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.

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.

Bilirubin, the principal and biologically most relevant bile pigment was, until recently, considered a waste product of haem catabolism. However, current data suggest that bile pigments possess biological potential, related to their antioxidant and anti-mutagenic effects. In this context, it is now assumed that bile pigments and their derivatives exert these effects via multiple mechanisms, including discrete anti-oxidative and physico-chemical interactive effects. The major scientific focus so far has concentrated on the compounds' antioxidant action, and mechanistic investigations of possible mutagentetrapyrrole interaction are lacking. Therefore we tested structurally related bile pigments/derivatives (bilirubin/-ditaurate/-dimethyl ester, biliverdin/-dimethyl ester, urobilin, stercobilin and protoporphyrin) for anti-genotoxicity in the Salmonella reverse mutation assay (strains TA98, TA102), together with the synthetic mutagen 2,4,7-trinitro-9H-fluoren-9-one (TNFone). To explore possible structural interactions, molecular systems of chlorin e6 porphyrin/bilirubin/biliverdin with TNFone were assayed using circular dichroism. These data consistently revealed, at suprastoichiometric concentrations, that tetrapyrroles interact with TNFone. Addition of TNFone to chlorin e6 porphyrin, bilirubin-albumin and biliverdinalbumin led to a marked change in pigment spectra, providing evidence for tight tetrapyrrole-mutagen interaction. This conclusion was also supported by substantial, TNFone-induced decrease of bilirubin oxidation in the bilirubin-albumin system. This outcome was reflected in a bacterial model, in which most tetrapyrroles and especially protoporphyrin, significantly attenuated TNFone-induced mutagenesis. These data indicate that aromatic, tetrapyrrolic molecules interact with TNFone, providing a novel mechanism to suggest the anti-mutagenic effects of bile pigments in vivo are related to their physicochemical interaction with genotoxins.

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