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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.

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.

Prostate cancer is among the most commonly diagnosed malignancies in men and its incidence increases with age. In addition to family history, race, and ethnicity, various external risk factors may contribute to its increased risk. Arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) are pervasive environmental contaminants and several experimental and epidemiological studies linked exposure to these metal(loid)s with an increased risk of prostate cancer. However, research on the impact of low-level exposure is scanty and largely inconclusive. Prostate specific antigen (PSA), combined with a digital rectal examination of prostate, has been routinely used for early detection of prostate cancer in spite of its insufficient specificity for clinically important cancers. Moreover, several studies in presumably healthy men indicated that various external factors can affect PSA levels, including metal(loid) exposure. Therefore, we aimed to evaluate the role of environmental exposure to As, Cd, Pb and Hg in prostate cancer risk and their impact on serum PSA. Blood As, Cd, Pb, and Hg and serum PSA were measured in 62 prostate cancer patients and 30 control men with no occupational exposure to metals. Prostate cancer patients had significantly higher serum PSA and blood Hg than control subjects. The results of Spearman’s correlation and a simple linear regression analysis in all 92 subjects showed a positive association between blood Hg and PSA. However, after control for the impact of the remaining elements in the models of multiple regression, this association was lost. Although we did not confirm a linear association between measured metal(loid)s and PSA in conditions of environmental exposure, further investigation in larger cohorts, taking into consideration possible synergistic or non-linear mixture effects, are warranted.

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.

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 use of pyrethroid and neonicotinoid insecticides has been increasing during the past decade. Although considered as a good alternative to organophosphate pesticides, there are reports indicating their adverse health effects. Our aim was to assess the effects of exposure to low doses of α-cypermethrine or imidacloprid on oxidative stress parameters and concentration of essential elements in testes and epididymis of adult Wistar rats. Rats were orally treated with doses comparable to currently proposed health-based reference values (α-cypermethrine: 0.02 (ADI), 0.15 (OEL), 0.80 (5×OEL) or 2.20 (REL) mg/kg b.w./day ; imidacloprid: 0.06 (ADI), 0.80 (10×AOEL) or 2.25 (1/200 LD50) mg/kg b.w./day) for 28 consecutive days. Exposure to α-cypermethrine or imidacloprid significantly decreased body weight gain in treated groups compared to the controls. The weight of testes was significantly lower in animals treated with the highest dose of imidacloprid, while treatment with α-cypermethrine had no effect on testicular/epididymal weight. Both insecticides significantly increased the level of reduced glutathione in epididymis and the activities of epididymal glutathione peroxidase and superoxide dismutase. Additionally, treatment with α-cypermethrine increased the activity of glutathione peroxidase in testes at the highest applied dose. A significantly lower concentration of Fe, Mo and Se, and higher concentration of Ca was measured in testicular tissue of animals treated with α-cypermethrine, while treatment with imidacloprid resulted in higher concentration of Mo and Na in testes compared to control animals. The fact that such low doses had the potential to produce measurable biological effects calls for further evaluation of these widely used insecticides.

Testicular germ cell tumours (TGCT) are the most common malignancy in men aged 15–35 years and its incidence is increasing worldwide. Tumour markers commonly used in the diagnosis and prognosis of TGCT have limited sensitivity and specificity and additional, minimally invasive biomarkers for disease management are needed. Metabolomic-based profiling of amino acids in urine may identify changed metabolites as potential biomarkers of testicular cancer. Impaired metal(loid) homeostasis may increase susceptibility to cancer development. We compared a quantitative profile of 30 amino acids and 18 metal(loid)s in urine samples between 86 men with newly diagnosed TGCT and 68 control subjects, aged 18 to 55 years. The concentration of free amino acids was determined by GC-MS using Phenomenex EZ:faast kit for sample preparation, multi-element analysis was performed by ICP-MS, and all results were corrected for creatinine levels. Testicular cancer patients had significantly higher levels of aspartic acid (Asp), manganese (Mn) and zinc (Zn), and significantly lower levels of threonine (Thr), serine (Ser), histidine (His) and cobalt (Co) when compared to control subjects. Linear discriminant analysis using all 30 amino acids successfully separated subjects with TGCT from control subjects and the discrimination rate was 86%. Metabolic pathway analysis (MetaboAnalyst 4.0) showed that Asp, Thr, Ser, and His were related to alterations in 12 metabolic pathways and those with the highest impact were metabolism of alanine, aspartate and glutamate (0.26) and glycine, Thr and Ser (0.23). Our results indicated that disorders in the citric acid cycle, the urea cycle and amino acid metabolism play a role in the pathogenesis of testicular cancer. The impaired urinary profile of Mn, Co and Zn may be highly relevant with regard to their role in amino acids synthesis, antioxidant defense and genetic repair processes. More research is needed to verify our findings

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).

Maternal cigarette smoking may impair the pro/antioxidant balance, affect bodily element disposition and disturb nutrient and oxygen transport to the developing foetus. The aim of this study was to assess the effect of active maternal smoking during pregnancy on superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and metallothionein (MT), cadmium (Cd), and zinc (Zn) concentrations in the samples of maternal and cord blood and placenta of healthy mother-newborn pairs collected after term vaginal delivery. We compared results for non-smokers and smokers (n = 37 each), grouped based on cotinine urine levels as a biomarker of tobacco smoke exposure (100 ng/mL in active smokers). Activity of SOD was 14 % lower in maternal plasma, 91 % higher in placenta and 12 % lower in cord plasma of smokers compared to non-smokers, whereas GPx and MT did not differ between the groups. These results accompanied decreased maternal blood Zn and increased placental Zn in smokers who also had significantly higher Cd in maternal blood and placenta than non-smokers. Cord blood Cd was low and did not differ between the groups. Although active maternal smoking contributed to Cd levels in maternal blood and placenta and Zn in placenta, there was no induction of increased placental MT synthesis. Overexpression of placental SOD can increase the susceptibility of offspring to oxidative stress and risk of disease development, even later in life. The study was funded by the Croatian Science Foundation (grant HRZZ-IP-2016-06-1998).

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.

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.

Dosadašnja epidemiološka istraživanja raka testisa pokazala su da je u uvjetima profesionalne izloženosti elementima poput As, Cd, Ni i Pb značajno povećan rizik za razvoj ove bolesti. Literaturni podaci ukazali su također na štetno djelovanje pojedinih elemenata u uvjetima niske do umjerene razine izloženosti, osobito kada je ta izloženost dugotrajna. S obzirom na to da su elementi široko rasprostranjeni u čovjekovu okolišu, čovjek im je neizbježno izložen. Stoga je važno razmatrati i utjecaj elemenata u uvjetima ekološke, relativno niske razine izloženosti, kao i utjecaj istovremene izloženosti različitim elementima. Cilj ovog istraživanja bio je izmjeriti koncentraciju Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Pt, Se, Sn i Zn u punoj krvi, serumu i urinu muškaraca s germinativnim tumorima testisa koji nisu profesionalno izloženi metalima i dobivene vrijednosti usporediti s onima izmjerenima u zdravih muškaraca. Profil elemenata u krvi, serumu i urinu i učinak koji oni mogu imati na razine tumorskih biljega do sada nisu proučavani u muškaraca s rakom testisa. Uspješnost diskriminacije ispitanika s rakom testisa u odnosu na kontrolne ispitanike temeljem profila svih mjerenih elemenata iznosila je preko 95% za sve tri biološke matrice (krv, serum i urin). Rezultati linearne diskriminantne analize (LDA) izdvojili su izmjerene koncentracije Ca, Co, Cu, Fe, K, Mg, Na i Zn u krvi, Ca, Cu, Na i Ni u serumu, te koncentraciju Cd, Co, Fe i Mn u urinu kao najjače prediktore bolesti. Utvrđena je značajna pozitivna korelacija između koncentracije α-fetoproteina (AFP) i Cu, β- humanog korionskog gonadotropina (β-HCG) i Cu te povezanost laktat dehidrogenaze (LDH) s koncentracijom Cu, Ca i K u serumu ispitanika s rakom testisa. Utvrđena je statistički značajna inverzna povezanost između koncentracije Fe u serumu s razinama AFP i LDH, kao i značajna inverzna povezanost između LDH i Se. S obzirom na važnu ulogu ispitivanih elemenata u mehanizmima koji određuju konačan ishod štetnog djelovanja različitih agenasa na ljudsko zdravlje, poremećaj homeostaze toksičnih i esencijalnih elemenata mogao bi dovesti do povećane osjetljivosti organizma i doprinijeti razvoju raka testisa, čak i u uvjetima umjerene do niske razine izloženosti.

Nastanak tumorskih stanica povezan je s brojnim biokemijskim i molekularnim promjenama u organizmu čije uočavanje ima veliki potencijal u ranoj dijagnostici i liječenju. Tumori, kao abnormalne nakupine tkiva, oduzimaju organizmu hranjive tvari, kisik, hormone i ostale životno bitne sastojke u svrhu vlastitog rasta te tako utječu na metabolizam stanice i regulaciju njene funkcije. Poznavanje i razumijevanje metaboličkih razlika između normalnih stanica i stanica raka prepoznato je kao moguća meta za ubijanje tumorskih stanica. Cilj ovog istraživanja bio je usporediti kvantitativni profil slobodnih aminokiselina i elemenata u urinu ispitanika s tumorom testisa u odnosu na njihove vrijednosti u kontrolnih ispitanika. Koncentracije slobodnih aminokiselina izmjerene su primjenom GC-MS-a nakon priprave uzoraka korištenjem komercijalnog seta reagensa Phenomenex EZ:faast, a koncentracije elemenata izmjerene su primjenom ICP-MS-a. U ispitanika s tumorom testisa izmjerene su značajno povišene koncentracije asparaginske kiseline, mangana i cinka, a značajno snižene koncentracije treonina, serina, histidina i kobalta u odnosu na koncentracije izmjerene u kontrolnih ispitanika. Ovi rezultati upućuju na to da razvojem tumora u testisima dolazi do poremećaja u ciklusu limunske kiseline i ciklusu uree. Naime, asparaginska kiselina ima važnu ulogu u ciklusu limunske kiseline i ciklusu uree. Dosadašnja istraživanja su pokazala da asparaginska kiselina sudjeluje u proliferaciji, iako ne svih vrsta tumora. Kobalt je neophodna komponenta kobalamina (vitamina B12) i ima vrlo važnu ulogu u formiranju aminokiselina, dok kobalaminski enzimi kataliziraju reakcije nastanka međuprodukata koji ulaze u ciklus limunske kiseline. Mangan ima ključnu ulogu u mnogim staničnim procesima, uključujući metabolizam lipida, proteina i ugljikohidrata te je sastavni dio enzima arginaze uključenih u sintezu uree i metabolizam piruvata. Dobiveni rezultati mogu poslužiti kao temelj za daljnja istraživanja o ulozi međuprodukata navedenih ciklusa u tumorigenezi što može pridonijeti eventualnom otkrivanju novih bioloških biljega tumora testisa.