Embryotoxic Concentrations Research Articles

Effect of Phlorethol from the Brown Alga Costaria costata (Turn.) Saund. (Order Laminariales) on Gametes and Embryogenesis of the Sea Urchin Strongylocentrotus intermedius (A. Agassiz, 1864)

The effect of a fraction of phlorethol (CcPh), one of the polyphenolic compounds isolated from the brown alga Costaria costata (Turn.) Saund (Laminariales), on gametes and embryogenesis of the sea urchin Strongylocentrotus intermedius (A. Agassiz, 1864) was studied. Using a model of developing sea urchin embryos, it was shown that phlorethol at concentrations ≤700 µg/mL did not exert cytotoxic effect on early developmental stages (from zygote to 16-celled stage blastomere). The embryotoxic effect of phlorethol, causing death of 50% of embryos, was expressed in a concentration (I) range of 50 I ≤ 100 µg/mL only after 24 h of incubation, in the blastula stage. When exposed to concentrations of ≥100 µg/mL, embryos that survived to the blastula stage did not develop further and died after 36 h of incubation. Under the effect of phlorethol, the fertilizing capacity of sea urchin spermatozoa and oocytes reduced. The concentration of phlorethol inhibiting fertilization by 50% (IC50) when acting on sperm was 1.28±0.38 µg/mL and when acting on oocytes was 3.83±0.82 µg/mL. Thus, phlorethol reduces the fertilizing capacity of sea urchin gametes at concentrations much lower than embryotoxic concentrations and can potentially find practical application as part of new contraceptive drugs for mammals and humans.

The influence of some promising fused azaisocytosine-containing congeners on zebrafish (Danio rerio) embryos/larvae and their antihaemolytic, antitumour and antiviral activities

The present study was aimed at broadening the profile of toxicity and biological activity of promising fused azaisocytosine-containing congeners (I–VI) possessing medical applicability and important pharmacokinetic properties. For this purpose, the in vivo zebrafish test was applied for evaluating embryotoxic effects of test compounds, whereas the ex vivo model of oxidatively-stressed rat erythrocytes was developed for assessing their antihaemolytic activities. Additionally, the MTT-based assays suitable for assessing cytotoxic and antiviral activities of I–VI were employed. The influence of compounds I–VI on zebrafish embryos/larvae was carefully investigated in relation to lack or presence of various substituents at the phenyl moiety. The least embryotoxic proved to be the parent compound (I) and its para-methyl (II) and ortho-chloro (III) derivatives. Simultaneously, they revealed the minimum embryotoxic concentrations higher than that of aciclovir, what makes them safer than this pharmaceutic. Moreover, most of test compounds showed protective effects (better or comparable to that of ascorbic acid) on oxidatively-stressed erythrocytes. All the investigated compounds were effective at inhibiting the growth of human solid tumours of pharynx (FaDu) and tongue (SCC-25). The majority of molecules showed good selectivity indices. The most selective proved to be II showing in normal Vero cells over a 5-fold and an almost 3-fold decreased cytotoxicity relative to that in tumour SCC-25 and FaDu cells, respectively. Additionally, a 3,4-dichloro derivative (VI) was shown to possess concentration-dependent inhibitory effects on the replication of Herpes simplex virus type 1 and simultaneously at active concentrations was found to be nontoxic for normal Vero cells.

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework.

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50–3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.

Effects of 13 developmentally toxic chemicals on the migration of rat cephalic neural crest cells in vitro.

The inhibition of neural crest cell (NCC) migration has been considered as a possible pathogenic mechanism underlying chemical developmental toxicity. In this study, we examined the effects of 13 developmentally toxic chemicals on the migration of rat cephalic NCCs (cNCCs) by using a simple in vitro assay. cNCCs were cultured for 48 h as emigrants from rhombencephalic neural tubes explanted from rat embryos at day 10.5 of gestation. The chemicals were added to the culture medium at 24 h of culture. Migration of cNCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of cNCCs between 24 and 48 h of culture. Of the chemicals examined, 13-cis-retinoic acid, ethanol, ibuprofen, lead acetate, salicylic acid, and selenate inhibited the migration of cNCCs at their embryotoxic concentrations; no effects were observed for acetaminophen, caffeine, indium, phenytoin, selenite, tributyltin, and valproic acid. In a cNCC proliferation assay, ethanol, ibuprofen, salicylic acid, selenate, and tributyltin inhibited cell proliferation, suggesting the contribution of the reduced cell number to the inhibited migration of cNCCs. It was determined that several developmentally toxic chemicals inhibited the migration of cNCCs, the effects of which were manifested as various craniofacial abnormalities.

Simplein vitromigration assay for neural crest cells and the opposite effects of all‐trans‐retinoic acid on cephalic‐ and trunk‐derived cells

Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all-trans-retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 μM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.

Proteomic analysis of ethanol-induced embryotoxicity in cultured post-implantation rat embryos

Protein expression changes were examined in day 10.5 rat embryos cultured for 24 hr in the presence of ethanol by using two-dimensional electrophoresis and mass spectrometry. Exposure to ethanol resulted in quantitative changes in many embryonic protein spots (16 decreased and 28 increased) at in vitro embryotoxic concentrations (130 and 195 mM); most changes occurred in a concentration-dependent manner. For these protein spots, 17 proteins were identified, including protein disulfide isomerase A3, alpha-fetoprotein, phosphorylated cofilin-1, and serum albumin. From the gene ontology classification and pathway mapping of the identified proteins, it was found that ethanol affected several biological processes involving oxidative stress and retinoid metabolism.

Concentrations of Polycyclic Aromatic Hydrocarbons (PAHs) in the Eggs of Predatory Birds in Britain

The eggs of gannets from two Scottish colonies (Ailsa Craig, Bass Rock) of golden eagles from the Hebrides and Highlands and of merlin eggs from the Scottish borders were analyzed for 52 PAHs, including 2-7 ring parent and alkylated PAHs. Phenanthrene was the most abundant PAH in gannet eggs from Ailsa Craig, and methylnaphthalenes predominated in the eggs from other locations and species. Most PAHs were detected in eggs but none were at likely embryotoxic concentrations. The sum concentrations for all the PAHs analyzed (3.1-5.7 ng g(-1) wet wt.) and for the U.S. EPA 16 priority PAHs (2.0-4.3 ng g(-1) wet wt.) did not differ significantly between species or locations. This uniform, low-level accumulation suggests background exposure to diffuse sources. PCA indicated that 3 ring parent and alkylated PAHs predominated in the eggs of merlins and gannets from Ailsa Craig and Hebridean golden eagles; other eggs had a more mixed profile. Source signature diagnostics largely suggested a petrogenic origin for the PAHs in the merlin eggs that we analyzed but otherwise gave equivocal results and further work is needed to determine which diagnostics can be successfully applied to PAHs in eggs.

The Effects of Nonylphenol and Octylphenol on Embryonic Development of Sea Urchin (Paracentrotus lividus)

In this study, embryotoxic and genotoxic effects of nonylphenol (NP) and octylphenol (OP), which are the derivates of alkylphenol (APs), were evaluated using the gametes and embryos of the sea urchin Paracentrotus lividus. The sperm and eggs of sea urchins were exposed to increasing concentrations of NP (0.937-18.74 microg/L) and OP (5-160 microg/L) under static conditions. The endpoints were sperm fertilization success, quantitative and morphologic changes in mitotic activity, larval malformations, developmental arrest, and embryonic/larval mortality. A dose-response-related reduction (approximately 20%) was observed in fertilization success and significant increases in the number of larvae with skeleton malformations at the pluteus stage of the contaminated sperms. The spermiotoxic and embryotoxic concentrations were determined as 0.937 microg/L for NP and 4.685 microg/L for OP for this species. The embryotoxicity of NP and OP is concentration dependent, and significant growth reduction at the early life stages and an increase in larval malformations as skeleton deformities at the pluteus stage were observed. Cytogenetic analysis of embryos showed a decreasing curve in mitotic indexes (number of mitosis per embryo) with increasing concentrations of NP and OP. It can be concluded that NP and OP adversely affect the reproduction and embryonic developmental stages of the P. lividus and this is of great ecological importance because of the hazard at the population level.

Effects of ethylene glycol and metabolites on in vitro development of rat embryos during organogenesis

The aim of this study was to investigate in vitro the relative impact of ethylene glycol, a major industrial chemical, and its individual metabolites on the embryonic development of rats. Rat whole embryos were exposed for 48 h (day 9.5–11.5 of gestation) to ethylene glycol (EG) and its metabolites glycolaldehyde (GAl), glycolic acid (GA), glyoxylic acid (GXA), glyoxale (GXAl) and oxalic acid (OXA) at increasing concentrations. Embryotoxic concentrations were achieved within the following range: ethylene glycol (100–200 m m), glycolic acid (3 m m), glyoxal (6 m m), oxalic acid (1–3 m m), glyoxylic acid (0.3–1 m m), glycolaldehyde (0.1–0.2 m m). The pattern of dysmorphogenesis with all compounds including EG showed a general embryotoxicity with diffusely distributed cell necroses with no specific target tissues selectively affected. The results obtained in this study emphasize the hypothesis that the metabolites and not ethylene glycol itself are responsible for the embryotoxicity of ethylene glycol in rats.

Developmental toxicity of indium in cultured rat embryos.

Developmental toxicity of indium was examined using rat embryo culture with reference to toxicokinetics. Rat embryos at day 9.5 of pregnancy were cultured for 48 h under various exposure conditions to indium trichloride. Indium was embryotoxic to cultured rat embryos at concentrations ranging from 25 to 50 microM for 24 h exposure according to the embryonic age, and the exposure concentration was more critical than the exposure time. The embryotoxic concentrations were comparable to the serum concentration at a developmentally toxic dose by intravenous administration in an in vivo experiment. It was considered from these results that the developmental toxicity of indium is a direct effect on the embryo or yolk sac and that weak developmental toxicity of indium by oral administration was due to low exposure concentrations in the embryo.

Effects of glutathione depletion on selenite- and selenate-induced embryotoxicity in cultured rat embryos.

Effects of depletion of reduced glutathione (GSH) on selenium (Se) embryotoxicity in cultured rat embryos were examined. Rat embryos at day 9.5 of gestation were cultured for 48 h in the presence of Se as either sodium selenite at 10 and 20 microM or sodium selenate at 30 and 100 microM. Embryonic GSH was depleted by the addition of 0.1 mM of L-buthionine-[S,R]-sulfoximine (BSO) without embryotoxicity, i.e., significant growth retardation and malformation of the embryos. Selenite at 10 microM or selenate at 100 microM significantly increased the incidence of malformation of the embryos. The incidence of selenite-induced malformation of the embryos at 20 microM was significantly decreased with BSO. On the contrary, the incidence of selenate-induced malformation at 30 microM was significantly increased with BSO. It was noted that the major malformed regions of the embryos by the embryotoxic concentration of BSO alone were the same to those affected by selenite or selenate. It was considered from these results that embryonic GSH was involved in the embryotoxicity of selenite and selenate. The embryotoxicity of selenate may not be mediated through the reduction to selenite. It was suggested that the formation of selenodiglutathione and the oxidative stress were involved in the embryotoxicity of selenite and selenate, respectively.

Spermiotoxicity and embryotoxicity of heavy metals in the echinoid Paracentrotus lividus

AbstractSpermio‐ and embryotoxicities of Cu, Ag, Cd, and Hg were investigated in Paracentrotus lividus, the dominant echinoid species of the Mediterranean. Spermiotoxicity was studied by assessing the effects of sperm exposure on fertilization rate (FR) as well as on the induction of transmissible damages to the offspring. Embryotoxicity was studied by assessing developmental defects in larvae exposed to the tested metals throughout their development. Sperm exposures resulted in significant decreases of FR, depending on both metal concentration and duration of the exposure. Lowest spermiotoxic concentrations recorded when sperm were exposed for 75 min to the metals were 10−7 M Hg(II), 10−6 M Ag(I), 10−5 M Cu(II), and 10−5 M Cd(II). Tested metals did not exert any transmissible damage to spermatozoa that could result in larval malformations in the offspring, even for concentrations that dramatically reduced FR. Single‐element exposures of embryos for 72 h resulted in developmental defects whose occurrence and severity showed a steep dose dependence, indicating that once a threshold is reached, any further increase in toxicant concentration rapidly enhances the impairment of target function(s). Those observations suggest the involvement of a saturable protective mechanism. Lowest observed embryotoxic concentrations of the metals were 10−7 M Hg(II), 2.5 × 10−7 M Ag(I), 5 × 10−7 M Cu(II), and 10−5 M Cd(II) and are in the range of concentrations reported in heavily polluted marine environments. Thus, the possibility of impairment of echinoid development actually exists in metal‐contaminated marine environments, possibly threatening echinoid populations in those environments.

Evaluation of di(2‐ethylhexyl)phthalate‐induced embryotoxicity in rodent whole‐embryo culture

Di(2-ethylhexyl)phthalate (DEHP) is a commonly used plasticizer. Human exposure has been documented, and therefore it is important to investigate the toxic potential of this compound. When DEHP was administered in vivo, it was found to be a developmental toxicant in rodents. The purpose of this investigation was to determine if DEHP was directly embryotoxic to rat embryos. Embryos were cultured for 44 h beginning on gestational d 10. Embryos were cultured in rat serum to which DEHP was added to attain final concentrations within the 0.01-2.0% range. DEHP decreased growth and development at all tested concentrations higher than 0.5%. These results suggest that the parent compound itself is able to alter normal embryonic growth and development; however the high embryotoxic concentrations are unlikely to be attained in vivo.

Chloroquine accumulation and alterations of proteolysis and pinocytosis in the rat conceptus in vitro

The teratogenicity of chloroquine (CQ) has been hypothesized to result from its effects on lysosomal function, specifically the ability of the visceral yolk sac (VYS) to capture and degrade external macromolecules. Using the rat whole embroyo culture system, we evaluated the ability of CQ to accumulate in conceptal tissues and its effects on aspects of VYS function known to be important in the uptake and processing of nutrients. When CQ was added directly to the culture medium, it was found to accumulate rapidly in conceptal tissues, particuarly the VYS. Tissue concentrations of CQ in the embryo proper reached approximately 10-fold those in the medium, whereas concentrations in the VYS exceeded by 100-fold the medium concentration within a 4-hr exposure on gestational day (GD) 10. Embryotoxic concentrations of CQ (10–30 μM) enhanced the activity of lysosomal cysteine proteinases measured in vitro under optimum pH conditions in both embryonic and VYS homogenates after a 26-hr treatment from GD 10–11. A different pattern of response in enzyme activity was observed between embryos and VYSs that could be attributed to the preferential accumulation of CQ in the VYS. Nonembryotoxic concentrations of CQ (1–7.5 μM) induced a concentration-dependent increase in VYS enzyme activity that peaked in conceptuses exposed to 20 μM CQ (an intermediate embryotoxic concentration). The enhanced cysteine proteinase activity was time dependent and appeared to increase gradually in conceptuses exposed to 10–20 μM CQ during the 26-hr culture period. This was in contrast to the rapid accumulation of CQ in conceptal tissues seen on gestational day 10. Protein content in the VYS was increased significantly after a 9-hr exposure of whole conceptuses to CQ (20 μM), indicating an inhibition of VYS proteolytic activity in situ. After 24 hr of exposure to 20μM CQ, VYS protein content was not significantly different from control, but embryonic protein was reduced significantly by 20%. These observations are consistent with a model of reversible inhibition of VYS proteolysis by CQ followed by a compensatory increase in lysosomal proteinase activity. VYS fluid-phase pinocytosis was also assessed after CQ exposure and found to be inhibited only in the highest CQ concentration tested (30 μM). Lower concentrations of CQ that were still embryotoxic (10–20 μM) did not affect VYS fluid-phase pinocytosis, suggesting that inhibition of this activity is not primarily responsible for CQ embryotoxicity.

Inhibition of mouse embryonic, yolk sac, and limb-bud functions by the methyl isocyanate metabolite S-(N-methylcarbamoyl)glutathione.

We previously reported that S-(N-methylcarbamoyl)glutathione (SMG), a conjugate formed by the reversible reaction between methyl isocyanate and glutathione, inhibited the development of mouse embryos in culture. The present study was done to determine whether SMG produced embryotoxicity by inhibiting yolk sac functions. For this purpose we determined the effects of an embryotoxic concentration of SMG on mouse yolk sac uptake mechanisms and lysosomal proteolysis as well as on the incorporation of [3H]leucine in mouse embryonic and limb-bud proteins. After 5 h of culture, SMG inhibited the uptake of [14C]sucrose and 125I-labelled bovine serum albumin in isolated day 15 yolk sacs to 62 and 77% of control, respectively. Lysosomal proteolysis was not inhibited, as judged by the release of trichloroacetic acid soluble radioactivity into the culture media. Uptake and incorporation of [3H]leucine from free [3H]leucine or from [3H]leucine-labelled protein in SMG-treated day 9 embryos were inhibited, respectively, to 61 and 25% of the control uptake during a 16-h labelling period. SMG also inhibited the incorporation of free [3H]leucine into limb-bud proteins. SMG-induced inhibition of 125I-labelled bovine serum albumin uptake by yolk sacs was partially prevented by the thiol donors N-acetylcysteine and glutathione but not by acivicin (gamma-glutamyl transpeptidase inhibitor) and aminooxyacetic acid (cysteine conjugate beta-lyase inhibitor). These data suggest that SMG suppresses embryonic growth primarily by an inhibition of nutrient uptake by the yolk sac. We postulate that this inhibition is due to tissue carbamoylation by methyl isocyanate released from SMG.

Genetic differences in heat-induced tolerance to cadmium in cultured mouse embryos are not correlated with changes in a 68-kD heat shock protein.

Heat-induced cross-tolerance to cadmium was investigated in two inbred strains of mice, BALB/c and SWV, using a whole embryo culture system. Embryos were exposed to a pretreatment of 5 min at 43 degrees C and subsequently to an embryotoxic concentration of cadmium, 1.75 microM. The two types of embryos responded differently to the heat pretreatment, as cross-tolerance was induced in SWV but not in BALB/c mice. In SWV embryos, prior exposure to 43 degrees C for 5 min essentially eliminated the negative effects of cadmium on embryonic development and growth. However, in BALB/c embryos, no protection was observed. The variation in development of cross-tolerance in embryos from the two strains of mice was not correlated with differences in the induction of a 68-kD heat-shock protein (hsp68). There was a rapid increase in this protein in both strains after the initial heat exposure but not excess induction in the SWV strain that developed tolerance. The induction of hsp68 is therefore not sufficient to elicit cross-tolerance, and other mechanisms are likely to be important in the protective response of the embryo.

Identification of cyclophosphamide-DNA adducts in rat embryos exposed in vitro to 4-hydroperoxycyclophosphamide

Cyclophosphamide and other bifunctional alkylating agents are potent animal teratogens inducing a variety of malformations. Although cyclophosphamide-induced DNA damage is implicated as a primary mechanism underlying the teratogenesis initiated by cyclophosphamide, additional insights into the complex nature of the teratogenic process have been hampered by the inability to analyze the primary teratogenic lesions, i.e., cyclophosphamide-DNA adducts. Using tandem mass spectrometry, we show that the monofunctional adduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (NOR-G) and bifunctional adduct N,N-bis[2-(7-guaninyl)ethyl]amine (G-NOR-G) can be detected in the DNA of organogenesis-stage rat embryos after an in vitro exposure to an embryotoxic concentration of activated cyclophosphamide, i.e., 4-hydroperoxycyclophosphamide.

Effects of 4-hydroperoxycyclophosphamide (4-OOH-CP) and 4-hydroperoxydechlorocyclophosphamide (4-OOH-deCICP) on the cell cycle of post implantation rat embryos.

In this study, we used preactivated forms of cyclophosphamide (CP) and dechlorocyclophosphamide (deClCP) to examine the effects of phosphoramide mustard (PM) and acrolein, respectively, on the cell cycle of postimplantation rat embryos. The percentage distribution of cells in the G1/G0, S, and G2/M phases of the cell cycle was determined by flow-cytometric analysis. At embryotoxic concentrations, 4-OOH-CP (PM) induced major cell cycle perturbations whereas 4-OOH-deClCP (acrolein) caused no major perturbation of the cell cycle. These data support the hypothesis that the mechanism of the embryotoxic action of PM involves alkylation of DNA, whereas the mechanism of action of acrolein does not. The primary effect of PM on the cell cycle was an initial delay in the S phase followed by a G2/M arrest. At low embryotoxic concentrations of 4-OOH-CP, there was apparent reversal of the G2/M arrest; at higher embryotoxic concentrations there was little recovery from the G2/M arrest. The high level of cell death found at higher drug concentrations suggests that prolonged G2/M arrest leads to cell death. Using radiolabeled CP and cell sorting, it was determined that PM predominantly alkylated DNA in the S phase of the cell cycle. Overall, the data from this study support the hypothesis that DNA cross-links, induced by the alkylation of DNA by PM, induce cell cycle perturbations. Furthermore, these cell cycle alterations may be one of the early steps in the mechanism leading to the embryotoxicity of PM.

Embryotoxicity of the intercalating agents m‐AMSA and o‐AMSA and the epipodophyllotoxin VP‐16 in postimplantation rat embryos in vitro

The intercalating agent, m-AMSA, and the epipodophyllotoxin, VP-16, both topoisomerase II-reactive anticancer agents, are also embryotoxic agents in rat embryos cultured in vitro. Quantifying the embryotoxic effects of these drugs revealed that the no observed adverse effect level (NOAEL) for m-AMSA is 10 nM, the embryotoxic concentration range is 50-500 nM, and complete lethality is observed at 1 microM. In contrast, the NOAEL for o-AMSA, an inactive isomer of m-AMSA, is 1.0 microM, the embryotoxic concentration range is 10-100 microM, and complete lethality occurs at 200 microM. Based upon the concentrations of drugs required to produce 50% embryotoxicity or 50% malformed embryos, m-AMSA exhibits a 200-500-fold-higher embryotoxicity compared to o-AMSA. VP-16 exhibits a NOAEL of 1.0 microM, an embryotoxic concentration range of 2-5 microM, and complete lethality at 10 microM. Compared to m-AMSA, VP-16 is approximately 10-fold less embryotoxic. At appropriate concentrations, all three drugs were dysmorphogenic resulting in embryos that were characterized by hypoplasia of the prosencephalon with associated microopthalmia and dilation of the rhombencephalon. and dilation of the rhombencephalon. As a prelude to future studies focusing on the mechanism of drug-induced embryotoxicity, we have used established biochemical and immunologic methods to identify and quantify topoisomerase II in rat embryos. In addition, we have demonstrated that the embryo topoisomerase II can be inhibited by both m-AMSA and VP-16. Finally, we have used a human cDNA probe to detect topoisomerase II mRNA in the rat embryo. Thus, the combination of the in vitro whole embryo culture and these biochemical/molecular assays should allow us to explore the role of a specific nuclear target, i.e., topoisomerase II, in the teratogenic effects of some commonly employed chemotherapeutic agents.

Relationship of DNA damage and embryotoxicity induced by 4‐hydroperoxydechlorocyclophosphamide in postimplantation rat embryos

4-Hydroperoxydechlorocyclophosphamide (4-OOHdeCl-CP) is a preactivated analogue of cyclophosphamide (CP) that undergoes an elimination reaction to yield acrolein and the nonalkylating derivative of phosphoramide mustard (PM), i.e., dechlorophosphoramide mustard. We used this analogue to assess the role of acrolein in CP-induced embryotoxicity. Embryotoxicity was assessed using day 10 rat embryos cultured in vitro. 4-OOHdeC1-CP was embryotoxic over a concentration range of approximately 75-150 microM and produced complete embryolethality at concentrations of 175 microM and above. This analogue induced abnormal development characterized by tail defects at low drug concentrations and microencephaly or prosencephalic hypoplasia at high concentrations. Using the technique of alkaline elution, we also assessed DNA damage induced by embryotoxic concentrations of drug. When embryos were cultured in serum-containing medium during drug exposure, no DNA damage was detected, even at embryolethal drug concentrations. However, if cellular glutathione (GSH) was depleted with buthionine sulfoximine (BSO) before drug exposure and embryos were cultured in serum-free medium during drug exposure, DNA damage, primarily DNA single-strand breaks, was detected, but only at embryolethal concentrations. Using radiolabeled CP, we showed that acrolein does reach the embryo; however, more acrolein is incorporated into the yolk sac. Binding studies revealed that acrolein binds preferentially to cellular protein, whereas PM binds preferentially to DNA. These results suggest that, unlike the case with PM, the embryotoxic target for acrolein is protein and not DNA. Furthermore, our results indicate that acrolein may mediate its effects on the embryo via the yolk sac.