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The current work was undertaken to settle the debate about the toxicity of artificial sweeteners (AS), particularly aspartame and saccharin. Twenty-five, 7-week-old male Wistar albino rats with an average body weight of 101 ± 4.8 g were divided into a control group and four experimental groups (n = 5 rats). The first and second experimental groups received daily doses equivalent to the acceptable daily intake (ADI) of aspartame (250 mg/Kg BW) and four-fold ADI of aspartame (1000 mg/Kg BW). The third and fourth experimental groups received daily doses equivalent to ADI of saccharin (25 mg/Kg BW) and four-fold ADI of saccharin (100 mg/Kg BW). The experimental groups received the corresponding sweetener dissolved in water by oral route for 8 weeks. The activities of enzymes relevant to liver functions and antioxidants were measured in the blood plasma. Histological studies were used for the evaluation of the changes in the hepatic tissues. The gene expression levels of the key oncogene (h-Ras) and the tumor suppressor gene (P27) were also evaluated. In addition to a significant reduction in the body weight, the AS-treated groups displayed elevated enzymes activities, lowered antioxidants values, and histological changes reflecting the hepatotoxic effect of aspartame and saccharin. Moreover, the overexpression of the key oncogene (h-Ras) and the downregulation of the tumor suppressor gene (P27) in all treated rat groups may indicate a potential risk of liver carcinogenesis, particularly on long-term exposure.

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.

ABSTRACT. 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 (PPARα) 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 PPARα functions. We treated Wistar rats with an AhR ligand, Sudan III (S.III), and/or PPARα ligand, Clofibric Acid (CA), for 3 days. We analysed the expression of one of the PPARα-target gene products, CYP4A protein and its mRNA. We also tested HepG2 cells with the afore-mentioned treatments and evaluated their effects on PPAR α and RXRα 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 PPARα. In HepG2 cells, PPARα and RXRα protein expression was decreased by S.III treatment in a dose dependent manner. Our results suggest that AhR has an inhibitory effect on PPAR α function and a new pathway by which AhR ligands could disturb lipid metabolism. KEY WORDS: AhR, CYP4A, PPARα rat, RXRα.J. Vet. Med. Sci. 66(11): 1377-1386, 2004 Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor deeply involved in the maintenance of lipid and glucose homeostasis [39] and is mainly expressed in liver, muscle and heart [3,40]. Activation of PPARα in liver, muscle and heart increases the expression and activity of lipoprotein lipase (LPL), an enzyme involved in lipolysis [35], and therefore increases the clearance of TG-rich lipoproteins, and circulating TG levels. PPARα agonist, fibrates, inhibits ApoCIII, which act as a natural inhibitor of LPL activity [5]. Treatment of diabetic db/db mice with a PPARα agonist normalizes circulating free fatty acids,TG, and glucose levels [2]. Moreover PPARα influences the expression of numerous genes implicated in major pathways of amino acid metabolism, and fasting PPARα-null mice have higher plasma urea concentration compared to wild type [19] PPARα agonists, fibrates, have been in use for over 40 years for the treatment of dyslipidemia, mainly due to their actions of lowering TG levels, raising HDL [9], and decreasing levels of LDL [40]. The CYP4A sub-family members which have a role in fatty acid and prostaglandin hydroxylation, are abundantly expressed in the liver and kidney [38]. The members of this sub-family are well known to be regulated by PPARα, which binds with RXRα to form heterodimers. These hetero-dimers then bind to enhancer elements PPRE on responsive genes including CYP4A family [17]. 20-hydroxyeicosatetraenoic acid is a major arachidonic acid metabolite of CYP4A isoforms in the microvasculature of the rat kidney and acts as a potent vasoconstrictor and mediator of the myogenic response [24]. Coll...

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The macrophages from Nramp1 congenic mice and tumor necrosis factor (TNF)-alpha(-/-) mice were used to examine the functions of Nramp1 and Tnfa genes in nitric oxide (NO) production and Salmonella typhimurium infection. It was confirmed that the level of inducible NO synthase (iNOS)-mediated NO production in Nramp1(r) peritoneal macrophages was generally higher than that of Nramp1(s) macrophages after stimulation by interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) alone or in combination. Nramp1 mRNA expression in both Nramp1 congenic macrophages was constitutive notwithstanding cytokine stimulation. During infection with S. typhimurium strain 6203, Nramp1(r) macrophages produced a lower amount of NO because of an initial strong reaction and unsustained iNOS gene expression as compared with Nramp1(s) macrophages. An inhibitory effect of the Nramp1(r) gene on bacterial replication was also observed during the early stage of S. typhimurium infection, whereas the effect of TNF-alpha occurred later. NO production and iNOS expression in TNF-alpha(-/-) macrophages were not detected from the start of the bacterial infection or at 24 h after infection. We also observed that S. typhimurium strain 6203 grew more profoundly without TNF-alpha, especially in Nramp1(s) macrophages. These data, therefore, demonstrate that there is cooperation of the Nramp1 and Tnfa genes in NO production and a growth inhibitory effect in response to S. typhimurium infection.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. The peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear receptor super-family of ligand-activated transcription factors and it functions as an obligate heterodimer with retinoid X-receptor alpha RXRalpha. The aim was to investigate whether the negative cross-talk recently proposed by the present authors between AhR and PPARalpha on CYP4A and CYP1A has any impact on other cytochrome P450 enzymes. Treatment of male Wistar rats with a PPARalpha ligand clofibric acid (CA) induced CYP2B1/2 and CYP3A proteins, activities, and the mRNA expression of CYP2B1, CYP2B2, CYP3A1 and CYP3A2, and suppressed CYP2C11 protein, activities and mRNA expression. AhR ligand Sudan III (S.III) treatment decreased basal and CA-induced CYP2B, CYP3A and CYP2C11 protein, activities and mRNA expression. To the best of the authors' knowledge, this is the first study showing the presence of mutual effects of AhR and PPARalpha on CYP2B and CYP3A and an additive inhibitory effect on CYP2C11 in the livers of male rats.