Comparative embryotoxicities of butyl benzyl phthalate, mono-n-butyl phthalate and mono-benzyl phthalate in mice and rats: in vivo and in vitro observations

The embryotoxic effects of butyl benzyl phthalate (BBP) and its two main metabolites mono-n-butyl (MBP) and mono-benzyl (MBzP) phthalate were evaluated in OF1 mice and Sprague-Dawley rats, in vivo and in whole embryo culture. In vivo, pregnant mice and rats received a single oral dose (0.9-5.4 mmol/kg) of either of these compounds on GD 8 and 10, respectively, and their fetuses were examined externally on GD 18 and 21, respectively. In mice, BBP, MBP and MBzP caused concentration-related embryolethality and malformations. In rats, MBP and MBzP did not show developmental toxicity. Some teratogenicity and a slight increase in post-implantation loss were observed after BBP administration, but mice were more susceptible to its toxic effects than were rats. In vitro, GD 8 mouse embryos and GD 10 rat embryos were cultured for 46 h in the presence of the test compounds (0.5 to 3-5mM). The cultured mouse embryos did not appear intrinsically more sensitive to MBP and MBzP, than the rat embryos. Altogether, these results suggest that the species sensitivity observed in vivo after an oral administration of BBP, MBP or MBzP during early organogenesis, might be due to maternal factors, i.e. toxicity and/or kinetics.     

In vitro embryotoxicity of petroleum creosote monitored via mouse preimplantation embryo culture.

A mouse preimplantation embryo culture system was utilized to characterize the in vitro embryotoxicity of petroleum creosote (PC), a complex mixture of aliphatic and polycyclic aromatic hydrocarbons. ICR mouse embryos, collected on d 3.5 of gestation (blastocyst stage), were exposed for 1 h to varying concentrations of petroleum creosote in serum-supplemented culture medium. Parallel embryo cultures were exposed to PC in medium supplemented with rodent hepatic S9 microsomal fractions to monitor the role of bioactivation in PC-induced embryotoxicity. Embryos were subsequently cultured in control medium for 72 h and observed for viability as well as specific, time-dependent developmental end points--hatching and attachment to the culture dish at 48 h, and trophoblastic outgrowth with a distinct inner cell mass at 72 h. Embryonic viability varied in inverse proportion to PC concentration. Petroleum creosote caused embryolethal effects at concentrations of 33 micrograms/ml of culture medium and 54 micrograms/ml. Embryotoxicity was not observed at 22 micrograms/ml. Culture supplementation with rodent hepatic S9 fractions did not modify, either qualitatively or quantitatively, the embryotoxicity of PC in vitro. These findings implicate PC as a prenatal toxicant and support environmental and human health concerns regarding PC exposure from PC-containing chemical waste sites.     

Biotransformation of cyclophosphamide in post-implantation rat embryo culture using maternal hepatocytes in co-culture

The post-implantation rat embryo culture technique is employed to study embryotoxic effects of xenobiotic compounds in the absence of the maternal compartment. For compounds biotransformed in vivo the embryo culture technique must be adapted in order to mimick the in vivo effects. In the present study the possibility of co-culturing metabolically active maternal hepatocytes suspended in the standard culture system with rat serum as a medium was investigated. Cyclophosphamide (CP) was used as a model compound as it needs bioactivation to display embryotoxicity. Morphologic and histologic effects were studied. Neither hepatocytes nor CP alone affected embryo development, whereas in the presence of hepatocytes embryotoxicity was observed at 30 micrograms/ml CP. Embryotoxicity was decreased in the additional presence of metyrapone, a monoxygenase inhibitor. Hepatocyte suspensions prepared via slicing or perfusion of livers were equally effective. In conclusion, co-culture of embryos and suspended hepatocytes can be performed under optimal conditions for embryo development and in the presence of biotransforming activity.     

In vitro effects of folate derivatives on valproate-induced neural tube defects in mouse and rat embryos

The anticonvulsant drug valproic acid (VPA), produces neural tube defects in mouse and rat embryos treated in vivo or in vitro. The mechanism for the drug's embryotoxic effect is unknown, but 5-formyltetrahydrofolate has been reported to decrease the incidence of VPA-induced neural tube defects in mice treated in vivo. In the present study we have examined the ability of 5-formyltetrahydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate and folic acid to protect against VPA-induced neural tube defects in CD-1 mouse or CD rat embryos grown in a whole embryo culture system. Mouse embryos with 2–5 somite pairs were cultured for 48 hr beginning on gestation day 8; presomite stage rat embryos were cultured beginning on gestation day 9 (for both species gestation day 0 was taken as the day a vaginal sperm plug was found). VPA at 1.2 m (rats) or 1.8 m (mice) produced a high incidence of open neural tubes. None of the folate derivatives in concentrations up to 100 μg/ml was able to decrease the incidence of VPA-induced defects in either species. These data suggest that folate is not involved in the mechanism of VPA-induced neural tube defects.     

Development and sister chromatid exchange of mouse morulae and blastocysts cultured in rat serum containing active metabolites of cyclophosphamide

To establish an in vitro test system in which sera from animals treated with various chemicals can be tested for embryotoxic effects during the preimplantation period, mouse morulae and blastocysts were cultured in the presence of rat serum (RS) from animals which had been treated with cyclophosphamide (CPA). Development during in vitro culture for 96 h, cell number, chromosomal aberrations and sister chromatid exchange (SCE) were the end-points tested in exposed embryos. SCE frequency was the most sensitive parameter, indicating embryotoxic effects in preimplantation mouse embryos after only 1 h of exposure to RS-CPA.     

Change of embryotoxic susceptibility to di-n-butyltin dichloride in cultured rat embryos

Di-n-butyltin dichloride (DBTCl), which is commonly used as heat and light stabilizer for polyvinyl chloride (PVC) plastics, is a teratogen in vivo. In the present study, the toxic effects were investigated of DBTCl on cultured rat embryos during three different stages of organogenesis. Rat embryos explanted on gestational day (GD) 8.5, GD 9.5, and GD 11.5 were cultured for 68, 46, and 48?h and were exposed to a range of DBTCl concentrations for the first 24, 46, and the last 46?h of culture, respectively. Significant decreases in the placental diameter at ?10?ng/ml and in the number of somite pairs and the morphological score at 30?ng/ml were noted in embryos cultured from GD 8.5. Significant decreases in the yolk sac diameter and the crown-rump length at 100?ng/ml, in the number of somite pairs at ?50?ng/ml, and in the morphological score at ?30?ng/ml were found in embryos cultured from GD 9.5. No adverse effects on these parameters were detected in embryos cultured from GD 11.5 even at 300?ng/ml. Dysmorphogenesis in embryos cultured from GD 8.5, GD 9.5, and GD 11.5 was observed at ?10, ?50, and 300?ng/ml, respectively. Incomplete turning and craniofacial defects in embryos cultured from GD 8.5 and GD 9.5 and defects of the forelimb buds and tail in embryos cultured from GD 11.5 were frequently observed. These results show that in vitro exposure to DBTCl interferes with normal development of embryos during three different stages of organogenesis and that susceptibility to the embryo-toxicity, including the dysmorphogenic potential of DBTCl, varies with developmental stage.     

Oxidative stress status and development of late organogenesis stage rat whole embryos cultured from gestational days 13.5 to 14.5.

A new method for culturing rodent whole embryos at the late organogenesis stage was developed using a roller-bottle system with intermittent gassing. Rat embryos were cultured for 24 h from gestational day (GD) 13.5 to 14.5. Growth and metabolic comparisons were made between in vivo embryos and embryos of the same GD cultured under various media and conditions. Crown-rump length, head length and protein content were used as growth indicators. Biologic markers such as embryonic tissue concentration of glutathione (GSH), glutathione disulfide (GSSG) and lipid peroxidation were used as assessments of metabolic activity in terms of oxidative stress. Embryos cultured with media consisting of either 15% or 20% male rat serum and balanced with Dulbecco's Modified Eagle Medium (DMEM) were found to most closely match in vivo embryos. 6 h gassing intervals and 5 mL medium volume/embryo provided optimal conditions for cultured embryos. By shortening the 24 h embryo culture period to 12 h, embryonic haemorrhaging was avoided. Moreover, the 12-h cultured embryos showed similar redox GSH/GSSG ratios and similar GSH content to the in vivo embryos, which was not observed in the embryos cultured under 24 h culture conditions. The present work demonstrates the utility of late organogenesis stage embryo culture as a model for the assessment of in vivo embryonic growth and oxidative stress indices.     

Specific teratogenic action of 2,4,5-trichlorophenoxyacetic acid on mouse and rat whole-embryo cultures

The effects of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were studied at concentrations ranging from 0.17 to 3.52 m on post-implanted mouse and rat embryos cultured for 48 hr, either in the absence of any metabolic activation system or in the presence of mouse and rat S-9 mix. 2,4,5-T proved to be a potential teratogen, at 0.88 and 1.76 m , on mouse embryos in the absence of any metabolic activation system. In the presence of mouse S-9 mix, 2,4,5-T showed a high teratogenic potential at 0.17, 0.88 and 1.76 m . In contrast, in the presence of rat S-9 mix, 2,4,5-T induced structural defects only at 1.76 m . At 3.52 m , 2,4,5-T was 100% embryolethal with or without S-9 mix. On rat embryos, 2,4,5-T was potentially teratogenic only in the presence of mouse S-9 mix, causing a significant increase in dysmorphogenic effects at 0.17, 0.88 and 1.76 m . With or without rat S-9 mix, 2,4,5,-T was only embryolethal to rat embryos at 1.76 and 3.52 m . The abnormalities mainly involved the forebrain, the midbrain and the branchial arches. These results are consistent with the known in vivo embryotoxic action of this compound.     

In vitro embryotoxicity and teratogenic potential of etretinate and etretin

The embryotoxicity and teratogenic potential of etretinate and its main metabolite, etretin, with or without enzymatic drug activation, were investigated in vitro using the rat whole embryo culture system. The metabolizing system used was either a rat liver homogenate (S-9 mix) or esterase (carboxylic-ester hydrolase). Groups of 8–14 rat embryos (explanted on day 9.5 of pregnancy) were cultured for 48 hr with 0, 3, 10, 30 or 100 μg etretinate/ml with or without S-9 mix, or with 0, 0.03, 0.1, 0.3, 1, 3 or 10 μg etretinate/ml in the presence of 0.5 or 1 U esterase/culture flask. Yolk-sac diameters and vascularization and crown-rump and head lengths were unaffected by etretinate treatment alone but somite numbers and morphological scores were reduced at 30 and 100 μg etretinate/ml. The number of embryos with morphological anomalies was increased in the presence of 10 μg etretinate/ml but not significantly so. At higher concentrations the incidence of embryos with anomalies was 100%. Activation of etretinate in the presence of S-9 mix led to increased teratogenic activity of the test compound in vitro, although embryonic crown-rump and head lengths were comparable to control measurements up to the highest concentrations tested. When it was co-incubated with esterase, etretinate became 100 times more active, and its effects occurred in the same concentration range as the embryotoxic effects of etretin added to the culture directly. Without activation etretinate was approximately 100 times less active than etretin in producing general embryotoxicity as well as anomalies in the in vitro culture. The anomalies found after in vitro exposure of rat embryos to etretinate or etretin in this study resembled to a great extent the malformations found in vivo in several species including man. The pattern of anomalies was specific for the compounds used and was similar to that induced by other retinoids. The concentrations of etretin eliciting teratogenic effects in vitro were in the range of etretin peak plasma levels during etretinate therapy in man. Our results indicate that the whole-embryo culture system is a sensitive and valuable model for assessing the embryotoxic and teratogenic potential of retinoids in vitro.     

In vitro teratogenicity of acetylsalicylic acid on rat embryos: studies with various culture conditions

Rat embryos taken at day 9.5 of gestation were exposed in vitro to acetylsalicylic acid (aspirin) using various culture conditions. It was observed that embryos were sensitive to aspirin emulsified in olive oil at concentrations greater than or equal to 150 micrograms/ml. Between 43% and 66% of the embryos exhibited multiple malformations depending on the culture medium, 100% homologous rat serum or Waymouth medium supplemented with 50% rat serum, respectively. At concentrations greater than or equal to 400 micrograms/ml aspirin induced further toxic effects on embryo growth and differentiation. When gelatin was used as the drug-delivery system, aspirin at concentrations of greater than or equal to 150 micrograms/ml induced some malformations (mainly irregular somite shapes) in 57% of the embryos cultured in Waymouth medium, but in only 13% of the embryos grown in 100% serum. At concentrations which were greater than or equal to 400 micrograms/ml aspirin induced dysmorphogenic effects in all embryos, without any concomittant toxicity.     

Sex difference in Aroclor 1254 induction of rat hepatocytes: Consequences for in vitro embryotoxicity and mutagenicity of cyclophosphamide

The sequential culture of rat hepatocytes and post-implantation rat embryos has been proposed as a model for the in vitro testing of pro-teratogens. Comparing this model with a model in which embryos and hepatocytes are cultured simultaneously a striking difference in sensitivity was noted. To address the question of whether this difference could be explained by different sex and/or Aroclor 1254 pretreatment of the rats providing the hepatocytes, an experiment was designed with four groups: male Aroclor 1254 pretreated (M₁), male untreated, pregnant female Aroclor 1254 pretreated (F₁) and pregnant female untreated rats. Hepatocytes were incubated in the presence of cyclophosphamide (CP) and rat embryos were cultured in the media derived from the hepatocyte culture (i.e. the sequential culture model). Additionally, the CP concentrations of the media were analysed and subsequently the media were tested in a bacterial mutagenicity test (Salmonella typhimurium TA1535). With a CP concentration of 300 μ , M₁ produced maximum embryotoxicity and mutagenicity after 4 hr of hepatocytes incubation. All other groups showed no or only a slight increase in embryotoxicity and mutagenicity for all hepatocyte incubations. M₁ was also quickest to eliminate CP from the medium. These results indicate that despite a strong increase in total cytochrome P-450 in both sexes as a result of Aroclor 1254 pretreatment, and in the absence of a significant difference in total cytochrome P-450 between M₁ and F₁, Aroclor 1254 pretreatment has a much more pronounced effect in male rats than in pregnant female rats with regard to the production of embryotoxic and mutagenic metabolites of CP.     

Effects of 3-methylcholanthrene and phenobarbital on the capacity of embryos to bioactivate teratogens during organogenesis

Pregnant Sprague-Dawley rats were divided into four groups and given ip injections of 3-methylcholanthrene (MC) in corn oil, corn oil only, phenobarbital (PB) in Hank's balanced salt solution (HBSS), or HBSS only. Maternal animals were killed on Day 10 of gestation, and embryos from each group were explanted in medium containing cyclophosphamide (CP), 2-acetylaminofluorene (AAF), or dimethylsulfoxide vehicle. After a 24-hr culture period, embryos from dams treated with HBSS, corn oil, or PB/HBSS exhibited no increase in abnormalities (as compared with controls) when either CP or AAF were added to the media. However, embryos transplacentally preexposed to MC and subsequently treated during culturing with AAF (but not CP) exhibited striking increases in malformation incidence. Commonly observed malformations included abnormally open neural tubes, abnormal flexure rotation, and prosencephalic defects. Homogenates of Day 10 embryos transplacentally preexposed to MC exhibited readily measurable oxidative biotransformation of AAF as assessed with HPLC. Biotransformation of AAF by embryos from the other three groups was virtually undetectable. Incorporation of exogenously supplemented bioactivating systems from livers of mature animals indicated that postmitochondrial supernatant fractions (S-9) from male, MC-pretreated rats effectively catalyzed the conversion of AAF (but not CP) to embryotoxic metabolites. Conversely, hepatic S-9 from adult, male, PB-pretreated rats was highly effective in converting CP (but not AAF) to embryotoxic metabolites. The results indicated the inducerspecific occurrence of embryonic bioconversion of AAF to embryotoxic metabolites via MC-inducible, P-450-dependent, embryonic enzyme systems.     

Developmental toxicity of trichloroethylene,tetrachloroethylene and four of their metabolites in rat whole embryo culture

The embryotoxicity of trichloroethylene (TRI) tetrachloroethylene (PER), and of four of their oxidative metabolites i.e. trichloroacetic acid, dichloroacetic acid, chloral hydrate, and trichloroacetyl chloride, was studied in vitro, using the rat whole embryo culture system. Embryos from Sprague-Dawley rats were explanted on gestational day 10 (plug day=day 0) and cultured for 46 h in the presence of the test chemical. All of the tested chemicals produced concentration-dependent decreases in growth and differentiation and increases in the incidence of morphologically abnormal embryos. TRI and PER produced qualitatively similar patterns of abnormalities, while TRI and/or PER metabolites, each elicited clearly distinguishable dysmorphogenic profiles. The presence of hepatic microsomal fractions in the culture medium produced marked decreases in TRI- and PER-induced embryotoxic effects, including mortality, severity of malformations, and delayed growth and differentiation.     

Effects of mono-n-butyl phthalate on the development of rat embryos: in vivo and in vitro observations

The present study was conducted to further characterize the embryotoxic effects mono-n-butyl phthalate, a major metabolite of the plasticizer di-n-butyl phthalate, and evaluate its role in the developmental toxicity of di-n-butyl phthalate. The embryotoxic effects of mono-n-butyl phthalate were compared to those of the parent compound di-n-butyl phthalate after a single oral administration of 1.8, 3.6, 5.4, or 7.2 mmol/kg di-n-butyl phthalate or mono-n-butyl phthalate to Sprague-Dawley rats on gestational day 10 (Day 10). Embryos were evaluated for growth and development on Day 12. Both chemicals induced concentration-dependent developmental toxicity (i.e. decreased growth and malformations) which became apparent at 3.6 mmol/kg. Di-n-butyl phthalate and mono-n-butyl phthalate were approximately equally potent and produced qualitatively similar dysmorphogenic effects. Macroscopically, the most common malformations involved the prosencephalon, the optic system, and the mandibular and maxillary processes. In addition, the embryotoxic potential of mono-n-butyl phthalate was evaluated in vitro using the rat whole embryo culture system. Day 10 embryos were cultured for 48 hr in the presence of 0.5 to 5 mM mono-n-butyl phthalate and were then evaluated as the embryos grown in utero. Mono-n-butyl phthalate was a potent direct acting embryotoxicant, causing concentration-related growth retardation and dysmorphogenesis. The spectrum of morphological defects observed in mono-n-butyl phthalate-exposed embryos in vitro was comparable to those seen in vivo in the embryos at the same developmental stage after maternal administration of di-n-butyl phthalate or mono-n-butyl phthalate. These data provide additional evidence in support of the hypothesis that mono-n-butyl phthalate may be the active species for the developmental toxicity of ingested di-n-butyl phthalate in rats.     

利用小鼠全胚胎培养研究环磷酰胺致畸作用

本文应用小鼠全胚胎培养技术 ,通过妊娠 d8分别 ip5,1 0 ,1 5和 2 0 mg·kg-1环磷酰胺 ( CP) ,研究了该药对器官原基形成期胚胎的致畸作用 ,并对其致畸机理作了初步探索 .给药 4h后取胚胎进行培养 ,于 d 1 0 .5收获胚胎 ,测量其卵黄囊直径 ,头长及颅臀长并记录其大体形态的变化 .结果表明 ,1 5mg· kg-1组尾畸形率最高 ;2 0 mg· kg-1组生长迟缓率最高 .电镜观察所显示的细胞凋亡的形态学变化及 DNA琼脂糖凝胶电泳的结果均提示 CP致畸作用可能与其诱导的细胞凋亡有关 .     

Cytogenetic studies on preimplantation mouse embryos exposed to methylnitrosourea in vivo

Since exposure of mice to methylnitrosourea (MNU) during the preimplantation period can induce malformations and an increased postnatal death rate, direct embryotoxic effects were studied in preimplantation embryos shortly after treatment of pregnant mice on days 2 and 3 of gestation with single i.p. injections of 2.5, 5.0, and 10.0 mg/kg MNU. Embryos exposed to MNU for 24 h after treatment on day 2 showed a significant reduction of cell number and induction of sister chromatid exchange (SCE) frequency, but no structural chromosomal aberrations or inhibition of development during culture. Embryos exposed to MNU in vivo for 3 h on day 3 showed significantly reduced cell numbers, a significant inhibition of development in culture, and an increase in structural chromosome aberrations. Due to the high cytotoxicity of MNU, determination of SCE was not possible. The results indicate that MNU reaches preimplantation mouse embryos shortly after maternal treatment and that malformations seen at term and postnatal effects are probably induced by the direct action of MNU on early embryos. Furthermore, the importance of the time interval chosen for evaluation of toxicologic endpoints in preimplantation embryos is demonstrated.     

Lack of embryotoxicity of homocysteine thiolactone in mouse embryos in vitro

Recent work from humans and chick embryos has suggested that homocysteine may play a role in producing neural tube defects (NTDs). In an effort to determine if homocysteine is able to produce NTDs in mammalian embryos, mouse embryos were explanted on GD 8 and cultured for 44 h. When either homocysteine or homocysteine thiolactone was added to the culture medium, treated embryos developed as well as controls and had closed neural tubes. Homocysteine thiolactone was also microinjected into the amniotic sac of mouse embryos. Again, development proceeded normally with no significant increase in the number of embryos with open neural tubes at the end of the culture period. HPLC analysis of embryonic thiols 24 h after microinjection revealed a significant increase in embryonic cystathionine levels. These data suggest that homocysteine does not produce NTDs in mouse embryos cultured in vitro and that early organogenesis-stage embryos are able to metabolize homocysteine.     

Action of allopurinol and aspirin on rat whole-embryo cultures.

The effects of allopurinol (HPP) at concentrations ranging from 0.33-1.83 mM and of aspirin (ASA) from 0.28-2.22 mM, were studied on the rat whole-embryo culture system. Embryos were explanted at day 10 of gestation and cultured for 48 h, either in the absence or in the presence of rat and human S9. HPP proved to be potentially embryolethal and teratogenic without any S9, while it was embryolethal with rat S9 and dysmorphogenic with human S9. ASA showed an embryolethal and teratogenic potency without any S9 samples. These responses were increased in the presence of rat S9, while ASA embryolethality was predominant with human S9. These results obtained on rat embryos in culture suggest a correlation between the species origin of the biotransforming system and the known teratogenicity of HPP in sensitive animal models. However, ASA elicited responses not in agreement with the known teratogenic response in rodents.     

Successful in vitro growth of rat two-cell embryos to blastocysts using a simple chemically defined medium

The aim of the present study was to formulate a simple chemically defined medium for the in vitro growth of rat two-cell embryos to blastocysts. Embryos from day 2 pregnant rats were retrieved and placed in paraffin oil-covered droplets of "rat two-cell embryo culture medium" (R2ECM) containing combinations of various serum supplements, glucose, L-glutamine, and cultured up to 96 h in a CO(2) incubator. Embryos cultured in the basic medium (R2ECM), as well as those supplemented either with fetal bovine serum (FBS) or male rat serum (MRS) did not develop beyond the two- to four-cell stage. In R2ECM with 0.3% bovine serum albumin (BSA) and 7.5 mM glucose, 44% of embryos reached the blastocyst stage by 96 h in culture, and the blastulation rate increased to about 83% when 1 mM of L-glutamine was added. To evaluate the effects of varying doses of glucose, two-cell embryos were cultured in R2ECM supplemented with 0.3% BSA, 1 mM L-glutamine, and 2.5, 5.0, or 7.5 mM of glucose. The percentage of embryos reaching the blastocyst stage for 2.5, 5.0, and 7.5 mM glucose was 64.6%, 65.3%, and 82.9%, respectively. The present study showed that the modified medium (R2ECM) is a simple chemically defined medium that is capable of supporting in vitro growth of rat two-cell embryos to blastocysts in high proportion (greater than 80%) without the need for change of medium within 96 h of culture.