The embryolethality and teratogenicity of acrolein in cultured rat embryos

Acrolein, a three-carbon unsaturated aldehyde, is teratogenic to rats in vivo following intraamniotic administration but has been reported not to be teratogenic in vitro in the rat whole embryo culture system. In this study the effects of acrolein on rat embryos cultured in the standard medium consisting of rat serum were assessed over a narrow-concentration range. Additionally, a comparison was done of the effects of culture in a serum medium vs. a serum-free medium. In the serum medium acrolein caused 100% embryolethality at 140 microM and was found to be teratogenic in the concentration range of 80-120 microM. In the serum-free medium acrolein was 100% embryolethal at 20 microM and was teratogenic in the range of 5-15 microM. The EC50 for malformations in the serum medium was 137 microM, whereas that for embryolethality was 115 microM; the EC50s for malformations and embryolethality in the serum-free medium were 2.8 microM and 8.3 microM, respectively. Malformations were observed in the brain, facial area, and heart in addition to blebs and twisted or kinked bodies. Decreases in yolk sac diameter, crown-rump length, head length, number of somites, morphological score, and protein content were observed within the teratogenic ranges in each type of medium. Thus acrolein is teratogenic and embryolethal in vitro as well as in vivo. Dissociation between embryolethality and teratogenicity was seen in the serum-free medium. The slope of the acrolein log concentration-response curve in the serum-free medium was twice that in the serum medium, indicating that acrolein may have a different mechanism of action in this medium.     

Inhibition of NADPH-cytochrome P450 reductase by cyclophosphamide and its metabolites

Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. $\text{In vitro}$ CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.     

A teratogenicity study on hydroxyurea and diphenylhydantoin in cats

Hydroxyurea, an antitumor drug and known teratogen in rat, miniature swine and dog, and diphenylhydantoin, a teratogen in mouse and rat, were assessed for teratogenic effects in cat. Pregnancies were induced, by synchronizing gonadotropin-stimulated estrus and ovulation with natural copulations. Hydroxyurea at 50 or 100 mg/kg, and sodium diphenylhydantoin at 1 or 2 mg/kg dosages, were administered orally in single daily doses from gestation days 10-22. Appropriate controls given empty capsules, were included for each drug. Cats were necropsied on gestation day 43. Fetuses were examined for external, visceral and skeletal malformations. Hydroxyurea at 50 mg/kg dose produced a low teratogenic activity and at 100 mg/kg a high incidence of non-pregnancy and resorptions with, consequently, fewer live fetuses. Diphenylhydantoin gave no clear evidence of teratogenicity at any test dose but was embryolethal at the maternally toxic dose of 2 mg/kg. So far, studies conducted suggest that the cat is a useful species for screening drugs and chemicals for their teratogenic potential.     

Lethal and teratogenic effects of long-term low-intensity radio frequency radiation at 428 MHz on developing chick embryo

Exposure of developing chick embryos to 428 MHz radio frequency (RF) radiation at a power density of 5.5 mW/cm2 for more than 20 days resulted in embryolethal and/or teratogenic effects and delayed hatching. These adverse biological effects were not due to any thermal effect of the RF radiation. We have demonstrated teratogenicity in the chick embryo as a result of protracted low-dose RF irradiation.     

Study of embryotoxic and teratogenic effects of amphotericin B and a methyl derivative of amphotericin B in rats after their intravenous and intra-amniotic administration]

The embryotoxic action of amphotericin B and its methyl derivative was compared in rats after their intravenous and intraamniotic administration. The concentrations of amphotericin B and its methyl derivative in the amniotic cavity on days 13, 14 and 15 of pregnancy were 1.5 and 36 micrograms/ml, respectively. When administered intravenously during the preimplantation period the antibiotics had no embryotoxic action. Intravenous administration of amphotericin B in a dose of 500 micrograms/kg and its derivative in a dose of 2000 micrograms/kg during organ genesis induced a decrease in the craniocaudal size. In a dose of 3000 micrograms/kg administered intravenously the methyl derivative of amphotericin B induced an increase in postimplantation death rates. Administration of amphotericin B to the amniotic cavity had no damaging action. Administration of the methyl derivative on day 15 of pregnancy led to anomalous development of the lower extremities and slower ossification. The threshold doses by the embryotoxic action for intravenous administration are 500 micrograms/kg for amphotericin B and 2000 micrograms/kg for the methyl derivative. Administration of the antibiotics to the amniotic cavity revealed potential teratogenic properties of the amphotericin B methyl derivative.     

Embryotoxic effects of sodium metavanadate administered to rats during organogenesis

Pregnant Sprague-Dawley rats were given orally a daily dose of 0, 5, 10 or 20 mg NaVO3/kg from the sixth through the fourteenth day of pregnancy. Fetal examinations were performed on day 20 of gestation. Sodium metavanadate was neither embryolethal nor teratogenic in rats when administered orally at 20 mg/kg/day or lower. Nevertheless, this dose was embryotoxic.     

Acrolein induces a cellular stress response and triggers mitochondrial apoptosis in A549 cells

Acrolein is a highly reactive, α,β-unsaturated aldehyde that is an omnipresent environmental pollutant. Humans are exposed to acrolein in food, vapors of overheated cooking oil, cigarette smoke and by combustion of organic products. Acrolein is a toxic by-product of lipid peroxidation resulting from oxidative stress, which is implicated in pulmonary, cardiac and neurodegenerative diseases. Low dose exposure to toxic compounds often leads to adaptive responses. If the adaptive response does not counteract the adverse exposure, death processes such as apoptosis will eliminate the cell. This study investigates the activation of antiapoptosis survival factors in relation to the induction of cell death by apoptosis, following exposure to low doses of acrolein, in A549 human lung cells. Exposure to acrolein (<15 μM, 30 min) activated the survival factor AKT, which led to phosphorylation of Bad and induction of antiapoptosis proteins cIAP1/2. Acrolein (10–50 μM, 30–60 min) increased reactive oxygen species and caused mitochondrial membrane hyperpolarisation. Inhibition by the antioxidants catalase, polyethylene glycol-catalase, sodium pyruvate and MnTBAP showed that acrolein-induced reactive oxygen species were responsible for mitochondrial membrane hyperpolarisation. Acrolein (3–27 μM, 30–60 min) activated early stage processes in the mitochondrial pathway of apoptosis, such as Bax translocation to mitochondria, cytochrome c release, caspase-9 activation, and translocation of apoptosis-inducing factor to the nucleus. Acrolein (10–50 μM) triggered later stage processes such as activation of caspases-3, -7 and -6, phosphatidylserine externalization and cleavage of poly(ADP)ribose polymerase after longer times (2 h). These events were inhibited by polyethylene glycol-catalase, showing that apoptosis was mediated by overproduction of reactive oxygen species by acrolein. The novel findings show that antiapoptosis processes dominate at low dose (<15 μM)/shorter exposure times to acrolein, whereas proapoptotic processes dominate at higher dose (10–50 μM)/longer exposure times. Acrolein induced apoptosis through the mitochondrial pathway that was mediated by reactive oxygen species.     

Embryotoxic effects of doxorubicin and N-trifluoroacetyladriamycin-14-valerate (AD-32)

Anthracyclines are mutagenic, carcinogenic, and also cardiotoxic. Concern has been shown over the use of anthracycline anticancer drugs during pregnancy as these may be teratogenic to the human fetus. We have performed a series of experiments using the chick embryo to investigate and compare the toxic and teratogenic effects of doxorubicin (DX) and its new analog N-trifluoroacetyladriamycin-14-valerate (AD-32). DX and AD-32, dissolved in 1:1 emulphor/ethanol and diluted to 1:4 with water, were injected into the air sac of white leghorn chick eggs at dose levels of 1-20 micrograms (DX) and 5-100 micrograms (AD-32) per egg. Eggs received a single injection of the drugs on days 1 and 2 of incubation. Control eggs were injected with an equivalent volume of the drug vehicle (0.05 ml per egg). Both of the drugs caused embryonic death, stunted growth, and various gross morphological malformations. Surviving embryos were sacrificed when they reached 13 days of incubation. The LD50 values for days 1 and 2 were as follows: (DX, 2.5 micrograms/egg on day 1 and 0.9 microgram/egg on day 2; AD-32, 10.6 micrograms/egg on day 1 and 11.8 micrograms/egg on day 2). Stunting of growth, a common anomaly with both drugs, decreased sharply from day 1 to day 2. The studies demonstrate that both DX and AD-32 are toxic and teratogenic during the period of early organogenesis in the chick embryos. However, the toxic and teratogenic potentials of DX and AD-32 differ quantitatively. AD-32 is a more potent teratogen than DX when injected on day 1.     

Developmental toxicity of artesunate in the rat: comparison to other artemisinins, comparison of embryotoxicity and kinetics by oral and intravenous routes, and relationship to maternal reticulocyte count

BACKGROUND: The antimalarial, artesunate, is teratogenic and embryolethal in rats, with peak sensitivity on Days 10 and 11 postcoitum (pc). METHODS: We compared the developmental toxicity of structurally related artemisinins, dihdyroartemisinin (DHA), artemether (ARTM), and arteether (ARTE) to that of artesunate after oral administration to rats on Day 10 pc. In separate studies, embryolethality was characterized after single intravenous (IV) administration of artesunate on Day 11 pc, and toxicokinetic parameters following oral and IV administration were compared. Lastly, to determine whether maternal hematologic effects occurred at doses that affect embryonic erythroblasts, artesunate was orally administered on Day 11 pc at a dose that caused 100% embryolethality. RESULTS: All artemisinins caused the same pattern of embryolethality and fetal cardiovascular and skeletal abnormalities as previously shown for artesunate. In the IV study, marked postimplantation loss occurred at 1.5 and 3 mg/kg artesunate, but not at 0.75 mg/kg. Among the toxicokinetic parameters evaluated, only the DHA AUC_0‐t was similar at embryolethal oral and IV doses of artesunate. An embryolethal dose of artesunate caused a 15% decrease in maternal reticulocyte counts and no other hematologic effects. CONCLUSIONS: Several structurally related artemisinins cause similar developmental toxicity, suggesting an artemisinin class effect. Equally embryotoxic oral and IV treatments of one artemisinin compound (artesunate) produced similar systemic exposure to the artesunate metabolite, DHA, suggesting that DHA may be the proximate developmental toxicant. Embryolethal doses of artesunate only caused minor changes in maternal reticulocyte counts indicating that adult hematology parameters are not as sensitive as embryonic erythroblasts. Birth Defects Res (Part B) 83:397–406, 2008. © 2008 Wiley‐Liss, Inc.     

Mevalonate supplementation in pregnant rats suppresses the teratogenicity of mevinolinic acid, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme a reductase

Mevinolin is a fungal metabolite, and in the hydroxyacid form, mevinolinic acid, it is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-Co A) reductase, an enzyme essential in cholesterol biosynthesis. Oral administration of 800 mg/kg/day of mevinolin to rats from days 6 through 17 of gestation produced fetal malformations of the vertebrae and ribs in 29% of the litters, and there was a treatment-related increase in the incidence of gastroschisis. Mevinolinic acid at 60 and 90 mg/kg/day also produced fetal malformations of the vertebrae and ribs, and these teratogenic manifestations were markedly suppressed by coadministration of the product of HMG-Co A reductase, mevalonic acid, at a dosage level of 500 mg/kg b.i.d. A diet supplemented with 0.5% or 1.0% cholesterol had no effect on the teratogenicity of mevinolinic acid. Teratology studies in rats with a dihydroxyheptanoic acid derivative of mevinolin, a compound 1/700 as potent as mevinolinic acid as an inhibitor of HMG-Co A reductase, and dihydromevinolinic acid, an inhibitor of this enzyme comparable in activity to mevinolinic acid, indicated that the teratogenicity of these compounds was related to their relative enzyme inhibitory activity. The dihydroxyheptanoic acid derivative was not teratogenic at doses as high as 150 mg/kg b.i.d.; in contrast, when dihydromevinolinic acid was administered at 50 and 100 mg/kg/day, its potency as a teratogenic agent was comparable to that of mevinolinic acid. These studies demonstrated that inhibitors of HMG-CoA reductase produced terata in rats and that the teratogenic effects could be antagonized by coadministration of the enzyme product, mevalonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryotoxicity of phenyl ketone analogs of cyclophosphamide

Phenylketophosphamide and phenylketoisophosphamide are preactivated acyclic ketone analogs of cyclophosphamide and isophosphamide with antitumor activity. These compounds undergo an elimination reaction to yield phosphoramide or isophosphoramide mustard and phenyl vinyl ketone. In this study, the embryotoxicity of phenylketophosphamide, phenylketoisophosphamide, and phenyl vinyl ketone were determined. Embryotoxicity was assessed in vitro in whole rat embryos cultured on day 10.5 of gestation in the absence and presence of an activating system derived from maternal liver. Both phenylketophosphamide and phenylketoisophosphamide were embryotoxic in the absence of metabolic activation. Moreover, there was no enhancement of this embryotoxicity in the presence of an activating system. A 10-microM concentration of phenylketophosphamide produced 100% malformed embryos, while this concentration of phenylketoisophosphamide was not teratogenic. At 25 microM phenylketoisophosphamide, all the surviving exposed embryos were malformed. Phenylketophosphamide was embryolethal to more than 50% of the exposed embryos at a concentration of 50 microM. In contrast, a concentration of phenylketoisophosphamide of 100 microM was required to produce significant embryolethality. Phenyl vinyl ketone was not embryotoxic at any of the concentrations tested. The major malformation observed, a hypoplastic prosencephalon, and the growth retardation effects were not only similar for phenylketophosphamide and phenylketoisophosphamide, but also similar to those previously reported for "activated" cyclophosphamide. Unlike the results with cyclophosphamide, where both phosphoramide mustard and the aldehydic metabolite of cyclophosphamide, acrolein, are toxic, the embryotoxic effects of phenylketophosphamide and phenylketoisophosphamide are mediated only by the mustard metabolite.     

Teratogenic evaluation of terpenoid derivatives

Eight terpenoid derivatives with juvenile hormone activity were tested for teratogenicity. Three of the derivatives were teratogenic when administered to mice as a single intraperitoneal injection of 1 mg/gm on either day 9 or day 10 of pregnancy. However, intubation at 5 mg/gm of the two most teratogenic derivatives resulted in one of the derivatives marginally increasing the incidence of abnormal embryos above peanut oil-intubated controls; the other intubated derivative was without adverse affect. Of the remaining five compounds, two with high juvenile hormone activity were without teratogenic effect when injected at 1 mg/gm. Since teratogenic effects were observed only at a dose above likely exposure levels and by a route of administration not possible in the environment, support is provided for the potential use of terpenoid derivatives in the control of insect pests.     

Mechanism of acrolein-induced vascular toxicity.

Acrolein, an environmental pollutant and a lipid peroxidation product, is implicated in vascular pathogenesis. Although evidence indicates a link between vascular pathogenesis and acrolein, no direct studies relating to effects of acrolein on vascular function and responses are known. This study investigated the effects of acrolein on vascular function to understand the underlying mechanism of acrolein-induced vascular responses. Male Sprague-Dawley rats were treated with acrolein (2 or 4 mg/kg; i.p.) for 3 or 7 days. Urine and blood samples were collected. Changes in systolic blood pressure (SBP) and responses to acetylcholine and phenylephrine were determined. Acrolein (4 mg/kg, 7 days) significantly increased SBP by 25%, phenylephrine vasoconstriction by 2-fold, but decreased urinary excretion of nitrite by 25%. Acrolein inhibited generation of cyclic guanosine 3'5'-monophosphate (cGMP) by 98%, and did not alter expression of nitric oxide synthase (eNOS). Acrolein increased the generation of lipid hydroperoxide in plasma and aortic tissue by 21% and 124% respectively, increased glutathione-S-transferase (GST) and glutathione peroxidase (GSH-Px) activities. Acrolein up-regulated the expression of GST by 2 fold. These data suggest that induced SBP and altered vasoconstriction/vasodilatation in acrolein treated rats may be due to reduced availability of NO via increased free radical generation and reduced antioxidant defense.     

Direct fetal injections of diethylstilbestrol and 17 beta-estradiol: a method for investigating their teratogenicity

The synthetic estrogen, diethylstilbestrol (DES), causes urogenital malformations in humans, primates, and rodents. This study was designed to determine whether these effects of DES are related to its estrogenicity. Therefore, DES (0.1, 1, and 10 micrograms) or the natural estrogen, estradiol (E2) (10 and 100 micrograms) was injected directly into day 19 rat fetuses. In the 6- to 7-week-old female offspring exposed to DES, a dose-related incidence of cleft phallus, hypospadias, and incomplete coiling of oviducts was observed. The single fetal injection of E2 elicited similar urogenital malformations, but was approximately 100-fold less potent than DES. A single subcutaneous dose of either DES (0.025, 0.25, or 2.5 mg/kg) or E2 (2.5 or 25 mg/kg) to dams on day 19 of pregnancy induced a spectrum of malformations similar to that following fetal injection. The offspring of treated dams, but not those injected directly as fetuses, had nonfunctioning ovaries (no corpora lutea) yet vaginal signs of estrous were present. It is concluded that DES can act directly in the fetus and its teratogenicity does not require maternal mediation. Since a high dose of E2 produced similar malformations when given to fetuses, it appears that excess estrogen during prenatal life is teratogenic. Thus, at least those endpoints of the teratogenicity of DES that were measured are accounted for by its estrogenic activity.     

Inhibition of rat liver glutathione S-transferase isoenzymes by acrolein

The in vitro effect of the toxin and teratogen, acrolein, on the fetal rat liver glutathione S-transferase isoenzyme, YcYfetus, was investigated and compared with acrolein's effect on some of the adult rat liver glutathione S-transferase isoenzymes. Acrolein was found to inhibit all the isoenzymes investigated and double-reciprocal plots suggest that inhibition is either noncompetitive or mixed-type noncompetitive. It is therefore attractive to suggest that should a similar situation arise in vivo, it may provide one mechanism for the teratogenicity of acrolein.     

Difference in teratogenic potency of ethylenethiourea in rats and mice: relative contribution of embryonic and maternal factors

Ethylenethiourea (ETU) is a potent teratogen in the rat but not in the mouse or any other species tested. Embryotoxic and teratogenic effects are produced in mice only after exposure to 10-40 times the teratogenic dose of ETU in rats. This study was undertaken to determine whether the difference in sensitivity between rats and mice is due to differences within the embryo, to maternal metabolic differences, or both. Comparably staged rat and mouse embryos (gestation day 10.5 and 8.5, respectively) with intact extra-embryonic membranes were maintained under identical conditions in whole embryo culture and exposed to static concentrations of ETU for 48 hours. The teratogenic effects of ETU were qualitatively similar in both species, characterized by excessive fluid accumulations in embryonic structures. The most common abnormalities were distended neural tube, especially in the hindbrain, and clear blisters on the caudal region. At least two times as much ETU was required to produce a similar incidence of abnormalities in mice as in rats. Thus, there is some intrinsic difference in the quantitative response of rat and mouse embryos to ETU, but it is insufficient to account for the in vivo discrepancy. The role of maternal metabolism in modifying the teratogenicity of ETU was assessed by adding hepatic S-9 fractions from Aroclor 1254-induced rats and mice to whole embryo culture. Rat S-9 had no effect on ETU teratogenicity. Mouse S-9 virtually eliminated the formation of abnormalities typical of ETU in both rat and mouse embryos. Decreased exocoelomic fluid osmolality, a physiological effect produced by ETU, also was not observed in embryos exposed to ETU and mouse S-9. ETU-typical defects were observed in embryos exposed to ETU and mouse S-9 which had been treated with carbon monoxide to inactivate its monooxygenase system, indicating that the mouse S-9 was metabolizing ETU. A surprising result was that adding mouse S-9 to embryo cultures containing ETU resulted in the formation of abnormalities (principally open neural tube) that were not observed in in vitro rat or mouse embryos exposed to ETU alone, or in mouse embryos in vivo. We believe that the most likely cause of these abnormalities is a putative ETU metabolite, which is rapidly excreted by the dam in vivo, but accumulates to teratogenic concentrations in vitro.     

Effects of phosphoramide mustard and acrolein, cytotoxic metabolites of cyclophosphamide, on mouse limb development in vitro

Phosphoramide mustard and acrolein are toxic and reactive metabolites of the widely used anticancer drug and known teratogen cyclophosphamide. To study the mechanism(s) involved and to determine which of the active metabolites of cyclophosphamide is responsible for the production of limb malformations, the effects of exposure of cultured limb buds to phosphoramide mustard and acrolein were investigated. Fore- and hindlimbs were excised from ICR mice on day 12 of gestation and cultured in roller bottles for 6 days. Limbs were exposed to either phosphoramide mustard or acrolein (10 or 50 micrograms/ml) for the first 20 hours of the culture period. Exposure to phosphoramide mustard produced limb reduction malformations in both the fore- and hindlimbs; total limb bone area was greatly reduced, while the relative contribution of the paw to this area in forelimbs was increased. There was a fourfold reduction in both DNA and RNA; protein content was reduced only by one-half. Alkaline phosphatase activity was significantly decreased in fore- and hindlimbs exposed to phosphoramide mustard, whereas creatine phosphokinase activity was only reduced in hindlimbs in the limbs exposed to the higher concentration of phosphoramide mustard. Exposure to acrolein also produced malformed limbs with a mangled appearance; however, total limb bone area and the relative contribution of the long bones versus paw structures were not altered. Acrolein exposure had little effect on growth parameters such as DNA (decreased only in hindlimbs exposed to 50 micrograms/ml), RNA (increased in hindlimbs exposed to 50 micrograms/ml), or protein content. Alkaline phosphatase and creatine phosphokinase activities were not altered in acrolein-exposed fore- or hindlimbs. Thus, phosphoramide mustard and acrolein have dramatically different effects on developing limbs in vitro; this observation may indicate that they have different targets and/or mechanisms of action as teratogens in the limb. The effects of phosphoramide mustard are very similar to those of "activated" cyclophosphamide (4-hydroperoxycyclophosphamide).     

Teratogenic action of an inhibitor of cholesterol synthesis in Wistar and Sprague-Dawley rats]

Sprague-Dawley rats are sensitive to the teratogenic action of AY 9944, an inhibitor of cholesterol synthesis, but the dose of inhibitor necessary to induce the same rate of characteristic malformations is twice as large for Sprague-Dawley as for Wistar rats. This variation is probably related to differences in levels of blood cholesterol in the strains and demonstrates a relationship between teratogenicity and metabolic disturbances.     

Embryolethality in rats caused by retinoic acid

Retinoic acid (25 mg/kg) administered by gavage to rats at 216 hours of gestation killed almost all conceptuses by 288 hours and all by full term. The embryolethal dose50 was 12.3 mg/kg. Embryos died from damage to the allantois leading to agenesis or hypogenesis of the chorioallantoic placenta. Suppression of cell division in, disturbed fluid entry into, and impaired normal vascularization of the allantois underlay the abnormality, the predominant element depending principally on when exposure occurred. Hydremia (Br. J. Exp. Pathol., 57:525-541, '76) affected over 50% of the sacs. Relating data from the literature to resorption patterns suggested that 25 mg/kg of retinoic acid raised a lethotoxic level (approximately 2 micrograms/ml) of retinoate in the plasma for about 5 hours. This, together with asynchronous development, was used to help explain why groups of embryos responded to the teratogen for 18 hours longer than single embryos and why exposure 18 hours before the allantois on average appears, killed some young. Comparison showed that retinoic acid was 4.8 times as embryolethal as retinol. Otherwise, each behaved qualitatively similarly, suggesting that either retinol acts through retinoic acid or via a common path entered independently by each. Placental agenesis is well documented in Soviet literature. It is almost unknown in Western teratology even though it is probably the most important cause of embryonal death following teratogenic procedures around the time of placentation.