Effects of Ovariectomy and Exercise Training on Mineral Status in a High-Fat Diet-Induced Obesity Rat Model.

Osteoporosis is a growing public health issue for an aging society. Previous studies have found both beneficial and detrimental effects of obesity on bone health. The purpose of this study was to investigate the impact of estrogen deficiency and physical activity on bone and blood concentrations of macrominerals (Ca, P, and Mg) and microminerals (Zn, Se, Cu, and Fe) in a high-fat diet-induced obesity rat model. Forty-eight female Wistar rats were divided into six groups: sham-operated and ovariectomized rats that received a standard diet (SD), high-fat diet (HFD), or HFD accompanied by physical exercise. The effect of ovariectomy on bone minerals varied with diet. Ovariectomy significantly decreased femoral Ca and Mg in sedentary rats receiving a SD; femoral Se, Cu, Zn, and Fe in sedentary rats on HFD; and plasma Fe in both sedentary rats on SD and exercising rats on HFD. The interaction of ovariectomy and diet had the strongest impact on Mg and Se concentrations in femur. In ovariectomized rats, HFD showed to have a protective effect on bone mineralization (femoral Ca and Mg), and a negative one on antioxidant microminerals (femoral Se, Cu, and Zn). Physical activity reduced the decline of Se, Cu, Zn, and Fe in the femur of ovariectomized rats on HFD. In the current state of knowledge, it is difficult to suggest if decreased femoral levels of antioxidant microminerals may contribute to the pathophysiology of osteoporosis in obese individuals or just reflect the mineral status in the body.

Correction to: Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In the original publication, the starting point in time for the three feeding trials.

Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In 2012, a controversial study on the long-term toxicity of a Roundup herbicide and the glyphosate-tolerant genetically modified (GM) maize NK603 was published. The EC-funded G-TwYST research consortium tested the potential subchronic and chronic toxicity as well as the carcinogenicity of the glyphosate-resistant genetically modified maize NK603 by performing two 90-day feeding trials, one with GM maize inclusion rates of 11 and 33% and one with inclusion rates of up to 50%, as well as a 2-year feeding trial with inclusion rates of 11 and 33% in male and female Wistar Han RCC rats by taking into account OECD Guidelines for the testing of chemicals and EFSA recommendations on the safety testing of whole-food/feed in laboratory animals. In all three trials, the NK603 maize, untreated and treated once with Roundup during its cultivation, and the conventional counterpart were tested. Differences between each test group and the control group were evaluated. Equivalence was assessed by comparing the observed difference to differences between non-GM reference groups in previous studies. In case of significant differences, whether the effects were dose-related and/or accompanied by changes in related parameters including histopathological findings was evaluated. It is concluded that no adverse effects related to the feeding of the NK603 maize cultivated with or without Roundup for up to 2 years were observed. Based on the outcome of the subchronic and combined chronic toxicity/carcinogenicity studies, recommendations on the scientific justification and added value of long-term feeding trials in the GM plant risk assessment process are presented.

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90 days (EU 7th Framework Programme project GRACE).

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insecticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various different animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadically altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance.

One-year oral toxicity study on a genetically modified maize MON810 variety in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GRACE (GMO Risk Assessment and Communication of Evidence; www.grace-fp7.eu ) project was funded by the European Commission within the 7th Framework Programme. A key objective of GRACE was to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of a 1-year feeding trial with a GM maize MON810 variety, its near-isogenic non-GM comparator and an additional conventional maize variety are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 452. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after a chronic exposure.

Health effects of selected nanoparticles in vivo: liver function and hepatotoxicity following intravenous injection of titanium dioxide and Na-oleate-coated iron oxide nanoparticles in rodents.

The study determined the effect of intravenous administration of acutely toxic or sub-lethal doses of Na-oleate-coated Fe3O4 (OC-Fe3O4) nanoparticles (NPs) on liver structure and function in Wistar rats, compared to titanium dioxide (TiO2) NPs and saline-injected controls. The acute study, using a modified OECD 425 progressive dosing procedure, found LD50 values of 59.22 and 36.42 mg/kg for TiO2 and OC-Fe3O4 NPs, respectively. In the sub-lethal study, rats were either injected with saline (negative controls), a sub-lethal reference (0.592 mg/kgTiO2 NPs, equal to 1% of LD50 on a body weight basis) or OC-Fe3O4 NPs in doses equivalent to 0.1, 1 or 10% of the LD50, respectively (corresponding to 0.0364, 0.364 and 3.64 mg Fe3O4/kg body weight). Animals were sampled 24 h, 1, 2 and 4 weeks post-injection for adverse effects. Mitochondrial respiration was significantly increased 2 weeks after injection of 10% OC-Fe3O4 NPs compared to controls, but the effect was transient. Cholesterol and triacylglycerol concentrations in the liver tissue did not increase in any treatment. There were some disturbances to antioxidant enzymes after OC-Fe3O4 NPs treatment in the livers of animals 1 week post-exposure; with the most sensitive changes occurring in glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities. Lipidosis and mild necrosis with changes in sinusoid space were also observed in histological sections of the liver. Overall, these data suggest that the liver likely retains functional integrity with acute and sub-lethal doses of OC-Fe3O4 NPs, albeit with some stimulation of redox defences and evidence of some tissue injury.

Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu ) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event.

Impact of ovariectomy, high fat diet, and lifestyle modifications on oxidative/antioxidative status in the rat liver.

AIM: To estimate the impact of high fat diet and estrogen deficiency on the oxidative and antioxidative status in the liver of the ovariectomized rats, as well as the ameliorating effect of physical activity or consumption of functional food containing bioactive compounds with antioxidative properties on oxidative damage in the rat liver. METHODS: The study was conducted from November 2012 to April 2013. Liver oxidative damage was determined by lipid peroxidation levels expressed in terms of thiobarbituric acid reactive substances (TBARS), while liver antioxidative status was determined by catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) activities, and glutathione (GSH) content. Sixty-four female Wistar rats were divided into eight groups: sham operated and ovariectomized rats that received either standard diet, high fat diet, or high fat diet supplemented with cereal selenized onion biscuits or high fat diet together with introduction of physical exercise of animals. RESULTS: High fat diet significantly increased TBARS content in the liver compared to standard diet (P=0.032, P=0.030). Furthermore, high fat diet decreased the activities of CAT, GR, and GST, as well as the content of GSH (P<0.050). GPx activity remained unchanged in all groups. Physical activity and consumption of cereal selenized onion biscuits showed protective effect through increased GR activity in sham operated rats (P=0.026, P=0.009), while in ovariectomized group CAT activity was increased (P=0.018) in rats that received cereal selenized onion biscuits. CONCLUSION: Feeding rats with high fat diet was accompanied by decreased antioxidative enzyme activities and increased lipid peroxidation. Bioactive compounds of cereal selenized onion biscuits showed potential to attenuate the adverse impact of high fat diet on antioxidative status.

Effects of high fat diet, ovariectomy, and physical activity on leptin receptor expression in rat brain and white fat tissue.

AIM: To evaluate in a rat animal model whether ovariectomy, high fat diet (HFD), and physical activity in the form of running affect leptin receptor (Ob-R) distribution in the brain and white fat tissue compared to sham (Sh) surgery, standard diet (StD), and sedentary conditions. METHODS: The study included 48 female laboratory Wistar rats (4 weeks old). Following eight weeks of feeding with standard or HFD, rats were subjected to either OVX or Sh surgery. After surgery, all animals continued StD or HFD for the next 10 weeks. During these 10 weeks, ovariectomy and Sh groups were subjected to physical activity or sedentary conditions. Free-floating immunohistochemistry and Western blot methods were carried out to detect Ob-R in the brain and adipose tissue. RESULTS: StD-ovariectomy-sedentary group had a greater number of Ob-R positive neurons in lateral hypothalamic nuclei than StD-Sh-sedentary group. There was no difference in Ob-R positive neurons in arcuatus nuclei between all groups. Ob-R distribution in the barrel cortex was higher in HFD group than in StD group. Ob-R presence in perirenal and subcutaneous fat was decreased in StD-ovariectomy group. CONCLUSION: HFD and ovariectomy increased Ob-R distribution in lateral hypothalamic nuclei, but there was no effect on arcuatus nuclei. Our results are first to suggest that HFD, ovariectomy, and physical activity affect Ob-R distribution in the barrel cortex, which might be correlated with the role of Ob-R in election of food in rats.

Pathophysiological rat model of vascular dementia: magnetic resonance spectroscopy, microimaging and behavioral study.

Chronic cerebral hypoperfusion and aging can be related to vascular dementia manifested by the decline in cognitive abilities and memory impairment. The identification of specific biomarkers of vascular disorder in early stages is important for the development of neuroprotective agents. In the present study, a three-vessel occlusion (3-VO) rat model of vascular dementia in the middle-aged rat brain was used to investigate the effect of global cerebral hypoperfusion. A multimodal study was performed using magnetic resonance spectroscopy, MR-microimaging, histology and behavioral tests. Our measurements showed a signal alteration in T2-weighted MR images, the elevation of T2 relaxation times and histologically proven neural cell death in the hippocampal area, as well as mild changes in concentration of proton and phosphorus metabolites. These changes were accompanied by mild behavioral alterations in the open field and slightly decreased habituation. The analysis of the effects of vascular pathology on cognitive functions and neurodegeneration can contribute to the development of new treatment strategies for early stages of neurodegeneration.

Comprehensive assessment of nephrotoxicity of intravenously administered sodium-oleate-coated ultra-small superparamagnetic iron oxide (USPIO) and titanium dioxide (TiO2) nanoparticles in rats.

As a main excretory organ, kidney is predisposed to direct/indirect injury. We addressed the potential nephrotoxic effects following expositions of healthy rats to nanoparticle (NP) loads relevant to humans in a situation of 100% bioavailability. Up to 4 weeks after administration, a single iv bolus of oleate-coated ultra-small superparamagnetic iron oxide NPs (in dose of 0.1%, 1.0% and 10.0% of LD50) or TiO2 NPs (1.0% of LD50) did not elicit decline in renal function, damage to proximal tubules, alterations in: renal histology or expression of pro-inflammatory/pro-fibrotic genes, markers of systemic or local renal micro-inflammation or oxidative damage. Antioxidant enzyme activities in renal cortex, mildly elevated at 24 h, completely restored at later time points. Data obtained by multifaceted approach enable the prediction of human nephrotoxicity during preclinical studies, and may serve as comparison for alternative testing strategies using in vitro and in silico methods essential for the NP-nephrotoxicity risk assessment.

Neuronal activation in the central nervous system of rats in the initial stage of chronic kidney disease-modulatory effects of losartan and moxonidine.

The effect of mild chronic renal failure (CRF) induced by 4/6-nephrectomy (4/6NX) on central neuronal activations was investigated by c-Fos immunohistochemistry staining and compared to sham-operated rats. In the 4/6 NX rats also the effect of the angiotensin receptor blocker, losartan, and the central sympatholyticum moxonidine was studied for two months. In serial brain sections Fos-immunoreactive neurons were localized and classified semiquantitatively. In 37 brain areas/nuclei several neurons with different functional properties were strongly affected in 4/6NX. It elicited a moderate to high Fos-activity in areas responsible for the monoaminergic innervation of the cerebral cortex, the limbic system, the thalamus and hypothalamus (e.g. noradrenergic neurons of the locus coeruleus, serotonergic neurons in dorsal raphe, histaminergic neurons in the tuberomamillary nucleus). Other monoaminergic cell groups (A5 noradrenaline, C1 adrenaline, medullary raphe serotonin neurons) and neurons in the hypothalamic paraventricular nucleus (innervating the sympathetic preganglionic neurons and affecting the peripheral sympathetic outflow) did not show Fos-activity. Stress- and pain-sensitive cortical/subcortical areas, neurons in the limbic system, the hypothalamus and the circumventricular organs were also affected by 4/6NX. Administration of losartan and more strongly moxonidine modulated most effects and particularly inhibited Fos-activity in locus coeruleus neurons. In conclusion, 4/6NX elicits high activity in central sympathetic, stress- and pain-related brain areas as well as in the limbic system, which can be ameliorated by losartan and particularly by moxonidine. These changes indicate a high sensitivity of CNS in initial stages of CKD which could be causative in clinical disturbances.

Promoter-context as a determinant of glucocorticoid receptor-responsiveness to activation of p38 and JNK mitogen-activated protein (MAP) kinases.

MAP kinases JNK and p38 play an important role in many immune and inflammatory processes, whereas glucocorticoids exert immunosuppressive and anti-inflammatory activities. We found previously that activation of p38 or JNK inhibits glucocorticoid receptor (GR)-mediated transcriptional activation of a mouse mammary tumor virus (MMTV) promoter-driven luciferase construct in HeLa cells. It appears that this effect is DNA regulatory element-specific, since p38 or JNK activation stimulates GR-dependent transcription from TAT3-ADH promoter-luciferase construct in the same cells. The apparent promoter-specificity of this action suggests that not all glucocorticoid-activated genes are negatively regulated by p38 or JNK. Using different MMTV/TAT3 chimeric reporters we demonstrate that the presence of other accessory binding sites of the MMTV construct contributes to the inhibitory effect of activated p38 or JNK on the MMTV-driven transcriptional activity; and diminishes, but does not reverse the stimulation observed using the TAT GREs from the TAT3-ADH promoter-luciferase construct. On the other hand, comparison of the effects of GRE sequences, either in isolation or in the context of the MMTV LTR accessory binding sites, demonstrates that the responsiveness of the GR depends on the GRE sequence; indicating that in addition to transcription factors bound nearby, interaction with the DNA itself modulates GR activity.

Regular moderate exercise reduces advanced glycation and ameliorates early diabetic nephropathy in obese Zucker rats.

Advanced glycation end products (AGEs) play a key role in the pathogenesis of diabetes and its complications, including the diabetic nephropathy. The renoprotective effects of exercise are well known; however, the mechanisms remain elusive. Here we examined whether a regular moderate exercise in obese Zucker rats (OZR), a model of diabetes- and obesity-associated nephropathy, will affect the development of early renal injury in OZR possibly via alteration of AGEs formation. The OZR were left without exercise (sedentary) or subjected to 10 weeks intermittent treadmill running of moderate intensity. Compared with sedentary OZR, kidneys of running OZR had significantly less glomerular mesangial expansion and tubulointerstitial fibrosis. Running OZR had significantly lower plasma AGEs-associated fluorescence and N(epsilon)-carboxymethyllysine. Correspondingly, renal AGEs and N(epsilon)-carboxymethyllysine content were lower in running OZR. Systemically, exercise increased aerobic metabolism, as apparent from urinary metabolite profiling. No differences in plasma glucose, insulin, or lipid profile were found between the 2 groups. Apart from lower advanced oxidation protein products (a marker of myeloperoxidase activity), no other marker of inflammation was altered by exercise, either systemically or locally in kidneys. No indication of changed oxidative status was revealed between the groups. Exercise in OZR decreased advanced glycation. This might represent the early event of exercise-induced renoprotection in diabetic nephropathy in OZR. If confirmed in clinical studies, regular moderate exercise could represent an easy and effective nonpharmacologic approach to reduce advanced glycation.

A novel way of liver preservation improves rat liver viability upon reperfusion.

BACKGROUND/AIM: Currently, the liver is cold-preserved at 0 approximately 4 degrees C for experimental and clinical purposes. Here, we investigated whether milder hypothermia during the initial phase of the preservation period was beneficial for liver viability upon reperfusion. METHODS: In the first set of experiments, rat livers were preserved either conventionally in clinically used histidine-trypthopan-ketoglutarate (HTK) solution (Group A: 45 min and Group B: 24 h) or by slow cooling HTK solution (from 13 degrees C to 3 degrees C) during the initial 45 min of preservation (Group C: 24 h). In the second set of experiments, additional groups of livers were evaluated: Group BB--preservation according to Group B and Group CC--preservation according to Group C. Further, some livers were preserved at 13 degrees C for 24 h. Livers were then reperfused using a blood-free perfusion model. RESULTS: Bile production was approximately 2-fold greater in Group C compared to Group B. Alanine transaminase (ALT) and aspartate transaminase (AST) release into perfusate were 2 approximately 3-fold higher in Group B compared to Group C. No significant differences were found in ALT and AST release between Group C and Group A. Livers in Group CC compared to Group BB exhibited significantly lower portal resistance, greater oxygen consumption and bromosulfophthalein excretion into bile and lower lactate dehydrogenase (LDH) release into perfusate. Histological evaluation of tissue sections in Group BB showed parenchymal dystrophy of hepatocytes, while dystrophy of hepatocytes was absent in Group CC. Livers preserved at 13 degrees C for 24 h exhibited severe ischemic injury. CONCLUSION: These results suggest that the conventional way of liver preservation is not suitable at least for rat livers and that slow cooling of HTK solution during the initial phase of cold storage can improve liver viability during reperfusion.

Copper impairs biliary epithelial cells and induces protein oxidation and oxidative DNA damage in the isolated perfused rat liver.

Copper is one of the major metals causing environmental contamination. Previous studies showed that copper induced toxic effects in isolated perfused rat liver models and these effects were associated with lipid peroxidation. Here we investigated whether effects of copper (at concentrations of 0.01, 0.03, and 0.1 mM of Cu(2+) in Krebs-Henseleit buffer perfusing the isolated rat liver for 60 min), were associated with biliary epithelial cell injury, as well as protein oxidation and oxidative DNA damage. The highest concentration of copper in perfusate (0.1 mM) did not allow complete evaluation of all parameters because it blocked portal flow within 30 min of perfusion, indicating severe microcirculatory disturbances. Further, copper decreased secretion of bile and it increased lactate dehydrogenase, aspartate transaminase, and alanine transaminase leakage into perfusate as well as liver weight in a dose-dependent manner. Biliary gamma-glutamyltransferase, an index of biliary epithelial cell integrity increased similarly at 0.01 and 0.03 mM copper concentrations in perfusate. Compared to controls, 0.01 and 0.03 mM concentrations of copper increased the amount of thiobarbituric acid reacting substances, a marker of lipid peroxidation, tissue protein carbonyl groups, an index of protein oxidation, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidative DNA damage. The results suggest that toxic effects of copper in the isolated perfused rat liver may involve biliary epithelial cells and they are associated with lipid peroxidation, protein oxidation, and oxidative DNA damage.