The effect of repeated administration of several chelators on the distribution and excretion of cadmium

The effect of repeated administration of several chelators on the distribution and excretion of cadmium (Cd) was determined in male Swiss Webster mice. Radioisotopic Cd (¹⁰⁹Cd, 1 mg Cd/kg) was administered iv; 48 hr later (after maximal induction of hepatic metallothionein) daily chelation therapy was initiated. Mice received one of the following ip treatments for 5 days: saline, diethyldithiocarbamic acid (DDC), nitrilotriacetic acid (NTA), 2,3-dimercaptopropanol (BAL), d,l-penicillamine (PEN), 2,3-dimercaptosuccinic acid (DMSA), ethylenediaminetetraacetic acid (EDTA), or diethylenetriaminepentaacetic acid (DTPA). Mice were housed in metabolic cages, and urine and feces were collected daily for 7 days. After 5 days of chelation therapy (7 days after Cd), the mice were killed and various organs removed. DTPA, EDTA, DMSA, and BAL significantly increased the excretion of Cd into urine. DDC significantly increased the fecal elimination of Cd and altered tissue concentrations of Cd. The concentration of Cd in DDC-treated mice was increased in testes, muscle, and brain and decreased in kidney, spleen, and blood. The observed increase in urinary excretion of Cd when certain chelators were administered after the induction of hepatic metallothionein suggests that increasing the duration of chelation therapy may decrease the concentrations of Cd in tissues and hence, reduce the toxicity of the metal.     

Subcellular distribution of gold in rat renal cortex cells: Limitations of electron microscopy and subcellular fractionation techniques

Electron microscopic and subcellular fractionation studies of renal cortex tissue from male Sprague-Dawley rats given gold sodium thiomalate (16 mg Au/kg) were undertaken to investigate the subcellular distribution of gold in kidney tissue. Although gold was present in the nuclear fractions prepared by differential centrifugation, electron microscopic examination indicated extensive contamination by aurosomes, which pellet in the nuclear fraction due to their greater density. No gold could be detected in the nuclei of intact tissue preparations by energy dispersive X-ray analysis (EDA). This observation does not preclude the possible presence of dispersed gold at levels below the 500 μm limit of detection for EDA. Nuclei from control animals, which were free of contamination by lysosomes, took up gold(I) from an incubation medium to the extent of 2.0 μg Au/mg of nuclear protein, independent of the gold concentration. When the nuclei were lysed, the gold was associated with the membrane fraction.     

Identification of cobalt protoporphyrin IX formation in vivo following cobalt administration to rats

An electron paramagnetic resonance method for the identification and quantitation of cobalt protoporphyrin IX has been developed to provide definitive evidence for the formation of the cobalt chelate of protoporphyrin IX in vivo. Both authentic and enzymatically generated cobalt protoporphyrin exhibited a characteristic primary resonance at g = 2.32 at 100° Kelvin (K) in the reduced state. At low concentrations, signal averaging was found to improve considerably the signal to noise ratio and thereby allow for the measurement of low concentrations of cobalt protoporphyrin. It was possible to determine concentrations of cobalt protoporphyrin IX as low as 0.5 μM in biologic preparations, and the signal height of the e.p.r. resonance was linear with cobalt protoporphyrin concentrations up to at least 8.0 μM. Following the administration of cobaltous chloride to rats, cobalt protoporphyrin IX was demonstrated in livers at times when alterations in heme biosynthesis are known to occur. Cobalt protoporphyrin IX at the level of 4.8 nmoles/g liver was detected 120 min after the administration of cobaltous chloride at a dose of 60 mg/kg of body weight. This finding supports the concept that cobalt protoporphyrin IX is rapidly produced in vivo after the administration of cobaltous chloride and may be responsible for the observed inhibition of hepatic heme biosynthesis.     

The nature of the substrate-selective interaction between rat liver mitochondrial monoamine oxidase and oxygen

The activity of rat liver monoamine oxidase was increased in an uncompetitive manner with increasing concentrations of oxygen. However, the value of the Michaelis constant for oxygen, estimated from determinations of enzyme activity with 6 oxygen concentrations and 5–6 amine substrate concentrations, was dependent upon the amine substrate used to assay for activity. In an attempt to determine the nature of these differences, the value of the Michaelis constants for oxygen have been related to the k_cat (rate constant of the limiting step in the overall enzyme-catalysed reaction) values of the enzyme towards the different amine substrates. The results are consistent with the hypothesis that the two forms of monoamine oxidase in rat liver are not independent enzyme forms, but interact one with the other in some way.     

Effects of repeated administration of oxytetracycline on kidney, liver, and liver mixed-function oxidases in the rat.

Repeated intraperitoneal administration of oxytetracycline to male rats had minor effects at doses of 20 and 40 mg/kg for 14 days. The 100-mg/kg doses resulted in changes in microsomal O-dealkylation and epoxidation, animal growth and kidney histology. Rats showed a dose-related depression of O-dealkylating activity early in the dosing schedule followed by a return to control values, while the activity of the epoxidation reaction was depressed late in the dosing schedule at the high dose only. Animals on the high dose also showed evidence of renal disease and a loss of weight as compared to controls.     

Influence of tansy ragwort (Senecio jacobaea), dietary copper, and feed restriction on the subcellular distribution of minerals in rat liver

Young male rats were pair-fed diets containing 0 or 5% tansy ragwort (TR), with each diet containing 0 or 50 micrograms/g copper. Other animals given TR-free diets were also maintained as ad libitum controls. After 5 weeks of feeding these diets, the rats were killed for the determination of the trace metal contents of liver fractions. TR with no dietary copper supplement increased the calcium content of all hepatic fractions except nuclei and debris, but had no effect on the subcellular concentrations of copper, zinc, or iron. When 50 micrograms/g copper was included in the TR diet, the subcellular distribution of copper, iron, and calcium was altered. This alteration was shown by an increase (as compared to pair-fed controls given copper-supplemented diet with no TR) in the copper content of all fractions except cytosol, the iron content of all fractions, and the calcium content of mitochondria, microsomes, lysosomes, and cytosol. Feed restriction (pair-fed versus ad libitum controls) using diets with no additional copper produced an increase in the copper and calcium contents of mitochondria; the copper, zinc, and calcium contents of cytosol; and the calcium content of lysosomes. Addition of 50 micrograms/g copper to the diets abolished these effects of feed restriction. These findings suggest that the subcellular distribution of copper, zinc, iron, and calcium in rat liver is subject to perturbation by pyrrolizidine alkaloid-containing diet, dietary copper, and feed restriction.     

Effect of sulfur mustards on lysosomes from rat liver in vitro

Treatment of lysosomes from a heavy mitochondrial (3500g) rat liver fraction with either a bifunctional or a monofunctional sulfur mustard resulted in slight release of three marker enzymes. Aryl sulfatase appeared to be released at lower concentrations of alkylating agents than either β-glucuronidase or acid phosphatase. Since release was observed only at extremely high concentrations of sulfur mustard, direct lysosomal labilization probably is not involved in the cytotoxic action of sulfur mustards.     

Evaluation of liver weight changes following a single oral administration of carbon tetrachloride in rats

To evaluate liver weight changes resulting from the primary toxic effect of carbon tetrachloride (CCl4), male and female rats were assigned to a control group, CCl4-treated groups administered a single po dose of 100 microliters (males only) or 200 microliters CCl4/100 g body wt, or a "hold" group in which the body weight change of the CCl4-treated group was duplicated by imposing restrictions of movement and feeding. Five rats from each group were killed at 1, 2, 3, 4, and 6 days after treatment, and body weight, food intake, and liver weight were tabulated. It was found that presentation of liver weight data in absolute terms may lead to erroneous conclusions, since the absolute liver weights of the CCl4-treated males showed changes similar to those of the control group, as if there had been no effect of CCl4 on liver weight. Furthermore, the results for the hold group (males and females) showed that there was not a linear relationship between liver weight and body weight during the period of rapid decrease in body weight, suggesting that comparisons made in terms of relative organ weights do not necessarily take proper account of differences in body weight. From the profile of the percentage absolute liver weight increases (delta W) of the CCl4-treated group from the levels in the hold group, it was concluded that CCl4 does induce an increase in liver weight following a single dose, and the effect on liver weight reached a maximum at 24 to 48 hr. These results demonstrate the need for great care in evaluating the direct effect of chemicals on organ weight when rapid changes in body weight are also occurring.     

Changes in some rat hepatic microsomal components induced by prolonged administration of chloroquine

Alterations in microsomal drug metabolizing enzymes and phospholipids following prolonged exposure to chloroquine have been investigated. The levels of microsomal aminopyrine-N-demethylase, aniline hydroxylase and both microsomal and cytosolic glutathione-S-transferase are reduced in treated rats. Microsomal epoxide hydrolase is unaffected by the treatment. An increase in the cholesterol-phospholipid ratio and a decrease in the phosphocholine-sphingomyelin ratio occur. There is a general reduction in the total microsomal phospholipid level though the percentage content of sphingomyelin is higher in all cases. The reduction in microsomal phospholipid level is probably due to a reduced incorporation of acetate into microsomal phospholipids as shown in this study using radioactive sodium acetate.     

Pharmacodynamics of methyl mercury in the rainbow trout (Salmo gairdneri): tissue uptake, distribution and excretion

The tissue distribution, rate of uptake and concentration of ²⁰³Hg-labeled methylmercury was investigated in 20 different tissues/organs over a period of 100 days following a single intragastric dose of 0.5 mg Hg/kg body weight. Mercury content was analyzed by gamma scintillation spectrometry. After 1 hr, mercury concentration factors >0.1 were detected in the blood, heart, liver, spleen and kidney (a concentration factor (CF) of 1.0 equals mercury concentration in dose). Highest mercury concentrations (CF > 7.0) were observed in the blood (at 7 days) and spleen (at 14 days). After 100 days, the CF of the blood was >2.0 and the CF values of the spleen, kidney and liver were >1.0. Maximum CF values were reached in the skeletal muscle, brain and lens after 34, 56 and >90 days, respectively. Maximum values were reached in most other tissues/organs at approximately 7 days. Skeletal muscle appeared to function as a reservoir for methyl mercury and accumulated 50% of the dose from 34 to 100 days post administration. Methyl mercury accumulation in the brain was limited to 0.1% of the dose. The rate of mercury excretion appeared to be biphasic as a result of a slow elimination from the skeletal muscle relative to the other tissues/organs. Employing both the slow and fast rate, the half-retention time for methyl mercury in rainbow trout was estimated to be >200 days.     

Substrate-selective activation of rat liver mitochondrial mono amine oxidase by oxygen

The activation by oxygen of the activity of monoamine oxidase (MAO) in preparations of rat liver mitochondrial membrane vesicles has been studied. The increase of activity with the substrates 5-hydroxytryptamine, tyramine, β-phenethylamine and benzylamine were in all cases uncompetitive. However, the degree of activation depended upon the particular substrate used. An attempt has been made to explain these results by use of an assumption that MAO activity can be divided into two forms unrelated to MAO-A and MAO-B. These two forms, tentatively called MAO-1 and MAO-2, differ in their Michaelis constants for oxygen.     

The isolation of rat lung cells for the purpose of studying drug metabolism

A method was developed for the isolation of viable cells from rat lungs. The cells were a heterogeneous population, which maintained a high percentage viability for up to 3 hr on incubation at 37°. Reduced cofactor levels (NADH, NADPH) decreased on incubation, but ATP remained constant. The cells were active in the metabolism of the two xenobiotics studied. A model compound, 7-ethoxycoumarin, was O-deethylated and subsequently conjugated with glucuronic acid and sulphate, whilst a pharmaceutical agent, N-acetylcysteine, was de-acetylated.     

Effects of heavy metals on corticosteroid production in cultured rat adrenocortical cells

Application of 10(-4) M Zn, 10(-5) M Ba, 10(-6) M Se, 10(-6) M Cd, 10(-6) M Hg, and 10(-6) M Mn did not affect the adrenocorticotrophic hormone (ACTH)-induced steroid production in cultured adrenocortical cells. The application of 10(-5) M Pb significantly reduced the ACTH-induced steroid production in cultured cells. However, Pb did not reduce the steroidogenesis induced by dibutyryl cyclic AMP (Dbc-AMP), suggesting that the plasma membrane is the site where Pb interferes with steroid production. The morphological changes induced by the addition of ACTH or Dbc-AMP plus the test metals were similar to those induced by ACTH or Dbc-AMP alone.     

In vivo effect of methylmercury on protein synthesis in brain and liver of the rat

Rats were given daily sc injections of methylmercury chloride, 10 mg/kg for 7 consecutive days. The manifestation of the neurological syndrome in the rat and the accumulation of mercury in rat tissues resembled the observations of previous investigators. The incorporation in vitro of [¹⁴C]leucine into brain protein began to decrease during the latent period of intoxication and declined to 56% of the control values at the symptomatic period. The incorporation of [¹⁴C]leucine into liver protein was also inhibited to a similar extent at the symptomatic period following a remarkable stimulation at the early stage after the onset of administration of methylmercury. The impairment of protein synthesis in the brain and liver at the symptomatic period was confirmed by the incorporation in vivo of a ¹⁴C-labeled amino acid mixture into proteins of these tissues. The decrease in the [¹⁴C]leucine incorporation in the liver of poisoned rats was largely affected by nutritional deficiency due to decreased food intake, but that in the brain resulted from the direct effect of methylmercury on this tissue.     

The effects of the administration of fenoprofen or indomethacin to rat dams during late pregnancy,with special reference to the ductus arteriousus of the fetuses and neonates

Examination of a series of normal neonatal rats showed that the ductus arteriosus did not begin closing until approximately 16 to 20 min after birth and was usually completely closed by 85 to 98 min after birth. Pregnant rats were orally dosed twice daily for 1.5 to 5 days with 50 mg/kg of fenoprofen calcium, 1.6 mg/kg of indomethacin, or vehicle (1% methylcellulose), beginning 18 days after mating. The fenoprofen calcium treatment resulted in premature (in utero) closure of the ductus arteriosus in 4 to 10% of the offspring of dams treated for 3.5 to 5 days. The indomethacin treatment caused premature (in utero) ductal closure in 1 to 3% of the offspring after 4.5 to 5 days. Both treatments also caused a high incidence of maternal mortality after 4.5 to 5 days (38 to 50% for fenoprofen calcium and 13 to 25% for indomethacin) and increased the duration of gestation or blocked parturition. Treatment of rat dams with 4 mg of progesterone daily from 18 through 22 days after mating blocked parturition, but the ductus arteriosus was not closed in any of the fetuses that were retained in utero. This suggests that the in utero closure of the ductus found in fetuses of dams treated with fenoprofen calcium or indomethacin was not a consequence of retention of the fetuses in the uterus.     

An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells

A short-term in vitro assay for teratogens has been evaluated and shown to have a high predictability (greater than 90%). Cultured cells derived from midbrain (CNS) and limb buds (LB) of 34 to 36 somite rat embryos were exposed to 46 compounds (27 teratogens, 19 nonteratogens) in a blind trial. Rat liver post mitochondrial supernatant fraction plus cofactors were included in the cultures to provide metabolizing enzymes. Differentiation of neurons from CNS cells and chondrocytes from LB cells was measured after 5 days of culture. Inhibition of differentiation (assessed by reduction of number of foci) was the indicator of potential teratogenicity. Variation between experiments was limited. In repeat experiments with two direct-acting teratogens, aldrin and diphenylhyantoin, interexperiment variability was low (coefficient of variation; range 7 to 24%). Of 27 teratogens 25 (93%) and only 2 of 19 nonteratogens (11%) inhibited differentiation (CNS or LB). Inhibition of differentiation in one cell type alone was less predictive (CNS: 85%, LB: 82%).     

Effect of thioacetamide-induced hepatic necrosis on the regioselective metabolism of S-warfarin by rat liver mixed-function oxidase enzymes

The biotransformation of S-warfarin was examined using liver microsomes prepared from rats 6-96 hr after treatment with a necrotizing dose (5.6 mmoles/kg) of thioacetamide. Four catalytically distinct classes of enzyme activity were observed which declined in activity with different half-lives after thioacetamide intoxication. S-Warfarin 7-hydroxylase activity was destroyed with a half-life of 16.6 +/- 3.1 hr. 6-Hydroxylase activity was destroyed with a half-life of 25.3 +/- 3.0 hr. 4'-Hydroxylase activity was destroyed with a half-life of 34.6 +/- 4.8 hr, which paralleled the loss of total hepatic cytochrome P-450 with a half-life of 33.4 +/- 3.6 hr. Production of an unidentified metabolite was not affected by thioacetamide intoxication during the first 48 hr. The ratio of rates of product formation were used as an alternative method to test the homogeneity of distinct enzyme catalytic activities. The ratio of measured responses (e.g. chromatographic peak heights) was used directly to determine the product ratios, provided that the rate of formation of each product was directly proportional to the experimentally measured response for each product. The use of product response ratios to discriminate between catalytic activities was inherently more precise because calibration errors were eliminated. Differences in the rates of destruction of warfarin hydroxylases provided further evidence of the multiplicity of hepatic mixed-function oxidases and suggested topographical differences in their location within the liver lobule.     

Effects of a polybrominated biphenyl mixture in the rat and mouse. II. Lifetime study

This study was undertaken to characterize the long-term toxic and carcinogenic potential of a polybrominated biphenyl (PBB) mixture in rats and mice of both sexes. Fischer 344 rats and B6C3F1 mice were given 125 po doses of PBB over a 6-month period at 0 (control), 0.1, 0.3, 1.0, 3.0, and 10.0 mg/kg body weight/day (5 days/week) and observed for an additional 23 months for rats and 24 months for mice (lifetime observation). The treatments (0.3 mg/kg or higher dosages) shortened the survival time in male rats whereas no such effect was observed in treated females. There was also evidence of shortened survival time in mice treated with 10.0 mg/kg PBB. As observed by uv light, hepatic porphyrin markedly increased at the 6-month observation, then tended to decrease, primarily in mice, following cessation of exposure. Significantly higher incidences of atypical hepatocellular foci, neoplastic nodules, hepatocellular carcinomas, and cholangiocarcinomas were observed in exposed rats. The incidence of hepatocellular carcinoma was also increased in both male (95%) and female (88%) mice (highest dose level) compared with control male (48%) and female (0%) mice. The incidence of hepatic neoplasms appeared to be dose dependent in both species. Liver tumors were observed primarily in those groups of animals to which PBB was given in doses sufficient to induce readily observable hepatic toxicity. Under the conditions of this experiment, polybrominated biphenyl mixture (Firemaster FF-1) was carcinogenic for Fischer 344 rats and B6C3F1 mice of both sexes. Lesions included neoplastic nodules, hepatocellular carcinomas, and cholangiocarcinomas in rats and hepatocellular carcinomas in mice. Other manifestations of toxicity included porphyrogenic effects and hepatotoxicity. A significantly higher incidence of chronic progressive nephropathy was observed in male rats of the 1.0, 3.0, and 10.0 mg/kg dosage groups when compared with control males. Gastric ulcers and hyperplastic gastropathy of the glandular portion of the stomach were observed more frequently in male rats, primarily in the high dosage groups.