Tom S. Miya

A mechanism for cadmium- and zinc-induced tolerance to cadmium toxicity: Involvement of metallothionein☆☆☆

Abstract Mouse-derived metallothionein was found to consist of two distinct cadmium-binding proteins possessing similar absorption and metal-binding properties. A sensitive technique for analyzing proteins, pulse polarography, was successfully employed for the analysis of metallothionein. Mice exhibited tolerance to Cd toxicity after pretreatment with either Cd or Zn acetate. Cd doses of 0, 1.0, and 3.2 mg/kg, 48 hr prior to Cd challenge, resulted in LD50 values of 4.5, 6.0, and 8.2 mg/kg, respectively. The hepatic metallothionein content increased with the pretreatment dose of cadmium. The LD50 values for cadmium were 5.7 and 10.3 mg/kg, respectively, for the control animals and for animals given three doses of 6.5 mg/kg of Zn at 12, 30, and 48 hr prior to the Cd challenge. Zn pretreatment induced formation of a Zn-binding protein the molecular weight of which appears to be identical with that of Cd-induced metallothionein. Cd-induced liver metallothionein contained Cd and Zn, whereas metallothionein resulting from Zn injections contained only Zn. Cd or Zn pretreatment of mice challenged with 10–12 mg of Cd/kg 3 hr prior to sacrifice resulted in an in vivo displacement by Cd of Zn from both Cd- and Zn-metallothionein. These results suggest that tolerance to Cd seen in pretreated mice is a result of the subsequent in vivo displacement of Zn from, and the chelation of Cd to, metallothionein. 14C-amino acid incorporation into liver metallothionein was found to be higher 12 hr after the administration of Cd than after 48 hr. The total amount of metallothionein protein in the liver at 48 hr was twice that found a 12 hr, whereas metallothionein-bound Cd was increased only slightly.

Correlation of hepatic metallothionein concentrations with acute cadmium toxicity in the mouse

Abstract Dose-dependent induction of metallothionein by cadmium was measured by gel filtration of liver supernatants prepared from mice 48 hr following single doses of 0.2–2.0 mg of Cd/kg (ip). Hepatic metallothionein concentrations increased in proportion to the cadmium pretreatment dose. A dose-dependent tolerance to acute cadmium toxicity was simultaneously evidenced by elevated LD50 values in cadmium-pretreated mice. A positive correlation between dose-related increases in hepatic metallothionein and cadmium LD50 suggests a protective function for metallothionein. The data suggest that a threshold dose of cadmium must be exceeded in order to induce concentrations of metallothionein adequate to ameliorate acute cadmium toxicity.

Cadmium inhibition of hepatic drug metabolism in the rat

Pretreatment of rats and mice of either sex with a single 2.0 mg/kg dose of cadmium acetate (840 μg Cd ion) potentiates the action of subsequently administered hexobarbital. Three days after cadmium administration to the male rat the duration of response to hexobarbital, but not that to barbital was significantly prolonged. Plasma concentrations of hexobarbital at awakening were not different from controls, indicating that cadmium treatment had not altered central nervous system sensitivity to the barbiturate. Such treatment of male rats 3 days prior to sacrifice leads to a significant inhibition of the metabolism of aminopyrine, hexobarbital, p-nitroanisole and zoxazolamine in microsomal subfractions obtained from these animals; microsomal cytochrome P-450 is reduced to 50% of control values by the cadmium treatment. Inhibition of the metabolism of these substrates by cadmium was also achieved when the metal was added to isolated microsomes in concentrations ranging from 5 × 10−4 to 5 × 10−7 m.

Effects of cadmium on nucleic acid and protein synthesis in rat liver

In male rats, cadmium (0.5 or 2.5 mg Cd 2+ /kg, ip) distributes among the major hepatic organelles, with the cytoplasmic fraction showing the greatest accumulation. Cadmium (0.34, 1.70, or 3.41 mg Cd 2+ /kg, ip) administration showed a biphasic effect with time on RNA synthesis in vivo . Upon its addition to isolated hepatic nuclei cadmium inhibited RNA synthesis. Administration of cadmium (0.34 or 1.70 mg Cd 2+ /kg, ip) to partially hepatectomized rats, decreased DNA synthesis. It was suggested from (DNA) melting profile measurements that cadmium may interact directly with DNA. The incorporation of l -[ 14 C]phenylalanine by a microsomal preparation from rats pretreated with cadmium (0.34, 1.70, or 3.41 mg Cd 2+ /kg, ip) was increased relative to controls. When hepatic microsomes were preincubated to destroy mRNA, the polyuridylic acid-directed incorporation of l -[ 14 C]phenylalanine was significantly inhibited by the cadmium pretreatment. This activity was likewise inhibited when cadmium was added to preincubated microsomes from control animals. The mRNA directed incorporation of amino acid by hepatic microsomes from control animals showed a significant biphasic effect dependent upon the concentration of added cadmium. At the doses of cadmium used in this study histologic examination of liver indicated focal necrosis, hemorrhage, inflammation, and congestion in some treatment groups.

Distribution and metabolism of barbital-14C in tolerant and nontolerant rats

Abstract Tolerance to barbital, as evidenced by a significant decrease in sleeping time, was induced in rats by chronic barbital administration. In an effort to elucidate the mechanism of tolerance, barbital- 14 C distribution and metabolism were compared between tolerant and nontolerant rats. Levels of radioactivity in brain, plasma, and urine did not significantly differ between the two groups. This indicated that tolerance to barbital cannot be ascribed to decreased drug absorption, to increased excretion of the drug, or to a decreased permeability of the blood-brain barrier. Tolerant rats metabolize barbital in a manner qualitatively similar to that of nontolerant rats, as indicated by the recovery of the same three barbital metabolites from urine of either tolerant or control animals. The quantity of barbital metabolites excreted by the tolerant rats was not significantly higher than control values.

Metabolism and tolerance studies with chlordiazepoxide-2-14C in the rat

Abstract Male rats made tolerant to chlordiazepoxide exhibit increased rates of tissue disappearance and excretion of 14 C-labeled chlordiazepoxide. A cross-tolerance is evident to hexobarbital, pentobarbital, and meprobamate, but not to phenobarbital. Stimulation by chlordiazepoxide of ascorbic acid excretion, and the ability of dl -ethionine to block the drug-induced stimulation of liver weight, liver protein content, and hepatic N -demethylase activity, suggest that the mechanism of tolerance development involves stimulation of hepatic microsomal enzymes responsible for drug metabolism.

Kinetics of cadmium-induced hepatic and renal metallothionein synthesis in the mouse.

Abstract Rates of hepatic and renal metallothionein synthesis were estimated by measuring the incorporation of [ 3 H]cysteine into metallothionein prepared from mice at various times following a single intraperitioneal injection of cadmium acetate (2 mg of Cd/kg). Tissue metallothionein concentrations were measured indirectly as a function of the total cadmium-binding capacity of the isolated metallothionein. Maximal incorporation of [ 3 H]cysteine into hepatic metallothionein occurred 6–12 hr following cadmium exposure, while renal metallothionein synthesis was maximal after 3 hr. Incorporation of [ 3 H]cysteine into metallothionein as well as metallothionein concentrations was greater in the liver than in the kidney. It is concluded that the liver is the primary site of cadmium-induced metallothionein synthesis.

Cadmium: inability to induce hypertension in the rat.

Abstract Cadmium has been suggested as a potential etiologic agent in human hypertension and has been reported to induce hypertension in experimenta animals. Cadmium acetate was administered ip to Sprague-Dawley derived and hooded Long-Evans rats at several dose levels and under a variety of treatment schedules and conditions. Under none of these circumstances was hypertension produced. These findings indicate that cadmium does not act as a proximate hypertension-inducing agent under all experimental conditions.

STRESS MODIFICATION OF DRUG RESPONSE.

Rats stressed by unilateral hind-leg ligation show a significantly decreased pharmacological response to the effects of hexobarbital, meprobamate, and pentobarbital. This effect is dependent upon an intact pituitaryadrenal axis, since it is absent in both hypophysectomized and adrenalectomized animals. It can be simulated by intravenous administration of adrenocorticotrophic hormone or corticosterone. The effect of stress on drug response is not noted with barbital, a compound which is not metabolized; this effect is inhibited by treatment of the animal with a drug metabolism inhibitor such as SKF 525-A (diethylaminoethyl 2,2-diphenylpentanoate). Compounds which can stimulate adrenocorticotrophic hormone secretion or act directly on the adrenals to produce corticosteroids should thus be able to stimulate their own metabolism or that of other drugs.

Cadmium potentiation of drug response—Role of the liver☆

Abstract Three days after a single cadmium dose (2 mg/kg), the duration of response to subsequently administered hexobarbital or zoxazolamine was potentiated. Duration of sleep was prolonged (225 per cent) in cadmium-treated rats, but the awakening plasma hexobarbital levels were similar in both the cadmium-treated animals and saline-treated controls. Moreover, cadmium-treated animals exhibited significantly higher plasma hexoharbital levels when sacrificed prior to awakening at a time corresponding to the mean duration of sleep in control rats, thus suggesting that the cadmium effect may be mediated by a decline in the plasma disappearance of hexobarbital. Duration of hexobarbital sleep was not changed in cadmium-treated hepatectomized rats compared to hepatectomized control animals, indicating the necessity of an intact liver for the cadmium effect to be elicited. In addition, pretreatment with cadmium significantly inhibited metabolism of hexobarbital in vitro in the isolated perfused rat liver (65 per cent). After the cadmium treatment zoxazolamine-induced paralysis was prolonged (185 per cent), but the plasma drug levels measured upon recovery were significantly lower. Significantly higher plasma levels of zoxazolamine were found in cadmium-treated rats when they were sacrificed prior to recovery at a time corresponding to the mean duration of paralysis in control rats. These results thus infer that the cadmium interaction with zoxazolamine may involve both an alteration in drug disposition as well as a change in tissue responsiveness.