Analysis of pharmacokinetic profile and ecotoxicological character of cefepime and its photodegradation products.

An important problem is the impact of photodegradation on product toxicity in biological tests, which may be complex and context-dependent. Previous studies have described the pharmacology of cefepime, but the toxicological effects of its photodegradation products remain largely unknown. Therefore, photodegradation studies were undertaken in conditions similar to those occurring in biological systems insilico, in vitro, in vivo and ecotoxicological experiments. The structures of four cefepime photodegradation products were determined by UPLC-MS/MS method. The calculated in silico ADMET profile indicates that carcinogenic potential is expected for compounds CP-1, cefepime, CP-2 and CP-3. The Cell Line Cytomotovity Predictor 2.0 tool was used to predict the cytotoxic effects of cefepime and related compounds in non-transformed and cancer cell lines. The results indicate that possible actions include: non-small cell lung cancer, breast adenocarcinoma, prostate cancer and papillary renal cell carcinoma. OPERA models were used to predict absorption, distribution, metabolism and excretion (ADME) endpoints, and potential bioactivity of CP-2, cefepime and CP-4. The results obtained in silico show that after 96h of exposure, cefepime, CP-1, CP-2, and CP-3 are moderately toxic in the zebrafish model, while CP-4 is highly toxic. On the contrary, cefepime is more toxic to T. platyurus (highly toxic) compared to the zebrafish model, similar to products CP-4, CP-3 and CP-2. In vitro cytotoxicity studies were performed by MTT assay and in vivo acute embryo toxicity studies using Danio rerio embryos and larvae. In vitro showed an increase in the cytotoxicity of products with the longest exposure period i.e. for 8 h. Additionally, at a concentration of 200 µg/mL, statistically significant changes in metabolic activity were observed depending on the irradiation time. In vivo studies conducted with Zebrafish showed that both cefepime and its photodegradation products have only low toxicity. Assessment of potential ecotoxicity included Microbiotests on invertebrates (Thamnotoxkit F and Daphtoxkit F), and luminescence inhibition tests (LumiMara). The observed toxicity of the tested solutions towards both Thamnocephalus platyurus and Daphnia magna indicates that the parent substance (unexposed) has lower toxicity, which increases during irradiation. The acute toxicity (Lumi Mara) of nonirradiated cefepime solution is low for all tested strains (<10%), but mixtures of cefepime and its photoproducts showed growth inhibition against all tested strains (except #6, Photobacterium phoreum). Generally, it can be concluded that after UV-Vis irradiation, the mixture of cefepime phototransformation products shows a significant increase in toxicity.

Elucidating the molecular mechanisms underlying the induction of autophagy by antidepressant-like substances in C57BL/6J mouse testis model upon LPS challenge.

The treatment of depression with pharmaceuticals is associated with many adverse side effects, including male fertility problems. The precise mechanisms by which these agents affect testicular cells remain largely unknown, but they are believed to induce cellular stress, which is sensed by the endoplasmic reticulum (ER) and the Golgi apparatus. These organelles are responsible for maintaining cellular homeostasis and regulating signal pathways that lead to autophagy or apoptosis. Therefore, in this study, we aimed to investigate the autophagy, ER, and Golgi stress-related pathways in mouse testis following treatment with antidepressant-like substances (ALS) and ALS combined with lipopolysaccharide (LPS). We found that most ALS and activated proteins are associated with the induction of apoptosis. However, when imipramine (IMI) was combined with NS-398 (a cyclooxygenase-2 inhibitor) after LPS administration, we observed a marked increase in the BECLIN1, Bcl-2, ATG16L, and LC3 expression, which are marker proteins of autophagosome formation. The expression of the BECN1 and ATG16L genes was also high compared to the control, indicating the induction of autophagy processes that may potentially protect mouse testicular cells from death and regulate metabolism in the testis. Our findings may provide a better understanding of the stress-related effects of specific ALS on the testis. Video Abstract.

Synthesis of Novel 2-(Cyclopentylamino)thiazol-4(5H)-one Derivatives with Potential Anticancer, Antioxidant, and 11ß-HSD Inhibitory Activities.

In this study, a series of nine new 2-(cyclopentylamino)thiazol-4(5H)-one derivatives were synthesized, and their anticancer, antioxidant, and 11ß-hydroxysteroid dehydrogenase (11ß-HSD) inhibitory activities were tested. Anticancer activity has been assessed using the MTS (MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay against human colon carcinoma (Caco-2), human pancreatic carcinoma (PANC-1), glioma (U-118 MG), human breast carcinoma (MDA-MB-231), and skin melanoma (SK-MEL-30) cancer cell lines. Cell viability reductions, especially in the case of Caco-2, MDA-MB-231, and SK-MEL-30 lines, were observed for most compounds. In addition, the redox status was investigated and oxidative, but nitrosative stress was not noted at a concentration of 500 µM compounds tested. At the same time, a low level of reduced glutathione was observed in all cell lines when treated with compound 3g (5-(4-bromophenyl)-2-(cyclopentylamino)thiazol-4(5H)-one) that most inhibited tumor cell proliferation. However, the most interesting results were obtained in the study of inhibitory activity towards two 11ß-HSD isoforms. Many compounds at a concentration of 10 µM showed significant inhibitory activity against 11ß-HSD1 (11ß-hydroxysteroid dehydrogenase type 1). The compound 3h (2-(cyclopentylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one) showed the strongest 11ß-HSD1 inhibitory effect (IC50 = 0.07 µM) and was more selective than carbenoxolone. Therefore, it was selected as a candidate for further research.

The Effect of Copper Nanoparticles and a Different Source of Dietary Fibre in the Diet on the Integrity of the Small Intestine in the Rat.

The aim of the study was to verify the hypothesis regarding the effect of recommended (6.5 mg/kg) or enhanced (13 mg/kg) level of CuNPs in the diet in combination with different types of dietary fibre-cellulose (control), inulin, pectin or psyllium-on selected biological parameters of intestinal integrity in rats. Rats were randomly divided into 10 groups. The first two groups were fed a control diet that contained cellulose, and a mineral mixture with standard or enhanced content of CuCO3. Experimental groups were fed a diet supplemented with CuNPs (6.5 or 13 mg/kg) and combined with different types of fibre (cellulose, pectin, inulin or psyllium). After the feeding period, blood and small intestine samples were collected for further analysis. Replacing CuCO3 by CuNPs in the diet positively reduced the level of lactic acid and apoptosis markers in the small intestine; however, it also resulted in the intensification of DNA oxidation. The most beneficial effect on DNA repair mechanisms is related to inulin, while pectin has the greatest ability to inhibit inflammatory processes that induce the apoptotic death of cells in the small intestine. Our results suggest that dietary fibre supplementation protects the small intestine against potentially harmful, oxidative effects of CuNPs by intensifying the intestinal barrier.

Gurgem-7 toxicity assessment: Regulation of cell survival or death by traditional Mongolian prescription.

Surgical treatments and chemotherapy are the most commonly used methods of colorectal cancer treatment (CRC), unfortunately, these therapies have many side effects. Moreover, despite advances in primary and adjuvant treatments, the survival time in CRC patients is still unsatisfactory. Treatment options for patients with CRC continue to advance and recent research has shown that colorectal cancer is sensitive to plant-derived substances. The use of natural compounds contained in herbal extracts for the treatment of colon cancer or as adjunctive therapy for CRC gives patients a wide range of treatment options. In this study, we evaluate the potential toxicity of the Mongolian preparation - Gurgem-7 composed of Crocus sativus, Veronica officinalis, Capsella bursa-pastoris, Arctostaphylos uva-ursi, Calendula officinalis, Gentiana lutea, and Terminalia chebula. Therefore, the aim of this study was to determine its biological activities, biochemical and molecular features in vitro and composition analysis by HPLC-ESI-QTOF-MS/MS platform. We identified 18 metabolites and 8 of them were quantified. Majority of the secondary metabolites belonged to the group of phenolic constituents with taxifolin, chlorogenic acids' family, hydroxysafflor yellow A and hydroxybenzoic acid as leading compounds. In turn, our in vitro results suggest that the preparation inhibits cell metabolic activity through oxidative stress, numerous DNA damage and cell cycle arrest. Simultaneously enzymatic and non-enzymatic cell protection mechanisms mediated by TP53/Keap1 and Nrf2/HO-1 pathways may be activated in a cell-specific manner in vitro. In conclusion, we provide preliminary molecular evidence of the toxic properties of Gurgem-7 preparation to Caco-2 and CT26. WT cells related to insufficient action of their repair and adaptive mechanisms to stress conditions.

Screening and Structure-Activity Relationship of D2AAK1 Derivatives for Potential Application in the Treatment of Neurodegenerative Diseases.

Neurodegenerative and mental diseases are serious medical, economic and social problems. Neurodegeneration is referred to as a pathological condition associated with damage to nerve cells leading to their death. Treatment of neurodegenerative diseases is at present symptomatic only, and novel drugs are urgently needed which would be able to stop disease progression. We performed screening of reactive oxygen species, reactive nitrogen species, glutathione and level intracellular Ca2+. The studies were assessed using one-way ANOVA of variance with Dunnett's post hoc test. Previously, we reported D2AAK1 as a promising compound for the treatment of neurodegenerative and mental disorders. Here, we show a screening of D2AAK1 derivatives aimed at the selection of the compound with the most favorable pharmacological profile. Selected compounds cause an increase in the proliferation of a hippocampal neuron-like cell line, changes in the levels of reactive oxygen and nitrogen forms, reduced glutathione and a reduced intracellular calcium pool. Upon analyzing the structure-activity relationship, we selected the compound with the most favorable profile for a neuroprotective activity for potential application in the treatment of neurodegenerative diseases.

Trade-offs between male fertility reduction and selected growth factors or the klotho response in a lipopolysaccharide-dependent mouse model.

The increasing number of depression cases leads to a greater need for new antidepressant treatment development. It is postulated that antidepressants may harm male fertility, but the cellular mechanism is still poorly understood. The role of growth factors and klotho protein in maintaining normal male reproductive function is well documented. Hence, the study aimed to investigate the effect of the antidepressant drug - imipramine (tricyclic AD), and other substances with antidepressant potential (ALS), administered in combination or in combination with LPS (an animal model of depression) on gene expression and protein synthesis of IGF-2 (insulin-like growth factor 2), TGF-ß1 (transforming growth factor ß1), NGF (nerve growth factor), KGF (keratinocyte growth factor) and protein synthesis of VEGF-A (vascular endothelial growth factor A), IGF-IR (insulin-like growth factor receptor 1), EGFR (epidermal growth factor receptor) and klotho in the testis of mice. Mice were injected intraperitoneally with selected ALS and LPS or 10% DMSO (controls) (n = 7/group) once a day for 14 days. Animals were decapitated and testes collected for RNA and protein purification. PCR and western blot methods were employed for the evaluation of growth factors and klotho expression. The results obtained indicated a decreased level of most of the analyzed genes and proteins, except KGF; its expression increased after treatment with MTEP and IMI administrated individually and after NS-398, and IMI in combination with LPS. Our results may suggest that the tested ALS and LPS can contribute to a reduction of male fertility, but NS-398, IMI, and IMI+NS-398 may also act as stimulants after LPS.

In vitro and in vivo evaluation of antioxidant and neuroprotective properties of antipsychotic D2AAK1.

The susceptibility of neurons to free radical toxicity partially underlies the pathomechanism of neurodegenerative diseases. On the other hand, excitotoxicity also contributes to neurodegeneration. Our previous studies demonstrated the unique properties of D2AAK1 as a potent multi-target ligand of aminergic G protein-coupled receptors (GPCRs) which dose-dependently stimulates growth, survival of neurons, and promotes their integrity. The aim of our study was to investigate the potential neuroprotective and antioxidant properties of D2AAK1. Here we show that D2AAK1 activates cellular and molecular neuroprotective mechanisms, prevents cells from excitotoxicity and free radicals. Furthermore, D2AAK1 induced no genotoxic events in neuronal cells in vitro. Most importantly, D2AAK1 protects neurons from the effects of high temperatures by molecular chaperones activation. The D2AAK1 effects on selected organs was further evaluated in mice and no pathological changes were observed after chronic administration. In the light of our experiments, D2AAK1 can be further developed into a potential treatment for neurodegenerative diseases, in particular related to memory impairment. In summary, D2AAK1 has promising properties for potential treatments of neurodegenerative diseases.

Cancer on-target: Selective enhancement of 3-bromopyruvate action by an electromagnetic field in vitro.

Cancer is one of the leading causes of death in the modern world. Nowadays, most often treatment methods used in clinical oncology are drug therapies applied as monotherapy or combined therapy. Additionally, recent studies focus on developing approaches with the use of a drug in combination with other factors, not only chemical, to improve the probability and magnitude of therapeutic responses and reduce the possibility of chemoresistance. Such a promising factor seems to be an electromagnetic field (EMF) application. Here, we tested the effect of continuous or pulsed EMF on human cancer cells of different origin treated or not with 3-bromopyruvate, a small and powerful alkylating agent with a broad spectrum of anticancer activities. We provide strong evidence suggesting that ELF-EMF potentiates the anti-cancer activity of 3BP in human cancer cells through inhibition of TNFα secretion leading to irreversible p21/p27-dependent G2/M cell cycle arrest and finally cancer cell death. Our findings suggest a novel approach combining pharmacotherapy with ELF-EMF. In conclusion, electromagnetic field seems to be a potential modulator of anti-cancer efficacy of 3BP while combined therapy offers off-target activity. These features contribute to the development of innovative therapeutic strategies for cancer treatment.

Molecular Consequences of Depression Treatment: A Potential In Vitro Mechanism for Antidepressants-Induced Reprotoxic Side Effects.

The incidence of depression among humans is growing worldwide, and so is the use of antidepressants. However, our fundamental understanding regarding the mechanisms by which these drugs function and their off-target effects against human sexuality remains poorly defined. The present study aimed to determine their differential toxicity on mouse spermatogenic cells and provide mechanistic data of cell-specific response to antidepressant and neuroleptic drug treatment. To directly test reprotoxicity, the spermatogenic cells (GC-1 spg and GC-2 spd cells) were incubated for 48 and 96 h with amitriptyline (hydrochloride) (AMI), escitalopram (ESC), fluoxetine (hydrochloride) (FLU), imipramine (hydrochloride) (IMI), mirtazapine (MIR), olanzapine (OLZ), reboxetine (mesylate) (REB), and venlafaxine (hydrochloride) (VEN), and several cellular and biochemical features were assessed. Obtained results reveal that all investigated substances showed considerable reprotoxic potency leading to micronuclei formation, which, in turn, resulted in upregulation of telomeric binding factor (TRF1/TRF2) protein expression. The TRF-based response was strictly dependent on p53/p21 signaling and was followed by irreversible G2/M cell cycle arrest and finally initiation of apoptotic cell death. In conclusion, our findings suggest that antidepressants promote a telomere-focused DNA damage response in germ cell lines, which broadens the established view of antidepressants' and neuroleptic drugs' toxicity and points to the need for further research in this topic with the use of in vivo models and human samples.

IGF-1 as selected growth factor multi-response to antidepressant-like substances activity in C57BL/6J mouse testis model.

Insulin-like growth factor (IGF-1) affects almost all cells in the body. Extremely important functions of this growth factor have been demonstrated in the brain and the reproductive system of both, females and males. Also, it is considered as a pro-inflammatory cytokine adjusting tissue homeostasis. However, it seems to play a special role in the male reproductive system and it may be disturbed by the application of antidepressants with different mechanisms of drug action during therapy. To date, the effect of antidepressant-like substances (ALS) on the course of physiological processes in male testicular cells is poorly understood. Therefore, the purpose of the research was to determine the presence, localizationof IGF-1R (insulin-like growth factor 1 ß receptor) and mRNA gene expression of IGF-1R and IGF-1 after administration of 3-[(2-methyl-1,3-tiazol-4-yl)ethynyl]-pyridine (MTEP) and N-[2-(Cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398) in the different scheme in the testis of mice. Imipramine was used as a reference drug having a documented interaction with the mGluR5 receptors. The immunohistochemical analyses showed the localization of IGF-1R in Sertoli, Leydig, and germinal cells after all used substances. Differences in receptor localization were observed depending on the drugs applied and the type of analyzed cells. In contrast, there was a significant increase in IGF-1 gene expression after IMI + NS-398 and in IGF-1R after MTEP + NS-398 and IMI + NS-398 administration. It can, therefore, be assumed that the use of a combination of NS-398 with some ALS may run different mechanisms of action and affect the regulation of reproductive function in mouse testis through maintaining homeostasis at the molecular and immunological levels related to IGF.

Risk of wild fungi treatment failure: Phallus impudicus-induced telomere damage triggers p21/p53 and p16-dependent cell cycle arrest and may contribute to male fertility reduction in vitro.

The multifunctional characteristics of Phallus impudicus extract encourage to conduct research for its potential use in medical applications. Well, science is constantly seeking new evidence for the biological activity of extracts of natural origin. Drugs of natural origin should not cause any side effects on the physiological functions of the human body; however, this is not always successful. In this study, we used in vitro approach to evaluate the toxicity of alcohol Phallus impudicus extract on spermatogenic cells. We show, for the first time, cytotoxic properties of Phallus impudicus treatment associated with a decrease in cellular metabolic activity, dysregulation of redox homeostasis and impairment of selected antioxidant cell protection systems. As a consequence, p53/p21- and p16-mediated cell cycle arrest followed by p27 activation is initiated. The observed changes were associated with telomere shortening and numerous DNA damage at the chromosome ends (altered expression pattern of TRF1 and TRF2 proteins), as well as upregulation of cleaved caspase-3 with a decrease in Bcl-2 expression, suggesting induction of apoptotic death. Therefore, these results provide molecular evidence for mechanistic pathways and novel adverse outcomes linked to the Phallus impudicus treatment towards men's health and fertility reduction.

The Antipsychotic D2AAK1 as a Memory Enhancer for Treatment of Mental and Neurodegenerative Diseases.

The treatment of memory impairments associated with the central nervous system diseases remains an unmet medical need with social and economic implications. Here we show, that a multi-target ligand of aminergic G protein-coupled receptors with antipsychotic activity in vivo (D2AAK1) stimulates neuron growth and survival and promotes neuron integrity. We focused on the multilevel evaluation of the D2AAK1-related effects on neurons in terms of behavioral, cellular, molecular, and biochemical features in vivo and in vitro, such as memory-related responses, locomotor activity, tissue sections analysis, metabolic activity, proliferation level, neurons morphology, and proteins level involved in intracellular signaling pathways. In silico studies indicate that activation of calcium/calmodulin-dependent protein kinase I (CaMKI) may underline some of the observed activities of the compound. Furthermore, the compound increases hippocampal neuron proliferation via the activation of neurotrophic factors and cooperating signals responsible for cell growth and proliferation. D2AAK1 improves memory and learning processes in mice after both acute and chronic administration. D2AAK1 also causes an increase in the number of hippocampal pyramidal neurons after chronic administration. Because of its neuroprotective properties and pro-cognitive activity in behavioral studies D2AAK1 has the potential for the treatment of memory disturbances in neurodegenerative and mental diseases.

Focus on the Role of Klotho Protein in Neuro-Immune Interactions in HT-22 Cells Upon LPS Stimulation.

Neuroinflammation is defined as the activation of the brain's innate immune system in response to an inflammatory challenge and is considered to be a prominent feature of neurodegenerative diseases. The contribution of overactivated neuroglial cells to neuroinflammation and neurodegenerative disorders is well documented, however, the role of hippocampal neurons in the neuroinflammatory process remains fragmentary. In this study, we show for the first time, that klotho acts as a signal transducer between pro-survival and pro-apoptotic crosstalk mediated by ER stress in HT-22 hippocampal neuronal cells during LPS challenge. In control HT-22 cells, LPS treatment results in activation of the IRE1α-p38 MAPK pathway leading to increased secretion of anti-inflammatory IL-10, and thus, providing adaptation mechanism. On the other hand, in klotho-deficient HT-22 cells, LPS induces oxi-nitrosative stress and genomic instability associated with telomere dysfunctions leading to p53/p21-mediated cell cycle arrest and, in consequence, to ER stress, inflammation as well as of apoptotic cell death. Therefore, these results indicate that klotho serves as a part of the cellular defense mechanism engaged in the protection of neuronal cells against LPS-mediated neuroinflammation, emerging issues linked with neurodegenerative disorders.

Towards Age-Related Anti-Inflammatory Therapy: Klotho Suppresses Activation of ER and Golgi Stress Response in Senescent Monocytes.

Immunosenescence in monocytes has been shown to be associated with several biochemical and functional changes, including development of senescence-associated secretory phenotype (SASP), which may be inhibited by klotho protein. To date, it was believed that SASP activation is associated with accumulating DNA damage. However, some literature data suggest that endoplasmic reticulum and Golgi stress pathways may be involved in SASP development. Thus, the aim of this study was to investigate the role of klotho protein in the regulation of immunosenescence-associated Golgi apparatus and ER stress response induced by bacterial antigens in monocytes. We provide evidence that initiation of immunosenescent-like phenotype in monocytes is accompanied by activation of CREB34L and TFE3 Golgi stress response and ATF6 and IRE1 endoplasmic reticulum stress response, while klotho overexpression prevents these changes. Further, these changes are followed by upregulated secretion of proinflammatory cytokines, which final modification takes place exclusively in the Golgi apparatus. In conclusion, we provide for the first time evidence of klotho involvement in the crosstalk on the line ER-Golgi, which may, in turn, affect activation of SASP. This data may be useful for a novel potential target for therapy in age-related and chronic inflammatory conditions.

Autophagy as a consequence of seasonal functions of testis and epididymis in adult male European bison (Bison bonasus, Linnaeus 1758).

The European bison is still an animal endangered with extinction, so by learning factors that regulate its reproduction, we can contribute to the survival of this species. On the other hand, autophagy is a dynamic, lisosomal, and evolutionary conserved process which is essential for animal cell survival, homeostasis, and differentiation. This process was demonstrated in many species and in many organs; however, information on the metabolic course of autophagy in the male reproductive system in seasonally reproducing species is lacking. Therefore, in this study, we examined for the first time several autophagy-related factors (mTOR, ULK1, Atg13, PI3K, beclin1, beclin2, Atg14, Atg5, Atg16L, LC3) in testicular and epididymal tissues obtained from adult male individuals of the European bison. We compared the level of gene expression, protein synthesis, and localization of autophagy-related factors between June, September, and December (before, during, and after reproductive activity, respectively). We confirmed that the induction of autophagy was at the highest level in the period after reproductive activity, i.e., in December, when a significant increase in the gene and protein expression was observed for the majority of these factors, probably to ensure cellular protection. However, autophagy was also clearly marked in September, during the intense spermatogenesis, and this may indicate a great demand for autophagy-related proteins required for the normal development of reproductive cells. Obtained results seem to confirm that autophagy pathway, as a consequence of seasonal reproduction, may control the normal course of spermatogenesis in the male European bison.

Klotho-mediated changes in the expression of Atg13 alter formation of ULK1 complex and thus initiation of ER- and Golgi-stress response mediated autophagy.

In the previous paper of our group, we have demonstrated that one of the crucial factors involved in the crosstalk between autophagy and apoptosis is klotho protein. We have shown that klotho silencing in normal human fibroblasts intensifies lipopolysaccharide (LPS)-induced p-eIF2a-mediated stress of endoplasmic reticulum and thus leads to retardation of prosurvival autophagy and induction of apoptotic cell death. In this study, we have performed a detailed step-by-step analysis of autophagy flux-related genes' expression and endoplasmic reticulum and Golgi stress related pathways in order to determine the exact mechanistic event when autophagy is inhibited in klotho-deficient cells on account of apoptosis initiation. We provide evidence that klotho-silencing in LPS-treated cells results in differential course of ER- and Golgi-mediated stress response. Further, we show that in klotho-deficient cells formation of ULK1 complex is inhibited and thus autophagy initiation is blocked on the account of apoptosis activation, while in the control cells cytoprotective autophagy is activated. Finally, in klotho-deficient cells formation of ULK1 complex is prevented by downregulated expression of Atg13. Thus, this study suggests a novel targeting pathway for efficient elimination of autophagy-deficient cells.

Neuronal life or death linked to depression treatment: the interplay between drugs and their stress-related outcomes relate to single or combined drug therapies.

Depression is a serious medical condition, typically treated by antidepressants. Conventional monotherapy can be effective only in 60-80% of patients, thus modern psychiatry deals with the challenge of new methods development. At the same moment, interactions between antidepressants and the occurrence of potential side effects raise serious concerns, which are even more exacerbated by the lack of relevant data on exact molecular mechanisms. Therefore, the aims of the study were to provide up-to-date information on the relative mechanisms of action of single antidepressants and their combinations. In this study, we evaluated the effect of single and combined antidepressants administration on mouse hippocampal neurons after 48 and 96 h in terms of cellular and biochemical features in vitro. We show for the first time that co-treatment with amitriptyline/imipramine + fluoxetine initiates in cells adaptation mechanisms which allow cells to adjust to stress and finally exerts less toxic events than in cells treated with single antidepressants. Antidepressants treatment induces in neuronal cells oxidative and nitrosative stress, which leads to micronuclei and double-strand DNA brakes formation. At this point, two different mechanistic events are initiated in cells treated with single and combined antidepressants. Single antidepressants (amitriptyline, imipramine or fluoxetine) activate cell cycle arrest resulting in proliferation inhibition. On the other hand, treatment with combined antidepressants (amitriptyline/imipramine + fluoxetine) initiates p16-dependent cell cycle arrest, overexpression of telomere maintenance proteins and finally restoration of proliferation. In conclusion, our findings may pave the way to better understanding of the stress-related effects on neurons associated with mono- and combined therapy with antidepressants.

Klotho-Mediated Changes in Shelterin Complex Promote Cytotoxic Autophagy and Apoptosis in Amitriptyline-Treated Hippocampal Neuronal Cells.

Amitriptyline, antidepressant frequently prescribed for treatment of depressive disorders and several neuropathic and inflammatory diseases, has been shown to cause neurotoxic effects. This effect has been partially linked with increased oxidative stress and apoptosis initiation; however, the exact mechanism is still unknown. Klotho protein due to its neuroprotective characteristics seems to be involved in the amitriptyline-mediated neurotoxicity. In this study, we have evaluated the effect of klotho silencing on mouse hippocampal cells exposed to amitriptyline. We show, for the first time, that klotho silencing intensified in hippocampal neurons amitriptyline-induced imbalance in oxido-nitrosative and mineral homeostasis, genomic instability associated with telomere dysfunction what resulted in p16- and p53/p21-mediated cell cycle arrest and activation of autophagy and apoptotic cell death in consequence. Therefore, these results indicate that klotho serves as a part of the cellular defense mechanism engaged in the protection of neurons against amitriptyline-mediated toxicity.

Seasonal expression of insulin-like growth factor 1 (IGF-1), its receptor IGF-1R and klotho in testis and epididymis of the European bison (Bison bonasus, Linnaeus 1758).

The European bisons are the largest mammals of Europe that are still in danger of extinction. The species conservation is associated with their continuous reproduction, and bisons are characterized by the well-pronounced seasonality of reproductive processes. However, the exact mechanisms regulating their reproduction still remain unknown. Our previous studies indicated the involvement of some of the growth factors in the regulation of male seasonal reproductive activities in bison, showing expression patterns that seemed to be regulated by the length of the daylight. In the present study, using RT-PCR and Western blot approaches, we verified the expression and possible relationship between the insulin-like growth factor (IGF-1), its receptor (IGF-1R), and klotho in testis and epididymis of the European bison in pre- and post-reproductive periods, i.e., in June and in December. The observed expression of IGF-1 and IGF-1R mRNA in testis and epididymis was higher in June than in December. At the same time, klotho mRNA expression in both testis and epididymis did not differ between the analyzed seasons. However, along with the higher levels of IGF-1R protein observed in June, klotho protein levels for the membrane form and for the secrete form were higher in December than in June. Finally, the messenger and protein expression profiles presented herein indicate the importance of both the IGF-system and klotho in reproductive processes in the European bison, implying their involvement in the regulation of seasonal testicular activity in males of this threatened species.