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The aim of this study was to investigate the oxidative and apoptotic potential of fluoxetine, a widely used antidepressant in Turkey and the world, and of its metabolite norfluoxetine on a model non-target organism, Daphnia magna to see how exposure to this group of antidepressants (specific serotonin reuptake inhibitors) could affect the aquatic environment in which they end up. Juvenile D. magna specimens were chronically exposed to fluoxetine and norfluoxetine alone and in combination at concentrations found in the aquatic environment (0.091 and 0.011 μg/L, respectively) and to their 10-fold environmental concentrations for 21 days. Another group of 17-day-old animals were subacutely exposed to 100-fold environmental concentrations for four days. After exposure, we measured their glutathione peroxidase (GPx) and cholinesterase (ChE) activities, thiobarbituric acid-reactive substances (TBARS), and total protein content spectrophotometrically, while mitochondrial membrane potential (MMP) was analysed by fluorescence staining, and cytochrome c and ERK1/2 protein content by Western blotting. This is the first-time cytochrome c and ERK1/2 were determined at the protein level in D. magna. We also measured their carapace length, width, and caudal spine length microscopically. At environmental concentrations fluoxetine and norfluoxetine caused an increase in ChE activity and brood production. They also caused a decrease in juvenile carapace length, width, and caudal spine length and depolarised the mitochondrial membrane. At 10-fold environmental concentrations, GPx activity, lipid peroxidation levels, cytochrome c, and ERK1/2 protein levels rose. The most pronounced effect was observed in D. magna exposed to norfluoxetine. Norfluoxetine also decreased brood production. Similar effects were observed with subacute exposure to 100-fold environmental concentrations. However, total protein content decreased. All this confirms that fluoxetine and norfluoxetine have oxidative and apoptotic potential in D. magna. Daphnia spp. have a great potential to give us precious insight into the mechanisms of environmental toxicants, but there is still a long way to go before they are clarified in these organisms.

Do danas su provedena mnoga istraživanja usmjerena ka inhibiciji enzima kolinesteraza. Među ranijim primjerima je istraživanje benzilaminskih oksazola koje uključuje njihovu sintezu, izolaciju i karakterizaciju. Ovaj rad svojevrsni je nastavak takvih istraživanja, a provedena je sinteza novih amino derivata tiostilbena. Početni spoj 1 sintetiziran je Wittgovom reakcijom, a daljnje aminacije provedene su Buchwald-Hartwigovom aminacijom. Korišteni su benzilni amini koji su u položaju 3 supstituirani metilnom i metoksi-skupinom, te fluorom i heterociklički furanski i tiofenski amin. Svi su spojevi (2, 3, 4, 5, 6) sintetizirani najprije uz BrettPhos kao ligand, a kasnije su sve reakcije ponovljene s ligandom XPhosom, osim one s metilnim supstituentom čiji su prvotni rezultati bili zadovoljavajući. Svi su spojevi okarakterizirani 1H NMR metodom te je u svim dobivenim uzorcima detektirana očekivana masa masenom spektrometrijom (MS). Ukupna iskorištenja su određena na temelju 1H NMR spektara. To date, many studies have been conducted aimed at inhibiting the enzyme cholinesterases. One of earlier examples is the study of benzylamine oxazoles which includes their synthesis, isolation and characterization. This work is a continuation of such research, and the synthesis of new amino derivatives of thiostilbene deivatives was performed. Initial compound 1 was synthesized by the Wittg reaction, and further aminations were performed by Buchwald-Hartwig amination. Benzyl amines substituted in position 3 by methyl and methoxy group and fluorine. Also heterocyclic furan and thiophene amines were used. All compounds (2, 3, 4, 5, 6) were synthesizedat first with BrettPhos as a ligand, and later all reactions were repeated with the XPhos ligand, except that with a methyl substituent whose initial results were satisfactory. All compounds were characterized by 1H NMR spectroscopy and expected masses were also de-tected my mass spectrometry (MS) in all samples. Overall yields were estimated based on 1H NMR spectra.

Earlier research has evidenced the oxidative and neurotoxic potential of imidacloprid, a neonicotinoid insecticide, in different animal species. The primary aim of this study was to determine how metabolic modulators piperonyl butoxide and menadione affect imidacloprid’s adverse action in the liver and kidney of Sprague-Dawley rats of both sexes. The animals were exposed to imidacloprid alone (170 mg kg-1) or in combination with piperonyl butoxide (100 mg kg-1) or menadione (25 mg kg-1) for 12 and 24 h. Their liver and kidney homogenates were analysed spectrophotometrically for glutathione peroxidase, glutathione S-transferase, catalase, total cholinesterase specific activities, total glutathione, total protein content, and lipid peroxidation levels. Imidacloprid displayed its prooxidative and neurotoxic effects predominantly in the kidney of male rats after 24 h of exposure. Our findings suggest that the observed differences in prooxidative and neurotoxic potential of imidacloprid could be related to differences in its metabolism between the sexes. Co-exposure (90-min pre-treatment) with piperonyl butoxide or menadione revealed tissue-specific effect of imidacloprid on total cholinesterase activity. Increased cholinesterase activity in the kidney could be an adaptive response to imidacloprid-induced oxidative stress. In the male rat liver, co-exposure with piperonyl butoxide or menadione exacerbated imidacloprid toxicity. In female rats, imidacloprid+menadione co-exposure caused prooxidative effects, while no such effects were observed with imidacloprid alone or menadione alone. In conclusion, sex-, tissue-, and duration-specific effects of imidacloprid are remarkable points in its toxicity., Rezultati ranijih istraživanja pokazali su oksidativni i neurotoksični potencijal imidakloprida, neonikotinoidnog insekticida, u različitih životinjskih vrsta. Primarni je cilj ovog istraživanja bio utvrditi kako modulatori metabolizma piperonil butoksid i menadion utječu na nepovoljne učinke imidakloprida na jetra i bubrege muških i ženskih štakora soja Sprague-Dawley. Životinje su 12 h odnosno 24 h bile izložene samo imidaklopridu (170 mg kg-1) ili njegovoj kombinaciji s piperonil butoksidom (100 mg kg-1) odnosno menadionom (25 mg kg-1). U homogenatima jetara i bubrega spektrofotometrijski su utvrđene razine glutation peroksidaze, glutation S-transferaze, katalaze, specifične aktivnosti ukupne kolinesteraze, ukupni glutation, ukupni proteini te razine lipidne peroksidacije. Imidakloprid se pokazao prooksidativnim i neurotoksičnim uglavnom u bubrezima muških štakora nakon 24-satne izloženosti. Naši rezultati upućuju na to da su razlike u prooksidativnom i neurotoksičnom djelovanju imidakloprida povezane sa spolnim razlikama. Predtretmanom piperonil butoksidom odnosno menadionom (90 min prije davanja imidakloprida) otkriveno je da imidakloprid djeluje na ukupnu aktivnost kolinesteraze specifično za pojedina tkiva. Povišena aktivnost kolinesteraza u bubrezima mogla bi odražavati prilagodbu na oksidativni stres uzrokovan imidaklopridom. Piperonil butoksid odnosno menadion u jetrima muških štakora samo su pogoršali toksičnost imidakloprida. U ženki je djelovanje imidakloprida s menadionom bilo prooksidativno; takvo se djelovanje nije vidjelo nakon primjene samo imidakloprida odnosno samo menadiona. Vjerujemo da je promjenjivo djelovanje imidakloprida s obzirom na spol, tkivo i trajanje izloženosti važno za daljnja istraživanja njegove toksičnosti.

Alzheimerova bolest je najučestaliji oblik demencije u svijetu. Demencija je karakterizirana višestrukim kognitivnim deficitima koji uključuju gubitak pamćenja. Trenutno su inhibitori kolinesteraza još uvijek najbolja farmakoterapija za bolesnike s Alzheimerovom bolešću. Upravo zbog toga, neophodne su učinkovite i brze metode za provjeru potencijalnih inhibitora. Damašćanska crnjika (Nigella damascena L.) i crni kumin (Nigella sativa L.) su vrste biljka iz porodice Ranunculaceae. U ovom radu vršilo se određivanje sposobnosti inhibicije eteričnih ulja i njihovih komponenti ovih dviju vrsta na djelovanje kolinesteraza tankoslojnom kromatografijom i metodom po Ellmanu. TLC bioautografija je pogodna za ovakve uzorke jer se istovremeno vršilo odjeljivanje komponenti eteričnog ulja i detekcija inhibitora uz reagens Fast Blue B sol. N. damascena je pokazala bolju sposobnost inhibicije acetilkolinesteraze u odnosu na N. sativu, dok je N. sativa kupljena u Švicarskoj inhibirala kolinesteraze u većem postotku od N. sative s hrvatskog područja. Inhibicija butirilkolinesteraza je bila nešto slabija u odnosu na acetilkolinesterazu.

Formulation components of organophosphate insecticidal preparations might affect their toxic action in animals. The objective of this study was to examine and compare the acute toxicity and cholinesterase inhibition in seven to 14-day-old chicks dosed orally with dichlorvos and diazinon in standard veterinary and agricultural formulations. The acute (24 h) oral median lethal doses (LD50) of the formulations were determined using the up-and-down method. Respective LD50 of dichlorvos of the veterinary and agricultural formulations in chicks were 11.1 mg kg-1 and 6.51 mg kg-1 and those of diazinon 6.4 mg kg-1 and 6.7 mg kg-1. Plasma and brain cholinesterase activities were measured by electrometry after in vivo and in vitro exposure to organophosphates. The chicks showed signs of cholinergic toxicosis within one hour of dosing. Dichlorvos (8 mg kg-1) and diazinon (4 mg kg-1) in the veterinary and agricultural formulation signifi cantly reduced both plasma and brain cholinesterase activities in the chicks. The veterinary formulation of dichlorvos reduced plasma ChE by 60 % and agricultural by 40 % and brain ChE by 93 % and 87 %, respectively. In contrast, ChE inhibition by diazinon in the agricultural formulation of diazinon was stronger than by the veterinary formulation; 72 % vs. 64 % in plasma and 97 % vs. 80 % in the brain, respectively. The highest in vitro inhibitions were observed with dichlorvos in the agricultural formulation (50 %) in the brain samples and with diazinon in the agricultural formulation (52 %) in the plasma samples. While they exist, differences between formulations cannot be taken as a rule and further investigations should inventory the toxicity of standard veterinary and agricultural organophosphate formulations in addition to the known data for pure forms., Sastojci formulacija mogu djelovati na toksičnost organofosfatnih insekticidnih pripravaka u životinja. Cilj je ovog istraživanja bio ispitati i usporediti akutnu inhibiciju kolinesteraza u pilića starih 7 do 14 dana koji su na usta primili organofosfatne insekticide diklorvos i diazinon u odgovarajućim formulacijama za veterinarsku odnosno poljoprivrednu primjenu. Dvadesetčetverosatna akutna letalna doza (LD50) diklorvosa bila je 11,1 mg kg-1 u veterinarskim odnosno 6,51 mg kg-1 u poljoprivrednim formulacijama, a diazinona 6,4 mg kg-1 odnosno 6,7 mg kg-1. Do kolinergičke toksikoze u pilića došlo je jedan sat nakon primjene. Nakon izlaganja organofosfatima in vivo i in vitro izmjerena je aktivnost kolinesteraza u plazmi i mozgu s pomoću elektrometrije. Oralne doze diklorvosa (8 mg kg-1) odnosno diazinona (4 mg kg-1) putem veterinarskih odnosno poljoprivrednih formulacija značajno su smanjile aktivnost kolinesteraza u plazmi i mozgu pilića. In vitro su također oba organofosfata inhibirala aktivnost kolinesteraza, bez obzira na formulaciju. Poljoprivredna formulacija diklorvosa izazvala je najjaču inhibiciju (50 %) u mozgu pilića, dok je poljoprivredna formulacija diazinona najjače inhibirala (52 %) aktivnost u plazmi. Ovo istraživanje pokazuje da različite formulacije organofosfatnih insekticida mogu dovesti do različita otrovanja i različito djelovati na kolinesteraznu aktivnost u mozgu i plazmi.

The antidotal effect of diphenhydramine against acute toxicosis induced by the organophosphorus insecticide dichlorvos in male rats was evaluated and compared with that of the standard antidote atropine. Dichlorvos at 45 mg/kg orally induced cholinergic toxicity in the rats with cholinesterase inhibition in the plasma (62%), erythrocytes (59%) and whole brain (37%) in comparison with the control group. Intraperitoneal administration of diphenhydramine or atropine at 20 mg/kg 5 minutes after the dichlorvos dosing significantly decreased the incidences of toxic manifestations and gradually decreased the severity of toxicosis during a 60-minute observation period. The antidotal effect of diphenhydramine appeared to be comparable with that of the atropine. However, addition of diazepam did not improve the antidotal efficacy of diphenhydramine. The reductions in blood and brain cholinesterase activities occurred in all the dichlorvos-treated rats regardless of antidotal therapy. The data indicate the antidotal efficacy of diphenhydramine against dichlorvos-induced poisoning in rats in a manner comparable with that of atropine., Protuotrovni učinak difenhidramina protiv trovanja uzrokovanog diklorvosom u štakora uspoređen je sa standardnim djelovanjem atropina. Diklorvos u dozi 45 mg/kg primijenjen oralno uzrokovao je kolinergično trovanje u štakora inhibicijom kolinesteraze plazme (62%), funkcije eritrocita (59%) te inhibicijom funkcije mozga (37%) u odnosu na kontrolnu skupinu. Intraperitonejska primjena difenhidramina ili atropina u dozi 20 mg/kg pet minuta nakon unošenja otrova znatno je smanjila toksični učinak i u tijeku 60 minuta postupno smanjivala opasnost od trovanja. Antidotni učinak difenhidramina u potpunosti je usporediv s djelovanjem atropina. Dodatna primjena diazepama nije sinergistički djelovala na učinak difenhidramina. Smanjenje aktivnosti kolinesteraze u krvi i mozgu uočeno je u svih životinja bez obzira na primjenu antidota.

The toxic effect of methomyl was studied in rats after a single or repeated oral administration. Rats treated with a single dose of methomyl (3, 5, or 7 mg/kg) showed significant increase (P, Istraživani su učinci karbamatnog insekticida metomila u štakora. Nakon jednokratne peroralne doze od 3, 5 ili 7 mg/kg nađeno je značajno povećanje sveukupne aktivnosti laktat dehidrogenaze (LDH) nakon prvog dana tretmana. Najveća je aktivnost utvrđena trećeg dana nakon tretmana metomilom u dozi od 7 mg/kg. Enzimska aktivnost postepeno se smanjivala i sedmoga se dana normalizirala. Posebno je zapaženo povećanje aktivnosti izoenzima LDH-3 i LDH-4. U posebnim je pokusima utvrđeno značajno smanjenje težine slezene i vijabilnosti splenocita u štakora tretiranih sa 6 mg/kg i 8 mg/kg metomila i to nakon prvog i nakon trećeg dana tretmana. Toksični učinci na slezenu mogli su se spriječiti pretretiranjem sa 60 mg/kg N-acetilcisteina. Prema rezultatima ovih pokusa čini se da bi toksični učinak metomila na slezenu mogao biti izravan učinak metomila na stanice slezene a ne putem kolinergičnog mehanizma. Važnost ovakvog citotoksičnog učinka i mehanizme citotoksičnosti i njihove povezanosti s reaktivnim oksidativnim procesima pri staničnom oštećenju valja još istraživati.

Indicator tubes from field kits for detection of organophosphorus compounds in the air were found on the territory of Croatia, in barracks liberated during the war years of 1991/1992, and were analysed during 1993/95. The detection kit consisted of glass indicator tubes with two sealed vials within each tube, a device for opening the tubes and breaking the vials, and an air sampling pump. The tubes were marked with serial numbers and expiry dates (1974-1992). but the description as to their contents was unavailable. The aim of this study was to identify the contents of the indicator tubes and to establish whether they were still suitable for detection of organophosphorus compounds. One vial was found to contain a cholinesterase (EC 3.1.1.7) preparation, while the other vial contained a non-thiocholine substrate and a pH-sensitive indicator (most probably cresol red). Applying ODVP as an organophosphorus cholinesterase inhibitor, it was found that all sets of indicator tubes were suitable for use regardless of the indicated expiry dates. Furthermore, the same tubes were found suitable for detection of organophosphorus compounds in water., U tijeku 1991/92. pronađeni su na teritoriju Hrvatske u oslobođenim vojarnicama test-paketi za detekciju orqanofostornih spojeva u zraku. Test-paketi nađeni su bez oznake podrijetla i bili su pohranjeni u vojarnicama u nepoznatim okolnostima. Test-paketi sadrže indikatorske cjevčice, alat za otvaranje cjevčice i lomljenje zataljenih staklenih ampula unutar cjevčica, te crpku za zrak. Unutar indikatorske cjevčice nalaze se dvije zataljene ampulice i sloj apsorbensa. U toku 1993.-1995. testirano je 14 setova indikatorskih cjevčica na kojima su bili označeni serijski brojevi i godina uporabljivosti (1974. do 1992.]. Utvrđeno je da se u jednoj ampulici nalazi preparat butirilkolinesteraze (EC 3.1.1.8), a da druga sadrži indikator koji reagira na pramjenu pH (vjerojatno krezolno crvenilo) i supstrat, koji nije ester tiokolina. U svim je setovima zabilježena aktivnost enzimskog preparata prema propioniltiokolinu kao supstratu, a u dva seta izmjerena je i aktivnost prema benzoilkolinu, koji je specifični inhibitor butirilkolinesteraze. Uporabljivost indikatorskih cjevčica iskušana je s dimetilnim diklorovinilfosfatom (DDVP), koji je organofosforni inhibitor butirilkolinesteraze. Kada se, bez prisutnosti inhibitora, sadržaj ampulice s preparatom butirilkolinesteraze pomiješa sa sadržajem druge ampulice, pojavljuje se ljubičasta boja, koja nakon 1-2 min prelazi u žutu. U prisutnosti DDVP-a svim je setovima cjevčica bilo potrebno mnogo dulje vrijeme za promjenu boje nego kada je test izveden bez prisutnosti inhibitora kolinesteraze. Provedeno testiranje pokazalo je da su reagencije unutar cjevčica ostale stabilne tijekom pohranjivanja, te da su bile uporabljive dulje nego što je označen njihov rok valjanosti. Pokazano je također da se indikatorske cjevčice mogu upotrijebiti i za dokazivanje organofosfornih spojeva i drugih direktnih inhibitora kolinesteraze u vodi.

Na osnovi kinetike inhibicije kolinesteraze razrađen je test za određivanje organofosfata u vodi pod terenskim uvjetima. U radu su prikazane potrebne reagencije, uvjeti analize i očitanje rezultata. Donja granica osjetljivosti testa je slijedeća (mg/L): soman 0.00012, VX 0.0059, sarin 0.0099, tabun 0.026 i DDVP 0.050.