In vitro anti-mutagenic, antioxidant and antigenotoxic properties of bile pigments and their derivatives

Gallenpigmente und ihre Derivate sind seit Jahrzehnten Gegenstand wissenschaftlicher Forschung, spezifischer und fokussierter allerdings werden sie erst seit wenigen Jahren untersucht. Wurden die Verbindungen zu Beginn hauptsächlich als unnütze oder gar toxische Neben- und Endprodukte des Häm-Katabolismus betrachtet, werden sie heute zunehmend in Zusammenhang stehend mit Gesundheitsförderung und Krankheitsprävention gesehen. Daten aus in vivo Studien legen vasoprotektive, anti-inflammatorische, anti-virale und anti-cancerogene Eigenschaften nahe, und in vitro Experimente attestieren Gallenpigmenten anti-genotoxische und anti-apoptotische Aktivität. Trotz solch vielversprechender Resultate bleiben exakte zugrundeliegende Wirkmechanismen weitestgehend unbekannt, und sind Gegenstand aktueller Forschungen. Speziell die Prävention weltweit zunehmender kardiovaskulärer Erkrankungen und Krebs, ist von großem medizinischem sowie ökonomischem Interesse, und unterstreicht die Wichtigkeit fundamentaler in vitro Grundlagenforschung in dem Bereich. Speziell die Erforschung natürlicher, endogen synthetisierter Verbindungen wie beispielsweise des Bilirubins bezüglich seiner Wirkmechanismen, stellt eine Voraussetzung dar um mögliche gesundheitlich vorteilhafte physiologische Effekte moderat erhöhter Bilirubinspiegel abzuschätzen. Daher war es das Ziel dieser Studie 1) bestehende Daten zu in vitro anti-mutagenen, anti-oxidativen und anti-genotoxischen Effekten um bis dato ungetestete Tetrapyrrole zu erweitern, und 2) zugrundeliegende Wirkmechanismen ihrer Effekte zu erforschen. Die Studie wurde im Rahmen des FWF-Projekts „The physiological relevance of bile pigments“ (Projekt Nr. P21162-B11) durchgeführt, und konzentrierte sich auf in vitro Bakterien- und Zellkulturmodelle. Zusammenfassend zeigte die Mehrzahl der getesteten Gallenpigmente und Derivate anti-genotoxische Effekte gegen eine Vielzahl unterschiedlicher Mutagene im Salmonella Assay. Weiters bestätigte sich ein vermutetes anti-oxidatives Potential in den durchgeführten Tests zur anti-oxidativen Kapazität der Testsubstanzen, und es zeigte sich im Comet assay DNA-Schädigung in Krebszellen, was ein toxisches Verhalten von Gallenpigmenten in entarteten Zellen im in vitro Modell nahelegt. Diese Resultate bestätigen zum Einen bereits bestehende Ergebnisse bezüglich eines anti-mutagenen/anti-oxidativen Potentials von Tetrapyrrolen, und stellen zum Anderen durch die Erweiterung der Testbatterie um bislang ungetestete Tetrapyrrole und die Anwendung ergänzender Testverfahren, komplett neue Ergebnisse aus dem Bereich der Mutationsforschung dar.Primary bile pigments including bilirubin and biliverdin as well as structurally related tetrapyrroles have been explored for decades, more closely however only during the recent years. Initially exclusively discussed as being toxic waste products of haem catabolism, bile pigments are increasingly considered as being beneficial in terms of health promotion and disease prevention. Data from in vivo studies suggest vasoprotective, anti-inflammatory, anti-viral and anti-cancer properties, and in vitro research attests anti-genotoxic and anti-apoptotic activity to these compounds. Despite such promising reports, underlying mechanisms of action remain essentially unknown and are a matter or current research. Especially the prevention of worldwide increasingly chronic disorders such as cardiovascular disease and cancer is of great medical and economic importance, and thus fundamental in vitro research in this field is importantly required. Especially testing endogenous compounds such as bilirubin in that regard and estimating the pigments’ underlying mechanisms of action in vitro, represent meaningful approaches towards deflecting potential physiological effects. Therefore, the aim of this study was 1) to enlarge the existing body of evidence on in vitro anti-mutagenic, antioxidant and anti-genotoxic properties with data on newly tested tetrapyrroles, and 2) to explore possible underlying mechanisms of action in vitro. The study was performed within the framework of the FWF project “The physiological relevance of bile pigments” (grant number P21162-B11), and focused on in vitro cell- and bacterial culture models. In summary, the majority of the tested bile pigments (especially protoporphyrin and the bilirubinoids) acted in an anti-genotoxic manner against synthetic (2,4,7-trinitro-9H-fluoren-9-one), food-borne (aflatoxin B1 and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and pro-oxidant (tertiary-butyl hydroperoxide) mutagens in the Salmonella reverse mutation assay. The obtained results specifically implicate protoporphyrin as a potent anti-mutagen in both bacterial tester strains, whose activity was most likely based on structural mutagen interaction with synthetic and food-borne mutagens. Especially bilirubin, biliverdin and bilirubin ditaurate showed moderate to high anti-oxidant properties in the performed antioxidant capacity assays versus trolox and iron, and for the first time in vitro TEAC and ORAC antioxidant capacities were reported for urobilin, stercobilin and protoporphyrin. Especially these intestinally abundant compounds also induced DNA-damage in cancer cells, detected using the comet assay, which implies a toxic effect in the applied malignant cell lines HepG2 and Caco2. In summary, this outcome strongly supports existing data on anti-mutagenic/anti-oxidative activity and adds entirely novel evidence to known in vitro effects of bile pigments and derivatives. Although results from in vitro studies cannot be directly transferred to the physiological condition, an antioxidant/anti-genotoxic behavior of tetrapyrroles could have implications for pathogenetic processes associated with prerequisite oxidative stress and/or mutagenesis. Such data represent the crucial basis for estimating potential effects of elevated bile pigment levels in the human organism

Immune Privilege Furnishes a Niche for Latent Infection

FUNDING University of Aberdeen Development Trust/Saving Sight in Grampian: grant number RG16220-10Peer reviewedPublisher PD

A Role for Folate in Microbiome-Linked Control of Autoimmunity

Acknowledgments The ideas developed in this article were derived from work supported by Fight for Sight, The Eye Charity [CSO project grant award: 3031-3032] awarded to HMW and by the Development Trust of the University of Aberdeen (Saving Sight in Grampian) [grant codes: RG-12663 and RG-14251.Peer reviewedPublisher PD

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

Anti-Genotoxic Potential of Bilirubin In Vivo:Damage to DNA in Hyperbilirubinemic Human and Animal Models

The bile pigment bilirubin is a known antioxidant and is associated with protection from cancer and cardiovascular disease (CVD) when present in too strong concentrations. Unconjugated bilirubin (UCB) might also possess anti-genotoxic potential by preventing oxidative damage to DNA. Moderately elevated bilirubin levels are found in individuals with Gilbert syndrome and more severe in the hyperbilirubinemic Gunn rat model. This study was therefore aimed to assess the levels of oxidative damage to DNA in Gilbert syndrome subjects and Gunn rats compared to matched controls. Seventy-six individuals (age- and sex-matched) were allocated into Gilbert syndrome (UCB =17.1 孯l/L; n = 38) or control groups (UCB < 17.1 孯l/L; n = 38). In addition, 40 Gunn rats were used to support the results of the human trial. Single-cell gel electrophoresis (SCGE) assay measuring standard conditions (strand breaks, apurinic/apyrimidinic sites) and formamidopyrimidine glycosylase (FPG)-sensitive sites was conducted in human peripheral blood mononuclear cells (PBMC) and rat PBMCs, colon, and hepatocytes. Furthermore, urinary 8-oxo-2'-deoxyguanosine (8oxodGuo, DNA oxidation) and 8-oxo-guanosine (8oxoGuo, RNA oxidation) were measured in humans. The Gilbert syndrome and Gunn rat groups had significantly higher UCB levels (P < 0.001) than the corresponding controls. No further differences in damage to DNA or RNA were detected between the two groups, except higher strand breaks (PBMCs) in Gunn rats when compared with controls. However, when demographic effects were analyzed, lower 8oxodGuo concentrations were detected in the human group with a BMI =25 kg/m2 (1.70 ᠰ.67 vs. 1.38 ᠰ.43 nmol/mmol creatinine, P < 0.05), although this group showed lower UCB levels than normal weight subjects. This study suggests that the disease preventative effect of UCB is unrelated to DNA oxidation/strand breaks in human and animal models of hyperbilirubinaemia.No Full Tex