Marlies Wallner

Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases

Bilirubin, the principal tetrapyrrole, bile pigment and catabolite of haem, is an emerging biomarker of disease resistance, which may be related to several recently documented biological functions. Initially believed to be toxic in infants, the perception of bilirubin has undergone a transformation: it is now considered to be a molecule that may promote health in adults. Data from the last decade demonstrate that mildly elevated serum bilirubin levels are strongly associated with reduced prevalence of chronic diseases, particularly cardiovascular diseases (CVDs), as well as CVD-related mortality and risk factors. Recent data also link bilirubin to other chronic diseases, including cancer and Type 2 diabetes mellitus, and to all-cause mortality. Therefore, there is evidence to suggest that bilirubin is a biomarker for reduced chronic disease prevalence and a predictor of all-cause mortality, which is of important clinical significance. In the present review, detailed information on the association between bilirubin and all-cause mortality, as well as the pathological conditions of CVD, cancer, diabetes and neurodegenerative diseases, is provided. The mechanistic background concerning how bilirubin and its metabolism may influence disease prevention and its clinical relevance is also discussed. Given that the search for novel biomarkers of these diseases, as well as for novel therapeutic modalities, is a key research objective for the near future, bilirubin represents a promising candidate, meeting the criteria of a biomarker, and should be considered more carefully in clinical practice as a molecule that might provide insights into disease resistance. Clearly, however, greater molecular insight is warranted to support and strengthen the conclusion that bilirubin can prevent disease, with future research directions also proposed.

Protection from age-related increase in lipid biomarkers and inflammation contributes to cardiovascular protection in Gilbert's syndrome

Recent epidemiological and clinical data show protection from CVD (cardiovascular disease), all-cause mortality and cancer in subjects with GS (Gilberts syndrome), which is characterized by a mildly elevated blood bilirubin concentration. The established antioxidant effect of bilirubin, however, contributes only in part to this protection. Therefore we investigated whether mildly elevated circulating UCB (unconjugated bilirubin) is associated with altered lipid metabolism. The study was performed on GS and age- and gender-matched healthy subjects (n=59 per group). Full lipoprotein profile, TAG (triacylglycerols), Apo (apolipoprotein)-A1, Apo-B, lipoprotein(a), the subfractions of LDL (low-density lipoprotein) and selected pro-inflammatory mediators were analysed. A hyperbilirubinaemic rodent model (Gunn rats, n=40) was investigated to further support the presented human data. GS subjects had significantly (P<0.05) improved lipid profile with reduced total cholesterol, LDL-C (LDL-cholesterol), TAG, low- and pro-atherogenic LDL subfractions (LDL-1+LDL-2), Apo-B, Apo-B/Apo-A1 ratio and lower IL-6 (interleukin 6) and SAA (serum amyloid A) concentrations (P=0.094). When the control and GS groups were subdivided into younger and older cohorts, older GS subjects demonstrated reduced lipid variables (total cholesterol and LDL-C, TAG and LDL-C subfractions, Apo-B/Apo-A1 ratio; P<0.05; Apo-B: P<0.1) compared with controls. These data were supported by lipid analyses in the rodent model showing that Gunn rat serum had lower total cholesterol (2.29±0.38 compared with 1.27±0.72 mM; P<0.001) and TAG (1.66±0.67 compared with 0.99±0.52 mM; P<0.001) concentrations compared with controls. These findings indicate that the altered lipid profile and the reduced pro-inflammatory status in hyperbilirubinaemic subjects, particularly in the older individuals, probably contribute additionally to the commonly accepted beneficial antioxidant effects of bilirubin in humans.

Effects of unconjugated bilirubin on chromosomal damage in individuals with Gilbert`s syndrome measured with the micronucleus cytome assay

Circulating unconjugated bilirubin (UCB) has been reported to protect against lung and colorectal cancer. The present study aimed to explore, for the first time, whether mildly elevated circulating UCB, as found in Gilbert`s syndrome (GS), is associated with changes of DNA damage. A random 76 individuals, matched for age and gender, were recruited from the general population and allocated into the GS group (UCB ≥ 17.1 µM; n = 38) or control group (UCB <17.1 µM; n = 38). Chromosomal and cytological changes were determined in lymphocytes and buccal cells using the cytokinesis-block micronucleus cytome assay (CBMN) and buccal micronucleus cytome assay (BMcyt). No significant differences were found between GS subjects and the control group in the CBMN and BMcyt determined endpoints. Subsequently, when age dependency of effects were analysed, lower formation of buccal micronucleated cells (by 73.3%) and buccal nuclear buds (by 70.9%) in the GS subgroup ≥ 30 years were found, compared to the GS subgroup <30 years. These findings suggest DNA protection in epithelial tissue of older individuals with GS.

Haem catabolism: a novel modulator of inflammation in Gilbert's syndrome.

Moderately elevated unconjugated bilirubin concentrations protect against inflammatory diseases and are present in individuals with Gilberts syndrome. This study examined the relationship between circulating haem oxygenase catabolites, unconjugated bilirubin, carboxy haemoglobin, iron and inflammatory parameters.

In vitro antioxidant capacity and antigenotoxic properties of protoporphyrin and structurally related tetrapyrroles

Abstract The antioxidant properties of protoporphyrin IX and related tetrapyrroles are poorly characterized. Therefore, eight tetrapyrroles, five of which are produced in vivo, were tested to assess their antioxidant capacities in the Salmonella reverse mutation, TEAC, FRAP and ORAC assays. Tertiary-butyl hydroperoxide (tert-BOOH) in the presence or absence of metabolic activation (±S9) was added to Salmonella strain TA102 together with the test compounds. In the absence of metabolic activation, the order of effectiveness was protoporphyrin > biliverdin > bilirubin ditaurate > bilirubin > biliverdin dimethyl ester > stercobilin > bilirubin dimethyl ester > urobilin. In the presence of S9, the effectiveness was reversed, with urobilin > biliverdin dimethyl ester > bilirubin dimethyl ester > stercobilin > biliverdin > bilirubin > bilirubin ditaurate > protoporphyrin. In the antioxidant capacity assays FRAP, TEAC and ORAC, mainly bilirubin, bilirubin ditaurate, biliverdin and protoporphyrin showed antioxidant activity. This study reports that previously untested tetrapyrroles of related structure prevent oxidatively induced genotoxicity, and for some, novel underlying mechanisms of antioxidant action were revealed. These results support the physiological importance and biological relevance of tetrapyrroles including protoporphyrin that might act as antioxidants, protecting from oxidatively induced DNA damage, particularly in the tissues/organs where they accumulate.

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 μmol/L; n = 38) or control groups (UCB < 17.1 μmol/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 ± 0.67 vs. 1.38 ± 0.43 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. Cancer Prev Res; 6(10); 1056–63. ©2013 AACR.

Features of an altered AMPK metabolic pathway in Gilbert's Syndrome, and its role in metabolic health.

Energy metabolism, involving the ATP-dependent AMPK-PgC-Ppar pathway impacts metabolic health immensely, in that its impairment can lead to obesity, giving rise to disease. Based on observations that individuals with Gilbert’s syndrome (GS; UGT1A1*28 promoter mutation) are generally lighter, leaner and healthier than controls, specific inter-group differences in the AMPK pathway regulation were explored. Therefore, a case-control study involving 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. By utilising intra-cellular flow cytometry (next to assessing AMPKα1 gene expression), levels of functioning proteins (phospho-AMPK α1/α2, PgC 1 α, Ppar α and γ) were measured in PBMCs (peripheral blood mononucleated cells). In GS individuals, rates of phospho-AMPK α1/α2, -Ppar α/γ and of PgC 1α were significantly higher, attesting to a boosted fasting response in this condition. In line with this finding, AMPKα1 gene expression was equal between the groups, possibly stressing the post-translational importance of boosted fasting effects in GS. In reflection of an apparently improved health status, GS individuals had significantly lower BMI, glucose, insulin, C-peptide and triglyceride levels. Herewith, we propose a new theory to explain why individuals having GS are leaner and healthier, and are therefore less likely to contract metabolic diseases or die prematurely thereof.

Longer telomeres in chronic, moderate, unconjugated hyperbilirubinaemia: insights from a human study on Gilbert's Syndrome.

Bilirubin (BR) is a natural endogenous compound with a potent bioactivity. Gilbert’s Syndrome (GS) is a benign hereditary condition of increased unconjugated bilirubin (UCB) in serum and serves as a convenient model for studying the effects of BR in humans. In absence of liver disease, increased UCB levels are inversely associated to all-cause mortality risk, especially from cardiovascular diseases (CVDs). On the other hand, telomere malfunction is linked to a higher risk of CVDs. To our knowledge, there is no data on whether UCB is linked to telomere length in healthy or diseased individuals In the present study we have observed a relationship between mildly increased serum UCB and telomere length. We used an in vivo approach, assessing telomere length in PBMCs from individuals with GS (n = 60) and matched healthy controls (n = 60). An occurrence of longer telomeres was observed in male individuals chronically exposed to increased UCB, as well as in Gunn rats, an animal model of unconjugated hyperbilirubinaemia. Previously identified differences in immunomodulation and redox parameters in individuals with GS, such as IL-6, IL-1β and ferric reducing ability of plasma, were confirmed and proposed as possible contributors to the occurrence of longer telomeres in GS.

Extracellular and intracellular anti-mutagenic effects of bile pigments in the Salmonella typhimurium reverse mutation assay.

In vitro anti-genotoxic properties of bile pigments have been explored and confirmed recently. Despite these reports mechanisms to explain DNA protection by endogenous bile pigments remain unclear. Surprisingly, the quantification of cellular pigment absorption which could represent a fundamental prerequisite for intracellular (e.g., anti-mutagenic) effects, has not been explored. Therefore, we aimed to measure the amounts of un-/conjugated bilirubin as well as biliverdin absorbed into colonies of Salmonella typhimurium, utilising HPLC analyses, and to observe whether intracellular compound concentrations could predict anti-genotoxic effects. HPLC analyses confirmed that bacterial bile pigment absorption was concentration-dependent. Plate bile pigment concentrations were inversely associated with genotoxicity of all tested mutagens, irrespective of strain and test conditions. However, protection against frame-shift mutation in strain TA98 most strongly depended on the bacterial absorption of bilirubin and biliverdin, which indicates that bile pigments can protect by intercepting mutations extracellularly and specifically inhibit frame-shift mutations intracellularly.

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.