Induction of proinflammatory cytokines and caspase-1 by leptin in monocyte/macrophages from holstein cows.

The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.

Effect of lycopene and beta-carotene on peroxynitrite-mediated cellular modifications.

Peroxynitrite formed by the reaction of superoxide and nitric oxide is a highly reactive species with a role in various pathological processes such as cancer, chronic inflammation, and cardiovascular and neurological diseases. In the present study, the effect of the carotenoids, lycopene and beta-carotene, on peroxynitrite-mediated modifications in plasmid DNA as well as cellular DNA and proteins were investigated. In pUC18 plasmid DNA, these carotenoids strongly inhibited DNA strand breaks caused by peroxynitrite generated from 3-morpholinosydnonimine (SIN-1). SIN-1 was also used to determine effects on DNA damage and protein tyrosine nitration in Chinese hamster lung fibroblasts. SIN-1 dose-dependently increased nitration of proteins in cells above basal levels as determined by Western blotting. This nitration was inhibited in the presence of the uric acid as well as lycopene. Physiological concentrations (0.31-10 microM) of lycopene and beta-carotene also had protective effects on DNA damage, as measured by the comet assay. Lycopene significantly reduced DNA damage particularly, in the median range of concentrations (2.5 microM). The protective effects of lycopene and beta-carotene could be due to their scavenging of reactive oxygen (ROS) and/or nitrogen species (RNS) as they reduce the amount of intracellular ROS/RNS produced following treatment with SIN-1 by as much as 47.5% and 42.4%, respectively. The results obtained in this study suggest that carotenoids may alleviate some of the deleterious effects of peroxynitrite and possibly other reactive nitrogen species as well in vivo.

Nitric oxide enhances catechol estrogen-induced oxidative stress in LNCaP cells.

Catechol estrogens (CEs), such as 4-hydroxyestradiol (4-OHE2), undergo redox cycling during which reactive oxygen species (ROS) such as superoxide (O2*-) and the chemically reactive estrogen semiquinone (CE-SQ) and quinone (CE-Q) intermediates are produced. The quinone's putative mutagenicity may be enhanced by ROS and/or reactive nitrogen species. High concentrations of nitric oxide (NO) present during inflammatory conditions may react with (O2*-) to form peroxynitrite (ONOO-), a potent oxidant implicated in many pathological conditions. In this study, the possible generation of peroxynitrite from the interaction of CEs and NO and its effect on plasmid DNA and intact cells were investigated. A combination of 4-OHE2 and NO increased the level of single strand breaks (SSB) in plasmid DNA by more than 60% compared to vehicle controls in a metal-free buffer system. 4-OHE2 alone or NO alone had no effect. Results obtained from use of different antioxidants and ROS scavengers suggested a role of peroxynitrite in oxidative stress. In cells, 4-OHE2 or NO alone induced dose-dependent DNA damage as assessed by single cell gel electrophoresis. Co-treatment with 4-OHE2 and NO had an additive effect at lower doses. Generation of intracellular ROS was measured by the oxidation of carboxy-2',7'-dichlorofluorescein diacetate to the fluorescent compound carboxy-2',7'-dichlorofluorescein. NO alone, in oxygenated media, generated little ROS whereas 4-OHE2 produced approximately 70% increase in fluorescence. When added together 4-OHE2 and NO, produced a 2-fold increase in ROS. The generation and involvement ofperoxynitrite to this increase was implied since uric acid inhibited it. Generation ofperoxynitrite was also observed by use of dihydrorhodamine 123. Therefore, we conclude that combined treatments with 4-OHE2 and NO generated peroxynitrite seen from increased fluorescence and its inhibition by uric acid or combined SOD and catalase treatments. Results reported here suggest a role of peroxynitrite in causing damage to biomolecules when CEs and NO are present simultaneously. This may have biological relevance as high concentrations of NO formed during inflammatory conditions may exacerbate cancers due to estrogens.

Lycopene and beta-carotene ameliorate catechol estrogen-mediated DNA damage.

The consumption of fruits and vegetables is associated with a reduced risk of various ailments, including cancer and cardiovascular diseases. Carotenoids, such as lycopene and beta-carotene, are natural constituents of edible plants and may protect against disease. In this study, the influence of lycopene and beta-carotene on DNA damage caused by catechol-estrogens in vitro is examined. One possible mechanism by which catechol estrogens such as 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol, which cause DNA damage in naked plasmid DNA as well as in cells, contributing to the process of carcinogenesis, is through the generation of reactive oxygen species. It was found that both carotenoids at concentrations ranging from 0.25 to 10 microM significantly inhibit strand breakage induced by 4-OHE2/copper sulphate by up to approximately 90% in plasmid DNA with beta-carotene being slightly more effective. No prooxidant or cytotoxic effects were observed at the concentrations tested. These carotenoids had a similar, though reduced effect on DNA damage as measured by the comet assay, in Chinese hamster lung fibroblasts. The results obtained show that both lycopene and beta-carotene, most probably and mainly through their potent antioxidant properties, are able to inhibit catechol-estrogen-mediated DNA damage.

Antigenotoxic effect of Pleurotus cornucopiae extracts on the mutagenesis of Salmonella typhimurium TA98 elicited by benzo[a]pyrene and oxidative DNA lesions in V79 hamster lung cells.

Pleurotus cornucopiae (PC) mushroom with a brilliant yellow pileus is found in the field and known in Japan as Tamogi dake mushroom. The purpose of this paper is to investigate the mechanism of the antimutagenic effect of PC mushroom using both the Ames test and Comet assay. We have found a strong inhibitory effect of both aqueous and organic PC extracts on the mutagenicity elicited by benzo[a]pyrene (B[a]P). This inhibition was dose-dependent in reaction mixtures containing cytosolic and microsomal fractions (S-9) from untreated rat liver as well as in those containing S-9 from aryl hydrocarbon receptor (Ah) ligand of Sudan III-treated rats. Sudan III was a potent inducer of cytochrome P450 1A (CYP1A) activity. We treated rats with Sudan III to enhance the metabolic activation of B[a]P by the S-9 fraction. To explain whether this antimutagenicity was due to the inhibition of CYP1A activity that metabolically activates B[a]P, we tested the effects of the extracts on activities of CYP1A1 and CYP1A2, represented by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD), respectively. Both aqueous and organic extracts inhibited EROD activity at all dose levels, while the inhibitory effect was only observed at high doses with regard to MROD activity. Furthermore, pre-treatment of Chinese hamster V79cells with PC extracts significantly reduced H2O2-induced-DNA damage, indicating that PC extracts provide a protective effect against oxidative DNA damage. These results indicate that whole-mushroom extracts contain compounds that may inhibit the metabolic activation of B[a]P by CYP1A1 as well as prevent oxidative DNA damage.

Down regulation of hepatic PPARalpha function by AhR ligand.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachloro dibenzo-p-dioxin (TCDD) and related compounds. Peroxisome proliferator activated receptor alpha (PPARalpha) is a nuclear receptor involved in the maintenance of lipid and glucose homeostasis. In this study we hypothesized that one of the possible mechanisms for the effect of TCDD and its related chemicals on fat metabolism could be through down regulation of PPARalpha functions. We treated Wistar rats with an AhR ligand, Sudan III (S.III), and/or PPARalpha ligand, Clofibric Acid (CA), for 3 days. We analysed the expression of one of the PPARalpha-target gene products, CYP4A protein and its mRNA. We also tested HepG2 cells with the afore-mentioned treatments and evaluated their effects on PPARalpha and RXRalpha protein. Treatment of Wistar rats with S.III was found to down regulates CYP4A protein expression and reduced its induction with CA. It also decreased mRNA expressions of CYP4A1, CYP4A2, CYP4A3 and PPARalpha. In HepG2 cells, PPARalpha and RXRalpha protein expression was decreased by S.III treatment in a dose dependent manner. Our results suggest that AhR has an inhibitory effect on PPARalpha function and a new pathway by which AhR ligands could disturb lipid metabolism.

PPARalpha-dependent modulation of hepatic CYP1A by clofibric acid in rats.

Fibrates, hypolipidemic drugs, have been reported to suppress the metabolic activities of cytochrome P450 1A1 and 1A2 in rats but the mechanism has not been elucidated. In the present study we tested the hypothesis that the inhibitory effect of fibrates on arylhydrocarbon receptor (AhR) function may be due to their stimulatory effects on PPARalpha. Sudan III (S.III) treatment induced CYP 1A1 and CYP 1A2 protein expression, mRNA and their metabolic activities, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD), in Wistar rats higher than those in the control. Co-treatment of rats with S.III and clofibric acid (CA) caused a 40-50% decrease in the induced levels of CYP1A1 and CYP1A2 protein, mRNA expression and their metabolic activities and reduced AhR protein expression. When we treated HepG2 cells with S.III and/or CA, no suppressive effect on S.III-induced CYP1A1 protein expression due to CA was found. HepG2 cells were transiently transfected with increasing concentrations of PPARalpha mammalian expression vector and exposed to the same treatment. CA co-treatment with S.III decreased AhR protein and S.III-induced CYP1A1 protein expression with increasing dose of PPARalpha transfected into HepG2 cells. Our results demonstrate that the suppressive effect of fibrates on CYP1A is PPARalpha-dependent and suggest that PPARalpha has an inhibitory effect on AhR function.