Therefore, we aimed to quantify the absorption of BR, BV and conjugated BR (ditaurate; BRDT) into two distinct strains of Salmonella typhimurium (S. typhimurium) via HPLC analyses. It was hypothesised that BPs would be absorbed in a dose-dependent manner into bacteria, and that extracellular check details (plate) and intracellular (absorbed) BP concentrations would broadly protect against genotoxicity mediated by various mutagens. The Salmonella reverse mutation assay is an in vitro test assessing the mutagenic potential of chemicals. Bacterial wild-type reversion in the presence of mutagens, allowing growth and colony

formation represents its fundamental, technical basis. Experiments were conducted as previously published ( Maron and Ames, 1983), and included 48 h of BP incubation at 37 °C. In some assays, S9 liver homogenate (S9 microsomal fraction from Aroclor-treated rats) was used as an enzymatic activation system. Bile pigment concentrations were tested in triplicate, negative/positive controls

were tested in each assay (n = 6). Experiments were repeated again on a different day and results were then pooled (n = 6 minimum). Two strains of S. typhimurium were tested: TA102, susceptible to oxidative damage, reverts by cross-linking agents, TA98 detects frame-shift mutations and base-pair deletions ( Mortelmans and Zeiger, 2000). Strains were kindly provided Z-VAD-FMK chemical structure by Dr. Bruce N. Ames and were attested to their genetic integrity and spontaneous mutation rate ( Mortelmans and Zeiger, 2000) in our laboratory. Unconjugated bilirubin 1Xα (CAS# 635-65-4), conjugated bilirubin (ditaurate; CAS# 635-65-4) and biliverdin 1Xα (CAS# 55482-27-4) were purchased from Frontier Scientific Europe, UK. Chemical structures can be the found online (Supplementary material 1). Pigment purity (>98%) and solubility were measured using HPLC and spectrophotometry. The S9 liver homogenate was obtained from MP Biomedicals, France. All other reagents and mutagens were purchased from Sigma Aldrich, Austria (unless otherwise noted),

were of the highest analytical grade available, and stored according to instructions. Bile pigment solutions were prepared in DMSO, protected from light, and used immediately. Composition and preparation of all necessary solutions can be found elsewhere ( Bulmer et al., 2007). To assess different possibilities of anti-genotoxic action (e.g., structural interactions, radical scavenging, complex formation), four different mutagens were applied at their respective appropriate concentrations ( Table 1): 2,4,7-trinitro-9H-fluoren-9-one (J & K Ltd., China; TNFone), tertiary-butyl hydroperoxide (Merck; t-BOOH), aflatoxin B1 (AfB1) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Toronto Research Chemicals, Canada; PhIP). Based on preceding investigations (Bulmer et al., 2007), BRDT and BV were tested at concentrations of 0.01, 0.05, 0.1, 0.5, 1 and 2 μmol/plate (equals 3.4, 17.2, 34.5, 172.4, 349 and 689.6 μM).