Diana J. Oakes

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.

The Relationship Between Cleft Lip, Maxillary Hypoplasia, Hypoxia and Phenytoin

Cleft lip (CL) is a common malformation that has both genetic and exogenous causes. The main pharmaceutical cause is exposure to phenytoin during early facial development in the 5th to 6th weeks of gestation. Phenytoin also causes CL if administered to pregnant rats during the period of early facial development. Evidence is presented that in the pregnant rat, a teratogenic dose of phenytoin slows the early embryonic heart and causes a prolonged period of embryonic hypoxia. It is proposed that this hypoxia, through an undefined downstream mechanism, leads to the development of CL. The involvement of hypoxia in the pathogenesis of CL is in agreement with studies in mouse strains with a spontaneous rate of CL in which exposure to hypoxia has been shown to increase the rate and hyperoxia to decrease the rate. Other exogenous risk factors during pregnancy for human CL include maternal cigarette smoking, residence at high altitude and exposure to corticosteroids. It is suggested that these exposures all involve an increased risk of embryonic hypoxia. It has been proposed that phenytoin affects the embryonic heart by inhibition of the human-ether-a-go-go (HERG) potassium channel. Phenytoin also inhibits sodium and calcium channels and these properties may also be involved in the observed effect on the embryonic heart. Phenytoin-induced bradycardia leading to embryonic hypoxia may be an important mechanism by which phenytoin causes birth defects.

Testicular changes induced by chronic exposure to the herbicide formulation, Tordon 75D® (2,4-dichlorophenoxyacetic acid and picloram) in rats

The second most used herbicide in the Vietnam war was Agent White, which contained the active components 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram). The herbicide formulation Tordon 75D is similar in terms of its active components to Agent White and is currently used by the agricultural industry in Australia. As part of an investigation into the possible adverse effects of this herbicide on male reproductive performance, groups of five male rats were gavaged 5 days a week for 9 weeks with either 0.125 ml/kg (low dose), 0.25 ml/kg (middle dose), or 0.5 ml/kg (high dose) Tordon 75D or water (controls). The high dose corresponded to 150 mg/kg body weight 2,4-D and 37.5 mg/kg picloram acid equivalents. At the end of the treatment period, the testes were collected, weighed, and examined histologically and blood samples were taken to determine serum testosterone. Groups of high dose animals were also examined after 1, 2, and 4 weeks treatment. The 9 weeks treatment with Tordon 75D caused severe reduction in testicular weight in some high dose animals. Histologically, the small testes showed shrunken tubules with germ cell depletion. This damage was still evident in some rats following a 21 weeks recovery period suggesting that the testicular damage was permanent. Testicular damage was not due to endocrine disruption as there were no significant differences in the serum concentration of testosterone in control animals compared to Tordon 75D-treated animals. Blood levels associated with the high dose were determined in a separate study and were much higher than those likely to be obtained by occupational exposure to this herbicide.

The Teratogenic Effect of Dofetilide during Rat Limb Development and Association with Drug-Induced Bradycardia and Hypoxia in the Embryo

BACKGROUND Dofetilide is an antiarrhythmic drug that blocks the cardiac repolarizing current IKr ((IKr, rapid component of the delayed rectifying potassium current). Previous studies have shown that (a) IKr is essential for normal cardiac function of the embryonic heart and (b) dofetilide is teratogenic in rodents. This study was undertaken to examine the mechanism by which dofetilide causes limb defects on gestational day 13 (GD 13) in the rat. METHODS Rats were treated with dofetilide (single oral dose, 5 mg/kg) on GD 13 and embryonic heart rates assessed by ultrasound (Vevo770, VisualSonics, Toronto, Ontario, Canada) 2 hr later. Fetuses were examined for malformations on GD 20. In a separate experiment, dofetilide treatment of GD 13 rats was followed 2, 4, 12, or 24 hr with iv dosing with the hypoxia marker, pimonidazole (60 mg/kg). Embryos were collected and heart rates were assessed in vitro and hypoxia in embryo limbs analyzed. RESULTS A teratogenic dose of dofetilide at a susceptible stage of development (GD 13) resulted in a period of bradycardia and arrhythmia of the embryonic heart and hypoxia in the developing limbs (GD 13) resulting in limb malformations (GD 20). CONCLUSIONS Drugs that induce periods of bradycardia and/or arrhythmia of the embryonic heart and cause the embryo to become hypoxic are potential human teratogens.

Model predicting the teratogenic potential of retinyl palmitate, using a combined in vivo/in vitro approach

Retinyl palmitate (RP) is a known laboratory animal teratogen inducing abnormalities of the second visceral arch when administered on day 9 of gestation in the rat. However, there are significant problems when attempting to extrapolate this result to the human. A combined in vivo/in vitro model was developed to assist in human risk assessment. The in vitro teratogenic threshold concentration of a number of retinyl palmitate metabolites was established. Serum concentrations of retinyl palmitate metabolites following a single teratogenic dose of RP in the pregnant rat were also measured. These dosed sera were also used to culture rat embryos. Our hypothesis was that malformations would only be induced by the dosed sera in vitro if the threshold concentration(s) of one or more metabolites was exceeded. Using this approach, it was determined that the teratogenicity of the sera were best predicted by serum retinol levels, with some indication that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid could be involved in some cases. The available human data suggest that threshold concentrations of these retinoids were unlikely to be exceeded following vitamin A supplements of 25,000 IU/day. While the proposed model does not take into account species differences, protein binding, and transfer to the embryo, it does have potential for human risk assessment.

Antipsychotic drugs cause bradycardia in GD 13 rat embryos in vitro

This study investigated the effects of antipsychotic drugs on heart function of gestational day (GD) 13 rat embryos in vitro since they all block the I(Kr)/hERG potassium ion channel in addition to their main pharmacological effect on neurotransmitters. The results showed that all the tested antipsychotic drugs caused bradycardia of the rat embryonic heart in a concentration-dependent manner. However, with the possible exception of haloperidol the tested drugs did not cause arrhythmias typically seen with the highly selective I(Kr)/hERG blocking drug dofetilide. For six of the eight drugs tested the effects on the embryonic rat heart were only seen at free drug concentrations that were much greater than those likely to occur in pregnant women taking antipsychotic medication. However, the safety margins for haloperidol and quetiapine were lower.

The Effect of Dofetilide on the Heart Rate of GD11 and GD13 Rat Embryos, in vivo, Using Ultrasound.

BACKGROUND There are a wide range of drugs including antidepressants, anticonvulsants and antipsychotics that cause embryonic bradycardia in vitro but it is unknown if they have a similar effect in vivo. One way to verify whether these in vitro findings are replicated in vivo is by the use of ultrasound examination of dosed pregnant rats. We tested this by examining the effect of dofetilide on embryonic heart rate (HR) in vivo using ultrasound. METHODS Rats were dosed with dofetilide (4 or 2.5 mg/kg) on GD11 or (5 or 2.5 mg/kg) on GD13 and embryonic HR assessed by ultrasound, 2 and 24 hr later. Fetuses were examined for malformations on GD20. RESULTS HR of control rat embryos showed a wide range at each gestational day. Dosing with dofetilide on GD11 caused severe bradycardia (∼ 60% reduction) 2 hours after dosing with recovery after 24 h of >60% of LD but death and slow HR among the HD embryos. At term, 32% of the LD surviving fetuses had hypoplastic upper lip while >90% of HD embryos had died. On GD13, embryonic HR was reduced in a dose-dependent manner with >85% of LD and HD recovered by 24 hr. At term, all LD fetuses were normal while 29% of HD fetuses had limb defects. CONCLUSIONS Ultrasound is a useful technique to investigate the effect of maternally administered drugs on the embryonic HR in the rat. The results may provide more information about the safety of these drugs in pregnancy leading to better risk assessment for the human.

The effect of prenatal exposure to a repeat high dose of toluene in the fetal rat

Solvent abuse during pregnancy results in a clinical pattern of adverse outcomes including deafness. The aim of this project was to determine whether high doses of toluene for a short duration during pregnancy produced adverse outcomes in the fetal rat. Pregnant rats were given either 1250 mg/kg of toluene or peanut oil by gavage from gestation day 16-19. The study demonstrated that administration of toluene at the dose used did not result in significant maternal toxicity. However, some maternal renal pathology was noted. There was no significant difference in placental or fetal weights nor was there a significant difference in the number of external or skeletal malformations of fetuses between treatment and control groups. Treated fetuses had an increased frequency and severity of enlarged renal pelveses. A pattern of accelerated development in the upper mid-turn and sometimes apical turns in the treated fetal cochleas was observed. This accelerated development suggests that toluene may induce excessive cell death resulting in premature maturation of the cochlea.

Early Gestational Hypoxia and Adverse Developmental Outcomes: Hypoxia and Adverse Developmental Outcomes

Hypoxia is a normal and essential part of embryonic development. However, this state may leave the embryo vulnerable to damage when oxygen supply is disturbed. Embryofetal response to hypoxia is dependent on duration and depth of hypoxia, as well as developmental stage. Early postimplantation rat embryos were resilient to hypoxia, with many surviving up to 1.5 hr of uterine clamping, while most mid‐gestation embryos were dead after 1 hour of clamping. Survivors were small and many had a range of defects, principally terminal transverse limb reduction defects. Similar patterns of malformations occurred when embryonic hypoxia was induced by maternal hypoxia, interruption of uteroplacental flow, or perfusion and embryonic bradycardia. There is good evidence that high altitude pregnancies are associated with smaller babies and increased risk of some malformations, but these results are complicated by increased risk of pre‐eclampsia. Early onset pre‐eclampsia itself is associated with small for dates and increased risk of atrio‐ventricular septal defects. Limb defects have clearly been associated with chorionic villus sampling, cocaine, and misoprostol use. Similar defects are also observed with increased frequency among fetuses who are homozygous for thalassemia. Drugs that block the potassium current, whether as the prime site of action or as a side effect, are highly teratogenic in experimental animals. They induce embryonic bradycardia, hypoxia, hemorrhage, and blisters, leading to transverse limb defects as well as craniofacial and cardiovascular defects. While evidence linking these drugs to birth defects in humans is not compelling, the reason may methodological rather than biological. Birth Defects Research 109:1358–1376, 2017.© 2017 Wiley Periodicals, Inc.

Counselling regarding paternal exposures: Can we do better?

MotherSafe is a free telephone‐based counselling service for the general public and healthcare providers concerned about exposures during pregnancy and breastfeeding. Calls relating to paternal exposures are less common, but can cause distress to the person concerned. This review seeks to identify the key concerns and what information is available to address these concerns.