Hepa 1c1c7 Research Articles

Peganum harmalaL. is a Candidate Herbal Plant for Preventing Dioxin Mediated Effects

Dioxins are widespread environmental contaminants that have been linked with a variety of deleterious effects on human health including increased cancer rates. The detrimental effects of 2,3,7,8-tetrachlorodibenzo- P-dioxin (TCDD, one of the most common environmental dioxins) are mediated via the aryl hydrocarbon receptor (AhR). AhR is a transcription factor that regulates the expression of the carcinogen-activating enzyme, cytochrome P450 1a1 (Cyp1a1). In the present study, we examined the ability of the methanolic extract of Peganum harmala L. (Zygophyllaceae) fruiting tops to affect TCDD-activated AhR-mediated signal transduction in mouse hepatoma Hepa 1c1c7 cells. Our results showed that Peganum harmala extract significantly inhibited the TCDD-mediated induction of Cyp1a1 at mRNA, protein, and activity levels. A similar pattern of inhibition at the catalytic activity level was observed with the other AhR ligands tested. The ability of the extract to inhibit Cyp1a1 was strongly correlated with its ability to inhibit AhR-dependent luciferase activity and electrophoretic mobility shift assays. Harmine and harmaline were found to be the dominant components of the plant extract with a relative abundance of 7 and 4.85 % (w/w), respectively. In addition, both of the active alkaloids showed an inhibitory effect on TCDD-induced Cyp1a1 activity level. We concluded that Peganum harmala L. can interfere with AhR ligands-mediated effects.

Hydrogen Peroxide Is a Second Messenger in Phase 2 Enzyme Induction by Cancer Chemopreventive Dithiolethiones

The ability of three dithiolethione cancer chemopreventives, oltipraz 1, anetholedithione (ADT) 2, 1,2-dithiole-3-thione (D3T) 3, and the major metabolite, 4, of 1, to induce the cytoprotective enzyme NQO1 in Hepa 1c1c7 cells and the inhibition of this induction by catalase are demonstrated. The ability of 1, 3, and 4 to form O(2)(*) has been reported, and it is here demonstrated that 2 decomposes in the presence of GSH to form, upon addition of the nitrone spin trap DMPO, the DMPO-OH adduct that is detectable by EPR. Decomposition of 2 in the presence of GSH elicits, upon the addition of hydroethidine and excitation at 510 nm, fluorescence at 580 nm that is diminished by the addition of superoxide dismutase. The compound 4, is a product of the reduction of 1, and it is demonstrated that 2 and 3 decompose in the presence of reductants such as thiolates and NaBH(4), followed by addition of CH(3)I, to form the dimethylated products of reductive cleavage of the S(1)-S(2) bond. The same products are isolated subsequent to lysis in buffer containing CH(3)I of Hepa 1c1c7 cells treated with 2 or 3. Reductive cleavage of 2 and 3 in aqueous ethanol by NaBH(4) in an argon atmosphere, followed by acidic destruction of remaining borohydride and neutralization and introduction of O(2) results in the reformation of 2 and 3 to the extent of 80 and 33%, respectively. The data in toto are consistent with a model in which dithiolethiones, generally, undergo reductive cleavage in Hepa 1c1c7 cells, thereby resulting in the generation of O(2)(*) that dismutates to H(2)O(2), that subsequently, by direct or indirect means, effects the nuclear translocation of transcription factor Nrf2, that upregulates phase 2 enzyme expression.

Induction of quinone reductase activity by psoralidin isolated from Psoralea corylifolia in mouse hepa 1c1c7 cells

Quinone reductase (QR) is a protective phase II enzyme against mutagens and carcinogens which is inducible by a number of chemical compounds in plants. This study was carried out to investigate effects of the fractions from the seeds of Psoralea corylifolia on the induction of QR with Hepa 1c1c7 murine hepatoma cell line. The ethyl acetate-soluble fraction of the methanolic extract from the seeds was found to induce QR and the concentration of 1.5 fold QR induction (1.5 FIC) was 1.2 mug/mL. We obtained as an active compound, psoralidin, isolated from the ethyl acetate-soluble fraction after further sequential fractionation with column chromatography and 1.5 FIC of psoralidin was 0.5 mug/mL. The seeds of Psoralea corylifolia and psoralidin might be a candidate for developing QR inducers.

Peptide-mediated targeting of hepatocytes via low density lipoprotein receptor-related protein (LRP)

It has previously been reported that a peptide sequence of T7 phage protein p17 mediates uptake of its cargo by liver parenchymal cells. The aim of this study was to identify the phage-binding receptor. The involvement of LRP was confirmed by the observations that phage binding to Hepa 1c1c7 cells was inhibited by the LRP-binding receptor-associated protein, LRP-deficient mouse embryonic fibroblasts bound phage with lower efficiency than their wild-type counterparts, and using mouse models with ablated LRP liver expression. The identification of LRP as a cognate receptor for this sequence offers a new ligand-receptor combination for hepatocyte delivery of therapeutic agents.

Down-regulation of the detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 by vanadium in Hepa 1c1c7 cells

Recent data suggest that vanadium (V 5+) compounds exert protective effects against chemical-induced carcinogenesis, mainly through modifying various xenobiotic metabolizing enzymes. In fact, we have shown that V 5+ down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism. However, incongruously, there is increasing evidence that V 5+ is found in higher amounts in cancer cells and tissues than in normal cells or tissues. Therefore, the current study aims to address the possible effect of this metal on the regulation of expression of an enzyme that helps maintain endogenous antioxidants used to protect tissues/cells from mutagens, carcinogens, and oxidative stress damage, NAD(P)H:quinone oxidoreductase 1 (Nqo1). In an attempt to examine these effects, Hepa 1c1c7 cells and its AhR-deficient version, c12, were treated with increasing concentrations of V 5+ in the presence of two distinct Nqo1 inducers, the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V 5+ inhibits the TCDD- and SUL-mediated induction of Nqo1 at mRNA, protein, and catalytic activity levels. At transcriptional level, V 5+ was able to decrease the TCDD- and SUL-induced nuclear accumulation of Nrf2 and the subsequent binding to antioxidant responsive element (ARE) without affecting Nrf2 protein levels. Looking at post-transcriptional level; we found that V 5+ did not affect Nqo1 mRNA transcripts turn-over rates. However, at the post-translational level V 5+ increased Nqo1 protein half-life. In conclusion, the present study demonstrates that V 5+ down-regulates Nqo1 at the transcriptional level, possibly through inhibiting the ATP-dependent activation of Nrf2.

Sulforaphane induces CYP1A1 mRNA, protein, and catalytic activity levels via an AhR-dependent pathway in murine hepatoma Hepa 1c1c7 and human HepG2 cells

Recent reports have proposed that some naturally occurring phytochemicals can function as anticancer agents mainly through inducing phase II drug detoxification enzymes. Of these phytochemicals, isothiocyanates sulforaphane (SUL), present in broccoli, is by far the most extensively studied. In spite of its positive effect on phase II drug metabolizing enzymes, its effect on the phase I bioactivating enzyme cytochrome P450 1a1 (Cyp1a1) is still a matter of debate. As a first step to investigate this effect, Hepa 1c1c7 and HepG2 cells were treated with various concentration of SUL. Our results showed that SUL-induced CYP1A1 mRNA in a dose- and time-dependent manner. Furthermore, this induction was further reflected on the protein and catalytic activity levels. Investigating the effect of SUL at the transcriptional level revealed that SUL increases the Cyp1a1 mRNA as early as 1h. The RNA polymerase inhibitor actinomycin D (Act-D) completely abolished the SUL-induced Cyp1a1 mRNA. Furthermore, SUL successfully activated AhR transformation and its subsequent binding to the XRE. At the post-transcriptional level, SUL did not affect the levels of existing Cyp1a1 mRNA transcripts. This is the first demonstration that the broccoli-derived SUL can directly induce Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which SUL induces this enzyme.

Monodictyochromes A and B, Dimeric Xanthone Derivatives from the Marine Algicolous Fungus Monodictys putredinis

Investigations of the marine-derived fungus Monodictys putredinis led to the isolation of two novel dimeric chromanones (1, 2) that consist of two uniquely modified xanthone-derived units. The structures were elucidated by extensive spectroscopic measurements including NOE experiments and CD analysis to deduce the configuration. The compounds (1, 2) were examined for their cancer chemopreventive potential and shown to inhibit cytochrome P450 1A activity with IC(50) values of 5.3 and 7.5 μM, respectively. In addition, both compounds displayed moderate activity as inducers of NAD(P)H:quinone reductase (QR) in cultured mouse Hepa 1c1c7 cells, with CD values (concentration required to double the specific activity of QR) of 22.1 and 24.8 μM, respectively. Compound 1 was slightly less potent than compound 2 in inhibiting aromatase activity, with IC(50) values of 24.4 and 16.5 μM.

MG-132 inhibits the TCDD-mediated induction of Cyp1a1 at the catalytic activity but not the mRNA or protein levels in Hepa 1c1c7 cells

Previous studies have shown that 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced degradation of aryl hydrocarbon receptor (AhR) is inhibited by MG-132, a potent inhibitor of the 26S proteasome. Therefore, the current study aims to address the effect of MG-132 on the AhR-regulated gene, cytochrome P450 1a1 ( Cyp1a1), using murine hepatoma Hepa 1c1c7 cells. Our results showed that MG-132 at the highest concentration tested, 10 μM significantly increased the Cyp1a1 at mRNA, protein and catalytic activity levels through a transcriptional mechanism. On the other hand, MG-132 further potentiated the TCDD-mediated induction of Cyp1a1 at mRNA but not at protein level. In contrast, MG-132 significantly inhibited the TCDD-mediated induction of Cyp1a1 catalytic activity. In addition, we showed that the decrease in Cyp1a1 catalytic activity is not Cyp specific, as MG-132 significantly inhibited Cyp2b1 and total cytochrome P450 catalytic activities. These results prompted us to examine the effect of MG-132 on total cellular heme content and heme oxygenase-1 (HO-1) mRNA, a rate limiting enzyme of heme degradation. Our results showed that MG-132 significantly induced HO-1 mRNA in a concentration-dependent manner. Furthermore, MG-132 potentiated the induction of HO-1 mRNA by TCDD in a concentration-dependent manner. The induction of HO-1 mRNA level coincided with a decrease in total cellular heme content. In conclusion, the present study demonstrates for the first time that MG-132, despite of increasing Cyp1a1 mRNA expression, it decreases its activity probably through decreasing its heme content.

Limited contribution of isoflavones to hepatocellular phase II enzyme-inducing activity of soybean ( Glycine max) extracts

Soy flour was successively extracted with ethyl acetate, ethanol, and water and tested for quinone reductase (QR) induction in the Hepa 1c1c7 cell line. The ethanol extract was found to be the most potent, with a CD value (concentration required to double QR specific activity) of 460 μg/ml. Phase separation of the ethanol extract and a phospholipid removal procedure yielded a fraction that doubled QR at 80 μg/ml. Reverse phase medium pressure chromatography of the purified ethanol extract yielded 13 fractions. Constituents of selected fractions were identified using mass spectrometry and NMR analysis. Fractions 2, 8, and 10 contained 47% of the recovered mass and were the least active of the isolates. These three fractions contained isoflavone glycosides. The latest eluting fractions 11–13 were found to be the most potent, and contained the majority of recovered inducing units (IUs), a measure of potency.

NF‐κB and AP‐1 are key signaling pathways in the modulation of NAD(P)H:Quinone oxidoreductase 1 gene by mercury, lead, and copper

We have previously shown that Hg(2+), Pb(2+), and Cu(2+), significantly modulate the expression of NAD(P):quinone oxidoreductase 1 (Nqo1) in Hepa 1c1c7 cells through oxidative stress-dependent mechanisms. In the current study, we examined the role of redox-sensitive transcription factors, NF-kappaB and AP-1 signaling pathways in the modulation of Nqo1 by heavy metals. Our results show that the depletion of cellular GSH using L-buthionine-(S,R)-sulfoximine further potentiated the heavy metal-mediated induction of Nqo1 at the mRNA and activity levels. The NF-kappaB activator, PMA, significantly abolished the metal-mediated effects on Nqo1 mRNA and activity. In parallel, the NF-kappaB inhibitor, PDTC, further potentiated the Pb(2+)- and Hg(2+)-mediated induction of Nqo1 mRNA and activity levels, respectively. Inhibition of AP-1 upstream signaling pathway such as JNK by SP600125 significantly suppressed heavy metal-mediated induction of Nqo1 mRNA and activity levels. In contrast, inhibition of ERK by U0126 further potentiated heavy metal-mediated effects on Nqo1 mRNA, while only potentiated Hg(2+)-mediated induction of Nqo1 activity. Furthermore, p38 MAPK inhibitor, SB203580 further potentiated Pb(2+)- and Cu(2+)-mediated effects at the mRNA levels, whereas did not alter the activity levels. These results clearly demonstrate that activation of NF-kappaB negatively regulates the expression of Nqo1 by heavy metals, whereas AP-1 signaling pathways differentially modulates the heavy metal-mediated effects.

Down-Regulation of the Carcinogen-Metabolizing Enzyme Cytochrome P450 1a1 by Vanadium

Vanadium (V(5+)), a heavy metal contaminant with important toxicological consequences, has received considerable attention as an anticancer agent, although the mechanisms remain unknown. As a first step to investigate these mechanisms, we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of the aryl hydrocarbon receptor (AhR)-regulated gene: cytochrome P450 1a1 (Cyp1a1) at each step of the AhR signal transduction pathway, using Hepa 1c1c7 cells. Our results showed a significant reduction in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 mRNA, protein and activity levels after V(5+) treatments in a dose-dependent manner. Investigation of the effect of coexposure to V(5+) and TCDD at transcriptional levels revealed that V(5+) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Furthermore, despite not affecting the direct activation of the cytosolic AhR by TCDD and subsequently transforming it to a DNA-binding form, V(5+) inhibited the nuclear accumulation of liganded AhR and subsequent formation of the AhR/aryl hydrocarbon nuclear translocator (Arnt)/xenobiotic responsive element (XRE) complex. Importantly, the V(5+)-mediated inhibition of AhR/Arnt/XRE complex formation coincided with a significant decrease in ecto-ATPase activity. Looking at the post-transcriptional and post-translational effects of V(5+) on existing Cyp1a1 mRNA and protein levels, we showed that V(5+) did not affect Cyp1a1 mRNA or protein stability, thus eliminating possible role of V(5+) in modifying Cyp1a1 gene expression through these mechanisms. This study provides the first evidence that V(5+) down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism.

Arsenite and cadmium, but not chromium, induce NAD(P)H:quinone oxidoreductase 1 through transcriptional mechanisms, in spite of post-transcriptional modifications

NAD(P)H:quinone oxidoreductase (Nqo1)-mediated detoxification of quinones plays a critical role in cancer prevention. Metals alter the carcinogenicity of AhR ligands, such as TCDD, by modulating the induction of Nqo1, but the mechanism(s) remain unresolved. To decipher the molecular mechanisms involved in the alteration of Nqo1, we analyzed the effect of the metals As3+ (5 microM), Cd2+ (5 microM), and Cr6+ (25 microM) on the transcriptional activation of the Nqo1 gene and post-transcriptional modifications, in Hepa 1c1c7 cells. Both As3+ and Cd2+ induced Nqo1 mRNA in a time-dependent manner and potentiated TCDD-induced Nqo1 mRNA. Cr6+ on the other hand, completely inhibited the induction of Nqo1 mRNA by TCDD. The induction of Nqo1 mRNA by the metals was completely inhibited with the DNA transcription inhibitor actinomycin-D, indicating a requirement for de novo mRNA synthesis for the induction. Furthermore, the protein synthesis inhibitor cycloheximide decreased Nqo1 mRNA induction, suggesting a role for a labile protein in the transcriptional induction of Nqo1 mRNA by metals. Surprisingly, all three metals decreased Nqo1 mRNA stability while having no effect on Nqo1 protein half-life. Meanwhile, As3+ and Cd2+ induced constitutive Nqo1 activity and potentiated the induction by increasing concentrations of TCDD. On the other hand, Cr6+ inhibited inducible Nqo1 activity. It is apparent that metals alter Nqo1 expression at the transcriptional level, through a labile protein-mediated pathway.

Xanthones with quinone reductase-inducing activity from the fruits of Garcinia mangostana (Mangosteen)

Bioactivity-guided fractionation of a dichloromethane-soluble extract of Garcinia mangostana fruits has led to the isolation and identification of five compounds, including two xanthones, 1,2-dihydro-1,8,10-trihydroxy-2-(2-hydroxypropan-2-yl)-9-(3-methylbut-2-enyl)furo[3,2-a]xanthen-11-one (1) and 6-deoxy-7-demethylmangostanin (2), along with three known compounds, 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthone (3), mangostanin (4), and alpha-mangostin (5). The structures of compounds 1 and 2 were determined from analysis of their spectroscopic data. All isolated compounds in the present study together with eleven other compounds previously isolated from the pericarp of mangosteen, were tested in an in vitro quinone reductase-induction assay using murine hepatoma cells (Hepa 1c1c7) and an in vitro hydroxyl radical antioxidant assay. Of these, compounds 1-4 induced quinone reductase (concentration to double enzyme induction, 0.68-2.2microg/mL) in Hepa 1c1c7 cells and gamma-mangostin (6) exhibited hydroxyl radical-scavenging activity (IC50, 0.20microg/mL).

Induction of the NAD(P)H:quinone oxidoreductase 1 by ketoconazole and itraconazole: A mechanism of cancer chemoprotection

Studies in many carcinogen-induced animal models and cell lines demonstrated that azole antifungal drugs are therapeutically effective against different types of cancer. Yet, the molecular mechanisms involved are still poorly understood. Therefore, we examined the ability of three structurally different antifungal drugs, ketoconazole (KTZ), itraconazole (ITZ), and fluconazole (FLZ) to induce the expression of NAD(P)H:quinone oxidoreductase 1 (Nqo1), an enzyme known to play an important role in xenobiotic and carcinogen detoxifications. We showed that KTZ and ITZ, but not FLZ, induced Nqo1 mRNA and enzymatic activity levels in a concentration- and time-dependent manner in wild-type but not aryl hydrocarbon receptor (AhR)-deficient Hepa 1c1c7 cells. Furthermore, KTZ and ITZ increased Nqo1 de novo RNA synthesis without significantly affecting the levels of existing RNA, suggesting a transcriptional mechanism is involved. This study provides the first evidence for the ability of KTZ and ITZ to induce the Nqo1 gene expression through an AhR-dependent mechanism.

Role of Nitric Oxide in Downregulation of Cytochrome P450 1a1 and NADPH: Quinone Oxidoreductase 1 by Tumor Necrosis Factor‐α and Lipopolysaccharide

We previously demonstrated that tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS) downregulate aryl hydrocarbon receptor (AhR)-regulated genes, such as cytochrome P450 1a1 (Cyp1a1) and NADPH: quinone oxidoreductase 1 (Nqo1) gene expression, yet the mechanisms involved remain unknown. The correlation between the inflammation-mediated suppression of AhR-regulated genes and the TNF-alpha or LPS-induced nitric oxide (NO) production especially in murine hepatoma Hepa 1c1c7 cells has been questioned; therefore we investigated whether NO is involved in the modulation of Cyp1a1 and Nqo1 by TNF-alpha or LPS in Hepa 1c1c7 cells. A significant dose-dependent increase in the inducible nitric oxide synthase (NOS2) expression and NO production were observed by various concentrations of TNF-alpha (1, 5, and 10 ng/mL) and LPS (1 and 5 microg/mL) which was completely inhibited by a NOS2 inhibitor, L-N6-(1-iminoethyl) lysine (L-NIL) (1 mM). Furthermore, TNF-alpha and LPS significantly induced NOS2 expression. Both TNF-alpha and LPS repressed the beta-naphthoflavone (betaNF)-mediated induction of Cyp1a1 and Nqo1 at mRNA and activity levels. The downregulation of Cyp1a1, but not Nqo1, was significantly prevented by L-NIL. However, proxynitrite decomposer, iron tetrakis (N-methyl-4'-pyridyl) porphyrinato (FeTMPyP) (5 microM) did not affect TNF-alpha- and LPS-mediated downregulation of Cyp1a1 and Nqo1 at mRNA and activity levels. These results show that NO, but not peroxynitrite, may be involved in TNF-alpha- and LPS-mediated downregulation of Cyp1a1 without affecting the downregulation of Nqo1.

Phytochemical profiling and phase II enzyme-inducing properties of Thunbergia laurifolia Lindl. (RC) extracts

Thunbergia laurifolia Lindl. (Acanthaceae) or Rang Chuet (RC) is described in traditional medicine for protection against dietary and environmental toxicants. This work, therefore, investigated RC's phytochemical profile, antimutagenic activity, and xenobiotic detoxification potential in its extracts. RC extracts were prepared by infusion with water, ethanol, acetone and subsequently assayed for major phytochemical constituents. Total phenolic content was 24.33, 5.65, and 1.42 μg gallic acid equivalent (GAE) per mL for water, ethanol and acetone extract, respectively. HPLC analysis identified caffeic acid and apigenin as primary constituents of water extracts. Acetone and ethanol extracts contained primarily chlorophyll a and b, pheophorbide a, pheophytin a, and lutein. Treatment of Hepa 1C1C7 cells with standardized RC extracts resulted in a dose-dependent increase in QR specific activity for all extracts. Acetone extract (92 μg GAE/mL) increased QR activity 2.8-fold, while ethanol (120 μg GAE/mL) and water (1000 μg GAE/mL) extracts increased QR activity by 1.35- and 1.56-fold, respectively. The RC extracts were subsequently assayed for mutagen and antimutagenic activity by bacterial reverse mutagenesis assay. All three RC extracts exhibited strong dose-dependent antimutagenic activity inhibiting 2-aminoanthracene induced mutagenesis up to 87% in Salmonella typhimurium TA 98. These results support the traditional medicinal use of RC for detoxification and suggest the potential role of both phenolic acids and natural chlorophyll constituents in modulating these effects.

Glucocorticoid-Enhanced Expression of Dioxin Target Genes through Regulation of the Rat Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) are ligand-activated transcription factors and members of the basic helix-loop-helix Period-aryl hydrocarbon nuclear translocator-single minded and nuclear hormone receptor superfamilies, respectively. Besides their individual role as activators of specific gene transcription, also interplay between both transcription factors can be an important mechanism of regulation. In this study, we report that GR can strongly activate AhR-mediated transcription and consequent gene expression in rat H4IIe cells. Reporter gene assays showed an enhanced effect of dexamethasone on the dioxin response mediated by GR in rat H4IIe cells and mouse Hepa 1c1c7 cells, but not in human HepG2 cells and human T47D cells. These deviations between the rodent and human cell lines were confirmed by CYP1A1 enzyme activities. In addition, quantitative reverse transcription-PCR showed enhanced GR-mediated effects of dexamethasone on endogenous 2,3,7,8-tetrachlorodibenzo-[p]-dioxin target genes as well in rat H4IIe cells, but not in human HepG2 and human T47D cells. Surprisingly, AhR itself was upregulated by combined dioxin/glucocorticoid exposure in rat H4IIe cells but not in the human cells which could be explained by the presence of two putative glucocorticoid response elements in the rat AhR promoter, but not in the human AhR promoter. This GR-mediated expression of dioxin target genes through upregulation of the AhR in rat but not in human cells opens the possibility that dioxin responses in rodent-based models for toxicity differ from humans and provides new insight into the interactions of stress-related pathways, biological effects of dioxin-like compounds and may possibly have implications for risk assessment.

Transcriptional activation and posttranscriptional modification of Cyp1a1 by arsenite, cadmium, and chromium

Heavy metals alter the carcinogenicity of AhR ligands by modulating the induction of the Cyp1a1 enzyme, but the mechanism(s) remain unresolved. In this study, the effect of the heavy metals, As 3+, Cd 2+, and Cr 6+, on the transcriptional and posttranscriptional regulation of the Cyp1a1 gene was investigated in Hepa 1c1c7 cells. A time-dependent study showed that all metals significantly induced the basal Cyp1a1 mRNA, but only As 3+ and Cd 2+ further potentiated the inducible Cyp1a1 mRNA level. Alternately, Cr 6+ inhibited the TCDD-mediated induction of Cyp1a1 mRNA. As 3+ potentiated the induction of Cyp1a1 mRNA by TCDD only at 3 h and 6 h after treatment. Cd 2+ on the other hand further potentiated the induction up to 24 h after treatment. The metal-mediated induction of Cyp1a1 mRNA was further potentiated by the protein synthesis inhibitor, cycloheximide and the 26S proteasome inhibitor, MG-132, but completely inhibited by the RNA transcription inhibitor, actinomycin-D, implying a transcriptional regulation of the Cyp1a1 gene by the heavy metals. Not surprisingly, Cd 2+ and Cr 6+ activated the DNA-binding capacity of the AhR for the xenobiotic responsive element, as measured by the electrophoretic-mobility shift assay while all three metals induced AhR-dependent luciferase reporter gene expression in transiently transfected Hepa 1c1c7 cells. On the other hand, only As 3+ increased the Cyp1a1 mRNA half-life while Cd 2+ and Cr 6+ increased the Cyp1a1 protein half-life, suggesting the involvement of posttranslational modifications. A significant decrease in TCDD-mediated induction of Cyp1a1 activity was associated with an increase in HO-1 mRNA levels and a concomitant decrease in cellular heme content after all metal treatments. We clearly demonstrated that As 3+, Cd 2+, and Cr 6+ increase Cyp1a1 mRNA levels at the transcriptional and posttranscriptional levels while decreasing Cyp1a1 activity at the posttranslational level.

Isolation and identification of potential cancer chemopreventive agents from methanolic extracts of green onion ( Allium cepa)

Phase II xenobiotic metabolizing enzymes confer amelioration of risk arising from potentially carcinogenic chemicals derived both endogenously, and exogenously, from food and the environment. In this study, efforts were made to isolate and identify potentially cancer preventive constituents from methanolic extracts of green onion ( Allium cepa) directed by the quinone reductase (QR) induction bioassay using murine hepatoma (Hepa 1c1c7) cells. Crude methanolic extracts of green onion tissue were solvent-partitioned, and subsequently fractionated by flash chromatography, thin layer chromatography and high pressure preparative liquid chromatography to afford pure QR-inducing isolates. Multiple isolates were found active at inducing QR. One newly identified compound, 5-hydroxy-3-methyl-4-propylsulfanyl-5H-furan-2-one ( 3), and four known compounds: 5-(hydroxymethyl) furfural ( 1), acetovanillone ( 2), methyl 4-hydroxyl cinnamate ( 4) and ferulic acid methyl ester ( 5), were isolated and identified as active agents.

Potential Cancer Chemopreventive in Vitro Activities of Monomeric Xanthone Derivatives from the Marine Algicolous Fungus Monodictys putredinis

Investigation of the fungal strain Monodictys putredinis isolated from the inner tissue of a marine green alga led to the isolation of four new monomeric xanthones and a benzophenone. All structures were elucidated by extensive spectroscopic measurements. The relative configuration of compound 1 was determined by X-ray crystal structure analysis, while for 2 and 3 configurations were confirmed by NOE experiments. Absolute configurations for compounds 1-3 were deduced by comparing experimental circular dichroism spectroscopic data with those calculated employing quantum-chemical time-dependent density functional theory (TDDFT). The compounds were examined for their cancer chemopreventive potential. Xanthone 2 was shown to inhibit cytochrome P450 1A activity with an IC50 value of 3.0 microM. Compounds 2 and 3 displayed moderate activity as inducers of NAD(P)H:quinone reductase (QR) in cultured mouse Hepa 1c1c7 cells, with CD values (concentration required to double the specific activity of QR) of 12.0 and 12.8 microM, respectively. Compound 3 showed weak inhibition of aromatase activity.