Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry.

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.

A comparative study of artificial ceroid/lipofuscin from different tissue materials of rats.

The artificial ceroid/lipofuscin pigments originated from different organ tissues, including liver, brain, heart, and kidney of rats, and biomaterials were studied with improved fluorometric techniques. With all tissue materials exposed under ultraviolet (UV) light, a series of similar fluorescent colors were observed under microfluorometer. Analogous fluorescence spectra were also demonstrated with a three-dimensional (3-D) front-surface fluorometric technique despite of the tissue differences. Measured with 3-D fluorometry, relatively simple lipofuscin-like fluorophores were observed from the reactions of malondialdehyde (MDA) with critical biological macromolecules, such as bovine serum albumin (BSA) and DNA. Our results demonstrated that the biomaterials from different tissues have a similar fate under accelerated oxidative/carbonyl stresses but may be differentiated by a fluorescence intensity ratio.

Ceroid/lipofuscin formation in cultured human fibroblasts: the role of oxidative stress and lysosomal proteolysis.

The mechanisms involved in the accumulation of ceroid/lipofuscin within non-dividing cells are not totally understood. Oxidative stress, as well as diminished activity of lysosomal proteolytic enzymes, are known to induce ceroid/lipofuscin accumulation in a variety of cell types. In order to clarify the roles of oxidative stress and lysosomal proteolysis in ceroidogenesis/lipofuscinogenesis, and to study the fate of already formed ceroid/lipofuscin, confluent cultures of AG-1518 human fibroblasts were exposed to oxidative stress (40% ambient oxygen) and/or treated with the thiol protease inhibitor leupeptin for 2 weeks. Both oxidative stress and protease inhibition caused accumulation of ceroid/lipofuscin per se (estimated by fluorescent, confocal and electron microscopy). The combined effect of these factors was, however, almost three times as large as the sum of their isolated effects. The pigment accumulated progressively as long as the oxidative stress and/or protease inhibition acted; was not eliminated after re-establishment of normal conditions; and decreased in amount after subsequent passage. The results suggest that (i) ceroid/lipofuscin forms within secondary lysosomes due to peroxidative damage of autophagocytosed material, and (ii) it is not substantially eliminated from non-dividing cells by degradation or exocytosis.

Diet-induced lipofuscin and ceroid formation in growing pigs.

Seven piglets, aged 2 weeks at the beginning of the experiment, were fed a diet lacking antioxidants (vitamin E and selenium) and enriched with thermally oxidated cod liver oil (the peroxide value of which was 310 meqv/kg). From one month before to 4 weeks after farrowing (weaning) the sow also received the experimental diet. One pig died at 9 days of age, due to degeneration of the parenchymal tissues. The oxidative stress produced was aggravated in four of the seven remaining pigs by giving them an injection of iron-dextran (300 mg) one day before the animals were killed for necropsy at the age of 2 months. The three other piglets were given an injection of a control saline solution. The nutritional myodegeneration produced ('white muscle disease') was characterized by a pale, yellowish colour and translucence of skeletal muscle, with degeneration, including swelling of the muscle fibres and slackening of the fibrillar pattern. The arrangement of nuclei in chains indicated repair. Ultrastructurally, skeletal muscle showed vacuolization of the sarcoplasmic reticulum, irregularities in the mitochondria and focal dissolution of myofibrils. The sarcoplasmic reticulum displayed condensation and lamellar formations. Hyaline degeneration of the heart muscle occurred both in iron-treated and control pigs, and in six animals a yellowish pigment resembling lipofuscin and ceroid was observed. Ultrastructurally, the ceroid deposits appeared as myelin-like figures. The livers were pale and brittle, four with centrilobular degeneration. By light microscopy, lipidosis was distinguished in four and ceroid accumulations in three pigs. Thiobarbituric-acid-reactive substances, creatine kinase activity and iron increased significantly in the serum as a result of the injection of iron-dextran.

Leupeptin causes an accumulation of lipofuscin-like substances in liver cells of young rats.

Leupeptin, a thiol protease inhibitor, has previously been shown to cause a dense accumulation of substances resembling age pigment and called ceroid-lipofuscin, in brain cells of young rats. Thus far, however, attempts to produce age pigments in hepatocytes of normal young rats with protease inhibitor(s) have not been successful. The present study provides the first demonstration that leupeptin induces lipofuscin-like substances in normal young rat hepatocytes. Male Fischer-344 rats (age 4-6 weeks) were continuously infused with leupeptin or saline i.p. for 2 weeks by an osmotic minipump (dosage, 1-50 mg/100 g per day). Liver tissues were then examined by light, fluorescence and electron microscopy. Both hepatocytes and non-parenchymal cells of livers treated with leupeptin, but not saline, showed a dense accumulation of pigments which stained deeply with toluidine blue, were PAS-positive and were brightly autofluorescent. After UV excitation the pigments had an emission spectrum with a broad peak at 480-540 nm extending to 650 nm resembling the spectrum of age pigment from livers of normal aged rats. Electron microscopic examination revealed numerous lipofuscin-like deposits with heterogeneous morphology in the cytoplasm of both hepatocytes and non-parenchymal cells; lipid and myelin-like bodies were also present in hepatocytes. The results indicate that the perturbation of proteolytic activity in liver by leupeptin causes an accumulation of substances which by several criteria resemble lipofuscin. These results thus provide further support for the 'Protease Inhibitor Model of Lipofuscin Formation' as well as a potential experimental model for studying hepatocellular aging processes.

Ceroid accumulation by murine peritoneal macrophages exposed to artificial lipid-containing particles: the role of the hydrophilic component.

Murine resident peritoneal macrophages were maintained in cell culture in a medium containing 10% lipoprotein-deficient foetal calf serum to which various artificial lipid-containing particles were added. These had a core of oxidizable lipid, generally cholesteryl linoleate, and were stabilized in aqueous suspension by one of a variety of poly-L-amino acids, proteins or polysaccharides. Most particles, except those containing poly-L-lysine or poly-L-arginine (both strongly basic), were readily taken up by the macrophages to form typical ceroid inclusions, the morphological form of which was determined by the nature of the core lipid. The hydrophilic stabilizing component seemed largely irrelevant in this respect. The role of the latter appears largely to be to allow the cellular uptake of lipid, although it may also participate in ceroid formation.

Macrophages and ceroid in human atherosclerosis.

It is proposed that atherosclerosis is a chronic inflammatory disease in which monocyte-derived macrophages are doing harm and smooth muscle cells are essentially reparative. Activities of macrophages that might be contributory to the development of atherosclerosis are tabulated. Observations and experiments are described that suggest macrophages may be contributing to lipoprotein oxidation within the plaque and that individual humans vary in their macrophage oxidative capacity. The possibility of intervention with antioxidants is discussed.

Protection factors against free radical-induced ceroidogenesis.

The most important products of the combustion process are SO2, NOx, CO2 and the heavy metals. When these substances come into contact with the biotic components of the ecosystems they produce an oxidative damage by means of a free radical mechanism. One of the significant natural sources of these oxides and metals are the volcanic emissions that contribute, either locally or more diffusely, to enrich the atmosphere with these substances. The area of Campi Flegrei (Naples, Italy) is an experimental model fit for studying the contemporary effect of the aforesaid oxidative agents, because it is characterized by a continuous fumarolic activity, particularly in the area of the widest crater (Solfatara). We have made so two experiments utilizing rats and earthworms (Octolasium complanatum) to evaluate the following aspects in phylogenetically very different organisms: 1. the combined effect of the atmospheric pollutants, 2. the effect of the only heavy metals (Cu, Ni, Mn), 3. the protection action played by reduced glutathione in rats. The reduced glutathione being either a substrate of the glutathione proxidase or an oxyradicals scavenger, is one of the main protection agents against the above stress. Because many papers suggest that the mentioned atmospheric pollutants damage both animal and vegetable organisms by their oxidative properties, the reduced glutathione seems to be able to counteract efficaciously the damaging activity studied in terms of age pigments production.

Ceroidogenic effect of ionizing radiation.

The effect of the exposure of Torpedo marmorata to a single dose of 400 cGy of ionizing radiation and doses of 200 cGy/week for 4 weeks has been studied. The parameters measured were the TBA reactivity and lipofuscin production, a fluorescent pigment that is thought to derive from the reaction of malonaldehyde, a peroxide-degradation product, with free amino-groups. Acute irradiation was found to generate an increase of peroxidative damage in some tissues. This effect was inhibited by acetyl homocysteine-thiolactone, a drug that increases the activity of tissue superoxide dismutase. Chronic irradiation produces a severe increase in the generation of lipofuscin.

Lipopigments in veterinary pathology: pathogenesis and terminology.

The lipopigments are a heterogenous group of pigments whose pathogenesis and terminology is confused. Whereas there is epidemiological and observational evidence that ceroid is derived from degeneration and peroxidation of unsaturated lipid, the assumption that all so-called lipopigments are similarly formed, is questioned. In particular, recent studies have distanced the pathogenesis of the pigment found in the ceroid-lipofuscinoses from that perceived for ceroid. The importance of protein rather than lipid in the pathogenesis of the pigment of ceroid-lipofuscinosis and of age pigment from the equine thyroid is noted. In the former the essential feature is storage of the DCCD binding protein subunit c of mitochondrial ATP synthase. There is a need for more analytical studies on isolated pigments which are generally more soluble than anticipated by the literature. It is proposed that the term ceroid be limited to a family of pathological pigments where lipid degeneration and peroxidation is implied from observational and/or epidemiological factors. The term age pigment is unequivocal and preferred for age related pigment not obviously complicated by other factors. The terms lipofuscin and lipopigment retain a usefulness as generic terms, particularly where the nature of the pigment is uncertain. The term ceroid-lipofuscinosis for the inherited storage diseases of children and animals is misleading. The term "proteolipid proteinosis" has been suggested to define this group of diseases but this is perhaps premature until their full pathogenesis is known.

Effects of lovastatin and leupeptin on ceroidogenesis of vitamin E-deficient and -supplemented young rats.

Previous studies in young normal rats have shown that intracerebral administration of the proteinase inhibitor, leupeptin, caused a rapid accumulation of lipofuscin-like pigment in lysosomes of brain cells (Ivy et al., 1984a). On the other hand, we have recently found that the administration of lovastatin, an inhibitor of HMG-CoA reductase, reduced the ceroid-like pigment and dolichol contents in the crushed epididymal fat pad of rats (Porta et al., 1988). In order to study now the possible modulating effects of these enzyme inhibitors on ceroidogenesis associated with vitamin E deficiency, two main groups of weanling Wistar female rats were respectively fed ad libitum a vitamin E-deficient basal diet, or the same diet supplemented with 16 mg% of dl-alpha-tocopherol acetate. The vitamin E-deficient and -supplemented rats were further subdivided and received for 8 weeks their diets alone or with 2, 1, or 0.5 g of lovastatin/kg of diet. Other subgroups were treated with constant peritoneal infusion of 0.5 mg/day of leupeptin by means of osmotic minipumps (Alzet 2002) consecutively implanted at days 15, 30, and 45. Lovastatin treatment to vitamin E-deficient rats was associated with dose-dependent toxicity, resulting in 100%, 75%, and 50% mortality at concentrations of 2, 1, and 0.5 g/kg diet, respectively. This mortality was mainly due to extensive hepatic necrosis. Food intake and growth rates were reduced, while the relative weights of liver, kidneys, spleen, heart and brain, as well as the serum levels of GPT and GOT were significantly increased over the values of the untreated vitamin E-deficient control rats. The volumetric densities of ceroid pigment and the dolichol contents in liver and kidneys were not significantly modified. Lovastatin toxicity was partially prevented by vitamin E supplementation. However, in these supplemented rats, lovastatin treatment did not modify the volumetric densities of hepatic and renal ceroid, although the contents of hepatic and renal dolichol were significantly increased. No correlations could be found between levels of hepatic or renal ceroid and total dolichol content in vitamin E-deficient and supplemented rats. Leupeptin treatment to vitamin E-deficient rats only slightly reduced food intake and growth rates, and did not significantly modify the relative organ weights or the serum levels of cholesterol, GOT and GPT. Although in both vitamin E-deficient and -supplemented rats the leupeptin treatment consistently showed a tendency to increase the volumetric densities of hepatic and renal ceroid pigment, the differences with the control untreated rats were not statistically significant.(ABSTRACT TRUNCATED AT 400 WORDS)

So-called membranocystic lesion (MCL)--a new variant of ceroid type lipopigment.

Structures very close morphologically to the so-called membranocystic formations of the Nasu-Hakola's disease and identical in histochemical properties with them were found in several other metabolically unrelated conditions such as cerebrotendinous xanthomatosis (perivascularly in the brain) and in human atheromatous plaques. This with some other literary data points to unspecific nature of the membranocystic lesion (MCL) which also has been resisting satisfactory classification in terms of pathobiochemistry. Evidence is presented suggesting the MCL is lipopigment in nature. This is based on its lipid histochemical properties dominated by prominent autofluorescence and marked sudanophilia resistant to lipid extraction procedures. Ultrastructural pattern of the MCL was membranous, being dominated by mostly individual trilaminar membranes about 15 nm thick which could be also occasionally identified in various intralysosomal ceroid type lipopigments. It is supposed that the MCL lipopigment is formed mainly extracellularly from the lipid rich debris.

Phospholipases and the molecular basis for the formation of ceroid in Batten disease.

Lysosomal ceroid/lipofuscinosis storage in human, canine, and ovine forms of neuronal ceroidlipofuscinosis is predominantly in neurons and retinal pigment epithelial cells. Despite problems in identifying individual storage materials, it is believed that non-enzymic oxidation of unsaturated fatty acids in phospholipids and inhibition of lysosomal proteolysis, leading to massive deposition of autofluorescent pigment, is the cause of the disease. We have, therefore, studied cellular phospholipases and find a marked deficiency of lysosomal phospholipase A1 (PLA1) in canine NCL brain. Other lysosomal hydrolases, and cytosolic/mitochondrial forms of phospholipase A2 are completely normal. We believe that the PLA1 deficiency leads to transient lysosomal storage of phospholipids containing peroxy fatty acids which are then chemically converted to hydroxynonenal, a potent inhibitor of a thiol-dependent enzymes. Inhibition of proteases is believed to be intrinsic to the formation of lipofuscin. An inherited deficiency of a thiol protease (the lysosomal cathepsin H) in two siblings with NCL can also lead to build up of peptides which are then cross-linked and converted into ceroid-containing curvilinear bodies. Thus there is evidence for molecular and genetic heterogeneity in Batten disease.

Lipofuscin and ceroid formation: the cellular recycling system.

Lipofuscin, age pigment, is a dark pigment with a strong autofluorescence seen with increasing frequency with advancing age in the cytoplasm of postmitotic cells. By bright-field light microscopy lipofuscin appears as irregular yellow to brown granules ranging in size from 1-2 nm in diameter. The fluorescent spectra of lipofuscin in situ generally show excitation maxima at about 360 nm and a yellowish emission maxima at 540-650 nm. Ultrastructurally the granules, localized in residual body-type lysosomes, are extremely heterogeneous and vary from one cell type to another, and frequently within a single cell. The pigment granules usually contain numerous liquid droplets embedded in an electron-dense matrix. The granules stain positively for neutral lipids but are not soluble in polar or non-polar lipid solvents. Lipofuscin contains about 50 percent by weight of proteinaceous substances, a lesser fraction of lipid-like material, and probably less than one percent by weight fluorophore(s); it is enriched in metals such as Al, Cu, and Fe, and in dolichols. Free radical reactions and the proteolytic system are implicated in lipopigment formation. Thus the rate of lipopigment formation is increased by vitamin E deficiency and by increased intake of polyunsaturated fatty acids as well as by protease inhibitors such as leupeptin. Free radical reactions and proteolysis are involved in the continual turnover of cellular components. Cellular damage from free radical reactions, and others such as hydrolysis, has been present since the beginning of life. The evolution of more complex cells necessitated development of defenses - DNA repair processes, antioxidants, etc. - against damaging reactions as well as the removal and replacement of altered parts, and of those no longer needed by the cells. Proteins "marked" for disposal by oxidation damage, or other means such as conjugation with ubiquitin, are apparently rendered more hydrophobic so that they are "recognized" for degradation by the lysosomes and the proteinases and peptidases of the cytosol and mitochondria. Oxidatively altered lipids are removed by enzymes such as phospholipase A2. The products of the degradation processes are reused by the cells. Normally the recycling of damaged components works extremely well. There may be some slow slippage with advancing age as the rate of free radical damage increases while protease activity decreases. As a result a gradually increasing fraction of lysosomal "food" may be converted to non-digestible forms, lipofuscin, before it can be broken down to reusable components. Ceroid is apparently formed when the disposal system is "overloaded" or impaired.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipid peroxidation and storage of fluorescent products by macrophages in vitro as a model of ceroid-like pigment formation.

Storage of fluorescent products, ceroid-like pigments was observed in P388D1 cells, an established macrophage-like cell line, when the cells were cultured in the presence of rat liver phosphatidylcholine liposomes containing polyunsaturated fatty acids. Some fluorescent products accumulated in the cells were extractable in organic solvents, ethanol/ether (3:1, v/v), while others were insoluble in organic solvents, but soluble in detergent. The fluorescent products dissolved in organic solvents or in detergent had a fluorescence maximum at 430 nm when excited at 360 nm. The formation of the ceroid-like pigments was inhibited, at least in part, by antioxidants, such as alpha-tocopherol and butylated hydroxytoluene (BHT). The fluorescence intensity of the pigments was quenched in alkaline media and restored by adjustment of pH to neutrality. These findings indicate that liposome uptake by macrophages causes the formation of ceroid-like pigments, and that the fluorescent chromophores of the pigments are Schiff base structures derived from the reaction of lipid peroxides from the exogenous phospholipids and subcellular amino compounds.

Ceroid accumulation by murine peritoneal macrophages exposed to artificial lipoproteins: ultrastructural observations.

Murine resident peritoneal macrophages were maintained in cell culture in a medium containing 10% lipoprotein-deficient fetal calf serum to which various artificial lipoprotein particles (coacervates of lipid and bovine serum albumin) had been added. The uptake and intracellular fate of these particles was studied by electron microscopy. The appearance of material accumulating within the cells varied according to the nature of the lipid component of the ingested particles. Lipids which are readily oxidised (cholesteryl linoleate, cholesteryl arachidonate, trilinolein) were associated with the formation of ceroid within membrane-bound structures. Less readily oxidized lipids (cholesteryl oleate, triolein) were not associated with ceroid accumulation but instead the cells contained numerous nonmembrane-bound lipid inclusions. The appearances of the ceroid within the murine peritoneal macrophages are similar to those of ceroid in macrophages in human atherosclerotic lesions.

Ceroid accumulation by murine peritoneal macrophages exposed to artificial lipoproteins.

Murine resident peritoneal macrophages were maintained in cell culture in a medium containing 10% lipoprotein-deficient fetal calf serum to which various artificial lipoproteins (lipid-bovine serum albumin complexes) had been added. Ceroid accumulated in cells exposed to artificial lipoproteins containing cholesteryl esters or acylglycerols possessing polyunsaturated fatty acid residues, but not in cells exposed to lipoproteins containing less readily oxidized lipids. Oxidation of cholesteryl linoleate before its incorporation into artificial lipoprotein accelerated ceroid production. Incorporation of free radical scavengers into cholesteryl linoleate-containing artificial lipoproteins impaired ceroid formation. The results are discussed in terms of the mechanisms by which the ceroid might have been produced and its significance for human atherogenesis.

The production of ceroid by mouse peritoneal macrophages in vitro.

Murine resident peritoneal macrophages accumulated lipid droplets and subsequently insoluble, ceroid-like material when cultured in vitro in a medium containing 33% fetal calf serum. At least some of this insoluble lipid was membrane-bound and by light microscopy it often appeared as 'rings' with a hollow centre. It is suggested that the production of ceroid may be the consequence of the uptake of lipids from the extracellular medium and the activity of the macrophage's membrane-bound oxidative microbicidal mechanisms. The results indicate that macrophages are capable of rendering lipids insoluble, supporting the suggestion that this might occur in the atherosclerotic plaque.

Peroxide formation, vitamin E and myocardial damage in the rat.

Rats were fed diets containing 10% cod liver oil with or without dietary tocopherol for 16 or 32 weeks. Adipose tissue, skeletal muscle and myocardium were isolated. They were examined histologically and analyzed for autoxidation products and a number of energy metabolites. After 16 weeks small amounts of peroxides were present in adipose tissue as determined by the thiocyanate method. Ceroid pigment and slightly defective striation were observed in myocardium and to a lesser extent in skeletal muscle of the tocopherol deficient group. The ATP content was also significantly lower in this group. After 32 weeks, adipose tissue of the tocopherol deficient group contained large amounts of ceroid and had a high content of peroxides and other autoxidation products. More ceroid and a significantly higher peroxide estimate in lipid extracts were found in myocardium of the tocopherol deficient group as compared to the controls. Similar but weaker signs of peroxide occurrence and ceroid formation were obtained in skeletal muscle. The significance of the findings for myocardial function is discussed.

Effects of parenteral nutrition with lipids on the human liver. An electron-microscopic study.

The ultrastructural findings in the Kupffer cells of a liver biopsy of a 5-week-old baby, who had received repeated i.v. administration of Intralipid (10%) in a dose of 20 ml/kg body weight/day for 12 days, are presented. Heterophagocytosis of free lipid droplets occurs by invagination of the Kupffer cell membrane. Large, solitary or conglomerated fat droplets surrounded by a membrane (lipophagosomes) are present within the hypertrophic and most active Kupffer cells. Small, round or elongated dark bodies (primary lysosomes) are linked with and/or seem to fuse with these lipophagosomes. In addition, smaller phagosomes almost completely surrounded by a dark small rim and a less electron-dense center probably represent (further stages of the lysosomal breakdown. The latter is also expressed by the condensation from the periphery towards the center of the original lipid droplets enclosed within the lysosomes. Angular inclusion bodies, build up of lipid remnants and a granular matrix of medium electron density, constitute far advanced stages of lysosomal digestion and represent the development or transformation of the phagocytosed lipids into lipogenous pigments. On the electron-microscopic level a distinction between lipofuscin granules and ceroid pigment can hardly be made at that stage. These pigment granules can be found in various amounts within the Kupffer cells and can stay there for a long time. This accumulation probably inhibits the clearance capacity of the cellular elements of the hepatic reticuloendothelial system. The hepatocytes and spaces of Disse seem not to be directly involved in the process of fat phagocytosis and breakdown as summarized above.