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Codeine and morphine are alkaloids from the opioid family widely used as pain relief drugs or antitussive cough suppressant. Both drugs are found naturally in the poppy plant, Papaver somniferum. If misused or abused, neurobehavioral alterations, respiratory depression, constipation, sedation, tolerance, nausea, vomiting, itch, dry mouth and addiction may occur. While the current knowledge related to toxic effects of these opioids is limited, the aim of this study was to determine if they could induce a DNA damaging effect and oxidative stress response in human neuroblastoma SH-SY5Y cells. 24-h treatment with morphine and codeine at 1.56 μmol/L resulted with 80.84±4.14 and 85.82±3.85 % viable cells, respectively. These concentrations allowed genotoxicity testing using the alkaline comet assay, which resulted in a significant increase of primary DNA damage after both treatments versus negative control (median tail intensity 0.22 % DNA). At the same tested concentration, codeine exerted a slightly higher genotoxic potency towards SH-SY5Y cells (median tail intensity 2.25 %) than morphine (median tail intensity 1.07 % DNA). Differences in the observed DNA damage pattern could be attributed to specific interactions of the tested compounds with DNA, which affect their behavior during alkaline denaturation and electrophoresis. Codeine induced a significant decrease in glutathione (GSH) and a significant increase in the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), whereas morphine induced a significant decrease in reactive oxygen species (ROS) production and SOD, and a significant increase in GPx and CAT activity when compared to controls. There was no significant impact of the investigated drugs on the levels of lipid peroxidation (MDA). Our results warrant further investigation to obtain insights into the mechanisms of toxic effects of codeine and morphine. Funding: This research was supported by the programme of cooperation between the Institute for Medical Research and Occupational Health (Zagreb, Croatia) and the University North (Varaždin, Croatia) and the Croatian Science Foundation’s grant number HrZZ- UIP-2017-05-7260.

Introduction: Ketamine is a dissociative anaesthetic widely used in clinical practice, while its subanaesthetic administration has been explored for treatment of depression and chronic pain. Due to its hallucinogenic and dissociative effects, ketamine is also used as a recreational drug. Agents used for the induction of anaesthesia have been shown to promote oxidative stress, which has been implicated in the aetiology of various chronic diseases. Research of oxidative/antioxidative properties of ketamine has so far produced ambiguous findings, while data on genotoxicity are insufficient to reach a conclusion on its genotoxic potential. Methods: In order to investigate the unexplored effects of ketamine on cell level, hepatocytes HepG2 were exposed to ketamine at concentrations of 0.09, 0.37 and 1.49 mg/L for 24 h, relevant in case of analgesia, anesthesia or drug-abuse. Cell viability was studied using the MTS assay. Ketamine toxicity was further evaluated using markers of DNA damage and oxidative stress according to a specific method protocol. Lipid peroxidation level [malondialdehyde (MDA)], reactive oxygen species (ROS) production and glutathione (GSH) levels were measured as biomarkers of oxidative stress, while primary DNA damage was determined using the alkaline comet assay. Results: The selected concentrations of ketamine did not decrease cell viability below 90 %. Although no changes in ROS levels were visible after the treatment, a significant decrease in concentration of MDA and GSH was observed at all three ketamine concentrations in comparison to control cells. Ketamine treatment at all of the tested concentrations resulted in a significant increase of primary DNA damage versus the negative control. We also found significant concentration- dependent differences in DNA damage after exposure to ketamine. The observed DNA damage pattern could be associated with specific interactions of ketamine and/or its metabolites with DNA (most likely intercalations), as well as with a repair- induced onset of additional breaks, which affected the amounts of lesions detectable by alkaline comet assay. Conclusion: The findings regarding genotoxic potency of ketamine detected in vitro, together with the observed imbalance in the oxidative- antioxidant equilibrium detected in HepG2 cells call for additional studies on a wider range of doses and other experimental models. Future studies at DNA level should focus on the use of specific modifications of the comet assay and related methods that will prove the presumed mechanisms of interaction of the tested substance with the DNA molecule. Funding: This research was supported by the programme of cooperation between the Institute for Medical Research and Occupational Health (Zagreb, Croatia) and the University North (Varaždin, Croatia) and the Croatian Science Foundation’s grant number HrZZ- UIP-2017-05-7260.

Background: Ketamine is a dissociative anaesthetic used in human and veterinary practice. The psychotropic properties of ketamine have been the main reason for its use also as a recreational drug in a variety of social settings, as well as in drug-facilitated sexual assault, owing to its dissociative and sedative properties. Ketamine abuse displays neurobehavioral alterations, such as depression, anxiety, cognition deficits, and schizophrenia. The exact mechanism of the neuronal damage following ketamine use is still unclear, but reactive oxygen species (ROS) may have an important role in the pathogenesis of neurological diseases. Also, data on genotoxic potentials are scarce and insufficient and do not allow a reasonable evaluation of its role in neurological impairment. Aim: The aim of this study was to determine the DNA damaging effect and oxidative stress response in human neuroblastoma SH-SY5Y cells treated with ketamine. Method: After a 24- hour treatment with a ketamine concentration that reduced cell viability up to 30 % and allowed genotoxicity testing, SH-SY5Y cells were subjected to a alkaline comet assay and biochemical assays that estimate oxidative stress. Results: For the assessments of ketamine toxicity, we selected the concentration of 6.25 μmol/L, which resulted in 78.59 ± 2.24 % of viable cells. Results of the alkaline comet assay showed significant increases of %DNA in tail, tail length and total comet area after ketamine treatment, compared to untreated cells. When compared to negative controls, the treated cells had a significant increase of malondialdehyde (MDA) level and significant decrease of glutathione (GSH) level as well as decreased catalase (CAT) and superoxide dismutase (SOD) activity. An increase in ROS level and decrease in glutathione peroxidase (GPx) activity were also observed, but the change was not significant. Discussion and Conclusion: Increased levels of DNA damage and the significant imbalance in the oxidative-antioxidant equilibrium detected in SH- SY5Y cells call for further studies on a wider range of doses and other experimental models. Funding: This study was supported by the programme of cooperation between the Institute for Medical Research and Occupational Health (Zagreb, Croatia) and the University North (Varaždin, Croatia) and the Croatian Science Foundation grant number HrZZ- UIP-2017-05-7260.

Irinotecan is one of the most commonly used cytostatics for the treatment of advanced colon cancer. The incidence of this type of cancer is a growing public health problem in all developed countries. Side effects caused by the use of irinotecan significantly reduce the quality of life of treated patients. Although they can be reduced to some extent by taking supportive therapies under medical supervision, patients treated with irinotecan often take various dietary supplements to reduce the effects of acute and delayed cholinergic syndrome and improve the general condition of the body. Due to the growing public opinion about the high antitumor potential of products with Δ-9-tetrahydrocannabinol (THC), for which the relevant scientific literature does not offer credible evidence, some cancer patients often take unregistered preparations containing up to 90% THC. Previous research has shown that the use of some bioactive substances can cause adverse interactions and disrupt the metabolism and pharmacokinetics of cytostatics. Considering the fact that the metabolic pathways of irinotecan and THC in the body overlap: (i) at the level of the first two phases of metabolic biotransformation in which the enzymes CYP3A4 and UDP- glucuronyltransferase are key ; (ii) bacterial β- glucuronidase is involved in the metabolism of SN- 38 (the active form of irinotecan) and THC ; (iii) both compounds have an inhibitory effect on the enzyme acetylcholinesterase (AChE), it is assumed that the intake of high concentrations of THC in the body during treatment with irinotecan could worsen the side effects. This lecture will present an investigation on the potential interactions of cytostatic irinotecan and THC in an experimental mouse model in terms of biochemical toxicity indicators (cholinesterase activity and markers of oxidative stress). Given the general lack of knowledge about the potential interactions of the test compounds, and the fact that the use of THC preparations is popular, results of conducted study could provide a number of insights useful for practitioners in medical toxicology and possible application in clinical practice.

Lysergic acid diethylamide (LSD) is a classic hallucinogen, widely abused for decades, while phencyclidine (PCP) has increased in popularity in recent years, especially among the adolescents. Very little is known about the general toxicity of these compounds, especially about their possible neurotoxic effects at the cell level. The aim of this study was to address these gaps by assessing the toxic effects of 24-hour exposure to LSD and PCP in the concentration range of 0.39–100 µmol/L in the human neuroblastoma SH-SY5Y cell line. After cell viability was established, cells treated with concentrations that reduced their viability up to 30 % were further subjected to the alkaline comet assay and biochemical assays that enable estimation of oxidative stress-related effects. Treatment with LSD at 6.25 µmol/L and with PCP at 3.13 µmol/L resulted with 88.06±2.05 and 84.17±3.19 % of viable cells, respectively, and led to a significant increase in primary DNA damage compared to negative control. LSD also caused a significant increase in malondialdehyde level, reactive oxygen species (ROS) production, and glutathione (GSH) level, PCP significantly increased ROS but lowered GSH compared to control. Treatment with LSD significantly increased the activities of all antioxidant enzymes, while PCP treatment significantly increased superoxide dismutase (SOD) and glutathione peroxidase (GPx) but decreased catalase (CAT) activity compared to control. Our findings suggest that LSD has a greater DNA damaging potential and stronger oxidative activity than PCP in SH-SY5Y cells., Dietilamid lizerginske kiseline (LSD) klasični je halucinogen koji se desetljećima naširoko zlorabio, dok je posljednjih godina porasla popularnost fenciklidina (PCP), osobito među adolescentima. Do sada se vrlo malo zna o općoj toksičnosti ovih spojeva, posebice o njihovim mogućim neurotoksičnim učincima na staničnoj razini. Ovo istraživanje procijenilo je toksične učinke LSD-a i PCP-a na staničnu liniju humanoga neuroblastoma SH-SY5Y, koja je bila izložena ispitivanim spojevima u rasponu koncentracija 0,39–100 µmol/L tijekom 24 sata. Stanice tretirane odabranim koncentracijama koje su smanjile vijabilnost stanica do 30 % potom su podvrgnute alkalnom komet-testu i biokemijskim testovima koji omogućuju procjenu oksidacijskoga stresa. Tretman s LSD-om od 6,25 µmol/L i PCP-om s 3,13 µmol/L rezultirao je s 88,06±2,05 % odnosno 84,17±3,19 % vijabilnih stanica. Ove koncentracije omogućile su ispitivanje genotoksičnosti, što je rezultiralo značajnim povećanjem primarnog oštećenja DNA nakon tretmana LSD-om i PCP-om u odnosu na negativnu kontrolu. LSD je izazvao značajno povećanje razine malondialdehida u usporedbi s kontrolom, za razliku od PCP-a. Dok je LSD inducirao značajno povećanje proizvodnje reaktivnih kisikovih vrsta (ROS) i razine glutationa (GSH), tretman PCP-om uzrokovao je značajno povećanje proizvodnje ROS-a, ali smanjenje razine GSH-a u usporedbi s kontrolom. Tretman stanica LSD-om značajno je povećao aktivnosti svih antioksidacijskih enzima u usporedbi s kontrolom. Tretman PCP-om značajno je povećao aktivnosti glutation peroksidaze (GPx) i superoksid dismutaze (SOD), ali je aktivnost katalaze (CAT) bila značajno niža nego u odgovarajućoj kontroli. Zaključno, LSD je imao veći potencijal oštećenja DNA i pokazao je jaču oksidacijsku aktivnost od PCP-a u stanicama SH-SY5Y.

Introduction: Amphetamine-like stimulants (ATS) are widely abused substances that impair dopaminergic and serotonergic functions. The mechanisms of their toxicity in human neuronal cells are not clear yet, but they may involve the formation of reactive oxygen species. The aim of this study was to determine the induction of oxidative stress parameters and the levels of DNA instability in human neuroblastoma SH-SY5Y cells treated with selected concentrations of four different ATS during 24 hours. Methods: SH-SY5Y cells were treated with ATS at concentrations that did not decrease viability below 75 %. A new psychoactive substance mephedrone was tested at 6.25 μM, while so-called classical ATS were applied as follows: 3, 4- methylenedioxymethamphetamine (MDMA, ecstasy) at 0.625 μM, methamphetamine at 0.625 μM, and amphetamine at 3.13 μM. After the treatment, cells were trypsinized, washed in phosphate-buffered saline, resuspended in complete medium and prepared for further analysis. The ATS-toxicity was evaluated using markers of DNA damage and oxidative stress. Lipid peroxidation level (malondialdehyde (MDA)), reactive oxygen species (ROS) production, glutathione (GSH) levels, and activities of antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase) were measured as biomarkers of oxidative stress, while primary DNA damage was determined using alkaline comet assay. Results: When compared to controls, cells treated with methamphetamine had a significant increase in MDA, cells treated with mephedrone had a significant increase in ROS, GSH and GPx, whereas cells treated with amphetamine had a significant increase in ROS, and a significant decrease in GSH. The range of DNA damage measured with the comet assay after treatment (based on the median values obtained for tail intensity) was: amphetamine > methamphetamine > mephedrone > MDMA. Conclusions: Significant induction of oxidative stress parameters and increased level of DNA damage detected at cell level call for further studies to clarify the toxicological risks associated with ATS consumption.

Background and purpose We focused on the, yet undescribed, therapy effect of the stable gastric pentadecapeptide BPC 157 in hippocampal ischemia/reperfusion injuries, after bilateral clamping of the common carotid arteries in rats. The background is the proven therapy effect of BPC 157 in ischemia/reperfusion injuries in different tissues. Furthermore, there is the subsequent oxidative stress counteraction, particularly when given during reperfusion. The recovering effect it has on occluded vessels, results with activation of the alternative pathways, bypassing the occlusion in deep vein thrombosis. Finally, the BPC 157 therapy benefits with its proposed role as a novel mediator of Roberts’ cytoprotection and bidirectional effects in the gut‐brain axis. Materials and Methods Male Wistar rats underwent bilateral clamping of the common carotid arteries for a 20‐min period. At 30 s thereafter, we applied medication (BPC 157 10 µg/kg; or saline) as a 1 ml bath directly to the operated area, that is, trigonum caroticum. We documented, in reperfusion, the resolution of the neuronal damages sustained in the brain, resolution of the damages reflected in memory, locomotion, and coordination disturbances, with the presentation of the particular genes expression in hippocampal tissues. Results In the operated rats, at 24 and 72 hr of the reperfusion, the therapy counteracted both early and delayed neural hippocampal damage, achieving full functional recovery (Morris water maze test, inclined beam‐walking test, lateral push test). mRNA expression studies at 1 and 24 hr, provided strongly elevated (Egr1, Akt1, Kras, Src, Foxo, Srf, Vegfr2, Nos3, and Nos1) and decreased (Nos2, Nfkb) gene expression (Mapk1 not activated), as a way how BPC 157 may act. Conclusion Together, these findings suggest that these beneficial BPC 157 effects may provide a novel therapeutic solution for stroke., Pentadecapeptide BPC 157 is a novel mediator of Roberts cytoprotection and has bidirectional effects on the gut‐brain axis. BPC 157 effectively ameliorated ischemia/reperfusion injuries and as such is a possible new therapeutic solution in ischemic/reperfusion injuries.

AIM: To provide new insights in treatment of colitis and ischemia and reperfusion in rats using stable gastric pentadecapeptide BPC 157. ----- METHODS: Medication [BPC 157, L-NAME, L-arginine (alone/combined), saline] was bath at the blood deprived colon segment. During reperfusion, medication was BPC 157 or saline. We recorded (USB microscope camera) vessel presentation through next 15 min of ischemic colitis (IC-rats) or reperfusion (removed ligations) (IC + RL-rats); oxidative stress as MDA (increased (IC- and IC + RL-rats)) and NO levels (decreased (IC-rats); increased (IC + RL-rats)) in colon tissue. IC + OB-rats [IC-rats had additional colon obstruction (OB)] for 3 d (IC + OB-rats), then received BPC 157 bath. ----- RESULTS: Commonly, in colon segment (25 mm, 2 ligations on left colic artery and vein, 3 arcade vessels within ligated segment), in IC-, IC + RL-, IC + OB-rats, BPC 157 (10 μg/kg) bath (1 mL/rat) increased vessel presentation, inside/outside arcade interconnections quickly reappeared, mucosal folds were preserved and the pale areas were small and markedly reduced. BPC 157 counteracted worsening effects induced by L-NAME (5 mg) and L-arginine (100 mg). MDA- and NO-levels were normal in BPC 157 treated IC-rats and IC + RL-rats. In addition, on day 10, BPC 157-treated IC + OB-rats presented almost completely spared mucosa with very small pale areas and no gross mucosal defects; the treated colon segment was of normal diameter, and only small adhesions were present. ----- CONCLUSION: BPC 157 is a fundamental treatment that quickly restores blood supply to the ischemically injured area and rapidly activates collaterals. This effect involves the NO system.

We studied the toxic effects of glyphosate in vitro on HepG2 cells exposed for 4 and 24 h to low glyphosate concentrations likely to be encountered in occupational and residential exposures [the acceptable daily intake (ADI ; 0.5 μg/mL), residential exposure level (REL ; 2.91 μg/mL) and occupational exposure level (OEL ; 3.5 μg/mL)]. The assessments were performed using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell proliferation, alkaline comet assay and cytokinesis-block micronucleus (CBMN) cytome assay. The results obtained indicated effects on cell proliferation, both at 4 and 24 h. The levels of primary DNA damage after 4-h exposure were lower in treated vs. control samples, but were not significantly changed after 24 h. Using the CBMN assay, we found a significantly higher number ofMNand nuclear buds at ADI and REL after 4 h and a lower number of MN after 24 h. The obtained results revealed significant oxidative damage. Four-hour exposure resulted in significant decrease at ADI [lipid peroxidation and glutathione peroxidase (GSH-Px)] and OEL [lipid peroxidation and level of total antioxidant capacity (TAC)], and 24-h exposure in significant decrease at OEL (TAC and GSH-Px). No significant effects were observed for the level of reactive oxygen species (ROS) and glutathione (GSH) for both treatment, and for 24 h for lipid peroxidation. Taken together, the elevated levels of cytogenetic damage found by the CBMN assay and the mechanisms of primary DNA damage should be further clarified, considering that the comet assay results indicate possible cross-linking or DNA adduct formation.

Background: Today, we are faced with the increasing use of illegal highly concentrated Δ9 -tetrahydrocannabinol (THC) preparations as supportive therapies for various malignancies and neurological disorders. Due to the fact that some, such as cannabis oils and butane hash oil, may contain over 80% of THC, their consumers can become intoxicated or experience various detrimental effects. Objective: The aim of this study was to assess the toxic effects of THC in male Wistar rats orally exposed to 7 mg/kg b.w. THC, which is comparable to the dose find in illicit preparations. Methods: The rats were sacrificed 24 h after treatment and plasma and brain samples were collected and stored for biochemical analyses. We determined the extent of oxidative stress as well as changes in activities of plasma and brain cholinesterases (ChE) in THC-treated and control rats. Results: Acute oral administration of 7 mg/kg b.w. THC did not cause changes in oxidative stress biomarkers in rat plasma, but did cause a significant elevation of TBARS and GSH concentration and a drop in SOD activity was observed in brain tissue. Cholinesterase activities were not affected either in the plasma or in brain tissue. Conclusion: The present study contributes to existing knowledge with evidence that acute exposure to a high THC dose induced oxidative stress in the brain, but did not affect changes in ChE activity.

Currently we are faced with an ever-growing use of &Delta, 9-tetrahydrocannabinol (THC) preparations, often used as supportive therapies for various malignancies and neurological disorders. As some of illegally distributed forms of such preparations, like cannabis oils and butane hash oil, might contain over 80% of THC, their consumers can become intoxicated or experience various detrimental effects. This fact motivated us for the assessments of THC toxicity in vivo on a Wistar rat model, at a daily oral dose of 7 mg/kg which is comparable to those found in illicit preparations. The main objective of the present study was to establish the magnitude and dynamics of DNA breakage associated with THC exposure in white blood and brain cells of treated rats using the alkaline comet assay. The extent of oxidative stress after acute 24 h exposure to THC was also determined as well as changes in activities of plasma and brain cholinesterases (ChE) in THC-treated and control rats. The DNA of brain cells was more prone to breakage after THC treatment compared to DNA in white blood cells. Even though DNA damage quantified by the alkaline comet assay is subject to repair, its elevated level detected in the brain cells of THC-treated rats was reason for concern. Since neurons do not proliferate, increased levels of DNA damage present threats to these cells in terms of both viability and genome stability, while inefficient DNA repair might lead to their progressive loss. The present study contributes to existing knowledge with evidence that acute exposure to a high THC dose led to low-level DNA damage in white blood cells and brain cells of rats and induced oxidative stress in brain, but did not disturb ChE activities.

There is growing interest regarding the use of herbal preparations based on Cannabis sativa for medicinal purposes, despite the poorly understood interactions of their main constituent &Delta, 9-tetrahydrocannabinol (THC) with conventional drugs, especially cytostatics. The objective of this pilot study was to prove whether the concomitant intake of THC impaired liver function in male Wistar rats treated with the anticancer drug irinotecan (IRI), and evaluate the toxic effects associated with this exposure. IRI was administered once intraperitoneally (at 100 mg/kg of the body weight (b.w.)), while THC was administered per os repeatedly for 1, 3, and 7 days (at 7 mg/kg b.w.). Functional liver impairments were studied using biochemical markers of liver function (aspartate aminotransferase&mdash, AST, alanine aminotransferase&mdash, ALP, alkaline phosphatase&mdash, AP, and bilirubin) in rats given a combined treatment, single IRI, single THC, and control groups. Using common oxidative stress biomarkers, along with measurement of primary DNA damage in hepatocytes, the degree of impairments caused at the cellular level was also evaluated. THC caused a time-dependent enhancement of acute toxicity in IRI-treated rats, which was confirmed by body and liver weight reduction. Although single THC affected ALP and AP levels more than single IRI, the levels of liver function markers measured after the administration of a combined treatment mostly did not significantly differ from control. Combined exposure led to increased oxidative stress responses in 3- and 7-day treatments, compared to single IRI. Single IRI caused the highest DNA damage at all timepoints. Continuous 7-day oral exposure to single THC caused an increased mean value of comet tail length compared to its shorter treatments. Concomitant intake of THC slightly affected the levels of IRI genotoxicity at all timepoints, but not in a consistent manner. Further studies are needed to prove our preliminary observations, clarify the underlying mechanisms behind IRI and THC interactions, and unambiguously confirm or reject the assumptions made herein.

Terbuthylazine belongs to the chloro-s-triazine group of herbicides and acts primarily as a photosynthesis inhibitor. The mechanisms of action related to its exposure, relevant both in animals and humans, are still insufficiently investigated. This comprehensive study focused on the outcomes of terbuthylazine exposure at cell level in vitro, and a mice model in vivo. Experiments in vitro were conducted on whole human peripheral blood, isolated lymphocytes, and HepG2 cells exposed for 4 h to terbuthylazine at 8.00, 0.80, and 0.58 ng/mL, which is comparable with current reference values set by the European Commission in 2011. Terbuthylazine cytotoxicity was evaluated using dual fluorescent staining with ethidium bromide and acridine orange on lymphocytes, and CCK-8 colorimetric assay on HepG2 cells. The levels of DNA damage were measured using alkaline and hOGG1-modified comet assays. The potency of terbuthlyazine regarding induction of oxidative stress in vitro was studied using a battery of standard oxidative stress biomarkers. The in vivo experiment was conducted on Swiss albino mice exposed to terbuthlyazine in the form of an active substance and its formulated commercial product Radazin TZ-50 at a daily dose of 0.0035 mg/kg bw for 14 days. Following exposure, the DNA damage levels in leukocytes, bone marrow, liver, and kidney cells of the treated mice were measured using an alkaline comet assay. In vitro results suggested low terbuthylazine cytotoxicity in non-target cells. The highest tested concentration (8.00 ng/mL) reduced lymphocyte viability by 15%, mostly due to apoptosis, while cytotoxic effects in HepG2 cells at the same concentration were negligible. Acute in vitro exposure of human lymphocytes and HepG2 cells to terbuthylazine resulted in low-level DNA instability, as detected by the alkaline comet assay. Further characterization of the mechanisms behind the DNA damage obtained using the hOGG1-modified comet assay indicated that oxidative DNA damage did not prevail in the overall damage. This was further confirmed by the measured levels of oxidative stress markers, which were mostly comparable to control. Results obtained in mice indicate that both the active substance and formulated commercial product of terbuthylazine produced DNA instability in all of the studied cell types. We found that DNA in liver and kidney cells was more prone to direct toxic effects of the parent compound and its metabolites than DNA in leukocytes and bone marrow cells. The overall findings suggest the formation of reactive terbuthylazine metabolites capable of inducing DNA cross-links, which hinder DNA migration. These effects were most pronounced in liver cells in vivo and HepG2 cells in vitro. To provide a more accurate explanation of the observed effects, additional research is needed. Nevertheless, the present study provides evidence that terbuthylazine at concentrations comparable with current reference values possesses toxicological risk because it caused low-level DNA instability, both at cellular and animal organism level, which should be further established in forthcoming studies.

We investigated the effect of low-dose exposure to glyphosate on 3 months old male Wistar rats that received orally for 28-day doses of 0.1 mg kg-1 b.w. of the acceptable operator exposure level-(AOEL), 0.5 mg kg-1 b.w. consumers acceptable daily intake (ADI), 1.75 mg kg-1 b.w. of the chronic population-adjusted dose (cPAD), 10 mg kg-1 b.w. (100xAOEL) and the same volume of phosphate buffer without glyphosate (negative control). Positive control received 300 mg kg-1 b.w. of ethyl methanesulphonate (EMS) over the last three days of experiment.The samples of whole blood, plasma and liver samples were analyzed for the levels of plasma cholinesterase activity, oxidative stress (lipid peroxidation (thiobarbituric reactive substances, TBARS), glutathione(GSH), glutathione peroxidase (GSH-Px), reactive oxidative species (ROS)) and DNA damage in whole blood and small (non-parenchymal cells) and medium (parenchymal cells-hepatocytes) liver cells.The exposed animals gained less weight than control. The cPAD and the 100xAOEL groups' liver weight increased. 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. Exposure lowered TBARS values in all exposed groups, with significant liver changes in the AOEL, ADI, and cPAD groups and plasma changes in the AOEL and cPAD group. Exposure also produced significant primary DNA damage in the leukocytes and liver cells. 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.This work was financially supported by Project Organic Pollutants in Environment – Markers and Biomarkers of Toxicity (OPENTOX), funded by the Croatian Science Foundation (grant number 8366).

The use of cannabinoid-based preparations by cancer patients raises concern whether delta-9-tetrahydrocannabinol (THC) can modulate or even compromise the effectiveness of concurrently administered anticancer drugs. Irinotecan (IRI) is a type of drug that causes various severe adverse effects such as diarrhoea, gastrointestinal toxicity and myelosuppression. Newer reports point to hepatotoxicity being an underestimated but important IRI side effect as well. This study focused on the evaluation of potentially detrimental interactions of IRI and THC in the liver of Wistar rats. Male rats were concomitantly exposed to IRI (at 100 mg/kg b.w., administered once i.p.) and THC (administered repeatedly for 1, 3 and 7 days per os at 7 mg/kg b.w.). Single IRI, THC and control groups were studied in parallel. We estimated changes in body and liver weight caused by the treatments, evaluated the level of primary DNA damage in hepatocytes using the alkaline comet assay, and evaluated the level of lipid peroxidation and catalase (CAT) activity. Changes in body weights and liver weights observed after the treatments suggest that all of the compounds at the tested doses and the applied experimental schedule produced acute toxicity and diminished the overall fitness of the exposed compared to control rats. It was most pronounced in rats administered a combined treatment. DNA damage was the most pronounced after 3-day treatment, and its highest level was observed in hepatocytes of single IRI-treated rats, followed by those given combined treatment. In contrast to 3-day, 7-day treatment with single THC slightly impaired hepatocyte DNA integrity. Rats given combined treatment demonstrated increased lipid peroxidation and higher CAT levels than those administered single IRI, at both time points, which may indicate that combined treatment induced more intense oxidative stress. Our findings provide evidence regarding a significant synergic enhancement of IRI toxicity caused by THC intake, which was confirmed using all of the applied biomarkers. Nevertheless, since we tested only one IRI and THC dose, further studies are required to further clarify their mutual interactions.

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 acetilkolinesteraze

In this 28 day-study, we evaluated the effects of the insecticide chlorpyrifos orally administered to Wistar rats at doses 0.160, 0.015, and 0.010 mg/kg b. w./day. Following treatment, total cholinesterase activity and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were measured. Oxidative stress responses were evaluated using a battery of endpoints to establish lipid peroxidation, changes in total antioxidant capacity, level of reactive oxygen species (ROS), glutathione (GSH) level and activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. Using HPLC-UV DAD analysis, levels of the parent compound and its main metabolite 3,5,6-trichloro-2-pyridinol in plasma and brain tissue were measured. The genotoxic effect was estimated using alkaline comet assay in leukocytes and brain tissue. The exposure did not result in significant effects on total cholinesterase, AChE and BChE activity in plasma and brain tissue. Lipid peroxidation slightly increased both in plasma and brain tissue. Total antioxidant capacity, ROS and GSH levels were marginally influenced by the exposure. Treatment led to significant increases of GSH-Px activity in blood, SOD activity in erythrocytes and a slight increase of catalase activity in plasma. HPLC-UV DAD analysis revealed the presence of both the parent compound and its main metabolite in the plasma of all of the experimental animals and brain tissue of the animals treated at the two higher doses. All of the tested doses of chlorpyrifos were slightly genotoxic, both to leukocytes and brain tissue. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Terbuthylazine is a selective pre- and post- emergency chloro-triazine herbicide used for a broad spectrum of weed control. We evaluated the potential of low doses of terbuthylazine to induce oxidative stress and cytogenetic damage in peripheral blood samples of adult male Wistar rats. Following 28-day repeated oral exposure at 0.004 mg/kg b.w./day, 0.4 mg/kg b.w./day and 2.29 mg/kg b.w./day, parameters of lipid peroxidation, total antioxidant capacity, and activities of antioxidant enzymes were measured in blood samples. Alkaline comet assay on leukocytes and erythrocyte micronucleus assay were used to measure DNA damage. In addition, the concentration of terbuthylazine and its metabolite in urine and plasma were determined using high performance liquid chromatography with UV diode-array detector (HPLC-UV-DAD). The fraction of terbuthylazine excreted in urine was negligible and was not found in plasma. Deethylterbuthylazine was only compound detected in plasma samples. Exposure to terbuthylazine did not induce significant lipid peroxidation products. The significant changes in antioxidant enzyme activities and the elevated total antioxidant capacity indicated that terbuthylazine at experimental conditions applied has potential to disturb oxidative/antioxidant balance. Results regarding the alkaline comet assay as well as micronucleated reticulocyte frequency indicated that treatment led to low-level DNA instability. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Terbuthylazine is a chloro-triazine herbicide that acts primarily as an inhibitor of photosynthesis. It is used mainly in the production of maize but is also applied in the cultivation of various fruits, and vegetables, as well as in forestry. We evaluated the in vivo effects of terbuthylazine following a 28- day oral administration in adult male Wistar rats at 2.300 mg/kg b.w./day, 0.400 mg/kg b.w./day, and 0.004 mg/kg b.w./day. After treatment, oxidative stress responses were measured in the blood of both the terbuthylazine-treated and the control rats using biomarkers of lipid peroxidation, activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. To establish DNA damaging effects, we applied an alkaline comet assay on the leukocytes of the exposed and control animals. Exposure to terbuthylazine slightly disturbed lipid peroxidation, which was not dose-dependent. Treatment also led to increases of GSH-Px activity in blood, which was statistically significant at 0.400 mg/kg b.w./day. The observed increase of SOD activity in erythrocytes was most prominent at the highest dose tested. On the other hand, plasma SOD activity dropped significantly at two lower doses. Catalase activity in plasma decreased, which was statistically significant at the highest dose. Terbuthylazine produced measurable effects on leukocyte rat DNA. Based on the values measured for comet tail length and tail intensity, which were both lower at the two higher concentrations, it can be concluded that one of possible mechanisms of terbuthylazine’s effect on the DNA molecule includes intercalation. This may in turn slow down the migration of DNA during electrophoresis. Taken together, our findings suggest that the 28-day exposure of rats to very low doses of terbuthylazine, which were within toxicology reference values, resulted in oxidative stress responses and low-level DNA instability. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios. This work was financially supported by Project No. 8366 Organic Pollutants in Environment - Markers and Biomarkers of Toxicity (OPENTOX), funded by the Croatian Science Foundation.

K048 is a member of K-oximes, a new oxime class that has recently been confirmed effective against poisoning by the nerve agent tabun and several pesticides. The toxicity profile of the K048 oxime has not been fully characterized and its optimal therapeutic dose has not yet been established. Earlier studies report excellent results with K048 in reactivating tabun-phosphorylated AChE and in the therapy of tabun-poisoned mice. It possesses a low acute toxicity and exerts an acceptable toxicity profile on isolated human peripheral blood lymphocytes in vitro. Intraperitoneal administration of K048 in rats resulted in an LD50 of 238.3 mg/kg. In this in vivo study, we investigated cholinesterase (ChE) activity and oxidative stress marker levels (lipid peroxidation and superoxide dismutase activity) in the plasma of exposed rats after administering the compound at 25% of its LD50. Lymphocyte viability was evaluated using an acridine orange/ethidium bromide in situ fluorescent assay. The levels of primary DNA damage in rat white blood cells were measured using the alkaline comet assay. The compound applied at 25% of its LD50 did not significantly affect ChE activity and lipid peroxidation and did not cause significant changes in the SOD activity in plasma. The cytotoxicity profile of K048 in the tested dose was also acceptable, and it did not possess significant DNA-damaging potential. The obtained results are promising for further evaluations of the K048 oxime, which should include tests on a broader concentration range and longer incubation times.

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.

This study evaluated the cyto- and genotoxic effects of three pesticides: α-cypermethrin, chlorpyrifos and imidacloprid applied in vitro to human lymphocytes and HepG2 cells for exposure times of 4 and 24 h at concentrations corresponding to OEL, ADI and REL. Assessments were made using oxidative stress biomarkers and the alkaline comet, cytokinesis-block micronucleus cytome and cell viability assays. Low doses of all three pesticides displayed DNA damaging potential, both in lymphocytes and HepG2 cells. At the tested concentrations, all three compounds induced lymphocyte apoptosis, though α-cypermethrin and chlorpyrifos were generally more cyto- and genotoxic than imidacloprid. At the tested concentrations, oxidative stress biomarkers were not significantly altered, and the effects mediated indirectly through free radicals may not have a key role in the formation of DNA damage. It is likely that the DNA damaging effects were caused by direct interactions between the tested compounds and/or their metabolites that destabilized the DNA structure. The tested pesticides had the potential for MN, NB and NPB formation and to disturb cell cycle kinetics in both cell types. There were also indications that exposure to α-cypermethrin led to the formation of crosslinks in DNA, though this would require more detailed study in the future.

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)

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.

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 ug/kg b.w.) and FB1 (200 ng/kg b.w. and 50 ug/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.

It is generally known that irreversible inhibition of acetylcholinesterase (AChE) by organophosphorous (OP) agents causes the vast majority of symptoms associated with their acute toxicity. Even if their acute effects exhibiting cholinergic disturbances have been extensively described, questions about their effects on molecular level following both acute and chronic exposure remain largely unanswered. Studies in experimental animals and tissue cultures, as well as in acutely or chronically exposed humans, have implicated a strong linkage between OP pesticide poisoning and the induction of oxidative stress. Since we have no such knowledge on OP nerve agents, the present study was conducted to analyze the biomarkers of oxidative damage to lipids, proteins and DNA in the tissue samples of tabun poisoned rats. Temporal and spatial distribution of the AChE activity in blood and brain of rats exposed to sublethal dose of tabun (75 % of its LD50) was determined by the Ellman assay. We used lipid peroxidation (LPO), activity of superoxide dismutase (SOD) and primary DNA damage determined by comet assay as markers of oxidative stress. The strong inhibitory effect (~90 %) of tabun was observed in the plasma during the first hour of exposition while the highest degree of inhibition (75 %) in the brain as a target organ was observed 1 h after tabun exposure, which is consistent with the culmination of the symptoms of poisoning. Enzyme activity in plasma and brain even 24 h after a single exposure to tabun did not fully recovered. Increased LPO was observed in plasma up to 6 h after tabun exposure, whereas increased level of primary DNA damage, activity of SOD and LPO were noticed in the brain during 24 h exposure to tabun. These results indicate that tabun poisoning caused excessive formation of free radicals which could associate oxidative stress with the neurotoxicity of tabun. Further understanding on how oxidative stress may be mediating toxicity of nerve agents can contribute in designing new and more efficient therapeutic strategies.

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.

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.

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.