Effects of high sucrose diet, gemfibrozil, and their combination on plasma paraoxonase 1 activity and lipid levels in rats

We investigated the influence of high sucrose diet (HSD) after 3 or 5 weeks of administration on paraoxonase 1 (PON1) activity in plasma of normolipidemic rats and the relationship between serum PON1 activity, triacylglycerides (TGs), HDL and total cholesterol vs. the control group of rats fed normal, control diet (CD). Because the data about the influence of gemfibrozil (GEM) on PON1 activity are controversial, we also investigated its effects (administration in the 4th and 5th week in rats on HSD and CD) on plasma PON1 activity and lipid levels in normolipidemic rats, and in rats with hypertriglyceridemia caused by HSD. Our results obtained in rats on HSD show a significant increase of plasma TGs levels by 47% (P<0.05) after 5 weeks of treatment, and PON1 activity by 32% and 23% (P<0.05) after 3 and 5 weeks, but without change in lipid levels vs. rats on CD. In the rats on CD and HSD, GEM caused a significant decrease of PON1 activity by 44% and 33%, while a significant decrease of TGs level by 38% (P<0.05) was measured only in rats on CD. The effects of GEM on total cholesterol, HDL and LDL in both groups of rats were typical for its action on lipoprotein metabolism. Because GEM in the rat liver stimulates proliferation of peroxisomes, β oxidation, and production of H2O2, it is possible that the oxidative stress induced by GEM damages hepatocytes and lowers the synthesis of PON1

Qualitative and Quantitative Gamma-Hydroxybutyrate Analysis

Gama-hidroksibutirat (GHB) supstancija je koja se prirodno sintetizira u organizmu sisavaca. Pretpostavlja se da posjeduje svojstva neurotransmitora. Prvi put GHB je sintetiziran in vitro 1960. godine. Pri tom su otkriveni njegovi sedativni i hipnotički učinci na središnji živčani sustav pa se GHB u 60-im godinama prošlog stoljeća počinje rabiti kao anestetik, a naknadno i kao sredstvo za jačanje mišića. Međutim, nakon što je dokazano da izaziva ovisnost te uzrokuje brojne opasne nuspojave, GHB se povlači iz uporabe i stavlja na popis zabranjenih supstancija. Unatrag zadnjih desetak godina GHB se počinje zlouporabljati kao psihoaktivno sredstvo. Ilegalno se nabavlja u obliku tekućine ili bijelog praha. Jednostavno se priprema iz svog prekursora gama-butirolaktona (GBL). Zbog rastućeg broja prometnih nesreća i kriminalnih djela počinjenih pod njegovim utjecajem GHB zadnjih desetak godina postaje važan analitički objekt forenzičkih i biomedicinskih laboratorija. U ovom radu prikazan je kratak pregled analitičkih metoda razvijenih za etekciju i kvantifikaciju GHB-a u ilegalnim pripravcima i mokraći.Gamma-hydroxybutyrate (GHB) is a naturally occurring compound present in the brain and peripheral tissues of mammals. It is a minor metabolite and precursor of gamma-aminobutyric acid (GABA). Just as GABA, GHB is believed to play a role in neurotransmission. GHB was first synthesized in vitro in 1960, when it revealed depressive and hypnotic effects on the central nervous system. In 1960s it was used as an anaesthetic and later as an alternative to anabolic steroids, in order to enhance muscle growth. However, after it was shown that it caused strong physical dependence and severe side effects, GHB was banned. For the last fifteen years, GHB has been abused for its intoxicating effects such as euphoria, reduced inhibitions and sedation. Illicitly it is available as white powder or as clear liquid. Paradoxically GHB can easily be manufactured from its precursor gamma-butyrolactone (GBL), which has not yet been banned. Because of many car accidents and criminal acts in which it is involved, GHB has become an important object of forensic laboratory analysis. This paper describes gas and liquid chromatography, infrared spectroscopy, microscopy, colourimetry and nuclear magnetic resonance as methods for detection and quantification of GHB in urine and illicit products

Effects of Oxime K203 and Oxidative Stress in Plasma of Tabun Poisoned Rats

The highly toxic nature of tabun has been known for many years, but there are still serious limitations to antidotal therapy. In this study, we used rats as an experimental model to evaluate the efficiency of bispyridinium para-oxime K203 as therapy against tabun poisoning as well as to examine if induction of oxidative stress is linked to organophosphate toxicity. K203 showed high potency in counteracting tabun poisoning. Either alone or in combination with atropine, this oxime significantly increased cholinesterase activity at 0.5 and 1 h compared to untreated rats poisoned with tabun. Simultaneous measurements of markers of oxidative stress (lipid peroxidation and superoxide dismutase) showed that tabun poisoning, but also therapy (oxime alone or oxime plus atropine) applied immediately after tabun poisoning, could generate free radical species that may cause oxidative stress in rats. (doi: 10.5562/cca1811

Usporedno određivanje učinkovitosti bispiridinijevih oksima pri trovanju paraoksonom

The inability of standard therapy to provide adequate protection against poisoning by organophosphorus compounds (pesticides and nerve agents) motivated us to search for new, more effective oximes. We investigated the pharmacotoxicological properties of six experimental K-oximes (K027, K033, K048, K074, K075, and K203) in vivo. The therapeutic efficacy of K-oximes (at doses of 5 or 25 % of their LD50) combined with atropine was assessed in paraoxon-poisoned mice and compared with conventionally used oximes HI-6 and TMB-4. The bisoxime K074 was the most toxic (LD50=21.4 mg kg-1) to mice, while monoxime K027 was the least toxic (LD50=672.8 mg kg-1). With the exception of K033, all of the tested K-oximes showed better therapeutic efficiency than HI-6 and TMB-4. K027 and K048 stood out by demonstrating low acute toxicities and ensuring protective indices ranging from 60.0 to 100.0 LD50 of paraoxon. Taking into account that these two oximes showed a similar therapeutic efficacy regardless of the applied doses, our results suggest that K027 and K048 could be antidotes for paraoxon intoxication.Činjenica da standardna terapija ne omogućuje dovoljnu zaštitu pri otrovanju organofosfornim spojevima (pesticidima i živčanim bojnim otrovima) potaknula nas je na istraživanje novih, učinkovitijih oksima. U uvjetima in vivo ispitali smo farmakotoksikološka svojstva šest eksperimentalnih K-oksima (K027, K033, K048, K074, K075 i K203). Terapijski učinak kombinacije K-oksima (primjenjenih u dozi 5 ili 25 % njihove LD50) i atropina testiran je na miševima otrovanim paraoksonom i uspoređen s konvencionalnim oksimima HI-6 i TMB-4. Bisoksim K074 je bio najtoksičniji (LD50=21.4 mg kg-1) za miševe, dok je monooksim K027 bio najmanje toksičan (LD50=672.8 mg kg-1). Osim K033, svi K-oksimi pokazali su bolji terapijski učinak u miševa trovanih paraoksonom u odnosu na HI-6 i TMB-4. Iz skupine testiranih oksima istaknuli su se K027 i K048 koji su pokazali nisku akutnu toksičnost i osigurali protektivne indekse u rasponu od 60.0 do 100.0 LD50 paraoksona. Uzmemo li u obzir da su ta dva oksima pokazala sličan terapijski učinak bez obzira na primjenjenu dozu, prikazani rezultati upućuju na K027 i K048 kao perspektivne antidote u terapiji trovanja paraoksonom

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

Učinci istodobne primjene THC-a i irinotekana na rast tumora i biokemijske markere na singeničnom modelu raka debelog crijeva u miševa

Clinical treatment with the antineoplastic drug irinotecan (IRI) is often hindered by side effects that significantly reduce the quality of life of treated patients. Due to the growing public support for products with Δ9-tetrahydrocannabinol (THC), even though relevant scientific literature does not provide clear evidence of their high antitumour potential, some cancer patients take unregistered preparations containing up to 80 % THC. This study was conducted on a syngeneic colorectal cancer mouse model to test the efficiency and safety of concomitant treatment with IRI and THC. Male BALB/c mice subcutaneously injected with CT26 cells were receiving 60 mg/kg of IRI intraperitoneally on day 1 and 5 of treatment and/or 7 mg/kg of THC by gavage a day for 7 days. Treatment responses were evaluated based on changes in body, brain, and liver weight, tumour growth, blood cholinesterase activity, and oxidative stress parameters. Irinotecan’s systemic toxicity was evidenced by weight loss and high oxidative stress. The important finding of this study is that combining THC with IRI diminishes IRI efficiency in inhibiting tumour growth. However, further studies, focused on more subtle molecular methods in tumour tissue and analytical analysis of IRI and THC distribution in tumour-bearing mice, are needed to prove our observations.Kliničko liječenje antineoplastičnim lijekom irinotekanom (IRI) često je otežano nuspojavama koje značajno smanjuju kvalitetu života liječenih bolesnika. Zbog sve veće javne potpore proizvodima s Δ9-tetrahidrokanabinolom (THC), iako relevantna znanstvena literatura ne daje jasne dokaze o njihovu visokom antitumorskom potencijalu, oboljeli od raka uzimaju neregistrirane pripravke koji sadržavaju i do 80 % THC-a. Ova studija provedena je na modelu singeničnoga tumora debelog crijeva u miševa kako bi se testirala učinkovitost i sigurnost istodobnog tretmana irinotekanom i THC-om. Mužjaci BALB/c miševa kojima su supkutano injicirane CT26 stanice primili su 60 mg/kg IRI-ja intraperitonealno prvi i peti dan i/ili 7 mg/kg THC-a oralno svaki dan tijekom sedam dana. Učinkovitost tretmana procijenjena je na temelju promjena u težini tijela, mozga i jetre, rasta tumora, aktivnosti kolinesteraza u krvi i parametara oksidacijskoga stresa. Sistemska toksičnost irinotekana potvrđena je smanjenjem težine miševa i povećanjem parametara oksidacijskoga stresa. Značaj je rezultata ove studije u smanjenoj učinkovitosti IRI-ja u inhibiciji rasta tumora tijekom istodobnog uzimanja s THC-om. Međutim, potrebna su daljnja istraživanja usmjerena na suptilnije molekularne metode u tumorskom tkivu i analitička analiza distribucije IRI-ja i THC-a u miševa s tumorom kako bi se dokazala naša opažanja

Učinak ketamina na vijabilnost, primarna oštećenja DNA i parametre oksidacijskog stresa u stanicama HepG2 i SH-SY5Y

Ketamine is a dissociative anaesthetic used to induce general anaesthesia in humans and laboratory animals. Due to its hallucinogenic and dissociative effects, it is also used as a recreational drug. Anaesthetic agents can cause toxic effects at the cellular level and affect cell survival, induce DNA damage, and cause oxidant/antioxidant imbalance. The aim of this study was to explore these possible adverse effects of ketamine on hepatocellular HepG2 and neuroblastoma SH-SY5Y cells after 24-hour exposure to a concentration range covering concentrations used in analgesia, drug abuse, and anaesthesia (0.39, 1.56, and 6.25 μmol/L, respectively). At these concentrations ketamine had relatively low toxic outcomes, as it lowered HepG2 and SH-SY5Y cell viability up to 30 %, and low, potentially repairable DNA damage. Interestingly, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) remained unchanged in both cell lines. On the other hand, oxidative stress markers [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)] pointed to ketamine-induced oxidant/antioxidant imbalance.Ketamin je disocijativni anestetik koji se koristi za izazivanje opće anestezije u određenim medicinskim postupcima kod ljudi, kao i u anesteziji laboratorijskih životinja. Zbog svojih halucinogenih i disocijativnih učinaka koristi se i kao rekreacijska droga. Anestetici također mogu prouzročiti toksične učinke na staničnoj razini i, utječući na preživljavanje stanica, izazvati oštećenje DNA te neravnotežu oksidansa i antioksidansa. Cilj ove studije bio je istražiti moguće štetne učinke ketamina na hepatocelularne HepG2 i neuroblastoma SH-SY5Y stanice nakon 24-satne izloženosti širokom rasponu koncentracija, uključujući koncentracije relevantne u slučajevima korištenja u analgeziji, zlouporabi droga i anesteziji (0,39, 1,56 odnosno 6,25 μmol/L). Naši rezultati pokazali su da je ketamin u ovim ispitivanim koncentracijama izazvao relativno nisku citotoksičnost, budući da je do 30 % smanjio preživljenje stanica HepG2 i SH-SY5Y, ali je uočen neznatan porast razine primarnih oštećenja DNA. Zanimljivo je da su razine reaktivnih kisikovih vrsta (ROS), malondialdehida (MDA) i glutationa (GSH) ostale nepromijenjene u objema staničnim linijama. S druge strane, markeri oksidacijskog stresa [suporeksid dismutaza (SOD), glutation peroksidaza (GPx), katalaza (CAT)] upućivali su na oksidacijsko-redukcijsku neravnotežu izazvanu ketaminom

Oksidacijski stres, aktivnost kolinesteraza i primarna oštećenja u jetri, krvi i plazmi Wistar štakora nakon 28-dnevnog izlaganja glifosatu

In this 28 day-study, we evaluated the effects of herbicide glyphosate administered by gavage to Wistar rats at daily doses equivalent to 0.1 of the acceptable operator exposure level (AOEL), 0.5 of the consumer acceptable daily intake (ADI), 1.75 (corresponding to the chronic population-adjusted dose, cPAD), and 10 mg kg-1 body weight (bw) (corresponding to 100 times the AOEL). At the end of each treatment, the body and liver weights were measured and compared with their baseline values. DNA damage in leukocytes and liver tissue was estimated with the alkaline comet assay. Oxidative stress was evaluated using a battery of endpoints to establish lipid peroxidation via thiobarbituric reactive substances (TBARS) level, level of reactive oxygen species (ROS), glutathione (GSH) level, and the activity of glutathione peroxidase (GSH-Px). Total cholinesterase activity and the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were also measured. The exposed animals gained less weight than control. Treatment resulted in significantly higher primary DNA damage in the liver cells and leukocytes. Glyphosate exposure significantly lowered TBARS in the liver of the AOEL, ADI, and cPAD groups, and in plasma in the AOEL and cPAD group. AChE was inhibited with all treatments, but the AOEL and ADI groups significantly differed from control. Total ChE and plasma/liver ROS/GSH levels did not significantly differ from control, except for the 35 % decrease in ChE in the AOEL and ADI groups and a significant drop in liver GSH in the cPAD and 100xAOEL groups. AOEL and ADI blood GSH-Px activity dropped significantly, but in the liver it significantly increased in the ADI, cPAD, and 100xAOEL groups vs. control. All these findings show that even exposure to low glyphosate levels can have serious adverse effects and points to a need to change the approach to risk assessment of low-level chronic/sub-chronic glyphosate exposure, where oxidative stress is not necessarily related to the genetic damage and AChE inhibition.U okviru 28-dnevnog pokusa istražili smo učinke herbicida glifosata na modelu odraslih mužjaka Wistar štakora koji su oralno dobivali testirani spoj u subletalnim dnevnim dozama: 0,1 od prihvatljive razine izloženosti operatera (0,1xAOEL), 0,5 od prihvatljivog dnevnog unosa za potrošače (0,5xADI), 1,75 (odgovara kroničnoj populacijskoj prilagođenoj dozi, cPAD) i 10 mg kg-1 tjelesne težine na dan (odgovara 100xAOEL). Tijekom pokusa praćeni su sistemski toksični učinci. Nakon završetka svih tretmana svakoj je pokusnoj životinji izmjerena tjelesna težina i težina jetre te su uspoređene s polazišnim vrijednostima. Alkalnim komet-testom izmjerena je razina primarnih oštećenja DNA u leukocitima i jetrenim stanicama. Primjenom metoda za procjenu oksidacijskog stresa izmjerene su razine lipidne peroksidacije (TBARs), reaktivnih kisikovih vrsta (ROS) i glutationa (GSH) te aktivnost enzima glutation peroksidaze (GSH-Px). Izmjerene su i aktivnosti ukupnih kolinesteraza (ChE), acetilkolinesteraze (AChE) i butirilkolinesteraze (BChE). Izloženi štakori imali su manje priraste težine od kontrolnih. Izloženost glifosatu uzrokovala je značajne poraste razine primarnih oštećenja DNA u jetrenim stanicama te malo manje u leukocitima. U svim izloženim skupinama izmjerene su niže vrijednosti TBARs u odnosu na kontrolu, sa značajno nižim vrijednostima u AOEL, ADI i cPAD skupinama u uzorcima jetre te u AOEL i cPAD skupinama u uzorcima plazme. Aktivnost AChE bila je smanjena u svim tretmanima, s najnižom stopom nakon izlaganja dozi ADI. Aktivnost BChE blago je smanjena nakon izlaganja ADI, a povećana nakon izlaganja dozama cPAD i 100xAOEL. Ukupna aktivnost ChE te razine ROS/GSH u plazmi / jetri nisu se značajno razlikovale od kontrole, osim značajnog smanjenja jetrenog GSH nakon izlaganja dozama cPAD i 100xAOEL te 35-postotnog smanjenja aktivnosti ChE nakon izlaganja dozama AOEL i ADI. Aktivnost GSH-Px u krvi značajno je smanjena u AOEL i ADI tretmanu, a aktivnost GSH-Px u uzorcima jetre značajno je povećana u skupinama ADI, cPAD i 100xAOEL prema kontroli. Dobiveni rezultati pokazuju da čak i izloženost vrlo niskim dozama glifosata može izazvati mjerljive toksične učinke te upućuje na potrebu za promjenom pristupa procjeni rizika zbog kronične/subkronične izloženosti niskim dozama glifosata gdje oksidacijski stres ne mora nužno korelirati s razinom oštećenja DNA i inhibicijom acetilkolinesteraz

Qualitative and Quantitative Gamma-Hydroxybutyrate Analysis

Gama-hidroksibutirat (GHB) supstancija je koja se prirodno sintetizira u organizmu sisavaca. Pretpostavlja se da posjeduje svojstva neurotransmitora. Prvi put GHB je sintetiziran in vitro 1960. godine. Pri tom su otkriveni njegovi sedativni i hipnotički učinci na središnji živčani sustav pa se GHB u 60-im godinama prošlog stoljeća počinje rabiti kao anestetik, a naknadno i kao sredstvo za jačanje mišića. Međutim, nakon što je dokazano da izaziva ovisnost te uzrokuje brojne opasne nuspojave, GHB se povlači iz uporabe i stavlja na popis zabranjenih supstancija. Unatrag zadnjih desetak godina GHB se počinje zlouporabljati kao psihoaktivno sredstvo. Ilegalno se nabavlja u obliku tekućine ili bijelog praha. Jednostavno se priprema iz svog prekursora gama-butirolaktona (GBL). Zbog rastućeg broja prometnih nesreća i kriminalnih djela počinjenih pod njegovim utjecajem GHB zadnjih desetak godina postaje važan analitički objekt forenzičkih i biomedicinskih laboratorija. U ovom radu prikazan je kratak pregled analitičkih metoda razvijenih za etekciju i kvantifikaciju GHB-a u ilegalnim pripravcima i mokraći.Gamma-hydroxybutyrate (GHB) is a naturally occurring compound present in the brain and peripheral tissues of mammals. It is a minor metabolite and precursor of gamma-aminobutyric acid (GABA). Just as GABA, GHB is believed to play a role in neurotransmission. GHB was first synthesized in vitro in 1960, when it revealed depressive and hypnotic effects on the central nervous system. In 1960s it was used as an anaesthetic and later as an alternative to anabolic steroids, in order to enhance muscle growth. However, after it was shown that it caused strong physical dependence and severe side effects, GHB was banned. For the last fifteen years, GHB has been abused for its intoxicating effects such as euphoria, reduced inhibitions and sedation. Illicitly it is available as white powder or as clear liquid. Paradoxically GHB can easily be manufactured from its precursor gamma-butyrolactone (GBL), which has not yet been banned. Because of many car accidents and criminal acts in which it is involved, GHB has become an important object of forensic laboratory analysis. This paper describes gas and liquid chromatography, infrared spectroscopy, microscopy, colourimetry and nuclear magnetic resonance as methods for detection and quantification of GHB in urine and illicit products