[WONNA RAHDEN-STAROŃ, HANNA CZECZOT, MAŁGORZATA PIEŃKOWSKA, TERESA SZYMCZYK-WASILUK
B A C T E R IA L M U T A G E N IC IT Y O F D IT H IO C A R B A M A T E F U N G IC ID E T H IR A M
AKTYWNOŚĆ MUTAGENNA TIURAMU W TESTACH BAKTERYJNYCH Department of Biochemistry, Medical School
02-097 Warszawa, 1 Banacha Str., Poland Head: prof. dr hab. T. Szymczyk-Wasiluk
In the present work, within a project o f re-evaluation o f authorized pesticides coordinated by P ZH (National Institute o f Hygiene) we aimed at looking for a mechanism o f induction o f chromosomal aberrations by thiram. We checked its ability to damage bacterial DNA.
INTRODUCTION
Thiram (tetram ethyl-bis-thiocarbam yl disulphide) (CAS № 137-26-8) is an im p o r tant m em ber o f the dithiocarbam ic acid derivatives extensively used as fungicide in agriculture as well as in the ru b b e r industry as accelerators and source o f sulphur. That is why it is a subject o f in tere st for genotoxic studies for m ore than tw enty years. The main achievem ent o f these studies has been the finding th a t thiram induces the developm ent o f chrom osom al aberrations in eukaryotic cells. It was initially shown to occur in m u rin e bone m arrow cells [2] and mice sperm [30] and then confirm ed by sister-chrom atide exchange in hum an lymphocytes (SC E ) [19,20,21], by bone m arrow micronucleus test (P C E ) [6] and in Chinese H am ster cell lines [17]. In in vivo systems thiram was positive in the sex-linked recessive lethal m utations and in th e w hite/w hite (w/w) som atic m u tatio n and recom bination test (S M A R T ) in Drosophila melanogaster [4,7].
Thiram is a very reactive com pound with a strong m etalbinding characteristics and capable to in teract with p ro tein SH -group [8]. It has been also shown to induce base substitutions and, at a lower degree, fram eshift in several bacterial reversion tests [6,
29] and to activate the excision repair system in S. typhim urium [29]. A lthough the above findings could explain som e genotoxic p ro p erties of thiram , they are not sufficient to suggest a m echanism of the induction of chrom osom al abnorm alities caused by this com pound.
The m ain reason of chrom osom al abnorm alities induced by chem ical com pounds ls a direct interaction o f chem icals with D N A resulting in its dam age (for review see: [18] and [24]). L ooking for th e m echanism o f th iram ’s action on chrom osom es we checked its ability to dam age bacterial D N A using several bacterial strains. A m e s te ste r
Effect o f thiram on survival o f E.coli AB1157, B H 2 0 fp g and B H 190 fpguvrA. T he bacteria w ere grown at 37°C in LB b ro th supplem ented w hen req u ired w ith 40 ng/m l kanamycyn sulphate.
T hiram o r M M S w ere a d d ed at the ap p ro p riate co ncentrations and th e cell su sp en sion was incubated for 60 m in at 37°C with agitation. A t th e end o f incubation the suspension was diluted and placed on n u trien t agar. T h e plates w ere incubated overnight at 37°C.
T he test fo r alkylation in th e 06position o f guanine was p erfo rm ed by the m ethod described by Jeggo et. al., [11, 12] with m odifications described below. All m edia and salt solutions used in the test w ere p rep a red according to Miller [16]. T h e p ro ce d u re of the test was as follows: the log-phase (А«ю = 0.2) bacterial cultures w ere sep arated from the LB m edium by centrifugation. 5 ml o f th e m inim al m edium su p p lem en ted with M63 salts and thiram (0 - 100 |ig/m l) was add ed to the pellet. H igher doses of thiram w ere toxic fo r E. c o li. In case o f m etabolic activation th e sam e concen tratio n s of thiram w ere preincubated for 20 m inutes with 100 ц1 o f S9 fraction in 0.5 ml o f S9 mix, and, subsequently, w ere d iluted to 5 ml with the m inim al m edium su p p lem en ted with M63 salts.
In both cases, after 30 m inutes of incubation, bacterial cultures w ere centrifuged, washed with 5 ml o f M9 salts, and suspended in 5 ml o f M9. Sam ples w ere diluted accordingly and placed on m edia selecting for arg+ revertants. A sm all am o u n t (1 Hg/ml) of arginine was added to the top agar. A fter 48-60 hours o f incubation at 370 С the nu m b er o f revertants p e r 107 bacterial cells was estim ated and co m p ared for AB1157 and AB1157ada3 trains. M N N G (0.5 |ig/m l) was used as a positive control.
RESULTS
In o rd e r to reveal possible dam ages introduced by thiram in D N A we used fo u r bacterial strains. W e found no effect of tested fungicide at nontoxic concen tratio n s in any o f th e above strains. T h e results ilustrating the lack o f ability o f thiram to in tro d u ce cross-links o r oxidative dam ages in S. typhim urium TA 102 strain as well as to induce the SOS system in E .c o li PQ 37 are p resen ted in Tabs I and II, respectively. In th e latter case the activity o f alkaline p hosphatase which is syntetized constitutively in the used strain was tre a te d as a control for the overall p ro tein syntesis. T hiram was found to be non deleterio u s to alkaline p hosphatase activity at the tested range o f co n cen tration (T able II ).
Figs 1A an d В show th at the sensitivity o f bacteria to thiram was not m odified in the fpg strain as com pared w ith th e wild type. In both strains a sim ilar inhibition o f survival was o b tain ed (e.g. a b o u t 70% at 420 mM thiram ). This was in c o n trast with the sensitivity to M M S w here a clear difference betw een a survival o f th e m u tan t and wild E. coli strain was revealed (eg. 1% and 45% , respectively, a t 6 m M M M S). M odifications o f purines, if they occured, could be excised by U vrA B C nucleases [18]. To exclude this possibility we em ployed in this test additionaly a double m u tan t fpg' uvrA'. H ow ever, th e survival o f fpguvrA m utant was n o t significantly decreased as com pared to th e fpg m u ta n t o r the wild- type strain ( Fig. 1A).
T he ability o f thiram for D N A m ethylation in the 06-guanine position was investi gated com paring the m utagenic activity o f thiram in th e AB1157 strain and
T a b l e I . Mutagenic evaluation of thiram in Ames test with S. typhimurium strain TA102. Określenie aktywności mutagennej tiuramu testem Amesa w szczepie S. ty phimurium TA102
Revertants expressed as x ± SD, average of 12 plates. 50 ц1 S9/plate were added NT - not tested
AB1157a<ia3strain o f E.coli. T h e results are p re se n ted in T able III. N o increase in the n u m b e r o f arg+ revertants in the АВ1157яг/яЗ strain in relation to the A B1157 strain was observed, in the presence or absence o f the S9 fraction, pointing to inability of thiram and its m etabolites to m ethylate D N A in the 06-guanine position. A s a positive control M N N G was used, which induced 12 tim es m ore arg+ rev ertan ts in A B\\51ada3 strain th an in AB1157.
DISCUSSION
S. typhim urium TA 102 strain enabled th e d etection of oxidative and cross-linking m utagens [13]. E. coli PQ 37 was used to reveal the induction o f the SO S response, a set o f functions specifically induced by a n u m b er o f types o f D N A dam age [10, 28]. T h e strain contained an uvrA m utation which abolished excision repair, a process essentially active on lesions affecting D N A conform ation [25]. E. c o lifp g and fp g uvrA m u tan ts allow ed to check o r a possibible effect on m odification o f purines. T h e Fpg p ro te in o f E.coli was initially identified as a D N A glycosylase which excided the im idasole ring-opened form of N -m ethylguanine residues in D N A (Fapy) [3]. T he biological im p o rtan ce o f Fapy-D N A glycosylase activities is sugested by the fact th at the enzym e has been conserved in both prokaryotes [5] and eukaryotes [14]. T h e Fpg pro tein excises im idasole ring-opend purines and nicks D N A at apurinic/apirym idynic sites [4]. E. coli A B1157 and A B1157at/a3 non-dam aged and dam aged function o f ada gene, respectively, allow to detect D N A alkylation. This test is based on th e ability of bacterial cells to th e „ad ap tatio n reactio n ” caused by agents. Low doses o f alkylating
T a b l e I I . Effect of thiram and its metabolites on the SOS system induction in PQ 37 strain of E. coli K-12
Wpływ tiuramu i jego metabolitów na indukcję systemu SOS w szczepie E. coli K-12 PQ 37
Up - international units of p-galactosidase activity UP - international units of alkaline phosphatase activity
IF - induction factor is the ratio of the activities, p-galactosidase/alkaline phosphatase, di vided by its value at concentration О of the compound tested.
Each value is an average from 5 independent experiments SD. NT-not tested.
agents induce th e synthesis o f 06-m ethylguanine-D N A m ethyltransferase coded by the ada gene [27]. T his enzym e transfers alkyl residues from 06-guanine o n to its own protein repairing in this way the m utagenic dam age o f D N A and reducing the n u m b er of m utations induced by alkylating agents. W e found no effect o f thiram at nontoxic concentrations in any o f the above strains.
The results o f this w ork exclude D N A cross-links, oxidative dam age o f D N A , opening of im idazole ring in purines and events leading to activation o f the SOS system as well as adaptive response as direct reasons o f chrom osom al abnorm alities induced by thiram .
T hiram -induced dam ages in D N A have been revealed as the activity o f the excision repair system in S. typhim urium [29]. Such activity can reflect various changes in D N A introduced by the tested agent (for review see: [26]). Rannug and R annug [23] have suggested th a t genotoxicity o f thiram could be caused by its ability to induce oxidative damages. O u r results do not confirm this suggestion, in agreem en t with th e observation of Crebelli et al. [6], who did n o t identified thiram m utagen o f oxidative type. O u r results excludes a covalent reactio n of thiram with D N A which could be the m ost serious reaso n o f chrom osom e aberrations, if occurred. This type o f D N A dam age
T a b l e I I I . Effect of thiram and its metabolites on arg+ mutation induction in AB1157 and AB5557ada3 strains of E. coli K-12
Wpływ tiuramu i jego metabolitów na indukcję mutacji arg+ w szczepach E. coli K-12 AB1157 i AB5557ada3
a - average of 12 plates b - 100 ц1 S9 per probe (5ml) NT-not tested
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