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.

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.

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.

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.

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.

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.

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.

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.

Klotho modulates ER-mediated signaling crosstalk between prosurvival autophagy and apoptotic cell death during LPS challenge.

Bacterial endotoxins have been shown to induce prosurvival autophagy or apoptosis in fibroblasts and thus impair the wound healing process. Endoplasmic reticulum has been proposed as a molecular switch between these processes and klotho protein possessing pleiotropic characteristics seems to be involved in both processes, however the exact molecular mechanism is unknown. In this study, we have evaluated the effect of klotho silencing on human fibroblasts exposed to a non-toxic dose of lipopolysaccharide in terms of in vitro wound healing ability. We show for the first time, that klotho silencing in fibroblasts intensified lipopolysaccharide-induced oxidative stress and inflammatory response, what resulted in genomic instability, p-eIF2a-mediated ER stress, retardation of prosurvival autophagy, induction of apoptotic cell death and finally in impaired wound closure. Therefore, our data suggest that klotho serves as a part of cellular defense mechanism engaged in providing protection against bacterial infections during wound healing by modulating ER-signaling crosstalk between autophagy and apoptosis.

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.

Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes.

Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells.

Aloe arborescens juice prevents EMF-induced oxidative stress and thus protects from pathophysiology in the male reproductive system in vitro.

More and more studies suggest that prolonged exposure to EMF may cause adverse biological effects and point directly to a significantly negative correlation between EMF and human health, especially men fertility. In our previous study, we reported that this could be related to the EMF-induced reactive oxygen species formation, followed by DNA damage, cell cycle arrest and apoptosis induction. In this study, we decided to expand our research by the search for substances which would prevent EMF-induced damage in spermatogenic cells. Such an agent seems to be Aloe arborescens Mill. juice, which was shown to possess a wide range of protective properties. The administration of aloe extract helps among others to prevent the formation of free radicals by various biochemical pathways. Therefore, the main aim of our study was to provide a significant knowledge concerning the mechanism involved in the multi-pathway cytoprotective response of aloe juice against EMF. The study was carried out in an in vitro mouse spermatogenesis pathway cell lines (GC-1 spg and GC-2 spd). Our results suggest that the aloe juice has many positive effects, especially for the cellular antioxidant systems by reducing the intracellular reactive oxygen species pool induced by EMF. In consequence, aloe juice prevents DNA damage, cell cycle arrest and therefore the viability and metabolic activity of both cell line tested are preserved. In conclusion, our study provides new insight into the underlying mechanisms through which aloe juice prevents spermatogenic cells from cytotoxic and genotoxic events.

Vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF-2) as potential regulators of seasonal reproductive processes in male European bison (Bison bonasus, Linnaeus 1758).

Growth factors: vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor (FGF-2) were reported to affect normal physiological reproductive processes in human, domestic and free living animals. Moreover, some reports suggest that VEGF-A and FGF-2 may be directly involved in the control of the annual reproductive cycle of seasonally breeding animals but detailed knowledge is still missing. Our study aimed to demonstrate the expression of mRNA and protein for both factors in the tissues of testis and epididymis (caput, corpus, cauda) at different periods of the year (March, June, November, December) in European bison as a model of seasonally breeding animal. Results suggest, that VEGF-A expression was more pronounced in testis than in epididymis and the highest expression was noted in December and June. Surprisingly, the highest protein accumulation was observed in June at the same level in all tissues analyzed. On the other hand, the highest FGF-2 mRNA expression was noted in testis in June and in epididymis in March. However, no differences in protein expression of FGF-2 were found between analyzed groups. The results indicate that both factors are necessary for proper functioning of the reproductive system and their levels differ seasonally. Perhaps, it is linked to increased need of these factors in the testis as well as epididymis during preparation for the reproductive functions. Moreover, VEGF-A and FGF-2 not only may regulate reproductive functions by affecting vascularization and cell nutrition, but it also may be possible that they possess protective functions by stabilizing the reproductive cells. Therefore, obtained results provide new insight into mechanisms underlying seasonal breeding of the male European bison.

Extremely low frequency variable electromagnetic fields affect cancer and noncancerous cells in vitro differently: Preliminary study.

The exposure to extremely low frequency electromagnetic field (ELF-EMF) may result in various changes at the cellular level. To identify the effect of ELF-EMF exposure on viability of cells, cancer cells (U87-MG; 143B) and noncancerous cells (BJ; HEK) in exponential growth phase were exposed or sham-exposed to different values of frequency (2, 20, 30, 50 and 60 Hz), different shapes (sinusoidal, square and triangular) and time of exposure (0.5, 1, 2, 3 h) to electromagnetic field. After exposure, viability of cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We found a different effect of exposition of cancer and noncancerous cells to ELF-EMF on viability of cells. This preliminary study revealed that electro magentic field(EMF) might serve as a potential tool for manipulating viability of cells.

Genome organization and DNA methylation patterns of B chromosomes in the red fox and Chinese raccoon dogs.

The molecular structure of B chromosomes (Bs) is relatively well studied. Previous research demonstrates that Bs of various species usually contain two types of repetitive DNA sequences, satellite DNA and ribosomal DNA, but Bs also contain genes encoding histone proteins and many others. However, many questions remain regarding the origin and function of these chromosomes. Here, we focused on the comparative cytogenetic characteristics of the red fox and Chinese raccoon dog B chromosomes with particular attention to the distribution of repetitive DNA sequences and their methylation status. We confirmed that the small Bs of the red fox show a typical fluorescent telomeric distal signal, whereas medium-sized Bs of the Chinese raccoon dog were characterized by clusters of telomeric sequences along their length. We also found different DNA methylation patterns for the B chromosomes of both species. Therefore, we concluded that DNA methylation may maintain the transcriptional inactivation of DNA sequences localized to B chromosomes and may prevent genetic unbalancing and several negative phenotypic effects.

Chromium nanoparticles improve bone turnover regulation in rats fed a high-fat, low-fibre diet.

The aim of the study was to investigate the effect of returning to a balanced diet combined with chromium picolinate (CrPic) or chromium nanoparticles (CrNPs) supplementation at a pharmacologically relevant dose of 0.3 mg/kg body weight on the expression level of selected genes and bone turnover markers in the blood and bones of rats fed an obese diet. The results of the study showed that chronic intake of a high-fat obesogenic diet negatively affects bone turnover by impairing processes of both synthesis and degradation of bones. The switch to a healthy diet proved insufficient to regulate bone metabolism disorders induced by an obesogenic diet, even when it was supplemented with chromium, irrespective of its form. Supplementation with CrPic with no change in diet stimulated bone metabolism only at the molecular level, towards increased osteoclastogenesis (bone resorption). In contrast, CrNPs added to the high-fat diet effectively regulated bone turnover by increasing both osteoblastogenesis and osteoclastogenesis, with these changes directed more towards bone formation. The results of the study suggest that unfavourable changes in bone metabolism induced by chronic intake of a high-fat diet can be mitigated by supplementation with CrNPs, whereas a change in eating habits fails to achieve a similar effect.

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.

Polymorphism of cytogenetic markers in wild and farm red fox (Vulpes vulpes) populations.

Analysis of the origin of domestic animals is of wide interest and has many practical applications in areas such as agriculture and evolutionary biology. Identification of an ancestor and comparison with the domesticated form allows for an analysis of genetic, physiological, morphological and behavioral effects of domestication. Because fox breeding has been an ongoing process for over a century, differences are expected between farm and wild populations at the chromosomal level. The aim of this work was to analyse polymorphisms at the chromosomal level in foxes raised on farms and those living in the wild. Blood samples and lung tissue served as the experimental material and were obtained after slaughter of 35 foxes, including 28 breeding animals and 7 wild animals. The classical cytogenetic method was used including AgNOR technique, as well as molecular methods such as fluorescence in situ hybridization (FISH), and primed in situ labeling (PRINS). Analysis of the number of B chromosomes showed the presence of polymorphisms in foxes from both studied populations, but there was no correlation between the number of B chromosomes and the origin and gender of particular animals. An analysis ofactive nucleolar organizers showed the presence of a large number of polymorphisms and a tendency towards reduction of the number of NORs in the captive-raised population.