Effects of diphenylhydantoin and phenobarbital on voltage-clamped myelinated nerve.

Diphenylhydantoin (DPH) and phenobarbital (PB) have a selective action in blocking spontaneous activity in nerves made hyperexcitable by lowering the calcium concentration of the bathing medium (Rosenberg, P. and Bartels, E. 1967 J. Pharmacol. Exp. Ther. 155, 532-544.). To investigate this further, we examined the action of DPH and PB on voltage-clamped single myelinated nerves at two different calcium concentrations. In 1.8 mM calcium Ringer, DPH reduced the sodium permeability (PNa) without affecting the potassium conductance (GK) or the voltage-dependent time constants of sodium activation (taum) and inactivation (tauh), and potassium activation (taun). PB was similar to DPH except that in addition to reducing PNa, it shifted taum in the direction of depolarization. When the calcium concentration was lowered to 0.36 mM, the curves relating taum and taun to membrane potential were shifted in the direction of hyperpolarization, as expected. However, the addition of DPH or PB reduced or abolished these shifts. It is suggested that both DPH and PB stabilize hyperexcitable membranes by an action on the parameter m, and that this may contribute to their antiepileptic action.     

Effect of diphenylhydantoin on blood pressure of spontaneously hypertensive rats

Diphenylhydantoin (DPH) has been shown to elicit direct peripheral vasodilatory effects in anaesthetised animals. Since spontaneously hypertensive (SH) rats exhibit many features similar to human essential hypertension, the effect of DPH on blood pressure of these rats was studied. DPH given orally for 5 days elicited dose-dependent fall in systolic blood pressure in conscious SH rats. In addition, repeated administrations of DPH increased the noradrenaline concentration in the hypothalamus. These results suggest that the central noradrenergic mechanisms might be involved in the hypotensive action of DPH in SH rats, probably at the supramedullary level.     

Convulsant action of diphenylhydantoin overdose in young rats

Acute toxicity of diphenylhydantoin (DPH) was studied in 241 male albino rats aged 7, 12, 18, 25 and 90 days. Single intraperitoneal dose of DPH (from 200 to 1000 mg/kg) induced only ataxia and loss of righting reflex in 25-day-old and adult rats. In rats aged 18 days or less ataxia of hindlimbs was also marked. In all these age groups generalized convulsions appeared; they were formed by wild running followed by a clonic phase. The dose of DPH necessary for elicitation of seizures was lowest in 7-day-old rats (75 mg/kg) and increased with age up to 200 mg/kg in 18-day-old rats. The 1000 mg/kg dose was lethal for 25- and 12-day-old rats, but not for 7-day-old ones. The uneven development of excitatory and inhibitory action of DPH is suggested.     

Lack of mutagenicity of diphenylhydantoin in in vitro short-term tests

The mutagenicity of diphenylhydantoin (DPH) and its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), has been re-evaluated by the Ames test using Salmonella typhimurium and, for DPH only, by an in vitro cytogenetic test with human lymphocytes and a turbidimetric assay of tubulin polymerization. As negative results were obtained in all test systems used here, one has to conclude that DPH is devoid of mutagenic properties.     

The effect of parahydroxylation of diphenylhydantoin on metaphase accumulation.

DPH has a colchicine-like action on metaphase arrest of cultured human lymphocytes. The first step in detoxification of DPH increased its power to accumulate metaphases 3-fold. This hydroxy derivative [5-[4-hydroxyphenyl]-5-phenylhydantoin, HPPH] 3.6 X 10(-4) M was equivalent colchicine 1 x 10(-5) M in its power to inhibit metaphase completion. The effect of HPPH on mitosis was reversible; colchicine effect was not reversed and vincristine effect was partially reversed by washing drug from the medium. Hydroxylation of DPH did not change its inhibition of DNA synthesis and enhanced inhibition of protein synthesis to a minor degree. Detoxification increased the colchicine-like action of DPH.     

Humoral immune dysfunction as a result of prenatal exposure to diphenylhydantoin: correlation with the occurrence of physical defects

The effect of prenatal exposure to diphenylhydantoin (DPH) on postnatal immune function of offspring was studied using a longitudinal experimental design and in vivo immunoassays. Maternal Balb/c mice were dosed by gavage on gestation days 9 through 18 with 0, 20, 40, or 60 mg/kg DPH. Humoral immune function was assessed by measuring the serum antibody levels to type III pneumococcal polysaccharide 5 days after immunization by radioimmunoassay. Cell-mediated immune function was assessed by measuring the delayed-type hypersensitivity response to the contact allergen oxazolone using a micrometer method. A dose-related suppression of humoral immune function was observed in male and female offspring at 25 days but not at 15 weeks of age. Cell-mediated immunity was not affected by prenatal DPH exposure at 25 days or 15 weeks of age. Offspring developed purulent eye exudates at 12 days of age; the incidence and persistence was related to DPH dose. The immunosuppressive effect of DPH on humoral immune function was significantly greater in offspring born with open eye defect than in similarly treated but physically normal offspring. The results suggest that prenatal exposure to DPH may adversely affect the normal development and expression of humoral immune function, particularly in those offspring with other manifestations of DPH's developmental toxicology.     

Withdrawal rates of DDT from chickens treated with diphenylhydantoin.

Male and female chickens of a broiler-type strain were fed, from 1 day old to 5 weeks of age, diets containing 0, 2.5, or 15.0 p.p.m. (mg/kg) 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT). Then the diets with pesticide were withdrawn and the chickens were fed dietary levels of diphenylhydantoin (DPH) at 0, 100, or 250 p.p.m. Adipose-tissue and liver samples were obtained on days 0, 10, 20, and 30 following withdrawal of diets with pesticides to determine DPH effect on DDT, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) levels. DPH had no effect on the concentration of DDT and DDE in adipose tissue; their levels declined at a rate having a half-life value of 16 days. DDD was not detected in adipose tissue. DDT accounted for 87% of the adipose residues on day 0, but 66% of the residues at day 30. DPH had no effect on the concentrations of DDT and DDE in livers of chickens fed 15.0 p.p.m. DDT, but did significantly reduce the levels of DDD by 28 and 54% for levels of 100 and 250 p.p.m. DPH, respectively. The similarity of these data to studies on dairy cows and humans, and the dissimilarity to data from rat studies were discussed.     

Augmentation of thalamo - motor cortico - cerebellar epileptogenesis by taurine and its antagonism by diphenylhydantoin.

An acute penicillin focus was established in the motor cortex of cats. Surface recordings were obtained from the penicillin focus; focal potentials were recorded from the ipsilateral thalamic VL nucleus and the contralateral cerebellar Purkinje layer. The interictal spike relationships were 1:1:1 at these recording sites. The effects of systemically administered taurine were compared with those of DPH which was administered either systemically or topically onto the penicillin focus. Two epileptic attributes have been explored: thalamo-corticocerebellar ictal episodes (their incidence, durations and “tonic-clonic” manifestations), and interictal excitatory potentials in the Purkinje layer. The data show that taurine and DPH exhibited disparate effects on these parameters of epilepsy: 1. the incidence and durations of icti, and the proportions of “tonic-clonic” bursts were increased by taurine and decreased by DPH; and 2. the interictal excitatory potentials in the Purkinje layer were enhanced by taurine and reduced by DPH. Inasmuch as DPH effects are potent antiepileptic, taurine appears to be, at least a short-term, potent epileptogenic in this experimental model of epilepsy.     

The effect of diphenylhydantoin on the frequency of micronuclei in bone-marrow polychromatic erythrocytes of mice

Using the micronucleus test to evaluate the mutagenic effect of 5,5-diphenylhydantoin (DPH) on bone marrow polychromatic erythrocytes, male Balb-C mice were treated with the drug in single and multiple injection tests. A significant increase in the frequency of micronucleated polychromatic erythrocytes (MPE), P less than 0.05, was found when the mice received a single injection of DPH at doses of 0.5 and 1.0 mg/kg, and this frequency did not increase at higher doses. When mice were treated 3 times, at 24-h intervals, with 1.0 mg/kg of DPH, a significant increase in MPE was also observed (P less than 0.05) but this was lower than when they received a single injection of the same dose. A cytotoxic effect of NaOH, 0.1 N, which was used as solvent, was also observed either when alone or when DPH (1.0 mg/kg) was injected 3 times. This effect was comparable to the one produced by mitomycin C (MMC) at a dose of 0.5 mg/kg.     

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.     

Chromosomal damage in epileptics on monotherapy with carbamazepine and diphenylhydantoin

Summary We analyzed the leukocyte chromosomes of 10 epileptic probands on monotherapy with carbamazepine (CP), of 14 epileptic probands on monotherapy with diphenylhydantoin (DPH) and of 20 clinically normal probands (controls, CO). In the CP and in the DPH group we found a significant elevation of exchange-type aberrations as compared to the CO group. In the CP group we found predominantly chromatid translocations, in the DPH group exclusively dicentric chromosomes.     

The effect of diphenylhydantoin and cortisol on the cell cycle

Normal human lymphocytes cultured in the presence of phytohemagglutinin were blocked in G0G1 when diphenylhydantoin (DPH) or cortisol 3.6 X 10(-4) M was added at the beginning of culture. The suppression of culture growth was analyzed by flow cytometry and confirmed by [3H]thymidine incorporation and mitotic rate analysis. The correlation of these measurements with flow cytometry was good for DNA synthesis and excellent for mitosis. There was an additive effect on the G0G1 retention of cells when both drugs were present in the culture. These data may partially explain the suppression of cell-mediated immunity which occurs in DPH-treated patients.     

Effect of diphenylhydantoin administered during gestation and lactation on the motor development and cerebellar histology of the young rat]

Rattus norvegicus females were treated by diphenylhydantoin (D.P.H.), all along pregnancy and lactation. 4 groups were constituted: a 100 mg DPH/kg/day group, a 50 mg DPH/kg/day group; a placebo group (treated with pure water), and control group. D.P.H. was given twice a day by a gastric tube. The cerebellar Purkinje cells studied through light microscopy and transmission electron microscopy in young rats (25 days old) showed no visible alteration. 2 motorcoordination tests were applied to the young rats, during their 2nd and 3rd weeks of post-natal life. Young rats of DPH 100, DPH 50 and placebo groups showed a backwardness relatively to control. This backwardness may be attributed to the maternal forced feeding stress, but not to a specific action of the DPH.     

The effect of diphenylhydantoin upon degradation of sulphated macromolecules in cat palatal mucosa in vitro

The effect of diphenylhydantioin (DPH) upon the degradation of in vivo [35S]-sulphate-labelled proteoglycans was studied in cat palatal mucosa during organ culture. 8-week-old cats were injected intraperitoneally with [35S]-sulphate and 24 hours later the palatal mucosa was taken to organ culture. The release of radioactivity into the culture medium was taken as a measure of degradation of sulphated macromolecules, presumably proteoglycans, and the release of hydroxyproline as an indicator for collagen degradation. A parallel decrease in the release of radioactivity and in the release of hydroxyproline was observed when the culture was done in the presence of DPH (20 mg/l). Chromatography of the culture medium upon Sephadex G-25 revealed that the reduced release of radioactivity was due to a reduction of macromolecular degradation products leaving the amounts of free sulphate in the medium unchanged. The results were interpreted using a two compartment theory for proteoglycan degradation, extracellular breakdown of the protein core resulting in the production of macromolecular degradation products and intracellular lysosomal degradation resulting in free sulphate as the identifiable product. The results indicate that DPH inhibited the extracellular enzymatic degradation of proteoglycans without influencing their intracellular degradation.     

Interaction of ouabain and diphenylhydantoin (DPH) with the cardiovascular actions of prostaglandin B2 and prostaglandin A2

Several reports have appeared indicating that ouabain may interact at sites on smooth muscle susceptible to activation by prostaglandins. This study reports on the interaction between ouabain (0) and diphenylhydantoin (DPH) with the cardiovascular actions of prostaglandin B₂ (PGB₂) and prostaglandin A₂ (PGA₂). Fifteen μg/kg i.v. of 0 enhanced the pressor response of the canine hindpaw to norepinephrine and tyramine but did not affect the pressor responses to sympathetic nerve stimulation (SNS), PGB₂ or PGA₂. PGB₂-induced bronchoconstriction (mediated solely by stimulation of smooth muscle) was reduced by ouabain. In a separate group of animals not receiving PG before 0, 0 reduced (p<0.05) the pressor responses to PGB₂. DPH enhanced the cutaneous pressor responses to SNS, NE, PGB₂ and PGA₂ but did not affect the bronchoconstrictor response to PGB₂. These data are consistent with the following conclusions: 1) Ouabain antagonizes the smooth muscle contractions produced by PGB₂. 2) The presence of PGB₂ antagonizes the prostaglandin inhibitory effects of ouabain suggesting that PGB₂ may compete for similar sites or allosterically interact with ouabain in smooth muscle. 3) DPH induced enhancement of PGB₂ and PGA₂ induced vasoconstriction may reflect DPH induced enhancement of adrenergic neurotransmitter release or inhibition of transmitter reuptake.     

Teratogenic effects of dosage levels and time of administration of carbamazepine, sodium valproate, and diphenylhydantoin on craniofacial development in the CD-1 mouse fetus

The objective of this investigation was to study the teratogenic effects of dosage levels and time of administration of three anticonvulsant drugs (carbamazepine [CMZ], sodium valproate [NaV], and diphenylhydantoin [DPH]) on craniofacial development in the CD-1 mouse fetus. Pregnant females were intubated on each of days 8-10, 11-13, 14-16, and 8-16 of gestation with the following dose levels for each drug: 375, 563, 938 mg/kg CMZ; 225, 338, 563 mg/kg NaV; 50, 75, 125 mg/kg DPH. Appropriate control groups were maintained for each drug. On gestation day 17, pregnant females were killed and implantation sites were recorded as live, dead, or resorbed. All live fetuses were examined for craniofacial defects. Results of examination of 1,398 fetuses indicated that CMZ, NaV, and DPH were teratogenic and embryotoxic at all dose levels. This study indicated that the observed decrease in mean fetal weight was drug-, dose-, and time-dependent. There was a drug-, dose-, and time-dependent increase observed in the number of dead fetuses, whereas the number of resorbed fetuses was observed to be only time-dependent. The observed frequencies of hydrocephalies, secondary palatal clefts, and submucous palatal clefts were significant for all three factors (drug, dose, and time) whereas the observed frequencies of hematomas and exencephalies were significant only for drug and time. Cleft lips were observed only in the highest dose level of DPH. Uterine horn distribution of defects indicated that fetuses located at the proximal end of the horns were less subject to major defects than those fetuses located at the distal end of the uterine horns. Fetuses with craniofacial hematomas were found in the proximal one-third of the uterine horn, resorbed fetuses, and fetuses with submucous palatal clefts in the middle one-third of the uterine horns and dead fetuses and fetuses with exencephalies, cleft lips, and secondary palatal clefts were localized in the distal one-third of the uterine horns. In comparing the effect of drug, dosage, and time on the development of craniofacial malformations in the CD-1 mouse fetus, CMZ was the least teratogenic and embryotoxic of the three anticonvulsant drugs employed in this study.     

Direct evidence that an arene oxide is a metabolic intermediate of 2,2',5,5'-tetrachlorobiphenyl.

Radiolabeled arene oxide was recovered from incubations containing [³H]-2,2′,5,5′-tetrachlorobiphenyl (³H-TCB), unlabeled 2,2′,5,5′-tetrachlorobiphenyl-3,4-oxide (TCBAO), 3,3,3-trichloropropene-1,2-oxide (TCPO), NADPH, and liver microsomes from phenobarbital-induced rats. No labeled arene oxide was generated in the absence of NADPH, nor during the metabolism of unlabeled TCB in the presence of [³H]-H₂O. The recovered oxide (radiolabeled and carrier) was characterized by mobility on silica gel and by conversion to 3- and 4-hydroxy-TCB. Formation of a dihydrodiol metabolite was apparently blocked by inhibition of epoxide hydrase. These data provide the first direct evidence that arene oxides are intermediates of halogenated biphenyl metabolism.     

Membranes modification of differentiating proerythroblasts. Variation of 1,6-diphenyl-1,3,5-hexatriene lifetime distributions by multifrequency phase and modulation fluorimetry

The fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) in K562 cell membranes has been studied using multifrequency phase and modulation fluorimetry. The DPH decay data collected at various modulation frequencies were analysed by assuming either a model of discrete exponential components or a model of continuous lifetime distribution. The fits showed smaller values of the reduced chi square using the model of continuous lifetime distribution. The K562 cell membranes dynamics were investigated during the cell differentiation along the erythroid pathway. By using the continuous lifetime distribution method for the analysis of the DPH decay, marked variations were observed during the four initial days of the erythroid differentiation. Namely, the width of the DPH lifetime distribution increased by a factor of about two, while the center value of the distribution remained constant. By using the discrete exponential components model for the analysis of the DPH decay no variations were observed during the K562 differentiation.     

Bacterial Nitration of 4-Chlorobiphenyl

In the course of a study dealing with the biodegradation of 4-chlorobiphenyl by strain B-206, we noticed that the gram-negative bacterium accumulated different metabolic intermediates depending on the nitrogen source of the medium. Hence, in the presence of nitrate, strain B-206 produced four compounds which were identified as 2- and 4-hydroxy-4′-chlorobiphenyl and 2- and 4-hydroxy-mononitro-4′-chlorobiphenyl. The accumulation of these compounds in the culture medium indicated the presence of a monooxygenase in strain B-206 leading to the production of arene oxide intermediates. The possible transformation of 4-chlorobiphenyl to an arene oxide by this bacterial strain is a matter of concern because of the high reactivity of these arene oxides with biological material.     

The hypothalamic and neurohypophysial vasopressin content as influenced by diphenylhydantoin in dehydrated rats.

Rats dehydrated up to 8 days were treated with diphenylhydantoin given intraperitoneally in daily doses of 10 mg/100 g of the initial body weight. The single dose of diphenylhydantoin diminished the vasopressin content in the hypothalamus and neurohypophysis of normally hydrated rats. Under conditions of severe dehydration (8 days), DPH treatment resulted in a more marked decrease of vasopressin in the hypothalamo-neurohypophysial system.