Specificity of Deoxyribonucleic Acid Intercalating Compounds

Compounds with planar triple ring systems such as acridine orange, 9-amino acridine, 9-amino-1,2,3, 4-tetrahydroacridine (tacrine), 6,9-diamino-2-ethoxyacridine lactate monohydrate (DE-acridine), 6-chloro-9-(3'-diethylamino-2'-hydroxypropylamino)-2-methoxyacridine 2 HCI (CDM-acridine), quinacrine, 6-chloro-9(4'diethylamino1'-methylbutylamino) .2-methoxy. 1, 10-diazaanthracene (CDM 1, 10-diazaanthracene), thionine, azure A, methylene blue, and pyronine Y when applied to excised pea pods were potent inducers of phenylalanine ammonia lyase or of pisatin, or of both. Compounds with an array of structural variation around the planar three-ring system were tested for their ability to induce these responses in pea tissue. In general, dimethylamino, diethylamnino, or amino substitutions at position 2 and 6 or an amino (with or without an aliphatic side chain) substitution at position 9 of the three-ring system augmented induction potential. Methyl green, methylene blue, 2, 7-diaminofluorene, nile blue, neutral red, pyrogallol red, ethidium bromide, nogalamycin, quinine, chloroquine, spermine, 8-azaguanine, gliotoxin, chromomycin A3, actinomycin D, and mitomycin C were also potent inducers. The inhibition of phenylalanine ammonia lyase induction by the application of actinomycin D (300 micrograms per milliliter) or 6-methylpurine (1 milligram per milliliter) within 1 hour after inducer application indicated that newly synthesized RNA is necessary for induction. Phenylalanine ammonia lyase induction was also inhibited by cycloheximide (150 micrograms per milliliter). Various chemicals with the potential to intercalate into DNA molecules have been used extensively to study DNA structure and mutagenic, antiviral, antimitotic, anticarcinogenic, antimalarial activity (1, 30, 37, 48). We recently reported that the genes responsible for the production of the isoflavonoid pisatin are expressed when DNA intercalating compounds 1 Scientific Paper 3474, College of Agriculture, Washington State University, Project 1844. 2 Present address: Universitat Dusseldorf, Institut Fur Allgemeine Biologie, 4000 Dusseldorf, Mettmannerstr. 16-18 West Germany. I The term "inducer" in this paper will refer to an agent which can cause an increase in extractable phenylalanine ammonia lyase and pisatin. I Abbreviation: PAL: phenylalanine ammonia lyase. such as ethidium bromide, actinomycin D, or acridine orange are administered in optimal concentrations to excised pea pods. The observation that certain intercalating compounds are potent inducers' (41) of pisatin production, has enticed us to test related compounds and further elucidate the chemical characteristics which mediate induction in this system. We have also assayed the induction of phenylalanine ammonia lyase (EC 4.3.1.5), a key enzyme in the biosynthesis of pisatin. The present report compares 9-aminoacridine, acridine orange, ethidium bromide, methyl green, thionine, crystal violet, pyronine Y, and nile blue with various other structurally related compounds as inducers of pisatin and PAL.4 MATERIALS AND METHODS Materials. Several compounds were generous gifts from the following scientists: nogalamycin, Gunther S. Fonken, Upjohn Co.; actinomycin D, Walter B. Gall, Merck Sharp and Dohme; myracil D, Erich Hirschberg, Columbia University; 5-diazouracil, Harry B. Wood, Jr., Cancer Chemotherapy National Service Center. Sources of the remaining compounds were as follows: quinine dihydrate, nile blue sulfate, pyrogallol red, 2,7-diaminofluorene, acridine, acridane, gallocyanine, tacrine, cresyl fast violet, neutral violet, 9-amino acridine HCl, CDM 1, l0-diazaanthracene, DE-acridine, CDM acridine, 10-thioxanthenone, Aldrich Chemical Co.; pyronine Y, acridine red, lacmoid, brilliant cresyl blue, crystal violet, azure A and B, thionine, Allied Chemical and Dye Corp.; methylene blue, neutral red, National Aniline and Chemical Co.; methylene green, auramine 0, Hartman-Leddon Co.; ethidium bromide, 8azaguanine, spermine, chromomycin A,, acridine orange, Calbiochem; quinacrine *HCI, Nutritional Biochemical Corp.; chloroquine, cordycepin, Sigma Chemical Co.; Michler's ketone, K and K Laboratories, Inc.; proflavine 2HCl, Mann Research Laboratories; malachite green, methyl green, basic fuchsin, fast green, Fisher Scientific Co.; brilliant green, methyl blue, Matheson Coleman and Bell Co. Methods. Experiments quantitating pisatin production and PAL activity utilized 1 g of fresh immature pea pods (1-2 cm long, harvested while still enclosed in the blossom). The general procedure, which involved splitting the pods and administering 1.5 ml of the aqueous solutions of the inducer compounds to the exposed pea endocarp, has been described in detail previously (41). Standardized methods for the isolation and quantitation of pisatin (41) and for assaying PAL activity (20) have been described. The compounds tested as inducers of pisatin or PAL were dissolved in water, and the solution was adjusted to pH 6.0 to 7.0, which approximates the pH of excised pea tissue. For 346 www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1971 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 47, 1971 SPECIFICITY OF DNA INTERCALATING COMPOUNDS