Sheldon D. Murphy

Effect of glutathione depletion on tissue deposition of methylmercury in rats

Diethylmaleate and other compounds capable of being metabolized to mercapturic acid derivatives were administered to rats in order to deplete tissue stores of glutathione (GSH). Deposition of mercury was significantly decreased in kidney and erythrocytes of depleted animals when [203Hg]methylmercuric chloride was administered during the period of maximal depletion of GSH. Increasing doses of diethylmaleate resulted in steadily decreasing GSH concentrations in brain, kidney, erythrocytes, and liver which were accompanied by decreases in mercury deposition in kidney and brain. Increasing the time interval between diethylmaleate and subsequent methylmercury administration resulted in correspondingly greater concentrations of mercury in kidney that were associated with recovery of renal GSH concentrations. Disodium maleate was also effective in lowering GSH values and reducing mercury deposition in kidney and erythrocytes. A second dose of disodium maleate, administered 24 hr after the first, was less effective than the first in depleting renal GSH and reducing mercury deposition. However, a second dose of diethylmaleate was equally as effective as the first. A high degree of correlation (r = 0.85) was found between renal mercury accumulation and renal GSH concentration when data from all depletion experiments were considered. The results suggest that renal GSH may be a determinant in the deposition of mercury in the kidney.

The influence of age on the toxicity and metabolism of methyl parathion and parathion in male and female rats

Abstract To determine the mechanisms responsible for the variations in toxicity of methyl parathion and parathion, the in vitro metabolism of these insecticides and cholinesterase sensitivity to their respective oxygen analogs methyl paraoxon and paraoxon were studied in male and female rats of several ages. For rats of five ages studied (1, 12–13, 23–24, 35–40, and 56–63 days), there was a gradual decrease in susceptibility to poisoning by both insecticides with increasing age up to 35–40 days for both sexes. Age differences in susceptibility were not related to differences in sensitivity of cholinesterase to inhibition by paraoxon or methyl paraoxon in vitro. Oxidative formation of the oxygen analogs, oxidative aryl cleavage, and glutathione-dependent dealkylation and dearylation of methyl parathion and parathion were assayed in liver homogenates of male and female rats of the five ages. Rates of enzymatic detoxification of their corresponding oxygen analogs by A-esterase, glutathione- S -aryl-, and - S -alkyl-transferase and inactivation by binding were also investigated. Correlation coefficients for rates of metabolism versus LD50 values for the different ages were calculated. In general, changes in LD50 values with age for methyl parathion and parathion correlated better with changes in rates of reactions which represented detoxification pathways for methyl paraoxon and paraoxon than for reactions which represented direct metabolism of the parent insecticides. Both male and female rats became much less sensitive to the acute lethal effects of methyl paraoxon and paraoxon with increasing age. This is consistent with a hypothesis that changes in LD50 values of methyl parathion and parathion with age are due to changes in rates of metabolism of the oxygen analogs.

Comparative toxicity, anticholinesterase action and metabolism of methyl parathion and parathion in sunfish and mice☆☆☆

Abstract The in vitro metabolism of methyl parathion ( O,O -dimethyl O-p -nitrophenyl phosphorothioate) and parathion ( O,O -diethyl O,p -nitrophenyl phosphorothioate) and the sensitivities of the target cholinesterases to inhibition by their oxygen analogs were studied in sunfish ( Lepomis gibbosus ) and mice to determine the basis for the low toxicity of methyl parathion in sunfish (LD50 > 2500 mg/kg). The LD50 values of parathion and methyl parathion in mice were 13.5 and 11 mg/kg, respectively, and the times to death were much shorter for both compounds in mice than in fish. Low sensitivity of fish cholinesterases to paraoxon as compared to mice accounted for the 10-fold lower toxicity of parathion in fish (LD50, 110 mg/kg). By contrast, sunfish had similar cholinesterase sensitivities to methyl paraoxon and paraoxon. Differences in rates of oxidative formation of the oxygen analog or oxidative cleavage to p -nitrophenol and the corresponding dialkyl thiophosphate could not account for the selective resistance of sunfish to methyl parathion toxicity. Fish and mouse liver homogenates catalyzed a glutathione (GSH)-dependent metabolism of methyl parathion and methyl paraoxon but not of parathion or paraoxon. Additionally, hydrolysis of methyl paraoxon by fish liver homogenates exceeded that for parathion by 5-fold, while methyl paraoxon hydrolysis in mice was 1 2 of that of paraoxon. Apparently, a longer time to death in fish provided the opportunity for GSH-dependent and hydrolytic detoxification, which favored methyl parathion and methyl paraoxon relative to parathion and paraoxon. Although in mice the GSH-dependent enzymes also favored detoxification of methyl parathion and methyl paraoxon, this is apparently of less importance because of their high cholinesterase sensitivity and because cleavage and hydrolysis favored parathion and paraoxon.

Tolerance to anticholinesterase compounds in mammals

Administration of multiple, sublethal doses of organophosphorus insecticides induces the development of tolerance to their toxicity. Among the different hypotheses investigated to explain the mechanism of this phenomenon, the one which has received the greatest experimental support is a downregulation of the muscarinic cholinergic receptors. Subsensitivity to cholinergic agonist has been demonstrated in vivo and in vitro in isolated organ preparations. Receptor binding experiments using muscarinic antagonists and agonists revealed a decrease of cholinergic receptors in central and peripheral tissues. Tolerance to another class of acetylcholinesterase inhibitors, carbamates has also been demonstrated. Differences from and similarities to organophosphate tolerance are discussed.

Effect of 18 HR fast and glutathione depletion on 1,1-dichloroethylene-induced hepatotoxicity and lethality in rats

Abstract Four-hour inhalation exposure to 1,1-dichloroethylene (1,1-DCE, vinylidene chloride) was more injurious to 18-hr (overnight) fasted rats than to rats fed ad libitum. The estimated 24 hr LC50 for fed rats was 15,000 ppm while the same value for fasted rats was 600 ppm. The minimum lethal concentration was 200 ppm for fasted rats and 10,000 ppm for fed rats. Serum alanine α-ketoglutarate transaminase (AKT) elevation occurred at 150 ppm in fasted rats, but in the fed rats, a significant elevation was only seen at 2000 ppm and higher. Elevated serum AKT preceded hepatic necrosis and death. This fed-fasted difference in serum AKT elevation was also demonstrable in an isolated perfused rat liver system. The AKT elevation in perfusate from livers of fasted rats was consistent with the time course of injury seen in vivo. Increased susceptibility to hepatic injury appeared to be related to decreased hepatic glutathione concentration associated with fasting (18 hour). Diethylmaleate, a material which results in a decreased hepatic glutathione concentration was administered in vivo and in vitro. This treatment potentiated the hepatic injury in fed rats and in livers taken from fed rats and subsequently perfused.

Differential alterations of cholinergic muscarinic receptors during chronic and acute tolerance to organophosphorus insecticides

Male mice treated for 2 weeks with the anticholinesterase insecticide disulfoton (O,O-diethyl S-[2-(ethylthio)-ethyl] phosphorodithioate; 10 mg per kg per day) became tolerant to the hypothermic and antinociceptive effects of disulfoton itself and of oxotremorine, a muscarinic cholinergic agonist. Homogenates of brain and ileum from tolerant animals exhibited reduced binding of the specific muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB). In forebrains of tolerant animals, the number of receptors (Bmax) was decreased 40% with no change in the affinity constant. Acetylcholinesterase (AChE) activity was 15% of control. Forty-eight hours after a single injection of disulfoton (10 mg/kg) mice were more resistant than their controls to the hypothermic and antinociceptive effects of a second administration of the same insecticide and of oxotremorine. Tolerance was not present 96 hr after a single administration of disulfoton. A single injection of disulfoton produced 74, 65 and 27% inhibition of AChE activity after 4, 48 and 96 hr respectively. Four hours after a second injection at 49 and 96 hr, 73 or 72% inhibition was found. [3H]QNB binding of animals treated with a single injection of disulfoton and of controls did not differ at either time point. An increase in the Ki for inhibition of [3H]QNB binding by unlabeled oxotremorine was observed in forebrain from mice killed 48 hr after a single injection of disulfoton, indicating a decreased affinity of the muscarinic receptor for agonists. Binding of [3H]oxotremorine-M was decreased significantly 48 hr after a single injection of disulfoton and after chronic treatment. It is suggested that a differential down-regulation of muscarinic receptors occurs in acute and chronic tolerance, involving agonist and antagonist binding sites and depending on duration of exposure.

Kinetic analysis of species difference in acetylcholinesterase sensitivity to organophosphate insecticides.

Abstract Previous in vitro studies have elucidated species differences in the concentration of organophosphates required for 50% inhibition (IC 50 ) of brain acetylchoinesterase (AChE). In this study, we tested the hypothesis that the differences in IC 50 s between species might be due to variability in the kinetic parameters of AChE from different species; i.e., different affinities ( K a ) to the binding of organophosphate inhibitors and/or different rates of phosphorylation of the enzyme. Brain AChEs from four classes of animals were used: mammalian (monkey, rat, and guinea pig), avian (chicken), piscine (catfish), and amphibian (frog). The kinetic parameters of inhibition of five organophosphates were determined. The observed differences in IC 50 values can be satisfactorily explained on the basis of different affinities and/or different rates of phosphorylation of AChEs from various sources. For example, in the IC 50 study, chicken brain AChE was 103-fold more sensitive to inhibition by methyl paraoxon than frog brain AChE. This difference could be explained on the basis that chicken brain AChE had a 7.1-fold higher affinity and a 15.5-fold faster rate of phosphorylation. The correlation of observed p I 50 (−log[IC 50 ]) with p I 50 calculated from kinetic data was almost ideal. The data from studies on homologs of paraoxon and Gutoxon indicated that increasing the alkyl chain from methyl to ethyl results in a 2.4-fold increase in the apparent affinity, while the rate of phosphorylation was not significantly changed. These kinetic studies suggest that both affinity and rate of phosphorylation are important in determining the sensitivity of AChEs from different species.

Effect of diethylmaleate and other glutathione depletors on protein synthesis.

The alpha, beta-unsaturated carbonyl compound diethylmaleate (DEM) depletes glutathione (GSH) from liver and other tissues, and for this reason it is often used in toxicological research to study the GSH-mediated metabolism of xenobiotics. In addition to GSH depletion, however, DEM has been shown to have other nonspecific effects, such as alteration of monooxygenase activities or glycogen metabolism. In this study we found that DEM (1 ml/kg) inhibited protein synthesis in brain and liver, following in vivo administration to mice. Protein synthesis was measured as the incorporation of [3H]valine into trichloroacetic acid-precipitable material. Administration of DEM also decreased body temperature by 2-3 degrees. By increasing the environmental temperature from 22 degrees to 35 degrees the hypothermic effect of DEM was prevented, without affecting its ability to deplete GSH from brain and liver. Furthermore, when mice were maintained at 35 degrees, DEM still caused a significant decrease in protein synthesis, suggesting that this effect was only partially due to hypothermia. To test whether inhibition of protein synthesis was related to GSH depletion, groups of animals were dosed with the alpha, beta-unsaturated carbonyl phorone (diisopropylidenacetone) or the specific inhibitor of GSH synthesis, buthionine sulfoximine (BSO). Phorone decreased GSH in liver and brain; however, it had no effect on protein synthesis. BSO decreased GSH levels in liver and kidney, but not in brain, and did not have any effect on protein synthesis in any of these tissues, nor did it cause any hypothermia. Furthermore, when hepatic GSH content was decreased by in vivo administration of DEM or BSO, there was no inhibition of protein synthesis measured in vitro. These results indicate that, at the dose normally used to deplete GSH from various tissues. DEM also exerts an inhibitory effect on protein synthesis, which appears to be only partially due to its hypothermic effect, and is independent from GSH depletion. BSO, which, in our experimental conditions, lacks this and other nonspecific effects, might be a good alternative for studies aimed at characterizing the role of GSH in the metabolism and toxicity of chemicals.

Comparative anticholinesterase action of organophosphorus insecticides in vertebrates.

Abstract Parathion, malathion, and Guthion were poor inhibitors of mammalian, avian, or piscine brain cholinesterases in vitro . The oxygen analogs of these insecticides were potent inhibitors. In general, avian brain cholinesterases were more sensitive to paraoxon than the cholinesterases of mammalian brains. Fish brain cholinesterases were much more resistant than mammalian. This was consistent with the relative susceptibilities of mice, sunfish, and chickens to poisoning by parathion and paraoxon. Avian brain cholinesterases were much more resistant to inhibition by Gutoxon than mammalian brain cholinesterases, and this agreed with the relatively high resistance of chickens to poisoning by Guthion and Gutoxon. Sunfish were more susceptible than mice to poisoning by Gutoxon, but their brain cholinesterase was only twice as sensitive as mouse brain cholinesterase to inhibition by Gutoxon. Differences in the sensitivities of brain cholinesterases of different species to inhibition by malaoxon were less marked. Additional studies are necessary to obtain a quantitative evaluation of the relative importance of rates of metabolism and sensitivity of cholinesterases to inhibition as determinants of species differences in susceptibility to poisoning by organophosphate insecticides. The results of this investigation indicate, however, that for some compounds species differences in the sensitivities of the cholinesterases to inhibition are sufficiently large to modify the influence of differences in rates of metabolism.

Reduced [3H]quinuclidinyl benzilate binding to muscarinic receptors in disulfoton-tolerant mice

The organophosphate, acetylcholinesterase inhibitor, disulfoton, O,O-diethyl S-[2-(ethylthio)ethyl]phosphorodithioate, was given daily for 2 weeks to male mice at two different dosages. Clinical signs of poisoning disappeared in 5 days after the beginning of the treatment, i.e., the animals developed apparent tolerance to disulfoton toxicity. Tolerant mice were less sensitive to a lethal dose of carbachol and exhibited a decrease of [3H]quinuclidinyl benzilate ([3H]QNB) binding in forebrain, hindbrain, and ileum. Scatchard analysis of saturation experiments revealed a decrease in the density of receptors (Bmax) in the disulfoton-treated mice, as compared with controls. No significant changes in affinity were found, except in the ileum. A time-course study showed a good parallelism between the decrease of [3H]QNB binding and the development of tolerance. Twenty-one days after the end of the disulfoton treatment AChE activity was still inhibited, but [3H]QNB binding had returned to normal levels. The recovery of [3H]QNB binding appears to be faster in ileum than in forebrain and hindbrain. These findings indicate that the development of tolerance to chronic organophosphate treatment is, at least partially, due to a reduction in the number of cholinergic receptors.