The anti-mutagenic properties of bile pigments

Bile pigments, including bilirubin and biliverdin, are endogenous compounds belonging to the porphyrin family of molecules. In the past, bile pigments and bilirubin in particular were thought of as useless by-products of heme catabolism that can be toxic if they accumulate. However, in the past 20 years, research probing the physiological relevance of bile pigments has been mounting, with evidence to suggest bile pigments possess significant antioxidant and anti-mutagenic properties. More specifically, bile pigments are potent peroxyl radical scavengers and inhibit the mutagenic effects of a number of classes of mutagens (polycyclic aromatic hydrocarbons, heterocyclic amines, oxidants). Coincidentally, persons with elevated circulating bilirubin concentrations have a reduced prevalence of cancer and cardio-vascular disease. Despite the encouraging in vitro anti-mutagenic effects of bile pigments, relatively little research has been conducted on their inhibitory capacity in bacterial and cultured cell assays of mutation, which might link the existing in vitro and in vivo observations. This is the first review to summarise the published data and it is our hope it will stimulate further research on these potentially preventative compounds.     

Interaction of bilirubin and biliverdin with reactive nitrogen species

Bilirubin (BR) and biliverdin (BV), two metabolites produced during haem degradation by haem oxygenase, possess strong antioxidant activities toward peroxyl radical, hydroxyl radical and hydrogen peroxide. Considering the importance attributed to nitric oxide (NO) and its congeners in the control of physiological and pathophysiological processes, we examined the interaction of BR and BV with NO and NO-related species in vitro. Exposure of BR and BV to agents that release NO or nitroxyl resulted in a concentration- and time-dependent loss of BR and BV, as assessed by high performance liquid chromatography. Peroxynitrite, a strong oxidant derived from the reaction of NO with superoxide anion, also showed high reactivity toward BR and BV. The extent of BR and BV consumption largely depended on the NO species being analysed and on the half-lives of the pharmacological compounds considered. Of major importance, BR and BV decomposition occurred also in the presence of pure NO under anaerobic conditions, confirming the ability of bile pigments to scavenge the gaseous free radical. Increasing concentrations of thiols prevented BR consumption by nitroxyl, indicating that bile pigments and thiol groups can compete and/or synergise the cellular defence against NO-related species. In view of the high inducibility of haem oxygenase-1 by NO-releasing agents in different cell types, the present findings highlight novel anti-nitrosative characteristics of BR and BV suggesting a potential function for bile pigments against the damaging effects of uncontrolled NO production.     

In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

Inhibitory effect of curcumin and its natural analogues on genotoxicity of heterocyclic amines from cooked food

Curcumin (C) and its natural analogues demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC), known for their potent anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic effects, were tested for their possible inhibitory effects against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor induced rat liver S9 homogenate. In the present investigations, curcumin as well as its two natural analogues i.e., dmC and bdmC were found to be highly effective in suppressing genotoxicity of all the tested cooked food mutagens in a dose-dependent manner, in both the frame shift (TA98) as well as base pair mutation sensitive (TA100) strains of S. typhimurium. However, bdmC appeared to be a relatively less active antimutagen compared to C and dmC. More than 80% inhibition of mutagenicity was observed at 200 microg/plate in case of C and dmC in both TA98 and TA100 against all tested cooked food mutagens. Where as, bdmC showed 39-79% inhibition in TA100 and 60-80% inhibition in TA98, at a dose of 200 microg/plate. These findings warrant further biochemical, enzymatic and in vivo investigations in animal models as well as in humans to establish the chemoprotective effect of these agents against mutagenic heterocyclic amines found in cooked food.     

Effect of bilirubin infusion on excretion of bilirubin and biliverdin in rabbits

Intravenous infusion of bilirubin (BR) at 171 micrograms/min/kg into rabbits resulted in biliary concentration of BR increasing from 3.8 (control) to 243 mg/dl and BR excretion increasing from 1.7 to 66 micrograms/min/kg. BR infusion resulted in biliary concentrations of biliverdin (BV) increasing from 9.1 to 30 g/dl and BV excretion increasing from 4.2 to 8.2 micrograms/min/kg. BR infusion produced a progressive decline in bile flow. BV was the predominant bile pigment in control rabbits fed either an alfalfa-based or chlorophyll-free diet. These results imply that rabbits can oxidize BR to BV.     

Inhibitory effect of dibenzoylmethane on mutagenicity of food-derived heterocyclic amine mutagens

Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in a widely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate. DBM has been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has recently been shown to be even more effective than curcumin in suppressing the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. But there are no reports regarding its antimutagenic properties against cooked food mutagens. Results of the present investigations clearly indicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibit mutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well as the base pair mutation sensitive (TA100) strains of S. typhimurium. These highly potent inhibitory effects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigations strongly warrant further studies of its efficacy as a cancer chemopreventive agent.     

Synergistic interaction between vitamin E and the bile pigments bilirubin and biliverdin

The oxidation of soybean phosphatidylcholine (PC) liposomes initiated with a lipid-soluble azo compound within the liposomal membranes has been studied in the absence and presence of membrane-bound vitamin E and water-soluble bile pigments. In the absence of vitamin E, lipid peroxidation proceeded linearly and without delay. Low micromolar amounts of bilirubin ditaurine (BR-DT, a model compound of conjugated bilirubin) or biliverdin (BV) inhibited the oxidation of PC significantly and in a concentration-dependent way. In contrast, neither taurine, ascorbic acid nor reduced glutathione inhibited significantly under these conditions. Both bile pigments were consumed during their protective action. Vitamin E incorporated into the liposomal membranes suppressed the oxidation initially almost completely, thereby producing an induction period. In the combined presence of vitamin E and either of the two bile pigments at 10 microM each, this induction period was increased by at least 200%. In contrast, when 10 microM vitamin E was combined with an equimolar concentration of reduced glutathione, the induction period increased by only about 30%. BR-DT and BV both spared the consumption of vitamin E during the oxidation of PC liposomes. These results demonstrate that conjugated bilirubin and BV located in the aqueous phase can directly scavenge lipid radicals to some extent. Furthermore, both bile pigments can act synergistically with membrane-bound vitamin E to prevent lipid peroxidation initiated in the lipid phase, most likely through regeneration of the vitamin from its chromanoxyl radical.     

The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[alpha]pyrene in Salmonella typhimurium strains

The comutagenic and desmutagenic effect of quercetin on the mutagenicity of typical mutagens e.g. 2-acetylaminofluorene (AAF), 4-nitroquinoline-1-oxide (4NQO) and benzo[alpha]pyrene (B[a]P), in Salmonella typhimurium TA98, TA100 and TA98/1,8 DNP6 were examined. In the mixed application of AAF with quercetin in the presence of mammalian metabolic activation system (S9 mix), the numbers of revertants in TA98 increased by as much 2.2-5.0-fold compared with the sum of those in the separate applications of AAF and quercetin. A 1.4-2.7-fold increase was observed in TA100. Quercetin did not affect the mutagenicity of 4NQO, and depressed that of B[a]P. Dose-response curves for mutagenicity of quercetin with or without AAF (5 micrograms/plate) were examined. The results suggest that quercetin, present in a molarity of up to 1.5 times that of AAF, is apparently effective in enhancing the mutagenicity of AAF, because a linear dose-response curve was observed in the range of 0-5 micrograms/plate quercetin with AAF although quercetin alone was not mutagenic in the same range. Dose-response curves for mutagenicity of quercetin with or without 5 micrograms/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was reduced to about 60% of the sum of separate application at doses ranging from 25 to 100 micrograms/plate of quercetin. Since enhancement and depression of mutagenicity by quercetin were observed for indirect mutagens, AAF and B[a]P, respectively, in the presence of S9 mix, quercetin may affect the metabolic pathway of these mutagens.     

In vitro anti-mutagenic effect of magnolol against direct and indirect mutagens

Magnolol, a component of the bark of Magnolia obovata, has been reported to possess various biological activities, such as anti-carcinogenicity, anti-promotion activity and anti-oxidative activity. These findings suggest potential for this compound in cancer chemoprevention. Interestingly, there have been no reports to date on the potential anti-mutagenic activity of magnolol, involving inhibition of initiation processes of the primary stage of carcinogenicity. In this study, anti-mutagenic activity of magnolol against mutagenicity induced by direct mutagens [1-nitropyrene (1-NP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)] and indirect mutagens [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), benzo(a)pyrene (B(a)P), 2-aminoanthracene (2-AA) and 7,12-dimethylbenz[a]anthracene (DMBA)] were investigated using the bacterial mutagenicity test (Ames test). Results show that magnolol strongly inhibits mutagenicity induced by indirect mutagens, but does not affect direct mutagens. To elucidate the mechanism of this effect against indirect mutagens, effect of magnolol on CYP1A1- and CYP1A2-related enzyme activities of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) were investigated. Magnolol strongly and competitively suppressed these enzyme activities, suggesting it inhibited mutation induced by indirect mutagens through suppression of CYP1A1 and CYP1A2 activity.     

Vibrational and electronic circular dichroism study of bile pigments: Complexes of bilirubin and biliverdin with metals

Complexation of bilirubin (BR) and biliverdin (BV) with biogenic and toxic metals (Mn, Cu, Cd, Co, Fe, Ni, Zn, and Ag) has been studied by means of electronic circular dichroism (ECD) and vibrational circular dichroism (VCD). Poly-l-lysine and β-cyclodextrin in water were chosen as matrices capable of recognizing the single stereoconformer of the pigments with defined M-helicity. Such systems allow structural changes caused by complexation of pigments with metals in aqueous solution at pH 10-11 to be followed using chiroptical methods, which are intrinsically sensitive to spatial structure. These and other spectroscopic techniques have revealed that BV and BR form monomeric complexes with Cd, Cu, and Zn and dimeric complexes with Mn. The stabilities of the complexes with Fe, Ni, Co, and Ag are remarkably lower. The sign of the ECD and VCD patterns of the complexed BV does not change for the chelates of any of the studied metals other than Zn, this exception being interpreted in terms of manifestation of the opposite helicity of BV in its chelate with Zn. In the case of BR, the observed inversion of ECD signal after complexation, together with the analysis of VCD spectra, reveals that a flattening of the molecule takes place, i.e., an increase in the angle between the pyrrinone chromophores without an inversion of helicity. This chiral stereoselectivity, which is very specific in the case of the Zn chelates, is discussed in connection with the specific inhibition of Zn-required enzymes by bile pigments.     

Evaluation of mutagenic and antimutagenic activities of alpha-bisabolol in the Salmonella/microsome assay

alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens.     

Antimutagenic activity of Terminalia chebula (myroblan) in Salmonella typhimurium.

Antimutagenicity of water and chloroform extracts of dried myroblan Terminalia chebula was determined against two direct acting mutagens, sodium azide and 4-nitro-o-phenylenediamine (NPD) in strains TA100 and TA1535, and TA97a and TA98 of Salmonella typhimurium respectively and S9-dependent mutagen 2-aminofluorene (2-AF) in TA97a, TA98 and TA100 strains. Water extract reduced NPD as well as 2-AF induced his+ revertants significantly but did not have any perceptible effect against sodium azide included his+ revertants in TA100 and TA1535 strains of S. typhimurium. The pre-incubation studies, where the extract was incubated at 37 degrees C for 30 min with the said mutagen prior to plating, enhanced the inhibitory effect. Autoclaving the water extract reduced the inhibitory effect but the reduction in the effect was not significant. No inhibitory effect was observed in any of the strains and against any of the test mutagens with chloroform extract.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Heterogeneity of bile pigments formed by the breakdown of haemoglobin in the isolated male-dog kidney.

Normothermic perfused isolated male-dog kidneys formed radioactive bile pigments by the breakdown of radioactive haemoglobin prepared from [2-14C]glycin. After column chromatographic separation and preparation of dipyrrolic azopigments, 86.3 +/- 2.2% of the bile pigments seemed to be conjugated bilirubin. Thin-layer chromatographic separation of the azopigments of ethyl anthranilate revealed a good correlation between photometric scanning, radiochromatographic scanning and the radioactivity of the azopigments scraped off the thin-layer glass plates and counted in a liquid scintillation counter. Although the same heterogenity of the azopigments was observed as in dog bile, the isolated male-dog kidney formed significantly less alpha2- and significantly more gamma-fractions.     

Induction of light emission by luminescent bacteria treated with UV light and chemical mutagens

Intensity of light emission by luminescent bacteria in response to UV irradiation and chemical mutagens was tested. We demonstrated that luminescence of six strains of marine bacteria (belonging to four species: Photobacterium leiognathi, P. phosphoreum, Vibrio fischeri and V. harveyi) is significantly increased by UV irradiation relatively shortly after dilution of cultures. Such a stimulation of luminescence was abolished in cells treated with chloramphenicol 15 min before UV irradiation, indicating that effective gene expression is necessary for UV-mediated induction of light emission. These results suggest that stimulation of luminescence in UV-irradiated bacterial cells may operate independently of the quorum sensing regulation. A significant induction of luminescence was also observed upon treatment of diluted cultures of all investigated strains with chemical mutagens: sodium azide (SA), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine x 2HCl (ICR-191), 4-nitro-o-phenylenediamine (NPD), 4-nitroquinolone-N-oxide (NQNO), 2-aminofluorene (2-AF), and benzo[alpha]pyrene. These results support the proposal that genes involved in bioluminescence belong to the SOS regulon. The use of bacterial luminescence systems in assays for detection of mutagenic compounds is discussed in the light of this proposal.     

Subsequent products after antioxidant actions of beta-carotene and alpha-tocopherol have no Salmonella mutagenicity

Beta-carotene and alpha-tocopherol are important antioxidants biologically, but whether their oxidized products are toxic or not remains to be discovered. Here, we chromatographically separated 5 pure products or isomeric mixtures from reaction mixtures of beta-carotene and reactive oxygens, and 17 lipid-radical scavenging products of alpha-tocopherol. The products were tested for mutagenicity using Salmonella typhimurium TA98, TA100, TA102, and TA104, in the presence and absence of S9. None showed mutagenicity against any of the four strains, or cytotoxicity that influenced the survival of the bacteria. Lipid-peroxides have been known to increase the formation of mutagens from dietary procarcinogens such as heterocyclic amines. So, we also measured the activity to increase 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) mutagenicity. The products from beta-carotene and alpha-tocopherol did not increase, but rather several of them suppressed, the mutagenicity of Trp-P-2. Thus, the products of beta-carotene and alpha-tocopherol formed after the antioxidant actions were not genotoxic.     

Inactivation of potent pyrolysate mutagens by chlorinated tap water

The potent mutagens 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2, 62450-07-1), 2-amino-6-methyldipyrido-[1,2-a:3', 2'-d]imidazole (Glu-P-1, 67730-11-4) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ, 76180-96-6), isolated from pyrolysates of tryptophan and glutamic acid and from broiled sardines, respectively, were effectively degraded by chlorinated tap water with a concomitant loss of mutagenicity toward Salmonella typhimurium TA98 and TA100. The half-life of 10 microM IQ in the presence of 1.5 ppm of residual chlorine was less than 10 sec; those of Glu-P-1 and Trp-P-2 were 0.5-1 and 2-3 min, respectively. This means that a glass of chlorinated tap water (150 ml) containing 1.5 ppm of residual chlorine can break down about 200 micrograms of these pyrolysate mutagens within a couple of minutes.     

Changes in bilirubins in human prenatal development.

1. A densitometric method has been developed for the quantification of azodipyrroles derived from dog bile pigments treated with diazotized ethyl anthranilate. 2. This method was used to estimate the bilirubins in bile and meconium from foetuses of 14-36 weeks gestation. 3. The proportion of the bilirubins in foetal bile changed during gestation. (a) No bile pigments were found until 14 weeks. (b) Between 14 and 15 weeks bilirubin-IX beta was the only bile pigment detected. (c) At 16-17 weeks some unconjugated bilirubin-IX alpha was found in the bile, but up to 20 weeks bilirubin-IX beta was the predominant bilirubin in the bile. (d) At about 20 weeks glucose, xylose, and an unidentified bilirubin-IX alpha monoconjugate were found in the bile. (e) Between 20 and 23 weeks bilirubin-IX alpha glucuronide appeared in the bile. (f) At 30 weeks monoconjugates of bilirubin-IX alpha were the predominant bilirubins in the bile. (g) Only in full-term foetuses was bilirubin-IX alpha monoglucuronide the major bilirubin derivative.     

Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.     

Photochemical oxidation of 2-aminofluorene: correlation between the induction of direct-acting mutagenicity and the formation of nitro and nitroso aromatics

The kinetics of near ultraviolet light-mediated phototransformation of 2-aminofluorene (2-AF) was studied using high performance liquid chromatography (HPLC) and the Ames/Salmonella mutagenicity bioassay. Employing tester strains TA98, TA1538, and the nitroreductase-deficient TA98NR without the addition of exogenous metabolic enzymes, we were able to detect and discriminate between the UVA exposure-dependent formation of two stable photoproducts, 2-nitrosofluorene (2-NOF) and 2-nitrofluorene (2-NO2F). Mutagenicity of irradiated 2-AF solutions (using dimethyl sulfoxide as a solvent) in the various tester strains indicates the rapid formation of the photo-labile 2-NOF, after which 2-NO2F accounts for the preponderance of mutagenic activity. Continued UVA irradiation (greater than 72 h at 6.8 J/m2/s) of 2-AF results in the formation of greater than 30 photoproducts resolvable on HPLC, several of which, in addition to 2-NOF and 2-NO2F, are mutagenic on Salmonella but are chemically undefined to date. Prolonged irradiation ultimately destroys the photo-induced mutagenicity of 2-AF. However, UVA-induced 2-AF photoproducts are stable for several weeks when stored in sealed vials in the dark. Light potentiated oxidation of aromatic amines constitutes an alternative mechanism for the transformation of aromatic amines into proximate mutagens/carcinogens.