Trichothecene resistance in wheat: Development of molecular markers for PDR-type ABC transporter genes.

Infection withFusarium graminearum andF. culmorum not only causes severe yield and quality losses, the most relevant concern is the contamination of cereal foods and feeds with trichothecenes (e.g. deoxynivalenol, DON). The ability to synthesize trichothecenes has been shown to be a virulence factor ofF. graminearum on wheat and, on the other hand, toxin resistance is most likely an important component of field resistance. Our hypothesis is that pleiotropic drug resistance mediated by PDR-type ABC transporter proteins (acting as membrane located drug efflux pumps) is a relevant mechanism of DON resistance not only in yeast but also in wheat. Goal of this project is the development of molecular markers for this gene family for use in marker-assisted plant breeding programs. The technical difficulties caused by the large size of the PDR-family are discussed.

Protective effects of fruits and vegetables against in vivo clastogenicity of cyclosphosphamide or benzo[a]pyrene in mice.

Seven fruits and 10 vegetables commonly consumed in Germany were investigated for their anticlastogenic potencies against cyclophosphamide (CP) and benzo[a]pyrene (BaP) in the in vivo mouse bone marrow micronucleus assay. We detected protective effects in 76.5% and 70.6% of the samples, respectively, and more or less distinct quantitative differences between the various plant materials and the two clastogens investigated. With respect to CP, moderate activities were exerted by sweet cherries, strawberries, cucumber, radish and tomatoes, average activities by bananas, oranges, peaches, asparagus and red beets and strong activities by yellow red peppers and especially spinach. Apples (cultivar Jona Gold), brussels sprouts, cauliflower and onions were inactive. With respect to BaP, we found moderate activities in strawberries, brussels sprouts and radish, average activities in sweet cherries, oranges, peaches, asparagus, red beets, cucumber and spinach and strong activities in bananas and kiwi. Apples, cauliflower, onions, tomatoes and yellow-red peppers were inactive. When oranges were fractionated according to previously described schemes (Edenharder et al., 1995), anticlastogenic activities against CP were exerted by materials extracted with n-hexane, acetone and 2-propanol and in the terminal residue, but not in the dichloromethane and water phases. With respect to BaP, materials extracted with acetone showed strong anticlastogenicity while the 2-propanol fraction, the aqueous phase and the terminal residue were less potent. The n-hexane and the dichloromethane fractions were inactive. In red beets, all fractions showed anticlastogenicity against CP and BaP as well. However, the n-hexane and dichloromethane fractions were most potent with respect to CP, while for BaP the aqueous phase and the terminal residue were most effective. These result suggest the presence of various (groups of) anticlastogenic compounds with different chemical structure.

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.

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).

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.

Electron diffraction studies of the peptidoglycan of bacterial cell walls.

Peptidoglycan of the gram-positive bacterium Lactobacillus plantarum was adsorbed onto hydrophilic crystalline films of graphitic oxide. Destruction by radiation damage was reduced by using a device which permitted scanning of the specimen for several hours through a focussed electron beam of low current density (2.5 X 10(-5) A/cm2). In additition to the sharp Debye-Scherrer rings caused by graphitic oxide, peptidoglycan causes a diffuse Debye-Scherrer ring in the region of 4.5 A. This can be interpreted as the packing periodicity of the peptidoglycan within the planes of the peptidoglycan sacculi.