Robert R. Miller

Homocysteine-induced changes in brain membrane composition correlate with increased brain caspase-3 activities and reduced chick embryo viability

In adult systems, high homocysteine (HoCys) levels inhibit methylation reactions and can induce apoptosis in the central nervous system. In embryos, exogenous HoCys is teratogenic and is associated with neural tube defects. Because, methylation inhibitors and inducers of apoptosis can influence membrane composition, we have studied whether or not embryonic exposure to HoCys influenced membrane phospholipid levels, membrane fatty acid composition, and Caspase-3 activities in embryonic chick brains. Embryonic exposure to HoCys caused reduced brain phosphatidylcholine levels and increased levels of brain phosphatidylethanolamine. Exogenous HoCys also promoted decreased levels of long-chain, unsaturated membrane fatty acids and increased levels of saturated short-chain membrane fatty acids. These HoCys-induced brain membrane changes correlated with HoCys-induced increases in brain Caspase-3 activities, HoCys-induced reductions in brain mass, HoCys-induced reductions in embryo mass, and HoCys-induced reductions in the percentage of embryos that survived to 11 days of development (theoretical stage 37). Thus, HoCys-induced changes in brain membrane composition correlated with HoCys-induced apoptosis and reduced embryo viability.

A postcryogenic comparison of membrane fatty acids of elephant spermatozoa.

Cryogenic protocols have been successful in storing spermatozoa collected from African elephants (Loxodonta africana). However, these same protocols and modifications of these protocols have failed to preserve spermatozoa collected from Asian elephants (Elephas maximus) [Buice et al., 1995, Proceedings of the Association of Zoo Veterinary Technicians, Baltimore, MD; O’Brien et al., 1997, Second International Elephant Research Symposium, Springfield, MO]. Because the success or failure of cryogenic freezing may rely on differences in membrane composition, a postcryogenic comparison of the membrane fatty acid composition of spermatozoa isolated from African and Asian elephants was studied. The spermatozoa of African elephants possessed significantly higher levels of docosahexaenoic acid (22:6, n-3) and docosapentaenoic acid (22:5, n-6) as compared to the spermatozoa of Asian elephants. Meanwhile, the spermatozoa of Asian elephants had higher levels of myristic acid (14:0), arachidonic acid (20:4, n-6), and docosatetraenoic acid (22:4, n-6) as compared to the spermatozoa of African elephants. The most abundant membrane fatty acid was docosahexaenoic acid (22:6, n-3). The percentage of membrane docosahexaenoic acid (22:6, n-3) in the spermatozoa of African elephants was 68.13 ± 0.52 as compared to 42.88 ± 0.87 in the spermatozoa of Asian elephants (t = 31.48, P ≤ 0.0001). Zoo Biol 19:461–473, 2000.

α-Tocopherol and γ-tocopherol attenuate ethanol-induced changes in membrane fatty acid composition in embryonic chick brains

Background This project investigated whether or not EtOH-induced reductions in the levels of long-chain polyunsaturated membrane fatty acids could be attenuated by exogenous exposure to either α-tocopherol, γ-tocopherol, or diallyl sulfide (DAS). Methods At 0 days of development, fertile chicken eggs were injected with a single dose of either saline supplemented with various concentrations of EtOH, α- or γ-tocopherol and EtOH, or DAS and EtOH. At 18 days of development, brains were isolated and subjected to membrane analyses. Results When exposed to EtOH, concentrations ranging from 0–60.50 μm/Kg egg, dose-dependent decreases in the levels of brain 18:0, 18:1 (n-9), 18:2 (n-6), 18:3 (n-3), and 20:4 (n-6) were observed. These ethanol–induced changes in membrane fatty acid composition correlated with ethanol-induced reductions in brain mass, brain protein levels, acetylcholine esterase (AChE) activities and correlated with increased lipid hydroperoxide levels. Exposure to either 2.5 μm α-tocopherol/Kg egg and 6.050 mm EtOH/Kg egg, or 2.5 μm α-tocopherol/ Kg egg and 6.050 mm EtOH/Kg egg attenuated EtOH-induced changes in membrane fatty acid composition, brain mass, brain protein levels, AChE activities, and lipid hydroperoxide levels. Embryonic exposure to the cytochrome p450-2E1 inhibitor, diallyl sulfide (DAS), also attenuated EtOH-induced decreases in long-chain, unsaturated membrane fatty acids. However, embryonic exposure to DAS promoted abnormally low brain mass. Conclusion EtOH-induced reductions in the levels of brain long-chain polyunsaturated fatty acid are caused by lipid peroxidation. Teratology 62:26–35, 2000.

Embryonic Exposure to Exogenous - and γ-Tocopherol Partially Attenuates Ethanol-induced changes in Brain Morphology and Brain Membrane Fatty Acid Composition

Abstract Previous studies demonstrated that embryonic exposure to ethanol (EtOH) promoted a reduction in brain mass, a reduction in brain neuron densities, and a reduction in membrane long-chain polyunsaturated fatty acids (PUFAs) in embryonic chick brains. These EtOH-induced reductions in brain membrane PUFAs may be the result of lipid peroxidation because embryonic exposure to exogenous - or γ-tocopherol partially attenuated EtOH-induced reductions in membrane PUFAs. In this paper, we report that embryonic exposure to exogenous - or γ-tocopherol attenuated EtOH-induced decreases in endogenous levels of -tocopherol in both embryonic chick brains and liver. Embryonic exposure to exogenous - or γ-tocopherol also partially attenuated EtOH-induced reductions in brain neuron densities within the cerebral hemispheres of embryonic chick brains. Finally, embryonic exposure to exogenous - or γ-tocopherol also partially attenuated EtOH-induced reductions in long-chain PUFAs in 2-day old neonatal chick brains.

Hyperglycemia-induced membrane lipid peroxidation and elevated homocysteine levels are poorly attenuated by exogenous folate in embryonic chick brains

Injection of L-glucose (9.29 micromol/kg egg) into the air sac of fertile chicken eggs during the first 3 days of embryonic development (E(0-2)) has been reported to cause hyperglycemia and membrane lipid peroxidation in embryonic chick hepatic membranes. These observations have now been extended into embryonic chick brains at 11 days of development (theoretical stage 37). L-glucose caused a 1.7-fold increase in serum D-glucose levels (p< or =0.05), a 1.4-fold decrease in the % living embryos (p< or =0.05), a 1.1-fold decrease in embryonic masses (p< or =0.05), and a 1.4-fold decrease in embryonic brain masses (p< or =0.05) as compared to controls. L-glucose also caused a 3.8-fold increase in brain lipid hydroperoxide (LPO) levels (p< or =0.05) and complex changes in the relative fatty acid composition of brain membranes. Consistent with the hypothesis of hyperglycemia-induced increases in lipid peroxidation were decreased docosahexaenoic acid (DHA: 22: 6, n-3) levels as compared to controls (p< or =0.05). However, hyperglycemia-induced increased docosapentaenoic acid (DPA: 22:5, n-6) levels, decreased arachidonic acid (20; 4, n-6) levels, decreased linoleic acid (18:2, n-6) levels, and increased levels of several saturated short-chain membrane fatty acids were also observed as compared to controls (p< or =0.05). l-glucose caused a 12-fold increase in brain homocysteine levels, a 2.5-fold decrease in S-adenosylmethionine levels, and a 2-fold increase in S-adenosylhomocysteine levels as compared to controls (p< or =0.05). These hyperglycemia-induced alterations were poorly attenuated by exogenous folic acid (181.2 micromol/kg egg).

Resveratrol can only partially attenuate ethanol-induced oxidative stress in embryonic chick brains

Abstract Ethanol (EtOH) exposure promotes increased levels of reactive oxygen species that degrade unsaturated long-chain membrane fatty acids within embryonic chick brains and is associated with apoptosis and reduced embryo viability. In vitro studies have demonstrated that resveratrol, a known antioxidant, attenuated EtOH-induced damage. In order to test whether or not resveratrol can attenuate EtOH-induced embryonic damage, fertile chicken eggs were injected daily with EtOH (6.05 mmol/kg egg) and various concentrations of trans-resveratrol (0–29.5 mmol/kg egg) during the first three days of embryonic development. At 11 days of embryonic development, viable embryos were collected, brains isolated, and brain membrane fatty acid composition analyzed. Embryonic EtOH exposure promoted fewer viable embryos at 11 days of development as compared to controls. Embryonic EtOH exposure also promoted reduced levels of unsaturated long-chain membrane fatty acids, increased levels of saturated short-chain membrane fatty acids, and elevated brain lipid hydroperoxides (LPO) levels. Embryonic exposure to moderate (2.95 nmol/kg egg) and high (29.5 nmol/kg egg) levels of trans-resveratrol attenuated EtOH-induced changes in brain membrane fatty acid composition but failed to attenuate EtOH-induced increases in brain LPO levels and increased brain Casp-3 activities.

Ethanol- and nicotine-induced membrane changes in embryonic and neonatal chick brains

In order to study the effects of EtOH and/or nicotine on brain membrane fatty acid composition, various concentrations of EtOH and/or nicotine were injected into the air sac of chicken eggs at 0 days of incubation. Controls were injected with saline. Experimental groups were injected with either 200 micromol EtOH/kg egg, 100 micromol nicotine/kg egg, 200 micromol nicotine/kg egg, 200 micromol EtOH/kg and 100 micromol nicotine/kg egg, or 200 micromol EtOH/kg and 200 micromol nicotine/kg egg. In all experimental groups, EtOH- and nicotine-induced decreases in brain long-chain polyunsaturated membrane fatty acids were observed in stage 44 embryos, stage 45 embryos, and neonatal chicks. These EtOH- and nicotine-induced decreases in brain membrane polyunsaturated fatty acids correlated with elevated levels of brain lipid hydroperoxides and reduced brain acetylcholinesterase (AChE; EC. 3.1.1.7) activities.

A DEVELOPMENTAL PROFILE OF THE EFFECTS OF ETHANOL ON THE LEVELS OF CHICK BRAIN PHOSPHOLIPIDS

The effects of embryonic exposure to ethanol on brain phospholipid levels were studied by injecting various concentrations of ethanol, ranging from 0 to 149 microns kg-1 egg, into fertile chicken eggs at 0 days of incubation. At 7, 9, 11, 15 and 18 days of incubation, brains were collected and the levels of total phospholipids and various phospholipid classes were measured. Although embryonic exposure to ethanol failed to influence total phospholipid levels, ethanol-induced changes in the levels of individual phospholipid classes were observed. Ethanol-induced increases in the levels of phosphatidylethanolamine (PE) and phosphatidylserine (PS) and ethanol-induced decreases in the levels of phosphatidylcholine (PC) were observed at 9, 11, 15 and 18 days of incubation. Ethanol-induced decreases in brain sphingomyelin (SP) levels were observed at 7 and 18 days of development. These ethanol-induced changes in brain phospholipid levels preceded detectable alcohol dehydrogenase (ADH) activities in both brain and liver.

Reduced de novo synthesis of 5-methyltetrahydrofolate and reduced taurine levels in ethanol-treated chick brains.

In previous studies, exogenous ethanol (3 mmol EtOH/kg egg) caused a 1.6-fold increase in chick brain homocysteine (HoCys) levels at 11 days of development and the mixture of 3 mmol EtOH/kg egg and 34 micromol folic acid/kg egg attenuated EtOH-induced increases in chick brain HoCys levels. Because HoCys is converted to methionine utilizing the methyl donor, 5-methyltetrahydrofolate (5-methyl THF), we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 micromol 5-methyl THF/kg egg inhibited chick brain 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH; EC 1.5.1.6) activities and brain N5, N10-methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) activities at 11 days of development. Three daily dosages of 3 mmol EtOH/kg egg (E0-2) caused approximately a 7-fold reduction in brain 10-FTHF DH activities and approximately a 1.9-fold reduction in brain MTHFR activities as compared to controls at 11 days of development (p<or=0.05). Because HoCys is also removed by the transsulfuration pathway, which synthesizes taurine, we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 micromol 5-methyl THF/kg egg influenced chick brain taurine levels. In EtOH-treated and EtOH and 5-methyl THF-treated embryos, brain taurine levels decreased by approximately 5.5-fold and 6.2-fold as compared to controls, respectively (p<or=0.05). Exogenous 5-methyl THF failed to attenuate EtOH-induced decreased brain taurine levels at 11 days of development.

Exogenous folate ameliorates ethanol-induced brain hyperhomocysteinemia and exogenous ethanol reduces taurine levels in chick embryos.

The effects of exogenous ethanol and/or folic acid on endogenous homocysteine (HoCys) and SAM (S-adenosylmethionine)/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<or=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 mumol 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; E(0)) and two daily dosages of EtOH (E(0-1)) failed to influence brain and hepatic 10-FTHF DH activities when compared to controls. However, three daily dosages of EtOH (E(0-2)) 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<or=0.05). Three daily EtOH dosages (E(0-2)) caused reduced taurine levels in both brain and hepatic tissues (p<or=0.05). Meanwhile, a single EtOH dosage (E(0)), two daily EtOH dosages (E(0-1)), and three daily EtOH dosages (E(0-2)), caused reduced hepatic taurine levels as compared to controls (p<or=0.05).