The bifunctional aldehydes glyoxal and malondialdehyde, known to be toxic products of the radiolysis of sugar and other food constituents, interact with DNA in vitro and in vivo as demonstrated by thermal denaturation profiles, chromatographic behavior, and incomplete degradation of the reaction product by deoxyribonuclease. After irradiation of DNA in diluted radioactive glucose solution followed by incubation a t slightly acid pH, the radioactivity is found in the core of the enzymic digest only. Attempts to locate the binding site proved that the radiolvtic compound is associated preferentially with the guanine and cytidine moiety of the DNA \ore.As previously reported [l], the toxic effect of glyoxal on human fibroblasts is caased by a blocking of DNA replication and protein synthesis. Perhaps more important than glyoxal, which is formed during irradiation in small quantities only, is the next higher homologue, malondialdehyde. This compound is associated with oxidative deterioration of food lipids and is found in the oxidation products of unsaturated fatty acid esters and squalene [2,3]. It is also produced in relatively high amounts during the irradiation of glucose, glycerine, and glutamic acid in aqueous solution [4-51. Both dialdehydes react with protein, presumably with the s-amino lysine and N-terminal amino aspartic acid groups [6,7]. To elucidate the possible mode of action on DNA, experiments were undertaken in vivo and in vitro with glyoxal, malondialdehyde, and with glucose solutions irradiated under conditions leading to the formation of these compounds. The results support the hypothesis of direct interaction. MATERIAL AND METHODSGlyoxal was a 300/, (w/v) aqueous solution of the trimer (Merck) ; malondialdehyde was prepared from the bis-diacetal (Fluka) by shaking it with an excess of an aqueous suspension of Dowex 50 ( x 2 ) in the Hf form. After appropriate dilution, samples were stored at pH 5 in a frozen state.Highly polymerized DNA (calf thymus) was obtained from Worthington, deoxyribonucleotides from Calbiochem. DNA was denatured by heating its solution in dilute standard salt-citrate solution in a bath of boiling water for 15 minutes and pouring Enzymes. Deoxyribonuelease (EC 3.1.4.5) ; ribonuclease (EC 2.7.7.16); snake venom phosphodiesterase (EC 3.1.4.1).Definition. Dilute standard salt-eitrate solution = 16 mM sodium chloride -1.5 mM trisodium citrate, pH 6.8.into an equal volume of dilute standard salt-citrate solution frozen a t -20". Deoxyribonuclease and ribonuclease were products of the Worthington Laboratories ; snake venom phosphodiesterase was obtained from Calbiochem. Labelled compounds were supplied by New England Nuclear.Sephadex G-25 (fine) wss a product of Pharmacia (Uppsala), and Gas Chrom P was donated by Serva Laboratories.Human fibroblasts (Institute of Histology, University of Vienna) were routinely cultivated as monolayers to a density of 2.6-3 x 106.The technique is described elsewhere [l]. To determine the influence of malondialdehyde on cell number, DNA and prote...

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Tertiare Mannich-Basen des Pyrrols (I) setzen sich mit 9cylamino-malonestern (11) unter Abspaltung von Amin und Verkniipfung der beiden Ileste um. Ein Austausch der Dialkylamino-methyl-Oruppe der Mannich-Baae gegen das reaktionsfahige H-.4tom des Esters wird nicht beobachtet. Austausch-Reaktion tritt jedoch regel-mkBig bei der in zwei Stufon verlaufenden Kondensation von Pyrrol mit tertiaren Munnich-Basen, Acylamino-malonestern (V), ein. Die hierbei primar entstehende Mannich-Base dea Pyrroh (I) kondensiert sich in zweiter Stufe mit dem gebildeten Acylamino-malonester (11) in der angegebenen Weise. Auf die Wahrschcinlichkcit eines analogen Reaktionsverlaufes bei der Tryptophan-Synthese aus Indol und Piperidinomethyl-formamino-malonester wird hingewiesen. Im Zusammenhang mit der Synthese des Pyrrylalanins, iiber die anderen Ortes bcrichtet werden soll, haben wir Kondensations-Reaktionen tertiarer Mannich-Basen in der Pyrrol-Reihe untersucht. Wir sind dabei u. a. zu Ergebnissen gekommen, die in Anbetracht der Analogic von Indol und Pyrrol Ruckschliisse auf den bisher nicht geklarten Reaktionsverlauf einer von A. B u t e n a n d t , H . H e l l m a n n und E. Renz') aufgefundenen Kondensation zwischen Indol und tertiiiren Esterbasen gestatten durfte.Wie bereits W. H e r z , I<. D i t t m e r und S. Cristo12) beschricben haben, IaiBt sich die in der Indol-Iteihe zur Synthese des Tryptophans3) angewandte Kondensations-Reaktion zwischen tertiaren Mannich-Basen des Indols und ~

It is now generally accepted that the integrity and biological function of DNA is reflected in its capacity to act as a template in the DNA-dependent RNA polymerase (E.C.2.7.7.6) system [ 1, 21. Using this system, Ono et al.[l] investigated the effects of ultraviolet light, and Phillips et al. [3] those of hydroxylamine on the template properties of polycytidine. As previously reported [4, 51 , the bifunctional aldehydes glyoxal and malondialdehyde (MDA), both formed during the radiolysis of aqueous sugar solutions and glutamic acid [6, 71, strongly inhibit the growth of cells in culture by blocking DNA replication in particular. Moreover, mutagenic effects of irradiated sucrose solutions were observed in cells by Shaw and Hayes [8], presumably attributable to the dialdehydes formed. It has been further demonstrated that MDA interacts with the guanine and cytidine bases of the DNA in vitro, preventing total DNA degradation by DNase due to incomplete strand separation of the DNA helix [9] . As the RNA polymerase seems to read the template DNA accurately and sequentially [lo] , it could be assumed that DNA, after treatment with MDA, would show altered template activity in this system.Therefore, experiments were performed with rats to investigate the template activity of liver DNA after *

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