The effect of cadmium chloride and hydrogen peroxide on the lipid peroxidation and fractional composition of lipids in hepatocytes of rats

The isolated hepatocytes were incubated in the medium, containing cadmium chloride or hydrogen peroxide. Influence of the latter on the intensity of lipid peroxidation and contents of some lipids fractions, as well as viability of hepatocytes in these conditions has been studied. It is shown that under such cultivation conditions the activation of lipid peroxidation in the hepatocytes takes place. Its activation in presence of cadmium chloride was one of the factors of the membranes damage. The changes in the content of some fractions of lipids were similar both under the incubations of the cells with cadmium chloride and hydrogen peroxide. This allows one to suppose that cadmium chloride causes changes in the lipid composition of membranes as a result of intensification of lipid peroxidation.     

Role of cadmium in activating nuclear protein kinase C and the enzyme binding to nuclear protein.

Specific effects of cadmium on nuclear protein kinase C activity were found with 3T3/10T1/2 mouse fibroblast and rat liver nuclei. Treatment of the mouse fibroblasts in culture with 12-O-tetradecanoylphorbol-13-acetate resulted in the stimulation of nuclear protein kinase C activity in a "fixed" pool which is defined by its resistance to chelator extraction, whereas the chelator extractable enzyme activity, defined as the "labile" pool was unaffected. Cadmium was found to potentiate the effect of the phorbol ester, directed specifically to nuclei, since the particulate protein kinase C activity was not changed under similar treatment. In a reconstituted system consisting of rat liver nuclei and rat brain protein kinase C, cadmium stimulated the binding of the enzyme to a 105-kDa nuclear protein. The binding of a 105-kDa protein to protein kinase C is attributed strictly due to the cadmium effect, whereas a 50-kDa protein binding to protein kinase C was only enhanced by cadmium. We propose a mechanistic model, where cadmium substitutes zinc in the regulatory domain of protein kinase C rendering the putative protein-protein binding site exposed.     

Effect of anthocyanins on selected biochemical parameters in rats exposed to cadmium

Cadmium is a dangerous occupational and environmental toxin. It accumulates in the human organism mainly in liver and kidneys. Cadmium half-life is about 10 years, so the symptoms of cadmium intoxication may occur several years after the exposure. Until now in treating intoxication with this metal chelating compounds have been used, burdened with numerous undesirable symptoms. In our investigations anthocyanins from Aronia melanocarpa were used to reduce the harmful results caused by cadmium. Administering anthocyanins with cadmium chloride resulted in a statistically significant decrease of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity, concentration of bilirubin and urea in blood serum and decreased cadmium cumulation in liver and kidneys in relation to animals receiving cadmium chloride only.     

The Retention of Cadmium and Selenium Influence in Fowl and Chickens of F1 Generation

The retention of cadmium and selenium influence on Cd retention in the muscle, liver and kidneys of hens, chickens and in eggs was studied. Cadmium (Cd) as cadmium chloride (CdCl(2)) and selenium (Se) as sodium selenite (Na(2)SeO(3)) were added to feed at dosages: group 0-control, group 1-20 mg/kg Cd, group 2-30 mg/kg Cd + 4 mg/kg Se. The birds were exposed to Cd for 8 weeks. Cadmium level in hens and cocks was found highest in the kidneys, followed by the liver and muscle. Se supplementation resulted in Cd increase in the muscle tissue and in the reduction of Cd content in the liver and in significant decrease in the kidneys (p < 0.05). A higher Cd level in the yolk and lower in the white was noted in both experimental groups. Nonsignificant increase of Cd in eggs was noted in experimental groups with Se supplementation. Level of cadmium in organs of 7-day-old chicks hatched from Cd-treated hens in both experimental groups was low but the tendency to accumulate preferentially the Cd in the liver and kidneys was recorded. Supplementation of selenium in hens and cocks was not reflected in the decrease of Cd in these two organs of F(1) chickens but was reflected in increase in the muscle. In spite of relatively high Cd levels in the organs of layers no layer-egg-chickens transfer was observed. It was confirm that kidneys and liver are organs more attacked by dietary cadmium than muscle. Supplementation of low dose of Se resulted in decrease of cadmium deposition in analyzed organs.     

Effect of cellular Ca2+ loading on alpha 1-agonist or protein kinase C activators-mediated stimulation of phosphorylase "a" in liver cells

Long chain unsaturated fatty acids stimulate phosphorylase "a" activity in liver cells. Similar degree of activation was achieved by increasing cellular Ca2+ content or by treatment with agents other than oleate, like 1,2-diolein or phorbol esters, sharing in common their ability to activate protein kinase C. In Ca2+-loaded liver cells only phenylephrine was capable of inducing a further stimulation of phosphorylase "a" activity. It is concluded that: 1) The state of activation of protein kinase C may play a role in the hormonal control of liver glycogen metabolism; 2) alpha 1-agonist-mediated activation of phosphorylase "a" can occur by a mechanism which is not related to a Ca2+-dependent activation of protein kinase C.     

Activity and distribution of protein kinase C in liver during the acute-phase response

Activity and subcellular distribution of protein kinase C were estimated in liver cytosol and membrane fractions of rats carrying a turpentine-induced inflammation. Protein kinase C activity increases significantly 8 h after treatment in the membrane fraction, with concurrent reduction in the cytosol; 10 h after treatment the membrane-associated activity returns to normal, without concomitant recovery of that detected in the cytosol. The specific binding of phorbol dibutyrate to the liver membrane fraction increases but overall the effect is less evident and delayed in time. The changes are associated to alterations in the phosphorylation pattern of some liver proteins. Liver protein kinase C activity and intracellular distribution seem to be affected by a treatment which is known to induce an acute-phase response in the liver cells.     

Subcellular distribution of metallothionein and cadmium in the liver and kidneys of bank voles (Clethrionomys glareolus) exposed to dietary cadmium

Metallothionein (MT) and cadmium (Cd) contents were determined in the subcellular fractions of the liver and kidneys of bank voles exposed for 6 weeks to elevated levels of dietary Cd-40 and 80 g g^-1 dry weight. Hepatic and renal MT was detected exclusively in the cytosol, while Cd was found in the cytosol (73–79% of the total content), nuclei (14–18%) and particulates (4–9%). The concentration of MT in the cytosol as well as Cd content in the particular subcellular fractions appeared to be a dose-dependent. The absence of MT in the nuclear and particulate fractions implied that Cd present in these compartments was not bound to the protein that is considered to provide protection against the toxic metal. Therefore, it is assumed that this component of intracellular Cd could be responsible for the histopathological changes that occurred in the liver (granuloma and focal hepatocyte swelling) and kidneys (focal degeneration of proximal tubules) of bank voles exposed to the higher level of dietary Cd.     

Biological function of metallothionein. VI. Metabolic interaction of cadmium and zinc in rats

The accumulation and depletion of cadmium in liver and kidney metallothionein (MT) and the effects of dietary zinc deficiency on cadmium metabolism were studied in rats. The accumulation of cadmium in liver MT started to plateau after 80 days, but there was a linear accumulation of this element in kidney MT over the entire 300-day experiment. Cadmium in MT fractions was depleted very slowly when rats were changed to a diet without cadmium. The accumulation of cadmium in MT also caused zinc to accumulate in this protein, even in rats fed zinc-deficient diets. However, the reverse situation was found not to be true; zinc did not cause cadmium to accumulate in MT. Dietary zinc deficiency limited the binding of injected¹⁰⁹Cd to MT of liver, but not of kidneys or testes. However, zinc-deficient rats fed cadmium in their diets metabolized cadmium similarly to zinc-supplemented rats, suggesting that zinc deficiency does not limit the ability of cadmium to stimulate MT synthesis.     

Age dependent changes in metallothionein and accumulation of cadmium in horses

1. Analysis of livers and kidneys from 28 horses for cadmium, zinc and metallothionein showed low cadmium content in liver. There was a gradual increase in cadmium content in kidney with age. 2. Metallothionein values varied with zinc content in the liver and with cadmium content in the kidney; copper values did not vary in either tissue. 3. Metallothionein was localized mainly in the cytoplasms in liver and kidney of horses by immunohistochemistry.     

In vivo and in vitro effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius Ham. (rosy barb).

1. Enzyme modulation by cadmium in selected organs of the fish, Barbus conchonius (rosy barb), was investigated in vivo (48 hr exposure to 12.6 mg/l cadmium chloride) and in vitro (10(-6) M cadmium chloride). 2. The acetylcholinesterase (AchE) activity was depressed in the gills but stimulated in the skeletal muscles and brain in vivo. The hepatic, branchial, and renal acid phosphatase (AcP) activity decreased marginally in vivo but it was significantly increased in the gut and ovary. In vitro, except for the liver, the AcP activity was depressed in the selected organs. Collaterally, gut alkaline phosphatase (AlP) was significantly inhibited but a pronounced stimulation was noted in the kidneys and ovary in vivo. In vitro, the AlP activity was conspicuously elevated in the kidneys and gut, and moderately in the gills. 3. Cadmium inhibited the glutamate-oxaloacetate and glutamate-pyruvate transaminases (GOT and GPT) in the liver, gills and kidneys in vivo. In vitro, the GOT and GPT activities were decreased in the liver, gills and kidneys. The lactic dehydrogenase (LDH) was significantly stimulated by Cd in the heart in vivo but in vitro the metal inhibited the enzyme in the gills. 4. Enzymes in the liver, followed by those in the kidneys and gills seem to be most seriously affected by Cd poisoning in this fish.     

Choline deficiency activates phospholipases A2 and C in rat liver without affecting the activity of protein kinase C

There is evidence to suggest that liver tumor promoters exert their effect through the interference of signal transduction in hepatic cells. Both phospholipase A(2) and phospholipase C play important roles in the generation of second messengers and in the activation of Ca(2+), phospholipid-dependent protein kinase C. Using male Sprague-Dawley rats, we investigated whether liver tumor-promoting regimens of a choline-deficient diet and phenobarbital alter the activities of phospholipase A(2) and phospholipase C in the liver, and extended the study to determine the effect of a choline-deficient diet on protein kinase C activities. Feeding a choline-deficient diet for 1 week increased the activities of both phospholipase A(2) (50%) and phospholipase C (22%), and the activities of both enzymes were more than doubled after 4 weeks. Feeding a phenobarbital diet resulted in a slight decrease in phospholipase A(2) activities at 4 weeks but no significant changes in PLC activities. The protein kinase C activities and its distribution between soluble and particulate fractions remained unchanged after 1, 2, and 4 weeks feeding of a choline-deficient diet. Thus, activation of both phospholipase A(2) and C is distinct for a choline-deficient diet, not shared by phenobarbital diet. Increased activities of these enzymes were not associated with the activation of protein kinase C under the present experimental condition.     

Low ambient temperature decreases cadmium accumulation in the liver and kidneys of the bank vole (Clethrionomys glareolus)

The importance of photoperiod and ambient temperature on the accumulation of cadmium in the liver and kidneys of bank voles was determined in the present study. Males and females, aged 1 month, were given 3.0 g Cd ml^–1 drinking water and divided into four groups according to photoperiod (16 h light/8 h dark and 8 h light/16 h dark) and ambient temperature (20 or 5°C); liver and kidneys were removed for cadmium as well as copper, iron and zinc analyses at the end of 6 weeks. Bank voles exposed to 5°C in both photoperiods consumed approximately 30% less water containing cadmium than those kept at 20°C. However, the total accumulation of cadmium in the liver and kidneys of males and females exposed to the low temperatures was 4.3–4.8 and 2.2–3.3 times less than that in animals maintained at room temperature in the long and short photoperiod, respectively. Simultaneously, the low temperature brought about an increase in the copper concentrations in the liver (12–43%) and kidneys (47–78%), giving rise to an inverse correlation between the cadmium accumulation and the tissue copper concentration. In contrast to cadmium and copper, the concentrations of iron and zinc were affected primarily by photoperiod. These findings indicate that ambient temperature is an important determinant of cadmium retention in the bank vole. It appears that low temperature decreases tissue cadmium accumulation not only by reducing cadmium intake but also through changes in copper metabolism.     

Protein kinase C located in rat liver nuclei. Partial purification and biochemical and immunochemical characterization

In the rat liver homogenate, maximal protein kinase C activity was found at two calcium concentrations (1.75 and 3.5 mM). Subcellular fractionation of the liver homogenate revealed that the protein kinase C activity requiring 1.75 mM calcium was present only in the cytosolic and particulate subcellular fractions. The protein kinase C activity requiring 3.5 mM calcium concentration was mainly located in the rat liver nuclei preparation. About 19% of the liver homogenate protein kinase C activity requiring 3.5 mM calcium was present in the nuclei. Goat anti-rat brain protein kinase C antibodies revealed a single immunoreactive band at 80-82 kDa in the rat liver nuclear, particulate, or cytosolic fractions. Based on the ratio of plasma membrane marker enzyme activity determined in the nuclear preparation, the purity of the isolated nuclei was ascertained. Rat liver nuclear protein kinase C activity has been partially purified. The purification steps sequentially employed were Triton X-100 extraction of isolated nuclei, DEAE-cellulose chromatography, Phenyl-Superose, and Mono Q (fast protein liquid) chromatography. The final purification step revealed, by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein bands at 80 and 66 kDa, respectively. These findings provide definitive data regarding the nuclear location of protein kinase C. The nuclear location of protein kinase C may lead to an understanding of the molecular pathway involved in signal transduction from the plasma membrane to the nucleus.     

Uptake of Exogenous PhosphatidyJserine by Human Neuroblastoma Cells Stimulates the Incorporation of [methyl-l4C]Cholne into Phosphatidylcholine

The phosphatidylserine (PtdSer) content of human cholinergic neuroblastoma (LA-N-2) cells was manipulated by exposing the cells to exogenous PtdSer, and the effects on phospholipid content, membrane composition, and incorporation of choline into phosphatidylcholine (PtdCho) were investigated. The presence of liposomes containing PtdSer (10-130 microM) in the medium caused time- and concentration-dependent increases in the PtdSer content of the cells, and smaller and slower increases in the contents of other membrane phospholipids. The PtdSer levels in plasma membrane and mitochondrial fractions prepared by discontinuous sucrose density gradient centrifugation increased by 50 and 100%, respectively, above those in control cells after 24 h of exposure to PtdSer (130 microM). PtdSer caused a concomitant, concentration-dependent increase of up to twofold in the incorporation of [methyl-14C]choline chloride into PtdCho at a choline concentration (8.5 microM) compatible with activation of the CDP-choline pathway, suggesting that the levels of PtdSer in membranes may serve as a stimulus to regulate overall membrane composition. PtdSer caused a mean increase of 41% in PtdCho labeling, but the phorbol ester, phorbol 12-myristate 13-acetate (PMA), which stimulates PtdCho synthesis in a number of cell lines, increased [14C]PtdCho levels by only 14% in LA-N-2 cells, at a concentration (100 nM) which caused complete translocation of the calcium- and phospholipid-dependent enzyme protein kinase C to the membrane. The translocation was inhibited by prior exposure of the cells to PtdSer. Treatment with PMA for 24 h diminished protein kinase C activity by 80%, but increased the labeling of PtdCho in both untreated and PtdSer-treated cells. These data suggest that uptake of PtdSer by LA-N-2 cells alters both the phospholipid composition of the membrane and synthesis of the major membrane phospholipid PtdCho; the latter effect does not involve activation of protein kinase C.     

NF-κB acts downstream of EGFR in regulating low dose cadmium induced primary lung cell proliferation

Apart from cytotoxicity cadmium has no special attributes towards cell’s physiological function. The role of cadmium with respect to cell growth is still under debate. Mitogen activated protein kinase and Ca²⁺/calmodulin dependent protein kinase dependent pathways are the two elaborately studied concerning cadmium induced cell proliferation. Low concentration of cadmium chloride (2.5 μM) was applied to mice primary lung epithelial cells and cell proliferation was measured both by cell cycle analysis and Brdu incorporation assay. Effects of differential dose of cadmium chloride on lung epithelial cells were evaluated morphologically by atomic force microscopy. RT-PCR and western blot altogether corroborated the specific signalling pathways concerning cadmium induced lung cell proliferation. Cadmium induced lung epithelial cells which over-expressed EGFR, were transfected with siEGFR, revealed downstream molecules and RNAi induced EGFR silencing. Use of siEGFR effectively prevents expression of proinflammatory and cell proliferative markers. Moreover N-acetyl cysteine and ascorbic acid mediated inhibition of EGFR and downstream signalling molecules indicate the involvement of reactive oxygen species. Exposure to low concentration of cadmium promotes the growth of primary mice lung epithelial cell by EGFR signalling. We have also transfected the primary lung epithelial cell with siRNA against the regulatory subunit of nuclear factor-κB (NF-κB) and the data shows that cadmium induced lung cell proliferation is the effect of EGFR mediated NF-κB activation.     

Nuclear activation and translocation of mitogen-activated protein kinases modulated by ethanol in embryonic liver cells

Activation and nuclear translocation of mitogen activated protein (MAP) kinases in ethanol-treated embryonic liver cells (BNLCL2) was investigated. The relative amount of MAPK proteins, MAP kinase activity and MAPK/LDH (lactate dehydrogenase) ratios were determined in nuclear and cytosolic fractions before and after serum stimulation. In ethanol-treated cells, serum-stimulated MAPK activation was potentiated in both cytosolic and nuclear fractions. Levels of both the p42 and p44 MAPK proteins increased in nuclear fractions from cells treated with ethanol alone for 24 h. Serum-stimulated nuclear translocation of both p42 and p44 MAPK was potentiated in ethanol-treated cells. Nuclear fractions from ethanol-treated cells had a modest increase in MAP kinase activity concurrent with the increased MAPK protein levels. The ratio of MAPK/LDH increased in nuclear fractions with increasing concentrations of ethanol and after serum stimulation. This further confirmed the nuclear translocation of MAPK and also demonstrated that it is not a non-specific effect of ethanol. These results demonstrate, for the first time, that in BNLCL2 liver cells ethanol treatment has dual effects. First, ethanol triggered nuclear translocation of MAPK without causing its activation. Second, it potentiated serum-stimulated activation and translocation of MAPK in the nucleus. These findings provide a novel mechanism through which ethanol may affect cellular and nuclear processes in liver cells.     

Influence of vitamin C on cadmium absorption and distribution in rats

The purpose of the present study was to evaluate the effect of a dietary vitamin C supplement on cadmium absorption and distribution in an animal model. An aqueous solution of cadmium chloride (labelled with cadmium-109) was given by gavage to male Wistar rats for 28 days at a daily dose corresponding to 10 mg Cd/kg diet (1.0-1.2 mg Cd/kg b.w.). The animals assigned to groups 1 and 2 (45 animals per group) received a standard laboratory diet LSM, and tap water or tap water supplemented with ascorbic acid (1.5 mg/l), respectively. The radioactivity of the samples was measured using a liquid scintillation counter (tissue samples) and a gas-flow automatic counter (ashed carcasses). The fractional uptake of cadmium-109 in the carcass and organs was evaluated within 32 days after treatment by dividing the cadmium-109 activity in the whole sample by the total activity of cadmium-109 administered for 28 days. Results were compared using AUC (areas under the concentration time curve) values. The vitamin C supplement decreased the carcass cadmium burden and the cadmium content in the liver, kidneys, testicles and muscles; the highest decreases were found in the testicles, the lowest ones in the muscles. In addition, the rats supplemented with vitamin C revealed an improved body weight gain during the experimental period.     

Concentrations of cyclic AMP-dependent protein kinase subunits in various tissues.

The concentrations of the regulatory (R) and catalytic (C) subunits of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase(s) were measured in extracts of skeletal muscle, heart, liver, kidney, and brain. These concentrations were also estimated for the particulate fraction from brain, the only tissue in which a major part of the total activity was not readily extracted in a soluble form. Values for R were determined by measuring the amount of cyclic [3H]amp bound to protein in these tissue fractions under specified conditions; it was assumed that 1 mol of cyclic AMP binds to 1 mol of R. Values for C were determined from measurements of the specific protein kinase activity of the fractions utilizing the turnover number of pure C in the calculations. Turnover numbers for C were found to be identical for this subunit obtained in the pure form from rabbit skeletal muscle, rabbit liver, and beef heart. The methods used for measuring C were evaluated by kinetic studies and through the use of the specific heatstable protein inhibitor of cyclic AMP-dependent protein kinase(s). R and C were found to exist in a 1:1 molar ratio in all of the tissue fractions that were studied. the absolute concentrations of R and C ranged from 0.23 mumol/kg wet weight for liver to 0.78 mumol/kg wet weight for brain. For brain this value was based on the amount of each subunit in the particulate as well as the soluble fraction. For other tissues the values were based solely on the subunit content of the latter fraction. It was noted that the molar concentrations of R are close to those of cyclic AMP under basal conditions in the various tissues.     

Some features of the bacterial membrane studied with the aid of a new fractionation technique

Membranes of Bacillus megaterium and other bacteria were bound to crystals of cadmium lauroylsarcosinate and were resolved into nine lipoprotein fractions by elution with potassium chloride and sodium deoxycholate solutions. The fractions differed widely in protein/lipid ratio. Some major protein species were probably common to all the fractions, but the phospholipid composition showed some variation. Electron-microscopic examination after negative staining revealed that material in certain fractions was in the form of particles of diameter about 12nm, and other fractions consisted of amorphous aggregates. The composition of the membrane in terms of the nine fractions was influenced markedly by the conditions of culture of the bacteria. Radioactive-labelling experiments suggested that some fractions served as precursors of others, and one fraction appeared to contain the attachment points of the DNA.