David A. Beckman

Reproductive and teratologic effects of electromagnetic fields

The reproductive risks of electromagnetic fields (EMF) were evaluated based on an extensive review of the scientific literature pertaining to human epidemiologic studies, secular trend data, in vivo animal studies and in vitro studies, and biologic plausibility. The epidemiologic studies involving the reproductive effects of EMF exposures to human populations have included populations exposed to: (1) video display terminals (VDTs), and (2) power lines and household appliances. The clinical use of diagnostic MRI (magnetic resonance imaging) has been increasing, but there are few reports or studies of pregnant women or individuals of reproductive age who have been exposed to MRI, and whose reproductive performance has been evaluated. The population that has been studied most frequently are women exposed to VDTs, but their EMF exposures are extremely low and frequently are at the level of the ambient EMF in a house or office. The results of epidemiologic studies involving VDTs are generally negative for the reproductive effects that have been studied. Based on the number of studies, the exposure levels, and the fairly consistent results, it can be argued that VDT epidemiologic studies should no longer be given priority. There have been fewer studies concerned with the reproductive risks of power lines, electric substations, and home appliances. In some publications, positive findings for reproductive risks were reported, but the more consistent findings indicate that EMF, even at these higher exposures, do not generate a measurable increase in reproductive failures in the human population. When compared to other fields of human epidemiology, it is obvious that these studies have many difficulties. Exposures are rarely determined. Studies frequently involve small sample sizes and the investigators rarely have a combined expertise in EMF physics, engineering, and reproductive biology. Because of the allegation that there may be particular windows of frequency, wave shape, and intensity that may be deleterious, it is impossible to disregard low frequency EMF exposures as having no deleterious reproductive effects. Yet the epidemiologic data that are available would point in that direction. Secular trend data analysis of birth defect incidence data indicate that increasing generation of electric power during this century is not associated with a concomitant rise in the incidence of birth defects. There are over 70 EMF research projects dealing with animal and in vitro studies that are concerned with some aspect of reproduction and growth. Unfortunately, a large proportion of the embryology studies utilized the chick embryo and evaluated the presence or absence of teratogenesis after 48 to 52 hours of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Sources of amino acids for protein synthesis during early organogenesis in the rat. 1. Relative contributions of free amino acids and of proteins

Rat conceptuses on the 10th day of gestation were cultured for 27 h in whole rat serum. An addition of either [3H]leucine or [3H]leucine-labelled rat serum proteins was made once during the culture period, and the acid-soluble and acid-insoluble radioactivities of embryo and visceral yolk sac measured at harvesting. The extent of radiolabel incorporation into embryonic and yolk-sac proteins increased linearly with the duration of exposure of the conceptus to the radiolabelled leucine or radiolabelled serum proteins, indicating roughly constant rates of incorporation, per unit mass of tissue, throughout the culture period. The incorporation rates, expressed as clearances, were 0.73 and 0.78 microliter/mg tissue protein/h for embryo and yolk sac, respectively, when the source was [3H]leucine; and 1.8 and 1.3 microliters/mg tissue protein/h, for embryo and yolk sac, respectively, when the source was [3H]leucine-labelled serum proteins. It is estimated, from the known leucine and protein concentrations in serum, that protein contributed over 99 per cent of the leucine supplied to the conceptus for its protein synthesis. In parallel experiments, measurements were made on cultures conducted in the presence of an antiserum against rat visceral yolk sac (100 micrograms/ml). Antiserum profoundly inhibited incorporation of radioactivity into embryo and yolk-sac proteins, when the source was 3H-labelled protein, a result consistent with the known ability of the antiserum to inhibit pinocytosis in the yolk sac. Antiserum also decreased incorporation from [3H]leucine in the yolk sac, suggesting that a proportion of the free leucine entering the yolk sac does so by pinocytosis. The failure of antiserum to affect incorporation of [3H]leucine into the embryo probably indicates that leucine can enter the embryo without the mediation of yolk-sac pinocytosis. The primacy of protein, as a source of amino acids for the organogenesis-stage embryo, is consistent with the serious effects, in terms of embryonic death and malformation, that result from the interruption of amino acid supply when either pinocytosis or lysosomal proteolysis in the yolk sac is inhibited.

Sources of amino acids for protein synthesis during earlyorganogenesis in the rat. 3. Methionine incorporation

Rat conceptuses at 9.5 days post-conception were cultured for 27 h in whole rat serum. [3H]Methionine, or rat serum proteins containing [3H]methionine, was introduced at 24 or 6 h before the termination of the culture. The total clearance of radioactivity into the embryo and the visceral yolk sac from the two sources was measured; also the extent to which the accumulated radioactivity was acid-insoluble. Similar experiments, but using [3H]leucine, were performed for comparison. The results indicate that free amino acid and protein can both serve as sources of amino acids for incorporation into the embryo and yolk sac, and it is estimated that in vivo over 95 per cent of the methionine (and the leucine) incorporated into these tissues arises from protein captured and digested by the yolk sac. Almost all the leucine accumulated into the conceptus is present as protein, but a larger fraction of the methionine accumulated is found in acid-soluble form. When the amino acids were delivered in the form of plasma proteins, the incorporation of methionine was two to three times more efficient than that of leucine, an observation most readily explained by leucine being provided in excess of requirements. In the light of reports that an adequate concentration of free methionine is important for the normal development of rat embryos in vitro, it is concluded that, although most of the amino acid required by the embryo is supplied as protein, the small fraction supplied as free amino acid may be critical for methionine but probably not for leucine.

Sources of amino acids for protein synthesis during earlyorganogenesis in the rat. 4. Mechanisms before envelopment of the embryo by the yolk sac

It was previously shown that uptake and digestion of protein by the visceral yolk sac supplies almost all of the amino acid needed by the 9.5-11.5-day rat conceptus cultured in vitro. Our aim was to test the hypothesis that protein uptake and digestion may not be as important as an amino acid source in the 8.5-9.5-day period, a stage of development before the yolk sac placenta envelops the embryo and before the vitelline circulation is established. Eight and a half-day rat conceptuses were cultured in serum supplemented with trace amounts of free [3H]leucine, [3H]leucine-containing serum proteins, free [3H]methionine or [3H]methionine-containing serum proteins. The incorporation of radiolabelled amino acid into acid-soluble and acid-insoluble fractions of the conceptus was determined. Leucine from either source was incorporated principally into proteins of the conceptus, but a greater proportion of the methionine incorporated was found in the low molecular weight fraction. It is estimated that 88 per cent of the leucine and 96 per cent of the methionine used by the conceptus was derived from protein in the culture serum; free amino acid comprised a minor supply source. We conclude that, despite the different anatomic relationships, the majority of amino acid incorporated into newly synthesized proteins of the conceptus very early in organogenesis is supplied by the digestion of protein in extraembryonic tissue, most likely the visceral yolk sac.

Sources of amino acids for protein synthesis during early organogenesis in the rat. 2. Exchange with amino acid and protein pools in embryo and yolk sac

Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine and harvested after 24 or 48 h. Hydrolysates of the acid-precipitable fraction of embryo or yolk-sac homogenates were prepared and subjected to paper chromatography. Liquid scintillation counting of the separated amino acids showed that leucine was the only amino acid with above-background radioactivity. This established that radiolabel was not transferred from leucine to other amino acids in the cultured rat conceptus. Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine, as above. After 19 h, some conceptuses were harvested; other conceptuses were rinsed, transferred to culture medium without [3H]leucine, and after a further 24 h of culture the embryos and yolk sacs were harvested. A comparison of the protein-associated radioactivity of embryo and yolk sac before and after culturing for the further 24-h period showed that these structures quantitatively conserve radiolabelled leucine incorporated into their proteins. Further experiments involved culturing the rat conceptus for 24 h as above but in the presence of either [3H]leucine or [3H]leucine-labelled serum proteins. After harvesting the conceptuses, the specific radioactivity of [3H]leucine was determined in the acid-soluble and acid-precipitable fractions prepared from embryo and yolk-sac homogenates. The specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos or yolk sacs from conceptuses grown in the presence of radiolabelled protein was about 120 per cent of that in the culture medium, while that in the acid-precipitable fractions was about 70 per cent of that in the culture medium. By comparison, the specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos and yolk sacs from conceptuses grown in the presence of free [3H]leucine was only 3-4 per cent of that in the culture medium, while that in the acid-precipitable fraction was about 1 per cent of that in the culture medium. If our data on the fate of leucine incorporated into newly synthesized proteins of the early post-implantation rat embryo can be extrapolated to the other amino acids, they suggest that once amino acids have been incorporated into newly synthesized protein in embryo or yolk sac, they are not exchanged to any detectable extent with amino acid pools outside the conceptus. The results also provide independent confirmation of our earlier conclusion that the only significant source of amino acids utilized by the 10th-day embryo is protein taken up by the yolk sac and digested intracellularly.(ABSTRACT TRUNCATED AT 400 WORDS)

The contribution of environmental teratogens to embryonic and fetal loss.

Environmental causes of human malformations account for approximately 10% of malformations, and less than 1% of all human malformations are related to prescription drug exposure, chemicals, or radiation. Malformations caused by drugs and other therapeutic agents are important, however, because these exposures are preventable. As we better understand the mechanisms of teratogenesis from all etiologies, we may learn how best to predict and test for teratogenicity and apply this knowledge to the prevention of human birth defects. Spontaneous abortions account for a much higher percentage of reproductive failure than do congenital malformations, but a very high proportion of aborted fetuses are malformed or defective. Therefore, the consequences of abortion are much different than the consequences of having a survival malformed infant, from both the medical and psychologic perspectives. We are less certain about the contribution of environmental, physical, and chemical agents to the incidence of spontaneous abortion. Although we realize that spontaneous abortions are a significant medical and emotional burden to a family, a surviving malformed infant can be greater burden to that family. We do not have an accurate picture of the contribution of environmental agents to the incidence of spontaneous abortion. At the present time, it would appear that only a very small proportion of abortions can be attributed to exposure to environmental toxicants during pregnancy (Tables 1 and 2). Conversely, it is the scientific and medical communitys responsibility to prevent the introduction and use of agents that cause unwanted embryonic and fetal loss. Because the teratogenic and abortigenic effects of environmental agents differ, one cannot conclude that an agent is an abortifacient because it is teratogenic, nor can one conclude that an agent is not an abortifacient because it is not teratogenic (Table 2).

Leucine sources for the rat fetus.

Fetal and maternal plasma were assayed for the concentration of free leucine, acid-insoluble radioactivity and acid-soluble radioactivity at intervals after an intravenous bolus injection of [3H]leucine into anaesthetized pregnant rats at 17.5 days post-conception. The concentrations of total free leucine and of free [3H]leucine in maternal and fetal plasma were effectively unchanged from 5 to 180 min post-injection. Plasma free leucine concentrations in the fetus were five times those in the mother. The concentration of free [3H]leucine in fetal plasma was similar to that in maternal plasma. Thus the specific radioactivity of free leucine in fetal plasma is substantially lower than that in maternal plasma, indicating that a significant portion of the free leucine in plasma of the 17.5-day rat fetus comes from a source other than the free leucine in the maternal plasma. The data are consistent with a major contribution of amino acids coming from the degradation of extraembryonic protein in the yolk sac. Other possible sources of unlabelled leucine are discussed.

Pinocytosis in the rat visceral yolk sac: Potential role in amino acid nutrition during the fetal period

At 9.5 to 10.5 days post-conception (p.c.) the rat embryo receives amino acids primarily by pinocytosis of maternal proteins by the visceral yolk sac (VYS) endodermal cells and their subsequent digestion in lysosomes. As this mechanism may also contribute to the nutritional support of the rapidly growing fetus later in gestation, this study determines the potential of the VYS to contribute to the amino acid needs of the rat fetus on 13.5, 15.5 and 17.5 days p.c. Wistar rats were killed on 11.5, 13.5, 15.5, 17.5 and 19.5 days p.c. and the protein content of fetuses, VYSs and serum was determined. Additional VYSs were isolated on the same days p.c. and the uptake of [14C]sucrose, a measure of fluid-phase pinocytosis, was determined. The rate of protein accretion by the fetus and the capacity of the VYS to pinocytose and digest proteins were calculated on 13.5, 15.5 and 17.5 days p.c. There was a decrease in pinocytic capacity, per mg protein, over the period of development studied but, owing to the growth of the tissue, the total capacity of the yolk sac to pinocytose and digest proteins increased from 13.5 to 17.5 days p.c. Comparison of the VYSs capacity to pinocytose and digest proteins and the rate of protein accretion by the fetus suggests that the VYS could support a significant proportion of the increase in protein content of the growing fetus. This conclusion is reinforced by the limited capacity of the maternal plasma to supply free amino acids to the fetus.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental toxicity of cysteamine in the rat: Effects on embryo–fetal development

The reproductive and developmental safety of cysteamine has become an important issue to children with cystinosis because renal transplants and treatment with cysteamine reduce the complications associated with cystinosis and increase the lifespan of the affected children. In addition, there is the potential to decrease the severity or the incidence of renal Fanconi syndrome with administration of cysteamine to pregnant women carrying fetuses with cystinosis, and to ease significantly the burden of this disease throughout their lives. If cysteamine increases significantly the risk of fetal death, growth retardation or birth defects at doses used to treat women with cystinosis, treatment of the affected female should cease during pregnancy and would not be considered for fetal treatment. The goal of this study was to assess the developmental safety of exposure in utero to cysteamine in the rat. Pregnant rats were given cysteamine (as phosphocysteamine) from day 6.5 through day 18.5 postconception and fetuses were assessed for survival, growth, and structural abnormalities on day 20.5. Cysteamine was administered orally in doses of 0, 37.5, 75, 100, or 150 mg/kg/day. Cysteamine produced dose-dependent developmental toxicity with an apparent no adverse effect observed level of 75 mg/kg/day. Specific malformations were associated with this effect (cleft palate, kyphosis), as well as intrauterine growth retardation and fetal death at 100-150 mg/kg/day, without signs of maternal toxicity. Investigations continue into the mechanism for the developmental toxicity of cysteamine.

Amino acid requirements in the early post-implantation rat conceptus

Amino acid analysis has been performed on hydrolysates of embryos/fetuses, visceral yolk sacs and ectoplacental cones/placentae from early post-implantation rat conceptuses. The increments in each amino acid between 10.5 and 11.5 days, between 11.5 and 12.5 days, and between 12.5 and 13.5 days, are expressed as percentages of the total amino acid increment. These three profiles are very similar to each other and also strongly resemble the amino acid composition of hydrolysates of rat serum. The results are discussed in the context of the hypothesis that a transudate of plasma is responsible for the amino acid nutrition of the embryo at this stage of gestation, and that inhibition of this pathway can lead to the induction of congenital defects. The results suggest that an inhibition of pinocytosis or lysosomal proteolysis would affect the supply of all protein-derived amino acids to approximately the same extent: there is no indication that the supply of any particular amino acid would be particularly vulnerable.