Mutagenic activation of 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole(Trp-P-1) and 3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2) by primary cultures of adult rat hepatocytes: effect of Aroclor induction in vitro

The mutagenic activation of tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, was studied in a Salmonella TA98/hepatocyte mutagenesis assay. Adult rat hepatocytes in primary culture were either untreated or induced by the addition of Aroclor 1254 (2 micrograms/ml) 18-20 h before the mutagenesis test which was performed at day 1 and at day 2 after the isolation of hepatocytes. The mutagenic activation of Trp-P-1 and Trp-P-2 was studied as a function of the time of incubation and of the concentration of chemical. Trp-P-1 and Trp-P-2 incubated for 20 min in the presence of untreated hepatocytes and bacteria gave rise to a weak number of revertants which doubled the level of spontaneous mutants. Aroclor-induced hepatocytes became highly competent in mutagenic activation of tryptophan pyrolysis products and the induction ratio reached 4.9 and 7.1 for Trp-P-1 and Trp-P-2, respectively, after 60 min of incubation, on day 2 of the experiment. It should be noted that the induction ratio was higher on day 2 than on day 1. When conditions were standardized, i.e. Aroclor-induced hepatocytes on day 2, final concentration of cellular protein about 1 mg/ml, 20 min of incubation, the Salmonella/hepatocyte assay produced a linear concentration-dependent mutagenic response for Trp-P-1 and Trp-P-2. By comparing the results obtained with Aroclor-induced hepatocytes and Aroclor-induced liver S9 fraction in the Salmonella test, it could be estimated that hepatocytes were 3 times less active than the S9 fraction with regard to mutagenic activation of both Trp-P-1 and Trp-P-2.     

Comparative genotoxic effects of the cooked-food-related mutagens Trp-P-2 and IQ in bacteria and cultured mammalian cells

As part of a major study to evaluate the mutagenicity of chemicals produced during the cooking of foods, we examined the responses of bacteria and cultured Chinese hamster cells to the compounds Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and IQ (2-amino-3-methylimidazo[4,5-f]quinoline), constituents identified in cooked beef and fish. In the Ames/Salmonella tester strain TA1538, both compounds were confirmed to be extremely potent mutagens that were active at levels below 1 ng/plate in the presence of hamster-liver S9 microsomal fraction. 50-fold higher doses of both compounds were required for mutagenicity in the uvr+ tester strain TA1978. Trp-P-2 also behaved as a strong mutagen in CHO cells using the standard exogenous activation with hamster-liver S9 fraction. At concentrations below 1 microgram/ml it produced dose-dependent increases in cell killing, mutations at the hprt and aprt loci, sister-chromatid exchanges, and chromosomal aberrations. An excision-repair-deficient strain was about 2-fold more sensitive than the normal CHO cells with respect to these genotoxic effects of Trp-P-2. IQ had unexpectedly weak activity for all genetic endpoints in the CHO cells, and it produced clear-cut responses only in the repair-deficient cells and only above a concentration of 10 micrograms/ml. The toxicity that was observed with IQ was not affected by the repair capacity of the cells and was not associated with chromosomal aberrations, indicating that damage to cellular structures other than nuclear DNA was likely the predominant pathway for cell killing. Because the culture conditions normally used for CHO cell exposure were shown to be competent in producing bacterial mutagenicity with IQ, it was concluded that the active metabolite of IQ was present in the medium but was somehow ineffective in reaching the DNA of CHO cells and/or reacting with it. These results suggest that the relative mutagenic potency of compounds in Salmonella may bear no direct relationship to relative mutagenicity in CHO cells, emphasizing precaution in attempting to extrapolate microbial data to mammalian somatic cells. This study illustrates the use and merits of a multi-endpoint assay for genetic damage in CHO cells, the utility of using CHO cells that are defective in excision repair of DNA, and the importance of comparative testing between bacterial and mammalian systems.     

Role of hemin in the inhibition of mutagenic activity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and other aminoazaarenes

The mechanism of inhibition by hemin of the mutagenic activities of food pyrolysate aminoazaarenes, particularly that of Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole), was investigated. Hemin efficiently inhibited the metabolic activation by S9 of Trp-P-2, as demonstrated by high-performance liquid chromatographic analysis of the reaction mixtures in which Trp-P-2 had been treated with S9 in the presence or absence of hemin. N-Hydroxy-Trp-P-2, an activated form of Trp-P-2 having direct mutagenicity on Salmonella typhimurium TA98, undergoes spontaneous oxidative degradation in its aqueous solution, and the presence of hemin in the solution accelerated the degradation significantly. The presence of excess hemin with N-hydroxy-Trp-P-2 completely abolished the mutagenic activity of this mutagen towards Salmonella. A UV-visible spectroscopic study has suggested the formation of a complex between hemin and N-hydroxy-Trp-P-2/Trp-P-2. In support of this view, the fluorescence spectrum of a Trp-P-2 solution was quenched efficiently by the addition of hemin. These observations indicate that this complex formation plays a role in the observed multiple actions of hemin. Similar inhibitory actions of hemin on several other direct-acting aminoazaarene mutagens are also described, as well as the inhibition activities of protoporphyrin, chlorophyllin, biliverdin and bilirubin.     

Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)) into its nitroso derivative

Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins. We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence. We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mutagenicity of Trp-P-2(NHOH)) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture. Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d] imidazole (Glu-P-1(NHOH)). A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed. Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH). Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin. We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules.     

Role of the uvr gene in the SOS function inducing activity of two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, in Escherichia coli K12

Two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 were assayed in the SOS-chromotest using PQ 37 (uvr A) and PQ 35 (uvr+) E. coli K12 strains, in the presence of S9 fraction from Aroclor-induced rats. Both compounds were able to induce the expression of SOS functions in uvr A bacteria, in the following order: Trp-P-1 less than Trp-P-2 less than aflatoxin B1, at low concentrations (less than 125 ng/assay). In this range, the induction of SOS functions was significantly decreased in the uvr+ strain. This implies that the uvr gene product plays an important role in the repair of genotoxic damage induced by Trp-P-1 and Trp-P-2. At higher concentrations (125-500 ng/assay), Trp-P-1 became more efficient in inducing SOS functions than Trp-P-2 and excision repair was less efficient than at low concentration.     

Modified metabolism of a carcinogen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by liver S9 from Schistosoma japonicum-infected mice.

Schistosoma japonicum infection has been associated with an increased incidence of liver and colorectal cancers in humans. To explore the mechanisms underlying this association, we investigated the carcinogen-metabolizing properties of liver S9 preparations from S. japonicum-infected mice and compared them with those of S9 from uninfected animals. When the carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was incubated with these S9s and the products were analyzed by high-performance liquid chromatography, we observed that the S9 from infected mice had a lower ability to convert Trp-P-2 into 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), an activated form of promutagenic Trp-P-2, than the S9 from uninfected mice. We found that both of these S9 preparations have a high ability to reduce Trp-P-2(NHOH) into Trp-P-2; however, the infected-mouse S9 showed a significantly greater reducing power than the control S9. This difference appears to be responsible for the observed lower mutagen-activating potential of the infected mouse S9. These results suggest that hepatic enzyme activities of S. japonicum-infected mice are quantitatively different from those of normal mice.     

Metabolic activation of hepatocarcinogens in chronic hepatitis B

S9 fraction pools of liver biopsy samples, collected from 129 patients in two consecutive studies, were comparatively assayed for their ability to activate aflatoxin B1 (AFB1) and a tryptophan pyrolysate product (Trp-P-2) in a miniaturized Salmonella mutagenicity test system. Metabolic activation was not affected to a significant extent by most of the monitored variability factors, such as sex, alcohol, cigarette smoking and liver histology (minimal changes, chronic persistent (CPH) or active (CAH) hepatitis, CAH steatosis, or cirrhosis). Conversely, a significant enhancement of activation was observed for AFB1 in cases of mild CAH and especially for Trp-P-2 in hepatitis B virus carriers, irrespective of their histologic diagnosis.     

Comparative mutagenic efficiencies of the DNA adducts from the cooked-food-related mutagens Trp-P-2 and IQ in CHO cells

The relationship between DNA-adduct formation and mutagenicity of two heterocyclic aromatic amines associated with cooked foods was determined in a CHO cell strain lacking nucleotide excision repair. Cells were exposed to tritiated IQ (2-amino-3-methylimidazo[4,5-f]quinoline) or Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) supplemented with hamster S9 microsomal fraction for metabolic activation. DNA from nuclei was isolated by DNAase-mediated elution from polycarbonate filters after RNAase and proteinase treatment. The presumed metabolites of both compounds bound to DNA in a dose-dependent fashion. Although the dose required to produce 50% cell killing was 15 times higher for IQ than Trp-P-2, the amount of radioactive material bound to DNA at that dose was about 10-fold lower with IQ. When mutations at the hprt and aprt loci were compared with the estimated levels of adducts, the calculated mutagenic efficiency of the adducts was about 4 mutations per 1000 adducts for both compounds, assuming a target sequence of 1000 base pairs for either locus. We conclude that IQ is acting as a weak mutagen in this system because its extracellular metabolites either do not reach or do not react efficiently with the DNA of the CHO cells.     

Protection against the bacterial mutagenicity of heterocyclic amines by purpurin, a natural anthraquinone pigment.

Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) is a naturally occurring anthraquinone pigment found in species of madder root. We have found that the presence of purpurin in bacterial mutagenicity assays is responsible for a marked inhibition of mutagenicity induced by food-derived heterocyclic amines. Purpurin was found to be a better inhibitor of Trp-P-2-dependent mutagenicity than either epigallocatechin gallate or chlorophyllin both of which are well-established anti-mutagenic components of diet. Inhibition of Trp-P-2(NHOH) mutagenicity by purpurin was dependent upon pH. It was a better inhibitor in neutral than acidic conditions. Purpurin was protective against the direct mutagen Trp-P-2(NHOH) in both the presence and the absence of hepatic S9 but required pre-incubation. Finally, purpurin was responsible for the inhibition of human CYP1A2 and human NADPH-cytochrome P450 reductase and a decrease in the bioactivation of Trp-P-2 by these enzymes when they were expressed in Salmonella typhimurium TA1538ARO. However, inhibition of Trp-P-2(NHOH)-dependent mutations suggests purpurin also has a direct effect on this mutagen in addition to inhibiting its formation by CYP1A2.     

Structural Elucidation of Rotihibin B by Tandem Mass Spectrometry

The mutagenicity and desmutagenicity of extracts of soybeans heated at 225 ± 5°C were investigated by the Ames test. The soybeans were refluxed in water, methanol, or diethylether for 2h. The aqueous and methanol extracts (2–4 mg/plate) of the heated soybeans exhibited strong desmutagenic activity of 43–92% against heterocyclic amines (Trp-P-1, Glu-P-2, IQ, MeIQx, PhIP), while no mutagenicity was observed. The desmutagenicity of the heated soybean extracts remained even after denaturation by 0.1 N HCI in vitro and absorption by the rat small intestine. The desmutagenic mechanism for heated soybeans was evaluated, and it was verified that the soybean extract exhibited its desmutagenicity by blocking the mutagenicity of activated Trp-P-1, and not by inhibiting the S9 enzyme system.     

Detection of Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, in dialysis fluid of patients with uremia

In order to estimate the exposure levels of mutagenic and carcinogenic heterocyclic amines in humans, we developed a high-performance liquid chromatography method to detect 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in dialysis fluid of patients with uremia. Using this methods, dialysis fluid of 12 patients who had received hemodialysis treatment or continuous ambulatory peritoneal dialysis was examined. Trp-P-1 was detected in dialysate of all uremic patients (727 +/- 282 pmoles, n = 12). In patients who had been treated with continuous ambulatory peritoneal dialysis, the average amount of Trp-P-1 found in whole dialysate (6 l) per day was 710 +/- 203 pmoles (mean +/- S.D., n = 8). Moreover, Trp-P-2 could be detected in 5 out of 12 patients (206 +/- 85 pmoles, n = 5). These results indicate that patients with uremia are actually exposed to carcinogenic tryptophan pyrolysis products. The average exposure level of Trp-P-1 in uremic patients apparently exceeded 710 pmoles (150 ng) per day.     

Enhancement of the mutagenicity of IQ and MeIQ by nitrite in the Salmonella system.

The fried food mutagens IQ, MeIQ, Glu-P-1 and Trp-P-2 were treated with nitrite at pH 3.0 for 1 h at 37 degrees C. The resulting reaction mixtures were tested for mutagenicity towards Salmonella typhimurium TA97, TA98, TA100 and TA1535. Glu-P-1 and Trp-P-2 were readily converted to weak or non-mutagenic deaminated compounds, whereas IQ and MeIQ were converted to extremely strong mutagenic derivatives in both the presence and the absence of rat liver S9 mix. The mutagenicity of MeIQ in TA98 was enhanced by nitrite up to 3-fold, while that of nitrosated MeIQ was further enhanced by S9 mix up to 15-fold. The nitrosation products of MeIQ were resolved into 7 bands by TLC on silica gel plate. Bands I, III, V and VI were highly mutagenic to both TA98 and TA100. The experimental results suggest that the non-enzymatic formation of direct-acting mutagens from indirect-acting mutagens such as IQ or MeIQ might be physiologically important, especially with regard to the etiology of human gastrointestinal tract tumors.     

Mutagenicity of thiol compounds in Escherichia coli WP2 tester strain IC203, deficient in OxyR: effects of S9 fractions from rat liver and kidney

Low doses of -cysteine (CYS), cysteinyl-glycine (CYSGLY) and reduced glutathione (GSH) activated by γ-glutamyl transpeptidase (GGT) were mutagenic in strain IC203 (oxyR), whereas higher doses were required to observe a weak mutagenicity in the oxyR⁺ strain WP2 uvrA/pKM101 (denoted IC188). This indicates that thiol mutagenesis is suppressed by OxyR-regulated antioxidant defenses and confirms its oxidative character. The mutagenesis by low doses of CYS, CYSGLY and GSH+GGT detected in IC203 was abolished by rat liver S9, through the activity of catalase, as well as by the metal chelator diethyldithiocarbamate (DETC), supporting the dependence of this mutagenesis on H₂O₂ production, probably in thiol autoxidation reactions in which transition metals are involved. Surprisingly, low DETC concentrations greatly potentiate the mutagenicity of low CYS doses. Mutagenesis by high doses of CYS and CYSGLY occurred in both IC203 and IC188 in the presence of liver S9, and was resistant to inhibition by catalase, although it was prevented by DETC. Mutagenesis by GSH activated by rat kidney S9, rich in GGT, was detected in IC203 and IC188 only at high doses since catalase and glutathione peroxidase, both present in kidney S9, might inhibit its induction by low GSH doses. In the presence of liver S9, almost deficient in GGT, GSH was not mutagenic. The mutagenicity of a high GSH dose occurring in the presence either of GGT plus liver S9 or of kidney S9 was weakly prevented by DETC.     

Mutagenicity modulating effect of quercetin on aromatic amines and acetamides

The effect of quercetin on the mutagenicity of 32 kinds of aromatic amines and their acetamides were investigated using Salmonella typhimurium TA98 with a mammalian metabolic activation system (S9 mix). Quercetin enhanced the mutagenicity of the tricyclic aromatic amines (aminofluorene, aminoanthracene and aminophenanthrene) and their acetamides by 1.2-5.9-fold. Whereas, quercetin depressed the mutagenicity of aniline derivatives, biphenyl derivatives, and bi- and tetra-cyclic amino derivatives. The modulation of mutagenicity of Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2 (heterocyclic amines) by quercetin were liable to be affected by the content of S9 in the S9 mix. It seems that quercetin does not have the same effect as norharman, because quercetin did not enhance the mutagenicity of aniline. It is suggested that the modulation of the mutagenicity of aromatic amines and acetamides is caused by the modulation of the balance between the mutagenic activation and inactivation in the metabolism of these amines and acetamides in the presence of quercetin. In this modulation, quercetin may participate through its effects on the promotion of N-hydroxylation and the inhibition of arylhydroxylation and transacylation. The presence of tricyclic aromatic rings of amines and acetamides is a structural requirement for the mutagenicity enhancement by quercetin.     

Identification of mutagenic heterocyclic amines (IQ, Trp-P-1 and AalphaC) in the water of the Danube river

Three mutagenic heterocyclic amines, 2-amino-3-methylimidazo-[4, 5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), were isolated and identified in water from the Danube River in Vienna. Heterocyclic amines were extracted from river water by the blue rayon hanging method, and analyzed by gas chromatography with a nitrogen-phosphorous detector (GC-NPD) and GC-mass spectrometry (GC-MS) after conversion into their N-dimethylaminomethylene derivatives. Identity of IQ, Trp-P-1 and AalphaC in the river water was confirmed by GC-MS. The contents of IQ, Trp-P-1 and AalphaC were estimated by GC-NPD at 1.78+/-0.17, 0.14+/-0.02 and 0.44+/-0.02 ng/g blue rayon equivalent (n=3), respectively. The total amounts of these amines accounted for 26% of the mutagenicity of blue rayon extracts evaluated by the Ames test using TA98 with metabolic activation.     

Regional mutagenicity of heterocyclic amines in the intestine: mutation analysis of the cII gene in lambda/lacZ transgenic mice

Transgenic mouse assays have revealed that the mouse intestine, despite its resistance to carcinogenesis, is sensitive to the mutagenicity of some heterocyclic amines (HCAs). Little is known, however, about the level and localization of that sensitivity. We assessed the mutagenicity of four orally administered (20 mg/kg per day for 5 days) HCAs-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) hydrochloride, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) acetate-in the intestine of male MutaMice. Two weeks after the last administration, we isolated epithelium from the small intestine, cecum, and colon and analyzed lacZ and cII transgene mutations. PhIP increased the lacZ mutant frequency (MF) in all the samples, and in the small intestine, cII and lacZ MFs were comparable. In the cII gene, G:C to T:A and G:C to C:G transversions were characteristic PhIP-induced mutations (which has also been reported for the rat colon, where PhIP is carcinogenic). In the small intestine, PhIP increased the cII MF to four-fold that of the control, but IQ, MeIQ, and Trp-P-2 did not have a significant mutagenic effect. In the cecum, cII MFs induced by IQ and MeIQ were 1.9 and 2.7 times those in the control, respectively. The MF induced by MeIQ in the colon was 3.1 times the control value. Mutagenic potency was in the order PhIP>MeIQ>IQ; Trp-P-2 did not significantly increase the MF in any tissue. The cecum was the most susceptible organ to HCA mutagenicity.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces caspase-dependent apoptosis in mononuclear cells

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the tryptophan pyrolysates, is a dietary carcinogen and is formed in cooked meat and fish in our daily diet. Trp-P-1 will affect the cells in the blood circulation system before it causes carcinogenicity in target organs such as the liver. In this study, the cytotoxicity of Trp-P-1 was investigated in mononuclear cells (MNCs) from blood. Trp-P-1 (10-15 microM) decreased cell viability and induced apoptosis characterized both by morphological changes and by DNA fragmentation 4 h after treatment. DNA fragmentation was also observed following treatment at 1 nM after 24 h in culture. This result suggested that apoptosis would occur in the body following unexpected intake of foods containing Trp-P-1. To determine the mechanism of apoptosis, we investigated the activation of the caspase cascade in MNCs. Trp-P-1 (10-15 microM) activated the caspase cascade, i.e. the activity of caspase-3, -6, -7, -8 and -9 increased dose-dependently using peptide substrates, the active forms of caspase-3, -8 and -9 were detected by immunoblotting, and cleavage of poly(ADP-ribose) polymerase and protein kinase C-delta as the intracellular substrates for caspases was observed. A peptide inhibitor of caspase-8 completely suppressed activation of all other caspases, while an inhibitor of caspase-9 did not. These results indicated that caspase-8 may act as an apical caspase in the Trp-P-1-activated cascade.     

Glutathione and glutathione S-transferases in the salmonella mammalian-microsome mutagenicity test

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH.

The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.     

Antimutagenic Acitivity against Trp-P-1 of the Edible Thai Plant,Oroxylum indicum Vent.

A methanolic extract of Oroxylum indicum strongly inhibited the mutagenicity of Trp-P-1 in an Ames test. The major antimutagenic constituent was identified as baicalein with an IC₅₀ value of 2.78±0.15 μM. The potent antimutagenicity of the extract was correlated with the high content (3.95±0.43%, dry weight) of baicalein. Baicalein acted as a desmutagen since it inhibited the N-hydroxylation of Trp-P-2.