Potentiated Toxicity of 2-sec-Butylphenyl Methylcarbamate (BPMC) by O,O-Dimethyl O-(3-Methyl-4-nitrophenyl)phosphorothioate (Fenitrothion) in Mice; Relationship between Acute Toxicity and Metabolism of BPMC

Potentiated Toxicity of 2-sec-Butytpbenyl Methylcarbamate (BPMC)by O,O-Dimethyl O-(3-Methyl-4-nitrophenyl)phosphorothioate (Fenitrothion)in Mice; Relationship between Acute Toxicity and Metabolismof BPMC. TAKAHASHI, H., MIYAOKA, T., TSUDA, S., AND SHIRASU,Y. (1984). Fundam. Appl. Toxicol. 4, 718–723. Fenitrothionof oral subtoxic dose (100 mg/kg; 4 hr pretreatment) decreasedacute oral LD50 of BPMC from 360 to 66 mg/kg in male mice. Thetreatment prolonged the hexobarbital sleeping time and increasedthe plasma BPMC concentrations. The BPMC toxicity and its plasmaconcentrations were significantly reduced by phenobarbital treatment(80 mg/kg/day, 2 days, ip). This treatment diminished the effectsof fenitrothion on BPMC toxicity and plasma BPMC concentrations.BPMC was metabolized by mixed-function oxidases of the liverin vitro. The metabolism of BPMC was competitively inhibitedby the addition of fenitrothion (5 µg/ml). Fenitrothionremained in the liver (7 µg/g liver). These results suggestthat competitive inhibition of BPMC metabolism by fenitrothionmay, at least in part, play a role in inhibition of BPMC detoxication,resulting in potentiation of its toxicity.     

Potentiation of Acute Toxicity of 2-sec-Butylphenyl N-Methylcarbamate (BPMC) by Fenthion in Mice

Potentiation of Acute Toxicity of 2-sec-Butylphenyl N-methylcarbamate(BPMC) by Fenthion in Mice. MIYAOKA, T., TAKAHASHI, H., TSUDA,S., AND SHIRASU, Y. (1984). Fundam. Appl. Toxicol. 4, 802–807.This study was undertaken to determine whether interactionsof toxicologic importance might occur during combined exposureof male mice to 2-sec-butylphenyl N-methylcarbamate (BPMC) andO,O-dimethyl O-(3-methyl-4-methylthiophenyl)phosphorothioate(fenthion). The equitoxic (administration of BPMC and fenthionresulted in only a 1.6-fold potentiation compared to the expectedLD50. However, 1 hr oral pretreatment with 3 (1/100 LD50), 7.5,15, and 30 mg/kg of fenthion resulted in 5-, 9-, 12-, and 15-foldpotentiation of the acute oral toxicity of BPMC, respectively.This fenthion pretreatment caused significant increases in theBPMC plasma concentrations and in the area under the concentration-timecurve. The increase of the plasma concentrations of BPMC dependedupon the fenthion dose and was associated with that of the potencyratio. The pretreatment of fenthion prolonged the hexobarbitalsleeping time. The plasma concentrations of BPMC after the administrationof a dose of LD2.5 (20 mg/kg) in fenthion-pretreated mice wereclearly lower than those of a dose of LD2.5 (195 mg/kg) in nontreatedmice. These results suggested that fenthion pretreatment causedthe potentiation of the acute toxicity of BPMC partially bythe inhibition of detoxification of BPMC.     

Contribution of Monoaminergic Nervous System in Potentiation of 2-sec-Butylphenyl N-Methylcarbamate (BPMC) Toxicity by Malathion in Male Mice

Contribution of Monoaminergic Nervous System in Potentiationof 2-sec-Butylphenyl N-Methylcarbamate (BPMC) Toxicity by Malathionin Male Mice. TAKAHASHI, H., TANAKA, J., TSUDA, S., and SHIRASU,Y. (1987). Fundam. Appl. Toxicol. 8, 415–422. Malathion-inducedmarked potentiation of BPMC toxicity (about fivefold) was analyzedby measuring LD50 as an index of acute toxicity. The acute lethalityof BPMC was decreased by muscarinic blockers (atropine, methylatropine,or trihexyphenidyl) or a monoamine oxidase inhibitor (pargyline)and increased by a monoamine depleter (reserpine) or a dopaminergicblocker (haloperidol). The potentiation observed with BPMC andmalathion was decreased by the muscarinic blockers, monoaminedepleters (reserpine, -methyl-p-tyrosine), an -noradrenergicblocker (phen-tolamine), or haloperidol. The acute toxicitiesof other N-methylcarbamates MPMC (3,4-di-methylphenyl N-methylcarbamate),MTMC (3-methylphenyl N-methylcarbamate), NAC (1-naphthyl N-methylcarbamate),and XMC (3,5-dimethylphenyl N-methylcarbamate) were potentiatedby malathion to a lesser degree than that of BPMC. Atropineprotected against the lethalities of all N-methylcarbamates.Reserpine or haloperidol potentiated the lethalities of N-methylcarbamateswith a similar tendency toward malathion. When the inhibitoryeffect of each N-methylcarbamate on brain acetylcholinesterase(AChE) was compared with its LD50, among five N-methylcarbamatesBPMC had particularly strong anti-AChE activity. This characteristicof BPMC was not observed after the treatment with reserpine.These results suggest that BPMC may act not only on cholinergicnerves as an anti-AChE, but also on monoaminergic nerves whichantagonize the lethal cholinergic effect. Malathion might inhibitthe effect of BPMC on the monoaminergic nerves, thereby markedlypotentiating the lethal effect of BPMC.     

Immunotoxicological Insignificance of Fenitrothion in Mice and Rats

Fenitrothion was administered orally to mice or rats in dailydoses of up to of the LD50 for 14 days, and numbers of splenic plaque-forming cells against sheep redblood cells (SRBC-PFC), one of the most common immune parameters,were measured. Splenic SRBC-PFC number was suppressed by fenitrothiononly in rats which received 30 mg/kg body weight (bw) of thecompound. Other immune parameters, including the arthus reaction,delayed-type hypersensitivity, and activities of macrophagesand natural killer cells in rats, were not influenced by fenitrothion.Adrenal hyperfunction manifesting as increased organ weightand elevated plasma corticosterone level was noted along withstrong cholinergic signs in rats which received 30 mg/kg bwof fenitrothion. At lower doses such as 3 or 0.3 mg/kg bw offenitrothion, rats had no strong cholinergic signs, adrenalhyperfunction, or evidence of immunosuppression despite significantsuppression of systemic cholinesterase (ChE) activities. Inmice, no suppression of SRBC-PFC number or mixed lymphocytereaction was noted even at the highest dose (40 mg/kg bw) offenitrothion, at which significant suppression of systemic ChEactivities but no cholinergic signs were noted. These findingsstrongly suggest that the im-munosuppressive effect of fenitrothionnoted in rats was due to systemic, potent cholinergic stressand that fenitrothion has no immunotoxicity in mice and rats.     

Contribution of monoaminergic nervous system in potentiation of 2-sec-butylphenylN-methylcarbamate (BPMC) toxicity by malathion in male mice

Malathion-induced marked potentiation of BPMC toxicity (about fivefold) was analyzed by measuring LD50 as an index of acute toxicity. The acute lethality of BPMC was decreased by muscarinic blockers (atropine, methylatropine, or trihexyphenidyl) or a monoamine oxidase inhibitor (pargyline) and increased by a monoamine depleter (reserpine) or a dopaminergic blocker (haloperidol). The potentiation observed with BPMC and malathion was decreased by the muscarinic blockers, monoamine depleters (reserpine, α-methyl-p-tyrosine), an α-noradrenergic blocker (phentolamine), or haloperidol. The acute toxicities of other N-methylcarbamates MPMC (3,4-dimethylphenyl N-methylcarbamate), MTMC (3-methylphenyl N-methylcarbamate), NAC (1-naphthyl N-methylcarbamate), and XMC (3,5-dimethylphenyl N-methylcarbamate) were potentiated by malathion to a lesser degree than that of BPMC. Atropine protected against the lethalities of all N-methylcarbamates. Reserpine or haloperidol potentiated the lethalities of N-methylcarbamates with a similar tendency toward malathion. When the inhibitory effect of each N-methylcarbamate on brain acetylcholinesterase (AChE) was compared with its LD50, among five N-methylcarbamates BPMC had particularly strong anti-AChE activity. This characteristic of BPMC was not observed after the treatment with reserpine. These results suggest that BPMC may act not only on cholinergic nerves as an anti-AChE, but also on monoaminergic nerves which antagonize the lethal cholinergic effect. Malathion might inhibit the effect of BPMC on the monoaminergic nerves, thereby markedly potentiating the lethal effect of BPMC.     

The Effect of Chlorphentermine Pretreatment on the Toxicity of Nitrogen Dioxide in Mice

The Effect of Chlorphentermine Pretreatment on the Toxicityof Nitrogen Dioxide in Mice. HASTINGS, C. E., JR., DENICOLA,D. B., REBAR, A. H., TUREK, J. J., BORN, G. S., AND KESSLER,W. V. (1987). Fundam. Appl Toxicol 9, 69–81. ChlorphentermineHCl (CP) was used to induce preexisting alveolar alterationsresembling a pulmonary lipidosis in mice to study these effectson the severity and duration of nitrogen dioxide (NO²) toxicity.Results indicated that a daily dose of 120 mg/kg for 14 daysproduced consistent histopathologic changes characterized byan accumulation of large foamy macrophages. Male Swiss-Webstermice were divided into a control and three treatment groups.Group 1 received 120 mg/kg CP po daily for 2 weeks followedby exposure to air for 48 hr. Group 2 received 20 ppm NO² for48 hr via whole-body inhalation, and group 3 received 120 mg/kgCP daily for 2 weeks followed by 20 ppm NO² for 48 hr. The fourthgroup served as a nontreated control and received water in placeof CP and air in place of NO². All groups were compared by morphologicevaluation of pulmonary tissues at the light and electron microscopiclevels at Days 0, 1, 3, 5, and 7 after the 48-hr exposure toair or NO². In a second experiment using the same treatmentgroups, thin-section light microscopy was used to count thenumber of type I and type II cells and macrophages. NO² exposurealone caused deaths in 20.8 and 18.5% of the mice in the twostudies, but no deaths were seen in the combination groups fromboth experiments. Histopathologic evaluation showed a typicalcellular response to the NO² exposure, but differences werenoted between the two groups receiving NO² on this treatment.There was increased type II cell hyperplasia and terminal bronchiolitison Days 0 and 1 but less on Days 3 to 7 in the combination groupcompared to the NO² alone group. CP treatment prior to NO² exposurecaused less terminal bronchiolar epithelial hyperplasia andless pulmonary edema than was seen in the NO² along group. TheCP treatment appeared to protect against the lethal effectsof NO² at the concentration and time of exposure used and alteredthe cellular repair mechanism that occurs in response to NO²toxicity. CP treatment prior to NO² exposure caused significantlyless loss of type I cells and less increase in type II cellsdue to NO² damage. The combination treatment also caused anincrease in macro phages greater than that seen in either individualtreatment, and this number remained increased through 5 dayspost-NO² exposure, whereas the NO² alone caused a steady increasein macro phages following the exposure until Day 3. Overall,prior treatment with CP offered partial protection against NO²toxicity.     

Acute Inhalation Toxicity of T-2 Mycotoxin in Mice

Acute Inhalation Toxicity of T-2 Mycotoxin in Mice. Creasia,D. A., THURMAN, J. D., JONES, L. J., III, NEALLEY, M. L., YORK,C. G., WANNEMACHER, R. W., Jr., and BUNNER, D. L. (1987). Fundam.Appl. Toxicol. 8, 230–235. Experiments were conductedto study the acute inhalation toxicity of T-2 mycotoxin in bothyoung adult and mature mice. For a 10-min aerosol exposure,the 24-hr LC50 of T-2 mycotoxin in young adult mice was 0.08± 0.04 mg T-2/liter air and that for mature mice was0.325 ± 0.1 mg T-2/liter air. Deaths among mice exposedto the higher aerosol concentrations used in this study (i.e.,1.5 to 2.4 mg T-2/liter air) occurred in less than 5 hr. Generalclinical symptoms in these animals immediately postexposurewere tremors, lethargy, stilted gait, and, in some animals,prostration. In experiments separate from the concentration-responsestudies, total deposition of T-2 aerosol and selective retentionof T-2 in the respiratory tract and nasal turbinates were determinedanalytically from ³H-labeled T-2. When total deposition of T-2was quantitated, there was excellent agreement between thatamount of T-2 deposited and that amount of T-2 predicted fromcalculations based on aerosol size and animal minute volume.Based on the aerosol deposition data, the LD50 values of T-2mycotoxins was 0.24 mg/kg for young adult mice and 0.94 mg/kgfor mature mice. For mice, inhalation of T-2 mycotoxin is atleast 10 times more toxic than systemic administration (LD50 4.5 mg/kg) and at least 20 times more toxic than dermal administration(LD50 > 10 mg/kg).     

Dynamic Epigenetic Changes Involved in Testicular Toxicity Induced by Di‐2‐(Ethylhexyl) Phthalate in Mice

Abstract: The aim of this study was to analyse epigenetic (specifically, DNA methylation) change in testes induced by maternal exposure to di‐2‐(ethylhexyl) phthalate (DEHP) in mice. Testicular dysgenesis syndrome was induced in foetuses by maternal exposure to DEHP. High‐performance liquid chromatography was performed to analyse DNA methylation status, and expression levels of the DNA methyltransferases were examined by quantitative real‐time polymerase chain reaction and western blotting. DEHP significantly had more than 10% relative increase in the global DNA methylation and also increased DNA methyltransferases’ expression. Changes in DNA methylation may play an important role in abnormal testicular function caused by environmental factors such as maternal exposure to DEHP, which may be one possible mechanism of DEHP‐mediated testicular toxicity.     

Short-Term PCB (Aroclor 1254) Toxicity on Few Phosphatases in Mice Brain

The present communication reports the dose and duration dependent toxicity of a PCB, Aroclor 1254, to a few ion dependent ATPases, Acid phosphatase, Alkaline phosphatase and Glucose-6-phosphatase in the whole brain tissue of mice. Two groups of mice were subjected to two sublethal doses (0.1 and 1 mg kgbw⁻¹ day⁻¹) of PCB orally and exposed for 4, 8 or12 days. A separate control group received the corn oil vehicle for the same exposure times. The observed results indicated exposure duration dependent changes in the enzymatic levels in the brain. The results suggest that the alteration in the enzymatic activity was possibly due to imposed oxidative stress generated by Aroclor 1254 on membrane-bound ion-dependent ATPases and other phosphatases in the brain tissue.     

Developmental Toxicity Evaluation of Dietary Di(2-ethylhexyl) phthalate in Fischer 344 Rats and CD-1 Mice

Developmental Toxicity Evaluation of Dietary Di(2-ethylhexyl)phthalatein Fischer 344 Rats and CD-I Mice. TYL, R. W., PRICE, C. J.,MARR, M. C. AND KJMMEL, C. A. (1988). Fundam. Appl. Toxicol.10, 395–412. Di(2-ethylhexyl)phthalate (DEHP), a widelyused plasticizing agent, was evaluated for developmental toxicityin timed-pregnant Fischer 344 rats (22-25 dams/dose) and CD-Imice (24-30 dams/dose). DEHP was administered in the diet ongestational Days (gd) 0 through 20 at 0.0,0.5, 1.0, 1.5, or2.0% (rats) and on gd 0 through 17 at 0.00, 0.025,0.05, 0.10,or 0.15% (mice). At termination (gd 20, rats; gd 17 mice), allfetuses were examined for external, visceral, and skeletal malformationsand variations. In rats, maternal toxicity and reduced fetalbody weight per litter were observed at 1.0, 1.5, and 2.0%.Increased resorptions and decreased number of live fetuses/litterwere observed at 2.0%. Maternal food consumption was reducedand water consumption was increased in all DEHP groups. Thenumber and percentage of fetuses malformed per litter were unaffectedby treatment. In mice, maternal toxicity, increased resorptionsand late fetal deaths, decreased number of live fetuses, andreduced fetal body weight per litter were observed at 0.10 and0.15%. Maternal food and water consumption exhibited a dose-relatedupward trend with food consumption significantly increased at0.15%. The number and percentage of fetuses malformed per litter(open eye, exophthalmia, exen-cephaly, short, constricted, orno tail, major vessel malformations, fused or branched ribs,and fused or misaligned thoracic vertebral centra) were elevatedat 0.05,0.10, and 0.15% DEHP. In conclusion, DEHP was not teratogenicat any dose tested in Fischer 344 rats when administered inthe feed throughout gestation but did produce maternal and otherembryofetal toxicity at 1.0, 1.5, and 2.0%. In contrast, DEHPadministration throughout gestation in CD-I mice resulted inan increased incidence of malformations at doses which producedmaternal and other embryofetal toxicity (0.10 and 0.15%) andat a dose (0.057percnt;) which did not produce significant maternaltoxicity. No treatment-related embryofetal toxicity includingteratogenicity was observed in mice at 0.025% Or in rats at0.5% DEHP.     

Toxicity of Anguidine in Mice

Toxicity of Anguidine in Mice. CONNER, M. W., DECAMARGO, J.,PUNYARIT, P., RIENGROPITAK, S., ROGERS, A. E., AND NEWBERNE,P. M. (1986). Fundam Appl Toxicol. 7, 153-164. A characteristicof the trichothecene mycotoxin, anguidine, is its extreme toxicityto organs with populations of rapidly dividing cells. In preparationfor evaluation of compounds that may protect against anguidinetoxicity, we measured the LD50 of anguidine administered bygastric gavage (ig) or intraperitoneal injection (ip) and studiedthe dose- and time-dependent effects of anguidine on lymphohematopoieticorgans, intestine, and testis, and measured hematocrit and peripheralblood leukocyte counts in male CD-I mice. The ig LD50 at 96hr was 15.5 mg/kg; after ip administration the LD50 at 96 hrwas 20.0 mg/kg. Characteristic changes caused by sublethal dosesof anguidine were cell depletion and necrosis in lymphohematopoieticorgans, multifocal necrosis of intestinal epithelium, and diffusenecrosis of germinal epithelium followed by progressive tubuledegeneration in the testes. There was leukocytosis due to bothlymphocytosis and neutro-philia in the first few hours followinganguidine exposure, followed by lymphopenia, neutropenia. andanemia by 3 days. After lethal doses, the intestinal necrosiswas transmural, and there was extensive necrosis of lymphohematopoieticorgans. There was rapid recovery after sublethal anguidine exposureof all anguidine-sensitive organs except for testis where decreasedweights and abnormal spermatogenesis persisted for the 2-weekobservation period. Our results suggest that intestinal necrosisis an important cause of death following anguidine exposure.Atrophy of seminiferous tubules may have some value as an indicatorof prior anguidine exposure, but the testicular changes arenot unique to this compound.     

络合钙在小鼠体内的药代动力学研究

目的：测定络含钙在小鼠体内的血药浓度,对其进行药代动力学研究。方法：口服给予高、低两个剂量的络合钙,于给药后不同时间采用原子吸收法测定血钙浓度,并与维丁钙片进行比较,对所得参数用计算机进行房室模型模拟。结果：高、低络含钙组以及维丁钙组小鼠的血钙浓度明显升高,高剂量组的血钙浓度明显高于维丁钙组（P≤0．01）。所得的动力学参数经计算机模拟表明络合钙在体内的药代动力学符合一室模型。结论：络合钙口服可明显提高小鼠的血钙水平。     

LC-MS法测定Beagle犬血浆中人参皂苷20(R)-Rh2及其药代动力学研究

目的建立测定人参皂苷20(R)-Rh2血浆药物浓度的液相色谱-电喷雾离子化-质谱联用的分析方法(LC-ESI-MS),探讨其在Beagle犬体内的药代动力学研究中的应用.方法Beagle犬6只,随机分为2组,采用单剂量双周期自身交叉设计,分别给犬单剂量静注(0.1 mg·kg-1)或灌胃(1 mg·kg-1)20(R)-Rh2,用LC-MS法测定给药后的血浆中药物浓度,计算其药代动力学参数,以及在Beagle犬体内的绝对生物利用度.结果20(R)-Rh2在 0.5～200 ng·ml-1浓度范围内线性关系良好(r2=0.9998),样品在血浆中的提取回收率大于70%,批内和批间的RSD均小于15%,静注后主要药代动力学参数T1/2,CL,AUC0-∞分别为 8.0±2.8 h、0.1±0.03 L·kg-1·h、857.0±209.6 ng·h·ml-1;口服后主要药代动力学参数Tmax,Cmax,T1/2,AUC0-∞分别为 2.6±1.3 h、371.0±199.6 ng·ml-1、5.8±2.6 h、1215.7±598.6 ng·h·ml-1.绝对生物利用度为(16.1±11.3)%.结论该法专属性强,灵敏度高,可用于20(R)-Rh2的体内定量分析.人参皂苷20(R)-Rh2在Beagle犬体内的绝对生物利用度较低.     

Correction for Non-Ideal Tracer Pharmacokinetic Disposition by Disposition Decomposition Analysis (DDA)

Purpose. Pharmacokinetic (PK) studies assume that the tracer's PK is equivalent to the parent compound. This assumption is often violated. The aim of this work is to present a method enabling the ideal tracer PK, i.e. the PK of the parent compound, to be predicted from the non-ideal tracer. Methods. The procedure uses a disposition decomposition-recomposition (DDR) that assumes that the labeling mainly changes the elimination kinetics while the distribution kinetics is not significantly affected. In the DDR procedure an elimination rate constant correction factor (k_COR) is determined from a simultaneously fitting to plasma concentration data resulting from an i.v. injection of both the tracer and the parent compound. The correction factor is subsequently used to predict the ideal tracer PK behavior from the disposition function (i.v. bolus response) of the non ideal tracer. Results. The DDR method when applied to plasma level data of erythropoietin (r-HuEPO) and its iodinated tracer (^l25I-r-HuEPO) from a high (4000U/kg) and a low (400U/kg) dosing of r-HuEPO in newborn lambs (n = 13) resulted in excellent agreements in the elimination rate corrected dispositions in all cases (r = 0.995, SD = 0.0095). The correction factor did not show a dose dependence (p > 0.05). The correction factors were all larger than 1 (kCOR = 1.94, SD = 0.519) consistent with a reduction in the EPO elimination by the iodination labeling. Conclusions. The DDR tracer correction methodology produces a better differentiation of the PK of endogenously produced compounds by correcting for the non-ideal PK behavior of chemically produced tracers.     

Exploration of the Plausible Mechanism of Ethambutol Induced Ocular Toxicity by Using Proteomics Informed Physiologically Based Pharmacokinetic (PBPK) Modeling

Pharmaceutical Research - Ethambutol (EMB) is a first-line anti-tubercular drug that is known to cause optic neuropathy. The exact mechanism of its eye toxicity is unknown; however, proposition is...     

Pharmacokinetic study of 2-(2'-methyl-3'-chloro-anilino)-lysine nicotinate (L-104)

The pharmacokinetic study of a new analgesic-antiinflammatory agent, 2-(2'-methyl-3'-chloro-anilino)lysine nicotinate (L-104), was preformed in the rat and the dog. When administered p.o. in rats at a dose of 40 mg/kg, the serum peak was 131.3 microgram/ml +/- 10.3 at 15 min post application, and the elimination t 1/2 was 1.20 h. For the same dose, given i.v., the biological t 1/2 was 1.38 h, the AUC 337.18 microgram/ml/h and the Vdss 0.232 l/kg. When administered i.v. in dogs of 10 mg/kg, the biological t 1/2 was 0.96 h +/- 0.14, the AUC 73,066 microgram/ml/h and the Vdss 0.178 l/kg. In the rat, for a dose of 40 mg/kg given p.o., 79.1% of tritium-labelled L-104 were excreted within 72 h, 56.0% of them through the kidneys. In the dog, at an i.v. dose of 10 mg/kg, the whole of the drug was excreted at the end of 72 h, corresponding 52.6% urinary excretion. The main metabolite in the rat was 2-(2'-methyl-3'-chloro-4'-hydroxy-anilino)-nicotinic acid (72.2%), whereas in the dog it was 2-(2'-methyl-3'-chloro-anilino)-5-hydroxy-nicotinic acid (35.2%).     

Reproductive Toxicity of Tricresyl Phosphate in a Continuous Breeding Protocol in Swiss (CD-1) Mice

Reproductive Toxicity of Tricresyl Phosphate in a ContinuousBreeding Protocol in Swiss (CD-I) Mice. CHAPIN, R. E., GEORGE,J. D., AND LAMB, J. C. IV. (1988). Fundam. Appl. Toxi-col. 10,344-354. The effects of a mixture of tricresyl phosphate isomerson reproductive performance in Swiss (CD-I) mice were evaluatedusing a continuous breeding protocol. Tricresyl phosphate (TCP)was mixed into the feed at 0, 0.05, 0.1, and 0.2% by weight.Although the fertility index was not changed in the animalsconsuming the high-concentration feed, the number of littersper pair decreased in a dose-related fashion, and the proportionof pups born alive, and their weight, was significantly decreasedin the high-dose group. A crossover mating trial found impairedfertility in both males and females exposed to 0.2% TCP, witha greater effect in females. Histopathology of the Fo pairsrevealed dose-related seminiferous tubule atrophy, and decreasedtestis and epididymal weights in the high-dose males, whilethe female reproductive tract showed no histopathologic changes.There were dose-related changes in the adrenals of both sexes,and body weight was depressed in both sexes at the highest concentration.The last litter born in the 98-day breeding phase was rearedto age 74 days and then mated within the control and two ofthe treatment groups (0.0,0.05, and 0.1 % TCP; there were toofew offspring in the 0.2% group). There was a decrease in thefertility index in the 0.1% TCP group, and a decreased proportionof liveborn and number of liveborn pups per litter. In the F,males at necropsy, sperm concentration and morphology were normalat termination, although motility was decreased in both the0.05% and the 0.1% groups compared to controls. These data showthat TCP impaired fertility in both sexes of mice in the Fogeneration and affected sperm motility at even the lowest dosein F, males.     

Shiga Toxin Type 2dact Displays Increased Binding to Globotriaosylceramide in vitro and Increased Lethality in Mice after Activation by Elastase

Shiga toxin type 2dact (Stx2dact), an Stx2 variant originally identified from Escherichia coli O91:H21 strain B2F1, displays increased cytotoxicity after activation by elastase present in intestinal mucus. Activation is a result of cleavage of two amino acids from the C-terminal tail of the A₂ subunit. In this study, we hypothesized that activation leads to increased binding of toxin to its receptor on host cells both in vitro and in vivo. To test this theory, Stx2dact was treated with elastase or buffer alone and then each toxin was assessed for binding to purified globotriaosylceramide (Gb3) in an enzyme-linked immunosorbent assay, or cells in culture by immunofluorescence, or flow cytometry. Elastase- and buffer-treated Stx2dact were also evaluated for binding to mouse kidney tissue and for relative lethality in mice. We found that activated Stx2dact had a greater capacity to bind purified Gb3, cells in culture, and mouse kidney tissue and was more toxic for mice than was non-activated Stx2dact. Thus, one possible mechanism for the augmented cytotoxicity of Stx2dact after activation is its increased capacity to bind target cells, which, in turn, may cause greater lethality of elastase-treated toxin for mice and enhanced virulence for humans of E. coli strains that express Stx2dact.     

Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin

Shiga toxin (Stx) is an AB₅ ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b–g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease. However, Stx2c is about 10-fold less toxic than Stx2d in vivo despite only two amino acid differences in the A subunit at positions 291 and 297. We made mutations at these two sites to create intermediate toxins between Stx2c and Stx2d, and determined the 50% cytotoxic dose on Vero cells before and after heat treatment, and the 50% lethal dose in mice of the toxins. We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability. We also assessed the secondary structure of Stx2c and Stx2d by circular dichroism (CD) spectroscopy. The CD studies suggest that Stx2c has a less-ordered secondary structure than Stx2d. We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.