Binding characteristics of methyl parathion to photosynthetic membranes of Chlorella

When methyl parathion is added at a given concentration to exponentially growing Chlorella cultures containing different numbers of cells, the inhibition of cell number, packed cell volume, pigment content, or photosynthesis has been found to be a function of the cell number, the inhibition being decreased with increased number of cells. Inhibition of photosynthesis has been studied further with a view to characterizing the mechanism of inhibition with a simple assumption that methyl parathion binds to a specific component in Chlorella cells to form an inhibitory complex. The concentration of methyl parathion causing 50% inhibition (I₅₀) of photosynthetic O₂ evolution increases linearly with increasing concentration of chlorophyll in the culture medium. With whole cells the inhibition constant (K_i) is 15 times greater than that with cell-free photosynthetic membranes. This shows that the cell wall acts as a permeability barrier. The relation between the I₅₀ and K_i values and the analyses of the Hill plot of the inhibition curves reveal one binding site per 0.5 chlorophyll molecule and a cooperative binding of methyl parathion with at least three binding sites per binding molecule. Mild treatment of the photosynthetic membranes with trypsin makes the photosynthetic electron transport insensitive to the insecticide, suggesting that the binding component is proteinaceous in nature and the binding sites are located on the external surface of the membrane. The reversal of methyl parathion inhibition is parallel to that of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (diuron) inhibition during trypsin treatment suggesting that the binding proteins for these two inhibitors are similar.     

Penetration of insecticides through the foregut of the honeybee (Apis mellifera L.)

The rates of penetration of ¹⁴C-labeled insecticides (parathion, carbaryl, and dieldrin) through the foregut of the honeybee (Apis mellifera L.) were measured in vitro and in vivo. Uptake of the insecticides from the lumen of the foregut into foregut tissue was directly proportional to insecticide lipophilicity, but penetration through the foregut was not. Of the three insecticides studied, parathion appeared to possess the optimal physicochemical characteristics required for penetration. The uptake of carbaryl and release of dieldrin by the foregut tissues may limit their respective penetration rates. Insecticide penetration was found to be inversely proportional to the sucrose concentration in the lumen of the foregut in both in vitro and in vivo studies. The oral toxicity of carbaryl showed a similar dependence on the sucrose concentration of the solution in which the insecticide was fed. The data presented indicate that the honeybee foregut is permeable to lipophilic compounds and strongly suggest that this permeability may contribute substantially to the toxicity of orally ingested insecticides in this insect.     

Organophosphate degradation by insecticide-resistant and susceptible populations of mosquitofish (Gambusia affinis)

Phosphorothionate and phosphate degradation was investigated as a factor which could influence the tolerance of organochlorine compound-resistant and susceptible mosquitofish (Gambusia affinis) to parathion and methyl parathion. The greater toxicity of methyl parathion than parathion can be attributed in part to a higher rate of degradation of methyl paraoxon than paraoxon (7-fold), but not to any difference in phosphorothionate dearylation. Resistant fish possess higher levels of microsomal mixed-function oxidases which can degrade methyl parathion (1.3-fold); these higher levels could contribute to the increased methyl parathion tolerance by this population over the susceptible population. Environmentally induced tolerance to parathion in the resistant population may be the result of increased levels of parathion degradation by induced mixed-function oxidases which can dearylate parathion. The increased tolerance of either insecticide by the resistant population is not caused by degradation of the phosphates by phosphotriesterases.     

Insect glutathione S-transferase: Separation of transferases from fat bodies of American cockroaches active on organophosphorus triesters

Several glutathione S-transferases which catalyze the conjugation of reduced glutathione with organophosphorus triesters were separated from fat bodies of adult female American cockroaches, Periplaneta americana (L.). Two transferases (I, V) were active on diazinon and three transferases (II, III, IV) were active on methyl parathion. The transferase (I) active on the pyrimidinyl moiety of diazinon was distinguishable from the other transferases on the O-methyl portion of methyl parathion, as shown by chromatographic properties, and additionally it was almost inactive or less active on 3,4-dichloronitrobenzene, methyl iodide, p-nitrobenzyl chloride, trans-cinnamaldehyde, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Transferase II had high activities with “aryl” and “aralkyl” compounds, transferase III with “epoxide” and “alkene,” and transferase IV with “alkyl,” “aryl,” and “aralkyl” compounds. This indicated that the transferases had overlapping substrate specificities. The molecular weight was 35,000–37,000 for both of the enzymes active on methyl parathion and diazinon. The pH optima with methyl parathion and diazinon were about 8.5 and 6.5, respectively. At a glutathione concentration of 5 mM, Michaelis constants were 0.28 and 0.13 mM for methyl parathion and diazinon, respectively.     

Uptake of dieldrin,lindane, and DDT by isolated rat hepatocytes

Studies of the uptake of dieldrin, lindane, and DDT by isolated rat hepatocytes in Krebs-Henseleit salt solution containing 2.34% bovine serum albumin have shown that the partition equilibrium of these hydrophobic organochlorine compounds is established very rapidly between the hepatocytes and the extracellular medium by reversible uptake and release processes. In the hepatocyte suspension system, these compounds are in dynamic partition equilibrium between the hepatocytes and the Krebs-Henseleit salt solution, and also between the bovine serum albumin molecules and the solution. The respective rate constants for the uptake and release processes were 0.83 and 2.52 × 10^?2 sec^?1 in a 2-ml suspension containing 10⁶ cells. It has thus been demonstrated that the transport of hydrophobic organochlorine compounds between the hepatocytes and the extracellular medium is a much faster process than the metabolic transformation reaction in hepatocytes.     

Glucose metabolism in hepatopancreas and gill of Lamellidens marginallis during methyl parathion toxicity

Changes in glucose metabolism were studied in hepatopancreas and gill of freshwater mussel, Lamellidens marginalis, exposed to a sublethal concentration (8 ppm) of methyl parathion. A slight decrease in glycogen and pyruvate and an increase in lactate levels were observed. An increase in phosphorylase and aldolase suggested increased formation of trioses during methyl parathion toxicity. The decrease in lactate dehydrogenase activity and increase in lactate content indicated reduced mobilization of pyruvate into the citric acid cycle. Glucose-6-phosphate dehydrogenase activity was increased, suggesting enhanced oxidation of glucose by the HMP shunt pathway. Citric acid cycle enzymes such as isocitrate, succinate, and malate dehydrogenases were found to be decreased, suggesting abnormality in mitochondrial oxidative metabolism as a consequence of methyl parathion toxicity. The decreased cytochrome c oxidase and Mg²⁺-ATPase, apart from citric acid cycle enzymes, indicated impaired energy synthesis as a result of reduced aerobic oxidation of glycose. The increase in acid and alkaline phosphatase activities suggested enhanced breakdown of phosphate to release energy in view of inhibition of the ATPase system during methyl parathion stress. The changes were more pronounced in hepatopancreas as compared to gill of mussel exposed to methyl parathion.     

Hormonal responses and tolerance to cold of female quail following parathion ingestion

Thirty-week-old female bobwhite quail (Colinus virgininus), maintained at 26 ± 1°C, were provided diets containing 0,25, or 100 ppm parathion ad libitum. After 10 days, birds were exposed to mild cold (6 ± 1°C) for 4, 8, 12, 24, or 48 hr. Brain acetylcholinesterase activity was inhibited in a dose-dependent manner in birds receiving 25 and 100 ppm parathion. Body weight, egg production, and plasma luteinizing hormone and progesterone concentrations were reduced in birds receiving 100 ppm parathion compared with other groups. Cold exposure did not alter plasma corticosterone levels in the 0- and 25-ppm parathion groups, but a two- to fivefold elevation of plasma corticosterone was observed in birds fed 100 ppm parathion. These findings indicate that (i) short-term ingestion of parathion can impair reproduction possibly by altering gonadotropin or steroid secretion, and (ii) tolerance to cold may be reduced following ingestion of this organophosphate.     

Glutathione-dependent degradation of parathion and its significance for resistance in the housefly

Glutathione-dependent degradation of parathion was studied in six strains of houseflies to find out whether it might be important as a cause of resistance. When supernatant fractions of high-speed centrifuged homogenates were fortified with glutathione and incubated with parathion, water-soluble products were formed. The rate of parathion detoxication was highest in a malathion-resistant strain (c. 4 μg parathion degraded per abdomen per hour), lowest in a susceptible strain, and intermediate in some other organophosphate-resistant strains. In one of the latter strains, E₁, the gene for glutathione-dependent degradation is located on the second chromosome, closely linked with gene cm⁺. This is the same chromosome on which gene a for low ali-esterase activity and hydrolytic detoxication of paraoxon is located. It is not likely that the gene for glutathione-dependent degradation is identical with gene a, since it is also present in strain Nie which lacks gene a, and, therefore, the presence of a separate gene which is called gene g is postulated.     

Uptake of [U-14C]Lindane (γ-hexachlorocyclohexane) following exposure of isolated hepatocytes for short intervals of time

The initial steps of the uptake of lindane by the liver were studied by using isolated rat hepatocytes. [U-¹⁴C]Lindane served as labeled indicator during the metabolite production. Results indicated a very fast uptake of the pesticide by the cells. During the first minute there were rapid exchanges between cells and medium; the rate of the uptake did not follow linear evolution. The metabolite production by the cells was time dependent. Up to eight metabolites were visualized during the first minute after the lindane addition. The time course of the uptake was concentration dependent. Profile evolution suggested that the cell metabolism was overloaded over 150 μM lindane. The depletion of hepatocytes in glutathion changed the partition of the radioactivity among the produced metabolites. At 37°C, the N₂ gassing decreased the quantities of cyclohexane rings in the cells. This effect was reversed by preliminary addition of SKF 525A to the incubation medium; SKF 525A had no effect under O₂ aeration. Isomers α and β of HCH, bromsulfophtalein interfered with the lindane transformation by the cells. Thus, isolated hepatocytes represented a sensitive intermediate preparation between perfused liver and homogenate experiments. They will be used for further comparison of the in vivo and in vitro metabolite formation corresponding to the elimination of lindane by the biliary pathway.     

Interstrain comparison of glutathione-dependent reactions in susceptible and resistant houseflies

Glutathione S-alkyl- and S-aryltransferase activities and the glutathione-dependent reactions involved in the metabolism of diazinon, parathion, DDT and γ-BHC were determined in two susceptible and three resistant housefly strains. The relative rate of formation of desethyl diazinon and desethyl parathion and the degradation of γ-BHC paralleled the activities of the alkyl and aryltransferases in the various strains of houseflies suggesting that a single enzyme might be involved. DDT-dehydrochlorinase showed different relative rates among the strains indicating that the dechlorination was catalyzed by a different enzyme. The enzyme responsible for the conjugation of the pyrimidinyl moiety of diazinon appears to be different from the one which catalyzes the conjugation of the p-nitrophenyl moiety of parathion. The dearylation reactions were not mediated by the glutathione S-aryltransferase in the various housefly strains.     

Metabolism and absorption of methyl parathion by tobacco budworms resistant or susceptible to organophosphorus insecticides

Factors involved in insecticide resistance were evaluated by using ¹⁴C-labeled methyl parathion and aldrin to compare rates of absorption and metabolism by Heliothis virescens (F.) larvae that were resistant (R) and susceptible (S) to methyl parathion. Tests with third-stage R and S larvae suggested that the rate of insecticide absorption from the cuticular surface was not a major resistance factor. Further evidence for this assumption was demonstrated by the resistance of R larvae to injected and orally administered doses of methyl parathion. Smaller amounts of unmetabolized methyl parathion and aldrin were recovered from S larvae, an indication that differences in metabolism were probably related to the resistance.     

Significance of phosphorylation in organophosphate-mediated β-glucuronidase release from the rat liver

The elevation of rat blood β-glucuronidase caused in vivo by O,O-dialkyl O-phenyl phosphates and phosphorothioates correlated well with the electron-withdrawing tendency (σ^?) of leaving group substituents indicating the importance of a phosphorylation mechanism in the enzyme release. Hydrophobic bonding of these compounds may facilitate the phosphorylation since hydrophobicity (π) of substituents also correlated with the enzyme release. SKF 525-A decreased the elevation of β-glucuronidase by parathion through the suppression of paraoxon production. Pretreatment of rats with phenobarbital or DDE resulted in lower and delayed enzyme release caused by parathion.     

Intestinal metabolism and absorption of carbaryl studied with a portal fistula

These data were obtained by use of a total and continuous portal vein fistula which virtually eliminated vascular redistribution of compounds absorbed from the gastrointestinal tract to nondigestive tissues (i.e., liver). The method allows direct measurement of the compounds absorbed, which is especially important in metabolism studies of ingested toxic compounds. These studies demonstrated that in vivo metabolism did occur within the intestine during the process of absorption of the pesticide carbaryl (naphthyl N-methylcarbamate) and naphthol, the hydrolysis product of the pesticide. Portal absorption of naphthol from a liquid diet (46 ± 4% of dose/120 min) was slower than from Ringer medium (75 ± 1%/120 min); portal absorption accounted for 82 ± 8 and 83 ± 4%, respectively, of the ¹⁴C absorbed from the intestine. The proportion of hydrophilic ¹⁴C-metabolites (water soluble) in portal blood varied from 6 to 89% and was a function of the substrate, dose vehicle (liquid diet vs Ringer), and time of portal fluid collection. Metabolism in the small intestine before absorption was confirmed for both substrates. The principal lipophilic constituent in portal fluid was the unmetabolized substrate for both carbaryl and naphthol; the principal ampholyte metabolite was naphthyl glucuronide. Although these in vivo data are qualitatively similar to evidence from previous in vitro studies, this in vivo evidence demonstrated that the extent of metabolism (and possibly detoxication) was considerably less than would be predicted from in vitro studies and indicates that the hazard of ingestion of carbaryl and other lipophilic toxic agents may be greater than realized.     

Interaction of insecticides with the Ca2+-pump activity of sarcoplasmic reticulum

The organophosphorus insecticides, parathion and azinphos (10^?5-10^?4M), significantly stimulate the Ca²⁺-pump activity of sarcoplasmic reticulum, while malathion has a limited effect. The rates of Ca²⁺ translocation and ATP hydrolysis are both stimulated and, apparently, the $\text{Ca}{}^{\mathrm{2+}}\text{ATP}$ ratio is improved. Parathion and azinphos maximally increase this ratio by 26 and 14%, respectively. The organochlorine compounds, DDT and aldrin, also stimulate the Ca²⁺ pump, and lindane has a reduced effect. These effects are smaller than those observed for parathion and azinphos. The order of effectiveness is similar to the toxicity of the compounds to mammals and can be described as follows: parathion > azinphos > DDT ≈ aldrin > malathion ≈ lindane.     

Action of pesticides on earthworms. Part II: Elimination of parathion by the earthworm Eisenia foetida (Savigny)

The earthworm, Eisenia foetida, eliminated parathion and carbofuran at first order rates when continually rinsed in water after treatment with the pesticides. This experiment was also carried out on Lumbricus rubellus for comparison. Carbofuran which is more soluble in water, was eliminated quicker than parathion. The later rate of elimination was very similar for the two species, but immediately after injection the rate was much higher in E. foetida. The metabolism of 1-ethyl¹⁴C labelled parathion and paraoxon (diethyl 4-nitrophenyl phosphate) was studied in E. foetida. The worm was able to convert parathion to paraoxon by a rather slow process although this metabolite could not be detected in the worms due to its rapid transformation to diethyl hydrogen phosphate. Indirectly, paraoxon can be postulated as a parathion metabolite because of a progressive depression of cholinesterase level observed after treatment with parathion. Small amounts of diethyl hydrogen phosphate were detected as a metabolite of parathion; this is also an indication of paraoxon formation. During the 30 h following injection of parathion, only 4.4% of the applied dose was recovered as water-soluble metabolites (2.8% in the worms and 1.6% in the sand surrounding them), while 52% was recovered as unmetabolised parathion. Because of inefficient injection, only 70-59% of the dose thought to be injected was recovered. Therefore the part of the actual applied dose that remained unmetabolised was probably even greater (88%). Five days after injection of parathion, 15 and 9.3 % of the recovered radioactivity in the surrounding sand and in the worm extracts, respectively, was identified as O,O-diethyl O-hydrogen phosphorothioate, 3.7 and 7.0% as diethyl hydrogen phosphate, 8.8 and 3.3% as O-ethyl O-4-nitrophenyl O-hydrogen phosphorothioate (desethylparathion) and/or O-4-aminophenyl O,O-diethyl phosphorothioate, while 70.3 and 80.4% was unmetabolised parathion. Paraoxon was very quickly hydrolysed to diethyl hydrogen phosphate in vivo and in vitro. The in-vitro hydrolysis was associated with a microsomal fraction and was not inhibited by ethylenediaminetetra-acetic acid or 4-(chloromercuri)benzoic acid, and incompletely by aldicarb. Cholinesterase and arylesterase were therefore excluded as enzymes responsible for the activity.     

Paraoxon formation and hydrolysis by mammalian liver

In vitro studies of the desulfuration of parathion at 37°C by hepatic tissue from males and females of nine mammalian species revealed sex and species variation in initial rates of parathion desulfuration and arylesterase-catalyzed hydrolysis of the oxygen analogue, paraoxon. Double reciprocal plots of initial rates of parathion activation for representative males and females of each species gave K_m values ranging from 0.2 × 10^?4?1.0 × 10^?4M parathion. Guinea pigs and rats were the only animals showing sex differences in activation, males possessing higher desulfurating abilities than the corresponding females. Based upon the sex possessing the higher desulfurating ability, the species pattern of decreasing activity was hamster > guinea pig > mouse > rat > rabbit > bovine > dog > porcine > cat. Studies of paraoxon hydrolysis indicated that only rats showed sex differences in hydrolysis, males possessing higher arylesterase activity than females. The species pattern of decreasing hydrolytic activity was in the order mouse > bovine > rat > guinea pig > rabbit > hamster > cat > dog > porcine.     

Comparative toxicity,absorption, and metabolism of chlorpyrifos and its dimethyl homologue in methyl parathion-resistant and -susceptible tobacco budworms

Chlorpyrifos (Dowco 179) and its dimethyl homologue, chlorpyrifosmethyl (Dowco 214), were used to study the influence of the O,O-dialkyl group of organophosphorus insecticides on toxicity, absorption, and metabolism among larvae of the tobacco budworm [Heliothis virescens (F.)] from strains that were resistant (R) and susceptible (S) to methyl parathion. In toxicity tests, chlorpyrifos and chlorpyrifosmethyl were more toxic than methyl parathion to 3rd-stage R larvae but less toxic to S larvae. Chlorpyrifosmethyl was more toxic (3–4 ×) than chlorpyrifos to both strains of larvae, and the results of absorption studies indicated that the toxicity differential of the homologues may be explained in part by the more rapid absorption of the dimethyl form. Studies of the in vivo metabolism of both Dowco compounds indicated that each was degraded mainly by the cleavage of the pyridylphosphate linkage. In vitro tests demonstrated that the NADPH-dependent microsomal oxidases were of primary importance in detoxification, while glutathione (GSH)-dependent mechanisms (aryl- and alkyltransferases) present in the soluble cell fractions were of lesser importance. O-dealkylation occurred only with chlorpyrifosmethyl. The R larvae demonstrated greater capability in detoxifying both compounds in the comparative in vivo and in vitro studies of metabolism, but the differences were more apparent during the 5th instar than during the 3rd instar.     

Toxicity in the brain of organophosphate insecticides: Comparison of the toxicities of metabolites with parent compounds using an intracerebral injection method

The toxicity in the brain of several parathion, fenthion, and fensulfothion insecticides and their toxic metabolites was determined by a technique of directly injecting the compounds into the region of the third ventricle of conscious mice, an area rich in cholinesterase activity. The results were compared on a body weight basis to the toxicity of these compounds when given by ip and oral routes. The results show that there is a direct relationship between the relative inhibition of cholinesterase activity in the brain by the organophosphates (e.g., methyl paraoxon, Sumioxon, and some members of the fenthion series) and the toxicity of these compounds in the brain. Methyl paraoxon and Sumioxon were found to be very toxic in the brain, Sumioxon being three to four times less toxic than methyl paraoxon. This is of the same order of effect of these compounds in inhibiting cholinesterases. It is concluded that any selective effects of Sumithion compared with methyl parathion must be due to the greater rate of metabolism of Sumithion to less toxic metabolites as well as to the lower toxicity of the oxon metabolite and not due to the relative rates of penetration of the toxic oxygen metabolites as previously suggested [J. Miyamoto, Agr. Biol. Chem.28, 422 (1964)]. A gas-liquid chromatographic method was employed to assess the distribution in the brain following intracerebral injection of the parathion-type compounds. The results suggest that there may be intracerebral metabolism of thionophosphates in vivo.     

Hydrolysis of paraoxon and malaoxon in three strains of Myzus persicae with different degrees of parathion resistance

Homogenates of three strains of Myzus persicae, A, R, and E, with an LD₅₀ for topically applied parathion of 9, 93, and 263 ng per aphid, showed an in vitro hydrolytic degradation of paraoxon of 2.3, 4.7, and 8.6 pmol/mg aphid/h, respectively. These values represent V_max; K_m was <10^?7M. The three strains showed a malaoxon degradation of 2.4, 11.9, and 18.8 pmol/mg/h at 10^?6M substrate concentration. V_max for R and E was 21 and 27 pmol, respectively and K_m 7 and 4 × 10^?7M. Activity in strain A was too low to estimate these entities. The breakdown product of paraoxon was mainly diethyl phosphoric acid, that of malaoxon mainly dimethyl phosphoric acid. No hydrolysis of the carboxylester groups of malaoxon was found. Hydrolysis of paraoxon and malaoxon was inhibited by isopropyl and n-propyl paraoxon and by the salioxon-analog K₂. The two latter compounds were shown to act as synergists with parathion when added in amounts that caused little mortality when given alone. The hydrolytic enzyme is soluble and retains its activity during incubations of several hours. It is likely that it is responsible for at least part of the resistance. Resistance was maintained without selection over a period of three years. There was no correlation between degree of resistance and carboxylesterase activity of the strains.     

Excretion of unchanged organophosphorus compounds after their consumption by larvae of the Egyptian cotton leafworm,Spodoptera littoralis Boisd

The ingestion and excretion of sublethal doses of phosfolan, monocrotophos, parathion, and leptophos were studied in larvae of the Egyptian cotton leafworm, Spodoptera littoralis Boisd. Styropor (foamed polystyrene) lamellae treated with insecticide-sucrose mixtures were fed to the larvae and recovery of the undecomposed insecticides from the feces could be estimated by gas-liquid chromatography, without any cleanup of the sample. The insecticidal residues on Styropor were found to be stable for 5 days.As regards the percentage of insecticide recovered from the feces, two groups could be distinguished: (a) leptophos, practically complete; parathion, 59–67%; b) phosfolan, 10–23%; monocrotophos, 4–7%. A tentative hypothesis was advanced that both oral toxicity of the four compounds for S. littoralis larvae and their subsequent recovery in the feces were related to water solubility.