Cellular cadmium distribution in the common winkle,Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Summary Various tissues of common winkles,Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Autometallographical localisation of Cu and Zn within target cell compartments of winkles following exposure to Cu&Zn mixtures

This investigation attempts to determine the usefulness of autometallography to localise particular metals in certain key tissues of molluscs exposed to metal mixtures. For this purpose, winkles (Littorina littorea) removed from shell were exposed to very high concentrations of either copper (Cu), zinc (Zn) or a mixture of both metals (Cu&Zn) dissolved in sea-water for short periods of time. Protein-bound metals were detected by autometallography as black silver deposits (BSD) on histological sections of gills, foot, mantle, digestive gland/gonad complex, stomach and kidney. Copper was localised within cytoplasmic granules of gill ciliated cells, nephrocytes and stomach epithelial cells as well as within digestive cell lysosomes. Zinc was essentially found in the basal lamina (histological sense) of gill, stomach, kidney and digestive gland epithelia. BSD were also evidenced in cytoplasmic granules of pore cells present in parenchymal connective tissue of mantle, foot, gill, digestive gland and stomach. Copper and zinc concentrations were additionally calculated for the whole soft body as well as for certain organs by atomic absorption spectrophotometry (AAS). According to AAS, a synergistic phenomenon would contribute to increase the rate of Cu and Zn accumulation in presence of each other. However, after exposure to Cu&Zn autometallography did not evidence any synergistic phenomenon, and Cu and Zn were localised in their respective accumulation sites. In conclusion, autometallography might indicate the presence of certain metals in the environment irrespective of factors, such as "metal-metal interaction-like" phenomena, affecting metal concentrations in soft tissues.     

Cadmium bioaccumulation and detoxification in the gill and digestive gland of the Antarctic bivalve Laternula elliptica

Exposure to a sublethal concentration of cadmium (Cd; 50 microg L(-1)) resulted in significantly increased Cd concentrations in the gill and digestive gland of the Antarctic bivalve Laternula elliptica. Continuous accumulation of Cd in the two organs during the 14-day exposure period was associated with sequestration of Cd to both the soluble cytosolic and insoluble particulate cell fractions. However, the contribution of each cell fraction to Cd sequestration differed between the two organs; in the gill, a larger portion of Cd was associated with the insoluble fraction, while in the digestive gland, both the soluble and insoluble fractions sequestered similar amounts of Cd. Metal-binding components in the insoluble cell fraction were not identified in this study. On the other hand, a metallothionein-like protein (MTLP) was the major Cd-detoxifying component in the soluble cell fraction of the gill and digestive gland. The amount of MTLP increased linearly with exposure time and the amount of Cd accumulated in the tissue, which suggests a potential utility of MTLP as a biomarker for exposure to Cd and possibly other metals.     

Heavy Metal Accumulation and Distribution in Organs of the Mussel Crenomytilus grayanus from Upwelling Areas of the Sea of Okhotsk and the Sea of Japan

The subcellular and cytosolic distribution of Zn, Fe, Ni, Cu, Mn, Cd, and Pb in the digestive gland and kidney of the mussel Crenomytilus grayanus from upwelling areas of the Sea of Okhotsk and the Sea of Japan was studied. Cd, Zn, Pb, and Ni have accumulated in the kidneys of mussels from these areas. When the concentrations of both essential and toxic metals in the mussel organs had increased significantly, their redistribution into the cytosol took place. Gel chromatography of cytosolic proteins in the kidneys of mussels sampled in the area with a strong stationary upwelling revealed metallothioneins. This is uncommon for bivalve mollusks from unpolluted areas. High molecular weight proteins act as preliminary ligands for Cd.     

Cellular cadmium distribution in the common winkle, Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Various tissues of common winkles, Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Comparative Analysis of Subcellular Distribution of Heavy Metals in Organs of the Bivalve Mollusks <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> and <Emphasis Type="Italic">Modiolus modiolus</Emphasis> in a Continuously Polluted Environment

The distribution of Zn, Fe, Ni, Cu, Mn, Cd, and Pb in subcellular fractions, and of Cd, Zn, and Cu in cytoplasm proteins of the kidney and digestive gland of the mussels Crenomytilus grayanus and Modiolus modiolus, sampled from contaminated and conditionally clean areas, was studied. It was found that, in a contaminated environment, the organs of mussels were more highly enriched with metals. It was shown that essential trace elements were accumulated mostly in the cytosol of organs of both molluscan species from contaminated areas, whereas in the background areas the trace elements were associated mostly with membrane structures in Gray's mussel, C. grayanus, and with the cytosol in M. modiolus, the northern horse mussel. The lead was bound mostly to membrane structures in organs of both mussel species at all stations. The method of gel chromatography enabled us to isolate metallothionein-like proteins from the kidney of the northern horse mussel sampled in contaminated areas, whereas their concentration in the kidney of Gray's mussels was lower than the limiting error of the method. It is supposed that in the kidney of Gray's mussel the synthesis of metallothionein-like proteins was quenched by the integrated effect of the accumulated metals.     

Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.     

Effects of dibutyl phthalate and di‐ethylhexyl phthalate on acetylcholinesterase activity in bagrid catfish,Pseudobagrus fulvidraco (Richardson)

Acetylcholinesterase (AChE) activity is a well‐known biomarker for exposure to organophosphate or carbamate compounds in aquatic organisms. However, the effect of dibutyl phthalate (DBP) and di‐ethylhexyl phthalate (DEHP), widely used as a plasticizer, on the change of AChE activity is not yet known. Bagrid catfish Pseudobagrus fulvidraco were administrated with 100, 500 and 1000 mg kg^?1 diet of DBP or DEHP and the effects on AChE activity were assessed in the liver, gill, kidney, heart, brain, muscle and eye of the exposed fish. All tissues contained different background AChE activity in non‐treated bagrid catfish: the highest was observed in the brain, followed by muscle, heart, and kidney. The enzyme activities in various tissues were significantly inhibited after exposure to DBP or DEHP in a concentration‐dependent manner, especially in brain and muscle. A similar, but less pronounced, inhibition was also observed in liver and kidney when exposed to DBP and DEHP. Although AChE activity in gill and heart was also affected by DBP and DEHP, the decrease in these organs was least marked in these organs. Exposure to 1000 mg kg^?1 led to mortalities of 8.0% with DBH and 14% with DEHP; both seemed to be ascribable to phthalate toxicity. This study is the first report that the measurement of AChE activity in bagrid catfish is a valuable biomarker of DBP and DEHP exposure. This biomarker could be incorporated into a battery of biomarkers to strengthen the confidence with which ecotoxicologists can assess the impact of phthalate ester pollution in the aquatic environment.     

Autometallographic localization of protein-bound copper and zinc in the common winkle,Littorina littorea: A light microscopical study

Summary Copper (Cu), zinc (Zn) and calcium (Ca) were demonstrated histochemically by means of conventional stains (rubeanic acid for copper, dithizone for zinc, and cobalt nitrare for calcium) and by autometallography in various tissues of winkles (Littorina littorea) sublethally exposed to either copper or zinc dissolved in sea water. Rubeanic acid and dithizone procedures exhibited poor sensitivity: there was no positive reaction after fixation tissues with Bouin's fixative, and only a weak reaction after ethanol fixation. Autometallography, however, produced a positive reaction with both fixatives in the form of black silver deposits in some key cell types. In winkles not exposed to either copper nor zinc, autometallographically demonstrated metals were found in the connective tissue pore cells, the lysosomes of digestive cells, the basal lamina of the digestive tubule epithelium, and cytoplasmic granules in the epithelial cells of the stomach wall. In addition, in winkles exposed to copper, metal deposits were present in some apical cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, and the circulating haemocytes. In winkles exposed to zinc, metal deposits were found in the basal cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, the apex and basal lamina of the nephrocytes in the kidney, and the connective tissue layer surrounding the blood vessels. Additionally, calcium was demonstrated histochemically in the cytoplasm of digestive cells, the cytoplasm of the epithelial cells of the stomach wall, the mucocytes of gills, the basal lamina of the kidneys, the haemocytes, the calcium and pore cells of connective tissue, and the oocyte cytoplasm. Metals were not detected by any procedure in sperm cells, in the cytoplasmic granules of oocytes, or in the basophilic cells in the digestive tubules. In conclusion, autometallography is a highly sensitive method and provides an excellent tool to localize protein-bound copper and zinc in molluscan tissues, and its use in combination with conventional histochemical or chemical methods is highly recommended.     

Localization of Metals in the Gastropod Littorina saxatilis (Prosobranchia: Littorinoidea) from a Polluted Site

Organ heavy metal levels, and ultrastructural localization were examined in the marine prosobranch mollusc Littorina saxatilis from a metal-contaminated site. Copper and zinc are localized in the digestive gland, stomach and kidney. The digestive gland does not appear to be a significant site of iron accumulation. High levels of copper and zinc in the stomach may indicate significant uptake from the food. Relatively high heavy metal levels in several organs were reflected in the presence of a variety of metal-containing aggregates (granules) within the tissues. In common with other invertebrates two broad classes of granule were present. The first appears to be proteinaceous, with a high sulphur content. This type contained copper and iron and was found in the stomach, kidney, pore cells, and rectum. The second type was inorganic and mineralized, containing phosphorus. They contained calcium, zinc and manganese, and were observed in the kidney and digestive gland. Differences in the number of metals were observed between the various granule types, possibly reflecting specific pathways involved in metal sequestration.     

Compensatory regulation of acid–base balance during salinity transfer in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In seawater-acclimated rainbow trout (Oncorhynchus mykiss), base secretion into the intestine is a key component of the intestinal water absorption that offsets osmotic water loss to the marine environment. Acid–base balance is maintained by the matched excretion of acid equivalents via other routes, presumably the gill and/or kidney. The goal of the present study was to examine acid–base balance in rainbow trout upon transfer to more dilute environments, conditions under which base excretion into the intestine is predicted to fall, requiring compensatory adjustments of acid excretion at the gill and/or kidney if acid–base balance is to be maintained. Net acid excretion via the gill/kidney and rectal fluid, and blood acid–base status were monitored in seawater-acclimated rainbow trout maintained in seawater or transferred to iso-osmotic conditions. As predicted, transfer to iso-osmotic conditions significantly reduced base excretion into the rectal fluid (by ~48%). Transfer to iso-osmotic conditions also significantly reduced the excretion of titratable acidity via extra-intestinal routes from 183.4 ± 71.3 to −217.5 ± 42.7 μmol kg⁻¹ h⁻¹ (N = 7). At the same time, however, ammonia excretion increased significantly during iso-osmotic transfer (by ~72%) so that the apparent overall reduction in net acid excretion (from 419.7 ± 92.9 to 189.2 ± 76.5 μmol kg⁻¹ h⁻¹; N = 7) was not significant. Trout maintained blood acid–base status during iso-osmotic transfer, although arterial pH was significantly higher in transferred fish than in those maintained in seawater. To explore the mechanisms underlying these adjustments of acid–base regulation, the relative mRNA expression and where possible, activity of a suite of proteins involved in acid–base balance were examined in intestine, gill and kidney. At the kidney, reduced mRNA expression of carbonic anhydrase (CA; cytosolic and membrane-associated CA IV), V-type H⁺-ATPase, and Na⁺/HCO₃ ⁻ co-transporter were consistent with a reduced role in net acid excretion following iso-osmotic transfer. Changes in relative mRNA expression and/or activity at the intestine and gill were consistent with the roles of these organs in osmotic rather than acid–base regulation. Overall, the data emphasize the coordination of acid–base, osmoregulatory and ionoregulatory processes that occur with salinity transfer in a euryhaline fish.     

A comparison of the sequestration of cadmium and zinc in the tissues of rainbow trout (Salmo gairdneri) following exposure to the metals singly or in combination

Rainbow trout were exposed to either cadmium (9 micrograms/l) or zinc (100 micrograms/l) in their aquarium water. They were then transferred to water containing concentrations of cadmium (54 micrograms/l) that would have otherwise proved fatal to the majority of the fish without the pretreatment. Most of the fish survived under both sets of conditions. However, two different mechanisms seem to be involved in the protection of the animals against the toxic manifestations of cadmium. In both cases, more than 99% of the total body load of cadmium was found in the liver, kidney and gills of the animals. Analysis of the metal-binding proteins in these organs was carried out. In the fish exposed to the two concentrations of cadmium, the toxic metal was found only in association with two low mol. wt specific binding proteins despite the presence of zinc- (and copper)-containing isometallothioneins in all three organs. On the other hand, cadmium was distributed between these binding-proteins and metallothioneins in the liver, kidney and gill of the trout pretreated with zinc before their exposure to cadmium.     

A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.     

Comparative metabolism of phospholipids of osmoregulation effector organs in European eel (Anguilla anguilla)]

The phospholipid composition from various organs of the fresh water eel, such as gill, kidney, gut, liver and muscle, were determined by thin-layer chromatography. The major phosphatides found in these tissues were PC, PE and SPH and minor constituents PS, PI, DPG, AP and also LPC in the gut. A greater percentage of PS and SPH occurs in the osmoregulatory effector organs such as gill, kidney, and gut. From in vivo comparative kinetic studies of the 32P incorporation into the phospholipids, between 6 and 48 hours, certain remarkable features of phospholipid metabolism have been found in these tissues. A low uptake of inorganic 32P into the tissue lipid phosphorus was observed in the eel at 15 degrees C. The specific activity of the lipid phosphorus increased continuously in all tissues during 48 hours after 32P injection. During this experimental period, phosphatidic acid and phosphatidyl inositol fractions were labelled most rapidly in gill, kidney and gut, while the specific activity of the phosphatidyl choline fraction remained low in these organs. In liver, the rate of PC formation appears to be faster than the PI and PE biosynthesis. In gill and gut, the PE showed greater turnover than the PC as measured by 32P incorporation. In the eel, an euryhalin fish, the DPG of osmoregulatory effector organs has a high specific activity at all times. PS showed only a high specific activity in the gill. Labelling of SPH occured slowly in the various tissues only becoming evident after 24 hours. The results are compared with those published for other poikilotherm and homeotherm vertebrates. Relative differences between the turnover of various tissue phosphatides are discussed with of reference to the general scheme on phospholipid biosynthesis and to the physiological functions of the various organs.     

Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream (Sparus aurata) after exposure to dietary or waterborne copper

The high affinity copper transporter 1 (Ctr1), metallothionein (MT) and glutathione reductase (GR) are essential for copper uptake, sequestration and defense respectively. Following rearing on a normal commercial diet (12.6+/-0.2 mg kg(-1) Cu), sea bream were fed an experimental control diet lacking mineral mix (7.7+/-0.3 mg kg(-1) Cu), an experimental diet enhanced with Cu (135+/-4 mg kg(-1) Cu) or an experimental diet (7.7+/-0.3 mg kg(-1) Cu) whilst exposed to Cu in water (0.294+/-0.013 mg L(-1)). Fish were sampled at 0, 15 and 30 days after exposures. Fish fed the Cu-enhanced experimental diet showed lower levels of expression of Ctr1 in the intestine and liver compared to fish fed control experimental diets, whilst Ctr1 expression in the gill and kidney was unaffected by excess dietary Cu exposure. Waterborne-Cu exposure increased Ctr1 mRNA levels in the intestine and the kidney compared to experimental controls. Excess dietary Cu exposure had no effect on levels of metallothionein (MT) mRNA, and the only effect of dietary excess Cu on glutathione reductase (GR) mRNA was a decrease in the intestine. Both MT mRNA and GR were increased in the liver and gill after waterborne-Cu exposure, compared to levels in fish fed experimental control low Cu diets. Thus, Ctr1, MT and GR mRNA expression in response to excess Cu is dependent on the route of exposure. Furthermore, the tissue expression profile of sea bream Ctr1 is consistent with the known physiology of copper exposure in fish and indicates a role both in essential copper uptake and in avoidance of excess dietary and waterborne copper influx.     

Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias)

We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.     

Comparative study on the inhibition of acetylcholinesterase activity in the freshwater fish Cyprinus carpio by mercury and zinc.

The effects of sublethal concentrations of mercury (0.1mg/l) and zinc (6 mg/l) on acetylcholinesterase activity and acetylcholine content of gill, kidney, intestine, brain, liver and muscle of the freshwater fish Cyprinus carpio at 1, 15 and 30 days of exposure were studied. A significant suppression in acetylcholinesterase activity was recorded in all the organs from both mercury and zinc intoxicated fish at all the exposure periods. Concurrently, a significant increase in the content of acetylcholine in the organs was observed. These changes observed in the organs of mercury treated fish in different exposure periods were in the order 1 greater than 15 less than 30 days and in zinc treated fish 1 greater than 15 greater than 30 days. Further, these changes were greater in magnitude in the brain, liver and muscle (non-osmoregulatory organs) than in the gill, kidney and intestine (osmoregulatory organs) in both metal media.     

Localization of mitochondrial DNA encoded cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase subunits I and II in rat pancreatic zymogen granules and pituitary growth hormone granules

Cytochrome c oxidase (COX) complex is an integral part of the electron transport chain. Three subunits of this complex (COX I, COX II and COX III) are encoded by mitochondrial (mit-) DNA. High-resolution immunogold electron microscopy has been used to study the subcellular localization of COX I and COX II in rat tissue sections, embedded in LR Gold resin, using monoclonal antibodies for these proteins. Immunofluorescence labeling of BS-C-1 monkey kidney cells with these antibodies showed characteristic mitochondrial labeling. In immunogold labeling studies, the COX I and COX II antibodies showed strong and specific mitochondrial labeling in the liver, kidney, heart and pancreas. However, in rat pancreatic acinar tissue, in addition to mitochondrial labeling, strong and specific labeling was also observed in the zymogen granules (ZGs). In the anterior pituitary, strong labeling with these antibodies was seen in the growth hormone secretory granules. In contrast to these compartments, the COX I or COX II antibodies showed only minimal labeling (five- to tenfold lower) of the cytoplasm, endoplasmic reticulum and the nucleus. Strong labeling with the COX I or COX II antibodies was also observed in highly purified ZGs from bovine pancreas. The observed labeling, in all cases, was completely abolished upon omission of the primary antibodies. These results provide evidence that, similar to a number of other recently studied mit-proteins, COX I and COX II are also present outside the mitochondria. The presence of mit-DNA encoded COX I and COX II in extramitochondrial compartments, provides strong evidence that proteins can exit, or are exported, from the mitochondria. Although the mechanisms responsible for protein exit/export remain to be elucidated, these results raise fundamental questions concerning the roles of mitochondria and mitochondrial proteins in diverse cellular processes in different compartments.