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

Hair analysis provides reliable information on long-term nicotine exposure, depending on hair length. The ability of hair to preserve substances such as nicotine incorporated into its shaft may be compromised by the chemical treatment of hair that can damage the structure of the hair and affect the accurate detection of nicotine in it. The aim of this study was to investigate whether different hair chemical treatments (bleaching, dyeing, and wash-out colouring) reduce hair nicotine levels. Hair nicotine content was determined in non-treated and chemically treated hair segments in 20 samples using the headspace solid-phase microextraction (HS-SPME) method followed by gas chromatography-mass spectrometry (GC-MS). Nicotine levels were lower in chemically treated hair in comparison to hair without treatment. The study also showed that different types of chemical treatment had different effect on hair nicotine levels. While bleaching influenced nicotine levels the most (23–88% decrease), certain other types of hair treatment (wash-out coloration) did not have a significant effect (less than an 8% decrease). Dying reduced hair nicotine levels up to 40%, depending on the colouring formula. Given that there is a limited number of similar studies, this one could provide a direction for other studies concerning the influence of diverse chemical treatments on drug levels in hair. These data also suggest that the effect of chemical cosmetic treatment on hair has to be taken into account when interpreting results of drug abuse standardly analysed in hair, which are mainly basic compounds such as nicotine.

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 used to induce general anaesthesia in certain medical procedures. Considering the dissociative and sedative properties of ketamine, it has also been used as a recreational drug in a variety of social settings. Ketamine abuse displays neurobehavioral alterations such as depression, anxiety, cognition deficits, and schizophrenia. While the exact mechanism is still unclear, some findings suggest that oxidative stress and genotoxic properties may be implicated in ketamine- induced neurological impairment. Methods: The aim of this study was to explore the effects of exposure to ketamine depending on selected concentrations used during general anaesthesia, analgesia or drug-abuse available from the literature. Human neuroblastoma SH-SY5Y cells were treated with ketamine at concentrations of 0.09, 0.37 and 1.49 mg/L. Cell viability was studied using the MTS assay. We measured biomarkers of oxidative stress [malondialdehyde (MDA), reactive oxygen species (ROS) production and glutathione (GSH) levels] and primary DNA damage (using the alkaline comet assay) as the endpoints that can be disrupted following ketamine use. Results: After a 24-hour treatment, the applied concentrations reduced cell viability up to 30 %. Although no changes in the concentration of MDA and GSH were observed, a significant decrease in ROS levels was determined after exposure to 0.37 and 0.09 mg/L of ketamine in comparison to control cells. Ketamine treatment at all of the tested concentrations resulted in a significant increase of primary DNA damage versus negative control. However, no significant differences between the applied concentrations were found. Conclusion: Considering that SH-SY5Y cells originated from neural tissue, the findings regarding genotoxic potency of ketamine in vitro call for concern, especially in light of the possible neurotoxic effects that may be relevant in in vivo conditions. Their extent and outcomes could be further elucidated using other molecular biology and cytogenetic methods and experimental models. 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.

α-cypermethrin belongs to pyrethroids, one of the most used groups of insecticides that can act as endocrine disruptive chemicals (EDCs), known to impact gene expression at the epigenetic level. Testicular exposure to EDCs in foetal development may impair PGC differentiation causing decreased sperm quality and number and increased apoptosis of germ cells, as well as infertility in adults. However, such an effect has still not been established for α- cypermethrin. One of the most studied epigenetic mechanism is DNA methylation, which plays a major role in the de novo silencing of retrotransposon sequences in germ cells, as well as in the establishment of sex-specific imprinting. In this study, we investigated the effect of prenatal exposure to α-cypermethrin on DNA methylation patterns in rat sperm of F1 offspring. Pregnant female Wistar rats were divided into 6 groups. The first three experimental groups (α- cyp1, α-cyp10 and α-cyp19) were treated per os from the 6th up to 21st gestational day with three different doses of α- cypermethrin (1, 10 and 19 mg/kg bw/day). In the same period, the positive control group (PC) was treated with diethylstilbestrol, solvent control group (SC) with corn oil and negative control group (NC) with water. DNA was isolated from the sperm of F1 generation pubertal pups. Subsequently, the methylation level of 3 different CpG sites within repetitive element LINE-1 was analysed for the assessment of global methylation by pyrosequencing method. Additionally, methylation analysis was performed for 7 different CpG sites within the differentially methylated regions (DMRs) of the imprinted genes Igf2 and H19. The results showed no significant difference in the methylation levels of examined DNA sequences of LINE-1 between the experimental groups and the controls. Also, exposure to α-cypermethrin had no effect on the methylation levels of the examined DNA sequences of Igf2 and H19. This study implies to the possibility that prenatal exposure to α- cypermethrin does not affect the de novo DNA methylation process in the rat sperm of F1 offspring and indicates a need for further research of epigenetic changes caused by this endocrine disruptor at the genome-wide level.

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.

The placenta is a temporary multifunctional organ with a key role in the maintenance of pregnancy and necessary for foetal growth and development. Exposure to endocrine disruptive chemicals (EDCs) during vulnerable period of life such as pregnancy can affect placental development and function as well as pregnancy outcome. Due to the high abundance of hormone receptors, the placenta is a highly sensitive organ to EDCs. Pyrethroids are the fourth most used group of insecticides worldwide, with proven endocrine disruptive effects. Alpha-cypermethrin (α-cyp) is a type II synthetic pyrethroid insecticide whose effect as an EDC on placental development has still not been explored. Thus, the aim of this investigation was to evaluate the effects of gestational exposure to α-cyp on developmental parameters in placentas of female rats by determination and quantification of protein expression of proliferating and apoptotic markers using an immunohistochemical method and assessment of histopathological changes by classical histopathological analyses. Pregnant Wistar rats were orally exposed to α-cyp at 1, 10 and 19 mg/kg bw/day, diethylstilbestrol (positive control), corn oil (solvent control) and water (negative control) from the 6th to the 21st day of gestation (DG). The gravid uterus was dissected under general anaesthesia at 21st DG and placentas were sampled. Immunohistochemical analysis of phosphorylated histone H3 expression revealed a statistically significant decrease in cell proliferation in α-cyp treated groups at 10 and 19 mg/kg bw/day. Analysis of the expression of cleaved caspase-3 showed that, at the tested concentrations, α-cyp does not cause apoptosis. Numerous histopathological changes were detected in placentas of rats exposed to α-cyp, but their occurrence was not statistically significant in comparison with controls. Further research is needed to better understand the effects of α- cypermethrin as an endocrine disruptor on developmental parameters in placentas of female rats exposed during gestation.

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.

Pyrethroids are today one of the most used groups of insecticides worldwide that can act as endocrine disruptive chemicals. α-cypermethrin is a type II synthetic pyrethroid insecticide. Exposure to endocrine disruptive chemicals can lead to changes in regulation of gene expression at the epigenetic level, not entailing a change in the DNA sequence, but such effect of α- cypermethrin is still unknown. DNA methylation is one of the most studied epigenetic mechanism, being a key player in de novo silencing of retrotransposon sequences in germ cells, as well as in the establishment of sex-specific imprinting. In this study, we investigated the effect of prenatal exposure to α-cypermethrin on DNA methylation patterns in foetal testes of Wistar rats. Pregnant rat dams were treated per os from the 6th until 21st gestational day with a solution of α-cypermethrin in three doses (1, 10 and 19 mg/kg bw/day, respectively), diethylstilbestrol as positive control, corn oil as solvent control and water as negative control. The foetal rat testes were isolated on the 21st day of gestation and the methylation level of LINE-1 repetitive elements was analysed in the testicular DNA for the assessment of global methylation by bisulfite pyrosequencing. Additionally, quantitative methylation analysis was performed for Igf2 and H19 imprinted genes. We observed a lack of significant differences in average CpG methylation levels of examined DNA sequences of repetitive elements LINE-1 between the treated groups and the control. Also, we found that exposure to α-cypermethrin had no effect on the average CpG methylation levels within the differentially methylated regions (DMRs) of the Igf2 and H19 imprinted genes under these experimental conditions. It seems that prenatal α- cypermethrin exposure does not affect de novo DNA methylation process in the foetal rat testes. These results call for further researches of epigenetic changes caused by α-cypermethrin at the genome-wide level.

Irinotecan (IRI) is one of the most important cytostatics used in the treatment of advanced colon cancer. Some of its side effects such as acute and delayed cholinergic syndrome deteriorate the quality of life in patients. To improve their overall wellbeing, patients often use various supplements, including illegal ∆9- tetrahydrocannabinol (THC) preparations, sometimes containing more than 80% THC [1]. Our study investigated the interactions of IRI and THC at the cellular and organ level of Wistar rats by integrating biochemical, molecular biology and analytical methods. The obtained results provide evidence of a synergistic increase in the acute toxicity of IRI tested at a single intraperitoneal dose of 100 mg/kg under the influence of THC taken per os at a daily dose of 7 mg/kg for one, three and seven days. Concomitant administration of IRI and THC enhances the degree of oxidative stress and genotoxic effects of IRI in liver cells [2]. In rats given the combined treatment, a lowering of leukocyte counts was observed compared with the group treated with single IRI [3]. Concomitant administration of THC with irinotecan enhanced the urinary excretion of THC compared with the group of rats treated with single THC, also suggesting a possible interaction of the tested compounds [4]. From the results obtained, we concluded that concomitant administration of IRI with high doses of THC leads to adverse pharmacokinetic and pharmacodynamic effects. Although the results obtained cannot be directly extrapolated to humans, they indicate a number of undesirable and potentially dangerous side effects as well as the need for extreme caution in the administration of THC in oncology patients. References 1. Kopjar, N. et al, Clinical Drug Investigation, 2020, 40, 775- 787. 2. Lucić Vrdoljak, A. et al., Molecules, 2018, 23, 1332. 3. Prester, Lj. et al., Chemico- Biological Interactions, 2018, 294, 128-134. 4. Fuchs, N. et al. Arhiv Hig Rada Toksikol, 2019, 70, 325-331. Acknowledgment – This study was funded through the programme of cooperation between the Institute for Medical Research and Occupational Health (Zagreb, Croatia) and the University North (Varaždin, Zagreb).

Psychoactive substance abuse has been increasing worldwide over recent decades and is a significant burden to human health. The majority of these substances are taken orally, but also by smoking, inhalation or injection. Lysergic acid diethylamide (LSD) is a classical hallucinogen widely abused for decades, while the popularity of phencyclidine (PCP or “angel dust”) has increased in recent years, especially among adolescents. Both substances produce significant psychoactive, neurochemical, and behavioral effects. Up to now, very little has been known on the general toxicity of these compounds, especially their possible neurotoxic effects at cell level. This study assessed the toxic effects of LSD and PCP on human neuroblastoma cell line SH-SY5Y. To establish cytotoxic effects, cells were first exposed to the tested compounds at a concentration range of 0.039 – 100 µM for 24 h. Using MTS assay, we determined concentrations that reduced cell viability by up to 30%. Cells treated with these selected concentrations were then subjected to the alkaline comet assay, along with a battery of biochemical assays that enable the estimation of oxidative stress-related effects (lipid peroxidation, induction of reactive oxygen species – ROS, glutathione level, and activities of enzymes glutathione peroxidase – GPx, superoxide dismutase – SOD, and catalase – CAT). Treatment with LSD at 6.25 µM resulted in 88.06±2.05 % viable cells, while for PCP treatment with 3.13 µM in 84.17±3.19 % viable cells. These concentrations allowed for genotoxicity testing, which resulted in a statistically significant increase of comet tail length and tail intensity after LSD treatment, and a statistically significant increase of comet tail length after PCP treatment versus negative control. At the same tested concentrations, LSD caused a marked increase of malondialdehyde level, compared to control, contrary to PCP. While LSD induced significant increases in ROS production and glutathione level, PCP treatment led to significant decreases of these parameters compared to control. Treatment with LSD significantly enhanced activities of all antioxidant enzymes compared to control. Treatment with PCP resulted in significantly increased GPx and SOD activities, but CAT activity was significantly lower than in the respective control. Taken together, LSD had a greater DNA damaging potential and exerted stronger oxidative activity than PCP in SH-SY5Y cells. To our knowledge, this is the first study that investigated all of the above-mentioned outcomes of LSD and PCP in vitro, and its results could contribute to a better understanding of the toxicity profiles of both compounds. 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).

Colorectal cancer patients treated with irinotecan (IRI) often manifest acute cholinergic syndrome, which is the reason behind the growing use of legally prescribed cannabis preparations as well as illicit ones that can contain very high Δ9- tetrahydrocannabinol (THC) levels. IRI-induced cholinergic syndrome originates from the inhibition of acetylcholinesterase (AChE), while butyrylcholinesterase (BChE) plays an important role in the hydrolytic metabolism of this prodrug. On the other hand, existing reports of THC effects on cholinesterases are inconclusive. In this study, we aimed to investigate how the concomitant use of a high THC dose with IRI affects cholinesterase activities in blood of mice with experimental colon cancer induced by injection of CT26 cell line. Male BALB/c mice were assigned randomly to five study groups: (i) negative control, (ii) cancer control, (iii) irinotecan (i.p. 60 mg/kg ; 1st and 5th day of the experiment), (iv) THC (p.o. 7 mg/kg ; repeatedly for 7 days), (v) IRI + THC. ChE activity in blood was assayed by the spectrophotometric Ellman method, using acetylthiocholine as a substrate and inhibitors to distinguish between AChE and BChE activities. The results showed a time-dependent increase in total ChE and BChE activities in all experimental groups, including cancer control. In contrast, AChE activities decreased, with some minor fluctuations in the THC and IRI+THC group. Although there was a lack of statistically significant differences, the time course of changes for AChE and BChE activities suggested that these esterases may not only be indicators of metabolic status but also functionally important in neoplastic cell transformation.

Opiates such as morphine and codeine are substances often misused to improve the performance of racing horses during competitions and are therefore on the International Federation for Equestrian Sports' list of banned substances. However, a positive antidoping test may be due to the consumption of feed (mainly alfalfa or oats) contaminated by poppy seeds containing the alkaloids morphine and codeine. In order to determine whether a positive antidoping test is the result of an intentional abuse of opiates or ingestion of feed contaminated by poppy seeds, we optimised conditions for the extraction of morphine and codeine from dehydrated alfalfa and developed and validated a gas chromatographic-mass spectrometric method for the simultaneous determination of both analytes. The most efficient extraction of morphine and codeine from dehydrated alfalfa was achieved using a citrate buffer pH4 followed by solid phase extraction. The method showed good linearity (R2>0.9980) in the tested concentration range (50-1600 ng/g), as well as good precision (RSD

Colorectal cancer patients treated with irinotecan (IRI) often manifest acute cholinergic syndrome, which is the reason behind the growing use of legally prescribed cannabis preparations as well as illicit ones that can contain very high Δ9- tetrahydrocannabinol (THC) levels. IRI-induced cholinergic syndrome originates from the inhibition of acetylcholinesterase (AChE), while butyrylcholinesterase (BChE) plays an important role in the hydrolytic metabolism of this prodrug. On the other hand, existing reports of THC effects on cholinesterases are inconclusive. In this study, we aimed to investigate how the concomitant use of a high THC dose with IRI affects cholinesterase activities in blood of mice with experimental colon cancer induced by injection of CT26 cell line. Male BALB/c mice were assigned randomly to five study groups: (i) negative control, (ii) cancer control, (iii) irinotecan (i.p. 60 mg/kg ; 1st and 5th day of the experiment), (iv) THC (p.o. 7 mg/kg ; repeatedly for 7 days), (v) IRI + THC. ChE activity in blood was assayed by the spectrophotometric Ellman method, using acetylthiocholine as a substrate and inhibitors to distinguish between AChE and BChE activities. The results showed a time-dependent increase in total ChE and BChE activities in all experimental groups, including cancer control. In contrast, AChE activities decreased, with some minor fluctuations in the THC and IRI+THC group. Although there was a lack of statistically significant differences, the time course of changes for AChE and BChE activities suggested that these esterases may not only be indicators of metabolic status but also functionally important in neoplastic cell transformation.

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.

α-cypermethrin (α-cyp) is a synthetic pyrethroid insecticide widely used in and around households, as well as in agriculture and veterinary applications due to its rapid degradation rate and low mammalian toxicity. Due to the fact that pyrethroids are one of the most used groups of insecticides worldwide and can act as endocrine disrupting chemicals, we investigated the effects of prenatal exposure to α- cyp on endocrine disruption parameters in rat mothers and foetuses at term. Pregnant Wistar rats were orally exposed from the 6th day of gestation (DG) to the 21st to α-cyp at 1, 10, and 19 mg/kg bw/day, diethylsilbestrol as positive control, corn oil as solvent control, and water as negative control. At the 21st DG, gravid uterus and ovaries were dissected under general anaesthesia and parameters of endocrine disruption were monitored according to adequate OECD protocols. No differences were found in the number of corpora lutea in the ovaries. The number of implants, live or dead foetuses, as well as of early and late resorptions did not change between groups. No changes in the number and weight of male and female foetuses were noticed. Male to female foetal sex ratio (% per litter) was approximately 70 to 30 % in the rats treated with α-cyp at 1 and 10 mg/kg bw/day and diethylsilbestrol, while about 40 to 60 %in negative and solvent control. Our findings suggest that α-cyp at the applied exposure scenarios and doses did not act as an endocrine disruptor in mother rats and foetuses.

Colon cancer is one of the most common cancers and causes of death worldwide. The aim of this study was to investigate the effects of the concomitant intake of Δ9-tetrahydrocannabinol (THC) and cytostatic irinotecan (IRI) on tumour growth and body and organ weights in mice with experimental colon cancer induced by an injection of the CT26 cell line. Male BALB/c mice (N=5 per group) were randomly assigned to the following groups: IRI (60 mg/kg b.w., i.p., 1st and 5th day of the experiment), THC (7 mg/kg b.w., p.o., consecutively during the 7-day experimental period), combination of IRI and THC, solvent control (sesame oil, p.o., consecutively during the 7-day experimental period), and cancer control (without treatment). Weight and tumour measurements were performed every other day throughout the experiment. Mice were sacrificed on the seventh day and tissue samples were dissected and weighed. Body weights decreased significantly in IRI and IRI-THC treated mice on the third day as well as in IRI-THC treated mice on the seventh day compared to THC-treated and cancer control mice. There were no significant changes in brain and liver relative weights between the experimental groups. The tumours grew constantly over time with significant differences in growth between the third and seventh day in all groups. Differences in tumour size between experimental groups were not observed. The present study showed that IRI exposure affected body weights when applied alone or in combination with THC and that neither single IRI, single THC or combined exposure affected tumour growth under these experimental conditions.

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.

Kanabis (lat. Cannabis sativa L., konoplja) je jedna od najviše istraživanih biljaka u povijesti znanosti. No, unatoč brojnim znanstvenim radovima o načinu djelovanja aktivnih tvari kanabisa (kanabinoida kojih je više od 100), još uvijek postoje prijepori oko postavljanja granice između primjerene medicinske primjene i rekreativne (narkotičke) uporabe kanabisa. Psihoaktivna tvar svojstvena kanabisu, čiji sadržaj u suhoj tvari služi zakonodavstvu kao osnovni razlikovni kriterij između industrijske i medicinske konoplje, jest delta-9-tetrahidrokanabinol (THC). Konzumiran u malim količinama THC uzrokuje blagi osjećaj euforije, dok se pri konzumaciji veće količine javljaju simptomi intoksikacije: izobličenje slike, dezorijentiranost, gubitak koordinacije te pojačan podražaj osjetila. Kronični učinci konzumacije pripravaka na bazi kanabisa, uz razvoj ovisnosti, najviše se očituju kao slabljenje kognitivnih funkcija i otežano pamćenje (Rocchetti i sur., 2013). S obzirom na navedene štetne učinke, liječnik će propisati primjenu pripravka kanabisa samo pri određenim medicinskim indikacijama za koje konvencionalni tretmani i lijekovi ne postižu željeni učinak ili izazivaju previše nuspojava (EMCDDA, 2018). Često se događa da pacijenti, posebice onkološki bolesnici, posežu za neregistriranim (ilegalnim) pripravcima kanabisa koji mogu sadržavati visoki udjel THC-a (čak i do 90 %) (Guzmán, 2018). Uzevši u obzir činjenice da se metabolički putevi THC-a i jednog od najčešće primjenjivanih citostatika za liječenje uznapredovalog karcinoma debelog crijeva, irinotekana, u organizmu međusobno preklapaju, proveli smo istraživanje na animalnom modelu kako bi utvrdili u kojoj mjeri primjena visokih koncentracija THC-a može uzrokovati neželjena međudjelovanja te poremetiti metabolizam i farmakokinetiku konvencionalnih lijekova (Prester i sur., 2018 ; Lucić Vrdoljak i sur., 2018 ; Kopjar i sur., 2019).

Background: Due to growing public interest in the antitumor potential of Δ9- tetrahydrocannabinol (THC) and its possible role as an agent for alleviating side-effects of chemotherapy with irinotecan (IRI), some oncology patients have begun to reach for highly concentrated THC preparations. Since IRI, THC and their metabolites are subjected to glucuronic acid conjugation via the same enzyme (UGT1A1), co-administered THC could reduce IRI metabolism and clearance of its metabolites (particularly SN-38), leading to enhanced toxicity. Objective: The aim of the study was to compare the urinary elimination of free (non- conjugated) THC and its metabolites [11-hydroxy- Δ9-THC (THC- OH) and 11-nor-9-carboxy-Δ9-THC (THC-COOH)] in IRI+THC treated rats with rats treated only with THC. Methods: Male Wistar rats (N = 5 per group) were administered a combination of a single dose of IRI (100 mg/kg b.w, i.p.) and THC (7 mg/kg b.w., p.o.) or THC only. Urine samples were collected during the first 24 h after the treatment. THC, THC-OH and THC-COOH concentrations were determined by gas chromatography-mass spectrometry. Results: Enhanced urinary THC-OH and THC-COOH excretion was noted in rats administered combined treatment compared to single THC treatment. Conclusion: Increased non-conjugated THC metabolite urinary concentrations in combined treatment might point to the competition of THC and IRI for the same enzyme in phase II metabolism. Further studies are needed to elucidate the increase of the free fraction of THC metabolites in the concomitant use of IRI and THC.

The global use of preparations based on Cannabis sativa, both prescribed and illicit, is increasing among cancer patients. Generally, drug-drug or herb-drug interactions can result in therapeutic failure and it has been estimated to be the cause of death in approximately 4% of cancer patients. Interactions of cannabinoids with conventional chemotherapeutics are still poorly recognized. Irinotecan (IRI) is an anticancer drug whose use may lead to severe toxicities (predominantly neutropenia and diarrhoea) that require co-medications. The main active constituent of C. sativa, delta-9- tetrahydrocannabinol (THC), possesses a wide range of pharmacologic effects that might be of therapeutic benefit during therapy with IRI but its adverse psychotropic effects, as well as unknown toxic effects, must be borne in mind. Furthermore, there is a complex overlapping between IRI and THC metabolic pathways: both compounds are subject to the first pass hepatic metabolism mediated by the CYP3A4 enzyme ; their metabolites are subject to glucuronidation via the UGT1A1 enzyme ; the transport of parent compounds and their metabolites depends on the same or related transporters ; both compounds are subject to enterohepatic recirculation ; both compounds bind to plasma proteins ; both compounds affect mitochondrial oxidative phosphorylation and fatty acids metabolism. We present the results of a pilot study conducted on healthy male Wistar rats as a preliminary assessment of IRI and THC interactions. The experimental schedule included a single intraperitoneal application of IRI (at 100 mg/kg), while THC was administered per os repeatedly for 1, 3, and 7 days (at 7 mg/kg). The concomitant use of THC with IRI caused a prominent body and liver weight reduction and affected several clinical chemistry parameters in rats. Enhanced urinary THC excretion in animals treated with THC and IRI was noticed compared to THC alone. THC intake caused a significant synergic enhancement of IRI genotoxicity and oxidative stress responses.

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.

Irinotecan (IRI) is one of the most important antineoplastic drugs used in the chemotherapy of metastatic colorectal cancer. By binding to topoisomerase I-DNA complex it prevents religation of the DNA strand, causing double-strand DNA breakage and cell death. Considering the well- established sensitivity of the alkaline comet assay for detection of DNA damage in single cells, this study aimed to clarify (I) dynamics of DNA instability in liver cells, leukocytes and brain cells of male Wistar rats that were intraperitoneally administered single IRI dose of 100 mg/kg b.w., and (II) to establish the differences between levels of DNA damage produced in the studied cell types. Outcomes of the IRI exposure were assessed at first, fourth and eight post-treatment day. Corresponding control groups of rats were studied in parallel. To establish the overall toxic effects, we also estimated changes in total body weight, liver and brain weights of experimental animals. IRI treatment diminished total body weight of rats at each time-point of interest and also affected liver and brain weights compared to controls. The highest level of primary DNA damage was detected on the fourth post- treatment day in liver and brain cells, and on the eight post-treatment day in leukocytes. Observed pattern of DNA damage could be related both to induction of lesions and their repair, which obviously created additional DNA damage detectable by the alkaline comet assay. Brain cells were the most vulnerable cell type following IRI exposure. Differences in the cell susceptibility could be also related to inherent defence mechanisms in tested tissues which has to be proven in the forthcoming studies.

Irinotecan (IRI) represents one of the most important antineoplastic drugs used in the chemotherapy of metastatic colorectal cancer. Its use is accompanied by various adverse effects such as diarrhoea, myelosuppression and hepatotoxicity. To diminish side-effects, some cancer patients use Cannabis sativa preparations, rich on delta-9-tetrahydrocannabinol (THC). The potentially harmful interactions resulting from co-administration of chemotherapy with biologically based complementary therapies have not yet been explained well, which motivated us to conduct a study on a Wistar rat model using a THC dose equal to the one found in commonly used illicit preparations. Male rats were concomitantly exposed to IRI (at 100 mg/kg b.w., administered once i.p.) and THC (administered repeatedly for 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 brain weight caused by the treatments and evaluated the level of primary DNA damage in the brain cells of exposed and control rats using alkaline comet assay. Combined treatment slightly diminished brain weights compared to controls after both exposure times. The 3-day treatment resulted in significantly increased levels of DNA damage in brain cells of rats given single IRI, which worsened after concomitant exposure to THC. Single THC caused a minor increase of DNA damage compared to control rats. After the 7-day treatment, levels of DNA damage in rats given single IRI and IRI+THC slightly lowered, but were still significantly higher than in control rats. Prolonged exposure to THC resulted in a further increase of DNA damage. The obtained results speak against the concomitant use of THC with IRI, due to the possible enhancement of brain damage, but these findings have to be proven in forthcoming studies using much wider dose ranges of both compounds.

There is considerable interest for natural products that contain cannabinoids in combination with conventional antineoplastic therapy.However, cannabinoid-drug interaction may be associated with the risk of clinically relevant toxicities. Irinotecan (CPT-11) is one of the most important anticancer drugs used to treat different solid tumors and metastatic colorectal cancer but its use is associated with clinically important toxic effects particulary leucopenia and delayed diarrhea. Here, we report on the acute clinical effects of irinotecan + Δ9-THC on Wistar rats. Serial blood samples were obtained up to seven days after dosing and were analyzed for complete blood count and inflammatory markers (CRP and TNF-α). Both irinotecan and irinotecan + Δ9-THC caused moderate leucopenia but a greater decrease in leukocyte count (10exp9/L) was observed in the irinotecan + Δ9-THC treated group (1.4 ± 0.3) compared to the irinotecan-treated alone (2.7 ± 0.8). This study suggests that a single irinotecan dose of 100 mg/kg b.w. was sufficient to induce moderate leucopenia in Wistar rats without diarrheal symptoms and without an increase in circulating pro-inflammatory mediators. Since concomitant Δ9-THC administration to irinotecan.treated rats might produce higher immunosuppression than irinotecan alone, further studies are needed to investigate the influence of medicinal cannabinoids on hematological toxicity.

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.

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.

Organophosphate compounds are among the most frequently used pesticides worldwide. In this study, we evaluated the in vivo effects of the insecticide chlorpyrifos, which was orally administered to male Wistar rats at 0.160 mg/kg, 0.015 mg/kg, and 0.010 mg/kg for 28 days. Following treatment, the level of lipid peroxidation and acetylcholinesterase (AChE) activity was estimated in plasma and brain. To establish the potential DNA damaging effects of the exposure, we applied an alkaline comet assay on the white blood cells and brain tissue of the exposed and control animals. We found that 28-day exposure resulted in dose-dependent changes in AChE activity, which was significantly more depressed in the brain. Lipid peroxidation, presented as TBARS concentration, was elevated both in plasma and in the brain. All of the tested doses of chlorpyrifos were slightly genotoxic, both to the white blood cells and brain tissue. Taken together, our findings confirmed the AChE-inhibiting potency of chlorpyrifos and indicated that its toxicity was mediated through free radicals, which contributed to DNA instability. Considering that our study focused on very low doses of chlorpyrifos within toxicology reference values our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Tembotrione is the youngest member of the triketone herbicide family, used for postemergence weed control in corn. Although tembotrione’s impacts on the environment and human and animal health have been determined to be acceptable with regard to regulations, the biomarkers of exposure to this compound have not been strictly defined. To our knowledge, no studies using molecular biology and biochemistry methods have been performed evaluating tembotrione’s cytotoxic and oxidative potential in mammals. Therefore, we evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione corresponding to the occupational exposure limit (0.0012 μg/ml), residential exposure level (0.002 μg/ml), acceptable daily intake (0.17 μg/ml), and reference concentration (3 μg/ml) on human hepatocellular carcinoma cell line HepG2 by assessing the extent of lipid peroxidation and total anti-oxidant capacity as well as the DNA damage using the alkaline comet assay. Viability of HepG2 cells after treatment with tembotrione was assessed using CCK-8 colorimetric assay. Our results have shown that low concentrations of tembotrione possess DNA damaging potential even though the viability of HepG2 cells was not significantly impaired. Considering that biomarkers of oxidative stress were not significantly altered at the tested concentrations, the indirect effects mediated through free radicals probably did not play an important role in the formation of DNA damage. Taking into account that similar investigations on the toxicity of low pesticide doses are rare, the present study points to the use of other model systems and methods for clarifying a compound’s toxic mode of action.

Terbuthylazine (TB) belongs to the chloro- triazine group of herbicides, and primarily acts as an inhibitor of photosynthesis. It is used as a substitute for atrazine that is banned in EU countries. Workers in industrial and residential settings could be exposed to TB, especially if pesticide is handled without appropriate safety precautions. Considering that its toxicity profile, especially genotoxic and carcinogenic potential is not fully described yet, this study focused on the evaluation of in vitro toxicity of TB on human peripheral blood lymphocyte model. Assessments were made using the biomarkers of lipid peroxidation and oxidative stress, quantitative fluorescent assay for simultaneous identification of apoptotic and necrotic cells, alkaline comet assay, and hOGG1-modified comet assay. Lymphocytes were treated for 4 h at doses of TB equal to its occupational exposure level, acceptable daily intake, and residential exposure level. Following TB treatment, we observed slight changes in the levels of thiobarbituric acid reactive substances, and total antioxidant capacity in lymphocytes. Cell viability decreased in a concentration- dependent manner. Apoptosis dominated over necrosis. The levels of primary DNA damage were concentration-dependent, both in terms of comet tail length and DNA % in comet tail. However, the results of hOGG1 comet assay did not indicate significant contribution of oxidative lesions to overall DNA damage. Taken together, our results suggest that TB has low cyto-/genotoxic potential to lymphocytes but to clarify the mechanisms involved in its toxicity further studies including more sensitive techniques to detect physiological and metabolic changes at the cell level should be undertaken.

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.

Pyridinium oximes are used to successful antidotes poisoning by various organophosphate compounds mainly due to their ability to protect unphosphorylated acetylcholinesterase (AChE) and/or reactivate phosphorylated AChE. Previous studies with the K048 oxime (N-(4-(4-hydroxyiminomethylpyridinio)butyl)-4-carbamoyl-pyriminium dibromide), a representative of a new generation of pyridinium oximes, have suggested its acceptable cytotoxicity and low potential for infliction of primary DNA damage in human peripheral blood lymphocytes in vitro and rat blood cells in vivo. In the present study, we evaluated the genotoxic potency of K048 oxime on the human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line HaCaT in vitro. Since both cell lines are known for non-neuromuscular AChE expressions, this study also aimed to clarify the mutual relationships between DNA damage and AChE activity. Both cell lines were cultivated in RPMI growth medium supplemented with glutamine, heat-inactivated fetal bovine serum, and antibiotics. Prior to the treatment, cells were seeded in 6-well plates (3x105 cells/ml). The K048 oxime was tested in the concentration range 0.0073 to 2mM, which was well below the IC50 value, established previously. After 30 minutes of treatment, aliquots of cells were used for the preparation of agarose microgels according to standard protocol for the alkaline comet assay. Slides were analysed under fluorescent microscope using the Comet Assay IV analysis system (Perceptive Instruments Ltd., UK). The level of DNA damage was evaluated based on comet tail length, tail intensity and tail moment. Our results show that both cell lines expressed a low level of spontaneous DNA damage. The short exposure time used here was obviously not critical for the induction of severe DNA lesions. The most prominent finding was time concentration- dependent decrease of genotoxicitym which was observed in both cell lines, Primary DNA damage was slightly increased as compared to the negative control only at two lowest concentrations tested. This observation points to a potential antioxidative efficacy of the tested compound, which has to be elucidated in future investigations. Taken together, our results confirm high biocompatibility for the K048 oxime, which has already been recognized

Institut za medicinska istraživanja i medicinu rada (IMI) od početka svog rada 1949. g. okuplja znanstvenike različitih struka koji proučavaju utjecaj općeg i radnog okoliša na zdravlje ljudi s različitih znanstvenih aspekata (biomedicina i zdravstvo, prirodne i društvene znanosti). Aktualna istraživanja IMI-a, kao i u ranijim razdobljima, u skladu su s očekivanjima i planovima Svjetske zdravstvene organizacije, tj. aktualnog „Globalnog akcijskog plana za zdravlje radnika 2008-2017“. Istraživanja obuhvaćaju različite aspekte određivanja vrsti i razina kemijskih, bioloških i fizikalnih štetnosti u radnom okolišu, te biomonitoringa kao metode procjene izloženosti i zdravstvenih učinaka okolišnih štetnosti, s naglaskom na pokazatelje ranih subkliničkih učinaka, te korištenje neinvazivnih metoda sakupljanja uzoraka. Novija istraživanja usmjerena su i na povezanost zdravstvenih učinaka okolišnih štetnosti s osobnim čimbenicima koji mogu značajno modificirati okolišne učinke. Određene genetske varijante mogu značajno mijenjati rizik pojave nekog zdravstvenog učinka te na taj način možemo izdvojiti skupine radnika koje su posebno osjetljive na određene štetnosti. Provode se i istraživanja kojima se procjenjuje učinkovitost mjera zaštite na radu, studije praćenja radnika sa profesionalnim bolestima, te istraživanja novih antidota za liječenje profesionalnih otrovanja. IMI od 2013.g. ponovno sudjeluje u edukaciji specijalizanata iz medicine rada i sporta, što će, vjerujemo, povećati njihovu uključenost i unaprijediti znanstvena istraživanja zdravlja radnika u Hrvatskoj.

Aim: Tabun is a highly toxic chemical warfare agent which causes fatal intoxication due to inhibition of acetylcholinesterase (AChE). Recent knowledge confirms the use of pyridinium oximes as causal antidotes for the protection/reactivation of AChE inhibited by organophosphorus compounds. K048 oxime showed an acceptable genotoxic profile both in vitro and in vivo models. Here we studied its potential neuroprotective effects in vivo using the alkaline comet assay in brain cells of rats which were given K048 oxime to relieve symptoms of tabun-poisoning. Methods: Altogether sixty male adult Wistar rats were used in the experiment. They were divided in five groups according to the treatment: (1) tabun-poisoned (75% LD50, s.c.), (2) K048-treated (25% LD50 i.p.), (3) atropine-treated (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), (4) concomitant treatment with K048 (25% LD50, i.p.) and atropine (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), and (5) negative controls given saline only (2 mL kg-1, i.p.). Rats were sacrificed 1, 6 and 24 h after the treatment, and comet assay on brain cells was performed. Results: We found that tabun itself caused a most notable increase of primary DNA damage in rat brain cells 24 h after its administration. K048 oxime had an acceptable genotoxicity profile at each time- point studied. Minor differences in the genotoxicity observed between three time-points could be explained by the metabolism of the compound. Concomitant administration of K048 with atropine changed its genotoxicity profile in the brain cells, especially 6 h and 24 after treatment, which has to be further studied. Conclusions: Taken together, K048 effectively counteracted the poisoning of rats by tabun, both alone and in combination with atropine. Observed neuroprotective ability speaks in favour of K048 as a promising molecule for therapeutic treatment against intoxication with organophosphorus compounds.

Aim: Oximes play an important role in the elimination of toxic effects caused by chemical warfare agents, especially in the cases resistant to the atropine treatment alone. K048 oxime as one of the promising antidote in humans recently showed an acceptable genotoxic profile. Present study is focused on potential hepatoprotective effects of K048 effects in vivo. Methods: We selected a model of tabun- poisoned rats, which were given K048 oxime to relieve symptoms of tabun-poisoning. The levels of primary DNA damage in liver cells were studied using the alkaline comet assay 1, 6 and 24 h after the treatments. Sixty male adult Wistar rats were divided in five groups according to the treatment: (1) tabun-poisoned (75% LD50, s.c.), (2) K048-treated (25% LD50 i.p.), (3) atropine-treated (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), (4) concomitant treatment with K048 (25% LD50, i.p.) and atropine (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), and (5) negative controls given saline only (2 mL kg-1, i.p.). Results: Tabun itself caused the highest level of primary DNA damage in hepatocytes 24 h after its administration. K048 oxime had an acceptable genotoxicity profile at each time point. Its hepatoprotective effects were obvious at each time point studied, and in all cases the levels of primary DNA damage measured in oxime-treated groups were lower or similar as compared to negative control. Minor differences in the genotoxicity observed between the time-points studied could be explained by the metabolism of the compound. Concomitant administration of K048 with atropine changed its genotoxicity profile in the hepatocytes, especially 6 h after treatment. Conclusions: K048 effectively counteracted the poisoning of rats by tabun, both alone and in combination with atropine. Hepatoprotective effects speak in favour of K048 as a promising molecule for therapeutic treatment against fatal poisoning with chemical warfare agents.