Various responses to copper and manganese exposure of Carassius auratus gibelio from two populations

The aim of this study was to compare the effect of pro-oxidants copper (Cu(2+), 0.005 and 0.050mg L(-1)) or manganese (Mn(2+), 0.17 and 1.7mg L(-1)) on Carassius auratus gibelio from polluted (B) and unpolluted (Z) sites after exposure for fourteen days. Fish from site B showed high levels of lipid peroxidation (TBARS concentration), lower levels of metallothionein (MT)-related metal, total glutathione (GSH), its redox index, superoxide dismutase (Cu,Zn- and Mn-SOD), glutathione-S-transferase (GST) and cholinesterase (ChE) activities and also higher MT-related thiol concentration in the liver and gills. A common effect of exposure was related to genotoxicity, a decrease in GSH and an increase in microsomal ethoxyresorufin O-deethylase activity in the liver. However, the systems of oxidative stress and biotransformation were more efficient in fish from the polluted site, while the responsivity of MTs in this fish was impaired. Principle Component Analysis separated the subgroups from the unpolluted site and fish loaded by lesser concentrations of metals on the one side, and fish from the polluted site jointly with fish exposed to higher concentrations of metals on the other side. The main distinguishing indices of sites and exposures selected by classification and regression tree (CART) analysis were MT characteristics and genotoxicity.     

Joint effects of dimethoate and heavy metals on metabolic responses in a grasshopper (Chorthippus brunneus) from a heavy metals pollution gradient

We studied how an exposure to an additional stressing factor-dimethoate, might affect detoxifying ability of grasshoppers collected at 5 meadow sites located along a heavy metal pollution gradient. Activities of esterases and enzymes linked with glutathione (GSH) metabolism were assayed 24 h after topical treatment with 0.32 microg dimethoate per insect. Inhibition of acetylcholinesterase (AChE) reaches nearly 50% of the value stated in untreated insects, without significant site-dependent differences. The pesticide also caused a significant decrease in activities of glutathione peroxidase (GPx) followed by a decrease in GSH levels in grasshoppers from all assayed groups, demonstrating high sensitivity of glutathione-dependent metabolism to the additional stressing factor. In the case of glutathione reductase (GR) and carboxylesterases (CarE) the fall of activity was shown especially in insects from less polluted meadows and the reference site. Glutathione reductase (GR) activity in individuals treated with dimethoate did not decrease only in insects from the most contaminated site I. This might suggest the trade-off mechanisms adapting grasshoppers to life in seriously polluted environments.     

Chromosomal aberrancy and the level of fluctuating asymmetry in black-striped mouse (Apodemus agrarius): effects of disturbed environment

Analysis of chromosomal aberrations in bone marrow cells and determination of the level of fluctuating asymmetry (FA) for eight bilateral cranial traits were used to estimate effects of disturbed environment on natural populations of small mammals. During the spring and fall of 1994-2000 (except in 1999) black-striped mice (Apodemus agrarius) were collected from a polluted industrial area (Pancevo) and from an unpolluted reference site (Cer) in Serbia and Montenegro. Mice from the polluted area had significantly higher mean number of aberrant cells per individual than those in the reference site (p<0.01). Both sexes had the greater number of lesions in the polluted site than in the reference one. Also, there were no significant differences between sexes, neither within the same site/season nor between sites/seasons. Chi-square analysis of the difference between lesions per individual and aberrant cells per individual among sites showed that no site deviated from the expected difference (p=1.000). Developmental stability, assessed as FA, was reduced in the polluted area for two traits (width of lower jaw and length of the diastema lower jaw). Juvenile animals from the polluted area had significantly higher levels of FA compared to those from the reference site for three traits (length of the foramina incisiva, distance between incisor and the third upper molar, and length of the upper molars row). Juvenile animals from the polluted area had significantly higher levels of FA for three traits (length of the foramina incisiva, length of the lower molars row and width of lower jaw) compared to adult ones. In the polluted area seasonal differences were detected for two traits (length of the foramina incisiva and length of the upper molars row). Finally, Apodemus agrarius may be an important species for environmental quality evaluation studies based on an assay using a combination of end-points.     

Heavy metal pollution in Antarctica: a molecular ecotoxicological approach to exposure assessment

Trematomus bernacchii sampled from two sites at Ross Island, Antarctica, were assessed for condition (gonadal and hepatic somatic indices and condition factor), pathological state (liver and gill histology), hepatic metal (Zn, Cu, Cd and Ni) concentrations, and metallothionein induction by quantitative competitive (qc) RT-PCR. Fish from a polluted site (Winter Quarters Bay) had pathological anomalies including necrosis and periductal inflammation in their livers, and X-cell disease, epithelial hyperplasia, lamellar fusion and aneurysms in their gills. Such anomalies were less common in fish from a relatively pristine site (Backdoor Bay, Cape Royds). Fish from both sites had similar liver concentrations of Zn, Cu and Cd and qcRT-PCR revealed similar levels of hepatic metallothionein mRNA. Ni in the livers of fish from Winter Quarters Bay were higher than those in fish from Backdoor Bay, but the differences were not great enough to affect hepatic metallothionein mRNA significantly. Despite the polluted state of Winter Quarters Bay waters, it seems that the heavy metals present may have only limited impact on the health status of fish collected from this locality. This may reflect a low bioavailability of the heavy metals in Ross Island marine sediments and suggests that other factors, such as relatively high levels of organic pollutants (PAHs, PCBs) or pathogens from the nearby sewage outlet, may play a more significant role in the aetiology of pathological conditions in fish from Winter Quarters Bay.     

Heme oxygenase-1 mRNA levels,metallothionein mRNA levels,lipid peroxidation and microsomal CYP1A activities in rats treated with 3,3-dichlorobenzidine and some other inducers of P450

The effect of 3,3-dichlorobenzidine (DCB), a potent inducer of CYP1A, on the levels of heme oxygenase-1 mRNA and metallothionein mRNAs was examined in the kidney, liver and lung of rats administered a single ip dose (157 μmol/kg) of the compound. DCB treatment increased heme oxygenase-I mRNA abundance in the kidney significantly from barely detectable levels in untreated animals; the maximum increase in the liver and lung was 24-fold and 4-fold, respectively. Hepatic microsomal heme oxygenase activity was also induced by DCB. In contrast with DCB, 2 other P450 inducers, β-naphthoflavone (β-NF) and phenobarbital did not elevate tissue HO-1 rnRNA levels. DCB pretreatment also elevated metallothionein mRNA levels in the kidney, liver and lung, with the effect in the lung being the least pronounced. In contrast with HO-1 mRNA, metallothionein mRNA was increased by the other P450 inducers examined. In vivo lipid peroxidation and in vitro NADPH-dependent microsomal lipid peroxidation were increased in the liver of DCB-treated rats but not in those of phenobarbital- or β-naphthoflavone-treated rats. Treatment with DCB or β-NF did not alter total hepatic microsomal P450 content, as measured spectrophotometrically, but induced the activity of CYP1A2. In contrast, the activity of CYP1A1 was induced to a lesser extent by DCB than by β-NF. The data show that DCB induces HO-1 as weD as P450 1A, confirm stimulation of lipid peroxidation by the compound, and suggest oxidative stress as a mechanism of HO-1 induction by the compound.     

Joint effects of parasitism and pollution on oxidative stress biomarkers in yellow perch Perca flavescens

Yellow perch Perca flavescens were collected from a contaminated site and a reference site in the St. Lawrence River, Quebec, Canada. Fish were assessed for oxidative stress (lipid peroxidation and reduced glutathione levels) and parasitism by the nematode Raphidascaris acus and metacercariae of the digenean Apophallus brevis. Lipid peroxidation is not only considered a measure of oxidative stress, but of stress in general, and thus serves as an indicator of fish health. Fish from the contaminated site exhibited higher levels of lipid peroxidation than those from the reference site, independent of parasitic infections. However, fish infected with R. acus at the contaminated site tended to have higher levels of lipid peroxidation than uninfected fish at the same site, whereas no difference was observed between infected and uninfected fish at the reference site. Yellow perch infected with > 10 metacercariae of A. brevis expressed higher levels of lipid peroxidation than those infected with < 10 metacercariae at both the contaminated and the reference sites. No differences were found in levels of reduced glutathione in liver or muscle in relation to site or either parasite species. Results support the use of lipid peroxidation as a biomarker of water contamination. They further suggest that lipid peroxidation may be used as a biomarker of pathological effects caused by parasitism. Most importantly, results demonstrate that contaminants and parasites occurring together exacerbate oxidative stress in fish, suggesting that parasitized fish in polluted environments are in a poorer state of health than uninfected fish.     

Population-related molecular responses on the effect of pesticides in Carassius auratus gibelio

The aim of this study was to evaluate population-related peculiarities of the adaptive responses of Carassius auratus gibelio. In order to do this, male specimens from polluted (B) and clean (Z) sites were exposed to commercial pesticides thiocarbamate Tatoo (9.1 μg·L(-1)and 91 μg·L(-1)) or tetrazine Apollo (2 μg·L(-1) and 10 μg·L(-1)) during fourteen days. The control fish from site B was distinguished by weakness of antioxidant defence (measured from superoxide dismutase and catalase activities, redox index of glutathione (GSH), superoxide anion (O(2)) and lipid peroxidation levels), imbalance of the concentrations of protein metallothionein (MT-SH) and MT-related metals (MT-Me) and neurotoxicity. Differences in glutathione-S-transferase activity in the liver and vitellogenin-like proteins in the serum were also showed between B and Z control groups. Common effects of pesticides were related to a decrease in GSH, an increase in O(2) production, ethoxyresorufin O-deethylase activity and hepatosomatic index. Apollo provoked particular elevation of MT-SH/MT-Me ratio. Population-related difference in the response was the activation of antioxidant defence in fish from site B and its inhibition in fish from site Z. The genotoxic effect of exposures was more expressed in fish from site B. Principal component analysis combine all exposed groups from site Z and control group from site B in one set, and separated each exposed group from site B. The main distinguishing index of each population selected by classification and regression tree analysis was MT-SH.     

The effects of reactive oxygen species on amphibian aging

To clarify the role of reactive oxygen species (ROS) in the aging process of amphibians, antioxidant enzyme activity and indexes of ROS damage were investigated biochemically using the livers of 3- and 10-year-old Rana nigromaculata frog males and females. Findings revealed no significant difference in survival rate between males and females. Antioxidant enzyme activity displayed an age-related decline. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activity in 10-year-old liver decreased 40-80% from 3-year-old liver levels. In contrast, urate oxidase activity in the 10-year-old liver increased more than 200% from 3-year-old liver levels. At the same time levels of ROS damage, including the concentration of inorganic peroxide and thiobarbituric acid reactive substances (TBARS), greatly increased with age. Liver catalase from 10-year-old frogs proved to be more susceptible to aminotriazole and urea, losing approximately 80% of its original activity after 30 min of treatment. It seems likely that liver catalase in older frogs has diverged from liver catalase in younger frogs through oxidative modification. These findings suggest that a decrease in the activity of antioxidant enzymes over time results in increased levels of ROS damage in the livers of older frogs.     

Stress-induced activation of the AMP-activated protein kinase in the freeze-tolerant frog Rana sylvatica

Survival in the frozen state depends on biochemical adaptations that deal with multiple stresses on cells including long-term ischaemia and tissue dehydration. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the metabolic re-sculpting that occurs during freezing. AMPK activity and the phosphorylation state of translation factors were measured in liver and skeletal muscle of wood frogs (Rana sylvatica) subjected to anoxia, dehydration, freezing, and thawing after freezing. AMPK activity was increased 2-fold in livers of frozen frogs compared with the controls whereas in skeletal muscle, AMPK activity increased 2.5-, 4.5- and 3-fold in dehydrated, frozen and frozen/thawed animals, respectively. Immunoblotting with phospho-specific antibodies revealed an increase in the phosphorylation state of eukaryotic elongation factor-2 at the inactivating Thr56 site in livers from frozen frogs and in skeletal muscles of anoxic frogs. No change in phosphorylation state of eukaryotic initiation factor-2alpha at the inactivating Ser51 site was seen in the tissues under any of the stress conditions. Surprisingly, ribosomal protein S6 phosphorylation was increased 2-fold in livers from frozen frogs and 10-fold in skeletal muscle from frozen/thawed animals. However, no change in translation capacity was detected in cell-free translation assays with skeletal muscle extracts under any of the experimental conditions. The changes in phosphorylation state of translation factors are discussed in relation to the control of protein synthesis and stress-induced AMPK activation.     

Separation and quantitation of metallothionein isoforms from liver of untreated rats by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry

A sensitive method for determination of metallothionein (MT) isoform levels in rat liver by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry was developed. Critical steps in sample preparation, like MT extraction, MT saturation with Cd and protein separation, were optimized. This method is capable of measuring levels of 2.0 μg/g liver for metallothionein-1 (MT-1) and 1.3 μg/g liver for metallothionein-2 (MT-2), respectively, with a high recovery of 103% on average. The method described, thus, proved suitable for analyzing metallothionein isoform concentrations even in untreated animals. The ratio of MT-1 to MT-2 was found to be 1:1 on average. MT decomposition during storage was very high in whole livers, but could be reduced by about 80% when extracted liver samples were used.     

Biochemical biomarkers in gills of mangrove oyster Crassostrea rhizophorae from three Brazilian estuaries

Responses of biochemical biomarkers were evaluated in gills of immature adult mangrove oyster Crassostrea rhizophorae collected in three estuarine regions along the Brazilian coast. In each region, ten oysters were collected in one reference site (R) located far from pollution sources, and in two polluted sites (P-I and P-II sites) located in another water body with similar characteristics. P-I site is located close to recognized pollution sources while P-II site is in the same water body, but far from pollution sources. At the Paranaguá Bay (Southern Brazil), polluted sites receive domestic, harbor and phosphate fertilizer plant discharges. High lipid peroxides (LPO) content was observed in winter oysters from the P-I site. In summer, higher catalase activity was observed in these oysters. In the Piraquê region (Southeastern Brazil), polluted sites receive domestic and agricultural effluents. Lower total oxyradical scavenging capacity (TOSC) towards peroxyradicals was observed in summer oysters from both P-I and P-II sites. In the Itamaracá region (Northeastern Brazil), polluted sites receive paper mill and caustic soda and chlorine factories effluents. Increased glutathione S-transferase (GST) activity was observed in oysters from the P-I site in both summer and winter. At Paranaguá Bay (higher latitude), no seasonal differences were observed in oysters from the R site, suggesting that temperature was not an important factor influencing biomarkers levels. Lower GST activity was observed in oysters from the R site of the Itamaracá Bay (lower latitude) in winter and summer. Taken together, data obtained point to responses of biomarkers in oysters from polluted sites of the three estuarine regions analyzed, indicating the need for future monitoring of the biological effects of contaminants in these environments. They also point to the relevance to consider both season and latitude as factors influencing biomarker responses in environmental contamination monitoring programs.     

镉致黑斑蛙肝脏氧化损伤与金属硫蛋白含量的变化

为观察镉对黑斑蛙(Rana nigromaculata)肝脏脂质过氧化产物和金属硫蛋白含量的影响,将黑斑蛙暴露于10.0mg·mL-1浓度的镉溶液中30d,分别测定了黑斑蛙在暴露4、10d和30d时肝脏组织中镉(Cd)、还原型谷胱甘肽(GSH)、金属硫蛋白(MT)和过氧化产物丙二醛(MDA)的含量。实验结果表明,黑斑蛙肝脏中镉的积累量、GSH和MT含量均随着镉暴露时间的延长而显著升高,具有明显的时间-效应关系;在镉暴露的第10天,肝MDA含量明显高于对照组。提示镉可对黑斑蛙肝脏造成氧化损伤,而GSH、MT含量的升高则可能是机体抗氧化损伤的机理之一。     

The use of biomarkers of oxidative stress in zebra mussel <Emphasis Type="Italic">Dreissena polymorpha</Emphasis> (Pallas, 1771) for chronic anthropogenic pollution assessment of the Rybinsk Reservoir

Biomarkers of oxidative stress such as catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR) activity, and malondialdehyde and reduced glutathione content, as well as heavy metal concentrations (HM: Pb, V, Cr, Mn, Co, Ni, Cu, Zn, and Cd), were studied in Dreissena polymorpha tissues. Mussels were collected on three sites located on the Rybinskoe Reservoir different in levels of anthropogenic pressure: the most polluted sites were 1 and 2 and site 3 was relatively clean. Mussels from sites 1 and 2 had higher concentrations of HM (Pb, V, Cr, Mn, Ni, Cu, and Mn) and their response to the pollutant action was manifested in increased processes of lipid peroxidation (LPO), the activation of CAT, and elevated level of GHS.     

Histopathological biomarkers and genotoxicity in gill and liver tissues of Nile tilapia Oreochromis niloticus from a polluted part of the Nile River,Egypt

Fish health is affected by water pollution. Oreochromis niloticus collected during summer 2014 from El-Serw, a polluted site on the Nile River, were compared with fish from a reference site, El-Zamalek. Histopathological changes were detected in gill and liver tissue samples using light and electron microscopy. In addition, the degree of DNA damage was measured using the comet assay. To indicate the severity of water pollution at the two sites, physico-chemical properties and heavy metal concentrations were investigated. Gill damage, including lamellar cell hyperplasia and aneurysm, was observed in the fish samples from the polluted site. The livers of fish from the polluted area showed necrosis and an increase in melanomacrophage centres. Histochemical results confirmed a marked rise of gill mucopolysaccharides and a reduction of carbohydrate stored in hepatocytes. Electron microscopy revealed clear alterations in gill and liver tissue of fish from the polluted site. The comet assay showed highly significant DNA damage in tilapia collected from the polluted site, compared to those from the reference site. Histopathological biomarkers and the comet assay may therefore be sensitive indicators of exposure to mixtures of aquatic pollutants in Nile tilapia.     

Zinc Supplementation Attenuates Metallothionein and Oxidative Stress Changes in Kidney of Streptozotocin-Induced Diabetic Rats

Zinc is an element that under physiological conditions preferentially binds to and is a potent inducer of metallothionein under physiological conditions. The present study was conducted to explore whether zinc supplementation morphologically and biochemically protects against diabetic nephropathy through modulation of kidney metallothionein induction and oxidative stress in streptozotocin-induced diabetic rats. Thirty-two Wistar albino male rats were equally divided into four groups. The first group was used as untreated controls and the second group was supplemented with 30?mg/kg/day zinc as zinc sulfate. The third group was treated with streptozotocin to induce diabetes and the fourth group was treated with streptozotocin and supplemented with zinc as described for group 2. The blood glucose and micro-albuminuria levels, body and kidney weights were measured during the 42-day experimental period. At the end of the experiment, the kidneys were removed from all animals from the four groups. Diabetes resulted in degenerative kidney morphological changes. The metallothionein immunoreactivity level was lower and the kidney lipid peroxidation levels were higher in the diabetes group than in the controls. The metallothionein immunoreactivity levels were higher in the tubules of the zinc-supplemented diabetic rats as compared to the non-supplemented diabetic group. The zinc and metallothionein concentrations in kidney tissue were higher in the supplemented diabetic group compared to the non-supplemented diabetes group. The activity of glutathione peroxidase did not change in any of the four groups. In conclusion, the present study shows that zinc has a protective effect against diabetic damage of kidney tissue through stimulation of metallothionein synthesis and regulation of the oxidative stress.     

Phosphamidon, methylparathion and dichlorvos impact on tissue oxidative metabolism in penaeid prawn, Metapenaeus monoceros

Changes in oxidative metabolism of hepatopancreas and muscle tissues of penaeid prawn, Metapenaeus monoceros was studied, following its exposure to selected organophosphorous insecticides phosphamidon, dichlorovos and methylparathion. The OPI are found to inhibit the activity levels of acetylcholinesterase, succinate dehydrogenase, isocitrate dehydrogenase, pyruvate dehydrogenase, lactate dehydrogenase and cytochrome-c-oxidase and cause accumulation of acetylcholine in the hepatopancreas and muscle tissues. These changes in the activity levels of selected oxidative enzymes during insecticide exposure in these tissues of prawn indicates the shift in the metabolic emphasis from aerobic to anaerobic conditions and is interpreted as a functional adaptation to insecticide induced metabolic stress. These observed changes at cellular level pave way for successful survival of prawns in insecticide polluted environ.     

Coal-smoke pollution modifies physio-chemical characteristics of tissues during the ontogeny of <Emphasis Type="Italic">Peristrophe bicalyculata</Emphasis>

Coal-smoke emissions of a thermal power plant affected the physio-chemical status of Peristrophe bicalyculata (Reth) Nees, as observed at the pre-flowering, flowering and post-flowering stages of plant growth. The nitrate level was raised while nitrate reductase activity, and the soluble protein content of leaf declined heavily at the polluted site during different stages of plant growth, compared to the control. The rate of photosynthesis also decreased under the pollution stress. Sugar level in root, stem and leaves increased with growing age of the plant but was always lower at the polluted site than at the reference site. In roots, the difference was marginal till flowering stage and quite conspicuous afterwards; stems showed a reverse pattern of variation. Sulphur content was higher at the polluted site in all the organs and at each stage of the plant life. The Zn and Fe concentrations were reduced in all plant parts under the pollution stress. Copper content in root was consistently low at the polluted site. In the stem and leaves, however, it was almost equal on both the sites at the pre-flowering stage but showed a wide difference during the later part of plant ontogeny.