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Exogenous folate ameliorates ethanol-induced brain hyperhomocysteinemia and exogenous ethanol reduces taurine levels in chick embryos
Comparative Biochemistry and Physiology, Part C 150 (2009) 107–112
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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c
Exogenous folate ameliorates ethanol-induced brain hyperhomocysteinemia and exogenous ethanol reduces taurine levels in chick embryos Robert K. Barnett, Stephanie L. Booms, Tracy Gura, Mara Gushrowski, Robert R. Miller Jr. ⁎ Hillsdale College, Biology Department, 278 N. West Street, Hillsdale, MI 49242-1205, USA
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Article history: Received 24 November 2008 Received in revised form 26 March 2009 Accepted 28 March 2009 Available online 1 April 2009 Keywords: Ethanol Homocysteine S-adenosylmethionine S-adenosylhomocysteine Folate Taurine 10-Formyltetrahydrofolate dehydrogenase Chick
a b s t r a c t The effects of exogenous ethanol and/or folic acid on endogenous homocysteine (HoCys) and SAM (Sadenosylmethionine)/SAH (S-adenosylhomocysteine) levels in chick brains were studied at 11 days of development. Embryonic EtOH (3.0 mmol/kg egg) exposure caused a 1.6-fold increase in brain HoCys levels and a 9-fold decrease in brain SAM/SAH levels as compared to controls (p≤0.05). Brain HoCys and SAM/SAH levels returned to control values when injected with a mixture of EtOH and folic acid (3.0 mmol EtOH/kg egg and 34 μmol folic acid/kg egg). The effects of exogenous EtOH on the remethylation pathway, as measured by 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH) activities, and the transsulfuration pathway, as measured by taurine levels, were studied at 18 days of development. A single dosage of EtOH (3.0 mmol/kg egg; E0) and two daily dosages of EtOH (E0–1) failed to influence brain and hepatic 10-FTHF DH activities when compared to controls. However, three daily dosages of EtOH (E0–2) caused approximately a two-fold increase in brain 10-FTHF DH activities and a three-fold increase in hepatic 10-FTHF DH activities as compared to controls (p≤0.05). Three daily EtOH dosages (E0–2) caused reduced taurine levels in both brain and hepatic tissues (p≤0.05). Meanwhile, a single EtOH dosage (E0), two daily EtOH dosages (E0–1), and three daily EtOH dosages (E0–2), caused reduced hepatic taurine levels as compared to controls (p≤0.05). © 2009 Elsevier Inc. All rights reserved.
1. Introduction Ethanol (EtOH) and homocysteine (HoCys) are both teratogenic in chick embryos and impair brain development. Embryonic EtOH exposure caused reduced brain masses, reduced brain neuron densities (Miller et al., 1996, 2000, 2003a; Miller, 2004), and elevated brain caspase-3 activities (Miller et al., 2003a; Miller, 2004). EtOH-impaired brain development correlated with EtOH-induced increased brain lipid hydroperoxide levels and EtOH-induced reduced brain membrane long-chain polyunsaturated fatty acids (PUFAs) levels (Miller et al., 1996, 2000, 2003a,b; Miller, 2004). Walcher and Miller (2008) reported that exogenous EtOH also caused elevated HoCys levels, reduced S-adenosylmethionine (SAM) levels, and increased S-adenosylhomocysteine (SAH) levels within brains at theoretical stage 37 (11 days of chick development; Hamburger and Hamilton, 1951). These EtOH-induced reductions in SAM/SAH levels correlated with EtOH-induced reduced brain masses (r=0.62, p≤0.0001) and with EtOH-induced increased brain caspase-3 activities (r=−0.39, p≤0.05) (Walcher and Miller, 2008). In adults, EtOH caused elevated HoCys levels in actively drinking alcoholics (Bleich et al., 2003, 2005; Sakuto and Abbreviations: DTT, Dithiotreitol; EtOH, Ethanol; EDTA, Ethylenediamine-tetraacetic acid; 10-FTHF DH, Formyltetrahydrofolate dehydrogenase; HEPES, N-[2-hydroxyethyl] piperazine-N′ [2-ethanesulfonic acid]; HoCys, Homocysteine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, 5-Methyl tetrahydrofolate 5-Methyl THF; THF, Tetrahydrofolate. ⁎ Corresponding author. Hillsdale College, Biology Department, Dow 213, 278 N. West St., Hillsdale, MI 49242-1205, USA. Tel.: +1 517 607 2393; fax: +1 517 607 2252. E-mail address: [email protected] (R.R. Miller). 1532-0456/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2009.03.005
Suzuki, 2005; Wilhelm et al., 2006) and hyperhomocysteinemia in early alcohol-abstinent patients correlated with impaired cognitive abilities (Wilhelm et al., 2006). Exogenous HoCys also impairs chick brain development (Rosenquist et al., 1996, 1999) and promoted reduced brain masses, and elevated brain caspase-3 activities (Miller et al., 2003b, 2006). Exogenous HoCys caused elevated brain lipid hydroperoxide levels, reduced brain membrane long-chain PUFAs levels, and increased saturated shortchain fatty acids levels within chick brain membranes (Miller et al., 2003b, 2006). Because HoCys is an antagonist of the glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) (Lipton et al., 1997), Rosenquist et al. (1999) hypothesized that HoCys-induced teratogenesis in chick embryos may be caused by HoCys-induced inhibition of the glycine-binding site of the chick NMDAR. To challenge this hypothesis, Rosenquist et al. (1999) demonstrated that exogenous glycine attenuated HoCys-induced neural crest cell defects and neural tube defects in embryonic chicks. Miller et al. (2006) demonstrated that exogenous glycine partially attenuated HoCys-induced increases in embryonic chick lipid hydroperoxide levels in both brain and liver tissues, partially attenuated HoCys-induced decreases in brain membrane long-chain polyunsaturated fatty acids, and partially attenuated HoCys-induced increases in brain and liver caspase-3 activities. HoCys metabolism intersects two pathways. These pathways include the remethylation of HoCys to methionine, which requires the transfer of a methyl group from either betaine or 5-methyltetrahydrofolate (5-methyl THF) via vitamin B12 (cobalamine), and the transsulfuration of HoCys to cystathionine and subsequent conversion to taurine (Selhub, 1999).
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Interest in the remethylation pathway exists because folate deficiencies have not only been observed in alcoholics (Cravo et al., 1996; Halsted, 1992; Hebert et al., 1963), but also have been associated with hyperhomocysteinemia (Selhub, 1999). In adult rats, EtOH inhibited 10formyltetrahydrofolate dehydrogenase (10-FTHF DH; EC 1.5.1.6) activities (Im et al., 1999; Min et al., 2005). This cytoplasmic enzyme irreversibly catalyzes the conversion of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate (THF). In the glycine cleavage system within the remethylation pathway, T-protein requires THF as a coenzyme and synthesizes N5, N10-methylene-tetrahydrofolate which is converted to the methyl donor 5-methyl THF (Kikuchi, 1973; van der Put et al., 2001). Low 5-methyl THF levels could not only inhibit the remethylation of HoCys to methionine, but also alter SAM/SAH levels because methionine is used to produce SAM, SAM is converted to SAH, and SAH is hydrolyzed to HoCys (Selhub, 1999). The relative contribution of the remethylation pathway in promoting hyperhomocysteinemia in EtOH-treated chick brains is questionable. If an active glycine cleavage system within the remethylation pathway exists in embryonic chicks, one would predict that exogenous glycine should ameliorate EtOH-induced increases in HoCys, EtOH-induced decreases in SAM, and EtOH-induced increases in SAH. However, Walcher and Miller (2008) reported that exogenous glycine failed to attenuate EtOH-induced increased brain HoCys levels. However, exogenous glycine partially ameliorated EtOH-induced decreased SAM levels, increased SAH levels, and decreased SAM/SAH levels in embryonic chick brains (Walcher and Miller, 2008). In the transsulfuration pathway, HoCys condenses with serine to form cystathionine. This irreversible reaction is catalyzed by a pyridoxial5′-phosphate (vitamin B6)-containing enzyme, cystathionine β-synthetase. Cystathionine is hydrolyzed by another pyridoxial-5′-phosphatecontaining enzyme, γ-cystathioninase, thus forming α-ketobutyrate and cysteine and excess cysteine is converted to taurine (Selhub, 1999). ten Busch et al. (1997) reported that EtOH-treated chick embryos had lower allantoic taurine levels at 13 days of development (theoretical stage 39; Hamburger and Hamilton, 1951) as compared to controls. This observation is of interest because Kerai et al. (1998) reported that dietary taurine attenuated EtOH-induced hepatic steatosis and hepatic lipid peroxidation in Sprague–Dawley rats and Nonaka et al. (2001) reported that exogenous taurine attenuated HoCys-induced inhibition of extracellular-superoxide dimutase m-RNA expression in cultured rat vascular smooth muscle cells. Thus, the objectives of these studies were as follows. First, did exogenous folic acid attenuate EtOH-induced increased HoCys levels, EtOH-induced decreased SAM levels, and EtOH-induced increased SAH levels in embryonic chick brains? Secondly, did exogenous EtOH alter 10-formyltetrahydrofolate dehydrogenase activities (10-FTHF DH; EC 1.5.1.6) in embryonic chick brains and/or livers? Finally, did exogenous EtOH promote reduced taurine levels in embryonic chick brains and/or livers?
and avian saline (0.72% NaCl; w/v) during the first three days of development (E0–2). Meanwhile, experimental eggs were injected with approximately 25 μL of either a 1:1 mixture of 95% EtOH and avian saline, pH 7.2 (3.0 mmol/EtOH/kg egg); or a 1:1 mixture of 181 μM folic acid in avian saline with H2O (34 μmol folic acid/kg egg), or a 1:1 mixture of 95% EtOH and 181 μM folic acid in avian saline (3.0 mmol EtOH/kg egg and 34 μmol folic acid/kg egg) during the first three days of development (E0–2). The dosage of 34 μmol folic acid/kg egg (15 mg/kg) was selected because Gareskog et al. (2006) injected 15 mg folic acid/kg (34 μmol folic acid/kg) into pregnant dames and reported that this dosage ameliorated hyperglycemia-induced oxidative stress in rat embryos. Like exogenous EtOH and HoCys, hyperglycemic chick embryos also exhibited reduced brain masses, elevated brain caspase-3 activities, undergo brain membrane lipid peroxidation, and have elevated brain HoCys levels (Cole et al., 2008; Miller et al., 2005). All embryos were incubated in a forced air incubator (model 1202; GQF Manufacturing, Savannah, GA) at 37.5 °C and turned every 4 h with a relative humidity ranging from 80–90%. At 11 days of incubation (theoretical stage 37; Hamburger and Hamilton, 1951), chick embryos were decapitated, brains excised, and stored at −80 °C, until subsequent brain HoCys, SAM, and SAH analyses. Theoretical stage 37 (11 days of development) was selected because we have previously measured brain HoCys, SAM, and SAH levels at this developmental stage (Cole et al., 2008; Walcher and Miller, 2008). In order to determine if exogenous EtOH influenced either 10-FTHF DH activities or taurine levels within chick brain and hepatic tissues, control eggs were injected with approximately 25 μL of a 1:1 mixture of H2O and avian saline (0.72% NaCl; w/v) into the air sac at 0 days of development. Meanwhile, experimental eggs were injected with approximately 25 μL of a 1:1 mixture of 95% EtOH and avian saline (3.0 mmol/EtOH/kg egg) into the air sac at 0 days of development (E0), at 0 and 1 days of development (E0–1), and at 0, 1, and 2 days of development (E0–2). All embryos were incubated in a forced air incubator (model 1202; GQF Manufacturing, Savannah, GA) at 37.5 °C and turned every 4 h with a relative humidity ranging from 80–90%. At 18 days of incubation (theoretical stage 44; Hamburger and Hamilton, 1951), chick embryos were decapitated, brains and livers excised, and stored at −80 °C until subsequent 10-FTHF DH or taurine assays. Because we are unaware that 10-FTHF DH activities have been characterized across various stages of chick development, we selected theoretical stage 44 (18 days of development) over theoretical stage 37 (11 days of development) because 18 days of chick development is only three days away from hatching and embryos at this late stage should have higher enzyme activities and be near neonatal 10-FTHF DH activities as compared to earlier stage 37 embryos (11 days of development). Taurine levels in theoretical stage 44 (18 days of development) embryos were measured so we could correlate taurine levels (a transsulfuration product) to 10-FTHF DH activities (a remethylation pathway enzyme).
2. Materials and methods 2.2. Embryo viability All experiments were performed using fertile, specific pathogen-free white Leghorn chicken eggs (Gallus gallus) purchased from Hy-Vac International (Adel, IA, USA). Chickens raised at this commercial hatchery ingest a standardized diet causing minimal differences in yolk composition from generation to generation. Discovery Bio-wide Pore C 18 HPLC columns were purchased from Supelco, Inc., Bellefonte, PA, USA). The disodium salt of (6R, S), (6R)-10-formyltetrahydrofolate was the generous gift of Merck Epova Ag. (Schaffhausen, Switzerland). All other reagents were purchased from the Sigma-Aldrich Group (St. Louis, MO, USA) and Thermo-Fisher (Itasca, IL, USA).
Embryo viability was indirectly monitored by measurements of embryo masses, brain masses, and % living embryos at 11 days of development (theoretical stage 37) and at 18 days of development (theoretical stage 44; Hamburger and Hamilton, 1951). Living embryos were defined as possessing a beating heart during the dissections and injections were performed on 3 different occasions (N=3) where 16 to 20 fertile eggs were injected per treatment group per set of injections for theoretical stage 37 and 5 different occasions (N=5) where 16 to 20 fertile eggs were injected per treatment group per set of injections for theoretical stage 44.
2.1. In ova treatments 2.3. Endogenous levels of brain HoCys, SAM, and SAH In order to determine if exogenous folic acid ameliorated EtOHinduced changes in brain HoCys, SAM, and/or SAH levels, the air sac of control eggs was injected with approximately 25 μL of a 1:1 mixture of H2O
Isolation and quantification of HoCys were achieved by modifying the technique of Yi et al. (2000). Embryonic brains were homogenized
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in 500 μL of ice-cold 50 mM HEPES buffer (pH 7.4), 1 mM DTT, 0.1 mM EDTA and non-soluble material was removed by centrifugation at 10,000 ×g (5 min at 4 °C). Supernatants were deproteinated by the addition of 400 μL of 0.4 M perchloric acid (PCA). The proteincontaining precipitates were removed by centrifugation at 10,000 ×g (5 min at 4 °C) and the pH was readjusted to approximately 6.5 to 7.5 by adding 15 μL of 6 M NaOH to each supernatant. HoCys and methionine derivatives were prepared as previously described (Miller et al., 2006; Walcher and Miller, 2008; Yi et al., 2000). The separation and quantification of HoCys derivative were achieved by high-pressure liquid chromatography using a 15 cm×4.6 mm Discovery Bio-Wide Pore C 18 HPLC column (5 μm beads) (Supelco, Inc., Bellefonte, PA, catalog number 568222-U) on a SCM 1000 high-pressure liquid chromatography system (HPLC) equipped with a FL-3000 fluorescence detector (Thermo Electron, Corp, Austin, TX). The mobile phase was 50 mM sodium monophosphate, 1 mM octane sulfonic acid, 2% acetonitrile, and adjusted to pH 2.7 with 85% phosphoric acid. The isocratic flow rate was 1 mL/min (1800 to 2100 psi). The excitation wavelength of the FL-3000 fluorescence detector was 230 nm and the emission wavelength was 418 nm. Using these HPLC conditions, the retention time of HoCys was 2.254±0.037 min (Miller et al., 2006). HoCys concentrations within biological samples were determined from a standard curve prepared from known concentrations of D-, L-homocysteine (0 to 10 μmol HoCys) (Sigma-Aldrich Group; St. Louis, MO). Brain HoCys levels were measure in 14 to 19 embryos per group (N=14 to 19) and these embryos were dedicated solely to the measurement of HoCys. The separation and quantification of SAM and SAH were achieved by modifying the technique of Yi et al. (2000). Embryonic brains were homogenized in 500 μL of ice-cold 50 mM HEPES buffer (pH 7.4), 1 mM DTT, 0.1 mM EDTA and non-soluble material is removed by centrifugation at 10,000 ×g (5 min at 4 °C). Aliquots of 200 μL were deproteinated by adding 40 μL of 40% tricloroacetic acid followed by incubation on ice for 30 min. The protein-containing precipitates were removed by centrifugation at 15,000 ×g (15 min at 4 °C) and supernatants were filtered through 0.2 μm Millex-FG13 PTFE filters (Sigma Chemical Co., St. Louis, MO, catalog number Z227536). The separation and quantification of SAM and SAH were achieved by HPLC using a 15 cm×4.6 mm Discovery Bio-Wide Pore C 18 HPLC column (5 μm beads) (Supelco, Inc., Bellefonte, PA, catalog number 568222-U) using a UV–VIS detector (UV-2000 detector; Thermo Electron, Corp, Austin, TX) set at a wavelength of 254 nm. The mobile phase was 50 mM sodium mono-phosphate, 10 mM 1-heptane sulfonic acid, 7. 5% methanol, and adjusted to pH 3.4 with 85% phosphoric acid. The isocratic flow rate was 1 mL/min (1800 to 2100 psi). Using these HPLC conditions, the retention time for SAH was 1.912±0.007 min and the retention time for SAM was 2.221 ± 0.061 min (Miller et al., 2006). SAH and SAM concentrations within biological samples were determined from standard curves prepared from known concentrations of SAH and SAM (0 to 350 nmol) (Sigma-Aldrich Group; St. Louis, MO). Brain SAM and SAH levels were measured in 18 to 23 embryos (N=18 to 23) per group and these embryos were dedicated solely to the measurement of SAM and SAH levels. 2.4. 10-FTHF DH activities in brain and liver tissues Embryonic chick brains and livers were homogenized in 400 μL of 50 mM Tris–HCl, pH 7.7 and non-soluble material was removed by centrifugation at 10,000 ×g for 5 min at 4 °C. Aliquots from each supernatant were removed and total protein levels were measured (Bradford, 1976). Aliquots from each supernatant were removed and 10-FTHF DH activities were measured according to Kutchback and Stokstad (1971). Each 1 mL reaction mixture contained 50 mM Tris– HCl, pH 7.7, 100 mM 2-mercaptoethanol, 100 μM NADP+, and 12 μM (6R, S)-10-FTHF. The conversion of 10-FTHF to THF was spectrophotometrically measured by absorption at 300 nm and a millimolar extinction coefficient (corrected for the contribution of NADPH) of
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22.6 cm− 1 was used (Min et al., 2005). Addition of NADP+ to the reaction mixture provides measurement of both dehydrogenase and hydrolase activities. Hydrolase activities were measured in the absence of NADP+ and were subtracted from total dehydrogenase and hydrolase activities to provide 10-FTHF DH activities (Krupenko et al., 2001) and are expressed as µmol of THF formed/min/mg protein. Brain 10-FTHF DH activities were measured in 6 to 8 embryos per group (N = 6 to 8) and hepatic 10-FTHF DH activities were measured in 5 to 6 dependent embryos (N = 5 to 6) per group. All embryos used in the measurement of 10-FTHF DH activities were dedicated solely to the measurement of 10-FTHF DH activities. 2.5. Brain and hepatic taurine levels Brain and hepatic taurine levels were measured by modifying the technique of Waterfield (1994). Brains and livers were homogenized and deproteinized in 500 μL of cold 0.2 M 5-sulfosialic acid and nonsoluble material was removed by centrifugation at 11,500 ×g for 2 min at 4 °C. After 40 μL of 100 μM homoserine was added to each supernatant as an internal standard, supernatants were layered onto dual-bed columns containing 1 mL of Dowex 1 × 4 resin over 3 mL of Dowex-50W-8 resin in 12 mL reservoirs (Restek Corp, Bellfonte, PA, USA; catalog number 26013) fitted with 1.5 cm frits (Restek Corp, Bellfonte, PA; catalog number 26019). Columns were pre-washed with 12 mL of 1 M HCl. Taurine and homoserine were then eluted from the columns with 4 mL of H2O. OPA (o-pthaladehyde) derivatives were then synthesized according to Waterfield (1994) and filtered through 0.2 μm Millex-FG13 PTFE filters (Sigma-Aldrich Group; St. Louis, MO, USA; catalog number Z227536). Aliquots of 20 μL were injected onto a 15 cm × 4.6 mm Discovery Bio-Wide Pore C 18 HPLC column (5 μm beads) (Supelco, Inc., Bellefonte, PA, catalog number 568222-U) a SCM 1000 high-pressure liquid chromatography system (HPLC) equipped with using dual detectors including a FL-3000 fluorescence detector and a UV–VIS detector (UV-2000 detector; Thermo Electron, Corp, Austin, TX) set at a wavelength of 250 nm. The excitation wavelength of the FL-3000 fluorescence detector was 350 nm and the emission wavelength was 476 nm (Mou et al., 2002). The mobile phase was 0.05 M NaH2PO4, pH 5.4 mixed with a 1:1 solution of methanol–water (43: 57, v/v). The isocratic flow rate was 1 mL/min (1900 to 2100 psi). Using these HPLC conditions, the retention time of homoserine (internal standard) was 3.683 ± 0.299 min and the retention time of taurine was 4.504 ± 0.287 min. Brain taurine levels were measured in 6 to 8 embryos (N = 6 to 8) per group and hepatic taurine levels were measured in 5 to 6 dependent embryos. These embryos were dedicated solely to the measurement of taurine levels. 2.6. Statistical analyses Embryo viability is partially reported as % living embryos. Because percentages may form binomial distributions rather than normal distributions, all % living embryo data were subjected to arcsine transformations prior to analysis of variance (ANOVA) followed by posthoc Tukey tests (Honestly Significant Differences tests) (Zar, 1999). All other data sets were not subjected to arcsine transformation; these data sets were analyzed by ANOVA followed by post-hoc Tukey tests (Honestly Significant Differences tests) and the level of significance was p≤0.05 (Zar, 1999). 3. Results 3.1. Embryo viability Three consecutive EtOH dosages (E0–2) caused significant reductions in % living embryos, reduced embryo masses, and reduced embryonic brain masses as compared to controls at theoretical stage 37 (Table 1).
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Table 1 Effect of exogenous EtOH and/or folate on chick embryo viability at theoretical stage 37. Treatments
% living embryos
Embryo mass (g)
Brain mass (mg)
Controls
82.47 ± 1.52 N=3 58.67 ± 5.72a N=3 78.20 ± 12.99 N=3 53.11 ± 11.39a N=3 7.36 3, 8 0.01
3.031 ± 0.198 N = 23 2.421 ± 0.288a N = 19 2.725 ± 0.189a N = 26 2.386 ± 0.200a N = 17 33.39 3, 81 0.0001
591 ± 83 N = 19 451 ± 105a N = 14 529 ± 89 N = 17 551 ± 89 N = 17 6.67 3, 70 0.0005
EtOH Folate EtOH and folate ANOVA
F= df = p≤
Data represented as mean ± standard deviation. a Experimental group significantly differs as compared to control group at p ≤ 0.05.
Table 3 Effect of exogenous EtOH and/or folate on brain HoCys, SAM, and SAH levels at theoretical stage 37. Treatments
nmol HoCys/mg brain
nmol SAM/mg brain
nmol SAH/mg brain
SAM/SAH
Controls
307 ± 163 N = 19 500 ± 174a N = 13 335 ± 201 N = 22 393 ± 175 N = 14 3.37 3, 64 0.02
30 ± 14 N = 23 5 ± 7a N = 19 8 ± 12a N = 21 9 ± 15a N = 18 16.82 3, 77 0.0001
572 ± 179 N = 23 772 ± 214a N = 19 632 ± 196 N = 21 706 ± 293 N = 18 3.23 3, 77 0.03
0.063 ± 0.040 N = 23 0.007 ± 0.010a N = 19 0.027 ± 0.051 N = 21 0.035 ± 0.040 N = 18 5.01 3,77 0.003
EtOH Folate EtOH and folate ANOVA F = df = p≤
Data represented as mean ± standard deviation. a Experimental group significantly differs as compared to control group at p ≤ 0.05.
Exogenous folate failed to ameliorate EtOH-induced reductions in % living embryos and promoted reduced embryo masses. Both EtOHtreated and EtOH and folate-treated embryos had significantly reduced % living embryos and reduced embryo masses as compared to controls (Table 1). In theoretical stage 44 embryos, a single dosage of EtOH (E0), two daily dosages of EtOH (E0–1), and three daily dosages of EtOH (E0–2) all caused significant reductions in embryo masses and brain masses as compared to controls (Table 2). Two daily dosages of EtOH (E0–1) and three daily dosages of EtOH (E0–2) also caused significant reductions in % living embryos and liver masses as compared to controls (Table 2). Clearly, embryonic EtOH exposure reduces chick embryo viability.
3.2. Endogenous levels of brain HoCys, SAM, and SAH While folate-treated embryos had control brain HoCys levels, three daily EtOH dosages (E0–2) caused significant increases in brain HoCys levels, reduced SAM levels, and increased brain SAH levels when compared to controls at theoretical stage 37 (Table 3). Statistically significant EtOH-induced reductions in brain SAM levels and EtOHinduced increased brain SAH levels were reflected in EtOH-induced reductions in SAM/SAH levels as compared to controls. Exogenous folate ameliorated EtOH-induced increases in brain HoCys levels because folate and EtOH-treated embryos had control brain HoCys levels, while EtOHtreated embryos had significantly elevated brain HoCys levels. While both folate-treated and EtOH and folate-treated embryos had significantly lower SAM levels as compared to controls, SAH and SAM/SAH levels were at control levels.
3.3. 10-FTHF DH activities in brain and liver tissues While a single dosage of EtOH (E0), two daily dosages of EtOH (E0–1), and three daily dosages of EtOH (E0–2) all failed to influence brain and hepatic 10-FTHF hydrolase activities (date not presented), three daily dosages of EtOH (E0–2) caused significantly increased brain and hepatic 10-FTHF DH activities as compared to controls at theoretical stage 44 (Table 4). The three daily dosages of EtOH (E0–2) caused approximately a two-fold increase in brain 10-FTHF DH activities as compared to controls (p ≤ 0.05) and approximately a three-fold increase in hepatic 10-FTHF DH activities as compared to controls (p ≤ 0.05; Table 4). Neither a single dosage of EtOH (E0) nor two daily dosages of EtOH (E0–1) influenced 10FTHF DH activities as compared to controls in both brain and hepatic tissues. 3.4. Brain and hepatic taurine levels While chronic EtOH (E0–2) exposure promoted reduced taurine levels in both brain and hepatic tissues (Table 4), hepatic tissues were more responsive as compared to brain. In hepatic tissues, all EtOH injection schemes, that is, a single EtOH dosage (E0), two daily EtOH dosages (E0–1), or three daily EtOH dosages (E0–2), caused significantly reduced taurine levels as compared to controls (Table 4). These EtOHinduced reductions in hepatic taurine levels ranged from 26 to 31% as compared to controls. When hepatic taurine levels were correlated with dependent hepatic masses in all groups, the Pearson productmoment coefficient (r) was −0.54 [F = (1, 20) 8.07; p b 0.01]. Only chronic EtOH (E0–2) exposure caused a significant 32% reduction in
Table 2 Effect of exogenous EtOH on chick embryo viability at theoretical stage 44. Treatments
% living embryos
Embryo mass (g)
Brain mass (mg)
Liver mass (mg)
Controls
88.80 ± 3.77 N=5 77.12 ± 8.36 N=5 70.80 ± 5.07a N=5 51.42 ± 16.11a N=5 14.74 3, 16 0.0001
22.836 ± 1.512 N = 20 20.467 ± 1.456a N = 20 20.555 ± 1.515a N = 20 16.714 ± 3.751a N = 20 25.43 3, 76 0.0001
708 ± 50 N = 20 584 ± 66a N = 20 478 ± 81a N = 13 381 ± 97a N = 15 63.72 3, 64 0.0001
483 ± 75 N = 10 405 ± 59 N = 11 361 ± 93a N = 11 478 ± 81a N = 13 7.11 3, 39 0.0006
EtOH–E0 EtOH–E0–1 EtOH–E0–2 ANOVA
F= df = p≤
EtOH–E0 eggs were injected with 3.0 mmol EtOH/kg egg on 0 days of development. EtOH–E0–1 eggs were injected with 3.0 mmol EtOH/kg egg on 0 and 1 days of development. EtOH–E0–2 eggs were injected with 3.0 mmol EtOH/kg egg on 0, 1, and 2 days of development. Data represented as mean ± standard deviation. a Experimental group significantly differs as compared to control group at p ≤ 0.05.
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Table 4 Effect of EtOH on 10-FTHF dehydrogenase activities and taurine levels in brain and hepatic tissues at theoretical stage 44. Treatments
Brain 10-FTHF dehydrogenase activities (µmol/ min/mg protein)
nmol taurine/g brain
Hepatic 10-FTHF dehydrogenase activities (µmol/ min/mg protein)
nmol taurine/g liver
Controls
561 ± 174 N=7 593 ± 156 N=6 785 ± 420 N=8 1198 ± 597a N=8 3.99 3, 25 0.02
1582 ± 472 N=8 1182 ± 346 N=8 1169 ± 173 N=9 1084 ± 113a N=6 3.79 3, 25 0.02
263 ± 103 N=4 271 ± 159 N=5 158 ± 94 N=5 774 ± 335a N=4 8.93 3, 14 0.002
1678 ± 281 N=5 1237 ± 268a N=6 1219 ± 184a N=5 1157 ± 244a N=6 4.83 3. 18 0.01
EtOH–E0 EtOH–E0–1 EtOH–E0–2 ANOVA
F= df = p≤
EtOH–E0 eggs were injected with 3.0 mmol EtOH/kg egg on 0 days of development. EtOH–E0–1 eggs were injected with 3.0 mmol EtOH/kg egg on 0 and 1 days of development. EtOH–E0–2 eggs were injected with 3.0 mmol EtOH/kg egg on 0, 1, and 2 days of development. Data represented as mean ± standard deviation. a Experimental group significantly differs as compared to control group at p ≤ 0.05.
brain taurine levels as compared to controls. While a single EtOH dosage (E0) and two daily EtOH dosages (E0–1) caused reduced brain taurine levels as compared to controls, the differences were insignificant (Table 4). Brain taurine levels failed to significantly correlate to brain masses. 4. Discussion Embryonic EtOH exposure caused elevated brain HoCys levels and reduced brain SAM/SAH levels as compared to controls at theoretical stage 37 (11 days of development) (Table 3). EtOH-induced increased brain HoCys levels and EtOH-induced decreases in brain SAM/SAH levels were attenuated by exogenous folate because EtOH and folatetreated embryos had control brain HoCys levels and control brain SAM/ SAH levels (Table 3). EtOH-induced increased brain HoCys levels correlated with reduced embryo viability as measured by EtOH-induced reductions in embryo masses, and brain masses (Table 1). When brain HoCys levels were correlated to embryo masses in all dependent groups, the Pearson product-moment coefficient (r) was −0.58 [F = (1, 58) 30.29; p b 0.0001] and when brain HoCys levels were correlated to brain masses in all dependent groups, the correlation coefficient (r) was −0.32 [F = (1, 58) 6.19; p b 0.01]. As predicted by the metabolism of SAM to SAH and SAH to HoCys (Selhub,1999), brain HoCys levels correlated to SAM/SAH levels in all independent groups [r = −0.28; F = (1, 58) 5.25; p b 0.03]. While three daily dosages of (34 μmol folic acid/kg; 15 mg of folic acid/kg egg) attenuated EtOH-induced increased brain HoCys levels and EtOH-induced reductions in brain SAM/SAH levels (Table 2), exogenous folate failed to ameliorate EtOH-induced reduced % living embryos levels and EtOH-induced reduced embryo masses at stage 37 (Table 1). Xu et al. (2006) reported that daily maternal ingestion of 60 mg of folic acid/kg and 1.0 mg of vitamin B12 (cobalamine)/kg ameliorated many of the EtOH-induced adverse effects in mice fetuses. The folic acid dosage of Xu et al. (2006) (60 mg/kg) is higher than the dosage we used (15 mg folic acid/kg egg) and the EtOH dosage of Xu et al. (2006) (5 g/kg or 108.7 mmol EtOH/kg) is greater than the EtOH dosage we used (3.0 mmol EtOH/kg egg). Thus, species-specific response differences exist. This report and a previous report (Walcher and Miller, 2008) have demonstrated that embryonic EtOH exposure caused elevated HoCys levels in developing chick brains at theoretical stage 37. Thus, the key question now is how does EtOH exposure promote hyperhomocysteinemia? Reduced ingestion of food, common in alcoholics, can cause folate deficiencies (Cravo et al., 1996; Eichner and Hillman, 1973) and folate deficiencies can cause elevated levels of HoCys (Selhub, 1999). However,
this mechanism seems unlikely in our study because chick embryos develop in a closed system where yolk folate levels remain relatively constant. However, folate deficiencies can result from decreased intestinal absorption rates and/or increased excretion rates of folates as observed in adult humans, rats, and pigs (Halsted, 1992; McMartin et al., 1989; Reisenauer et al., 1989). This last mechanism is possible in chick embryos and deserves further study. Another possible hypothesis in explaining hyperhomocysteinemia is EtOH-impaired remethylation of HoCys to methionine that requires methyl group donation from either betaine, or 5-methyltetrahydrofolate (5-methyl THF) via vitamin B12 (cobalamine). Dietary betaine supplementation ameliorated EtOH-induced hepatic steatosis (Kharbanda et al., 2007) in adult rats and EtOH-induced and HoCys-induced decreased phosphatidylcholine levels and increased phosphatidylethanolamine levels have been reported in embryonic chick brains (Miller et al., 1998; 2003b). Recently, Ji et al. (2008) created transgenic mice by insertion of a vector containing the structural gene coding for human betainehomocysteine methyltransferase (BHMT). When these transgenic mice and wild-type mice were infused with EtOH for a period of four weeks, plasma alanine transaminase activities increased by 5-fold in wild-type mice and only 2.7-fold in the transgenic mice; plasma HoCys levels increased by 7-fold in wild-type mice and only 2-fold in the transgenic mice. Another possible control point within the remethylation pathway is 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH). Im et al. (1999) and Min et al. (2005) reported EtOH inhibited 10-FTHF DH activities in adult rats. This enzyme irreversibly catalyzes the conversion of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate (THF). In the glycine cleavage system, T-protein requires THF as a coenzyme and synthesizes N5, N10-methylene-tetrahydrofolate which is later converted to 5-methyl tetrahydrofolate (5-methyl THF) (Kikuchi, 1973; van der Put et al., 2001). Because 5-methyl THF is a methyl donor used in the conversion of HoCys to methionine (Miller and Kelly, 1996), a failure to replenish 5-methyl THF levels could cause hyperhomocysteinemia. Surprisingly, we are reporting that three daily dosages of EtOH (E0–2) caused elevated 10-FTHF DH activities in both brain and hepatic tissues as compared to controls in theoretical stage 44 (18 days of development) chick embryos (Table 4). This late (stage 44) increase in 10-FTHF DH activities may be compensatory in nature and an attempt to reduce earlier (stage 37) hyperhomocysteinemia (Table 3). Elevated brain HoCys levels at stage 37 (11 days of development) are of interest because the earliest time for detectable alcohol dehydrogenase (ADH) activities in embryonic chick liver is 9 to 10 days of development (Wilson et al., 1984) and ADH activities are very low in both brain and hepatic tissues at 11 days of development (stage 37) (Miller et al., 1998). At 15 and
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18 days of development (stage 44), ADH activities increase by 3- to 6fold in both brain and hepatic chick tissues (Miller et al., 1998). Thus, the late (stage 44) EtOH-induced increase in 10-FTHF DH activities (Table 4) reported here may be occurring after EtOH has been metabolized from surviving chick tissues. It is now imperative to measure 10-FTHF DH activities at earlier developmental stages and correlate these activities to endogenous HoCys levels and ADH activities within chick tissues. We hypothesize that EtOH exposure may inhibit 10-FTHF DH activities in stage 37 embryos and 10-FTHF DH activities may correlate with EtOHinduced increases in brain and hepatic HoCys levels in stage 37 embryos. EtOH-induced inhibition of the transsulfuration pathway may also explain EtOH-induced hyperhomocysteinemia. In the transsulfuration pathway, HoCys is ultimately metabolized to taurine (Selhub, 1999). ten Busch et al. (1997) reported that EtOH-treated chick embryos had lower taurine levels in the allantoic fluid at 13 days of development (theoretical stage 39; Hamburger and Hamilton, 1951) as compared to controls. We extended the observations of ten Busch et al. (1997) and are reporting that chronic EtOH (E0–2) exposure caused reduced taurine levels as compared to controls in stage 44 chick brains (Table 4). Meanwhile, a single EtOH dosage (E0), two daily EtOH dosages (E0–1), or three daily EtOH dosages (E0–2) caused reduced hepatic taurine levels as compared to controls in stage 44 embryos (Table 4). When the transsulfuration pathway is impaired, as in homozygous cystathionine β-synthetase defect, HoCys is diverted to the remethylation pathway causing momentary elevated SAM levels which can cause feedback inhibition of the remethylation enzyme, methylenetetrahydrofolate reductase (Selhub, 1999). As both the remethylation and transsulfuration pathways slow, HoCys levels rise. 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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c
Exogenous folate ameliorates ethanol-induced brain hyperhomocysteinemia and exogenous ethanol reduces taurine levels in chick embryos Robert K. Barnett, Stephanie L. Booms, Tracy Gura, Mara Gushrowski, Robert R. Miller Jr. ⁎ Hillsdale College, Biology Department, 278 N. West Street, Hillsdale, MI 49242-1205, USA
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Article history: Received 24 November 2008 Received in revised form 26 March 2009 Accepted 28 March 2009 Available online 1 April 2009 Keywords: Ethanol Homocysteine S-adenosylmethionine S-adenosylhomocysteine Folate Taurine 10-Formyltetrahydrofolate dehydrogenase Chick
a b s t r a c t The effects of exogenous ethanol and/or folic acid on endogenous homocysteine (HoCys) and SAM (Sadenosylmethionine)/SAH (S-adenosylhomocysteine) levels in chick brains were studied at 11 days of development. Embryonic EtOH (3.0 mmol/kg egg) exposure caused a 1.6-fold increase in brain HoCys levels and a 9-fold decrease in brain SAM/SAH levels as compared to controls (p≤0.05). Brain HoCys and SAM/SAH levels returned to control values when injected with a mixture of EtOH and folic acid (3.0 mmol EtOH/kg egg and 34 μmol folic acid/kg egg). The effects of exogenous EtOH on the remethylation pathway, as measured by 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH) activities, and the transsulfuration pathway, as measured by taurine levels, were studied at 18 days of development. A single dosage of EtOH (3.0 mmol/kg egg; E0) and two daily dosages of EtOH (E0–1) failed to influence brain and hepatic 10-FTHF DH activities when compared to controls. However, three daily dosages of EtOH (E0–2) caused approximately a two-fold increase in brain 10-FTHF DH activities and a three-fold increase in hepatic 10-FTHF DH activities as compared to controls (p≤0.05). Three daily EtOH dosages (E0–2) caused reduced taurine levels in both brain and hepatic tissues (p≤0.05). Meanwhile, a single EtOH dosage (E0), two daily EtOH dosages (E0–1), and three daily EtOH dosages (E0–2), caused reduced hepatic taurine levels as compared to controls (p≤0.05). © 2009 Elsevier Inc. All rights reserved.
1. Introduction Ethanol (EtOH) and homocysteine (HoCys) are both teratogenic in chick embryos and impair brain development. Embryonic EtOH exposure caused reduced brain masses, reduced brain neuron densities (Miller et al., 1996, 2000, 2003a; Miller, 2004), and elevated brain caspase-3 activities (Miller et al., 2003a; Miller, 2004). EtOH-impaired brain development correlated with EtOH-induced increased brain lipid hydroperoxide levels and EtOH-induced reduced brain membrane long-chain polyunsaturated fatty acids (PUFAs) levels (Miller et al., 1996, 2000, 2003a,b; Miller, 2004). Walcher and Miller (2008) reported that exogenous EtOH also caused elevated HoCys levels, reduced S-adenosylmethionine (SAM) levels, and increased S-adenosylhomocysteine (SAH) levels within brains at theoretical stage 37 (11 days of chick development; Hamburger and Hamilton, 1951). These EtOH-induced reductions in SAM/SAH levels correlated with EtOH-induced reduced brain masses (r=0.62, p≤0.0001) and with EtOH-induced increased brain caspase-3 activities (r=−0.39, p≤0.05) (Walcher and Miller, 2008). In adults, EtOH caused elevated HoCys levels in actively drinking alcoholics (Bleich et al., 2003, 2005; Sakuto and Abbreviations: DTT, Dithiotreitol; EtOH, Ethanol; EDTA, Ethylenediamine-tetraacetic acid; 10-FTHF DH, Formyltetrahydrofolate dehydrogenase; HEPES, N-[2-hydroxyethyl] piperazine-N′ [2-ethanesulfonic acid]; HoCys, Homocysteine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, 5-Methyl tetrahydrofolate 5-Methyl THF; THF, Tetrahydrofolate. ⁎ Corresponding author. Hillsdale College, Biology Department, Dow 213, 278 N. West St., Hillsdale, MI 49242-1205, USA. Tel.: +1 517 607 2393; fax: +1 517 607 2252. E-mail address: [email protected] (R.R. Miller). 1532-0456/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2009.03.005
Suzuki, 2005; Wilhelm et al., 2006) and hyperhomocysteinemia in early alcohol-abstinent patients correlated with impaired cognitive abilities (Wilhelm et al., 2006). Exogenous HoCys also impairs chick brain development (Rosenquist et al., 1996, 1999) and promoted reduced brain masses, and elevated brain caspase-3 activities (Miller et al., 2003b, 2006). Exogenous HoCys caused elevated brain lipid hydroperoxide levels, reduced brain membrane long-chain PUFAs levels, and increased saturated shortchain fatty acids levels within chick brain membranes (Miller et al., 2003b, 2006). Because HoCys is an antagonist of the glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) (Lipton et al., 1997), Rosenquist et al. (1999) hypothesized that HoCys-induced teratogenesis in chick embryos may be caused by HoCys-induced inhibition of the glycine-binding site of the chick NMDAR. To challenge this hypothesis, Rosenquist et al. (1999) demonstrated that exogenous glycine attenuated HoCys-induced neural crest cell defects and neural tube defects in embryonic chicks. Miller et al. (2006) demonstrated that exogenous glycine partially attenuated HoCys-induced increases in embryonic chick lipid hydroperoxide levels in both brain and liver tissues, partially attenuated HoCys-induced decreases in brain membrane long-chain polyunsaturated fatty acids, and partially attenuated HoCys-induced increases in brain and liver caspase-3 activities. HoCys metabolism intersects two pathways. These pathways include the remethylation of HoCys to methionine, which requires the transfer of a methyl group from either betaine or 5-methyltetrahydrofolate (5-methyl THF) via vitamin B12 (cobalamine), and the transsulfuration of HoCys to cystathionine and subsequent conversion to taurine (Selhub, 1999).
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Interest in the remethylation pathway exists because folate deficiencies have not only been observed in alcoholics (Cravo et al., 1996; Halsted, 1992; Hebert et al., 1963), but also have been associated with hyperhomocysteinemia (Selhub, 1999). In adult rats, EtOH inhibited 10formyltetrahydrofolate dehydrogenase (10-FTHF DH; EC 1.5.1.6) activities (Im et al., 1999; Min et al., 2005). This cytoplasmic enzyme irreversibly catalyzes the conversion of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate (THF). In the glycine cleavage system within the remethylation pathway, T-protein requires THF as a coenzyme and synthesizes N5, N10-methylene-tetrahydrofolate which is converted to the methyl donor 5-methyl THF (Kikuchi, 1973; van der Put et al., 2001). Low 5-methyl THF levels could not only inhibit the remethylation of HoCys to methionine, but also alter SAM/SAH levels because methionine is used to produce SAM, SAM is converted to SAH, and SAH is hydrolyzed to HoCys (Selhub, 1999). The relative contribution of the remethylation pathway in promoting hyperhomocysteinemia in EtOH-treated chick brains is questionable. If an active glycine cleavage system within the remethylation pathway exists in embryonic chicks, one would predict that exogenous glycine should ameliorate EtOH-induced increases in HoCys, EtOH-induced decreases in SAM, and EtOH-induced increases in SAH. However, Walcher and Miller (2008) reported that exogenous glycine failed to attenuate EtOH-induced increased brain HoCys levels. However, exogenous glycine partially ameliorated EtOH-induced decreased SAM levels, increased SAH levels, and decreased SAM/SAH levels in embryonic chick brains (Walcher and Miller, 2008). In the transsulfuration pathway, HoCys condenses with serine to form cystathionine. This irreversible reaction is catalyzed by a pyridoxial5′-phosphate (vitamin B6)-containing enzyme, cystathionine β-synthetase. Cystathionine is hydrolyzed by another pyridoxial-5′-phosphatecontaining enzyme, γ-cystathioninase, thus forming α-ketobutyrate and cysteine and excess cysteine is converted to taurine (Selhub, 1999). ten Busch et al. (1997) reported that EtOH-treated chick embryos had lower allantoic taurine levels at 13 days of development (theoretical stage 39; Hamburger and Hamilton, 1951) as compared to controls. This observation is of interest because Kerai et al. (1998) reported that dietary taurine attenuated EtOH-induced hepatic steatosis and hepatic lipid peroxidation in Sprague–Dawley rats and Nonaka et al. (2001) reported that exogenous taurine attenuated HoCys-induced inhibition of extracellular-superoxide dimutase m-RNA expression in cultured rat vascular smooth muscle cells. Thus, the objectives of these studies were as follows. First, did exogenous folic acid attenuate EtOH-induced increased HoCys levels, EtOH-induced decreased SAM levels, and EtOH-induced increased SAH levels in embryonic chick brains? Secondly, did exogenous EtOH alter 10-formyltetrahydrofolate dehydrogenase activities (10-FTHF DH; EC 1.5.1.6) in embryonic chick brains and/or livers? Finally, did exogenous EtOH promote reduced taurine levels in embryonic chick brains and/or livers?
and avian saline (0.72% NaCl; w/v) during the first three days of development (E0–2). Meanwhile, experimental eggs were injected with approximately 25 μL of either a 1:1 mixture of 95% EtOH and avian saline, pH 7.2 (3.0 mmol/EtOH/kg egg); or a 1:1 mixture of 181 μM folic acid in avian saline with H2O (34 μmol folic acid/kg egg), or a 1:1 mixture of 95% EtOH and 181 μM folic acid in avian saline (3.0 mmol EtOH/kg egg and 34 μmol folic acid/kg egg) during the first three days of development (E0–2). The dosage of 34 μmol folic acid/kg egg (15 mg/kg) was selected because Gareskog et al. (2006) injected 15 mg folic acid/kg (34 μmol folic acid/kg) into pregnant dames and reported that this dosage ameliorated hyperglycemia-induced oxidative stress in rat embryos. Like exogenous EtOH and HoCys, hyperglycemic chick embryos also exhibited reduced brain masses, elevated brain caspase-3 activities, undergo brain membrane lipid peroxidation, and have elevated brain HoCys levels (Cole et al., 2008; Miller et al., 2005). All embryos were incubated in a forced air incubator (model 1202; GQF Manufacturing, Savannah, GA) at 37.5 °C and turned every 4 h with a relative humidity ranging from 80–90%. At 11 days of incubation (theoretical stage 37; Hamburger and Hamilton, 1951), chick embryos were decapitated, brains excised, and stored at −80 °C, until subsequent brain HoCys, SAM, and SAH analyses. Theoretical stage 37 (11 days of development) was selected because we have previously measured brain HoCys, SAM, and SAH levels at this developmental stage (Cole et al., 2008; Walcher and Miller, 2008). In order to determine if exogenous EtOH influenced either 10-FTHF DH activities or taurine levels within chick brain and hepatic tissues, control eggs were injected with approximately 25 μL of a 1:1 mixture of H2O and avian saline (0.72% NaCl; w/v) into the air sac at 0 days of development. Meanwhile, experimental eggs were injected with approximately 25 μL of a 1:1 mixture of 95% EtOH and avian saline (3.0 mmol/EtOH/kg egg) into the air sac at 0 days of development (E0), at 0 and 1 days of development (E0–1), and at 0, 1, and 2 days of development (E0–2). All embryos were incubated in a forced air incubator (model 1202; GQF Manufacturing, Savannah, GA) at 37.5 °C and turned every 4 h with a relative humidity ranging from 80–90%. At 18 days of incubation (theoretical stage 44; Hamburger and Hamilton, 1951), chick embryos were decapitated, brains and livers excised, and stored at −80 °C until subsequent 10-FTHF DH or taurine assays. Because we are unaware that 10-FTHF DH activities have been characterized across various stages of chick development, we selected theoretical stage 44 (18 days of development) over theoretical stage 37 (11 days of development) because 18 days of chick development is only three days away from hatching and embryos at this late stage should have higher enzyme activities and be near neonatal 10-FTHF DH activities as compared to earlier stage 37 embryos (11 days of development). Taurine levels in theoretical stage 44 (18 days of development) embryos were measured so we could correlate taurine levels (a transsulfuration product) to 10-FTHF DH activities (a remethylation pathway enzyme).
2. Materials and methods 2.2. Embryo viability All experiments were performed using fertile, specific pathogen-free white Leghorn chicken eggs (Gallus gallus) purchased from Hy-Vac International (Adel, IA, USA). Chickens raised at this commercial hatchery ingest a standardized diet causing minimal differences in yolk composition from generation to generation. Discovery Bio-wide Pore C 18 HPLC columns were purchased from Supelco, Inc., Bellefonte, PA, USA). The disodium salt of (6R, S), (6R)-10-formyltetrahydrofolate was the generous gift of Merck Epova Ag. (Schaffhausen, Switzerland). All other reagents were purchased from the Sigma-Aldrich Group (St. Louis, MO, USA) and Thermo-Fisher (Itasca, IL, USA).
Embryo viability was indirectly monitored by measurements of embryo masses, brain masses, and % living embryos at 11 days of development (theoretical stage 37) and at 18 days of development (theoretical stage 44; Hamburger and Hamilton, 1951). Living embryos were defined as possessing a beating heart during the dissections and injections were performed on 3 different occasions (N=3) where 16 to 20 fertile eggs were injected per treatment group per set of injections for theoretical stage 37 and 5 different occasions (N=5) where 16 to 20 fertile eggs were injected per treatment group per set of injections for theoretical stage 44.
2.1. In ova treatments 2.3. Endogenous levels of brain HoCys, SAM, and SAH In order to determine if exogenous folic acid ameliorated EtOHinduced changes in brain HoCys, SAM, and/or SAH levels, the air sac of control eggs was injected with approximately 25 μL of a 1:1 mixture of H2O
Isolation and quantification of HoCys were achieved by modifying the technique of Yi et al. (2000). Embryonic brains were homogenized
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in 500 μL of ice-cold 50 mM HEPES buffer (pH 7.4), 1 mM DTT, 0.1 mM EDTA and non-soluble material was removed by centrifugation at 10,000 ×g (5 min at 4 °C). Supernatants were deproteinated by the addition of 400 μL of 0.4 M perchloric acid (PCA). The proteincontaining precipitates were removed by centrifugation at 10,000 ×g (5 min at 4 °C) and the pH was readjusted to approximately 6.5 to 7.5 by adding 15 μL of 6 M NaOH to each supernatant. HoCys and methionine derivatives were prepared as previously described (Miller et al., 2006; Walcher and Miller, 2008; Yi et al., 2000). The separation and quantification of HoCys derivative were achieved by high-pressure liquid chromatography using a 15 cm×4.6 mm Discovery Bio-Wide Pore C 18 HPLC column (5 μm beads) (Supelco, Inc., Bellefonte, PA, catalog number 568222-U) on a SCM 1000 high-pressure liquid chromatography system (HPLC) equipped with a FL-3000 fluorescence detector (Thermo Electron, Corp, Austin, TX). The mobile phase was 50 mM sodium monophosphate, 1 mM octane sulfonic acid, 2% acetonitrile, and adjusted to pH 2.7 with 85% phosphoric acid. The isocratic flow rate was 1 mL/min (1800 to 2100 psi). The excitation wavelength of the FL-3000 fluorescence detector was 230 nm and the emission wavelength was 418 nm. Using these HPLC conditions, the retention time of HoCys was 2.254±0.037 min (Miller et al., 2006). HoCys concentrations within biological samples were determined from a standard curve prepared from known concentrations of D-, L-homocysteine (0 to 10 μmol HoCys) (Sigma-Aldrich Group; St. Louis, MO). Brain HoCys levels were measure in 14 to 19 embryos per group (N=14 to 19) and these embryos were dedicated solely to the measurement of HoCys. The separation and quantification of SAM and SAH were achieved by modifying the technique of Yi et al. (2000). Embryonic brains were homogenized in 500 μL of ice-cold 50 mM HEPES buffer (pH 7.4), 1 mM DTT, 0.1 mM EDTA and non-soluble material is removed by centrifugation at 10,000 ×g (5 min at 4 °C). Aliquots of 200 μL were deproteinated by adding 40 μL of 40% tricloroacetic acid followed by incubation on ice for 30 min. The protein-containing precipitates were removed by centrifugation at 15,000 ×g (15 min at 4 °C) and supernatants were filtered through 0.2 μm Millex-FG13 PTFE filters (Sigma Chemical Co., St. Louis, MO, catalog number Z227536). The separation and quantification of SAM and SAH were achieved by HPLC using a 15 cm×4.6 mm Discovery Bio-Wide Pore C 18 HPLC column (5 μm beads) (Supelco, Inc., Bellefonte, PA, catalog number 568222-U) using a UV–VIS detector (UV-2000 detector; Thermo Electron, Corp, Austin, TX) set at a wavelength of 254 nm. The mobile phase was 50 mM sodium mono-phosphate, 10 mM 1-heptane sulfonic acid, 7. 5% methanol, and adjusted to pH 3.4 with 85% phosphoric acid. The isocratic flow rate was 1 mL/min (1800 to 2100 psi). Using these HPLC conditions, the retention time for SAH was 1.912±0.007 min and the retention time for SAM was 2.221 ± 0.061 min (Miller et al., 2006). SAH and SAM concentrations within biological samples were determined from standard curves prepared from known concentrations of SAH and SAM (0 to 350 nmol) (Sigma-Aldrich Group; St. Louis, MO). Brain SAM and SAH levels were measured in 18 to 23 embryos (N=18 to 23) per group and these embryos were dedicated solely to the measurement of SAM and SAH levels. 2.4. 10-FTHF DH activities in brain and liver tissues Embryonic chick brains and livers were homogenized in 400 μL of 50 mM Tris–HCl, pH 7.7 and non-soluble material was removed by centrifugation at 10,000 ×g for 5 min at 4 °C. Aliquots from each supernatant were removed and total protein levels were measured (Bradford, 1976). Aliquots from each supernatant were removed and 10-FTHF DH activities were measured according to Kutchback and Stokstad (1971). Each 1 mL reaction mixture contained 50 mM Tris– HCl, pH 7.7, 100 mM 2-mercaptoethanol, 100 μM NADP+, and 12 μM (6R, S)-10-FTHF. The conversion of 10-FTHF to THF was spectrophotometrically measured by absorption at 300 nm and a millimolar extinction coefficient (corrected for the contribution of NADPH) of
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22.6 cm− 1 was used (Min et al., 2005). Addition of NADP+ to the reaction mixture provides measurement of both dehydrogenase and hydrolase activities. Hydrolase activities were measured in the absence of NADP+ and were subtracted from total dehydrogenase and hydrolase activities to provide 10-FTHF DH activities (Krupenko et al., 2001) and are expressed as µmol of THF formed/min/mg protein. Brain 10-FTHF DH activities were measured in 6 to 8 embryos per group (N = 6 to 8) and hepatic 10-FTHF DH activities were measured in 5 to 6 dependent embryos (N = 5 to 6) per group. All embryos used in the measurement of 10-FTHF DH activities were dedicated solely to the measurement of 10-FTHF DH activities. 2.5. Brain and hepatic taurine levels Brain and hepatic taurine levels were measured by modifying the technique of Waterfield (1994). Brains and livers were homogenized and deproteinized in 500 μL of cold 0.2 M 5-sulfosialic acid and nonsoluble material was removed by centrifugation at 11,500 ×g for 2 min at 4 °C. After 40 μL of 100 μM homoserine was added to each supernatant as an internal standard, supernatants were layered onto dual-bed columns containing 1 mL of Dowex 1 × 4 resin over 3 mL of Dowex-50W-8 resin in 12 mL reservoirs (Restek Corp, Bellfonte, PA, USA; catalog number 26013) fitted with 1.5 cm frits (Restek Corp, Bellfonte, PA; catalog number 26019). Columns were pre-washed with 12 mL of 1 M HCl. Taurine and homoserine were then eluted from the columns with 4 mL of H2O. OPA (o-pthaladehyde) derivatives were then synthesized according to Waterfield (1994) and filtered through 0.2 μm Millex-FG13 PTFE filters (Sigma-Aldrich Group; St. Louis, MO, USA; catalog number Z227536). Aliquots of 20 μL were injected onto a 15 cm × 4.6 mm Discovery Bio-Wide Pore C 18 HPLC column (5 μm beads) (Supelco, Inc., Bellefonte, PA, catalog number 568222-U) a SCM 1000 high-pressure liquid chromatography system (HPLC) equipped with using dual detectors including a FL-3000 fluorescence detector and a UV–VIS detector (UV-2000 detector; Thermo Electron, Corp, Austin, TX) set at a wavelength of 250 nm. The excitation wavelength of the FL-3000 fluorescence detector was 350 nm and the emission wavelength was 476 nm (Mou et al., 2002). The mobile phase was 0.05 M NaH2PO4, pH 5.4 mixed with a 1:1 solution of methanol–water (43: 57, v/v). The isocratic flow rate was 1 mL/min (1900 to 2100 psi). Using these HPLC conditions, the retention time of homoserine (internal standard) was 3.683 ± 0.299 min and the retention time of taurine was 4.504 ± 0.287 min. Brain taurine levels were measured in 6 to 8 embryos (N = 6 to 8) per group and hepatic taurine levels were measured in 5 to 6 dependent embryos. These embryos were dedicated solely to the measurement of taurine levels. 2.6. Statistical analyses Embryo viability is partially reported as % living embryos. Because percentages may form binomial distributions rather than normal distributions, all % living embryo data were subjected to arcsine transformations prior to analysis of variance (ANOVA) followed by posthoc Tukey tests (Honestly Significant Differences tests) (Zar, 1999). All other data sets were not subjected to arcsine transformation; these data sets were analyzed by ANOVA followed by post-hoc Tukey tests (Honestly Significant Differences tests) and the level of significance was p≤0.05 (Zar, 1999). 3. Results 3.1. Embryo viability Three consecutive EtOH dosages (E0–2) caused significant reductions in % living embryos, reduced embryo masses, and reduced embryonic brain masses as compared to controls at theoretical stage 37 (Table 1).
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Table 1 Effect of exogenous EtOH and/or folate on chick embryo viability at theoretical stage 37. Treatments
% living embryos
Embryo mass (g)
Brain mass (mg)
Controls
82.47 ± 1.52 N=3 58.67 ± 5.72a N=3 78.20 ± 12.99 N=3 53.11 ± 11.39a N=3 7.36 3, 8 0.01
3.031 ± 0.198 N = 23 2.421 ± 0.288a N = 19 2.725 ± 0.189a N = 26 2.386 ± 0.200a N = 17 33.39 3, 81 0.0001
591 ± 83 N = 19 451 ± 105a N = 14 529 ± 89 N = 17 551 ± 89 N = 17 6.67 3, 70 0.0005
EtOH Folate EtOH and folate ANOVA
F= df = p≤
Data represented as mean ± standard deviation. a Experimental group significantly differs as compared to control group at p ≤ 0.05.
Table 3 Effect of exogenous EtOH and/or folate on brain HoCys, SAM, and SAH levels at theoretical stage 37. Treatments
nmol HoCys/mg brain
nmol SAM/mg brain
nmol SAH/mg brain
SAM/SAH
Controls
307 ± 163 N = 19 500 ± 174a N = 13 335 ± 201 N = 22 393 ± 175 N = 14 3.37 3, 64 0.02
30 ± 14 N = 23 5 ± 7a N = 19 8 ± 12a N = 21 9 ± 15a N = 18 16.82 3, 77 0.0001
572 ± 179 N = 23 772 ± 214a N = 19 632 ± 196 N = 21 706 ± 293 N = 18 3.23 3, 77 0.03
0.063 ± 0.040 N = 23 0.007 ± 0.010a N = 19 0.027 ± 0.051 N = 21 0.035 ± 0.040 N = 18 5.01 3,77 0.003
EtOH Folate EtOH and folate ANOVA F = df = p≤
Data represented as mean ± standard deviation. a Experimental group significantly differs as compared to control group at p ≤ 0.05.
Exogenous folate failed to ameliorate EtOH-induced reductions in % living embryos and promoted reduced embryo masses. Both EtOHtreated and EtOH and folate-treated embryos had significantly reduced % living embryos and reduced embryo masses as compared to controls (Table 1). In theoretical stage 44 embryos, a single dosage of EtOH (E0), two daily dosages of EtOH (E0–1), and three daily dosages of EtOH (E0–2) all caused significant reductions in embryo masses and brain masses as compared to controls (Table 2). Two daily dosages of EtOH (E0–1) and three daily dosages of EtOH (E0–2) also caused significant reductions in % living embryos and liver masses as compared to controls (Table 2). Clearly, embryonic EtOH exposure reduces chick embryo viability.
3.2. Endogenous levels of brain HoCys, SAM, and SAH While folate-treated embryos had control brain HoCys levels, three daily EtOH dosages (E0–2) caused significant increases in brain HoCys levels, reduced SAM levels, and increased brain SAH levels when compared to controls at theoretical stage 37 (Table 3). Statistically significant EtOH-induced reductions in brain SAM levels and EtOHinduced increased brain SAH levels were reflected in EtOH-induced reductions in SAM/SAH levels as compared to controls. Exogenous folate ameliorated EtOH-induced increases in brain HoCys levels because folate and EtOH-treated embryos had control brain HoCys levels, while EtOHtreated embryos had significantly elevated brain HoCys levels. While both folate-treated and EtOH and folate-treated embryos had significantly lower SAM levels as compared to controls, SAH and SAM/SAH levels were at control levels.
3.3. 10-FTHF DH activities in brain and liver tissues While a single dosage of EtOH (E0), two daily dosages of EtOH (E0–1), and three daily dosages of EtOH (E0–2) all failed to influence brain and hepatic 10-FTHF hydrolase activities (date not presented), three daily dosages of EtOH (E0–2) caused significantly increased brain and hepatic 10-FTHF DH activities as compared to controls at theoretical stage 44 (Table 4). The three daily dosages of EtOH (E0–2) caused approximately a two-fold increase in brain 10-FTHF DH activities as compared to controls (p ≤ 0.05) and approximately a three-fold increase in hepatic 10-FTHF DH activities as compared to controls (p ≤ 0.05; Table 4). Neither a single dosage of EtOH (E0) nor two daily dosages of EtOH (E0–1) influenced 10FTHF DH activities as compared to controls in both brain and hepatic tissues. 3.4. Brain and hepatic taurine levels While chronic EtOH (E0–2) exposure promoted reduced taurine levels in both brain and hepatic tissues (Table 4), hepatic tissues were more responsive as compared to brain. In hepatic tissues, all EtOH injection schemes, that is, a single EtOH dosage (E0), two daily EtOH dosages (E0–1), or three daily EtOH dosages (E0–2), caused significantly reduced taurine levels as compared to controls (Table 4). These EtOHinduced reductions in hepatic taurine levels ranged from 26 to 31% as compared to controls. When hepatic taurine levels were correlated with dependent hepatic masses in all groups, the Pearson productmoment coefficient (r) was −0.54 [F = (1, 20) 8.07; p b 0.01]. Only chronic EtOH (E0–2) exposure caused a significant 32% reduction in
Table 2 Effect of exogenous EtOH on chick embryo viability at theoretical stage 44. Treatments
% living embryos
Embryo mass (g)
Brain mass (mg)
Liver mass (mg)
Controls
88.80 ± 3.77 N=5 77.12 ± 8.36 N=5 70.80 ± 5.07a N=5 51.42 ± 16.11a N=5 14.74 3, 16 0.0001
22.836 ± 1.512 N = 20 20.467 ± 1.456a N = 20 20.555 ± 1.515a N = 20 16.714 ± 3.751a N = 20 25.43 3, 76 0.0001
708 ± 50 N = 20 584 ± 66a N = 20 478 ± 81a N = 13 381 ± 97a N = 15 63.72 3, 64 0.0001
483 ± 75 N = 10 405 ± 59 N = 11 361 ± 93a N = 11 478 ± 81a N = 13 7.11 3, 39 0.0006
EtOH–E0 EtOH–E0–1 EtOH–E0–2 ANOVA
F= df = p≤
EtOH–E0 eggs were injected with 3.0 mmol EtOH/kg egg on 0 days of development. EtOH–E0–1 eggs were injected with 3.0 mmol EtOH/kg egg on 0 and 1 days of development. EtOH–E0–2 eggs were injected with 3.0 mmol EtOH/kg egg on 0, 1, and 2 days of development. Data represented as mean ± standard deviation. a Experimental group significantly differs as compared to control group at p ≤ 0.05.
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Table 4 Effect of EtOH on 10-FTHF dehydrogenase activities and taurine levels in brain and hepatic tissues at theoretical stage 44. Treatments
Brain 10-FTHF dehydrogenase activities (µmol/ min/mg protein)
nmol taurine/g brain
Hepatic 10-FTHF dehydrogenase activities (µmol/ min/mg protein)
nmol taurine/g liver
Controls
561 ± 174 N=7 593 ± 156 N=6 785 ± 420 N=8 1198 ± 597a N=8 3.99 3, 25 0.02
1582 ± 472 N=8 1182 ± 346 N=8 1169 ± 173 N=9 1084 ± 113a N=6 3.79 3, 25 0.02
263 ± 103 N=4 271 ± 159 N=5 158 ± 94 N=5 774 ± 335a N=4 8.93 3, 14 0.002
1678 ± 281 N=5 1237 ± 268a N=6 1219 ± 184a N=5 1157 ± 244a N=6 4.83 3. 18 0.01
EtOH–E0 EtOH–E0–1 EtOH–E0–2 ANOVA
F= df = p≤
EtOH–E0 eggs were injected with 3.0 mmol EtOH/kg egg on 0 days of development. EtOH–E0–1 eggs were injected with 3.0 mmol EtOH/kg egg on 0 and 1 days of development. EtOH–E0–2 eggs were injected with 3.0 mmol EtOH/kg egg on 0, 1, and 2 days of development. Data represented as mean ± standard deviation. a Experimental group significantly differs as compared to control group at p ≤ 0.05.
brain taurine levels as compared to controls. While a single EtOH dosage (E0) and two daily EtOH dosages (E0–1) caused reduced brain taurine levels as compared to controls, the differences were insignificant (Table 4). Brain taurine levels failed to significantly correlate to brain masses. 4. Discussion Embryonic EtOH exposure caused elevated brain HoCys levels and reduced brain SAM/SAH levels as compared to controls at theoretical stage 37 (11 days of development) (Table 3). EtOH-induced increased brain HoCys levels and EtOH-induced decreases in brain SAM/SAH levels were attenuated by exogenous folate because EtOH and folatetreated embryos had control brain HoCys levels and control brain SAM/ SAH levels (Table 3). EtOH-induced increased brain HoCys levels correlated with reduced embryo viability as measured by EtOH-induced reductions in embryo masses, and brain masses (Table 1). When brain HoCys levels were correlated to embryo masses in all dependent groups, the Pearson product-moment coefficient (r) was −0.58 [F = (1, 58) 30.29; p b 0.0001] and when brain HoCys levels were correlated to brain masses in all dependent groups, the correlation coefficient (r) was −0.32 [F = (1, 58) 6.19; p b 0.01]. As predicted by the metabolism of SAM to SAH and SAH to HoCys (Selhub,1999), brain HoCys levels correlated to SAM/SAH levels in all independent groups [r = −0.28; F = (1, 58) 5.25; p b 0.03]. While three daily dosages of (34 μmol folic acid/kg; 15 mg of folic acid/kg egg) attenuated EtOH-induced increased brain HoCys levels and EtOH-induced reductions in brain SAM/SAH levels (Table 2), exogenous folate failed to ameliorate EtOH-induced reduced % living embryos levels and EtOH-induced reduced embryo masses at stage 37 (Table 1). Xu et al. (2006) reported that daily maternal ingestion of 60 mg of folic acid/kg and 1.0 mg of vitamin B12 (cobalamine)/kg ameliorated many of the EtOH-induced adverse effects in mice fetuses. The folic acid dosage of Xu et al. (2006) (60 mg/kg) is higher than the dosage we used (15 mg folic acid/kg egg) and the EtOH dosage of Xu et al. (2006) (5 g/kg or 108.7 mmol EtOH/kg) is greater than the EtOH dosage we used (3.0 mmol EtOH/kg egg). Thus, species-specific response differences exist. This report and a previous report (Walcher and Miller, 2008) have demonstrated that embryonic EtOH exposure caused elevated HoCys levels in developing chick brains at theoretical stage 37. Thus, the key question now is how does EtOH exposure promote hyperhomocysteinemia? Reduced ingestion of food, common in alcoholics, can cause folate deficiencies (Cravo et al., 1996; Eichner and Hillman, 1973) and folate deficiencies can cause elevated levels of HoCys (Selhub, 1999). However,
this mechanism seems unlikely in our study because chick embryos develop in a closed system where yolk folate levels remain relatively constant. However, folate deficiencies can result from decreased intestinal absorption rates and/or increased excretion rates of folates as observed in adult humans, rats, and pigs (Halsted, 1992; McMartin et al., 1989; Reisenauer et al., 1989). This last mechanism is possible in chick embryos and deserves further study. Another possible hypothesis in explaining hyperhomocysteinemia is EtOH-impaired remethylation of HoCys to methionine that requires methyl group donation from either betaine, or 5-methyltetrahydrofolate (5-methyl THF) via vitamin B12 (cobalamine). Dietary betaine supplementation ameliorated EtOH-induced hepatic steatosis (Kharbanda et al., 2007) in adult rats and EtOH-induced and HoCys-induced decreased phosphatidylcholine levels and increased phosphatidylethanolamine levels have been reported in embryonic chick brains (Miller et al., 1998; 2003b). Recently, Ji et al. (2008) created transgenic mice by insertion of a vector containing the structural gene coding for human betainehomocysteine methyltransferase (BHMT). When these transgenic mice and wild-type mice were infused with EtOH for a period of four weeks, plasma alanine transaminase activities increased by 5-fold in wild-type mice and only 2.7-fold in the transgenic mice; plasma HoCys levels increased by 7-fold in wild-type mice and only 2-fold in the transgenic mice. Another possible control point within the remethylation pathway is 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH). Im et al. (1999) and Min et al. (2005) reported EtOH inhibited 10-FTHF DH activities in adult rats. This enzyme irreversibly catalyzes the conversion of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate (THF). In the glycine cleavage system, T-protein requires THF as a coenzyme and synthesizes N5, N10-methylene-tetrahydrofolate which is later converted to 5-methyl tetrahydrofolate (5-methyl THF) (Kikuchi, 1973; van der Put et al., 2001). Because 5-methyl THF is a methyl donor used in the conversion of HoCys to methionine (Miller and Kelly, 1996), a failure to replenish 5-methyl THF levels could cause hyperhomocysteinemia. Surprisingly, we are reporting that three daily dosages of EtOH (E0–2) caused elevated 10-FTHF DH activities in both brain and hepatic tissues as compared to controls in theoretical stage 44 (18 days of development) chick embryos (Table 4). This late (stage 44) increase in 10-FTHF DH activities may be compensatory in nature and an attempt to reduce earlier (stage 37) hyperhomocysteinemia (Table 3). Elevated brain HoCys levels at stage 37 (11 days of development) are of interest because the earliest time for detectable alcohol dehydrogenase (ADH) activities in embryonic chick liver is 9 to 10 days of development (Wilson et al., 1984) and ADH activities are very low in both brain and hepatic tissues at 11 days of development (stage 37) (Miller et al., 1998). At 15 and
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18 days of development (stage 44), ADH activities increase by 3- to 6fold in both brain and hepatic chick tissues (Miller et al., 1998). Thus, the late (stage 44) EtOH-induced increase in 10-FTHF DH activities (Table 4) reported here may be occurring after EtOH has been metabolized from surviving chick tissues. It is now imperative to measure 10-FTHF DH activities at earlier developmental stages and correlate these activities to endogenous HoCys levels and ADH activities within chick tissues. We hypothesize that EtOH exposure may inhibit 10-FTHF DH activities in stage 37 embryos and 10-FTHF DH activities may correlate with EtOHinduced increases in brain and hepatic HoCys levels in stage 37 embryos. EtOH-induced inhibition of the transsulfuration pathway may also explain EtOH-induced hyperhomocysteinemia. In the transsulfuration pathway, HoCys is ultimately metabolized to taurine (Selhub, 1999). ten Busch et al. (1997) reported that EtOH-treated chick embryos had lower taurine levels in the allantoic fluid at 13 days of development (theoretical stage 39; Hamburger and Hamilton, 1951) as compared to controls. We extended the observations of ten Busch et al. (1997) and are reporting that chronic EtOH (E0–2) exposure caused reduced taurine levels as compared to controls in stage 44 chick brains (Table 4). Meanwhile, a single EtOH dosage (E0), two daily EtOH dosages (E0–1), or three daily EtOH dosages (E0–2) caused reduced hepatic taurine levels as compared to controls in stage 44 embryos (Table 4). When the transsulfuration pathway is impaired, as in homozygous cystathionine β-synthetase defect, HoCys is diverted to the remethylation pathway causing momentary elevated SAM levels which can cause feedback inhibition of the remethylation enzyme, methylenetetrahydrofolate reductase (Selhub, 1999). As both the remethylation and transsulfuration pathways slow, HoCys levels rise. 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