Circadian Rhythm of Liver Parameters (Cellular Structures, Mitotic Activity, Glycogen and Lipids in Liver and Serum) During Three Consecutive Cycles in Phenobarbital-Treated Rats

The circadian rhythm of gastric content, serum alkaline phosphatase (alk.P.), serum lipids, body weight (wt), relative (rel.) liver wt, cellular structures (by light- and electron-microscopy), mitotic activity of hepatocytes, glycogen content, protein and lipids in liver was studied in 180 male Sprague-Dawley rats orally treated at 0830-1030 with 50 mg/kg phenobarbital (PB) for 7 days. Thereafter, five PB-treated males and five controls each were studied at 4-hr intervals at 0600, 1000, 1400, 1800, 2200 and 0200 on 3 consecutive days. The lighting schedule in the colony was 12:12 = light/dark (light from 0600 to 1800). Following the rhythm of gastric emptying, the rel. liver wt showed a clear circadian rhythm with a peak at 0800. The rel. liver wt was raised in PB-treated rats at all times of the day. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which exhibited a clear rhythm with the peak also at 0800, but lowered values were found in PB-treated rats. The mitotic activity of hepatocytes was significantly increased in PB-treated rats but displayed the same circadian rhythm as controls with peaks at noon and troughs at midnight. The well-known hypertrophy of the smooth endoplasmic reticulum in PB-treated rats was not found at 0600, but was fully developed at 1400 and 2200. PB-treatment increased significantly the liver content of cholesterol, triglycerides and phospholipids. Liver cholesterol showed a clear circadian rhythm with peaks at 1800. No rhythm of liver protein, triglycerides and phospholipids was observed. In serum, levels of cholesterol were significantly elevated, those of triglycerides and alk.P. significantly lowered, while those of phospholipids were not affected by the treatment. The three serum lipids, alk.P. and beta-lipoprotein exhibited a clear circadian rhythm, while serum glucose and non-esterified fatty acids did not.     

Circadian Rhythm of Liver Parameters (Cellular Structures,Mitotic Activity,Glycogen and Lipids in Liver and Serum) During Three Consecutive Cycles in Phenobarbital-Treated Rats

The circadian rhythm of gastric content, serum alkaline phosphatase (alk.P.), serum lipids, body weight (wt), relative (rel.) liver wt, cellular structures (by light- and electron-microscopy), mitotic activity of hepatocytes, glycogen content, protein and lipids in liver was studied in 180 male Sprague-Dawley rats orally treated at 0830-1030 with 50 mg/kg phenobarbital (PB) for 7 days. Thereafter, five PB-treated males and five controls each were studied at 4-hr intervals at 0600, 1000, 1400, 1800, 2200 and 0200 on 3 consecutive days. The lighting schedule in the colony was 12:12 = light/dark (light from 0600 to 1800). Following the rhythm of gastric emptying, the rel. liver wt showed a clear circadian rhythm with a peak at 0800. The rel. liver wt was raised in PB-treated rats at all times of the day. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which exhibited a clear rhythm with the peak also at 0800, but lowered values were found in PB-treated rats. The mitotic activity of hepatocytes was significantly increased in PB-treated rats but displayed the same circadian rhythm as controls with peaks at noon and troughs at midnight. The well-known hypertrophy of the smooth endoplasmic reticulum in PB-treated rats was not found at 0600, but was fully developed at 1400 and 2200. PB-treatment increased significantly the liver content of cholesterol, triglycerides and phospholipids. Liver cholesterol showed a clear circadian rhythm with peaks at 1800. No rhythm of liver protein, triglycerides and phospholipids was observed. In serum, levels of cholesterol were significantly elevated, those of triglycerides and alk.P. significantly lowered, while those of phospholipids were not affected by the treatment. The three serum lipids, alk.P. and beta-lipoprotein exhibited a clear circadian rhythm, while serum glucose and non-esterified fatty acids did not.     

Effect of nickel(II) chloride on iron content in rat organs after oral administration

The effect of oral administration of nickel(II) chloride on iron content in serum and certain body organs of rats was investigated. The male adult rats were given 300 and 1200 ppm Ni in drinking water for 90 d. The iron content in serum, liver, kidney, lung, spleen, and brain was analyzed 30 and 90 d postexposure. The hemoglobin, hematocrit, and body and organ weights were also measured. Nickel given in drinking water led to a pronounced increase in iron content in serum and the liver, as compared to control rats. This effect was related to Ni concentration in the water. There was not great time-dependent difference in the iron content as a response to continuous nickel treatment, except the lung of 1200-ppm Ni-treated rats. In relation to hematological parameters, Ni supplementation did not affect any of them. Body weight significantly decreased, and lung weight was significantly increased in 1200-ppm Ni-treated rats. The results of this study indicate that nickel ingestion (300 and 1200 ppm in the drinking water) induces the iron uptake by serum and some organs of rats. The highest amount of iron was found in the liver of all exposed animals, and the time-dependent difference in iron content was observed in the lung of 1200-ppm Ni-treated rats.     

Circadian Rhythm of the Liver of Male Rats Pre-Treated with Phenobarbital—ii. Hexobarbital Sleeping Time and Lipids Content in Liver and Serum

The circadian rhythm of hexobarbital sleeping time and lipids content in liver and serum were studied in 226 male Sprague-Dawley rats pretreated daily at 0800-0900 with 70 mg/kg (study 1 or 3) or 50 mg/kg (study 2) phenobarbital (PB) orally for 7 days. Thereafter, eight (study 1) or five (study 2 and 3) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through the following day. The lighting schedule in the colony was 12:12 ± light:dark (light from 0600 to 1800). The hexobarbital sleeping times of PB-pretreated rats were generally shortened compared to the controls and no circadian rhythm was observed. PB-treatment increased slightly the liver content of cholesterol, and significantly that of triglycerides and phospholipids. Liver cholesterol and phospholipids showed circadian rhythms with peaks during the dark phase. No circadian rhythm of liver triglycerides existed. In serum, levels of triglycerides and phospholipids were slightly lowered by PB-treatment, while levels of cholesterol and beta-lipoprotein were not influenced. Serum values did not exhibit circadian rhythms.     

Postnatal development of TRH and TSH rhythms in the rat

We previously observed that under a 12-hour light/12-hour dark schedule (lights off at 19.00 h), adult male Sprague-Dawley rats showed a circadian rhythm for serum thyroid-stimulating hormone (TSH) with a zenith near midday. In the present work, the ontogenesis of serum TSH rhythm was determined as well as pituitary TSH variations. In addition, hypothalamic and blood TRH were measured in these rats aged 15, 25, 40 and 70 days when sacrificed. As from the first age studied (15 days), a hypothalamic thyrotropin-releasing hormone (TRH) circadian rhythm was present. The mesor and the amplitude of this hypothalamic TRH rhythm increased while the rats were growing up, in contrast with the decrease observed for these parameters as far as blood TRH circadian rhythm is concerned. The time of the acrophase moved from 17.32 h in the 15-day-old rats to 13.57 h in the 70-day-old rats, being constantly in phase opposition with the blood TRH acrophase. The low amplitude pituitary TSH circadian rhythm detected in the young rat disappeared in the adult while, in contrast, the serum TSH rhythm became consistent to reach the well-characterized circadian midday peak in the 70-day-old rats.     

Influence of ionizing radiation, application of iron ions and their chelate complexes on the oxidative status of blood serum of rats

Influence of ionizing radiation, ions of iron and their chelate complexes on the oxidative status of blood serum of rats has been investigated. Animals were irradiated by gamma-rays 60Co at a dose of 4 Gy. Ions of iron and iron chelates with nitrilotriacetic acid and citric acid were introduced into animals intra-abdominally at a doze of 10 mg of iron on 1 kg of body weight. The oxidative status of blood serum was determined according to the estimated content of oxidizing peroxide equivalents which oxidize ferrous iron in ferric iron with the subsequent estimation of ferric iron by means of xylenol orange. We also estimated the total content of iron in blood serum using ferrozine as an indicator. The oxidative status was defined 24 and 96 hours after irradiation and 2 hours after introduction of iron ions and their chelates. The research conducted has shown that the concentration of oxidizing peroxide equivalents in serum and the total iron concentration increase 1.47 times and 1.63 times correspondingly 24 hours after irradiation. The increase in the content of oxidizing peroxide equivalents and iron owing to Fenton's reaction can lead to the appearance of OH* radical and raise the level of damage of nuclear and membrane structures in irradiated cells. 2 hours after introduction of iron ions and their chelates, the content of oxidizing peroxide equivalents increased in the blood serum of irradiated and non-irradiated rats, and the maximum effect was observed when introducing ferrous iron and its chelate with citric acid.     

The impact of iron content in a diet high in fat,fructose, and salt on metabolic state and mineral status of rats

The purpose of the study was to assess the influence of dietary iron content on lipid and carbohydrate metabolism and on zinc and copper status in rats fed with a diet high in fat, fructose, and salt. Wistar rats were fed with diets high in fat, fructose, and salt, containing differing amounts of iron, namely, deficit, normal, and high levels. After 6 weeks, the animals were weighed and killed. The liver, heart, and pancreas were collected, as were blood samples. The total cholesterol, triglycerides, fasting glucose, and insulin levels in the serum were measured. The iron, zinc, and copper concentrations in tissues and serum were determined. It was found that in rats fed with the iron-deficit diet, cholesterol and glucose profiles improved. Both deficit and excess iron in the diet decreased insulin concentration in rats and disturbed iron, zinc, and copper status. High-iron level in the diet decreased the relative mass of the pancreas. In conclusion, the decrease in serum insulin concentration observed in rats fed with the modified diet high in iron was associated with iron and copper status disorders, and also, with a relatively diminished pancreas mass. A deficit of iron in the diet improved lipid and carbohydrate metabolism in rats.     

Determination of nonheme iron using inductively coupled plasma-atomic emission spectrometry

A technique for the rapid and accurate estimation of nonheme iron using inductively coupled plasma-atomic emission spectrometry is described. Yttrium was used as an internal standard. An external calibration method was used. The standards were prepared in a matrix composed of 2.5N HCl in 10% (w/v) trichloroacetic acid. The supernatant and coagulum fractions of liver nonheme iron were separated by the method of Drysdale and Ramsay with minor modification. The data determined by this procedure was compared and found to be agreement with data determined by the method of Hallgren. To evaluate the iron status of rats, hemoglobin and liver nonheme iron were determined. Hemoglobin and all of the nonheme iron fractions of the rats fed an iron-deficient diet were significantly lower than those of the rats fed an iron-sufficient diet. The blood content in the liver was estimated to be 80 microL/g from the blood iron concentration, and the difference between total and nonheme iron concentration in liver.     

The effect of cobalt on the uptake of plasma iron by the liver.

1. After an intraperitoneal injection of 59Fe the recovery of radioactivity in the liver, but not in other tissues, was increased in cobalt-pretreated rats. There was no proportional increase in the radioactivity recovered from liver haem. 2. Rats were injected intravenously with serum containing protein-bound 59Fe and 125I-labelled albumin as a marker. At various times after injection the specific radioactivities of iron in plasma and of non-haem iron in liver were determined; corrections were applied for the content of plasma in samples of liver. In cobalt-pretreated rats there was a more rapid loss of 59Fe radioactivity from the plasma and a corresponding increase in the uptake of 59Fe into liver non-haem iron. 3. The results are discussed in relation to the possible sites of action of cobalt, and the possibility is considered that only a fraction of the liver non-haem iron may be involved.     

Iron metabolism and erythropoiesis in the prenatal and early postnatal periods of the rat

Spectrophotometric studies have been made on iron balance in the liver, spleen, bone marrow, and blood serum of 15-, 17- and 20-day rat embryos, as well as 1-90 days old rats. It was shown that foetal period is the main one for the formation of iron-accumulating function of the liver. Anaemic period in 14-28-day rat puppies results from insufficient hemoglobin synthesis, rather than from iron deficiency in the organism. The latter is observed in 35-60-days old rats. Maturation of the spleen as the organ involved in reutilization of iron was noted in 42-60-day rats.     

Green tea activity and iron overload induced molecular fibrogenesis of rat liver

Iron overload toxicity was shown to associate with chronic liver diseases which lead to hepatic fibrosis and subsequently the progression to cancer through oxidative stress and apoptotic pathways. Green tea potential activity as chelating, anti-oxidative, or anti-apoptotic mechanisms against metal toxicity was poorly clarified. Here, we are trying to evaluate the anti-oxidant and anti-apoptotic properties of green tea in the regulation of serum hepcidin levels, reduction in iron overloads, and improve of liver fibrosis in iron overloaded experimental rats. Three groups of male adult rats were randomly classified into three groups and treated as follows: control rats, iron treated rats for two months in drinking water followed by either vehicle or green tea extract (AGTE; 100 mg/kg) treatment for 2 more months. Thereafter, we studied the effects of AGTE on iron overload-induced lipid peroxidation, anti-oxidant depletion, liver cell injury and apoptosis. Treatment of iron-overloaded rats with AGTE resulted in marked decreases in iron accumulation within liver, depletion in serum ferritin, and hepcidin levels. Iron-overloaded rats had significant increase in malonyldialdehyde (MDA), a marker of lipid peroxidation and nitric oxide (NO) in liver when compared to control group. Also, significant change in cytochrome c and DNA content as apoptotic markers were reported in iron treated rats. The effects of iron overload on lipid peroxidation, NO levels, cytochrome c and DNA content were significantly reduced by the intervention treatment with AGTE (P < 0.001). Furthermore, the endogenous anti-oxidant capacities/levels (TAC) in liver were also significantly decreased in chronic iron overload and administration of AGTE restored the decrease in the hepatic antioxidant activities/levels. Also, hepatic hepcidin was shown to be significantly correlated with oxidative and apoptotic relating biomarkers as well as an improvement in liver fibrosis of iron treated rats following AGTE treatment. In-vitro analysis showed that, the improvement in iron toxicity of the liver depend mainly on antioxidant and protective ability of green tea polyphenolic compounds especiallyepigallocatechin-3-gallate (EGCG). Our study showed that green tea extract (GTE) ameliorates iron overload induced hepatotoxicity, apoptosis and oxidative stress in rat liver via inhibition of hepatic iron accumulation; improve of liver antioxidant capacity, and down regulation of serum hepcidin as well as reduction in the release of apoptotic relating proteins.     

The circadian rhythm of the adrenal glucocorticoid function in female rats selected for the reaction of the estrous cycle to constant illumination

Studies have been made of the circadian rhythms of a glucorticoid hormone, corticosterone, in the adrenals and blood serum in female Wistar rats from two substrains selected for high (ESTH) and low (ESTL) ability to develop permanent oestrus under constant illumination. Significant changes in parameters of the circadian rhythm of the hormone were observed in animals of the 26th generation of selection. Total alleviation of corticosterone rhythm in the blood was on observed in ESTL rats, while in ESTH animals maximum level of the hormone in the blood was shifted to the dark time. Comparison of a high corticosterone content of the adrenals in ESTL rats with a low concentration in the blood plasma indicates the increase in metabolic clearance of the hormone in animals from this strain. It is suggested that the decreased corticosteroid production in the adrenals of ESTH rats facilitates the development of permanent oestrus under constant illumination.     

Sex Differences in Iron Status and Hepcidin Expression in Rats

Studies have shown that men and women exhibit significant differences regarding iron status. However, the effects of sex on iron accumulation and distribution are not well established. In this study, female and male Sprague-Dawley rats were killed at 4 months of age. Blood samples were analyzed to determine the red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Hct), and mean red blood cell volume (MCV). The serum samples were analyzed to determine the concentrations of serum iron (SI), transferrin saturation (TS), ferritin, soluble transferrin receptor (sTfR), and erythropoietin (EPO). The tissue nonheme iron concentrations were measured in the liver, spleen, bone marrow, kidney, heart, gastrocnemius, duodenal epithelium, lung, pallium, cerebellum, hippocampus, and striatum. Hepatic hepcidin expression was detected by real-time PCR analysis. The synthesis of ferroportin 1 (FPN1) in the liver, spleen, kidney, and bone marrow was determined by Western blot analysis. The synthesis of duodenal cytochrome B561 (DcytB), divalent metal transporter 1 (DMT1), FPN1, hephaestin (HP) in the duodenal epithelium was also measured by Western blot analysis. The results showed that the RBC, Hb, and Hct in male rats were higher than those in female rats. The SI and plasma TS levels were lower in male rats than in female rats. The levels of serum ferritin and sTfR were higher in male rats than in female rats. The EPO levels in male rats were lower than that in female rats. The nonheme iron contents in the liver, spleen, bone marrow, and kidney in male rats were also lower (56.7, 73.2, 60.6, and 61.4 % of female rats, respectively). Nonheme iron concentrations in the heart, gastrocnemius, duodenal epithelium, lung, and brain were similar in rats of both sexes. A moderate decrease in hepatic hepcidin mRNA content was also observed in male rats (to 56.0 % of female rats). The levels of FPN1 protein in the liver, spleen, and kidney were higher in male rats than in female rats. There was no significant change in FPN1 expression in bone marrow. Significant difference was also not found in DcytB, DMT1, FPN1, and HP protein levels in the duodenal epithelium between male and female rats. These data suggest that iron is distributed differently in male and female rats. This difference in iron distribution may be associated with the difference in the hepcidin level.     

Foot-shock Stress-Induced Regional Iron Accumulation and Altered Iron Homeostatic Mechanisms in Rat Brain

Like in other organs, iron in the brain plays an important role in various biological processes. Previous studies have shown that systemic iron homeostasis in mammalians was changed under specific stress conditions. The present study aimed to investigate effects of stress on brain iron homeostasis in rats using a foot-shock stress model. Young adult male Sprague-Dawley rats were randomly assigned to foot-shock stress group subjected to 30 min of cutaneous foot-shock (0.80 mA, 1 pulse/s, 300 ms duration) daily for 1 week or control group left undisturbed. Then, the rats were sacrificed and iron concentration in serum, liver, and some brain regions were measured using atomic absorption spectrophotometry. Expression of ferritin, Transferrin receptor (TfR), divalent metal transporter 1 (DMT1, with or without iron-responsive element), lactoferrin (Lf), and iron regulatory protein 1 (IRP1) in rat hippocampus were determined using western blot analysis. The results showed that stress induced decreased serum iron concentration, increased liver iron content, and elevated iron contents in specific brain regions including hippocampus, striatum, and frontal cortex. In the hippocampus, stress caused decreased expression of ferritin, increased expression of TfR and IRP1, and no change in expression of DMT1 or Lf. Results of this study demonstrated that foot-shock stress induced region specific iron accumulation and altered iron homeostatic mechanisms in the brain in addition to a changed systemic iron homeostasis characterized by decreased serum iron concentration and increased liver iron content. And, elevated IRP1 expression might be associated with the increased TfR and decreased ferritin expression, leading to subsequent iron accumulation and possible increased vulnerability to oxidative damage in hippocampus.     

Circadian rhythm of serum and tissue lipids in fed and fasted rats

The oscillations of the free fatty acid concentration in the serum and white (epididymal) adipose tissue, of triglycerides in the serum and liver, of total serum, liver and adrenal cholesterol and of serum phospholipids were studied at 3-hour intervals for a period of 24 hours in fed male Wistar rats and in animals fasted for 24 hours (both adapted to an illumination regimen of 12 hours' light and 12 hours' darkness. The rhythm--studied by means of the cosinor analysis--was present in most of the given parameters; it was not recorded in the liver triglycerides and serum phospholipids of fasted rats and in the adrenal cholesterol of fed animals. Apart from the circadian rhythm, many parameters distinctly displayed an ultradian rhythm, mainly an approximately 12-hour period. In general, one day's starvation did not significantly affect the course of the circadian oscillations of the given indicators of rat lipid metabolism.     

Influence of multistrain probiotic and iron supplementation on iron status in rats

ObjectiveThe impact of multistrain probiotics on iron (Fe) metabolism under Fe-deficient diet conditions remains unknown. The study aimed to compare the effect of 6 weeks simultaneous and exclusive oral multistrain probiotic and iron supplementation on selected parameters of Fe metabolism in rats on an Fe-deficient diet.MethodsForty rats were assigned to five groups, with eight animals in each, and for 6 weeks received: the CC group- a standard diet, the DD group- an Fe-deficient diet, the DPB group- an Fe-deficient with a multispecies probiotic, the DFE group- an Fe-deficient diet supplemented with iron, the DPBFE group- an Fe-deficient diet with iron and a multispecies probiotic. The Fe content in blood and tissues; serum concentration of erythroferrone, ferritin (Ft), homocysteine, hepcidin (HEPC) and lactoferrin; liver content of divalent metal transporter 1 (DMT1), transferrin receptor protein 1 (TfR1) and 2 (TfR2) and ZRT/IRT-like protein 14 (ZIP14) and faecal microbiota were assessed.ResultsIn DPBFE group, unlike in DPB and DFE groups, duodenal Fe content was higher compared to DD group. Similarly, serum Ft level was higher in DPBFE group, but not in DPB and DFE groups, compared to DD group.ConclusionsSix weeks simultaneous oral multistrain probiotic and Fe supplementation, but not exclusive probiotic or Fe intake, increases duodenal Fe absorption in rats and presents higher effectiveness in increasing tissue Fe stores.     

Circadian rhythms of blood components in dementia senilis

In eleven patients with dementia senilis circadian rhythms of hematocrit, hemoglobin, erythrocytes, leukocytes, serum iron and transferrin were studied by means of the cosinor method. An evident circadian rhythm was observed for all blood components, except leukocytes. There were no significant differences between patients with dementia senilis and healthy aged persons as far as hematocrit, hemoglobin, erythrocytes, and leukocytes are concerned. Acrophase for serum iron and transferrin occurs a little earlier and mesor is lower than in healthy aged persons.     

Effect of salvipholin on the carbohydrate and lipid metabolism in the rat liver in alloxan diabetes]

Peroral administration of salvipholin in a dose of 50 mg/kg to intact male rats (the body weight 180-220 g) had a positive effect on the carbohydrate-lipid metabolism in the liver and blood serum of animals. Administration of the same dose to rats with alloxan diabetes induced a significant decrease in the content of glucose, free fat acids, triglycerides and lysophospholipids of the liver and blood serum. The level of these components has sharply increased after subcutaneous alloxan administration in a dose of 150 mg/kg, the content of glycogen and pyruvic acid being normalized and insulin deficiency removed. These changes are closely related to salvipholin-promoted restoration of phospholipid spectra of the blood serum and liver of experimental animals disturbed under conditions of insulin deficiency. The possible mechanism of the salvipholin action is analyzed.     

Preventive Effects of Zinc Against Psychological Stress-Induced Iron Dyshomeostasis, Erythropoiesis Inhibition, and Oxidative Stress Status in Rats

Psychological stress (PS) could cause decreased iron absorption and iron redistribution in body resulting in low iron concentration in the bone marrow and inhibition of erythropoiesis. In the present study, we investigated the effect of zinc supplementation on the iron metabolism, erythropoiesis, and oxidative stress status in PS-induced rats. Thirty-two rats were divided into two groups randomly: control group and zinc supplementation group. Each group was subdivided into two subgroups: control group and PS group. Rats received zinc supplementation before PS exposure established by a communication box. We investigated the serum corticosterone (CORT) level; iron apparent absorption; iron contents in liver, spleen, cortex, hippocampus, striatum, and serum; hematological parameters; malondialdehyde (MDA); reduced glutathione (GSH); and superoxide dismutase (SOD). Compared to PS-treated rats with normal diet, the PS-treated rats with zinc supplementation showed increased iron apparent absorption, serum iron, hemoglobin, red blood cell, GSH, and SOD activities; while the serum CORT; iron contents in liver, spleen, and regional brain; and MDA decreased. These results indicated that dietary zinc supplementation had preventive effects against PS-induced iron dyshomeostasis, erythropoiesis inhibition, and oxidative stress status in rats.     

Gangliosides in blood serum of normal rats and Morris hepatoma 5123tc-bearing rats.

The effects of malignancy upon blood serum ganglioside patterns were investigated. Lipids extracted from the blood serum of Morris hepatoma 5123tc-bearing rats were characterized by severalfold increases in the content of hematosides, monosialogangliosides and disialogangliosides, as compared with lipids extracted from the serum of normal rats. However, the content of trisialogangliosides in lipids extracted from the serum of cancer-bearing rats substantially decreased. In general, the change in the profile of gangliosides in blood serum reflects, but is less pronounced, than that observed in the comparison of Morris hepatoma tissue to normal liver tissue.