Kvalitativna procjena eliminacije TCP-a i TAMORF-a iz organizma štakora metodom GC-MS

Nerve agents are highly toxic organophosphorus (OP) compounds. They inhibit acetylcholinesterase (AChE), an enzyme that hydrolyses acetycholine (ACh) in the nervous system. Pathophysiological changes caused by OP poisonings are primarily the consequence of surplus ACh on cholinergic receptors and in the central nervous system. Standard treatment of OP poisoning includes combined administration of carbamates, atropine, oximes and anticonvulsants. In order to improve therapy, new compounds have been synthesised and tested. Tenocyclidine (TCP) and its adamantane derivative 1-[2-(2-thienyl)-2-adamantyl] morpholine (TAMORF) have shown interesting properties against soman poisoning. In this study, we developed a qualitative GC-MS method to measure elimination of TCP and TAMORF through rat urine in order to learn more about the mechanisms through which TCP protects an organism from OP poisoning and to determine the duration of this protective effect. GC-MS showed that six hours after treatment with TCP, rat urine contained only its metabolite 1-thienylcyclohexene, while urine of rats treated with TAMORF contained both TAMORF and its metabolites.Živčani bojni otrovi po strukturi su organofosforni (OP) spojevi, čija je zajednička značajka ireverzibilna inhibicija acetilkolinesteraze (AChE), enzima koji hidrolizira acetilkolin (ACh) u živčanom sustavu. Patofi ziološka zbivanja koja nastaju pri otrovanju OP-spojevima primarno su posljedica akumuliranog ACh na kolinergičkim receptorima i u središnjem živčanom sustavu. Još uvijek nesavršen, standardni tretman liječenja otrovanja OP-spojevima uključuje kombiniranu primjenu estera karbamata, atropina, oksima i antikonvulziva. Kako bi se unaprijedila uobičajena terapija, osobito kod otrovanja somanom, ispituju se antidotski učinci mnogih spojeva. Tenociklidin (TCP) i njegov adamantanski derivat TAMORF pokazali su zanimljiva svojstva pomoćne terapije pri otrovanju somanom. Kako bi se proširile dosadašnje spoznaje o načinu na koji tenociklidini štite organizam od trovanja OP-spojevima te također o trajanju njihova antidotskog učinka, u ovom radu razvijena je GC-MS-metoda za praćenje eliminacije TCP-a i TAMORF-a iz organizma. Rezultati GC-MS-analize pokazali su da šest sati nakon tretiranja štakora TCP-om mokraće sadržavaju metabolit TCP-a 1-tienilcikloheksen, dok šest sati nakon tretiranja štakora TAMORF-om mokraće sadržavaju i TAMORF i njegove metabolite. Drugim riječima, šest sati nakon tretmana TCP se potpuno metabolizira, dok se TAMORF metabolizira djelomično, a djelomično ostaje nepromijenjen

Odnos strukture i aktivnosti u reaktivaciji tabunom fosforilirane ljudske acetilkolinesteraze bispiridinijevim para-aldoksimima

We investigated interactions of bispyridinium para-aldoximes N,N’-(propano)bis(4-hydroxyiminomethyl) pyridinium bromide (TMB-4), N,N’-(ethano)bis(4-hydroxyiminomethyl)pyridinium methanosulphonate (DMB-4), and N,N’-(methano)bis(4-hydroxyiminomethyl)pyridinium chloride (MMB-4) with human erythrocyte acetylcholinesterase phosphorylated by tabun. We analysed aldoxime conformations to determine the flexibility of aldoxime as an important feature for binding to the acetylcholinesterase active site. Tabun-inhibited human erythrocyte acetylcholinesterase was completely reactivated only by the most flexible bispyridinium aldoxime - TMB-4 with a propylene chain between two rings. Shorter linkers than propylene (methylene or ethylene) as in MMB-4 and DMB-4 did not allow appropriate orientation in the active site, and MMB-4 and DMB-4 were not efficient reactivators of tabun-phosphorylated acetylcholinesterase. Since aldoximes are also reversible inhibitors of native acetylcholinesterase, we determined dissociation constants and their protective index against acetylcholinesterase inactivation by tabun.Proučavali smo interakcije bispiridinijevih para-oksima N,N’-(propano)bis(4-hidroksiiminometil)piridinijeva bromida (TMB-4), N,N’-(etanano)bis(4-hidroksiiminometil)piridinijeva metanosulfonata (DMB-4) i N,N’- (metano)bis(4-hidroksiiminometil)piridinijeva klorida (MMB-4) s ljudskom eritrocitnom acetilkolinesterazom fosforiliranom tabunom. Da bismo odredili fleksibilnosti aldoksima, što je važna osobina kod njihova vezanja u aktivno mjesto acetilkolinesteraze, analizirali smo i konformacijske odlike aldoksima. Ljudska acetilkolinesteraza inhibirana tabunom bila je potpuno reaktivirana samo najfleksibilnijim bispiridinijevim aldoksimom – TMB-4. Aldoksimi MMB-4 i DMB-4 nisu bili efikasni reaktivatori acetilkolinesteraze fosforilirane tabunom jer je kod tih spojeva lanac koji povezuje dva prstena kraći od propilena (metilen u MMB-4 i etilen u DMB-4), što ne dopušta povoljnu orijentaciju tih aldoksima unutar aktivnog mjesta enzima. S obzirom na to da su aldoksimi i reverzibilni inhibitori nativne acetilkolinesteraze, odredili smo njihove disocijacijske konstante, kao i zaštitu acetilkolinesteraze od inhibiranja tabunom reverzibilnim vezanjem aldoksima

Mehanizam toksičnosti i detoksikacije organofosfornih spojeva s naglaskom na istraživanja u Hrvatskoj

This review comprises studies on the mechanisms of toxicity and detoxication of organophosphorus (OP) compounds done in Croatia in different research areas. One area is the synthesis of antidotes against OP poisoning and their in vivo testing in experimental animals. In vitro studies included in this review focus on the mechanisms of reversible inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), protection of cholinesterases from inhibition by OPs, and reactivation of phosphylated cholinesterases. The third area comprises distribution profiles of BChE and paraoxonase (PON) phenotypes in selected population groups and the detection of OPs and metabolites in humans. Finally, methods are described for the detection of OP compounds in human blood and other media by means of cholinesterase inhibitionPrikazana su istraživanja vođena u Hrvatskoj na različitim područjima mehanizma toksičnosti i detoksikacije organofosfornih (OP) spojeva. Jedno je područje sinteza antidota protiv otrovanja OP spojevima i testiranje in vivo antidota na eksperimentalnim životinjama. Istraživanja in vitro odnose se na mehanizam reverzibilne inhibicije acetilkolinesteraze (AChE) i buturilkolinesteraze (BChE), zaštitu kolinesteraza od inhibicije OP spojevima te reaktivaciju fosfiliranih kolinesteraza. Treće je područje distribucija fenotipova BChE i paraoksonaze (PON) u odabranim populacijama te detekcija OP spojeva i njihovih metabolita u ljudima. Na kraju su opisane metode detekcije OP spojeva u ljudskoj krvi i drugim medijima koje se osnivaju na inhibiciji kolinesteraza

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells