Comparative determination of the efficacy of bispyridinium oximes in paraoxon poisoning.

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.

Evaluation of the cyto/genotoxicity profile of oxime K048 using human peripheral blood lymphocytes: an introductory study.

This study investigates the effects of oxime K048 (730, 200, and 7.3nM) on the viability and chromosome stability of human peripheral blood lymphocytes (PBLs) after a 30min exposure in vitro. Cytotoxicity was tested by a viability assay with ethidium bromide and acridine orange. For the evaluation of the genotoxic potential, we used comet assays, cytokinesis-blocked micronucleus (CBMN) assay, and chromosome aberration (CA) analysis. We found acceptable cytotoxicity for K048 (9.7±2.1% non-viable PBL at highest concentration vs. 7.3±2.5% in control; apoptosis dominated over necrosis). Overall primary DNA damage was low and not significantly different from controls. The hOGG1-comet assay showed a slight increase in the level of oxidative DNA damage. In oxime treated PBLs, we found 13-19 MN compared to 15 MN in control cultures. The frequencies and types of CA in oxime-treated PBLs did not significantly differ from controls. K048 showed acceptable biocompatibility at the level of cell viability and chromatin/chromosome integrity. Since no increase in secondary genome damage was detected, the primary DNA lesions may have resulted from treatment-induced cell stress, subsequently becoming repaired and not fixed as chromosome aberrations. The toxicity profile of K048 should be further studied and compared with other clinically relevant oximes.

Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes.

In the present paper we show a comprehensive in vitro, ex vivo and in vivo study on hydrolytic detoxification of nerve agent and pesticide OPs (organophosphates) catalysed by purified hBChE (human butyrylcholinesterase) in combination with novel non-pyridinium oxime reactivators. We identified TAB2OH (2-trimethylammonio-6-hydroxybenzaldehyde oxime) as an efficient reactivator of OP-hBChE conjugates formed by the nerve agents VX and cyclosarin, and the pesticide paraoxon. It was also functional in reactivation of sarin- and tabun-inhibited hBChE. A 3-5-fold enhancement of in vitro reactivation of VX-, cyclosarin- and paraoxon-inhibited hBChE was observed when compared with the commonly used N-methylpyridinium aldoxime reactivator, 2PAM (2-pyridinealdoxime methiodide). Kinetic analysis showed that the enhancement resulted from improved molecular recognition of corresponding OP-hBChE conjugates by TAB2OH. The unique features of TAB2OH stem from an exocyclic quaternary nitrogen and a hydroxy group, both ortho to an oxime group on a benzene ring. pH-dependences reveal participation of the hydroxy group (pKa=7.6) forming an additional ionizing nucleophile to potentiate the oxime (pKa=10) at physiological pH. The TAB2OH protective indices in therapy of sarin- and paraoxon-exposed mice were enhanced by 30-60% when they were treated with a combination of TAB2OH and sub-stoichiometric hBChE. The results of the present study establish that oxime-assisted catalysis is feasible for OP bioscavenging.

Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases.

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.

A conjugate of pyridine-4-aldoxime and atropine as a potential antidote against organophosphorus compounds poisoning.

A conjugate of pyridine-4-aldoxime and atropine (ATR-4-OX) was synthesized and its antidotal efficiency was tested in vitro on tabun- or paraoxon-inhibited acetylcholinesterase (AChE) of human erythrocytes as well as in vivo using soman-, tabun- or paraoxon-poisoned mice. Its genotoxic profile was assessed on human lymphocytes in vitro and was found acceptable for further research. ATR-4-OX showed very weak antidotal activity, inadequate for soman or tabun poisoning. Conversely, it was effective against paraoxon poisoning both in vitro and in vivo. All animals treated with 5 % or 25 % LD(50) doses of the new oxime survived after administration of 10.0 or 16.0 LD(50) doses of paraoxon, respectively. Based on the persistence of toxicity symptoms in mice, the atropine moiety had questionable effects in attenuating such symptoms. It appears that ATR-4-OX has a therapeutic effect related to the reactivation of phosphylated AChE, but not to receptor antagonization.

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, ß oxidation, and production of H2O2, it is possible that the oxidative stress induced by GEM damages hepatocytes and lowers the synthesis of PON1.

In vivo experimental approach to treatment against tabun poisoning.

Organophosphorus compounds pose a potential threat to both military and civilian populations. Since post-exposure therapy has its limitations, our research was focused on the possibility of improving pretreatment in order to limit the toxic effects of tabun. We determined the protective index of various combinations of atropine, oximes (K074, K048, and TMB-4), and pyridostigmine given to mice before tabun intoxication. Although the tested oximes showed very good therapeutic efficacy in tabun-poisoned mice, the given pretreatments improved therapy against tabun poisoning. These regimens ensured survival of all animals up to 25.2 LD(50) of tabun. Our results indicate that even pretreatment with atropine alone is sufficiently effective in enhancing the survival of mice poisoned by multiple doses of tabun, if oxime therapy follows. K048 is our oxime of choice for future research, as it shows better protective and reactivating potency.

The antidotal efficacy of the bispyridinium oximes K027 and TMB-4 against tabun poisoning in mice.

A toxic effect of highly toxic nervous agents is irreversible inhibition of vitally important enzyme acethylcholinesterase (AChE). Inhibition of AChE results in accumulation of acetylcholine (ACh) at the synaptic cleft of the cholinergic neurons, 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. In this paper a bispyridinium compound K027 [1-(4-hydroxyiminomethylpyridinium)-3-(-4-carbamoylpyridinium) propane dibromide] was tested as potential antidote in tabun poisoned mice. Oxime TMB-4 was included for comparison. The therapeutic efficacy of applied antidotal regimens was tested as pretreatment given 15 min before tabun poisoning and/or as therapy given 1 min after tabun poisoning. Using oxime K027 (25% of its LD(50)) plus atropine as both, pretreatment and therapy, we showed that this combination can protect mice 8 times better than the therapy alone. Under these experimental conditions we confirmed good antidotal efficacy of K027. Moreover, its low acute toxicity is as much as beneficial effect in contrast to high toxicity of currently used TMB-4.

Effect of occupational exposure to multiple pesticides on translocation yield and chromosomal aberrations in lymphocytes of plant workers.

Employees handling pesticides are simultaneously exposed to different active substances. Occurring multiple chemical exposures may pose a higher risk than it could be deduced from studies evaluating the effect of a single substance. This study comprised 32 pesticide plantworkers exposed to carbofuran, chlorpyrifos, metalaxyl, and dodine and an equal number of control subjects. Groups were matched by age (43.8 +/- 10.16 vs 41.8 +/- 7.42, respectively), sex (14 females; 18 males), and smoking (11 smokers; 21 nonsmokers). Chromosome aberration and translocation frequencies were determined using a standard aberration assay and fluorescent in situ hybridization (FISH) by applying painting probes for chromosomes 1, 2, and 4. Although significant, an observed increase in chromatid breaks (5.2 +/- 2.49) compared to controls (2.1 +/- 0.87), p(PostHoc) = 0.000001 is biologically irrelevant. Genomic frequency of translocations was also significantly elevated (exposed 0.0165 +/- 0.0070; control 0.0051 +/- 0.0023, P(PostHoc) = 0.000004). The distribution of translocations among chromosomes 1, 2, and 4 did not differ from control subjects. It corresponded to the distribution of DNA content among selected chromosomes indicating randomness of DNA damage. A good translocation yield correlation within years spent in pesticide production indicates that multiple pesticide exposure may pose a risk to genome integrity. However, for more accurate health risk assessments, the use of probes for some other groups of chromosomes should be considered.

Irinotecan side effects relieved by the use of HI-6 oxime: in vivo experimental approach.

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.

Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro.

In the present study we evaluated the genotoxic and oxidative potential of glyphosate on human lymphocytes at concentrations likely to be encountered in residential and occupational exposure. Testing was done with and without metabolic activation (S9). Ferric-reducing ability of plasma (FRAP), thiobarbituric acid reactive substances (TBARS) and the hOGG1 modified comet assay were used to measure glyphosate's oxidative potential and its impact on DNA. Genotoxicity was evaluated by alkaline comet and analysis of micronuclei and other nuclear instabilities applying centromere probes. The alkaline comet assay showed significantly increased tail length (20.39 microm) and intensity (2.19%) for 580 microg/ml, and increased tail intensity (1.88%) at 92.8 microg/ml, compared to control values of 18.15 mum for tail length and 1.14% for tail intensity. With S9, tail length was significantly increased for all concentrations tested: 3.5, 92.8, and 580 microg/ml. Using the hOGG1 comet assay, a significant increase in tail intensity was observed at 2.91 microg/ml with S9 and 580 microg/ml without S9. Without S9, the frequency of micronuclei, nuclear buds and nucleoplasmic bridges slightly increased at concentrations 3.5 microg/ml and higher. The presence of S9 significantly elevated the frequency of nuclear instabilities only for 580 microg/ml. FRAP values slightly increased only at 580 microg/ml regardless of metabolic activation, while TBARS values increased significantly. Since for any of the assays applied, no clear dose-dependent effect was observed, it indicates that glyphosate in concentrations relevant to human exposure do not pose significant health risk.

Evaluation of oxime k203 as antidote in tabun poisoning.

We studied bispyridinium oxime K203 [(E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-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 efficient 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 (K(i) = 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.

Cholinesterase-inhibiting and genotoxic effects of acute carbofuran intoxication in man: a case report.

Carbofuran belongs to the group of N-methylcarbamate insecticides used for the control of soil-dwelling and foliar-feeding insects in various crops; its consumption totals approximately 20,000 tonnes per year. Although the neurological effects on human beings have been well documented, little is known on its impact on the genome. A 38-year-old, healthy male worker employed in a carbofuran production facility accidentally inhaled the dust of the active ingredient carbofuran. Thirty minutes later, he experienced weakness, fatigue, perspiration, breathing difficulties, cephalalgia, disorientation, abdominal pain and vomiting. Blood samples were taken to measure cholinesterase activity, and to perform the alkaline comet assay and micronucleus assay combined with pancentromeric probes. Analyses were repeated 72 hr after intoxication and compared with the results obtained from regular monitoring conducted 10 days prior to the accident. Cholinesterase activity showed the highest correlation with the number of apoptotic cells, comet assay tail length, and number of long-tailed nuclei, suggesting that these are the genomic end-points primarily affected by carbofuran intake. Only a weak correlation was detected for the total number of micronuclei, centromere-containing micronuclei and nuclear buds. Since those end-points increased significantly 72 hr after the accident, they could be considered as late biomarkers of the effects of carbofuran intoxication. The results of this report suggest that, in the interests of higher standards in risk assessment and health hazard protection, periodical medical examination of carbamate-exposed populations should include genotoxicity testing in addition to the assessment of cholinesterase activity.

New bispyridinium oximes: in vitro and in vivo evaluation of their biological efficiency in soman and tabun poisoning.

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still a challenge for the scientific community. This study investigated the interactions of four bispyridinium oximes with human erythrocyte acetylcholinesterase (AChE) and their effects on soman- and tabun-poisoned mice. Oximes HI-6 and TMB-4 were used for comparison. These oximes inhibited AchE with inhibitory potency (IC(50)) ranging from 0.02 to 1.0 mM. The best reactivating potency (%R) was obtained with K074, when AChE was inhibited by tabun. The protective potency (P(50)) of all oximes in human erythrocyte AChE inhibited by soman and tabun could not be determined. In tabun-poisoned mice very good antidotal efficacy was obtained with K027, K048, and K074, which makes them interesting for future investigation. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.

Oximes: Reactivators of phosphorylated acetylcholinesterase and antidotes in therapy against tabun poisoning.

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. Three of our tested pyridinium oximes K027, K048, K074, along with TMB-4, were the most promising for AChE reactivation. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Herein, we showed that a promising treatment in tabun poisoning by selected oximes and atropine could be improved if oximes are also used in pretreatment. 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.

Tenocyclidine treatment in soman-poisoned rats--intriguing results on genotoxicity versus protection.

This study 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 (1/4) and (1/2) of LD(50) 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 of soman. Plasma activities of the enzyme after a joint treatment with TCP and soman were significantly increased at 30 min (P<0.001) and 24 h (P=0.0043), as compared to soman alone. TCP and atropine, given as therapy, were more effective than TCP administered alone as a pretreatment. The above therapy significantly increased activities of the enzyme at 30 min (P=0.046) and 24 h (P<0.001), as compared to controls treated with (1/4) LD(50) of soman alone. Using the alkaline comet assay, acceptable genotoxicity of TCP was observed. However, the controversial role of TCP in brain protection of soman-poisoned rats should be studied further.

Comparative evaluation of acetylcholinesterase status and genome damage in blood cells of industrial workers exposed to carbofuran.

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 nucleoplasmic 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.

Evaluation of HI-6 oxime: potential use in protection of human acetylcholinesterase inhibited by antineoplastic drug irinotecan and its cyto/genotoxicity in vitro.

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 intoxication with different chemicals or drugs that inhibit the enzyme. Pyridinium or bispyridinium aldoximes (oximes) are able to recover the activity of the 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 the antineoplastic drug irinotecan as well as its cyto/genotoxicity in vitro were investigated. HI-6 was effective in protection of AChE and increased its activity up to 30%; the residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme previously inhibited by 50% irinotecan (4.6 microg/ml) applied at 1/4 of the IC50 value. The tested concentrations of HI-6 exhibited acceptable genotoxicity towards white blood cells, as estimated by the alkaline comet assay, DNA diffusion assay and cytogenetic endpoints (structural chromosome aberrations and cytokinesis-block micronucleus assay). The results obtained warrant the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

The involvement of oxidative stress in ochratoxin A and fumonisin B1 toxicity in rats.

The aim of this study was to find out whether very low doses of nephrotoxic and hepatotoxic mycotoxins ochratoxin A (OTA) and fumonisin B1 (FB1) induce oxidative stress in rat kidney and liver and whether their effect is synergistic. Rats were treated orally with OTA (5 ng/kg b.w. and 50 microg/kg b.w.) and FB1 (200 ng/kg b.w. and 50 microg/kg b.w.), or their combinations. Malondialdehyde (MDA) and protein carbonyls (PCs) concentration in kidney was affected with lower dose of OTA than in liver (p<0.05). FB1 did not affect MDA and PCs concentrations in the liver, while in the kidney both FB1 doses increased MDA concentration (p<0.05). The combination of the lower doses of OTA+FB1 increased the MDA and PCs concentration both in the liver and the kidney, compared to controls and animals treated with respective doses of mycotoxins (p<0.05). The combinations of mycotoxins reduced the catalase activity only in the kidney when compared to controls (p<0.05). In contrast to the increased kidney concentrations of MDA and PCs even with very low doses of OTA and FB1, the activity of catalase and SOD does not change. Combinations of OTA+FB1 affected almost all parameters, which indicates their potential to produce oxidative damage.

Irinotecan toxicity to human blood cells in vitro: relationship between various biomarkers.

Toxic effects of the antineoplastic drug irinotecan on human blood cells at concentrations of 9.0 microg/ml and 4.6 microg/ml were evaluated in vitro. Using the alkaline and neutral comet assay significantly increased levels of primary DNA damage in lymphocytes were detected. The induction of apoptosis/necrosis, as determined by a fluorescent assay, was also notably increased. Cytogenetic outcomes of the treatment were assessed by the analysis of structural chromosome aberrations and fluorescence in situ hybridization. A significantly higher incidence of chromatid breaks and complex quadriradials was observed. Painted chromosomes 1, 2 and 4 were equally involved in translocations, but only the chromosome 1 was involved in the formation of quadriradials. Sister chromatid exchange analysis was performed in parallel with the analysis of lymphocyte proliferation kinetics. The higher concentration of irinotecan caused almost seven-time increase, while the lower one caused a five-time increase of the basal sister chromatid exchange frequency, accompanied with significant lowering of the lymphocyte proliferation index. Using the cytokinesis-block micronucleus assay, a dose-dependent increase in micronucleus frequency along with the formation of nuclear buds and nucleoplasmic bridges was noticed. Inhibitory effects of irinotecan on enzyme acetylcholinesterase (AChE) were studied in erythrocytes. An IC(50) value of 5.0 x 10(-7) was established. Irinotecan was found to be strong inhibitor of the acetylcholine hydrolysis and to cause a continuous decrease of catalytic activity of AChE. The results obtained on a single donor may contribute to the understanding of irinotecan toxicity, but further in vitro and in vivo studies are essential in order to clarify remaining issues, especially on possible inter-individual variability in genotoxic responses to the drug.