Plasma Carboxylesterase Activities Research Articles

Novel Genetically Modified Mouse Model to Assess Soman-Induced Toxicity and Medical Countermeasure Efficacy: Human Acetylcholinesterase Knock-in Serum Carboxylesterase Knockout Mice.

The identification of improved medical countermeasures against exposure to chemical warfare nerve agents (CWNAs), a class of organophosphorus compounds, is dependent on the choice of animal model used in preclinical studies. CWNAs bind to acetylcholinesterase and prevent the catalysis of acetylcholine, causing a plethora of peripheral and central physiologic manifestations, including seizure. Rodents are widely used to elucidate the effects of CWNA-induced seizure, albeit with a caveat: they express carboxylesterase activity in plasma. Carboxylesterase, an enzyme involved in the detoxification of some organophosphorus compounds, plays a scavenging role and decreases CWNA availability, thus exerting a protective effect. Furthermore, species-specific amino acid differences in acetylcholinesterase confound studies that use oximes or other compounds to restore its function after inhibition by CWNA. The creation of a human acetylcholinesterase knock-in/serum carboxylesterase knockout (C57BL/6-Ces1ctm1.1LocAChEtm1.1Loc/J; a.k.a KIKO) mouse may facilitate better modeling of CWNA toxicity in a small rodent species. The current studies characterize the effects of exposure to soman, a highly toxic CWNA, and evaluate the efficacy of anti-seizure drugs in this newly developed KIKO mouse model. Data demonstrate that a combination of midazolam and ketamine reduces seizure duration and severity, eliminates the development of spontaneous recurrent seizures, and protects certain brain regions from neuronal damage in a genetically modified model with human relevance to organophosphorus compound toxicity. This new animal model and the results of this study and future studies using it will enhance medical countermeasures development for both defense and homeland security purposes.

Efficacy of Midazolam and Ketamine Combination Therapy over Midazolam Monotherapy against Soman Exposure in Carboxylesterase Knockout Mice

IntroductionChemical warfare nerve agents (CWNAs) inhibit acetylcholinesterase (AChE), which leads to status epilepticus (SE), spontaneous recurrent seizures (SRS) and severe neuropathology when treatment is delayed. In addition to binding to AChE, some organophosphorus (OP) CWNAs such as soman (GD) also inhibit carboxylesterase (CaE), which acts as a bioscavenger and can thus reduce the severity of the toxicity of OP agent exposure. Unlike humans, rodents have plasma CaE activity. The CaE knockout (ES1−/−) mouse specifically lacks plasma CaE and might better model human GD exposure compared to wildtype rodents. Delayed treatment of CWNA‐induced SE with midazolam leads to benzodiazepine‐refractory SE. We evaluated combination therapy of midazolam and the NMDA antagonist ketamine in male and female mice for efficacy against soman‐induced lethality, SE and epileptogenesis.MethodsMice implanted with telemetry transmitters for electroencephalography (EEG) seizure identification were exposed to 82 μg/kg GD (~4LD50) and treated with an admix (ip) of atropine sulfate (4 mg/kg) and HI‐6 (50 mg/kg) 1 min after exposure, and with midazolam (3 mg/kg; ip) alone or in combination with ketamine (30 mg/kg, ip) at 40 min after seizure onset. Mice were continuously recorded to evaluate initial seizure duration and SRS and then euthanized 2 weeks after exposure for neuropathology assessment.ResultsDelayed treatment with midazolam resulted in poor survival, was unable to rapidly terminate behavioral or EEG seizure activity and did not prevent the development of SRS or neuronal loss following GD exposure. Combination therapy of midazolam and ketamine resulted in improved outcome, including an increase in survival from 30% to 78% in female and from 57% to 77% in males compared to midazolam monotherapy. In addition, the incidence of epileptogenesis was lower in mice treated with ketamine and midazolam dual therapy compared to midazolam monotherapy.ConclusionsThis study demonstrates that delayed treatment of SE with midazolam is not fully protective against the GD‐induced epileptogenesis and neuropathology, exemplifying the need for adjunct treatment to midazolam to prevent or reduce effects of GD‐induced SE. Ketamine combination with midazolam improves outcome.Support or Funding InformationResearch was supported by the CounterACT Program, NIH OD, and the NINDS (Grant 1R21NS103820‐01 to LA Lumley‐Lange) and by DTRA‐JSTO. Erica Kundrick, Katie Walker, and Sean O'Brien were supported in part by an appointment to the Research Participation Program for the U.S. Army Medical Research and Materiel Command administered by the Oak Ridge Institute for Science and Education through an agreement between the U.S. Department of Energy and U.S. Army Medical Research and Materiel Command.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Carboxylesterase, Butyrylcholinesterase, Acetylcholinesterase, Paraoxonase, and Albumin Pseudoesterase in Guinea Pig Plasma through Nondenaturing Gel Electrophoresis.

Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis-p-nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with α- or β-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.

Kinetics of hydrolysis of 1,4-benzodiazepine derivative by carboxylesterases in mice organism

Chemical modification of the physiologically active substances and creation of prodrugs is one of the ways for pharmacotherapy optimization. The aim of the work was determination of the kinetic parameters ofnonspecific esterases that catalyze hydrolysis of new hypnotic drug Levana (1,4-benzodiazepine derivative). The experiments were carried out using the 14C-labelled Levana and its active metabolite - 3-hydrixyphenazepam. In vitro it was shown that Levana undergoes spontaneous hydrolysis even in buffer solution (pH 7.4), though in plasma and homogenates of brain and liver this process is more intensive (conventional Vmax was 6.9 ± 0.5, 19 ± 4 and 12 ± 1 mM/(h·mg of protein, correspondingly). The samples mentioned differ by activity of tissue esterases being most active in the liver (conventional K 0.45 ± 0.04 mM for the liver and 47 ± 11 mM for the brain). In plasma carboxylesterase activity (for Levana) is the lowest (conventional K 129 ± 10 mM). In vivo it was shown that Levana more easily permeates brain-blood barrier (compared to 3-hydroxyphenazepam), that leads to higher concentrations (after hydrolysis) of its metabolite in brain tissue. Also it is quantitatively estimated as the increase of concentration (brain/blood) ratio ~1.4 times.

Production of ES1 Plasma Carboxylesterase Knockout Mice for Toxicity Studies

The LD(50) for soman is 10-20-fold higher for a mouse than a human. The difference in susceptibility is attributed to the presence of carboxylesterase in mouse but not in human plasma. Our goal was to make a mouse lacking plasma carboxylesterase. We used homologous recombination to inactivate the carboxylesterase ES1 gene on mouse chromosome 8 by deleting exon 5 and by introducing a frame shift for amino acids translated from exons 6 to 13. ES1-/- mice have no detectable carboxylesterase activity in plasma but have normal carboxylesterase activity in tissues. Homozygous ES1-/- mice and wild-type littermates were tested for response to a nerve agent model compound (soman coumarin) at 3 mg/kg sc. This dose intoxicated both genotypes but was lethal only to ES1-/- mice. This demonstrated that plasma carboxylesterase protects against a relatively high toxicity organophosphorus compound. The ES1-/- mouse should be an appropriate model for testing highly toxic nerve agents and for evaluating protection strategies against the toxicity of nerve agents.

Selective inhibition of butyrylcholinesterase in vivo in horses by the feed-through larvacide Equitrol ®

The organophosphate insecticide tetrachlorvinphos (TCVP, Rabon) is the active ingredient in “feed-through” larvacides (e.g., Equitrol ®) for fly control around horse stables. As with other organophosphates, TCVP elicits toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and cholinergic signs. Relatively little is known, however, on the effects of TCVP-containing larvacides on acetylcholinesterase or other esterases in horses. Previous in vitro studies indicated that horse plasma cholinesterase activity was substantially (>10,000-fold) more sensitive than erythrocyte cholinesterase activity to inhibition by TCVP. In the current study, we examined the relative proportion of acetylcholinesterase and butyrylcholinesterase activities in horse plasma and muscle, and evaluated the in vivo effects of Equitrol ® on target and non-target esterases following oral feeding in horses. In vitro inhibition studies suggested that essentially all cholinesterase activity in horse plasma was butyrylcholinesterase, while muscle contained >90% acetylcholinesterase activity. For in vivo studies, adult, male horses (364–590 kg; n = 3/treatment group) were given either sweet feed alone or sweet feed supplemented with Equitrol ® daily for 21 consecutive days at the recommended rate. Clinical signs (vital signs, abdominal auscultation, ophthalmic exam, body temperature) were recorded on a daily basis. Heparinized blood samples were taken at days −1, 1, 3, 7, 21, 28, and 42 while muscle (semimembranosus) biopsies were taken under aseptic conditions on days −1 and 21. No signs of overt toxicity were noted at any time during the study. Plasma cholinesterase activity was significantly inhibited (33%) in larvacide-treated horses as early as one day after treatment and peak inhibition (69–71%) was noted at days 7 and 21. Following cessation of dosing, plasma cholinesterase activity recovered (46% and 83% of control on days 28 and 42, respectively). Neither erythrocyte cholinesterase activity nor plasma carboxylesterase activity was affected by larvacide treatment in vivo. Muscle cholinesterase activity was highly variable among individual horses (pre-treatment range: 0.50–4.92 nmole/min/mg protein), but there was no suggestion of a treatment-related reduction in muscle cholinesterase activity. These in vivo results confirm our previous in vitro studies indicating marked differential sensitivity of horse plasma and erythrocyte cholinesterase to inhibition by TCVP. Furthermore, the results suggest that recommended dosing levels of the TCVP-containing larvacide in horses are unlikely to affect acetylcholinesterase activities or disrupt cholinergic neurotransmission in target tissues.

Tumor-Targeted Enzyme/Prodrug Therapy Mediates Long-term Disease-Free Survival of Mice Bearing Disseminated Neuroblastoma

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

Interactive toxicity of chlorpyrifos and parathion in neonatal rats: Role of esterases in exposure sequence-dependent toxicity

We previously reported that sequence of exposure to chlorpyrifos and parathion in adult rats can markedly influence toxic outcome. In the present study, we evaluated the interactive toxicity of chlorpyrifos (8 mg/kg, po) and parathion (0.5 mg/kg, po) in neonatal (7 days old) rats. Rats were exposed to the insecticides either concurrently or sequentially (separated by 4 h) and sacrificed at 4, 8, and 24 h after the first exposure for biochemical measurements (cholinesterase activity in brain, plasma, and diaphragm and carboxylesterase activity in plasma and liver). The concurrently-exposed group showed more cumulative lethality (15/24) than either of the sequential dosing groups. With sequential dosing, rats treated initially with chlorpyrifos prior to parathion (C/P) exhibited higher lethality (7/23) compared to those treated with parathion before chlorpyrifos (P/C; 1/24). At 8 h after initial dosing, brain cholinesterase inhibition was significantly greater in the C/P group (59%) compared to the P/C group (28%). Diaphragm and plasma cholinesterase activity also followed a relatively similar pattern of inhibition. Carboxylesterase inhibition in plasma and liver was relatively similar among the treatment groups across time-points. Similar sequence-dependent differences in brain cholinesterase inhibition were also noted with lower binary exposures to chlorpyrifos (2 mg/kg) and parathion (0.35 mg/kg). In vitro and ex vivo studies compared relative oxon detoxification of carboxylesterases (calcium-insensitive) and A-esterases (calcium-sensitive) in liver homogenates from untreated and insecticide pretreated rats. Using tissues from untreated rats, carboxylesterases detoxified both chlorpyrifos oxon and paraoxon, while A-esterases only detoxified chlorpyrifos oxon. With parathion pretreatment, A-esterases still detoxified chlorpyrifos oxon while liver from chlorpyrifos pretreated rats had little apparent effect on paraoxon. We conclude that while neonatal rats are less capable than adults at detoxifying many organophosphorus insecticides including chlorpyrifos and parathion, toxicant-selective differences in detoxification play a role in sequence-dependent toxicity in both neonatal and adult rats with these two insecticides.

Efficacy of Trimedoxime in Mice Poisoned with Dichlorvos, Heptenophos or Monocrotophos

The aim of the study was to examine antidotal potency of trimedoxime in mice poisoned with three direct dimethoxy-substituted organophosphorus inhibitors. In order to assess the protective efficacy of trimedoxime against dichlorvos, heptenophos or monocrotophos, median effective doses and efficacy half-times were calculated. Trimedoxime (24 mg/kg intravenously) was injected 5 min. before 1.3 LD50 intravenously of poisons. Activities of brain, diaphragmal and erythrocyte acetylcholinesterase, as well as of plasma carboxylesterases were determined at different time intervals (10, 40 and 60 min.) after administration of the antidotes. Protective effect of trimedoxime decreased according to the following order: monocrotophos > heptenophos > dichlorvos. Administration of the oxime produced a significant reactivation of central and peripheral acetylcholinesterase inhibited with dichlorvos and heptenophos, with the exception of erythrocyte acetylcholinesterase inhibited by heptenophos. Surprisingly, trimedoxime did not induce reactivation of monocrotophos-inhibited acetylcholinesterase in any of the tissues tested. These organophosphorus compounds produced a significant inhibition of plasma carboxylesterase activity, while administration of trimedoxime led to regeneration of the enzyme activity. The same dose of trimedoxime assured survival of experimental animals poisoned by all three organophosphorus compounds, although the biochemical findings were quite different.

PLASMA B-ESTERASE ACTIVITIES IN EUROPEAN RAPTORS

B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and Strigidae families were characterized by a BChE contribution that dominated the total ChE activity, while in the species of the Falconidae family, AChE activity dominated. With the exception of the barn owl, CbE activity (eserine-insensitive alpha-naphthyl acetate esterase [alpha-NAE] activity) in all species was almost absent or very low. The values obtained in this study for ChE, AChE, and BChE activities and the AChE:BChE ratios for buzzard, kestrel, barn owl, and tawny owl provide a good estimate of the normal values in free-living individuals of these European species. They can be used as a baseline to evaluate the effect of anticholinesterase insecticides in the field.

GLUCOSE FEEDING EXACERBATES PARATHION-INDUCED NEUROTOXICITY

Excessive dietary intake of sugars could alter various biotransformation processes and the pharmacological and toxicological properties of numerous xenobiotics. In the present study, the effects of glucose supplementation were examined on the neurotoxicity of the organophosphorus (OP) pesticide parathion (PS) and its active metabolite, paraoxon (PO), a potent inhibitor of acetylcholinesterase (AChE). Rats (n = 6-12/treatment group) were given free access to tap water or 15% glucose (w/v) in tap water beginning 7 d prior to OP toxicant exposure. Food, caloric intake, and body weight were measured daily. Animals were challenged with either PS (4.5, 9, or 18 mg/kg, sc) or PO (0.3, 0.5, or 0.7 mg/kg, sc) and clinical signs of neurotoxicity (i.e., autonomic dysfunction, involuntary movements) were recorded daily for the following 13 d. Glucose feeding was associated with a dramatic drop (~50%) in feed intake and an increase (~20%) in total caloric consumption over the 7 d prior to OP exposure. Functional toxicity associated with PS exposure was increased in glucose-fed (GF) rats, but the glucose diet had no apparent effect on clinical signs of toxicity following PO treatment. Glucose feeding increased the magnitude of AChE inhibition in the frontal cortex and plasma at lower dosages (i.e., 4.5 and 9 mg/kg) 3 d following PS treatment. Time-course studies (3, 7, and 11 d after PS exposure, 18 mg/kg, sc) indicated significantly greater brain and plasma AChE inhibition in glucosefed animals at later time points. In contrast, glucose feeding had no effect on the degree of AChE inhibition following PO exposure. Neither liver microsomal oxidative desulfuration of PS, nor liver or plasma paraoxonase, nor liver or plasma carboxylesterase activities were measurably affected by glucose feeding. Downregulation of muscarinic receptors 7 d after PS exposure (18 mg/kg, sc) was more extensive in GF rats. It is postulated that excessive glucose consumption decreases the intake of other dietary components, in particular amino acids, limiting the de novo synthesis of AChE and consequent recovery of synaptic transmission. Due to the shorter duration of inhibition following PO exposure, spontaneous reactivation of AChE may be more important than de novo protein synthesis in recovery of function, and thus with the effects of glucose feeding on its toxicity. Individuals that derive a large proportion of their calories from sugars may be at higher risk of acute toxicity from organophosphorus pesticides such as PS.

Carboxylesterase and A-esterase activities during maturation and aging: relationship to the toxicity of chlorpyrifos and parathion in rats.

Chlorpyrifos (CPF) and parathion (PS), two common organophosphorus (OP) pesticides, exhibit higher acute toxicity in younger animals compared to adults. Maturational differences in detoxification via carboxylesterases (CEs) and A-esterases (AEs) have been suggested as contributors to the higher sensitivity of younger animals to OP toxicants. AEs (e.g., chlorpyrifos oxonase and paraoxonase) catalytically inactivate while CEs stoichiometrically eliminate OP anticholinesterases. While earlier studies have reported a relationship between the toxicity of some OP pesticides and the maturational profile of AEs and CEs, little information exists on the relative OP-toxicant sensitivity and detoxification capacities of aged animals. In the present study, we investigated the relationship between toxicity of CPF and PS and the activity of CEs and AEs in liver, plasma, and lung of neonatal (7 day), juvenile (21-day), adult (3-month), and aged (24-month) Sprague Dawley rats. CE sensitivity in vitro to chlopyrifos oxon and paraoxon was also evaluated across age groups. Neonatal and juvenile rats were more sensitive than adults to the acute lethality of both CPF and PS. Aged rats exhibited similar sensitivity to CPF but were markedly more sensitive than adults to PS. Levels of CEs and AEs in neonatal and juvenile rats were significantly lower than in adult tissues. Aged rats showed similar levels of AEs in all tissues and CEs in liver and lung, but plasma CE levels were significantly lower (50%) when compared to the adult rats. There were no significant age-related differences in in vitro sensitivity of CEs to either chlorpyrifos oxon or paraoxon in any tissues. In general, acute sensitivity (MTD) was highly correlated with age-related differences in both esterase activities across all 3 tissues with CPF, but only plasma carboxylesterase activity was highly correlated with sensitivity to parathion. The results suggest that both carboxylesterase and A-esterase activities can be correlated with acute sensitivity to CPF and PS, but that age-related differences in CE activity are probably more important in differential toxicity. Furthermore, plasma carboxylesterase activity may play a more pivotal role in the differential sensitivity to PS.

Avian plasma carboxylesterase activity as a potential biomarker of organophosphate pesticide exposure

AbstractPlasma cholinesterase (ChE) and carboxylesterase (CaE) activities were measured in pigeons (Columba hvia), captive and wild American kestrels (Falco sparveius), red‐tailed hawks (Buteo jamaicensis), Swainson's hawks (Buteo swainsoni), Cooper's hawks (Accipiter cooperit), and red‐shouldered hawks (Buteo lineatus). The effect of organophosphate (OP) insecticides on pigeon ChE and CaE activities was determined. Pigeon plasma CaE activity showed distinct groups averaging 12.4 and 138 nmol/min/ml plasma. The CaE activity for raptors was less than 20 nmol/min/ml plasma. Both ChE and CaE activities were inhibited by 0.4 mg/kg ethyl parathion, but CaE activity was not inhibited by 10 mg/kg methidathion. There was no difference in the blood ChE activities of pigeons with low or high blood CaE after treatment with parathion. In general, CaE activity was less severely depressed and recovery was less predictable than was ChE activity.

Differences in plasma carboxylesterase activity: relevance to anticholinesterase sensitivity

Differences in plasma carboxylesterase activity: relevance to anticholinesterase sensitivity

The effect of carboxylesterase inhibition on interspecies differences in soman toxicity

Subcutaneous administration of 2 mg/kg cresylbenzodioxaphosphorin oxide (CBDP) produced complete inhibition of carboxylesterase activity in plasma and lung of mice, rats, guinea pigs and rabbits, without inhibition of acetylcholinesterase activity in either brain or diaphragm. This CBDP treatment also reduced the subcutaneous soman LD 50 in these species by 48–90% in comparison to the soman LD 50 in control animals. The interspecies differences in the soman LD 50 values that were seen in control animals were absent in CBDP-treated animals. The soman LD 50 values in control animals were 125 μg/kg (mouse), 116 μg/kg (rat), 32.3 μg/kg (guinea pig) and 22.8 μg/kg (rabbit), whereas the soman LD 50 values in CBDP-treated animals from these species were clustered in a narrow dose range (11.8–15.6 μg/kg) and were not significantly different. This suggests that the amount of CBDP-sensitive carboxylesterase available for detoxification of soman in each species may be an important determinant of interspecies differences in soman toxicity.

Diethylsuccinate carboxylesterase activity in sheep poisoned by copper

Plasma diethylsuccinate carboxylesterase activity in plasma was elevated within 7 days in 4 sheep which received 4 mg Cu per kg body weight in gelatin capsules daily for 100 days followed by the same amount of Cu as a drench until the haemolytic crisis commenced. Plasma aspartate amino-transferase and sorbitol dehydrogenase activities in plasma were not elevated in these animals until sixty days after the Cu treatment began. Acivities of all 3 enzymes in the liver homogenates from copper-treated sheep were lower ( P < 0·01) than in control animals.

Biochemical and histochemical alterations following acute soman intoxication in the rat

Rats injected with a nonlethal acute dose (100 μg/kg, sc) of soman (pinacolyl methylphosphonofluoridate) exhibited signs of anticholinesterase toxicity beginning at 5–15 min with increasing severity and lasting for 4–6 hr. Generalized tremors and seizure activity indicated comparatively greater involvement of the central cholinergic system than peripheral neuromuscular effects. During peak toxicity, all the brain regions tested showed more than 95% inhibition of acetylcholinesterase (AChE) activity. The cortex area was maximally affected (99% inhibition). Among skeletal muscles, soleus AChE was most severely affected (94%) and extensor digitorum longus (EDL) the least (72%). Inhibition of EDL AChE occurred at a much slower rate than in brain and other muscles. Significant recovery of AChE activity was seen by 48–72 hr after soman treatment in both brain and skeletal muscles. By Day 7, recovery was virtually complete in skeletal muscles but not in brain, although significant recovery had occurred by this time. Muscle fiber necrosis developed within 6 hr in the soleus and diaphragm, while no necrotic fibers were found in the EDL. The 16 S AChE molecular form showed the fastest recovery of the AChE isozymes in all three muscles. Full recovery was seen after 7 days in soleus and was increased to greater than control activity in diaphragm and EDL. The inhibition pattern of butyrylcholinesterase (BuChE) activity was similar to that described for AChE activity, but the recovery was comparatively faster. Carboxylesterase activity in plasma was decreased to less than 10% of control within 1 hr and recovered to 53% of control within 24 hr. No significant inhibition was seen in hepatic carboxylesterase activity. It can be concluded that soman-induced acute toxicity is directly related to the rate and degree of AChE inhibition. A significant amount of soman binds to non-AChE enzymes with serine sites such as BuChE and carboxylesterases.

Effects of acute and chronic dieldrin administration on liver and plasma esterases of the rat

The administration of a single oral dose of dieldrin, 40 mg/kg, or the feeding of a diet containing 50 ppm of dieldrin for up to 8 weeks to male and female rats produced changes in plasma and liver esterases that were dependent on the type of esterase, the duration of exposure, and the sex of the animal. Two hours after oral administration of dieldrin, plasma carboxylesterase activity declined in males but not in females; hepatic microsomal carboxylesterase activity was also slightly lower in dieldrin-treated males, but not females. Forty-eight and 120 hr after dieldrin administration, hepatic microsomal carboxylesterase activity was elevated in rats of both sexes. After 8 weeks of dieldrin feeding (50 ppm), both plasma and microsomal carboxylesterase activities were significantly elevated in males and females. In most instances, elevations in the activities of hepatic microsomal esterases were due to dieldrin-induced increases in microsomal protein content and not to increased esterase specific activity. Electrophoresis of solubilized microsomal membranes from control and dieldrin-treated animals revealed no sex differences or major dieldrin-induced alterations on esterase isozyme patterns. Plasma cholinesterase activity was significantly reduced in females fed 50 ppm of dieldrin in the diet for 8 weeks. Since the level of activity of this enzyme is regulated in part by estrogen, depression of cholinesterase activity may result from an increased rate of steroid metabolism in dieldrin-treated female rats. Plasma cholinesterase activity of male rats was unaffected by either acute or chronic dieldrin administration.

Impairment of hepatic microsomal and plasma esterases of the rat by disulfiram and diethyldithiocarbamate

Twenty-four hr after oral administration (0.2 to 2.0 g/kg) of disulfiram (DS) to male rats. significant impairment of hepatic microsomal carboxylesterase and plasma carboxyl- and cholinesterase was observed. Plasma esterase activities returned to control values between 48 and 72 hr after a single oral dose of DS (2.0 g/kg), but microsomal carboxylesterase activity was still significantly lower in treated animals at both times. Daily administration of DS (0.1 or 0.4 g/kg) resulted in decreased microsomal carboxylesterase activity after 2 days. However, continued administration of DS for a total of 12 days did not produce further depression of microsomal esterase activity. Microsomal and plasma carboxylesterase activities were also decreased 24 hr after oral administration (1.0 to 2.0 g/kg) of sodium diethyldithiocarbamate (DDTC), the reduced metabolite of DS. Hepatic microsomal esterases that migrated rapidly toward the anode during polyacrylamide disc gel elcctrophoresis were the most sensitive to impairment by DS or DDTC. Esterase activity in the lung was also impaired after DS or DDTC administration, whereas esterases of the heart, kidney and testis were essentially unaffected. Incubation in vitro of liver microsomes with DS decreased microsomal carboxylesterase activity, while incubation with DDTC had little effect. Plasma carboxylesterase was inhibited in vitro to a greater extent than microsomal esterases by both DS and DDTC. Diethylamine and CS 2, the decomposition products of DDTC, were essentiallv inactive as esterase inhibitors in vitro.

A simplified bioassay for organophosphate detoxification and interactions

In the simplified bioassay 0.5 nmol malaoxon or 0.15 nmol paraoxon was incubated with mouse brain with or without addition of a second, detoxifying tissue. After 25 min acetylcholine was added to the flasks and the brain cholinesterase activities remaining were determined manometrically. The difference in brain cholinesterase activities was a measure of the organophosphate detoxifying capacity of the second tissue, and was proportional to the quantity of detoxifying tissue added. Malaoxon detoxification was greatest in mouse liver, but lung, kidney and plasma also possessed considerable activity. Hepatic subcellular fractionation studies demonstrated that malaoxon detoxification was greatest in the microsomes. Administration of TOTP (triorthotolyl phosphate) and other carboxylesterase inhibitors also inhibited malaoxon detoxification in the simplified bioassay. EDTA, an inhibitor of paraoxonase but not of paraoxon binding did not inhibit paraoxon detoxification in the simplified bioassay. TOTP, an inhibitor of paraoxon binding but not of paraoxonase, inhibited paraoxon detoxification in the simplified bioassay. These results suggest that the simplified bioassay measures paraoxon binding rather than hydrolysis by paraoxonase. TOTP (125 mg/kg, 18 hr) pretreatment potentiated the anticholinesterase action of paraoxon in vivo. A single pretreatment with aldrin (16 mg/kg, po, 4 days earlier) increased the liver: body weight ratio in the mouse to 130% of control, and increased liver and plasma paraoxon and malaoxon detoxification and liver and plasma carboxylesterase activities. A corresponding decrease was observed in the acute toxicity of paraoxon and malaoxon administered 4 days after aldrin. The simplified bioassay for determining detoxification of malaoxon or paraoxon in vitro may be useful in screening tests for detecting other synergists and antagonists of organophosphate toxicity.