Tissue-specific oxidative stress responses in fish exposed to 2,4-D and azinphosmethyl

Species- and tissue-specific defenses against the possibility of oxidative stress and lipid peroxidation were compared in adult fish, Oreochromis niloticus and Cyprinus carpio, exposed to 2,4-dichlorophenoxyacetic acid (2,4-D), azinphosmethyl and their combination for 96 h. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activities were monitored in kidney, brain and gill. In all exposure groups there was a marked increase in SOD activity in gill tissues in both fish species, while it was at the control level in other tissues. The highest elevation of SOD activity by combined treatment was observed in C. carpio. Individual and combined treatments caused an elevation in catalase and GPx activities in kidney of C. carpio. Catalase activity was unaffected in brain of O. niloticus, while GPx activity was decreased after all treatments. Glutathione S-transferase (GST) activity was higher than the control levels in kidney of both fish exposed to pesticides. No significant changes were observed in malondialdehyde level in kidney and brain of C. carpio. Our results indicate that the toxicities of azinphosmethyl and 2,4-D may be related to oxidative stress. Also, the results show that SOD activity in gill and GST activity in kidney may be used as biomarkers for pollution monitoring and indicate that the activities of certain biomarkers in C. carpio are more sensitive to pesticides than those in O. niloticus.     

Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.     

N‐nitrosodimethylamine changes the expression of glutathione S‐transferase in the liver of male mice: The role of antioxidants

The present study investigated the protective effect of gossypol, selenium, zinc, or glutathione (GSH) against dimethylnitrosamine (DMN)‐induced hepatotoxicity in the livers of male mice. The expression and the activity of glutathione S‐transferase (GST), levels of GSH, and free radicals (malondialdehyde (MDA)), as well as the activity of glutathione reductase were determined after the treatment of mice for seven consecutive days with low or high doses of gossypol, selenium, zinc, or GSH. In experimental groups, DMN was administered as a single dose for 2 h after the repeated dose treatments of mice for seven consecutive days with each antioxidant. DMN reduced the expression and inhibited the activity of GST. However, repeated treatments of mice with low‐dose gossypol or high dose of either selenium or GSH followed by a single dose of DMN induced the expression and the activity of GST. In contrast, low‐dose treatments of mice with zinc, selenium, or GSH followed by a single dose of DMN reduced the expression and the activity of GST compared to either control or DMN‐treated groups. In addition, high‐dose treatment with either gossypol or selenium markedly induced the levels of GSH compared to either control or DMN‐treated groups. Interestingly, pretreatment of mice with high dose of either gossypol or selenium for seven consecutive days followed by a single dose of DMN decreased the levels of MDA, whereas DMN induced such levels. It is concluded that high dose of either gossypol or selenium is a stronger protector than zinc and GSH in ameliorating the toxic effects of DMN. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:389–395, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20255     

Uptake, transport and metabolism of 14C-2,4-dichlorophenoxyacetic acid (14C-2,4-D) in cucumber (Cucumis sativus L.) explants

The uptake, distribution and metabolism of 2,4-D using 14C-labelled 2,4-dichlorophenoxyacetic acid (14C-2,4-D) was studied in isolated hypocotyl and cotyledon explants of cucumber (Cucumis sativus L.) in vitro. Cotyledons had a higher uptake capacity than hypocotyls; the uptake in cotyledons increased linearly up to 20 h, while in hypocotyls only up to 8 h. The distribution of 14C-activity in both organs was basipetal, but more pronounced in cotyledons. The 2,4-D taken up by cotyledons was metabolized very rapidly: only 7% of 14C-activity was associated with free 2,4-D after 20 h exposure. Hypocotyls metabolized 2,4-D more slowly: after 20 h 50% of the 14C-activity was still associated with free 2,4-D. After short incubation periods (2–5 h) 2,4-D-aspartate was a predominant metabolite, after longer incubation (8–20 h) 2,4-D-glucosyl ester prevailed. Roots or callus were formed on the base of cotyledons depending on the length of exposure to 2,4-D.     

The toxicity of 2,4-D to Cyprinus carpio var. Communis in relation to the seasonal variation in the temperature

The paper deals with the variations in the toxicity of 2,4-D to Cyprinus carpio at different temperatures during different seasons of the year. The higher temperature has higher toxicity and vice versa at the same concentration. The TLm values indicate that there is a 7–7.5 fold increase in toxicity with the rise in temperature from 17°C in February (winter) to 39°C in May (summer). These results have significance in manipulating the 2,4-D doses for the eradication of aquatic weeds in different seasons.     

Reduced glutathione esters—antidotes to toxicity. Cytotoxicity induced by hydrogen peroxide, 1-chloro-2,4-dinitrobenzene,and menadione in murine P388D1 macrophages in vitro

Repletion of depleted cellular reduced glutathione (GSH) levels in oxidative stress and exposure to arylating agents is a strategy for the development of antidotes to chemical toxicity. The effect of GSH, reduced glutathione ethyl monoester (GSHEt), and reduced glutathione ethyl diester (GSHEt₂) on the cytotoxicity of hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), and menadione to P388D₁ macrophages in vitro was investigated. The median toxic concentration TC₅₀ values of the toxicants were hydrogen peroxide 24 ± 2 mM (N = 19), CDNB 63 ± 6 μM (N = 18), and menadione 30 ± 4 μM (N = 22). Reduced glutathione, GSHEt, and GSHEt₂ were poor antidotes to hydrogen peroxide toxicity. Indeed, the observed antidote effects were attributed to the nonenzymatic reaction of the GSH derivatives with hydrogen peroxide in the extracellular medium. Reduced glutathione ethyl diester was a more potent antidote of CDNB- and menadione-mediated toxicity than GSHEt and GSH. For cell incubations with the approximate median toxic concentration TC₅₀ values of hydrogen peroxide, CDNB, and menadione, the respective median effective antidote concentration EC₅₀ values were GSHEt 23.8 ± 4.1 mM (N = 9), 3.6 ± 0.6 mM (N = 11), and 226 ± 93 μM (N = 12); and GSHEt₂ 20.4 ± 1.9 mM (N = 6), 603 ± 2 μM (N = 9), and 7.6 ± 2.3 μM (N = 12). Reduced glutathione ethyl diester was a potent antidote to CDNB- and menadione-induced toxicities but not to hydrogen peroxide-induced toxicity under acute intoxication conditions. © 1996 John Wiley & Sons, Inc.     

Time- and dose-dependent biom arker responses in flounder (Platichthys flesus L.) exposed to benzo[a]pyrene, 2,3,3',4,4', 5-hexachlorobiphenyl (PCB-156) and cadmium

Responses in flounder (Platichthys flesus) towards benzo [a]pyrene (BaP), 2,3,3',4,4',5-hexachlorobiphenyl (PCB-156), and cadmium (Cd) were investigated in time-course and dose-response studies of selected biomarkers. Measurements of biliary fluorescent BaP metabolites and hepatic concentrations of PCB-156 and cadmium showed that the injected toxicants were rapidly m obilized from the muscle to the liver, but a depot effect was indicated in the highest dose groups of BaP and PCB-156 (12 mg kg^-1 bodyweight). Clearest biomarker responses were found in the induction of hepatic cytochrome P450 1A (CYP1A) enzymes as a response towards BaP and PCB-156 exposure. Maximum induction of CYP1A dependent 7-ethoxyresorufin O-deethylase (EROD) activity was observed after 2 and 8 days in BaP and PCB-156-treated flounder, respectively. Positive dose-effect relationships were observed towards both compounds, but the CYP1A induction was more persistent with PCB exposure than with BaP exposure. In Cd-exposed fish, the hepatic level of metallothionein responded more slowly with highest levels observed after 16 days in the time-study. In the combined BaP + Cd treatment, the CYP1A induction was only slightly suppressed. Aspartate aminotransferase in serum appeared to be responsive towards BaP, but also towards the acetone vehicle in controls in the first part of the exposure period. Hematocrit as well as hepatic activities of aldrin epoxidase, glutathione S-transferase, and UDP-glucuronyl transferase were not responsive to any treatm ent in the present study. In general, the results demonstrate that selected biom arkers in flounder are responsive to PAH, PCB, and heavy metal pollutant exposure, indicating the applicability of this species in future environmental pollution monitoring programmes.     

Morphometry of muscle fibre types in the carp (Cyprinus carpio L.)

Summary Ultrastructural parameters of muscle fibre types of the carp (Cyprinus carpio L.) were measured and compared with their contractile properties. In red fibres, which are slower than pink fibres, the relative length of the junction between the T system and the sarcoplasmic reticulum (T-SR junction) is smaller and the Z lines are thicker than in pink fibres. Pink fibres have a smaller relative length of T-SR junction than white fibres from the axial muscles. The two types of red fibres present in carp muscle also differ in their relative lengths of T-SR junction. Significant differences in the relative areas of the SR were not found.The relative volume of myofibrils in red fibres is two-thirds that in pink fibres, a difference that is not reflected in the maximal isometric tetanic tensions of these types. Red fibres, which are less easily fatigued than pink fibres, have larger relative volumes of subsarcolemmal and intermyofibrillar mitochondria. Small pink fibres have a larger relative volume of subsarcolemmal mitochondria than large pink fibres, but have a similar relative volume of intermyofibrillar mitochondria. Small and large pink fibres differ in the relative volumes of their membrane systems, but have similar relative lengths of T-SR junction.     

Biochemical and morphological changes in carp (Cyprinus carpio L.) liver following exposure to copper sulfate and tannic acid

As a consequence of human activity various toxicants reach the aquatic ecosystems; humics may interact with them and may change their toxicity. Many fish are exposed to a considerable concentration of humics and pollutants. Because of paucity of data on the biochemical action of tannins in the presence of the fungicide CuSO4 a comparative study was undertaken. The alterations of redox-parameters in carp liver were monitored and tissue necrosis was followed by measuring the plasma transaminase activities and by electron microscopy. Tannic acid, a representative phenolic/humic compound, exerted prooxidant effects in carp, which may be partially due to formation of prooxidant intermediates/end-products via its biotransformation. Alternatively, tannic acid may partially inhibit the antioxidant enzymes of fish. The response to CuSO4 was more severe. Although tannic acid alone acted as a prooxidant in fish, electron micrographs demonstrated that it reduced the necrotizing effect of copper, which may be due to the complexing activity of tannic acid with the biomolecules of the hepatocytes and to the H2O2-degrading activity of tannin-CuSO4 combination. Our results indicate that the heavy metal-detoxifying capacity of tannin may be significant; however, tannin-exposure alone or combined with metals may be toxic for fish due to enzyme inhibition and oxidative stress induction.     

Texture analysis in liver of common carp (Cyprinus carpio) sub-chronically exposed to perfluorooctanoic acid

An operator-neutral, objective method was implemented to comparatively assess liver pathology in 30 specimens of common carp (Cyprinus carpio): 20 after experimental flow-through exposure to two perfluorooctanoic acid (PFOA) dosages (10 fish exposed to 200 ng l⁻¹ and 10 fish exposed to 2 mg l⁻¹) for 56 days and 10 unexposed (negative control). The method relies on texture analysis as a complementary approach to traditional histopathology and chemical dosage analysis performed previously on the same experimental material. Texture features data were analyzed by means of Redundancy Analysis (RDA), Linear Discriminant Analysis (LDA) and Canonical Variates Analysis (CVA). LDA resulted in the correct classification of 80% of cases (24 out of 30 cases) with a sensitivity of 83.3% and a specificity of 83.3. In particular, four male samples from the low dosage group (200 ng l⁻¹) were misclassified as unexposed fish and two female samples from the unexposed group were misclassified as low dosage exposed. Nevertheless, PFOA liver chemical dosage analysis results were the same both in unexposed and in low dosage group fish, all below the limit of detection. No sample from the high dosage group (2 mg l⁻¹) has ever been misclassified. Interestingly, texture features correlated with the PFOA concentrations detected in the liver of each sampled fish. In the present study the technique of texture analysis was combined with techniques of multivariate exploratory data analysis (RDA, LDA/CVA). This approach resulted in a robust, sufficiently sensitive and specific means to study PFOA-induced liver pathology. The new method can discriminate between unexposed and two PFOA exposed groups with better confidence and in a more affordable way, compared to chemical quantification of liver PFOA. The texture features correlated well with liver PFOA concentrations and objectively quantified degenerative liver morphology. In conclusion the overall approach may be a suitable candidate as a reliable and broad-ranging method for biomarker analysis of exposure and effect.     

Expression of titin isoforms in red and white muscle fibres of carp (Cyprinus carpio L.) exposed to different sarcomere strains during swimming

Titin (also known as connectin) is a striated-muscle-specific protein that spans the distance between the Z- and M-lines of the sarcomere. The elastic segment of the titin molecule in the I-band is thought to be responsible for developing passive tension and for maintaining the central position of thick filaments in contracting sarcomeres. Different muscle types express isoforms of titin that differ in their molecular mass. To help to elucidate the relation between the occurrence of titin isoforms and the functional properties of different fibre types, we investigated the presence of different titin isoforms in red and white fibres of the axial muscles of carp. Gel electrophoresis of single fibres revealed that the molecular mass of titin was larger in red than in white fibres. Fibres from anterior and posterior axial muscles were also compared. For both white and red fibres the molecular mass of titin in posterior muscle fibres was larger than in anterior muscle fibres. Thus, the same fibre type can express different titin isoforms depending on its location along the body axis. The contribution of titin to passive tension and stiffness of red anterior and posterior fibres was also determined. Single fibres were skinned and the sarcomere length dependencies of passive tension and passive stiffness were determined. Measurements were made before and after extracting thin and thick filaments using relaxing solutions with 0.6 mol · l⁻¹ KCl and 1 mol · l⁻¹ KI. Tension and stiffness measured before extraction were assumed to result from both titin and intermediate filaments, and tension after extraction from only intermediate filaments. Compared to mammalian skeletal muscle, intermediate filaments developed high levels of tension and stiffness in both posterior and anterior fibres. The passive tension-sarcomere length curve of titin increased more steeply in red anterior fibres than in red posterior fibres and the curve reached a plateau at a shorter sarcomere length. Thus, the smaller titin isoform of anterior fibres results in more passive tension and stiffness for a given sarcomere strain. During continuous swimming, red fibres are exposed to larger changes in sarcomere strain than white fibres, and posterior fibres to larger changes in strain than anterior fibres. We propose that sarcomere strain is one of the functional parameters that modulates the expression of different titin isoforms in axial muscle fibres of carp. Accepted: 7 May 1997     

Effect of GA3 and 2,4-D foliar application on the anatomy of date palm (Phoenix dactylifera L.) seedling leaf

Two concentrations (10-5M and 10-3M) of both GA3 and 2,4-D were used as foliar spray to evaluate the response of date palm (Phoenix dactylifera L.) cv. Khedri seedlings. They affected some of the anatomical characteristics of the first leaf emerging after the beginning of the spray. The high concentration of GA3 increased the size of the midrib and its vascular bundle numbers. Both low and high concentrations of 2,4-D inhibited the formation of the midrib. 2,4-D in both low and high concentrations decreased the number of vessels in both protoxylem and metaxylem and also decreased their diameters, where as GA3 in low and high concentrations have less effect on the number of vessels and its diameters. GA3 in high concentration increased the number of vascular bundles in 1mm long of the leaf blade, while 2,4-D in low and high concentrations decreased their numbers. 10-3M of 2,4-D increased the size and layers of special hypodermal cells.     

Protein expression profiling of glutathione S-transferase pi null mice as a strategy to identify potential markers of resistance to paracetamol-induced toxicity in the liver

GST pi (GSTP) is a member of the glutathione S-transferase (EC 2.5.1.18; GST) family of enzymes that catalyse the conjugation of electrophilic species with reduced glutathione and thus play an important role in the detoxification of electrophilic metabolites. Deletion of GSTP in mice has previously been shown to lead to enhanced susceptibility to chemical-induced skin carcinoma, consistent with its known metabolic functions. A decreased susceptibility to paracetamol hepatotoxicity has also been observed, which has not been fully explained. One possibility is that deletion of the GSTP gene locus results in compensatory changes in other proteins involved in defence against chemical stress. We have therefore used complementary protein expression profiling techniques to perform a systematic comparison of the protein expression profiles of livers from GSTP null and wild-type mice. Analysis of liver proteins by two-dimensional electrophoresis confirmed the absence of GSTP in null mice whereas GSTP represented 3-5% of soluble protein in livers from wild-type animals. There was a high degree of quantitative and qualitative similarity in other liver proteins between GSTP null and wild-type mice. There was no evidence that the absence of GSTP in null animals resulted in enhanced expression of other GST isoforms in the null mice (GST alpha, 1.48%, GST mu, 1.68% of resolved proteins) compared with the wild-type animals (GST alpha, 1.50%, GST mu, 1.40%). In contrast, some members of the thiol specific antioxidant family of proteins, notably antioxidant protein 2 and thioredoxin peroxidases, were expressed at a higher level in the GSTP null mouse livers. These changes presumably reflect the recently described role of GSTP in cell signalling and may underlie the protection against paracetamol toxicity seen in these animals.     

2,4-D丁酯对罂粟(Papaver somniferum L.)保护酶活性及脂质过氧化作用的影响

罂粟(Papaver somniferum L.)花蕾期用不同浓度的2,4-D丁酯对植株进行处理,测定了4d中叶片相对含水量(RWC)、丙二醛(MDA)含量、膜相对透性及保护酶(SOD、POD)活性变化情况.结果表明:在1ml/L、2ml/L、4ml/L浓度处理下对罂粟叶片RWC影响不显著,8ml/L处理下的RWC随处理时间的延长呈显著下降趋势,第4天比第1天下降了48.7%.在各处理下MDA含量和膜相对透性的变化趋势基本一致,1ml/L、2ml/L、4ml/L处理前期下降,后期升高.8ml/L处理对MDA含量和膜相对透性的影响显著的高于其它处理,随处理时间的延长呈先升高后降低的趋势.保护酶在处理条件下随时间延长其活性有明显的变化:POD活性变化波动较大,除8 ml/L外其它处理在第2、第4天有两次上升峰,8 ml/L峰值出现在第2天,之后下降在第4天达最低.SOD活性先降低后升高,都高于对照,所有处理SOD活性在第3d最低,第4天8 ml/L处理的活性显著的高于其它处理.表明了高浓度(8 ml/L)的2,4-D能使膜脂过氧化而产生较大的损伤,其它浓度处理对膜脂过氧化及膜透性影响相对较小,表明8ml/L的2,4-D是灭杀罂粟的最适用量.     

Host plant defenses of black (Solanum nigrum L.) and red nightshade (Solanum villosum Mill.) against specialist Solanaceae herbivore Leptinotarsa decemlineata (Say)

Black nightshade (Solanum nigrum, S. nigrum L.) and red nightshade ( Solanum villosum, S. villosum Mill.) are medicinal plants from the Solanaceae family that synthesize glycoalkaloids and other secondary metabolites. To recognize the potential insecticide activity of these compounds, leaf extracts (containing glycoalkaloid and methanol fractions) were tested for enzyme inhibition, antifeedant activity and toxicity. For in‐vitro glutathione S‐transferase (GST) inhibition activity, we used insecticide‐resistant Colorado potato beetle, Leptinotarsa decemlineata ( L. decemlineata; Say) midgut and fat‐body homogenate. In‐vivo toxicity and the antifeedant activity were performed using larval bioassays. The methanol extracts had greater GST inhibitory activity compared to the glycoalkaloids, as well as greater 2nd instar larvae mortality and antifeedant activity. Furthermore, the green leaf volatile compound, cis‐hex‐3‐enyl acetate, at the concentration of 5 ppm, caused 50% mortality of 2nd instar larvae. Our findings suggest the potential usefulness of S. nigrum and S. villosum extracts to control L. decemlineata.     

Gradual Depletion of 2,4-D in the Culture Medium for Indirect Shoot Regeneration from Leaf Explants of Camellia sinensis (L.) O. Kuntze

Adventitious shoot regeneration via callus phase from in vitro leaf explants is reported for the first time in tea. Callus was obtained on Murashige and Skoog medium supplemented with varied concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5, 5.0, 7.5 and 10.0 mg/l). Rhizogenesis was observed at all concentrations of 2,4-D. Adventitious shoot buds developed indirectly on leaf explants after prolonged culture for 16 weeks on medium supplemented with 10.0 mg/l 2,4-D. GC analysis of the medium and the tissues at different stages of development showed that specific levels of 2,4-D in the tissue were responsible for morphogenesis. Shoot buds developed on rhizogenic calli, only when 2,4-D declined to undetectable or negligible concentrations in the tissue probably due to detoxification and metabolism. Alternatively, shoot buds could also be evoked when rhizogenic calli were transferred to medium supplemented with low concentration of 2,4-D (1.5 mg/l). The adventitious nature of the shoots was confirmed through histological studies.     

Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications

Summary Two protein antigens, horseradish peroxidase (HRP) and ferritin, have been administered to the digestive tract of carp. Electron-microscopical observations reveal considerable absorption of both antigens in the second segment of the gut (from 70 to 95% of the total length) and also, although to a lesser extent, in the first segment (from 0 to 70% of the total length). Even when administered physiologically with food, a large amount of ferritin is absorbed by enterocytes in the second gut segment.HRP and ferritin are processed by enterocytes in different ways. HRP seems to adhere to the apical cell membrane, probably by binding to receptors, and is transported in vesicles to branched endings of lamellar infoldings of the lateral and basal cell membrane. Consequently, most of the HRP is released in the intercellular space where it contacts intra-epithelial lymphoid cells. Only small amounts of HRP become localized in secondary lysosomes of enterocytes. Ferritin does not bind to the apical cell membrane; after uptake by pinocytosis, it is present in small vesicles or vacuoles that appear to fuse with lysosome-like-bodies. In the second segment, intact ferritin ends up in the large supranuclear vacuoles (after 8 h), where it is digested slowly. Although no ferritin is found in the intercellular space, ferritin-containing macrophages are present between the epithelial cells, in the lamina propria and also to a small extent in the spleen. The transport of antigens from the intestinal lumen, through enterocytes, to intra-epithelial lymphoid cells or macrophages may have immunological implications, such as induction of a local immune response and prospectives for oral vaccination.     

Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.     

Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.