Effects of atrazine and chlorpyrifos on acetylcholinesterase and Carboxylesterase in brain and muscle of common carp

The aim of this study was to investigate the effects of chlorpyrifos (CPF), atrazine (ATR) and the mixture of them on acetylcholinesterase (AChE) and carboxylesterase (CbE) in brain and muscle of common carp, respectively. 220 carps were averagely divided into 11 groups according to the different treatments and concentration, including the exposure and recovery experiments. The activities of AChE and CbE of the brain and muscle were determined at the end of the exposure and the recovery. The results showed that in the control group, the specific enzymatic activities in the brain were higher than that in the muscle. The activities of AChE and CbE in the exposure groups were significantly lower than that in the control group except for the CbE activity in the ATR low-dose group. There was a negative dose-response relationship between the activities of AChE and CbE and the pesticides concentration. The activities of AChE and CbE in the recovery groups were significantly higher than that in the exposure group except for the CbE activity in the ATR low-dose group, AChE activity in the high-dose group of the mixture of ATR and CPF, and AChE activity of the brain in the CPF high-dose group. The results suggested that: (1) brain AChE may be considered as a very sensitive and early biomarker of exposure to CPF, ATR, or the mixture of ATR and CPF, (2) brain CbE may be used as a secondary biomarker for evaluating the exposure to CPF, ATR, or the mixture of ATR and CPF and (3) the change of the AChE and CbE activities caused by the mixture of ATR and CPF was more sensitive than that caused by the ATR or CPF alone.     

EEG spectra,behavioral states and motor activity in rats exposed to acetylcholinesterase inhibitor chlorpyrifos

Exposure to organophosphates (OP) has been associated with sleep disorders such as insomnia and "excessive dreaming." The central mechanisms of these effects are not well understood. OPs inhibit acetylcholinesterase (AChE) activity, leading to a hyperactivity of the brain cholinergic systems that are involved in sleep regulation. We studied alterations in the EEG, behavioral states, motor activity and core temperature in rats orally administered with 10 or 40 mg/kg of the OP insecticide chlorpyrifos (CHP). Occipital EEG, motor activity and core temperature were recorded with telemetric transmitters. Behavioral sleep-wake states were visually scored. Both doses of CHP produced alterations of the EEG (decrease in power of sigma/beta and increase in slow theta and fast gamma bands) characteristic of arousal. EEG alterations were consistent with behavioral changes such as an increase in wakefulness and a decrease in sleep. Waking immobility was a prevalent behavior. We did not detect any overt signs of CHP toxicity, such as an abnormal posture or gait, suggesting that reduced locomotion can be a result of central effects of CHP (such as activation of cholinergic motor inhibitory system) rather than peripheral (such as an impairment of neuromuscular function). Changes in the EEG and behavior occurred independently of the decrease in core temperature. Increased wakefulness together with reduced motor activity after exposure to CHP seems to be a result of hyperactivity in brain cholinergic neuronal networks.     

Histopathological changes in gill, liver and kidney of neotropical fish Colossoma macropomum exposed to paraquat at different temperatures

This work focused on the histological alterations in gill, liver and kidney of fish Colossoma macropomum exposed to different temperatures (18°C, 29°C, 35°C) with 10mg/L of herbicide Paraquat (PQ), during 21 days. The fish exhibited histopathological changes in these tissues; the most important alteration in gills was telangiectasis. Liver showed debris accumulation inside cytoplasm hepatocytes, karyolysis, karyohesis and a decrease in the size of sinusoids. Hyperplasia of melanomacrophagic centers (MMC) and an increase in basophils were observed in kidney. The lesion inducing by PQ and the damage in tissue depended of temperature exposure fish. The severity of lesions clearly differed among organs with the liver showing the most extensive damages followed in order by the kidney and gills. In PQ/18°C group it was observed the changes in the pattern of lesions, with kidney showing higher damage followed gills and liver.     

Effect of amoxicillin exposure on brain,gill, liver,and kidney of common carp (Cyprinus carpio): The role of amoxicilloic acid

Amoxicillin (AMX) is one of the most commonly prescribed antibiotics around the world due to its broad‐spectrum activity against different bacterial strains as well as its use as a growth promoter in animal husbandry. Although residues of this antibacterial agent have been found in water bodies in diverse countries, there is not enough information on its potential toxicity to aquatic organisms such as the common carp Cyprinus carpio. This study aimed to evaluate AMX‐induced oxidative stress in brain, gill, liver and kidney of C. carpio. Carp were exposed to three different concentrations of AMX (10 ng/L, 10 μg/L, 10 mg/L) for 12, 24, 48, 72, and 96 h, and the following biomarkers were evaluated: lipid peroxidation (LPX), hydroperoxide content (HPC), protein carbonyl content (PCC) and activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Amoxicillin and its main degradation product amoxicilloic acid (AMA) were determined by high performance liquid chromatography coupled with electrochemical detection and UV detection (HPLC‐EC‐UV). Significant increases in LPX, HPC, and PCC (P < 0.05) were found in all study organs, particularly kidney, as well as significant changes in antioxidant enzymes activity. Amoxicilloic acid in water is concluded to induce oxidative stress in C. carpio, this damage being highest in kidney. The biomarkers used are effective for the assessment of the environmental impact of this agent on aquatic species. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1102–1120, 2017.     

Effects of carbofuran and deltamethrin on acetylcholinesterase activity in brain and muscle of the common carp

This work investigated the effect from exposure to insecticides carbofuran and deltamethrin on acetylcholinesterase (AChE) activity in the brain and muscle of common carp (Cyprinus carpio). Both pesticides were evaluated through two separate experiments, and carp were exposed in a semi‐static system to three different concentrations of carbofuran (10, 50, and 100 μg/L) and deltamethrin (0.08, 0.4, and 0.8 μg/L) during a month with sampling times at 0, 4, 15, and 30 days (n = 7 from each aquarium). AChE activity was significantly inhibited in both organs of carps exposed to carbofuran at all sampling times depending on dose and time, reaching inhibition values of 73.5 and 67.1%, in brain and muscle tissues respectively, after 30 days with the highest concentration. On the contrary, AChE activity was not significantly affected after deltamethrin exposure at all concentrations and times of the assay. This study shows that the measurement of brain and muscle AChE activity in Cyprinus carpio is a useful biomarker of carbamates exposure and/or effects, but has no application with pyrethroids. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 386–393, 2014.     

Comparison of the protective effects of antioxidant compounds in the liver and kidney of Cd‐ and Cr‐exposed common carp

The aim of this study was to see whether the taurine (TAU), alpha‐lipoic acid (LA), curcumin (CUR), and N‐acetylcysteine (NAC) protection against oxidative stress caused by heavy metals is owed to the metal‐decreasing or antioxidative effect. In this context, liver and kidney tissues of common carp (Cyprinus carpio carpio L.) were exposed in vivo to model toxicants cadmium (Cd) and chromium (Cr). The tissues were dissected 96 h after intraperitoneal injection of the metals and antioxidant substances. Cd and Cr levels were determined in the liver using the ICP‐OES, but we could not obtain enough kidney tissue to make the same measurements in the kidney. The enzymatic activities of SOD, CAT, and GPx, and the GSH redox status and lipid peroxidation levels were analyzed using spectrophotometric methods. Of all investigated antioxidants, only NAC decreased metal levels in the liver. Cd had little effect on oxidative stress parameters, while Cr showed a weak prooxidative effect. Cotreatment with TAU/LA/CUR/NAC and Cr significantly increased liver SOD activity. Chromium induced kidney SOD and CAT, but all antioxidants lowered CAT activity. Cadmium reduced liver and increased kidney GSSG. NAC increased liver GSH, but the increase did not correlate with decrease in Cd. Curcumin given with Cd increased kidney and decreased liver lipid peroxidation, whereas TAU with Cr increased lipid peroxidation in both tissues. N‐Acetylcysteine was the most effective antioxidative agent, owing to its metal‐decreasing function as well as to its effects on the GSH redox status. We believe that the investigated antioxidant substances which may have been involved in the reduction of Cr caused an increase in SOD activity and a decrease in CAT activity. Changes in the GSSG levels in both tissues might be an adaptive response to the prooxidative potential of Cd. Because of their respective tissue‐ and metal‐dependent prooxidative effects, CUR and TAU deserve particular attention in regard to their use against metal toxicity, Cr in particular. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 129–137, 2014.     

Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity

The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (≈ 50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the use of organophosphorus pesticides in the natural habitat of the axolotl.     

Effect of long-term exposure to simazine on brain and muscle acetylcholinesterase activity of common carp (Cyprinus carpio)

Several water-contamination incidents with simazine have occurred in the province of Badajoz (Spain), due to its excessive use for controlling weeds in olive trees and vineyards. Simazine residues were also detected in drinking water, increasing public health concern. However, little is known on the effects that low levels of simazine pose to environment organisms. We investigated if residues of simazine in the natural waters would affect brain and muscle acetylcholinesterase activity in common carps captured in areas in which simazine residues were detected at average levels of 4.5 microg/L. Results confirmed depression on brain and muscle acetylcholinesterase activity of 20% and 29%, respectively, in carps inhabiting one of the simazine-contaminated ponds, termed "Molinos de Matachel." To assess the biological significance of this finding, we developed a controlled laboratory study in which carps were exposed to simazine at 45 microg/L (10-fold that of the natural water levels) for 90 days. The results obtained in the field study were not confirmed in our laboratory experiment, since carps did not show evidence or brain or muscle acetylcholinesterase activity depression for the duration of the experiment, and therefore, we can conclude that acetylcholinesterase depression found in carps collected in "Molinos de Matachel" should be ascribed to other compounds or mixtures of xenobiotics.     

大鼠缺血再灌注肾组织一氧化氮合成酶mRNA表达的研究

目的 探讨大鼠缺血再灌注肾组织一氧化氮合成酶（NOS）mRNA表达的特点,分析缺血再灌注肾损伤的分子机制。方法 建立大鼠肾缺血再灌注模型。采用组织细胞原位杂交及图像分析技术对不同实验条件下各型NOS（eNOS,nNOS及iNOS)mRNA表达的定位及含量进行检测。并对肾组织NOS总活性及血肌酐（Cr)值进行生化测定。结果（1）正常情况下,eNOS,nNOS及iNOS在正常肾组织中均有表达,cNOS/iNOS比值为2.29;eNOS和nNOS主分布于肾小球及血管内皮;iNOS仅分布于皮质远,近曲小管上皮。（2）缺血时,肾组织NOS总活性显著下降,三种NOSmRNA在皮,髓质及小球中的表达均下调,以eNSO最明显,cNSO/iNOS比值降为2.01。（3）再灌注后,三种NOSmRNA的表达明显上调,以iNOSmRNA最明显,cNOS/iNOS比值降为1.77,eNOS及nNOS上调部位仅限于肾皮,髓质血管,而小球及小管中则表现为下调,尤以nNOSmRNA在小球中的下调最明显。结论 （1）缺血再灌注后,皮质肾小管上皮中iNOSmRNA的高表达是导致肾缺血再灌注损伤的重要分子机制。（2）cNOS/iN-OS比值与缺血再灌注过程中肾功能的变化密切相关,该比值的恒定对肾血流量和肾小球滤过率（GFR）的调节可能具有重要意义。     

Alterations in the expression of intestinal enzymes in rats exposed to nickel

The effect of feeding nickel (50 mg kg(-1) body weight) daily for 7 days was studied on the development of various brush border enzymes across the crypt-villus axis. The activities of brush border maltase (P < 0.05), lactase (P < 0.05), alkaline phosphatase (P < 0.05) and leucine amino peptidase (P < 0.05) were augmented in purified brush borders, whereas sucrase, trehlase (P < 0.01) and glutamyl transpeptidase (P < 0.05) were reduced in nickel fed animals compared with controls. Kinetic and heat inactivation studies with brush border sucrase and alkaline phosphatase confirmed these findings. Western blot analysis of alkaline phosphatase showed a strong signal for the enzyme protein but a reduced level of sucrase antigen in nickel fed rat intestine compared with the controls. These findings suggest that the expression of various brush border enzymes along the crypt-villus axis is modulated in rat intestine exposed to nickel, which may disrupt the digestive functions of the intestinal tissue.     

The combined toxicity assessment of carp (Cyprinus carpio) acetylcholinesterase activity by binary mixtures of chlorpyrifos and four other insecticides

Mixtures of organophosphate (OP) and carbamate (CB) insecticides are commonly detected in freshwater habitats. These insecticides inhibit the activity of acetylcholinesterase (AChE) and have potential to interfere with behaviors that may be essential for survival of species. Although the effects of individual anticholinesterase insecticides on aquatic species have been studied for decades, the combined toxicity of mixtures is still poorly understood. In the present study, we assessed whether pesticides in a mixture act in isolation (resulting in additive AChE inhibition) or whether components interact to produce either antagonistic or synergistic toxicity. Brain AChE inhibition in carp (Cyprinus carpio L.) exposed to a series of concentrations of the OP (chlorpyrifos, malathion and triazophos) as well as the CB (fenobucarb and carbosulfan) were measured. The concentration addition (CA) model and the isobole method were used to determine whether toxicological responses to binary mixtures of pesticides. In 50:50 % effect mixtures, the observed combined toxicity of chlorpyrifos and malathion was significantly higher than observed and was considered as synergistic. For equivalent dose mixtures, when chlorpyrifos mixed with fenobucarb or malathion, the observed toxicities were significantly higher than predicted, suggesting synergistic joint actions. The rest five binary combinations exhibited concentration additive or slight antagonistic joint actions. The CA model and the isobole method provided estimates of mixture toxicity that did not markedly underestimate the measured toxicity, therefore these methods are suitable to use in ecological risk assessments of pesticide mixtures.     

In vivo and in vitro liver and gill EROD activity in rainbow trout (Oncorhynchus mykiss) exposed to the beta-blocker propranolol

The conservation of common physiological systems across vertebrate classes suggests the potential for certain pharmaceuticals, which have been detected in surface waters, to produce biological effects in nontarget vertebrates such as fish. However, previous studies assessing the effects of such compounds in fish have not taken into account the potential for metabolism and elimination. This study aimed to assess if propranolol, a β-adrenergic receptor antagonist or β-blocker, could modulate EROD activity (indicative of CYP1A activity) in rainbow trout (Oncorhynchus mykiss) gills and liver. For this, an in vivo time course exposure with 1 mg/L was conducted. Additionally, using measured in vivo plasma concentrations, an in vitro exposure at human therapeutic levels was undertaken. This allowed comparison of in vitro and in vivo rates of EROD activity, thus investigating the applicability of cell preparations as surrogates for whole animal enzyme activity analysis. In vitro exposure of suspended liver and gill cells at concentrations similar to in vivo levels resulted in EROD activity in both tissues, but with significantly higher rates (up to six times in vivo levels). These results show that propranolol exposure elevated EROD activity in the liver and gill of rainbow trout, and that this is demonstrable both in vivo (albeit nonsignificantly in the liver) and in vitro, thus supporting the use of the latter as a surrogate of the former. These data also provide an insight into the potential role of the gill as a site of metabolism of pharmaceuticals in trout, suggesting that propranolol (and feasibly other pharmaceuticals) may undergo "first pass" metabolism in this organ.     

Assessment of the acute toxicity of chlorpyrifos and cypermethrin to Heteropneustes fossilis and their impact on acetylcholinesterase activity

In the present study, the acute toxicity of chlorpyrifos (an organophosphate, OP) and cypermethrin (a pyrethroid) pesticides was estimated for 96?h in Heteropneustes fossilis. The LC₅₀ for chlorpyrifos (CPF) and cypermethrin was found to be 1.90?mg/L and 0.085?mg/L, respectively. The acetylcholinesterase (AChE, EC 3.1.1.7) activity in Heteropneustes fossilis exposed to both the insecticides was assayed in brain, muscle and gills. In general, tissue specific as well as dose-dependent decrease in the AChE activity was exhibited by both pesticides. In response to the increasing concentrations of chlorpyrifos and cypermethrin as well, a significant decrease in the activity of AChE was found in brain while muscle and gills exhibited lesser inhibition. Thus, the brain was the main target organ for both insecticides, followed by muscle and gills. Between the two pesticides chlorpyrifos acted as more potent AChE inhibitor than cypermethrin since more intense changes in behavioral pattern was observed with the chlorpyrifos. These changes indicate that the effects of these pesticides are at neural as well as neuromuscular level.     

Alterations of glycosaminoglycans in human liver and kidney tumors

Glycosaminoglycans were investigated in surgically removed human liver and kidney tumours by applying biochemical methods. Four liver adenoma, 6 focal nodular hyperplasia and 9 primary hepatocellular carcinoma samples were compared with normal liver from autopsy cases and also with liver tissue adjacent to PHC. The studies on kidney included 14 renal cell carcinoma and 4 wilms' tumour samples. Three findings emerged from the quantitative and qualitative characterization of the tumours with epithelial origin. 1) The rise in the amount of total GAG was not limited to the malignant lesion. Similar increase was observed in benign liver tumours and also in the tissue adjacent to liver or kidney malignant tumours. 2) The dominant type of the GAG subclasses varies with the histology of the tumours. In benign liver tumours dermatan sulfate, in PHC and renal cell carcinoma chondroitin sulfate, but in Wilms' tumour hyaluronate was the prominent GAG subclass. 3) In all tumour-affected tissues dermatan and chondroitin sulfates had lower degree of sulfation. However, in the histologically different tumours various disaccharides showed reduced level of sulfation. The GAG alteration in renal cell carcinoma was compared with the prognostic factors of each individual case. This analysis showed a good correlation between HS/CS ratio and the prognostic factors of the kidney tumour cases.     

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.     

Antioxidant enzymes activity and lipid peroxidation in liver and kidney of rats exposed to cadmium and ethanol.

The oxidative status of liver and kidney of rats co-exposed to cadmium (50 mg Cd/l in drinking water) and ethanol (5 g EtOH/kg body weight/24 h, intragastrically) for 12 weeks was studied. The activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) as well as the concentration of malondialdehyde (MDA), as an indicator of lipid peroxidation, were measured in homogenates of the liver and kidney. Concentrations of zinc (Zn), copper (Cu), iron (Fe) and Cd in the serum or blood, and their content in the liver and kidney as well as EtOH concentration in the whole blood were assayed. Daily Cd intake in the Cd and Cd+EtOH groups was similar and ranged from 2.39 to 4.88 mg/kg body weight/24 h and from 2.64 to 4.14 mg/kg body weight/24 h, respectively. After the administration of EtOH alone, the activity of SOD increased in the kidney and decreased in the liver, whereas the activity of CAT decreased in both these organs, and MDA concentration increased in the liver and was unchanged in the kidney. The exposure to 50 mg Cd/l led to a decrease in the activities of SOD in the liver and CAT in the liver and kidney, and an increase in the kidney activity of SOD and MDA concentration in both these organs. In the rats co-exposed to Cd and EtOH, the kidney activity of SOD and the liver concentration of MDA were lower, whereas the kidney activity of CAT was higher compared to the Cd group. The concentration of Fe in the serum and its content in the liver of rats treated with EtOH increased, whereas the concentrations of Zn and Cu in the serum and the content of Zn, Cu and Fe in the kidney and that of Zn and Cu in the liver were unchanged. In the liver and kidney of rats treated with Cd alone, the content of Fe was decreased and that of Zn and Cu was enhanced. After EtOH administration to Cd-exposed rats, a decrease in Cu serum concentration and its liver content and an increase in Fe concentration in the serum and its content in the liver and kidney, compared to the group exposed to Cd alone, were noted. Moreover, EtOH decreased the blood Cd concentration and its accumulation in the liver and kidney of these animals. EtOH alone decreased Cd content in the liver and increased in the kidney, however the whole content of Cd in these organs was unchanged compared with control. The results of this study indicate that despite the ability of Cd and EtOH to induce the oxidative stress the effect in the liver and kidney is not intensified at simultaneous exposure to both substances. The changes in the studied indicators of oxidative stress (SOD, CAT and MDA) observed in the kidney and especially in the liver of the rats co-exposed to Cd and EtOH may result from an independent effect of Cd and/or EtOH and also from their interaction. The interactive effect may involve, among others, changes in Cd accumulation and content of Zn, Cu and Fe in these organs and their concentration in serum. Since the rats treated with Cd and Cd+EtOH had reduced drinking fluids intake that might result in dehydratation, the effect of the both xenobiotics on the oxidative status of the body may be not solely due to Cd and/or EtOH, but also the modyfing influence of accompanying alterations such as reduced water intake and dehydratation. The results of the study allow us to hypothesize that Cd-exposed alcohol misusers are not at enhanced risk of liver and kidney damage due to lipid peroxidation.