In vivo and in vitro effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius Ham. (rosy barb).

1. Enzyme modulation by cadmium in selected organs of the fish, Barbus conchonius (rosy barb), was investigated in vivo (48 hr exposure to 12.6 mg/l cadmium chloride) and in vitro (10(-6) M cadmium chloride). 2. The acetylcholinesterase (AchE) activity was depressed in the gills but stimulated in the skeletal muscles and brain in vivo. The hepatic, branchial, and renal acid phosphatase (AcP) activity decreased marginally in vivo but it was significantly increased in the gut and ovary. In vitro, except for the liver, the AcP activity was depressed in the selected organs. Collaterally, gut alkaline phosphatase (AlP) was significantly inhibited but a pronounced stimulation was noted in the kidneys and ovary in vivo. In vitro, the AlP activity was conspicuously elevated in the kidneys and gut, and moderately in the gills. 3. Cadmium inhibited the glutamate-oxaloacetate and glutamate-pyruvate transaminases (GOT and GPT) in the liver, gills and kidneys in vivo. In vitro, the GOT and GPT activities were decreased in the liver, gills and kidneys. The lactic dehydrogenase (LDH) was significantly stimulated by Cd in the heart in vivo but in vitro the metal inhibited the enzyme in the gills. 4. Enzymes in the liver, followed by those in the kidneys and gills seem to be most seriously affected by Cd poisoning in this fish.     

In vivo and in vitro effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius ham. (Rosy Barb)

1. Enzyme modulation by cadmium in selected organs of the fish, Barbus conchonius (rosy barb), was investigated in vivo (48 hr exposure to 12.6 mg/1 cadmium chloride) and in vitro (10⁻⁶M cadmium chloride).2. The acetylcholinesterase (AchE) activity was depressed in the gills but stimulated in the skeletal muscles and brain in vivo. The hepatic, branchial, and renal acid phosphatase (AcP) activity decreased marginally in vivo but it was significantly increased in the gut and ovary. In vitro, except for the liver, the AcP activity was depressed in the selected organs. Collaterally, gut alkaline phosphatase (A1P) was significantly inhibited but a pronounced stimulation was noted in the kidneys and ovary in vivo. In vitro, the AIP activity was conspicuously elevated in the kidneys and gut, and moderately in the gills.3. Cadmium inhibited the glutamate-oxaloacetate and glutamate-pyruvate transaminases (GOT and OPT) in the liver, gills and kidneys in vivo. In vitro, the GOT and GPT activities were decreased in the liver, gills and kidneys. The lactic dehydrogenase (LDH) was significantly stimulated by Cd in the heart in vivo but in vitro the metal inhibited the enzyme in the gills.4. Enzymes in the liver, followed by those in the kidneys and gills seem to be most seriously affected by Cd poisoning in this fish.     

Sublethal effects of an organophosphorus insecticide (RPR-II) on biochemical parameters of tilapia, Oreochromis mossambicus

The effect of exposure to sublethal concentrations (0.017 mg L(-1), 1/10 of LC50) of the novel organophosphate (OP) insecticide, 2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester (RPR-II) on biochemical parameters in Oreochromis mossambicus was studied during exposure for 3, 7, 15, 30 and its recovery response after seven days. Acetylcholinesterase (AChE) activity of brain, gill and muscle was inhibited by 67%, 77% and 73% respectively on day-30. The plasma and kidney alanine aminotransferase (AlaAT), and aspartate aminotransferase (AspAT) activity increased, while decreases were observed in gill and liver. Increases in acid phosphatase (AcP), and alkaline phosphatase (AP) activities were observed in plasma, gill, and kidney, and reductions of 20% and 61% in liver AcP and AP, respectively. Depletion of glycogen was observed in all tissues, an indication of typical stress related response of the fish with pesticide. Lactate dehydrogenase (LDH) activity decreased in liver and muscle, indicating tissue damage but a significant increase in LDH activity in gill and brain was observed. Depletion of glutathione (GSH) was observed in all tissues, thereby enhancing lipid peroxidation resulting in cell damage. The induction in hepatic glutathione-S-transferase (GST) levels indicates protection against the toxicity of xenobiotic-induced lipid peroxidation. There was a significant recovery in the above biochemical parameters, in all tissues of fish after a recovery period of seven days. These results revealed that the OP insecticide RPR-II is highly toxic and affects the intermediary metabolism of O. mossambicus.     

Effects of cadmium toxicity upon the in vivo and in vitro activity of proteins and five enzymes in blood serum and tissue homogenates of Mugil cephalus

This study reports changes in total protein and certain liver, heart, gill and serum enzymes after exposing fish or tissue homogenates and serum to Cd, i.e. in vivo and in vitro effects. Five enzymes were selected for assay; aspartate amino transferase (AAT), alanine amino transferase (A1AT), alkaline phosphatase (A1P), acid phosphatase (ACP) and lactic dehydrogenase (LDH). Total protein content in the exposed fish showed an increase in the liver, gill and serum while there was no change in heart protein. The sensitivity of the five assayed enzymes to Cd varied in different tissues. Gill AAT, A1AT were the most sensitive. Liver A1P and heart LDH showed the maximum inhibition at higher Cd concentrations.     

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.     

Effect of estragole on glucocorticoid induction of tyrosine aminotransferase and tryprophan oxygenase in the rat and mouse liver

A single intraperitoneal injection of Estragole (300 mg/kg) to female ICR mice 19 hours prior to Dexamethasone induction decreased induced activities of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) nearly to 50% of the control values. In these mice, activities of the marker enzymes of liver damage: alanine aminotransferase (ALAT) and aspartate aminotransferase (AAT) increased in the blood 1.7-2.3-fold as compared with the untreated controls. By contrast, carbon tetrachloride (100 mg/kg) increased the blood AIAT and AsAT activities 135- and 30-fold as compared with the control, but inhibited the TAT and TO induction much less than Estragole did. Estragole seems to inhibit the glucocorticoid induction of these hepatic enzymes not via the unspecific toxic damage of the liver.     

Mercury chloride increases hepatic alanine aminotransferase and glucose 6-phosphatase activities in newborn rats in vivo

This work investigated the in vivo and in vitro effects of HgCl2 and ZnCl2 on metabolic enzymes from tissues of young rats to verify whether the physiological and biochemical alterations induced by mercury and prevented by zinc are related to hepatic and renal glucose metabolism. Wistar rats received (subcutaneous) saline or ZnCl2 (27 mg/kg/day) from 3 to 7 days old and saline or HgCl2 (5.0 mg/kg/day) from 8 to 12 days old. Mercury exposure increased the hepatic alanine aminotransferase (～6-fold) and glucose 6-phosphatase (75%) activity; zinc pre-exposure prevented totally and partially these mercury alterations respectively. In vitro, HgCl2 inhibited the serum (22%, 10 μM) and liver (54%, 100 μM) alanine aminotransferase, serum (53%) and liver (64%) lactate dehydrogenase (10 μM), and liver (53%) and kidney (41%) glucose 6-phosphatase (100 μM) from 10- to 13-day-old rats. The results show that mercury induces distinct alterations in these enzymes when tested in vivo or in vitro as well as when different sources were used. The increase of both hepatic alanine aminotransferase and glucose 6-phosphatase activity suggests that the mercury-exposed rats have increased gluconeogenic activity in the liver. Zinc prevents the in vivo effects on metabolic changes induced by mercury.     

Enzymes of cultured human fibroblasts. IV. Enzyme activity in trisomy C strains]

The activity of five enzymes (AIP, AcP, GOT, LDH, MDH) was investigated in four cell strains derived from spontaneous abortuses with C-trisomy (three cell strains with trisomy 7, one--with trisomy 9). Significant differences in the activity of three enzymes were revealed. In all the strains AIP activity was lower and GOT activity--higher than in diploid strains. Lowering of AcP level was found in three strains (two cell strains with trisomy 7, one--with trisomy 9). The data obtained are evaluated as a result of disturbed regulatory interrelations in an abnormal genome.     

Subcellular distribution of aminotransferases, and pyruvate branch point enzymes in gill tissue from four bivalves

Aspartate aminotransferase (AAT), alanine aminotransferase (ALAT), malic enzyme (ME), malate dehydrogenase (MDH), pyruvate kinase (PK), and phosphoenolpyruvate carboxykinase (PEPCK) activities in cytosolic and mitochondrial fractions of gill tissue from Modiolus demissus (ribbed mussel), Mytilus edulis (sea mussel), Crassostrea virginica (oyster) and Mercenaria mercenaria (quahog) were determined using enzyme assay and starch gel electrophoresis combined with subcellular fractionation. AAT showed distinct mitochondrial and cytosolic isozymes in gills of all these animals. Although ALAT showed distinct mitochondrial and cytosolic isozymes in the gills of oysters, sea mussels and quahogs, only the mitochondrial ALAT was evident in ribbed mussel gill tissue. PK and PEPCK were cytosolic in all these preparations. ME was found only in the mitochondrial fraction of ribbed mussel and quahog gill tissue whereas sea mussel gills showed distinct cytosolic and mitochondrial ME isozymes. With oyster gills, the "cytosolic ME" was electrophoretically identical to the mitochondrial ME indicating that in vivo, the ME is probably mitochondrial. MDH showed distinct cytosolic and mitochondrial isozymes in all bivalve gills tested.     

Possible mechanism for the decrease of mitochondrial aspartate aminotransferase activity in ischemic and hypoxic rat retinas.

Glutamate is believed to be an excitatory amino acid neurotransmitter in the retina. Enzymes for glutamate metabolism, such as glutamate dehydrogenase, ornithine aminotransferase, glutaminase, and aspartate aminotransferase (AAT), exist mainly in the mitochondria. The abnormal increase of intracellular calcium ions in ischemic retinal cells may cause an influx of calcium ions into the mitochondria, subsequently affecting various mitochondrial enzyme activities through the activity of mitochondrial calpain. As AAT has the highest level of activity among enzymes involved in glutamate metabolism, we investigated the change of AAT activity in ischemic and hypoxic rat retinas and the protection against such activity by calpain inhibitors. We used normal RCS (rdy+/rdy+) rats. For the in vivo studies, we clamped the optic nerve of anesthetized rats to induce ischemia. In the in vitro studies, the eye cups were incubated with Locke's solution saturated with 95% N2/5% CO2. The activity of cytosolic AAT (cAAT) was about 20% of total activity, whereas mitochondrial AAT (mAAT) was about 75% in rat retina. Ninety minutes of ischemia or hypoxia caused a 20% decrease in mAAT activity, whereas cAAT activity remained unchanged. To examine the contribution of intracellular calcium ions to the degradation of mAAT, we used Ca2+-free Locke's solution containing 1 mM EGTA, ryanodine (Ca2+ channel blocker), and thapsigargin (Ca2+-ATPase inhibitor). In the present study, thapsigargin in Ca2+-free Locke's solution, but not ryanodine in this solution, was found to prevent AAT degradation. AAT degradation was also prevented by calpain inhibitors (Ca2+-dependent protease inhibitor) such as calpeptin at 1 nM, 10 nM, 0.1 microM, 1 microM and 10 microM, and by calpain inhibitor peptide, but not by other protease inhibitors (10 microM leupeptin, pepstatin, chymostatin). Additionally, we determined the subcellular localization of calpain activity and examined the change of calpain activity in ischemic rat retinas. Our results suggest that decreased activity of mAAT in ischemic and hypoxic rat retinas might be evoked by the degradation by calpain-catalyzed proteolysis in mitochondria.     

Endogenous generation of sulfur dioxide in rat tissues

While sulfur dioxide (SO₂) has been previously known for its toxicological effects, it is now known to be produced endogenously in mammals from sulfur-containing amino acid l-cysteine. l-cysteine is catalyzed by cysteine dioxygenase (CDO) to l-cysteinesulfinate, which converts to β-sulfinylpyruvate through transamination by aspartate aminotransferase (AAT), and finally spontaneously decomposes to pyruvate and SO₂. The present study explored endogenous SO₂ production, and AAT and CDO distribution in different rat tissue. SO₂ content was highest in stomach, followed by tissues in the right ventricle, left ventricle, cerebral gray matter, pancreas, lung, cerebral white matter, renal medulla, spleen, renal cortex and liver. AAT activity and AAT1 mRNA expression were highest in the left ventricle, while AAT1 protein expression was highest in the right ventricle. AAT2 and CDO mRNA expressions were both highest in liver tissue. AAT2 protein expression was highest in the renal medulla, but CDO protein expression was highest in liver tissue. In all tissues, AAT1 and AAT2 were mainly distributed in the cytoplasm rather than the nucleus. These observed differences among tissues endogenously generating SO₂ and associated enzymes are important in implicating the discovery of SO₂ as a novel endogenous signaling molecule.     

Distribution of glutamine hexosephosphate aminotransferase in rat tissues; changes with state of differentiation

The activity of glutamine hexosephosphate aminotransferase (L-glutamine: D-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was determined with an improved assay method in some three dozen rat tissues: adult, developing and neoplastic. The highest activities (20–200 units/g) were seen in colon, mammary (during late lactation), submaxillary, sublingual and parotid glands, placenta and liver. The activity increased strikingly along the length of the intestine; glucose feeding inhibited it in ileum and colon. In liver and intestine the activity increased with age but in brain, muscle, heart and kidney the activity was considerably higher during fetal (7.1–12.8 units/g) than in adult life (0.8–3.5 units/g). Renal, mammary and muscle tumors (but not hepatomas) had much higher activities (4–20.5 units/g) than the cognate normal adult tissue.The distribution pattern among tissues indicates that glutamine hexosephosphate aminotransferase is of general importance to all growing, undifferentiated tissues and of special importance to the differentiated function of particular adult organs. The latter are organs which engage in glycoprotein secretion. The results support the assumption that glutamine hexosephosphate aminotransferase activity is essential for glycoprotein synthesis.     

Toxicity studies of nonylphenol on rosy barb (Puntius conchonious): a biochemical and histopathological evaluation

Acute and subacute toxicity of the nonylphenol (NP) on fish was investigated in laboratory toxicity tests with rosy barb (Puntius conchonious). The acute toxicity of NP to rosy barb was determined in semi-static bioassays. Median lethal concentration (LC50) for 96 h was 1.72 microM. The effects of sublethal concentrations of NP (0.17, 0.34 and 0.68 microM) on the structures and biochemical parameters [alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanin aminotransferase (ALT)] in gills, liver and kidney of rosy barb were studied after 14 days. The results showed that NP caused alteration of the structure in organs, as evidenced by the hyperplasia of epithelium and the fusion of secondary lamellae in the gills, the disappearance of the cell membrane and the cell necrosis in the liver as well as haemorrhages in the kidney. In addition, the functional enzyme activities were also changed. The increase trend in ALP activity in organs of fish treated with NP was recorded. The levels of AST and ALT in gills, liver and kidney were stimulated to rise at the lower concentration and fall at the higher concentration NP treatment compared to controls. This study suggests that NP can alter of the structures and biochemical parameters within non-endocrine tissue of fish and these changes may be mediated via destroying membrane structure and inducing cell necrosis.     

Diphenyl diselenide reverses cadmium-induced oxidative damage on mice tissues

The concept that selenium-containing molecules may be better antioxidants than classical antioxidants, has led to the design of synthetic organoselenium compounds. In the present investigation subchronic deleterious effects of cadmium-intoxication in mice and a possible protective effect of diphenyl diselenide (PhSe)2 (5 micromol/kg) were studied. Male adult Swiss albino mice (25-35 g) received CdCl2 (10 micromol/kg, subcutaneously), five times/week, for 4 weeks. A number of toxicological parameters in blood, liver, kidney, spleen and brain of mice were examined including delta-aminolevulinic acid dehydratase (delta-ALA-D) activity, lipid peroxidation and ascorbic acid content, the parameters that indicate tissue damage such as plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, creatinine and lactate dehydrogenase (LDH) were also determined. The results demonstrated that cadmium caused inhibition of delta-ALA-D activity in liver (24%), kidney (33%) and spleen (73%) and (PhSe)2 therapy was effective in restoring enzyme activity in all tissues. A reduction in ascorbic acid content was observed in kidney (11%) and spleen (10.7%) of cadmium-treated mice and (PhSe)2 was only effective in improving this reduction in kidney. An increase of lipid peroxidation induced by cadmium was noted in liver (29%) and brain (28%) tissues and (PhSe)2 therapy was effective in restoring TBARS levels in both tissues. We also observed an increase on plasma LDH (1.99-times), AST (1.93-times) and ALT (4.24-times) activities. (PhSe)2 therapy was effective in restoring AST activity at control level. (PhSe)2 did not present toxic effects when plasma parameters were evaluated. The results suggest that the administration of an antioxidant (PhSe)2, during cadmium intoxication may provide beneficial effects by reducing oxidative stress in tissues.     

The possible metabolic diversions adapted by the cockroach, Periplaneta americana to counteract the toxicity of fenvalerate

Effects of sublethal doses of fenvalerate through topical application were monitored in the central nervous system (CNS) of P. americana. A decrease in total and soluble proteins with an increase in free amino acids, alanine aminotransferase (AlAT) and aspartate aminotransferase (AAT) was observed during fenvalerate toxicity. Further the levels of glycogen, pyruvate and activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) dropped significantly. Lactate content and lactate dehydrogenase (LDH) activity also showed an elevation following fenvalerate toxicity.     

Carbohydrate and nitrogenous metabolism condition in the rat tissue under experimental rhabdomyolysis

Some effects of glycerol injection on indices of the condition of the thiol-disulfide system as well as carbohydrate and nitrogen metabolism in rats in vivo were studied. A decrease was revealed in levels of non-protein SH-groups in the liver, kidney and heart, as well as of protein SH-groups in the kidney and heart of rats following glycerol injection. That might be connected with SH-group oxidation under the excessive arrival of free haem into tissues under rhabdomyolysis. A decrease in glycogen and increase in tyrosine aminotransferase activity in the liver were observed. Activation of nitrogenous metabolism following glycerol injection is indicated by the increase of aminotransferase activity in organs, and concentration of blood urea. High concentration of creatinine in the rat serum can reflect malfiltration in kidneys.     

Control of ketogenesis from amino acids. III. In vitro and in vivo studies on ketone body formation lipogenesis and oxidation of tyrosine by rats.

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferse (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rat limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate. For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1- 14C]tyrosine. Then their respiratory 14CO2 and the incorporation of 14C into total lipids of liver were measured. The amounts of radioactivity in CO2 and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U- 14C]acetate the radioactivities in CO2 and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studied gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

鲮鱼冷休克及其死亡的某些生化因素

采用分光光度法和淀粉凝胶电泳法初步研究了鲮鱼冷休克前后脑乙酰胆碱酯酶(AchE)活力和三种酶的同工酶类的动态变化。并根据鲮鱼在冷休克期间(7—6℃)脑AchE活力显著降低和肝脏组织乳酸脱氢酶(LDH)同工酶活性明显升高以及苹果酸脱氢酶(MDH)、酯酶(EST)同工酶类出现酶活性变化的情况,讨论了导致鲮鱼耐寒能力差的某些生化因素。同时提出脑AchE可以作为评定鲮鱼冷休克期间中枢神经系统受害程度的一种生化指标。       

Divergent effects of alpha1-antitrypsin on neutrophil activation, in vitro

alpha1-Antitrypsin (AAT) is a major circulating serine proteinase inhibitor in humans. The anti-proteinase activity of AAT is inhibited by chemical modification. These include inter- or intramolecular polymerisation, oxidation, complex formation with target proteinases (e.g., neutrophil elastase), and/or cleavage by multi-specific proteinases. In vivo, several modified forms of AAT have been identified which stimulate biological activity in vitro unrelated to inhibition of serine proteinases. In this study we have examined the effects of native and polymerised AAT and C-36 peptide, a proteolytic cleavage product of AAT, on human neutrophil activation, in vitro. We show that the C-36 peptide displays striking concentration-dependent pro-inflammatory effects on human neutrophils, including induction of neutrophil chemotaxis, adhesion, degranulation, and superoxide generation. In contrast to C-36 peptide, native and polymerised AAT at similar and higher concentrations showed no effects on neutrophil activation. These results suggest that cleavage of AAT may not only abolish its proteinase inhibitor activity, but can also generate a powerful pro-inflammatory activator for human neutrophils.     

The mechanisms of the neutrophil suppression of creatine kinase and aspartate aminotransferase activities in rat skeletal muscles]

Abdominal neutrophils effect on rat skeletal muscle m. soleus was investigated in vitro. The incubation was carried out in Hanks balanced solution within 24 hrs. It was a release of proteins from m. soleus 1 hr later. Creatine kinase (CK) and aspartate aminotransferase (AAT) activities increase was detected in incubation medium. The neutrophils released their proteins quicker than muscles. A dramatic inhibition of CK and AAT activities took place during coincubation of m. soleus and neutrophils. Zymosan-activated cells had a higher inhibition potency in comparison to nonactivated neutrophils. Analysis of proteinase and myeloperoxidase activities in incubation medium has given evidence that CK and AAT inhibition by non-activated neutrophils mainly depends on cell-secreted proteinases. Zymosan-activated neutrophil inhibition of CK and AAT consists of proteinases and myeloperoxidase effects. AAT appeared to be more resistant than CK to the damage by neutrophils. The used approach failed to demonstrate the direct damage effect of neutrophils on m. soleus, but the described enzyme inhibition mechanism can take place in vivo during leukocyte infiltration of skeletal muscles after intensive muscular activity.