Biomedical subjects

B A Fowler

Publications and source records attributed to B A Fowler.

At least 73 records · Page 4Linked to original sources

PubMed1985

Effects of lead inclusion bodies on subcellular distribution of lead in rat kidney: the relationship to mitochondrial function.

The effect of Pb pretreatment on the subcellular binding of a tracer dose of 203Pb was studied in kidneys of rats in which intranuclear and cytoplasmic inclusion bodies had been induced by a single ip injection of Pb acetate (50 mg Pb/kg) 6 days earlier. Results of subcellular fractionation studies in rats injected iv with 203Pb 24 hr prior to sacrifice demonstrated that 203Pb activity was about 1.5 times higher in kidney homogenates and mitochondrial fractions of control compared to Pb-pretreated rats. Cytosolic 203Pb activity in control rats was 5 times higher than that in Pb-pretreated rats. In contrast, Pb pretreatment increased the 203Pb binding capacity to the nuclear and inclusion body fractions by 7 and 20 times, respectively, compared with controls. Pb pretreatment decreased total mitochondrial 203Pb binding but resulted in a higher proportion of 203Pb bound to the inner membrane and matrix fractions relative to the controls. After in vitro incubation of control renal mitochondria with 203Pb the binding to inner and outer membranes and matrix fractions increased with increasing concentration of unlabeled lead added to the incubation. Deposits on the inner membrane of isolated mitochondria from Pb-pretreated rats were observed by isotonic ammonium molybdate negative staining and these mitochondria also showed decreased respiratory control ratios (RCRs). Succinate-mediated respiration rates and membrane binding of the fluorescent probe ethidium bromide were not affected by Pb pretreatment. These data indicate that lead influences its own subcellular distribution in the kidney following Pb pretreatment as shown by the increase of nuclear and inclusion body binding and increase of mitochondrial inner membrane and matrix binding of lead. The mitochondrial inner membrane shows a preferential affinity for lead following in vivo treatment which can be correlated with impairment of a specific inner membrane function (depressed RCRs). Lead exposure did not alter the activity of the mitochondrial membranes to undergo energy linked conformational changes.

Animals↗

PubMed1985

Effects of lead on the heme biosynthetic pathway in rat kidney.

Exposure of rats to lead in drinking water at concentrations of 500 ppm and 1000 ppm for 3 and 6 months resulted in elevated blood lead levels, formation of kidney intranuclear inclusion bodies, and increased urinary excretion of uroporphyrin and coproporphyrin. The erythrocytic Zn-protoporphyrin was increased in the highest dose group. No significant effects on body weight gain or kidney weight were observed. Renal activity of delta-aminolevulinic acid synthetase (ALAS) was not significantly affected by lead treatment. The renal activity of delta-aminolevulinic acid dehydrase (ALAD) was moderately increased and ferrochelatase activity was significantly decreased. The relatively small effects of chronic lead exposure on renal heme biosynthesis suggests that intracellular complexation of lead with high-affinity renal lead binding proteins (PbBP) and formation of intranuclear inclusions in proximal tubule cells protects this highly sensitive pathway in kidney from lead inhibition in vivo. These data also suggest that the observed increases in urinary porphyrin excretion are primarily due to lead effects on the erythropoietic system.

5-Aminolevulinate Synthetase↗

PubMed1985

High-affinity lead binding proteins in rat kidney cytosol mediate cell-free nuclear translocation of lead.

The PbII binding characteristics of the previously reported (Oskarsson et al., 1982) PbII binding proteins of rat kidney cytosol were investigated further. Saturation and Scatchard analysis of 203Pb binding in whole cytosol and in 40% saturated ammonium sulfate precipitated fractions disclosed a class of relatively high-affinity sites with an apparent Kd of approximately 50 nM and binding capacities of approximately 41 and 9 pmol/mg of protein, respectively. Two 203Pb binding proteins with approximate molecular masses of 63K and 11.5K daltons and a high molecular weight component (greater than 200K) were isolated by Sepharose-6B column chromatography. The time course of association of 203Pb with cytosol and the 63K protein showed maximum binding at 18 hr which was stable up to 25 hr at 4 degrees C. The approximate half-time dissociation rate (T 1/2) of specifically bound 203Pb to the 63K protein was 100 min at 4 degrees C whereas the 11.5K protein showed little dissociation of specifically bound ligand at this temperature. Saturation analysis of the three isolated proteins disclosed low capacity, high-affinity sites with similar apparent Kd values to the cytosol assay. Sucrose density gradient analysis of kidney cytosol showed approximate sedimentation coefficients of 2S, 4.6S and 7S for the 11.5K, 63K and the high molecular weight proteins, respectively. Competitive binding studies with cytosol demonstrated displacement of 203Pb by PbII, CdII and ZnII ions but not CaII ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Mechanism of renal lead-binding protein reversal of delta-aminolevulinic acid dehydratase inhibition by lead.

The bioavailability of lead in kidney is mediated in part by binding to endogenous high-affinity cytosolic lead-binding proteins (PbBP), which are not detectable in liver. Addition of semipurified 11,500 dalton PbBP to liver delta-aminolevulinic acid dehydratase (ALAD) reaction mixtures reverses inhibition of this enzyme by lead and thus provides an explanation for the relative insensitivity of renal ALAD to lead inhibition in vivo and in vitro. This effect results in part from a marked increase in binding of 203Pb to the PbBP relative to control liver cytosol (no PbBP) as demonstrated by Sephadex G-150 gel filtration chromatography. Zinc is known to activate ALAD and is an endogenous component of the PbBP fraction (6 microM in reaction mixtures). Zinc activated hepatic and renal ALAD over a range of 1.5 to 50 microM and also reversed the IC50 lead-inhibited activity. Studies of zinc release and/or displacement from PbBP under ALAD assay conditions (37 degrees C, + glutathione, pH 6.8) were conducted utilizing Sephadex G-25 chromatography. Fifteen to twenty-five percent of the zinc in the PbBP fraction was released, and this value was not markedly influenced by addition of IC50 lead, temperature (4 degrees C) or absence of glutathione; however, zinc release was primarily dependent upon the pH of the reaction mixture. These data indicate that the PbBP fraction attenuates lead inhibition of ALAD in vitro both by chelating lead and apparently serving as a zinc donor for this enzyme under optimal conditions of the ALAD assay.

Animals↗

PubMed1984

Studies on the mechanisms of thallium-mediated inhibition of hepatic mixed function oxidase activity. Correlation with inhibition of NADPH-cytochrome c (P-450) reductase.

Thallium (TlCl3) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome c (P-450) reductase and microsomal mixed function oxidase activities within 2-4 hr following treatment. These changes occurred independently of apparent effects on microsomal heme or cytochrome P-450 content, both of which remained unchanged with respect to control levels despite transient inhibition of delta-aminolevulinic acid (ALA) synthetase and induction of heme oxygenase. These results are consistent with the recognized properties of thallium as both a flavoprotein antagonist and sulfhydryl inhibitor and differ uniquely from the action of other metals which impair mixed function oxidase activity through compromise of heme biosynthesis and heme depletion. The potential utility of thallium compounds in further evaluating the functional characteristics of NADPH-cytochrome c (P-450) reductase and its role in microsomal oxidative processes is suggested from these observations.

5-Aminolevulinate Synthetase↗

PubMed1984

Intracellular metabolism and effects of circulating cadmium-metallothionein in the kidney.

The mechanism of cadmium-metallothionein (CdMT)-mediated nephrotoxicity is being studied in rats using an acute dose regimen. Results of metabolism studies have shown that injected CdMT is rapidly degraded by the kidney with the release of Cd2+ into the cell cytoplasm. Ultrastructural studies indicate that an increase in the number of small lysosomes is the first measurable effect of CdMT in the kidney at 1 hr. This is followed by an increase in the number of small vesicles at 4 hr. It is proposed that these effects are the result of decreased primary lysosome formation and an inhibition of the fusion of pinocytotic vesicles with cell lysosomes by Cd. Functional alterations measured 8 hr after CdMT injection include an increase in urine volume and increased excretion of the low molecular weight protein, RNAase. Prior induction of renal MT by Zn pretreatment prevents the induction of polyuria and low molecular weight proteinuria by CdMT. These data provide further evidence that CdMT nephrotoxicity occurs as a result of Cd2+ toxicity within the cell.

Animals↗

PubMed1984

Regulation of lead inhibition of delta-aminolevulinic acid dehydratase by a low molecular weight, high affinity renal lead-binding protein.

The bioavailability of Pb in kidney is mediated in part by binding to high affinity cytosolic Pb-binding proteins (PbBP) of 11,500 (11.5K) and 63,000 (63K) daltons, which are not found in liver. Renal delta-aminolevulinic acid dehydratase (ALAD) is also markedly more resistant to Pb inhibition than hepatic ALAD in vivo. This study was undertaken to evaluate further the differences in sensitivity of renal and hepatic ALAD to Pb and to determine if inhibition of hepatic ALAD by Pb could be reversed by addition of partially purified PbBP from kidney to liver cytosol. Rat liver or kidney cytosol was incubated with Pb over a concentration range of 0.1 to 10 microM. Renal ALAD was 7.5 times more resistant to Pb inhibition than that in liver. Kidney cytosol and 203Pb were incubated before Sephadex G-75 or G-150 column chromatography to isolate the 11.5K and 63K PbBP, respectively. Inhibition of hepatic ALAD activity by Pb was partially reversed by a single addition of semipurified 11.5K PbBP in the presence of 0.1 to 0.4 microM Pb, but no protective effect was observed at higher concentrations of Pb. This effect was not observed with the 63K PbBP added at an equivalent high affinity binding capacity or bovine serum albumin added at an 8-fold higher total protein concentration. Reversal of Pb-induced inhibition of hepatic ALAD activity was dependent on the concentration of 11.5K PbBP in the reaction mixture. Kinetic analysis of either hepatic or renal ALAD activity at an IC50 concentration of Pb indicated a noncompetitive inhibition pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Lead accumulation by rat renal brush border membrane vesicles.

Pb++ accumulation by rat renal cortical brush border membrane vesicles was evaluated by in vitro incubation with rapid filtration technique. Pb++ uptake was time- and concentration-dependent, with apparent saturation of binding sites at 100 to 200 microM (5 sec initial rate experiments). Equilibrium binding studies (60-min incubation) showed that the ratio of bound Pb++ to free Pb++ was constant at 1.25 +/- 0.07 between 0.01 to 10 microM Pb, with decreasing bound to free ratios at higher concentrations. Osmotic experiments showed that Pb++ uptake was due primarily to membrane binding rather than intravesicular accumulation. Electrochemical gradients of NaCl, KCl or protons did not increase vesicle uptake of Pb++. Incubation of vesicles with a number of amino acids did not stimulate Pb++ uptake although two (cysteine and glutathione) and the chelators EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) completely blocked this process. Competition studies with a number of other metals (at 10 microM and 1 mM) showed that only Sn++ or Sn +, La , Fe++ or Fe and Cu++ produced significant reductions in Pb++ uptake whereas Mg++, Ca++, Zn++, Cd++ and Hg++ were without effect on this process. Release of 203Pb from preloaded vesicles was accelerated in the presence of either cysteine or Sn +. Prior in vivo exposure to Pb (3 mg of Pb/kg i.v.) reduced Pb uptake to 70% of that of vesicles prepared from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Cadmium-Metallothionein nephropathy: relationships between ultrastructural/biochemical alterations and intracellular cadmium binding.

Cadmium metallothionein (CdMT) nephrotoxicity was studied in rats injected i.p. with a single nonlethal dose of CdMT (0.6 mg of Cd per kg). Within 8 hr of CdMT injection, urine volume and urine sodium excretion were increased and sodium dodecyl sulfate gel electrophoresis of urine proteins showed that elevated levels of low molecular weight proteins were present in the urines of CdMT-treated rats. Urine RNAase activity was also elevated, approximately 7-fold, by CdMT but not by zinc metallothionein (ZnMT) or lysozyme at equivalent protein doses, demonstrating that a proteinuria indicative of proximal tubule cell dysfunction develops as an early response to CdMT exposure. Ultrastructural alterations were also present in animals injected with CdMT but not ZnMT or lysozyme. The earliest alterations occurred in the lysosome compartment of the cell. By 1 hr, the number of small lysosomes in renal proximal convoluted tubule cells increased significantly with no changes in other organelle compartments. By 4 and 8 hr, there was a further increase in lysosome number with a concomitant decrease in size and a marked increase in the number of small clear apical vacuoles. Lysosomal cathepsin D activity was decreased at 4 and 8 hr after CdMT injection, and in vitro studies indicated that this effect was not due to a direct inhibition of the enzyme by Cd++ or CdMT. Thus, both lysosome size and protease activity were rapidly altered by CdMT exposure. Studies of Cd binding in the kidney suggest that non-MT-bound Cd is an important factor in CdMT-associated toxicity. Approximately 97% of the Cd present in the cytoplasm at 1 hr was non-MT-bound. Prior induction of renal MT by treatment with zinc (20 mg of Zn per kg as ZnSO4, i.p. 16 hr before CdMT injection) markedly reduced non-MT binding of Cd++ in kidneys of treated animals and inhibited the alterations in urine volume and low molecular weight protein reabsorption induced by CdMT. These data suggest that acute CdMT exposure provides an excellent system for studying the mechanism of cadmium tubular proteinuria and that the intracellular renal MT pool plays a key role in regulating this process.

Animals↗

PubMed1983

Role of ultrastructural techniques in understanding mechanisms of metal-induced nephrotoxicity.

The combined use of ultrastructural morphometry and X-ray microanalysis in conjunction with biochemical analysis is one approach to elucidating mechanisms of metal nephrotoxicity at the cellular level. Ultrastructural morphometry conducted on proximal tubule cells of rats exposed to low levels of methyl mercury for prolonged periods of time showed statistically significant increases in the volume densities of the lysosomal and mitochondrial compartments. These findings were associated with marked changes in lysosomal marker enzymes and mitochondrial heme biosynthesis enzymes leading to the detection of a renal porphyrinuria that occurred before changes in standard tests of renal function. Ultrastructural morphometry, X-ray microanalysis, and biochemical studies of the low-molecular-weight tubular proteinuria produced by injection of cadmium metallothionein (CdMT) showed a rapid proximal tubule cell lysosome uptake and degradation of the CdMT complex, which led to a subsequent decrease in the numerical density (Nv) and average diameter of lysosomes and to an increase in the Nv of apical pinocytolic vesicles with time. The data indicate disruption of the normal primary lysosome-pinocytolic vesicle fusion process and related development of tubular proteinuria. Ultrastructural techniques may provide information useful in elucidating mechanisms of ongoing metal-induced nephrotoxic processes when consideration is given to sampling strategies for morphometric analysis and the inherent detection limits, elemental volatility, translocation effects, and limitations of quantification for X-ray microanalysis in soft biological tissues.

Animals↗

PubMed1983

Alteration of hepatic microsomal structure and function by indium chloride. Ultrastructural, morphometric, and biochemical studies.

The effects of indium-chloride (InCl3) on hepatocyte structure and function were studied in male rats injected with doses of 0, 10, 20, or 40 mg of InCl3/kg and killed after 16 hours. Fragmentation and degranulation of the rough endoplasmic reticulum and increased numbers of In- and Fe-containing autophagic lysosomes were the most marked cellular changes observed by electron microscopy. Morphometric analyses of hepatocytes disclosed a maximal 4-fold increase in the volume density of the lysosome compartment and a 2-fold decrease in the volume density of the vacuole compartment. Surface densities of the mitochondrial cristae and rough endoplasmic reticulum were increased by 1.5-fold, whereas the surface densities of the smooth endoplasmic reticulum showed a maximal increase of 7-fold. These structural changes were associated with inhibition of microsomal aniline hydroxylase by as much as 50% and ethoxyresorufin-O-deethylase by as much as 30% but no change in aminopyrine demethylase activity. Microsomal acid phosphatase activity was also decreased to 74% of control, whereas beta-glucuronidase was unchanged. Mild inhibition of mitochondrial respiratory function but no changes in marker enzyme activities were noted. Lysosomal marker enzyme activities were also unaffected, with the exception of acid phosphatase, which was maximally decreased to 55% of control. The data indicate that acute InCl3 injection produces a primary effect on hepatocyte endoplasmic reticulum structure with attendant changes in both heme- and nonheme-dependent biochemical functions. These findings suggest that altered regulation of hepatic microsomal heme metabolism by indium and other metals occurs as part of a general process involving degradative changes in the endoplasmic reticulum structure due to membrane damage with subsequent lysosomal autophagy of nonfunctional components.

Animals↗

PubMed1982

Production of low molecular weight cadmium-binding proteins in rabbit lung following exposure to cadmium chloride.

Low molecular weight cadmium-binding proteins were studed in lung tissue from rabbits exposed to aerosols of CdCl2. Lungs obtained from animals exposed by inhalation to aerosols of 800 or 1600 micrograms/m3 CdCl2 for 2-hr periods/day, every other day for a 5-day period, were found to contain at least three low molecular weight cadmium-binding proteins, two of which were similar electrophoretically and spectrally to rabbit liver metallothionein. The third protein(s), which accounted for the majority of the cadmium in the soluble fraction of the tissue, did not bind to an anionic exchange gel and did not appear to be a polymerized form of metallothionein. Translocation studies of lung cadmium suggest a long half-life for cadmium in lung tissue following inhalation exposure, due perhaps to the high affinity of cadmium for specific lung cadmium-binding proteins. A small but significant redistribution of lung cadmium did occur to both kidney and liver tissue with time.

Aerosols↗

PubMed1982