Teratological and cytogenetical evaluation of two antihistamines (pipethiadene and pizotifen maleate) in mice

The teratogenic and cytogenetic effects of two drugs with antihistaminic properties, Pipethiadene and Pizotifen maleate, were investigated. Three groups of pregnant mice were treated daily with oral doses (0.24, 0.6 and 1.2 mg/kg) of these drugs from day 4 to day 16 of gestation. The following parameters were investigated: reproductive health of the dams, external, skeletal and visceral malformations of fetuses and frequencies of micronuclei and chromosome aberrations in bone marrow cells of dams. Oral administration of Pipethiadene or Pizotifen maleate produced no teratogenic effects. No elevation was observed in the frequencies of micronuclei and chromosome aberrations. However, the significant reduction of fetal weight after all doses of Pipethiadene or Pizotifen maleate was found to correlate well with the decreased values of the mitotic indices of bone marrow cells of mice, suggesting a potential embryotoxic effect of the tested substances.     

Benzene-induced micronuclei formation in mouse fetal liver blood, peripheral blood, and maternal bone marrow cells

The transplacental cytogenetic effects of benzene were studied by using the micronucleus test of polychromatic erythrocytes (PCE) found in both fetal liver and fetal peripheral blood, and were compared with PCE from maternal bone marrow. Timed-pregnant mice received single intraperitoneal doses of benzene (0, 109, 219, 437, or 874 mg/kg bw) on the 14th day of gestation and were sacrificed 21 hr after injection. Benzene elicited a significant increase (P less than 0.01) in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in fetal liver blood cells (0.55 to 1.36%, control 0.18%) at doses of 219 to 874 mg/kg, and in fetal peripheral blood cells (0.49 to 0.58%, control 0.25%) and maternal bone marrow cells (0.53 to 0.70%, control 0.10%) at doses of 437 and 874 mg/kg. The data demonstrate that benzene is a moderate transplacental clastogenic agent, and that the mouse transplacental micronucleus test using fetal liver blood cells is a potentially more sensitive indicator of the genotoxicity of benzene than either fetal peripheral blood or maternal bone marrow cells.     

Transplacental genotoxicity of triethylenemelamine, benzene, and vinblastine in mice.

Transplacental cytogenetic effects of triethylenemelamine (TEM), benzene, and vinblastine on maternal mice and their fetuses have been investigated using micronucleus and sister chromatid exchange (SCE) as genetic endpoints. CD-1 mice were treated on day 14 and 15 of gestation with TEM (0.125, 0.25, and 0.5 mg/kg), benzene (439,878, and 1,318 mg/kg), and vinblastine (0.5, 1, and 2 mg/kg) by intraperitoneal injection at 24 hr intervals, and sacrificed 40 hr after the first injection. Erythrocytic precursor cells in maternal bone marrow and fetal livers (2-4) from each pregnant mouse were used for the micronucleus and/or the SCE analyses. Significant dose-related increases in both micronuclei and SCE were found in maternal bone marrow and fetal liver following TEM treatment. Benzene at the highest dose (1,318 mg/kg) also caused a significant increase in micronuclei and SCE in both maternal bone marrow and fetal liver cells. The embryonic genotoxic effect of TEM was much higher than that of benzene for both genetic endpoints, and the frequency of micronuclei induced by benzene was higher in fetal liver than in maternal bone marrow cells. Vinblastine, a spindle poison, induced micronuclei but not SCE. Micronuclei induction by vinblastine was 7 fold greater in maternal bone marrow than in fetal liver cells. All three chemicals were cytotoxic in maternal bone marrow cells, but not in fetal liver cells except for TEM, which showed a weak cytotoxicity in fetal liver cells in the micronucleus assay. These results indicate that TEM, benzene, and vinblastine are transplacental genotoxicants in mice.     

Induction of micronuclei in mouse bone marrow after combined X-rays-cyclophosphamide and X-rays-mitomycin C treatments.

The induction of micronuclei in polychromatic erythrocytes of bone marrow of Pzh:SWISS mice after combined treatment with X-rays and cyclophosphamide (CP) or X-rays and mitomycin C (MMC) were investigated. Combinations of high (1.00 Gy + 100 mg/kg bw CP and 1. 00 Gy + 5.25 mg/kg bw MMC) and low (0.25 Gy + 25 mg/kg bw CP and 0. 25 Gy + 1.75 mg/kg bw MMC) doses were used. Both chemicals enhanced the mutagenic effects caused by irradiation. After combined treatment with high doses of X-rays + CP and X-rays + MMC at different sample times increases in frequency of micronuclei were observed. Mutagenic effects were found also after treatment with two low doses, when irradiation alone produced no effects. The effects of combined treatments are generally similar to the additive effect of the single treatments.     

Sister chromatid exchanges and chromosomal aberrations induced by mitomycin C in mouse lymphocytes carrying a leukemogenic virus

The induction of chromosomal damage (sister chromatid exchanges (SCEs), chromosomal aberrations, and micronuclei) in T lymphocytes from mouse spleen was analyzed after treatment in vivo with different concentrations of mitomycin C (MMC). Lymphocytes were derived from BALB/Mo mice, which carry an endogenous type C retrovirus (Moloney murine leukemia virus, M-MuLV), and from BALB/c mice (controls, M-MuLV-free). Chromosomal damage was determined in vitro on lymphocytes stimulated with concanavalin A (Con A) and incubated for two generation cycles with bromodeoxyuridine (BUdR). The baseline frequency of SCEs was significantly higher in untreated BALB/Mo than in BALB/c lymphocytes. The frequencies of SCEs were significantly increased by increasing doses of MMC in both BALB/c and BALB/Mo T lymphocytes. Treatment with a low dose of MMC (0.3 mg/kg) produced an additive effect on SCE frequency in BALB/Mo lymphocytes, which was gradually suppressed by increasing the MMC concentration (3-5 mg/kg). Indeed, the levels of SCEs became significantly lower in BALB/Mo than in BALB/c lymphocytes at the highest MMC concentration tested (10 mg/kg), indicating that a negative synergistic effect was eventually produced. Chromosomal aberrations (breaks and total aberrations) were significantly increased by the highest MMC doses (5-10 mg/kg) and were more frequent in BALB/Mo than in BALB/c lymphocytes at 10 mg/kg MMC. The frequencies of micronuclei were increased by all MMC doses and were significantly higher in BALB/Mo than in BALB/c lymphocytes at 10 mg/kg MMC. These results are referred to interferences of M-MuLV and MMC with the function of enzymes, such as DNA topoisomerases, involved in the mechanism of SCE production.     

N-alkyl-N-nitrosourea induced secondary structural changes in DNA from rat embryos and fetal brains in vivo.

N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) are gestational stage dependent teratogens and transplacental carcinogens capable of inducing neurogenic tumours in rats. Intravenous treatment of gravid Wistar rats showed that MNU is teratogenic but ENU is a transplacental carcinogen and may be a teratogen when administered on day 12 of gestation. Twenty-four hours after single doses of 2, 5, or 10 mg MNU/kg on day 12, dose dependent decreases in embryonic wet weight and total embryonic DNA were observed. Rats similarly treated with 2 and 5 mg MNU/kg showed dose dependent decreases in fetal brain DNA synthesis, DNA content, and wet weight 9 days later. Administration of single ENU doses of 1.5, 3, 6, 12, 48, and 80 mg/kg to day 12 embryos resulted in a dose dependent reduction in [methyl-14C]-thymidine (14C-TdR) incorporation into DNA after 24 h although total DNA amounts and embryonic wet weights were unaffected. Benzoylated DEAE-cellulose (BD-cellulose) chromatography fractionates DNA on the basis of secondary structure by stepwise elution of double-stranded DNA with 1.0 M NaCl solution (SE-DNA) followed by elution of DNA containing single-stranded regions with caffeine solution (CE-DNA). Day 13 embryonic and day 21 fetal brain DNA was monitored by in vivo labelling with [methyl-3H]-thymidine on days 6 and 7 of gestation. Significant reduction in percentages of CE-DNA (%CE-DNA) 24 h after treatment of day 12 embryos with 2, 5, or 10 mg MNU/kg were attributed to the necrotic effect of MNU. Day 12 treatment with MNU produced no change in %CE-DNA values of day 21 fetal brains. A teratogenic dose of 80 mg ENU/kg to day 12 embryos resulted in significantly increased %CE-DNA values compared to controls but no changes were observed after 1.5 to 48 mg/kg. Analysis of the distribution of %CE-DNA values from the 80 mg ENU/kg treated litter showed that the increase in %CE-DNA was due to a second distinct population of embryos with higher %CE-DNA values than controls. Incorporation of 14C-TdR into embryonic and fetal brain DNA demonstrated the effects of treatment with these compounds on DNA synthesis in vivo. The relative %CE-DNA is expressed as the ratio of the percentage of caffeine-eluted 14C-labelled DNA to %CE-DNA (i.e., %CE-14C-DNA:%CE-3H-DNA). In the majority of control embryos the 14C-specific activity of CE-DNA was higher than the 14C-specific activity of SE-DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Micronucleus formation by benzene, cyclophosphamide, benzo(a)pyrene, and benzidine in male, female, pregnant female, and fetal mice

Male, female, pregnant female, and fetal ICR mice were compared for their acute sensitivity to four single doses of model carcinogens, as measured by micronucleus formation in polychromatic erythrocytes 24 h after treatment in adult bone marrow and fetal liver at days 17-19 of gestation. Cyclophosphamide caused a dose-responsive increase in micronuclei in all groups, without a consistent difference based on gender or pregnancy. At doses of 50 and 75 mg/kg given orally to the pregnant female, the fetuses were three to six times as sensitive as was the mother. Benzo(a)pyrene showed a similarly increased sensitivity of the fetus relative to the other groups, although it is a much weaker clastogen. Benzidine did not cause an increase in micronuclei in any group, although it was thought that the fetal liver might have been sensitive enough to detect it, relative to adult bone marrow. Benzene caused much less response in females than in males and almost no response in pregnant females and their fetuses, even though pregnant females metabolized at least half as much of the total dose as did the males (as measured by the presence of urinary metabolites of benzene). No single metabolite of benzene in the urine was consistently correlated with micronucleus formation in the bone marrow. Several factors must be interacting in different ways for different chemicals to influence their clastogenicity.     

Experimental model for evaluating the effects of genetic and exogenous factors on transplacental mutagenesis

Cyclophosphamide (CP)-induced micronuclei were evaluated infemales of two strains of mice (C57BL and CD1), in F₁ hybridfemales and in fetuses (day 13 of gestation) obtained from differentcrosses. F₁ adult hybrids from a cross between the strain witha high level of induced micronuclei (C57BL) and the strain witha low response (CD1) exhibited micronuclei values closer tothe latter. CD1/CD1 fetuses showed a higher susceptibility thanC57BL/C57BL ones. Heterozygous fetuses from reciprocal crosses,whatever the maternal genotype, showed the same sensitivity,which is very close to that of C57BL/C57BL fetuses. Phenobarbital(PB) pre-treatment modified the mutagenic response to CP dependingon the genotype of the treated animal. These results demonstratethat the response to a pro-mutagen requiring metabolic activationdepends to a large extent on the genetic background of the targetanimals, and that mother-fetus interactions in transplacentalmutagenesis seem to depend more on the fetal than on the maternalgenotype. ¹To whom correspondence should be addressed     

Quinoline-induced chromosome aberrations and sister chromatid exchanges in rat liver

Induction of chromosome aberrations and sister chromatid exchanges (SCEs) was studied in hepatocytes of F344 rats exposed in vivo to the hepatocarcinogen quinoline (Q). Hepatocytes were isolated 4–48 hr after a single dose of 200 mg/kg body weight or 24 hr after 28 repeated doses (once a day) of 25–200 mg/kg body weight/day by gastric intubation, and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 hr. A single dose of Q induced chromosome aberrations in up to 22% of metaphase cells, and SCEs with a frequency of up to 1.27 per chromosome 12 hr after the dose, while the control values were 1% and 0.63 per chromosome, respectively. Treatment with 28 repeated doses of Q induced significant chromosome aberrations and SCEs dose-dependently. Cytogenetic damage induced in the liver by repeated doses of Q was greater than induced by a single dose. Furthermore, Q induced replicative DNA synthesis in the liver, but failed to induce micronucleus formation in the bone marrow. The noncarcinogen 8-hydroxyquinoline was also examined and found to be essentially non-genotoxic to rat liver. These results show that Q is a genotoxic carcinogen to rat liver and the present method of in vivo cytogenetic assay should be useful for evaluating the genotoxicity of hepatocarcinogens. Environ. Mol. Mutagen. 30:459–467, 1997 © 1997 Wiley-Liss, Inc.     

TGFbeta2 in embryos with inborn anomalies: effect of maternal immunopotentiation

PROBLEM: TGFbetas are among the main immunoregulatory molecules contributing to successful embryonic development. Besides, our and other studies revealed that maternal immunopotentiation has a potential to increase the resistance of the embryo to the teratogenic insult. This work was designed to evaluate: (1) whether the formation of teratogen-induced anomalies is accompanied by an altered pattern of TGFbeta2 expression in embryonic cells and (2) whether maternal immunopotentiation modifies the pattern of TGFbeta2 expression in embryos responding to the teratogenic insult. METHOD OF STUDY: Experiments were performed in embryos of ICR mice exposed to 15 and 40 mg/kg of a reference teratogen, cyclophosphamide (CP) on day 12 of gestation. A group of mice was immunopotentiated with xenogeneic rat splenocytes 21 hr before the beginning of mating. Embryos were examined for the occurrence of gross structural anomalies 24 and 72 hr after CP treatment. Then, immunohistohemistry and in situ hybrydization assays were used to evaluate the expression of TGFbeta2 protein and mRNA in the brain, face, limbs and liver of these embryos. RESULTS: No external anomalies were observed in embryos examined 24 hr after CP treatment. Embryos examined 72 hr after CP treatment at 40 mg/kg exhibited agnathia, micrognathia, kinky tail, phocomelia, but no signs of dismorphogenesis were observed in the liver at the organ level. A significant increase in the expression of TGFbeta2 mRNA was observed in cells, residing in the brain, face and limbs but not in the liver of CP-exposed embryos tested 24 hr after CP injection in both doses. The level of TGFbeta2 protein in these embryos did not differ from that of controls. In embryos tested 72 hr after CP injection in the high dose both TGFbeta2 protein and mRNA expression were found to be elevated. Maternal immunopotentiation while enhancing the embryo's resistance to CP practically abolished an elevated expression of the TGFbeta2 mRNA detected in tested organ structures of embryos of non-immunopotentiated CP treated mice 24 hr after CP injection in both the low and the high doses. Also, a significant decrease in the level of TGFbeta2 mRNA expression was observed in embryos of immunopotentiated mice examined 72 hr after CP treatment. CONCLUSIONS: The results of this work show a possible involvement of TGFbeta2 in the formation of teratogen-induced structural anomalies and suggest that the stimulation of the maternal immune system may realize its protective effect by normalizing the level of TGFbeta2 expression in teratogen-targeted embryonic structures.     

Comparative teratogenic activities of two retinoids: effects on palate and limb development

Two closely related retinoids, all-trans and 13-cis retinoic acids, were assessed for their relative activities as teratogens in ICR mice by monitoring the frequency with which either isomer produced discrete dysmorphogenesis of the embryonic limb and the secondary palate. A single oral dose of all-trans retinoic acid at 100 mg/kg on either day 11.5 or 12.0 of gestation (plug day = day one) was maximally effective; more than 90% of the treated embryos developed reduction defects of the limb bones and an equally high percentage also had cleft palate. The limb development was most sensitive on day 11.5 of gestation while the peak susceptibility for palatal clefts began on day 12.0. Under identical experimental conditions, treatment with 100 mg/kg 13-cis retinoic acid produced no apparent teratogenic effects. By assessing the relative incidence of readily identifiable malformations of the limb and palate associated with various doses of the two isomers, we found that 13-cis retinoic acid was four to eight times less embryopathic than all-trans retinoic acid. Since the mechanism of teratogenic action of retinoids is still far from clear, it is suggested that further studies on causative factors will be greatly assisted by the use of these two closely related retinoids, which substantially differ from each other in their teratogenic potency.     

Transplacental mutagenicity of N-ethyl-N-nitrosourea at the hprt locus in T-lymphocytes of exposed B6C3F1 mice

Previous studies have compared age-related differences in total mutagenic burden in mice of differing age (preweanling, weanling, or young adult) after single intraperitoneal (i.p.) injections of ethylnitrosourea (ENU). The purpose of the present investigation was to determine the effects of time elapsed since treatment on the frequency of hprt mutant T-cells (Mf) from mice treated transplacentally with single acute vs. multiple split doses of ENU. To this end, pregnant C57BL/6 mice (n = 13-16/group), which had been bred to C3H males, were given i.p. injections of 40 mg ENU/kg bw in a single dose on day 18 of gestation, in a split dose of 6 mg ENU/kg bw on days 12 through 18 of gestation, or DMSO vehicle alone. Groups of pups were necropsied on days 10, 13, 15 (single dose only), 17, 20, 40, and 70 postpartum for T-cell isolations and hprt Mf measurements using the T-cell cloning assay. The time required to reach maximum Mfs in T-cells isolated from thymus of transplacentally treated animals was 2 weeks, the same time span as previously observed after ENU treatment of adult, weanling, and preweanling mice. Mfs in T-cells isolated from spleens of control animals averaged 2.1 +/- 0.3 (SE) x 10(-6). In spleens of mice treated transplacentally with ENU in a single dose, Mfs reached a maximum at 15 days postpartum [84.7 +/- 15.8 (SE) x 10(-6)] and decreased to lower but still elevated levels at 40 days postpartum. In spleens of mice treated transplacentally with ENU in a split dose, Mfs reached a maximum at 13 days postpartum [74.0 +/- 16.3 (SE) x 10(-6)] and decreased to background levels at 40 days postpartum. The areas under the curves describing the change in hprt Mfs over time for ENU-treated vs. control mice estimate the mutagenic potency for transplacental single- and split-dose exposures to be 1.9 and 0.8 x 10(3), respectively. Comparison of the mutagenic potency estimates for mice exposed to ENU in utero to 4-week-old mice given a similar dose of the same lot number of ENU indicates that the mouse is more susceptible to ENU-induced mutagenesis during fetal life.     

Comparative teratogenicity of triamcinolone acetonide, triamcinolone, and cortisol in the rat

Pregnant rats were injected im with 0.5 mg/kg triamcinolone acetonide (TAC) on day 12, 13, or 14 of gestation and the fetuses were examined for cleft palate on day 20. All three TAC-treated groups showed an increased proportion of fetuses with cleft palate compared to an untreated control group. Only the group treated on day 13 showed a significant increase in the proportion of litters affected. This indicates that day 13 of gestation is the most sensitive day for cleft palate induction by TAC in the rat. Pregnant rats were then treated on day 13 of gestation with either TAC, triamcinolone (TA), or cortisol. TAC was 59 times as potent as TA in inducing cleft palate, with ED50 values of 1.1 mg/kg and 65 mg/kg respectively. Cortisol induced a significant increase in cleft palates at 500 mg/kg, but the efficacy of this compound was too low to calculate an ED50 and relative teratogenic potency value. Other developmental abnormalities including umbilical hernias, resorption, and fetal death resulted from TAC treatment. Fetal growth retardation was produced by all three compounds. The rank order of teratogenic potency was determined to be TAC greater than TA greater than cortisol.     

Cytogenetic analysis of peripheral blood lymphocytes of children treated with nitrofurantoin for recurrent urinary tract infection.

The objective of this study was to determine whether nitrofurantoin, used for long-term antimicrobial prophylaxis of urinary tract infection, may induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) in lymphocytes of treated children. Ninety-nine blood samples were taken from 69 children aged from 0.2 to 13 years and suffering from urinary tract infection. The treatment consisted of oral administration of nitrofurantoin at doses of 5-8 mg/kg/day for the first 7 days and at doses of 1-2 mg/kg/day for the rest of the treatment period. Blood was sampled before the start of the nitrofurantoin therapy and after 1, 3, 6 and 12 months of the therapy. Analysis of variance showed no statistically significant increase in CA and SCE frequencies in lymphocytes of children treated with nitrofurantoin for 1-12 months. However, a significant increase in SCE rates was determined in lymphocytes of those patients whose blood samples were available both before and after treatment with nitrofurantoin (6.21 +/- 0.28 and 7.30 +/- 0.39 SCE/cell, respectively, P = 0.0315, Student's paired t-test). Moreover, there was a statistically significant correlation (r = 0.6603, P = 0.0270) between cumulative dose of nitrofurantoin and SCE frequency in lymphocytes of children after 1 month of the therapy. Also, in vitro experiments indicated that nitrofurantoin was able to induce both CAs and SCEs in human lymphocytes. Positive findings with chromosome aberrations and SCEs in vitro and suggestive results with SCEs in vivo indicate that further, much larger follow-up studies are needed to elucidate the genetic safety of the therapeutic use of nitrofurantoin.     

In vivo and in vitro inhibition of hepatic microsomal drug metabolism by ketoconazole.

Ketoconazole (KC), a broad spectrum antifungal drug, has been recognized recently as a cause of hepatic injury. The mechanism of the adverse reaction remains unclear: a metabolic idiosincrasy has been suggested. However as a substituted imidazole, KC might be expected to interfere with the hepatic microsomal mixed function oxidases. Ethylmorphine N-demethylase (E-DM) and aniline hydroxylase (A-OH) activities were determined in rat liver microsomes in the presence of increasing amounts of KC. Both were inhibited in an exponential fashion. The E-DM inhibition was almost complete at concentrations greater than 250 microM and was of the mixed type. A much weaker effect was observed for A-OH. A significant inhibition of E-DM was also observed when KC was administered in vivo to rats either orally for 7 days at the dose of 100 mg/kg/day (P less than 0.02) or intraperitoneally for 4 days at the dose of 50 or 100 mg/kg day (P less than 0.01 or P less than 0.001 respectively). A-OH activity was significantly reduced (P less than 0.01) only after ip administration of 100 mg/kg/day of the drug for 4 days. Neither the amount of cytochrome P-450 nor NADPH cytochrome c reductase activity were affected at the doses considered. These data show that KC interferes with hepatic oxidative drug metabolism and suggest that this mechanism might be involved in the unwanted side effects of therapy with KC.     

In vivo and in vitro inhibition of hepatic microsomal drug metabolism by ketoconazole

Ketoconazole (KC), a broad spectrum antifungal drug, has been recognized recently as a cause of hepatic injury. The mechanism of the adverse reaction remains unclear: a metabolic idiosincrasy has been suggested. However as a substituted imidazole, KC might be expected to interfere with the hepatic microsomal mixed function oxidases. Ethylmorphine N-demethylase (E-DM) and aniline hydroxylase (A-OH) activities were determined in rat liver microsomes in the presence of increasing amounts of KC. Both were inhibited in an exponential fashion. The E-DM inhibition was almost complete at concentrations greater than 250 microM and was of the mixed type. A much weaker effect was observed for A-OH. A significant inhibition of E-DM was also observed when KC was administered in vivo to rats either orally for 7 days at the dose of 100 mg/kg/day (P less than 0.02) or intraperitoneally for 4 days at the dose of 50 or 100 mg/kg day (P less than 0.01 or P less than 0.001 respectively). A-OH activity was significantly reduced (P less than 0.01) only after ip administration of 100 mg/kg/day of the drug for 4 days. Neither the amount of cytochrome P-450 nor NADPH cytochrome c reductase activity were affected at the doses considered. These data show that KC interferes with hepatic oxidative drug metabolism and suggest that this mechanism might be involved in the unwanted side effects of therapy with KC.     

Methotrexate increases valproic acid-induced developmental toxicity, in particular neural tube defects in mice.

The hypothesis that valproic acid-induced dysmorphogenesis may be due to an interference of this drug with folate metabolic pathways was further investigated by a study of a possible interaction of valproic acid (VPA) and the established folate antagonist methotrexate (MTX). The dihydrofolate reductase inhibitor MTX (1.25 and 2.5 mg/kg, i.p.) was injected 15 min prior to VPA (300 and 400 mg/kg, s.c.) in day 8 pregnant NMRI mice. Fetuses were examined for exencephaly, resorption, and fetal weight retardation on day 18 of gestation. MTX produced no exencephaly or reduction in fetal weight, and the 2.5-mg/kg dose caused 56% resorption. Higher doses (5-20 mg/kg) produced embryolethality and fetal weight retardation, but no exencephaly. VPA (300 and 400 mg/kg) administration resulted in 3.4% and 12.6% exencephaly and 9% and 19% resorptions, respectively. Coadministration of MTX with VPA significantly increased VPA-induced resorption and exencephaly rates as well as fetal weight retardation. Exencephaly induced by VPA 400 mg/kg was increased to 29.5% and 24.1% (P < 0.01 and P < 0.05) when given with 1.25 and 2.5 mg/kg MTX, respectively. MTX (2.5 mg/kg i.p.) did not alter transplacental VPA (400 mg/kg, s.c.) pharmacokinetics. These results support the view that VPA-induced teratogenesis may be mediated by interaction with folate metabolism.     

Clastogenic effects of transplacental exposure of mouse embryos to nitrogen mustard or cyclophosphamide

Embryos from day 12 pregnant Swiss mice given intraperitoneal injections of nitrogen mustard (HN2) or cyclophosphamide (CP) were evaluated for chromosomal aberrations. Both agents induced dose-dependent increases in the frequency of cells with aberrations observed in embryos from females treated 6 hr before sacrifice. The highest frequencies of cells with aberrations were observed when females were injected 15 or 18 hr before sacrifice on day 12. A teratogenic dose of HN2 (1.0 mg/kg) induced significantly higher frequencies of damaged cells than a teratogenic dose of CP (20 mg/kg). Cytogenetic analysis of rodent embryos from pregnant females exposed to xenobiotic agents may be an effective screening test for evaluation of genetic effects induced by transplacental exposure.     

Cell cycle alterations and cell death in cyclophosphamide teratogenesis

Litters of pregnant mice treated with cyclophosphamide (CP) exhibit malformations of the limbs ranging from oligodactyly to amelia. Previous studies have indicated that cell death occurs in limb buds shortly after maternal exposure. We have investigated the relationship of cell death, cell cycle perturbation, and embryo/fetal toxicity in the mouse using vital staining and flow cytometry (FCM). CP (20, 30, and 40 mg/kg) was investigated via intraperitoneal administration to Swiss-Webster mice on day 10 of gestation. At 4, 8, or 28 hours later, embryos were removed. Cell death was identified with Nile blue sulphate (NBS). Two embryos per litter were stained with NBS, and the remaining embryos were frozen at -70 degrees C prior to FCM analysis. After thawing, the forelimb buds were removed for the isolation of nuclei. Tissues were dissociated through a wire mesh followed by cytolysis with 0.1% nonidet P-40 in PBS with 0.5 mg/ml RNase. Nuclei were stained with the fluorescent nucleic acid probe propidium iodide and analyzed (10,000 nuclei per sample) for propidium iodide fluorescence by FCM. NBS revealed a dose-related increase in cell death by 8 hours after dosing. CP-induced cell death was greatest in areas of rapid cell proliferation (DNA synthesis). FCM analysis revealed retardation of progression through the S-phase of the cell cycle by 4 hours post-exposure at all doses. This retardation occurred earlier in S-phase with increasing dose and persisted through 8 hours. At 28 hours, cell cycle histograms were normal in the low-dose embryos, but remained perturbed in the intermediate- and high-dose embryos. On day 17 of gestation, the last group of dams was killed. A high incidence of fetal malformations, including limb defects, occurred at the 20 mg/kg dose, and fetal mortality was observed at 30 and 40 mg/kg. The pattern and magnitude of cell death correlated with cell cycle perturbation and fetal toxicity at term, suggesting a relationship between cell cycle perturbation, cell death, and malformations produced by CP.