Fluoranthene enhances p53 expression and decreases mutagenesis induced by benzo[a]pyrene.

Fluoranthene (Fla) is the most abundant polycyclic aromatic hydrocarbon (PAH) in diesel particulate extracts. Benzo[a]pyrene (BaP) is genotoxic and is a prototype of PAH carcinogens. Fla's toxicity and mutagenicity are minor relative to BaP's. Our data showed that Fla enhanced BaP-induced p53 expression and promoted BaP-induced cell death. In contrast, Fla decreased BaP-induced mutagenesis. Fla had almost no influence on the cell cycle. However, the effect of cotreatment with BaP (1µM) and Fla (10µM) in regulating the cell cycle was greater than that of BaP (2µM) alone. It is known that BaP activates the aryl hydrocarbon receptor (AhR), and, in turn, the AhR induces cytochrome P450 (Cyp)1a1 expression. The expression of Cyp1a1 corresponds well with the induction of apoptosis and mutagenesis by BaP. Fla did not activate the AhR or antagonize BaP's induction of AhR activity and Cyp1a1 expression. Therefore, the actions of Fla on BaP's toxicity were independent of the AhR signal and Cyp1a1. In summary, results indicated that Fla directs BaP-treated cells into death rather than mutagenesis, consequently preventing cells from being transformed. The novel cooperation between Fla and BaP provides valuable information for how to increase expression of the p53 tumor suppressor.

1,10-phenanthroline stabilizes mRNA of the carcinogen-metabolizing enzyme, cytochrome P450 1a1.

1,10-phenanthroline (phen), flufenamic acid, and indomethacin are inhibitors of aldo-keto reductases 1C1 (AKR1C1), but only phen decreased the benzo[a]pyrene (BaP)-induced cytochrome P450 1a1 (Cyp1a1) protein level. Therefore the decrease in the BaP-induced Cyp1a1 protein level was not due to inhibition of Akr1c1, but to phen itself. Phen decreased the BaP-induced Cyp1a1 promoter activity and protein expression, and in contrast, it increased Cyp1a1 mRNA, resulting from an increase in mRNA stability. Phen is also known as a transition metal ion-chelator. Along with the phen study, we also found that Zn(2+), Fe(2+) and Cu(2+) increased Cyp1a1 mRNA and protein stability. Our results show that phen stabilized the mRNA of Cyp1a1, although it decreased cell viability. In addition, Zn(2+) and Fe(2+) highly neutralized phen's suppression of Cyp1a1 protein expression, but they only slightly neutralized phen's promotion of mRNA stability and suppression of cell viability, and had no effect on phen's suppression of promoter activity. Phen's effect on Cyp1a1 expression was reversible, which indicates that phen is non-covalently linked to its target. This report elucidates a new role for phen of stabilizing Cyp1a1 mRNA, and provides information for further studies on mRNA stabilization.

1-Nitropyrene stabilizes the mRNA of cytochrome P450 1a1, a carcinogen-metabolizing enzyme, via the Akt pathway.

Cytochrome P450 1a1 (Cyp1a1) is a phase I xenobiotic-metabolizing enzyme, the expression of which is mainly driven by the aryl hydrocarbon receptor (AhR). Cyp1a1 messenger (m)RNA is labile. Our study indicates that 1-nitropyrene (1-NP) highly induced Cyp1a1 protein expression, although its induction of AhR transactivation activity was negligible. The fact that the nuclear receptors, CAR, FXR LXR, or PXR, did not induce Cyp1a1 expression indicates that they do not mediate 1-NP's action. When the AhR transcript was degraded by small hairpin (sh)RNA-AhR, 1-NP-induced Cyp1a1 expression largely decreased. In addition, 1-NP did not induce Cyp1a1 in AhR pathway-deficient mutant cells, which indicates that the AhR is essential for 1-NP's action. When Cyp1a1's turnover was examined, 1-NP was able to stabilize the 1-NP- and benzo[a]pyrene (BaP)-induced Cyp1a1 mRNA, but not protein. 1-NP-induced Cyp1a1 mRNA stabilization was mediated by Akt, but not by p38 MAPK, MEK1/2, or JNK. Among aryl hydrocarbons with four annealed phenyl rings, including pyrene, 1-NP, fluoranthene, 3-nitrofluoranthene, chrysene, and 6-nitrochrysene, only 1-NP was able to stabilize Cyp1a1 mRNA. 1-NP's action was gene specific. In conclusion, stabilizing Cyp1a1 mRNA greatly contributed to 1-NP-induced Cyp1a1 expression, which provides new insight into gene regulation by the AhR ligand and mRNA stabilization.

The inhibitory effect of Gynostemma pentaphyllum on MCP-1 and type I procollagen expression in rat hepatic stellate cells.

AIM OF THE STUDY: Gynostemma pentaphyllum is a popular folk medicine that has been used for treatment of hepatitis in Asia. Our previous study demonstrates that Gynostemma pentaphyllum n-butanol extract inhibits the onset and improves the recovery of CCl(4)-induced liver fibrogenesis in rats and inhibits PDGF-induced rat hepatic stellate cells (HSCs) proliferation. In this study, the effect of Gynostemma pentaphyllum extract on cytokines and type I procollagen expression was analyzed. MATERIALS AND METHODS: Rat HSCs were treated with PDGF, Gynostemma pentaphyllum n-butanol extract, RP-18-Gyp fraction, rapamycin or vehicle. Rat cytokine antibody array chip or ELISA kit was used for cytokines detection. Intracellular protein expression was detected by Western blotting, mRNA expression was analyzed by RT-PCR. RESULTS: RP-18-Gyp fraction is the more purified gypenosides fraction from Gynostemma pentaphyllum n-butanol extract. In cell proliferation, the inhibitory effect of 200 microg/ml RP-18-Gyp fraction is similar to 500 microg/ml Gynostemma pentaphyllum n-butanol extract. Furthermore, both of them have the ability of decreasing monocyte chemoattractant protein-1 (MCP-1) mRNA expression and protein release and inhibiting type I procollagen protein expression. CONCLUSIONS: Both of Gynostemma pentaphyllum n-butanol extract and its more purified RP-18-Gyp fraction have the biological activities in the inhibition of cell proliferation, MCP-1 release and type I procollagen expression in rat HSCs. These data could provide the evidence to support for the traditional use of Gynostemma pentaphyllum in hepatitis.

Crosstalk between activated forms of the aryl hydrocarbon receptor and glucocorticoid receptor.

Pyrene, benzo[a]pyrene (BaP), and indeno[1,2,3-cd]pyrene (IND) are poly cyclic aromatic hydrocarbons (PAHs) with four to six annealed phenyl rings. Dexamethasone (Dex) is a synthetic agonist of glucocorticoids. The aryl hydrocarbon receptor (AhR) ligands, BaP and IND, did not directly activate the glucocorticoid receptor (GR), and Dex did not activate the AhR either. Whenever BaP and IND were added to Dex-treated cultures, they were present with Dex for longer periods, and higher enhancement of Dex-induced transactivation of the GR was found, which indicates that the freshly activated AhR is essential for synergistic interactions with the activated GR. The degree of enhancement of Dex-induced transactivation of the GR by PAHs, BaP approximately IND>pyrene, paralleled the potency of PAHs in activating the AhR. This synergistic interaction was more distinct in ovarian granulosa cells (HO23) than in HepG2, 293T, or HeLa cells. In contrast, Dex suppressed AhR-mediated expressions, including AhR and cytochrome P450 (CYP) 1 A1 expressions. Dex also counteracted the BaP-induced decrease in cell viability. Crosstalk between the AhR and GR was independent of their expression levels. We concluded that the AhR functionally cross-reacts with the GR, through which transactivation activity of the GR is further enhanced, and in contrast, transactivation activity of the AhR is inhibited. This report shows the significance of in vitro endocrine-related results, which provide a clue for molecular studies of an interactive mechanism between the AhR and GR, and should be confirmed by future in vivo studies.

Aldo-keto reductase 1C2 is essential for 1-nitropyrene's but not for benzo[a]pyrene's induction of p53 phosphorylation and apoptosis.

It is reported that diesel exhaust particles contain more 1-nitropyrene (1-NP) than benzo[a]pyrene (B[a]P), both of which are potent carcinogenic compounds. In this study, we show that 1-NP is more potent in reducing cell viability than B[a]P, pyrene, nitrobenzene, and nitromethane. Aldo-keto reductases (AKRs) are enzymes which metabolize polycyclic aromatic hydrocarbons into active metabolites that form PAH-DNA-adducts causing mutagenesis of DNA. We found that the AKR1C2 inhibitor, ursodeoxycholic acid (UA), inhibited 1-NP-induced, but not B[a]P-induced, phosphorylation of p53 and cleavage of poly (ADP-ribose) polymerase (PARP). 1-NP-induced apoptosis was also suppressed by UA, as detected by Hoechst 33342 staining, flow cytometric analysis of subG0/G1 phase and annexin V binding to phosphatidylserine. The AKR1C1 and 1C4 inhibitor, 1,10-phenanthroline (Phen), inhibited the toxic effects of both 1-NP and B[a]P. In contrast, the AKR7A1 and 7A5 inhibitors, succinate and citrate, did not influence the toxic effects of 1-NP or B[a]P. In addition, several metabolic and signaling pathways were analyzed, these were used to compare the results of the toxic effect of AKRs on 1-NP and B[a]P. Through the application of kinase inhibitors, results indicated that p38-MAPK, but not ERK1/2 or JNK, was essential for mediating both 1-NP's and B[a]P's induction of the phosphorylation of p53 and cleavage of PARP. Neither ellipticine, a CYP1A1 inhibitor, nor 2,6-diisopropylphenol, a CYP1A2 and 2B1 inhibitor, blocked the toxic effects of 1-NP and B[a]P, which indicates that neither CYP1A1, 1A2, nor 2B1 is essential for the transformation of 1-NP and B[a], into toxic metabolites. AKR1C2 was constitutively expressed in HepG2 cells and was not regulated by 1-NP or B[a]P. In conclusion, this is the first report on AKRs' actions toward nitro-PAH in cells. The metabolic and signaling pathways for the toxic effects of both 1-NP and B[a]P are similar except that AKR1C2 plays differential role between them. The results provide valuable information for further investigations on AKRs.

Peptidoglycan enhances transcriptional expression of CCAAT/enhancer-binding protein delta gene in mouse macrophages.

Peptidoglycan-activated gene expression is mediated through various transcription factors including CCAAT/enhancer-binding protein delta (C/EBPdelta). The purpose of the present study is to elucidate the mechanism of PGN-activated C/EBPdelta gene. PGN stimulated C/EBPdelta protein and mRNA expression in mouse macrophages RAW 264.7 cells. Analysis of C/EBPdelta promoter activity by luciferase reporter assay indicated that PGN-induced C/EBPdelta gene activation is partially mediated by the -345 to +24 bp of C/EBPdelta gene promoter. The in vitro protein-DNA binding assay showed that Sp1, c-Rel and c-Jun are the major protein binding to this PGN-response element of C/EBPdelta promoter, and the binding of c-Rel and c-Jun is increased after PGN treatment. All of these binding activities were abolished when Sp1-, NF-kappaB/APRE-, CRE-sites were mutated. Furthermore, analysis of this promoter region by site-directed mutants constructed in luciferase reporter vector indicated that two Sp1-sites, one NF-kappaB/APRE-site and one CRE-site are prominent for PGN-induced gene expression. In addition, when Sp1, c-Rel or c-Jun transcription factors were overexpressed in cells, all of them enhanced C/EBPdelta promoter activity. In summary, we suggest that Sp1, c-Rel and c-Jun transcription factors play important roles in activation of C/EBPdelta gene promoter under the stimulation of PGN. Given the importance of C/EBPdelta in inflammatory disease, these results reveal a clue as a potential therapeutic target for suppression of C/EBPdelta expression under PGN stimulation.