Ludwig Müller

Consequences of cadmium toxicity in rat hepatocytes: mitochondrial dysfunction and lipid peroxidation.

Cadmium (Cd) (10-100 microM) decreased the ATP/ADP ratio and enhanced lipid peroxidation (LPO) (measured as thiobarbituric acid reactants) in incubated rat hepatocytes. Analysis of the subcellular distribution of Cd indicated its preferential attachment to the inner membranes of mitochondria. Incubation of isolated mitochondria with 0.005-0.05 microM Cd resulted in increased formation of formazans from nitroblue tetrazolium salts, indicating enhanced membrane permeability to succinate. These Cd-concentrations also diminished mitochondrial ATP. LPO in mitochondria strongly increased only after Cd-exposures above 1 microM Cd. Similarly, in Cd-treated hepatocytes decreases in ATP/ADP ratios corresponded to increases in LPO stimulation only at 30 and 60 min but not at 15 min of incubation when ATP/ADP ratios were already affected. Moreover, neither hepatocellular ATP/ADP decrease nor mitochondrial formazan formation due to Cd were prevented by (+)-cyanidanol-3, an effective inhibitor of Cd-induced LPO. These data suggest that even low Cd-concentrations in the hepatocyte disturb the integrity of its mitochondrial membranes concomitantly impairing the hepatocellular energy supply. LPO, only observed at higher Cd-concentrations, is not responsible for these adverse Cd-effects.

Studies on the efficacy of lipoate and dihydrolipoate in the alteration of cadmium2+ toxicity in isolated hepatocytes

Lipoate (thioctic acid) is presently used in therapy of a variety of diseases such as liver and neurological disorders. However, nothing is known about the efficacy of lipoate and its reduced form dihydrolipoate in acute cadmium (Cd2+) toxicity which involves severe liver disturbances. Therefore, we investigated the effects of these redox compounds on Cd2(+)-induced injuries in isolated rat hepatocytes. The cells were coincubated with 150 microM Cd2+ and either 1.5-6.0 mM lipoate or 17-89 microM dihydrolipoate for up to 90 min and Cd2+ uptake as well as viability criteria were monitored. Both exposure regimens diminished Cd2+ uptake in correspondence to time and concentration. They also ameliorated Cd2(+)-induced cell deterioration as reflected by the decrease in Cd2(+)-induced membrane damage (leakage of aspartate aminotransferase), by the lessening of the Cd2(+)-stimulated lipid peroxidation (TBA-reactants) and by the increase in Cd2(+)-depleted cellular glutathione (GSH + 2 GSSG). Half-maximal protection was achieved at molar ratios of 9.9 to 19 (lipoate vs. Cd2+) and 0.25 to 0.74 (dihydrolipoate vs. Cd2+), indicating a 19.5 to 50.6 lower protective efficacy of lipoate as compared to dihydrolipoate. Lipoate induced an increase in extracellular acid-soluble thiols different from glutathione. It is suggested that dihydrolipoate primarily protects cells by extracellular chelation of Cd2+, whereas intracellular reduction of lipoate to the dihydro-compound followed by complexation of both intra- and extracellular Cd2+ contributes to the amelioration provided by lipoate.

Cadmium-induced alteration of the energy level in isolated hepatocytes

The incubation of isolated hepatocytes with 10-100 microM cadmium (Cd) decreased the ATP/ADP-ratio, the ATP/AMP-ratio and the adenylate energy charge and enhanced the lactate/pyruvate-ratio in a time- and concentration-dependent manner. Likewise, the cellular oxygen-consumption was decreased at early incubation times, when the cell membrane was still intact, as judged by the Trypan Blue-exclusion test. Similarly, Cd-concentrations above 0.3 nmol/mg mitochondrial protein inhibited the succinate- and malate/pyruvate-stimulated respiration of isolated mitochondria. This critical concentration was also reached in mitochondria of hepatocytes, when exposed to 25 microM Cd for less than 30 min. We therefore suggest that the inhibition of cellular respiration and the breakdown of cellular energy generation is mediated by a severe disturbance of mitochondrial respiratory functions due to Cd. This effect seems to be an early event in Cd-toxicity in isolated hepatocytes.

Consequences of cadmium toxicity in rat hepatocytes: effects of cadmium on the glutathione-peroxidase system.

Cadmium (Cd; 10-100 microM) produced in isolated hepatocytes the formation of thiobarbituric acid-reactants (lipid peroxidation; LPO) and a decrease in SH groups in a time- and concentration-dependent manner. However, glutathione peroxidase (GSH-Px) was not affected indicating that LPO does not originate from GSH-Px injury. In contrast, in presence of Cd a time- and concentration-dependent decrease of glucose-6-phosphate dehydrogenase (G6PDH) and, most strongly, of glutathione reductase (GSSG-R), both members of the GSH-Px system, was observed. These effects could not be inhibited by (+)-cyanidanol-3, a compound known as inhibitor of LPO. Therefore, our data show that Cd-induced LPO is not a function of GSH-Px injury and inhibition of GSSG-R and G6PDH-activity is not a function of LPO. They also indicate that Cd-induced depletion of cellular SH groups are associated with toxic effects of Cd on GSSG-R and G6PDH different from LPO-induction.

Absorption and distribution of cadmium (Cd), copper and zinc following oral subchronic low level administration to rats of different binding forms of cadmium (Cd-acetate, Cd-metallothionein, Cd-glutathione)

Male Wistar rats received by gavage saline or about 25 micrograms cadmium (Cd)/kg/day as Cd-acetate (Cd-Ac), Cd-metallothionein (Cd-MT) or Cd-glutathione (Cd-GSH) 5 times per week for 28 times. At all treatments 0.2-0.3% of the totally administered Cd dose was found in liver, kidneys, small intestine and pancreas, whereas none of the Cd forms applied resulted in a Cd accumulation in testes. Cd in small intestine was not increased by Cd-MT. However, it was raised by Cd-Ac and even more by Cd-GSH. A smaller increase in hepatic and renal Cd resulted from Cd-GSH than from Cd-Ac or Cd-MT. Cd in pancreas increased after Cd-GSH but not after Cd-Ac or Cd-MT. Copper (Cu) rose in small intestine and testes but decreased in kidneys independent of either Cd treatment. Concomitantly, zinc (Zn) was decreased in small intestine and testes. The tissue concentration of metallothionein (MT) was only marginally increased by all treatments. The highest value (80%) above controls) was found in small intestine after Cd-GSH. Intestinal Cd as well as testicular Cu were related to the tissue MT. Therefore, the distribution of Cd between various organs depends on the Cd form applied. There is some relationship to the distribution of Cu and Zn.

Protective effects of DL-α-lipoic acid on cadmium-induced deterioration of rat hepatocytes

Abstract The suitability of DL -α-lipoic acid (LA) to serve as an antidote in cadmium (Cd) toxicity in rat hepatocytes was investigated. Isolated hepatocytes were exposed to 200 and 450 μM Cd in the presence of 0.2, 1.0 and 5.0 mM LA, respectively. After 30 min of incubation various criteria of cell viability were monitored. Lipoic acid markedly diminished Cd uptake. Concomitantly, Cd-induced membrane injury, as reflected by the leakage of aspartate aminotransferase and sorbitol dehydrogenase (SDH) was decreased. Moreover, LA protected against intracellular toxic responses to Cd, such as a decrease in cellular SDH activity, a decrease in cellular acid soluble thiols, especially in total glutathione, a decrease in cellular urea and an increase in thiobarbituric acid (TBA) reactants, as a measure of lipid peroxidation. Most protective effects were seen in hepatocytes challenged with the lower Cd concentration and coincubated with 5 mM LA. In contrast, at 450 μM Cd even the highest LA concentration applied either did only reverse Cd-effects incompletely (SDH-response, TBA-reactants) or did not protect at all (Cd uptake, enzyme leakage, loss of glutathione). The data indicate that DL -α-lipoic acid serves as a protective tool against Cd-induced membrane damage and cell dysfunction in hepatocytes. This stands as long as Cd exposure is low enough to permit interaction with LA prior to interaction with cell structures.

Uptake and distribution of aluminium in rat hepatocytes and its effect on enzyme leakage and lactate formation.

Aluminium (Al) chloride (10-200 microM) increased the Al content in hepatocytes isolated from fed male rats in a time- and concentration-dependent manner. After 60 min of incubation with 100 microM Al about 45% of cellular Al was found each in the mitochondrial and the postmitochondrial fraction of hepatocytes, whereas about 5% of Al sedimented with nuclei and cell debris. Concomitantly, the leakage of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased in the presence of Al time- and concentration-dependently, but only to a moderate extent. Aluminium (10-200 microM) also accelerated the formation of lactate by hepatocytes. No significant differences were found in Al uptake and distribution and its effect on LDH leakage and lactate formation when the metal ion was given as AlCl3, Al(NO3)3 or Al(lactate)3. Al concentrations (AlCl3) exceeding 250 microM severely disturbed the determination of LDH, AST and lactate in a cell free system. The data suggest only a moderate toxicity of Al compounds to isolated hepatocytes, when given in amounts approximating (patho)physiological conditions.

Differential sensitivity of integrity criteria as indicators of cadmium-induced cell damage.

The reliability of the stainability by Trypan Blue (TB) to detect membrane damage in hepatocyte suspensions of different TB stainability and in freshly isolated cells exposed to 50 microM cadmium (Cd) for 60 min was assessed by comparison with the leakage of lactate dehydrogenase (LDH) and NADH-oxidation. The cellular nitrophenyl-phosphate (NPP) uptake and the digitonin-induced fluorescence of 8-anilinonaphthalene-sulfonate-1 (dig-ANS-F) were introduced as integrity parameters of hepatocytes and compared with TB stainability. Cd was without effect on all these parameters. LDH-leakage was a poor criterion, whereas NADH-oxidation and dig-ANS-F were as sensitive as TB stainability. The NPP-uptake was more sensitive than TB stainability to indicate toxic Cd-effects when the plasma membrane still seemed to be intact. Because of the enhanced extracellular NPP-degradation, which resulted from the release of alkaline phosphatase, NPP-uptake was useful as a measure of Cd-induced damage only under certain conditions. These results confirm TB stainability as a simple and reliable criterion for plasma membrane integrity and therefore viability of hepatocytes also in Cd-toxicity.

Effects of cadmium in rat heptocytes: Interaction with aluminum

The influence of aluminium (Al) chloride on toxic responses to cadmium (Cd) chloride of hepatocytes isolated from fed rats were investigated. Hepatocytes exposed to 50-200 microM Al took up the Al-ion more efficiently when the cells were simultaneously incubated with 50 microM Cd for 60 min. Aluminium (50-200 microM) could not prevent the release of about 50% of lactate dehydrogenase (LDH) from cells due to 50 microM Cd. Aluminium itself increased LDH leakage only to a moderate extent, indicating the low toxicity of Al to hepatocytes. Cadmium diminished the total hepatocellular thiols (protein + non-protein) and, even more pronounced, the acid soluble thiols after 60 min of incubation. However, this response to Cd was inhibited by simultaneous exposure to 50-200 microM Al in an Al concentration-dependent manner. Concomitantly, the Cd-dependent lipid peroxidation (LPO; measured as thiobarbituric acid reactants) at 60 min was decreased by Al, which itself did not enhance basal LPO in hepatocytes. These data show that Al partly protects hepatocytes from Cd-induced depletion of acid soluble thiols (i.e. reduced glutathione) and from stimulation of LPO. However, Al did not prevent the Cd-induced damage of the cell membrane.

Influence of paracetamol (acetaminophen) on cadmium-induced lipid peroxidation in hepatocytes from starved rats

Isolated hepatocytes form thiobarbituric acid (TBA)-reactants, a parameter of lipid peroxidation (LPO), when exposed to cadmium (Cd). Paracetamol (Para) inhibits this response, dependent on time and concentration. Cd-induced SH-group decline, however, and enhanced plasma membrane permeability to Trypan Blue (TB) were not inhibited by Para. This indicates that Cd-induced cell damage and Cd-induced LPO are independent events. Experiments using cells with enhanced and lowered metabolism of Para after phenobarbital- and acetone- or heat-and metyrapone treatment, respectively, suggest a potentially inhibitory effect of both Para and its metabolite(s) on LPO. The antioxidative potency of Para turned out to be small compared to that of the radical scavenger (+)-cyanidanol-3.