Author/Editor     Jenko-Brinovec, Špela; Plaper, Andreja; Zalar, Stane
Title     Introduction of in vitro toxicological methods in the pharmaceutical factory Krka
Translated title     Uvajanje in vitro toksikoloških metod v tovarni zdravil Krka
Type     članek
Source     Farm Vestn
Vol. and No.     Letnik 51, št. 3
Publication year     2000
Volume     str. 459-66
Language     eng
Abstract     Two in vitro tests for toxicological studies were introduced into and compared by our company: Ames test (AT), which is a classical in vitro mutagenicity method, and Stress Promoters Induction Test, which is a new bacterial assay that was employed to improve the understanding of the toxicity mechanisms of tested compounds. In the latter test, the regulatory sequences of several stress genes are placed in front of the lac Z structural reporter gene whose product B-D-galactosidase can be readily measured. Mutagenicity and toxicity of a new nonsedating antihistamine that had previously shown moderate toxicity in animal studies were examined with both tests. None of the tested concentrations was mutagenic, but the antihistamine was toxic to Salmonella typhimurium tester strains in concentrations above 0.83 mM (1mg/plate). The mechanism of antihistamine toxicity was determined with the Stress Promoters Induction Test. On the basis of transcription from micF, osmY, dinD and soi28 promoters, it was presumed that the antihistamine acts on DNA topology. This was later confirmed with an in vitro test on gyrase activity. The obtained results met our expectations about mechanistic potential of the new method that is based on measuring stress response events in organisms.
Summary     V Krki smo uvedli dva in vitro toksikološka Test Indukcije Stresnih Promotorjev (TISP), ki omogoča ugotavljanje mehanizmov toksičnosti/mutagenosti testiranih spojin. TISP vključuje seve Escherichia coli s kromosomskimi fuzijami različnih stresnih promotorjev z genom lacZ. Ob indukciji stresnega promotorja nastaja B-galaktozidaza, katere aktivnost merimo s fotometrom. Z AT in TISP smo preverili mutagenost in toksičnost novega antihistaminika, ki je v predhodnih toksikoloških študijah na živalih pokazal zmerno toksičnost. V AT antihistaminik ni deloval mutageno, pač pa je bil v koncentracijah nad 0,83 mM (1 mg/ploščo) toksičen za testne seve S. typhimurium. Mehanizem toksičnega delovanja antihistaminika smo ugotovili s TISP. Na osnovi povečanega prepisa gena lacZ od promotorjev micF, osmY, dinD in soi28 smo predpostavili, da testirana učinkovina vpliva na topologijo DNA. Predpostavko smo potrdili s testiranjem girazne aktivnosti in vitro. Dobljeni rezultati so potrdili naša pričakovanja, da metoda, ki temelji na merjenju stresnega odgovora v organizmih, omogoča razumevanje mehanizmov toksičnega delovanja kemikalij.
Descriptors     TOXICITY TESTS
SALMONELLA TYPHIMURIUM
MUTAGENICITY TESTS
HISTAMINE H1 ANTAGONISTS
ESCHERICHIA COLI
MUTAGENS
CLONING, MOLECULAR
BETA-GALACTOSIDASE
DNA TOPOISOMERASE (ATP-HYDROLYSING)