Intracellular distribution of iron (and associated elements) in various cell types of larvae and juveniles of the sea lamprey (Petromyzon marinus)

The intracellular distribution of iron and other elements was examined in various cell types in larvae and juveniles of the sea lamprey (Petromyzon marinus) using transmission electron microscopy and energy dispersive x-ray microanalysis. The objective was to establish whether there are cell-type specific relationships between iron and other elements in the iron-rich organs and tissues (adipose tissue, opisthonephric kidneys, dorsal integument, fat column, liver, and posterior intestine) of these two life cycle periods. Iron was localized within either dense bodies (presumptive lysosomes, siderosomes) or in the cytoplasmic matrix of many cell types where it was viewed as haemosiderin/ferritin and ferritin, respectively. Presumptive lysosomes of adipocytes of the nephric folds, dorsal integument, and fat column possessed iron and sulphur and this elemental association was also prevalent in the epithelia of the larval proximal tubules and in the posterior intestine and epidermis of both life periods. Macrophages of the larval haemopoietic tissue (posterior intestine) and of the juvenile opisthonephros, which were described as melanomacrophages because of their granules, possessed iron, sulphur, and calcium. This elemental association was also noted in the presumptive lysosomes of the iron-loaded hepatocytes of the juvenile liver while no elements could be detected in these cells in the larval organ. The variations and similarities in elemental associations between the cell types in each life period and at different life periods is discussed in the context of specific cell functions related to the prevention of iron toxicity. These functions are sequestration of iron and storage as the less toxic haemosiderin (larval adipocytes, macrophages, juvenile hepatocytes) or as part of a process of elimination of excesses of this metal (posterior intestine, dorsal epidermal cells). Due to its unique ability to deal with copious amounts of iron at all periods of the life cycle, the lamprey serves as an important model for studies of iron loading in vertebrates.     

Iron loading in the livers of metamorphosing lampreys,Petromyzon marinus L.

Iron loading of hepatocytes was followed through the stages (1-7) of metamorphosis in lamprey (Petromyzon marinus L.) using light- and electron-microscopic histochemistry. Iron is present in ferric and ferrous forms in the hepatocytes of larval lampreys in levels that can only be detected in the electron microscope. During the initial stages (1-3) of metamorphosis iron begins to increase in the cytoplasmic matrix and in dense bodies but it is not apparent in the light microscope until stage 4. The increased accumulation of iron through the subsequent stages (5-7) of metamorphosis coincides with the advanced degeneration and ultimate disappearance of bile canaliculi and bile ducts. The absence of a bile canaliculus is concurrent with the beginning of staining of lateral cell borders for ferrous iron and with intense concentrations of ferric iron throughout the cytoplasmic matrix and within cytoplasmic dense bodies. By the end of metamorphosis the hepatocytes resemble iron-loaded hepatocytes in pathological and experimentally induced situations in other vertebrates. The iron loading of hepatocytes during metamorphosis is discussed with respect to both the concomitant atresia of the biliary tree and alteration of several aspects of blood morphology and chemistry. Since iron loading occurs synchronously in the hepatocytes of a given population of metamorphosing lampreys, this organism should prove to be a useful experimental system for investigation on cellular mechanisms of iron loading in vertebrates.     

Distribution of55Fe in juvenile adult lampreys,Petromyzon marinus L., following oral intubation of the isotope

Summary The capacity of a sanguivorous lamprey,Petromyzon marinus L., to deal with ingested iron was studied over time using autoradiography and scintillation counting of solubilized tissue samples after intubation of the oesophagus with a single dose of⁵⁵ferrous citrate. A highly efficient mechanism for absorption in the anterior intestine was recognized with 17% of the intubated radioactivity absorbed into the body after only 5 min, 66% by 3 h, and almost 80% by 21 h. Iron concentration in the epithelial cells of the anterior intestine may be a factor in restricting iron absorption during spontaneous feeding. A decline in total body radioactivity over the 15 days following iron intubation probably results from transport of the metal in the blood and release of radioiron from the mucous cells of the posterior intestine. The kidneys appear to play a smaller but still significant role in iron loss. Gradual increases in radioiron concentration (cpm g^–1 wet weight) and percent of total body radioactivity occur in the liver (2 to 26%), carcass (14 to 37%), and integument (4 to 12%) during the course of the experiment, indicating that these are the chief sites of iron storage during times of metal excess. However, eventually integument may also be a site of iron excretion. Significant fluctuations in radioiron concentration (cpm ml^–1) in whole blood during the 15 day period can be correlated with transport of the metal to sites of storage and excretion, and maybe with incorporation into haemoglobin and with erythropoietic activity. Feeding adult lampreys represent a valuable system, with both general and unique characters, for studying iron metabolism in vertebrates.     

Quantitative studies on the skin of the paired species of lampreys,Lampetra fluviatilis (L.) and Lampetra planeri (BLOCH)

The number of mucous, club, and granular cells in the epidermis, and the number of rows of subcutaneous adipose cells, as well as the thickness of the epidermis and the dermal collagen layer, have been recorded for the larval and metamorphosing stages of the anadromous parasitic lamprey, Lampetra fluviatilis, and for the larval, metamorphosing, and adult stages of the nonparasitic lamprey, Lampetra planeri. In L. fluviatilis, the mucous cells predominated in all stages but were more abundant in fully metamorphosed individuals than in larvae. During metamorphosis, the number of granular cells increased continuously, whereas the club cells showed little change. Although lampreys do not feed during metamorphosis, there was an increase in the thickness of the epidermis and in the dermal collagen sheath; the latter increase probably foreshadows the increase in activity by the adults. Simultaneously, there is a reduction in the subcutaneous fat layer, which can be attributed to mobilization of lipid as an energy source. Changes similar to those just described for L. fluviatilis were also found in metamorphosing L. planeri. However, the pattern altered markedly during adult stages in this nonparasitic species. There were marked declines in the number of cells, in the thickness of the epidermis, in the width of the collagen sheath, and in the quantity of subcutaneous fat.     

Sulphur, thiols, and disulphides in the fish epidermis, with remarks on keratinization

Energy dispersive x-ray (EDX) analysis and qualitative and quantitative histochemistry were applied to study the distribution and contents of sulphur, thiols and disulphides in the epidermis of the river lamprey Lampetra fluviatilis , the lesser spotted dogfish Scyliorhinus canicula and the brown trout Salmo trutta fario . Thiols generally reacted weakly throughout the entire epidermis, whereas disulphide reactions were more distinct and differentiated. In the river lamprey, the concentrations of -S-S- groups clearly increased in the developing mucous cells from the stratum basale to the stratum superficiale; skein cells and granular cells reacted negatively to weakly. In the lesser spotted dogfish, amounts of disulphides appeared at moderate concentrations, and only goblet cells displayed a strong reaction. In the brown trout, filament cells showed low concentrations or weak reactions of disulphides, goblet cells and the most outer superficial cells stained strongly. Sulphur distribution and contents generally supported the histochemical observations in normal epidermis cells (absolute sulphur contents: 41–59 mM), only the brown trout showed high amounts of sulphur in the stratum basale (81 mM). The findings corroborate the view that there is an inverse correlation between keratinization and mucous secretion in normal fish epidermis. The sometimes distinct contents of disulphides in the outer mucous layer indicate that this system could endure higher mechanical stress than predictable from its large amounts of neutral glycoproteins.     

Tissue levels of bilirubin and biliverdin in the sea lamprey, Petromyzon marinus L., before and after biliary atresia

1. Liver, intestine, kidney, muscle and epidermis from larvae, juvenile adults and upstream migrants of the sea lamprey, Petromyzon marinus L., were assayed for the presence of biliverdin and bilirubin. Urine was also examined for these bile pigments in juveniles and upstream migrants. 2. Bilirubin concentration increased dramatically in the liver and caudal intestine following loss of larval bile ducts while biliverdin levels were highest in the liver of upstream migrants and rose sharply in the caudal intestine immediately following the atresia. 3. Small amounts of bile pigment were present in larval kidneys but high concentrations were found in this organ in upstream migrants. The urine of the latter possessed biliverdin. 4. Mucus of the epidermis may be a vehicle for transport and release of bilirubin in upstream migrants. 5. These data indicate that lampreys utilize different avenues for bile pigment storage and elimination over the course of their life cycle.     

Morphology of the intestine of prefeeding and feeding adult lampreys,Petromyzon marinus (L): The mucosa of the posterior intestine and hindgut

The epithelium of the posterior intestine and hindgut of recently metamorphosed adult lampreys (Petromyzon marinus L.) prior to and during spontaneous feeding was examined using light and electron microscopy. These two regions differ slightly in their general morphology but possess the same mucosal cell types. Included are caveolated absorptive and mucous cells, which are not present in more cephalic regions of the intestine, and ciliated and enteroendocrine cells. During feeding, the caveolated cells undergo dramatic transformation in their structure, namely, through the acquisition of numerous heterophagic vacuoles. Due to their morphology and to the fact that there are low amounts of lipid, it is suspected that caveolated cells are primarily involved in the absorption of protein components from the ingested host blood and body fluids. Iron in caveolated cells may result from the degradation of ingested heme or reflect the excretion of bile products at this location in the intestine. Mucous cells are likely responsible for lubrication of the luminal surface and may be important as a stem cell for the mucosal epithelium.     

Molecular and cellular bases of iron metabolism in humans

Iron is a microelement with the most completely studied biological functions. Its wide dissemination in nature and involvement in key metabolic pathways determine the great importance of this metal for uniand multicellular organisms. The biological role of iron is characterized by its indispensability in cell respiration and various biochemical processes providing normal functioning of cells and organs of the human body. Iron also plays an important role in the generation of free radicals, which under different conditions can be useful or damaging to biomolecules and cells. In the literature, there are many reviews devoted to iron metabolism and its regulation in proand eukaryotes. Significant progress has been achieved recently in understanding molecular bases of iron metabolism. The purpose of this review is to systematize available data on mechanisms of iron assimilation, distribution, and elimination from the human body, as well as on its biological importance and on the major iron-containing proteins. The review summarizes recent ideas about iron metabolism. Special attention is paid to mechanisms of iron absorption in the small intestine and to interrelationships of cellular and extracellular pools of this metal in the human body.     

Increased Iron in the Substantia Nigra Compacta of the MPTP-Lesioned Hemiparkinsonian African Green Monkey: Evidence from Proton Microprobe Elemental Microanalysis

Abstract: The association of free radicals and particularly free iron in the pathogenesis of idiopathic Parkinson's disease and MPTP-induced parkinsonism remains controversial. Whereas the actual cause of dopamine cell death in the substantia nigra compacta (SNc) remains unknown, disturbances in lipid peroxidation and subsequent mitochondrial and cell membrane disruption has been demonstrated. In a genetically susceptible host, abnormal elimination of oxygen and trace metal free radicals may further damage dopamine cells. Using a unilaterally MPTP-treated African Green monkey, which showed obvious contralateral hemiparkinsonism, the total free iron concentration was measured. Iron, Fe²⁺ and Fe³⁺, but not other trace elements, was significantly elevated in the SNc compared with the opposite unlesioned side, which was similar to separate control animals. Iron content in the SNc, periaqueductal gray area, and crus cerebri was 228–270 ppm. Normal control SNc was 285 (±59) ppm, whereas iron levels of 532 (±151) ppm were found in the MPTP-lesioned SNc. These animals were drug naive and not on long-term levodopa maintenance. Proton microprobe elemental analysis was matched against adjacent immunocytochemically stained tissue slices to ensure the cells studied were in the SNc. Iron was found not only in the degenerating dopamine cells themselves but also in the surrounding matrix and glial cells. Whether free iron that is not bound to neuromelanin is responsible for dopamine cell death as suggested by these experiments remains to be proved.     

Copper and iron concentrations in Ascophyllum nodosum (Fucales, Phaeophyta) from different sites in Ireland and after culture experiments in relation to thallus age and epiphytism

In laboratory experiments, copper concentrations in plants of Ascophyllum nodosum (L.) Le Jolis (Fucales, Phaeophyta) increased with the concentrations in the culture media and were highest in younger, meristematic thallus parts. After initial accumulation in high-copper medium and subsequent transfer to clean seawater for 5 days, no release of copper could be detected. Iron concentrations in A. nodosum tissue were not related to concentrations in the culture medium. Differences between copper concentrations in plants from different sites in areas with high yachting activity in Strangford Lough, Northern Ireland, could be explained by differences in water motion and human activity, in particular the application of copper-releasing antifouling paints to leisure boats. Iron concentrations were also highest in plants from the sheltered, polluted site but did not differ significantly between the other two sites. No differences in copper nor iron concentrations were found between different-aged thallus parts of plants from any site. X-ray microanalysis revealed that most of the iron detected was located in epiphytic pennate diatoms on the A. nodosum surface. In thallus areas without diatoms, iron levels were below the detection limit for X-ray microanalysis. Mapping for copper indicated that most of the accumulated copper was located in cells near and immediately below the thallus surface. "Epidermis"-shedding occurred in plants from the culture experiments and also in freshly-collected material and may have resulted in a loss of metal ions accumulated by surface cells and by epiphytic diatoms. The results suggest that A. nodosum could be used as a biological indicator for copper but not for iron, and that young, apical plant parts are most sensitive to changes in metal concentrations in the water.     

The relationship between nitrogen output and changes in mucous cell function in the amphibious teleost Blennius pholis L. during aerial exposure

The numbers of mucous cells in the epidermis of the head and body and in the surface of the gill arch of the amphibious blenny, Blennius pholis L., were estimated on fish immersed in sea water and after 4 h aerial exposure. During emersion there appeared to be a considerable reduction in the frequency of epithelial mucous cells in the areas studied, although counts for the head epidermis were somewhat variable. A concomitant decline in the number of cells thought to be actively secreting was also recorded in tissue samples from both the head and gills, while in the body epidermis the potential for mucus-secretion was maintained close to the levels observed in immersed fish.
Histochemical studies revealed epidermal mucous cells containing either sialylated acid mucopolysaccharides or neutral mucins, or a mixture of these, in both the head and the body, whereas in the gill arch epithelia there were, in addition, cells containing sulphated acid mucopolysaccharides. After emersion, a disproportionate loss of cells containing neutral and sialylated mucus from the gill epithelia resulted in an increase in the proportion of secreting cells staining positively for sulphated acid mucopolysaccharides.
The results of this study are discussed in relation to nitrogenous excretion during aerial exposure.     

Structural characteristics of the epidermal mucosa in yellow and silver European eel, Anguilla anguilla (L.)

Histological observations were made on the distribution and concentration of the superficial mucous cells and on the thickness of the epidermis in immature yellow and in male and female silver eels. In all groups there was a decrease in the concentration of mucous cells from the anterior regions to the posterior regions, while the epidermis was thickest in the median parts of the body. Besides these common characteristics, a sexual dimorphism was shown: in silver eels the epidermis is thicker than in immature yellow eels; female silver eels have both the thickest epidermis and the greatest concentration of superficial mucous cells.
Results are discussed in the light of knowledge on the influence of environmental and endocrine factors on the structure and function of epidermal mucosa in teleosts.     

Metal localization in water hyacinth roots from an urban wetland

PCA, principal components analysis
MDS, multidimensional scaling
STEM, scanning transmission electron microscopy

Metal localization within and around roots of water hyacinth ( Eichhornia crassipes ) growing in a wetland receiving urban run-off was studied by energy dispersive X-ray microanalysis of sections from freeze-substituted roots. Sampling randomly from an order of magnitude gradient in metal concentrations (Cu and Pb) allowed us the opportunity to identify general patterns of metal localization. Iron was present at high levels at the root surface, and this may have been a root plaque as described for wetland plants with roots anchored in flooded soils. Iron levels decreased centripetally across the root and were higher in cell walls than within cells. Trace metals (Cu, Zn and Pb) were not localized at the root surface. In contrast with iron, trace metal levels increased centripetally across the root, tended to be higher inside cells and were highest within cells in the stele. Variability of localization was high for all metals analysed. Multivariate statistical analyses (principal components analysis and multidimensional scaling) were useful for identifying overall patterns in elemental distribution.     

Concentrations of iron correlate with the extent of protein, but not lipid, oxidation in advanced human atherosclerotic lesions

Previous studies have provided compelling evidence for the presence of oxidized proteins and lipids in advanced human atherosclerotic lesions. The catalyst responsible for such oxidation is unknown and controversial. We have previously provided evidence for elevated levels of iron in lesions. In this study we hypothesized that if iron ions catalyzed protein and lipid oxidation in the artery wall, then there should be a positive correlation between these parameters. Iron concentrations in ex vivo healthy human arteries and advanced carotid lesions were quantified by electron paramagnetic resonance spectroscopy. Four specific side-chain oxidation products of proteins, and the lipid oxidation products 7-ketocholesterol and cholesterol ester alcohols and hydroperoxides, were quantified by HPLC in the same samples used for the iron measurements. Parent amino acids, cholesterol, and cholesterol esters were also quantified. Statistically elevated levels of iron, cholesterol, cholesterol esters, 7-ketocholesterol, and cholesterol ester alcohols and hydroperoxides were detected in advanced lesions compared with healthy control tissue. Iron levels correlated positively and strongly with all four markers of protein oxidation, but not with either marker of lipid oxidation. These data support the hypothesis that elevated levels of iron contribute to the extent of protein, but not lipid, oxidation in advanced human lesions.     

Categorization of the mitochondria-rich cells in the gill epithelium of the freshwater phases in the life cycle of lampreys

The distribution and ultrastructure of the mitochondria-rich (MR) cells in the gills of larval (ammocoetes) and adult lampreys (Petromyzon marinus and Geotria australis) have been studied. One type of MR cell, which is found only in ammocoetes, occurs in groups on and between gill lamellae. Freeze-fracture replicas show that the apical membrane of this ammocoete MR cell contains globular particles. The second type of MR cell, which is present in both ammocoetes and adults in freshwater, is located between lamellae and at the base of the filament. This cell usually occurs singly and is typically intercalated between ammocoete MR cells in larval lampreys and between pavement cells and pavement and chloride cells in adult lampreys. It contains rod-shaped particles in either the apical membrane (subtype A) or, far less frequently, the lateral membrane (subtype B) and in membranes of cytoplasmic vesicles and tubules. These features characterize this intercalated MR cell as a member of a group of MR cells that are also found in urinary epithelia of tetrapods and the amphibian epidermis, where they are involved in H⁺ and HCO₃ ^- secretion. Because this type of MR cell disappears when the young adult lamprey enters the sea and reappears immediately after the fully grown adult re-enters freshwater on its spawning run, it is presumably essential for osmoregulation in freshwater. On the basis of electrophysiological studies on frog skin, it is proposed that the subtype A of the branchial intercalated MR cell of lampreys provides the driving force for the Na⁺ uptake by active H⁺ secretion. By analogy with urinary epithelia, the subtype B cells may exchange Cl^- for HCO₃ ^-.     

Cell renewal in the epidermis of the loach Misgurnus anguillicaudatus (Cypriniformes)

Cell kinetics of the epidermal cells of normal juvenile loach ( Misgurnus anguillicaudatus ) were studied with autoradiography. Fish were labelled with single tritiated thymidine injections and killed at regular time intervals. Three cell types are identified by light microscopy, namely the epithelial cells, the club cells and the mucous cells. Epithelial cells are the only cell type that is involved in cell proliferation and, like the epithelial cells in the epidermis of other teleosts, proliferation of these cells occurs at all epidermal layers. The club cells and the mucous cells seem to be differentiated from the epithelial cells. Based on the time-course study of the labelling index and the grain count halving method, the generation time of the epithelial cells is estimated to be 4 days. From the labelling index of double injections, the duration of the S phase is determined as 8.3 h. Significant cell loss from the outermost layer and cell translocation from the lower layer to the upper layer within 4 days are inferred from the fluctuations of the labelling index curve. The renewal of these cells in the tissue seems rapid in comparison to the epidermis of terrestrial vertebrates.     

Cellular responses in the skin of carp maintained in organically fertilized water

Carp were maintained for a month in well-oxygenated water fertilized with organic manure. During the experiment the thickness of the epidermis increased from 140 to 180 urn. Fish from pulluted water were dark, an adaptation to the dark, turbid water. The number of cytoplasmic extensions from the dermal pigment cells increased continuously from 6 per unit length in control specimens to over 80 in the experimental specimens at the end of the month. Apart from background adaptation, this activity of the pigment cells may be a stress reaction. Holocrine secretion of mucous cells was pronounced, with a progressive reduction on the first day after the transfer, to almost total disappearance of this cell type from the epidermis after 3 days. A thick mucous coat became visible on the outside of the epidermis. Eight days after the transfer, a slightly subnormal mucous cell count was observed, indicating the development of newly differentiated mucous cells. This subnormal cell count lasted until the end of the experiment. The pavement cells actively secreted glycocalyx, while newly differentiated pavement cells with still-intact secretory granules replaced the exhausted cells at the epidermis surface throughout the experimental period. Granulocytes, both baso- and neutrophilic, as well as macrophages, infiltrated the epidermis; despite the high bacterial count in the water, no bacteria were observed either inside the skin or entangled in the mucous coat.     

THE UPTAKE OF IRON IN RABBIT SYNOVIAL TISSUE FOLLOWING INTRA-ARTICULAR INJECTION OF IRON DEXTRAN : A Light and Electron Microscope Study

Iron dextran (molecular weight 7,000) diffuses rapidly from the joint cavity through the synovium, along lymphatics and extracellular tissue spaces; articular cartilage is impermeable to iron dextran. There is also rapid cellular uptake by synovial lining cells, particularly of the vacuolar type; endoplasmic reticulum-containing lining cells rarely take up iron dextran. Cellular uptake is probably effected by pseudopodial folds projecting from the cell surface and enclosing extracellular material. Cells containing iron may degenerate and be ingested by phagocytes, and this may account for the concentration of iron in a smaller proportion of cells on or below the synovial surface in the later stages. At 6 to 18 hours after injection there is a mild inflammatory reaction and some synovial proliferation; from this stage onwards intracellular iron occurs in the form of haemosiderin. Granules of haemosiderin are present in the synovium 3 months after injection and possibly longer.     

Iron homeostasis in the lung

Iron is essential for many aspects of cellular function. However, it also can generate oxygen-based free radicals that result in injury to biological molecules. For this reason, iron acquisition and distribution are tightly regulated. Constant exposure to the atmosphere results in significant exposure of the lungs to catalytically active iron. The lungs have a mechanism for detoxification to prevent associated generation of oxidative stress. Those same proteins that participate in iron uptake in the gut are also employed in the lung, to transport iron intracellularly and sequester it in an inactive form within ferritin. The release of metal is expedited (as transferrin and ferritin) from lung tissue to the respiratory lining fluid for clearance by the mucocilliary pathway or to the reticuloendothelial system for long-term storage. This pathway is likely to be the major method for the control of oxidative stress presented to the respiratory tract.     

Waterborne zinc alters temporal dynamics of guppy Poecilia reticulata epidermal response to Gyrodactylus turnbulli (Monogenea)

The present study assessed the histological changes in the epidermis of Poecilia reticulata induced by the combined effects of an ectoparasite Gyrodactylus turnbulli and differing concentrations of waterborne zinc (Zn). Infected guppies were exposed to 0, 15, 30, 60, or 120 μg Zn l-1 and monitored over 3 wk during the exponential increase in parasite numbers on the fish. The fish epidermis responded within 3 d to G. turnbulli infection with a rapid increase in epidermal thickness and a modest increase in number, but not size or composition, of mucous cells. In contrast, in the presence of combined waterborne Zn and infection, mucous cell numbers declined rapidly. As the parasite numbers increased, the epidermis remained thicker than normal, and the number and size of mucous cells decreased. The addition of Zn led to a dramatic thickening of the epidermis during the exponential growth of the parasite population. Mucous cell numbers remained depressed. Temporal changes in mucous cell size were Zn concentration dependent. At 60 μg Zn l-1, cells returned to normal size as infection progressed, whereas they remained extremely small at 120 μg Zn l-1. Changes in mucin composition previously reported in response to Zn alone were subdued in the presence of the parasite except at 60 μg Zn l-1, where all cells contained only acidic mucins. Together these results demonstrate that, on exposure to both Zn and G. turnbulli infection, the epidermal response is initially a protective response to both stressors, and then mainly driven by the increased parasite burden.