Curtis D. Klaassen

Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes

Abstract The toxicity of cadmium may be due to alteration in membrane structure which may be caused by peroxidation of the composite lipids. Isolated hepatocytes provide a suitable system to examine the role of lipid peroxidation in a toxic response at the cellular level. Therefore, isolated rat hepatocytes were incubated with varying cadmium concentrations (50–400 μ m ) for up to 75 min. An increase in lipid peroxidation due to cadmium was observed. The integrity of the cell membrane, as measured by loss of intracellular potassium ion and leakage of aspartate aminotransferase, was adversely affected in the presence of cadmium. The lactate to pyruvate ratio of hepatocyte suspensions was increased upon incubation with cadmium. Several chelating compounds were found to reduce intracellular accumulation of cadmium, cellular toxicity and lipid peroxidation. However, amelioration of toxicity was not consistently associated with inhibition of the lipid peroxidation response. The antioxidant compounds, sodium diethyldithiocarbamate and N,N′-diphenyl-p-phenylene-diamine were found to inhibit the lipid peroxidation attributable to cadmium, but did not have any consistent protective effect against loss of intracellular potassium ion. The results of this study show that the toxicity induced by cadmium in isolated rat hepatocytes can be dissociated from the concurrently observed lipid peroxidation, which indicates that the toxic response is not caused by the lipid peroxidation.

Dose-response effects of various metal ions on rat liver metallothionein, glutathione, heme oxygenase, and cytochrome P-450

Adult male rats received ip injections of the maximum tolerable dose (MTD; μmol/kg/day, in parentheses following metal), or a fraction thereof, of Hg (5), Cd (20), Se (25), Ag (65), Cu (75), Co (100), Ni (120), Zn (200), Mn (250), Fe (300), Pb (400), or Cr (400) 36 and 12 hr before sacrifice. MTDs were estimated from previous studies, and at least three serial dilutions (12, 14, 18, etc.) of the MTDs were tested for each metal. The effects of metal treatment on hepatic heme oxygenase activity (HO), cytochrome P-450, reduced glutathione (GSH) and metallothionein (MT), and renal MT and GSH were determined. Nine metals increased HO at the MTD, but only Cd, Se, Mn, and Pb increased HO at lower doses. These four metals plus Ag and Cr depressed cytochrome P-450 levels at the MTD, but only Cd, Mn, and Pb depressed cytochrome P-450 at a lower dose. Se increased hepatic GSH at the two highest doses, but all other metals had little or no effect. Kidney GSH was increased by all metals except Cd, Ag, Cu, and Cr, to a maximum level of only 150% of control (Pb). Cd and Zn induced hepatic MT in a dose-related manner to 420 and 580% of control, respectively. On a molar basis, Cd was about eight times more potent than Zn in increasing hepatic MT concentration. Hg, Ni, Mn, Fe, Pb, and Cr also significantly increased hepatic MT, but only to 150–200% of control. Pb had a slight but significant effect on hepatic MT at all doses down to 1/16th the MTD. This effect of PB, as well as other metals having a small effect on MT, may be the result of the effects of stress on MT rather than the metal ion per se. Renal MT was effectively induced by Hg, Cd, Cu, Ni, Zn, and Pb. Relatively small amounts of Hg (0.62 μmol/kg/day) significantly increased renal MT when compared to the minimum effective dose of Cd (10 μmol/kg/day) or Zn (50 μmol/kg/day). In conclusion, metals have a number of effects on potential hepatic and renal biochemical defense mechanisms. Most of the metals lacked specificity, affecting a number but not all of the parameters examined. However, of the 12 metals examined, Zn was the most selective in that it produced marked increases in MT and little or no effect on the other parameters, whereas Cd had the broadest effect, altering all parameters except GSH.

Toxicity and distribution of cadmium administered to rats at sublethal doses

Male Sprague-Dawley rats were given a single oral dose of [ 109 Cd]cadmium chloride (0, 25, 50, 100, and 150 mg of Cd/kg), and plasma glucose, blood hemoglobin, hematocrit, hepatic cytochrome P -450 concentrations, aniline hydroxylase activity, and hexobarbital oxidase activity were measured 2 and 14 days after dosing. The plasma glucose, blood hemoglobin, and hematocrit values observed were the same in all rats. Hepatic cytochrome P -450 and aniline hydroxylase activity were also the same in all rats, however, the hexobarbital oxidase activity was lower in the rats receiving the higher doses. Testicular function measured 14 days after dosing was decreased in male rats given the higher doses of Cd (100 and 150 mg of Cd/kg). Systolic blood pressure, heart rate, body weight, urine, protein excretion, and motor activity were measured daily for 14 days after dosing. The systolic blood pressure and heart rate remained unchanged in dosed and control rats. The rats given the higher doses showed a decrease in body weight during the first 3–4 days; thereafter, all rats had the same growth rate as controls. Urine flow and protein excretion of dosed rats were approximately one-half the value of control rats for 2 days but the same thereafter. Daily motor activity for the first 2–3 days was lower in the dosed rats (50, 100, 150 mg of Cd/kg) than the control rats. The tissue concentration of cadmium after 2 days was highest in the liver, which contained most of the body burden of Cd, followed by intestine, kidney, pancreas, spleen, heart, lung, testes, muscle, brain, blood, and plasma. After 2 weeks most tissue concentrations decreased 50% with some exceptions, notably the liver, which remained unchanged at higher doses, and the kidney, which showed up to a three- to four-fold increase. The concentration of metallothionein in the liver was increased at 2 days and was approximately the same after 14 days. In contrast, the concentration of metallothionein in the kidney at 2 days was unchanged but at 14 days had increased. It appears that the redistribution of cadmium to the kidney with time is due to the longer time period required for the increase of metallothionein levels in the kidney.

The relationship of metallothionein to the toxicity of cadmium after prolonged oral administration to rats.

Abstract Male Sprague-Dawley rats were exposed to cadmium at concentrations of 10, 30, and 100 ppm in their drinking water for 24 weeks. The testicular function, blood pressure, heart rate, EKG, hematocrit, blood hemoglobin, plasma glucose, aniline hydroxylase, hexobarbital oxidase, cytochrome P -450, concentration of Cd in the tissues, concentration of metallothionein in the kidney and liver, organ weights, bone calcification, and histopathology of all the rats were recorded after 3, 6, 12, and 24 weeks. In addition, the weight gain, food and water intake, urine flow and protein excretion, and motor activity were measured weekly in the 24-week group. CNS function was assessed by measuring the motor activity. The hourly nocturnal and daily motor activities decreased with time for the 30- and 100-ppm rats when compared to the control rats. Renal injury was indicated by an increase in the concentration of protein in the urine with time for the 30- and 100-ppm rats when compared to the control rats. There was also slight and focal tubular necrosis in the 30- and 100-ppm rats by Week 24. The time- and dose-dependence of the concentration of the Cd in the intestine indicates that the suggested protective mechanism of intestinal metallothionein, where metallothionein sequesters dietary Cd in the mucosal cells and thus hinders the transfer of the metal to the systemic circulation, is quickly overloaded at concentrations of 10 ppm or less of Cd in the drinking water. The concentration of metallothionein and Cd in the kidney and liver increased with dose at all time intervals and increased with time at most doses. However, the rate of increase of the concentration of metallothionein and Cd in the liver and kidney was not the same. In the liver ratio of Cd to Cd-binding capacity of metallothionein reached a plateau with time, which may explain hepatic tolerance to Cd. On the other hand, in the kidney, were necrosis and dysfunction were observed, the ratio continued to increase with time.

Comparison of the effects of metals on cellular injury and lipid peroxidation in isolated rat hepatocytes

Various mechanisms, including increases in lipid peroxidation, have been proposed to account for metal-induced cellular injury. By comparing several metals in the same cell population, it is possible to determine whether a correlation exists between ability to produce cell injury and ability to alter parameters pertaining to a particular mechanism. Of particular interest in this study was the relation between metal-induced cytotoxicity and increases in lipid peroxidation. The effects of Cr, Mn, Zn, Ni, Pb, Se, V, Fe, Cd, Hg, Cu, at final concentrations of 1-1000 microM, on the viability of isolated hepatocytes were therefore examined by assessing the loss of intracellular K+ and aspartate aminotransferase (AST). Simultaneously, the ability of the metals to induce lipid peroxidation, as measured by an increase in thiobarbituric acid (TBA) reactants, was assessed. Hg and Cu required the lowest concentration to produce cellular injury, while Cd produced less dramatic changes in cell viability and Fe at 1000 microM produced only a small decrease in intracellular K+. The largest absolute increases in lipid peroxidation were found in the presence of V, followed by Fe and Hg, with Cd and Se causing the smallest increase in TBA reactants. These observations suggest that the lipid peroxidation associated with Cd and Hg is not necessarily responsible for the loss of cell viability induced by these two metals.

Inhibition of lipid peroxidation without prevention of cellular injury in isolated rat hepatocytes

Abstract The role of lipid peroxidation in tissue injury has been the subject of many investigations. Recently evidence has suggested that lipid peroxidation due to various chemicals is directly responsible for loss of hepatocyte viability. In the present study hepatocytes, isolated by a collagenase perfusion technique, were incubated in either a Tris buffer or Medium 199 with and without the antioxidant N,N′ -diphenyl- p -phenylenediamine (DPPD) (2.5 μ m ). In order to determine if there is a relationship between lipid peroxidation and cell injury, samples were taken over a 6-hr incubation period for estimation of concentration of thiobarbituric acid (TBA) reactants, diene conjugates of lipids, and loss of intracellular potassium ion (K + ) and aspartate aminotransferase (AST). The content of reduced glutathione (GSH) was also assessed. The same parameters were also investigated in cells, which were suspended in Medium 199, incubated with either sodium iodoacetamide (100 μ m ), diethyl maleate (4 m m ), or sodium vanadate (400 μ m ). The effects of DPPD (2.5 μ m ) on the responses induced by these chemicals were also studied. Medium 199 was found to be a superior incubation buffer with respect to the cellular viability characteristics, leakage of intracellular K + and AST. Furthermore, GSH was maintained at much higher concentrations and there was less evidence of lipid peroxidation when Medium 199 was used rather than Tris. However, in the presence of DPPD the increase in concentration of TBA reactants was inhibited but DPPD had no protective effect on the release of K + and AST or the decrease in GSH concentration. The three chemical treatments, sodium iodoacetamide, diethyl maleate, and sodium vanadate, each caused loss of cell viability, marked and rapid decrease in GSH, and increases in the estimates of lipid peroxidation. DPPD completely inhibited the lipid peroxidation due to sodium iodoacetamide, but only delayed and did not prevent the loss of K + and had no effect on the loss of GSH. Similarly, DPPD inhibited the lipid peroxidation due to diethyl maleate but did not cause a significant change in the decline of cell viability or alteration in the loss of GSH. The decrease in cell viability produced by vanadium was prevented by DPPD, although at 6 hr of incubation intracellular K + was significantly lower in hepatocytes incubated with DPPD plus vanadium than those with DPPD alone. Again DPPD inhibited the increase in lipid peroxidation but did not alter the loss of GSH with vanadium treatment. Overall the results of this study suggest that lipid peroxidation is not totally responsible for the loss of cellular viability associated with incubation in different mediums or in response to chemicals such as sodium iodoacetamide and diethyl maleate.

Cadmium uptake by isolated rat hepatocytes

Abstract Isolated hepatocytes have been employed to investigate the uptake of the heavy metal cadmium ( 109 Cd). A rapid initial phase of uptake was followed by a second slower phase, both of which exhibited a linear relationship between velocity of uptake and substrate concentration. The presence of potassium cyanide (1 m m ) and carbonyl cyanide- m -chlorophenyl hydrazone (2 μ m ) did not cause any change in the uptake of Cd (200 μ m ). This suggests that the uptake of Cd into isolated hepatocytes is not an active process. Incubation of 10 μ m Cd in the presence of zinc (50 and 130 μ m ) produced a decrease in the velocity of uptake of only the first phase. Pretreatment of rats with zinc, which elevates hepatic metallothionein, resulted in isolated hepatocytes which showed a greater uptake rate for the second phase when compared to hepatocytes isolated from control rats. The results therefore demonstrate that uptake of Cd by isolated rat hepatocytes is a biphasic response in which at least part of the first phase appears to be carrier mediated with no indication of involvement of a carrier in the second phase of uptake. This second phase may be related to binding of Cd to intracellular components, since elevation of hepatic metallothionein was found to increase the uptake velocity of only this phase.

Comparison of methods for estimating hepatic metallothionein in rats.

Male Sprague-Dawley rats were given a single oral dose of 109 CdCl 2 (25, 50, and 100 mg of Cd/kg) and the hepatic metallothionein (MT) was estimated by the amount of 109 Cd bound to MT with and without prior addition of excess 109 Cd and by the method of Piotrowski et al. (1973). The method of Piotrowski et al. (1973) was found to be rapid and reliable and gave results for MT similar to those obtained after saturation of hepatic MT with 109 Cd. However, employing 109 Cd bound to MT without prior saturation as an estimate of MT was not reliable.

Uptake of bile acids by isolated rat hepatocytes

Hepatic uptake of five common bile acids was examined in isolated rat hepatocytes. Taurocholic acid (TCA), glycocholic acid (GCA), cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) were studied. Uptake was extremely rapid and was linear for at least 45 sec for all bile acids tested at substrate concentrations from 1 to 400 micrometers. Both nonsaturable binding to the cell surface membrane and the initial rate of uptake (V0) of the dihydroxy bile acids (DCA and CDCA) were about ten times greater than those of the trihydroxy bile acids (TCA, GCA and CA) which correlates with the higher lipophilicity of the dihydroxy bile acids. Kinetic analysis demonstrated that uptake of these bile acids was due both to a saturable process and a linear process. The apparent diffusion constant (Dapp) of the unsaturable process for the dihydroxy bile acids was also ten times greater than that for the trihydroxy bile acids. After correction for the nonsaturable binding to the cell membrane and linear entry, the Km and Vmax for the uptake was determined. Conjugation with taurine decreased the Km of CA but not the Vmax, while glycine conjugation did not alter either parameter, suggesting that conjugation with taurine may increase its affinity for the transport system. The trihydroxy bile acids have a higher affinity but a lower transporting capacity for the saturable process than the dihydroxy bile acids. In vivo hepatic extraction appears to be more dependent on the affinity of the bile acid for the transport system than the capacity at which it can be transported.

Interaction of metal ions with cadmium-induced cellular toxicity.

Interactions between Cd and other metal ions are important from both nutritional and toxicological aspects. As Cd is toxic to isolated hepatocytes, these cells can be used to investigate the effects of other metals on Cd-induced cellular injury. Isolated hepatocytes were incubated at 37 degrees C with vehicle (saline); Cd (200 or 400 microM); or Cd plus Cr, Mn, Zn, Ni, Pb, Se, or Fe (200-1000 microM). Evidence of cellular injury was assessed by loss of intracellular K+ and aspartate aminotransferase from the hepatocytes. Effects on lipid peroxidation, as measured by concentration of the thiobarbituric acid reactants, were assessed. Uptake of 109Cd and interaction of the other metal ions with this accumulation were also quantitated. Cell injury due to Cd was consistently reduced by Cr, Mn, Zn, Pb, and Fe. Lipid peroxidation due to Cd was inhibited by Cr, Mn, and Zn. All the metals except Ni produced an increase in the amount of Cd accumulated by hepatocytes. There was no consistent relation between reduction of cellular toxicity and either inhibition of lipid peroxidation or uptake of Cd. These experiments show that (1) protective properties of some metals seen in vivo can be demonstrated at the cellular level and (2) protective effects of metals in general on Cd-induced cellular toxicity are not due to a decrease in either Cd uptake or lipid peroxidation.