Hepa1c1c7 Cells Research Articles

Natural potential therapeutic agents of neurodegenerative diseases from the traditional herbal medicine Chinese Dragon׳s Blood

Ethnopharmacological relevanceDragon׳s blood has been used as a famous traditional medicine since ancient times by many cultures. It is a deep red resin, obtained from more than 20 different species of four distinct genera. Red resin of Dracaena cochinchinensis S.C. Chen, known as Chinese Dragon׳s Blood or Yunnan Dragon׳s Blood, has been shown to promote blood circulation, alleviate inflammation, and to treat stomach ulcers, diarrhea, diabetes, and bleeding. This study investigated an effective approach to identify natural therapeutic agents for neurodegeneration from herbal medicine. The dichloride extract and isolated effective constituents of Chinese Dragon׳s Blood showed quinone oxidoreductase 1 (NQO1) inducing activity and anti-inflammatory effect significantly, which are therapy targets of various neurodegenerative diseases. Materials and methodsMultiple chromatography and spectra analysis were utilized to afford effective constituents. Then Hepa 1c1c7 and BV-2 cells were employed to assay their NQO1 inducing and anti-inflammatory activities, respectively. ResultsBioactivities guided isolation afforded 21 effective constituents, including two new polymers cochinchinenene E (1), cochinchinenene F (2) and a new steroid dracaenol C (16). The main constituent 3 (weight percent 0.2%), 5 (weight percent 0.017%), 4 (weight percent 0.009%), 9 (weight percent 0.094%), 10 (weight percent 0.017%) and 8 (weight percent 0.006%) are responsible for the anti-inflammatory activities of Chinese Dragon׳s Blood. While, new compounds 1, 2 and known compounds 5, 11 showed good NQO1 inducing activities. The brief feature of the activities and structures was discussed accordingly. ConclusionOverviewing the bioactivities and phytochemical study result, 4'-hydroxy-2,4-dimethoxydihydrochalcone (3) and pterostilbene (5) as effective constituents of Chinese Dragon׳s Blood, were found to be potential candidate therapeutic agents for neurodegenerative diseases.

Novel prenylated bichalcone and chalcone from Humulus lupulus and their quinone reductase induction activities

A new prenylated chalcone xanthohumol M (1), a novel prenylated bichalcone humulusol (2) and six known chalcones (3–8) were found from Humulus lupulus. Their structures were determined by spectroscopic methods. All the chalcones' electrophilic abilities were assessed by GSH (glutathione) rapid screening, and their QR (quinone reductase) induction activities were evaluated using hepa 1c1c7 cells. The results of electrophilic assay and QR induction activity assay were quite well. New compounds 1 and 2, along with some known prenylated chalcones, displayed certain QR induction activity.

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.

Soybean-derived glyceollins induce apoptosis through ROS generation

Glyceollins, which are synthesized from daidzein in soybeans infected with fungi, have been shown to have anti-fungal effects and antioxidant properties. However, the anti-proliferative mechanism of glyceollins against tumor cells is unknown. Glyceollin-induced apoptosis was evidenced by a decrease in cell viability and mitochondrial membrane potential, and an increase in early redistribution of plasma membrane phosphatidylserine, the sub G1 phase, and DNA fragmentation in hepa1c1c7 cells. Western blot analysis showed that treatment of the hepa1c1c7 cells with the glyceollins decreased the expression of pro-caspase-3, Bcl-2, and cell cycle-related proteins, but increased the expression of p21 and p27, and cytochrome C release into cytosol. At a concentration of 6 μg mL(-1) or higher, glyceollins significantly stimulated the production of reactive oxygen species (ROS), which appear to be responsible for the apoptotic activity of the compounds. Our present study demonstrated that the high dose of glyceollins possibly caused apoptosis in mouse hepatoma cells through the production of ROS, suggesting the potential to exploit glyceollins as anti-tumorigenic agents.

In vitro and in vivo hepatoprotective effect of ganodermanontriol against t-BHP-induced oxidative stress

Ethnopharmacological relevanceGanoderma lucidum (Fr.) Karst. (Ganodermataceae) is a mushroom which is used as a traditional remedy in the treatment of human diseases such as hepatitis, liver disorders, hypercholesterolemia, arthritis, bronchitis and tumorigenic diseases. This study targets the evaluation of hepatoprotective activity of ganodermanontriol, a sterol isolated from Ganoderma lucidum, and the investigation of its mechanism of action in Hepa1c1c7 and murine liver cells upon tert-butyl hydroperoxide (t-BHP)-induced inflammation. t-BHP was utilized to stimulate an anti-inflammatory reaction in the hepatic cell lines and murine hepatic tissue examined. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were used to estimate the expression of ganodermanontriol (GDT)-induced proteins, including heme oxidase-1 (HO-1) and mitogen-activated protein kinases (MAPKs) as well as the corresponding mRNA. Luciferase assays were conducted to evaluate the interaction between NF-E2-related factor-2 (Nrf-2), the antioxidant response element (ARE), and the promoter region of the HO-1 gene and subsequent gene expression. Biochemical markers for hepatotoxicity were monitored to assess whether GDT protected the cells from the t-BHP-mediated oxidative stimuli. ResultsGDT induced HO-1 expression via the activation of Nrf-2 nuclear translocation and the subsequent transcription of the HO-1 gene in vitro and in vivo, which seemed to be regulated by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p38 signaling pathways. GDT exhibited in vitro and in vivo hepatoprotective activity as determined by the lowered levels of hepatic enzymes and malondialdehydes and the elevated glutathione levels. ConclusionsThis study validates the ethnopharmacological application of Ganoderma lucidum as a treatment for hepatic disorders. GDT induced in vitro and in vivo anti-inflammatory activity in t-BHP-damaged hepatic cells through the expression of HO-1, and in which PI3K/Akt and p38 kinases are involved. Our study motivates further research in the exploration of potent hepatoprotective agents from Ganoderma lucidum.

Topical Application of PPADS Inhibits Complement Activation and Choroidal Neovascularization in a Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. AMD patients have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and retinal pigment epithelium (RPE). MAC forms pores in cell membranes. Low levels of MAC result in an elevation of cytokine release such as vascular endothelial growth factor (VEGF) that promotes the formation of choroidal neovascularization (CNV). High levels of MAC result in cell lysis and RPE degeneration is a hallmark of advanced AMD. The current standard of care for CNV associated with wet AMD is intravitreal injection of anti-VEGF molecules every 4 to 12 weeks. Such injections have significant side effects. Recently, it has been found that membrane pore-forming proteins such as α-haemolysin can mediate their toxic effects through auto- and paracrine signaling and that complement-induced lysis is amplified through ATP release followed by P2X receptor activation. We hypothesized that attenuation of P2X receptor activation may lead to a reduction in MAC deposition and consequent formation of CNV. Hence, in this study we investigated topical application of the purinergic P2X antagonist Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) as a potential treatment for AMD. We found that 4.17 µM PPADS inhibited formation of HUVEC master junctions and master segments by 74.7%. In a human complement mediated cell lysis assay, 104 µM PPADS enabled almost complete protection of Hepa1c1c7 cells from 1% normal human serum mediated cell lysis. Daily topical application of 4.17 mM PPADS for 3 days attenuated the progression of laser induced CNV in mice by 41.8% and attenuated the deposition of MAC at the site of the laser injury by 19.7%. Our data have implications for the future treatment of AMD and potentially other ocular disorders involving CNV such as angioid streaks, choroidal rupture and high myopia.

The effect of bicarbonate on menadione-induced redox cycling and cytotoxicity: potential involvement of the carbonate radical

We have investigated the effect of NaHCO3 on menadione redox cycling and cytotoxicity. A cell-free system utilized menadione and ascorbic acid to catalyze a redox cycle, and we utilized murine hepatoma (Hepa 1c1c7) cells for in vitro experiments. Experiments were performed using low (2 mmol/L) and physiological (25 mmol/L) levels of NaHCO3 in buffer equilibrated to physiological pH. Using oximetry, ascorbic acid oxidation, and ascorbyl radical detection, we found that menadione redox cycling was enhanced by NaHCO3. Furthermore, Hepa 1c1c7 cells treated with menadione demonstrated cytotoxicity that was significantly increased with physiological concentrations of NaHCO3 in the media, compared with low levels of NaHCO3. Interestingly, the inhibition of superoxide dismutase (SOD) with 2 different metal chelators was associated with a protective effect against menadione cytotoxicity. Using isolated protein, we found a significant increase in protein carbonyls with menadione-ascorbate-SOD with physiological NaHCO3 levels; low NaHCO3 or SOD-free reactions produced lower levels of protein carbonyls. In conclusion, these findings suggest that the hydrogen peroxide generated by menadione redox cycling together with NaHCO3-CO2 are potential substrates for SOD peroxidase activity that can lead to carbonate-radical-enhanced cytotoxicity. These findings demonstrate the importance of NaHCO3 in menadione redox cycling and cytotoxicity.

Sulforaphane Accelerates Acetaldehyde Metabolism by Inducing Aldehyde Dehydrogenases: Relevance to Ethanol Intolerance

Many East Asians are highly intolerant to even modest alcohol consumption. These individuals accumulate acetaldehyde, the primary metabolite of ethanol, because of a genetic polymorphism of aldehyde dehydrogenase (ALDH) that metabolizes acetaldehyde to nontoxic acetate. The aim of these studies is to upregulate ALDH by dietary means, thereby reducing acetaldehyde toxicity. Sulforaphane [SF, 1-isothiocyano-4-(methylsulfinyl)butane] derived from its glucosinolate precursor contained in cruciferous vegetables and related inducers of the Keap1/Nrf2/ARE pathway were assessed for their potencies to induce ALDH in murine hepatoma Hepa1c1c7 cells. Inducer potencies for ALDH were compared with those for NQO1, a prototypical cytoprotective enzyme present downstream of the Keap1/Nrf2/ARE pathway. SF (5 or 20 µmol/day) was fed to CD-1 mice for 7 days prior to a single administration of ethanol, and then ALDH induction in organs and pharmacokinetics of acetaldehyde was examined. In addition to SF, other electrophiles, including many Michael reaction acceptors, induce ALDH. Potencies of these agents as inducers parallel their activities in inducing NQO1, and are also dependent on Nrf2. In mice, in vivo, feeding of SF induced tissue ALDH and dramatically increased (doubled) the rate of elimination of acetaldehyde arising from the administration of ethanol. SF and other edible phytochemicals may ameliorate the alcohol intolerance of individuals who are polymorphic with respect to ALDH.

The aryl hydrocarbon receptor interacts with nuclear factor erythroid 2-related factor 2 to mediate induction of NAD(P)H:quinoneoxidoreductase 1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin

NAD(P)H:quinoneoxidoreductase 1 (NQO1) belongs to a group of the aryl hydrocarbon receptor (AhR) battery of drug-metabolizing enzymes that are characteristically induced by both AhR agonists and nuclear factor erythroid 2-related factor 2 (Nrf2) activators. We have previously reported that induction of Nqo1 by the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in hepa1c1c7 cells involves Nrf2 (Ma et al., Biochem J 377, 205–213, 2004). Here we analyzed the molecular mechanism of induction. Induction required AhR and its DNA-binding partner Arnt because induction was not observed in AhR or Arnt-defective cells, but induction was restored upon reconstitution of the variant cells with functional AhR or Arnt. Induction also required Nrf2, as induction by benzo[a]pyrene was lost in the liver of Nrf2 knockout mice similarly to induction by butyl hydroxyanisol, demonstrating a cross-interaction between the AhR and Nrf2 pathways for induction in vivo. TCDD increased the protein level and induced the nuclear accumulation of Nrf2 with a delayed kinetics compared with activation of AhR. Chromatin immunoprecipitation revealed that TCDD recruited both AhR and Nrf2 to the Nqo1 promoter enhancer region containing a DRE and an ARE in time-dependent manners. Co-immunoprecipitation experiments revealed that, in addition to AhR-Arnt binding, TCDD induced an interaction between AhR and Nrf2 as well as Keap1. The findings reveal that TCDD induces multi protein complexes to mediate cross-interaction between the AhR and Nrf2 pathways, uncovering a novel mechanistic aspect of gene regulation by environmental chemicals through AhR and Nrf2.

Detection of AHR in in vitro-generated Th17 cells using novel monoclonal antibodies (P1191)

Abstract Several studies have shown an increase in the mRNA levels of AHR in both mouse and human Th17 cells, however, protein levels in Th17 cells have been difficult to assess due to lack of reagents for flow cytometry. We developed novel monoclonal antibodies to enable detection of AHR by flow cytometry. The antibodies were characterized by western blot, flow cytometry, and immunocytochemistry. By western blot, both antibodies produced bands of approximately 95-100 kDa in lysates prepared from transiently transfected HEK-293T cells and HepG2 or Hepa1c1c7 cells (for human and mouse AHR, respectively). The anti-mouse AHR antibody also recognized a band of 95 kDa in liver lysates prepared from C57Bl/6 mice but not from AHR knockout mice. High levels of AHR protein were detected in cells polarized under Th17 conditions for 3 days, and these levels decreased on days 6 and 10, while AHR was not detected if Th17 were polarized from splenocytes from AHR knockout mice. Analysis of AHR expression by immunocytochemistry showed that these antibodies recognized AHR in the cytoplasm of untreated cells. Upon treatment of cells with FICZ, a high affinity ligand for AHR, AHR translocated to the nucleus. These antibodies will enable the examination of expression levels of AHR by flow cytometry and immunocytochemistry to provide insight into the role of AHR in many biological systems.

Abstract 4795: N-hydroxylation of 4-aminobiphenyl (ABP) and associated oxidative stress may influence ABP carcinogenicity in the mouse liver.

Abstract Liver cancer is the 3rd most common cause of death from cancer worldwide due to poor prognosis and lack of treatment options. A prominent sex difference is observed in human liver cancer such that men have a 3 to 5 fold higher incidence than women even after accounting for known etiological factors. In a tumor study carried out previously in our laboratory, two doses of the human carcinogen 4-aminobiphenyl (ABP) given to mice on postnatal days 8 and 15 resulted in the formation of liver tumors at 1 year. Moreover, female mice were dramatically protected from liver tumors compared to males, which parallels the sex difference found in human liver cancer. Our goal is to elucidate the molecular mechanisms behind the sex differences in ABP carcinogenesis in mouse liver with the hope of extrapolating to the human condition. Oxidative stress may play an important role in human liver carcinogenesis since all major etiological factors for human liver cancer, including viral hepatitis, alcohol, obesity and chemical carcinogens have been shown to be associated with oxidative stress. To determine whether oxidative stress is involved in ABP-mediated carcinogenesis in the mouse, we first assessed the ability of ABP to generate oxidative stress in the mouse Hepa1c1c7 hepatoma cell line. ABP did not produce reactive oxygen species (ROS) or oxidative DNA damage in Hepa1c1c7 cells. However, N-hydroxy-ABP (HOABP), an in vivo metabolite of ABP, was a potent inducer of both ROS and oxidative DNA damage. Furthermore, HOABP-induced oxidative stress was dose-dependent and could be blocked by co-treatment with the antioxidant N-acetylcysteine. These results suggest that HOABP may be a potential source of oxidative stress in mouse liver. We subsequently characterized the kinetics of ABP N-hydroxylation by mouse liver microsomes, and found that more than one cytochrome P450 (CYP) appears to be involved in the reaction. As predicted by the traditional model of ABP bioactivation, the high affinity ABP N-hydroxylation reaction is mediated by CYP1A2, while at least one additional low affinity site belonging to an as yet unidentified CYP is responsible for significant ABP N-hydroxylation at higher concentrations of ABP that would be expected following the doses of ABP used in our tumor study. However, no sex difference was found in either high or low affinity ABP N-hydroxylation activities between liver microsomes from male and female postnatal (day 15) or adult mice. Future studies will focus on quantifying both acute and chronic changes in oxidative stress and antioxidant levels in the mouse liver following carcinogenic doses of ABP, and correlating such changes to tumor formation. Citation Format: Shuang Wang, Kim S. Sugamori, Denis M. Grant. N-hydroxylation of 4-aminobiphenyl (ABP) and associated oxidative stress may influence ABP carcinogenicity in the mouse liver. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4795. doi:10.1158/1538-7445.AM2013-4795

Potential Chemopreventive Activity of a New Macrolide Antibiotic from a Marine-Derived Micromonospora sp.

Agents capable of inducing phase II enzymes such as quinone reductase 1 (QR1) are known to have the potential of mediating cancer chemopreventive activity. As part of a program to discover novel phase II enzyme-inducing molecules, we identified a marine-derived actinomycete strain (CNJ-878) that exhibited activity with cultured Hepa 1c1c7 cells. Based on this activity, a new macrolide, juvenimicin C (1), as well as 5-O-α-l-rhamnosyltylactone (2), were isolated from the culture broth of a Micromonospora sp. Compound 1 enhanced QR1 enzyme activity and glutathione levels by two-fold with CD values of 10.1 and 27.7 μM, respectively. In addition, glutathione reductase and glutathione peroxidase activities were elevated. This is the first reported member of the macrolide class of antibiotics found to mediate these responses.

Quinone Reductase Induction Activity of Phlorotannins Derived from Eisenia bicyclis in Hepa1c1c7 Cells

To assess the feasibility of phlorotannins from Eisenia bicyclis as cancer chemopreventative agents, we tested whether they induced quinone reductase (QR) in Hepa1c1c7 cells. The ethyl acetate (EtOAc) soluble fraction obtained from E. bicyclis exhibited a QR induction activity in Hepa1c1c7 cells. Successive column chromatography of the active EtOAc fraction resulted in the isolation of four phlorotannins. Their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopic techniques and characterized as phloroglucinol (1), dioxinodehydroeckol (2), dieckol (3), and fucofuroeckol-A (4). Among these compounds, fucofuroeckol-A (4) showed moderate QR induction activity, and dioxinodehydroeckol (2) exhibited potent QR induction potency with 2.05 ± 0.04 fold induction at a concentration of 50 µM compared to the dimethyl sulfoxide solvent-treated control cells. However, phloroglucinol (1) and dieckol (3) exerted no detectable QR induction activity in Hepa1c1c7 cells. These results suggest that dioxinodehydroeckol could serve as a useful cancer chemopreventive chemical.

Aryl Hydrocarbon Receptor Modulates NADPH Oxidase Activity via Direct Transcriptional Regulation of p40phoxExpression

A member of the NADPH oxidase subunits, p40(phox) plays an important role in the regulation of NADPH oxidase activity and the subsequent production of reactive oxygen species (ROS). In this study, we show that mouse p40(phox) is a novel transcriptional target of the aryl hydrocarbon receptor (AhR), known as a dioxin receptor or xenobiotic receptor, in the liver. Treatment of mice with 3-methylcholanthrene (3MC) increased p40(phox) gene expression in the liver, but this induction of p40(phox) gene expression was diminished by the deletion of the AhR gene in the liver. Consistent with the in vivo results, the expression of the p40(phox) gene was increased in 3MC-treated Hepa1c1c7 cells in an AhR-dependent manner. In addition, promoter analysis established p40(phox) as a transcriptional target of AhR. Studies using the RNA-interference technique revealed that p40(phox) is involved in the increase of NADPH oxidase activity and the subsequent ROS production in AhR-activated Hepa1c1c7 cells. Consequently, the results obtained here may provide a novel molecular mechanism for ROS production after exposure to dioxins.

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.

S-Alk(en)ylmercaptocysteine: Chemical Synthesis, Biological Activities, and Redox-Related Mechanism

S-Alk(en)ylmercaptocysteine (CySSR, R = methyl, ethyl, propyl, 1-propenyl, and allyl), which are the putative metabolites of Allium thiosulfinates, were chemically synthesized. CySSR, but not the corresponding monosulfide species S-alk(en)yl cysteine (CySR), were able to induce quinone reductase (QR, a representative phase II enzyme) in Hepa 1c1c7 cells and inhibit nitric oxide (NO, an inflammatory biomarker) formation in lipopolysaccharide (LPS)-activated RAW 264.7 cells. These results indicate the importance of the disulfide bond for the biological activities of CySSR. Glutathione (GSH) and N-acetylcysteine (NAC), but not other types of cellular antioxidants, suppressed multiple biological activities of CySSR in vitro. The inhibitory effects of GSH and NAC on the biological activities of CySSR were correlated with a glutaredoxin (Grx)-dependent intracellular reduction of CySSR to generate cysteine and RSH, which were secreted into the extracellular medium.

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.

Aryl Hydrocarbon Receptor Activation Attenuates Per1 Gene Induction and Influences Circadian Clock Resetting

Light-stimulated adjustment of the circadian clock is an important adaptive physiological response that allows maintenance of behavioral synchrony with solar time. Our previous studies indicate that the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8- tetrachlorodibenzo-p-dioxin attenuates light-induced phase resetting in early night. However, the mechanism of inhibition remains unclear. In this study, we showed that another potent AhR agonist-β-naphthoflavone (BNF)-significantly decreased light-induced phase shifts in wild-type (WT) mice, whereas AhR knockout mice had an enhanced response to light that was unaffected by BNF. Mechanistically, BNF blocked light induction of the Per1 transcript in suprachiasmatic nucleus and liver in WT mice, and BNF blocked forskolin (FSK)-induced Per1 transcripts in Hepa-1c1c7 (c7) cells. An E-box decoy did not affect BNF inhibition of FSK-induced Per1 transcripts in c7 cells. cAMP-response element (CRE)-dependent induction of Per1 promoter activity in response to FSK in combination with phorbol 12-tetradecanoate 13-acetate was suppressed in cells that expressed high levels of AhR (c7) compared with cells lacking functional AhR activity (c12). In addition, the inhibitory effect of BNF on FSK-induced Per1 was dependent on phosphorylation of JNK. Together, these results suggest that AhR activation inhibits light-induced phase resetting through the activation of JNK, negative regulation of CREs in the Per1 promoter, and suppression of Per1.

Cyprodinil as an activator of aryl hydrocarbon receptor

Cyprodinil is a pyrimidinamine fungicide, used worldwide by agriculture. It is used to protect fruit plants and vegetables from a wide range of pathogens. Benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are toxic environmental pollutants and are prototypes of aryl hydrocarbon receptor (AHR) ligands. Although the structure of cyprodinil distinctly differs from those of BaP and TCDD, our results show that cyprodinil induced nuclear translocation of the AHR, and induced the transcriptional activity of aryl hydrocarbon response element (AHRE). Cyprodinil induced the expression of cytochrome P450 (CYP) 1A1, a well-known AHR-targeted gene, in ovarian granulosa cells, HO23, and hepatoma cells, Hepa-1c1c7. Its induction did not appear in AHR signal-deficient cells, and was blocked by the AHR antagonist, CH-223191. Cyprodinil decreased AHR expression in HO23 cells, resulting in CYP1A1 expression decreasing after it peaked at 9h of treatment in HO23 cells. Dexamethasone is a synthetic agonist of glucocorticoids. Cyprodinil enhanced dexamethasone-induced gene expression, and conversely, its induction of CYP1A1 expression was decreased by dexamethasone in HO23 cells, indicating its induction of crosstalk between the AHR and glucocorticoid receptor and its role as a potential endocrine disrupter. In addition to BaP, TCDD, and an AHR agonist, β-NF, cyprodinil also phosphorylated extracellular signal-regulated kinase (ERK) in HO23 and Hepa-1c1c7 cells, indicating its deregulation of ERK activity. In summary, our results demonstrate that cyprodinil, similar to BaP, acts as an AHR activator, a potential endocrine disrupter, and an ERK disrupter.

Zerumbone, an electrophilic sesquiterpene, induces cellular proteo-stress leading to activation of ubiquitin–proteasome system and autophagy

Zerumbone, a sesquiterpene present in Zingiber zerumbet Smith, has been implicated as a promising chemopreventive agent. Interestingly, a number of studies have revealed that its potent bioactivities are dependent on the electrophilic moiety of its α,β-unsaturated carbonyl group, while our recent findings showed its chemical potential for binding to cellular proteins through a Michael reaction. In the present study, modifications of proteins by zerumbone led to their insolubilization in vitro. In living cell models, zerumbone induced ubiquitination and aggregation of cellular proteins, which demonstrated its substantial proteo-toxicity. On the other hand, it was also revealed that zerumbone possesses potential for activating intracellular proteolysis mechanisms of the ubiquitin–proteasome system and autophagy. Furthermore, it up-regulated expressions of pro-autophagic genes including p62, which is known as a cargo receptor of aggrephagy, the selective autophagic process for protein aggregates. Pretreatment of Hepa1c1c7 cells with zerumbone conferred a phenotype resistant to cytotoxicity and protein modifications by 4-hydroxy-2-nonenal, an endogenous lipid peroxidation product, in a p62-dependent manner. Together, these results suggest that protein modifications by zerumbone cause mild proteo-stress, thereby activating intracellular proteolysis machineries to maintain protein homeostasis. We consider these effects on proteolysis mechanisms to be hormesis, which provides beneficial functions through mild biological stresses.