Effect of Lathyrogenic Aminonitriles, Related Amines, and Copper-Complexing Agents on Conidial Germination of Molds.

Abstract

Dasler et aZ( 1,2) have postulated that diverse chemical substances inducing spinal curvatures and other lathyritic symptoms in experimental animals may exert their effects by means of formation of coordination complexes or chelates. Mager et aZ(3) have reported that low levels of copper ions are necessary for the toxic action of aminoacetonitrile on fibroblasts in vitro. The well-known medial lesions in aortas of lathyritic animals bear a marked resemblance to those produced by copper deficiency in chicks(4) or pigs (5).Common molds are reported to be affected by trace elements and ion-complexing agents. Aspergillus niger requires Cu+ + for sporulation (6). The germinations of Neurospora tetrasperma conidia and activated ascospores are inhibited by ethylenediaminetetracetic acid (0.01 M ) , and at least partial reversal of these inhibitions can be obtained with Cu+ + ( 7 ) . We have found conidial germination of A . niger to be inhibited by Betaaminopropionitrile (BAPN) (8). Therefore, a comparison of the effect of aminonitriles and Cu++-complexing agents on spore germination was undertaken using A . niger and N . crassa, a species closely related to N . t etrasperma.Materials. A . niger (strain AN-6275) and N . crassa (strains NC-11943, histidine requiring; NC-10334, proline requiring; NC-9279, wild type) were obtained from American Type Culture Collection. BAPN acid fumarate (BAPN Fu) was obtained from Abbott Laboratories; BAPN free base from Calbiochem ; propioni trile, e thylenediamine dihydrochloride and ethylamine hydrochloride from Distillation Products, Inc. (Eastman) ; Dglucosamine hydrochloride, L-histidine monohydrochloride, and L-proline from Nutritional Biochemicals Corp. ; imidazole from General Biochemicals ; a,d-dipyridyl, 8-hy-* This study was supported by research grants from Nat. Inst. of Arthritis and Metab. D'is., U.S.P.H.S. droxyquinoline, reagent grade inorganic salts, and sucrose from Fisher Scientific Co. Aminoacetonitrile hydrochloride (AAN) was synthesized in our laboratory.Methods. Molds were grown on Neurospora culture agar (Difco 0321-15) used at 2/3 strength. I t was supplemented with 0.005 M histidine or 0.02 M proline when growing NC-11943 or NC-10334, respectively. Deionized water, made by passing distilled water thru a Crystalab water demineralizer, was used for preparation of all solutions.A . niger spores were obtained from an agar slant culture grown at 26"Ct for 2 9 to 3 days. They were harvested by suspension in potassium phosphate buffer a t concentrations ranging from 0.025 M to 0.1 M. The spore suspensions were dispersed by means of a Vortex Jr. mixer and diluted with buffer to an optical density of 0.5 at 560 mp (Coleman Universal Spectrophotometer) . After a further 1 : 20 dilution with spore germination media, the suspensions were inoculated onto slides (0.1 ml of suspension per slide). The slides and cover slips had been previously washed with detergent, soaked in 0.6 N HC1 for a t least a day, and rinsed 4 times with deionized water. N . crassa spores were treated...