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The Journal of Nutrition Vol. 128 No. 2 February 1998,
pp. 459S-462S
The Burnham Institute, La Jolla, CA 92037
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ABSTRACT |
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Abstract
References
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Vitamin A (retinol) must be metabolized to an active retinoid ligand in order to fulfill all of its roles in vertebrate development. During retinoid signaling, retinol is first converted to retinal followed by conversion of retinal to the active ligand retinoic acid, which modulates nuclear retinoic acid receptors (RAR). The alcohol dehydrogenase (ADH) enzyme family may function in the metabolism of retinol, the alcohol form of vitamin A, as well as ethanol metabolism. Some members of the ADH family prefer retinol as a substrate over ethanol, and their ability to oxidize retinol is competitively inhibited by intoxicating levels of ethanol. Likewise, there exists an aldehyde dehydrogenase (ALDH) family containing several members preferring retinal as a substrate over acetaldehyde. The spatiotemporal expression patterns of ADH-IV and two forms of ALDH match the spatiotemporal detection of retinoic acid during mouse embryogenesis, i.e., no detection at 6.5 d of embryogenesis (E6.5), followed by detection at E7.5 in the primitive streak, and then detection in numerous tissues later in development. This suggests that certain forms of ADH and ALDH may cooperate to upregulate retinoic acid synthesis during development. Treatment of mouse embryos at E7.5 with an intoxicating amount of ethanol leads to a reduction in retinoic acid levels. At E7.5, two other mouse enzymes known to metabolize ethanol (ADH-I and P450 2E1) are not expressed, indicating that ADH-IV may be the only enzyme available at this stage to metabolize both ethanol and retinol. These findings suggest that ADH-IV participates in the initiation of retinoid signaling by functioning as a retinol dehydrogenase and that this can be inhibited by ethanol intoxication.
KEY WORDS: alcohol dehydrogenase · vitamin A · retinoic acid · embryogenesis · mice
In addition to its function in vision, vitamin A (retinol) regulates several other processes in vertebrate organisms, including embryogenesis, reproduction and epithelial differentiation. However, unlike vision, which is carried out by the vitamin A metabolite 11-cis-retinal bound to rhodopsin, most other functions of vitamin A in vertebrates are carried out by the conversion of retinol to retinoic acid, which functions as a ligand controlling a retinoic acid receptor (RAR) signaling pathway (Kastner et al. 1994 Our understanding of how retinol is physiologically activated to form the ligand for retinoid signaling is limited. Retinol is transported via the serum throughout the body at relatively high levels where it is available to essentially all cells for potential conversion to retinoic acid (Soprano and Blaner 1994 The pathway for conversion of retinol to retinoic acid involves first the oxidation of retinol to retinal, then the oxidation of retinal to retinoic acid. Studies on the conversion of retinol to retinoic acid in homogenates of rat embryos have shown that retinol oxidation is the rate-limiting step (Chen et al. 1995
To help identify the enzymes involved in retinol and ethanol metabolism during embryogenesis, mouse embryos at stages E6.5 through E8.5 have been analyzed for expression of all known members of the mouse ADH gene family (i.e., ADH-I, ADH-III and ADH-IV) as well as P450 2E1 (another ethanol-active enzyme) and two members of the mouse ALDH gene family (ALDH-I and RALDH-2) (Table 1).
The gene expression studies described above provide evidence that the enzymatic machinery needed to perform both oxidation steps in the conversion of retinol to retinoic acid exists by E7.5 of mouse embryogenesis, the stage when retinoic acid is first detectable (Fig. 2). By stage E7.5 all three retinoic acid receptors are already expressed in numerous tissues (Ang and Duester 1997
Ethanol intoxication during embryogenesis can lead to fetal alcohol syndrome characterized by a high incidence of craniofacial, central nervous system and cardiovascular defects (Jones and Smith 1973
VITAMIN A FUNCTIONS THROUGH
RETINOIC ACID
). In response to retinoic acid binding, RAR functions by directly interacting with DNA regulatory sequences leading to modulation of gene transcription. Generation of mice carrying mutations in three different receptors, RAR
, RAR
and RAR
, provides proof that they indeed control retinoid signaling because the defects observed are those seen during embryonic vitamin A deficiency, i.e., defects in development of the heart, eye, genitourinary tract, respiratory tract and cranial neural crest (Lohnes et al. 1994, Luo et al. 1996, Mendelsohn et al. 1994). A major challenge remaining in the study of vitamin A function is the understanding of how vitamin A metabolism is normally regulated to provide the ligand for this signaling pathway. This should provide insight into disease states in which abnormal vitamin A function is observed.
DETECTION OF RETINOIC ACID
IN MOUSE EMBRYOS
). However, it seems that retinoic acid is not equally produced by all cells at all stages of development but is instead produced in a unique spatiotemporal pattern. Studies performed using a tissue explant bioassay have shown that retinoic acid is undetectable in mouse embryos at 6.5 d of embryonic development (E6.5) during the beginning of gastrulation but that it is detectable at E7.5 during primitive streak formation and later (Ang et al. 1996). At E8.5-E9.5, retinoic acid is localized preferentially in the posterior trunk region as well as the craniofacial region, with very little detected in the brain (Ang et al. 1996). Expression in mouse embryos of a lacZ transgene linked to a retinoic acid response element is not observed at E6.5 but is observed at E7.5 in the posterior half of the embryo (Rossant et al. 1991). A posterior preference for retinoic acid synthesis has also been demonstrated in tissues of E7.75 mouse embryos cultured in the presence of labeled retinol (Hogan et al. 1992). Thus, retinoid signaling during embryogenesis may be initiated by upregulation of endogenous retinoic acid synthesis which seems to occur during primitive streak formation. Also, studies on vitamin A-deficient chicken and quail embryos indicate that retinoid signaling may not be necessary prior to gastrulation because the defects observed begin after primitive streak formation, i.e., defects in the cardiovascular system, somites, hindbrain, neurite outgrowth and cranial neural crest (Dersch and Zile 1993, Maden et al. 1996, Thompson et al. 1969).
ENZYMATIC PROPERTIES OF ALCOHOL DEHYDROGENASES
). Numerous enzymes able to catalyze retinol and retinal metabolism in vitro have been identified, including members of the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzyme families, as summarized in a recent review (Duester 1996). These enzyme families are of ancient origin and contain some members that are active primarily for ethanol metabolism and other members that are active primarily for retinoid metabolism (Fig. 1). It is likely that divergence of ancient forms of these enzymes has given rise to more recent forms that participate in retinoid metabolism needed for retinoid signaling, a relatively recent signaling pathway known to exist only in vertebrate animals and possibly other chordates (Shimeld 1996).
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Fig 1.
The dual function of alcohol dehydrogenase in ethanol and retinol metabolism, and implications for regulation of retinoic acid synthesis. Alcohol dehydrogenase (ADH) is traditionally associated with ethanol metabolism, as indicated in the top metabolic pathway in which ethanol is oxidized to acetaldehyde by ADH, followed by oxidation of acetaldehyde to acetic acid by aldehyde dehydrogenase (ALDH). However, both ADH and ALDH can participate in retinoid metabolism by oxidizing retinol to retinaldehyde to retinoic acid, as indicated in the lower pathway. Retinoic acid binds a nuclear retinoic acid receptor and transcriptionally regulates genes involved in several biological processes. Because ADH can use either ethanol or retinol as a substrate, ethanol will act as a competitive inhibitor of ADH-catalyzed retinol oxidation. This may lead to a reduction in retinoic acid synthesis, hence an alteration in gene regulation via the retinoid signaling pathway.
, Boleda et al. 1993, Yang et al. 1994, Kedishvili et al. 1995). Molecular modeling studies have shown that the active site of ADH-IV is quite large and easily accommodates retinol (MW) [molecular weight = 286.5] (Kedishvili et al. 1995, Moreno et al. 1996), indicating that the active site of this enzyme is not designed to bind simply a two-carbon alcohol substrate such as ethanol (MW = 46). A comparison of the efficiencies of these ADH for ethanol oxidation indicates that ADH-I is the most efficient for this substrate, with ADH-IV about 10 times less efficient and ADH-III nearly inactive. Although not appearing to function in either ethanol or retinol metabolism, ADH-III is quite efficient as a glutathione-dependent formaldehyde dehydrogenase using S-hydroxymethylglutathione (MW = 337) as a substrate (Koivusalo et al. 1989). This is a property not shared by the other ADH family members, indicating that the various classes of ADH have evolved to perform quite different functions. ADH-III is the only ADH family member conserved in all organisms, i.e., bacteria, fungi, plants and animals, thus indicating that it is the progenitor of the ADH family (Danielsson and Jörnvall 1992). This progenitor evidently evolved to bind a large alcohol substrate (i.e., S-hydroxymethylglutathione), with divergent forms later evolving to bind the comparably-sized substrate retinol as well as the much smaller substrate ethanol.
). Also, an additional class of mouse ALDH known as RALDH-2 functions as an efficient retinal dehydrogenase (Zhao et al. 1996). The oxidation of acetaldehyde is performed most efficiently by ALDH-II (Dockham et al. 1992). Thus, for both the ADH and ALDH families, the forms most efficient for retinol metabolism differ from the forms most efficient for ethanol metabolism.
, Julià et al. 1986), and treatment of mouse embryos at stage E7.5 with an intoxicating amount of ethanol leads to a reduction in retinoic acid detection (Deltour et al. 1996). This latter finding suggests that an ADH is involved in the synthesis of retinoic acid at stage E7.5 of embryogenesis.
EMBRYONIC EXPRESSION PATTERNS OF RETINOL- AND ETHANOL-METABOLIZING ENZYMES
View this table:
Table 1.
Temporal appearance of retinoic acid as well as mRNAs for retinol- and ethanol-metabolizing enzymes
). ADH-I is the major liver ethanol-metabolizing enzyme as well as an inefficient retinol dehydrogenase, but it does not seem to contribute to embryonic ethanol or retinol metabolism during E6.5-E8.5 due to its lack of expression. ADH-III is very inefficient with ethanol as a substrate (cannot be saturated at molar levels) and inactive with retinol, and thus it is not thought to contribute to either ethanol or retinol metabolism in vivo and would not be expected to do so in embryos despite its expression at all stages. ADH-IV is inefficient with ethanol as a substrate but is very efficient as a retinol dehydrogenase in vitro. Thus, the expression pattern of the mouse ADH gene family suggests that ADH-IV is the only known form that could participate at stage E7.5 of embryogenesis in both retinol metabolism to produce retinoic acid as well as ethanol metabolism during intoxication.
). This finding is significant because it, along with the expression studies on ADH, provides evidence that ADH-IV may be the target of ethanol action in stage E7.5 mouse embryos as described above. The relative inefficiency of ADH-IV for ethanol metabolism could be the reason why a low level of ethanol (10 mmol/L) does not lead to a significant reduction in retinoic acid detection in E7.5 embryos, whereas a high level (100 mmol/L) does (Deltour et al. 1996). On the basis of the enzymatic properties of ADH-IV, ethanol would need to reach a high level before it would be expected to compete effectively with retinol as a substrate for this enzyme.
). Thus, ADH-IV and ALDH-I could potentially cooperate to convert retinol to retinoic acid in these tissues. In addition, zymography assays have demonstrated that RALDH-2 enzyme activity is present in mouse embryos as early as E8.0 (McCaffery et al. 1993). In situ hybridization analysis has shown that RALDH-2 expression is initially similar to that of ALDH-I with no detection at E6.5 followed by detection at E7.5 in the primitive streak mesoderm, but diverges from that of ALDH-1 since it is not expressed in the cranial mesenchyme by E8.5 (Niederreither et al. 1997). Thus, ALDH-1 and RALDH-2 may play overlapping roles in retinoic acid synthesis during primitive streak formation but play distinct roles in retinoic acid synthesis later in development. In particular, RALDH-2 is expressed in the mouse embryonic spinal cord, which is known to be a site of retinoic acid synthesis (McCaffery and Dräger 1994, Zhao et al. 1996).
ADH-IV AS AN EMBRYONIC RETINOL DEHYDROGENASE
, Ruberte et al. 1991). The simultaneous expression of ADH-IV, ALDH-I and RALDH-2 at stage E7.5, when retinoic acid is first detectable, provides a compelling argument that these enzymes are designed to function in the upregulation of retinoic acid synthesis so as to enable retinoid signaling to commence.
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Fig 2.
Model for the initiation of retinoid signaling during mouse embryogenesis and its disruption by ethanol intoxication. When gastrulation initiates at E6.5 and mesoderm formation begins, retinoic acid is not detectable using a very sensitive bioassay. At E7.5, retinoic acid is now easily detectable using the bioassay. Also at E7.5, the mRNAs for ADH-IV, ALDH-I, and RALDH-2 are first detectable, suggesting that these enzymes may be responsible for upregulation of retinoic acid synthesis at this stage. Ethanol reduces the level of embryonic retinoic acid at E7.5, thus potentially inhibiting the initiation of retinoid signaling and downstream events such as cranial neural crest cell survival.
). Loss-of-function studies using vitamin A-deficient chick embryos have shown that embryogenesis proceeds apparently normally until about stages 10-13 (comparable to stage E8.5 in the mouse), when embryos have progressed beyond the initiation of gastrulation and are developing cranial neural crest cells that contribute to craniofacial bone and cartilage, the central nervous system, and the cardiovascular system (Thompson et al. 1969). Further studies using a vitamin A-deficient quail embryo model have also demonstrated apparently normal early embryonic development, followed by cranial neural crest cell death and defects in the cardiovascular system, hindbrain and neurite outgrowth (Dersch and Zile 1993, Maden et al. 1996). The cardiovascular defects in quail embryos can be rescued by administration of retinoic acid to fertilized eggs prior to heart development (Dersch and Zile 1993). Thus, it is reasonably certain that avian embryos have an essential requirement for retinoic acid by the time cranial neural crest cell differentiation has begun. These general principles are likely to apply to mammalian embryos as well. Expression of ADH-IV and ALDH-I in the cranial mesenchyme of mouse embryos could provide a source of retinoic acid for cranial neural crest cell survival.
ADH-IV AS A TARGET FOR ETHANOL ACTION
). Some of these defects may be caused by a reduction in cranial neural crest cell survival following an ethanol insult during early embryogenesis (Kotch and Sulik 1992). The ability of ethanol to reduce the level of retinoic acid in mouse embryos at stage E7.5 (Deltour et al. 1996) suggests that the negative effects of ethanol upon embryonic development are due, at least in part, to a block in the initiation of retinoid signaling needed for neural crest cell survival (Fig. 2). This may be the result of ethanol-inhibition of retinoic acid synthesis catalyzed by ADH-IV, an enzyme able to metabolize both retinol and ethanol at this stage. Cells expressing ADH-IV may thus be the targets for the destructive effects of ethanol intoxication during early embryonic development.
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FOOTNOTES |
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LITERATURE CITED |
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Abstract
References
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and RAR1 reveal developmental functions for multiple RAR isoforms.
Mech. Dev.
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