all bilirubinoids including 13 significantly reduced the mutagenic 
 effects of 9 ( p < 0.01 ; figure 2a ) . both 4 and 5 also 
 reduced revertant counts significantly versus the positive control 
 ( p < 0.001 ; figure 2b ) , 
 with 4 being the more effective compound . compound 8 , however , was the most effective of all tetrapyrroles tested , 
 reducing the revertant counts in strain ta102 ( p < 
 0.001 ; figure 2c ) . compound 7 attenuated 
 mutation induced by 9 at all concentrations tested , with 
 similar results achieved for 6 ( p < 
 0.05 ; figure 2c ) . the overall order of effectiveness 
 based on ip0.5 values ( table 1 ) 
 was 8 > 1 = 2 > 4 > 3 > 6 = 7 > 5 . antimutagenic effects of ( a ) bilirubin ( 1 ) , bilirubin 
 ditaurate ( 2 ) , bilirubin dimethyl ester ( 3 ) ; ( b ) biliverdin ( 4 ) , biliverdin dimethyl ester ( 5 ) ; and ( c ) stercobilin ( 6 ) , urobilin ( 7 ) , and protoporphyrin ( 8) against metabolically 
 activated aflatoxin b1 ( afb1 , 9 ; 0.24  10 mol / plate)-induced mutagenesis in salmonella typhimurium strain ta102 . compound 7 was 
 the most antimutagenic of all compounds tested in strain ta98 and 
 reduced mutagenesis induced by 9 by up to 75% ( p < 0.05 ; figure 3c ) . significant 
 antimutagenic effects were also demonstrated for 3 , 5 , and 6 with a lower but still measurable effect 
 observed for 8 ( p < 0.05 ; figure 3a  c ) . the overall order of effectiveness 
 based on ip0.5 values ( table 1 ) 
 was 7 > 5 > 3 > 6 > 8 . in contrast to these compounds and 
 to the previous 
 ta102 strain , 1 , 2 , and 4 did 
 not attenuate mutagenesis provoked by 9 under the test 
 conditions used ( figure 3a ) , and 1 and 4 had a promutagenic effect ( p > 0.05 ) . antimutagenic effects of ( a ) bilirubin ( 1 ) , bilirubin 
 ditaurate ( 2 ) , bilirubin dimethyl ester ( 3 ) ; ( b ) biliverdin ( 4 ) , biliverdin dimethyl ester ( 5 ) ; and ( c ) stercobilin ( 6 ) , urobilin ( 7 ) , and protoporphyrin ( 8) against metabolically 
 activated aflatoxin b1 ( afb1 , 9 ; 0.8  10 mol / plate)-induced mutagenesis in salmonella typhimurium strain ta98 . the most effective tetrapyrrole 
 at inhibiting mutagenesis induced by 10 in ta98 was 8 , which attenuated the mutagenic effects by more than 90% 
 and resulted in near complete detoxification of the mutagen ( p < 0.001 ; figure 4c ) . all bilirubinoids 
 ( 13 ) were highly effective against 
 mutagenesis provoked by 10 ( p < 0.001 ; 
 figure 4a ) and attenuated its effects by over 
 60% . compounds 4 and 5 also reduced revertant 
 counts over the entire range of concentrations tested ( p < 0.001 ; figure 4b ) as did 6 and 7 ( p < 0.05 ; figure 4c ) . the overall order of effectiveness based on 
 ip0.5 values ( table 1 ) was 8 > 1 > 4 > 3 > 2 > 6 > 5 > 7 . antimutagenic effects of ( a ) bilirubin ( 1 ) , bilirubin 
 ditaurate ( 2 ) , bilirubin dimethyl ester ( 3 ) ; ( b ) biliverdin ( 4 ) , biliverdin dimethyl ester ( 5 ) ; and ( c ) stercobilin ( 6 ) , urobilin ( 7 ) , and protoporphyrin ( 8) against metabolically 
 activated 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine 
 ( phip , 10 ; 0.1  10 mol / plate)-induced 
 mutagenesis in salmonella typhimurium strain ta98 . 1 : 
 unconjugated bilirubin , 2 : bilirubin ditaurate , 3 : bilirubin dimethyl 
 ester , 4 : biliverdin , 5 : biliverdin dimethyl 
 ester , 6 : stercobilin , 7 : urobilin , 8 : protoporphyrin . afb1 : aflatoxin b1 ( 9 ) , phip : 
 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ( 10 ) . ip0.5 : percentage positive 
 control inhibition at 0.5 mol / plate ( highest common sample 
 dose per plate ) :  indicates mutagen attenuating effect , + 
 amplifying effect . results from the bacterial model of this study demonstrate 
 that 
 tetrapyrroles inhibit the mutagenic effects of 9 in both s. typhimurium strains ta102 and ta98 . the tetrapyrroles 
 used , however , vary greatly in the planarity of their structure , aromaticity , 
 and extent of conjugation and in the presence or absence of free carboxylic 
 acid groups . the irregularity in effectiveness of the various tetrapyrroles 
 tested across the two strains , therefore , suggests that the presence 
 of a single , unifying mechanism of action for this inhibition is unlikely . previous studies have suggested that the planar , aromatic nature of 9 allows for tetrapyrrole complex formation through -stacking 
 interactions , inhibiting covalent and noncovalent 
 interaction between dna and 9 . similarly , complex formation 
 in the gut between porphyrins and food - borne mutagens , followed by 
 electrophile scavenging and complexing , has been suggested . if this is the major mechanism of the inhibition of mutagenesis , 
 the more conjugated , planar tetrapyrroles such as 4 and 8 would be expected to be the most potent inhibitors of those 
 tetrapyrroles tested . while 8 showed the highest potency 
 in the 9-ta102 experiment , derivatives of 1 were the next most potent , and these are both not planar nor fully 
 conjugated . simple -electron - mediated complexation is therefore 
 unlikely to be the sole explanation for the antimutagenic effects 
 of tetrapyrroles . proposed that the exocyclic 
 amine groups of poly-/heterocyclic 
 mutagens covalently bind to free cooh groups in chlorophyllin . in the 9-ta102 study , 1 and 4 exhibited significantly higher inhibition activity 
 than their respective dimethyl esters ( 3 and 5 ) , suggesting that the cooh groups present in 1 and 4 may be key to their activity . it is difficult 
 to draw a solid conclusion , as 6 and 7 both 
 contain cooh groups and were less potent inhibitors of mutation 
 than 3 in this assay . however , the reduced potency of 6 and 7 may be related to the progressive loss 
 of aromaticity of their terminal pyrrole rings . despite the 
 potencies of 1 and 4 in the 9-ta102 experiment , these two compounds were promutagenic 
 in the ta98 strain . this promutagenic effect is likely due to 1 and 4 competing with afb1 for the same phase - ii 
 metabolism enzymes , thus resulting in 
 increased concentrations of reactive intermediates of 9 . this has a relatively higher impact on strain ta98 , because its 
 dna is particularly sensitive to g - adduct formation . from a physiological perspective , this result has potential 
 important ramifications for individuals with gilbert s syndrome , 
 who experience impaired ugt1a1 activity and mildly elevated concentrations of 1 , based on a less efficient glucuronidation . the observation that compound 8 was the most effective 
 agent against 9 using strain ta102 and was moderately 
 effective in strain ta98 confirms the previously reported potent antigenotoxic 
 activity of this substance . however , 
 this is the first investigation to demonstrate an inhibitory effect 
 against food - derived genotoxins . a previous study has suggested that 8 is a strong reducing agent based on its extended -electron 
 system , allowing for covalent binding 
 capacity with biomolecules . this supports the hypothesis 
 that 8 inhibits mutagenesis induced by 9 , by direct chemical reaction with the mutagen . as observed 
 previously in the 9-ta102 experiment of 
 this study , 8 most effectively inhibited the mutagenic 
 effects of 10 in strain ta98 . this suggests that 8 is a potent inhibitor of frame - shift mutations at concentrations 
 present within the intestine / enterocytes ( 0.214 m ) . considering that frame - shift mutations are an 
 important cause of carcinogenesis in gastric and colon cancer , these results could be of importance in explaining a possible protective 
 role for 8 , due to its enterohepatic distribution . with 
 reference to related tetrapyrroles , this hypothesis is supported by 
 a preceding study that did not include 8 , but showed that bacterial absorption of 1 and 4 protected against frame - shift mutation 
 in strain ta98 . this observation might explain in part the inverse 
 relationship between serum levels of 1 and colorectal 
 cancer in vivo . mass spectrometry ( low - resolution 
 esims ) analysis of the initial 
 experiments indicated that g ( mw = 151 g / mol ) under the test conditions 
 reacted with 11 ( mw = 120 g / mol ) to produce two detectable 
 products , with the major one ( > 90% by ms signal intensity ) giving 
 a [ m + h ] peak at 272 amu and the minor product ( < 10% ) 
 showing an m / z of 392 ( [ m + h ] ) . these correspond to covalent adducts of g with one and 
 two units of 11 , respectively . the conversion of g to 
 these adducts over a seven - day period was low , and a large quantity 
 of unreacted g was recovered . a control experiment confirmed that 
 g in the presence of silica gel alone was stable over seven days under 
 these conditions . in the presence of selected tetrapyrroles ( reactions 
 35 in table 2 ) , none of the g11 adducts were detected ; however , several compounds with 
 molecular weights corresponding to tetrapyrrole11 adducts were detected . in the case of 4 and 8 , peaks corresponding to unreacted g and tetrapyrrole were accompanied 
 by peaks assigned to adducts of tetrapyrrole and one to five units 
 of 11 . for compound 1 , evidence of the conversion 
 of 1 to 4 , followed by reaction of both 
 tetrapyrroles with one to four units of 11 , was observed . the addition of 11 to the tetrapyrroles was repeated 
 in the absence of g , yielding similar results . the extent of reaction 
 of tetrapyrroles with 11 was high over the seven - day 
 period of study , with only small amounts of unreacted tetrapyrroles 
 remaining , in contrast to the significantly less facile reaction between 
 g and 11 . table 2 summarizes the 
 positive ion esims analysis of the reaction products . the negative 
 ion esims analysis showed the corresponding negative ions and thereby 
 fully supported these data . these results confirmed that tetrapyrroles 
 react with activated aromatic epoxides to form covalent adducts more 
 efficiently and more rapidly than g reacts with the same epoxide . 1 : unconjugated bilirubin ; 4 : biliverdin ; 8 : protoporphyrin ; 11 : styrene 
 epoxide ( so ) . esims : low - resolution mass 
 spectrometry . to examine the possibility 
 of covalent adduct formation of reactive 
 epoxides of 9 and tetrapyrroles , novel reactions of 1 , 4 , and 8 with 11 the data clearly showed tetrapyrroles efficiently 
 react with 11 , with several units of 11 being 
 conjugated to each tetrapyrrole . while it was demonstrated that g 
 does react with 11 , it was significantly less efficient 
 than the reaction of tetrapyrroles with g. when g was placed in reaction 
 with 11 in the presence of any of the tetrapyrroles , 
 no g adduct was detected . while any compound with a cooh group 
 would have been expected to react with 11 via a nucleophilic 
 opening of 11 to provide the ester , this would only account 
 for two units of 11 per molecule of tetrapyrrole . the 
 data indicate that more units are being conjugated to the tetrapyrrole , 
 suggesting that there are other sites on the molecules that may be 
 reactive to epoxides . these preliminary data emphasize that the antimutagenic 
 properties of tetrapyrroles are partly a consequence of their ability 
 to react with an activated epoxide ( 11 ) more quickly 
 than dna bases , forming covalent adducts , and thus reducing cellular 
 dna damage . in summary , the experiments conducted in this investigation 
 demonstrate that endogenous tetrapyrroles exert antigenotoxic effects , 
 by preventing mutation caused by food - derived mutagens . this is the 
 first study to provide mechanistic insight showing that tetrapyrroles 
 react more readily than g with a model epoxide ( 11 ) to 
 form covalent adducts . therefore , the data suggest endogenous tetrapyrroles 
 may neutralize reactive epoxides before they react with dna and cause 
 mutation . although not directly transferable to the human condition , 
 this investigation could have important ramifications for maintaining 
 health . this is particularly pertinent to cancer prevention within 
 the digestive tract , as one of the main loci of tetrapyrrole abundance . low - resolution mass 
 spectrometric data were recorded in positive- and negative - electrospray 
 ionization modes , using a bruker esquire hct ( high - capacity 3d ion 
 trap ) instrument , with a bruker esi source . for ms analysis , the solutions 
 were diluted in meoh to a concentration of 100 nm . flash column 
 chromatography was performed on lomb silica gel 60 ( 0.040.06 
 mm ; 230400 mesh ) , which was also used as the activating agent 
 in the reactivity studies . the first experiments were designed 
 to assess the reactivity of selected tetrapyrroles ( 1 , 4 , and 8) and g with 11 . 
 compound 11 ( 5 mol ) was dissolved in chcl3 , and powdered silica gel ( 3 mg ) was added as a proton source 
 in the absence of h2o . guanine 
 ( 1 mol ) and each tetrapyrrole ( 1 mol ) were then added 
 separately , with the final reaction volume being 1 ml . each vial was 
 sealed under an inert atmosphere ( ar gas ) , protected from light , and 
 was allowed to stand at 37 c for one week ( reactions 35 
 in table 2 ) . control reactions containing g 
 and 11 with and without silica gel in chcl3 were also prepared in the same fashion ( reactions 1 and 2 in table 2 ) . the second experiment directly investigated reactions 
 between tetrapyrroles and 11 without g. compound 11 ( 5 mol ) , a selected tetrapyrrole ( 1 mol ) , 
 and silica gel ( 3 mg ) were again dissolved in chcl3 ( 1 
 ml ) , placed under ar , sealed , wrapped in foil , and allowed to react 
 at 37 c for one week ( reactions 68 in table 2 ) . control reactions containing a tetrapyrrole and 
 silica gel in the absence of 11 were also performed ( reactions 
 911 in table 2 ) . after incubation , 
 all reactions were subjected to flash column chromatography , initially 
 using 100% ch2cl2 as eluent , increasing the 
 percentage of meoh in the eluting solvent from 0% to 30% . the fractions 
 containing the compounds of interest eluted as mixtures in 5% meoh 
 to 30% meoh . these fractions were further purified by a second chromatographic 
 column using toluene / acetone ( from 0% acetone to 50% acetone ) . all 
 fractions from the chromatographic column were examined by low - resolution 
 esims , as described below . the salmonella reverse mutation 
 assay is a well - accepted 
 screening test to evaluate the mutagenic potential of chemicals in 
 vitro . mutagenic substances revert mutated 
 bacteria to their wild - type variant , which allows them to grow . genotoxicity 
 ( or vice versa antigenotoxic effects ) of compounds can therefore be 
 assessed by quantifying bacterial growth relative to controls . the 
 experiments performed followed the method of maron and ames and included 25 min of preincubation . s9 liver 
 homogenate ( s9 microsomal fraction from aroclor 1254 pretreated rats ; 
 mp biomedicals , illkirch , france ) was used as an enzymatic activation 
 system in all of the assays . bruce n. ames 
 ( university of california , 
 berkeley , ca , usa ) . prior to the tests being conducted , the genetic integrity 
 and spontaneous mutation rates of the strains were assessed . two distinct strains were adopted to consider 
 multiple mechanisms of protection from mutation : the dna repair proficient 
 strain ta102 ( hisg428 mutation ) , for the detection of a t base 
 pair damage and small deletions provoked by cross - linking agents , 
 which can be reverted by mutagens causing oxidative damage , and ta98 
 ( hisd3052 mutation ) , which detects primarily g  c base pair 
 and frame - shift mutations . the s. typhimurium strains 
 used in the experiments have different mutations in various genes 
 in the histidine operon ; each of these mutations is designed to be 
 responsive to mutagens that act via different mechanisms . the assays were performed in a dimly lit laboratory 
 under aseptic working conditions ( laminar airflow cabinet safemate 
 1.8 ; bioair euroclone , milan , it ) . samples were protected from light 
 throughout the experiments and were freshly prepared in amber vials 
 before each test . pbs ( 500 l ) or s9-mix ( 19.75 ml of distilled 
 h2o , 25 ml of pbs , 500 l of mgcl2 ( 0.85 
 m ) , 0.5 ml of kcl ( 1.65 m ) , 2 ml of nadp ( 90.8 mm ) , 250 l of 
 glucose-6-phosphate ( 1.08 m ) , and 2 ml of s9 ) , 100 l of overnight 
 culture , and 200 l of a selected tetrapyrrole solution ( in 
 dmso ) were mixed in sterile test tubes . for antimutagenic testing , 
 100 l of mutagen ( in dmso or dmso alone for controls ; table 1 ) was added to each tube . after 25 min of preincubation 
 ( 37 c , on a rotary shaker ) , 2 ml of molten top agar was added 
 to every tube . the mixtures were poured onto minimum glucose agar 
 plates that were incubated at 37 c for 48 h. his revertants were counted manually after having routinely checked 
 the background lawn under a microscope ( 40 magnification ; olympus 
 ch-2 ) . dmso final plate concentrations did not exceed 10% v / v . in the austrian laboratories , work with mutagens was performed according 
 to directive 2004/37/ec of the european parliament and the council 
 of april 29 , 2004 , on the protection of workers from the risks related 
 to exposure to carcinogens or mutagens at work ( sixth individual directive 
 within the meaning of article 16(1 ) of council directive 89/391/eec ) . in australia , australia worksafe by the national occupational health 
 and safety commission , approved criteria for classifying hazardous 
 substances ( 1994 ) , was followed . compounds 1 ( bilirubin ix , 
 cas # 635 - 65 - 4 ) , 2 ( bilirubin ditaurate , cas # 89771 - 93 - 7 ) , 3 ( bilirubin dimethyl ester , cas # 19792 - 68 - 8 ) , 4 ( biliverdin ix , cas # 55482 - 27 - 4 ) , 5 ( biliverdin 
 dimethyl ester , cas # 10035 - 62 - 8 ) , 6 ( stercobilin , cas # 
 34217 - 90 - 8 ) , 7 ( urobilin , cas # 28925 - 89 - 5 ) , and 8 ( protoporphyrin ix , cas # 553 - 12 - 8 ) were purchased from frontier 
 scientific , carnforth , lancashire , uk . compound purity ( > 98% ) and 
 solubility were assessed using hplc ( hitachi hplc , equipped with a 
 shimadzu spd - m20a detector , a fortis c18 reversed - phase 
 column ( 4.6  150 mm , 3 m ) , and a phenomenex c18 guard column ( 4  3 mm ) ) and visible spectrophotometry ( perkin - elmer 
 lambda 2 uv / vis spectrophotometer ) . all other reagents were from sigma - aldrich 
 at and au ( unless otherwise noted ) , were of the highest analytical 
 grade available , and were stored and used according to the manufacturer s 
 instructions . compound 10 was purchased from toronto 
 research chemicals , north york , on , ca . tetrapyrrolic compounds were 
 solubilized in dmso and protected from light using foil , and solutions 
 were used immediately . the composition and preparation of all necessary 
 reagents and solutions have been published elsewhere . following procedures 
 that have been 
 previously published , six doses of 2 , 4 , and the previously untested compounds 3 and 58 were investigated 
 at 0.01 , 0.05 , 0.1 , 0.5 , 1 , and 2 mol / plate ( 3.5714 
 m ) . compound 1 was tested over a range of five 
 doses , including 0.01 , 0.05 , 0.1 , 0.5 , and 0.75 mol / plate ( 3.5268 
 m ) . the lowest concentrations tested were selected based on 
 an ability to reduce mutagenicity by 50% and to reflect physiologically 
 relevant concentrations present within the enterohepatic circulation . the respective maximum sample doses were ascertained : ( 1 ) by testing 
 the maximum amount of dmso that did not result in bacterial cytotoxicity 
 ( 350 l / plate , ca . 12% v / v ) and ( 2 ) from the respective maximum 
 solubility of each pigment ( supernatant analysis : visible spectrophotometry : 13 , 455 nm ; 4 , 5 , 380 nm ; 6 , 7 , 400 nm ; 8 , 
 410 nm ) read on a perkin - elmer lambda 2 uv / vis spectrophotometer after 
 high - speed centrifugation ; hplc analysis was performed as published 
 previously . each sample was tested 
 in triplicate , and all of the assays were repeated independently . every test included three positive control plates ( mutagen only ) and 
 six negative control plates ( no mutagen , no pigment ) as well as three 
 no - treatment plates ( no mutagen , no pigment , no dmso ) . for a substance to be classified as genotoxic in the salmonella reverse 
 mutation assay , the number of revertant colonies on the test compound 
 plates had to exceed twice the number of colonies grown on the solvent 
 control plates ( negative control ) . in addition to investigating the antimutagenic effects of the tetrapyrroles 
 selected for study , the spontaneous mutagenic activities of these 
 compounds were tested ; for each sample the respective highest and 
 lowest concentrations were applied without mutagen addition . none 
 of the samples tested caused mutagenesis in either of the bacterial 
 strains ( p > 0.05 ) . aflatoxin b1 ( 9 ) induces both base - pair substitution 
 mutation and frame - shift 
 mutation in those strains of s. typhimurium carrying 
 the r - factor plasmid pkm101 , which applies to both of the selected tester strains . metabolism of 9 involves a series 
 of cytochrome enzymatic processes to generate the 8,9-epoxide , which 
 forms covalent dna and rna adducts . 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ( 10 ) is a potent mutagen in strain 
 ta98 , but is virtually inactive in strain ta102 . it is metabolized by multiple cyps , forming a 2-hydroxyamino 
 intermediate , mainly provoking frame - shift and base substitution mutations . 9 : aflatoxin b1 ( afb1 ) , 10 : 
 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ( phip ) . s9 : 
 + indicates metabolic activation 
 was used . data were analyzed using ibm spss 
 17.0 for windows . parametric 
 statistical analyses ( one - way analysis of variance and the scheff 
 post hoc test ) were performed on normally distributed data , and corresponding 
 nonparametric tests ( kruskal  wallis test , dunnet t3 post hoc 
 test ) on not normally distributed data . to assess the order of effectiveness 
 of the test compounds , data were analyzed using ip0.5 values 
 ( percentage positive control inhibition at 0.5 mol / plate test 
 compound ) , which were determined using the software derive 6 ( texas 
 instruments , dallas , tx , usa ) .
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 ( 0.012 mol / plate ; 3.5714 m ) 
 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 .