The antimutagenic and antioxidant potential of stercobilin and urobilin in the Ames Salmonella test

Das Ziel der vorliegenden Studie war die Abschätzung des antimutagenen bzw. antioxidativen Potentials von den beiden Gallenpigmenten Stercobilin und Urobilin im in vitro Ames Test. Für die Untersuchung wurden die Bakterienstämme Salmonella typhimurium TA98 und TA102 verwendet. Als mutagene Substanzen wurden 2, 4, 7- Trinitro-9-Fluorenon (TNFone), Aflatoxin B1 (AFB1), 2-Amino-1-methyl-6-phenylimidazo [4, 5,-b] Pyridin (PhiP) und tert-Butylhydroxyperoxide (t-BuOOH) herangezogen. Die Testkonzentrationen der Gallenfarbstoffe reichten von 0.001 bis 2 µmol/Platte (Urobilin) bzw. von 0.01 bis 2 µmol/Platte (Stercobilin). Um sicherzustellen, dass die beiden Gallenpigmente kein mutagenes Potential aufweisen, wurde dies in Mutagenitätstests überprüft. Die Tests wurden sowohl ohne (TNFone, t-BuOOH) als auch mit (PhiP, AFB1, t-BuOOH) metabolischer Aktivierung durchgeführt.The aim of the present study was to investigate the antimutagenic and antioxidant potential of the bile pigments, stercobilin and urobilin, in the Ames Salmonella test. The experiments in the bacterial system were designed with two Salmonella typhimurium tester strains, TA98 and TA102. Different mutagens including 2, 4, 7- trinitro-9-fluorenone (TNFone), aflatoxin B1 (AFB1), 2-amino-1-methyl-6-phenylimidazo [4, 5,-b] pyridine (PhiP) und tert-butylhydroxyperoxide (t-BuOOH) were used to confirm the formation of mutant revertants. Six doses of stercobilin (0.01, 0.05, 0.1, 0.5, 1 and 2 µmol/plate) and eight doses of urobilin (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 2 µmol/plate) were screened. In order to ensure non-mutagenic potential of bile pigments, mutagenicity assays were performed. Tests were conducted without (TNFone, t-BuOOH) and with (PhiP, AFB1 and t-BuOOH) metabolic activation

Bilirubin and related tetrapyrroles inhibit food-borne mutagenesis: a mechanism for antigenotoxic action against a model epoxide

Bilirubin exhibits antioxidant and antimutagenic effects in vitro. Additional tetrapyrroles that are naturally abundant were tested for antigenotoxicity in Salmonella. Un-/conjugated bilirubin (1 and 2), biliverdin (4), bilirubin and biliverdin dimethyl esters (3 and 5), stercobilin (6), urobilin (7), and protoporphyrin (8) were evaluated at physiological concentrations (0.01-2 μmol/plate; 3.5-714 μM) against the metabolically activated food-borne mutagens aflatoxin B1 (9) and 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (10). Compound 8 most effectively inhibited the mutagenic effects of 9 in strain TA102 and 10 in TA98. Compound 7 inhibited 9-induced mutagenesis in strain TA98 most effectively, while 1 and 4 were promutagenic in this strain. This is likely due to their competition with mutagens for phase-II detoxification. Mechanistic investigations into antimutagenesis demonstrate that tetrapyrroles react efficiently with a model epoxide of 9, styrene epoxide (11), to form covalent adducts. This reaction is significantly faster than that of 11 with guanine. Hence, the evaluated tetrapyrroles inhibited genotoxicity induced by poly-/heterocyclic amines found in foods, and novel evidence obtained in the present investigation suggests this may occur via chemical scavenging of genotoxic metabolites of the mutagens investigated. This may have important ramifications for maintaining health, especially with regard to cancer prevention

Metabolic link between phosphatidylethanolamine and triacylglycerol metabolism in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae triacylglycerols (TAG) are synthesized by the acyl-CoA dependent acyltransferases Dga1p, Are1p, Are2p and the acyl-CoA independent phospholipid:diacylglycerol acyltransferase (PDAT) Lro1p which uses phosphatidylethanolamine (PE) as a preferred acyl donor. In the present study we investigated a possible link between TAG and PE metabolism by analyzing the contribution of the four different PE biosynthetic pathways to TAG formation, namely de novo PE synthesis via Psd1p and Psd2p, the CDP-ethanolamine (CDP-Etn) pathway and lyso-PE acylation by Ale1p. In cells grown on the non-fermentable carbon source lactate supplemented with 5 mM ethanolamine (Etn) the CDP-Etn pathway contributed most to the cellular TAG level, whereas mutations in the other pathways displayed only minor effects. In cki1∆dpl1∆eki1∆ mutants bearing defects in the CDP-Etn pathway both the cellular and the microsomal levels of PE were markedly decreased, whereas in other mutants of PE biosynthetic routes depletion of this aminoglycerophospholipid was less pronounced in microsomes. This observation is important because Lro1p similar to the enzymes of the CDP-Etn pathway is a component of the ER. We conclude from these results that in cki1∆dpl1∆eki1∆ insufficient supply of PE to the PDAT Lro1p was a major reason for the strongly reduced TAG level. Moreover, we found that Lro1p activity was markedly decreased in cki1∆dpl1∆eki1∆, although transcription of LRO1 was not affected. Our findings imply that (i) TAG and PE syntheses in the yeast are tightly linked; and (ii) TAG formation by the PDAT Lro1p strongly depends on PE synthesis through the CDP-Etn pathway. Moreover, it is very likely that local availability of PE in microsomes is crucial for TAG synthesis through the Lro1p reaction

Sequential Synthesis and Methylation of Phosphatidylethanolamine Promote Lipid Droplet Biosynthesis and Stability in Tissue Culture and in Vivo*

Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt−/− mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt−/− animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability