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The wide use of cobalt (Co) in food, industry, and medical devices requires full elucidation of its biological effects on tissues and organs. The aim was to assess serum metabolic alterations in immature mice after subchronic exposure to CoCl2. Pregnant ICR mice were subjected to a daily dose of 75 mg cobalt chloride/kg body weight (CoCl2x6H2O) 2-3 days before they gave birth, and treatment continued until days 25 and 30 after delivery. The compound was dissolved in and obtained with regular tap water. ICP-DRC-MS analysis showed significantly elevated serum Co2+ and diverse alterations in metabolic parameters of 25- and 30-day-old pups after exposure to CoCl2. Cholesterol and urea levels were significantly elevated in day 25 mice while HDL-C and LDL-C were reduced. In day 30, Co-exposed mice LDL-C and triglycerides were significantly increased while the total cholesterol level remained unchanged. Alkaline phosphatase was significantly reduced in day 25 Co-exposed mice. Blood glucose level of Co-exposed mice remained close to the untreated controls. Total protein content was slightly increased in day 30 mice. Co-exposure reduced albumin content and albumin/globulin ratio but increased significantly globulin content. Co administration showed strong correlation with cholesterol, urea, and HDL-C in both day 25 and 30 mice. Inverse correlation was found with alkaline phosphatase and albumin for day 25 and with triglycerides, globulin, and total protein content in day 30 Co-exposed mice. Subchronic CoCl2 exposure of immature mice induced significant changes in key metabolic parameters suggesting possible further disturbances in energy metabolism, osteogenesis, and reproduction.

N-methyl pyrrolidone (NMP) is an FDA approved molecule used as an excipient in pharmaceutical industry. Besides having a central role in formulation of drugs, the most important function of any excipient is to guarantee the safety of the medicine during and after its administration. Several studies have shown that exposure to NMP and especially in rats produce a gonadotoxic effect leading to infertility. However, the mechanisms underlying the effect of NMP on male reproduction are unknown. The aim of this study was to assess the reproductive toxicity of NMP in male rats and to elucidate the underlying mechanism. Male Sprague Dawley rats were injected intraperitoneally, twice/ week, at a dose of 108 mg/ 100 g of body weight with NMP. Analysis of reproductive parameters revealed testicular atrophy in NMP treated animals compared to control animals. Germ cell composition within the seminiferous tubules was disturbed and manifested in an increase in number of cells with fragmented DNA. A subsequent decrease in number of spermatocytes and spermatids was observed. Alpha screen assay shows that NMP acts at the concentrations we applied in vivo as a low affinity inhibitor for BRDT (testis specific bromodomain protein). BRDT inhibition is mirrored by a significant decrease in the expression of early stage spermatocyte markers (lmna, aurkc and ccna1), during which BRDT expression predominates. A significant decrease in testosterone levels was also observed. Since NMP interferes with spermatogenesis on various levels, its use in humans must be carefully monitored.

In this study, we compare the effects of deferiprone (Def) and tetraethylammonium salt of salinomycinic acid (Sal) on lead (Pb)-induced toxicity in testes of Pb-exposed mice. Mature male ICR mice were allocated into four groups as follows: untreated control mice (ctrl)-received distilled water for 4 weeks; Pb-exposed mice (Pb)-subjected to 14-day Pb (II) nitrate administration at dose 80 mg/kg body weight (b.w.); Pb + Def group-Pb-exposed mice, treated with 20 mg/kg b.w. Def for 2 weeks; and Pb + Sal group-Pb-intoxicated mice, treated with 16 mg/kg b.w. Sal for 14 days. The results demonstrated that Pb exposure significantly increased blood and testicular Pb concentrations, decreased testicular calcium (Ca) content, significantly elevated testicular levels of magnesium (Mg), zinc (Zn), and selenium (Se) but did not significantly affect the endogenous contents of phosphorous (P) and iron (Fe) compared with untreated controls. Pb intoxication induced disorganization of the seminiferous epithelium. Def or Sal administration reduced blood Pb and testicular Pb concentrations in Pb-exposed mice compared with the Pb-intoxicated group. Mg, Zn, and Se concentrations in testes of Pb-exposed mice, treated with Def or Sal, remained higher compared with the untreated controls. Sal significantly increased testicular P concentration compared with untreated controls and significantly elevated the testicular Ca and Fe concentrations compared with the toxic control group. Both chelating agents improved testicular morphology to a great extent. The results demonstrate the potential of both compounds as antidotes for treatment of Pb-induced impairment of male reproductive function.

The IGFs are the major intratesticular factors regulating immature Sertoli cell proliferation and are, therefore, critical to establish the magnitude of sperm production. However, the intratesticular source of IGF production and the downstream signaling pathway mediating IGF-dependent Sertoli cell proliferation remain unclear. Single-cell RNA sequencing on mouse embryonic testis revealed a robust expression of Igf1 and Igf2 in interstitial steroidogenic progenitors, suggesting that IGFs exert paracrine actions on immature Sertoli cells. To elucidate the intracellular signaling mechanism that underlies the proliferative effects of IGFs on immature Sertoli cells, we have generated mice with Sertoli cell-specific deletion of the Pten gene, a negative regulator of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway, alone or together with the insulin receptor (Insr) and the IGF1 receptor (Igf1r). Although ablation of Pten appears dispensable for Sertoli cell proliferation and spermatogenesis, inactivation of Pten in the absence of Insr and Igf1r rescued the Sertoli cell proliferation rate during late fetal development, testis size, and sperm production. Overall, these findings suggest that IGFs secreted by interstitial progenitor cells act in a paracrine fashion to promote the proliferation of immature Sertoli cells through the IGF/PTEN/PI3K pathway.

Aims Experimental and clinical studies have demonstrated that components of renin-angiotensin system are elevated in the hippocampus in epileptogenic conditions. In the present work, we explored the changes in the expression of angiotensin II receptor, type 1 (AT1 receptor) in limbic structures, as well as the effect of the AT1 receptor antagonist losartan in a model of comorbid hypertension and epilepsy. Main methods The expression of AT1 receptors was compared between spontaneously hypertensive rats (SHRs) and Wistar rats by using immunohistochemistry in the kainate (KA) model of temporal lobe epilepsy (TLE). The effect of losartan was studied on AT1 receptor expression in epileptic rats that were treated for a period of 4 weeks after status epilepticus. Key findings The naive and epileptic SHRs were characterized by stronger protein expression of AT1 receptor than normotensive Wistar rats in the CA1, CA3a, CA3b, CA3c field and the hilus of the dentate gyrus of the dorsal hippocampus but fewer cells were immunostained in the piriform cortex. Increased AT1 immunostaining was observed in the basolateral amygdala of epileptic SHRs but not of epileptic Wistar rats. Losartan exerted stronger and structure-dependent suppression of AT1 receptor expression in SHRs compared to Wistar rats. Significance Our results confirm the important role of AT1 receptor in epilepsy and suggest that the AT1receptor antagonists could be used as a therapeutic strategy for treatment of comorbid hypertension and epilepsy.

Cobalt (Co) is an endocrine disruptor and a hypoxiamimicking agent. Its effect on spermatogenesis after prolonged administration remains unclear. The aim of the study was to assess chronic cobalt treatment on spermatogenesis. Pregnant mice were exposed to 75 mg (low dose) and/or 125 mg (high dose)/kg b.w. cobalt chloride (CoCl2×6H2O) with drinking water for 3 days before delivery and treatment continued until postnatal day 90. Age-matched control animals obtained regular tap water. At the end of the treatment, testes of control and Co-treated mice were excised and processed for histological and ICPMS analysis for metal content. Spermatozoa were isolated from both ductus deference and counted. Results showed a significant dose-dependent reduction in testicular index (testis weight to body weight ratio), up to 60% after treatment with 125 mg/kg and up to 34% after treatment with 75 mg/kg. Morphology of the testis was considerably affected in both doses of CoCl2 although in some tubules spermatogenesis was normal and completed. Diameter ofmany seminiferous tubules was significantly reduced and there was lack of lumen formation. We observed disorganization of seminiferous epithelium, islet of undifferentiated germ cells in luminal region of the tubules and presence of multinuclear germ cells. These findings corresponded to the significant reduction of sperm count by 2.15-fold for the low dose and 3.35-fold for the high dose. Chronic CoCl2 administration resulted in accumulation of the metal ions in the testes of the treated mice. The considerable impact of Co on male reproduction after chronic exposure shows negative correlation between metal concentration and male fertility.

Background and aim Sodium nitrite (NaNO2) is an inorganic salt with numerous applications in a variety of industries, as well as in medicine. Nevertheless, exposure to high levels of NaNO2 is toxic for animals and humans. Sodium nitrite intoxication is shown to decrease the activity of major antioxidant defence enzymes which is dependent on the maintenance of specific ion equilibrium. The aim of the present study was to investigate the effect of acute NaNO2 intoxication on the content of the essential metals iron (Fe), calcium (Ca) and zinc (Zn) in mouse spleen. Methods Mature male ICR mice were divided into four groups and subjected to acute NaNO2 exposure by a single intraperitoneal injection of 120 mg/kg body weight. Animals in each group were sacrificed at certain time interval after treatment (1 h, 5 h, 1 day and 2 days). Spleens were excised and processed for atomic absorption spectrometry analysis of Fe, Ca and Zn content. Results At the first hour after treatment, a decrease in Fe and Ca levels was observed. One day following NaNO2 administration, Zn concentration reached its lowest value and Ca levels remained lower, compared to the untreated controls. In contrast, Fe concentration increased on the first and second day after treatment. Conclusion The results of the present study demonstrate that acute NaNO2 intoxication provokes changes in the endogenous levels of Fe, Ca and Zn in mouse spleen. These findings suggest disruption of the ionic balance and impact on the activity of antioxidant defence enzymes.

The objective of the present study was to elucidate the effect of perinatal cobalt chloride (CoCl2) exposure on extramedullary erythropoiesis in suckling mice in relation to iron (Fe) content and transferrin receptor (TfR) expression. Pregnant ICR mice were subjected to a daily dose of 75 mg CoCl2/kg body weight 2-3 days prior and 18 days after delivery. Co exposure significantly increased erythrocyte count (RBC), and reduced the erythrocytic parameters mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in the offspring. Total iron-binding capacity (TIBC) was decreased while bilirubin values were ~ 1.2-fold higher in the metal-exposed mice. Perinatal CoCl2 treatment also induced pathohistological changes in target organs (spleen, liver, and kidneys) as altered organ weight indices, leukocyte infiltration, abundant Kupffer cells in the liver, increased mesangial cellularity, and reduced capsular space in the kidney. CoCl2 administration induced significant 68-, 3.8-, 41.3-, and 162-fold increase of Co content in the kidney, spleen, liver, and RBC, respectively. Fe content in the target organs of CoCl2-treated mice was also significantly elevated. Immunohistochemical analysis demonstrated that TfR1 was well expressed in the renal tubules, hepatocytes, the red pulp, and marginal zone of white pulp in the spleen. TfR2 showed similar expression pattern, but its expression was stronger in the spleen and liver samples of Co-treated mice compared with that of the untreated controls. The results demonstrate that exposure to CoCl2 during late pregnancy and early postnatal period affects body and organ weights and alters hematological and biochemical parameters, iron content, and TfR expression in target organs.

Developing brain is very sensitive to the influence of environmental factors during gestation and the neonatal period. The aim of the study is to assess cobalt and iron accumulation in the brain as well as changes in the expression of iron-regulatory proteins transferrin receptor 1, hepcidin, and ferroportin in suckling mice. Perinatal exposure to cobalt chloride increased significantly cobalt content in brain tissue homogenates of 18-day-old (d18) and 25-day-old (d25) mice inducing alterations in brain iron homeostasis. Higher degree of transferrin receptor 1 expression was demonstrated in cobalt chloride-exposed mice with no substantial changes between d18 and d25 mice. A weak ferroportin expression was found in 18-day-old control and cobalt-treated mouse brain. Cobalt exposure of d25 mice resulted in increased ferroportin expression in brain compared to the untreated age-matched control group. Hepcidin level in cobalt-exposed groups was decreased in d18 mice and slightly increased in d25 mice. The obtained data contribute for the better understanding of metal toxicity impact on iron homeostasis in the developing brain with further possible implications in neurodegeneration.

An in vivo experimental model for testing the effects of long-term chronic treatment with cobalt(II) compounds — cobalt chloride (CoCl2) and cobalt-EDTA (Co-EDTA) on mice at different stages of development was optimized. Pregnant mice and their progeny were treated with daily doses of 75 or 125 mg kg−1 body weight until postnatal day 90. The compounds were dissolved in regular tap water. Mice were sacrificed on days 18, 25, 30, 45, 60 and 90 after birth, which correspond to different stages of their development. Altered organ weight indices (calculated as a ratio of organ weight to body weight) of spleen, liver and kidneys, were found depending on the type of compound used, dose, duration of treatment, and the age of the animals. The results also showed significant accumulation of cobalt ions in blood plasma, spleen, liver and kidneys of the exposed mice. More Co(II) was measured in the organs of the immature mice (day 18, 25 and 30 pnd) indicating that they were more sensitive to treatment.

Vitamin A together with testosterone (T) and FSH play an essential role in regulating spermatogenesis. Androgens and Vitamin A act through specific nuclear receptors: AR, retinoic acid receptors (R...

Cobalt is an essential oligoelement for mammals. It is not a cumulative toxin but chronic exposure induces negative effects on the organism. Data from the literature evidenced that in experimental animals cobalt impaired male reproductive organs and fertility when applied chronically. The aim of our study is to follow the effect cobalt on pubertal male progeny of female mice treated with cobalt in late pregnancy and during suckling period. Macroscopic parameters as weight of male reproductive organs and organ/body weight ratio were established. Significant reduction in body weight and 20% decrease (non significant) of testicular and epididymal weight as well as in testis/body weight index was found. The impact of cobalt on male progeny could be explained with transplacental route of exposure and with possible transfer of cobalt into mothers' milk. The negative effect of cobalt was not seen in mid puberty (day 25) with the exception of epididymal weight which was not compensated suggesting that epi...

In this study, we present a comparative assessment of the effects of two polyether ionophorous antibiotics (monensin and salinomycin) on the concentrations of lead (Pb), cooper (Cu), zinc (Zn) and iron (Fe) in the kidneys, spleen, liver and brain of Pb-intoxicated animals. Our data demonstrated that the intoxication of ICR male mice with Pb salt resulted in a significant accumulation of Pb in all studied organs of the mice compared to the untreated control animals. The biodistribution of the toxic metal was in the order kidneys>spleen>liver>brain. The treatment of the Pb-intoxicated animals with tetraethylammonium salts of monensic and salinomycinic acids significantly decreased the concentration of the toxic metal ion compared to the toxic control. The effect varied in the interval 38% (for kidneys) to 52% (for brain) compared to the toxic control group (Pb). The tetraethylammonium salt of salinomycinic acid was more effective in reducing the Pb concentration in the brain of the Pb-treated mice compared to monensin. Pb-intoxication did not affect significantly the Zn endogenous concentration compared to the normal values. The treatment of ICR male mice with Pb-salt decreased the Cu concentration in the spleen and increased the Cu concentration in the liver compared to the untreated control animals. The detoxification of the Pb-intoxicated mice with tetraethylammonium salts of salinomycinic and monensic acids restored the Cu concentration in the spleen, but did not affect the Cu levels in the liver. The Pb-intoxication of the ICR mice resulted in a significant decrease of the Fe-concentration in the spleen and liver compared to the untreated control animals. The administration of the tetraethylammonium salts of salinomycinic and monensic acids to the Pb-treated animals restored the levels of Fe in both organs.