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Connecting Prenatal Alcohol, Its Metabolite Acetaldehyde, and the Fetal Brain
C H A P T E R
9 Connecting Prenatal Alcohol, Its Metabolite Acetaldehyde, and the Fetal Brain M. Gabriela Chotro, Mirari Gaztan˜aga and Asier Angulo-Alcalde Department of Basic Psychological Processes and Their Development, Faculty of Psychology, University of the Basque Country UPV/EHU, San Sebastian, Spain
INTRODUCTION
CYP2E1 enzyme metabolizes the remaining fraction, and saturation of the ADH enzyme after high or chronic alcohol consumption induces increased activity of CYP2E1. During alcohol oxidation through this second pathway, acetaldehyde is generated, together with other highly reactive molecules known as oxygen radicals. Catalases are the third oxidative pathway and do not measurably contribute to the hepatic metabolism of alcohol. Excess alcohol not metabolized by this first pass through the liver is distributed through the blood stream to all body tissues, where it can also be metabolized into acetaldehyde, although at a much lower rate than in the liver (Zakhari, 2006). Due to the low affinity and reduced metabolic rate of the placental ADH, the placenta does not represent an obstacle to alcohol, which enters freely into the fetal compartment (Fig. 9.2) (Blakley & Scott, 1984; Heller & Burd, 2014). Once in the fetal circulation, alcohol reaches all tissues and organs, including the developing brain, resulting in circulation levels similar to maternal plasma (Zorzano & Herrera, 1989). Alcohol will be eliminated from fetal blood, mostly unchanged, through urinary and pulmonary excretion into the amniotic fluid, where it accumulates at concentrations often above those found in blood, which makes it an alcohol reservoir (Clarke, Steenaart, & Brien, 1986). Further, since the fetus constantly incorporates amniotic fluid by swallowing, breathing, and transdermal absorption, fetal alcohol exposure is prolonged until the drug is completely cleared (Heller & Burd, 2014). Fetal hepatic ADH activity is minimal and the amount of CYP2E1 is very low compared to the adult liver (Boleda, Farres, Guerri, & Pares, 1992; Heller &
Fetal alcohol exposure has well-documented adverse effects on brain development, although it is still uncertain whether these effects are generated by alcohol or by its metabolite, acetaldehyde. Understanding the complex factors involved in the numerous effects induced by prenatal alcohol exposure, and unfolding the connection between them, may help in finding strategies for preventing these effects, or intervening in cases of fetal alcohol exposure. In this chapter, the role of acetaldehyde, the first metabolite of alcohol, is reviewed in connection with the known effects of alcohol exposure on fetal development. Special emphasis is placed on the behavioral effects of alcohol and acetaldehyde during prenatal exposure leading to heightened alcohol intake after birth.
ALCOHOL: FROM THE MOTHER TO THE FETUS Alcohol (ethanol) is a relatively small molecule, which, after oral ingestion, is absorbed primarily in the small intestines. From there it is carried by the portal venous system to the liver where it is eliminated, mainly through reversible oxidative metabolism, and transformed into acetaldehyde (Fig. 9.1). The main enzymes involved in alcohol detoxification in the liver are alcohol dehydrogenase (ADH), CYP2E1, and catalase. Of those, ADH is the primary enzyme accounting for 90% 95% of the hepatic oxidation of alcohol. The
Neuroscience of Alcohol. DOI: https://doi.org/10.1016/B978-0-12-813125-1.00009-X
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FIGURE 9.1 Oxidative pathways of alcohol metabolism. The enzymes alcohol dehydrogenase (ADH), cytochrome P450-2E1 (CYP2E1), and catalase all contribute to oxidative metabolism of alcohol. ADH, present in the fluid of the cell (cytosol), converts alcohol (ethanol) to acetaldehyde. This reaction involves an intermediate carrier of electrons, 1 nicotinamide adenine dinucleotide (NAD), which is reduced by two electrons to form NADH. Catalase, located in cell bodies called peroxisomes, requires hydrogen peroxide (H2O2) to oxidize alcohol. CYP2E1, present predominantly in the cell’s microsomes, assumes an important role at elevated ethanol concentrations. Acetaldehyde is metabolized mainly by aldehyde dehydrogenase 2 (ALDH2) in the mitochondria to form acetate and NADH. Source: Adapted from Zakhari, S. (2006). Overview: How is alcohol metabolized by the body? Alcohol Research & Health, 29(4), 245 254.
FIGURE 9.2 Fetal alcohol and acetaldehyde disposition. This scheme shows how alcohol ingested by the mother reaches the fetus and possible fetal disposition of acetaldehyde.
Burd, 2014). Therefore, complete elimination of alcohol from the fetus and amniotic fluid relies mainly on maternal metabolism. Consequently, the level of prenatal alcohol exposure, that is, alcohol concentration and its duration, will depend on the amount of alcohol ingested by the mother and the metabolic capacity of her liver.
ACETALDEHYDE IN THE FETAL ENVIRONMENT The first and main product of alcohol oxidation, acetaldehyde, is a highly toxic and reactive molecule that in normal conditions has a very short life and is rapidly and irreversibly metabolized into acetate by the enzymes acetaldehyde dehydrogenase (ALDH)
(Fig. 9.1). The ALDH enzymes are found in many tissues, but are at their highest concentration in the liver (Crabb, Matsumoto, Chang, & You, 2004). Thus, after maternal alcohol ingestion, alcohol and acetaldehyde escaping the liver’s first pass arrive through the maternal circulation to the placenta. Unlike alcohol, acetaldehyde does not reach the fetal environment either immediately or in significant amounts (Blakley & Scott, 1984; Zorzano & Herrera, 1989). Several studies with animals after maternal administration of alcohol found acetaldehyde in the placenta, but null or minimal amounts in fetal tissues or amniotic fluid (Hayashi, Shimazaki, Kamata, Kakiichi, & Ikeda, 1991; Zorzano & Herrera, 1989). ALDH enzymes have been found in the fetal liver of humans and other mammals (Cao, Tu, & Weiner, 1989; Fakhoury, deBeaumais et al., 2009), as well as in the placenta (Blakley & Scott, 1984),
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THE ROLE OF ACETALDEHYDE IN THE EFFECTS OF ALCOHOL ON FETAL BRAIN DEVELOPMENT
although in all cases the amount and activity of ALDH is lower than in adult liver. Hence, it seems that this protective barrier against acetaldehyde is effective, at least with the amounts of acetaldehyde accumulated in maternal circulation under normal conditions and after consumption of alcohol doses below 2 4 g/kg. However, when high levels of acetaldehyde circulate in maternal blood (above 80 μM/L), acetaldehyde can be detected in fetal tissues (Zorzano & Herrera, 1989). This threshold above which placental and fetal hepatic metabolic capacity of acetaldehyde are surpassed are found either after chronic alcohol consumption, extremely high alcohol intake, or when acetaldehyde metabolism is pharmacologically manipulated (Blakley & Scott, 1984; Boleda et al., 1992; Eriksson, 2001; Guerri & Sanchis, 1985). This evidence seems to indicate that while after maternal ingestion alcohol freely enters the fetal compartment, within a moderate range of alcohol intake the fetus is relatively protected from peripheral acetaldehyde either coming from the mother or produced by the fetal liver (Clarke et al., 1986). In any case, when high concentrations of acetaldehyde are found in maternal blood circulation, the amount entering the fetal compartment would always be considerably lower than that in maternal plasma (Zorzano & Herrera, 1989).
ACETALDEHYDE IN THE FETAL BRAIN In contrast, relatively high amounts of acetaldehyde have been reported in the fetal brain (HambyMason, Chen, Schenker, Perez, & Henderson, 1997). Given that the minimal amounts of acetaldehyde that may cross the placenta are metabolized by the fetal hepatic ALDH, as well as ALDH in the blood brain barrier (Zimatkin, 1991), acetaldehyde detected inside the fetal brain must necessarily be produced in situ from alcohol (HambyMason et al., 1997). Alcohol arrives freely to the fetal brain where it is transformed into acetaldehyde by catalases, the main enzymes responsible for this transformation in both adults and the developing brain, accounting for approximately 60% of alcohol’s oxidation (HambyMason et al., 1997; Zimatkin, Pronko, Vasiliou, Gonzalez, & Deitrich, 2006). Moreover, the concentration and activity of catalases in neonatal and fetal rat brains were found to be much higher than in adults, which could partially explain the higher amount of central acetaldehyde found in the fetus brain (Delmaestro & McDonald, 1987; HambyMason et al., 1997). As in the liver, CYP2E1 activity in the adult and fetal brain is induced by acute elevated alcohol ingestion or in response to chronic drinking, and it accounts for 20% 25% of alcohol
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brain detoxification (HambyMason et al., 1997). However, in addition to acetaldehyde, it generates oxygen radicals, which are very reactive and potentially harmful molecules (Brzezinski, Boutelet-Bochan, Person, Fantel, & Juchau, 1999; Tuma & Casey, 2003). Acetaldehyde is then metabolized irreversibly to acetate by the fetal brain-ALDH enzymes (Fakhoury et al., 2009). In summary, a greater accumulation of acetaldehyde is expected in the fetal brain compared to adults at similar blood alcohol concentrations when considering the combination of all three mentioned concomitant conditions: prolonged and continuous fetal exposure to alcohol accumulated in amniotic fluid, higher activity of fetal brain catalases, and the lower expression of the ALDH enzymes in the fetal brain. On the basis of these considerations, it appears that after maternal alcohol intake, the fetus will be exposed to alcohol in the blood, brain, and amniotic fluid, while exposure to acetaldehyde is almost exclusively limited to the brain.
THE ROLE OF ACETALDEHYDE IN THE EFFECTS OF ALCOHOL ON FETAL BRAIN DEVELOPMENT Human and animal studies have revealed that the central nervous system is particularly vulnerable to the deleterious effects of prenatal alcohol exposure. Given its complexity and development throughout gestation, and even long after birth, there is no safe prenatal period for the toxic effects of alcohol and its metabolites. Many of the effects of prenatal alcohol have been shown to be attributable to the action of its main oxidation product, acetaldehyde (Eriksson, 2001). Acetaldehyde was found to induce retarded neural development and malformations matching those observed after prenatal exposure to relatively high amounts of alcohol during different stages of gestation (Sreenathan, Padmanabhan, & Singh, 1982; Webster, Walsh, McEwen, & Lipson, 1983). More recent experiments conducted at a physiological level have also shown acetaldehyde to affect neural development by disrupting cellular differentiation, neuronal growth, myelination, and by enhancing the deleterious effects of alcohol (Coutts & Harrison, 2015; Giavini, Broccia, Prati, Bellomo, & Menegola, 1992). In addition, placental formation is also negatively affected by high concentrations of acetaldehyde, altering nutrition functions fundamental for normal neurodevelopment (Lui et al., 2014). After the placenta is formed, maternal acetaldehyde does not reach the fetus but is abundantly produced in the fetal brain (Clarke et al., 1986). These amounts of acetaldehyde formed locally after
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maternal alcohol ingestion seem sufficient to be functionally relevant and to induce a wide variety of harmful effects in this vulnerable developing system (HambyMason et al., 1997). The mechanisms by which alcohol and acetaldehyde produce their harmful effects are diverse and still under investigation. The presence of alcohol in the fetal brain induces higher activity of CYP2E1 leading to the production of acetaldehyde and oxygen radicals. Both are very reactive substances that interact with other complex molecules in the cell generating hybrid compounds called adducts, a single product resulting from direct covalent chemical additions (Tuma & Casey, 2003). This, combined with the lower antioxidant capacity of the developing brain (Shim & Kim, 2013), generates an imbalance in the redox state of the neural cells inducing oxidative stress. Fetal brain tissues are particularly vulnerable to oxidative stress, which damages all components of the cell including lipids, proteins, and DNA by generating toxic adducts and disrupting their normal functioning, and in the long term, inducing cell death, contributing to the neurodegeneration observed after prenatal alcohol exposure (Tong et al., 2011; Zakhari, 2006). Further, acetaldehyde readily forms adducts with other molecules such as proteins and neurotransmitters, impairing their normal functions and inducing neurotoxicity (Tuma & Casey, 2003). Acetaldehyde also reacts with endogenous neurogenic amines such as catecholamines, generating adducts. Salsolinol, the condensation product of acetaldehyde and dopamine, is one of the most studied due to its neurotoxic effects and its involvement in the motivational effects of alcohol (Hipolito et al., 2012; Peana et al., 2017) while it has been detected in the fetal brain after chronic prenatal alcohol exposure (Mao et al., 2013). This could represent another mechanism by which acetaldehyde produces neural damage after prenatal alcohol exposure (Quertemont, Tambour, & Tirelli, 2005).
CONNECTING THE BEHAVIORAL EFFECTS OF ALCOHOL AND ACETALDEHYDE IN THE FETUS Acetaldehyde, in addition to its neurotoxic consequences, has been found to produce behavioral effects. Since many of these coincide with those of alcohol it has been suggested—and more recently demonstrated—that acetaldehyde mediates several effects of alcohol on behavior. In adult humans and animals, acetaldehyde induces motor effects including motor stimulation at low doses and sedation at high doses; memory impairing effects at high doses; and anxiolytic effects at moderate doses, while motivational effects can be
aversive or appetitive. Other consequences such as anticonvulsive and analgesic effects have not yet been tested with acetaldehyde (Quertemont et al., 2005). For the motivational effects of acetaldehyde, many studies have shown that it has differential properties depending on the dose and site of action. Elevated concentrations of peripheral acetaldehyde have been found to be highly aversive, while central acetaldehyde induces appetitive effects and appears to be responsible for the reinforcing effect that sustains alcohol consumption (Correa et al., 2012; Quertemont et al., 2005). Due to difficulties in measuring and interpreting fetal behavior, data on the prenatal effects of alcohol on behavior are scarce, and those of acetaldehyde have not yet been tested. However, indirect evidence from a few studies on alcohol or during early postnatal development may help to shed some light on this, as yet, unexplored topic. There are no reports on the fetal motor effects of acetaldehyde, but the few studies with alcohol reveal an effect of sedation in fetuses; in humans, maternal ingestion of alcohol reduced fetal breathing movements when tested 0.5 3 hours later (McLeod et al., 1983) and similar effects were described in sheep (Smith et al., 1990). A reduction of general activity was also observed in rat fetuses 1 hour after maternal administration of 1 2 g/kg alcohol (Chotro & Spear, 1997), or 4 hours after a 4 g/kg dose (Smotherman et al., 1986). Alcohol intoxication in pregnant rats also reduced the fetal motor activation induced by acute hypoxia after umbilical cord compression (Smotherman & Robinson, 1987). Although there are no data showing fetal motor stimulation with alcohol, this effect has been reported in 8- to 12-dayold rat pups during the first 5 10 minutes of alcohol intoxication, while sedation was observed 15 20 minutes later (Arias, Mlewski, Molina, & Spear, 2009); and sequestering acetaldehyde eliminated the stimulating effect of alcohol (Pautassi, Nizhnikov, Fabio, & Spear, 2011). It is possible that alcohol stimulation would be detected in fetuses if tested during the first minutes of intoxication.
REINFORCING EFFECTS OF ALCOHOL AND ACETALDEHYDE IN THE FETUS With respect to the positive reinforcing properties of alcohol and acetaldehyde in early development, in newborn rats it was found that administration of low doses of alcohol paired with an odor induced a conditioned odor preference for that smell (Petrov, Varlinskaya, & Spear, 2003). Similar appetitive responses were obtained by injecting alcohol or acetaldehyde directly into the newborn brain, but not when inhibiting brain catalases or sequestering acetaldehyde, thus, confirming the
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PERINATAL LEARNING WITH ALCOHOL AND ACETALDEHYDE
appetitive properties of central acetaldehyde in neonate rats (March, Abate, Spear, & Molina, 2013; Nizhnikov, Molina, & Spear, 2007). In prenatal stages, alcohol also appears to exert appetitive effects. When alcohol is administered to the pregnant rat during the last days of gestation (17 20), the fetus is exposed simultaneously to the chemosensory (flavor) and pharmacological properties of alcohol. This prenatal experience results in increased postnatal alcohol consumption as well as enhanced liking for the taste of alcohol when tested in infancy and adolescence (Chotro & Arias, 2003). It is generally accepted that the near-term fetus can perceive chemosensory stimuli present in the amniotic fluid during swallowing and breathing the fluid, forming associations with their aversive or appetitive consequences, which modifies postnatal responses to those stimuli (Smotherman & Robinson, 1988). Accordingly, the enhanced acceptance of alcohol found after prenatal alcohol exposure was demonstrated to be a conditioned appetitive response acquired in utero (Chotro & Arias, 2003). This prenatal experience was also found to modulate postnatal learning by retarding the acquisition of aversions and facilitating learned preferences when alcohol was the reinforcer (Arias & Chotro, 2006a, 2006b). Recently, it was demonstrated that the reinforcing effects of acetaldehyde are crucial for this prenatal appetitive learning about alcohol, since sequestering acetaldehyde was able to prevent such learning (Gaztanaga, Angulo-Alcalde, Spear, & Chotro, 2017). Additional indirect support comes from studies in which the reinforcing effects of alcohol were suppressed when blocking the endogenous opioid system, particularly the μ-opioid receptors, during prenatal alcohol exposure (Chotro & Arias, 2003; Chotro, Arias, & Laviola, 2007; Diaz-Cenzano, Gaztanaga, & Chotro, 2014). This is particularly relevant since the reinforcing and stimulating effects of central alcohol-derived acetaldehyde appear to be mediated by the μ-opioid system (Font, Lujan, & Pastor, 2013). Acetaldehyde has been found to stimulate the release of β-endorphins and salsolinol appears to induce its effects interacting with the μ-opioid receptors in the posterior ventral tegmental area, an element of the “dopamine reward pathway” (Xie, Hipolito et al., 2012). In addition to acetaldehyde, high amounts of salsolinol were found in the fetal brain after chronic alcohol exposure (Mao et al., 2013), although its behavioral effects in early ontogeny have not yet been tested.
PERINATAL LEARNING WITH ALCOHOL AND ACETALDEHYDE Although the reinforcing effects of alcohol and acetaldehyde during prenatal and neonatal stages appear
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to be clear (March et al., 2013), no evidence of the aversive effects of alcohol have yet been found until postnatal day 11 of the rat (Arias & Chotro, 2006; Pautassi, Nizhnikov, & Spear, 2009). Moreover, the same alcohol doses that are aversive for adults or 2-week-old infants are appetitive when experienced either prenatally or during the first postnatal week (Arias & Chotro, 2006; Chotro, Arias, & Spear, 2009). This developmental change in the motivational effects of alcohol could be related to the metabolic status of the developing rat at each age. As explained, the rat fetus and neonate do not generate aversive peripheral acetaldehyde due to the low or null hepatic ADH activity that begins to increase after birth and reaches the adult value by day 20 (Boleda et al., 1992) and the fetus is relatively protected from maternal acetaldehyde. Conversely, the intense activity of brain catalase that produces high levels of reinforcing acetaldehyde at fetal and neonatal stages decreases gradually with development (Delmaestro & McDonald, 1987). Thus, it seems unlikely that the fetus and the neonate have the chance to perceive the negative consequences of peripheral acetaldehyde during alcohol intoxication, while their central appetitive effects are predominant. If the perceived motivational effects of alcohol intoxication are considered to be a result of the balance between peripheral and central levels of acetaldehyde (Correa et al., 2012), this suggests that fetuses and newborns will perceive exclusively the appetitive effects of acetaldehyde, and the likelihood of perceiving its aversive properties will increase during development. This implies that fetal learning during prenatal alcohol exposure will necessarily generate appetitive responses (Fig. 9.3). This associative mechanism could underlie the enhanced liking of alcohol odor observed in two subsequent studies with newborn babies and adolescents with a history of prenatal alcohol exposure (Faas, March, Moya, & Molina, 2015; Hannigan, Chiodo, Sokol, Janisse, & Delaney-Black, 2015). Similarly, this might explain the increased alcohol consumption observed in many animal studies with prenatal alcohol exposure, along with other neurodevelopmental effects (Chotro et al., 2007). In summary, when the pregnant mother consumes alcohol, the fetus is exposed to alcohol in the blood, brain, and amniotic fluid, and to centrally formed acetaldehyde. This produces a wide range of physiological and behavioral effects that not only alter fetal neurodevelopment, but also induce changes in alcohol acceptance, which can eventually lead to alcohol abuse problems. Clinical studies show a clear relationship between prenatal alcohol and alcohol abuse in adolescents and young adults, with prenatal alcohol exposure being a good predictor of alcohol problems (Baer, Sampson, Barr, Connor, & Streissguth, 2003). More
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FIGURE 9.3 Fetal alcohol learning after maternal alcohol consumption. This scheme shows possible associative mechanisms underlying increased alcohol intake and liking after prenatal alcohol exposure.
research is needed to better identify the relative importance of each intervening factor as well as the mechanisms underlying this complex interplay between the effects of alcohol and acetaldehyde.
KEY FACTS About Alcohol and Acetaldehyde in the Fetus • Alcohol consumed by the pregnant mother crosses the placenta and reaches the fetus in similar amounts to maternal plasma, and accumulates in the amniotic fluid. • Acetaldehyde, the first oxidation metabolite of alcohol, is mainly produced in maternal liver. • Low or null amounts of maternal acetaldehyde reach the fetus after maternal alcohol ingestion, this being metabolized by the placenta and fetal liver. • The fetus, due to its hepatic metabolic immaturity, cannot produce peripheral acetaldehyde from alcohol. • Acetaldehyde is produced from alcohol in the fetal brain, mainly by catalases. • Brain acetaldehyde may produce many of the neurodevelopmental and behavioral effects of alcohol.
SUMMARY POINTS • The role of alcohol-derived acetaldehyde in relation to the known effects of prenatal alcohol exposure is reviewed.
• Alcohol ingested by the pregnant mother easily reaches the fetus, while maternal acetaldehyde does not. • Acetaldehyde is produced in the fetal brain and may be responsible for many of the effects attributed to alcohol. • Brain acetaldehyde retards development and induces neural damage through the formation of toxic adducts. • Alcohol and acetaldehyde in early development induce comparable behavioral effects and the reinforcing properties of central acetaldehyde during alcohol exposure may underlie prenatal appetitive learning producing postnatal increased alcohol intake.
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plasma prostaglandin-e2 concentrations and fetal breathing movements. Journal of Developmental Physiology, 14(1), 23 28. Smotherman, W. P., & Robinson, S. R. (1987). Stereotypic behavioralresponse of rat fetuses to acute-hypoxia is altered by maternal alcohol-consumption. American Journal of Obstetrics and Gynecology, 157(4), 982 986. Smotherman, W. P., & Robinson, S. R. (1988). The uterus as environment: The ecology of fetal behavior. In E. M. Blass (Ed.), Handbook of behavioral neurobiology. developmental psychobiology and behavioral ecology. New York: Plenum Press. Smotherman, W. P., Woodruff, K. S., Robinson, S. R., Delreal, C., Barron, S., & Riley, E. P. (1986). Spontaneous fetal behavior after maternal exposure to ethanol. Pharmacology Biochemistry and Behavior, 24(2), 165 170. Sreenathan, R. N., Padmanabhan, R., & Singh, S. (1982). Teratogenic effects of acetaldehyde in the rat. Drug and Alcohol Dependence, 9 (4), 339 350. Tong, M., Longato, L., Nguyen, Q. G., Chen, W. C., Spaisman, A., & delaMonte, S. M. (2011). Acetaldehyde-mediated neurotoxicity: Relevance to FASD. Alcoholism—Clinical and Experimental Research, 35(6), 115A.
Tuma, D. J., & Casey, C. A. (2003). Dangerous byproducts of alcohol breakdown—Focus on adducts. Alcohol Research & Health, 27(4), 285 290. Webster, W. S., Walsh, D. A., McEwen, S. E., & Lipson, A. H. (1983). Some teratogenic properties of ethanol and acetaldehyde in c57bl/6j mice, implications for the study of the fetal alcohol syndrome. Teratology, 27(2), 231 243. Xie, G., Hipolito, L., Zuo, W., Polache, A., Granero, L., Krnjevic, K., et al. (2012). Salsolinol stimulates dopamine neurons in slices of posterior ventral tegmental area indirectly by activating mu-opioid receptors. Journal of Pharmacology and Experimental Therapeutics, 341(1), 43 50. Zakhari, S. (2006). Overview: How is alcohol metabolized by the body? Alcohol Research & Health, 29(4), 245 254. Zimatkin, S. M. (1991). Histochemical-study of aldehyde dehydrogenase in the rat CNS. Journal of Neurochemistry, 56(1), 1 11. Zimatkin, S. M., Pronko, S. P., Vasiliou, V., Gonzalez, F. J., & Deitrich, R. A. (2006). Enzymatic mechanisms of ethanol oxidation in the brain. Alcoholism—Clinical and Experimental Research, 30 (9), 1500 1505. Zorzano, A., & Herrera, E. (1989). Disposition of ethanol and acetaldehyde in late pregnant rats and their fetuses. Pediatric Research, 25(1), 102 106.
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9 Connecting Prenatal Alcohol, Its Metabolite Acetaldehyde, and the Fetal Brain M. Gabriela Chotro, Mirari Gaztan˜aga and Asier Angulo-Alcalde Department of Basic Psychological Processes and Their Development, Faculty of Psychology, University of the Basque Country UPV/EHU, San Sebastian, Spain
INTRODUCTION
CYP2E1 enzyme metabolizes the remaining fraction, and saturation of the ADH enzyme after high or chronic alcohol consumption induces increased activity of CYP2E1. During alcohol oxidation through this second pathway, acetaldehyde is generated, together with other highly reactive molecules known as oxygen radicals. Catalases are the third oxidative pathway and do not measurably contribute to the hepatic metabolism of alcohol. Excess alcohol not metabolized by this first pass through the liver is distributed through the blood stream to all body tissues, where it can also be metabolized into acetaldehyde, although at a much lower rate than in the liver (Zakhari, 2006). Due to the low affinity and reduced metabolic rate of the placental ADH, the placenta does not represent an obstacle to alcohol, which enters freely into the fetal compartment (Fig. 9.2) (Blakley & Scott, 1984; Heller & Burd, 2014). Once in the fetal circulation, alcohol reaches all tissues and organs, including the developing brain, resulting in circulation levels similar to maternal plasma (Zorzano & Herrera, 1989). Alcohol will be eliminated from fetal blood, mostly unchanged, through urinary and pulmonary excretion into the amniotic fluid, where it accumulates at concentrations often above those found in blood, which makes it an alcohol reservoir (Clarke, Steenaart, & Brien, 1986). Further, since the fetus constantly incorporates amniotic fluid by swallowing, breathing, and transdermal absorption, fetal alcohol exposure is prolonged until the drug is completely cleared (Heller & Burd, 2014). Fetal hepatic ADH activity is minimal and the amount of CYP2E1 is very low compared to the adult liver (Boleda, Farres, Guerri, & Pares, 1992; Heller &
Fetal alcohol exposure has well-documented adverse effects on brain development, although it is still uncertain whether these effects are generated by alcohol or by its metabolite, acetaldehyde. Understanding the complex factors involved in the numerous effects induced by prenatal alcohol exposure, and unfolding the connection between them, may help in finding strategies for preventing these effects, or intervening in cases of fetal alcohol exposure. In this chapter, the role of acetaldehyde, the first metabolite of alcohol, is reviewed in connection with the known effects of alcohol exposure on fetal development. Special emphasis is placed on the behavioral effects of alcohol and acetaldehyde during prenatal exposure leading to heightened alcohol intake after birth.
ALCOHOL: FROM THE MOTHER TO THE FETUS Alcohol (ethanol) is a relatively small molecule, which, after oral ingestion, is absorbed primarily in the small intestines. From there it is carried by the portal venous system to the liver where it is eliminated, mainly through reversible oxidative metabolism, and transformed into acetaldehyde (Fig. 9.1). The main enzymes involved in alcohol detoxification in the liver are alcohol dehydrogenase (ADH), CYP2E1, and catalase. Of those, ADH is the primary enzyme accounting for 90% 95% of the hepatic oxidation of alcohol. The
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FIGURE 9.1 Oxidative pathways of alcohol metabolism. The enzymes alcohol dehydrogenase (ADH), cytochrome P450-2E1 (CYP2E1), and catalase all contribute to oxidative metabolism of alcohol. ADH, present in the fluid of the cell (cytosol), converts alcohol (ethanol) to acetaldehyde. This reaction involves an intermediate carrier of electrons, 1 nicotinamide adenine dinucleotide (NAD), which is reduced by two electrons to form NADH. Catalase, located in cell bodies called peroxisomes, requires hydrogen peroxide (H2O2) to oxidize alcohol. CYP2E1, present predominantly in the cell’s microsomes, assumes an important role at elevated ethanol concentrations. Acetaldehyde is metabolized mainly by aldehyde dehydrogenase 2 (ALDH2) in the mitochondria to form acetate and NADH. Source: Adapted from Zakhari, S. (2006). Overview: How is alcohol metabolized by the body? Alcohol Research & Health, 29(4), 245 254.
FIGURE 9.2 Fetal alcohol and acetaldehyde disposition. This scheme shows how alcohol ingested by the mother reaches the fetus and possible fetal disposition of acetaldehyde.
Burd, 2014). Therefore, complete elimination of alcohol from the fetus and amniotic fluid relies mainly on maternal metabolism. Consequently, the level of prenatal alcohol exposure, that is, alcohol concentration and its duration, will depend on the amount of alcohol ingested by the mother and the metabolic capacity of her liver.
ACETALDEHYDE IN THE FETAL ENVIRONMENT The first and main product of alcohol oxidation, acetaldehyde, is a highly toxic and reactive molecule that in normal conditions has a very short life and is rapidly and irreversibly metabolized into acetate by the enzymes acetaldehyde dehydrogenase (ALDH)
(Fig. 9.1). The ALDH enzymes are found in many tissues, but are at their highest concentration in the liver (Crabb, Matsumoto, Chang, & You, 2004). Thus, after maternal alcohol ingestion, alcohol and acetaldehyde escaping the liver’s first pass arrive through the maternal circulation to the placenta. Unlike alcohol, acetaldehyde does not reach the fetal environment either immediately or in significant amounts (Blakley & Scott, 1984; Zorzano & Herrera, 1989). Several studies with animals after maternal administration of alcohol found acetaldehyde in the placenta, but null or minimal amounts in fetal tissues or amniotic fluid (Hayashi, Shimazaki, Kamata, Kakiichi, & Ikeda, 1991; Zorzano & Herrera, 1989). ALDH enzymes have been found in the fetal liver of humans and other mammals (Cao, Tu, & Weiner, 1989; Fakhoury, deBeaumais et al., 2009), as well as in the placenta (Blakley & Scott, 1984),
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THE ROLE OF ACETALDEHYDE IN THE EFFECTS OF ALCOHOL ON FETAL BRAIN DEVELOPMENT
although in all cases the amount and activity of ALDH is lower than in adult liver. Hence, it seems that this protective barrier against acetaldehyde is effective, at least with the amounts of acetaldehyde accumulated in maternal circulation under normal conditions and after consumption of alcohol doses below 2 4 g/kg. However, when high levels of acetaldehyde circulate in maternal blood (above 80 μM/L), acetaldehyde can be detected in fetal tissues (Zorzano & Herrera, 1989). This threshold above which placental and fetal hepatic metabolic capacity of acetaldehyde are surpassed are found either after chronic alcohol consumption, extremely high alcohol intake, or when acetaldehyde metabolism is pharmacologically manipulated (Blakley & Scott, 1984; Boleda et al., 1992; Eriksson, 2001; Guerri & Sanchis, 1985). This evidence seems to indicate that while after maternal ingestion alcohol freely enters the fetal compartment, within a moderate range of alcohol intake the fetus is relatively protected from peripheral acetaldehyde either coming from the mother or produced by the fetal liver (Clarke et al., 1986). In any case, when high concentrations of acetaldehyde are found in maternal blood circulation, the amount entering the fetal compartment would always be considerably lower than that in maternal plasma (Zorzano & Herrera, 1989).
ACETALDEHYDE IN THE FETAL BRAIN In contrast, relatively high amounts of acetaldehyde have been reported in the fetal brain (HambyMason, Chen, Schenker, Perez, & Henderson, 1997). Given that the minimal amounts of acetaldehyde that may cross the placenta are metabolized by the fetal hepatic ALDH, as well as ALDH in the blood brain barrier (Zimatkin, 1991), acetaldehyde detected inside the fetal brain must necessarily be produced in situ from alcohol (HambyMason et al., 1997). Alcohol arrives freely to the fetal brain where it is transformed into acetaldehyde by catalases, the main enzymes responsible for this transformation in both adults and the developing brain, accounting for approximately 60% of alcohol’s oxidation (HambyMason et al., 1997; Zimatkin, Pronko, Vasiliou, Gonzalez, & Deitrich, 2006). Moreover, the concentration and activity of catalases in neonatal and fetal rat brains were found to be much higher than in adults, which could partially explain the higher amount of central acetaldehyde found in the fetus brain (Delmaestro & McDonald, 1987; HambyMason et al., 1997). As in the liver, CYP2E1 activity in the adult and fetal brain is induced by acute elevated alcohol ingestion or in response to chronic drinking, and it accounts for 20% 25% of alcohol
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brain detoxification (HambyMason et al., 1997). However, in addition to acetaldehyde, it generates oxygen radicals, which are very reactive and potentially harmful molecules (Brzezinski, Boutelet-Bochan, Person, Fantel, & Juchau, 1999; Tuma & Casey, 2003). Acetaldehyde is then metabolized irreversibly to acetate by the fetal brain-ALDH enzymes (Fakhoury et al., 2009). In summary, a greater accumulation of acetaldehyde is expected in the fetal brain compared to adults at similar blood alcohol concentrations when considering the combination of all three mentioned concomitant conditions: prolonged and continuous fetal exposure to alcohol accumulated in amniotic fluid, higher activity of fetal brain catalases, and the lower expression of the ALDH enzymes in the fetal brain. On the basis of these considerations, it appears that after maternal alcohol intake, the fetus will be exposed to alcohol in the blood, brain, and amniotic fluid, while exposure to acetaldehyde is almost exclusively limited to the brain.
THE ROLE OF ACETALDEHYDE IN THE EFFECTS OF ALCOHOL ON FETAL BRAIN DEVELOPMENT Human and animal studies have revealed that the central nervous system is particularly vulnerable to the deleterious effects of prenatal alcohol exposure. Given its complexity and development throughout gestation, and even long after birth, there is no safe prenatal period for the toxic effects of alcohol and its metabolites. Many of the effects of prenatal alcohol have been shown to be attributable to the action of its main oxidation product, acetaldehyde (Eriksson, 2001). Acetaldehyde was found to induce retarded neural development and malformations matching those observed after prenatal exposure to relatively high amounts of alcohol during different stages of gestation (Sreenathan, Padmanabhan, & Singh, 1982; Webster, Walsh, McEwen, & Lipson, 1983). More recent experiments conducted at a physiological level have also shown acetaldehyde to affect neural development by disrupting cellular differentiation, neuronal growth, myelination, and by enhancing the deleterious effects of alcohol (Coutts & Harrison, 2015; Giavini, Broccia, Prati, Bellomo, & Menegola, 1992). In addition, placental formation is also negatively affected by high concentrations of acetaldehyde, altering nutrition functions fundamental for normal neurodevelopment (Lui et al., 2014). After the placenta is formed, maternal acetaldehyde does not reach the fetus but is abundantly produced in the fetal brain (Clarke et al., 1986). These amounts of acetaldehyde formed locally after
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maternal alcohol ingestion seem sufficient to be functionally relevant and to induce a wide variety of harmful effects in this vulnerable developing system (HambyMason et al., 1997). The mechanisms by which alcohol and acetaldehyde produce their harmful effects are diverse and still under investigation. The presence of alcohol in the fetal brain induces higher activity of CYP2E1 leading to the production of acetaldehyde and oxygen radicals. Both are very reactive substances that interact with other complex molecules in the cell generating hybrid compounds called adducts, a single product resulting from direct covalent chemical additions (Tuma & Casey, 2003). This, combined with the lower antioxidant capacity of the developing brain (Shim & Kim, 2013), generates an imbalance in the redox state of the neural cells inducing oxidative stress. Fetal brain tissues are particularly vulnerable to oxidative stress, which damages all components of the cell including lipids, proteins, and DNA by generating toxic adducts and disrupting their normal functioning, and in the long term, inducing cell death, contributing to the neurodegeneration observed after prenatal alcohol exposure (Tong et al., 2011; Zakhari, 2006). Further, acetaldehyde readily forms adducts with other molecules such as proteins and neurotransmitters, impairing their normal functions and inducing neurotoxicity (Tuma & Casey, 2003). Acetaldehyde also reacts with endogenous neurogenic amines such as catecholamines, generating adducts. Salsolinol, the condensation product of acetaldehyde and dopamine, is one of the most studied due to its neurotoxic effects and its involvement in the motivational effects of alcohol (Hipolito et al., 2012; Peana et al., 2017) while it has been detected in the fetal brain after chronic prenatal alcohol exposure (Mao et al., 2013). This could represent another mechanism by which acetaldehyde produces neural damage after prenatal alcohol exposure (Quertemont, Tambour, & Tirelli, 2005).
CONNECTING THE BEHAVIORAL EFFECTS OF ALCOHOL AND ACETALDEHYDE IN THE FETUS Acetaldehyde, in addition to its neurotoxic consequences, has been found to produce behavioral effects. Since many of these coincide with those of alcohol it has been suggested—and more recently demonstrated—that acetaldehyde mediates several effects of alcohol on behavior. In adult humans and animals, acetaldehyde induces motor effects including motor stimulation at low doses and sedation at high doses; memory impairing effects at high doses; and anxiolytic effects at moderate doses, while motivational effects can be
aversive or appetitive. Other consequences such as anticonvulsive and analgesic effects have not yet been tested with acetaldehyde (Quertemont et al., 2005). For the motivational effects of acetaldehyde, many studies have shown that it has differential properties depending on the dose and site of action. Elevated concentrations of peripheral acetaldehyde have been found to be highly aversive, while central acetaldehyde induces appetitive effects and appears to be responsible for the reinforcing effect that sustains alcohol consumption (Correa et al., 2012; Quertemont et al., 2005). Due to difficulties in measuring and interpreting fetal behavior, data on the prenatal effects of alcohol on behavior are scarce, and those of acetaldehyde have not yet been tested. However, indirect evidence from a few studies on alcohol or during early postnatal development may help to shed some light on this, as yet, unexplored topic. There are no reports on the fetal motor effects of acetaldehyde, but the few studies with alcohol reveal an effect of sedation in fetuses; in humans, maternal ingestion of alcohol reduced fetal breathing movements when tested 0.5 3 hours later (McLeod et al., 1983) and similar effects were described in sheep (Smith et al., 1990). A reduction of general activity was also observed in rat fetuses 1 hour after maternal administration of 1 2 g/kg alcohol (Chotro & Spear, 1997), or 4 hours after a 4 g/kg dose (Smotherman et al., 1986). Alcohol intoxication in pregnant rats also reduced the fetal motor activation induced by acute hypoxia after umbilical cord compression (Smotherman & Robinson, 1987). Although there are no data showing fetal motor stimulation with alcohol, this effect has been reported in 8- to 12-dayold rat pups during the first 5 10 minutes of alcohol intoxication, while sedation was observed 15 20 minutes later (Arias, Mlewski, Molina, & Spear, 2009); and sequestering acetaldehyde eliminated the stimulating effect of alcohol (Pautassi, Nizhnikov, Fabio, & Spear, 2011). It is possible that alcohol stimulation would be detected in fetuses if tested during the first minutes of intoxication.
REINFORCING EFFECTS OF ALCOHOL AND ACETALDEHYDE IN THE FETUS With respect to the positive reinforcing properties of alcohol and acetaldehyde in early development, in newborn rats it was found that administration of low doses of alcohol paired with an odor induced a conditioned odor preference for that smell (Petrov, Varlinskaya, & Spear, 2003). Similar appetitive responses were obtained by injecting alcohol or acetaldehyde directly into the newborn brain, but not when inhibiting brain catalases or sequestering acetaldehyde, thus, confirming the
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PERINATAL LEARNING WITH ALCOHOL AND ACETALDEHYDE
appetitive properties of central acetaldehyde in neonate rats (March, Abate, Spear, & Molina, 2013; Nizhnikov, Molina, & Spear, 2007). In prenatal stages, alcohol also appears to exert appetitive effects. When alcohol is administered to the pregnant rat during the last days of gestation (17 20), the fetus is exposed simultaneously to the chemosensory (flavor) and pharmacological properties of alcohol. This prenatal experience results in increased postnatal alcohol consumption as well as enhanced liking for the taste of alcohol when tested in infancy and adolescence (Chotro & Arias, 2003). It is generally accepted that the near-term fetus can perceive chemosensory stimuli present in the amniotic fluid during swallowing and breathing the fluid, forming associations with their aversive or appetitive consequences, which modifies postnatal responses to those stimuli (Smotherman & Robinson, 1988). Accordingly, the enhanced acceptance of alcohol found after prenatal alcohol exposure was demonstrated to be a conditioned appetitive response acquired in utero (Chotro & Arias, 2003). This prenatal experience was also found to modulate postnatal learning by retarding the acquisition of aversions and facilitating learned preferences when alcohol was the reinforcer (Arias & Chotro, 2006a, 2006b). Recently, it was demonstrated that the reinforcing effects of acetaldehyde are crucial for this prenatal appetitive learning about alcohol, since sequestering acetaldehyde was able to prevent such learning (Gaztanaga, Angulo-Alcalde, Spear, & Chotro, 2017). Additional indirect support comes from studies in which the reinforcing effects of alcohol were suppressed when blocking the endogenous opioid system, particularly the μ-opioid receptors, during prenatal alcohol exposure (Chotro & Arias, 2003; Chotro, Arias, & Laviola, 2007; Diaz-Cenzano, Gaztanaga, & Chotro, 2014). This is particularly relevant since the reinforcing and stimulating effects of central alcohol-derived acetaldehyde appear to be mediated by the μ-opioid system (Font, Lujan, & Pastor, 2013). Acetaldehyde has been found to stimulate the release of β-endorphins and salsolinol appears to induce its effects interacting with the μ-opioid receptors in the posterior ventral tegmental area, an element of the “dopamine reward pathway” (Xie, Hipolito et al., 2012). In addition to acetaldehyde, high amounts of salsolinol were found in the fetal brain after chronic alcohol exposure (Mao et al., 2013), although its behavioral effects in early ontogeny have not yet been tested.
PERINATAL LEARNING WITH ALCOHOL AND ACETALDEHYDE Although the reinforcing effects of alcohol and acetaldehyde during prenatal and neonatal stages appear
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to be clear (March et al., 2013), no evidence of the aversive effects of alcohol have yet been found until postnatal day 11 of the rat (Arias & Chotro, 2006; Pautassi, Nizhnikov, & Spear, 2009). Moreover, the same alcohol doses that are aversive for adults or 2-week-old infants are appetitive when experienced either prenatally or during the first postnatal week (Arias & Chotro, 2006; Chotro, Arias, & Spear, 2009). This developmental change in the motivational effects of alcohol could be related to the metabolic status of the developing rat at each age. As explained, the rat fetus and neonate do not generate aversive peripheral acetaldehyde due to the low or null hepatic ADH activity that begins to increase after birth and reaches the adult value by day 20 (Boleda et al., 1992) and the fetus is relatively protected from maternal acetaldehyde. Conversely, the intense activity of brain catalase that produces high levels of reinforcing acetaldehyde at fetal and neonatal stages decreases gradually with development (Delmaestro & McDonald, 1987). Thus, it seems unlikely that the fetus and the neonate have the chance to perceive the negative consequences of peripheral acetaldehyde during alcohol intoxication, while their central appetitive effects are predominant. If the perceived motivational effects of alcohol intoxication are considered to be a result of the balance between peripheral and central levels of acetaldehyde (Correa et al., 2012), this suggests that fetuses and newborns will perceive exclusively the appetitive effects of acetaldehyde, and the likelihood of perceiving its aversive properties will increase during development. This implies that fetal learning during prenatal alcohol exposure will necessarily generate appetitive responses (Fig. 9.3). This associative mechanism could underlie the enhanced liking of alcohol odor observed in two subsequent studies with newborn babies and adolescents with a history of prenatal alcohol exposure (Faas, March, Moya, & Molina, 2015; Hannigan, Chiodo, Sokol, Janisse, & Delaney-Black, 2015). Similarly, this might explain the increased alcohol consumption observed in many animal studies with prenatal alcohol exposure, along with other neurodevelopmental effects (Chotro et al., 2007). In summary, when the pregnant mother consumes alcohol, the fetus is exposed to alcohol in the blood, brain, and amniotic fluid, and to centrally formed acetaldehyde. This produces a wide range of physiological and behavioral effects that not only alter fetal neurodevelopment, but also induce changes in alcohol acceptance, which can eventually lead to alcohol abuse problems. Clinical studies show a clear relationship between prenatal alcohol and alcohol abuse in adolescents and young adults, with prenatal alcohol exposure being a good predictor of alcohol problems (Baer, Sampson, Barr, Connor, & Streissguth, 2003). More
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FIGURE 9.3 Fetal alcohol learning after maternal alcohol consumption. This scheme shows possible associative mechanisms underlying increased alcohol intake and liking after prenatal alcohol exposure.
research is needed to better identify the relative importance of each intervening factor as well as the mechanisms underlying this complex interplay between the effects of alcohol and acetaldehyde.
KEY FACTS About Alcohol and Acetaldehyde in the Fetus • Alcohol consumed by the pregnant mother crosses the placenta and reaches the fetus in similar amounts to maternal plasma, and accumulates in the amniotic fluid. • Acetaldehyde, the first oxidation metabolite of alcohol, is mainly produced in maternal liver. • Low or null amounts of maternal acetaldehyde reach the fetus after maternal alcohol ingestion, this being metabolized by the placenta and fetal liver. • The fetus, due to its hepatic metabolic immaturity, cannot produce peripheral acetaldehyde from alcohol. • Acetaldehyde is produced from alcohol in the fetal brain, mainly by catalases. • Brain acetaldehyde may produce many of the neurodevelopmental and behavioral effects of alcohol.
SUMMARY POINTS • The role of alcohol-derived acetaldehyde in relation to the known effects of prenatal alcohol exposure is reviewed.
• Alcohol ingested by the pregnant mother easily reaches the fetus, while maternal acetaldehyde does not. • Acetaldehyde is produced in the fetal brain and may be responsible for many of the effects attributed to alcohol. • Brain acetaldehyde retards development and induces neural damage through the formation of toxic adducts. • Alcohol and acetaldehyde in early development induce comparable behavioral effects and the reinforcing properties of central acetaldehyde during alcohol exposure may underlie prenatal appetitive learning producing postnatal increased alcohol intake.
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