Effect of various amounts of selenium on the metabolism of mercuric chloride in mice

Male ddY mice were given one injection of (1) mercury (mercuric chloride) simultaneously with various doses of selenium (sodium selenite), (2) mercury alone, or (3) various doses of selenium alone. The interaction between mercury and selenium in the liver and kidneys at 1, 5, 24, 120, and 240 hr after administration was investigated. The concentrations of mercury in the liver of mice receiving mercury and selenium simultaneously were higher than those after administration of mercury alone, while the concentrations of mercury in the kidney decreased markedly over a 1-120 hr period after administration, depending on the dose of selenium administered simultaneously with mercury. Clearly, selenium had a different effect on the accumulation of mercury in the liver and kidneys. Subcellular distribution studies revealed that mercury and selenium which were administered simultaneously were incorporated into the crude nuclear and mitochondrial fractions as stable complexes. The transport of these complexes to the kidneys seems to be limited. In addition, gel filtration of supernatant fractions of liver and kidney through a Sephadex G-75 column indicated that the proportion of mercury bound to metallothionein fraction decreased depending on the dose of selenium administered simultaneously with the mercury. This reduction was attributed to the decreased synthesis of mercury-thionein due to a reduction in the activity of Hg2+ which results from binding between mercury and selenium in the cells.     

Unilateral nephrectomy in the rat: effects on mercury handling and renal cortical subcellular distribution

As unilateral nephrectomy (UNX) is associated with enhanced mercuric chloride nephrotoxicity, studies were undertaken to characterize the effects of UNX on the tissue content, urinary excretion, and renal cortical subcellular distribution of mercury in the rat. Animals were studied immediately, 2 days or 14 days following UNX, during separate phases of compensatory renal hypertrophy. As compared to sham surgery controls, mercury content in renal cortex was higher in all UNX groups at 24 hr following injection and in the immediate and 2-day groups at 1 or 3 hr. However, UNX was not associated with any alteration in mercury content within outer or inner medulla, liver, plasma, or red blood cells. Subcellular distribution studies demonstrated that cytosolic mercury was uniformly elevated in all UNX groups at 1, 3, and 24 hr following injection while mercury bound to "metallothionein-like" proteins or free in the cytosol was increased only at 1 or 3 hr. Nuclear, mitochondrial, or microsomal mercury content was elevated in the animals studied immediately or 14 days after UNX at 3 or 24 hr following injection, while animals studied 2 days after UNX demonstrated a nearly uniform increase at 1, 3, and 24 hr. Single-kidney urinary mercury excretion was elevated in all UNX groups while excretion per gram kidney weight was increased only in the animals studied immediately or 2 days after surgery. These studies suggest that all phases of compensatory renal hypertrophy are associated with an enhanced content of mercury within the cell cytoplasm and in critical cellular organelles, which may explain the enhanced nephrotoxicity seen following UNX.     

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.     

Subcellular distribution of metformin in rat liver.

The subcellular localization of metformin was studied in livers of 18 h fasted rats treated orally with [14C]metformin 20 microCi kg-1, 50 mg kg-1. Sequential determination of 14C radioactivity showed that maximum concentrations of metformin in plasma (about 15 mumol L-1) and liver (about 50 mumol kg-1) were achieved at 30-60 min and approximately half-maximal concentration were achieved at 4 h. At 30 min and 4 h after administration of [14C]metformin, livers were removed and homogenized. Nuclear, mitochondrial and lysosomal, mixed membrane, and cytosolic fractions were separated by ultra-centrifugation. Distribution of 14C was similar at both time points, being greatest in the cytosolic fraction (78% of total radioactivity, and 60-69% of relative specific activity). Small amounts of 14C were associated with the other fractions. The total radioactivity and relative specific activity were respectively 2-3% and 7-8% in the nuclear fraction, 9-10% and 7-10% in the mitochondrial and lysosomal fraction, and 8-9% and 13-14% in the mixed membrane fraction. The higher concentration of metformin in liver compared with plasma suggests that metformin enters hepatocytes via a specific mechanism, and is distributed mainly within the cytosol.     

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 role of lysosomes in the hepatic accumulation and release of beryllium

The toxic metal beryllium (Be) can produce hepatic necrosis and is known to be concentrated by the liver after i.v. injection of particulate and soluble Be compounds. In the present study the accumulation and release of Be by a hepatic lysosome fraction of the rat has been examined after i.v. administration of sublethal doses of particulate Be phosphate (12.5 and 150 μmoles/kg) or the more hepatotoxic soluble BeSO₄ (12.5 and 25 μmoles/kg). Maximal lysosomal Be content is produced within 2 hr or 5 hr after the injection of Be phosphate or BeSO₄ respectively and in both cases this is followed by a gradual decrease in the lysosomal Be concentration of the next 7 days to 30–40 per cent of the maximal values, which is consistent with the loss of Be observed for whole liver. The release of Be from lysosomes has been examined by measurement in vitro of the liberation of Be into a suitable incubation medium from lysosomes prepared from Be treated animals. The results indicate that the liberation of Be was maximal for lysosomes isolated 5 hr after injection of either form of Be and was accompanied by a significant release of lysosomal β-glucuronidase activity, particularly at the higher doses of Be used, and therefore indicates that some loss of lysosomal integrity occurred. Rupture of the lysosomal membrane could not be demonstrated in vitro by incubation of lysosomes from untreated animals with externally added Be compounds. It is concluded, however, that any release of hydrolytic enzymes into the cytosol occurring in vivo may not be the major cause of cell necrosis produced by Be and it is suggested, therefore, that the role of lysosomes in Be hepatotoxicity is primarily in the intracellular accumulation and subsequent release of Be and that the main cytotoxic target for Be is probably extralysosomal.     

Enhanced accumulation of injected inorganic mercury in renal outer medulla after unilateral nephrectomy

The effects of unilateral nephrectomy on renal accumulation and intrarenal distribution of mercury following a single injection of mercuric chloride (HgCl2, 0.5 mumol Hg/kg body wt, ip) were evaluated in the rat. In rats injected with HgCl2 immediately after nephrectomy or 10, 28, or 43 days after nephrectomy, the accumulation of mercury in the renal outer medulla was significantly greater than in respective sham-operated control rats. The increased accumulation of mercury in renal outer medulla was evident at 24 hr after injection of HgCl2 and persisted for at least 72 hr. The effect appeared to be a phenomenon associated specifically with the renal outer medulla in that elevated concentrations of mercury in the outer medulla were observed in the absence of similar elevations in the renal cortex, inner medulla, liver, and blood or in the whole body mercury content. Urinary excretion of mercury was unaffected by nephrectomy. Thus, associated with unilateral nephrectomy are changes in the renal accumulation and intrarenal distribution of systemically administered inorganic mercury that persist long after the rapid phase of compensatory renal growth (0-7 days) is completed.     

Methyl mercury and selenium interaction in relation to mouse kidney gamma-glutamyltranspeptidase, ultrastructure, and function

The effects of methyl mercury (CH3Hg) and selenium (Se) on renal ultrastructure were investigated and correlated to changes in renal gamma-glutamyl transpeptidase (gamma-GTPase) activity, mercury (Hg) accumulation, and renal function (serum creatinine and urea nitrogen). Three experimental protocols were used to investigate CH3Hg and Se interactions of both Se-sufficient and Se-deficient mice involving ip injection of the following administered alone or in combination: CH3Hg (4.0 mg/kg) and Se (0.16 mg/kg) daily for 7 days, CH3Hg (1.0 mg/kg) and Se (0.08 mg/kg) daily for 20 days, and a single acute dose of CH3Hg (8.0 mg/kg). Acivicin (12 to 50 mg/kg), an antitumor glutamine antagonist, was also used as a highly effective specific inhibitor of the gamma-GTPase. Our results show that CH3Hg administered to Se-deficient mice for 7 or 20 days resulted in significant (p less than or equal to 0.05) but only moderate inhibition (20%) of gamma-GTPase activity and extensive renal ultrastructural damage. Acivicin-treated mice had significant inhibition of gamma-GTPase activity (80%) following a single injection while ultrastructural damage was substantial only after several days of administration. These results may indicate different modes of action of acivicin and CH3Hg. Acivicin inhibited gamma-GTPase prior to renal damage while CH3Hg produced greater pathological effects with only moderate gamma-GTPase inhibition. Renal damage from acute and chronic CH3Hg toxicity occurred after distinct neurological signs were present. Selenium administered to Se-deficient mice ameliorated both the neurotoxic effects and nephrotoxic action of CH3Hg. While Se and CH3Hg treatments caused some of the same ultrastructural pathology as the treatment with CH3Hg alone (cytoplasmic vacuolation, increased lysosomal profile, mitochondrial swelling, and extrusion of cellular masses into the tubular lumen), degeneration was not as extensive. Although the total doses administered during both the 7- and the 20-day studies were similar, mice from the chronic 20-day study showed greater ultrastructural pathological effects from CH3Hg. The primary effects of CH3Hg appeared to be on the lysosomal system, while acivicin exerted its effects on the mitochondrial and endoplasmic reticulum systems. The accumulation studies on Hg suggest that dietary Se may have only an initial protective effect against Hg accumulation in the kidney while injected Se offers longer protection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of mitochondrial neutral protease activity and the response of lysosomal enzymes to clofibrate feeding in rat liver.

The phosphate-inhibitable neutral protease activity of the heavy mitochondrial fraction of rat liver is of lysosomal origin. The activity is essentially due to the thiol proteinases of the lysosomes. Digitonin treatment of the mitochondrial fraction results in the release of about 85 per cent of the neutral protease activity and the residual activity has an alkaline pH optimum and is not inhibited by phosphate. Clofibrate feeding at 0.5 per cent level in the diet results in enhanced levels of lysosomal enzymes. The increase is however restricted to the lysosome-rich fraction such that the activities associated with the heavy mitochondrial fraction show a significant decrease. It is suggested that clofibrate inhibits engulfment of mitochondria by lysosomes and this results in enhanced mitochondrial protein content.     

The Importance of Organ Blood Mercury when Comparing Foetal and Maternal Rat Organ Distribution of Mercury after Methyl Mercury Exposure

Abstract Foetal rat brain has previously been shown to contain twice as much mercury as maternal brain, after methyl mercury injection in the mother rat. However when brain mercury is corrected for the mercury which is present in the blood of the brain, foetal rat brain will contain 4 to 5 times as much mercury as maternal brain (depending on the stage of gestation), 24 hours after methyl mercuric chloride injection in the mother. Even when methyl mercuric chloride was injected in the mother about 14 days before term, near-term foetal brain contains 1.4 times as much mercury as the maternal brain. Likewise when corrected for mercury in the blood of the organ, foetal rat liver contains from 2.0 to 2.6 times more mercury than the maternal liver, and the foetal kidney contains from 13 to 23 times less mercury than the maternal kidney. The amount of mercury in foetal blood is about 65 % of the mercury in maternal blood 24 hours after methyl mercuric chloride injection in the mother, but maternal and foetal blood contain equal amounts 14 days after the injection. Except for the foetal membranes, no inorganic mercury released by biotransformation of methyl mercuric chloride was detected in the foetal-placental unit.     

Comparison of organic and inorganic mercury distribution in suckling rat

Thiomersal is used as a preservative in vaccines given to small children. The metabolic product of thiomersal is ethylmercury and its distribution and kinetics are still not known, especially at this early age. The purpose of this study was to compare the body distribution of two forms of mercury: organic (thiomersal) and inorganic (mercury(2+) chloride) in very young, suckling rats. Mercury was applied subcutaneously three times during the suckling period on days 7, 9 and 11 of pups age, imitating the vaccination of infants. A single dose of mercury was equimolar in both exposed groups, i.e. 0.81 micromol Hg kg(-1). At 14 days of age the animals were killed and the total mercury analysed in blood and organs (kidney, liver and brain). The analytical method applied was total decomposition, amalgamation, atomic absorption spectrometry. The results showed that the level of mercury was higher in the liver and kidney of the inorganic mercury group than in the thiomersal exposed group. However, the brain and blood concentrations of mercury were higher in the thiomersal exposed group. These results need to be clarified by additional data on the kinetic pathways of ethylmercury compared with inorganic mercury.     

Partitioning of zinc and copper in fetal liver subfractions: Appearance of metallothionein-like proteins during development

The appearance and subsequent accumulation of cytosolic metallothionein (MT)-like proteins in developing fetal rat liver begin as early as 14 days postfertilization (P.F.). Fetal MT-like proteins increase steadily; while in contrast, maternal MT-like protein levels remain fairly constant throughout pregnancy. The accumulation of these proteins seems to be related to the differential partitioning of zinc and copper in several cellular subfractions: zinc accumulates in the fetal hepatic cytosol, while copper specifically localizes in the particulate fraction. We report an approximate 10-fold increase in the percentage of cytosolic zinc bound to MT-like proteins between 15 and 17 days (P.F.). Furthermore, from Day 17 (P.F.) until term, the percentage zinc associated with MT-like proteins remains relatively constant, with a slight increase from 21 to 23 days (P.F.). We are suggesting that this initial partitioning of zinc within the MT fraction may be one of the biochemical signals that elicits the shift from proliferation to differentiation during hepatic development in the rat.     

Effects of carbon monoxide exposure on the distribution of methylmercury in mice

Effects of long-term exposure to sublethal concentration (300-350 ppm) of carbon monoxide (CO) on the distribution of methylmercury (MeHg) in the blood and organs of mice were examined using 6th week-old ICR mice of both sexes. Firstly, female mice were exposed to CO immediately after single ip injection of CH3HgCl (1 mg/kg). At the earliest stage, brain mercury level was higher in CO mice than in control mice, while blood mercury level was lower in CO mice than in control mice. There were indications that compensatory hemoconcentration and resultant increase of mercury levels in the blood, brain and liver occurred in CO mice by the 8th day of CO exposure. Mercury in the blood, brain, liver and kidney decreased more rapidly in CO mice than in control mice for a short period after hemoconcentration had occurred. Secondly, male mice were pre-exposed to CO for 7 days, received single ip injection of CH3HgCl (1 mg/kg) and were re-exposed to CO for an additional 21 days. Hemoconcentration, increased mercury levels in the blood, brain and liver were observed in CO mice. Thirdly, male mice were pre-exposed to CO for 7 days, administered po with CH3HgCl (2 mg/kg) and re-exposed to CO for 24 hr. Mercury levels in the blood, brain and liver but not the kidneys were higher in CO mice than in control mice. The relationships between hemoconcentration and MeHg distribution in vertebrates were discussed.     

Distribution of mercury in metallothionein-null mice after exposure to mercury vapor: amount of metallothionein isoform does not affect accumulation of mercury in the brain

To examine the contribution of metallothionein (MT) to mercury accumulation in mouse tissues, 129 strain female mice and MT null mice were exposed to metallic mercury vapor at a sub-toxic level, and Hg levels in the brain, kidney and liver were determined on 1, 3 and 7 days after the exposure. After exposure to mercury vapor, significant Hg accumulation was observed in the brains of wild-type and MT-I/II null and MT-III null mice, as well as in the liver and kidneys. No strain difference was observed in the tissue Hg accumulations 24 hr after the exposure except for the kidneys, where the highest accumulation was found in MT-III null mice. Although the brains of MT-III null mice showed slightly higher Hg accumulation than the other two strains, no significant difference was observed except in the cerebrum on Day 7. Gel chromatograms of cerebrum soluble fractions revealed that a significant amount of Hg existed as an MT-bound form in all the mouse strains. On the other hand, MT-bound Hg was found as a minor fraction in soluble fractions of the kidneys and livers in wild-type and MT-III null mice. Despite a significant strain difference in total MT levels in the cerebrum, there was no difference among the three strains in the amount of Hg accumulated in the cerebrum and its distribution rates in MT fractions. The present study demonstrated that brain uptake of Hg⁰ and its accumulation as Hg²⁺ did not depend on the amount of MT isoform in the tissue, at least in the early phase.     

Defluorination of fluoroacetate in vitro by rat liver subcellular fractions.

An earlier report showing enhanced accumulation of ionic fluoride in bones of rats given fluoroacetate (FAc) suggested an in vivo defluorination of fluoroacetate. Rat liver an organ which shows minimal pathological and biochemical effects in FAc intoxication was found to possess defluorination activity in vitro. Subcellular fractionation of livers from male Sprague-Dawley rats was performed in 0.25 m sucrose and yielded the following fractions: whole homogenate, nuclear, mitochondrial, microsomal and 105,000 g supernatant. Defluorination activity was measured by incubating subcellular fractions and their boiled controls with FAc (1 hr, 37°C, pH 7.4, in 0.1 m Tris-HCl or 0.1% Triton X-100) and comparing the difference in ionic fluoride content at the end of the incubation. Defluorination activity based on protein content was consistently the highest in the 105,000 g supernatant fraction, the only fraction showing an activity increase over the original homogenate. The microsomal fraction had minimal activity. Defluorination activity in the 105,000 g supernatant as a function of time after fraction isolation at 4°C revealed a time-dependent reduction in activity. After 24 hr at 4°C, activity was almost completely lost. The time-dependent loss of activity at 4°C could be regained when glutathione (GSH) was added to this fraction at a final concentration of 5 mm. Furthermore, GSH significantly increased the defluorination activity in all subcellular fractions. A study of the optimum pH for defluorination activity in the 105,000 g supernatant fraction was performed in 0.1 m Tris-maleate buffer. Surprisingly, a complete loss of activity with this buffer resulted throughout the pH range studied (pH 5.6–8.0). Furthermore, defluorination activity of all subcellular rat liver fractions was completely inhibited by maleate and stimulated by glutathione. These results are consistent with the involvement of a sulfhydryl group in the defluorination of FAc in rat liver.     

Role of lycopene in recovery of radiation induced injury to mammalian cellular organelles

Whole body exposure of male rats to 7 Gy gamma irradiation increased lipid peroxidation in the liver resulting in biomembrane damage of subcellular structures and release of their enzymes. This is evidenced by increase of thiobarbituric acid-reactive substances (TBARS) in mitochondria, lysosomes and microsomes. This was associated with a decrease in activity of the enzymes specific for each subcellular fraction; namely, mitochondrial glutamate dehydrogenase (GDH), lysosomal beta-glucuronidase and microsomal glucose 6-phosphatase. This was paralleled by an increased activity of these enzymes in the cytosol. Rats were supplemented with lycopene, a carotenoid present in tomatoes (5 mg/kg weight/day), by gavage, for 7 days before exposure to 7 Gy gamma irradiation. This resulted in diminishing amount of TBARS recorded for each subcellular structure in the liver of irradiated animals. Significant amelioration in the decrease recorded for the activity of mitochondrial glutamate dehydrogenase, lysosomal beta-glucuronidase and microsomal glucose 6-phosphatase was observed. This was associated with significant amelioration in the increase recorded for the activity of these enzymes in the cytosol. It is postulated that lycopene could play an important role in the recovery of the integrity of biological membranes of the liver after radiation injury.     

Early loss of large genomic DNA in vivo with accumulation of Ca2+ in the nucleus during acetaminophen-induced liver injury

Hepatotoxic doses of acetaminophen cause early impairment of Ca2+ homeostasis in the liver. This in vivo study considers the nucleus as a possible site of lethal Ca2+ action by evaluating whether acetaminophen raises Ca2+ in this compartment, whether DNA becomes altered, and whether DNA changes occur early enough during injury to contribute causally to necrosis. Fed Swiss mice were treated with 600 mg/kg acetaminophen ip and livers and blood samples were collected over time. Total nuclear Ca2+ accumulation and fragmentation damage to DNA showed modest parallel increases between 2 and 6 hr, followed by greater than 200% rises at 12 hr mirroring the appearance of frank liver injury (ALT greater than 10,000 U/liter). However, agarose gel electrophoresis revealed extensive loss of large genomic DNA from 2 hr onward, accompanied by the appearance of periodic DNA fragments. Thus, acetaminophen raised nuclear Ca2+ concentrations and promoted DNA fragmentation in vivo. The considerable cleavage of DNA seen at late times probably resulted from cell death, whereas loss of large genomic DNA from 2 hr onward appeared at an early enough point in time to be a contributing factor in acetaminophen-induced liver necrosis.     

Formation of a toxic metabolite from gentamicin by a hepatic cytosolic fraction.

We have demonstrated recently that incubation of the aminoglycoside gentamicin with an hepatic post-mitochondrial fraction produces a compound toxic to sensory cells from the inner ear in short-term culture; in contrast, the parent aminoglycoside was non-toxic in vitro (Huang MY and Schacht J, Biochem Pharmacol 40: R11-R14, 1990). In the present study, we investigated the subcellular distribution of the enzymatic activity and the nature of the metabolite. Isolated outer hair cells from the guinea pig cochlea were used to assay for cytotoxicity. The enzyme(s) responsible for this novel reaction of aminoglycosides was exclusively localized to the cytosolic fraction of guinea pig liver. No activity was detected in nuclear, lysosomal/mitochondrial or microsomal preparations. Furthermore, the toxin-forming enzymatic activity was associated with the high molecular weight fraction of the cytosol and did not require low molecular weight components. Filtration of the toxin through molecular weight cut-off membranes showed a molecular size of approximately 500. This evidence is consistent with the toxin being a gentamicin derivative.