A comparison of cholinesterase activity after intravenous, oral or dermal administration of methyl parathion

Time-dependent changes in blood cholinesterase activity caused by single intravenous, oral or dermal administration of methyl parathion to adult female rats were defined. Intravenous and oral administration of 2.5 mg/kg methyl parathion resulted in rapid (<60 min) decreases in cholinesterase activity which recovered fully in vivo within 30-48 h. In contrast, spontaneous reactivation of cholinesterase in vitro was complete within 6 h at 37 degrees C. Dermal administration of methyl parathion caused dose-dependent inhibition of cholinesterase activity which developed slowly (> or =6 h) and was prolonged (> or =48 h). Time- and route-dependent effects of methyl parathion on cholinesterase activity in brain and other tissues generally paralleled its effects on activity in blood. In conclusion, pharmacodynamics of methyl parathion differ substantially with route of exposure. Recovery of cholinesterase in vivo after intravenous or oral exposure may partially reflect spontaneous reactivation and suggests a rapid clearance of methyl parathion or its active metabolite methyl paraoxon. The more gradual and prolonged inhibition of cholinesterase caused by dermal administration is consistent with disposition of methyl parathion at a site from which it or methyl paraoxon is only slowly distributed. Thus, dermal exposure to methyl parathion may pose the greatest risk for long-term adverse effects.     

Effects of single or repeated dermal exposure to methyl parathion on behavior and blood cholinesterase activity in rats

The effects of a single or repeated dermal administration of methyl parathion on motor function, learning and memory were investigated in adult female rats and correlated with blood cholinesterase activity. Exposure to a single dose of 50 mg/kg methyl parathion (75% of the dermal LD(50)) resulted in an 88% inhibition of blood cholinesterase activity and was associated with severe acute toxicity. Spontaneous locomotor activity and neuromuscular coordination were also depressed. Rats treated with a lower dose of methyl parathion, i.e. 6.25 or 12.5 mg/kg, displayed minimal signs of acute toxicity. Blood cholinesterase activity and motor function, however, were depressed initially but recovered fully within 1-3 weeks. There were no delayed effects of a single dose of methyl parathion on learning acquisition or memory as assessed by a step-down inhibitory avoidance learning task. Repeated treatment with 1 mg/kg/day methyl parathion resulted in a 50% inhibition of blood cholinesterase activity. A decrease in locomotor activity and impairment of memory were also observed after 28 days of repeated treatment. Thus, a single dermal exposure of rats to doses of methyl parathion which are lower than those that elicit acute toxicity can cause decrements in both cholinesterase activity and motor function which are reversible. In contrast, repeated low-dose dermal treatment results in a sustained inhibition of cholinesterase activity and impairment of both motor function and memory.     

Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1mg/kg per day for 1, 7 and 14 days), methapyrilene (100mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and (3) develop hypotheses regarding mechanisms of toxicity.     

Effect of N-acetylcysteine on the pharmacokinetics of acetaminophen in rats

Oral administration of N-acetylcysteine (163 mg/kg at zero time and 82 mg/kg 30 minutes later) to adult male Sprague-Dawley rats given an intravenous injection of acetaminophen, 150 mg/kg at zero time, increased the formation of acetaminophen sulfate and thereby enhanced the elimination of acetaminophen. Apparently, N-acetylcysteine is an in vivo source of inorganic sulfate since availability of the latter is rate-limiting in the formation of acetaminophen sulfate. Increased metabolic conversion of acetaminophen to its sulfate conjugate results in decreased formation of other metabolites of acetaminophen, presumably including the reactive metabolite responsible for the hepatotoxic effect of the drug. This may account, at least in part, for the protective effect of N-acetylcysteine against acetaminophen-induced hepatotoxicity.     

Protection of a Ceramide Synthase 2 Null Mouse from Drug-induced Liver Injury: ROLE OF GAP JUNCTION DYSFUNCTION AND CONNEXIN 32 MISLOCALIZATION*

Very long chain (C22-C24) ceramides are synthesized by ceramide synthase 2 (CerS2). A CerS2 null mouse displays hepatopathy because of depletion of C22-C24 ceramides, elevation of C16-ceramide, and/or elevation of sphinganine. Unexpectedly, CerS2 null mice were resistant to acetaminophen-induced hepatotoxicity. Although there were a number of biochemical changes in the liver, such as increased levels of glutathione and multiple drug-resistant protein 4, these effects are unlikely to account for the lack of acetaminophen toxicity. A number of other hepatotoxic agents, such as d-galactosamine, CCl₄, and thioacetamide, were also ineffective in inducing liver damage. All of these drugs and chemicals require connexin (Cx) 32, a key gap junction protein, to induce hepatotoxicity. Cx32 was mislocalized to an intracellular location in hepatocytes from CerS2 null mice, which resulted in accelerated rates of its lysosomal degradation. This mislocalization resulted from the altered membrane properties of the CerS2 null mice, which was exemplified by the disruption of detergent-resistant membranes. The lack of acetaminophen toxicity and Cx32 mislocalization were reversed upon infection with recombinant adeno-associated virus expressing CerS2. We establish that Gap junction function is compromised upon altering the sphingolipid acyl chain length composition, which is of relevance for understanding the regulation of drug-induced liver injury.     

Neurotoxicity in Snakebite—The Limits of Our Knowledge

Snakebite is classified by the WHO as a neglected tropical disease. Envenoming is a significant public health problem in tropical and subtropical regions. Neurotoxicity is a key feature of some envenomings, and there are many unanswered questions regarding this manifestation. Acute neuromuscular weakness with respiratory involvement is the most clinically important neurotoxic effect. Data is limited on the many other acute neurotoxic manifestations, and especially delayed neurotoxicity. Symptom evolution and recovery, patterns of weakness, respiratory involvement, and response to antivenom and acetyl cholinesterase inhibitors are variable, and seem to depend on the snake species, type of neurotoxicity, and geographical variations. Recent data have challenged the traditional concepts of neurotoxicity in snake envenoming, and highlight the rich diversity of snake neurotoxins. A uniform system of classification of the pattern of neuromuscular weakness and models for predicting type of toxicity and development of respiratory weakness are still lacking, and would greatly aid clinical decision making and future research. This review attempts to update the reader on the current state of knowledge regarding this important issue.     

Metabolomics and biomarker discovery: NMR spectral data of urine and hepatotoxicity by carbon tetrachloride, acetaminophen, and d-galactosamine in rats

The primary objective of this study was to discover biomarkers which are correlated with hepatotoxicity induced by chemicals using ¹H NMR spectral data of urine. A procedure of nuclear magnetic resonance (NMR) urinalysis using pattern recognition was proposed for early screening of the hepatotoxicity of CCl₄, acetaminophen (AAP), and d-galactosamine (GalN) in rats. The hepatotoxic compounds were expected to induce necrosis in hepatocytes. This was confirmed through blood biochemistry and histopathology. CCl₄ (1 ml/kg, po) or GalN (0.8 g/kg, ip) was single administered to Sprague–Dawley (S–D) rats and urine was collected every 24 h. Animals were sacrificed 24 h or 48 h post-dosing. AAP (2 g/kg, po) was administered for 2 days and then the animals were sacrificed 24 h after the last treatment. NMR spectroscopy revealed evidently different clustering between control groups and hepatotoxicant treatment groups in global metabolic profilings as indicated by partial least square (PLS)-discrimination analysis (DA). In targeted profilings, endogenous metabolites of allantoin, citrate, taurine, 2-oxoglutarate, acetate, lactate, phenylacetyl glycine, succinate, phenylacetate, 1-methylnicotinamide, hippurate, and benzoate were selected as putative biomarkers for hepatoxicity by CCl₄, AAP, and GalN. Comparison of our rat ¹H NMR PLS-DA data with histopathological changes suggests that ¹H NMR urinalysis can be used to predict hepatotoxicity induced by CCl₄, AAP, and GalN.     

Glutathione reductase and catalase as potential biomarkers for synergistic intoxication of pesticides in fish

Abstract

Synergy occurs when chemicals give pronounced effect on combination in contrast to their individual effect. The objective of this study was to investigate the synergistic effect of pesticides carbaryl (C) and methyl parathion (MP) on oxidative stress biomarkers viz catalase (CAT), glutathione reductase (GSSG-R) including different enzymes like lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acetyl cholinesterase (AChE) in different tissues of carps Catla catla. Fishes were exposed to 6.25?mg/L of MP and 2.3?mg/L of C in mixture (one-third of LC₅₀ value). CAT and GSSG-R were studied in gills, brain, liver and muscle of carp were found to be elevated significantly (p?<?0.005). LDH activity increased significantly (p?<?0.005) in synergistic group, there was a seven-fold (748%) increase in LDH activity in muscle compared to individual studies with same pesticides. Contrary to LDH, sudden decrease in SDH activity was accounted. Significant (p?<?0.005) decrease in AChE activity after initial 24?h was remarkable addressing to the shift in neurotransmission pathway in organism. Significant increase was observed in activity of CAT and GSSG-R in all tissues compared to control fishes in individual as well as synergistic (MP?+?C) group suggesting that CAT and GSSG-R can be a potential biomarker of oxidative stress when studied in combination.     

Tris-(2,3-Dibromopropyl) Isocyanurate,a New Emerging Pollutant,Impairs Cognition and Provokes Depression-Like Behaviors in Adult Rats

Tris-(2,3-dibromopropyl) isocyanurate (TDBP-TAZTO), an emerging brominated flame retardant, possesses the characteristics of candidate persistent organic pollutants and has displayed toxicity to fish and rodents. TDBP-TAZTO can pass through the blood brain barrier and accumulate in brain. However, the neurotoxicity of TDBP-TAZTO has not yet studied in rodents. We hypothesize that TDBP-TAZTO could induce the neurotoxicity in rat hippocampal neurons. The male adult rats were exposed to TDBP-TAZTO of 5 and 50 mg/kg by gavage, daily for 6 months. TDBP-TAZTO resulted in cognitive impairment and depression-like behaviors, which may be related with TDBP-TAZTO-induced hypothalamic-pituitary-adrenal axis hyperactivation, upregulation of inflammatory and oxidative stress markers, overexpression of pro-apoptotic proteins, downexpression of neurogenesis-related proteins in hippocampus, and hippocampal neurons damage in DG, CA1 and CA3 areas. Our findings suggested that TDBP-TAZTO induces significant hippocampal neurotoxicity, which provokes cognitive impairment and depression-like behaviors in adult rats. Therefore, this research will contribute to evaluate the neurotoxic effects of TDBP-TAZTO in human.     

Arjunolic acid,a triterpenoid saponin,prevents acetaminophen (APAP)-induced liver and hepatocyte injury via the inhibition of APAP bioactivation and JNK-mediated mitochondrial protection

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug and is safe at therapeutic doses but its overdose frequently causes liver injury. In earlier studies, we demonstrated that arjunolic acid (AA) has a protective effect against chemically induced hepatotoxicity. The purpose of this study was to explore whether AA plays any protective role against APAP-induced acute hepatotoxicity and, if so, what molecular pathways it utilizes for the mechanism of its protective action. Exposure of rats to a hepatotoxic dose of acetaminophen (700 mg/kg, ip) altered a number of biomarkers (related to hepatic oxidative stress), increased reactive oxygen species production, reduced cellular adenosine triphosphate level, and induced necrotic cell death. Arjunolic acid pretreatment (80 mg/kg, orally), on the other hand, afforded significant protection against liver injury. Arjunolic acid also prevented acetaminophen-induced hepatic glutathione depletion and APAP metabolite formation although arjunolic acid itself did not affect hepatic glutathione levels. The results suggest that this preventive action of arjunolic acid is due to the metabolic inhibition of specific forms of cytochrome P450 that activate acetaminophen to N-acetyl-p-benzoquinone imine. In addition, administration of arjunolic acid 4 h after acetaminophen intoxication reduced acetaminophen-induced JNK and downstream Bcl-2 and Bcl-xL phosphorylation, thus protecting against mitochondrial permeabilization, loss of mitochondrial membrane potential, and cytochrome c release. In conclusion, the data suggest that arjunolic acid affords protection against acetaminophen-induced hepatotoxicity through inhibition of P450-mediated APAP bioactivation and inhibition of JNK-mediated activation of mitochondrial permeabilization.     

Early manifestations and mechanism of the neurotoxic action of organophosphorus pesticides

The inhibition of neurotoxic esterase activity in chicken brain has been studied in vitro and in vivo. Aphos exposure, causing chicken paralysis, has demonstrated that the initial stage of delayed neurotoxicity was significant esterase activity inhibition (by 60-80%) within 3-24 hours after the pesticide administration. The inhibition of cholinesterase activity occurred both in the blood and sciatic nerve. The delayed conduction through peripheral nerves caused by demyelination has been revealed in the latent period (before the clinical signs of intoxication).     

Simultaneous measurement of gene expression for hepatotoxicity in thioacetamide-administered rats by DNA microarrays

DNA microarray technology was developed as a tool for simultaneously measuring a number of gene expression changes, and has been applied for investigations of toxicity assessments of chemicals. In this study, we used a typical hepatotoxicant, thioacetamide (TA), to find correlations between the extent of hepatotoxicity and certain gene expression patterns or specific gene expression profiles. TA was intraperitoneally administered at high (400 mg/kg), medium (150 mg/kg) or low (50 mg/kg) dose (four rats per group) and then the serum and liver were collected at the indicated time (6, 12, 24, 36 and 48 h). Serum biochemical markers were measured and hepatic mRNA expression profiles were analyzed by a DNA microarray. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased by TA-administration in a dose-dependent manner and reached the maximum at 24h. Hierarchical clustering analysis of all dosage groups revealed in 2 major clusters, distinguished by an early (6 and 12h) and a late (24, 36 and 48 h) phase. The early and late phase clusters were sorted in time- and dose-dependent manners. The major gene expression profile obtained by quality-threshold (QT) clustering analysis showed the same maximal toxic time as that estimated by the serum biochemical markers. The individual expression profiles of the candidate genes selected in our previous studies and the simultaneous gene expression patterns measured by five typical hepatotoxicants including TA also reflected the hepatotoxicity of TA. These findings suggest that the potential toxic effects appearing as gene expression changes are independent of the dosage of TA. This study suggested that the major gene expression profile estimated by QT clustering would be a sensitive marker of hepatotoxicity.     

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl-l-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.     

Reversible inhibition of cholinesterases by salts of pyrilium, thiopyrilium and selenopyrilium derivatives

Salts of pyrilium, thiopyrilium and selenopyrilium derivatives at pH 7.5 and temperature of 25 degrees C are studied for their effect on the catalytic activity of acetyl cholinesterase (EC 3.1.1.7) of human blood erythrocytes and butyryl cholinesterase (EC 3.1.1.8) of horse blood serum which is measured by the method of potentiometric titration. All enumerated salts are established to be strong reversible inhibitors of mixed-type cholinesterases, that is testified by small values of the inhibitory constants: competitive Ki, noncompetitive K'i and generalized K epsilon. Pyrilium and selenopyrilium salts inhibit acetyl cholinesterase of human blood erythrocytes to a higher extent than butyryl cholinesterase of horse blood serum, and thiopyrilium salts inhibit the latter to the highest extent. By the value of the inhibitory effect on acetyl cholinesterase of human blood erythrocytes thiopyrilium salts exceed the analogous pyrilium salts, whereas in experiments with butyl cholinesterase of horse blood serum there is an opposite dependence.     

Assessment of Environmental Conditions That Favor Hepatotoxic and Neurotoxic Anabaena spp. Strains Cultured under Light Limitation at Different Temperatures

Abstract Toxic cyanobacterial mass occurrences have caused animal poisonings worldwide and may pose a health hazard for humans. Strains of the genus Anabaena are either non-toxic or produce hepatotoxins, microcystins (MCYST), or neurotoxins (such as anatoxin-a). In order to study which growth conditions favor hepatotoxic vs neurotoxic strains and how production of toxins varies, we compared the responses of two microcystin- and two anatoxin-a-producing Anabaena strains in continuous turbidostat cultures, at different temperatures, under growth-limiting light levels. Growth rates consistently remained <0.8 divisions per 24 h. Differences were strain-specific and not associated with hepatotoxicity or neurotoxicity. Thus, differential adaptation of strains to temperature and to growth-limiting light levels cannot explain why, in some cyanobacterial water blooms, hepatotoxic strains, and in others, neurotoxic ones become dominant. A statistical analysis of field data showed that the most significant discriminating factors between different types of blooms were the concentrations of dissolved PO₄-phosphorus and NO₃-nitrogen. Anabaena blooms with unknown neurotoxicity associated with low PO₄-phosphorus and high NO₃-nitrogen concentrations. Among other Anabaena blooms, the hepatotoxic ones associated with the lowest, and most of the non-toxic ones with higher concentrations of PO₄-phosphorus. Anabaena blooms that contained anatoxin-a and hepatotoxic Microcystis blooms showed tendencies towards the highest concentrations of PO₄-phosphorus. Non-toxic blooms dominated by genera other than Anabaena occurred over a wide range of growth conditions. In turbidostat cultures, maximal production of microcystins correlated with maximal growth rates. Light regulated the production of MCYST-LR variants, and temperature affected the production of MCYST-RR variants. Anatoxin-a seemed to be produced most under temperatures and light levels slightly suboptimal for growth. Under low light, considerable amounts of extracellular anatoxin-a were detected while microcystins consistently remained intracellular. Received: 25 August 1997; Accepted: 2 December 1997     

Acetaminophen hepatotoxicity in vivo is not accompanied by oxidant stress

Hepatotoxic doses of acetaminophen in Fischer 344 rats did not increase biliary efflux of oxidized glutathione. Pretreatment of the animals with bis(2-chloroethyl)-N-nitrosourea inhibited hepatic glutathione reductase by 73 percent but did not potentiate the hepatotoxicity of acetaminophen and did not produce an increase in biliary efflux of oxidized glutathione in response to acetaminophen. Hepatic protein thiol content was not depleted by acetaminophen. A proposed role for oxidant stress mechanisms mediated either by reactive oxygen species or by the direct oxidant action of a reactive metabolite in acetaminophen-induced hepatotoxicity is unsubstantiated and unlikely.     

Construction of an engineered strain free of antibiotic resistance gene markers for simultaneous mineralization of methyl parathion and ortho-nitrophenol

Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP–0.1 mM ONP simultaneously. This method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental pollutants.