Carbofuran Metabolism and Toxicity in the Rat

Carbofuran Metabolism and Toxicity in the Rat. FERGUSON, P.W., DEY, M. S., JEWELL, S. A., AND KRIEGER, R. I. (1984). Fundam.Appl. Toxicol. 4, 14–21. The influence of carbofuran metabolismon acetylcholinesterase inhibition has been defined after lowdose (50 µg/kg, iv and oral) [carbonyl-¹⁴C]carbofuranexposures to male Sprague–Dawley Rats. Red blood cellacetylcholinesterase (RBC AchE) inhibition (83% at 2 min, 37%at 15 min for iv and oral, respectively, with recovery by 3hr), was correlated with carbofuran plasma concentrations (r= 0.97). Eight-hour sample collection indicated that ultimatecarbofuran fate (41–47% ^l4CO₂, 14–15% urine, <1%feces, and 30–31% carcass) was independent of exposureroute. Carbofuran absorption (peak plasma levels < 7 min),distribution, and elimination (t? = 29 ? 5 min) occurred rapidly.3-Hydroxycarbofuran, a significant oxidative metabolite of carbofuranwith anticholinesterase activity, was rapidly formed and subjectto enterohepatic circulation (plasma t? = 64 ? 5 min). Resultsindicated that rapid RBC AchE recovery closely paralleled carbofuranmetabolism and the primary in vivo disposition of 3-hydroxycarbofuranwas metabolic conjugation.     

Comparison of Cholinergic and Neuromuscular Toxicity following Acute Exposure to Sarin and VX in Rat

Comparison of Cholinergic and Neuromuscular Toxicity followingAcute Exposure to Sarin and VX in Rat. GUPTA, R. C, PATTERSON,G. T., AND DETTBARN, W-D. (1991). Fundam. Appl. Toxicol. 16,449–458. Male Sprague-Dawley rats injected with a sublethalsc dosage of 110 µg/ kg of sarin (isopropyl methylphosphonofluoridate),or 12 µg/kg of VX (S-{2-diisopropylaminoethyl) O-ethylmethylphosphonothioate), developed severe toxic signs within5–15 min after sarin and 20–50 min after VX lastingfor 5 to 7 hr. Myonecrotic lesions were seen in soleus and diaphragmmuscles within 1 hr. A maximum number of lesions had developedafter 24 hr, and lesions were also present in extensor digitorumlongus (EDL) at this time. Regeneration of muscle fibers wasslow since lesions were still evident past 7 days of treatment.Within 1 hr following VX, AChE activity was reduced to 8, 12,and 17% of control activity in soleus, diaphragm, and EDL, respectively,whereas with sarin the enzyme activity was reduced to 23, 48,and 82% of control. A still greater inhibition was seen 24 hrafter sarin when AChE activity was reduced to 19, 13, and 43%in these muscles. In skeletal muscles the different molecularforms of AChE, such as 16 S, 12 S, 10 S, and 4 S vary in locationand functional importance with the 16 S form highly concentratedat the neuromuscular junction. All forms in a given muscle wereequally sensitive to the inhibitors. In EDL, sarin was the leasteffective in reducing AChE or its molecular forms. In the brainstructures (cortex, brain stem, striatum, and hippocampus),AChE activity was reduced to 1–6% of control by sarinand VX with the exception that following VX striatal AChE wasreduced to only 41% of control activity. AChE activity in thebrain cortex following either of the agents was maximally affected(1%). A slow but significant recovery of brain AChE was evidentafter 24 hr and more so after Day 7. Butyrylcholinesterase (BuChE)activity was less sensitive to inhibition by both inhibitorscompared to AChE activity and showed a rapid recovery. Basedon the equitoxic doses (toxic signs of similar magnitude), VXwas found to be 10 times more toxic than sarin. The mechanismsof this disparity may be due to differences in rate of uptake,circulation, susceptibility to hydrolysis, and reactivity withnonspecific binding sites.     

Erythrocyte and Plasma Cholinesterase Activity in Male and Female Rhesus Monkeys before and after Exposure to Sarin

The rhesus monkey (Macaca mulatta) has a menstrual cycle similarto the human. Differences in hormone levels have been demonstratedbetween the sexes and in females during the menstrual cyclebut these differences in terms of organophosphorus toxicityhave not been explored. Plasma cholinesterase (ChE/BuChE) anderythrocyte (RBC) acetylcholinesterase (AChE) activity weremeasured before and after exposure to the organophosphorus compoundsarin (11 µg/kg, iv; 0.75 LD50) in six male and six femalerhesus monkeys. After baseline measurements were obtained, sarinwas administered to atropinized monkeys to determine in vivodifferences between the sexes in their response to sarin. Withthe baseline values, the intraanimal and intragroup BuChE/AChEvariations were found to be minimal. Following sarin intoxicationand 2-PAM treatment no significant differences were seen betweenthe sexes in the rate of reactivation of BuChE or AChE by 2-PAM.The rate of aging of sarin phosphonylated RBC AChE between thesexes was also similar. De novo regeneration of RBC AChE andplasma BuChE after sarin intoxication was different betweenthe male and female monkeys. The female plasma BuChE recoveryrate was 48% slower than the male recovery rate, while the early(first 63 days) RBC AChE recovery rate was 24.5% faster in thefemales. In conclusion, there probably are not any clinicallysignificant differences between male and female rhesus monkeysacutely intoxicated with sarin. However, on subsequent exposureclinical differences may be observed due to substantial differencesin the rate of de novo synthesis of both plasma BuChE and RBCAChE.     

Subchronic Oral and Intravenous (iv) Safety Evaluation and Pharmacokinetics in Rats and Dogs of Ipazilide Fumarate (Win 54, 177-4), an Antiarrhythmic Agent

Ipazilide fumarate (Win 54,177-4) is a chemically novel antiarrhythmicagent that prolongs ventricular refractoriness and possessesantiectopic activity. Subchronic (29 days) nonclinical safetyevaluation of ipazilide was conducted following oral and ivadministration in Sprague-Dawley rats (20–320 mg/kg oraland 1.25–10 mg/kg iv) and 14 and 28 days in beagle dogs(3–30 mg/kg oral and 2.5–20 mg/kg iv). The pharmacokineticparameters of ipazilide indicate that ipazilide is absorbed(t_max 1 hr) in fasted rats and dogs following single and repeatedoral administrations. The apparent elimination half-life inthe two species is approximately 1 hr (except in rats at a dosageof 320 mg/kg), suggesting rapid clearance. Increases in liverweights (rats 320 mg/kg) accompanied by the observation of centrilobularhypertrophy of hepatocytes were considered an expression ofan adaptive metabolic response of the liver to ipazilide andmay be associated with the induction of microsomal enzymes.Duodenal villous atrophy and epithelial hyperplasia (rats, 80and 320 mg/kg) were interpreted to represent an irritant responseto the drug. Local irritation was also observed at the injectionsite in rats and dogs. Dogs tolerated the oral and the iv administrationof ipazilide at dosages of up to 30 and 20 mg/kg, respectively.Despite emesis (oral dogs), which was reduced in frequency followingrepeated treatment over several weeks, plasma levels in treateddogs (i.e., C_max 4–5 µg/ml) were approximately twicethat required to convert spontaneous arrhythmias in the conscious dog model 24 hr after myocardial infarction. Moreover,plasma levels (C_max 6–7 µg/ml) of iv-treated dogswere approximately three times higher than the efficacious levelsin the dog model and did not cause adverse effects except emesis.Electrocardiographic changes (i.e., increased P wave and QRSdurations, and T wave alterations) in dogs were transient andrepresented an extension of the pharmacological effects of ipazilide.In summary, since ipazilide, at multiple therapeutic dosages,was well tolerated in rats and dogs, it may be considered anappropriate drug for clinical evaluation of safety and efficacyin humans as a potential antiarrhythmic agent. The safety profileof ipazilide in clinical trials is currently ongoing.     

Investigations of Amitraz Neurotoxicity in Rats: Effects on Motor Activity and Inhibition of Monoamine Oxidase

Investigations of Amitraz Neurotoxicity in Rats. III Effectson Motor Activity and Inhibition of Monoamine Oxidase. MOSER,V. C., AND MACPHAIL, R. C. (1989). Fundam. Appl. Toxicol 12,12–22. The formamidine pesticide amitraz (AMZ) producesmany behavioral and physiological changes in rats. We examinedthe dose effect and time course of AMZ on motor activity, monoamineoxidase (MAO) activity, and acetylcholinesterase (AChE) activityto evaluate possible neurochemical mechanisms for the behavioraleffects of AMZ. For motor activity studies, male Long-Evanshooded rats were tested in photocell activity measurement devices.AMZ produced dose-related decreases in motor activity of ratsallowed free access to food and rats maintained at a stablebody weight through food restriction. Lowest effective dosesof AMZ tested were 1–3 mg/kg, administered 20 min beforetesting. AMZ appeared to be about three times more potent infood-restricted rats, indicating that amount of body fat mayplay a significant role in the pharmacokinetics of AMZ. Motoractivity returned to control levels over 4–5 days afterdosing with 100–200 mg/kg AMZ, whereas recovery was evidentthe day after admin istration of low doses (1–30 mg#x002F;kg).Inhibition of MAO was measured in whole brain of rats sacrificedat various times after dosing with AMZ. Only > l00 mg/kgAMZ inhibited MAO, which was measurable within 2 hr of dosingand lasted up to 7 days. AMZ appeared to be more selective fortype B MAO when given in vivo, although MAO-A was also inhibitedat doses 300 mg/kg. However, no selectivity was indicated bythe IC50 values determined in vitro (IC50=31 and 28 µMfor MAO-A and MAO-B, respectively). AMZ produced only negligibleinhibition of AChE at the highest doses administered in vivoor at 10 mM in vitro. These data indicate that while AMZ doesinhibit MAO, the dose range over which it produces this actionis much higher than that which suppressed motor activity. ThusMAO inhibition is probably not responsible for AMZ-induced alterationsin motor activity.     

A physiologically based pharmacokinetic/pharmacodynamic model for carbofuran in Sprague-Dawley rats using the exposure-related dose estimating model.

Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl-N-methylcarbamate), a broad spectrum N-methyl carbamate insecticide, and its metabolite, 3-hydroxycarbofuran, exert their toxicity by reversibly inhibiting acetylcholinesterase (AChE). To characterize AChE inhibition from carbofuran exposure, a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model was developed in the Exposure-Related Dose Estimating Model (ERDEM) platform for the Sprague-Dawley (SD) rat. Experimental estimates of physiological, biochemical, and physicochemical model parameters were obtained or based on data from the open literature. The PBPK/PD model structure included carbofuran metabolism in the liver to 16 known metabolites, enterohepatic circulation of glucuronic acid conjugates, and excretion in urine and feces. Bolus doses by ingestion of 50 microg/kg and 0.5 mg/kg carbofuran were simulated for the blood and brain AChE activity. The carbofuran ERDEM simulated a half-life of 5.2 h for urinary clearance, and the experimental AChE activity data were reproduced for the blood and brain. Thirty model parameters were found influential to the model outputs and were chosen for perturbation in Monte Carlo simulations to evaluate the impact of their variability on the model predictions. Results of the simulation runs indicated that the minimum AChE activity in the blood ranged from 29.3 to 79.0% (as 5th and 95th percentiles) of the control level with a mean of 55.9% (standard deviation = 15.1%) compared to an experimental value of 63%. The constructed PBPK/PD model for carbofuran in the SD rat provides a foundation for extrapolating to a human model that can be used for future risk assessment.     

Concerted role of carboxylesterases in the potentiation of carbofuran toxicity by iso-OMPA pretreatment

Pretreatment of rats with the nonspecific esterase inhibitor iso-OMPA (1 mg/kg sc) 1 h prior to carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbamate, 0.5 mg/kg sc) administration potentiated carbofuran toxicity by more than threefold. Neither iso-OMPA nor carbofuran in the given doses produced any gross toxic signs. Rats receiving combined treatment, however, showed severe hypercholinergic signs (salivation, tremors, muscle fasciculations, and convulsions) within 5-10 min following carbofuran administration, and the severity was comparatively greater than that observed with an acute dose of carbofuran (1.5 mg/kg sc). Rats pretreated with iso-OMPA (0.5 mg/kg) died within 10-15 min following the acute dose of carbofuran (1.5 mg/kg). Each drug when given alone (1.0 mg/kg iso-OMPA, 0.5 mg/kg carbofuran) caused a significant (p less than .01) inhibition of carboxylesterase (CarbE) activity in brain structures (cortex, stem, striatum, and hippocampus), skeletal muscle (hemidiaphragm), liver, and plasma, whereas acetylcholinesterase (AChE) activity remained significantly (p greater than .01) unchanged. The maximal CarbE inactivation in plasma (less than 14% remaining activity) following either drug indicated a tremendous nonspecific binding to non-AChE serine-containing enzymes. iso-OMPA pretreatment markedly potentiated carbofuran's anticholinesterase activity both in neuronal and in nonneuronal tissues. It can be concluded that iso-OMPA pretreatment potentiates carbofuran toxicity either by preventing nonspecific binding of carbofuran to CarbE and/or possibly by inhibiting its detoxification.     

Survival and behaviour of captive Mallard broods exposed to carbofuran-sprayed vegetation: a field experiment

A field study was undertaken to determine the effects of exposure to vegetation sprayed with carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranylmethyl carbamate) on the survival and behavior of Mallard (Anas platyrhynchos) ducklings. Pairs of captive hens with newly hatched broods of five to eight ducklings were released into sprayed or control 200 m long walkways leading directly to 0.1 ha pond enclosures. Carbofuran was applied at 132 and 264 g a.i. ha^–1 during five and four replicate trials, respectively. Broods were observed during the walk, and activity was recorded systematically during four 2-h periods over the following two days. Dead or abandoned ducklings were retrieved and brain acetylcholinesterase (AChE) activity was determined. Carbofuran did not affect the rate of duckling loss on the day of exposure at either spray rate compared with that of controls (3/34 versus 3/38 ducklings and 3/31 versus 0/31 ducklings, respectively). Nevertheless, one and three of the abandoned or dead ducklings in the low and high spray-rate trials, respectively, exhibited depression of brain AChE activity associated with mortality caused by anticholinesterase compounds (16 to 47% of mean control activity). Although no significant differences were detected in duckling behaviour between treated and control broods at either exposure level, treated broods tended to spend a larger proportion of time hidden in emergent vegetation. The ingestion of carbofuran by ducklings walking through sprayed vegetation appears to be the critical mode of intake, with dermal absorption being minimal.     

Phosphotriesterase--A Promising Candidate for Use in Detoxification of Organophosphates

The effect of phosphotriesterase (PTE) on cholinesterase (ChE)activities was studied with exposures to different organophosphatesin mice. Paraoxon (PO) (1.0 mg/kg, ip) almost totally inhibitedserum ChE activity. This activity, however, recovered to thenormal level within 24 hr. The PTE pretreatment (16.8 U/animal,2.5 µg/10 g body wt, iv 10 min before the organophosphate)accelerated this reactivation. The same phenomenon was alsoseen in vitro. In vitro with human serum, there was only minimalreactivation of the inhibited ChE. PTE, however, reactivatedit significantly. The PTE-pretreated mice (168 U/animal, 30µg/10 g body wt, iv) tolerated even 50 mg/kg of PO withoutshowing any remarkable signs of intoxication. In PTE-untreatedanimals, however, PO doses as low as 1.0 and 1.5 mg/kg causedsevere signs of poisoning. PTE (16.8 U/animal, 4 µg/10g body wt, iv) reduced the inhibition of brain and serum ChEactivities after PO and diisopropyl fluorophosphate exposure.In sarin and soman intoxications, PTE decreased only slightlythe inhibition of ChE activities. The results indicate thatPTE pretreatment given iv prevents the inhibition of ChE activitiesafter certain organophosphates and it also hastens the recoveryof activities after PO poisoning.     

Inhibition of Hen Brain Acetylcholinesterase and Neurotoxic Esterase by Chlorpyrifos in Vivo and Kinetics of Inhibition by Chlorpyrifos Oxon in Vitro: Application to Assessment of Neuropathic Risk

Chlorpyrifos (CPS; O,O-diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate;Dursban) is a widely used broad-spectrum organophosphorus (OP)insecticide. Because some OP compounds can cause a sensory-motordistal axonopathy called OP compound-induced delayed neurotoxicity(OPIDN), CPS has been evaluated for this paralytic effect. Earlystudies of the neurotoxicity of CPS in young and adult hensreported reversible leg weakness but failed to detect OPIDN.More recently, a human case of mild OPIDN was reported to resultfrom ingestion of a massive dose (about 300 mg/kg) in a suicideattempt. Subsequent experiments in adult hens (the currentlyaccepted animal model of choice for studies of OPIDN) showedthat doses of CPS in excess of the LD₅₀ in atropine-treatedanimals inhibited brain neurotoxic esterase (NTE) and producedmild to moderate ataxia. Considering the extensive use of CPSand its demonstrated potential for causing OPIDN at supralethaldoses, additional data are needed to enable quantitative estimatesto be made of the neuropathic risk of this compound. Previouswork has shown that the ability of OP insecticides to causeacute cholinergic toxicity versus OPIDN can be predicted fromtheir relative tendency to inhibit the intended target, acetylcholinesterase(AChE), versus the putative neuropathic target, NTE, in braintissue. The present study was designed to clarify the magnitudeof neuropathic risk associated with CPS exposures by measuringhen brain AChE and NTE inhibition following dosing in vivo anddetermining the bimolecular rate constant of inhibition (k₁)for each enzyme by the active metabolite, CPS oxon (CPO), invitro. CPS administered to atropine-treated adult hens at 0,75, 150, and 300 mg/kg po in corn oil produced mean values forbrain AChE inhibition 4 days after dosing of 0, 58, 75, and86%, respectively, and mean values for brain NTE inhibitionof 0, 21, 40, and 77%, respectively. Only the high dose (sixtimes the unprotected LD₅₀ in hens) produced NTE inhibitionabove the presumed threshold of 70%, and these animals werein extremis from cholinergic toxicity at the time of euthanizationdespite continual treatment with atropine. When 150 mg/kg CPSpo in corn oil was given to atropine-treated hens on Day 0,inhibition on Days 1, 2,4, 8, and 16 for brain AChE was 86,82, 72, 44, and 29%, respectively, and for brain NTE was 30,28, 38, 29, and 6%, respectively. No signs of OPIDN were observedin any of the animals during the 16-day study period. Kineticstudies of the inhibition of hen brain AChE and NTE by CPO invitro demonstrated that CPO exhibits high potency and extraordinaryselectivity for its intended target, AChE. The k₁, values were15.5 µM^–1 min^–1 for AChE and 0.145 µM^–1min^–1 for NTE. The calculated fixed-time (20-min) I₅₀values were 2.24 nM for AChE and 239 nM for NTE, yielding anI₅₀ ratio for NTE/AChE of 107. These results may be comparedwith data compiled for other OP compounds with respect to NTE/AChEI₅₀ ratios and the corresponding doses required to produce OPIDNrelative to the LD₅₀. In general, NTE/AChE I₅₀ ratios greaterthan 1 indicate that the dose required to produce OPIDN is greaterthan the LD₅₀. Taken together, the results of this study indicatethat acute exposures to CPS would not be expected to cause OPIDNexcept under extreme conditions such as attempted suicides involvingmedically assisted survival of doses considerably in excessof the LD₅₀.     

Pharmacokinetic Fate of14C-Labeled Deoxynivalenol in Swine

Pharmacokinetic Fate of ¹⁴C-Labeled Deoxynivalenol in Swine.PRELUSKY, D. B., HARTIN, K. E., TRENHOLM, H. L., AND MILLER,J. D. (1988). Fundam. Appl Toxicol. 10,276-286. The pharmacokineticsof the trichothecene mycotoxin deoxynivalenol (DON) was investigatedin swine following intravenous (0.30 mg, 0.35 µCi/kg)and intragastric (0.60 mg, 0.60 µCi/kg) administrationof the ¹⁴C-labeled toxin. After iv dosing, plasma concentrationdata favored a three-compartment open model with half-life valuesfor the rapid distribution (), slower distribution (ß),and terminal elimination () phases of 5.8, 96.7, and 510.0 min,respectively. The apparent volume of distribution (V') was 1.34liter/kg, the volume of the central compartment (K_c) was 0.166liter/kg, and the plasma clearance was 1.81 ml/min/kg. DON wasrapidly cleared essentially unchanged (>95%), and was excretedprimarily in unne (86–104%), with minor elimination inbile (3–5%). Following intragastric dosing DON was veryrapidly absorbed, reaching near peak plasma levels within 15–30min. Levels remained elevated (63-325 ng/ml) for approximately9 hr, and began declining slowly (t ß= 7.1 hr) thereafter.The calculated systemic bioavailability (F) was between 48 and65%, although urinary and biliary recoveries indicated marginallygreater absorption actually occurred (54–85%). Overall,although DON was eliminated rapidly and completely within 24hr following a single iv or intragastric dose, data suggestthat residues may undergo temporary sequestration in a tissuedepot.     

An in Vitro Comparison of Rat and Chicken Brain Neurotoxic Esterase

An in Vitro Comparison of Rat and Chicken Brain Neurotoxic Esterase.NOVAK, R., AND PADLLA, S. (1986). Fundam. Appl. Toxicol. 6,464–471. A systematic comparison was undertaken to characterizeneurotoxic esterase (NTE) from rat and chicken brain in termsof inhibitor sensitivities, pH optima, and molecular weights.Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterasesshowed that rat esterases were more sensitive than chicken toparaoxon inhibition at concentrations <1 µM and superimposablewith chicken esterases at concentrations of 2.5–1000 µM.Mipafox titration of the paraoxon-resistant esterases at a fixedparaoxon concentration of 100 µM (mipafox concentration:0-1000 µM) resulted in a mipafox 150 of 7.3 µM forchicken brain NTE and 11.6 µM for rat brain NTE. NTE (i.e.,paraoxon-resistant, mipafox-sensitive esterase activity) comprised80% of chicken and 60% of rat brain paraoxon-resistant activitywith the specific activity of chicken brain NTE approximatelytwice that of rat brain NTE. The pH maxima for NTE from bothspecies was similar showing broad, slightly alkaline optimafrom pH 7.9 to 8.6. [³H]Diisopropyl phosphorofluoridate (DFP)-labeledNTE from the brains of both species had an apparent mol wt of160,000 measured by sodium dodecyl sulfate polyacrylamide gelelectrophoresis. In conclusion, NTE from both species was verysimilar, with the mipafox 150 for rat NTE within the range ofreported values for chicken and human NTE, and the inhibitorparameters of the chicken NTE assay were applicable for therat NTE assay.     

Effects of the Organophosphorus Compound, O-ethyl-N-dimethyl phosphoramidocyanidate (Tabun), on Flavor Aversions, Locomotor Activity, and Rotarod Performance in Rats

Effects of the Organophosphorus Compound, O-ethyl-N-dimethyl-phosphoramidocyanidate(Tabun), on Flavor Aversions, Locomotor Activity, and RotarodPerformance in Rats. ROMANO, J. A., JR., AND LANDAUER M. R.(1986). Fundam. Appl. Toxicol. 6,62–68. An assessmentof the behavioral effects of acutely administered anti-cholinesterasecompound O-ethyl-N-dimethyl phosphoramidocyanidate (tabun),to male rats was performed in three studies across five doselevels (100–198 µg/kg, sc). Doses of 122 µg/kgor greater produced conditioned flavor aversions. Spontaneouslocomotor activity was significantly decreased at doses of 122µg/kg or greater when compared to vehicle control levels.When administered doses of 168 µg/kg or greater, ratsexhibited significant decrements in rotarod performance. Inaddition, the LD50 of tabun was determined to be 240 µg/kg.Signs of cholinergic intoxication were not apparent until 144µg/kg tabun or more were given. Behavioral effects werethus obtained at doses that were between 54 and 71% of the LD50.     

Acute Cardiotoxicity of the Anti-HIV Dideoxynucleoside, F-ddA, in the Rat

2'-ß-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable,pu-rine dideoxynucleoside with in vitro anti-HIV activity, hasbeen selected by the NCI as a clinical trial candidate. A recentreport that high, single doses of F-ddA produce cardiotoxicityin rats prompted the present investigation whose objective wasto quantitate this effect and establish a relationship betweenthis toxicity and F-ddA plasma concentrations. Microscopic examinationof cardiac tissues for degenerative lesions established theeffects of F-ddA and ddA on three iv schedules [daily x 1 (2.5–250mg/kg); daily x 5 (125, 250 mg/kg), and BID x 1 (250 mg/kg)]as well as one oral schedule [BID x 1 (500 mg/kg)] using 8-to 12-week-old female Sprague-Dawley rats. For both F-ddA andddA, the group mean severity of the cardiac lesions was dose-dependentand proportional to the measured plasma concentrations of theundeaminated parent drugs. F-ddl and ddI, the respective deaminatedcatabolites of F-ddA and ddA, were essentially nontoxic in thisstudy (iv, 250 mg/kg, daily x 1 and daily x 5), since plasmaconcentrations exceeding 2 mM produced only minimal cardiaclesions. The cardiomyopathy of F-ddA was minimal to mild forall iv doses except 250 mg/kg (daily x 1) and usually was greaterthan that of ddA at any given dose. This is a consequence ofthe fact that F-ddA is deaminated 20 times more slowly thanddA, resulting in higher plasma concentrations of F-ddA relativeto ddA at any given time for any given dose. Neither F-ddA norddA was more cardiotoxic on a repeated iv schedule (daily x5) than when administered only once, suggesting that rat cardiotoxicityis related to C_max, rather than total exposure. In this mostsensitive species, the formation of cardiac lesions above thebackground level is associated with iv F-ddA administrationwhen the F-ddA plasma concentration approaches 300 µM,30–50 times the anticipated therapeutic level in humans.     

A Novel Tertiary Pyridostigmine Derivative [3-N,N-Dimethylcarbamyloxy)-1-Methyl-{Delta}3-Tetrahydropyridine]: Anticholinesterase Properties and Efficacy against Soman

In an effort to develop an effective centrally acting pretreatmentcompound against organophosphorus poisons, the tertiary pyridostigmine(Pyr) derivative 3-(N,N-dimethyI-carbamyloxy)-l-methyl-³-tetrahydropyridine(THP) was synthesized and studied for its anticholinesteraseproperties, as well as its efficacy against soman intoxicationin guinea pigs. Injection of THP (262 µg/kg, im) intoadult male guinea pigs caused inhibition of Wood (30%) and brain(25%) acetylcholinesterase (AChE), showing that THP penetratesthe blood-brain barrier. Pyr (131 µg/kg, im) caused AChEinhibition in the Hood (59%), but not in the brain. The inhibitorypotencies of THP and Pyr were compared by determining theirIC50 values for in vitroinhibition of both AChE (brain, erythrocyte)and pseudo-cholinesterase (plasma) in three mammalian species(guinea pig, rat, rabbit). THP, although effective in inhibitingboth types of cholinesterase, was in general less potent thanPyr. Pretreatment of guinea pigs with THP (262 µg/kg,im) plus Pyr (131 µg/kg, im). 30 min prior to subcutaneoussoman challenge, with no antimuscarink or oxime treatment, protected60% of the animals against 2 ? LD50 of soman. Neither THP norPyr alone was effective. The protective pretreatment regimendid not prevent convulsions, but shortened the recovery timein surviving animals (median recovery time 1.6 hr, comparedto 24 hr in control and other groups of animals pretreated withTHP or Pyr alone). A combination of THP and Pyr thus appearsto provide a means of evaluating the relative importance ofselective peripheral plus central vs peripheral AChE protectionagainst soman.     

Vitamin A Potentiation of Vinylidene Chloride Hepatotoxicity in Rats and Precision-Cut Rat Liver Slices

Pretreatment of large doses of vitamin A (VA) is known to potentiatethe hepatotoxicity of carbon tetrachloride. Therefore the effectsof 1-day VA pretreatment on VDC hepatotoxicity was examinedboth in vivo and in an in vitro system of precision-cut ratliver slices. Male Sprague-Dawley rats were pretreated with250,000 IU/kg VA by oral gavage. After 24 hr rats were administered50, 100, or 200 mg/kg VDC ip. Precision-cut liver slices wereprepared from VA pretreated rats 24 hr later and the liver sliceswere exposed for 2–8 hr to 0.025–1.0 µl VDCevaporated into the gas phase of the incubation vials. VA pretreatmentresulted in an enhancement of VDC toxicity, both in vivo andin vitro. There was a dose-dependent increase in plasma ALT24 hr after VDC treatment of rats and an increase in K⁺ leakagefrom liver slices after VDC exposure. Histological analysisof the liver or the liver slices revealed that VA+VDC treatmentresulted in centrilobular necrosis of the liver. When GdCl₃(10 mg/kg iv) was administered just before VA pretreatment ofrats, VDC toxicity was partially reversed as observed by a decreasein ALT in vivo and a decrease in the loss of K⁺ in vitro. Theseresults indicated that Kupffer cells, the resident macrophagesof the liver, were partially responsible for the VA-potentiatedVDC hepatotoxicity. One-day pretreatment of VA induced cytochromeP450IIE1 protein content as well as its enzymatic activity asmeasured by p-nitrophenol hydroxylation. Because VDC is bioactivatedby cytochrome P450IIE1, the increase in VDC hepatotoxicity afterVA may be due to an increased bioactivation of VDC in the liverand in precision-cut liver slices. Thus, more than one mechanismmay be involved in the VA enhancement of VDC hepatotoxicity.     

Inhibition of Human Serum Complement Activity by Diisopropylfluorophosphate and Selected Anticholinesterase Insecticides

Inhibition of Human Serum Complement Activity by Diisopropylfluorophosphateand Selected Anticholinesterase Insecticides. CASALE, G. P.,BAVARI, S., AND CONNOLLY, J. J. (1989). Fundam. Appl. Toxicol.12, 460–468. Activation of the human complement (C') system,a major line of defense against infections, requires the participationof serine esterases. Since the widely used anticholinesteraseinsecticides inhibit serine esterases, the present study evaluatedpotencies of carbaryl, carbofuran, dichlorvos, and paraoxonto inhibit C' activities of a panel of normal human sera. C'-mediatedlysis of sheep red cells was measured with a modified assay(1) incorporating suboptimal concentrations of sensitizing antibodyand (2) exhibiting increased sensitivity to serine esteraseinhibitors. Test chemicals were added to diluted sera 2 hr priorto incorporation into C' reaction mixtures. Potencies to inhibitC' and serum cholinesterase (CHE) were compared to potenciesof diisopropylfluorophosphate (DFP), a potent serine esteraseinhibitor and a standard probe for C' esterases. At 0.5 to 3.0mM carbaryl, carbofuran, dichlorvos, and DFP produced a dose-dependentinhibition of lysis, whereas paraoxon was not inhibitory. Ona molar basis, carbalyl was three times more potent than DFP,and inhibited lysis 15–25 and 26–45% at 1.0 and3.0 mM respectively. Carbofuran, dichlorvos, and DFP were equipotent.Mean IC50's for inhibition of CHE (a marker for occupationalexposure to organophosphates and carbamates)by DFP, paraoxon,dichlorvos, carbofuran, and carbaryl were 1.0?l0^–8 4.1?l0^–8,1.0?l0^–7 3.3?l0^–6and 1.8?l0^–5M, respectively.Potencies of the insecticides to inhibit CHE did not predictabsolute or relative potencies to inhibit serum C' activity.     

Kinetics of Respiratory Tract Absorption and Plasma Clearance of Horseradish Peroxidase in Guinea Pigs

Kinetics of Respiratory Tract Absorption and Plasma Clearanceof Horseradish Peroxidase in Guinea Pigs. CONNER, M. W., CHAUDHURI,I., ROGERS, A. E., and AMDUR, M. O. (1985). Fundam. Appl. Toxicol.5, 99–104. Horseradish peroxidase (HRP) absorption acrossthe wall of the upper airway, monitored by the amount detectedin the blood, is used to measure epithelial damage by toxins.Full details of kinetics of absorption and blood clearance havenot been reported previously. We measured the kinetics underthe experimental conditions used in testing toxins. HRP wasadministered to guinea pigs either by intraarterial injectionof a 7.5 µg bolus (plasma clearance) or by intratrachealinstillation of 1 mg (respiratory tract absorption). Plasmaconcentrations were monitored for 60 min. Plasma concentrationsof HRP rose linearly with time after intratracheal instillation,reaching 236 ± 51 ng/ml (mean ± SE) at 60 minafter instillation. HRP was cleared from the plasma rapidlyafter bolus injection. The elimination coefficient, k2, determinedfrom the biphasic log normal plot, was 0.322 min^–1. Thesedata were used to estimate the kinetics of absorption acrossthe respiratory epithelium. A single 3-hr exposure to an atmospherecontaining 2.5 mg/m³ of submicrometer zinc oxide particles increasedplasma concentration of HRP after intratracheal deposition (407± 63 ng/ml at 60 min) and had no effect on plasma clearance(k² = 0.342 min^–1). Therefore plasma concentrations ofHRP measured after intratracheal deposition can be used as asensitive indicator to evaluate the effects of inhalation ofa test atmosphere on epithelial permeability, if plasma clearancekinetics are not altered by the exposure.     

An Assessment of Comparative Acute Toxicity of Diisopropylfluorophosphate, Tabun, Sarin, and Soman in Relation to Cholinergic and GABAergic Enzyme Activities in Rats

An Assessment of Comparative Acute Toxicity of Diisopropylfluorophosphate,Tabun, Sarin, and Soman in Relation to Cholinergic and GABAergicEnzyme Activities in Rats. SIVAM, S. P., HOSKJNS, B., AND HO,I. K. (1984). Fundam. Appl. Toxicol. 4, 531–538. The scLD50s (µmol/kg) in rats for diisopropylfluorophosphate(DFP), Tabun, Sarin, and Soman were 14.5, 1.9, 1.4, and 0.88,respectively. The relative potency was as follows: DFP <Tabun < Sarin < Soman (1:7.6:10.4:16.4). The relativepotencies correlated with the in Vitro acetylcholinesterase(AChE) inhibition (in terms of the IC50) by these compounds,in whole brain homogenates or the purified bovine erythrocyteAChE. There was a dose versus time for mortality relationshipfor all four compounds; the average time for death decreasedwith increase in dose. However, there was no correlation betweentime for death and the extent of AChE inhibition. The striatalas well as other regional (medulla, diencephalon, cortex, andcerebellum) AChE activity was inhibited over 90% of the control,by the lethal doses of these compounds. None of the lethal orsublethal doses had any apparent effect on choline acetyhransferase(CAT) or GABA-trans-aminase activities. Glutamic acid decarboxylaseactivity was increased by Soman, Sarin, and Tabun at certainlethal doses but was not affected by DFP even at the lethaldose. The results indicate that (a) the acute toxicity of organophosphateacetylcholinesterase inhibitors is directly related to the inhibitionof AChE though there is a wide difference in their potency,(b) a substantial inhibition of AChE activity (over 90% of control)is necessary for lethality to ensue after an acute exposureand the margins between lethal and nonlethal doses are extremelysmall; and (c) qualitative differences seem to exist among thevarious organophosphates in affecting noncholinergic neurotransmitterenzymes.     

Percutaneous Absorption and Excretion of Xenobiotics after Topical and Intravenous Administration to Pigs

Interspecies comparisons suggest that the weaning pig is a suitablesurrogate for man in percutaneous absorption studies. Despiteknown anatomical and physiological similarities between porcineand human skin, very few investigations of percutaneous absorptionphenomena have been conducted in pigs. This study examined radiolabelexcretion patterns after intravenous (iv) and topical administrationof six ¹⁴C-radioIabeled compounds in weanling Yorkshire sows.Radiolabel recovery from excrement collected over 6 days followingiv doses in physiological saline (200 µg, 10 µCi)showed that malathion (M), parathion (P), caffeine (C), andbenzoic acid (B) were primarily excreted into urine (>80%),while greater fractions of testosterone (T, 72%) and progesterone(R, 35%) were fecally eliminated. Percutaneous absorption wasdetermined from total urine and fecal excretion of radiolabelafter topical application, corrected for incomplete excretionfollowing iv administration. Topical doses in ethanol (200 µg,10 µCi) were applied at a surface concentration of 40µg cm^–2 and penetrated in the following rank order(percentage dose): B (25.7%) > R (16.2%) > C (11.8%) >T (8.8%) > P (6.7%) > M (5.2%). Fecal clearances of radiolabel,expressed as a percentage of total excretion, were greater aftertopical administration for four of the six compounds (B, C,P, and T, p < 0.05). Calculations based on urinary excretionalone underestimated percutaneous absorption determined fromtotal excretion by 5–30%, although the difference betweenthe two estimates was statistically significant only for C (p< 0.05). These results suggest that percutaneous absorptionestimates based on urinary radiolabel excretion alone shouldbe interpreted with caution whenever compounds with unknownpenetration characteristics arc used. Factors known to affecthuman skin absorption, such as applied dose, anatomical region,sex, age, various vehicles and solvents, and differences incutaneous metabolism, should be more closely examined in allanimal species used to model percutaneous absorption phenomenain man.