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.     

Biotransformation of methyl parathion by glutathione S-transferases.

The organo(thio)phosphate esters are one of the most widely used classes of insecticides. Worldwide, organophosphate insecticides (OPs) result in numerous poisonings each year. In insects, glutathione S-transferases (GSTs) play an important role in OP resistance; limited data suggest that GST-mediated O-dealkylation occurs in humans as well. To characterize the capacity of mammalian GSTs to detoxify OPs, we investigated mammalian GST biotransformation of the widely used OP, methyl parathion (MeP). Cytosolic fractions isolated from rat, mouse, and ten individual adult human livers biotransformed 300 microM MeP at rates of 2.36, 1.76, and 0.70 (mean rate) nmol desmethyl parathion/min/mg, respectively. Our study focused on human GSTs; in particular, we investigated hGSTs M1-1 and T1-1, since deletion polymorphisms occur commonly in these genes. However, we found no correlation between hGSTM1/T1 genotypes and MeP O-dealkylation activities of the ten human liver cytosolic samples. We also measured MeP O-dealkylation activities of several purified recombinant GSTs belonging to the alpha (human GSTs A1-1 and A2-2, mouse GSTA3-3, rat GSTA5-5), mu (human GSTs M1a-1a, M2-2, M3-3, M4-4), pi (human GSTP1-1, mouse GSTs P1-1, P2-2), and theta (human GSTT1-1) classes. At 1 mM glutathione and 300 microM MeP concentrations, hGSTT1-1 and hGSTA1-1 exhibited the highest O-dealkylation activities: 545.8 and 65.0 nmol/min/mg, respectively. When expression level and enzymatic activity are considered, we estimate that hGSTA1-1 is responsible for the majority of MeP O-dealkylation in human hepatic cytosol. In target organs such as brain and skeletal muscle, where hGSTT1-1 is expressed, hGSTT1-1-mediated biotransformation of MeP may be important.     

Metabolic activation of the organophosphorus insecticides chlorpyrifos and fenitrothion by perfused rat liver.

The present study was undertaken to characterize the metabolic activation of the organophosphorus insecticides chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothionate] and fenitrothion [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphorothionate] by intact rat liver. Single-pass perfusions of rat livers with chlorpyrifos or fenitrothion to steady state conditions resulted in the appearance of their corresponding oxygen analogs in effluent. In addition, detoxification of chlorpyrifos oxon [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate] or fenitrooxon [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphate] by rat blood did not proceed at a rate rapid enough to prevent passage of at least some of these chemicals from liver to extrahepatic tissues, suggesting that hepatic biotransformation of chlorpyrifos and fenitrothion by rat liver results in their net activation. Although male rat livers produced more chlorpyrifos oxon and fenitrooxon from chlorpyrifos and fenitrothion, respectively, than did livers from female rats, the acute toxicities of chlorpyrifos and fenitrothion were greater in females than in males. Therefore, differences in hepatic activation of chlorpyrifos and fenitrothion in males and females cannot account for the sex differences in their acute toxicities in the rat. Finally, S-methyl glutathione and S-p-nitrophenyl glutathione were not detected in effluent or bile of livers perfused with fenitrothion, suggesting that glutathione-mediated biotransformation of this insecticide does not occur to any significant degree in intact liver.     

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

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.     

Biotransformation of paraoxon and p-nitrophenol by isolated perfused mouse livers

Single-pass perfusion in situ of mouse livers with the organophosphate paraoxon resulted in formation of p-nitrophenol (PNP), p-nitrophenyl sulfate (PNPS), and p-nitrophenyl-beta-D-glucuronide (PNPG). Following initiation of perfusion of paraoxon steady--state conditions were achieved in 15-25 min, at which time the extraction ratio was 0.55 (S.D. = 0.05). This suggests the capacity of mouse liver to biotransform paraoxon is not as great as previously reported. At all concentrations of paraoxon examined the amount of PNPS produced exceeded that of PNPG. However, as the concentration of paraoxon increased the relative proportion of PNP to PNPS and PNPG increased, indicating the capacity of liver to biotransform paraoxon exceeded the capacity to biotransform PNP. Single-pass perfusion in situ of mouse livers with PNP resulted in production of PNPS and PNPG. As with paraoxon, steady-state conditions were achieved in 15-25 min. The extraction ratio of PNP, as well as the metabolic profile, changed markedly with varying concentrations of PNP. At PNP reservoir concentrations of 4 microM or less the extraction ratio of PNP was 1, with all PNP metabolized to PNPS. As PNP concentrations increased (up to 75 microM) both unchanged PNP and PNPG appeared in the effluent. Thus the hepatic biotransformation of PNP was clearly dependent on substrate concentration.     

N(G)-nitro-L-arginine methyl ester potentiates anaphylactic venoconstriction in rat perfused livers

1. The effects of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on anaphylaxis-induced venoconstriction were examined in rat isolated livers perfused with blood-free solutions in order to clarify the role of NO in anaphylactic venoconstriction. 2. Rats were sensitized with ovalbumin (1 mg) and, 2 weeks later, livers were excised and perfused portally in a recirculating manner at a constant flow with Krebs'-Henseleit solution. The antigen (ovalbumin; 0.1 mg) was injected into the reservoir 10 min after pretreatment with L-NAME (100 micromol/L) or D-NAME (100 micromol/L) and changes in portal vein pressure (Ppv), hepatic vein pressure (Phv) and perfusate flow were monitored. In addition, concentrations of the stable metabolites of NO ( and ) were determined in the perfusate using an HPLC-Griess system. 3. The antigen caused hepatic venoconstriction, as evidenced by an increase in Ppv from a mean (SEM) baseline value of 7.7 +/- 0.1 cmH2O to a peak of 21.4 +/- 1.1 cmH2O at 3 min in D-NAME-pretreated livers. Pretreatment with L-NAME augmented anaphylactic venoconstriction, as reflected by a higher Ppv (27.4 +/- 0.8 cmH2O) after antigen than observed following D-NAME pretreatment. The addition of L-arginine, a precursor for the synthesis of NO, reversed the augmentation of anaphylactic venoconstricion by L-NAME. This suggests that hepatic anaphylaxis increased the production of NO, which consequently attenuated anaphylactic venoconstriction. However, perfusate NOx levels did not increase significantly after antigen in livers pretreated with either L-NAME or D-NAME. 4. In conclusion, L-NAME potentiates rat anaphylactic hepatic venoconstriction, suggesting that NO contributes to the attenuation of the venoconstriction. However, this functional evidence was not accompanied by corresponding changes in perfusate NOx concentrations.     

Biotransformation of organophosphorus compounds.

This study reviews current understanding of mechanisms of biotransformation of organophosphorus compounds (OPC). The first part of this article covers chemical aspects of biotransformation describing reactions that lead to activation or detoxication of OPC. The second part explains biochemical mechanisms of biotransformation describing the role of enzymes that are involved in this process. Among them are the enzymes that take part in metabolic activation of OPC such as cytochrome P450 system, NADPH-cytochrome P450 reductase and flavin-containing monooxygenases. Among enzymes participating in detoxication of OPC, the role of phosphoric triester hydrolases, carboxylesterases and glutathione redox system is explained. This article also deals with other aspects of detoxication of OPC such as protein binding and the role of tissue depots for these compounds.     

Biotransformation of 4-dimethylaminophenol in the isolated perfused rat liver and in the rat

Isolated rat livers were perfused with various concentrations of 4-dimethylaminophenol-[¹⁴C] (DMAP). During single pass perfusion with modified protein-free Krebs-Henseleit solution up to 70μM prehepatic 4-dimethylaminophenol (DMAP) were metabolized by the liver. The main route of biotransformation was conjugation. At steady state conditions glucuronide formation showed an apparent V_max of 8.5 μmoles × min^?1 × g protein^?1, and K_m of 562 μM, whereas sulfate formation had an apparent V_max of 1.2 and a K_m of 35. Thus, at low substrate concentration the sulfate conjugation outweighed glucuronidation whereas at high substrate concentration the ratio of conjugates was reversed. In contrast to DMAP-sulfate, some DMAP-glucuronide was stored by the liver and was released with a half life of about 15 min which showed positive correlation with the dose of DMAP during the washout period. Perfusion with human or rat erythrocytes demonstrated the other important path of biotransformation of DMAP within erythrocytes, namely thioether formation with glutathione and SH-groups of hemoglobin. The pattern of DMAP-conjugation was affected depending on the time of prehepatic exposure to erythrocytes, and the species of red cells. The results obtained from the isolated metabolic system resemble the hepatic part of the overall metabolism under in vivo conditions.     

Influence of methyl parathion on reproductive parameters in male rats

Exposure of human population to pesticides and industrial pollutants has considerably increased the risk of human health hazard. In the present study, therefore we have sought to investigate the toxic effects of Methyl Parathion on male reproductive system of rat. The tested dose was given orally to the rats for 30 days at the dose level of 30 mg/kg/day. Sex organs weight analysis, histochemical and histopathological changes and mating trials were the criteria used to evaluate the reproductive efficacy of the treated rats. The body weight of the animals did not show any significant change. However, Methyl Parathion caused significant decrease in the weight of testis, epididymis, seminal vesicle and ventral prostate with marked pathomorphological changes. Also, marked reduction in epididymial and testicular sperm counts in exposed males were noticed. Fertility test showed 80% −ve fertility in treated animals. A significant reduction in the sialic acid contents of testis, epididymis, seminal vesicle, ventral prostate and testicular glycogen were noticed, while the protein and cholesterol content were raised significantly. From the above-mentioned findings, it has been concluded that exposure to Methyl Parathion has deleterious effects on male reproductive system of rat. Therefore, application of such insecticide should be limited to a designed program.     

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.     

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.     

Human hepatic cytochrome P450-specific metabolism of the organophosphorus pesticides methyl parathion and diazinon

Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (K(m) and V(max)) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (K(m) = 1.25 μM; V(max) = 9.78 nmol · min(-1) · nmol P450(-1)), CYP2C19 (K(m) = 1.03 μM; V(max) = 4.67 nmol · min(-1) · nmol P450(-1)), and CYP1A2 (K(m) = 1.96 μM; V(max) = 5.14 nmol · min(-1) · nmol P450(-1)), and the bioactivation of diazinon was mediated primarily by CYP1A1 (K(m) = 3.05 μM; V(max) = 2.35 nmol · min(-1) · nmol P450(-1)), CYP2C19 (K(m) = 7.74 μM; V(max) = 4.14 nmol · min(-1) · nmol P450(-1)), and CYP2B6 (K(m) = 14.83 μM; V(max) = 5.44 nmol · min(-1) · nmol P450(-1)). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (K(m) = 16.8 μM; V(max) = 1.38 nmol · min(-1) · nmol P450(-1)) and 3A4 (K(m) = 104 μM; V(max) = 5.15 nmol · min(-1) · nmol P450(-1)), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (K(m) = 5.04 μM; V(max) = 5.58 nmol · min(-1) · nmol P450(-1)). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure.     

Effect of carbon tetrachloride on activation and detoxification of organophosphorus insecticides in the rat.

Carbon tetrachloride, 250 or 500 μl/kg, was administered po to rats 24 hr before the insecticide administration. After pretreatment with CCl₄ no signs of fenitrothion poisoning (75% LD50) were observed, but signs of chlorfenvinphos, disulfoton, or dichlorvos poisoning were similar to those observed in the control groups. The liver concentrations of fenitrothion and chlorfenvinphos were increased after pretreatment with CCl₄. The degradation of fenitrothion, chlorfenvinphos, and disulfoton by liver homogenates was diminished after CCl₄ pretreatment. Fenitrothion, parathion, disulfoton, ronnel, bromophos, chlorfenvinphos and bromfenvinphos cause type I substrate-cytochrome P-450 binding spectra. Difference spectra with dichlorvos, trichlorfon, or polfos were not detectable. These results suggest that most of the organophosphorus insecticides may be metabolized by mixed function oxidase systems and cytochrome P-450. The susceptibility of rats to poisoning by fenitrothion or derivative dialkyl aryl thiophosphates depends upon the activity of the mixed function oxidase system.     

Renal damage induced by the pesticide methyl parathion in male Wistar rats

Little information is apparently available regarding the nephrotoxic effects induced by pesticides. The aim of this study was to examine the influence of low doses of methyl parathion (MP) on the structure and function of the kidney of male Wistar rats. A corn oil (vehicle) was administered to control rats, whereas treated rats received MP at 0.56 mg/kg orally (1/25 of LD₅₀), every third day, for 8 weeks. At the end of each week following MP exposure, creatinine and glucose levels were measured in plasma, while glucose, inorganic phosphate, total proteins, albumin, and activity of γ-glutamyltranspeptidase (GGT) were determined in urine. Kidney histological study was also performed. Compared with control rats, MP significantly increased plasma glucose and creatinine levels accompanied by decreased urinary flow rate and elevated urinary excretion rates of glucose, phosphate, and albumin. Further, the activity of GGT in urine was increased significantly. The proximal cells exhibited cytoplasmic vacuolization, positive periodic acid Schiff inclusions, and brush border edge loss after 2 or 4 weeks following MP treatment. Finally, renal cortex samples were obtained at 2, 4, 6, and 8 weeks of MP treatment, and the concentrations of reduced glutathione (GSH) and glutathione peroxidase (GPx) activity were measured. The mRNA expression levels of BAX and tumor necrosis factor-α (TNF-α) were also determined (RT-PCR). MP significantly decreased renal GSH levels, increased GPx activity, as well as downregulated the mRNA expression of TNF-α and BAX. Densitometry analysis showed a significant reduction in TNF-α and BAX mRNA expression levels at 2 and 4 weeks following MP treatment. Low doses of MP produced structural and functional damage to the proximal tubules of male rat kidney.     

Biotransformation of methyl parathion by human foetal liver glutathione S-transferases: an in vitro study

The role of human foetal liver glutathione S-transferases in the detoxification of methyl parathion was investigated. Glutathione S-transferases were partially purified by affinity chromatography utilizing reduced glutathione as the ligand coupled to epoxy-activated Sepharose 4B. This resulted in the isolation of material with an average activity (mean +/- S.E.) of 58.90 +/- 4.83 mumol 1-chloro-2,4-dinitrobenzene conjugate formed/min per mg, representing a purification of 70-fold. These partially purified foetal liver transferases catalysed the metabolism of methyl parathion exclusively to desmethyl parathion via O-dealkylation. High-performance liquid chromatography, radiometric analysis of the enzymic reaction, and co-chromatography with reference standard on thin-layer chromatography confirmed the sole metabolite as desmethyl parathion. The range of foetal liver activity towards methyl parathion was from 30 to 122 nmol desmethyl parathion formed/min per mg. Analysis of the kinetic parameters of three partially purified foetal liver preparations with gestational ages of 14, 16 and 21 weeks resulted in Km values for methyl parathion of 0.24, 0.38 and 0.86 mM, respectively; whereas, the Km values assessed for glutathione were 0.20, 0.10 and 0.18 mM. The ability of human foetal liver glutathione S-transferases to catalyse the metabolism of methyl parathion exclusively to desmethyl parathion via O-dealkylation represents a major qualitative biochemical difference from the rat-liver isozymes.     

Activation and degradation of the phosphorothionate insecticides parathion and EPN by rat brain

Cytochrome P-450-dependent monooxygenases are known to activate phosphorothionate insecticides to their oxon (phosphate) analogs by oxidative desulfuration. These activations produced potent anticholinesterases, decreasing the I50 values to rat brain acetylcholinesterase almost 1000-fold (from the 10(-5) M range to the 10(-8) M range). Since the usual cause of death in mammals from organophosphorus insecticide poisoning is respiratory failure resulting, in part, from a failure of the respiratory control center of the brain, we investigated the ability of rat brain to activate and subsequently degrade two phosphorothionate insecticides, parathion (diethyl 4-nitrophenyl phosphorothioate) and EPN (ethyl 4-nitrophenyl phenylphosphonothioate). Microsomes from specific regions (cerebral cortex, corpus striatum, cerebellum, and medulla/pons) of the brains of male and female rats and from liver were incubated with the phosphorothionate and an NADPH-generating system. Oxon production was quantified indirectly by the amount of inhibition resulting in an exogenous source of acetylcholinesterase added to the incubation mixture as an oxon trap. The microsomal activation specific activity was low for brain when compared to liver [0.23 to 0.44 and 5.1 to 12.0 nmol.min-1.(g tissue)-1 respectively]. The mitochondrial fraction of the brain possessed an activation activity for parathion similar to that of microsomes [about 0.35 nmol.min-1.(g tissue)-1 for each fraction], but mitochondrial activity was slightly greater than microsomal activity for EPN activation [0.53 to 0.58 and 0.23 to 0.47 nmole.min-1.(g tissue)-1]. Whole homogenates were tested for their ability to degrade paraoxon and EPN-oxon (ethyl 4-nitrophenyl phenylphosphonate), quantitated by 4-nitrophenol production. Specific activity for oxon degradation in liver was greater than that in brain [31 to 74 and 1.1 to 10.7 nmole.min-1.(g tissue)-1 respectively]. Overall, the brain and liver had about 1.5- to 12-fold higher specific activities for degradation than activation depending on the compound used. These findings demonstrate that the brain possesses both phosphorothionate activation and oxon degradation abilities, both of which may be significant during exposures to organophosphorus insecticides.     

Differential mutagenic response of Salmonella typhimurium to the plant-metabolized organophosphorus insecticides, phoxim and azinphos methyl.

The plant cell/microbe coincubation assay was used to analyze organophosphorus insecticide activation. Salmonella typhimurium strains TA98 and TA100 were exposed to several concentrations of the pesticides phoxim and azinphos methyl with and without TX1 cell line of Nicotiana tabacum activation. When the bacterial strains were treated directly with phoxim, mutagenic activity increased significantly. In contrast, no mutagenic activity was detected with plant activation. Azinphos methyl inhibited the growth of Salmonella strains without plant activation. The coincubation with N. tabacum increased mutagenic activity significantly. These findings and those obtained in animals demonstrated that azinphos-methyl was an indirect mutagen or pro-mutagen activated by the plant metabolism.     

Biotransformation of methyl parathion by human foetal liver glutathione S-transferases: an in vitro study

1. The role of human foetal liver glutathione S-transferases in the detoxification of methyl parathion was investigated.

2. Glutathione S-transferases were partially purified by affinity chromatography utilizing reduced glutathione as the ligand coupled to epoxy-activated Sepharose 4B. This resulted in the isolation of material with an average activity (mean ± S.E.) of 58.90 ± 4.83 µmol 1 -chloro-2,4-dinitrobenzenceo njugate formed/min per mg. representing a purification of 70-fold.

3. These partially purified foetal liver transferases catalysed the metabolism of methyl parathion exclusively to desmethyl parathion via O-dealkylation.

4. High-performance liquid Chromatography, radiometric analysis of the enzymic reaction, and co-chromatography with reference standard on thin-layer chromatography confirmed the sole metabolite as desmethyl parathion.

5. The range of foetal liver activity towards methyl parathion was from 30 to 122 nmol desmethyl parathion formed/min per mg.

6. Analysis of the kinetic parameters of three partially purified foetal liver preparations with gestational ages of 14, 16 and 21 weeks resulted in K_M values for methyl parathion of 0.24, 0.38 and 0.86mM, respectively; whereas, the K_M values assessed for glutathione were 0.20, 0.10 and 0.18mM.

7. The ability of human foetal liver glutathione S-transferases to catalyse the metabolism of methyl parathion exclusively to desmethyl parathion via O-dealkylation represents a major qualitative biochemical difference from the rat-liver isozymes.