Potent antimutagenic and their anti-lipid peroxidative effect of kaikasaponin III and tectorigenin from the flower of Pueraria thunbergiana

The MeOH extract of Pueraria thunbergiana (Leguminosae) flowers and its fractions were subjected to Ames test to test the antimutagenicity. EtOAc fraction (1 mg/plate) decreased the number of revertants of Salmonella typhymurium TA100 by 95% against aflatoxin B, (AFB1). Phytochemical isolation of the EtOAc fraction afforded four isoflavonoids (tectorigenin, glycitein, tectoridin and glycitin) and one saponin (kaikasaponin III). Though the three isoflavonoids other than tectoridin showed significant antimutagenicity, the activity of kaikasaponin III was the most potent. Kaikasaponin III (1 mg/plate) decreased the number of revertants of S. typhymurium TA100 by 99% against AFB, but by 75% against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Tectorigenin (1 mg/plate) inhibited the AFB1-induced mutagenicity by 90% and MNNG-induced one by 76%. Glycitein and glycitin were less active than tectorigenin and kaikasaponin III. This result suggested that kaikasponin III prevents the metabolic activation of AFB1 and scavenge electrophilic intermediate capable of mutation. The two components with potent activities, tectorigenin and kaikasaonin III, significantly prevented the malondialdehyde formation caused by bromobenzene in the rat.     

Antimutagenic effect of plant flavonoids in theSalmonella assay system

The antimutagenic effects of 27 kinds of plant flavonoids on the mutagenicity of aflatoxin B1(AFB1) and N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) in Salmonella typhimurium TA100 were investigated. In the mixed applications of AFB1 (1 microgram/plate) with the flavonoids (300 micrograms/plate) in the presence of a mammalian metabolic activation system (S9 mix), chrysin, apigenin, luteolin and its glucoside, kaempferol, fisetin, morin, naringenin, hesperetin, persicogenin, (+)-catechin and (-)-epicatechin showed the antimutagenic effect against AFB1 with more than 70% inhibition rate. A little or no antimutagenicities except flavone against MNNG (0.5 microgram/plate) were observed. For the antimutagenicity of the flavonoids on AFB1, the flavonoid structure that contains the free 5-, 7-hydroxyl group seemed to be essential. However, saturation of the 2,3-double bond or elimination of the 4-keto group did not affect the activity.     

Mouse strain differences in glutathione S-transferase activity and aflatoxin B1 biotransformation

Previous studies have suggested that mice are resistant to the carcinogenic effects of aflatoxin B1 (AFB1) and that this resistance is largely the result of expression of an isoenzyme of glutathione S-transferase (GST) with high activity toward AFB1-8,9-epoxide. Significant interstrain differences in cytosolic GST activities toward a variety of substrates have been reported in mice. If such differences exist for the conjugation of AFB1-8,9-epoxide, then there may be significant mouse strain differences in susceptibility to AFB1-induced hepatocarcinogenicity. The hepatic microsomal and cytosolic biotransformation of AFB1 was studied in 8 different strains of mice fed a purified diet. GST-mediated conjugation of AFB1-8,9-epoxide with glutathione and GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide (CHP) were determined with cytosolic fractions from 8-10 pooled livers. Specific activities of cytochrome-P-450-mediated oxidation of AFB1 to aflatoxin Q1 (AFQ1), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1), as well as the reactive intermediate AFB1-8,9-epoxide, were determined with hepatic microsomal fractions from each mouse strain. No striking differences in specific activity between mouse strains were observed for any of the P-450- or GST-mediated enzymatic pathways measured, although some statistically significant differences were found. GST specific activities toward AFB1-8,9-epoxide, CDNB, DCNB, ECA and CHP ranged from 1.5-2.1, 2,830-5,370, 81-144, 38-69 and 32-73 nmol/mg protein/min, respectively. The rate of formation of AFB1-8,9-epoxide ranged from 208 to 465 pmol/mg protein/min. The specific activities of AFQ1,AFM1, and AFP1 formation by microsomes ranged from 36-70, 161-326, and 252-426 pmol/mg protein/min, respectively. Mice fed a standard rodent chow diet showed evidence of microsomal and cytosolic enzyme induction when compared to mice fed a purified diet. The lack of substantial differences in enzyme specific activities between mouse strains suggests that interstrain variations in the hepatocarcinogenic effects of AFB1 in mice should not be large.     

Inhibition of the mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine and 9-Aminoacridine by indenopyridines in the Salmonella typhimurium tester strain 1537 and E. coli

Abstract

The goal of the present research was to determine the protective potential of five newly synthesized indenopyridine derivatives against N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and 9-aminoacridine (9-AA) induced mutagenesis. MNNG sensitive Escherichia coli WP2uvrA and 9-AA sensitive Salmonella typhimurium TA1537 were chosen as the bacterial tester strains. All of the test compounds showed significant antimutagenic activity at various tested concentrations. The inhibition rates ranged from 25.6% (Compound 2 - 1?mM/plate) to 68.2% (Compound 1 - 2.5?mM/plate) for MNNG and from 25.7% (Compound 4 - 1?mM/plate) to 76.1% (Compound 3 - 2.5?mM/plate) for 9-AA genotoxicity. Moreover, the mutagenicity of the test compounds was investigated by using the same strains. None of the test compounds has mutagenic properties on the bacterial strains at the highest concentration of 2.5?mM. Thus, the findings of the present study give valuable clues to develop new strategies for chemical prevention from MNNG and 9-AA genotoxicity by using synthetic indenopyridine derivatives.     

The effect on performance and biochemical parameters when soil was added to aflatoxin-contaminated poultry rations.

The effects of silty clay loam soil on the performance and biochemical parameters of chicks were investigated when the soil was added to aflatoxin B1 (AFB1)-contaminated diets. One hundred 14-d-old White Leghorn chicks were fed a control ration (clean corn), a low aflatoxin-contaminated ration (120 ng AFB1/g), a high aflatoxin-contaminated ration (700 ng AFB1/g), or high aflatoxin-contaminated rations (700 ng AFB1/g) +10% or 25% soil. Body weight, feed consumption and blood samples were monitored weekly. Decreased feed consumption, body weight gain and efficiency of feed utilization, increased SGOT and LDH activities, and cholesterol and triglyceride concentrations, and decreased uric acid concentrations and ALP activity were observed in the chicks fed the high aflatoxin-contaminated ration without soil. Hepatomegaly was prominent in chicks fed the high aflatoxin-contaminated ration without soil, and some livers had extensive hepatocyte vacuolation, hepatocellular swelling, fatty change and hydropic degeneration, and stained positive for fat accumulation. Addition of soil reduced the detrimental effects of AFB1 for some parameters, although the reduction was less when 10% soil was fed compared with the 25% soil feeding.     

Inhibitory effects of neem seed oil and its extract on various direct acting and activation-dependant mutagens-induced bacterial mutagenesis

Abstract

Context: Azadirachta indica A. Juss (Meliaceae), commonly called neem is a plant native to the Indian sub-continent. Neem oil extracted from the seeds of neem tree has shown promising medicinal properties.

Objective: To investigate the possible anti-mutagenic activity of neem seed oil (NO) and its dimethylsulfoxide (DMSO) extract (NDE) on the mutagenicity induced by various direct acting and activation-dependant mutagens.

Materials and methods: The possible anti-mutagenic activity of NO (100–10?000?µg/plate) and NDE (0.1–1000?µg/plate) as well as the mechanism of anti-mutagenic activity was studied in an in vitro Ames Salmonella/microsome assay.

Results: NSO and NDE inhibited the mutagenic activity of methyl glyoxal (MG), in which case the extent of inhibition ranged from 65 to 77% and against 4-nitroquinoline-N-oxide (NQNO); it showed a 48–87% inhibition in the non-toxic doses. Similar response of NSO and NDE was seen against the activation-dependant mutagens aflatoxin B1 (AFB1, 48–88%), benzo(a)pyrene (B(a)P, 31–85%), cyclophosphamide (CP, 66–71%), 20-methylcholanthrane (20-MC, 37–83%) and acridine orange (AO, 39–72%) in the non-toxic doses. Mechanism-based studies indicated that NDE exhibits better anti-mutagenic activity in the pre- and simultaneous-treatment protocol against MG, suggesting that one or several active phytochemicals present in the extract covalently bind with the mutagen and prevent its interaction with the genome.

Discussion and conclusion: These findings demonstrate that neem oil is capable of attenuating the mutagenic activity of various direct acting and activation-dependant mutagens.     

Effect of repeated dietary exposure of aflatoxin B1 on brain biogenic amines and metabolites in the rat

Male Sprague-Dawley rats were treated po twice weekly for 3 weeks with a low (32.8 micrograms/kg) and high dose (327.9 micrograms/kg) of aflatoxin B1 (AFB1) in corn oil. A control group received corn oil only. At the end of the experiment the rats were killed, and the concentrations of the brain catecholamines, norepinephrine (NE) and dopamine (DA), catecholamine metabolites, 3-methoxy-4-hydroxymandelic acid (VMA), homovanillic acid (HVA), and dihydroxyphenylacetic acid (DOPAC), and the indoleamine serotonin (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were determined by high-pressure liquid chromatography in five brain regions. The major effects were found in striatal dopamine and serotonin concentrations, with decreases of 37 and 29%, respectively. A corresponding decline was observed in the dopamine metabolites, homovanillic acid (44%) and dihydroxyphenylacetic acid (30%). Concentrations of these neurotransmitters and metabolites were only marginally altered in cerebral cortex, cerebellum, hypothalamus, and medulla oblongata. It appears that a major effect of AFB1 is on dopaminergic pathways, possible by selectively perturbing the conversion of tyrosine to biogenic catecholamine neurotransmitters.     

GC-MS and GC-IRD studies on brominated dimethoxyamphetamines: regioisomers related to 4-Br-2,5-DMA (DOB)

A series of regioisomeric bromodimethoxyamphetamines have mass spectra essentially equivalent to the controlled drug substance 4-Br-2,5-dimethoxyamphetamine (4-Br-2,5-DMA; DOB); all have molecular weight of 274 and major fragment ions in their electron ionization mass spectra at m/z 44 and m/z 230/232. The trifluoroacetyl, pentafluoropropionyl and heptafluorobutryl derivatives of the primary regioisomeric amines were prepared and evaluated in gas chromatography-mass spectrometry (GC-MS) studies. The mass spectra for these derivatives did not show unique fragment ions for specific identification of individual isomers. However, the mass spectra do serve to divide the compounds into three groups, depending on their base peak. Gas chromatography with infrared detection (GC-IRD) provides direct confirmatory data for the identification of the designer drug 4-bromo-2,5-dimethoxyamphetamine from the other regioisomers involved in the study. The perfluoroacylated derivatives of the six regioisomeric bromodimethoxyamphetamines were successfully resolved on non-polar stationary phases such as a 100% dimethylpolysiloxane stationary phase (Rtx-1) and 50% phenyl - 50% methyl polysiloxane (Rxi-50).     

Mutagencity and antimutagencity studies of lipidic extracts from yellowtail fish (Seriola lalandi), lisa fish (Mugil cephalus) and cazón fish (Mustelus lunulatus).

Organic extracts from fresh and smoked yellowtail fish (Seriola lalandi), lisa fish (Mugil cephalus) and cazon fish (Mustelus lunulatus) were tested for mutagenicity using Ames Salmonella tester strains TA98 and TA100 with metabolic activation (S9). Also, the antimutagenicity of the organic extract from yellowtail fish was tested against aflatoxin B1 (AFB1). Yellowtail fish extract was sequentially fractionated by thin-layer chromatography (TLC) and each fraction was also tested for antimutagenicity. None of the fresh species showed mutagenicity. Extract from smoked yellowtail showed the highest mutagenic potential among the smoked species tested. Organic extract from fresh yellowtail reduced the number of revertants caused by AFB1 showing a dose response type of relationship. Sequential TLC fractionation of the antimutagenic extract produced four antimutagenic fractions from fresh yellowtail fish. These results that the lipidic fraction of the species tested contains at least four compounds with chemoprotective properties that reduce the mutagenicity of AFB1.     

The significance of glutathione conjugation for aflatoxin B1 metabolism in rainbow trout and coho salmon

Rainbow trout are known to be more susceptible to aflatoxin B1 (AFB1) hepatocarcinogenesis than coho salmon, or trout pre-fed the carcinogenesis inhibitors beta-naphthoflavone (beta NF), Aroclor 1254 or indole-3-carbinol. The study reported here examined the relationship between AFB1-glutathione (GSH) conjugation and AFB1 carcinogenesis in salmon, trout and trout pre-fed the three inhibitors. The AFB1-glutathione (AFB1-SG) conjugate was not detected in salmon bile and was present in trout bile in amounts representing less than 0.2% of the administered dose 24 hr after injection of [3H]AFB1. The major conjugates were glucuronides of aflatoxicol and aflatoxicol M1. In incubations of isolated liver cell fractions, less than 0.5% of the original AFB1 dose was recovered as AFB1-SG in salmon and trout preparations, compared to 25% in mouse-liver cell preparations. The GSH concentration in livers of the control trout was higher than that for coho salmon but lower than that for trout pre-fed beta NF. Liver GSH-transferase activity in control trout livers was much higher than in the control salmon livers, but was only 62% of that found for trout fed beta NF. There was no apparent relationship among the various groups between liver GSH concentrations, liver GSH-transferase activity, or biliary GSH conjugate, and the degree of carcinogenic response of AFB1. Thus current evidence does not indicate a major role for aflatoxin B1 epoxide-GSH detoxification in coho salmon, or rainbow trout fed any of the three anticarcinogens tested. These results in salmonid fish are contrary to those which suggest AFB1-SG conjugation as a major determinant of AFB1 carcinogenesis and its dietary modulation in rodent models.     

Application of adsorbent agents technology in the removal of aflatoxin B(1) and fumonisin B(1) from malt extract.

The commercially hydrated sodium calcium aluminosilicate (HSCAS) and the Egyptian montmorillonite (EM) had an excellent capability of adsorbing AFB(1) and FB(1) in an aqueous solution at different tested levels. The adsorption ratio of HSCAS ranged from 95.3% to 99.1% and 84.7% to 92.4% of the available AFB(1) and FB(1) respectively. EM showed an adsorption ratio ranged from 95.4% to 99.2% and 78.2% to 92.2% for AFB(1) and FB(1) respectively. Both adsorbents were effective at 0.5% level. Results of the ability of these adsorbents at level of 0.5% (w/v) to adsorb AFB(1) and FB(1) in malt extract spiked with 50, 100 and 200 ppb indicated that the capability of adsorbing of HSCAS ranged from 98.5% to 98.9% and 88.2% to 91.9% for AFB(1) and FB(1) respectively. Whereas, the capability of adsorbing of EM ranged from 98.1% to 98.7% and 88.2% to 92.5% for AFB(1) and FB(1) respectively.     

Interaction of fumonisin B(1) and aflatoxin B(1) in a short-term carcinogenesis model in rat liver

The co-existence of the fumonisin and aflatoxin mycotoxins in corn merited studies to investigate their possible synergistic toxicological and carcinogenic effects. When utilising a short-term carcinogenesis model in rat liver, both the compounds exhibited slow cancer initiating potency as monitored by the induction of foci and nodules stained positively for the placental form of gluthatione-S-transferase (GSTP(+)). However, when rats were treated in a sequential manner with AFB(1) and FB(1) the number and size of GSTP(+) lesions significantly increased as compared to the separate treatments. Histopathological analyses indicated that the individual treatments showed far less toxic effects, including occasional hepatocytes with dysplastic nuclei, oval cell proliferation and, in the case of FB(1), a few apoptotic bodies in the central vein regions. The sequential treatment regimen induced numerous foci and dysplastic hepatocyte nodules, and with oval cells extending from the periportal regions into the centrilobular regions. This would imply that, in addition to the cancer promoting activity of FB(1) of AFB(1)-initiated hepatocytes, the AFB(1) pre-treatment enhanced the FB(1) initiating potency, presumably by rendering the liver more susceptible to the toxic effects of FB(1). The co-occurrence of AFB(1) and FB(1) in corn consumed as a staple diet could pose an increased risk and should be included in establishing risk assessment parameters in humans.     

The determination of cocaine, benzoylecgonine, and cocaethylene in small-volume oral fluid samples by liquid chromatography-quadrupole-time-of-flight mass spectrometry

A quantitative analysis was developed for the determination of cocaine, benzoylecgonine, and cocaethylene in oral fluid using liquid chromatography-tandem mass spectrometry. After internal standardization and solid-phase extraction, chromatographic separation was achieved on a reversed-phase column by gradient elution. The reconstructed mass chromatograms of the collision-induced dissociation transitions of m/z 290 --> m/z 168 (benzoylecgonine), m/z 304 --> m/z 168+119 (2'-methylbenzoylecgonine), m/z 304 --> m/z 182 (cocaine), m/z 318 --> m/z 196 (cocaethylene), and m/z 318 --> m/z 182+119 (2'-methylcocaine) were used for quantitation. The developed method was adequately validated. Good linearity was obtained from 10 to 1000 microg/L. Extraction recoveries exceeded 85% for all compounds. Excellent total and within-run reproducibilities (CV% < 20%) and accuracy figures were obtained. The limit of detection (signal-to-noise ratio >/= 3) was 1 microg/L for all three compounds. As such, a method for drug abuse confirmation analysis in oral fluid, compatible with the present day saliva collecting devices, is obtained. The method was applied to real samples (n = 15) obtained from suspected drug users, of which seven proved positive. The concentrations found in the positive samples were between 10.2 and 200.6 microg/L for cocaine, < limit of quantification (LOQ) and 10.5 microg/L for cocaethylene, and < LOQ and 59.2 microg/L for benzoylecgonine.     

Characterization of dark liver pigment observed in rats after subchronic dosing of the beta3-adrenergic receptor agonist LY368842

Dark liver pigmentation was observed in F344 rats in a subchronic toxicology study after daily dosing of LY368842 glycolate. In addition, green-colored urine was observed in some animals. To identify the source of the pigment and its potential for toxic consequences, the liver pigment was isolated from the liver tissue of rats. The resulting material was a dark brown to black powder that was insoluble in water, organic solvents, or a tissue-solubilizing agent. Several techniques, such as chemical degradation, HPLC, tandem mass spectrometry (LC/MS/MS), (1)H NMR, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were employed to characterize the dark liver pigment. Following oxidative degradation of the isolated pigment, degradation products related to LY368842 were identified or tentatively identified using LC/MS/MS. Two degradation products had the same protonated molecular ion at m/z 505, which is 30 amu higher than that of LY368842. The major m/z 505 product has been identified as the indole-2,3-dione oxidative product based on (1)H NMR data and confirmed by an authentic standard. In addition, monohydroxylated product was also identified in the degradation mixture. These degradation products were consistent with the metabolites found in vivo in rats. MALDI-MS analyses of liver and urine pigment both identified a product with a protonated molecular ion at m/z 977, suggesting formation of indirubin-like and indigo-like pigments. The results obtained suggest that the oxidative metabolites of LY368842 played a key role in the formation of the liver and urine pigments.     

Antibacterial activity of aminals and hemiaminals of pyrazole and imidazole

Antibacterial activity of 1,1′-methylenedipyrazole (AM1), 1-hydroxymethylpyrazole (SAM1), 1,1′-methylenediimidazole (AM2), and 1-hydroxymethylimidazole (SAM2) has been tested against reference and clinical strains by both difusimetric and broth dilution methods. Overall, the minimal inhibitory concentrations of tested compounds ranged from 180 to 270?μg/ml, while the minimal bactericidal concentrations were between 360 and 720?μg/ml. Comparative assessment with phenol and formaldehyde shows that AM1, AM2, SAM1, and SAM2 have moderate to good antibacterial activity.     

Toward Revealing Microcystin Distribution in Mouse Liver Tissue Using MALDI-MS Imaging

Cyanotoxins can be found in water and air during cyanobacterial harmful algal blooms (cHABs) in lakes and rivers. Therefore, it is very important to monitor their potential uptake by animals and humans as well as their health effects and distribution in affected organs. Herein, the distribution of hepatotoxic peptide microcystin-LR (MC-LR) is investigated in liver tissues of mice gavaged with this most common MC congener. Preliminary matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging experiments performed using a non-automated MALDI matrix deposition device and a MALDI-time-of-flight (TOF) mass spectrometer yielded ambiguous results in terms of MC-LR distribution in liver samples obtained from MC-LR-gavaged mice. The tissue preparation for MALDI-MS imaging was improved by using an automated sprayer for matrix deposition, and liver sections were imaged using an Nd:YAG MALDI laser coupled to a 15 Tesla Fourier-transform ion cyclotron resonance (FT-ICR)-mass spectrometer. MALDI-FT-ICR-MS imaging provided unambiguous detection of protonated MC-LR (calculated m/z 995.5560, z = +1) and the sodium adduct of MC-LR (m/z 1017.5380, z = +1) in liver sections from gavaged mice with great mass accuracy and ultra-high mass resolution. Since both covalently bound and free MC-LR can be found in liver of mice exposed to this toxin, the present results indicate that the distribution of free microcystins in tissue sections from affected organs, such as liver, can be monitored with high-resolution MALDI-MS imaging.     

Inactivation of aflatoxin B1 in corn meal, copra meal and peanuts by chlorine gas treatment

More than 75% degradation of aflatoxin B1 (AFB1) was achieved after treatment of AFB1-spiked corn meal, spiked copra meal (the residue of the kiln-dried coconut kernels after mechanical expulsion of oil) and peanuts artificially infected with Aspergillus parasiticus, with 11, 16 and 35 mg chlorine gas per g meal or peanuts, respectively. At these chlorine gas treatment levels, extension of the exposure period of the corn meal and copra meal beyond 2.5 hr, and the peanuts beyond 1 day, did not increase the percentage degradation of AFB1. The mutagenicity of chlorine-treated copra meal and peanuts spiked with AFB1 was greatly reduced compared with untreated controls, as determined in Salmonella typhimurium strain TA98 in the presence of rat liver S-9 mix; the reduction in mutagenicity was found to be highly correlated with the reduction in AFB1 levels. Reactions of chlorine with AFB1 or constituents of the meals or peanuts did not appear to generate new mutagenic compounds. The moisture content of the meals and peanuts appeared to be an important factor affecting the degradation of AFB1 by chlorine gas.     

Unusually high concentrations in a fatal GHB case

The first case in France involving a fatal overdose resulting from the ingestion of gamma-hydroxybutyrate (GHB) is presented. GHB was tested by gas chromatography-mass spectrometry (GC-MS) after precipitation. Briefly, 20 microL of body fluids (blood, bile, urine, gastric contents, or vitreous humor) was pipetted in a glass tube, followed by 20 microL GHB-d6 and 45 microL acetonitrile. After vortex mixing and centrifuging, the supernatant was collected and evaporated to dryness. The residue was derivatized with BSTFA with 1% TMCS for 20 min at 70 degrees C. After injection on a 30-m HP5 MS capillary column, GHB (m/z 233, 204, and 147) and GHB-d6 (m/z 239) were identified by MS. GHB was also tested in pubic hair after incubation in 0.01 N NaOH, neutralization, acidification, extraction in ethyl acetate and derivatization with MTBSTFA, using GC-MS-MS. GHB was positive in all the tested specimens, with the following concentrations 2937, 33,727, 1800, and 2856 mg/L in femoral blood, urine, bile, and vitreous humor, respectively. This seems to be the highest blood concentration ever observed. Postmortem redistribution appears weak, as the concentration in cardiac blood was 3385 mg/L (cardiac blood/femoral blood ratio of 1.15). Oral route was suggested with GHB at 7.08 g in 100 mL of gastric contents. Pubic hair analysis clearly indicated chronic GHB abuse, with concentrations along the shaft in the range 19.4 to 25.0 ng/mg (in comparison with physiological concentrations < 2 ng/mg). Methylenedioxymethamphetamine was present in femoral blood at 144 ng/mL. These results are consistent with an acute fatal overdose of GHB.     

Pseudouridine, an antimutagenic substance in beer towards N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Previously we reported that beer is antimutagenic against several food-derived mutagens including heterocyclic amines. We describe here the isolation and identification of pseudouridine from beer as an antimutagenic substance against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All of the 17 samples of beer tested showed inhibition of the MNNG mutagenicity in Salmonella. Extensive fractionation through chromatography of the active components from a freeze-dried beer sample gave six antimutagenic fractions. One contained pseudouridine, as characterized by the UV spectra, nuclear magnetic resonance, and co-chromatography in HPLC. Pure pseudouridine inhibited the mutagenicity of MNNG in a dose-dependent manner. The amount of pseudouridine in the beer sample, estimated at about 0.4 mg/100 ml beer, can account for 3% of the total antimutagenicity of beer. Thus, the major active components in beer remain to be identified. The role of pseudouridine in inhibiting the mutagenicity of MNNG is to be studied further. Among analogs of pseudouridine, spongouridine, but not uridine, was antimutagenic against MNNG. The bacterial mutagenicity of another methylating agent N-methyl-N-nitrosourea was also inhibited by pseudouridine. Pseudouridine is the first example among nucleosides to be shown to possess an antimutagenic property.     

Metabolism of aflatoxin B1 by normal human bronchial epithelial cells

Aflatoxin B, (AFB1) is a potent hepatocarcinogen in animal models and a suspected carcinogen in humans. High concentrations of AFB, have been found in respirable grain dusts, and may therefore be a risk factor for human lung cancer in certain occupations. To study the potential for AFB, activation in human lung, cytochrome P-450 (CYP)-mediated activation and glutathione S-transferase (GST)-mediated detoxification of AFB1 were examined in cultured normal human bronchial epithelial (NHBE) cells. Cells were exposed to 0. 15 microM or 1.5 microM AFB, for 48 h and media was collected for metabolite analysis by high-performance liquid chromatography (HPLC). At 0. 15 microM, AFB1 was metabolized only to the detoxified metabolite aflatoxin Q1 (AFQ1). At 1.5 microM AFB1, both aflatoxin M1 (AFM1), and AFQ1 were produced. Cells pretreated with 50 degrees M 3-methylcholanthrene (3MC), a CYP 1A inducer, for 72 h prior to 0.15 microM AFB1, produced the activated AFB1 8,9-epoxide (AFBO). Similarly, microsomes prepared from 3MC-pretreated cells formed AFBO, but microsomes from noninduced cells did not. While AFB1-DNA adducts were not detected at low AFB1 concentrations in untreated NHBE, 3MC induction caused the production of AFB1-DNA adducts at 0.015 and 0.15 microM AFB1. Western immunoblots showed that the primary CYP isoforms responsible for AFB1 activation in the liver, 1A and 3A4, to be constitutively expressed in NHBE cells. Expression of CYP 1A was significantly increased in 3MC-pretreated cells, while CYP 3A4 expression increased slightly, but not to the extent of the 1A isoforms. The principal AFBO detoxifying enzyme, glutathione S-transferase (GST), was constitutively expressed in NHBE cells, and was increased approximately twofold by 3MC pretreatment. Cytosolic fractions from neither control nor 3MC-induced NHBE had measurable AFBO conjugating activity, indicating that these cells may lack AFB1-relevant GST activity. From these data, it appears that NHBE cells activate AFB1 inefficiently, but possess CYPs reportedly responsible for metabolism of AFB1. These data support earlier findings showing modest CYP-mediated AFB1 activation in human airways, but indicate that exposure to polycyclic aromatic hydrocarbons (PAHs), such as 3MC, which induce CYP(s) that specifically activate AFB1 may increase the harmful effects of AFB1 exposures in human airways.