Alan G. Fantel

Teratogenic bioactivation of cyclophosphamide in vitro

Abstract Day 10 rat embryos grown as cultured explants in vitro developed characteristic defects when culture media contained cyclophosphamide (6.25 micrograms per milliliter or more), an hepatic microsomal fraction, and cofactors for a monooxygenase system. Cyclophosphamide concentrations as high as 250 micrograms per milliliter were innocuous when either the microsomal material or cofactors were omitted from the medium. These experiments represent the first direct demonstration of bioactivation of a proteratogen.

Periconceptional multivitamin use in relation to the risk of congenital urinary tract anomalies.

To study the relation of maternal periconceptional vitamin use to the risk of a congenital urinary tract anomaly (CUTA), we conducted a case-control study using the Washington State Birth Defect Registry. We identified CUTA cases with no known chromosomal abnormality in seven counties in western Washington State occurring between January 1, 1990, and December 31, 1991. We randomly selected a sample, as controls, of all infants delivered in five large hospitals in King County who did not have a birth defect and who were born in the same year as the cases. About 55% of all infants in King County and a smaller proportion of infants in the other six counties are delivered in these five hospitals. We interviewed mothers of 118 cases and 369 controls to obtain information about their vitamin use during the pregnancy and during the year before the conception. After adjustment for maternal race, family income, county of maternal residence, and birth year, we found that women who used multivitamins during the first trimester had only 15% the risk of bearing a child with a CUTA compared with women who did not take vitamins [odds ratio (OR) = 0.15; 95% confidence interval (CI) = 0.05–0.43]. The reduction was smaller for use restricted to the second or third trimesters (OR = 0.31; 95% CI = 0.09–1.02). Among women who used vitamins during the first trimester, vitamin use before conception was not associated with any further reduction in the risk, nor did there appear to be an association with the amount or brand of vitamin used. Restricting the analysis to residents of King County did not change the results. Our results indicate that prenatal multivitamin use, particularly during the first trimester, may reduce the risk of a CUTA. Because all of the preparations taken by study participants contained many vitamins as well as folic acid, it was not possible to identify which one (or several) chemical(s) may have been responsible for the reduced risk of a CUTA.

Teratogenicity of cyclophosphamide metabolites: Phosphoramide mustard, acrolein, and 4-ketocyclophosphamide in rat embryos cultured in vitro

Abstract Phosphoramide mustard, acrolein, and 4-ketocyclosphosphamide, known stable metabolic products of bioactivated cyclophosphamide, were tested for their teratogenicity against rat embryos grown in vitro from Day 10 to Day 11 of gestation. Results indicate that phosphoramide mustard is the teratogenic metabolite of cyclophosphamide, since the effects of phosphoramide mustard exactly parallel those of bioactivated cyclophosphamide. These effects are reductions in total embryo protein content, crown-rump length and number of somites, characteristic malformations, and pattern of cell necrosis. Acrolein at a dose equimolar to a dose of bioactivated cyclophosphamide which produced malformations in 100% of treated embryos had no effect on any of the parameters measured. An equimolar dose of 4-ketocyclophosphamide had no effect on total embryo protein content or crown-rump length and number of somites, but consistently produced some malformed embryos. The kinds of malformations observed, however, are not seen in embryos treated with phospharamide mustard or bioactivated cyclophosphamide.

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.

Involvement of Mitochondria and Other Free Radical Sources in Normal and Abnormal Fetal Development

Shepard and Mackler have documented quantitative increases in mitochondrial cristae between gestational days 10 and 14 in rats accompanied by decreased glucose utilization and increased NADH oxidase activity. Findings show a shift from glycolytic to oxidative metabolism starting at around the time of implantation. Exposure to many substances that cause transient uteroplacental hypoperfusion, including cocaine, phenytoin, calcium channel blockers, and nitric oxide synthase (NOS) inhibitors, induce limb and central nervous system (CNS) malformations while sparing the heart. We have reported that isolated electron transport particles prepared from sensitive tissues show reduced NADH oxidase activities compared with insensitive heart. They also have significantly greater superoxide formation in association with significantly reduced superoxide dismutase activities. NOS inhibitors induce severe limb reductions in late gestation. Exposure is associated with hemorrhage and nitrotyrosine (NT) formation shortly after treatment. Hemorrhage, malformations, and NT formation can be significantly reduced by coadministration of PBN, allopurinol, or aminoguanidine. On the basis of these findings, we have proposed a role for the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the genesis of limb reduction defects. It is important to note that limb reduction defects occur in humans (∼0.22/1000) and have been associated with the agents listed above.

Organ Weight Standards for Human Fetuses

Five hundred fifty-eight fresh human embryos and fetuses were obtained from the universities of Washington and Michigan following spontaneous loss, elective termination, or neonatal death within 2 days of delivery. The body weights ranged from 1.5 to 1500 g. Each of these autopsied specimens was morphologically normal. Specimens from diabetic or hypertensive mothers were not included. Correlations between fetal body weight and weights of adrenal, brain, kidney, liver, lung, spleen, and thymus were established. For analysis, regression curves were calculated as quadratic equations of best fit by the weighted least squares. The relation of the weights of brain, heart, and liver to body weight appeared linear. The ratios of thymus, spleen, and kidney to body weight were nonlinear and gradually increased. The ratios of lung and adrenal weights to body weight were also nonlinear and gradually decreased. Ninety-five percent prediction intervals were generated for each of the eight organs using a computerized statistical package. The results compare closely with smaller studies in the literature.

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 Hanks 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.

Maternal smoking during pregnancy and the risk of congenital urinary tract anomalies.

To study maternal smoking during pregnancy and the risk of congenital urinary tract anomalies, we interviewed mothers of 118 affected infants born to residents of western Washington State during 1990 and 1991 and mothers of 369 control infants randomly selected from those without birth defects delivered during those years in five hospitals in King County, Washington. Maternal smoking was associated with an increased risk of congenital urinary tract anomalies in offspring (adjusted odds ratio [OR] = 2.3; 95% confidence interval [CI] = 1.2, 4.5). This risk was higher among light smokers (1-1000 cigarettes during the pregnancy) (OR = 3.7; 95% CI = 1.7, 8.6) than among heavy smokers (OR = 1.4; 95% CI = 0.6, 3.3). Our results corroborate previous findings and support the hypothesis of a causal relation.

Bioactivation of procarcinogens to mutagens in human fetal and placental tissues.

Abstract Enzyme systems in homogenates of human fetal hepatic, pulmonary, adrenal, renal and placental tissues catalyzed the conversion of benzo(a)pyrene, 7, 12-dimethylbenz(a)anthracene and N-2-fluorenyl-acetamide to intermediary metabolites that produced mutations in Salmonella typhimurium . The mutagenic metabolites generated from the three procarcinogen/promutagens displayed specificity with respect to the auxotrophic strains, chemicals and tissues investigated.

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.