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,3
UCLA School of Public Health, Los Angeles, CA 90095;
* UCLA Center for Human Nutrition, Los Angeles, CA 90095-1742;
Food, Nutrition and Consumer Sciences Department, California State Polytechnic University, Pomona, CA. 91768;
** Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, 37232-0146;
Zentrumsabteilung fur Lebensmitteltoxikologie, Tierarztliche Hochschule Hannover, D-30173, Hannover, Germany
4To whom correspondence should be addressed. E-mail: mdc{at}ucla.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • vitamin A • retinoid • methylation • neural tube • teratogenesis
Epidemiologic studies have demonstrated that dietary supplementation with folic acid at the time of conception prevents the recurrence of neural tube defects in women who have had a child with such a defect (1,2), and that this same treatment may prevent the primary occurrence of neural tube defects (3). There is debate concerning whether supplementation with folic acid is correcting a dietary deficiency or overcoming some inborn error of metabolism (4). However, although the magnitude of the effect is in question, it appears that some human neural tube defects are prevented by folic acid supplementation (5,6).
A rodent model of this human phenomenon would facilitate mechanistic experimentation. If a folic acid metabolite is functioning as a methyl-donor, then one of the possibilities is that it would contribute to the formation of methionine, which is required to form S-adenosyl methionine (SAM),4 the ubiquitous methyl donor for methyltransferase activity. If this enzymatic activity is required for neural tube closure, then a deficiency of methyl donors could contribute to abnormal development. For rat embryos grown in methionine-deficient serum, a deficit associated with inhibition of neural tube closure, three proteins that were normally methylated during neurulation were hypomethylated, which was associated with altered neuroepithelial morphology (7). Thus, data highlight the importance of the biochemical process of methylation in neural tube closure.
Nevertheless, dietary folate deficiency alone has not caused neural tube defects in mice (8). However, folates, which can be formed by endogenous microorganisms, provide only one source of methyl groups. Due to the interdependence of metabolic pathways of folic acid with choline and L-methionine, a deficiency in one of these methyl-donor compounds will affect the others (9). Consequently, the diet in this study was depleted of three methyl donors, i.e., folic acid, choline and supplemental L-methionine (10). Supplemental L-methionine refers to the addition of the individual amino acid to the diet supplementing the methionine that is in protein. Even with all three dietary deficiencies, the diet did not induce murine neural tube defects in preliminary studies; therefore, the methyl-group-deficient diet was supplemented with an agent known to induce this malformation in mice, namely, retinyl palmitate or vitamin A (11). Vitamin A derivatives (retinoids) are potent teratogenic agents in both laboratory animals (including mice, rats, guinea pigs, hamsters, rabbits, dogs, pigs, chicks and monkeys) and humans (12,13), and retinoids can induce neural tube defects as well as a variety of other malformations depending upon gestational age at the time of administration (14). It was hypothesized that the combination of two dietary insults suspected to be involved in the inhibition of neuropore closure would produce a higher percentage of fetuses with the malformation than either insult alone.
The current study utilized a whole-embryo culture system that is relatively sensitive to the induction of neural tube defects because the embryos are explanted at the time of neural tube closure and chemicals present in the culture medium have direct access to the embryo without traversing the maternal system (15). This system utilizes undiluted serum for the culture medium, and rat serum has been shown to support mouse embryonic growth and development (16). This culture process allows whole embryos to grow and develop in vitro to approximately the same extent as they do in vivo (17).
The morphological data indicate that in contrast to the hypothesis, retinoid-induced dysmorphogenesis is ameliorated by a diet with a methyl-donor deficiency. To explain these results, the culture medium was analyzed for various intermediates of the methylation, transsulfuration, retinoid and antioxidant pathways. The methylation and retinoid pathways were examined because the diets administered have a deficiency and an excess of these constituents, respectively. The transsulfuration pathway is a metabolic alternative to the methylation pathway. The levels of 
-tocopherol (-Toc) and glutathione (GSH) were measured because a previous investigation determined that antioxidants were reduced by this same diet (10). GSH is also a product of the transsulfuration pathway. Additionally, GSH depletion caused by oxidative stress in hepatocytes was demonstrated to induce 
-glutamyl transferase (GGT) (18); thus, the activity of the enzyme was measured in the liver of rats fed different diets. Antioxidant depletion may be involved in the formation of anterior neural tube defects in cultured rodent embryos because excess oxygen was shown to specifically induce these defects (19).
The quantity of retinoic acid receptor-ß2 mRNA has been measured as an indicator of active retinoids in the embryo. The limited tissue from cultured mouse embryos was the impetus to find a sensitive assay to detect embryonic retinoids. Studies had indicated that retinoic acid receptor (RAR)-ß was more sensitive to in vivo induction by retinoids than RAR- or - (20,21), and ß2/4 was more sensitive than ß1/3 (22). The induction of RAR-ß expression may reflect the concentration of morphogenetically active retinoids available to cells (23).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Simonsen albino rats, weighing between 70 and 90 g, were obtained from Simonsen Laboratories, Gilroy, CA. The rat and mouse experiments and procedures followed the institutional guidelines of the Chancellor’s Animal Research Committee of the University of California, Los Angeles. Rats were randomly assigned to one of the dietary groups for 36 d. They were kept in a room with a 12-h light:dark cycle, 30–70% humidity and constant temperature (22 ± 2°C).
Ten-wk-old ICR mice of both sexes were supplied by Charles River Laboratories, Hollister, CA. Mice were mated by placing a male with multiple females for the last 2 h of the dark cycle (0700–0900 h), and detection of a vaginal plug initiated day 0 at 0900 h, to provide concepti for whole-embryo culture as described by Sadler and New (16). The mice were housed under environmental conditions similar to those described for the rats.
Experimental diets.
In Experiment 1, the six diets shown in Table 1 were studied. In Experiment 2, performed to corroborate previous findings and to obtain further biochemical data on the culture medium, only the diets with low (0.016 g/kg of diet) and high (4.016 g/kg of diet) levels of retinyl palmitate were included because the medium dose effects were not significantly different from those of the low dose. Because a majority of the biochemical measurements were performed only in Experiment 2, many tables report values for only diets 1, 2, 5 and 6. Each diet was prepared by mixing the components for 3 h in a blender (Model CC-80, G. S. Blakeslee, Chicago, IL) and the diet was stored at 4°C. All dietary preparations were performed in a room with a dim yellow light to prevent photoisomerization and photodegradation.
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Experimental design.
At the time of detection of a vaginal plug, the female mice were switched from a nonpurified diet (Purina 5008, PMI Nutrition International, Brentwood, MO) to one of the six experimental diets. Each female was fed this diet for 8 d and then the embryos were removed from the uterus and grown in 100% rat serum for a period of 46 h. The serum was from a rat fed the same diet for 36 d as that consumed by the mice for the first 8 d of pregnancy. Thus, embryos received two exposures to one of the six diets; the first exposure was from the diet of the pregnant female and the second exposure was to the serum (or culture medium) derived from a rat fed the same diet. In each case, the embryos were exposed to only a single experimental diet. The mouse embryonic phenotype was determined after the embryo culture period. Chemical analyses were performed to allow biochemical differentiation of the sera from rats fed the different diets. Rat livers were analyzed for both folate levels and enzymatic activity of GGT. The quantity of RAR-ß2 mRNA in whole-mouse embryo was determined at the end of the culture period. The experimental design is diagrammed in Figure 1.
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After 36 d of exposure to the defined diets, the rats were anesthetized by inhalation of diethyl ether. Blood was collected using sterile technique from the dorsal aortic region located a few millimeters above the femoral bifurcation. Blood samples were obtained without anticoagulants, protected from light and immediately centrifuged at 2500 x g for 10 min at 4°C to obtain the sera. Livers were immediately frozen after collection, whereas sera were centrifuged and separated from RBC before freezing with liquid nitrogen and storage at -70°C. Sera were subsequently heat inactivated at 56°C for 30 min and immediately placed in a forced-air hood for 30 min to allow residual ether to volatilize. After heat inactivation, the sera were stored at -20°C until use in embryo culture.
Whole-embryo culture procedure.
Embryos were explanted at gestational day 8.0 in HBSS 24020 (Gibco BRL, Rockville, MD). The decidua and Reichert’s membrane were removed from the concepti, but the visceral yolk sac was left intact. Embryos with between 3 and 6 somites were selected for culture. The culture medium was 100% rat serum. Up to three embryos were grown in a 30-mL bottle with a Teflon stopper containing 1 mL of serum/embryo on a roller apparatus at 37.5 ± 1.0°C. The embryos were gassed at 0 and 12 ± 1 h with a mixture of 5% O2/5% CO2/90% N2, and at 24 ± 1 and 36 ± 1 h with 20% O2/5% CO2/75% N2. Culture was terminated at 46 ± 1 h, and the embryos were examined for viability, growth and development. To quantify the embryonic development, a morphological scoring system was used that evaluates 16 anatomical categories according to van Maele-Fabry et al. (25).
Analytical procedures.
The total folates in serum and liver were determined with a microbiological assay using Lactobacillus casei (ATCC 7469; Microbe No.9640, Difco Laboratories, MI) according to the technique of Tamura (26). L. casei are obligate-folate-dependent bacteria, and growth correlates with active forms of folate.
The homocysteine (Hcy), cysteine (Cys), and GSH levels in rat serum were determined in a single chromatographic analysis according to the method of Vester and Rasmussen (27) modified by using the internal standard cystamine (28). GGT was determined in rat liver microsomes by the method of Ngah et al. (29). The S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) assay was performed by HPLC using the method of Capdevila and Wagner (30). -Toc was determined in rat serum using the extraction method of Epler et al. (31). The extracts were injected onto a narrow-pore Bakerbond C18 analytical column (5 µm) and separated using an Agilent Technologies HPLC, Model 1050 (San Diego, CA) with detection at 295 nm.
Retinoids were analyzed using HPLC coupled to an automated solid-phase extraction technique (Varian AASP, Palo Alto, CA) described by Pearce et al. (32). Three different methods were applied. Method I was for detection of polar retinoids except 9,13-di-cis-retinoic acid (RA) from 350 µL (33). Due to the extraction method, this HPLC procedure did not provide quantitative recovery of retinol, retinyl esters or 9,13-di-cis-RA. Method II was used to provide quantitative recovery and analysis of retinol and retinyl esters according to Tzimas et al. (34). This method involved extraction of 100 µL plasma (35), but because of the lower sample volume, it was less sensitive for polar retinoids than Method I. Method III was designed to separate 9,13-di-cis-RA from the other acidic retinoids using an extraction volume of 200 µL (36).
A fourth method applied HPLC-MS techniques for rechromatography and peak identification of putative 9,13-di-cis-RA. The system (Method IV) consisted of a P4000 low pressure gradient system, an SCM 1000 membrane degasser, an AS3000 autosampler and a UV6000LP diode array detector, all from Thermoseparations Products (Egelsbach, Germany). The LCQ MS unit was from Finnigan MAT (Egelsbach). For this method, putative 9,13-di-cis-RA peaks from method III were collected, and an aliquot was injected directly into the HPLC, while the solvent was evaporated from the remaining aliquot using a vacuum concentrator (Alpha RVC, Christ, Osterode, Germany) set at 1 mbar and room temperature. The residue was redissolved in the eluent and an aliquot was injected into the HPLC. Chromatography was achieved using the parameters of HPLC method II. UV spectra as well as the MS and MS-MS (m/z 301) spectra were recorded.
RAR-ß 2 mRNA quantification.
Retinoic acid receptor-ß2 mRNA was quantified with the Hybspeed Ribonuclease Protection Assay kit (Ambion, Austin, TX) using cyclophilin as an internal control as described in Mao and Collins (37). Absolute quantification was obtained by generating a standard curve of known concentrations of target RAR-ß2 and is reported as femtograms (fg) RAR-ß2 mRNA/µg total RNA from three or four different flasks of embryos (with each flask having 3 embryos).
Statistical analysis.
Data are presented as means ± SEM. The data were analyzed by two-way ANOVA and the significance level was P < 0.05. If the ANOVA yielded a significant difference due to folate (F), vitamin A (V) or the combination (F x V), it was followed by comparisons of individual means using t tests with the Bonferroni correction. Analyses were conducted with the statistical data analysis program Intercooled Stata 7.0 (Stata, College Station, TX).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-Toc in rat serum was decreased by both the methyl-donor-deficient diet and the high level of retinyl palmitate, with a greater depression associated with the high retinoid level (Table 6). The decrease of -Toc by the two dietary alterations was not additive because the decrease was not significantly greater in rats fed the diet with both alterations than in those fed the diet with retinoid excess alone. The most abundant acidic retinoid in the serum from rats fed the diets with the high level of retinyl palmitate was 9,13-di-cis-retinoic acid (Fig. 2). Because the predominance of this retinoid was not anticipated, the chemical identity of 9,13-di-cis-retinoic acid was confirmed by the UV absorption spectra (see Fig. 3), and MS-MS analyses (Fig. 4and 5). Serum 9,13-di-cis-retinoic acid levels were increased significantly when the rats were fed diets with high levels of vitamin A, with the highest values in those fed the methyl-donor-supplemented high vitamin A diet. The concentration of all-trans-retinoic acid in the serums of all dietary groups was below the limit of detection (0.83 nmol/L; Table 7).
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70% more RAR-ß2 mRNA than embryos exposed to serum with low levels of retinyl palmitate but equivalent methyl-donor sufficient status (P = 0.085; compare Diet 1 with Diet 5 in Table 7). However, when the embryos were grown in serum with a high retinyl palmitate level and a deficiency of methyl-donor constituents (Diet 6 in Table 6), two flasks had high levels of RAR- ß 2 mRNA (comparable to the induced level of 17.9 fg/µg of total RNA) and two flasks had low levels of RAR-ß2 mRNA (comparable to the noninduced levels of 10.7 or 8.6 fg/µg of total RNA). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For many biochemical variables that were measured in the present study, the expected differences between the methyl-donor-sufficient and -deficient diets were affected by a high level of vitamin A. Perhaps hypervitaminosis A (with or without the deficiency of methyl-donor compounds) caused hepatotoxicity, leading to the release of liver cell intermediates. This could apply to the serum folate levels of rats fed diets that were deficient in methyl-donor constituents; these were three times higher when rats consumed the high dose of vitamin A. Thus, the hypothesis for this study, which was that excess vitamin A would exacerbate the effects of folate deficiency, was in error because the hypervitaminosis A ameliorated the effect of the folate deficiency. Another example in which hepatotoxicity may have a role is the GGT activity in the liver of rats fed the methyl-donor deficient diet; this activity was reduced when the diet was high in vitamin A (P = 0.007; Table 6). Also, for rats fed diets both sufficient and insufficient in methyl donors, the GSH levels in the serum tended to be greater (P = 0.246) when the vitamin A level was high (Table 6). Thus, it may be difficult to differentiate the metabolic effects from the toxic effects of retinoids in vivo.
In the aforementioned antagonism studies, as well as the current study, it was proposed that teratogenesis in mice was induced by exposure to the retinoid. To determine the ultimate teratogenic metabolite of the retinyl palmitate, the various forms of RA were quantified in rat serum. Acidic retinoids were examined because these molecules are more potent teratogens than the nonacidic counterparts, they are the primary ligands for the receptors, and the teratogenic response to retinoids has been attributed to ligands for the RAR [(42), reviewed in (13)]. The quantity of all-trans-RA in serum was below the limit of detection for all diets. Because the retinyl palmitate was an all-trans retinyl ester, we anticipated that the most direct acidic metabolite would be all-trans-RA. The plasma half-life (t1/2) of this compound, however, is relatively short when administered orally to rodents (t1/2 60 min) (43). The most abundant acidic retinoid isolated from rat serum was 9,13-di-cis-retinoic acid. This isomer was previously determined to be an endogenous retinoid (44), and was the predominant isomer in bovine periparturient plasma at concentrations of 13 nmol/L (45). However, the total amount of all of the acidic retinoids found in rat serum in the current study was relatively low; it was so low that it is difficult to conclude that the dysmorphogenesis was caused by the acidic retinoids in the culture medium alone. The relative potency of 9,13-di-cis-RA to induce teratogenesis has not been reported, but is worthy of further investigation. Because RA is purported to induce teratogenesis in rats using area-under-the-curve (AUC) as opposed to the peak concentration as the pharmacokinetic parameter, (46), the whole-embryo culture system may be a highly sensitive system because the time of exposure may be relatively long compared with the in vivo situation. Alternatively, the various forms of vitamin A in the culture medium may be biotransformed to acidic retinoids in the embryo. Previously, a relatively small amount (0.024 nmol/g) of all-trans-RA in the embryo was shown to induce dysmorphogenesis in 100% of the cases (47); however, 3.3 µmol/L RA was added to the culture medium to achieve these moderate embryonic levels. Thus, we propose that the embryonic dysmorphogenesis was caused at least in part by the high levels of retinyl esters and/or retinol in the culture medium (rat serum) that was biotransformed to acidic or other active retinoids in the embryo.
Due to the inability to measure directly the quantity of individual retinoids in the cultured embryos, the amount of total active retinoids in embryos was inferred from a determination of the level of RAR-ß2 mRNA (48). There was a relatively minimal 70% increase in mRNA level for this receptor, which is low compared with previous inductions caused by teratogenic doses of retinoids (48,49). This minimal induction, indicating a low level of active retinoid exposure, may be effective in inducing dysmorphogenesis because of the duration of exposure, or because the embryo culture system is more sensitive to perturbation. The methyl-deficient diet only partially decreased the level of RAR-ß2 mRNA induction in this study.
Several potential mechanisms could explain the inhibitory effects of the methyl-donor deficiency on teratogenesis of retinyl palmitate. First, the presence of Hcy prevents the metabolic formation of retinoic acid (50). The dietary deficiency of the methyl donors led to an increase in the Hcy level in the rat serum that was used as the culture medium (Table 6). This mechanism has potential importance if the induction of embryonic dysmorphogenesis is contingent upon retinoid biotransformation in the embryo as opposed to the retinoids found in the culture medium. Second, cells that overexpress the Ha-ras oncogene are less responsive to retinoids (51). A diet deficient in methionine, choline, folic acid and cyanocobalamin was shown to upregulate expression of the Ha-ras gene in mammary gland and liver by four- and sixfold compared with the control synthetic diet (52). Thus, the dietary deficiency of methyl-donor compounds may increase embryonic Ras function, which inhibits retinoid-induced cellular events and subsequently dysmorphogenesis. Third, cell lines with higher levels of intracellular reactive oxygen species (ROS) are less sensitive to the effects of RA than cell lines with lower levels of these species (53). Additionally, oxidants decreased and antioxidants increased RAR activity, and hypoxia enhanced the inhibition of proliferation by RA in A375 melanoma cells. In vitro, oxidant conditions lowered and reducing conditions enhanced the DNA binding of the RAR/retinoid X receptor heterodimer. Because the methyl-donor-deficient diet was associated previously with a decrease in antioxidant levels (10) and the diet decreased the -Toc level in this study (Table 6), and because dietary folate depletion caused oxidative stress even when -Toc and GSH (the two antioxidants measured in this study) were not affected (54), it could be hypothesized that the decrease in responsiveness to retinoids was due to an increased production of ROS.
These mechanisms are unconfirmed, and furthermore, a dietary deficiency in one methyl-donor compound may not contribute additively to the toxicity from a deficiency in another methyl donor. Evidence comes from different mutant mice with various neural tube defects, phenotypes that can be altered by administration of chemical antagonists. The axial defect (Axd) mouse mutant has an open lumbosacral neural tube (spina bifida) and a curly tail that can be antagonized by methionine but not by folinic acid or cyanocobalamin (55). Alternatively, the splotch (2H) mutant mouse has spina bifida that can be antagonized by folic acid or thymidine, but is exacerbated by methionine (56). Thus, methionine and folates may not be biochemically equivalent in these mutants. Furthermore, the curly tail (ct/ct) mutant mouse has a high percentage of tail flexion and lumbosacral myeloschisis that can be antagonized by inositol, but not by folic acid, folinic acid or methionine (57). Therefore, there are biochemical pathways other than the methylation pathway that are important in neural tube closure. This principle was demonstrated recently for choline, in which an inhibitor of phosphatidylcholine synthesis, a biochemical pathway that differs from the methylation pathway of choline, induced neural tube defects (58). Thus, the methyl-donor-deficient diet may affect multiple biochemical pathways during neurulation.
In conclusion, this study demonstrated that in a murine system, a dietary deficiency of folic acid, choline and supplemental methionine antagonized anterior neuropore closure defects and visceral arch hypoplasia caused by excess retinyl palmitate. This interaction may be used to test hypotheses regarding the molecular mechanisms by which retinoids induce dysmorphogenesis. Furthermore, the difficulty in producing a rodent model for human folate deficiency may result in part from the higher endogenous plasma level of folates in rodents than in humans (Table 2) (59). A result directly opposing the dominant finding in this study was reported recently in which retinoid-induced cleft palate was antagonized by administration of either folic acid or methionine (60).
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FOOTNOTES |
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2 Supported by Fogarty International Training Grant 3D43 TW00623, the UCLA Center for Occupational and Environmental Health, Dr. Marian Swendseid and the UCLA Academic Senate.
3 Current address: Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
5 Abbreviations used: AUC, area under the curve; Axd, axial defect; BLD, below the limit of detection; +FCM , containing folic acid, choline and L-methionine supplementation or methyl-donor sufficient; -FCM , devoid of folic acid, choline and supplemental L-methionine or methyl-donor deficient; GGT, -glutamyl transferase; GSH, glutathione; Hcy, homocysteine; RA, retinoic acid; RAR, retinoic acid receptor; ROS, reactive oxygen species; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine; t1/2, half-life; -Toc, -tocopherol.
Manuscript received 19 June 2003. Initial review completed 3 July 2003. Revision accepted 14 August 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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