Examination of the metabolism in vitro of parathion (diethyl p-nitrophenyl phosphorothionate) by rat lung and brain.

These studies have indicated there is present in rat lung microsomes a mixed-function oxidase enzyme system capable of metabolizing parathion to paraoxon and to diethyl phosphorothioic acid. In addition, analogous to previous results using rat liver microsomes, the sulfur atom released in the metabolism of parathion to paraoxon was found to covalently bind to lung microsomes. In contrast to liver, the metabolism of parathion by rat lung microsomes is not inducible by pretreatment of the animals with phenobarbital or 3-methylcholanthrene. The metabolism of parathion by lung microsomes is stimulated by NADPH and oxygen and is inhibited by carbon monoxide, anaerobic conditions, SKF-525A and piperonyl butoxide. There is also present in a particulate fraction of rat brain equivalent to microsomes an enzyme or enzyme system capable of metabolizing parathion to paraoxon and to diethyl phosphorothioic acid. The metabolism of parathion to paraoxon by rat brain microsomes is also accompanied by the release and covalent binding of the sulfur atom of parathion. The activity in rat brain microsomes is stimulated by oxygen and NADPH and inhibited by carbon monoxide, anaerobic conditions, SKF-525A and piperonyl butoxide. These data suggest that cytochrome P-450-containing mixed-function oxidase enzyme systems are responsible for the NADPH-stimulaled catalytic activity toward parathion found in rat lung and rat brain microsomes.     

Biotransformation of the organophosphorus insecticides parathion and methyl parathion in male and female rat livers perfused in situ.

Although numerous previous reports have characterized the mammalian biotransformation of the organophosphorus insecticides parathion and methyl parathion, questions still remain regarding the toxicological significance of certain metabolic pathways in vivo. The present study utilized rat liver perfusions in order to better characterize the hepatic biotransformation of parathion and methyl parathion in intact liver. Single-pass liver perfusions with parathion and methyl parathion over a range of perfusate concentrations of 10-80 microM resulted in the appearance of paraoxon and methyl paraoxon, respectively, in effluent. Furthermore, rat blood did not have the capacity to prevent transport of paraoxon and methyl paraoxon to extrahepatic tissues, suggesting that oxon produced hepatically can distribute to extrahepatic tissues. In addition, striking sex differences were noted in the metabolite profile of parathion and methyl parathion in perfused livers. However, these differences could not account for the observation that females are more susceptible to parathion, but less susceptible to methyl parathion, compared to males. And finally, S-methyl glutathione or S-p-nitrophenyl glutathione could not be detected in effluent or bile of livers from either sex perfused with methyl parathion, suggesting that glutathione-dependent detoxification of this insecticide does not occur to any significant degree in intact rat liver.     

Methyl parathion activation by a partially purified rat brain fraction

Organophosphorus pesticides are one of the most commonly used insecticide classes. They act through a potent inhibition of acetylcholinesterase (AChE). Many of them must undergo transformation into the corresponding oxon analogs to inhibit AChE. This study showed that a brain tissue subfraction transformed methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) in vitro. Methyl parathion activation was assayed by solvent extraction of the products followed by HPLC and GC-MS analyses and, indirectly, by the inhibition of AChE present in the incubation mixture. The lack of impairment of AChE after 2 h of incubation of the brain subfraction with methyl parathion and, alternatively, with NADPH, CO, SKF 525-A, piperonyl butoxide or nitrogen indicated that this brain subfraction transformed methyl parathion without the involvement of a mixed-function oxidative pathway.The results from HPLC analysis did not show a peak corresponding to methyl paraoxon (O,O-dimethyl O-p-nitrophenylphosphate), but showed the production of an unidentified peak which eluted nearby standard methyl parathion (retention times of 10.65 and 8.86 min, respectively). GC-MS analysis suggested that the unidentified product could be a methyl parathion isomer.     

Studies of the mechanisms of metabolism of diethyl p-nitrophenyl phosphorothionate (parathion) by rabbit liver microsomes

Based on the protein content of microsomes, the administration of 3-methylcholanthrene (3-MC) and phenobarbital (PB) to adult rabbits leads to an increased rate of metabolism of parathion (diethyl 4-nitrophenyl phosphorothionate) by rough-surfaced and whole microsomes but not by smooth-surfaced microsomes. Although prior administration of both PB and 3-MC increased the cytochrome P-450 content of the microsomes, when the rate of metabolism of parathion was calculated on the basis of the concentration of cytochrome P-450 in these microsomes, there is no difference in the rate of metabolism of parathion by rough-surfaced and smooth-surfaced microsomes from the untreated, 3-MC-treated and PB-treated animals. However, based on the cytochrome P-450 concentration, the rate of metabolism of parathion by whole microsomes from 3-MC and PB-treated animals is less than the rate with whole microsomes from untreated animals. Further studies have shown there is no correlation between the concentration of high spin or low spin cytochrome P-450 in any of the microsomal fractions or subfractions and the rate of metabolism of parathion to paraoxon or diethyl phosphorothionate.     

Effect of piperonyl butoxide on the metabolism of dimethyl and diethyl phosphorothionate insecticides

The effect of piperonyl butoxide on the acute toxicity of phosphorothionate insecticides was studied in male mice. One hour after piperonyl butoxide (400 mg/kg), the toxicity of the dimethyl phosphorothionates, methyl parathion and Guthion, was antagonized, whereas the toxicity of their respective diethyl homologs, parathion and Ethyl Guthion, was potentiated. Piperonyl butoxide did not appreciably alter the toxicity of the oxygen analogs of these compounds. Pretreatment with SKF 525-A (50 mg/kg) modified the toxicity of the phosphorothionates in a manner qualitatively similar to piperonyl butoxide pretreatment. Plasma concentrations of all four insecticides were increased three- to sevenfold in piperonyl butoxide-pretreated mice. This increase may result in a greater total oxon formation; however, reactivation in vitro of esterases inhibited in vivo was 5 to 10 times more rapid following methyl parathion or Guthion challenge than after their diethyl homologs. Although a greater total oxon formation-cholinesterase inhibition is possible for both dimethyl and diethyl phosphorothionates following piperonyl butoxide pretreatment, rapid reactivation of inhibited nerve tissue cholinesterases after dimethyl phosphorothionate challenge appears to compensate for further inhibition occurring at a decreased rate. The net result would be a reduction in dimethyl phosphorothionate toxicity. In contrast, slow reactivation of inhibited nerve tissue cholinesterases following diethyl phosphorothionate challenge appears unable to compensate for increased oxon formation-cholinesterase inhibition. The net result is a potentiation of the toxicity of the diethyl-substituted compounds.     

Activation of rat brain tryptophan hydroxylase by polyelectrolytes

The in vitro activity of rat brain tryptophan hydroxylase is increased 2-fold by heparin and 4-fold by dextran sulfate. Other polysaccharides including hyaluronic acid, chondroitin sulfate and dermatan sulfate, as well as the unsulfated polymer dextran, do not alter hydroxylase activity. The effect of heparin or dextran sulfate on tryptophan hydroxylase is manifested kinetically as a decrease in the apparent K_m of the enzyme for both substrates 6-methyl-tetrahydropterin (6-MPH₄) and tryptophan, as well as increases in the tV_max values. A variety of polyanions (DNA, glycogen, poly-d- and poly-l-glutamic acid) have no effect on tryptophan hydroxylase, whereas salts [NaCl, KCl, (NH₄)₂SO₄ and MgSO₄] inhibit the enzyme, indicating that the effects of heparin and dextran sulfate on tryptophan hydroxylase are not mediated by increases in ionic strength per se. Several lines of data suggest that tryptophan hydroxylase binds ionically to these polyelectrolytes: (1) the activation produced by heparin and dextran sulfate diminishes as the ionic strength of the assay medium increases, (2) tryptophan hydroxylase binds to heparin-substituted Sepharose 4B and is eluted by increasing the ionic strength of the eluant buffer, and (3) large molecular weight dextran sulfate (mol. wt = 500,000) dramatically shifts the elution profile of tryptophan hydroxylase from a K_av of 0.41 to a K_av of 0.10 on a Sepharose/CL-6B column. Taken together, these data suggest that the binding of certain polyelectrolytes to tryptophan hydroxylase may induce a conformational change in the enzyme which results in increased catalytic activity.     

Differential modulation of muscarinic receptors in the rat brain by repeated exposure to methyl parathion

The neurochemical and behavioral effects of repeated subdermal administration of methyl parathion (MP) at low doses were investigated. Adult male rats were treated repeatedly with either vehicle or MP subcutaneously (3 mg/kg/day) and observed for the signs of toxicity during the treatment period. The toxic sign, tremor, reached maximum right after 9th injection in MP-treated rats, and declined thereafter. Animals were sacrificed and brains were taken 1 week or 3 weeks after the daily treatment for measurement of acetylcholinesterase (AChE) activity and binding of radioligands, [3H]QNB (nonselective), [3H]pirenzepine (M1-selective), and [3H]AF-DX384 (M2-selective) to muscarinic receptors. With this treatment regimen, the AChE activity in the blood dropped quickly and maintained at 30% of the control level after 6 injections. After 3 weeks of treatment, MP caused 80-90% AChE inhibition and substantial reductions in [3H]QNB binding (9-33%), [3H]pirenzepine binding (9-22%) and [3H]AF-DX384 binding (6-38%) in different brain regions, including striatum, hippocampus, frontal cortex, thalamus and midbrain. After 1 week of treatment, the inhibition of AChE in brain regions was from 54 to 74%, whereas receptor densities were only marginally affected in a few regions. The timing of the changes in receptor population correlates well with the changes in behaviors during the repeated MP exposure. Our findings suggest that down-regulation of muscarinic receptors plays a role in the development of tolerance to MP. And, the regulations of muscarinic receptors were different among receptor subtypes and brain regions.     

Biotransformation of the insecticide parathion by mouse brain.

The acute toxicity of organothiophosphate insecticides like parathion results from their metabolic activation by cytochromes P450. The present study is directed towards the characterization of cytochrome-P450-dependent metabolism of parathion by various mouse brain regions. Intraperitoneal administration of [35S]parathion to mice led to covalently bound [35S]sulfur in various tissues, indicating their capacity to oxidatively desulfurate this insecticide. Liver contained the greatest amount of covalently bound sulfur, and brain the least. Among individual brain regions the olfactory bulb and hypothalamus possessed the highest levels of sulfur binding when expressed on a per milligram tissue basis. However, when expressed on a per brain region basis, sulfur binding was greatest within the cortex as a result of the large mass of this region, compared to the hypothalamus and olfactory bulb. Incubation of the 78,000 x g fraction of mouse brain with parathion resulted in formation of p-nitrophenol, although paraoxon could not be detected. However, given the current understanding of parathion metabolism by cytochromes P450, and given that paraoxon can rapidly disappear through phosphorylation of serine hydroxyl groups, it is reasonable to assume that at least some paraoxon was formed. Production of p-nitrophenol required NADPH and was inhibited by carbon monoxide. In vitro incubations of parathion with the 78,000 x g fraction of mouse brain indicated that the hypothalamus and olfactory bulb had the greatest capacity to produce p-nitrophenol. These results demonstrate that various mouse brain regions possess different capacities to metabolize parathion.     

Activation of rat brain adenylate cyclase by copper plus dithiothreitol

Cu(II), an indispensable metal ion for living cells, is known to oxidize SH groups to disulfide bonds in proteins (1). Many enzymes, such as adenylate cyclase (2–4), phosphodiesterase (5, 6) and ATPase (7), are inactivated by fairly high concentrations of Cu(II). In studies on the mechanism of inactivation of adenylate cyclase by low concentrations of Cu(II), we found a new effect of this metal ion on the activity of the enzyme. This paper reports the activation of adenylate cyclase in the membrane fraction of rat brain by Cu(II) in the presence of dithiothreitol (DTT).     

Effect of temperature on the mixed function oxidase-catalyzed metabolism of O,O-diethyl p-nitrophenyl phosphorothionate (parathion)

Using hepatic microsomes from both untreated and phenobarbital-treated rats, the energies of activation for the hepatic mixed function oxidase-catalyzed metabolism of parathion to paraoxon and to diethyl phosphorothioic acid were found to be significantly different. In addition, the energies of activation for the metabolism of parathion to paraoxon and to diethyl phosphorothioic acid were found to be decreased by pretreatment of rats with phenobarbital. A decrease in the energy of activation of NADPH-cytochrome P-450 reductase was not seen in phenobarbital-treated as compared to untreated animals.     

Mutagenic evaluation of the organophosphorus insecticides methyl parathion and triazophos in Drosophila melanogaster

The possible genotoxic effects of the organophosphorus insecticides methyl parathion and triazophos were evaluated by their ability to induce gene and chromosome mutations in male germ cells of Drosophila melanogaster. Sex-linked recessive lethal (SLRL), total and partial sex-chromosome losses (SCL), and non-disjunction (ND) assays were conducted. The routes of administration included adult feeding, injection, and larval feeding. Methyl parathion was unable to induce point mutations or chromosome mutations, although a small increase in the frequency of non-disjunction was detected after larval treatment. Triazophos induced point mutations when assayed in the SLRL test and induced a weak increase in the non-disjunction frequency, but gave negative results in the SCL test.     

Interactive toxicity of the organophosphorus insecticides chlorpyrifos and methyl parathion in adult rats

The acute interactive toxicity following exposure to two common organophosphorus (OP) insecticides, chlorpyrifos (CPF) and methyl parathion (MPS), was investigated in adult male rats. Oral LD1 values were estimated by dose-response studies (CPF = 80 mg/kg; MPS = 4 mg/kg, in peanut oil, 1 ml/kg). Rats were treated with both toxicants (0.5 or 1 x LD1) either concurrently or sequentially, with 4-h intervals between dosing. Functional signs of toxicity (1-96 h) and cumulative lethality (96 h) were recorded. Rats treated with CPF (1 x LD1) did not show any signs of toxicity although MPS (1 x LD1) elicited slight to moderate signs (involuntary movements) within 1-2 h. Concurrent exposure (LD1 dosages of both CPF and MPS) caused slight signs of toxicity only apparent between 24 and 48 h after dosing. When rats were treated sequentially with MPS first followed by CPF 4 h later, slight signs of toxicity were noted between 6 and 24 h, whereas reversing the sequence resulted in 100% lethality within 1 h of the second dosage. Following exposure to lower dosages (0.5 x LD1), the CPF first group showed higher signs of cholinergic toxicity compared with MPS first or concurrent groups. Cholinesterase inhibition in plasma, diaphragm, and frontal cortex was generally higher in rats treated sequentially with CPF first than in those treated initially with MPS from 4 to 24 h after dosing. Plasma and liver carboxylesterase inhibition at 4 h was also significantly higher in the CPF first (62-90%) compared with MPS first (22-43%) group, while at 8 and 24 h, there was no significant difference between any of the treatment groups. ChE inhibition assays to evaluate in vitro hepatic detoxification of oxons indicated that carboxylesterase (CE)- and A-esterase-mediated pathways are markedly less important for methyl paraoxon (MPO) than chlorpyrifos oxon (CPO) detoxification. CPF pretreatment blocked hepatic detoxification of methyl paraoxon while MPS pretreatment had minimal effect on hepatic CPO detoxification ex vivo. These findings suggest that the sequence of exposure to two insecticides that elicit toxicity through a common mechanism can markedly influence the cumulative action at the target site (acetylcholinesterase, AChE) and consequent functional toxicity.     

The interaction between phosphorothionate insecticides,pneumotoxic trialkyl phosphorothiolates and effects on lung 7-ethoxycoumarinO-deethylase activity

A number of phosphorothionate (P=S) insecticides, including bromophos and fenitrothion, prevent trialkyl phosphorothiolate (P=O)-induced lung toxicity and the resulting increase in lung weight normally observed at 3 days in the rat. Measurement of 7-ethoxycoumarinO-deethylase (7-EC) activity after both phosphorothionate and phosphorothiolate dosing revealed differing patterns of loss of enzyme activity. Depletion of 7-EC activity by phosphorothionates was maximal between 2 and 10 h after dosing, with recovery between 24 and 72 h. Phosphorothiolates, however, appear to cause two phases of loss of 7-EC activity, an initial fall of approximately 30% observed at 2 h and a secondary fall, maximal on day 3, with loss of 97% of activity, apparently associated with the pathological changes in the lung. It is suggested that oxidative metabolism of phosphorothionates known to occur at the P=S moiety, with suicidal loss of P-450, may then prevent oxidative activation of an S-methyl on the phosphorothiolates, the most likely site for production of a reactive intermediate capable of damaging the lung. Lung 7-EC in rat is sensitive to concentrations of the phosphorothionates bromophos and fenitrothion at 5–25 times less than those causing loss of liver 7-EC activity and at doses 125–600 times less than their LD₅₀s. If repeated in man this may have implications for personnel occupationally exposed to these compounds.     

Effect of selected insecticides on rat brain synaptosomal adenylate cyclase and phosphodiesterase

Previous reports from our laboratory and others clearly indicated that organochlorine insecticides such as chlordecone and DDT are potent inhibitors of ATPases involved in active ion transport. The present studies were initiated to study the effect of plictran, chlordecone, toxaphene, aldrin, dieldrin, endrin, isodrin, and telodrin on enzymes involved in cyclic AMP metabolism. Rat brain synaptosomes were prepared by Ficoll-sucrose gradient centrifugation method. Adenylate cyclase activity, which is involved in anabolism of cAMP, was determined using the radioactive method by measuring [32P]cAMP formed during hydrolysis of [32P]ATP. Phosphodiesterase activity, which is involved in the catabolism of cAMP, was estimated by measuring [3H]adenosine formed using [3H]cAMP as a substrate. Synaptosomal adenylate cyclase activity was inhibited significantly by plictran with an IC50 of 25 microM, and a maximum inhibition of 30% was observed with 50 microM chlordecone. Toxaphene, aldrin, dieldrin, endrin, isodrin, and telodrin did not affect the adenylate cyclase activity. Similarly, none of the insecticides studied inhibit the activity levels of synaptosomal phosphodiesterase. The significant inhibition of adenylate cyclase observed with plictran might be due to the tin component, since several heavy metals affect cAMP metabolism. The lack of inhibition of adenylate cyclase and phosphodiesterase with other compounds tested clearly supports our postulation that these organochlorine insecticides exert their neurotoxic action by the selective inhibition of ATPases in synaptosomes.     

Effects of insecticides on GABA-induced chloride influx into rat brain microsacs

The actions of different insecticides known to affect binding of ligands to gamma-aminobutyric acid (GABA) receptors were studied on the function of GABAA receptors in rat brain as assayed by GABA-induced 36Cl- influx into membrane microsacs. This flux was inhibited by the competitive antagonist bicuculline and the noncompetitive antagonist t-butylbicyclophosphorothionate, and the GABA effect was potentiated by the tranquilizer flunitrazepam and the depressant pentobarbital, as expected for effects on a GABAA receptor. The GABA-induced 36Cl- flux was inhibited by several cyclodienes and gamma-hexachlorocyclohexane (gamma-BHC) with the following order of decreasing potency: endosulfan I greater than endrin greater than endosulfan II greater than dieldrin greater than heptachlor epoxide greater than gamma-BHC greater than heptachlor. The noninsecticidal beta-BHC had no effect, while the IC 50 values for gamma-BHC and endrin were 1 and 0.2 microM, respectively. The four pyrethroids tested also inhibited the GABA-induced 36Cl- flux with the following decreasing potencies: 1R,cis,alpha S-cypermethrin greater than 1R,trans, alpha S-cypermethrin greater than fluvalinate greater than allethrin. Avermectin B1a was the only insecticide tested that, in the absence of GABA, stimulated 36Cl- flux in a dose-dependent manner, and this flux was inhibited by bicuculline. The stereospecific inhibition of the GABA-induced 36Cl- influx by the cyclodienes and gamma-BHC supports previously published data on their binding to mammalian brain GABAA receptor and suggests that these insecticides inhibit this receptor's function. It is also suggested that type II pyrethroids are potent inhibitors of the same receptor. However, avermectin B1a appears to act as a partial agonist of GABAA receptors.     

The role of glutathione in the detoxification of the insecticides methyl parathion and azinphos-methyl in the mouse

The dimethyl-substituted organothiophosphate insecticides methyl parathion and azinphos-methyl are thought to undergo glutathione-mediated detoxification in mammals. In the present study, depletion of hepatic glutathione in the mouse by pretreatment with diethyl maleate potentiated the acute toxicities of methyl parathion and azinphos-methyl, whereas depletion of hepatic glutathione by pretreatment with buthionine sulfoximine did not. Furthermore incubation of 50 microM methyl parathion with mouse hepatic microsomes for 5 min in the presence of 1 mM diethyl maleate led to significantly greater (p less than 0.05) production of methyl paraoxon, compared to incubations in the absence of diethyl maleate. Conversely, 1 mM diethyl maleate had no effect on metabolic activation of azinphos-methyl by mouse hepatic microsomes, while 10 mM inhibited slightly production of azinphos-methyl oxon from azinphos-methyl. These results suggest normal levels of hepatic glutathione are not required for detoxification of methyl parathion or azinphos-methyl in the mouse. Moreover the potentiation of the acute toxicity of methyl parathion following diethyl maleate pretreatment could result, at least in part, from enhanced production of methyl paraoxon. However, diethyl maleate likely acts through another mechanism(s) as well since it did not enhance the metabolic activation of azinphos-methyl in vitro. These data raise serious doubts about the participation of glutathione in the detoxification of methyl parathion and azinphos-methyl in vivo in the mouse.     

Regional effect of organochlorine insecticides on cholinergic muscarinic receptors of rat brain

In different brain regions of the rat we studied the effect of chronic feeding with the organochlorine insecticides p,p'-DDT and gamma-HCH on the cholinergic muscarinic receptors. Using [3H]quinuclidinyl benzylate binding to membranes from cerebral cortex, medulla pons, diencephalon, and cerebellum it was found that the two insecticides produced a decrease in the number of muscarinic receptor sites in cerebellum; while gamma-HCH also reduced these receptors in diencephalon. In both cases no changes in receptor affinity were observed. It is suggested that the chronic treatment with these organochlorine insecticides may cause an alteration in cholinergic transmission leading to a down regulation of the muscarinic receptor in certain brain regions.