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.     

Increased sister-chromatid exchange frequency in preimplantation mouse embryos after maternal cyclophosphamide treatment before implantation

The effect of a toxic agent in vivo on sister-chromatid exchange (SCE) frequency of preimplantation mouse embryos and bone marrow cells was determined using combined in vivo treatment and in vitro culture in the presence of 5-bromo-2-desoxyuridine (BrdU) for differential staining of the chromatids. In mice exposed to cyclophosphamide (CPA) on day 2 of pregnancy SCE frequency was increased dose-dependently both in embryos and bone marrow cells 1 h after treatment. It returned to control values in bone marrow cells obtained 24 h after exposure but was still significantly increased in the embryos. A closer time-related evaluation of SCE on day 2 of gestation showed a significant increase in SCE in bone marrow cells and in embryos obtained 20-60 min after CPA treatment. Furthermore, SCE frequency was the most sensitive toxicological endpoint to detect embryotoxic effects of CPA treatment before implantation, since it was significantly increased in embryos exposed to 5 mg/kg CPA on day 2 of pregnancy while embryolethality at term and both cytogenetical (structural chromosomal aberrations, micronuclei) and developmental parameters (cell number, differentiation in culture) before implantation did not indicate any toxic effect.     

Effect of tumour necrosis factor-alpha (TNF-alpha) on protein synthesis by mouse preimplantation stage embryos in vitro

Successful blastocyst implantation depends upon the synchronous dialogue between age- and stage-matched embryo and adequately primed maternal endometrium. Endometrial signals present in the uterine lumen influence the growth and the viability of preimplantation stage embryo. Thus, uterine secretion of embryotoxic cytokines may affect the preimplantation stage embryo. Our previous study in the rhesus monkey has indicated that tumor necrosis factor-alpha (TNF-alpha) is one such candidate present in the uterine lumen, which may act as an embryotoxic agent. In the present study, the effect of TNF-alpha on de novo protein synthesis by mouse morulae (n = 100) and blastocysts (n = 100) in vitro was investigated by 2D-polyacrylamide gel electrophoresis. A total of 35 and 40 protein spots were detected in lysates of control morulae and blastocysts, respectively. Exposure of embryos to TNF-alpha (50 ng/ml) reduced the number of protein spots to 15 and 17 compared to that of control morulae and blastocysts. Seven spots in morula and 13 protein spots in blastocyst flourograms showed quantitative changes in their expressions with exposure to TNF-alpha. Morulae and blastocysts exposed to TNF-alpha expressed 8 and 17 protein spots, respectively, that were not seen in control embryos. It appears from the present study that exposure of preimplantation stage embryos to TNF-alpha affects their protein synthesis both quantitatively and qualitatively.     

Recent progress in teratology. A survey of methods for the study of drug actions during the preimplantation period

To evaluate the effects of drug treatment during the first days of pregnancy new approaches have been developed which allow the study of teratogenic effects already before and around implantation, during organogenesis, and at term. The procedures used for the culture of preimplantation mouse embryos that were either pretreated in vivo or previously untreated and exposed to a toxic agent in vitro are presented in particular detail. In addition, an example is given which shows that it is possible to detect a DNA repair mechanism in preimplantation embryos maintained in vitro. The techniques of embryo transplantation and in vitro cultivation of embryos beyond implantation are outlined. The importance of the two methods in teratological research on embryos pretreated either in vivo or in vitro is discussed. Also presented are a survey of the literature and recent data obtained with rats and mice from our laboratory which prove that the action of drugs on the embryo and mother during the preimplantation period are more complex than is generally assumed.     

Direct effects of nicotine on rabbit preimplantation embryos

The effects of various concentrations of nicotine on the in vitro development of 1-cell rabbit preimplantation embryos and on the DNA-synthesis of 4-day-old rabbit blastocysts are investigated. Exposure of rabbit preimplantation embryos to concentrations of nicotine higher than 1 X 10(-3) M results in a marked decrease in the in vitro development and in DNA-synthesis. Concentrations of nicotine below 1 X 10(-3) M have no effect on these early embryos. It can be concluded that the concentrations of nicotine which exert a direct embryotoxic effect are higher than the concentrations that may be expected in the blood circulation of humans considered to be "normal smokers".     

In vitro culture of postimplantation hamster embryos

In vitro culture of intact rat and mouse embryos has been described extensively, but information on the culture of other species is sparse. The present study examined some culture requirements of early somite stage hamster embryos and assessed the embryotoxic effects of sodium salicylate (SS), a direct acting chemical and cyclophosphamide (CP), a proteratogen, on these embryos. Hamster embryos explanted on gestation days (GD) 8 and 9 were cultured in Waymouth's embryo-hepatocyte co-cultivation medium (WEHC), 70% McCoy's 5A medium-30% male rat serum (MMRS) or 100% male rat serum (MRS) for 24 hours under various oxygen concentrations. Embryos cultured GD 8 to 9 in the various media grew and differentiated much as they did in vivo, while embryos cultured GD 9 to 10 grew best in MMRS as compared to embryos at the same stage in vivo. Embryos exposed to SS in MMRS at concentrations of 250, 300, or 400 micrograms/ml showed dose related embryotoxicity which included CNS defects, absence of hind limb bud formation, and lack of axial rotation. Hamster embryos co-cultivated with pregnant hamster hepatocytes and treated with 2.5, 6.25 and 12.5 micrograms/ml of CP, showed dose-dependent toxicity when compared to co-cultivated controls. Hamster embryos develop extensively in culture over a 24 hour period. This system may therefore provide a valuable tool for evaluating the species differences of a variety of potential teratogens and embryotoxins and allow the comparison of these embryotoxic effects between rat, mouse and hamster during similar stages of organogenesis.     

Genotoxicity of selected pesticides in the mouse bone-marrow micronucleus test and in the sister-chromatid exchange test with human lymphocytes in vitro

Selected pesticides, (aldicarb, 1,3-dichloropropene, methidathion, parathion, triadimefon, vinclozolin) were tested for their clastogenic and aneugenic activities in the mouse bone-marrow micronucleus (MN) test in vivo and for their sister-chromatid exchange-inducing activities in human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system from rat-liver S9. 1,3-Dichloropropene significantly increased the frequencies of micronucleated polychromatic erythrocytes (PCE) in bone-marrow cells of female mice from 3.3 MN/1000 PCE to 15.3 MN/1000 PCE (187 mg per kg body weight). 1,3-Dichloropropene (100 μM) induced 16.0 SCE/metaphase after 24 h of incubation as compared with the basal rate of 11.2 SCE/metaphase (− S9) and of 15.4 SCE/metaphase as compared with 10.5 SCE/metaphase of the control (+ S9). These values were statistically significantly different from each other. The other pesticides tested did neither increase the rate of micronuclei significantly in polychromatic erythrocytes in male nor in female animals. Aldicarb and methidathion induced a significant increase in SCEs in human lymphocytes in vitro only without the metabolic activating system: aldicarb, 5 μm, 24 h incubation: 15.5 SCE/metaphase; control: 12.6 SCE/metaphase; methidathion, 100 μM, 24 h incubation: 15.8 SCE/metaphase, control: 11.1 SCE/metaphase. Parathion, triadimefon and vinclozolin did not have any SCE-inducing effects.     

Comparative developmental toxicities of aliphatic nitriles: in vivo and in vitro observations

The effects on embryonic development of a series of eight saturated (acetonitrile, propionitrile, and n-butyronitrile) and unsaturated (acrylonitrile, methacrylonitrile, allylnitrile, cis-2-pentenenitrile, and 2-chloroacrylonitrile) nitriles were compared in vitro using the whole embryo culture system. Day 10 rat embryos were cultured for 46 h in rat serum in the presence of either of these chemicals. All the tested chemicals produced concentration-dependent decreases in growth and differentiation and increases in the incidences of morphologically abnormal embryos. A wide range of embryotoxic potency was observed, with 2-chloroacrylonitrile and acetonitrile at the extremes (lowest effect levels of 50 microM and 40 mM, respectively). No common pattern could be drawn for all the eight nitriles tested in vitro, although there were some similarities between the malformations elicited by propionitrile and n-butyronitrile or between those elicited by the five unsaturated nitriles. Presence of a rat hepatic microsomal fraction and NADPH in the culture medium enhanced the embryotoxic effects of the five unsaturated nitriles tested but had no effects on saturated nitriles embryotoxicity. In addition to these in vitro experiments, pregnant rats were given a single oral dose of each compound on Day 10 of gestation and the embryos were evaluated on Day 12 of gestation, i.e., at a time of development corresponding to the developmental stage at the end of the whole embryo culture. All the nitriles investigated produced the characteristic defects developed by embryos exposed to sodium cyanide in utero or in culture. Our results provide further evidence that maternal production of cyanide may contribute to the developmental toxicity of saturated and unsaturated nitriles and suggest that distinct metabolites derived from microsomal metabolism of unsaturated nitriles may also play a role.     

Impact of chronic low-dose ethanol ingestion during sexual maturation of female mice on in-vitro and in-vivo embryo development

Little is known of the consequences of ethanol intake prior to fertilization on preimplantation embryo development. Recently we showed that chronic 10 and 5% w/v ethanol intake by young female mice reduces in vitro fertilization (IVF) rates. The purpose of the present work was to investigate whether the adverse effects of preconceptional low-dose chronic ethanol intake by sexually maturing female mice affects preimplantation embryo growth in vitro or in vivo in subsequent pregnancy. Prepubertal female mice were given 5% ethanol in their drinking water for 30 days. On day 27 and 29 of the ethanol treatment, females were superovulated. IVF-derived cultured embryos (in vitro development) or embryos obtained from oviducts and uteri (in vivo development) were evaluated. Whether analyzed on a per embryo or per dam basis, ethanol treatment was associated with a significant decrease in progression through embryo stages during the seven days of in vitro development and with an increase in morphologically abnormal embryos. Progression through embryo stages during four days of in vivo development was also inhibited by ethanol pretreatment of dams At 99 h post-hCG of in vivo development, there were fewer total, hatched, and expanded blastocysts, and a complete absence of implanting blastocysts among females treated with ethanol. In summary, low-dose chronic ethanol consumption of sexually maturing female mice prior to conception has adverse effects on preimplantation embryo development, both under in vitro and in vivo conditions, manifested as retarded development, embryo anormalities, and a reduction in expansion and hatching of the preimplantation blastocyst.     

Embryotoxic effects of Solanum lycocarpum St. Hill fruits consumption during preimplantation and organogenesis in rats

Solanum lycocarpum St. Hill is a common plant in the Brazilian savanna. This plant has an alkaloid with stereospecific configuration to the synthesis of steroid hormones. Since the plant may be consumed by pregnant animals, the present study was undertaken to determine the possible embryotoxic effects of S. lycocarpum fruit ingestion (3% added to the diet) during preimplantation (from the first to the sixth days of gestation) or during organogenesis in rats (from the sixth to the sixteenth days of gestation). Few differences were observed in food and water consumption without biological importance. The placental weight in the group that received the plant during the organogenesis period was decreased. An increase in sternebra abnormalities was observed in animals treated with the plant during organogenesis. Olfactory bulb hemorrhage was increased in the group that received the plant during preimplantation when compared to the control group. These results indicate that consumption of S. lycocarpum at 3% in diet during pregnancy cause slight toxicological effects. Other studies have to be conducted to better investigate the causes of toxicity and other toxic effects of higher levels of exposure to this plant.     

Cleavage and development in cultured preimplantation mouse embryos exposed to lidocaine

Two-cell preimplantation mouse embryos were exposed in vitro to lidocaine (0 to 1,000 μg/mL) for 72 h to determine the effects of this anesthetic on subsequent cleavage and development during prolonged exposures. Embryonic development was monitored each 24 h for 3 d. Lidocaine adversely affected the in vitro development of the mouse embryos, altering the distribution of the development stages at the evaluated culture tunes. The percentage of two-cell embryos that cleaved and developed to more advanced stages was decreased by the exposure to lidocaine. After 24 h of culture, two-cell embryos were arrested before completion of cellular division; this occured in 30% of the embryos at concentrations of 10 to 100 μg/mL and in 73.2% of the embryos with 1,000 μg/mL. After 48 h the blastomeres of the arrested embryos began to degenerate, showing lysis or fragmentation. At the lowest concentration, 14.9% of the arrested embryos exhibited the capacity to recover. These embryos continued their cleavage and normal development towards blastocyst formation. The cytotoxic effect and arrest at the two-cell stage were observed in a dose-dependent manner after 72 h of culture. We conclude that sensitivity to lidocaine embryotoxicity occurs during a window at the two-cell stage.     

Embryologic and cytogenetic effects of ethanol on preimplantation mouse embryos in vitro.

Ethanol and its primary metabolite acetaldehyde were studied in cultured preimplantation mouse embryos with respect to embryotoxicity, embryolethality, chromosome breaking activities, and ability to induce sister chromatid exchange (SCE). Analysis of differentiation and cell number of mouse morulae and blastocysts show that acetaldehyde is three orders of magnitude more toxic than ethanol, indicating that the metabolite is responsible for the embryotoxicity of ethanol in preimplantation embryos. Concentrations of ethanol that do not inhibit growth induce SCEs and chromosome aberrations. The SCE-inducing effect of ethanol disappears in the presence of 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH). These data suggest that preimplantation embryos are able to convert ethanol to acetaldehyde and that ADH is the enzyme involved. It is, furthermore, shown histochemically that mouse oocytes as well as morulae and blastocysts are able to oxidize ethanol in the presence of NAD+.     

Effects of 3H-thymidine on preimplantation mouse embryos in vitro

The effect of 3H-thymidine on in vitro development of preimplantation mouse embryos was studied. Two-cell and 4-8-cell embryos from B6CBA/F1 mice were continuously exposed to 3H-thymidine in medium containing 3H-thymidine in concentrations ranging from 10-500 nCi/ml. The effect of the radioactive precursor on embryo development to the blastocyst stage was studied by morphological observation, counting the blastocyst cell number and measuring 3H-thymidine incorporation. The continuous presence of 3H-thymidine significantly inhibited development of 2-cell and 4-8-cell embryos to the blastocyst stage. Embryos cultured from the 2-cell stage were more sensitive to 3H-thymidine than those exposed from the 4-8-cell stage. Even in morphologically normal blastocysts the cell number was significantly reduced. A 2 hr pulse of 100 nCi/ml 3H-thymidine at the blastocyst stage, did not affect the blastocyst formation or the blastocyst cell number and the amount of incorporated 3H-thymidine was sufficient to provide a reliable quantitation of DNA synthesis during the culture of preimplantation embryos in vitro. Continuous incubation with 3H-thymidine in order to measure DNA synthesis of preimplantation mouse embryos should be avoided when DNA synthesis is used as a means of evaluating toxic effect of an agent. Adverse radiation effects by 3H-thymidine on preimplantation mouse embryos during toxicity testing can be avoided by pulse labelling.     

Recovery of mouse embryos after short-term in vitro exposure to toxic nickel chloride

The development of preimplantation mouse embryos in vitro was adversely affected by the addition of nickel chloride (NiCl₂·6H₂O) to the culture medium. For day 3 (4–8 cell) embryos developmental cessation occurred after 48 h in culture, in NiCl₂·6H₂O-containing medium. However, transfer to NiCl₂·6H₂O-free medium after 5 min, 1 h, and 3 h exposure, resulted in regaining of the developmental capacity for a proportion of the exposed embryos.The in vivo development, in pseudopregnant recipients, of in vitro nickel-exposed embryos was not significantly different from that in control embryos.The results indicated that the effect of NiCl₂·6H₂O on the development of day 3 mouse embryos in vitro was reversible after a short exposure period.     

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.     

Nicotine and cotinine effects on development of two-cell mouse embryos in vitro

The independent and interactive effects of nicotine and cotinine on the development of cultured two-cell embryos were investigated. Cultures were maintained for 120 h and developmental stages of embryos were scored after 72 h and at the termination of culture. Concentrations of nicotine at or below 0.5 mM, and concentrations of cotinine at or below 0.008 mM, did not adversely affect development. In addition, neither nicotine nor cotinine produced synergistic effects at higher concentrations at which both independently impaired development. These data show, therefore, that nicotine and its major metabolite, cotinine, significantly interfere with preimplantation development of mouse embryos only at concentrations far in excess of those anticipated to be present in the blood of an "average" smoker. Thus, we conclude that the well documented adverse effects of smoking during pregnancy are unlikely to be attributable to a direct effect of nicotine or cotinine on the preimplantation embryo.     

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.     

Prevention of ochratoxin A‐induced oxidative stress‐mediated apoptotic processes and impairment of embryonic development in mouse blastocysts by liquiritigenin

Ochratoxin A (OTA), a mycotoxin constituent of a range of food commodities, including coffee, wine, beer, grains, and spices, exerts toxicological and pathological effects in vivo, such as nephrotoxicity, hepatotoxicity, and immunotoxicity. In a previous report, we highlighted the potential of OTA to induce apoptosis via reactive oxygen species (ROS) generation in mouse blastocysts that led to impaired preimplantation and postimplantation embryo development in vitro and in vivo. Here, we have shown that liquiritigenin (LQ), a type of flavonoid isolated from Glycyrrhiza radix, effectively protects against OTA‐mediated apoptosis and inhibition of cell proliferation in mouse blastocysts. Preincubation of blastocysts with LQ clearly prevented OTA‐triggered impairment of preimplantation and postimplantation embryonic development and fetal weight loss, both in vitro and in vivo. Detailed investigation of regulatory mechanisms revealed that OTA mediated apoptosis and embryotoxicity through ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase‐9 and caspase‐3, which were effectively prevented by LQ. The embryotoxic effects of OTA were further validated in an animal model in vivo. Intravenous injection of dams with OTA (3 mg/kg/day) led to apoptosis of blastocysts, impairment of embryonic development from zygote to blastocyst stage and decrease in day 18 fetal weight. Notably, preinjection of dams with LQ (5 mg/kg/day) effectively prevented OTA‐induced apoptosis and toxic effects on embryo development. Our collective results clearly demonstrate that OTA exposure via injection has the potential to damage preimplantation and postimplantation embryonic development against which LQ has a protective effect.     

Mutagenicity testing of doxylamine succinate, an antinauseant drug

Doxylamine succinate (DA), a compound which was formerly used as an antinauseant during pregnancy, showed no substantial mutagenicity in mouse embryos following transplacental exposure. A small dose-dependent induction of chromosomal aberrations was found in mouse embryos on day 11 of gestation. No induction of sister chromatid exchanges (SCE) was found in embryos on day 11 of gestation. A micronucleus test with fetal blood on day 17 of gestation was negative. Additionally, DA was negative in Chinese hamster bone marrow in vivo (micronuclei) and in human lymphocyte cultures in vitro (SCE).     

Role of biotransformation in the in vitro preimplantation embryotoxicity of naphthalene

The in vitro developmental toxicity of the bicyclic aromatic hydrocarbon naphthalene was characterized with a preimplantation mouse embryo culture system. Day 3 ICR mouse blastocysts were co-cultured with naphthalene for 1 h either alone or in media supplemented with an Aroclor-induced rat S-9 preparation and cofactors. Toxin-treated blastocysts were subsequently cultured in NCTC 109 media with 10% fetal bovine serum for 72 h to observe the developmental effects of exposure. Developmental parameters observed included viability, hatching, culture dish attachment and trophoblastic outgrowth with the presence of a distinct inner cell mass. At media concentrations up to 0.78 mM, naphthalene alone exhibited negligible toxic effects in culture; however naphthalene co-cultured with Aroclor-induced rat hepatic S-9 fractions exhibited concentration-dependent embryolethality with an approximate LC50 of 0.18 mM in media. Naphthalene also induced concentration-dependent embryotoxicity at all observed parameters in S-9-supplemented media at concentrations ranging from 0.20 to 0.78 mM. These findings document the role of biotransformation in naphthalene's embryotoxicity to early mouse blastocysts and implicate naphthalene as a potentially embryotoxic and abortifacient component of polycyclic aromatic hydrocarbon mixtures.