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Antioxidants and fetal protection against ethanol teratogenicity
Neurotoxicology and Teratology 25 (2003) 1 – 9 www.elsevier.com/locate/neutera
Review
Antioxidants and fetal protection against ethanol teratogenicity I. Review of the experimental data and implications to humans Raanan Cohen-Kerem*, Gideon Koren Motherisk Program, Division of Clinical Pharmacology and Toxicology, Department of Pediatrics, University of Toronto, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 Received 9 April 2002; received in revised form 1 August 2002; accepted 9 October 2002
Abstract Ethanol is the most common human teratogen, and heavy drinking during pregnancy can result in serious adverse outcomes to the fetus. The cellular mechanisms by which ethanol induces damage in utero are not well understood, while induction of oxidative stress is believed to be one putative mechanism. Our objective is to review the data of antioxidant effects in experimental models of fetal alcohol syndrome. Prior to the description of the available experimental data, we will briefly review the mechanisms leading to ethanol-induced oxidative stress. Ethanol-induced oxidative damage to the fetus could be attenuated by a variety of antioxidants as was documented in whole animal and tissue culture studies. Experiments, retrieved from the literature search, are described and criticized. Although experimental data are still limited, the application of a treatment strategy that includes antioxidants is justified since antioxidant treatment in human pregnancy for pre-eclampsia was demonstrated to be safe and effective. The available experimental evidence and the safety of vitamins C and E in pregnancy suggest that experimental use of antioxidants in alcohol-consuming mothers should be seriously considered to reduce fetal alcohol damage. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Antioxidant; Ethanol; Teratogenicity
1. Introduction Heavy drinking during pregnancy can result in serious adverse outcomes to the fetus. The Fetal Alcohol Syndrome (FAS) is a triad characterized by pre- and/or postnatal growth retardation, central nervous system damage, and typical facial dysmorphology [42]. The insult on the brain by ethanol affects intelligence, cognitive function, and behavior [1]. The incidence of FAS in developed countries is estimated to be as high as 0.97 cases per 1000 live births [2]. Huge efforts and extensive resources are required for the rehabilitation, education, and integration of these children in the community [28]. According to the Centers for Disease Control study on the prevalence of alcohol consumption among women of childbearing age, 50% appear to consume alcohol. Fortyfive percent are defined as light drinkers ( 30 drinks/ month), 3% as moderate (30 – 60 drinks/month), and 2% * Corresponding author. Tel.: +1-416-813-7500x4413; fax: +1-416813-7562. E-mail address: [email protected] (R. Cohen-Kerem).
as heavy drinkers ( 60 drinks/month). The rates of drinking in North America, Europe, and Australia are similar [3]. Although women tend to decrease their alcohol consumption during pregnancy [3,32], their actual level of drinking depends to a large extent on their drinking habits prior to conception. In many cases, the fetus is exposed to the teratogenic effects of ethanol during the critical period of organogenesis, before pregnancy is confirmed. Sixty percent of drinking women were not aware of their pregnancy until the fourth week after conception [30]. The fetus is more susceptible to ethanol than the mother: Offspring of rats that were fed nontoxic levels of ethanol presented with reduced growth and antioxidant activity in the liver while their mothers were not affected [5]. Mothers of children with FAS, who are typically heavy drinkers, do not present with signs of toxicity until late in the course of alcohol abuse when impairments of the central nervous system, liver, and pancreas prevail. The ways by which ethanol affects biochemical processes and cellular structures have been the focus of extensive research and they are still far from being completely understood. Various mechanisms have been pro-
0892-0362/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0892-0362(02)00324-0
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posed in explaining the teratogenicity of ethanol. We will focus on the induction of oxidative stress and the formation of oxygen radicals by ethanol, resulting in cellular injury [21]. The oxidative stress leads to peroxidation of lipids, nucleic acids, proteins, and carbohydrates. Sequelae of oxidative stress can be manifested by chromosomal abnormalities, enzymatic malfunction, and disruption of cellular membranes. The cell possesses antioxidant activity catalyzed by enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase, which are capable of neutralizing reactive oxygen species (ROS). Other molecules that contribute to antioxidant activity are vitamins such as vitamin C, vitamin E, and b-carotene. During the last decade, several groups of investigators have hypothesized that supplementation of antioxidants in FAS will attenuate ethanol-induced oxidative stress and thus reduce its fetal damage [4,18,24, 26,45,53,54,63,72]. 1.1. Objective Our objective is to review the data of antioxidant effects in experimental models of FAS. A critical review of data from experimental models is essential before considering treatment with antioxidants in pregnant alcoholic women in order to potentially prevent ethanol teratogenicity. 1.2. Methods of literature search We reviewed the world literature using MEDLINE and EMBASE databases. The search was based on the following keywords: pregnancy, ethanol, fetus, antioxidants, and animal. Studies were identified and reviewed for methods and endpoints. Included were studies that examined the effect of antioxidants on cell culture systems and studies on pregnancy outcome in animals exposed to ethanol following antioxidant supplementation. Reports of pregnancy outcome following exposure to ethanol that did not include in their methods intervention with antioxidants were excluded. In order to understand the rationale for treatment with antioxidants, a short section on the proposed mechanisms of ethanol-induced oxidative stress will precede the review of experimental evidence.
2. Mechanisms of ethanol-induced cellular injury In adults, ethanol toxicity has been demonstrated in many organs, but most affected are the brain and the liver. The most affected organ in FAS is the brain. The mechanisms of ethanol fetotoxicity are presently not completely understood and various mechanisms have been investigated. In 1981, Henderson et al. [35] reviewed the mechanisms leading to FAS, focusing on the mutagenicity of ethanol. A review by Michaelis [51] followed a decade later, in which the fetal consequences of ethanol-induced disruption of placental
transport of nutrients, of hypoxia, and of calcium-handling mechanisms were discussed. It is believed that ethanol is distributed into the lipid membrane of the cell, affecting the action of ion channels and proteins. Special emphasis was given to the amino acid-activated ion channels of excitation (glutamate) and inhibition (GABA) in the brain [33]. A recent observation that may shed light on how ethanol affects the developing brain is the triggering of widespread neuronal death in the developing rat forebrain by blocking the N-methyl-D-aspartate (NMDA) –glutamate receptor and activating GABAA receptors [39]. During the last 15 years, oxidative stress has been increasingly recognized as one of the mechanisms leading to ethanol toxicity [4]. Ethanol can induce oxidative stress directly and indirectly. The direct effect is achieved by formation of free radicals, including hydroxyl and hydroxyethyl groups, which are reacting with various cellular components. Formation of free radicals in the presence of ethanol was demonstrated in cell lines [21,24,34] and in animals [64]. The cellular damage caused by free radicals is a consequence of the peroxidation of lipids, [6,56,70], of nucleic acids [59], and alterations of enzyme activity [71] (Fig. 1). Another direct oxidative stress effect of ethanol is the formation of ROS. These are formed as biological products of molecular oxygen reduction [31] and they probably play a role in mediating programmed cell death [41]. The formation of ROS that is induced by cytochrome P-450 2E1 (CYP2E1) is mostly observed in the brain [56] and the liver [49,58], organs in which the enzyme is abundant. Formation of ROS is also induced by mitochondria in hepatocytes exposed to ethanol and is probably achieved by reduction in mitochondria-derived components of electron transport [11,12,14] (Fig. 1). Ethanol can also induce oxidative stress indirectly by reducing intracellular antioxidant capacity, such as the levels of glutathione peroxidase. In rats, chronic ethanol consumption decreased significantly cytosolic and mitochondrial glutathione peroxidase activities by 40% and 30%, respectively, and caused a parallel increase of the oxidative modification of proteins in hepatocytes [13,61]. The reason for the reduced activity of glutathione peroxidase might lie in the decreased mitochondrial pool size of glutathione because entry of cytosolic glutathione into mitochondria is impaired [29]. The direct and indirect mechanisms by which ethanol causes oxidative stress are summarized in Fig. 1. Other factors that may contribute considerably to the final outcome of damage caused by ethanol in FAS include hypoxia [9,36], formation of fatty acid ethyl esters [15,38], retinoic acid [22], L1 neural cell adhesion molecule [16], and endocrine-related mechanisms. Regarding hypoxia, it was shown that prolonged treatment of the isolated liver with alcohol increases utilization of and damage by oxygen [73]. Eventually, cellular damage induced by ethanol is probably the sum of all those mechanisms and of others that have not yet been investigated. Based on the assumption that
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Fig. 1. Summary of ethanol-induced oxidative stress mechanisms. Pathways of oxidative stress are divided into a direct and an indirect effect. The outcome of the indirect pathway is a reduction in glutathione peroxidase activity and the outcome of the direct pathway is peroxidation of lipids, nucleic acids, and proteins through free radicals and ROS. Hydroxyethyl and hydroxyl groups = oxygen free radicals. CYP2E1 = cytochrome P-450 2E1. = Excitatory effect, = Inhibitory effect.
oxidative stress is one of the major routes of ethanol-induced damage, it is reasonable to investigate interventions that may attenuate this damage. Such interventions may include supplementation with antioxidants, which are molecules that possess the ability to stabilize free oxygen radicals. Different antioxidants had been used in the studies reviewed here. Vitamin C is known as a potent scavenger of oxygen free radicals, such as superoxide and hydroxyl, and therefore, inhibits their activity [60]. It can also maintain intracellular glutathione levels [50]. Vitamin C interacts with vitamin E by recycling the a-tocopheryl radical to the stable a-tocopherol. Vitamin E is known to protect cellular membranes and low-density lipoproteins (LDL) from peroxidation [27] by acting as an electron donor, transforming the free radical to its stable form. Flavonoids are a class of water-soluble plant pigments that have antioxidative properties. During the last 50 years, their importance as ‘health supporters’ has been increased. The flavonoids are scavengers of oxidants by virtue of the number and arrangement of their phenolic hydroxyl groups [65].
3. Experimental evidence for reduced ethanol teratogenicity by antioxidants Experiments that show the fetal effects of antioxidants on oxidative stress induced by ethanol approached the issue in two different ways: (a) by cell cultures and (b) by animal studies. The use of experiments based on cell culture is an
excellent tool to study the molecular basis of alcoholinduced damage, allowing researchers to manipulate different experimental conditions. However, one should be cautious when extrapolating cell culture data to animals or humans since other mechanisms occur simultaneously in the living organ affecting the final outcome. Another problem is that oxygen-free radicals might not have the same destructive potential in cell cultures as they have in vivo. Some of the biochemical and physiological characteristics of such animal models resemble those of human pathophysiology, but genetic features may be different, suggesting that susceptibility to ethanol may vary between species, making extrapolation of results to humans not always possible. 3.1. Cell cultures Cell cultures originating from organs mostly affected by ethanol, such as the brain or the liver, may also aid in understanding the mechanism of damage by ethanol. We identified two studies investigating the protective effects of vitamin E and b-carotene in a hippocampal culture model [54,55]. Several studies have documented specific ethanol-induced damage to the hippocampus. The hippocampal formation and number of neurons are adversely affected by exposure to high doses of ethanol in neonatal rats [52], a phenomenon that had been studied previously in third-trimester equivalent rats [77]. Hippocampal regions CA1 and CA3 of ethanol-treated rats showed lower levels of cytochrome c oxidase mRNA expression that is related to
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oxidative stress, suggesting that brain damage is induced by oxidation [44]. Mitchell et al. [54,55] demonstrated a dosedependent decreased viability of hippocampal cell cultures following exposure to ethanol in concentrations ranging from 400 to 2400 mg/dl. The source of the cells were gestational Day 18 Long Evans hooded rat fetuses; exposing these cells to ethanol created a model for hippocampal response to alcohol exposure. Since ethanol-induced damage is probably due to causes other than just oxidative stress, the cultures were deprived of glucose and were exposed to an anoxic environment (95% N2 and 5% CO2 for 2 h) in order to imitate ischemia. Neuroprotection despite ischemia and hypoglycemia was achieved by adding vitamin E or b-carotene to the culture at all ethanol concentrations [54]. In all settings, neuronal viability was significantly higher in cultures maintained on vitamin E or b-carotene, favoring vitamin E in higher concentrations [55]. Overall, nutritional supplementation of vitamin E and b-carotene improved the survival of the hippocampal cell culture despite ethanol exposure at high concentrations. Unfortunately, the authors did not measure oxidative stress to demonstrate a mechanistic plausibility to link the protective effect to supplementation of antioxidants, since protection could also have been achieved through other mechanisms. The model imitates FAS conditions for hippocampal cells only partially, because it lacks the ethanol exposure period of the fetus between conception and the development of hippocampal tissue. Obtaining embryonic hippocampal cells from fetuses exposed to ethanol in utero can also reduce this experimental gap in ethanol exposure. Devi et al. [24], using hepatocyte cultures, had presented a similar system a few years earlier. Their culture system was based on liver cells obtained from Sprague – Dawley rat fetuses on Day 19 of gestation. The medium was supplemented with 2.0 mg/ml ethanol, which was reduced to 1.5 mg/ml after 24 h. Ethanol levels were one-tenth the levels in Mitchell’s study. The main outcome measure was cell replication and, in contrast to Mitchell’s studies, lipid peroxidation and glutathione levels were obtained as oxidative stress measures. Adding to the medium, prior to ethanol supplementation, the antioxidants N-acetylcysteine or S-adenosylmethionine prevented the reduction of glutathione and the lipid peroxidation. The latter was also prevented by pre-supplementation with vitamin E, but the reduction of glutathione was not prevented. 3.2. Animal studies Only a few animal studies have been conducted to investigate the ability of antioxidants to attenuate alcohol teratogenicity. While oxidative stress damage could be detected in several organs, the brain and the liver were the organs most commonly investigated. Researchers have been using several different antioxidants in their experiments: flavonoids, a-tocopherol (vitamin E),
folic acid, and b-carotene. To our knowledge, the first experiment of the effect of an antioxidant on ethanol-exposed fetuses was reported by Tanaka et al. [72]. As part of a study looking for supplementary agents which may prevent ethanol teratogenicity, they supplemented pregnant rats, fed 20% ethanol, with 0.03% vitamin E, and 10% ethanol-fed pregnant rats with 0.02% vitamin E. The outcome measurements of their study were fetal body and cerebral weights. The investigation failed to show a protective effect of vitamin E although increased levels of a-tocopherol and lipid peroxidase were found in the cerebrum of those rats. The flavonoids have been shown to be potent antioxidants. La Grange et al. [45] investigated the antioxidant effects of Silymarin – Phytosome in rats fed an ethanolenriched diet. This compound is a mixture of several flavonoids (silymarin) and phosphatidylcholine to allow enhanced absorption. Tissue gamma glutamyl transpeptidase (GGT) activity was used to quantify brain and liver damage in female rats and their offspring. The rats were randomly assigned to seven groups, five of which were fed a liquid diet containing 6.7% EtOH ( = 36% EtOH-derived calories). Higher activity of GGT was found in the maternal and fetal liver and brain when ethanol was added to the diet. Following administration of Silymarin – Phytosome, the GGT activity was significantly suppressed in fetal and maternal liver and maternal brain in a dose-dependent manner [45]. Although suppression of the adverse effects of ethanol was achieved with this antioxidant, outcome measures linking the adverse effect to oxidative stress were not obtained. Subsequently, the same group used a similar experimental design with Siliphos, which contains the flavonoid silybin and phosphatidylcholine. Silybin is considered to be the primary active constituent among the flavonoids [75]. The feed of the rats in this study contained 6.7% EtOH or an isocaloric amount of dextrin maltose. To the diet of one of the groups, 400 mg/kg Siliphos were added on the first day of pregnancy. Rats and their offspring receiving the EtOH diet combined with Siliphos did not exhibit any elevation in liver and brain GGT activity, and the fetal mortality was lower in comparison to nontreated rats that were fed with EtOH [26]. A second outcome measure in this study was the level of glutathione. Reduction in the level of glutathione may represent an indirect adverse effect of ethanol-induced oxidative stress. While general measures, such as body weight and the mortality rate of the pups had improved with Siliphos supplementation, glutathione levels remained as low as in the ethanol-only treated animals. It is not clear how the flavonoids prevent ethanol damage to the fetus. However, preventing ethanol-induced lipid peroxidation is probably an important part of this process as flavonoids had been shown to prevent lipid peroxidation in studies on atherosclerosis [66]. As in their first report, enriching the diet of the rats with an antioxidant appeared to be beneficial here as well; however, no association with a measurable marker of oxidative stress was demonstrated.
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Teratogenic effects of ethanol have also been demonstrated in the behavioral abnormalities of ethanol-exposed children [10]. A review of the prenatal exposure to ethanol in mice suggests that there is a decrease in locomotor activity and there are deficits in avoidance learning and operant performance [17]. Kim et al. [43] have shown that spatial cognitive abilities in rats are more vulnerable to ethanol than object recognition. Pregnant rats, fed 35% ethanol-derived calories, had daily subcutaneous injections of 400 mg/kg silymarin or received 400 mg/kg by the oral route. Following delivery, the offspring were started on a rat chow diet and after 90 days their social memory was tested and recorded as a social recognition score. The social recognition task for the rats consisted of sharing two periods of 5 min separated by a 60-min interval, with a strange male rat. Three different observers watched and scored the rats’ behavior in these periods. The score recorded for the ethanol-exposed pups was significantly lower than the score for the ‘‘ethanol/silymarin’’ rats or the control groups, implying a protective effect of silymarin on short-term memory [63]. The mechanism by which silymarin improves social learning was not determined in this study, as measurements of oxidative stress were not obtained. While this study presents encouraging results in terms of fetal protection, it is not clear whether the flavonoids or vitamin E protect the fetus as antioxidants or through other mechanisms. Folic acid acts also as an antioxidant. Cano et al. [18] studied the effects of folic acid on oxidative stress induced by ethanol. Two groups of pregnant rats were fed chronically with ethanol-supplemented tap water up to a mean of 8.9 ± 0.4 g ethanol/kg/day for 3 weeks. For four additional weeks, the rats were maintained on 16.6 ± 2.1 g ethanol/kg/ day. The rats in the control groups received supplements of folic acid (60 mg/day) during pregnancy. The study group received folic acid in a dose of 152 mg/day. The specific activity of glutathione reductase in liver and pancreas was used as a marker of oxidative stress. Its level was higher by 32 –34% in the female rats and by 24% in the progeny following an ethanol-containing diet. Reduction in glutathione reductase was significant in the group that received the high dosage of folic acid. The amount of carbonyl group proteins in the liver and pancreas was also used as an index for oxidative damage. Levels of carbonyl groups were reduced significantly in the liver of offspring supplemented with folic acid but were unchanged in their pancreas. The antioxidant activity of folic acid was assessed and proven by an assay that utilizes a free radical-sensitive fluorescent indicator protein [18]. This study showed an association between FAS in an animal model and oxidative stress measurements and mitigation of those measures by using an antioxidant. Treatment outcomes in the liver and in the pancreas were different and may reflect distinctive susceptibilities of different tissues. Brain damage is the major component of FAS; unfortunately, the authors did not study the protective effect of folic acid on the brain.
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Spong et al. [68] used mice as a model for FAS. The mice were injected with 25% ethanol in saline or saline alone and were supplemented with NAPVSIPQ (NAP) and/or SALLRSIPA (ADNF-9) before or after ethanol injection. NAP and ADNF-9 are peptides regulated by the vasoactive intestinal peptide and were shown to have antioxidant properties in brain cell culture. The outcome measures in their study were fetal death and fetal body and brain weight, and as an oxidative stress measurement, they used the ratio of reduced glutathione and oxidized glutathione. Pretreatment with NAP was associated with reduced fetal death, whereas ADNF-9 alone had no effect on fetal survival. The combination of the two, administered prior to ethanol injection, prevented growth restriction and the decrease in reduced glutathione. The combination also prevented reduction in brain weight when administered after ethanol injection. This study shows that treatment with an antioxidant prior to or after ethanol administration may reduce some of its adverse effects. Correlation with the oxidative stress process was demonstrated in one group only (NAP and ADNF-9 prior to ethanol) but not in the other groups where antioxidants were administered after injecting ethanol. A different approach to studying the effect of ethanol on the fetus is by exposing the fetus directly to ethanol. In mammals, this could be problematic because it can result in fetal loss, but it can be achieved in other species of animals such as fowl. Theoretically, adding ethanol to chicken eggs should yield similar results as introducing ethanol to the mammalian fetus through the placenta. Miller et al. [53] injected ethanol at day zero of incubation to the air sac of eggs in concentrations ranging from 0 to 302.5 mmol EtOH/kg egg. Injecting ethanol in various concentrations into fertile chicken eggs caused a doserelated decrease in cerebral long-chain polyunsaturated fatty acids: stearic, oleic, linoleic, linolenic, and arachidonic acids. Long-chain fatty acids, important components of cell membranes, are oxidized, a process that eventually leads to a reduction in brain mass. Vitamin E components, atocopherol or g-tocopherol, were injected concomitantly with ethanol into the egg’s air sac at concentrations of 2.5 mmol tocopherol per kg egg. Following this procedure, brain mass was not reduced, levels of brain protein were maintained, acetylcholine esterase activity was not reduced, and the levels of lipid hydroperoxide were not increased. The system in this study is not equivalent to those in mammals because of the different fetal environment and maternal – fetal interaction in utero, but it can demonstrate isolated effects of ethanol and antioxidants on the developing embryo. Miller et al. [53] have shown that the effect of ethanol was associated with oxidative stress, and supplementation with tocopherol reduced this effect significantly. Findings from these animal studies may be in contradiction to the work of Amini et al. [8] who monitored fetal oxidative stress in Quackenbush Special mice and found that, although increases in lipid peroxides and
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decreases in glutathione peroxidase and superoxide dismutase in the mother took place, only minor changes were observed in the embryo. The conclusion of their investigations was that the contribution of other fetotoxic effects of ethanol might be more substantial than ethanolinduced oxidative stress.
4. Discussion The term ‘‘oxidative stress’’ describes several biochemical reactions that lead to a final outcome that can be devastating in terms of fetotoxicity. In this review, we focused on fetal exposure to alcohol but oxidative stress is implicated also in the mechanisms of other pathological conditions such as diabetic pregnancy. Cederberg et al. [19] showed that supplementation of vitamin E and vitamin C to diabetic rats reduced fetal malformations and diminished the levels of oxygen radicals in the fetal liver. Several aspects of ethanol-induced damage in the fetus were studied in the research papers we reviewed. The adverse effects of ethanol on the liver and pancreas are among the main issues in the alcohol-addicted population. In contrast, FAS children do not present with liver damage as a main feature [3]; and reports regarding liver damage are anecdotal [46,57]. Using liver and pancreas as targets of ethanol injury provides proof for the ability of antioxidants to reduce damage. Reduced activity of GGT, which is increased in response to alcohol [40], and of glutathione reductase are good examples for that [18]. Brain damage due to ethanol exposure is a cardinal feature in FAS and in chronic alcoholism. Enhanced neurodegeneration [39] and increased dopamine deficiency [25] in combination with oxidative stress outcomes probably lead to the neurodevelopmental deficiencies that are encountered in FAS. In the available animal studies, one can clearly observe a protective effect of antioxidants against damage induced by ethanol. Despite the contradictory conclusions drawn from the study done by Amini et al. [8], it is fair to state that there is some strong evidence to support the assumption that concomitant administration of antioxidants and ethanol attenuates ethanol-induced fetotoxicity. Applying antioxidant treatment in pregnancy is not a new concept. Oxidative stress was found to play a role in the pathogenesis of pre-eclampsia. Elevation of lipid peroxidation products was highly correlated to the level of blood pressure and, in parallel, the severity of pre-eclampsia was found to be correlated to plasma antioxidant levels of glutathione peroxidase [74]. Following this theory, 283 women who had been identified as being at increased risk for pre-eclampsia were assigned randomly to vitamin E or vitamin C treatment, or to placebo. The dosage of vitamin C was 1000 mg/day and of vitamin E 400 IU/day. The treatment was provided during Weeks 16 –22 of gestation. Vitamin supplementa-
tion resulted in a 21% decrease of the PA1/PA2 ratio (the ratio of plasminogen-activator inhibitor 1 and 2 usually increases in pre-eclampsia) and the occurrence of preeclampsia was 17% in the placebo group compared to 8% of the group treated with vitamins. No adverse effects to the fetus were noted following this treatment. These results suggest that women who are at high risk for preeclampsia may benefit from diet supplementation with antioxidants [20]. Chappell et al. [20] used 1000 mg vitamin C since complete plasma saturation occurs with this daily dose [47]. The daily dose of vitamin C in the common multivitamin supplements in pregnancy is 50 –100 mg making the suggested dose 10 –20 fold higher [62]. Vitamin C in the appropriate doses is safe in pregnancy; chronic ingestion of high doses might cause renal stones in the mother [33]. Vitamin E was given in a high dose (400 IU) as well, since it was shown to decrease oxidation of LDL [23] and had some benefit in the treatment of patients with coronary artery disease [69]. The common multivitamin supplements in pregnancy contain 0– 30 IU [62], here as well the suggested dose is considerably higher than that recommended for pregnancy supplementation. Vitamin E is considered safe in pregnancy, although we did not find a study that examined the safety of high-dose vitamin E treatment in pregnancy. Chapell’s study was focused on pre-eclampsia and did not report on any adverse outcome associated with this highdose vitamin treatment. Pregnant women treated with high doses of vitamin C should be followed for renal stones formation throughout the treatment period. Several studies [45,54,55,63,72] failed to demonstrate clearly oxidative stress as a major mechanism of alcoholinduced damage, suggesting that the human application of treatment with antioxidants may not be easily justified. Other data [18,24,26,53] are more encouraging since a linkage between antioxidant activity and oxidative stress was established. Dietary supplementation with vitamin C and E in therapeutic doses ( 1 g/day and 2100 units/day, respectively) has not been shown to cause adverse effects in humans, neither to the mother nor to the fetus [7]. The question of the optimal dose still remains to be answered; high doses may not necessarily be desirable, and extrapolating from cancer studies, the use of antioxidants may interfere with apoptosis. In this process, oxygen-free radicals play a part in the cascade of programmed cell death [37]. Hence, the dose of vitamins will have to be considered as not to interfere with naturally occurring oxidative processes, which are probably important for the fetus. Antioxidant activity is found in numerous organic structures. Dietary supplements are required because the body does not have the capacity to synthesize vitamins and flavonoids. Other antioxidants do exist in the body but their levels are probably not sufficiently high to prevent the oxidative stress induced by large amounts of alcohol. Adequate levels of folic acid have been shown to prevent
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neural tube defects, but the desired level is achieved in most women only by supplementing folic acid or by fortifying the diet [76]. Finally, treating alcoholic women with antioxidants through food supplements such as vitamins is appealing because, in addition to the potential protective effect shown in the above studies, it may reverse other nutritional deficits common in this population [48,67]. Notwithstanding our focus on preventing oxidative fetal damage, a major effort must be invested in alcoholic prevention programs, thus improving maternal health while protecting the unborn child.
Acknowledgements We should like to thank Ingeborg C. Radde, MD, PhD, FRCP(C), for reading and commenting on this manuscript. RCK is supported by a Research Training Fellowship from the HSC Research Institute. GK is a Senior Scientist of the Canadian Institutes for Health Research (CIHR), Supported by a NET (New Emerging Team) grant from CIHR and by the Research Leadership in Better Pharmacotherapy during Pregnancy and Lactation.
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Review
Antioxidants and fetal protection against ethanol teratogenicity I. Review of the experimental data and implications to humans Raanan Cohen-Kerem*, Gideon Koren Motherisk Program, Division of Clinical Pharmacology and Toxicology, Department of Pediatrics, University of Toronto, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 Received 9 April 2002; received in revised form 1 August 2002; accepted 9 October 2002
Abstract Ethanol is the most common human teratogen, and heavy drinking during pregnancy can result in serious adverse outcomes to the fetus. The cellular mechanisms by which ethanol induces damage in utero are not well understood, while induction of oxidative stress is believed to be one putative mechanism. Our objective is to review the data of antioxidant effects in experimental models of fetal alcohol syndrome. Prior to the description of the available experimental data, we will briefly review the mechanisms leading to ethanol-induced oxidative stress. Ethanol-induced oxidative damage to the fetus could be attenuated by a variety of antioxidants as was documented in whole animal and tissue culture studies. Experiments, retrieved from the literature search, are described and criticized. Although experimental data are still limited, the application of a treatment strategy that includes antioxidants is justified since antioxidant treatment in human pregnancy for pre-eclampsia was demonstrated to be safe and effective. The available experimental evidence and the safety of vitamins C and E in pregnancy suggest that experimental use of antioxidants in alcohol-consuming mothers should be seriously considered to reduce fetal alcohol damage. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Antioxidant; Ethanol; Teratogenicity
1. Introduction Heavy drinking during pregnancy can result in serious adverse outcomes to the fetus. The Fetal Alcohol Syndrome (FAS) is a triad characterized by pre- and/or postnatal growth retardation, central nervous system damage, and typical facial dysmorphology [42]. The insult on the brain by ethanol affects intelligence, cognitive function, and behavior [1]. The incidence of FAS in developed countries is estimated to be as high as 0.97 cases per 1000 live births [2]. Huge efforts and extensive resources are required for the rehabilitation, education, and integration of these children in the community [28]. According to the Centers for Disease Control study on the prevalence of alcohol consumption among women of childbearing age, 50% appear to consume alcohol. Fortyfive percent are defined as light drinkers ( 30 drinks/ month), 3% as moderate (30 – 60 drinks/month), and 2% * Corresponding author. Tel.: +1-416-813-7500x4413; fax: +1-416813-7562. E-mail address: [email protected] (R. Cohen-Kerem).
as heavy drinkers ( 60 drinks/month). The rates of drinking in North America, Europe, and Australia are similar [3]. Although women tend to decrease their alcohol consumption during pregnancy [3,32], their actual level of drinking depends to a large extent on their drinking habits prior to conception. In many cases, the fetus is exposed to the teratogenic effects of ethanol during the critical period of organogenesis, before pregnancy is confirmed. Sixty percent of drinking women were not aware of their pregnancy until the fourth week after conception [30]. The fetus is more susceptible to ethanol than the mother: Offspring of rats that were fed nontoxic levels of ethanol presented with reduced growth and antioxidant activity in the liver while their mothers were not affected [5]. Mothers of children with FAS, who are typically heavy drinkers, do not present with signs of toxicity until late in the course of alcohol abuse when impairments of the central nervous system, liver, and pancreas prevail. The ways by which ethanol affects biochemical processes and cellular structures have been the focus of extensive research and they are still far from being completely understood. Various mechanisms have been pro-
0892-0362/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0892-0362(02)00324-0
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posed in explaining the teratogenicity of ethanol. We will focus on the induction of oxidative stress and the formation of oxygen radicals by ethanol, resulting in cellular injury [21]. The oxidative stress leads to peroxidation of lipids, nucleic acids, proteins, and carbohydrates. Sequelae of oxidative stress can be manifested by chromosomal abnormalities, enzymatic malfunction, and disruption of cellular membranes. The cell possesses antioxidant activity catalyzed by enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase, which are capable of neutralizing reactive oxygen species (ROS). Other molecules that contribute to antioxidant activity are vitamins such as vitamin C, vitamin E, and b-carotene. During the last decade, several groups of investigators have hypothesized that supplementation of antioxidants in FAS will attenuate ethanol-induced oxidative stress and thus reduce its fetal damage [4,18,24, 26,45,53,54,63,72]. 1.1. Objective Our objective is to review the data of antioxidant effects in experimental models of FAS. A critical review of data from experimental models is essential before considering treatment with antioxidants in pregnant alcoholic women in order to potentially prevent ethanol teratogenicity. 1.2. Methods of literature search We reviewed the world literature using MEDLINE and EMBASE databases. The search was based on the following keywords: pregnancy, ethanol, fetus, antioxidants, and animal. Studies were identified and reviewed for methods and endpoints. Included were studies that examined the effect of antioxidants on cell culture systems and studies on pregnancy outcome in animals exposed to ethanol following antioxidant supplementation. Reports of pregnancy outcome following exposure to ethanol that did not include in their methods intervention with antioxidants were excluded. In order to understand the rationale for treatment with antioxidants, a short section on the proposed mechanisms of ethanol-induced oxidative stress will precede the review of experimental evidence.
2. Mechanisms of ethanol-induced cellular injury In adults, ethanol toxicity has been demonstrated in many organs, but most affected are the brain and the liver. The most affected organ in FAS is the brain. The mechanisms of ethanol fetotoxicity are presently not completely understood and various mechanisms have been investigated. In 1981, Henderson et al. [35] reviewed the mechanisms leading to FAS, focusing on the mutagenicity of ethanol. A review by Michaelis [51] followed a decade later, in which the fetal consequences of ethanol-induced disruption of placental
transport of nutrients, of hypoxia, and of calcium-handling mechanisms were discussed. It is believed that ethanol is distributed into the lipid membrane of the cell, affecting the action of ion channels and proteins. Special emphasis was given to the amino acid-activated ion channels of excitation (glutamate) and inhibition (GABA) in the brain [33]. A recent observation that may shed light on how ethanol affects the developing brain is the triggering of widespread neuronal death in the developing rat forebrain by blocking the N-methyl-D-aspartate (NMDA) –glutamate receptor and activating GABAA receptors [39]. During the last 15 years, oxidative stress has been increasingly recognized as one of the mechanisms leading to ethanol toxicity [4]. Ethanol can induce oxidative stress directly and indirectly. The direct effect is achieved by formation of free radicals, including hydroxyl and hydroxyethyl groups, which are reacting with various cellular components. Formation of free radicals in the presence of ethanol was demonstrated in cell lines [21,24,34] and in animals [64]. The cellular damage caused by free radicals is a consequence of the peroxidation of lipids, [6,56,70], of nucleic acids [59], and alterations of enzyme activity [71] (Fig. 1). Another direct oxidative stress effect of ethanol is the formation of ROS. These are formed as biological products of molecular oxygen reduction [31] and they probably play a role in mediating programmed cell death [41]. The formation of ROS that is induced by cytochrome P-450 2E1 (CYP2E1) is mostly observed in the brain [56] and the liver [49,58], organs in which the enzyme is abundant. Formation of ROS is also induced by mitochondria in hepatocytes exposed to ethanol and is probably achieved by reduction in mitochondria-derived components of electron transport [11,12,14] (Fig. 1). Ethanol can also induce oxidative stress indirectly by reducing intracellular antioxidant capacity, such as the levels of glutathione peroxidase. In rats, chronic ethanol consumption decreased significantly cytosolic and mitochondrial glutathione peroxidase activities by 40% and 30%, respectively, and caused a parallel increase of the oxidative modification of proteins in hepatocytes [13,61]. The reason for the reduced activity of glutathione peroxidase might lie in the decreased mitochondrial pool size of glutathione because entry of cytosolic glutathione into mitochondria is impaired [29]. The direct and indirect mechanisms by which ethanol causes oxidative stress are summarized in Fig. 1. Other factors that may contribute considerably to the final outcome of damage caused by ethanol in FAS include hypoxia [9,36], formation of fatty acid ethyl esters [15,38], retinoic acid [22], L1 neural cell adhesion molecule [16], and endocrine-related mechanisms. Regarding hypoxia, it was shown that prolonged treatment of the isolated liver with alcohol increases utilization of and damage by oxygen [73]. Eventually, cellular damage induced by ethanol is probably the sum of all those mechanisms and of others that have not yet been investigated. Based on the assumption that
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Fig. 1. Summary of ethanol-induced oxidative stress mechanisms. Pathways of oxidative stress are divided into a direct and an indirect effect. The outcome of the indirect pathway is a reduction in glutathione peroxidase activity and the outcome of the direct pathway is peroxidation of lipids, nucleic acids, and proteins through free radicals and ROS. Hydroxyethyl and hydroxyl groups = oxygen free radicals. CYP2E1 = cytochrome P-450 2E1. = Excitatory effect, = Inhibitory effect.
oxidative stress is one of the major routes of ethanol-induced damage, it is reasonable to investigate interventions that may attenuate this damage. Such interventions may include supplementation with antioxidants, which are molecules that possess the ability to stabilize free oxygen radicals. Different antioxidants had been used in the studies reviewed here. Vitamin C is known as a potent scavenger of oxygen free radicals, such as superoxide and hydroxyl, and therefore, inhibits their activity [60]. It can also maintain intracellular glutathione levels [50]. Vitamin C interacts with vitamin E by recycling the a-tocopheryl radical to the stable a-tocopherol. Vitamin E is known to protect cellular membranes and low-density lipoproteins (LDL) from peroxidation [27] by acting as an electron donor, transforming the free radical to its stable form. Flavonoids are a class of water-soluble plant pigments that have antioxidative properties. During the last 50 years, their importance as ‘health supporters’ has been increased. The flavonoids are scavengers of oxidants by virtue of the number and arrangement of their phenolic hydroxyl groups [65].
3. Experimental evidence for reduced ethanol teratogenicity by antioxidants Experiments that show the fetal effects of antioxidants on oxidative stress induced by ethanol approached the issue in two different ways: (a) by cell cultures and (b) by animal studies. The use of experiments based on cell culture is an
excellent tool to study the molecular basis of alcoholinduced damage, allowing researchers to manipulate different experimental conditions. However, one should be cautious when extrapolating cell culture data to animals or humans since other mechanisms occur simultaneously in the living organ affecting the final outcome. Another problem is that oxygen-free radicals might not have the same destructive potential in cell cultures as they have in vivo. Some of the biochemical and physiological characteristics of such animal models resemble those of human pathophysiology, but genetic features may be different, suggesting that susceptibility to ethanol may vary between species, making extrapolation of results to humans not always possible. 3.1. Cell cultures Cell cultures originating from organs mostly affected by ethanol, such as the brain or the liver, may also aid in understanding the mechanism of damage by ethanol. We identified two studies investigating the protective effects of vitamin E and b-carotene in a hippocampal culture model [54,55]. Several studies have documented specific ethanol-induced damage to the hippocampus. The hippocampal formation and number of neurons are adversely affected by exposure to high doses of ethanol in neonatal rats [52], a phenomenon that had been studied previously in third-trimester equivalent rats [77]. Hippocampal regions CA1 and CA3 of ethanol-treated rats showed lower levels of cytochrome c oxidase mRNA expression that is related to
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oxidative stress, suggesting that brain damage is induced by oxidation [44]. Mitchell et al. [54,55] demonstrated a dosedependent decreased viability of hippocampal cell cultures following exposure to ethanol in concentrations ranging from 400 to 2400 mg/dl. The source of the cells were gestational Day 18 Long Evans hooded rat fetuses; exposing these cells to ethanol created a model for hippocampal response to alcohol exposure. Since ethanol-induced damage is probably due to causes other than just oxidative stress, the cultures were deprived of glucose and were exposed to an anoxic environment (95% N2 and 5% CO2 for 2 h) in order to imitate ischemia. Neuroprotection despite ischemia and hypoglycemia was achieved by adding vitamin E or b-carotene to the culture at all ethanol concentrations [54]. In all settings, neuronal viability was significantly higher in cultures maintained on vitamin E or b-carotene, favoring vitamin E in higher concentrations [55]. Overall, nutritional supplementation of vitamin E and b-carotene improved the survival of the hippocampal cell culture despite ethanol exposure at high concentrations. Unfortunately, the authors did not measure oxidative stress to demonstrate a mechanistic plausibility to link the protective effect to supplementation of antioxidants, since protection could also have been achieved through other mechanisms. The model imitates FAS conditions for hippocampal cells only partially, because it lacks the ethanol exposure period of the fetus between conception and the development of hippocampal tissue. Obtaining embryonic hippocampal cells from fetuses exposed to ethanol in utero can also reduce this experimental gap in ethanol exposure. Devi et al. [24], using hepatocyte cultures, had presented a similar system a few years earlier. Their culture system was based on liver cells obtained from Sprague – Dawley rat fetuses on Day 19 of gestation. The medium was supplemented with 2.0 mg/ml ethanol, which was reduced to 1.5 mg/ml after 24 h. Ethanol levels were one-tenth the levels in Mitchell’s study. The main outcome measure was cell replication and, in contrast to Mitchell’s studies, lipid peroxidation and glutathione levels were obtained as oxidative stress measures. Adding to the medium, prior to ethanol supplementation, the antioxidants N-acetylcysteine or S-adenosylmethionine prevented the reduction of glutathione and the lipid peroxidation. The latter was also prevented by pre-supplementation with vitamin E, but the reduction of glutathione was not prevented. 3.2. Animal studies Only a few animal studies have been conducted to investigate the ability of antioxidants to attenuate alcohol teratogenicity. While oxidative stress damage could be detected in several organs, the brain and the liver were the organs most commonly investigated. Researchers have been using several different antioxidants in their experiments: flavonoids, a-tocopherol (vitamin E),
folic acid, and b-carotene. To our knowledge, the first experiment of the effect of an antioxidant on ethanol-exposed fetuses was reported by Tanaka et al. [72]. As part of a study looking for supplementary agents which may prevent ethanol teratogenicity, they supplemented pregnant rats, fed 20% ethanol, with 0.03% vitamin E, and 10% ethanol-fed pregnant rats with 0.02% vitamin E. The outcome measurements of their study were fetal body and cerebral weights. The investigation failed to show a protective effect of vitamin E although increased levels of a-tocopherol and lipid peroxidase were found in the cerebrum of those rats. The flavonoids have been shown to be potent antioxidants. La Grange et al. [45] investigated the antioxidant effects of Silymarin – Phytosome in rats fed an ethanolenriched diet. This compound is a mixture of several flavonoids (silymarin) and phosphatidylcholine to allow enhanced absorption. Tissue gamma glutamyl transpeptidase (GGT) activity was used to quantify brain and liver damage in female rats and their offspring. The rats were randomly assigned to seven groups, five of which were fed a liquid diet containing 6.7% EtOH ( = 36% EtOH-derived calories). Higher activity of GGT was found in the maternal and fetal liver and brain when ethanol was added to the diet. Following administration of Silymarin – Phytosome, the GGT activity was significantly suppressed in fetal and maternal liver and maternal brain in a dose-dependent manner [45]. Although suppression of the adverse effects of ethanol was achieved with this antioxidant, outcome measures linking the adverse effect to oxidative stress were not obtained. Subsequently, the same group used a similar experimental design with Siliphos, which contains the flavonoid silybin and phosphatidylcholine. Silybin is considered to be the primary active constituent among the flavonoids [75]. The feed of the rats in this study contained 6.7% EtOH or an isocaloric amount of dextrin maltose. To the diet of one of the groups, 400 mg/kg Siliphos were added on the first day of pregnancy. Rats and their offspring receiving the EtOH diet combined with Siliphos did not exhibit any elevation in liver and brain GGT activity, and the fetal mortality was lower in comparison to nontreated rats that were fed with EtOH [26]. A second outcome measure in this study was the level of glutathione. Reduction in the level of glutathione may represent an indirect adverse effect of ethanol-induced oxidative stress. While general measures, such as body weight and the mortality rate of the pups had improved with Siliphos supplementation, glutathione levels remained as low as in the ethanol-only treated animals. It is not clear how the flavonoids prevent ethanol damage to the fetus. However, preventing ethanol-induced lipid peroxidation is probably an important part of this process as flavonoids had been shown to prevent lipid peroxidation in studies on atherosclerosis [66]. As in their first report, enriching the diet of the rats with an antioxidant appeared to be beneficial here as well; however, no association with a measurable marker of oxidative stress was demonstrated.
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Teratogenic effects of ethanol have also been demonstrated in the behavioral abnormalities of ethanol-exposed children [10]. A review of the prenatal exposure to ethanol in mice suggests that there is a decrease in locomotor activity and there are deficits in avoidance learning and operant performance [17]. Kim et al. [43] have shown that spatial cognitive abilities in rats are more vulnerable to ethanol than object recognition. Pregnant rats, fed 35% ethanol-derived calories, had daily subcutaneous injections of 400 mg/kg silymarin or received 400 mg/kg by the oral route. Following delivery, the offspring were started on a rat chow diet and after 90 days their social memory was tested and recorded as a social recognition score. The social recognition task for the rats consisted of sharing two periods of 5 min separated by a 60-min interval, with a strange male rat. Three different observers watched and scored the rats’ behavior in these periods. The score recorded for the ethanol-exposed pups was significantly lower than the score for the ‘‘ethanol/silymarin’’ rats or the control groups, implying a protective effect of silymarin on short-term memory [63]. The mechanism by which silymarin improves social learning was not determined in this study, as measurements of oxidative stress were not obtained. While this study presents encouraging results in terms of fetal protection, it is not clear whether the flavonoids or vitamin E protect the fetus as antioxidants or through other mechanisms. Folic acid acts also as an antioxidant. Cano et al. [18] studied the effects of folic acid on oxidative stress induced by ethanol. Two groups of pregnant rats were fed chronically with ethanol-supplemented tap water up to a mean of 8.9 ± 0.4 g ethanol/kg/day for 3 weeks. For four additional weeks, the rats were maintained on 16.6 ± 2.1 g ethanol/kg/ day. The rats in the control groups received supplements of folic acid (60 mg/day) during pregnancy. The study group received folic acid in a dose of 152 mg/day. The specific activity of glutathione reductase in liver and pancreas was used as a marker of oxidative stress. Its level was higher by 32 –34% in the female rats and by 24% in the progeny following an ethanol-containing diet. Reduction in glutathione reductase was significant in the group that received the high dosage of folic acid. The amount of carbonyl group proteins in the liver and pancreas was also used as an index for oxidative damage. Levels of carbonyl groups were reduced significantly in the liver of offspring supplemented with folic acid but were unchanged in their pancreas. The antioxidant activity of folic acid was assessed and proven by an assay that utilizes a free radical-sensitive fluorescent indicator protein [18]. This study showed an association between FAS in an animal model and oxidative stress measurements and mitigation of those measures by using an antioxidant. Treatment outcomes in the liver and in the pancreas were different and may reflect distinctive susceptibilities of different tissues. Brain damage is the major component of FAS; unfortunately, the authors did not study the protective effect of folic acid on the brain.
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Spong et al. [68] used mice as a model for FAS. The mice were injected with 25% ethanol in saline or saline alone and were supplemented with NAPVSIPQ (NAP) and/or SALLRSIPA (ADNF-9) before or after ethanol injection. NAP and ADNF-9 are peptides regulated by the vasoactive intestinal peptide and were shown to have antioxidant properties in brain cell culture. The outcome measures in their study were fetal death and fetal body and brain weight, and as an oxidative stress measurement, they used the ratio of reduced glutathione and oxidized glutathione. Pretreatment with NAP was associated with reduced fetal death, whereas ADNF-9 alone had no effect on fetal survival. The combination of the two, administered prior to ethanol injection, prevented growth restriction and the decrease in reduced glutathione. The combination also prevented reduction in brain weight when administered after ethanol injection. This study shows that treatment with an antioxidant prior to or after ethanol administration may reduce some of its adverse effects. Correlation with the oxidative stress process was demonstrated in one group only (NAP and ADNF-9 prior to ethanol) but not in the other groups where antioxidants were administered after injecting ethanol. A different approach to studying the effect of ethanol on the fetus is by exposing the fetus directly to ethanol. In mammals, this could be problematic because it can result in fetal loss, but it can be achieved in other species of animals such as fowl. Theoretically, adding ethanol to chicken eggs should yield similar results as introducing ethanol to the mammalian fetus through the placenta. Miller et al. [53] injected ethanol at day zero of incubation to the air sac of eggs in concentrations ranging from 0 to 302.5 mmol EtOH/kg egg. Injecting ethanol in various concentrations into fertile chicken eggs caused a doserelated decrease in cerebral long-chain polyunsaturated fatty acids: stearic, oleic, linoleic, linolenic, and arachidonic acids. Long-chain fatty acids, important components of cell membranes, are oxidized, a process that eventually leads to a reduction in brain mass. Vitamin E components, atocopherol or g-tocopherol, were injected concomitantly with ethanol into the egg’s air sac at concentrations of 2.5 mmol tocopherol per kg egg. Following this procedure, brain mass was not reduced, levels of brain protein were maintained, acetylcholine esterase activity was not reduced, and the levels of lipid hydroperoxide were not increased. The system in this study is not equivalent to those in mammals because of the different fetal environment and maternal – fetal interaction in utero, but it can demonstrate isolated effects of ethanol and antioxidants on the developing embryo. Miller et al. [53] have shown that the effect of ethanol was associated with oxidative stress, and supplementation with tocopherol reduced this effect significantly. Findings from these animal studies may be in contradiction to the work of Amini et al. [8] who monitored fetal oxidative stress in Quackenbush Special mice and found that, although increases in lipid peroxides and
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decreases in glutathione peroxidase and superoxide dismutase in the mother took place, only minor changes were observed in the embryo. The conclusion of their investigations was that the contribution of other fetotoxic effects of ethanol might be more substantial than ethanolinduced oxidative stress.
4. Discussion The term ‘‘oxidative stress’’ describes several biochemical reactions that lead to a final outcome that can be devastating in terms of fetotoxicity. In this review, we focused on fetal exposure to alcohol but oxidative stress is implicated also in the mechanisms of other pathological conditions such as diabetic pregnancy. Cederberg et al. [19] showed that supplementation of vitamin E and vitamin C to diabetic rats reduced fetal malformations and diminished the levels of oxygen radicals in the fetal liver. Several aspects of ethanol-induced damage in the fetus were studied in the research papers we reviewed. The adverse effects of ethanol on the liver and pancreas are among the main issues in the alcohol-addicted population. In contrast, FAS children do not present with liver damage as a main feature [3]; and reports regarding liver damage are anecdotal [46,57]. Using liver and pancreas as targets of ethanol injury provides proof for the ability of antioxidants to reduce damage. Reduced activity of GGT, which is increased in response to alcohol [40], and of glutathione reductase are good examples for that [18]. Brain damage due to ethanol exposure is a cardinal feature in FAS and in chronic alcoholism. Enhanced neurodegeneration [39] and increased dopamine deficiency [25] in combination with oxidative stress outcomes probably lead to the neurodevelopmental deficiencies that are encountered in FAS. In the available animal studies, one can clearly observe a protective effect of antioxidants against damage induced by ethanol. Despite the contradictory conclusions drawn from the study done by Amini et al. [8], it is fair to state that there is some strong evidence to support the assumption that concomitant administration of antioxidants and ethanol attenuates ethanol-induced fetotoxicity. Applying antioxidant treatment in pregnancy is not a new concept. Oxidative stress was found to play a role in the pathogenesis of pre-eclampsia. Elevation of lipid peroxidation products was highly correlated to the level of blood pressure and, in parallel, the severity of pre-eclampsia was found to be correlated to plasma antioxidant levels of glutathione peroxidase [74]. Following this theory, 283 women who had been identified as being at increased risk for pre-eclampsia were assigned randomly to vitamin E or vitamin C treatment, or to placebo. The dosage of vitamin C was 1000 mg/day and of vitamin E 400 IU/day. The treatment was provided during Weeks 16 –22 of gestation. Vitamin supplementa-
tion resulted in a 21% decrease of the PA1/PA2 ratio (the ratio of plasminogen-activator inhibitor 1 and 2 usually increases in pre-eclampsia) and the occurrence of preeclampsia was 17% in the placebo group compared to 8% of the group treated with vitamins. No adverse effects to the fetus were noted following this treatment. These results suggest that women who are at high risk for preeclampsia may benefit from diet supplementation with antioxidants [20]. Chappell et al. [20] used 1000 mg vitamin C since complete plasma saturation occurs with this daily dose [47]. The daily dose of vitamin C in the common multivitamin supplements in pregnancy is 50 –100 mg making the suggested dose 10 –20 fold higher [62]. Vitamin C in the appropriate doses is safe in pregnancy; chronic ingestion of high doses might cause renal stones in the mother [33]. Vitamin E was given in a high dose (400 IU) as well, since it was shown to decrease oxidation of LDL [23] and had some benefit in the treatment of patients with coronary artery disease [69]. The common multivitamin supplements in pregnancy contain 0– 30 IU [62], here as well the suggested dose is considerably higher than that recommended for pregnancy supplementation. Vitamin E is considered safe in pregnancy, although we did not find a study that examined the safety of high-dose vitamin E treatment in pregnancy. Chapell’s study was focused on pre-eclampsia and did not report on any adverse outcome associated with this highdose vitamin treatment. Pregnant women treated with high doses of vitamin C should be followed for renal stones formation throughout the treatment period. Several studies [45,54,55,63,72] failed to demonstrate clearly oxidative stress as a major mechanism of alcoholinduced damage, suggesting that the human application of treatment with antioxidants may not be easily justified. Other data [18,24,26,53] are more encouraging since a linkage between antioxidant activity and oxidative stress was established. Dietary supplementation with vitamin C and E in therapeutic doses ( 1 g/day and 2100 units/day, respectively) has not been shown to cause adverse effects in humans, neither to the mother nor to the fetus [7]. The question of the optimal dose still remains to be answered; high doses may not necessarily be desirable, and extrapolating from cancer studies, the use of antioxidants may interfere with apoptosis. In this process, oxygen-free radicals play a part in the cascade of programmed cell death [37]. Hence, the dose of vitamins will have to be considered as not to interfere with naturally occurring oxidative processes, which are probably important for the fetus. Antioxidant activity is found in numerous organic structures. Dietary supplements are required because the body does not have the capacity to synthesize vitamins and flavonoids. Other antioxidants do exist in the body but their levels are probably not sufficiently high to prevent the oxidative stress induced by large amounts of alcohol. Adequate levels of folic acid have been shown to prevent
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neural tube defects, but the desired level is achieved in most women only by supplementing folic acid or by fortifying the diet [76]. Finally, treating alcoholic women with antioxidants through food supplements such as vitamins is appealing because, in addition to the potential protective effect shown in the above studies, it may reverse other nutritional deficits common in this population [48,67]. Notwithstanding our focus on preventing oxidative fetal damage, a major effort must be invested in alcoholic prevention programs, thus improving maternal health while protecting the unborn child.
Acknowledgements We should like to thank Ingeborg C. Radde, MD, PhD, FRCP(C), for reading and commenting on this manuscript. RCK is supported by a Research Training Fellowship from the HSC Research Institute. GK is a Senior Scientist of the Canadian Institutes for Health Research (CIHR), Supported by a NET (New Emerging Team) grant from CIHR and by the Research Leadership in Better Pharmacotherapy during Pregnancy and Lactation.
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