TERATOLOGY 46:15-21 (1992)
Maternal Factors Affecting Teratogenic Response: A Need for Assessment CARL L. KEEN Departments of Nutrition and Internal Medicine, University of California, Davis, California 95616
A review of current literature suggests that maternal nutriABSTRACT tional status can be an important modulator of the developmental toxicity of a number of agents in the environment. While the provision of multivitamin/ multimineral supplements during the periconceptional period is often associated with improved pregnancy outcome, it has been difficult to identify specific nutrient deficiencies as causative factors of abnormal development in humans. One explanation for this is that nutrient deficiencies can arise through a number of means in addition to a simple dietary deficit of the nutrient. The hypothesis is proposed that one mechanism contributing to the embryotoxicity of a diverse group of insults is an alteration in the metabolism of select nutrients. Evidence is presented that zinc is one nutrient whose metabolism can be markedly influenced by a variety of insults. One consequence of this alteration can be a reduction in embryonic zinc uptake, the development of embryonic zinc deficiency and abnormal development. o 1992 Wiley-Liss, Inc. the use of multivitamin/multimineral supDespite intensive investigative efforts plements during pregnancy is associated over the past two decades, causative factors with a reduced risk of birth defects and can only be identified for approximately 35- other signs of abnormal development (Insti60% of the developmental defects reported tute of Medicine, '90). In the first section of in typical populations, with specific terato- this paper, the historical basis for considergenic agents, monogenic defects, and chro- ing maternal nutritional status as a predicmosomal abnormalities accounting for ap- tor for pregnancy outcome will be briefly proximately 8-lo%, 15-25%, and 15-28% outlined. In the subsequent section, the poof the defects, respectively (Shepard, '86). tential role of suboptimal maternal nutriWhile a number of potential reproductive tional status t o poor pregnancy outcome will insults have been identified, individual sus- be presented, using evidence for the hypothceptibility to these insults may vary consid- esis that maternal primary and secondary erably. This phenomenon is well recognized zinc (Zn) deficiencies may be relatively comand is presumed to involve the genetic back- mon factors underlying the induction of emground of the pregnant female and fetus, bryonic and fetal abnormalities, as well as the physical environment, and the presence other complications of pregnancy. or absence of other interactive insults. In EARLY STUDIES ON THE INFLUENCE OF the discussion below, the evidence that maMATERNAL DIET ON PREGNANCY OUTCOME ternal nutritional status is one component Although it is typically not listed as a maof the environment that may have a considjor contributory factor, it has long been recerable influence on the expression of some teratogens is reviewed. During the past de- ognized that maternal diet can have a sigcade, there has been increasing interest in nificant effect on pregnancy outcome. Based the idea that maternal nutritional status on an early analysis of data on the effects of may be a critical predictor for embryonic wartime starvation in Holland on pregand fetal development. For example, during the past few years, there has been considerable controversy over the hypothesis that Received May 29, 1991; accepted September 16, 1991. INTRODUCTION
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1992 WILEY-LISS, INC.
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nancy outcome, Smith (’47a,b)reported that severe overall undernutrition resulted in an increased risk for infertility, growth retardation and abortion. Stein et al. (’75) later extended the findings by Smith by noting that prenatal exposure to famine during early gestation was associated with an increased risk of central nervous system (CNS) abnormalities and increased perinatal mortality. By contrast, exposure to famine during late gestation primarily resulted in interuterine growth retardation (IUGR). It has been long recognized that maternal nutrition also affects pregnancy outcome even when caloric intake is adequate. For example, in the early studies by Burke et al. (’43)and Jeans et al. (‘551, poor dietary habits were associated with poor pregnancy outcome based on a number of criteria, including prematurity, low birthweight, and the frequency of congenital defects. Smithells et al. (’76) and Laurence et al. (’83) have also reported an association between poor maternal dietary habits and poor pregnancy outcome. While it can be argued that individuals characterized by poor dietary habits are often also characterized by other factors which can contribute to poor pregnancy outcome (e.g., low socioeconomic status, cigarette smoking, use of recreational drugs), there is evidence that poor maternal nutrition can contribute to poor pregnancy outcome independent of other factors. Primrose and Higgins (‘71) reported that nutrient supplements improved pregnancy outcome in a subset of women in Canada previously identified as being in poor nutritional status. Consistent with the study by Primrose and Higgins, Tolarova (’82),Smithells et al. (‘83, ’89), Laurence et al. (‘83),Winship et Mual. (’84), Baile and Lewenthal (’€34)’ linare et al. (’88), Bower and Stanley (’fig), and Nevin and Seller (’90) have reported that the use of vitamidmineral supplements during the periconceptional period can result in reductions in the predicted frequencies of birth defects in select populations. It is important t o note that while a positive effect of supplements was noted in the above studies, in a recent large retrospective study by Mills et al. (’89),the use of vitamin supplements during the periconceptional period was not associated with a lower frequency of birth defects compared with nonsupplemented controls. Taken together, the above reports would seem to argue for the use of periconceptional
vitamidmineral supplements, however, there is still considerable controversy over this issue. One of the arguments against the use of nutrient supplements is that for the general public there is a paucity of studies in which a primary maternal dietary deficiency of a nutrient has been linked to the induction of developmental defects. Thus, it has been argued that in the absence of primary maternal nutrient deficiencies (i.e., deficiencies due to inadequate concentrations of specific nutrients in the maternal diet), risks associated with recommending the use of supplements during pregnancy (potential mineral and vitamin toxicities and the possible belief by the individual that the use of supplements reduces their need to be concerned about their general diet) may outweigh the benefits gained from their use. However, in contrast to the idea that “poor” maternal diet represents the major insult in the above studies, an alternative (and more likely) possibility is that a compromised nutritional status of an individual increases the risk of adverse reactions to other potential reproductive hazards andlor that there is a synergistic interaction between a “hazard(s)” and a deficiency of one o r more nutritional factors resulting in poor pregnancy outcome. As an example of the above, the teratogenic potential of a number of compounds including ochratoxin A (Singh and Hood, ’85),nitrofen (Zeman et al., ’86) and benomyl (Ellis et al., ’87) has been reported to be exacerbated by concomitant maternal protein deficiency. Below, the hypothesis is presented that another nutrient that may modify the teratogenicity of some insults is Zn. ZN DEFICIENCY AND ABNORMAL DEVELOPMENT
The logic for using Zn as a nutrient to illustrate the potential role of maternal nutritional status as a factor which can influence the teratogenicity of some agents is based on two general observations, the first being that the average dietary intake of this nutrient by many women during pregnancy is significantly below its US.Recommended Daily Allowance (RDA) (15 mg for pregnant women) (Cherry et al., ’89).While it must be appreciated that the intake of a nutrient such as Zn below its RDA level does not by definition result in its deficiency (assuming that the RDA level is appropriate), it does
MATERNAL FACTORS AFFECTING TERATOGENIC RESPONSE
suggest that a subpopulation of pregnant women may be in marginal Zn status. Three lines of evidence support the idea that Zn deficiency can be a teratogenic agent in humans. First, women who suffer from the genetic disorder acrodermatitis enteropathica, a disorder of Zn metabolism that produces signs of Zn deficiency, are characterized by a high frequency of pregnancy complications including abnormal births unless they are given supplemental Zn (Brenton et al., '81; Hambidge et al., '75). Second, women with low serum Zn concentrations during early pregnancy tend to have a higher frequency of pregnancy complications as compared with women with higher serum Zn concentrations (Hinks et al., '89; Jameson, '76; Neggers et al., '90); similarly, term serum Zn concentrations tend to be lower in mothers who deliver infants with neural tube defects compared with controls (Baumah et al., '84; Cavdar et al., '88; Flynn et al., '811. Finally, support for the idea that maternal dietary Zn intake can be a significant factor in human pregnancy outcome has been provided by Kynast and Saling ('86) and Cherry et al. ('891, who reported that in prospective studies, Zn supplementation was associated with reductions in pregnancy complications. The teratogenicity of Zn deficiency in mammals is well established. Typical malformations associated with severe Zn deficiency in animal models include cleft lip and palate, brain and eye malformations, and numerous abnormalities of the heart, lung and urogenital systems. Fetuses from zincdeficient dams also show a high frequency of skeletal defects including cranial, vertebral, rib, sternal, long bone, and digit defects (Keen and Hurley, '89; Rogers et al., '85; and ref. cited therein). In addition to morphological abnormalities, biochemical and functional abnormalities in the young can also occur as a result of maternal Zn deficiency. These include defects in pancreatic function, lung metabolism and immune competence (Keen and Gershwin, '90; Keen and Hurley, '89). Mechanistically, Zn deficiency is thought to influence embryonic development by a number of means including: abnormal nucleic acid metabolism, reduced protein synthesis, reductions in the rate of tubulin polymerization, increased rates of peroxidative damage (protein, nucleic acid, and lipid), which can result in cell necrosis
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and cell death leading to asynchrony in development, and reduced binding of hormones and transcription factors dependent on "Zn finger" regions (Berg, '90; Harding et al., '87; Keen and Hurley, '89; Oteiza et al., '90). It is significant to note that Zn deficiency can affect development within a very short period of time, with congenital defects occurring in the fetuses of rats fed Zn-deficient diets for only 4 days during pregnancy (Hurley, '81). While the above reports provide evidence for the general hypothesis that maternal Zn status may be an important predictor for pregnancy outcome, in the absence of the use of supplements it has been difficult to correlate maternal dietary Zn intake to pregnancy outcome in well nourished populations (Swanson and King, '87). In other words, while several investigators have reported an association between low maternal plasma Zn concentrations and poor pregnancy outcome, it has been difficult to correlate maternal dietary Zn intake with maternal plasma Zn concentrations (Keen and Hurley, '89; Swanson and King, '87). One interpretation of the above is that embryonidfetal pathology by itself triggers a reduction in maternal plasma Zn concentrations perhaps via a cytokine response, in which cytokines such as interleukins 1 and 6 (IL-1 and IL-6) are synthesized in response to tissue injury, with one consequence being a stimulation of metallothionein synthesis in maternal liver with a subsequent sequestering of Zn in this tissue (see below). However the above mechanism would not explain the reported positive effects of Zn supplements on pregnancy outcome. An alternative explanation is that in addition to a primary dietary Zn deficiency, low maternal plasma Zn concentrations may in some situations reflect disease and/or environment-induced perturbations in maternal Zn metabolism. This idea is discussed in detail below. DISEASE AND STRESS-INDUCED DEFICIENCIES OF ZINC
That Zn deficiency can be a complication of some disease states is well established. For example, altered Zn status is a complication of diabetes in some subjects (Walter et al., '91), and diabetes-induced alterations in maternal Zn metabolism have been suggested as a potential biochemical lesion underlying the high incidence of teratogenic-
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ity associated with diabetes (Uriu-Hare et al., '85). In a pregnant diabetic rat model, maternal Zn supplementation was associated with an improvement in pregnancy outcome (Uriu-Hare et al., '89). A second "disease" state linked to disturbances in Zn metabolism is alcoholism, with a common finding being that of low plasma Zn concentrations (Halsted and Keen, '90). Based on the observation that there was an inverse relationship between plasma Zn concentrations and the expression of Fetal Alcohol Syndrome in pregnant alcoholic women, Flynn et al. ('81)have proposed that one biochemical lesion underlying the development of Fetal Alcohol Syndrome is embryonic Zn deficiency. Consistent with this idea, maternal alcohol feeding in rats is associated with lower than normal fetal Zn concentrations, and reduced placental Zn transport (Ghisham et al., '82; Suh and Firek, '82). However it should be noted that in contrast to diabetes, alcohol associated developmental toxicity has not been observed to be influenced by maternal dietary Zn intake in experimental animal models (ZidenbergCherr et al., '88). In addition to the possible direct effect of some diseases on Zn metabolism, Zn deficiency may also be induced during pregnancy as a result of certain drug therapies. In general, drug-mineral interactions can be separated into two categories: in the first, there is a direct interaction between the drug and the element; in the second, the drug indirectly affects the metabolism of the element. With regard t o Zn, the first category of interactions is exemplified by drugs which have the potential to chelate Zn and either decrease its absorption or increase its excretion. Examples of the above would be ethylenediaminetetraacetic acid, penicillamine, triethylenetetramine and acetazolamide. The teratogenicity of each of the above drugs has been linked in part to the development of fetal Zn deficiency (Hackman and Hurley, '83; Hirsch and Hurley, '78; Keen et al., '84; Swenerton and Hurley, '71). The second category of zinc-drug interactions is more difficult to predict, as a simple examination of the drug's chemical structure may not allow a prediction of its effect on Zn metabolism. Representatives of this category of interaction include 6-mercaptopurine, valproic acid, and urethane (Amemiya et al., '86, '88; Daston et al., '91;
Keen et al., '89). Each of the above drugs is teratogenic, and each has been shown t o result in marked reductions in fetal Zn uptake. For example, the flux of "Zn transport to the day 12 rat embryo can be reduced by 50% in valproic acid-treated dams compared with controls (Keen et al., '89). A common mechanism which seems to underlay the effect of each of the above drugs on fetal Zn metabolism is that the drug is associated with an induction in maternal liver metallothionein synthesis with a marked increase in metallothionein concentrations. Metallothionein is a lowmolecular-weight cysteine-rich cytosolic protein that binds divalent metals, with a high affinity for Zn. When pregnant animals are treated with any of the above drugs (valproic acid, 6-mercaptopurine, or urethane), the metabolic fate of ingested Zn is altered such that a higher than normal proportion of absorbed Zn remains in maternal liver associated with metallothionein. Although divalent metals such as Zn and cadmium are considered the classic inducers of metallothionein, a number of organic compounds (Klaassen and LehmanMcKeeman, '89) endogenous hormones (adrenocorticosteroids), and cytokines (particularly IL-1 and IL-6) (Cousins, %), can stimulate its synthesis. Consistent with the above inducers of this protein, one of the characteristics of metallothionein is that its synthesis in liver is typically increased in response to tissue injury and inflammation; in this regard, an increase in metallothionein synthesis can be considered part of the acute phase response. While the biological function(s) of metallothionein have not been agreed upon (Hamer, '861, it is recognized that one consequence of its synthesis in adults is the sequesterization of Zn in liver with a concomitant reduction of Zn concentrations in plasma (Cousins, '85). Given the recognition that (1)plasma is the major source of Zn for the developing embryoifetus; (2) Zn deficiency is teratogenic; and (3) a number of drugs can induce metallothionein synthesis in maternal liver, it seems reasonable to suggest that drug-induced embryonicifeta1 Zn deficiencies may be a common mechanism contributing to the developmental toxicity associated with a wide number of drugs. This hypothesis is supported by the observation that the developmental toxicity of one of the above drugs, 6-mercaptopurine, has
MATERNAL FACTORS AFFECTING TERATOGENIC RESPONSE
been reported to be ameliorated by maternal Zn supplementation (Amemiya et al., ’86; Hirsch and Hurley, ’78). It is significant to note that in addition to a diverse group of drugs (primarily those whose side effects include liver toxicity), a number of other environmental stressors including heat shock, starvation, cadmium, arsenic, and strenuous exercise are known inducers of metallothionein (Cousins, ’85). Several disease states, including diabetes and alcoholism, are also characterized by high liver metallothionein concentrations (Halsted and Keen, ’90;Uriu-Hare et al., ’89). Thus, it is possible that some of the negative reproductive effects associated with these stressors and stress conditions may also be due to the induction of embryonidfetal Zn deficiency. Indeed, the developmental toxicity of cadmium has been linked in part to the induction of fetal Zn deficiency (Daston, ’82; Hazelhoff-Roelfzema et al., ’89).At present it is not known whether there is a synergism between the influence of reduced food intake and stressinduced tissue pathology on metallothionein synthesis. However, given the observation that reduced food intake in animal studies is a common characteristic of “maternal toxicity,” it is evident that altered fetal Zn metabolism may be a consequence of several initiators of maternal toxicity independent of direct effects of the insult on metallothionein synthesis. If the hypothesis that the developmental toxicity of a number of insults is due in part to the induction of embryonidfetal Zn deficiency is correct, this may provide one explanation for the mechanisms of developmental toxicity often associated with “maternal toxicity” (Khera, ’84; ’85). According to this idea, in situations in which maternal toxicity results in a stimulation of maternal liver metallothionein synthesis (either due to a direct effect of the primary insult or due to a secondary effect of cytokines elaborated as a consequence of tissue injury), developmental toxicity can result in part as a consequence of embryonicifetal Zn deficiency. Two obvious implications can be drawn from the above hypothesis: first, the provision of Zn supplements to the mother may reduce the developmental toxic effects associated with a diverse range of maternal insults; and second, the Zn status of the mother at the time of the insult, and her reaction to the insult with respect to liver
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metallothionein synthesis, will be important predictors of the expression of the developmental insult. It should be noted that while the above discussion has focused on Zn, a stimulation of the acute phase response in an animal can also result in marked alterations in iron and copper metabolism (Cousins, ’85). The impact of these alterations on fetal development remains to be determined. SUMMARY
While there can be little argument with the idea that the consumption of a well balanced nutritious diet will minimize the possibility of embryonidfetal nutritional deficiencies, it must be recognized that the consumption of an “adequate” diet by the mother does not necessarily translate into “good” embryonidfetal nutrition. In this paper, using Zn as an example, the argument has been made that secondary nutrient deficiencies (i.e., those arising in the presence of what would normally be considered an adequate intake of the nutrient by the mother) may also represent a serious risk to the developing embryolfetus. The hypothesis that secondary embryonidfetal Zn deficiencies may be a common factor contributing t o the developmental toxicity associated with a number of compounds and environmental insults is proposed. Given the above, it is evident that an assessment of maternal nutritional status can be of value in predicting the relative risk of an individual to a reproductive hazard. The determination of the effect(s) of a reproductive insult on maternal delivery of nutrients to the embryo/ fetus may contribute to the identification of the biochemical lesions underlying the teratogenicity of the insult in question. Potential toxicant-nutrient interactions will probably not be detected in standard developmental toxicity assays, which, along with other types of toxicity tests, use animals of optimal health and nutritional status. Unfortunately, this does not always accurately reflect the health and nutrition of females of reproductive age in the human population. Further understanding of the role of compromised maternal nutritional status in determining the developmental outcome of exposure to toxicants during pregnancy is needed to improve our ability to carry out realistic and rational human risk assessments.
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C.L. KEEN ACKNOWLEDGMENTS
This work was supported in part by U.S. EPA cooperative agreement CR-816713 and by NIH grants HD01743 and HD14388. LITERATURE CITED Amemiya K., C.L. Keen, and L.S. Hurley (1986) 6-Mercaptopurine induced alterations in mineral metabolism and teratogenesis in the rat. Teratology, 34:321334. Amemiya, K., L.S. Hurley, and C.L. Keen (1988) The effect of 6-mercaptopurine on "Zn distribution in the pregnant rat. Teratology, 39:387-393. Baile, Y., and H. Lewenthal (1984) Effect of folic acid supplementation on congenital malformations due to anticonvulsive drugs. Eur. J. Obstet. Gynec. Reprod. Biol., 18:211-216. Berg, J.M. (1990) Zinc finger domains: Hypotheses and current knowledge. Annu. Rev. Biophys. Chem., 19: 405-421. Bower, C., and F.J. Stanley (1989) Dietary folate as a risk factor for neural-tube defects: Evidence from a case control study in Western Australia. Med. J . Aust., 150:613-619. Brenton, D.P., M.J. Jackson, and A. Young (1981) Two pregnancies in a patient with acrodermatis enteropathica treated with zinc sulphate. Lancet, 2;500502. Buamah, P.K., M. Russell, M. Bakes, W.A. Milford, and A.W. Skillen (1984) Maternal zinc status: A determination of central nervous system malformations. Br. J. Obstet. Gynaecol., 911788-790. Burke, B.S., V.A. Beal, S.B. Kirkwood, and H.C. Stuart (1943) The influence of nutrition during pregnancy upon the condition of the infant at birth. J. Nutr., 26569. Cavdar, A.O., M. Bahceci, N. Akar, J . Erten, G. Bahceci, E. Babacan, A. Arcasoy, and H. Yavuz (1988) Zinc status in prgenancy and the occurrence of anencephaly in Turkey. J . Trace Elem. Electrolytes Health Dis., 2:9-14. Cherry, F.F., H.H. Sandstead, P. Rojas, L.K. Johnson, H.K. Batson, and X. B. Wang (1989) Adolescent pregnancy: Associations among body weight, zinc nutriture, and pregnancy outcome. Am. J. Clin. Nutr., 50; 945-954. Cousins, R.J. (1985) Absorption, transport, and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev., 65: 238-309. Daston, G.P. (1982) Fetal zinc deficiency as a mechanism for cadmium-induced toxicity to the developing rat lung and pulmonary surfactant. Toxicology, 24: 55-63. Daston, G.P., G.J. Overmann, M.W. Taubeneck, L.D. Lehman-McKeeman, J.M. Rogers and C.L. Keen (1991) The role of metallothionein induction in maternally mediated developmental toxicity: Comparison of the effects of urethane and styrene in rats. Toxicol. Appl. Pharmacol., 110;450-463. Ellis, W.G., J.L. Semple, E.R. Hoogenboom, R.J. Kavlock and F.J. Zeman (1987) Benomyl-induced craniocerebral anomalies in fetuses of adequately nourished and protein-deprived rats. Teratogen., Carcinogen., Mutagen., 7:357-375. Flynn, A,, S.S. Martier, R.J. Sokol, S.I. Miller, N.L. Golden, and B.C. Villano (1981) Zinc status of pregnant alcoholic women: A determinant of fetal outcome. Lancet, lr572-574.
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Vitamin deficiencies and neural tube defects. Arch. Dis. Child., 51:944-948. Smithells, R.W., S. Sheppard, J . Wild, and C.J. Schorah (1989) Prevention of neural tube defect recurrences in Yorkshire: Final report. Lancet, 2:498-499. Stein, Z., M. Susser, G. Saeger, and F. Marolla (1975) Famine and Human Development. The Dutch Hunger Winter of 1944/45. Oxford University Press, New York. Suh, S.M., and A.F. Firek (1982) Magnesium and Zn deficiency and growth retardation in offspring of alcoholic rats. J. Am. Coll. Nutr., 1:193-201. Swanson, C.A., and J.C. King (1987) Zinc and pregnancy outcome. Am. J. Clin. Nutr., 46:763-771. Swenerton, H., and L.S. Hurley (1971) Teratogenic effects of a chelating agent and their prevention by zinc. Science, 73:62-64. Tolarova, M. (1982) Periconceptional supplementation with vitamins and folic acid to prevent recurrence of cleft lip. Lancet 2217. Uriu-Hare, J.Y., J.S. Stern, G.M. Reaven, and C.L. Keen (1985) The effect of maternal diabetes on trace element status and fetal development in the rat. Diabetes, 34:1031-1040. Uriu-Hare, J.Y., J.S. Stern, and C.L. Keen (1989) Influence of maternal dietary Zn intake on expression of diabetes-induced teratogenicity in rats. Diabetes, 38: 1282-1290. Walter, R.M., J.Y. Uriu-Hare, K.L. Olin, M.H. Oster, B.D. Anawalt, J.W. Critchfield, and C.L. Keen (1991) Copper, zinc, manganese and magnesium status and complications of diabetes mellitus. Diabetes Care 14: 1050-1056. Winship, K.A., D.A. Cahal, J.C.P. Weber, and J.P. Griffin (1984) Maternal drug histories and central nervous system anomalies. Arch. Dis. Child., 59:10521060. Zeman, F.J., E.R. Hoogenboom, C. Chase-Deesing, R.J. Kavlock, and J.L. Semple (1986) Effects on the fetus of maternal nitrofen exposure in the protein-deprived rat. Toxicology, 3855-68. Zidenberg-Cherr, S., P.A. Benak, L.S. Hurley, and C.L. Keen (1988) Altered mineral metabolism: A mechanism underlying the fetal alcohol syndrome in rats. Drug-Nutr. Interact., 5~257-274.
Maternal Factors Affecting Teratogenic Response: A Need for Assessment CARL L. KEEN Departments of Nutrition and Internal Medicine, University of California, Davis, California 95616
A review of current literature suggests that maternal nutriABSTRACT tional status can be an important modulator of the developmental toxicity of a number of agents in the environment. While the provision of multivitamin/ multimineral supplements during the periconceptional period is often associated with improved pregnancy outcome, it has been difficult to identify specific nutrient deficiencies as causative factors of abnormal development in humans. One explanation for this is that nutrient deficiencies can arise through a number of means in addition to a simple dietary deficit of the nutrient. The hypothesis is proposed that one mechanism contributing to the embryotoxicity of a diverse group of insults is an alteration in the metabolism of select nutrients. Evidence is presented that zinc is one nutrient whose metabolism can be markedly influenced by a variety of insults. One consequence of this alteration can be a reduction in embryonic zinc uptake, the development of embryonic zinc deficiency and abnormal development. o 1992 Wiley-Liss, Inc. the use of multivitamin/multimineral supDespite intensive investigative efforts plements during pregnancy is associated over the past two decades, causative factors with a reduced risk of birth defects and can only be identified for approximately 35- other signs of abnormal development (Insti60% of the developmental defects reported tute of Medicine, '90). In the first section of in typical populations, with specific terato- this paper, the historical basis for considergenic agents, monogenic defects, and chro- ing maternal nutritional status as a predicmosomal abnormalities accounting for ap- tor for pregnancy outcome will be briefly proximately 8-lo%, 15-25%, and 15-28% outlined. In the subsequent section, the poof the defects, respectively (Shepard, '86). tential role of suboptimal maternal nutriWhile a number of potential reproductive tional status t o poor pregnancy outcome will insults have been identified, individual sus- be presented, using evidence for the hypothceptibility to these insults may vary consid- esis that maternal primary and secondary erably. This phenomenon is well recognized zinc (Zn) deficiencies may be relatively comand is presumed to involve the genetic back- mon factors underlying the induction of emground of the pregnant female and fetus, bryonic and fetal abnormalities, as well as the physical environment, and the presence other complications of pregnancy. or absence of other interactive insults. In EARLY STUDIES ON THE INFLUENCE OF the discussion below, the evidence that maMATERNAL DIET ON PREGNANCY OUTCOME ternal nutritional status is one component Although it is typically not listed as a maof the environment that may have a considjor contributory factor, it has long been recerable influence on the expression of some teratogens is reviewed. During the past de- ognized that maternal diet can have a sigcade, there has been increasing interest in nificant effect on pregnancy outcome. Based the idea that maternal nutritional status on an early analysis of data on the effects of may be a critical predictor for embryonic wartime starvation in Holland on pregand fetal development. For example, during the past few years, there has been considerable controversy over the hypothesis that Received May 29, 1991; accepted September 16, 1991. INTRODUCTION
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1992 WILEY-LISS, INC.
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nancy outcome, Smith (’47a,b)reported that severe overall undernutrition resulted in an increased risk for infertility, growth retardation and abortion. Stein et al. (’75) later extended the findings by Smith by noting that prenatal exposure to famine during early gestation was associated with an increased risk of central nervous system (CNS) abnormalities and increased perinatal mortality. By contrast, exposure to famine during late gestation primarily resulted in interuterine growth retardation (IUGR). It has been long recognized that maternal nutrition also affects pregnancy outcome even when caloric intake is adequate. For example, in the early studies by Burke et al. (’43)and Jeans et al. (‘551, poor dietary habits were associated with poor pregnancy outcome based on a number of criteria, including prematurity, low birthweight, and the frequency of congenital defects. Smithells et al. (’76) and Laurence et al. (’83) have also reported an association between poor maternal dietary habits and poor pregnancy outcome. While it can be argued that individuals characterized by poor dietary habits are often also characterized by other factors which can contribute to poor pregnancy outcome (e.g., low socioeconomic status, cigarette smoking, use of recreational drugs), there is evidence that poor maternal nutrition can contribute to poor pregnancy outcome independent of other factors. Primrose and Higgins (‘71) reported that nutrient supplements improved pregnancy outcome in a subset of women in Canada previously identified as being in poor nutritional status. Consistent with the study by Primrose and Higgins, Tolarova (’82),Smithells et al. (‘83, ’89), Laurence et al. (‘83),Winship et Mual. (’84), Baile and Lewenthal (’€34)’ linare et al. (’88), Bower and Stanley (’fig), and Nevin and Seller (’90) have reported that the use of vitamidmineral supplements during the periconceptional period can result in reductions in the predicted frequencies of birth defects in select populations. It is important t o note that while a positive effect of supplements was noted in the above studies, in a recent large retrospective study by Mills et al. (’89),the use of vitamin supplements during the periconceptional period was not associated with a lower frequency of birth defects compared with nonsupplemented controls. Taken together, the above reports would seem to argue for the use of periconceptional
vitamidmineral supplements, however, there is still considerable controversy over this issue. One of the arguments against the use of nutrient supplements is that for the general public there is a paucity of studies in which a primary maternal dietary deficiency of a nutrient has been linked to the induction of developmental defects. Thus, it has been argued that in the absence of primary maternal nutrient deficiencies (i.e., deficiencies due to inadequate concentrations of specific nutrients in the maternal diet), risks associated with recommending the use of supplements during pregnancy (potential mineral and vitamin toxicities and the possible belief by the individual that the use of supplements reduces their need to be concerned about their general diet) may outweigh the benefits gained from their use. However, in contrast to the idea that “poor” maternal diet represents the major insult in the above studies, an alternative (and more likely) possibility is that a compromised nutritional status of an individual increases the risk of adverse reactions to other potential reproductive hazards andlor that there is a synergistic interaction between a “hazard(s)” and a deficiency of one o r more nutritional factors resulting in poor pregnancy outcome. As an example of the above, the teratogenic potential of a number of compounds including ochratoxin A (Singh and Hood, ’85),nitrofen (Zeman et al., ’86) and benomyl (Ellis et al., ’87) has been reported to be exacerbated by concomitant maternal protein deficiency. Below, the hypothesis is presented that another nutrient that may modify the teratogenicity of some insults is Zn. ZN DEFICIENCY AND ABNORMAL DEVELOPMENT
The logic for using Zn as a nutrient to illustrate the potential role of maternal nutritional status as a factor which can influence the teratogenicity of some agents is based on two general observations, the first being that the average dietary intake of this nutrient by many women during pregnancy is significantly below its US.Recommended Daily Allowance (RDA) (15 mg for pregnant women) (Cherry et al., ’89).While it must be appreciated that the intake of a nutrient such as Zn below its RDA level does not by definition result in its deficiency (assuming that the RDA level is appropriate), it does
MATERNAL FACTORS AFFECTING TERATOGENIC RESPONSE
suggest that a subpopulation of pregnant women may be in marginal Zn status. Three lines of evidence support the idea that Zn deficiency can be a teratogenic agent in humans. First, women who suffer from the genetic disorder acrodermatitis enteropathica, a disorder of Zn metabolism that produces signs of Zn deficiency, are characterized by a high frequency of pregnancy complications including abnormal births unless they are given supplemental Zn (Brenton et al., '81; Hambidge et al., '75). Second, women with low serum Zn concentrations during early pregnancy tend to have a higher frequency of pregnancy complications as compared with women with higher serum Zn concentrations (Hinks et al., '89; Jameson, '76; Neggers et al., '90); similarly, term serum Zn concentrations tend to be lower in mothers who deliver infants with neural tube defects compared with controls (Baumah et al., '84; Cavdar et al., '88; Flynn et al., '811. Finally, support for the idea that maternal dietary Zn intake can be a significant factor in human pregnancy outcome has been provided by Kynast and Saling ('86) and Cherry et al. ('891, who reported that in prospective studies, Zn supplementation was associated with reductions in pregnancy complications. The teratogenicity of Zn deficiency in mammals is well established. Typical malformations associated with severe Zn deficiency in animal models include cleft lip and palate, brain and eye malformations, and numerous abnormalities of the heart, lung and urogenital systems. Fetuses from zincdeficient dams also show a high frequency of skeletal defects including cranial, vertebral, rib, sternal, long bone, and digit defects (Keen and Hurley, '89; Rogers et al., '85; and ref. cited therein). In addition to morphological abnormalities, biochemical and functional abnormalities in the young can also occur as a result of maternal Zn deficiency. These include defects in pancreatic function, lung metabolism and immune competence (Keen and Gershwin, '90; Keen and Hurley, '89). Mechanistically, Zn deficiency is thought to influence embryonic development by a number of means including: abnormal nucleic acid metabolism, reduced protein synthesis, reductions in the rate of tubulin polymerization, increased rates of peroxidative damage (protein, nucleic acid, and lipid), which can result in cell necrosis
17
and cell death leading to asynchrony in development, and reduced binding of hormones and transcription factors dependent on "Zn finger" regions (Berg, '90; Harding et al., '87; Keen and Hurley, '89; Oteiza et al., '90). It is significant to note that Zn deficiency can affect development within a very short period of time, with congenital defects occurring in the fetuses of rats fed Zn-deficient diets for only 4 days during pregnancy (Hurley, '81). While the above reports provide evidence for the general hypothesis that maternal Zn status may be an important predictor for pregnancy outcome, in the absence of the use of supplements it has been difficult to correlate maternal dietary Zn intake to pregnancy outcome in well nourished populations (Swanson and King, '87). In other words, while several investigators have reported an association between low maternal plasma Zn concentrations and poor pregnancy outcome, it has been difficult to correlate maternal dietary Zn intake with maternal plasma Zn concentrations (Keen and Hurley, '89; Swanson and King, '87). One interpretation of the above is that embryonidfetal pathology by itself triggers a reduction in maternal plasma Zn concentrations perhaps via a cytokine response, in which cytokines such as interleukins 1 and 6 (IL-1 and IL-6) are synthesized in response to tissue injury, with one consequence being a stimulation of metallothionein synthesis in maternal liver with a subsequent sequestering of Zn in this tissue (see below). However the above mechanism would not explain the reported positive effects of Zn supplements on pregnancy outcome. An alternative explanation is that in addition to a primary dietary Zn deficiency, low maternal plasma Zn concentrations may in some situations reflect disease and/or environment-induced perturbations in maternal Zn metabolism. This idea is discussed in detail below. DISEASE AND STRESS-INDUCED DEFICIENCIES OF ZINC
That Zn deficiency can be a complication of some disease states is well established. For example, altered Zn status is a complication of diabetes in some subjects (Walter et al., '91), and diabetes-induced alterations in maternal Zn metabolism have been suggested as a potential biochemical lesion underlying the high incidence of teratogenic-
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C.L. KEEN
ity associated with diabetes (Uriu-Hare et al., '85). In a pregnant diabetic rat model, maternal Zn supplementation was associated with an improvement in pregnancy outcome (Uriu-Hare et al., '89). A second "disease" state linked to disturbances in Zn metabolism is alcoholism, with a common finding being that of low plasma Zn concentrations (Halsted and Keen, '90). Based on the observation that there was an inverse relationship between plasma Zn concentrations and the expression of Fetal Alcohol Syndrome in pregnant alcoholic women, Flynn et al. ('81)have proposed that one biochemical lesion underlying the development of Fetal Alcohol Syndrome is embryonic Zn deficiency. Consistent with this idea, maternal alcohol feeding in rats is associated with lower than normal fetal Zn concentrations, and reduced placental Zn transport (Ghisham et al., '82; Suh and Firek, '82). However it should be noted that in contrast to diabetes, alcohol associated developmental toxicity has not been observed to be influenced by maternal dietary Zn intake in experimental animal models (ZidenbergCherr et al., '88). In addition to the possible direct effect of some diseases on Zn metabolism, Zn deficiency may also be induced during pregnancy as a result of certain drug therapies. In general, drug-mineral interactions can be separated into two categories: in the first, there is a direct interaction between the drug and the element; in the second, the drug indirectly affects the metabolism of the element. With regard t o Zn, the first category of interactions is exemplified by drugs which have the potential to chelate Zn and either decrease its absorption or increase its excretion. Examples of the above would be ethylenediaminetetraacetic acid, penicillamine, triethylenetetramine and acetazolamide. The teratogenicity of each of the above drugs has been linked in part to the development of fetal Zn deficiency (Hackman and Hurley, '83; Hirsch and Hurley, '78; Keen et al., '84; Swenerton and Hurley, '71). The second category of zinc-drug interactions is more difficult to predict, as a simple examination of the drug's chemical structure may not allow a prediction of its effect on Zn metabolism. Representatives of this category of interaction include 6-mercaptopurine, valproic acid, and urethane (Amemiya et al., '86, '88; Daston et al., '91;
Keen et al., '89). Each of the above drugs is teratogenic, and each has been shown t o result in marked reductions in fetal Zn uptake. For example, the flux of "Zn transport to the day 12 rat embryo can be reduced by 50% in valproic acid-treated dams compared with controls (Keen et al., '89). A common mechanism which seems to underlay the effect of each of the above drugs on fetal Zn metabolism is that the drug is associated with an induction in maternal liver metallothionein synthesis with a marked increase in metallothionein concentrations. Metallothionein is a lowmolecular-weight cysteine-rich cytosolic protein that binds divalent metals, with a high affinity for Zn. When pregnant animals are treated with any of the above drugs (valproic acid, 6-mercaptopurine, or urethane), the metabolic fate of ingested Zn is altered such that a higher than normal proportion of absorbed Zn remains in maternal liver associated with metallothionein. Although divalent metals such as Zn and cadmium are considered the classic inducers of metallothionein, a number of organic compounds (Klaassen and LehmanMcKeeman, '89) endogenous hormones (adrenocorticosteroids), and cytokines (particularly IL-1 and IL-6) (Cousins, %), can stimulate its synthesis. Consistent with the above inducers of this protein, one of the characteristics of metallothionein is that its synthesis in liver is typically increased in response to tissue injury and inflammation; in this regard, an increase in metallothionein synthesis can be considered part of the acute phase response. While the biological function(s) of metallothionein have not been agreed upon (Hamer, '861, it is recognized that one consequence of its synthesis in adults is the sequesterization of Zn in liver with a concomitant reduction of Zn concentrations in plasma (Cousins, '85). Given the recognition that (1)plasma is the major source of Zn for the developing embryoifetus; (2) Zn deficiency is teratogenic; and (3) a number of drugs can induce metallothionein synthesis in maternal liver, it seems reasonable to suggest that drug-induced embryonicifeta1 Zn deficiencies may be a common mechanism contributing to the developmental toxicity associated with a wide number of drugs. This hypothesis is supported by the observation that the developmental toxicity of one of the above drugs, 6-mercaptopurine, has
MATERNAL FACTORS AFFECTING TERATOGENIC RESPONSE
been reported to be ameliorated by maternal Zn supplementation (Amemiya et al., ’86; Hirsch and Hurley, ’78). It is significant to note that in addition to a diverse group of drugs (primarily those whose side effects include liver toxicity), a number of other environmental stressors including heat shock, starvation, cadmium, arsenic, and strenuous exercise are known inducers of metallothionein (Cousins, ’85). Several disease states, including diabetes and alcoholism, are also characterized by high liver metallothionein concentrations (Halsted and Keen, ’90;Uriu-Hare et al., ’89). Thus, it is possible that some of the negative reproductive effects associated with these stressors and stress conditions may also be due to the induction of embryonidfetal Zn deficiency. Indeed, the developmental toxicity of cadmium has been linked in part to the induction of fetal Zn deficiency (Daston, ’82; Hazelhoff-Roelfzema et al., ’89).At present it is not known whether there is a synergism between the influence of reduced food intake and stressinduced tissue pathology on metallothionein synthesis. However, given the observation that reduced food intake in animal studies is a common characteristic of “maternal toxicity,” it is evident that altered fetal Zn metabolism may be a consequence of several initiators of maternal toxicity independent of direct effects of the insult on metallothionein synthesis. If the hypothesis that the developmental toxicity of a number of insults is due in part to the induction of embryonidfetal Zn deficiency is correct, this may provide one explanation for the mechanisms of developmental toxicity often associated with “maternal toxicity” (Khera, ’84; ’85). According to this idea, in situations in which maternal toxicity results in a stimulation of maternal liver metallothionein synthesis (either due to a direct effect of the primary insult or due to a secondary effect of cytokines elaborated as a consequence of tissue injury), developmental toxicity can result in part as a consequence of embryonicifetal Zn deficiency. Two obvious implications can be drawn from the above hypothesis: first, the provision of Zn supplements to the mother may reduce the developmental toxic effects associated with a diverse range of maternal insults; and second, the Zn status of the mother at the time of the insult, and her reaction to the insult with respect to liver
19
metallothionein synthesis, will be important predictors of the expression of the developmental insult. It should be noted that while the above discussion has focused on Zn, a stimulation of the acute phase response in an animal can also result in marked alterations in iron and copper metabolism (Cousins, ’85). The impact of these alterations on fetal development remains to be determined. SUMMARY
While there can be little argument with the idea that the consumption of a well balanced nutritious diet will minimize the possibility of embryonidfetal nutritional deficiencies, it must be recognized that the consumption of an “adequate” diet by the mother does not necessarily translate into “good” embryonidfetal nutrition. In this paper, using Zn as an example, the argument has been made that secondary nutrient deficiencies (i.e., those arising in the presence of what would normally be considered an adequate intake of the nutrient by the mother) may also represent a serious risk to the developing embryolfetus. The hypothesis that secondary embryonidfetal Zn deficiencies may be a common factor contributing t o the developmental toxicity associated with a number of compounds and environmental insults is proposed. Given the above, it is evident that an assessment of maternal nutritional status can be of value in predicting the relative risk of an individual to a reproductive hazard. The determination of the effect(s) of a reproductive insult on maternal delivery of nutrients to the embryo/ fetus may contribute to the identification of the biochemical lesions underlying the teratogenicity of the insult in question. Potential toxicant-nutrient interactions will probably not be detected in standard developmental toxicity assays, which, along with other types of toxicity tests, use animals of optimal health and nutritional status. Unfortunately, this does not always accurately reflect the health and nutrition of females of reproductive age in the human population. Further understanding of the role of compromised maternal nutritional status in determining the developmental outcome of exposure to toxicants during pregnancy is needed to improve our ability to carry out realistic and rational human risk assessments.
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C.L. KEEN ACKNOWLEDGMENTS
This work was supported in part by U.S. EPA cooperative agreement CR-816713 and by NIH grants HD01743 and HD14388. LITERATURE CITED Amemiya K., C.L. Keen, and L.S. Hurley (1986) 6-Mercaptopurine induced alterations in mineral metabolism and teratogenesis in the rat. Teratology, 34:321334. Amemiya, K., L.S. Hurley, and C.L. Keen (1988) The effect of 6-mercaptopurine on "Zn distribution in the pregnant rat. Teratology, 39:387-393. Baile, Y., and H. Lewenthal (1984) Effect of folic acid supplementation on congenital malformations due to anticonvulsive drugs. Eur. J. Obstet. Gynec. Reprod. Biol., 18:211-216. Berg, J.M. (1990) Zinc finger domains: Hypotheses and current knowledge. Annu. Rev. Biophys. Chem., 19: 405-421. Bower, C., and F.J. Stanley (1989) Dietary folate as a risk factor for neural-tube defects: Evidence from a case control study in Western Australia. Med. J . Aust., 150:613-619. Brenton, D.P., M.J. Jackson, and A. Young (1981) Two pregnancies in a patient with acrodermatis enteropathica treated with zinc sulphate. Lancet, 2;500502. Buamah, P.K., M. Russell, M. Bakes, W.A. Milford, and A.W. Skillen (1984) Maternal zinc status: A determination of central nervous system malformations. Br. J. Obstet. Gynaecol., 911788-790. Burke, B.S., V.A. Beal, S.B. Kirkwood, and H.C. Stuart (1943) The influence of nutrition during pregnancy upon the condition of the infant at birth. J. Nutr., 26569. Cavdar, A.O., M. Bahceci, N. Akar, J . Erten, G. Bahceci, E. Babacan, A. Arcasoy, and H. Yavuz (1988) Zinc status in prgenancy and the occurrence of anencephaly in Turkey. J . Trace Elem. Electrolytes Health Dis., 2:9-14. Cherry, F.F., H.H. Sandstead, P. Rojas, L.K. Johnson, H.K. Batson, and X. B. Wang (1989) Adolescent pregnancy: Associations among body weight, zinc nutriture, and pregnancy outcome. Am. J. Clin. Nutr., 50; 945-954. Cousins, R.J. (1985) Absorption, transport, and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev., 65: 238-309. Daston, G.P. (1982) Fetal zinc deficiency as a mechanism for cadmium-induced toxicity to the developing rat lung and pulmonary surfactant. Toxicology, 24: 55-63. Daston, G.P., G.J. Overmann, M.W. Taubeneck, L.D. Lehman-McKeeman, J.M. Rogers and C.L. Keen (1991) The role of metallothionein induction in maternally mediated developmental toxicity: Comparison of the effects of urethane and styrene in rats. Toxicol. Appl. Pharmacol., 110;450-463. Ellis, W.G., J.L. Semple, E.R. Hoogenboom, R.J. Kavlock and F.J. Zeman (1987) Benomyl-induced craniocerebral anomalies in fetuses of adequately nourished and protein-deprived rats. Teratogen., Carcinogen., Mutagen., 7:357-375. Flynn, A,, S.S. Martier, R.J. Sokol, S.I. Miller, N.L. Golden, and B.C. Villano (1981) Zinc status of pregnant alcoholic women: A determinant of fetal outcome. Lancet, lr572-574.
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