Zinc(II) Complexes - A New Group of Non-Mutagenic Herpes Simplex Virus Inhibitors

Former studies showed that complexes of Zn(II) with picolinic and with aspartic acids, Zn(pic)2 and Zn(asp)2, are able to inhibit HSV infection in cultured cells by affecting key steps of virus replication. As these complexes are candidates for a novel class anti-HSV drugs, further studies on their mutagenicity are of particular interest. In the present paper we present data showing that Zn(pic)2 and Zn(asp)2 do not express mutagenic effect in both prokaryotic (Salmonella typhimurium) and eukaryotic (Saccharomices cerevisiae) test systems.


Introduction
Systematic studies of the relationship between chemical structure and efficacy or toxicity of metal compounds began around 1931 when Collier and Kraus described the first systematic studies of tumour-inhibiting metal complexes (1). Among metal compounds used in medicine are zinc ones: ZnSO4 is reeommeneed for the treatment of conjunctivitis, laryngitis and vagynitis, while ZnO is used for topical application against skin diseases. The anti-herpes virus activity of zinc was firstly described by Falke in 1967 (2). Added to Herpes simplex virus (HSV) infected cells, ZnSO4 acts in dose-dependent manner, i. e. concentrations of 0.05 0.1mM inhibit the synthesis of HSV proteins (3,4); 0.2mM suppressed the activity of viral DNA polymerase (3)(4)(5)(6), while 15mM decrease the infectious activity of extraeellular virions interacting with viral glyeoproteins gB and gD (7). Previously reported studies have shown that complexes of Zn(II) with picolinie and aspartie acids are able to inhibit HSV infection in cultured cells by affecting key steps of virus replication (8,9). As these complexes are candidates for a novel class anti-HSV drugs, further studies on their mutagenieity are of particular interest. In the present paper we present data showing that both Zn(pie)2 and Zn(asp)2 do not express any mutagenie effect.

Materials and Methods
The mutagenicity of Zn(asp)2 and Zn(pic)2, in concentrations of 100, 10 and ltM, previously recognised as non-toxic f6r cultured cells (8,9) was evaluated. The mutagenicity of zinc compounds was evaluated on both prokaryotic Salmonella typhimurium, strains TA100 and TA98 (10), and eukaryotic Saccharomices cerevisiae, strains PV2 and PV3 (11) systems. S. typhimurium strains are auxotrophic by his and are characterised by gene mutations in hisG and hisD loci respectively (10). The genotype of the S. cerevisiae strain PV2 is: MATa hi$7-1 ade-47,5 1y2-67 can1 R,4D1 MATa his7-1 ade-475 1ys2-68 CAN1 radl-5 The normal allele of the RAD1 gene is required for excision repair. The PV3 strain has the same genotype but is homozygous at the radl-5 allele (11). As a result of a mutation in radl-5 the susceptibility of the PV3 strain to different agents, here including metal compounds, is increased. The prototrophics obtained by reversion of the ochre mutations of his7-1 and mitotic gene conversion ]n the lys2 genes were considered. The reversion by his was evaluated using selective medium without his. The mitotic gene conversion was evaluated using selective medium without lys (11). The reversion induced by Zn-compounds in S. typhimurium was evaluated without a microsomal fraction $9 (10). Analyses of the induction 0i" the reversions and mitotic gene conversion, as well as of the lethal effect of Zn(asp)2 and Zn(pic)2 in S. cerevisiae were evaluated by Inge-Vechtomov et al. (11). Cells from the logarithmic phase of growth cultured at 30C in a liquid YPD (DIFCO) medium with aeration were pelted, washed twice in potassium phosphate buf(er (PPB) (pH 7.4) and resuspended in the same buffer until the final concentration of 10"-10 cells/ml. Mixtures of 98 % cell suspension and 2% of Zncompound in different concentrations were incubated for 4h at 30C. Compounds were removed by washing in an ice-cold buffer, pelting and resuspending cells in lml PPB. The induction of reversions, as well as the mitotic gene conversion were studied on the 5th day Herpes Implex Virus Inhibitors.
after culturing cells at 30C onto two selective solid media. The viability was evaluated on the 3rd day after culturing at 30C onto YPD solid medium. Each experiment was in triplicate and the average number of colonies was statistically evaluated (11, I2). A frequency greeter than at least 2-fold over the control background was judged as a significant (13).

Results and Discussion
The his D 305 2 mutation in the S. typhimurium strain TA98 is localised in his D gene encoding histidine dehydrogenase. This is why TA98 is useful for detecting various frameshift mutagens. The mutation in the other strain, TA100, localised in the his G-46 gene encoding the enzyme participated in the biosynthesis of histidine and is suitable for detecting mutagens causing G C base pair substitutions (14).
The fact that under the action of 1001.tM and 10 l.tM of Zn(asp)2 and Zn(pic)_ no significant differences in the number of TA98 and TA100 revertants are obtained undoubtedly show that the zinc complexes do not induce any frameshift mutation in the TA98 strain, nor base-pair substitution in the strain TA100. This is why the effect of ll.tM was not tested (table 1). In contrast, the response of S. cerevisiae cells against Zn(pic) was strain specific (table 3).
Thus, the survival rate of PV2 cells was progressively reduced when the concentrations of the complex decreased. This is well manifested by the fact that ll.tM of Zn(pic)2 significantly reduced cell viability as compared to the control. In the concentration range 100-1tM, this complex significantly suppressed the surveillance of PV3 cells as well. The effect was doseindependent and the surviving rate was similar to that obtained after influence PV3 strain with Zn(asp). In our eukaryotic experimental system, the mutagenic effect of a particular compound is manifested by the appearance of prototrophic colonies, as a result of reversion of ohre mutation in the his7-1 locus. According to Parry (13) the tested compound is recognised as a mutagen when the number, of prototrophic colonies is at least two times higher than that in the control. As shown in tables 2 and 3, the number of prototrophic colonies induced by Zn(asp): and Zn(pic)_ is not significantly different as compared to the control and show that both zinc complexes did not induce reversion in his7-1 locus. Zn(pie)2 did not induce any gene conversion in the lys2 gene of both PV2 and PV3 strains as well. However, the number of prototrophie PV3 but not of PV2 colonies after action with l l.tM Zn(asp)2 was 2.3 times higher than that in the control. To summarise, the data show that the mutagenicity of a particular zinc complex depends i) on the specific characteristics of the complex itself: Zn(pic)_ is a mutagen neither for prokaryotic, nor for eukaryotic test systems, while Zn(asp)_ expressed a weak mutagenic effect in eukaotic S. cerevisiae strain PV3 only.
ii) on the specificity of the test system. Zn(asp)2 is not a mutagen for S. typhimurium strains, nor for S. cerevisiae strain PV2. The sensitivity of the S. cerevisiae strain PV3 to IM Zn(asp)2 _is.manifested by a weak mutagenic effect, obviously due to the disturbed reparation system of this strain.