Ameliorative Effect of Folic Acid on Pyrimethamine Teratogenesis in Pigs

Abstract The effects of oral dosing of folic acid (FA) on pyrimethamine (PYR) teratogenesis were examined in Goettingen minipigs. PYR (3.6 mg/kg/day) was orally administered in the feed to sows with or without FA (2.5, 10 or 50 mg/kg/day) for 12 days, (days 11 - 22 of gestation). Major malformations, such as cleft palate, cleft lips, micrognathia and clubfoot, were observed in all treated groups. The incidence of malformed fetuses decreased dose-dependently as the FA level was raised. This new finding suggests FA administration may prevent newborns from PYR induced malformations. This result contrasts in the observations in rats, in which PYR teratogenesis was potentiated by the concurrent oral dosing of FA. Neither PYR blood concentrations nor plasma protein binding was significantly affected by FA dosing in nonpregnant sows. These results suggest a mechanism other than pharmacokinetic modification was responsible for the protective effect of FA on PYR teratogenesis in minipigs.     

Correlation of Active Folate Availability with Effects of Folic Acid on Pyrimethamine Teratogenesis in Rats

Oral administration of pyrimethamine (PYR) 3.6 mg/kgBW/day to the pregnant rats from day 11 to day 15 of gestation caused malformations in all the fetuses, but concomitant intraperitoneal administration of folic acid (ip FA) 50 mg/kg/day inhibited the incidence of malformations by 25%. On the other hand, the same dose of oral FA (in feed) had an opposite effect on PYR teratogenesis; the incidences of malformed fetuses were 0% in the PYR 1.6 group, but 100% in the PYR with oral FA group. The plasma levels of 5-methyltetrahydrofolic acid (5MF), a principal transfer form of folate, were measured after single oral administration of PYR with or without FA (ip or oral) in non-pregnant female rats. The area under the plasma concentration-time curve (AUC) after the concomitant administration of PYR with ip FA was larger than that after PYR alone. This result indicated that the available 5MF for the peripheral tissues was increased by the concomitant administration of ip FA. The AUC after the concomitant administration of PYR with oral FA was smaller than that after PYR alone. These results suggested that ip FA increased the available 5MF to ameliorate, but oral FA decreased the availability to aggravate the PYR teratogenesis.     

Effects of Folic Acid and Folinic Acid on Pyrimethamine Teratogenesis in Rats

Abstract The effects of folic acid (FA) and folinic acid (FNA) on the teratogenicity of the antifolate drug pyrimethamine (PYR) were examined in rats. PYR and FA were orally administered with mashed feed. FNA was intraperitoneally injected. The drugs were administered for 5 days of day 11–15 of gestation. Six groups were made; G-I PYR 1.6 (mg/kg/day), G-II PYR 3.6, G-HI PYR 1.6 + FA 50, G-IV PYR 3.6 + FNA 12, G-V FA 50, G-VI control. No malformations were found in G-I, IV, V and VI. All fetuses in G-II and III had malformations. Main malformations in G-II and III were cleft palate and brachygnathia. Accordingly it might be demonstrated that the teratogenicity of PYR was potentiated by concurrent FA in rats.     

Effects of Folic Acid and Folinic Acid on Pyrimethamine Teratogenesis in Rats

ABSTRACT  The effects of folic acid (FA) and folinic acid (FNA) on the tera-togenicity of the antifolate drug pyrimethamine (PYR) were examined in rats. PYR and FA were orally administered with mashed feed. FNA was intraperitoneal-ly injected. The drugs were administered for 5 days of day 11–15 of gestation. Six groups were made; G-I PYR 1.6 (mg/kg/day), G-II PYR 3.6, G-HI PYR 1.6 + FA 50, G-IV PYR 3.6 + FNA 12, G-V FA 50, G-VI control. No malformations were found in G-I, IV, V and VI. All fetuses in G-II and HI had malformations. Main malformations in G-II and III were cleft palate and brachygnathia. Accordingly it might be demonstrated that the teratogenicity of PYR was potentiated by concurrent FA in rats.     

Potentiated Embryotoxicity of Pyrimethamine by Folic Acid in Mice

ABSTRACT Effects of folic acid (FA) and folinic acid (FNA) on the embryotoxicity of pyrimethamine (PYR), an antifolate drug, were examined in mice. PYR and FA were administered orally, and FNA was injected intraperitonially for 7 days from day 9 to 15 of pregnancy. The incidence of embryotoxicity including intrauterine deaths and malformations, was 33.5 % in the PYR 50 mg/kg/day group. However, all the fetuses were resorbed in the PYR 50 + FA 50 mg/kg/day group, and FA potentiated the embryotoxicity of PYR. On the contrary, FNA reduced the embryotoxicity of PYR (PYR 50 + FNA 10mg/kg/day; 8.5%). Plasma concentrations of PYR and 5-methyltetrahydrofolic acid (5MF), a principal form of folate in plasma, were determined after single oral administration of PYR 50 mg/kg with or without FA 50 mg/kg to non-pregnant mice. Plasma 5MF concentration decreased drastically in the group receiving PYR with FA, compared with the PYR alone group. The pharmacokinetics of PYR were not affected by co-administration of FA. Therefore, we consider that the potentiated embryotoxicity of PYR by oral FA results from a decrease of plasma 5MF concentration in dams.     

Modified Susceptibility to Teratogenesis in the Offspring of Male Mice Exposed to Mutagens

Preconceptual exposure of male mice to a mutagen can increase the incidence of malformations in the offspring. In mutagenized spermatogonial stem cells, genetic changes responsible for F₁ malformations were produced in a manner similar to that involved in the production of specific locus mutations. Irrespective of the kind of mutagens applied, the germ cell stage treated and the strain of mice used, resulting F₁ malformations showed type distributions that were indistinguishable from the spectrum of those occurring spontaneously. When a teratogen was applied at the organogenic stage, embryos whose sires were preconceptually exposed to a mutagen showed higher susceptibility to induced malformations than those derived from untreated males. Experimental results pertaining to these contentions are summarized and the possible implications are discussed.     

Effect of Folic Acid on Pharmacokinetics of Pyrimethamine in Rats

The effect on pyrimethamine (PYR) pharmacokinetics of folic acid (FA) which potentiated the PYR teratogenesis was studied in rats. Gavage administration of PYR 1.6 mg/kgBW/day from day 11 to day 15 of gestation did not cause any malformation, but with concomitant dosing of FA 50 mg/kg/day in feed caused malformations in 75% of fetuses. Neither the plasma concentration-time profile nor the plasma protein binding of the gavaged PYR was affected by the concomitant dosing of FA in the non-pregnant rats. The pregnant rats which received the gavaged PYR with or without FA in feed for 5 days (from day 11 to day 15 of gestation) were killed at 4 or 12 hrs after the last dosing of PYR, and their plasma and fetuses were subjected to determination of PYR. The PYR concentration in the maternal plasma was comparable in both groups. The PYR concentration in fetuses of the PYR alone group was significantly higher than that of the PYR with FA group 4 hr after but not 12 hr after the last dosing. These results indicated that the pharmacokinetics of PYR was not affected by the concomitant dosing of FA, suggesting that FA potentiated the PYR teratogenesis by other mechanisms than pharmacokinetic modifications.     

Effects of Pyrimethamine and Folic Acid on Plasma Level of 5-Methyltetrahydrofolic Acid in Rats

Abstract: The relationship between pyrimethamine (PYR) teratogenesis and the plasma level of 5-methyltetrahydrofolic acid (5MF), known as an active form of folate in plasma, was studied in rats using an HPLC-ECD method for 5MF determination. The rat received, for 5 days, in-feed PYR with or without folic acid (FA), which was previously reported as a potentiator of PYR teratogenesis. PYR alone caused a dose-dependent decrease in the plasma 5MF level after the 5 day dosings. A potentiated decrease in the 5MF level was observed after the concomitant dosing of PYR with FA. The concomitant dosing of PYR 1.6 mg/kgBW/day (subteratogenic dose of PYR alone) with FA 50 mg/kg/day, which was 100% teratogenic dose in our previous report, decreased the 5MF level more than that after the dosing of PYR 3.6 mg/kg/day alone (100% teratogenic dose of PYR alone).
The sequential changes of the plasma 5MF level after the single oral dosing of PYR 3.6 mg/kg with or without oral FA 50 mg/kg also revealed a strong potentiative effect of oral FA on the 5MF-level-lowering effect of PYR. These results indicate that a decrease in the plasma 5MF level may be related to PYR teratogenesis.     

Male-Mediated Developmental Toxicity: Enhanced Susceptibility to Induced Teratogenesis in the Offspring of Male Mice Treated with Ethylnitrosourea

Male ICR strain mice were injected intraperitoneally with ENU at 50 mg/kg daily for 5 days and mated to untreated virgin females of the same strain on days 64–80 after the last dose. Copulations during this period involved spermatogonial stem cells at the time of the last treatment. Subsequently, copulated females were injected intraperitoneally with ENU at 25–100 mg/kg on day 8 of gestation, at 50–200 mg/kg on day 12 of gestation or injected subcutaneously with triamcinolone acetonide at 1.25–10 mg/kg on day 12 of gestation. The uterine contents were examined on day 18 of gestation. Fetuses of dams treated on day 8 of gestation were inspected for external and skeletal abnormalities, and those of dams treated on day 12 were inspected for external abnormalities including cleft palate. Frequencies of microphthalmia and cleft palate in the group in which females mated with ENU-treated males were treated with ENU at 50 mg/kg on day 8 of gestation or with ENU at 50 mg/kg on day 12 of gestation, respectively, were significantly higher than those in the group in which females mated with phosphate buffer-treated males were treated with ENU at 50 mg/kg on day 8 or at 50 mg/kg on day 12. No significant increases in the frequency of cleft palate were observed in the groups in which females mated with ENU-treated males were treated with triamcinolone acetonide on day 12 of gestation as compared with groups in which females mated with phosphate buffer-treated males were treated with triamcinolone acetonide on day 12 of gestation. These results suggested the increased susceptibility to induced teratogenesis (congenital malformations induced by exposure of embryo/fetus during gestation) in the offspring derived from paternal germ cells treated with the potent mutagen ENU, but not the non-mutagen triamcinolone acetonide.     

Comparative Susceptibility of a Wild-Derived Strain of Mus musculus molossinus and Laboratory Mice to Teratogenesis by Ethylnitrosourea

ABSTRACT A wild-derived strain of Japanese house mice, Mus musculus molossinus (MSM), was compared with laboratory mice (DBA/2N, C57BL/6, C3H/He, BDF₁, and ICR) for the susceptibility to teratogenic effect of ethylnitrosourea (ENU). ENU was applied i.p. to mice in each strain on day 7 of gestation. On day 18 of gestation, uterine contents were examined and viable fetuses were inspected for external and skeletal malformations. The dose (mg/kg) required to induce 25% embryonic lethality (LD₂₅) was 46 in MSM and 28–54 in the laboratory mice. The frequencies of external and of skeletal malformations at LD₂₅ were, respectively, 8 and 47% in MSM, 29–97% and 65–95% in the laboratory mice. Thus, MSM was less susceptible to ENU teratogenesis compared to any of the laboratory mice used and the difference in the susceptibility between MSM and the laboratory mice was more marked for external malformations than for skeletal malformations. These results are compatible with the hypothesis that laboratory mice may have genetic traits that facilitate teratological assays of drugs.     

Micrencephaly in Rats Caused by Maternal Hyperthermia on Days 13 and 14 of Pregnancy1

ABSTRACT: Hyperthermia was induced in rats twice at an interval of eight hours on both days 13 and 14 of gestation by partial immersion in a water-bath. Deep rectal temperature was raised to 42.5°C for five minutes. Body weights of newborn rats were reduced, but by three weeks of age compensatory post-natal growth had eliminated the defect. In both newborn and 21-day-old rats, brain weight was significantly reduced compared with controls of similar body weight. No other anomalies were found.     

The Epidemiology of Lost Residual β-cell Function in Short Term Diabetic Children

ABSTRACT. Using the country-wide Swedish childhood diabetes register 526 children, who had had diabetes for 6–30 months were traced for measurements of 24-hour urinary C-peptide. Lost ft-cell function was defined as a 24-hour urinary C-peptide excretion per kg body weight less than 10% of the mean for healthy children (<0.025 nmol/kg). The estimated cumulative incidence of lost β-cell function was 0.64 at 30 months. The incidence of lost β-cell function did not differ by sex. Neither was there any significant variation in season at onset for cases with lost β-cell function. A significant age dependency was shown for the cumulative incidence of lost β-cell function with the highest incidence in the young age groups, i.e. a reversed age dependency compared to that of clinical onset. In contrast to the clinical onset of diabetes no significant geographical variation was found for lost β-cell function when comparing standardized morbidity ratios. The urinary C-peptide excretion was significantly correlated to age at onset but not to degree or duration of ketonuria at onset. It is concluded that there are striking differences when comparing the epidemiology of lost β-cell function to that of clinical onset in terms of age, sex, seasonal and geographical variations. The timing of clinical onset may thus partly be determined by factors different from those determining the rate of fall in β-cell function.     

Congenital Diaphragmatic Hernia Induced by Nitrofen in Mice and Rats: Characteristics as Animal Model and Pathogenetic Relationship between Diaphragmatic Hernia and Lung Hypoplasia

Abstract Congenital diaphragmatic hernia (CDH) and lung hypoplasia were induced in high frequency and dose-dependently in the offspring from dams, treated orally with 2,4-dichlorophenyl- p -nitrophenyl ether (nitrofen) during pregnancy in CD-I mice and CD rats. Both in mice and rats, CDH found in the fetal and neonatal periods was a posterolateral type of diaphragmatic hernia (DH) showing a distinct side-preponderance: the left-side preponderance in mice and right-side preponderance in rats. CDH in the offspring, surviving after weaning, was mostly of retrosternal type in mice and of pericardial type in rats. CDH induced experimentally in the present study can be regarded as an excellent animal model for human CDH in terms of both anatomical features and the time of appearance of different types of CDH as well as clinical symptoms.
Lung hypoplasia was observed in the offspring with and without CDH; though its severity was greater in those with CDH. The offspring with severe lung hypoplasia died of respiratory insufficiency during the neonatal period, regardless of the presence or absence of CDH. These findings suggest that lung hypoplasia is not a consequence of CDH, but that a common pathogenetic process in the early embryonic stage might involve both lung hypoplasia and CDH.     

Atrioventricular Septal Defect in Rat Fetuses Induced by Administration of Bisdiamine

ABSTRACT A high incidence of atrioventricular septal defect was induced in rat fetuses by administration of N, N -his (dichloroacetyl)-1, 8-octamethylenediamine, bisdiamine, to pregnant Donryu rats. Induction of atrioventricular septal defect was highly dependent on day of bisdiamine administration. When bisdiamine was administered singly on day 9.4 of gestation or consecutively on days 9 and 10 of gestation, the incidence of atrioventricular septal defect in fetuses increased. Survival rate decreased with increase in the amount of bisdiamine administered. Normalized incidence ⁵ for atrioventricular septal defect, which was calculated as a product of incidence and survival rate, was as high as 54.5% when 175 mg bisdiamine was administered on day 9.4 of gestation. Atrioventricular septal defect thus formed was of the complete type associated with either persistent truncus arteriosus or tetralogy of Fallot, indicating that the conotruncal portion was unexceptionally malformed. The present model is useful for analysis of development and pathogenesis of atrioventricular septal defect and also contributes to clarifying the normal development of atrioventricular valves and septa.     

Therapeutic Efficacy and Safety of GH in Japanese Children with Down Syndrome Short Stature Accompanied by GH Deficiency

In this study, we investigated the effects of GH treatment in children with Down syndrome who had been diagnosed with GH deficiency (GHD). A total of 20 subjects were investigated in this study. Fourteen Down syndrome children (5 boys and 9 girls) with short stature due to GHD were treated with GH at Okayama Red Cross General Hospital, and 6 Down syndrome children (4 boys and 2 girls) with short stature due to GHD were registered in the Pfizer International Growth Database (KIGS). Height SD score (SDS) increased throughout the three-year GH treatment period. The overall mean height SDS increased from –3.5 at baseline to –2.5 after 3 yr of treatment. The mean change in height SDS during these 3 yr was 1.1. In addition, height assessment of SD score based on Down syndrome-specific growth data in the Japanese population revealed that the height SDS (Down syndrome) also increased across the 3-yr GH treatment period. The mean change in height SDS (Down syndrome) during these three years was 1.3. GH therapy was effective for Down syndrome short stature accompanied by GHD, and no new safety concerns were found in this study.     

STUDIES OF URINARY TRACT INFECTIONS IN INFANCY AND CHILDHOOD X. Short or Long-term Treatment in Girls with First or Second-time Urinary Tract Infections Uncomplicated by Obstructive Urological Abnormalities

The aim of the present investigation, concerned with 279 girls with their first-time, or in a few cases second-time urinary tract infection, was to explore the possibility to improve the “1-year cure rate” by means of long-term sulfonamide therapy (2 months) instead of short-term sulfonamide therapy (10 days). A “1-year cure” was then defined as an eradication of the index infection and freedom from infection during the following year. The “1-year cure rate” was found to be equal in the two therapy groups i. e. 64 per cent. The material showed some characteristics which might permit some general statements about uncomplicated first or second-time urinary tract infections in girls. The material showed a marked predominance of the younger age groups. This finding is discussed with regard to the pathogenesis of urinary tract infection. The bacteria isolated at the initial infections were in 90 percent E. coli and also in 90 per cent sensitive to sulfonamide. Thus for the present there is no reason to abandon sulfonamide as the drug of choice in uncomplicated first-time infections in girls. The first recurrence had a tendency to appear shortly after the original infection rather than late. Observations suggest that recurrences, even those occurring immediately after cessation of therapy, were usually a reinfection and not a recrudescence. These findings are discussed in relation to the pathogenesis of recurrent urinary tract infections. The first recurrence was in 1/3 of the cases asymptomatic, in the other 2/3 symptomatic. During long term prophylaxis asymptomatic infections even outnumbered the symptomatic ones. Therapy thus may mask an infection. The bacteria isolated at the first recurrences after the index infection were in 30–90 per cent resistant to sulfonamide, the rate being related to the time interval between the cessation of therapy and the recurrence. The therapeutic consequences of the difference in sulfonamide sensitivity of the initial infection and the recurrences are discussed.