notable skin anti‐aging effect in animal model, thereby warranting further clinical efficacy study.Support or Funding InformationThis study was funded by BK21 Plus Program

In vitro and in vivo anti-inflammatory activities of mixed fruit and vegetable juice.

In this study, we attempted to develop fruit-based functional juice with anti-inflammatory activity for consumers living under heavy air pollution. At first, four different formulations for functional juice prepared by combining 10 kinds of fruits and other ingredients in various ratios were tested for NAD(P)H: quinoneoxidoreductase 1 (NQO1)-inducing activity in mouse hepatoma Hepa1c1c7 cells. The formulation showing the highest NQO1-inducing activity [named detoxification juice (DJ)] was investigated for its anti-inflammatory activity in a co-culture system of human lung carcinoma A549 cell line and a differentiated human monocytic cell line THP-1. DJ significantly reduced the LPS-induced production of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-1β, and tumor necrosis factor α (TNF-α). Furthermore, DJ administration suppressed the benzo[a]pyreneinduced elevation of plasma cytokine levels of C57BL/6 mice. However, the protective effect of DJ against inflammation in lung and trachea caused by air pollution needs to be assessed through clinical studies.

Effects of Benzyl Isothiocyanate and Its N-Acetylcysteine Conjugate on Induction of Detoxification Enzymes in Hepa1c1c7 Mouse Hepatoma Cells

The induction of detoxification enzymes by benzyl isothiocyanate (BITC) and its synthetic N-acetyl-L-cysteine (NAC) conjugate (NAC-BITC) was examined in Hepa1c1c7 murine hepatoma cells. BITC and NAC-BITC inhibited Hepa1c1c7 cell growth in a dose-dependent manner. Cell growth was 4.5~57.2% lower in Hepa1c1c7 cells treated with 0.1~10 μM BITC than in control-treated Hepa1c1c7 cells. The NAC-BITC treatment had a similar inhibitory pattern on Hepa1c1c7 cell growth; 0.5 μM and 10 μM NAC-BITC decreased cell growth by 13.6% and 47.4%, respectively. Treatment of Hepa1c1c7 cells with 0.1~2.0 μM BITC also elicited a dose-response effect on the induction of quinone reductase quinone reductase (QR) activity and QR mRNA expression. Treatment with 1 μM and 2 μM BITC caused 1.8- and 2.8-fold inductions of QR mRNA, respectively. By comparison, treatment with 1 μM and 2 μM NAC-BITC caused 1.6- and 1.9-fold inductions of QR mRNA, respectively. Cytochrome P450 (CYP) 1A1 and CYP2E1 induction were lower in 0.1~2 μM BITC-treated cells than in control-treated cells. CYP2E1 activity was 1.2-fold greater in 0.1 μM NAC-BITC-treated cells than in control-treated cells. However, the CYP2E1 activity of cells treated with higher concentrations (i.e., 1~2 μM) of NAC-BITC was similar to the activity of control-treated cells. Considering the potential of isothiocyanatesto prevent cancer, these results provide support for the use of BITC and NAC-BITC conjugates as chemopreventive agents.

Differential effects of triclosan on the activation of mouse and human peroxisome proliferator-activated receptor alpha

Triclosan is an anti-bacterial agent used in many personal care products, household items, medical devices, and clinical settings. Liver tumors occur in mice exposed to triclosan, a response attributed to peroxisome proliferator-activated receptor alpha (PPARα) activation; however, the effects of triclosan on mouse and human PPARα have not been fully evaluated. We compared the effects of triclosan on mouse and human PPARα using PPARα reporter assays and on downstream events of PPARα activation using mouse hepatoma Hepa1c1c7 cells and human hepatoma HepG2 cells. PPARα transcriptional activity was increased by triclosan in a mouse PPARα reporter assay and decreased in a human PPARα reporter assay. Concentrations of triclosan inhibiting 50% cell growth were similar in both human and mouse hepatoma cells. Western blotting analysis showed that triclosan increased acyl-coenzyme A oxidase (ACOX1), a PPARα target, in Hepa1c1c7 cells but decreased the level in HepG2 cells. Treatment of Hepa1c1c7 cells with triclosan enhanced DNA synthesis and suppressed transforming growth factor beta-mediated apoptosis. This did not occur in HepG2 cells. These data demonstrate that triclosan had similar cytotoxicity in Hepa1c1c7 and HepG2 cells, but differential effects on the activation of PPARα, the expression of ACOX1, and downstream events including DNA synthesis and apoptosis.

Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) analysis uncovers broad changes in chromatin structure resulting from hexavalent chromium exposure.

The ability of chromatin to switch back and forth from open euchromatin to closed heterochromatin is vital for transcriptional regulation and genomic stability, but its dynamic structure is subject to disruption by exposure to environmental agents such as hexavalent chromium. Cr(VI) exposure disrupts chromatin remodeling mechanisms and causes chromosomal damage through formation of free radicals, Cr-DNA adducts, and DNA-Cr-protein cross-links. In addition, acute, high-concentration, and chronic, low-concentration exposures to Cr(VI) lead to significantly different transcriptional and genomic stability outcomes. We used mouse hepatoma Hepa-1c1c7 cells to investigate how transcriptional responses to chromium treatment might correlate with structural chromatin changes. We used Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) analysis coupled with deep sequencing to identify regions of the genome that may switch between open and closed chromatin in response to exposure to varying Cr(VI) concentrations. At either Cr(VI) concentration, chromatin domains surrounding binding sites for AP-1 transcription factors become significantly open, whereas BACH2 and CTCF binding sites are open solely at the low and high concentrations, respectively. Parallel gene expression profiling using RNA-seq indicates that the structural chromatin changes caused by Cr(VI) affect gene expression levels in the target areas that vary depending on Cr(VI) concentration, but show no correlation between global changes in the overall transcriptional response and Cr(VI) concentration. Our results suggest that FAIRE may be a useful technique to map chromatin elements targeted by DNA damaging agents for which there is no prior knowledge of their specificity, and to identify subsequent transcriptomic changes induced by those agents.

The Ah Receptor Recruits IKKα to Its Target Binding Motifs to Phosphorylate Serine-10 in Histone H3 Required for Transcriptional Activation

Aryl hydrocarbon receptor (AHR) activation by xenobiotic ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is key to their toxicity. Following activation and nuclear translocation, AHR heterodimerizes with the AHR nuclear translocator (ARNT) and binds to AHR response elements (AhREs) in the enhancer of target genes, of which Cyp1a1 is the prototype. Previously, we showed that concomitant with AHR binding, histone H3 in the Cyp1a1 enhancer-promoter AhRE cluster became phosphorylated in serine-10 (H3S10), suggesting that the ligand-activated AHR recruited one or more kinases to the enhancer chromatin to phosphorylate this residue. To test this hypothesis, we used mouse hepatoma Hepa-1c1c7 cells and their c35 mutant derivative, lacking a functional AHR, to search for candidate kinases that would phosphorylate H3S10 in an AHR dependent manner. Using chromatin immunoprecipitation with antibodies to a comprehensive set of protein kinases, we identified three kinases, IκB kinase α (IKKα), mitogen and stress activated protein kinase 1 (MSK1), and mitogen and stress activated protein kinase 2 (MSK2), whose binding to the Cyp1a1 enhancer was significantly increased by TCDD in Hepa-1c1c7 cells and absent in control c35 cells. Complexes of AHR, ARNT, and IKKα could be coimmunoprecipitated from nuclei of TCDD treated Hepa-1c1c7 cells and shRNA-mediated IKKα knockdown inhibited both H3S10 phosphorylation in the Cyp1a1 enhancer and the induction of Cyp1a1, Aldh3a1, and Nqo1 in TCDD-treated cells. We conclude that AHR recruits IKKα to the promoter of its target genes and that AHR-mediated H3S10 phosphorylation is a key epigenetic requirement for induction of AHR targets. Given the role of H3S10ph in regulation of chromosome condensation, AHR-IKKα cross-talk may be a mediator of chromatin remodeling by environmental agents.

Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene

This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3–30 μM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10–30 μM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H2DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by 32P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.

Non-Specific Protein Modifications by a Phytochemical Induce Heat Shock Response for Self-Defense

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.

Reactive oxygen species scavenging activities of naturally occurring colorants

Twenty-five kinds of plant extract used as natural dyeing agents were tested for their reactive oxygen species (ROS) scavenging activities using several assay systems. Among plant extracts examined in this study the hot water extracts from logwood, wattle, pomegranate, dragon’s blood, and quebracho showed relatively strong antioxidant activity. In particular, the pigments extracted from pomegranate and wattle effectively suppressed intracellular generation of ROS induced by hydrogen peroxide in mouse hepatoma hepa1c1c7 cells as well as showed a strong oxygen radical scavenging activity in in vitro assay systems, suggesting their potential usefulness as health functional food ingredients and natural food colorants.