Connective tissue responses to some heavy metals. III. Silver and dietary supplements of ascorbic acid. Histology and ultrastructure.

Silver-loaded ion exchange resin beads implanted into loose connective tissue of the rat pinna produced a local reaction. Initially the lesion comprised local necrosis and tissue disruption with predominantly small round cell infiltration. The subsequent organization was delayed and disordered. Fibroblasts developed grossly dilated cisternae of the rough endoplasmic reticulum. The matrix contained poorly orientated collagen fibrils of varying size and ground substance appeared condensed and granular. Distorted collagen fibrils were identified within membrane-bound vacuoles in the cytoplasm of both fibroblasts and macrophages. Abnormalities of the silver lesion were indicative of disordered collagen biosynthesis. Silver interfered with the biosynthesis and assembly of matrix components of the connective tissue. The reaction to silver beads in rats maintained on a diet heavily supplemented with ascorbic acid approached that of the control (sodium-loaded bead) with respect to the time scale, tissue reaction and tissue organization. The collagen matrix which formed was more organized and of greater density than that in the rat maintained on a normal diet. However, the repair tissue retained some of the morphological features of the legacy of silver toxicity, in particular delayed repair and dense intracellular fibrils within fibroblasts and macrophages. The excess of ascorbic acid partially ameliorated the effect of silver, possibly by compensating catabolysis of ascorbic acid caused by the presence of the released silver.

Silver wire implant of 40 years' duration: influence on local tissues.

The retention of an implanted silver wire in human tissue 40 years after surgical fixation of the frontal bone was studied post mortem by histology, transmission electron microscopy, and energy-dispersive x-ray analysis. Corrosion products of the wire were associated with a chronic inflammatory response and were bound to certain connective tissue elements; they were deposited as discrete particles, comprising silver in association with sulphur, on collagen fibrils and vascular basement membranes. Bone structure appeared normal except close to the wire, where it was replaced by a loose connective tissue in which collagen bundles were disorganized.

Functional and morphologic effects of ioxilan, iohexol, and diatrizoate on endothelial cells.

The effects of a new contrast agent, ioxilan, on vascular endothelium were compared with those of iohexol and diatrizoate. Rabbit aortic rings were incubated in contrast medium (CM) (350 mgI/mL) or Krebs solution as a control agent, for 5 minutes. Scanning and transmission electron micrographs showed that iohexol and ioxilan produced irregularities in the cell borders and in some vesicles, whereas diatrizoate produced intercellular gaps and numerous vesicles containing myelin figures. The ability of the endothelial cells to release endothelium-derived relaxing factor was tested by measuring the dilator response to acetylcholine. Incubation of aortic rings in CM for 5 minutes caused no changes in responses. However, 15-minute contact with diatrizoate irreversibly reduced the dilator response to 49%, and contact with sucrose (2100 mOsm/kg) reduced it to 9%. After incubation for 60 minutes, iohexol reduced the dilator response to 43%, while ioxilan caused no change. Since the hydrophilicity of the nonionic compounds, ioxilan and iohexol, is similar, while ioxilan's osmolality is substantially lower, the endothelial changes detected by electron microscopy and induced by the CM are attributable to their chemical properties, whereas the loss of dilator response appears to be mediated by high osmolality.

Cationic radioisotope delivery to loose connective tissue in vivo using ion-exchange resin beads.

Ion-exchange resin beads, implanted into connective tissue, were used as the vehicle for the delivery of radiolabelled cations to determine the local distribution of lead, silver and cadmium in loose connective tissue. The system was devoid of systemic toxicity, permitted a predictable release, enabled location of released cations in respect to the site of bead implantation, afforded safety and could be used with several other cations. The radiolabels released into the tissue formed an immediate relationship with inflammatory cells and a more protracted relationship with the matrix elements of the local connective tissue. The lesion induced by the presence of the bead was complicated by the particular cation.

Connective tissue responses to some heavy metals. I. Sodium loaded ion exchange beads as a control: histology and ultrastructure.

Ion exchange resin beads (Amberlite IR-120) were implanted into loose connective tissue as the vehicle for cations in both control and experimental studies. Beads were loaded with sodium for control purposes or with the metal of interest for subsequent experimental studies. When single control beads were implanted in the loose connective tissue of the rat pinna, they were well accepted by tissue and permitted rapid healing. The bead implantation initially produced minimal inflammatory disruption and subsequent repair at the bead-tissue interface which rapidly matured leaving no fibro-collagenous capsule. Some minor geometric rearrangement occurred in the alignment of the connective tissue to compensate for the physical presence of the bead. This system provides a valid control for studies of the effects of metal cations on tissue inflammation and repair--free from anionic complications, surface phenomena or systemic interference.

Connective tissue responses to some heavy metals. II. Lead: histology and ultrastructure.

Lead loaded ion exchange resin beads implanted into the loose connective tissue of the rat pinna induced local lesions which differed widely from those of the control (sodium loaded) beads (Ellender & Ham 1987). These lesions were characterized by changes in the granulation tissue and the approximating connective tissue. Granulation tissue contained mononuclear phagocytes in various guises, and some cells with intranuclear inclusion bodies. The matrix of the granulation tissue contained collagen fibrils having a wide range of diameters suggestive of altered collagen biosynthesis. Foci of collagen mineralization occurred in zones of combined trauma and lead impregnation. Once mineralized they became enveloped by giant cells and epithelioid cells. Lead in damaged tissues is thought to modify the protective mechanism of calcification inhibition and the biosynthesis of the matrix.

Dialysis osteomalacia: a possible role for zirconium as well as aluminium.

Six patients with dialysis osteomalacia were studied before and after treatment with desferrioxamine. Before treatment, all six had severe osteomalacia with histochemical evidence of metals at the mineralization front, confirmed by energy dispersive X-ray microanalysis to include Al, Zr and Fe. Zr was not detected by histological staining in patients without dialysis osteomalacia. After treatment a decrease of Al and Zr was associated with improvement in clinical, biochemical, radiological and histological parameters. These observations suggest the possibility of a role for metals other than Al in the pathogenesis of dialysis osteomalacia.

Origin of glomerular crescents in rabbit nephrotoxic nephritis.

The origin of glomerular crescents was investigated in an accelerated model of rabbit nephrotoxic nephritis. In rabbits immunised against sheep gamma-globulin, the administration of sheep nephrotoxic serum provoked cellular crescent formation affecting 30 to 90 per cent. of glomeruli at 6 days. The crescents were composed predominantly of cells with ultrastructural features of macrophages. In animals depleted of circulating leukocytes by whole body irradiation, with or without shielding of the kidney, crescent formation was inhibited despite severe glomerular damage and fibrin deposition in Bowman's space. These findings support the hypothesis that glomerular crescents develop principally from the emigration and accumulation of bone marrow-derived mononuclear cells.

Macrophages and glomerular crescent formation. Studies with rat nephrotoxic nephritis.

A model of crescentic glomerulonephritis in the rat was developed, based upon an augmented form of nephrotoxic nephritis. The glomerular lesions were relatively mild, permitting an analysis of the morphologic events in crescent formation to an extent not possible in models in other species in which the sequence of changes is obscured by the severity of the inflammatory process. Monocytes and macrophages accumulated in glomerular capillary lumens and walls. Crescents were composed of cells with ultrastructural features indistinguishable from those of the intracapillary mononuclear cells. The findings support the view that the crescent cells are predominantly composed of macrophages derived from circulating monocytes.

Treatment of dialysis osteomalacia with desferrioxamine.

Two patients with severe dialysis osteomalacia, whose fractures and deformities had become progressively worse over 3 years, were treated with desferrioxamine (DFO). They improved rapidly and over 6 months were able to stop taking analgesics and return to normal activities. Pre-treatment bone biopsy samples contained extensive osteoid covering trabecular surfaces. Electron microscopy showed morphological abnormalities in the matrix. Treatment brought about a decrease in the extent and depth of osteoid, but matrix abnormalities remained. Energy-dispersive X-ray microanalysis showed the presence of several metals not seen in normal bone. The changes in trace metals after DFO treatment were not consistent. Atomic absorption analysis showed no significant change in the aluminium content of bone with treatment. DFO appears to be an effective treatment for severe dialysis osteomalacia.

N-hydroxyacetaminophen: a postulated toxic metabolite of acetaminophen.

The decomposition of N-hydroxyacetaminophen has been shown to occur via an initial first-order dehydration step to N-acetyl-p-benzoquinone imine with a rate constant at pH 7.6 of 8.66 x 10(-3) min-1 and a half-life of 80 min. This is followed by a complex reaction between the quinone imine and the N-hydroxy compound to ultimately yield p-nitrosophenol and acetaminophen. The glucuronide and sulfate conjugates of N-hydroxyacetaminophen have been observed as urinary metabolites of N-hydroxyacetaminophen. No N-hydroxylated metabolites were found among the metabolites of acetaminophen. These results have been interpreted to show that N-hydroxyacetaminophen is not a metabolite of acetaminophen. It is proposed that the hepatotoxicity and nephrotoxicity of acetaminophen are mediated by a direct oxidation of acetaminophen to the toxic reactive intermediate N-acetyl-p-benzoquinone imine by the cytochrome P450 dependent mixed-function oxidase system.

Studies on the mechanism of toxicity of acetaminophen. Synthesis and reactions of N-acetyl-2,6-dimethyl- and N-acetyl-3,5-dimethyl-p-benzoquinone imines.

N-Acetyl-2,6-dimethyl-p-benzoquinone imine and N-acetyl-3,5-dimethyl-p-benzoquinone imine were prepared from 2,6-dimethylacetaminophen and 3,5-dimethylacetaminophen by oxidation with lead tetraacetate. Reaction of N-acetyl-2,6-dimethyl-p-benzoquinone imine with hydrochloric acid gave 3'-chloro-2',6'-dimethyl-4'-hydroxyacetanilide, whereas ethanethiol, aniline, and ethanol gave tetrahedral adducts resulting from addition to the imine carbon. Water gave 2,6-dimethyl-p-benzoquinone. With N-acetyl-3,5-dimethyl-p-benzoquinone imine, water and aniline gave substitution on the imine carbon, yielding 2,6-dimethyl-p-benzoquinone and 3,5-dimethyl-N-phenyl-p-benzoquinone imine, respectively. Ethanethiol gave 3'.5'-dimethyl-2'-(ethylthio)-4'-hydroxyacetanilide. The toxicity of 2,6-dimethylacetaminophen and 3,5-dimethylacetaminophen was examined histologically in mice and rats. 3,5-Dimethylacetaminophen was slightly more nephrotoxic but showed a similar hepatotoxicity to acetaminophen. 2,6-Dimethylacetaminophen, like N-methylacetaminophen, showed very little tissue damage.

The nephrotoxicity of p-aminophenol. I. The effect on microsomal cytochromes, glutathione and covalent binding in kidney and liver.

p-Aminophenol administration lowered the microsomal cytochrome P-450 and b5 content and decreased the activity of NADPH cytochrome c reductase in kidney, but not in liver. Kidney GSH was depleted to 29% of the control value at 2 h, and only partly restored (50% of control) at 24 h. Liver GSH was transiently decreased, the lowest levels (77% of control) occurring at 30 min. The maximum level of covalently bound radioactivity was at two hours when 16.8% of the total radioactivity in kidney, 1.5% in liver and 3.6% in plasma was protein bound. At this time 81% of the total radioactivity in kidney and 95% of that in the liver was present in the soluble fraction.

The nephrotoxicity of p-aminophenol. II. The effect of metabolic inhibitors and inducers.

Inducers and inhibitors of the microsomal mixed function oxidase system have no consistent effect upon the nephrotoxicity of p-aminophenol, or on binding of the compound in vivo to cell protein. p-[ring-3H]Aminophenol was bound in vitro to kidney microsomal protein and to a lesser extent to liver. The binding was enhanced by preincubation of the p-aminophenol in air and inhibited by ascorbate, GSH, N2 and NADPH. These findings indicate that in contrast to paracetamol hepatoxicity which is dependent upon the mixed function oxidase system, that nephrotoxicity of p-aminophenol is dependent upon oxidation to a toxic metabolite by some other pathway. A similar metabolite may be responsible for the nephrotoxic action of phenacetin.

Visceral oedema and increased vascular permeability in early experimental hypertension.

Increased vascular permeability and visceral oedema without significant hypertension or arterial damage was found in clip-nephrectomized rats dying in the first post-operative week. Increased vascular permeability was demonstrated by intravenous colloidal carbon labelling, and was restricted to capillaries and venules. Rats surviving the first week became hypertensive, and visceral oedema with predominantly venular and capillary labelling continued, although carbon was now present in arteries also, associated with smooth muscle necrosis. These results suggest that the pressor and permeability effects of renal artery constriction are independent.

The effects of temporary fluid restriction in clip-nephrectomized rats.

Constriction of the remaining renal artery in unilaterally nephrectomized rats is associated with a high mortality in the early post-operative phase before hypertension has developed. The high mortality is accompanied by increased vascular permeability, mesenteric and pancreatic oedema. Temporary post-operative water restriction reduces this mortality and also modifies subsequent blood pressure elevation, heart weight and cardiac index in surviving animals.

The ultrastructural localization of the corrosion products of dental amalgam.

The ultrastructural localization of corrosion products released from dental amalgam implants in rat subcutaneous tissue was studied by means of a sulphide-silver technique. Implants stimulated a prolonged inflammatory response with delayed granulation tissue formation and slow and faulty collagen formation. The metallic corrosion products were found both in cells and in association with matrix components. Intracellular metal deposits were predominantly in the cytoplasmic matrix and in vacuoles in the early (19 day) lesions, but at 60 days there was metal in many nuclei, both viable and dead. Collagen fibrils had metal on the surface and vascular basement membranes had granules of bound metals.

Lead-loaded ion-exchange resin bead as a calcergen.

Several heavy metals have direct calcifying effects in connective tissues; most notable among them is lead (Pb), whether administered topically (1) or systemically with local injury (2). The mechanisms of metal-induced soft tissue calcification have been studied by injecting salts of known in vitro calcifying potential into subcutaneous connective tissue (3); of the metals used, only lead and holmium produced an early accumulation of minerals on collagen in vivo. Lead is also claimed to accelerate bone healing in the rabbit leg with no toxic effects (4). Lead-loaded ion-exchange resin beads, implanted into surgically prepared subcutaneous pouches in rats, rapidly induced subcutaneous calcification which has been studied by microradiography and electron-probe microanalysis.