William S. Webster

Teratogen update: Congenital rubella

It is apparent that there are many unanswered questions about the pathogenesis of CRS. For instance, the chance of embryonic infection decreases in the second semester only to increase again in the third trimester. This is presumably due to unspecified changes in the placenta. When the embryo is infected early in the first trimester it does not appear to have any conventional immunological response to prevent spread of the virus. Yet it has been suggested that only 1 in 10(3) to 10(5) of its cells become infected. If this is true, what controls the spread of the virus in the early embryo? Why does the virus not affect major morphogenetic processes? There is considerable evidence that the virus spreads through the vascular system of the infected fetus and the observed cardiovascular, CNS, and hearing defects may be primarily due to focal cytopathic damage to the walls of blood vessels and lining of the heart; subsequent organ infection and/or ischemia may lead to further damage. Damage to blood vessels is probably extensive throughout the fetus and may be the cause of the generalized growth retardation. The effects in the eye appear to be due to a direct cytopathic effect, particularly on the lens. The short susceptible period for cataract formation is consistent with the protective effect of the lens capsule. Deafness, cardiovascular and neurological damage, and retinopathy all occur when infection takes place in the first 16 weeks of gestation and are rare after this time, despite the absence of any obvious morphological or functional changes in the susceptible structures. This termination of susceptibility in the second trimester is consistent with development of the fetal immune response and increased transfer of maternal IgG. The effect on blood vessels in particular may be limited by antibody production, although existing endothelial infection and damage may be progressive. The fetus seems unable to rid itself of established intracellular virus. The causes of the well-established late manifestations remain unknown. If these serious late-appearing effects are due to prenatal damage, then it is possible that other human teratogens may also cause unexpected late symptoms. This should also be a concern in the area of animal reproductive toxicology testing.

The Toxic Effects of Cadmium on the Neonatal Mouse CNS

Mice (QS outbred strain) received a single subcutaneous injection of cadmium chloride in saline on postnatal day 1, 8, 15, or 22. Histological examination, 24 hours after cadmium exposure on day 1, revealed petechial hemorrhages, edema, and cellular pycnosis throughout much of the immature brain. Treatment on days 8 or 15 produced similar damage, particularly edema and pycnosis, but affected progressively less of the brain until, by day 22, the brain was apparently unaffected by cadmium. Some animals, allowed to survive six to eight weeks after cadmium injection, showed behavioral anomalies and persistent brain deficits. Electron microscopic examination of parietal cortex from animals exposed to cadmium on day 1 revealed that petechial hemorrhages first occurred two hours after treatment. The hemorrhages increased during the next six hours, and were accompanied by thinning and vacuolization of the capillary walls and widening of interendothelial gaps. In general, such changes were restricted to the partially differentiated capillaries. Degenerative changes in the brain cells were first seen about six hours after cadmium exposure

Initiation of phenytoin teratogenesis: Pharmacologically induced embryonic bradycardia and arrhythmia resulting in hypoxia and possible free radical damage at reoxygenation

The aim of this study was to investigate if phenytoin has the capacity to induce embryonic hypoxia mediated via adverse effects on the embryonic heart. Mouse embryos of different strains (CD-1, C57B1/6J and A/J) as well as Sprague Dawley (SD) rat embryos were cultured in vitro (in 75-80% rat serum) by the whole embryo technique. Effects on the heart were examined on gestational day 10 for mouse embryos and days 11 and 13 for rat embryos. Phenytoin was dissolved in water to give concentrations of 50-800 microM. In the mouse embryo studies, phenytoin caused a concentration-dependent decrease in embryonic heart rate in all three strains, with a slight decrease at 100 microM (2-7%) and a more pronounced effect at 200 microM (approximately 20%). Temporary or permanent cardiac arrest occurred in 86% of the CD-1 embryos at 500 microM, in 67% of the C57B1/6JM at 400 microM, and in all A/J embryos at 300 microM. Arrhythmias was observed in 8% in CD-1 embryos at 200 microM, in 18% at 150 microM in C57B1/6J embryos, and in 67% of the A/J embryos at 100 microM (lowest tested concentrations where arrhythmias occurred). In rat embryos, a concentration-dependent decrease in heart rate was observed on both days 11 and 13 at similar concentrations as in the mouse embryo studies. In a separate experiment, the effects on the heart rate of free phenytoin (not serum protein bound) were examined in rat embryos cultured in serum-free medium. Already at 12 microM a significant decrease in heart rate was observed. Altogether, the results support the hypothesis that phenytoin teratogenicity is initiated by pharmacologically induced embryonic hypoxia. A genetic susceptibility to the adverse effects of phenytoin on the embryonic heart may be of importance to explain strain and species differences in phenytoin teratogenicity.

Warfarin exposure and calcification of the arterial system in the rat.

There is evidence from knock‐out mice that the extrahepatic vitamin K‐dependent protein, matrix gla protein, is necessary to prevent arterial calcification. The aim of this study was to determine if a warfarin treatment regimen in rats, designed to cause extra‐hepatic vitamin K deficiency, would also cause arterial calcification. Sprague‐Dawley rats were treated from birth for 5–12 weeks with daily doses of warfarin and concurrent vitamin K1. This treatment causes an extrahepatic vitamin K deficiency without affecting the vitamin K‐dependent blood clotting factors. At the end of treatment the rats were killed and the vascular system was examined for evidence of calcification. All treated animals showed extensive arterial calcification. The cerebral arteries and the veins and capillaries did not appear to be affected. It is likely that humans on long‐term warfarin treatment have extrahepatic vitamin K deficiency and hence they are potentially at increased risk of developing arterial calcification.

Maternal hyperphenylalaninemia fetal effects

Thirty-four children of 11 mothers with untreated hyperphenylalaninemia had a pattern of malformation consisting of prenatal and postnatal growth retardation, microcephaly and central nervous system dysfunction, increased incidence of malformations, and a peculiar facial appearance. Maternal hyperphenylalaninemia appears to be teratogenic, with a variability related to the blood phenylalanine concentration.

Severe cervical dysplasia and nasal cartilage calcification following prenatal warfarin exposure

We present an infant who was exposed to warfarin throughout pregnancy and has warfarin embryopathy. When the child was examined radiologically at 20 months areas of calcification were visible in the septal and alar cartilages of the small external part of the nose. The location of this ectopic calcification is consistent with that seen in an animal model of the warfarin embryopathy. It supports the hypothesis that warfarin interferes with the prenatal growth of the cartilaginous nasal septum by inhibiting the normal formation of a vitamin K-dependent protein that prevents calcification of cartilage. The child also had severe abnormalities of the cervical vertebrae and secondary damage to the spinal cord. Cervical vertebral anomalies are a relatively common finding in the warfarin embryopathy and in the related Binder syndrome.

Prescription drugs and pregnancy

Prescribing drugs in pregnancy is an unusual risk-benefit situation. Drugs that may be of benefit or even life-saving to the mother can deform or kill the fetus. However, the risk to the fetus should not be exaggerated. There are only ~ 20 drugs or groups of drugs which are known to cause birth defects in humans. For one of these drugs to cause birth defects, a number of criteria must be fulfilled. The drug exposure must take place at a critical stage of pregnancy and the dose must be high enough to cause a threshold of exposure for an appropriate duration of time. For most of the known human teratogens, > 90% of pregnancies exposed during the first trimester result in normal offspring. Although only a few drugs are known to cause birth defects in humans, uncertainty about the safety of the majority may lead to underprescribing for pregnant women and women of childbearing age. Epidemiological studies of pregnancy outcome after specific drug exposures are often superficially reassuring, but most are severely limited in their power to detect adverse outcomes. Safety in animal studies may also be reassuring but species differences demand caution in this interpretation. Concerns about prescription drugs in the first trimester, when they can cause birth defects, are mostly quite different to concerns about use in the second and third trimesters. As the fetal organ systems mature, the fetus can be affected by the pharmacological activity of the drug in the same way as the mother. Many drugs have pharmacological effects on the fetus in the second and third trimesters but in most cases, they are well recognised and can be managed or avoided. The material presented in this paper is mostly concerned with the ‘risks’ associated with drugs in pregnancy. No attempt has been made to quantitate the possible benefits to the mother or fetus. Communicating the risk-benefit situation to the patient is always a challenge for physicians with limited time and sometimes limited knowledge. Fear of litigation is an unfortunate and an unwanted parameter in the assessment. Better knowledge of the parameters that determine teratogenicity may allow physicians to feel more confident in assessing the risks and benefits associated with prescribing in pregnancy.

Teratogenesis after acute alcohol exposure in cultured rat embryos

In order to investigate whether alcohol has teratogenic properties, rat embryos were cultured in vitro during their organogenetic period and exposed to ethanol at 200-800 mg% culture medium for the 48 hours of culture (0-24 somite stage) or to 600-800 mg% for 24 hours, or 6-hour periods. Exposure to alcohol throughout the entire 48-hour culture period or the first 24-hour period (0-12 somites) produced marked growth retardation, particularly of the head region in a dose-dependent manner, but did not prevent neural tube closure. Exposure to high levels of ethanol during specific 6-hour periods of early organogenesis (three to nine somites) prevented closure of the neural tube in 30% of cultured rat embryos, indicating a direct teratogenic action of ethanol. These results implied an effect of ethanol on embryonic development, independent of maternal metabolism. The 6-hour exposure experiments demonstrated that high doses of ethanol during specific periods of organogenesis can be teratogenic.

IN VITRO ASSESSMENT OF INDIVIDUAL AND INTERACTIVE EFFECTS OF AROMATIC HYDROCARBONS ON EMBRYONIC DEVELOPMENT OF THE RAT

There have been reports of disruption of embryonic development following exposure of pregnant women to aromatic hydrocarbons. In the present study, the embryotoxicity of toluene, xylene, benzene, styrene, and its metabolite, styrene oxide, was evaluated using the in vitro culture of postimplantation rat embryos. Possible interactions between toluene, xylene, and benzene were also studied using mixtures of these solvents. The results of the study showed that toluene, xylene, benzene, and styrene all have a concentration-dependent embryotoxic effect on the developing rat embryo in vitro. Styrene was embryotoxic at a lower concentration (1.00 mumol/mL) than benzene (1.56 mumol/mL), toluene (2.25 mumol/mL), or xylene (1.89 mumol/mL). The metabolite of styrene, styrene oxide, was embryotoxic at a concentration (0.038 mumol/mL). more than 20 times less than the parent compound. There was no evidence of a synergistic interaction between toluene, xylene, and benzene in causing embryotoxicity; the solvents interacted in an additive manner. The embryos were exposed to the solvents for 40 h of the organogenic period. When the levels of solvents found to be embryotoxic in the present study are compared to blood levels in the human following industrial exposure or solvent abuse, it appears unlikely that the threshold blood levels for embryotoxicity would be exceeded in the workplace. However, the possibility that exposure to solvents earlier or later or throughout the entire organogenic period might result in a different conclusion cannot be excluded.

Prothrombin and PIVKA-II levels in cord blood from newborn exposed to anticonvulsants during pregnancy.

Summary: Purpose: To determine whether anticonvulsant exposure during human pregnancy caused an increase of the abnormal form of prothrombin, known as PIVKA‐II (prothrombin induced by vitamin K absence for factor II), and a decrease in total prothrombin, in the blood of the newborn.