Fruits and vegetables protect against the genotoxicity of heterocyclic aromatic amines activated by human xenobiotic-metabolizing enzymes expressed in immortal mammalian cells.

Heterocyclic aromatic amines (HAAs) can be formed during the cooking of meat and fish at elevated temperatures and are associated with an increased risk for cancer. On the other hand, epidemiological findings suggest that foods rich in fruits and vegetables can protect against cancer. In the present study three teas, two wines, and the juices of 15 fruits and 11 vegetables were investigated for their protective effect against the genotoxic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). To closely mimic the enzymatic activation of these HAAs in humans, genetically engineered V79 Chinese hamster fibroblasts were employed that express human cytochrome P450-dependent monooxygenase (hCYP) 1A2 (responsible for the first step of enzymatic activation) and human N(O)-acetyltransferase (hNAT) 2*4 or human sulfotransferase (hSULT)1A1*1 (responsible for the second step of enzymatic activation): V79-hCYP1A2-hNAT2*4 for IQ activation and V79-hCYP1A2-hSULT1A1*1 for PhIP activation. HAA genotoxicity was determined by use of the comet assay. Black, green and rooibos tea moderately reduced the genotoxicity of IQ (IC(50)=0.8-0.9%), whereas red and white wine were less active. From the fruit juices, sweet cherry juice exhibited the highest inhibitory effect on IQ genotoxicity (IC(50)=0.17%), followed by juices from kiwi fruit, plum and blueberry (IC(50)=0.48-0.71%). The juices from watermelon, blackberry, strawberry, black currant, and Red delicious apple showed moderate suppression, whereas sour cherry, grapefruit, red currant, and pineapple juices were only weakly active. Granny Smith apple juice and orange juice proved inactive. Of the vegetable juices, strong inhibition of IQ genotoxicity was only seen with spinach and onion juices (IC(50)=0.42-0.54%). Broccoli, cauliflower, beetroot, sweet pepper, tomato, chard, and red-cabbage juices suppressed IQ genotoxicity only moderately, whereas cucumber juice was ineffective. In most cases, fruits and vegetables inhibited PhIP genotoxicity less strongly than IQ genotoxicity. As one possible mechanism of antigenotoxicity, the inhibition of activating enzymes was studied either indirectly with diagnostic substrates or directly by measuring CYP1A2 inhibition. Only sour cherry, blueberry, and black currant juices suppressed the first step of HAA enzymatic activation, whereas most plant-derived beverages inhibited the second step.

Characterization of NADP-dependent 12 beta-hydroxysteroid dehydrogenase from Clostridium paraputrificum.

Clostridium paraputrificum D 762-06 was found to contain an NADP-dependent 12 beta-hydroxysteroid dehydrogenase, already present in uninduced cells. Its specific activity could, however, be enhanced up to about 3-fold by the inclusion of bile acids with a 12-keto group or a 12 beta-hydroxy group in the growth medium. 3 alpha-Hydroxy-12-keto-5 beta-cholanoic acid was the most effective inducer. A pH optimum of 10.0 and a molecular weight of 126,000 were estimated by molecular sieve chromatography. The enzyme preparation reduced 12-keto groups in conjugated and unconjugated bile acids and oxidized a 12 beta-hydroxy function, but oxidative activity was only about 25% of the reductive one. Disubstituted bile acids showed lower Km values than the corresponding trisubstituted ones, the lowest Km values being those observed for 3,12- and 7,12-5 beta-cholanoic acids. No measurable activity against 12 alpha-hydroxyl groups could be detected. The enzyme was found to be heat-labile (95% inactivation at 50 degrees C for 10 min), but the activity was maintained for about 4 weeks when lyophilized preparations were stored at -20 degrees C. 12 beta-Hydroxysteroid dehydrogenase activity was also demonstrated in the membrane fraction after solubilization with Triton X-100, suggesting that it was originally a membrane-bound enzyme.

Characterization of NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus.

A human fecal isolate, characterized by morphological, physiological and biochemical data as a strain of Peptostreptococcus roductus, was shown to contain NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenases and a NADP-dependent 7 beta-hydroxysteroid dehydrogenase. All enzyme activities could be demonstrated in crude extracts and in membrane fractions. The 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were synthesized constitutively. Specific enzymatic activities were significantly reduced when bacteria were grown in the presence of 3-keto bile acids, while other bile acids were ineffective. For the 3 alpha (3 beta)-hydroxysteroid dehydrogenase, a pH optimum of 8.5 (9.5) and a molecular weight of 95,000 (132,000) was estimated. 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were heat-sensitive (about 75% inactivation at 50 degrees C for 10 min). The 7 beta-hydroxysteroid dehydrogenase was already present in uninduced cells, but specific activity could be enhanced up to more than 2.5-fold when bacteria were grown in the presence of 7-keto bile acids. Disubstituted bile acids were more effective than trisubstituted ones, ursodeoxycholic acid was ineffective as an inducer. A pH optimum of 10.0 and a molecular weight of about 82,000 were shown for the 7 beta-hydroxysteroid dehydrogenase. The enzyme preparation reduced the 7-keto group of corresponding bile acids. Again the affinities of disubstituted bile acids for the enzyme were higher than those of the trisubstituted bile acids, but no significant differences between conjugated and free bile acids were observed. The 7 beta-hydroxysteroid dehydrogenase was heat-sensitive (72% inactivation at 50 degrees C for 10 min), but was detectable at 4 degrees C for at least 48 h.

NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from a lecithinase-lipase-negative Clostridium species 25.11.c.

A lecithinase-lipase-negative Clostridium sp. 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenases. The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound. The 3 beta-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids. A pH optimum of 7.5 and a molecular weight of approx. 104,000 were estimated by molecular sieve chromatography. The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3 beta-hydroxyl function could not be demonstrated. The lowest Km values were found for disubstituted bile acids, trisubstituted and conjugated bile acids having higher Km values. 7 alpha-Hydroxysteroid dehydrogenase, but not 7 beta-hydroxysteroid dehydrogenase, was already present in uninduced cells. The specific activities, however, were greatly enhanced when cells were grown in the presence of chenodeoxycholic acid or 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid. Ursodeoxycholic acid with its 7 beta-hydroxyl group was ineffective as an inducer. Molecular weights of approx. 82,000 and 115,000 were found for the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase, respectively. In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive direction in vitro. Here, the pH optimum for the overall reaction was 8.5-8.7. 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4 degrees C, but were found to be heat-sensitive.

Genotoxicity of selected pesticides in the mouse bone-marrow micronucleus test and in the sister-chromatid exchange test with human lymphocytes in vitro.

Selected pesticides (aldicarb, 1,3-dichloropropene, methidathion, parathion, triadimefon, vinclozolin) were tested for their clastogenic and aneugenic activities in the mouse bone-marrow micronucleus (MN) test in vivo and for their sister-chromatid exchange-inducing activities in human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system from rat-liver S9. 1,3-Dichloropropene significantly increased the frequencies of micronucleated polychromatic erythrocytes (PCE) in bone-marrow cells of female mice from 3.3 MN/1000 PCE to 15.3 MN/1000 PCE (187 mg per kg body weight). 1,3-Dichloropropene (100 microM) induced 16.0 SCE/metaphase after 24 h of incubation as compared with the basal rate of 11.2 SCE/metaphase (-S9) and of 15.4 SCE/metaphase as compared with 10.5 SCE/metaphase of the control (+S9). These values were statistically significantly different from each other. The other pesticides tested did neither increase the rate of micronuclei significantly in polychromatic erythrocytes in male nor in female animals. Aldicarb and methidathion induced a significant increase in SCEs in human lymphocytes in vitro only without the metabolic activating system: aldicarb, 5 microM, 24 h incubation: 15.5 SCE/metaphase; control: 12.6 SCE/metaphase; methidathion, 100 microM, 24 h incubation: 15.8 SCE/metaphase, control: 11.1 SCE/metaphase. Parathion, triadimefon and vinclozolin did not have any SCE-inducing effects.

Free radical scavenging abilities of flavonoids as mechanism of protection against mutagenicity induced by tert-butyl hydroperoxide or cumene hydroperoxide in Salmonella typhimurium TA102.

Mutagenicity induced by tert-butyl hydroperoxide (BHP) or cumene hydroperoxide (CHP) in Salmonella typhimurium TA102 was effectively reduced by flavonols with 3',4'-hydroxyl groups such as fisetin, quercetin, rutin, isoquercitrin, hyperoxide, myricetin, myricitrin, robinetin, and to a lesser extent also by morin and kaempferol (ID50=0.25-1.05 micromol per plate). With the exception of isorhamnetin, rhamnetin, morin, and kaempferol, closely similar results were obtained with both peroxides. Hydrogenation of the double bond between carbons 2 and 3 (dihydroquercetin, dihydrorobinetin) as well as the additional elimination of the carbonyl function at carbon 4 (catechins) resulted in a loss of antimutagenicity with the notable exception of catechin itself. Again, all flavones and flavanones tested were inactive except luteolin, luteolin-7-glucoside, diosmetin, and naringenin. The typical radical scavenger butylated hydroxytoluene also showed strong antimutagenicity against CHP (ID50=5.4 micromol per plate) and BHP (ID50=11.4 micromol per plate). Other lipophilic scavengers such as alpha-tocopherol and N,N'-diphenyl-1,4-phenylenediamine exerted only moderate effects, the hydrophilic scavenger trolox was inactive. The metal chelating agent 1,10-phenanthroline strongly reduced mutagenicities induced by CHP and BHP (ID50=2.75 and 2.5 micromol per plate) at low concentrations but induced mutagenic activities at higher concentrations. The iron chelator deferoxamine mesylate, however, was less effective in both respects. The copper chelator neocuproine effectively inhibited mutagenicity induced by BHP (ID50=39.7 micromol per plate) and CHP (ID50=25.9 micrommol per plate), the iron chelator 2,2'-dipyridyl was less potent (ID50=6.25 mmol per plate against BHP, 0.42 mmol per plate against CHP). In the absence of BHP and CHP, yet not in the presence of these hydroperoxides, quercetin, rutin, catechin, epicatechin, and naringenin induced strong mutagenic activities in S. typhimurium TA102. Radical scavenging activities of flavonoids against peroxyl radicals generated from 2,2'-azo-bis(2-amidinopropane)dihydrochloride (AAPH) as measured in the haemolysis test, confirmed that in general flavonoids with di- or trihydroxy hydroxyl functions especially in positions 3', 4', 5' are effective radical scavengers. In this test system, however, luteolin was the most potent compound, followed by epicatechin and eriodictyol. Again, isorhamnetin was a potent inhibitor of lysis of red blood cells despite the presence of a 3'-OCH3 function. Radical scavenging activities of flavonoids against the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in principle obeyed the rules outlined above. Flavanones, tamarixetin, and rhamnetin, however, were only weakly active against DPPH, while isorhamnetin was again a potent compound. From these results we conclude that in the Salmonella/reversion assay with strain TA102 antimutagenic activities of flavonoids against the peroxide mutagens CHP and BHP are mainly caused by radical scavenging effects.

Modifying actions of solvent extracts from fruit and vegetable residues on 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (MeIQx) induced mutagenesis in Salmonella typhimurium TA 98.

The edible parts of 13 fruits--apples, apricots, bananas, blackberries, sweet cherries, red currants, white grapes, oranges, peaches, pears, plums, raspberries, and strawberries--and of 12 vegetables--asparagus, green beans, broccoli, brussels sprouts, red and white cabbage, carrots, cauliflower, onions, green peppers, spinach, and tomatoes--were squeezed in order to separate juices and residues. The residues were washed, lyophilized, and extracted sequentially with n-hexane, dichloromethane, acetone, and 2-propanol. Solvent extracted materials were tested in Salmonella typhymurium TA 98 for antimutagenicity against IQ and MeIQx. We found antimutagenic activities in 96% of the n-hexane extracts, 64% of the dichloromethane extracts, 44% of the acetone extracts, and 36% of the 2-propanol extracts. Since no or only minor differences were seen between the mutagens IQ and MeIQx investigations were continued with IQ only. Additional antimutagenic activities were detected in a total of 29.6% of extracts tested when an enzyme preparation with glycosidase-activities (fecalase) was included in the assay. These activities were found in originally inactive or less active dichloromethane, acetone, and 2-propanol extracts, and are therefore strongly suggestive for the liberation of antimutagenic aglycones from inactive glycosides. The existence of possibly a multitude of antimutagenic factors in fruits and vegetables was further substantiated by: (1) solvent partitioning of the n-hexane extracts of cauliflower, peaches, and spinach; (2) separation of the n-hexane and dichloromethane extracts of cauliflower, peaches, and spinach into acidid, neutral, and basic compounds; (3) chromatographic analysis of the n-hexane and dichloromethane extracts of spinach. Taken together, antimutagenic activities were present in 32 of 36 subfractions, corresponding to 88.9%. In the green vegetables beans, broccoli, and spinach the known antimutagen chlorophyll was proven to contribute considerably to antimutagenic potency. Other important contributions may be caused by various fibers: (I) antimutagenicity of fruit and vegetable solvent extracts was extensively heat stable; (II) heating surprisingly caused an increase of antimutagenic potencies or generated new antimutagenic activities in several solvent fractions, especially of broccoli, white and red cabbage. Indeed, mutagenicity induced by IQ was strongly reduced by lignin, weakly by alginic acid and pectin A, while cellulose, gum arabic, gum guar, and xylan were ineffective. With respect to the mechanisms of antimutagenicity binding of IQ by various fibers and inhibition of cytochrome P-450-dependent monooxygenases might be of major importance since no solvent fraction of any fruit or vegetable was able to reduce mutagenic activity induced by N-OH-IQ in S. typhimurium TA 98NR.

The influence of automobile exhausts on mutagenicity of soils: contamination with, fractionation, separation, and preliminary identification of mutagens in the Salmonella/reversion assay and effects of solvent fractions on the sister-chromatid exchanges in human lymphocyte cultures and in the in vivo mouse bone marrow micronucleus assay.

To test the assumption that automobile exhausts contribute to soil mutagenicity, two soils with low levels of mutagenic activities were exposed to traffic exhausts at a heavily charged junction of German motorways (Autobahnen) for 3, 7, 10, 13, 17, 21, and 26 weeks. Indeed, in the presence of a metabolic activation system from rat liver (S9), an average increase of 8 and 9 (4 and 12) revertants per gram per week was found in Salmonella typhimurium TA 98 (TA 100). In the absence of S9, meaningful measurements were impossible on account of a concurrent dose dependent increase of toxicity. No correlation between the increase of mutagenicity and the contents of polycyclic aromatic hydrocarbons (PAH) could be detected. In another series, soils sampled at the roadside and at distances of 10 and 50m of five roads near Mainz expressed 10-20-fold higher mutagenicity (revertants per gram) under identical test conditions as compared with the average of agricultural soils. Toxic effects, however, again confounded the results and no correlation between the distance from roads and the levels of mutagenicity could be demonstrated. Subsequently, Soxhlet-extraction with the solvent sequence dichloromethane, acetone, and toluene/diethylketone was found to be an optimum procedure for soils at roadsides. The mass balance of solvent fractionation of such soils revealed that <2% each belonged to organic acids and bases, approximately 4% to fractions designed polar neutrals, approximately 8% to polar aromatics, approximately 7% to dichloromethane solubles, and approximately 79% to cylohexane solubles, among them approximately 63% acetone soluble compounds. The major part of mutagenicity (55-65%) was present in the fraction of polar aromatics, followed by polar neutrals and the acetone subfraction of cyclohexane solubles ( approximately 10% each) summarizing the results obtained with S. typhimurium TA 98, TA 98NR, YG 1021, YG 1024, TA 100, YG 1026, and YG 1029 with and without addition of S9. The modified tester strains, either deficient in nitroreductase (TA 98NR) or overproducing nitroreductase (YG 1021, 1026) or O-acetyl-transferase (YG 1024, 1026), indicated a major contribution of nitroarenes to soil mutagenicity. With respect to mutagenic PAH, high pressure liquid chromatography (HPLC) revealed that >90% of dibenz[a,h]anthracene (4.18mg/kg soil), benzo[a]pyrene (1.96mg), benzofluoranthenes (0.14mg), and benz[a]anthracene (0. 18mg) were present in the acetone subfraction of cyclohexane solubles. Concentrations and mutagenic activities, however, did not correlate. Additional preparative and analytical HPLC of the solvent fractions of polar neutrals and polar aromatics, resulted in the tentative identification of 2-nitrofluorene. Analysis of the vertical profile of soil revealed an increase of mutagenicity per gram from the surface to a maximum at 5-15cm depth and a subsequent decrease with very little activity remaining deeper than 35cm. In human lymphocyte cultures, the fraction of polar aromatics, 0.01-0. 3microg/ml, induced 11.27+/-4.76-20.70+/-6.19 sister-chromatid exchanges (SCE) per cell in the absence of S9 (solvent control: 10. 16+/-4.83 SCE per cell) and 12.77+/-6.53-17.87+/-4.93 SCE per cell in the presence of S9 (solvent control: 8.37+/-3.92 SCE per cell). However, no activities could be detected in the fractions of polar neutrals and non-polar neutrals. Again, negative results were obtained in the in vivo mouse bone marrow micronucleus assay at 2000mg/kg p.o. with all fractions.

Antimutagenic effects of flavonoids, chalcones and structurally related compounds on the activity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and other heterocyclic amine mutagens from cooked food.

Sixty-four flavonoids were tested for their antimutagenic potencies with respect to IQ in Salmonella typhimurium TA98 and in part also towards MeIQ, MeIQx, Trp-P-2, and Glu-P-1 and in S. typhimurium TA100. Antimutagenic potencies were quantified by the inhibitory dose for 50% reduction of mutagenic activity (ID50). A carbonyl function at C-4 of the flavane nucleus seems to be essential for antimutagenicity: two flavanols and four anthocyanidines were inactive. Again, five isoflavons, except biochanin A, were inactive. Within the other groups of 21 flavones, 16 flavonols and 16 flavanones the parent compounds flavone, flavonol, and flavanone possessed the highest antimutagenic potencies (ID50: 4.1, 2.5, 5.5 nmoles). Increasing polarity by introduction of hydroxyl functions reduced antimutagenic potency. Reducing polarity of hydroxy flavonoids by methyl etherification, however, increased antimutagenic potency again. 6-Hydroxy- and 2'-hydroxy substituted flavonoids were considerably less potent antimutagens. Of 11 flavonoid glycosides tested all compounds except apigenin- and luteolin-7-glucoside (ID50:74, 115 nmoles) were inactive or only weakly antimutagenic. Rings C and A of the nucleus were not essential for antimutagenicity: chalcone and three derivatives were nearly as active as comparable flavones while antimutagenicity of benzylidenacetone was considerably reduced (ID50: 95 nmoles). Cinnamylaldehyde and cinnamoates, however, were inactive. A planar structure in the vicinity of the carbonyl group may also be important for antimutagenicity. Flavanones were less potent antimutagens than the corresponding flavones, but dihydrochalcones and 14 structurally related saturated aromatic carbonyl compounds were inactive. Fisetin and 6-hydroxyflavone were competitive inhibitors, but luteolin was a mixed type inhibitor. The inhibition mechanisms of flavone, kaempferol, morin, flavanone, and 2'-hydroxyflavanone were concentration dependent, being competitive at low concentrations and mixed or non-competitive (2'-hydroxyflavanone) at concentrations about the ID50 value. No fundamental differences between the two tester strains and no clear influence of mutagen structure on antimutagenic potency could be detected.

The inhibition by flavonoids of 2-amino-3-methylimidazo[4,5-f]quinoline metabolic activation to a mutagen: a structure-activity relationship study.

The mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Salmonella typhimurium TA98 is inhibited by flavonoids with distinct structure-antimutagenicity relationships (Edenharder, R., I. von Petersdorff I. and R. Rauscher (1993). Antimutagenic effects of flavonoids, chalcones and structurally related compounds on the activity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and other heterocyclic amine mutagens from cooked food, Mutation Res., 287, 261-274). With respect to the mechanism(s) of antimutagenicity, the following results were obtained here. (1) 7-Methoxy- and 7-ethoxyresorufin-O-dealkylase activities in rat liver microsomes, linked to cytochrome P-450-dependent 1A1 and 1A2 monooxygenases catalyzing oxidation of IQ to N-hydroxy-IQ (N-OH-IQ), were effectively inhibited by 16 flavonoids (IC50: 0.4-9.8 microM). Flavones and flavonols are in general more potent enzyme inhibitors than flavanones, isoflavones, and chalcones. Among flavones the presence of hydroxyl or methoxyl groups resulted in minor changes only. However, among flavonols and flavanones the parent compounds exerted the strongest inhibitory effects, which decreased in dependence on number and position of hydroxyl functions. Contrary to the results obtained in the Salmonella assay in the tests with alkoxyresorufins no extraordinary counteracting effects of isoflavones, of hydroxyl groups at carbons 6 or 2' or of the elimination of ring B (benzylideneacetone) were detected. (2) No effects of flavonoids on NADPH-dependent cytochrome P-450 reductase activity could be detected. (3) The effects of 30 flavonoids on mutagenicity induced by N-OH-IQ in S. typhimurium TA98NR were again structure dependent. The most striking feature was the, in principle, reverse structure-antimutagenicity pattern as compared to IQ: non-polar compounds were inactive and a 50% inhibition was achieved only by some flavones and flavonols (IC50: 15.0-148 nmol/ml top agar). Within the flavone and flavonol subgroups inhibitory effects increased in dependence on number and position of hydroxyl functions. Isoflavones and flavanones, however, as well as glycosides, were inactive. Hydroxyl groups at carbons 7, 3', 4', and 5' generated antimutagenic compounds, a hydroxyl function at C5 was ineffective, but hydroxyls at C3 and 6 as well as methoxyl groups at C3' (isorhamnetin) or 4' (diosmetin) generated comutagenic compounds. 4. Cytosolic activation of IQ to mutagenic metabolites as determined by experiments with the hepatic S105 fraction comprises about 10% of the mutagenicity after activation by the combined microsomal and cytosolic fractions (S9). The pattern of inhibition as produced by 20 flavonoids was closely similar to that observed with the S9 fraction. 5. In various experiments designed for modulation of the mutagenic response, it could be shown that further mechanisms of flavonoid interaction with the overall mutagenic process may exist, such as interactions with biological membranes (luteolin, fisetin) and effects on fixation and expression of.DNA damage (flavone, fisetin).

In vitro antimutagenic and in vivo anticlastogenic effects of carotenoids and solvent extracts from fruits and vegetables rich in carotenoids.

The water insoluble residues of some carotenoid-rich fruits and vegetables, such as apricots, oranges, brussels sprouts, carrots, yellow-red peppers, and tomatoes, were sequentially extracted with n-hexane, dichloromethane, acetone, and 2-propanol, and solvent extracted materials were tested for inhibition of mutagenicities induced by aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and cyclophosphamide (CP) in histidine-deficient strains of Salmonella typhimurium. Antimutagenic activities were found in many extracts, but especially in the n-hexane extracts. For example, in the case of oranges, 100 microg of this extract reduced the bacterial mutagenicity of AFB1, BaP, CP and IQ by 72, 67, 53, and 27%, respectively. Separation by semi-preparative HPLC of the n-hexane extracts of carrots, tomatoes, and oranges indicated that the antimutagenicity was mainly associated with the fractions of the hydrocarbon carotenoids (alpha-, beta-carotene, lycopene), the xanthophylls (beta-cryptoxanthin, lutein), and also the carotenolesters (oranges). When 16 reference carotenoids were investigated as described above, the following results were obtained: In the case of BaP, antimutagenic activity, quantified by dose-response curves, was exhibited by 8'-apo-beta-carotenal, alpha- and beta-carotene, beta-cryptoxanthin, lutein, retinal, and retinol (ID50-values: 20-100 nmol ml-1 top agar, 50-70% maximum inhibition at 1 micromol ml-1 top agar), while the maximum inhibition by torularhodin did not exceed 40%. Astaxanthin, 10'- and 12'-apo-beta-carotenal, bixin, canthaxanthin, ethyl-8'-apo-beta-caro-ten-8'-oate, lycopene, and zeaxanthin were inactive or at best marginally active (<20% inhibition). Closely similar results were obtained with AFB1. The bacterial mutagenicity of CP was strongly reduced by alpha- and beta-carotene, canthaxanthin, and retinol (ID50-values: 67-112 nmol ml-1 top agar, 50-63% maximum inhibition at 1 micromol ml-1 top agar), moderately by beta-cryptoxanthin, and lutein (45% and 28%, respectively), and only marginally or, not at all, by all remaining carotenoids. In the case of IQ, the carotenoids exhibited the weakest antimutagenic potency (7-43%, ID50-values of retinal and retinol: 160 and 189 nmol ml-1 top agar, 60% and 55% inhibition, respectively). The mutagenic activity of the proximal mutagen of IQ, N-OH-IQ, in S. typhimurium TA 98NR was not significantly reduced by any carotenoid tested. These observations as well as the inhibition of various cytochrome P-450 linked 7-alkoxyresorufin-O-dealkylase activities (EROD, MROD, PROD) by four selected carotenoids (retinol>beta-cryptoxanthin>beta-carotene>lutein, IC50-values: 19-109 microM), indicate that the inhibition of the metabolic activation of the different promutagens could cause antimutagenicity. Finally, it could be demonstrated that the number of BaP or CP induced micronuclei in polychromatic erythrocytes in bone-marrow of mice was reduced significantly by the carotenoids lycopene, canthaxanthin, lutein and beta-cryptoxanthin (25-46%). These results clearly show that carotenoids possess biological activities in vitro and in vivo distinct from their function as precursors of vitamin A or antioxidants suggesting effects on activation of promutagens.

Soil mutagens are airborne mutagens: variation of mutagenic activities induced in Salmonella typhimurium TA 98 and TA 100 by organic extracts of agricultural and forest soils in dependence on location and season.

As our hypothesis was that soil mutagens are airborne mutagens, possibly modified by soil microorganisms, we checked solvent extracts from agricultural and forest soils collected during late summer in the environment of Mainz, a region highly charged by anthropogenic air pollution, or near Bayreuth, a rural low charged region of Germany, or in a remote region of western Corsica without anthropogenic air pollution for the presence of mutagenicity in Salmonella typhimurium. Levels of mutagenic activities were quantified by calculation of revertants/g from the initial slope of dose-response curves applying tester strains S. typhimurium TA 98 and TA 100 in the absence and presence of an activation system from rat liver (S9). Three soils from Corsica did not induce mutagenicity under any test condition. However, most soils from Germany exhibited mutagenic activities, though preferentially in strain TA 98, but no statistically significant differences could be detected between 27 soils from the Mainz and nine soils from the Bayreuth regions. On the other hand, no correlation could be detected between the levels of mutagenic activities at any test condition and agricultural practice - rye growing, viniculture, fruit growing, meadow, and fallow - texture of soils - % composition of clay, slit, and sand - or the contents of organic matter. The only significant difference of mutagenicity was, however, found with S. typhimurium TA 98-S9 between forest soils of pH approximately 4.0 as compared with agricultural soils of pH approximately 7.0. The presence of antimutagens in soil as demonstrated by the course of dose-response curves of the three soils from Corsica may be another possible confounder. Calculation of mean values of mutagenic activities for all soils from Germany gave the following results: S. typhimurium TA 98: 69.7+/-153.2 (-S9); 63.0+/-176.3 (+S9); S. typhimurium TA 100:-144.7+/-399.4 (-S9); 43.3+/-172.0 (+S9) revertants/g of dry soil. In another series of experiments, soil mutagenicity in 10 rye fields near Mainz was monitored for 1 year. It became evident that low levels of mutagenic activities in late summer increased during autumn, reached a peak in late winter, and subsequently, decreased during spring and summer. These results agree with the hypothesis of an airborne origin of soil mutagens, deposition, and an adjacent transformation to non-mutagenic compounds by soil microorganisms.

Antimutagenic effects and possible mechanisms of action of vitamins and related compounds against genotoxic heterocyclic amines from cooked food.

Possible antimutagenic activity of 26 vitamins and related compounds - ascorbic acid, beta-carotene, cyanocobalamin, folic acid, nicotinic acid, nicotinamide, pantothenic acid, pyridoxale, pyridoxamine, pyridoxine, retinal, retinol, retinoic acid, retinyl acetate, retinyl palmitate, riboflavin, riboflavin 5'-phosphate, flavin adenine dinucleotide (FAD), alpha-tocopherol, alpha-tocopherol acetate, vitamins K(1), K(3), K(4), 1, 4-naphthoquinone, and coenzyme Q(10) - was tested against six heterocyclic amine (HCA) mutagens, i.e., 2-amino-3-methyl-imidazo[4, 5-f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in the Salmonella/reversion assay using tester strains Salmonella typhimurium TA 98 and TA 100. Retinol, retinal, riboflavin, riboflavin 5'-phosphate, FAD, vitamins K(1), K(3), K(4), 1, 4-naphthoquinone, and coenzyme Q(10) caused a concentration-dependent decrease in the mutagenicity of all six mutagens in both tester strains. Quantification of antimutagenic potencies by calculating ID(50)1000; vitamin K(1): 401-740; vitamin K(3) (menadione): 85-590; vitamin K(4): 45-313; 1,4-naphthoquinone: 170-290; coenzyme Q(10): 490-860. In general, there were no major differences between HCAs tested except in part with Trp-P-2 nor between the two tester strains. In enzyme kinetic experiments with Salmonella, retinol, vitamins K(3), and K(4) behaved as competitive inhibitors of IQ induced mutagenesis. However, at the highest concentration of menadione (200 nmol/plate) and of riboflavin 5'-phosphate (2000 nmol/plate), non-competitive inhibition was observed. At other concentrations of riboflavin 5'-phosphate and at all concentrations of FAD, meaningful interpretation of enzyme kinetics were not possible. Reduction of the activity of 7-ethoxy- and 7-methoxyresorufin-O-dealkylases with IC(50) values of 2.03-30.8 microM indicated strong inhibition of 1A1 and 1A2 dependent monooxygenases by menadione and retinol. Riboflavin 5'-phosphate and FAD were less effective (IC(50): 110-803.7 microM). Nicotinamide-adenine-dinucleotidephosphate (NADPH) cytochrome P-450 reductase was not affected by retinoids but stimulated by naphthoquinones and both riboflavin derivatives up to about 50 and 80%, respectively. Again, the mutagenic activity of N-hydroxy-2-amino-3-methyl-imidazo[4,5-f]quinoline (N-OH-IQ) in Salmonella was not suppressed by K-vitamins but marginally reduced by retinol, retinal, and FAD but distinctly by riboflavin 5'-phosphate. In various experiments designed for modulation of the mutagenic response, inhibition of metabolic activation of IQ to N-OH-IQ was found to be the only relevant mechanism of antimutagenesis of menadione while a weak contribution of an other way seemed possible for retinol and FAD.

Inhibition of clastogenicity of benzo[a]pyrene and of its trans-7,8-dihydrodiol in mice in vivo by fruits, vegetables, and flavonoids.

In the in vivo mouse bone marrow micronucleus assay, homogenates of spinach, artichoke, peaches, and blue grapes as well as commercial concentrates of these vegetables and fruits reduced induction of micronuclei by benzo[a]pyrene (BaP) by 43-50%. Concentrates of strawberries (31% reduction) and of cauliflower (20% reduction) were less potent. Inhibition of genotoxicity by spinach and peaches was not caused by any delay in maturation of micronucleated erythrocytes as shown by experiments with sampling times of 24, 48, and 72 h after dosing of BaP. Pre-treatment of the mice with spinach 48, 24, and 12h before application of BaP resulted in a 44% reduction of micronuclei while peaches generated only a marginal effect. A post-treatment procedure administering spinach or peaches 6h after dosing of BaP did not indicate any protective effects. When trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-OH) was applied for induction of micronuclei spinach and peaches reduced the number of micronuclei by 55 and 48%, respectively. Pre-treatment of mice with spinach 96, 72, and 60 h before sacrifice caused a decline of hepatic 7-ethoxyresorufin-O-dealkylase (EROD) and of 7-pentoxyresorufin-O-dealkylase (PROD) activities by factors of 2.2 and 1.4, respectively. However, statistical significance was not reached. On the other hand, peaches had no influence on hepatic EROD or PROD activities. The flavonoids quercetin and its glucoside isoquercitrin, administered orally in doses of 0.03 mmol/kg body weight simultaneously with intraperitoneally given BaP, reduced the number of micronuclei in polychromatic erythrocytes of the bone marrow of mice by 73 and 33%. Ten-fold higher concentrations, however, reversed the effects with a particular strong increase observed with isoquercitrin (+109%; quercetin: +16%).

Protection by beverages, fruits, vegetables, herbs, and flavonoids against genotoxicity of 2-acetylaminofluorene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in metabolically competent V79 cells.

Chinese hamster lung fibroblasts, genetically engineered for the expression of rat cytochrome P450 dependent monooxygenase 1A2 and rat sulfotransferase 1C1 (V79-rCYP1A2-rSULT1C1 cells), were utilized to check for possible protective effects of beverages of plant origin, fruits, vegetables, and spices against genotoxicity induced by 2-acetylaminofluorene (AAF) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Antigenotoxic activities of juices from spinach and red beets against AAF could be monitored with similar effectivity by the HPRT-mutagenicity test (IC(50)=0.64%; 2.57%) and alkaline single cell gel electrophoresis (comet assay; IC(50)=0.12%; 0.89%) which detects DNA strand breaks and abasic sites. Applying the comet assay, genotoxicity of PhIP could, however, be demonstrated only in the presence of hydroxyurea and 1-[beta-D-arabinofuranosyl]cytosine, known inhibitors of DNA repair synthesis. As expected, AAF and PhIP were unable to induce any genotoxic effects in the parent V79 cells. Genotoxic activity of PhIP was strongly reduced in a dose-related manner by green tea and red wine, by blueberries, blackberries, red grapes, kiwi, watermelon, parsley, and spinach, while two brands of beer, coffee, black tea, rooibos tea, morellos, black-currants, plums, red beets, broccoli (raw and cooked), and chives were somewhat less active. One brand of beer was only moderately active while white wine, bananas, white grapes, and strawberries were inactive. Similarly, genotoxicity of AAF was strongly reduced by green, black, and rooibos tea, red wine, morellos, black-currants, kiwi, watermelon, and spinach while plums, red beets, and broccoli (raw) were less potent. Broccoli cooked exerted only moderate and white wine weak antigenotoxic activity. With respect to the possible mechanism(s) of inhibition of genotoxicity, benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8-OH) and N-OH-PhIP were applied as substrates for the CYP1A family and for rSULT 1C1, respectively. Morellos, black-currants, and black tea strongly reduced the genotoxicity of BaP-7,8-OH, onions, rooibos tea, and red wine were less potent while red beets and spinach were inactive. On the other hand, red beets and spinach strongly inhibited the genotoxicity of N-OH-PhIP, rooibos tea was weakly active while all other items were inactive. These results are suggestive for enzyme inhibition as mechanism of protection by complex mixtures of plant origin. Taken together, our results demonstrate that protection by beverages, fruits, and vegetables against genotoxicity of heterocyclic aromatic amines may take place within metabolically competent mammalian cells as well as under the conditions of the Salmonella/reversion assay.

Development and application of test methods for the detection of dietary constituents which protect against heterocyclic aromatic amines.

This article describes the development and use of assay models in vitro (genotoxicity assay with genetically engineered cells and human hepatoma (HepG2) cells) and in vivo (genotoxicity and short-term carcinogenicity assays with rodents) for the identification of dietary constituents which protect against the genotoxic and carcinogenic effects of heterocyclic aromatic amines (HAs). The use of genetically engineered cells expressing enzymes responsible for the bioactivation of HAs enables the detection of dietary factors that inhibit the metabolic activation of HAs. Human derived hepatoma (HepG2) cells are sensitive towards HAs and express several enzymes [glutathione S-transferase (GST), N-acetyltransferase (NAT), sulfotransferase (SULT), UDP-glucuronosyltransferase (UDPGT), and cytochrome P450 isozymes] involved in the biotransformation of HAs. Hence these cells may reflect protective effects, which are due to inhibition of activating enzymes and/or induction of detoxifying enzymes. The SCGE assay with rodent cells has the advantage that HA-induced DNA damage can be monitored in a variety of organs which are targets for tumor induction by HAs. ACF and GST-P(+) foci constitute preneoplastic lesions that may develop into tumors. Therefore, agents that prevent the formation of these lesions may be anticarcinogens. The foci yield and the sensitivity of the system could be substantially increased by using a modified diet. The predictive value of the different in vitro and in vivo assays described here for the identification of HA-protective dietary substances relevant for humans is probably better than that of conventional in vitro test methods with enzyme homogenates. Nevertheless, the new test methods are not without shortcomings and these issues are critically discussed in the present article.

Isolation and characterization of structurally novel antimutagenic flavonoids from spinach (Spinacia oleracea).

Thirteen compounds, isolated from spinach (Spinacia oleracea), acted as antimutagens against the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in Salmonella typhimurium TA 98. The antimutagens were purified by preparative and micropreparative HPLC from a methanol/water (70:30, v/v) extract of dry spinach (commercial product) after removal of lipophilic compounds such as chlorophylls and carotenoids by solid-phase extraction (SPE). Pure active compounds were identified by instrumental analysis including FT-IR, (1)H and (13)C NMR, UV-vis spectroscopy, and mass spectrometry. All of these compounds were flavonoids and related compounds that could be attributed to five groups: (A, methylenedioxyflavonol glucuronides) 5,3'-dihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 1), 5,2',3'-trihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 2), 5-hydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 3); (B, flavonol glucuronides) 5,6,3'-trihydroxy-7,4'-dimethoxyflavonol 3-O-beta-glucuronide (compound 4), 5,6-dihydroxy-7,3',4'-trimethoxyflavonol 3-O-beta-glucuronide (compound 5); (C, flavonol disaccharides) 5,6,4'-trihydroxy-7,3'-dimethoxyflavonol 3-O-disaccharide (compound 6), 5,6,3',4'-tetrahydroxy-7-methoxyflavonol 3-O-disaccharide (compounds 7 and 8); (D, flavanones) 5,8,4'-trihydroxyflavanone (compound 9), 7,8,4'-trihydroxyflavanone (compound 10); (E, flavonoid-related compounds) compounds 11, 12, and 13 with incompletely elucidated structures. The yield of compound 1 was 0.3%, related to dry weight, whereas the yields of compounds 2-13 ranged between 0.017 and 0.069%. IC(50) values (antimutagenic potencies) of the flavonol glucuronides ranged between 24.2 and 58.2 microM, whereas the flavonol disaccharides (compounds 7 and 8), the flavanones (compounds 9 and 10), and the flavonoid-related glycosidic compounds 11-13 were only weakly active. The aglycons of compounds 7 and 8, however, were potent antimutagens (IC(50) = 10.4 and 13.0 microM, respectively).

Inhibition of the mutagenicity of 2-nitrofluorene, 3-nitrofluoranthene and 1-nitropyrene by flavonoids, coumarins, quinones and other phenolic compounds.

When 56 flavonoids, 32 coumarins, five naphthoquinones, 12 anthraquinones and five structurally-related compounds were tested for their antimutagenic potencies with respect to mutagenicities induced by 2-nitrofluorene (2-NF), 3-nitrofluoranthene (3-NFA) and 1-nitropyrene (1-NP) in Salmonella typhimurium TA98 distinct structure-activity relationships were detected. First, the tetracyclic nitroarenes 3-NFA and 1-NP were in general more effectively antagonized by potent antimutagenic flavonoids and coumarins than the tricyclic 2-NF, while there were only minor differences with quinones. Secondly, antimutagenicity of natural compounds of plant origin correlated with the aglyconic nature 10 of a total of 15 glycosides were inactive, four flavonoid glycosides exerted antimutagenicity but to a distinctly lower degree than the corresponding aglycones. Thirdly, within flavonoids, coumarins and anthraquinones positive correlations were found between antimutagenic potency and the polarity of a molecule with the existence of an optimum of activity within flavonols and anthraquinones. However, polarity seemed to be unimportant within the chalcone and naphthoquinone series. Among flavonoids, the parent compounds flavone and flavanone were inactive, but all flavones and many flavonoids with phenolic hydroxyl groups exerted antimutagenicity. Antimutagenic potency reached a maximum with the presence of four hydroxyl functions-luteolin, kaempferol-though the position of hydroxyls was also a determinant of antimutagenic potency. Methylation of phenolic hydroxyl groups, however, always reduced antimutagenicity. A carbonyl group at carbon 4 was essential for antimutagenicity: two catechins and anthocyanidins each were inactive. On the other hand, ring C of the flavane nucleus was not essential for antimutagenicity: chalcones and dihydrochalcones were potent antimutagens. Among coumarins, the parent compound showed antimutagenicity against 1-NP and 3-NFA, although dihydrocoumarin, methylcoumarins and compounds with bulky substituents were inactive. Otherwise, antimutagenic activity depended on the presence of polar hydroxyl, amino or carboxyl groups at carbons 3, 4 or 7 but was diminished by interactions of hydroxyl groups vicinal to carbon 7. Again, antimutagenic potencies were reduced by alkylation or acetylation. Among furanocoumarins xanthotoxin exerted strong and bergapten moderate antimutagenicity, while psoralen (except against 3-NFA), isopimpinellin and the furanochromanones visnagin and khellin were inactive. Among anthraquinones, the principles delineated here were valid again, resulting in potent antimutagenicity of most phenolic compounds and inactivity of anthraquinone itself. Among compounds structurally related to anthraquinones, anthrone, acridone and xanthone exerted antimutagenicity, anthrone being the most potent one, while thioxanthone and 9-fluorenone were inactive. All naphthoquinones were potent antimutagens irrespective of the presence of methyl or hydroxyl functions. Plumbagin, 2-methyl-5-hydroxynaphthoquinone, however, showed exceptional antimutagenicity.

Inhibition of the mutagenicity of 2-nitrofluorene, 3-nitrofluoranthene and 1-nitropyrene by vitamins, porphyrins and related compounds, and vegetable and fruit juices and solvent extracts.

When 21 vitamins including related compounds haemin, chlorophyllin, chlorophyll, biliverdin and bilirubin, as well as juices from five fruits and 25 vegetables and solvent extracts from the residues of fruits and vegetables were tested for their antimutagenic potencies with respect to mutagenicity induced by 2-nitrofluorene (2-NF), 3-nitrofluoranthene (3-NFA) and 1-nitropyrene(1-NP) in Salmonella typhimurium TA98 the following results were obtained. The tetracyclic nitroarenes 3-NFA and 1-NP were in general more effectively antagonized by potent antimutagenic compounds than the tricyclic 2-NF. beta-Carotene, retinol, retinal, retinoic acid, retinol palmitate, riboflavin 5'-phosphate, alpha-tocopherol, vitamins B12, C, K1 and K3 as well as biliverdin, bilirubin, chlorophyll, chlorophyllin and haemin exerted antimutagenicity against the nitroarenes cited previously. All other vitamins were inactive. While part of the juices were inactive, juices from cauliflower, carrots, chives, radishes and spinach exerted weak antimutagenic activities. However, weak to moderate co-mutagenic effects were seen with grapes, kiwi, pineapple, eggplant, celeriac, chicory greens, fennel leaves and radishes and strong effects with peppers which were not caused by the presence of growth-promoting factors. Most solvent fractions were inactive but fractions containing chlorophyll exerted antimutagenicity.

Effects of beta-carotene, retinal, riboflavin, alpha-tocopherol and vitamins C and K1 on sister-chromatid exchanges induced by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and cyclophosphamide in human lymphocyte cultures.

The vitamins and related compounds cited in the title were investigated for their abilities to modulate sister-chromatid exchanges (SCEs) induced by Trp-P-2 or cyclophosphamide (CP) in human peripheral lymphocyte cultures in the presence of an exogenous metabolizing system from rat liver. When inducer and test substances were given simultaneously, beta-carotene, retinal and alpha-tocopherol caused a dose-dependent decrease of SCE frequencies induced by Trp-P-2 and CP. Vitamin K1, however, brought about an identical effect with Trp-P-2 only, while with CP an initial decrease of SCEs was followed by a statistically significant re-increase at higher concentrations. Vitamin C was ineffective against Trp-P-2, but caused an overall increase of SCEs induced by CP. When blood cultures were preincubated with vitamins before the addition of CP or Trp-P-2, basically identical effects were observed with beta-carotene, retinal, alpha-tocopherol, vitamin K1 and vitamin C. Riboflavin decreased SCEs induced by Trp-P-2 in all treatment schedules, although statistically confirmed minima were observed in the dose-response curves, except in post-treatment experiments. On the other hand, riboflavin only reduced SCEs induced by CP when it was preincubated with lymphocytes. When vitamins were applied in a post-treatment schedule after removal of Trp-P-2 or CP, again, basically identical results against both genotoxins were observed with beta-carotene, retinal and alpha-tocopherol with vitamin K1, however, only with respect to Trp-P-2, and with vitamin C only with respect to CP. In the post-treatment schedule, vitamin K1 caused a decrease of SCE frequencies induced by CP, and vitamin C a decrease of SCEs induced by Trp-P-2.