Reactive oxygen species and DNA oxidation in fetal rat tissues.

It is well recognized that reactive oxygen species (ROS) are formed during the reperfusion of ischemic tissues and ROS may be pathogenic in adult tissues. Although there is little information on the formation and toxicity of ROS during prenatal life, a strong association has been made between limb and possibly brain malformations and uteroplacental ischemia during fetal stages of gestation. It has been proposed that these malformations result from attack by ROS formed during the resumption of placental perfusion. Studies reported here examined formation of ROS in teratogenically sensitive limb and brain and insensitive heart before and during the period of teratogenic sensitivity. Also examined was the formation of ROS following hypoxia and reoxygenation in fetal culture and DNA hydroxylation in sensitive and insensitive fetal tissues during uteroplacental ischemia and reperfusion in vivo. Rates of formation of superoxide anion radical and hydrogen peroxide declined with increasing gestational age whereas those for hydroxyl radical increased. Hydrogen peroxide generation was greatest in insensitive heart whereas hydroxyl radical formation was significantly lower in brain than in limb or heart, which had comparable rates. Hydrogen peroxide generation, which declined significantly in fetuses, but not in membranes with gestation, failed to respond to reoxygenation in vitro. Finally, there were significant increases in DNA hydroxylation in fetal hearts and limbs, but not in brains during uteroplacental ischemia but no further significant change could be detected after reperfusion.

Differential glutathione depletion by L-buthionine-S,R-sulfoximine in rat embryo versus visceral yolk sac in vivo and in vitro.

L-Buthionine-S,R-sulfoximine (L-BSO), a selective inhibitor of glutathione synthesis, exhibited the capacity to deplete embryonic and visceral yolk sac glutathione (GSH) after administration in vivo and also after addition of L-BSO to a whole embryo culture system. Administration of L-BSO to pregnant dams at 2.5 or 18 hr prior to the explantation of day 10 conceptuses resulted in greater than 55% GSH depletion relative to control values in both the yolk sac and the embryo proper. The levels of GSH returned to normal in all embryos and in the corresponding visceral yolk sacs pretreated at 2.5 hr (but not at 18 hr) after culturing for 24 hr in L-BSO-free medium. GSH content was significantly lower in yolk sacs but not in embryos of conceptuses cultured for 24 hr in medium containing 1 mM L-BSO. This treatment, however, significantly increased the amount of protein and DNA in the embryo. The differential sensitivity of the yolk sac versus embryo and the demonstration of the ability to modulate tissue levels of GSH during organogenesis promise to provide important new tools for the study of embryonic protective mechanisms in chemical teratogenesis.

Human embryonic cytochrome P450S: phenoxazone ethers as probes for expression of functional isoforms during organogenesis.

Human embryonic tissues were investigated during the period of organogenesis with a combination of substrate probes, selective inhibitors and immunoprobes in terms of their capacity to express functional P450 isoforms. A series of phenoxazone ethers utilized as substrate probes revealed that human embryonic hepatic, pulmonary, renal, adrenal and cardiac tissues each contained a complement of functional P450 isoforms when analyzed between days 50 and 60 of gestation. Preparations of each of these tissues contained isoforms capable of catalyzing O-demethylation, O-deethylation, O-depentylation and O-debenzylation of the respective phenoxazone ethers. Investigations with chemical inhibitors and inhibitory antibodies as well as comparisons with vector-expressed, human P450 isoforms suggested that isoforms of P450 subfamilies 1A, 2B, 2C or 3A were not major contributors to any of the observed reactions. The P450-dependent reactions studied exhibited several unexpected and unusual characteristics including a preference for NADH over NADPH as the initial electron donor. Results were consistent with the concept that conceptal-specific P450 isoforms participate in the human embryonic O-dealkylation/debenzylation probe reactions investigated.

The potential role of redox cycling as a mechanism for chemical teratogenesis.

A survey of the literature indicates that several chemicals whose reduced metabolites are capable of undergoing redox cycling in biological systems also possess significant teratogenic properties when tested in vivo. We have initiated investigations to determine whether the embryotoxic effects of such chemicals could result from their redox cycling properties and whether redox cycling could be an important mechanism in chemical teratogenesis. In order to obviate the potentially confounding influences of maternal factors, our initial studies have been performed with a whole embryo culture system with redox cycling agents added directly to the culture medium. Several representative redox cycling agents including doxorubicin, paraquat, a series of nitroheterocycles, nitrosofluorene, and diethylstilbestrol (converted metabolically to redox cycling quinone/semiquinone radicals) have been investigated thus far. The nitroheterocycles which bear nitro groups with comparatively high redox potentials produced a striking, asymmetric defect involving primarily the right half of the prosencephalic and mesencephalic regions. The effect was exacerbated under conditions of low O2 tension. Accumulated data to date strongly suggest that reduction of the nitro group is an essential feature in the embryotoxic mechanism. Quinones (doxorubicin, paraquat) and compounds metabolically converted to quinones (diethylstilbestrol) appeared to produce embryotoxic effects via mechanisms not associated with redox cycling. Nitrosofluorene embryotoxicity was markedly exacerbated by changes in both intra- and extracellular glutathione levels, but definitive dependence on a radical-mediated effect or redox cycling was not demonstrated.

Studies of the cellular distribution of superoxide dismutases in adult and fetal rat tissues.

Activities of three types of superoxide dismutase in tissue fractions were significantly lower in fetal and adult brain and fetal limb preparations than in fetal and adult heart preparations. An exception was the cytoplasmic fraction of adult brain that had levels of Cu, Zn-superoxide dismutase activity comparable to those in cytoplasmic fractions of heart. In addition, Mn superoxide dismutase activity appeared to be very low in all fetal mitochondrial matrix fractions and cytoplasmic fractions as well as in adult brain. Finally, the results of these studies emphasize the importance of two antioxidant defense systems in the tissues studied, one associated with the mitochondrial electron transport system and the other, the cytosolic Cu, Zn enzyme.

Teratogenicity of acetaldehyde in vitro: relevance to the fetal alcohol syndrome.

Day 10 rat embryos grown in vitro showed significant retardation in growth and development when culture media contained acetaldehyde. A concentration-response range for acetaldehyde-induced embryotoxicity was defined, from no effect at 5 microM to complete lethality at 100 microM. The relative teratogenicity of ethanol and acetaldehyde, and the potential roles of these compounds in producing the Fetal Alcohol Syndrome are discussed. Despite intensive investigation into alcohol teratogenicity, the mechanism that produces the Fetal Alcohol Syndrome (FAS) remains unknown. Observed anomalies may result from direct embryonic exposure to ethanol or one of its metabolites, or from some indirect effect such as altered placental function or maternal nutritional status. Use of in vitro techniques allows study of direct embryonic exposures in the absence of indirect influences. Under such conditions, ethanol has been found to exert direct embryotoxicity (1). Rat embryos, grown as cultured explants and subjected to ethanol concentrations of 32.5 or 65 mM, were retarded in growth and development when compared to untreated controls. In this paper, we report direct embrytoxic effects of acetaldehyde, the primary metabolite of ethanol, at concentrations as low as 25 microM. Acetaldehyde teratogenicity has not been extensively studied. Veghelyi et al. (2) and Lambert, Papp and Nishiura (3) employed a combination of ethanol and disulfiram (an inhibitor of acetaldehyde-oxidizing enzymes). Teratogenic effects exceeded expectations based upon assumption of an additive interaction between these two compounds, and were attributed to elevated maternal blood acetaldehyde. O'Shea and Kauffman (4,5) and Dreosti et al. (6) administered acetaldehyde to pregnant animals by injection. Treatment resulted in retarded growth and development, decreased DNA synthesis, and increased frequencies of malformation and resorption. While these studies imply a role for acetaldehyde in alcohol-induced teratogenesis, indirect effects through altered maternal or placental factors cannot be eliminated. We present here the first concentration-response data for direct embryonic exposure to acetaldehyde.

Cytochrome P450-dependent bioactivation of prodysmorphogens in cultured conceptuses.

These investigations were undertaken to determine the extent to which tissues of cultured rat conceptuses contain cytochrome P450 isoforms in sufficient quantities to significantly influence the capacity of certain chemicals to elicit dysmorphogenic effects in vitro. Investigations with highly sensitive probe substrates/inhibitors and with immunologic methods enabled the detection of at least four separate P450 isoforms in tissues of the visceral yolk sac, ectoplacental cone, and embryo proper. One of the isoforms was identified as P450IA1 and was found to be inducible by polycyclic aromatic hydrocarbons in all three tissues. Other isoforms exhibited properties differing from characterized adult rat hepatic isoforms. Each of the isoforms was detectable in conceptuses on gestational days 10, 11, 12, and 14 and was present in the highest concentrations in the visceral yolk sac. Conceptal P450IA1 catalyzed the conversion of dysmorphogenically inactive 2-acetylaminofluorene to 7-hydroxy-2-acetylaminofluorene, a proximate dysmorphogen. Investigations with microinjections suggested that visceral yolk sac hydroxylation was largely responsible for the bioactivation reaction in vitro. The same isoform exhibited no capacity to influence the dysmorphogenic activity of cyclophosphamide. The results demonstrated that tissues of cultured rat conceptuses may contain P450 isoforms in sufficient amounts to markedly influence the dysmorphogenic activity of substrates of the corresponding isoforms.

The teratogenicity of N(omega)-nitro-L-ariginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, in rats.

Exposure of gravid rats to the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water or by implanted osmotic minipumps significantly elevates maternal blood pressure, reducing uteroplacental perfusion. Administration by either route causes fetal growth retardation, but oral exposure also causes hind limb reduction malformations. The present study employed both oral and intraperitoneal routes to determine the period of sensitivity to developmental toxicity, dose-response, and possible fetotoxic mechanisms. Hind limb hemorrhage occurred only in litters from dams exposed to oral doses of 1 to 2 mg/mL from gestational days 15 through 17. In contrast to oral exposure, single intraperitoneal injections caused both fore and hind limb reductions at doses of 25 mg/kg and above administered on gestational day 16 and later. Many other exposures that reduce uteroplacental perfusion have been associated with vascular disruptive dysmorphogenesis. These exposures include phenytoin, calcium channel inhibitors, cocaine, and uterine vascular clamping. Limb hemorrhage induced by these exposures is usually limited to distal structures, typically phalanges, and the incidence of affected fetuses rarely exceeds 50%. By contrast, hemorrhage caused by L-NAME frequently involves entire limbs, extending into adjacent flank in severe instances, and 100% of fetuses from treated dams may be affected. The basis of this difference and the differing defect patterns associated with the various routes of exposure are unclear, but the generation of reactive oxygen species during resumption of normal perfusion may play a role in this vascular disruption.

A new technique for culturing rat embryos between gestation days 14 and 15.

A method for culturing rat embryos between days 14 and 15 has been developed. On gestation day 14 (morning of sperm and plug = day 0), laparotomies were performed on time-mated rats that had been anaesthetized with halothane. The embryos, with open attached yolk sacs and placentas, were cultured in 650-ml plastic flasks with 120 ml medium (0.7% bovine serum albumin in Earle's balanced salt solution with antibiotics, prewarmed to 37 degrees C and continuously flushed with water-saturated 95% O(2)/5% CO(2), pH 7.4) in a Dubnoff metabolic shaker with gentle agitation. Viability and morphology were assessed after 26 hr in culture. On day 15, embryonic growth and development were similar to that in in vivo controls, and viability was high (55%).

Embryonic and early fetal loss.

Embryonic and early fetal loss occurs in approximately one out of two pregnancies. Although this rate might appear unreasonably high, it is borne out by several types of studies and further confirmed by careful animal study. It now appears that karyotype deviation is common in early abortuses and probably underlies the high rate of gross abnormality seen in this group. In addition, environmental factors (radiation, smoking, anesthetics, and previous therapeutic abortion) may contribute to this wastage. Study and analysis of the abortus have allowed identification of a number of new embryonic and fetal syndromes. Careful description and classification combined with continued monitoring of abortuses shows promise both as an adjunct to reproductive counseling and as an important defense against teratogenic exposure. It is through this study that the etiology and ultimately the control of preventable pregnancy wastage will be understood and implemented.

Culture of whole rodent embryos in teratogen screening.

Rodent embryos can be grown in vitro during a period of rapid organogenesis. During a 24-48 hour culture period, growth and development closely approximate the values of embryos in utero at the same gestational stage. Embryos can be exposed to carefully controlled concentrations of test substances in a system which is free of maternal variables such as nutritional status or stress effects. Developmental abnormalities are limited to those systems developing during the culture period, but those available may show heightened sensitivity relative to the embryo in utero. Recently several systems have been developed which permit incorporation of either metabolites or metabolic enzymes in embryo culture. These materials can be obtained from species other than that of the test embryos. Because studies have shown the importance of maternal metabolism in the activation and inactivation of teratogens, it is hoped that these systems will enable us to understand the biochemical basis of species variability in teratogenic sensitivity and construct a teratogen screen which can reliably identify compounds which pose teratogenic hazards to humans.

Fetomaternal potassium relations in the rat on the twentieth day of gestation. II. Effects of maternal hypokalemia.

Time-corrected measurements of transplacental and transamniotic electrical potentials were made in potassium-deficient rats on the 20th day of gestation. These averaged 6.4 mV and 2.0 mV, respectively, in the deficient group as compared with 14.3 and 18.4, respectively, in the controls. The measured maternal potassium concentrations were 2.8 mEq/liter in the K+-restricted animals and 4.8 mEq/liter in the controls. These figures were substituted into the Nernst equation in order to predict fetal concentrations in plasma. Similar predictions were made for amniotic fluid assuming equilibrium with fetal plasma. Predicted concentrations in fetal plasma were 2.3 mEq/liter in the deficient animals and 2.8 mEq/liter in the controls. By contrast, observed time-corrected potassium concentrations in fetal plasma were 4.0 mEq/liter in deficient animals and 2.9 mEq/liter in controls. Amniotic fluid values were predicted to be 4.7 mEq/liter in deficient and 2.5 mEq/liter in control animals. These values were compared with the observed averages in amniotic fluid of 4.7 and 4.6 mEq/liter in the deficient and control groups, respectively.

Fetomaternal potassium relations in the fetal rat on the twentieth day of gestation.

Time-corrected measurements of transplacental and transamniotic potentials were made in the fetal rat on the 20th day of gestation. These averaged 14.3 mV and 18.4 mV, respectively. The measured maternal potassium concentration in plasma of 4.8 mEq/liter and these potential measurements were used in the Nernst equation to predict the fetal concentrations in plasma. The concentration in amniotic fluid was predicted, using the potentials and either the fetal or maternal plasma concentration. The predicted concentrations in fetal plasma and amniotic fluid were 2.8 and 2.5 mEq/liter, respectively. The sampling time-corrected potassium concentration in fetal plasma was 2.9 mEq/liter and the mean in amniotic fluid was 4.6 mEq/liter. The observed concentration in amniotic fluid is significantly higher than the predicted level.

The teratogenicity of cocaine.

Pregnancy rats and mice received high doses of cocaine hydrochloride by intraperitoneal injection. Despite treatment periods which included most of organogenesis, no increase in congenital abnormalities was observed. Rats showed significant reductions in maternal and fetal weights as well as increased resorption frequencies. Fetal edema was also found. Mice showed only decreased fetal weights with no increase in malformations.