Search

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.

Oximes are pharmacologically important nucleophylic agents acting as antidotes against poisoning by organophosphorus compounds. In this study, effect of K048 oxime on cell viability and chromosome stability in vitro was evaluated in primary cell cultures of isolated human peripheral blood lymphocytes. K048 oxime at concentrations of 730, 200, and 7.3 nmol/dm3 was tested in 30 minutes treatment. Cytotoxic effect was tested by using differential viability staining with a mixture of acridine orange and ethydium bromide, while in evaluation of genotoxic potential hOGG1 modified comet assay, cytokinesis-blocked micronucleus assay, and chromosome aberration analysis were used. Viability staining showed that oxime treatment does not induce apoptosis (8.0% highest concentration vs. 7.0% control) or necrosis indicating lack of its cytotoxic effect (9.6% highest concentration vs. 7.3% control). HOGG1 modified comet assay revealed dose dependant increase in the level of oxidative primary damage to DNA, significant at all doses tested. Expressed as % of DNA in comet tail, oxidative damage level in K048 treated lymphocytes ranged from 7.73-11.46% compared to 2.12% in control lymphocytes. However, oxime treatment did not affect micronucleus frequency nor induced significant increase in formation of structural chromosomal aberrations. Micronucleus frequency in oxime treated lymphocytes ranged from 13-19 per 3000 binucleated cells compared to 15 micronuclei per 3000 cells in control cultures. Chromatid breaks were detected in both, control and oxime treated lymphocytes with no difference in frequency, while formation of acentric fragments was observed only in lymphocytes treated with lowest K048 concentration (7.3 nmol/dm3). However, observed level of acentric formation (2 per 200 cells) was not statistically significant. In applied battery of cyto/gentoxicity assays, considering applied testing conditions K048 oxime showed acceptable biocompatibility at the level of cell viability and chromatin/chromosome integrity. Since no increase in secondary genome damage was detected, induced primary oxidative DNA damage might be result of cell stress induced by treatment and are rather repaired than fixed as chromosome damage.

Accidental exposures but also threat of the abuse of organophosphorous compounds (OP) leads to search for more effective treatment regimens against these poisonings. Toxic action of OP, including pesticides and nerve agents, is primarily attributed to irreversible inhibition of acetylcholinesterase (AChE). AChE inhibition results in acetylcholine accumulation in the synaptic cleft with consequences to the central and peripheral nervous system. The current treatment of OP poisonings includes a combination of an antimuscarinic agent (atropine) and an AChE reactivator (oxime). Recently a series of oximes have been synthesized by the copper-catalyzed azide-alkyne cycloadition reaction. The purpose of this investigation was to evaluate the toxicity and protection against OP compounds poisoning provided by three of those oximes: JAR-2-28B, JAR-2-153 and JAR-1-81A (synthesis not yet published). The therapeutic effect of oximes in mice was tested by intraperitoneal administration of oxime (5 or 25% of its LD50) and atropine (10 mg/kg) one minute after OP (given subcutaneously). We used conventional oximes HI-6 and TMB-4 to compare JAR’s efficacy expressed as protective index (PI) and maximal dose of poison (MDP). Tested oximes showed high acute toxicity (8.4 to 26.7 mg/kg b.w.) which is in accordance with an approach for in situ design of high affinity ligands for AChE. Pronounced dose-response relationship was observed against all nerve agent poisoning. Considering tabun intoxication, JAR oximes showed lower therapeutic efficacy than TMB-4. The same stands for soman, sarin and VX poisoning forwhich HI-6 was superior as therapeutic agent. JAR oximes showed relatively good efficiency in therapy of paraoxon poisoned mice regardless the dose. Combined with atropine they ensured survival of all animals against 6.3 to 10.0 LD50 of paraoxon. Despite their high toxicity, JAR oximes were able to provide protection against the lethal effects of OP probably due to their ability to reactivate organophosphate-inhibited AChE.

Rats have been used in study of temporal distribution of the cholinesterase activity after exposure to tabun and treatment with oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide, K048. In the event of poisoning by organophosphorus compounds (OP), immediate therapeutic treatment usually consists of combined administration of an anticholinergic drug, such as atropine, and an oxime-reactivator of acetylcholinesterase. However, the treatment is very limiting in case of nerve agent tabun poisoning. Since our recent in vitro and in vivo studies on mice showed that oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide (K048) is very promising as reactivator of tabun-inhibited human erythrocyte acetylcholinesterase and as antidote of tabun poisoned mice, we tested antidotal potency of K048 in tabun-poisoned rats. We measured acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in plasma after 0.5, 1, 6 and 24 h long oxime therapy. In view of the possible oxidative stress involved in OP poisoning, it has been decided to estimate the levels of oxidative stress (lipid peroxidation), and to see whether any difference exists in this parameter upon tabun poisoning and administrated therapy in rats. Male rats received a subcutaneous (s.c.) dose of 3/4 of the tabun LD50. Oxime was given intraperitoneally (i.p.) at doses of 1/4 of its LD50 after 1 min of tabun poisoning. Animals were divided into 5 groups of four animals receiving: tabun (I), oxime (II), atropine (III) tabun+oxime+atropine (IV) and saline as control group. Atropine was administered i.p. (10.0 mg/kg body weight) together with oxime. All animals were sacrificed after 0.5, 1, 6 and 24 h. Plasma was diluted 50 times for AChE and BChE activity measurement by Ellman assay at 25 °C using acetylthiocholine as substrate. AChE and BChE activity was determined using selective inhibitors BW284C51 and ethopropazine, respectively. As a measure of lipid peroxidation the concentration of thiobarbituric reactive substances (TBARS) was determined using modification of the method by Drury et al. (1997). Activity of AChE and BChE (control group and group II) ranged from 1.5-2.9 and 0.6-1.8  mol min-1 per g of protein, respectively, with no matter of sacrifice time. Activity of group I and III of both enzymes was below 0.3  mol min-1 per g of protein up to 6 h, while in 24 h both activities were completely restored. AChE activity of group IV in 30 min and 1 h was in same range as control confirming the K048 reactivation potency, while in 6 h the therapy by K048 was depleted - AChE activity was low as in group I. Treatment with K048 did not preserve BChE activity that is also in accordance with previous K048 in vitro study, due to limiting BChE reactivation by K048. The levels of plasma TBARS progressively increased up to 6 h for all treatment groups. The major increase was detected after tabun administration while only minor time-dependent differences were observed after treatment with K048. Although the levels of TBARS decreased up to 24 h, they remained above the range of the control for most of the treatments. The significant improvement was noticed in the AChE activity of tabun-poisoned rats with applied K048 therapy. Poisoning did not significantly induce the oxidative stress.

We investigated the influence of high sucrose diet (HSD) after 3 and 5 weeks of administration on PON1 activity in plasma of normolipidemic rats and to determine the relationship between serum PON1 activity, triacylglycerides (TG), HDL and total cholesterol vs. the control group (CD). Because the data about the influence of gemfibrozil (GEM) on PON1 activity are controversial, we also investigated its effects (the administration in the 4th and 5th week in rats on HSD and CD) on plasma PON1 activity and lipids level in normolipidemic rats, and in rats with hypertriglyceridemia caused by HSD. Our results obtained in rats on HSD show the significant increase of plasma TGs levels by 47% (p

The widespread use of organophosphorous compounds (OP) as pesticides and the misuse of OP nerve agents in military conflicts or terrorist attacks emphasize the need for effective antidotal preparedness. The toxic symptomatology of OP is caused by irreversible inhibition of the serine-protease acetylcholinesterase (AChE), a vitally important enzyme that hydrolyses the neurotransmitter acetylcholin. Generally, anticholinergics such as atropine and AChE reactivators - oximes are used as first aid antidotes for OP intoxications. However, reactivation by oximes is not possible in all cases of OP poisoning, especially in the case of nerve agent tabun. Recent in vitro and in vivo studies on mice draw attention to oxime [(E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide] (K203) as a very potent reactivator of tabun-inhibited AChE. In this study we used rats as experimental model to broaden our knowledge on antidotal potency of K203 as well as to examine impact of possible oxidative stress on OP toxicity and efficacy of applied therapy. Animals were injected subcutaneously with sublethal dose of tabun, and treated intraperitoneally 1min later with oxime K203 (5 or 25% of its LD50) alone or in combination with atropine. All animals were sacrificed after 0.5, 1, 6 and 24 h. Plasma samples were analyzed for cholinesterase (ChE) activity and levels of oxidative stress markers (the concentration of thiobarbituric reactive substances, TBARS, as a measure of lipid peroxidation and activity of an antioxidant enzyme superoxide dismutase, SOD). Either alone or in combination with atropine, K203 significantly increased ChE activity at 0.5 and 1 h, in respect to untreated rats poisoned with tabun. In addition, intraperitoneal administration of oxime alone as a control didn't disturb enzyme activity in plasma. Determined TBARS concentrations suggest that this acute treatments resulted with free radical-mediated lipid peroxidation. Considering SOD activity, a notable correlation exists between ChE depression and SOD elevation. Together with published data on K203's efficacy our results indicate the high potency of this oxime in counteracting tabun poisoning. Based on simultaneous measurements of TBARS and SOD we can conclude that the existence of oxidative stress have a role in OP toxicity.

We studied bispyridinium oxime K203 [(E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyimino methylpyridinium)- but-2-ene dibromide] with tabun-inhibited human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro, and its antidotal effect on tabun-poisoned mice and rats in vivo. We compared it with oximes K048 and TMB-4, which have proven the most effi cient oxime antidotes in tabun poisoning by now. Tabun-inhibited AChE was completely reactivated by K203, with the overall reactivation rate constant of 1806 L mol-1 min-1. This means that K203 is a very potent reactivator of tabun-inhibited AChE. In addition, K203 reversibly inhibited AChE (Ki = 0.090 mmol L-1) and BChE (Ki = 0.91 mmol L-1), and exhibited its protective effect against phosphorylation of AChE by tabun in vitro. In vivo, a quarter of the LD50 K203 dose insured survival of all mice after the application of as many as 8 LD50 doses of tabun, which is the highest dosage obtained compared to K048 and TMB-4. Moreover, K203 showed high therapeutic potency in tabun-poisoned rats, preserving cholinesterase activity in rat plasma up to 60 min after poisoning. This therapeutic improvement obtained by K203 in tabun-poisoning places this oxime in the spotlight for further development., Proučavali smo bispiridinijski oksim K203 [(E)-1-(4-karbamilpiridinij)-4-(4-hidroksiiminometilpiridinij)- but-2-ene dibromid] u uvjetima in vitro - studirajući njegove interakcije s ljudskom acetilkolinesterazom (AChE) i butirilkolinesterazom (BChe) inhibiranim tabunom te u uvjetima in vivo - određivanjem njegova antidotskog učinka na miševe i štakore otrovane tabunom. Radi usporedbe uključili smo rezultate dobivene s oksimima K048 i TMB-4 kao najučinkovitijim oksimima kod otrovanja tabunom. K203 je potpuno reaktivirao AChE inhibiranu tabunom sa sveukupnom brzinom reaktivacije od 1806 L mol-1 min-1 što ga svrstava u najučinkovitije reaktivatore AChE inhibirane tabunom. K203 je reverzibilno inhibirao AChE (Ki = 0,090 mmol L-1) i BChE (Ki = 0,91 mmol L-1) pokazujući svoja in vitro zaštitna svojstva od inhibicije tabunom. Terapija dozom K203 od ¼ njegove LD50 omogućila je preživljavanje svih miševa nakon otrovanja dozom tabuna od 8,0 LD50. Time je K203 pokazao bolju učinkovitost u usporedbi s K048 ili TMB-4. K tome, K203 je značajno zaštitio štakore od otrovanja tabunom kompenzirajući toksični učinak tabuna na aktivnost kolinesteraze i do 60 min nakon trovanja. Pokazano poboljšanje terapeutske učinkovitosti K203 ističe ovaj oksim pretečom za daljnji razvoj antidota u otrovanju tabunom.

Some compounds, although not primarily designed as supportive drugs in chemotherapy, are promising candidates for clinical use. The ability of HI-6 oxime to relieve the side effects of irinotecan was recently determined in vitro. In this animal study, we investigated the efficacy of HI-6 in vivo, when given as a pre-treatment and concomitantly with irinotecan. We evaluated the cholinesterase (ChE)/acetylcholinesterase (AChE) activity, the levels of oxidative stress markers, DNA damage and the radical scavenging capacity of HI-6. Both HI-6 and irinotecan inhibited ChE/AChE activity but showed different levels of ChE inhibition in plasma and AChE inhibition in the liver and brain tissue. We also observed a weak antioxidant capacity of HI-6, undiscovered until now, and found an acceptable genotoxicity profile in three types of somatic cells in rats. The in vivo erythrocyte micronucleus assay showed that HI-6 did not significantly change either the frequency of micronuclei or the ratio of polychromatic and normorchromatic erythrocytes. Taken together, our results provide a good argument in favour of HI-6 as a promising molecule for further studies and eventual use in humans.

Organophosphorus compounds (OP) are widely used as pesticides and unfortunately as nerve agents in chemical warfare. A toxic effects of highly toxic nervous warfare agents is irreversible inhibition of vitaly important acethylcholinesterase (AChE). Inhibition of AChE results in accumulation of acetylcholine (ACh ) at the synaptic cleft of the cholinergic neurones, leading to overstimulation of cholinergic receptors. The current standard treatment for poisoning by OP compounds includes the combined administration of cholinesterase reactivatior (oxime), a muscarinic cholinergic receptor antagonist (atropine sulphate) and pre-treatment with reversible carbamate AChE-inhibitor, such as pyridostigmine. In order to find the best antidote, new compounds have been synthesized and tested. In this paper a new bis-pyridinium compound K075 [1, 4-bis (hidroxyiminomethylpyridinium)-but-2- ene dibromide] was tested as potential antidote in tabun poisoned mice. Its antidotal effect was compared with TMB-4, the best-known antidote in tabun poisoning. In all experiments, oxime K075 in doses of ¼ or 5% of its LD50 was used for therapy one minute after tabun application or as pretreatment 15 min before tabun intoxication. The antidotal efficacy of tested compound was expressed as protection index (PI) and maximal dose of poison (MDP). Under these experimental conditions, our results showed K075 as good protector/reactivator of tabun inhibited AChE. In this study the best result was obtained with the therapeutic regimen consisting of ¼ LD50 of K075 as pretreatment and ¼ of LD50 of K075 plus atropine as treatment. The PI was 15.9 ; MDP was 7.9 LD50 of tabun with survival of all tested animals.

A toxic effect of highly toxic nervous warfare agents is irreversible inhibition of vitaly important enzyme acethylcholinesterase (AChE). Inhibition of AChE results in accumulation of acetylcholine (ACh) at the synaptic cleft of the cholinergic neurones, leading to overstimulation of cholinergic receptors. The highly toxic nature of tabun has been known for many years, but there are still serious limitations to the antidotal therapy. Tabun-phosphorylated AChE is resistant to oxime reactivation due to an electron pair located on the amidic group that makes the nucleophilic attack almost impossible . The current standard treatment for poisoning by OP compounds includes the combined administration of cholinesterase reactivatior (oxime), a muscarinic cholinergic receptor antagonist (atropine sulphate) and pre-treatment with reversible carbamate AChE-inhibitor, such as pyridostigmine. The inability of the standard therapy to provide adequate protection against tabun calls for a synthesis of new compounds with the characteristic oxime group. In this paper a new bis-pyridinium compound K027 [1-(4-hidroxyiminomethylpyridinium)-3-(-4-carbamoylpyridinium) propane] was tested as potential antidote in tabun poisoned mice. In all experiments, oxime K027 in dose of ¼ of its LD50 was used as pretreatment 15 min before tabun intoxication, and ¼ LD50 of oximes K027, K048 or TMB-4 together with atropine as therapy one minute after tabun poisoning. The antidotal efficacy of tested compound was expressed as protection index (PI) and maximal dose of poison (MDP). In this study the best result was obtained with the therapeutic regimen consisting of ¼ LD50 of K027 as pretreatment and ¼ of LD50 of K027 or K048 plus atropine as therapy. MDP was 7.9 LD50 of tabun with survival of all tested animals. Under these experimental conditions oxime K027 demonstrated an improvement in treatment over the therapy with oxime plus atropine or pretreatment alone. Herein we show that already promising treatment in tabun poisoning by oximes and atropine could be improved if oximes are also used in pretreatment.

Our recent in vitro and in vivo studies showed that the oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide (K048) shows great promise as a reactivator of tabun-inhibited human erythrocyte acetylcholinesterase (AChE) and as an antidote for tabun poisoned mice. Encouraging results of our experiments prompted us to expand this study by new aims: (i) to establish K048 efficacy against AChE inhibition by tabun in rats ; (ii) to establish the levels of oxidative stress (lipid peroxidation) and to see whether there are any differences in this parameter upon tabun poisoning and applied treatments in rats. AChE and butyrylcholinesterase (BChE) activity was measured in rat plasma and brain after 0.5, 1, 6 and 24 h long oxime therapy. In view of the possible oxidative stress involved in OP poisoning, lipid peroxidation was measured in the same samples to determine the concentration of thiobarbituric reactive substances (TBARS). Significant reactivation potency of oxime K048 in rat plasma was noticed after 30 and 60 min long oxime therapy (oxime + atropine) when AChE activity was in the range of the control group (rats treated with saline only). Efficiency of K048 decreased after 6 h, probably due to the relatively short oxime circulation time in the blood. Interestingly, after 24 h AChE activities of both poisoned and treated rats were high (in the range of the control group). Unlike in plasma, no significant therapeutic effect of K048 was observed on cholinesterase activity in the rat brain, probably due to the limited penetration of the oxime through the blood brain barrier. Further, treatment with K048 did not preserve BChE activity in plasma or in brain, which is in agreement with the previous K048 in vitro study, showing limited BChE reactivation by K048. The plasma TBARS levels remained above the range of the control for most of the treatments. The levels of rat brain TBARS increased up to 24 h in all groups. According to the remaining AChE activity in the brain of approximately 30 % 24 h after tabun application, OP toxicity seems to be associated with tissue oxidative stress. However, to confirm this concept, a good strategy for further studies would be to measure simultaneously a number of biomarkers representing oxidative damage.

Improving the efficacy of antidotal treatment of poisonings with organophosphorous compounds (OPc) is still challenge for the scientific community. OPc are widely used as pesticides, as drugs in the treatment of cholinergic disorders, and as nerve agents in chemical warfare. Irreversible inhibition of acetylcholinesterase (AChE) by these compounds results in acethylcholine (ACh) accumulation in the synaptic cleft with consequences to the central and peripheral nervous system. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhea, tremor, respiratory distress, convulsions, and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Presently, OPc poisoning is treated with a combination of an antimuscarinic agent, e.g. atropine and an AChE reactivator oxime. In this study a new compound atropine-4-oxime (synthesis not yet published), was tested as potential antidote in vivo in the therapy of soman, tabun and paraoxon intoxication in mice. Currently used oximes HI-6 and TMB-4 were included for comparison. The studied compounds (5 or 25 % of their LD50) plus atropine (10 mg/kg) were given intraperitoneally one minute after OPc (given subcutaneously). Their therapeutic efficacy was expressed as protective index (PI) and maximal dose of poison (MDP). Atropine-4-oxime showed very weak antidotal activity inadequate for soman or tabun poisoning. On the other hand, it was very effective at both applied doses upon paraoxon poisoning, showing survival of all animals after administration of 10.0, that is 15.9 LD50 of paraoxon. As well as HI-6 and TMB-4, atropine-4-oxime appear to be suitable for the antidotal treatment of acute paraoxon poisonings, regardless of its high toxicity.

To the present time, only four pyridinium oximes, TMB-4, 2-PAM, HI-6 and obidoxime, have found clinical application in the therapy of organophosphorus compounds poisoning. Treatment with these oximes still has disadvantages, in example ; they all have limited reactivation potency in nerve agent tabun poisoning. We tested different oximes varying in structure to find more effective oximes to reactivate tabun inhibited human erythrocyte AChE. Two of our tested pyridinium oximes, K048 and K074, were the most promising for AChE reactivation with the overall reactivation rate constants (kr) of 673 and 2375 M-1min-1, respectively. Oximes are also reversible inhibitors of AChE and in that way exhibit protection against phosphorylation by tabun. Therefore, we tested K048 and K074 in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. The highest TF and TD (22.5 and 10.0, respectively) were obtained when we applied K048 in a dose of 25 % of its LD50 in pretreatment and treatment. This is a considerable increase in protection of mice compared to K048 treatment alone (TF = 8.0, TD = 5.0). Whatever are the shortcomings of using the reversible inhibitors as protectors, we have shown that already promising treatment in tabun poisoning by oximes and atropine could be significantly improved if oximes are also used in pretreatment. This finding may provide a platform for further modifications and development of more potent protectors and reactivators in organophosphorus compounds poisoning.

Dacarbazine (DTIC) is a methylating agent, generally considered the most active antineoplastic drug for treating malignant melanoma. Clinical studies indicate that its photodegradation products cause adverse reactions when drug is administered to patients. As the molecular structure of DTIC involves carbamate moiety, possible impacts on the acetylcholinesterase (AChE) enzyme are also anticipated. Recent findings point to the role of AChE in cells undergoing apoptosis, but the underlying mechanisms remain to be clarified. Present in vitro study aimed at investigating the relationships between the genotoxicity and AChE activity in DTIC-treated human blood cells and to establish how they are influenced by photoactivation. The alkaline comet assay was used for the evaluation of primary DNA damage in leukocytes, while inhibitory potential of the drug on AChE was studied in erythrocytes by the Ellman method. Blood cells were treated with two therapeutically relevant concentrations of the drug (200 mg/m2 and 350 mg/m2) for 30 and 60 min, with and without photoactivation. The results of alkaline comet assay demonstrated a dose-dependent photogenotoxicity of DTIC, indicating that its degradation products possess higher DNA damaging potential then the parent drug. When photoactivated DTIC-treated cells were transferred in dark and incubated for 60 min, the reduction of DNA damage was noted. AChE activity in DTIC-treated erythrocytes, especially those incubated in dark, was lower than in negative controls, indicating the AChE- inhibiting ability of the agent. As observed, longer photoactivation of DTIC-treated cells was accompanied with the restoration of AChE activity, possibly due to the parent drug deactivation. The results sustain the concurrent evaluation of DNA damage and AChE activity, as both biomarkers used provide much better insight into the mechanisms underlying the drug toxicity, especially in studies on antineoplastic drugs having the potential to modulate AChE.

This study was aimed to evaluate the antidotal potency of tenocyclidine (TCP) that probably might protect acetylcholinesterase (AChE) in the case of organophosphate poisoning. TCP was tested alone as a pretreatment or in combination with atropine as a therapy in rats poisoned with ¼ and ½ of LD50 of soman. Possible genotoxic effects of TCP in white blood cells and brain tissue were also studied. Results were compared with previous findings on the adamantyl tenocyclidine derivative TAMORF. TCP given alone as pretreatment, 5 min before soman, seems to be superior in the protection of cholinesterase (ChE) catalytic activity in the plasma than in brain, especially after administration of the lower dose. Plasma activities of enzyme significantly increased as compared to corresponding controls 30 min (P < 0.001) and 24h (P = 0.0043). TCP and atropine, given as therapy, were more effective than TCP administered solely as a pretreatment. Activities of enzyme significantly increased as compared to controls 30 min (P = 0.046) and 24h (P < 0.001) when ¼ LD50 dose of soman was administered. Using the alkaline comet assay acceptable genotoxicity of TCP was observed. However, controversial role of TCP in brain protection of soman-poisoned rats should be further studied.

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still very important task. Organophosphates-nerve agents are characterized as compounds influencing cholinergic nerve transmission via inhibition of acetylcholinesterase (AChE), an extremely active enzyme that hydrolyses acetylcholin. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsions and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Pyridinium oximes are nowadays used as successful treatment of poisoning with many organophosphates. Reason for that lies in the mechanism of oxime activity, which is based on protection of unphosphorylated AChE and/or reactivation of phosphorylated AChE. The aim of this investigation was to define the biochemical parameters of three bispyridinium oximes K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K033 [1, 4-bis (2-hydroxyiminomethylpyridinium) butane dibromide] and K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide]. For each of the compounds it was determined the toxic effect on human erythrocytes AChE in vitro (IC50) and their reactivating (%R) and protective potency (P50) with respect to soman- and tabun- inhibited AChE. Earlier we described in vivo antidotal activity of tested oximes against soman and tabun in male mice. The new tested oximes were toxic to human erythrocytes AChE ; in decreasing order from K033, K048 to K027. IC50 ranged from 0.02 to 1.0 mM. The best reactivating potency was obtained with K048 when AChE was inhibited by tabun. The protective potency of all oximes on human erythrocytes AChE inhibited by soman and tabun could not be determined. Our results indicate a good affinity of the tested compounds for AChE. The oximes are reversible ihibitors of AChE in vitro and show pharmacological properties which are related to reactivation of AChE.

Paraoxonase (PON1) is serum esterase which is synthesized in the liver, and has unknown physiological function. PON1 has a clinical importance, because it plays a protective role in case of organophosphates intoxication. According to the many experimental data PON1 is included in lipid metabolism. It is suggested that PON1 is associated with high density (HDL)-cholesterol, and that HDL stimulates PON1 secretion from the liver. Also it has been shown that PON1 prevents the formation of oxidized LDL and protects phospholipids in HDL from oxidation. Hyperlipidemia and oxidative stress reduce PON1 activity. It has been suggested that statins which are used for the treatment of hyperlipidemia, can decrease or increase serum PON1 activity, and that this is the consequence of another, independent effect of statins, i.e. their antioxidant action. Because the most of these results were goten from human and animals in conditions of altered lipid’ s metabolism, it was of interest to investigate the effects of simvastatin (SIMV) and atorvastatin (ATOR) on PON1 activity in serum and liver of normolipidemic rats. Material and Methods. Forty six male Wistar rats (weight 170-200 g), were divided in two control groups (n=7 each) and four experimental groups. Two experimental groups were on SIMV treatment and the remaining two groups (n=8) on ATOR treatment. Both agents were given orally in the same doses (10 and 50 mg/kg/day) and for the period of 3 weeks. At the end of drug treatment animals from all groups were sacrificed. PON1 activity in plasma and liver was measured by the spectrophotometric method using synthetic diethyl-p-nitrophenyl phosphate (paraoxon) and moderator CaCl2. The activity toward paraoxon was determined by measuring the initial rate of substrate hydrolysis to p- nitrophenol. The plasma or liver enzyme activity was calculated from E405 of p-nitrophenol and expressed as μ mol of substrate hydrolysed per min per ml of serum or g of the tissue wet weight. Concentration of HDL-cholesterol and TGs in plasma were determined by enzymatic colorimetric methods and expressed as mmol/L of plasma. Data were analyzed by Kruskal-Wallis test and results were considered as significant with p

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both, the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of the intoxication with different chemicals or drugs that inhibits activity of enzyme. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. In this study we evaluated the in vitro efficacy of oxime HI-6 to protect and/or reactivate human erythrocyte AChE inhibited by anticancer drug irinotecan, which cause acute cholinergic syndrome after its administration. Moreover, using alkaline comet assay possible genotoxic effects of HI-6 were evaluated on human peripheral blood leukocytes. HI-6 increases the activity of AChE to 30% ; residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme by 40% when applied in a concentration of ¼ of the IC50 value. None of the tested concentrations of HI-6 was genotoxic as estimated by evaluation of three main comet parameters: tail length, tail intensity and tail moment. The results obtained sustain the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime found as the most effective reactivators have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE and studied their effects on tabun-poisoned mice. Using molecular mechanics we performed conformational analysis to determine the flexibility of the oxime molecules. Tabun-inhibited human erythrocyte AChE was completely reactivated by three flexible bispyridinium oximes with the oxime group in para-position and with the linker of three or four methylene groups. Although oximes with imidazolium ring or with the oxime group in the ortho-position on the pyridinium ring had similar electron density on the oxygen of the oxime group as para-bispyridinium oximes, they did not show significant reactivation potency. However, as inhibitors they had higher affinity for the native human erythrocyte AChE. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Without pretreatment the best antidotal efficacy was obtained with para-oximes that protected mice from 5-fold LD50 of tabun. Pretreatment by the oxime insured survival of all tested animals after the application of 10 LD50 of tabun. Herein we show that already promising treatment in tabun poisoning by oximes and atropine could be improved if oximes are also used in pretreatment. Furthermore, low toxicity of these oximes makes these compounds leading in the therapy of tabun poisoning. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both, the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of the intoxication with different chemicals or drugs that inhibits activity of enzyme. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. In this study we evaluated the in vitro efficacy of oxime HI-6 to protect and/or reactivate human erythrocyte AChE inhibited by anticancer drug irinotecan, which cause acute cholinergic syndrome after its administration. Moreover, using alkaline comet assay possible genotoxic effects of HI-6 were evaluated on human peripheral blood leukocytes. HI-6 increases the activity of AChE to 30% ; residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme by 40% when applied in a concentration of ¼ of the IC50 value. None of the tested concentrations of HI-6 was genotoxic as estimated by evaluation of three main comet parameters: tail length, tail intensity and tail moment. The results obtained sustain the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both, the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of the intoxication with different chemicals or drugs that inhibits activity of enzyme. Different substances, pyridinium or bispyridinium aldoximes (oximes) among them, are able for the recovery of inhibited enzyme. Since their adverse effects are not well elucidated, in this study the efficiency of HI-6 oxime in protection and/or reactivation of human erythrocyte (AChE) inhibited by antineoplastic drug irinotecan as well as its cyto / genotoxicity in vitro were investigated. HI-6 increases the activity of AChE to 30% ; residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme previously inhibited by irinotecan (4.6 mg/ml) by 50% when applied in a concentration of ¼ of the IC50 value. Tested concentrations of HI-6 exhibited acceptable genotoxicity on white blood cells, as estimated by the alkaline comet assay, DNA diffusion assay and cytogenetic endpoints (structural chromosome aberrations and cytokinesis-block micrunucleus assay). The results obtained sustain the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

Literature data on carbofuran genotoxicity in vitro and in vivo are very scarce. There are few papers indicating that occupational exposure to this AChE inhibiting insecticide might be connected to increased risk of developing non-Hodgkin’ s lymphoma and lung cancer. Other authors showed its genotoxicity in vitro. We used comet and CBMN micronucleus assay combined with centromere probes to evaluate genome damage in lymphocytes of workers employed in carbofuran production. Also, the level of AChE activity in blood and plasma was measured. Only few workers exhibited AChE activity below 85%. Comet assay parameters were slightly but significantly elevated compared to control subjects, especially the long-tailed nuclei ratio. We found poor correlation between AChE activity and comet assay parameters, but significant effect of smoking and alcohol intake on the latest. In binucleated lymphocytes of workers significantly increased number of micronuclei, nuclear buds, and nucleoplasmatic bridges was detected. Proportion of micronuclei with centromere, DAPI signal positive micronuclei was also elevated. Micronucleus assay parameters also appeared to be significantly influenced by duration of exposure to carbofuran. Together with published data on carbofuran’ s effect on health our results might indicate the need for further evaluations of its genotoxicity using a range of different cytogenetic techniques.

Tenocyclidine - TCP presents a broad spectrum of pharmacological activity including antidotal effect in organophosphorus compounds poisoning, radioprotective and anticancer effects. In present study in vitro interactions of TCP and its adamantane derivative - TAMORF with human erythrocyte acethylcholinesterase (AChE) were investigated. The genotoxicity and possible radioprotective activity on human white blood cells were assessed by the alkaline comet assay, viability testing and the analysis of the structural chromosome aberrations. The tested compounds were found to be weak inhibitors of AChE, for TCP IC50 =1x10-5 M and for TAMORF IC50 >1x10-3 M, without reactivating and protective effects on AChE inhibited by soman. Results indicate that TCP modified by the replacement of the cyclohexyl ring with an adamantly ring and piperidine with morpholine group (TAMORF) have lower toxicity. Both compounds have low cytotoxicity and radioprotective activity, but TAMORF also demonstrates cell growth inhibitory effects. To elucidate variations in their biological effectiveness observed in vitro and in vivo, additional analyses are needed. Since TAMORF was found to significantly inhibit cell growth and proliferation in vitro, it seems logically to consider it as a source molecule promising for additional modifications and development of more potent compounds with antitumor properties rather then radioprotector or antidote in organophosphorus poisoning.

Organophosphorus compounds are widely used as pesticides and unfortunately as nerve agents in chemical warfare. They are known inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7) an enzyme that hydrolizes the neurotransmitter acetylcholine in the nervous system. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsion and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many organophosphorus compounds. In this paper three new bis-pyridinium compounds: K033 [1, 4-bis(2-hydroxyiminomethylpyridinim)butane dibromide], K027 [1, 4-hydroksyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as potential antidotes in tabun poisoned mice. Their antidotal effect was compared with TMB-4 [1, 3-bis(4-hydrxyiminomethylpyridinium) propane dibromide], which is the best-known antidote in tabun poisoning. In all experiments, oxime K033 in doses of 1/4 or 5% of its LD50 was used for the pre-treatment 15 minutes before tabun-intoxication. Also, one or 5 minutes after tabun application experimental animals received oxime K027, K033 or K048 (5% or 1/4 of its LD50) plus atropine sulphate as therapy. The antidotal efficacy of tested compounds was expressed as therapeutic factor (TF) and therapeutic dose (TD). Under same experimental conditions, our experiment selected compound K048 as the most reactivator of tabun inhibited AChE. Namely, this study has shown that the therapeutic regimen consisting of K033 in dose 5% of its LD50 as preatretment and &frac14 ; of LD50 of K048 plus atropine as treatment had the highest TF and TD. The TF was 13.3 LD50 of tabun, TD was 10 LD50 of tabun and insurance survival of all tested animals. In conclusion, treatment with these new bis-pyridinium oximes seems to be a very good alternative for current treatment in tabun poisoning. For this reason, these and other similar compounds require further investigation.

Acetylcholinesterase (AChE: EC 3.1.1.7.) is an extremely active enzyme necessary for terminating the action of acetylcholine in cholinergic synapses. Inhibition of AChE enzyme by organophosphorus compounds (pesticides, nerve agents in chemical warfare) leads to an accumulation of neurotransmitter acetylcholine (ACh) resulting in an over-stimulation of the whole cholinergic system. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsion and seizures. Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many organophosphorus compounds. In this paper new bis-pyridinium oxime K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] was tested as potential antidote in vitro using human erythrocyte AChE inhibited by tabun and in vivo using tabun poisoned mice. Its antidotal effect was compared with TMB-4 [1, 3-bis (4-hydrxyiminomethylpyridinium) propane dibromide], which is the best-known antidote in tabun poisoning. K048 was poor inhibitor of human erythrocyte AChE in vitro (its IC50 value could not be determined). Its protective potency expressed as P50 was 3.3 x 10-3 M, and the reactivating potency (applied in final concentration of 1.4 x 10-3 M) was 98%. In all experiments on mice, oxime doses of ¼ or 5% of its LD50 were used for pre-treatment 15 minutes before tabun-intoxication and for treatment together with atropine one minute after tabun administration. The antidotal efficacy of tested compound was expressed as therapeutic factor (TF) and therapeutic dose (TD). The highest TF and TD (22.5 and 10.0, respectively) were obtained when K048 was used in dose of ¼ of its LD50 in both, pre-treatment and treatment with atropine. Herein we showed that already promising treatment in tabun poisoning by oximes and atropine could be improved if oximes are also used in pre-treatment. Low acute toxicity of K048 ; about three times lower than of TMB-4, makes it interesting for further investigation of tabun poisoning treatment. K048 may even provide a platform for further modifications and development of more potent protectors and reactivators in organophosphorus poisoning.

U Zborniku radova seminara DDD i ZUPP iz 2004. godine detaljnije smo predstavili Direktivu o biocidnim pripravcima (98/8/EC) kojom se u Europskoj Uniji uređuje proces registracije i stavljanja u promet biocidnih pripravaka. Za aktivne tvari u biocidnim pripravcima koje su već bile na tržištu prije stupanja na snagu Direktive o biocidima (14. 5. 2000.) predviđeno je da se na nivou EU provede proces procjene sigurnosti/djelotvornosti prije nego se uvrste na popise dozvoljenih aktivnih tvari, poznate kao Annex I, IA i IB. Nakon dovršene prve faze programa revizije, 2003. je u drugoj Uredbi o reviziji objavljen popis aktivnih tvari koje su obuhvaćene procesom identifikacije i notifikacijete kao takove ostaju na tržištu u desetogodišnjem periodu revizije, kao i popis aktivnih tvari koje se u određenim rokovima povlače s tržišta EU. Europska komisija također je objavila i Prvi zajednički izvještaj o provođenju odredbi direktive u 15 zemalja članica za razdoblje od 2000.d0 2003. Hrvatska, kao zemlja kandidat za članstvo u Uniji, u prijelaznom periodudo potpunog prihvaćanja zakonodavstva EU, može kao najjednostavnije rješenje zadržati postojeći sustavstavljanja biocida u promet, odnosno zadržati Zakon o otrovima. Ujedno je potrebno da se, čim se donese novi hrvatski Zakon o biocidima (i zakon o opasnim tvarima) počnu postupno primjenjivati i neke njegove odredbe, uz istovremenu prilagodbu i razvoj sustava koji će omogućiti njihovo potpuno provođenje.

Enzim acetilkolinesteraza (AChE) je izravni biomarker toksičnog djelovanja antikolinesteraznih spojeva kojima pripadaju brojni pesticidi po kemijskoj strukturi organofosforni spojevi i karbamati. Određivanje aktivnosti AChE u krvi radnika profesionalno izloženih antikolinesteraznim şpojevima jedina je mjera koja ukazuje na izloženost radnika tim spojevima, i način na koji se može uočiti potencijalna opasnost te spriječiti daljnja izloženost. Kroz petogodišnje razdoblje obrađeno u ovoj studiji radnici su kontrolirani jednom godišnje što svakako nije dovoljno s obzirom na nalaze koje smo našli kod određenog broja radnika. U njih su uz karakteristične simptome otrovanja, aktivnosti AChE pune krvi bile inhibirane 30% ili više. Međutim, pritužbe radnika nisu uvijek bile u skladu s inhibicijom enzima. Naime, u nekoliko osoba u kojih su bile prisutne subjektivne smetnje, inhibicija AChE nije bila značajno smanjena. Uvidom u ranija mjerenja i praćenja izloženosti radnika antikolinesteraznim spojevima u proizvodnji pesticida uočeno je da se kontrolom mjerenja aktivnosti AChE više puta unutar jedne godine može smanjiti broj otrovanih radnika i stupanj otrovanja. Također, upotrebom propisanih zaštitnih sredstava pri radu, i pridržavanjem higijenskih i sanitarnih odredaba moglo bi se pridonijeti još boljem stupnju zaštite radnika u proizvodnji pesticida.

Pesticidi su skupina potencijalno visoko - toksičnih proizvoda, kod kojih su mjere prevencije otrovanja najučinkovitije, jer se adekvatnim pakiranjem, skladištenjem, uporabom zaštitnih sredstava pri radu i konačno sigurnim zbrinjavanjem otpada mogu djelotvorno spriječiti štetni učinci na ljudsko zdravlje i okoliš. Osim ovih poznatih preventivnih mjera u Hrvatskoj se vrlo djelotvorna pokazala i tzv. primarna prevencija koja uključuje izdavanje dozvole za promet samo onim proizvodima za koje je detaljnim ispitivanjima potvrđena najmanja moguća škodljivost pri uporabi i minimalna izloženost opće populacije, odnosno povlačenje iz uporabe, drastično ograničenje i zabrana visokotoksičnih preparata za koje je procijenjeno da predstavljaju neprihvatljiv rizik za čovjeka i okoliš. Zbog toga su danas sve rjeđa otrovanja spojevima kao što su parakvat, lindan, dinitro-o-krezol (DNOC) ili organska živa, a u smanjenju je i broj otrovanja organofosfornim esterima, spojevima bakra, pa čak i antikoagulantnim rodenticidima, ako se promatraju samo otrovanja s pojavom težih simptoma. Agrokemijska industrija prati zakonske zahtjeve za sigurnim i niskotoksičnim pesticidima te na tržištu ima sve novijih proizvoda koji su sigurni za rukovanje i niskog rizika čak i kod slučajne ili namjerne ingestije visokih doza.

Like organophosphosphate insecticides, nerve agents phosphorylate and inactivate acetylcholinesterase (AChE), leading to accumulation of acetylcholine at nicotinic and muscarinic receptors, and other receptors in the central nervous system (CNS). Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many OPs. The aim of this study was to evalute the efficacy of three new bis-pyridinium compounds: [1-(4-hydroxyiminomethyl pyridinium)-3-(4-carbamoyl pyridinium)] propane dibromide ( K 027), [1-(4- hydroxyiminomethyl pyridinium)-4-(4-carbamoylpyridinium)] butane dibromide (K 048) and [1, 4-bis (2-hydroxyiminomethyl pyridinium)] butane dibromide (K 033), in combination with atropine in the therapy of soman intoxication in mice in vivo. Their acute intraperitoneal (i.p.) toxicity (LD50 with 95% confidence limits) was tested and observed for 24 hours. The therapeutic effect was expressed as the therapeutic factor (TF) with 95% confidence limits and as the therapeutic dose (TD). In vivo results show that the tested compounds are relatively toxic (their LD50 was from 33.4 to 672.8 mg/kg body weight). Generally, in vivo toxicity of these compounds and their antidotal efficacy expressed as the TF, TD and the ratio between dead and injected experimental animals were depended of type of the substituent in the pyridinium ring, and chains between pyridinium moieties.

The increased concern about terrorist use of nerve agents prompted us to search for new, more effective oximes against tabun and soman poisoning. We investigated the interactions of five bispyridinium oximes: K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide], K033 [1, 4-bis(2-hydroxyiminomethylpyridinium) butane dibromide], TMB4 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) propane dibromide] and HI-6 [(1-(2’ -hydroxyiminomethyl-1'-pyridinium)-3-(4''-carbamoyl-1''-pyridinium)-2-oxapropane dichloride)] with human erythrocyte acetylcholinesterase (AChE ; E.C. 3.1.1.7) and their effects on tabun and soman poisoned mice. All the oximes reversibly inhibited AChE, and the enzyme-oxime dissociation constants were between 17 and 180  M. Tabun-inhibited AChE was completely reactivated by TMB4, K027 and K048, with the overall reactivation rate constants of 311, 381 and 659 min-1M-1, respectively. The reactivation of tabun-inhibited AChE by K033 reached 50 % after 24 hours, while HI-6 failed to reactivate any AChE at all. Soman-inhibited AChE was resistant to reactivation by 1 mM oximes. All studied oximes protected AChE from phosphorylation by both soman and tabun. Our results obtained from in vivo experiments showed that the studied oximes were relatively toxic to mice ; K033 was the most toxic (LD50=33.4 mg/kg), while K027 was the least toxic (LD50=672.8 mg/kg). The best antidotal efficacy was obtained with K048, K027 and TMB4 for tabun poisoning, and HI-6 for soman poisoning. Therefore, the potency of the oximes to protect mice from 5-fold LD50 of tabun makes these compounds important in the therapy of tabun poisoning. Whatever its shortcomings, the combination of HI-6 and atropine has been the therapy of choice for soman poisoning.

This paper describes effect of glucocorticoide dexamethasone (DM) given intraperitoneally on the catalytic activity of buthyrylcholinesterase (BuChE) measured in plasma, liver and white adipose tissue of rats of both sexes. The effects of DM on the concentration of plasma lipids and lipoproteins were also tested. The rats were given multiple (2 and 4) pharmacological doses (0.4 and 3.0 mg kg-1 body mass) of DM. All animals were sacrificed 48 hours after the last dosing. The administration of DM significantly decreased the catalytic activity of BuChE in plasma and liver of all treated groups regardless of the sex. BuChE catalytic activity in white adipose tissue differed depending on dose and frequency of administration. In contrast to liver where both doses caused significant BuChE inhibition, in adipose tissue the lower DM dose did not inhibit BuChE activity, and the inhibition achieved by the higher dose was not as strong as in liver. This result corroborates an earlier hypothesis that BuChE is also synthesized in the adipose tissue. DM significantly increased plasma concentrations of triglycerides, total cholesterol and high-density lipoprotein (HDL) cholesterol and decreased low-density lipoprotein (LDL) cholesterol concentration. There was neither positive correlation between BuChE and triglycerides nor negative correlation between BuChE and HDL. Changes in lipid profile during DM treatment were not sex- and time-dependent.

Acethylcholinesterase (AChE) is an extremely active enzyme. Irreversible inhibition of AChE by organophosphorus compounds (OPs) results in the accumulation of endogenous acetylcholine in synaptic cleft and paralysis of nerve impulse transmission in the central and peripheral nervous system. In order to find the best treatment of organophosphate poisoning new compounds have been synthesized and tested. The aim of this study was to evalute the pretreatment efficacy of adamantyl tenocyclidine derivative TAMORF which antagonize the NMDA receptors and might protect AChE. Its antidotal effect was compared with physostigmine which also has a good prophylactic efficacy in soman poisoning. The therapeutic efficacy of TAMORF and physostigmine was tested on rats poisoned with two different sub-lethal dose of soman (&frac14 ; ; and &frac12 ; ; of LD50). Our results indicate that TAMORF when administered 5 min before intoxication clearly stopped soman-induced seizures and markedly improved respiration of animals. In contrast to TAMORF, physostigmine seems to be slightly effective in the elimination of soman-induced toxicity in rats. Catalytic activities of AChE in brain, especially after administration of higher dose of soman (&frac12 ; ; of LD50) were additionally lower throughout of the experiment, except after 24 h. Similar results with physostigmine in contrast to TAMORF were obtained in plasma also. In conclusion, treatment with TAMORF seems to be a good alternative for current pretreatment in soman intoxication.

Ochratoxin A (OTA) is a mycotoxin produced by several Aspergillus and Penicillium species. It is known as a worldwide common food contaminant. This toxin has received considerable attention because of its deleterious effects on human and animal health. It has immunotoxic, hepatotoxic, teratogenic, neurotoxic and carcinogenic properties in different experimental animals, with the kidney being its main target organ. The role of OTA as a possible causative agent involved in the etiology of human Endemic nephropathy has still not been ruled out. To study adverse effects of OTA on cellular level, three renal cell lines (CV-1, Hek293, LLC-PK1) were cultivated and exposed to the different OTA concentrations, ranging from 0.1 to 50 μ M, respectively. Metabolic activity was measured by using thiazolyl-tetrazolium bromide mitochondrial dehydrogenase activity test (MTT) over 72 hours. Remaining cell viability after treatment with 20 μ M OTA amounted to 18.2 % for CV-1 and 64.9 % for LLC-PK1. The same OTA concentration induced cell death of almost 43 % Hek293 cell population. Induction of apoptotic and necrotic cell death was measured by anexin V cell labeling and flow cytometry analysis after being exposed to treatment in the same OTA concentration range over 24 hours. Results suggest to apoptosis and necrosis induction in OTA dose dependent manner. Ochratoxin A (OTA) is a mycotoxin produced by several Aspergillus and Penicillium species. It is known as a worldwide common food contaminant. This toxin has received considerable attention because of its deleterious effects on human and animal health. It has immunotoxic, hepatotoxic, teratogenic, neurotoxic and carcinogenic properties in different experimental animals, with the kidney being its main target organ. The role of OTA as a possible causative agent involved in the etiology of human Endemic nephropathy has still not been ruled out. To study adverse effects of OTA on cellular level, three renal cell lines (CV-1, Hek293, LLC-PK1) were cultivated and exposed to the different OTA concentrations, ranging from 0.1 to 50 μ M, respectively. Metabolic activity was measured by using thiazolyl-tetrazolium bromide mitochondrial dehydrogenase activity test (MTT) over 72 hours. Remaining cell viability after treatment with 20 μ M OTA amounted to 18.2 % for CV-1 and 64.9 % for LLC-PK1. The same OTA concentration induced cell death of almost 43 % Hek293 cell population. Induction of apoptotic and necrotic cell death was measured by anexin V cell labeling and flow cytometry analysis after being exposed to treatment in the same OTA concentration range over 24 hours. Results suggest to apoptosis and necrosis induction in OTA dose dependent manner.

Although the basic mechanisms of toxicity of the highly toxic nerve agents has been known for a long time, there are still major problems with the effective treatment of acute poisoning by them, especially by soman. The objective of this study was to evaluate the efficacy of tamorf (a new synthesized adamantyl derivative containing morpholine group) on rats poisoned with soman. In this study rats given subcutaneously &frac14 ; ; and &frac12 ; ; LD50 of soman were treated intraperitoneally with tamorf (2.5 mg/kg b.w.) before or after soman injection. Catalytic activity of acethylcholinesterase (AChE) was measured in plasma and brain at different time periods from 10 minutes up to 24 hours after application of soman. Tamorf was applied alone or together with atropine sulphate (10.0 mg/kg b.w.). The best effect of tamorf in rats given &frac14 ; ; LD 50 of soman was obtained when it was given as pre-treatment together with atropine. Treatment with tamorf increased the catalytic activity of AChE in plasma and brain of all soman poisoned rats, except when given up to 10 minutes after &frac12 ; ; LD 50 of soman. Our results suggest that the treatment with tamorf together with atropine antagonise the acute toxicity of soman, and could be supplementary therapy in soman poisoning.

Osnovna namjera s kojom je Europski Parlament i Vijeće Europe 16. 2. 1998. donio Direktivu 98/8/EC, poznatu kao Direktiva o biocidnim pripravcima, bila je stvaranje jedinstvenog zakonskog okvira koji regulira sve aspekte stavljanja u promet biocidnih pripravaka, sa svrhom prvenstveno osiguravanjavisoke razine sigurnosti za zdravlje ljudi i okoliš, ali ujedno i omogućavanje pravilnog funkcioniranja zajedničkog europskog tržišta. Direktiva je opsežan dokument koji u 35 članakai za sada dovršenih 5 Priloga, uređuje proces registracije i stavljanja u promet biocidnih pripravaka, uzajamno priznavanje registracija unutar EU i uspostavlja pozitivnulistu aktivnih tvariza biocidne pripravke. Kako je u novi hrvatski Zakon o kemikalijama ugrađeno desetak odredbi koje se mogu tumačiti kao pokušaj usklađivanja s Direktivom o biocidnim pripravcima, osobito je važno da donositelji zakona i stručna javnosto tome imaju dovoljno informacija kako bi mogli kritički i odgovorno procijeniti najbolji način implementacije odredbi Direktive u Hrvatskoj. Pri tome treba težiti ne samo da se tekst Direktive mehanički ugradi u naše zakone nego omogućiti njegovu provedbu i predvidjeti sve povoljne i nepovoljne utjecaje na ekonomske i tržišne tokove u zemlji u ovom segmentu. Svrha ovog pregleda je prikaz nekih važnijih odredbi Direktive o biocidnim pripravcima i procesa revizije aktivnih tvari koji je započeo u EU, sa ciljem njihovog boljeg razumijevanja uključujući i učinke primjene u Hrvatskoj.