Dynamics of (Cd,Zn)-metallothioneins in gills, liver and kidney of common carp Cyprinus carpio during cadmium exposure

Cadmium concentrations, (Cd,Zn)-metallothionein (MT) concentrations, MT synthesis and the relative amounts of cadmium bound to (Cd,Zn)-MTs were determined in gills, liver and kidney of common carp Cyprinus carpio exposed to 0, 0.5 microM (0.06 mg.l(-1)), 2.5 microM (0.28 mg.l(-1)) and 7 microM (0.79 mg.l(-1)) Cd for up to 29 days. Cadmium accumulation was in the order kidney > liver > gills. Control levels of hepatic (Cd,Zn)-MT were four times higher compared to those of gills and kidney. No increases in (Cd,Zn)-MT concentrations were observed in liver during the exposure period. In comparison with control carp, (Cd,Zn)-MT concentrations increased up to 4.5 times in kidney and two times in gills. In both these organs, (Cd,Zn)-MT concentrations were linearly related with cadmium tissue levels and with the de novo synthesis of MTs. Hepatic cadmium was almost completely bound to (Cd,Zn)-MT, while percentages of non-MT-bound cadmium were at least 40% in gills and 25% in kidney. This corresponded with a total saturation of (Cd,Zn)-MT by cadmium in kidney and a saturation of approximately 50 and 60% in gills and liver, respectively. The final order of non-MT-bound cadmium was kidney > gills > liver. Our results indicate that cadmium exposure causes toxic effects, which cannot be correlated with the accumulated levels of the metal in tissues. Although cadmium clearly leads to the de novo synthesis of MT and higher (Cd,Zn)-MT concentrations, the role of this protein in the detoxification process is clearly organ-specific and its synthesis does not keep track with cadmium accumulation.     

Effects of copper on cortisol receptor and metallothionein expression in gills of Oncorhynchus mykiss

Effects of waterborne Cu (2.4 microM) on the expression of glucocorticoid receptor (GR) and metallothionein (MT) in the branchial epithelium of freshwater rainbow trout (Oncorhynchus mykiss) was studied by immunocytochemistry. After 5 days of Cu exposure, the number of GR-immunoreactive (GR-ir) cells in the gill epithelium had decreased, whereas the number of MT-ir cells had increased. Localization of GR in chloride cells was achieved by double staining for Na(+)/K(+)-ATPase; other cell types were identified on the basis of their topology. GRs were present in the chloride cells in both the filaments and lamellae, in respiratory cells in the lamellae, in pavement cells, basal layer cells and undifferentiated cells in the filaments. Co-localization of Na(+)/K(+)-ATPase and MT revealed chat MT was expressed in chloride cells, both in filaments and lamellae. Occasionally, MT immunoreactivity was found in pavement cells and in undifferentiated cells. By double staining for Na(+)/K(+)-ATPase and GR, for Na(+)/K(+)-ATPase and MT and for GR and MT, we can conclude that after 5 days of Cu stress there are chloride cells that express GR and MT, GR or MT alone or neither of the two proteins. This apparent functional heterogeneity of branchial chloride cells may reflect a limited window when chloride cell subpopulations show an adaptive response to Cu.     

Significance of increase in urinary metallothionein of rats repeatedly exposed to cadmium

Cadmium chloride was injected subcutaneously (s.c.) into female Wistar rats at a dose of 1 mg Cd/kg body weight, 5 times a week up to 10 weeks. At specified intervals, 24-h urine was collected and the excreted amounts of metallothionein (MT), cadmium, copper, zinc and several indicators of renal damage were determined. Concentrations of cadmium and MT in the livers and kidneys of rats were also determined. Both cadmium and MT in the livers and kidneys were increased upon cadmium exposure. The urinary MT excretion started to increase within a week after the start of exposure. This increased excretion preceded those of enzymes and total protein as well as histopathological abnormalities in the proximal tubular cells. After the occurrence of tubular damage that disturbs reabsorption of MT, MT in urine was drastically increased. These results indicate that urinary MT levels may be an indicator not only of cadmium exposure but also of tubular damage.     

Combined nephrotoxicity of methylmercury, lead, and cadmium in Pekin ducks: metallothionein, metal interactions, and histopathology

This report describes the metallothionein (MT) levels and accumulation of mercury, lead, and cadmium, as well as their interaction with tissue zinc, copper, and iron, and the histopathological changes in kidneys of ducks exposed to methylmercury chloride (MeHgCl), lead acetate (PbAc), and cadmium chloride (CdCl2), singly or in combination for 13 wk. Forty-eight female Pekin ducks, divided into 8 groups of 6 birds each, were fed diets containing no added metals (control), 8 mg MeHgCl/kg feed, 80 mg PbAc/kg feed, 80 mg CdCl2/kg feed, 8 mg MeHgCl + 80 mg PbAc/kg feed, 8 mg MeHgCl + 80 mg CdCL2/kg feed, 80 mg PbAc + 80 mg CdCl2/kg feed, and 8 mg MeHgCl + 80 mg PbAc + 80 mg CdCL2/kg feed. Cadmium (Cd) when administered alone or in combination caused a 60-fold increase in kidney MT levels, while methylmercury (MeHg) or lead (Pb) administration caused a threefold increase in kidney MT levels. No significant changes in kidney MT levels were observed when metals were administered concurrently when compared with single-treatment groups. Residue analysis revealed accumulation of administered metals in kidney tissue. However, lead administration resulted in accumulation of small amounts of this element in kidney tissue. Simultaneous administration of MeHgCl and PbAc significantly increased the accumulation of lead in kidney when compared with PbAc-treated group. Cadmium when administered alone or in combination caused an increase in the levels of zinc and copper in kidney. Administration of MeHgCl or PbAc either alone or in combination caused increased iron levels in kidney, while cadmium administration either alone or in combination caused decreased iron levels. Administration of cadmium either alone or in combination caused degenerative changes in kidney proximal tubules. The severity of degenerative lesions increased when cadmium was simultaneously administered with other metals. These results indicate that combined administration of MeHg, Pb, and Cd has no significant effect on kidney MT levels or on essential elements in kidney tissue when compared with single metal groups. However, there appears to be an increase in the severity of histopathologic changes.     

Cadmium tolerance in the Nile tilapia (Oreochromis niloticus) following acute exposure: assessment of some ionoregulatory parameters

The Nile tilapia (Oreochromis niloticus) can tolerate very high levels of waterborne cadmium. It has one of the highest 96 h LC50 recorded for a freshwater teleost fish (14.8 mg/L Cd; hardness 50 mg/L CaCO(3)). Cadmium is known to perturb ion balance in teleost fishes. However, in an acute time course experiment, plasma Na(+) concentrations were unaffected, and plasma Ca(2+) values only decreased after 96 h exposure in a dose-independent manner. Branchial Na(+)/K(+)-ATPase activity and alpha-subunit protein level expression in crude gill homogenates were not affected by Cd exposure during this period. Branchial chloride cell numbers, identified as Na(+)/K(+)-ATPase immunoreactive cells using immunohistochemistry, decreased 24 h after exposure but recovered thereafter. Histopathological changes did not follow a consistent pattern of variation with exposure time, and the alterations noted in gill epithelium were basically nonspecific to cadmium. Because of its tolerance, it can be concluded that the tilapia O. niloticus would not be a suitable test organism to evaluate sublethal toxicity of cadmium and the realistic impact of this pollutant in the environment. However, it certainly could contribute significantly to our understanding of the toxic mechanism of cadmium exposure in aquatic organisms. This is the first work to investigate the effect of waterborne pollutants on Na(+)/K(+)-ATPase alpha-subunit protein expression in fish gills.     

Morphological and biochemical changes in the gills of Tilapia (Oreochromis mossambicus) to ambient cadmium exposure

Tilapia, Oreochromis mossambicus, were reared in freshwater and exposed to 40, 80 and 160 ppb ambient cadmium for a period of 7 days to investigate the effects of short-term Cd exposure on the cellular function of gill chloride cells. Gills were sampled after 3 and 7 days exposure. The accumulated Cd concentration in gills was analyzed using a graphite furnace atomic absorption spectrophotometer while the morphological changes of pavement and chloride cells were examined using scanning and transmission electron microscopes. Gill Cd concentration was significantly increased in samples (10.03-44.36 ppb) at 3 and 7 days exposure. Accompanying this was an augmentation of microridge in pavement cells, and an increase in the density (1964-3603 /mm(2)) and apical membrane area (11.57-46.32 μm(2)) in chloride cells, indicating an adaptational modification of the cell morphology in assisting gaseous transfer and Ca(2+) uptake, respectively. However, biochemical analyses of the gill tissues enumerated a decrease in both the activities of alkaline and high-affinity Ca(2+)-ATPases. This indicated a reduction in the Ca(2+)-transport capacity per unit chloride cell, suggesting chloride cells being the primary target of Cd which subsequently lead to fish hypocalcemia.     

Cadmium influx and efflux across perfused gills of the shore crab, Carcinus maenas

Cadmium influx across perfused gills of the shore crab Carcinus maenas exposed to 9.0 μM cadmium in the external medium ranged from 0.6 to 2.0 nmol Cd(2+) g(-1) gill wet wt. per h. Cadmium efflux across perfused gills exposed to 9.0 μM cadmium in the internal medium ranged from 0.3 to 0.9 nmol Cd(2+) g(-1) gill wet wt. per h. There was no significant difference between cadmium influx and efflux across the gills. Cadmium influx across the gills was not detectable after exposure to lanthanum in the external medium, whereas cadmium efflux was unaffected by external as well as internal lanthanum. Cadmium influx was not affected when the internal medium was changed to Na-free medium, whereas efflux showed a fourfold increase when the gills were perfused with Na-free medium. Low pH in the external medium did not exert any significant effect on cadmium influx and efflux.     

Induction and binding of Cd, Cu, and Zn to metallothionein in carp (Cyprinus carpio) using HPLC-ICP-TOFMS.

The binding of Cd, Cu, and Zn to metallothionein in carp was studied under control and acute Cd exposure scenarios. Carp were exposed to different Cd concentrations for 96 h. Total (Cu, Cd, Zn)-MT levels were determined by the cadmium thiomolybdate saturation assay. Total tissue and cytosolic Cd, Cu, and Zn concentrations were determined by ICP-MS. The cytosolic metal speciation was determined by high pressure liquid chromatography (size-exclusion [SE] in combination with anion exchange [AE]) directly coupled to an inductively coupled plasma time of flight mass spectrometer (ICP-TOFMS). This coupled technique allows the chromatographic separation and online determination of the metals associated to the protein fractions separated. Very strong differences in the tissue compartmentalization and cytosolic speciation of the metals were observed. For example, over 30% of cytosolic zinc was bound to MT in liver while this was only 2% in the kidneys although total cytosolic levels were considerably higher. Induction of metallothionein during cadmium exposure was also tissue specific, displaying different response patterns in gills, liver, and kidney. Cadmium accumulated much stronger in liver and kidney compared to the gills and the latter also showed much lower MT levels. The renal MT-induction was more sensitive to Cd exposure than the hepatic MT induction since a significant increase of Cd-MT and total MT levels occurred at lower tissue Cd concentrations in the kidney in comparison to the liver, except for the highest Cd exposure level where a drastic 10-fold increase in hepatic Cd-MT was observed. At this Cd exposure level also an apparent spill over of zinc to the high molecular weight fraction was observed in the kidneys.     

Influence of cadmium-metallothionein pretreatment on tolerance of rat kidney cortical cells to cadmium toxicity in vitro and in vivo

Kidney cells were isolated from rats pretreated by daily subcutaneous doses of cadmium metallothionein (CdMT: 0.05-0.2 mg Cd/kg X 5) and from non-pretreated rats. Upon exposure to CdCl2 in vitro (0-200 micrograms Cd/ml), a concentration dependent decrease in viability was observed in the non-pretreated cells, while no such decrease occurred in the pretreated cells indicating that these cells were more resistant to the toxic action of cadmium. There was a higher in vitro uptake of Cd+2 and an increased metallothionein (MT) concentration in the pretreated cells (compared to non-pretreated cells). Subcellular distribution studies revealed that Cd was mainly recovered in the "cytosol" fraction. The higher total cadmium uptake in pretreated cells corresponded to an increase of Cd in "cytosol" and "nuclear" fractions. This observation may be explained by MT-binding of Cd in the cells and is in accordance with a possible protective effect of induced MT in the pretreated cells. In order to assess whether pretreatment-induced tolerance to cadmium toxicity--indicated by the cellular studies--could also be observed in vivo, some whole animal experiments were also performed. A dose-related proteinuria was observed in non-pretreated rats after a single subcutaneous administration of 109Cd-MT at doses of 0.05 and 0.4 mg Cd/kg. Urinary total Cd, 109Cd and MT was also increased in a dose-related fashion. Cadmium concentrations in kidney were dose related and reached 19 micrograms/g wet weight. In contrast, in animals repeatedly pretreated with CdMT according to 1), no proteinuria was observed after administration of the same single doses of 109CdMT. Total Cd. 109Cd and particularly MT-concentrations in urine were lower in such pretreated animals than in in non-pretreated ones in spite of the accumulation of higher tissue concentrations of total Cd (up to 80 micrograms/g). The pretreatment was thus shown to prevent some of the acute nephrotoxicity of CdMT, possibly by means of induction of MT synthesis.     

Effects of dietary cadmium exposure on tissue-specific cadmium accumulation, iron status and expression of iron-handling and stress-inducible genes in rainbow trout: influence of elevated dietary iron

Recent evidences suggest that dietary cadmium (Cd) uptake likely occurs via the dietary iron (Fe) uptake pathway in freshwater fish, at least in part. The present study investigated the interactive effects of dietary Cd and Fe in juvenile rainbow trout (Oncorhynchus mykiss). Fish were treated for four weeks with four different diets: normal Fe, high Fe, normal Fe plus Cd, and high Fe plus Cd. Physiological parameters, tissue-specific Fe and Cd level, plasma Fe status, and tissue-specific mRNA expression of transferrin, metallothioneins (MT-A and MT-B) and heat shock proteins 70 (HSP70a and HSP70b) were analyzed. Exposure to dietary Cd increased Cd burden in the following order: intestine>kidney>stomach>liver>gill>carcass. Interestingly, high dietary Fe reduced Cd accumulation in the stomach and intestine as well as in the wholebody of fish. Dietary Cd increased hepatic transferrin mRNA expression and total Fe binding capacity in the plasma, indicating the effect of Cd on Fe handling in fish. The mRNA expression of MTs and HSP70s was also increased in various tissues following dietary Cd exposure, however the response profile of different MT and HSP70 genes was not consistent among different tissues. In general, MT-A was more responsive to Cd exposure in the intestine and liver, whereas MT-B was more responsive in the kidney. Similarly, HSP70a expression was more sensitive to Cd exposure than HSP70b, particularly in the intestine. Interestingly, high Fe diet suppressed Cd-induced induction of transferrin, MT and HSP70 genes in various tissues. Overall, our study suggests that elevated dietary Fe can reduce Cd accumulation and ameliorate Cd-induced stress responses in freshwater fish.     

Metallothionein overexpression and resistance to toxic stress

Metallothionein (MT) protects against the harmful effects of a wide spectrum of stress factors. The most studied of these factors is cadmium, whose toxicity is reduced on sequestration by MT. However, there is poorer consensus in the literature about protection afforded by MT against stressors other than cadmium. In this study, a CHO-K1 cell line continuously overexpressing MT (MToex) was developed in order to evaluate the relative protection afforded by MT against different toxic agents. Cadmium was used as a positive control and, as expected, the MToex cells were more than 13-fold more resistant to the effects of cadmium chloride than were wild-type (WT) cells using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay (IC50 values of 10 and 132 microM for WT and MToex cells, respectively). In contrast, overexpression of MT afforded no protection against mercuric chloride, staurosporine and hydrogen peroxide (IC50 values of about 50, 11 and 925 microM, respectively). Cd and Hg uptake by MToex and WT cells exposed to 1-10 microM of metal chloride was similar and yet a significant amount of these metals was associated with the cytosol MT fraction in the MToex cells but not in the WT cells. From this study it can be concluded that while MT overexpression protects against Cd toxicity, it has no influence on Hg, staurosporine or hydrogen peroxide toxicity and it is proposed that this reflects mechanistic differences of toxicity or depletion of labile intracellular zinc by the presence of excess binding ligand in the form of MT.     

Induction of hepatic metallothionein in the immature rat following administration of cadmium

Hepatic metallothionein (MT) levels, as measured indirectly by total metal-binding capacity, are approximately 8.5-fold higher in the 7-day-old rat than in the 28-day-old rat where levels are barely detectable. To stydy how the presence of MT might influence the toxicity of cadmium, a single subcutaneous injection of cadmium chloride was administered at dose levels of 0, 1, 2, 3, and 6 mg Cd/kg body weight, 48 hr prior to sacrifice on Day 7 or Day 28. In both the 7- and 28-day-old groups, there was a dose-related increase in the amount of cadmium bound to MT. There were no significant age-related differences in the amount of cadmium bound to MT at the various doses, with the exception of the 6 mg/kg dose, where 7 day levels were higher. The 28-day-old rats responded to cadmium exposure with induction of MT and subsequent binding of both cadmium and zinc at all doses. The 7-day-old appeared to have sufficient levels of MT to handle cadmium doses at or below 3 mg/kg without induction; however, at the 6 mg/kg dose induction was observed. Despite the presence and inducibility of hepatic MT at Day 7, 30% of the rats treated with 6 mg Cd/kg died within 48 hr of exposure compared with only 4% at Day 28.     

Effects of long term sublethal Cd exposure in rainbow trout during soft water exposure: implications for biotic ligand modelling

The objectives of the study were to determine the physiological and toxicological effects of chronic cadmium exposure on juvenile rainbow trout in soft water. Particular attention focused on acclimation, on comparison to an earlier hard water study, and on whether a gill surface binding model, originally developed in dilute soft water, could be applied in this water quality to fish chronically exposed to Cd. Juvenile rainbow trout, on 3% of body weight daily ration, were exposed to 0 (control), 0.07, and 0.11 microg l(-1) Cd [as Cd(NO(3))(2).4H(2)O] in synthetic soft water (hardness=20 mg l(-1) as CaCO(3), alkalinity=15 mg l(-1) as CaCO(3), pH 7.2) for 30 days. Mortality was minimal for all treatments (up to 14% for 0.11 microg l(-1) Cd). No significant effects of chronic Cd exposure were seen in growth rate, swimming performance (stamina), routine O(2) consumption, or whole body/plasma ion levels. In contrast to the hard water study, no acclimation occurred in either exposure group in soft water, with no significant increases in 96-h LC(50) values. Cadmium accumulated in a time-dependent fashion to twice the control levels in the gills and only marginally in the liver by 30 days. No significant Cd accumulation occurred in the gall bladder or whole body. Cadmium uptake/turnover tests were run using radioactive 109Cd for acute (3 h) exposures. Saturation of the gills occurred for control fish but not for Cd-exposed fish when exposed to up to 36 microg l(-1) Cd for 3 h. Cd-exposed trout accumulated less 'new' Cd in their gills compared to controls and they internalized less 109Cd than control fish. This effect of lowered Cd uptake by the gills of acclimated trout was earlier seen for the fish acclimated to 10 microg l(-1) Cd in hard water. The affinity of the gill for Cd was greater in hard water (logK(Cd-gill)=7.6) than in soft water (logK(Cd-gill)=7.3) but the number of binding sites (B(max)=0.20 microg g(-1) gill) was similar in both media. In addition, there was a shift in affinity of the gill for Cd (i.e. lowered logK(Cd-gill)) and increased B(max) with chronic Cd exposure in both soft water and hard water. We conclude that the present gill modelling approach (i.e. acute gill surface binding model or Biotic Ligand Model) does work for soft and hard water exposures but there are complications when applying the model to fish chronically exposed to cadmium.     

Effects of Zn pre-exposure on Cd and Zn bioaccumulation and metallothionein levels in two species of marine fish

Zinc is an essential trace metal but also a potential toxicant to aquatic organisms. In this study, two juvenile marine fish species, the black sea bream Acanthopagrus schlegeli and the grunt Teraponjarbua, were pre-exposed to Zn either from waterborne (0.74-170 microg L-1) or dietary (39-5926 microg g-1) Zn for 1 or 3 weeks. The concentrations of Zn and metallothionein (MT) in the whole body of the black sea bream and in the gills, viscera and carcass of the grunt were then measured during this pre-exposure. Following the pre-exposure, both fish species were then exposed to 109Cd and 65Zn labeled food or water to quantify the dietary assimilation efficiency (AE) and the uptake rate of dissolved Cd and Zn. Zn concentrations in both fish species were enhanced after pre-exposure, but the increases were much less than the increase of ambient Zn pre-exposure concentration. Following Zn pre-exposure, MT concentrations in the viscera and carcass were significantly elevated, whereas the MT levels were not significantly elevated in the gills. Waterborne and dietary Zn exposure enhanced the uptake rates of dissolved Cd and Zn in both fish. The maximum increases of uptake rate constants of dissolved Cd and Zn were up to 1.9-2.8 and 2.1-2.6 times, respectively, in the seabream and grunt. In contrast, dietary assimilation efficiency of Cd and Zn was not significantly enhanced following Zn pre-exposure. A positive linear relationship was found between the uptake rate constants of dissolved metals and Zn or MT concentrations in the fish. The results suggested that Zn pre-exposure increased the potential of metal uptake from ambient water, but had little effect on dietary metal uptake. Furthermore, the Zn body concentration and metal uptake from the dissolved phase were significantly dependent on the fish body size.     

Influence of long-term exposure to dietary cadmium on growth, maturation and reproduction of goldfish (subspecies: Prussian carp Carassius auratus gibelio B.)

The influence of long-term exposure of goldfish to dietary cadmium (Cd) on its accumulation in tissues, growth, ovarian development, luteinizing hormone (LH) secretion and a response to hormonal stimulation of spawning were evaluated. The study was conducted on four groups of females for the period of 3 years, from the age of 10 weeks to second spawning. Four doses of Cd were applied in the feed: 0 (control group), 0.1, 1 and 10 mg Cd g(-1) of feed (wet weight). The highest dose of Cd (10 mg g(-1)) inhibited growth and caused several behavioural effects. In contrast, lower dose of Cd (1 mg g(-1)) stimulated fish growth. The doses of Cd from 0.1 to 1 mg Cd g(-1) did not influence ovarian development. The gonado-somatic index (GSI) and histological analysis of ovaries showed no differences in ovarian development between the control group and the groups receiving these doses of Cd. However, in the group receiving the highest Cd dose, GSI decreased. This was associated with persistent, long-lasting elevation of plasma LH levels. Ovulation did not occur in this group. Injections of salmon GnRH-analogue (sGnRHa) alone or with domperidone (a dopamine receptor antagonist) in sexually mature fish caused an increase of LH levels in all groups, although in the group fed with the highest Cd dose the effect was weaker than in the other groups. After the first spawning season, a negative effect of lower Cd doses (0.1 and 1mg Cd g(-1)) on ovarian recrudescence (rebuilding of ovaries) and on the response to the consecutive hormonal stimulation of spawning was observed (lower number of ovulating females). There was a significantly higher content of Cd in the livers of fish than in their muscles. The results of hormonal stimulation of spawning and histological analysis of ovaries suggest that in goldfish cadmium acts mainly at the level of ovary rather than on the pituitary gland. We suppose that in the natural environment cadmium present in the feed can play an important role in the accumulation of this element in fish tissues and can influence vital physiological processes.     

Hepatic and renal metallothionein induction by an oral equimolar dose of zinc, cadmium or mercury in mice.

The hepatic and the renal subcellular distribution of zinc, cadmium or mercury and induction of tissue metallothionein (MT) at 24, 48 and 72 h following an oral equimolar dose (15 micro;mol metal/kg) of zinc (II) chloride, cadmium (II) chloride or mercury (II) chloride in male albino mice were investigated. There was a moderate increase in hepatic and renal zinc levels mainly in their nuclear mitochondrial fraction (NMF) 24 h post zinc chloride administration. Subsequently, the hepatic zinc increased and the renal zinc declined with time. The zinc-induced hepatic MT level was maximum at 48 h, which decreased slightly thereafter, while there was no marked increase in renal MT level at any time interval. The cadmium was equally distributed in liver and kidney more in their supernatant cytosol fraction (SCF) than in their NMF at 24 h after a dose of cadmium chloride. The cadmium levels showed a decreasing trend in hepatic fractions and an increasing trend in renal fractions with time. The cadmium-induced hepatic and renal MT were substantial at 24 h post cadmium administration, the former decreased thereafter while the latter enhanced at 48 h before declining. The accumulation of mercury in kidney was 1.5 times that in liver, which was localised more in their SCF than in their NMF at 24 h in response to a dose of mercuric chloride. The mercury levels of hepatic and renal subcellular fractions started declining after 24 h and at 72 h they were significantly lower. The induction of hepatic and renal MT was maximum at 24 h after mercuric chloride administration, which declined thereafter concomitant with the decrease in their mercury levels. However, the MT levels in both the organs remained considerably higher than in normal animals at 72 h post exposure. The results show that the accumulation of metal in liver and kidney follows the order: Hg > Cd > Zn and the induction of MT follows Hg > Cd > Zn in liver and Cd > Hg > Zn in kidney. The alterations in zinc and copper homeostasis were more marked in liver than in kidney and follows the order: Hg > Cd > Zn.     

Gastrointestinal uptake and fate of cadmium in rainbow trout acclimated to sublethal dietary cadmium

Adult rainbow trout were pre-exposed to a sublethal concentration of dietary Cd (500 mg/kg dry wt.) for 30 days to induce acclimation. A gastrointestinal dose of radiolabeled Cd (276 microg/kg wet wt.) was infused into the stomach of non-acclimated and Cd-acclimated trout through a stomach catheter. Repetitive blood samples over 24 h and terminal tissue samples were taken to investigate the gastrointestinal uptake, plasma clearance kinetics, and tissue distribution of Cd. Only a small fraction of the infused dose (non-acclimated: 2.4%; Cd-acclimated: 6.6%) was internalized across the gut wall, while most was bound in the gut tissues (10-24%) or remained in the lumen (16-33%) or lost from the fish (approximately 50%) over 24 h. Cadmium loading during pre-exposure produced a profound increase of total Cd in the blood plasma (approximately 28-fold) and red blood cells (RBC; approximately 20-fold). The plasma Cd-time profiles consisted of an apparent rising (uptake) phase and a declining (clearance) phase with a maximum value of uptake in 4 h, suggesting that uptake of gastrointestinally infused Cd was very rapid. Acclimation to dietary Cd did not affect plasma Cd clearance (approximately 0.5 ml/min), but enhanced new Cd levels in the plasma (but not in the RBC), and resulted in a longer half-life for plasma Cd. Tissue total and new Cd levels varied in different regions of the gastrointestinal tract, and overall levels in gut tissues were much greater than in non-gut tissues, reflecting the Cd exposure route. Dietary Cd, but not the infused Cd, greatly increased total Cd levels of all gut tissues in the order posterior-intestine (640-fold) > cecae (180-fold) > mid-intestine (94-fold) > stomach (53-fold) in Cd-acclimated fish relative to na?ve fish. Among non-gut tissues in the Cd-acclimated fish, the great increases of total Cd levels were observed in the liver (73-fold), kidney (39-fold), carcass (35-fold), and gills (30-fold). The results provide some clear conclusions that may be useful for environmental risk assessment of dietary Cd exposure in fish.     

Ultrastructural changes in various organs of the fish Puntius gonionotus fed cadmium-enriched cyanobacteria

The accumulation and toxicity of cadmium in Puntius gonionotus fish that consumed the cyanobacterium Spirulina platensis contaminated with cadmium were studied. Fish were fed cadmium-contaminated cells for 4 weeks, after which cadmium accumulation in various organs was determined. The highest cadmium content was found in the kidney (56.0 microg Cd/g wet weight). Cadmium was not detected in the gill during the entire 4 weeks of cadmium feeding. Histopathological alteration of cells was observed in the gill, kidney, and liver. The results showed that dietary cadmium caused hypertrophy and edema of gill filaments. Coagulative necrosis and karyolysis of the nucleus were observed in the kidney. Vacuoles and hyaline droplets had accumulated in the epithelial cells of the proximal tubule. In the liver vacuolation of the cytoplasm, infiltration of macrophages, and focal necrosis were found. The ultrastructural changes that occurred in the cells of different organs were similar. These included a proliferation of vacuoles and lysosomes, formation of myelin bodies, degranulation, vesiculation, and dilation of rough endoplasmic reticulum, as well as swelling of mitochondria with loss of cristae.     

Effects of fasting on cadmium toxicity, glutathione metabolism, and metallothionein synthesis in rats

Acute oral toxicity of Cd (as cadmium chloride) was enhanced in rats fasted 24 hr, as shown by a markedly decreased LD50. To examine the relationship among Cd toxicity, hepatic glutathione (GSH), and metallothionein (MT) during fasting, rats were administered 75 mg Cd/kg orally 24 hr after fasting and euthanized after a further 4 or 24 hr for various assays. Serum glutamic-pyruvic transaminase activity 24 hr after Cd treatment was higher in fasted rats than in fed rats. Both total GSH and nonprotein sulfhydryl (NPSH) concentrations in liver decreased to 50% of control levels after 28 hr of fasting and returned to 75% of control values by 48 hr. Total hepatic GSH concentration in fed rats decreased 4 and 24 hr after Cd treatment, whereas that in fasted rats remained unchanged at 4 hr and decreased significantly at 24 hr. Cd uptake by the liver (both concentration and content) 24 hr after Cd treatment was higher in fasted rats than in fed rats. Hepatic MT concentration was markedly increased by Cd treatment and higher in fasted rats than in fed rats. There was no relationship between Cd toxicity and hepatic thiobarbituric acid (TBA) value, an indicator of lipid peroxidation. Fasting had no effect on hepatic GSH peroxidase and GSH reductase activities. These enzymes probably are not involved in Cd toxicity. On histological examination, focal degenerative and necrotic changes were observed from the midlobular to the pericentral region in the livers of fed rats 24 hr after Cd treatment. These changes were enhanced by fasting, diffusing from the pericentral to the periportal region. Histochemical examination revealed a heterogeneous distribution of GSH in the livers of fed rats, with strong staining of GSH in the periportal region. This heterogeneous distribution of GSH in liver was not observed in fed rats 4 hr after Cd treatment or in fasted rats at 24 hr. The present results suggest that hepatic GSH plays an important role in protection against Cd toxicity before the onset of MT synthesis. Animals in bad condition, such as that resulting from interruption of nutrient supply, cannot be protected against Cd toxicity even if the hepatic MT level is high.     

The modulation by metallothionein of cadmium-induced cytotoxicity in primary hepatocyte cultures

The cytotoxicity of CdCl2 and 2 isoforms of hepatic cadmium-metallothionein (CdMT I and II), was investigated using primary cultures of rat hepatocytes. The cell cultures were exposed to cadmium as CdCl2 or as either isoform of CdMT for a 20-h period at concentrations ranging from 50 to 500 ng Cd X ml-1. Cytotoxicity was assessed by determining the amount of lactic dehydrogenase released from the cells into the incubation medium and the incorporation of [3H] arginine into cell protein. The uptake of Cd by the cells was also measured. Cadmium chloride and both isoforms of CdMT were found to be toxic to hepatocytes although partial protection was afforded by the binding of cadmium to metallothionein (MT). At the higher exposure concentrations and in accordance with the toxicity data, the cells exposed to CdCl2 were found to accumulate more cadmium than those exposed to CdMT. The distribution of cadmium in the culture medium was examined using Sephadex G-75 chromatography. The speciation of cadmium is discussed in relation to its cytotoxicity.