Maleimide--biochemical, pharmacologic and toxicologic studies. Interaction with L-asparagine metabolism.

The effect of maleimide on the metabolism of l-asparagine has been examined in vitro and in viro. In vitro, maleimide is a potent and irreversible inhibitor of the l-asparagine synthetase [l-glutamine hydrolyzing. (EC 6.3.5.4)] from murine leukemia 5178Y/AR and from murine pancreas; this inhibition is accomplished via sulfhydryl blockade and can be prevented by suitable thiols. In vivo, maleimide was highly irritating locally, producing peritonitis and phlebitis after intraperitoneal and intravenous injection, respectively. The ld₅₀ in the mouse was 9 mg/kg by the intravenous and intraperitoneal routes; renal, hepatic, neurologic and hematologic toxicities were the principal effects of the drug in this species. l-Asparagine did not alter the lethal effects of maleimide. Maleimide at a dose of 6 mg/kg intraperitoneally did not begin to inhibit pancreatic or tumoral l-asparagine synthetase until 24 hr after its administration: maximum inhibition was delayed until 48–72 hr after dosing. Although maleimide was found to be a potent inhibitor of hepatic l-asparaginase (EC 3.5.1.1) and l-asparagine transaminase (EC 2.6.1.14) in vitro, at no time did the agent inhibit these enzymes in vivo. Additionally, maleimide failed to inhibit protein and nucleic acid synthesis in pancreas and liver, although it did exert a transient repressive effect on these processes in subcutaneous L5178Y/AR tumor. The drug was a potent cytotoxin to 12L1210 cells in culture, causing partial arrest in the G₂ phase of the cell cycle as well as general slowing of progression through the cell cycle. No therapeutic action was produced vs this tumor growing in the peritoneum of mice nor was the drug effective vs subcutaneous L5178Y or L5178Y/AR. However, when maleimide was used to wash the surgical wound created by extirpation of Lewis Lung Carcinoma growing in the muscles of the hind limb, the drug did inhibit local recurrence of tumor, most notably when used in conjunction with parenteral cyclophosphamide. It is concluded that maleimide is an inhibitor, in vitro, of the enzymes of l-asparagine metabolism because of its ability to form covalent bonds with critical sulfhydryl compounds.     

Azotomycin--toxicologic, biochemical and pharmacologic studies in mice

A biochemical basis was sought for the acute and cumulative toxicity of Azotomycin, (glutamine, N[1-[(1-carboxy-5-diazo-4-oxopentyl) carbamoyl]-5-diazo-4-oxopentyl]-, sodium salt), an antagonist of l-glutamine. Whereas mice can tolerate relatively large single doses of the drug, very small repeated doses are toxic. l-Asparaginase augments the toxicity of the latter regimen but not the former. Brain, liver, intestines and spleen are the organs principally damaged by Azotomycin. A systematic study of the synthesis and concentration of l-glutamine, l-asparagine and other metabolites in these organs has revealed that Azotomycin is also a powerful inhibitor of l-asparagine synthetase in vivo. A variety of murine organs have been shown to hydrolyze Azotomycin to yield 1 mole l-glutamic acid and approximately 2 moles 6-diazo-5-oxo-l-norleucine (DON). On this evidence it is suggested that DON, a classical antagonist of l-glutamine, is responsible for the toxicity of Azotomycin in these organs. When the analogue interfered more with the utilization than with the breakdown of l-glutamine, l-glutamine accumulated to a pathologic degree; this situation has been seen in liver. When both utilization and breakdown were curtailed to a similar degree, the steady state concentration of l-glutamine was maintained; this situation was seen in spleen, intestines and brain. In no case did Azotomycin inhibit the biosynthesis of l-glutamine.     

Synthesis, pharmacologic and toxicologic study of carbonic and carbamic acid esters

The results of the pharmacologic study of some synthesized derivatives of carbamic and carbonic acids show that the compounds are biologically active. They are less toxic than imipramine, and exert a marked excitation effect on the central nervous system. Some of the compounds (including PS-1, P-8-Y and PS-4) possess greater antidepressive action than imipramine.     

Reproduction toxicologic studies on rats following oral administration of benzopyrone preparations

The influence of the benzopyrone preparation Venalot (active substances: coumarin and troxerutin; in the following briefly called CT) on the fertility and teratogenicity as well as on the perinatal and postnatal development of a total of 3 generations was evaluated in a combined study. The 1-, 8-, 64-, and 128fold of the daily therapeutical doses for humans was suspended in tap water and administered orally by gavage to 95 male and 190 female SPF rats (Wistar) (test groups 2 to 5). 23 male and 46 female rats (test group 1) served as controls and were given tap water alone. The male animals were subjected to a pretreatment of 10, the female animals to one of 3 weeks. The treatment was continued during the phase of mating. The animals scheduled for cesarian section received the test substance until the day of the laparatomy (gestation day 20), those selected for littering throughout lactation (day 24 post partum). With the aid of a stepwise histological technique, the teratological examination could also disclose non lethal malformations of the organs. The treatment resulted in a decreased food consumption in the animals of group 5 and in a reduced gain of body weight as well as pathologic-anatomically and histologically demonstrable, definitely dose related hepatic lesions. The test substance had no effect on either the treated P generation nor the untreated F1 generation. As teratogenic effects could also not be demonstrated and the peri- and postnatal development of the filial generations 1 and 2 was undisturbed, the present study does not indicate a reproduction toxicological risk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Introduction/overview: gender-based differences in pharmacologic and toxicologic responses

Gender may be the most important factor in mammalian development and response to exogenous agents. From believing sex-related differences required sheltering women to protect their reproductive capacity (Victorians thought exercise, education, train travel, and certain music neuro- and reprotoxic to females) to legislating a status of essential equality of the sexes may have increased women's health issues. Men and women often respond differently to drugs. Inclusion of women in phase I/II clinical trials is insufficient to identify gender-based differences in response; rather, animal models should be the basis for predicting gender-based differences in pharmacologic and toxicologic effects. Unfortunately, current animal models do not consistently demonstrate such differences. Use of commonly used species (e.g., rats and dogs) does not necessarily result in relevant evaluation of an agent in a species at appropriate development (age), physiological state, anatomy, metabolism, or kinetics for estimation of human risks. The need to test agents in relevant animal models and advances in metabolic, pharmacokinetic, and pharmacodynamic capabilities challenge us to improve methods by using the most relevant models for estimating human risk. We need to be concerned about gender-related differences and the dynamics of gender-based growth and development over the entire life cycle. We must also consider potential interactions of dietary supplements and other exogenous agents that can act as drugs or modulate the potential effects of drugs differently in men, women, and developing children of both sexes. To this end, the health benefits of genistein and the effects of this dietary agent in a multigeneration study in rats will be described. It is envisioned that this symposium will assist in re-recognition of the importance of gender-related differences in use and response to pharmaceuticals and result in optimization of nonclinical testing procedures to identify benefits and risks for human use of these agents.     

Reproductive toxicity of cabergoline in mice, rats, and rabbits

Cabergoline, a new dopaminergic ergot derivative with potent long-lasting prolactin (PRL)-lowering properties, was assessed using standard reproductive studies in the mouse, rat, and rabbit with oral administration. Because of the compound's pharmacologic activity, several aspects remain incompletely explored in the rat, in which prolactin is the luteotrophic hormone. A fertility study in female rats was possible only at very low doses (0.5, 1, and 2 μg/kg/d) because higher doses completely inhibited implantation. In male rats no adverse effects were seen on male reproductive performance or on the offspring at doses up to 320 μg/kg/d given for 10 weeks prior to mating with untreated females. In a developmental toxicity study in rats treated from day 6 to day 15 of gestation at doses (6.25, 12.5, and 25 μg/kg/d) not exceeding the active dose for inhibition of egg nidation (ED₅₀ = 25 μg/kg), a high incidence of total litter loss occurred as a reflection of inhibition of egg nidation at the highest dose, but embryofetal development was not impaired in litters reaching term. An exploratory study at 30 or 1000 μg/kg/d with treatment from day 5 of gestation or later demonstrated that cabergoline did not affect the maintenance of pregnancy at 30 μg/kg/d given from day 7 or later, or at 1000 μg/kg/d given from day 9. Doses of 500, 2000, and 8000 μg/kg/d (treatment from day 6 to day 15 of gestation) did not inhibit egg nidation in mice and showed no adverse effects on intrauterine development Doses ranging from 5 to 8000 μg/kg/d administered from day 6 to day 18 of gestation in the rabbit were associated with maternal effects, including a reduction in body weight gain and food and water intake starting from 500 μg/kg/d and increased reactivity at the highest doses (4000 and 8000 μg/kg/d). Effects on intrauterine development were restricted to a reduction in mean fetal and placental weights at 4000 and 8000 μg/kg/d. In peri- and postnatal studies in rats (treatment from day 15 or 17 of gestation to weaning) cabergoline did not affect fetal development and parturition up to 100 μg/kg/d, but strongly inhibited milk secretion starting from 10 μg/kg/d, thus leaving unexplored the postnatal phase at higher doses. When neonatal rats (born from untreated dams) were treated directly with cabergoline at 10, 30, and 90 μg/kg/d from day 7 to 13 after birth, treatment was well tolerated up to the highest dose tested (90 μg/kg/d). It was concluded that cabergoline did not impair fertility in the male rat, was not teratogenic in mice and rabbits, did not affect the latter phase of gestation or parturition in the rat, and was not toxic when administered directly to neonatal rats.     

芍药甙在兔和大鼠体内的药动学研究

兔iv25mg·kg~(-1)芍药甙后,血药浓度—时间曲线符合二室模型。药动学参数为T_(1/2α)=5.93min,T_(1/2β)66.02min,V(?)=516.8ml·kg~(-1),CL=6.11ml·kg~(-1)·min~(-1)。兔ig250mg·kg~(-1)芍药甙,生物利用度为F=7.24％±4.15％,T_(max)=77.4min,C_(max)=21.57mg·L~(-1)大鼠ig550mg·kg~(-1)芍药甙,24h内粪、尿排泄量及iv55mg·kg~(-1)7h内胆汁排泄量分别占给药量的10.61％、1.08％、864％。离体肝脏灌流结果提示:芍药甙在肝内代谢少.     

Interspecies pharmacokinetic scaling of BSH in mice,rats, rabbits,and humans

Sodium mercaptoundecahydrododecaborate or BSH is an important compound for boron neutron capture therapy (BNCT). The total clearance and steady state volume of distribution of BSH in humans and in laboratory animals were analyzed as a function of species body weight using the allometric equation for interspecies scaling. Significant linear relationships were obtained between log CL_t (L h^?1) and log W(kg) (r=0.972; p=0.028) as well as log V_SS(L) and log W(kg) (r=0.999; p=0.0005). The corresponding allometric equations were CL_t=0.127 W^0.68 and V_SS=1.557 W^0.87, respectively. BSH clearance in various species was shown to be a constant fraction (0.26) of creatinine clearance, the relationship being independent of body weight. Thus BSH clearance in various species occurred at similar pace when measured by a physiological parameter (creatinine clearance) rather than chronological time. Interspecies scale-up of plasma concentration—time data for the four species using a complex Dedrick plot resulted in similar profiles. Our results indicate that the BSH data obtained in laboratory animals could be utilized to generate preliminary estimates of the pharmacokinetic parameters in humans. These parameters can serve as guidelines for better planning of clinical studies.     

Studies on the prenatal toxicity of N,N-dimethylformamide in mice, rats and rabbits.

Prenatal toxicity studies with N,N-dimethylformamide (DMF) in rabbits, rats and mice were carried out using the oral (gavage), dermal, inhalation and ip injection routes of administration. Administration of DMF by gavage led to an increase in malformations in rats and mice in the absence of overt maternal toxicity. The lowest-observable-effect level was 182 mg/kg body weight/day in mice and 166 mg/kg body weight/day in rats. After dermal administration a dose-dependent incidence of teratogenicity was observed in rats at 94-944 mg/kg/body weight/day in the absence of overt maternal toxicity. In rabbits dermal administration led to a steeper increase in the dose-response relationship and at 400 mg/kg body weight/day to a clear teratogenic effect in the presence of slight maternal toxicity. The 200 mg/kg body weight/day dose appeared to be the no-adverse-effect level. Inhalation in rats caused foetotoxicity and embryolethality at 287 ppm. A clear teratogenic effect was shown in rabbits at 450 ppm and a marginal effect at 150 ppm. The no-effect level for does and foetuses was 50 ppm. Ip injection in mice caused clear teratogenicity at 944 mg/kg body weight/day and slight embryotoxicity at 378 mg/kg body weight/day. The rabbit appears to be more sensitive than the rat to DMF-related prenatal toxicity and should, therefore, be used as the basis for the evaluation of teratogenic risk in humans.     

Teratological studies with dichlorvos vapour in rabbits and rats

Experimental manganese encephalopathy was produced in rabbits by intratracheal inoculation of manganese dioxide (400 mg). After a period of 18 to 24 months manganese inoculated rabbits developed paralysis of the hind limbs. There was widespread neuronal loss and neuronal degeneration in cerebral cortex, caudate nucleus, putamen, substantia nigra and cerebellar cortex. This was associated with neuroglial proliferation.There was marked reduction in the activity of acid phosphatase and adenosine triphosphatase in the degenerated neurones in manganese inoculated animals as compared to controls. No alteration was observed in the activity of alkaline phosphatase and 5-nucleotidase. It has been suggested that manganese may possibly have an inhibitory effect on acid phosphatase and adenosine triphosphatase in affected neurones by disturbing the catabolism of enzyme protein or by destroying lysosomes and mitochondria.     

Subchronic toxicologic studies using cyclamate-Na in dogs

2 male and 2 female beagle dogs were fed cyclamate-Na in daily doses of 0 (control), 150, 500, 1500 mg/kg of body weight with the diet. Liquid feces occurred only after feeding 1500 mg/kg/day during the first few days of the experiment. Haematological, clotting and clinical chemistry parameters were unchanged in the groups up to the level of 1500 mg/kg/day. No effects were noted in carbohydrate and fat metabolism, nor in ECG or blood pressure. Gross pathology and histopathology did not reveal any change in any dose group. Daily fed doses of cyclamate-Na up to 1500 mg/kg body weight were well tolerated for three month in the dog.     

DON, CONV and DONV-III. Pharmacologic and toxicologic studies.

The pharmacologie, toxicologic and oncolytic properties of the ketoamino acids DON (l-DON; 6-diazo-5-oxo-l-norleucine), CONV (l-CONV; 5-chloro-4-oxo-l-norvaline; 2-amino-5-chloro-levulinic acid) and DONV (l-DONV; 5-diazo-4-oxo-l-norvaline) were examined. DON was found to be the most active therapeutic agent of the three drugs against leukemia 1210 and also the most potent cytocidal agent against KB tumor cells in culture. The acute ld₅₀ values of the agents were dissimilar: CONV was the most toxic drug of the three after single intraperitoneal injections, and DONV the least toxic. Only DON showed evidence of prominent cumulative toxicity. In studies with isolated cells of leukemia 5178Y rendered resistant to l-asparaginase (L5178Y/AR), all three agents appeared to compete both with l-asparagine and with l-glutamine for transport into the cell. DONV competed most effectively with l-glutamine and CONV most effectively with l-asparagine. In mice, all three drugs were cleared from the plasma and excreted into the urine at a rapid rate. None was bound to the proteins of mouse plasma. After an intraperitoneal injection of 100 mg/kg, the concentration of DONV in the pancreas was approximately ten times that of CONV or DON; after comparable intravenous injections, only DONV could be identified in this tissue. Although the metabolism of all three ketoamino acids was found to be minor in degree, evidence is presented that they can be degraded in vitro by organ homogenates and also that purified enzymes can catalyze their transamination. In addition, DON was a good substrate for renal γ-glutamyl transferase (EC 2.3.2.2). In the case of DONV, some conversion to CO₂ by isolated tumor cells also was observed. From these and previous studies it is concluded that, of these analogs of l-glutamine and l-asparagine, DON is the most “l-glutamine-like” agent of the three, DONV the most “l-asparagine-like,” while CONV has important attributes of both amino acids.     

Effects of cobalt sulfate on prenatal development of mice, rats, and rabbits, and on early postnatal development of rats

The effects of cobalt sulfate administered to pregnant C57BI mice, OFA-SD rats, and New Zealand rabbits was studied on fetal and postnatal offspring. Cobalt concentration in the maternal blood was increased in proportion to the administered doses. Cobalt crossed the placenta and appeared in the fetal blood and amniotic fluid. Regardless of the administered dose of cobalt sulfate, cobalt concentration in the blood peaked 2 h after administration. Cobalt produced dose-dependent maternal toxicity and was found to be embryotoxic in all three species, as evidenced by elevated frequency of fetuses with body weight or skeletal retardation and embryolethality. Cobalt increased the frequency of major anomalies significantly in mice and rats, with anomalies of the eyes, kidneys, skull, spine, and sternum in mice, and anomalies of the urogenital system in rats. Cobalt sulfate was not teratogenic in rabbits. Intra-amnial administration of cobalt sulfate produced a dose-dependent increase of the frequency of dead fetuses, and weight retardation of the live fetuses. The direct cytotoxic effect probably plays a role in the embryotoxic and teratogenic effects of cobalt. The postnatal examinations revealed a decrease of the perinatal index in the treated group. The body weight of the pups in the treated group was lower during wk 1 of life, but no difference was found between the control and treated by the end of wk 2. Eye opening was completed in the usual time period in both groups, while time of appearance of the teeth, descending of the testes, shaping of ears, and development of hearing was delayed in the treated group. The development of muscle strength and of the locomotor system was delayed. All the functions studied (forward movement, swimming, righting reflex) normalized by postnatal d 21, with the exception of muscle strength. It was concluded that cobalt sulfate exposure decreases the perinatal viability of the fetuses, but the functions of the surviving fetuses with perinatal retardation become compensated by postnatal wk 2-3. The development of fetuses is undisturbed thereafter.     

Review of approaches to the recording of background lesions in toxicologic pathology studies in rats

Pathological evaluation of lesions caused directly by xenobiotic treatment must always take into account the recognition of background (incidental) findings. Background lesions can be congenital or hereditary, histological variations, changes related to trauma or normal aging and physiologic or hormonal changes. This review focuses on the importance and correct approach to recording of background changes and includes discussion on sources of variability in background changes, the correct use of terminology, the concept of thresholds, historical control data, diagnostic drift, blind reading of slides, scoring and artifacts. The review is illustrated with background lesions in Sprague Dawley and Wistar rats.