exogenous ethanol ( etoh ) causes elevated brain and hepatic homocysteine ( hocys ) levels , decreased brain and hepatic taurine levels , and increased apoptosis rates within embryonic chick brains and livers [ 13 ] .
exposure to either teratogen causes reduced brain masses , elevated brain lipid hydroperoxide ( lpo ) levels , elevated brain membrane lipid peroxidation intermediates , and elevated brain caspase-3 activities [ 48 ] .
hocys catabolism uses remethylation pathways and the transsulfuration pathway ( figure 1 ) . in remethylation pathways
, hocys is remethylated back to methionine by using either betaine homocysteine methyltransferase ( ec 2.1.1.15 ; non - folate - dependent remethylation ) , or the cobalamin - dependent enzyme , methionine synthase ( ec 2.1.1.13 ) , which uses 5-methyltetrahydrofolate as the methyl donor . in the transsulfuration pathway
, hocys is converted to cystathionine through the use of cystathionine -synthase ( ec 4.2.1.22 ) and cystathionine is ultimately converted into -ketobutyrate , reduced glutathione ( gsh ) , or taurine [ 9 , 10 ] ( figure 1 ) .
etoh - induced decreased taurine levels within embryonic chick brains [ 2 , 3 ] may be devastating because taurine 's oldest known function is regulating osmotic stress . during mammalian brain swelling , astrocytes release taurine into extracellular spaces creating a hypertonic environment and cause h2o efflux from brain cells [ 11 , 12 ] .
hypotonicity causes astrocytes and neurons to incorporate cysteine sulfinic acid and synthesize taurine through the use of cysteine sulfinic acid decarboxylase ( csad ; ec 4.1.1.29 ) [ 11 , 13 ] .
ironically , elevated levels of d / l - homocysteine , l - cysteate , l - glutamate , and l - aspartate are all potent inhibitors of na - dependent cysteine sulfanilic acid uptake in chick astrocytes , chick neurons , and rat astrocytes .
hence , etoh - induced reductions in brain taurine levels coupled with etoh - induced increased brain hocys levels [ 13 ] may alter neural osmotic regulation [ 11 , 13 ] .
taurine , and especially hypotaurine , has membrane - protective and antioxidant effects in a variety of systems [ 11 , 14 , 15 ] .
taurine supplementation ameliorated etoh - induced increased lipid peroxidation , alcohol - induced liver damage , and etoh - induced cytochrome p450 - 2e1 ( cyp 2e1 ) activities within sprague - dawley rats .
because cats have extremely low csad activities and primates have low brain csad activities , dietary taurine is essential for cats and is conditionally essential for primates [ 11 , 14 , 15 , 17 ] .
taurine deficiencies in cats cause destabilization and fragmentation of retina and tapetum membranes ; increased frequencies of retinopathy and blindness ; increased frequencies of spontaneous abortions and stillbirths ; altered immune systems where the proportions , morphology , and functions of leukocytes are altered ; and myocardial failure ( dilated cardiomyopathy ) [ 11 , 14 , 15 , 18 ] . neuroblasts and glioblasts cell division and migration within the cortex and cerebellum of cat brains is normally completed by the third neonatal week . in taurine - deprived kittens ,
cell division and migration within the cortex and cerebellum is delayed [ 15 , 18 ] .
taurine supplementation ameliorated reduced synaptic connections , reduced neuron and glial cell numbers , and increased apoptosis rates within the cortex of fetal rat brains experiencing intrauterine growth restriction .
consequently , taurine is important in embryonic and neonatal development . because etoh reduced taurine levels within embryonic chick brains [ 2 , 3 ] , the overall objective of this study was to determine if taurine supplementation alleviated etoh - induced oxidative stress within embryonic chick brains .
oxidative - stress measures included rates of apoptosis ( caspase-3 activities ) , homocysteine ( hocys ) levels , reduced glutathione levels ( gsh ) , membrane fatty acid composition , and lipid hydroperoxide ( lpos ) levels . unlike mammalian embryos , avian embryos are closed systems ( cleidoic egg ) that offer the ability to solely observe embryonic responses to a teratogen as compared to combined maternal responses , fetal responses , and maternal to fetal transport as seen within eutherian embryos .
all experiments used fertile , specific pathogen - free white leghorn chicken eggs ( gallus domesticus ) purchased from a commercial hatchery .
chickens raised at this commercial hatchery ingest a standardized diet causing minimal differences in yolk composition from generation to generation .
animal care was in compliance with the guide for the care and use of laboratory animals , 8th edition ( national academies press , washington dc , usa , 2011 ) .  
control eggs were injected with approximately 25  l of h2o or a 1  :  1 ( v / v ) mixture of 0.01  m taurine and h2o ( 4  mol taurine / kg egg ) into each air sac during the first three days of development ( e02 ) .
experimental eggs were injected with approximately 25  l of a 1  :  1 ( v / v ) mixture of 47.5% etoh and h2o , ( 1.5  mmol etoh / kg egg ) ; a 1  :  1 ( v / v ) mixture of 95% etoh and h2o ( 3  mmol etoh / kg egg ) ; a 1  :  1 ( v / v ) mixture of 47.5% etoh and 0.01  m taurine ( 1.5  mmol etoh / kg egg and 4  mol taurine / kg egg ) ; or a 1  :  1 ( v / v ) mixture of 95% etoh and 0.01  m taurine ( 3  mmol etoh & 4  mol taurine / kg egg ) into each air sac during the first three days of development ( e02 ) .
all embryos were incubated in a forced air incubator at 37.5c and turned every 4 hours with relative humidity ranging from 80 to 90% . at 11 days of incubation [ 20 , theoretical stage 37 ]
, blood was drawn from chorio - allantoic blood vessels and serum collected by centrifugation ( 1,000  g ) .
chick embryos were then decapitated , brains excised , and all serum and isolated brains stored at 80c , until subsequent biochemical analyses . because chicks normally hatch at 21 days of development , embryos at 11 days of development [ 20 , theoretical stage 37 ] have completed approximately 52% of total embryogenesis ( midembryogenesis ) and embryonic exposure to etoh and/or taurine during the first three days of development ( e02 ) represents approximately 14% of embryogenesis . theoretical stage 37 [ 20 , 11 days of development ] was selected because exogenous etoh ( 3  mmol etoh / kg egg ) caused elevated brain hocys levels ; increased brain caspase-3 activities ; increased brain lipid hydroperoxide levels ( lpos ) ; decreased brain membrane polyunsaturated fatty acids ( pufas ) levels ; and decreased brain taurine levels at theoretical stage 37 [ 13 , 68 ] .
the 4  mol of exogenous taurine / kg egg dosage was recently deemed to be  semi - safe . 
this dosage failed to impact brain masses , brain caspase-3 activities , or brain lipid hydroperoxide ( lpo ) levels within chick embryos at 18 days of development [ 20 , theoretical stage 44 ] ( unpublished data ) . in order to monitor apoptosis within brain homogenates ,
embryonic chick brains were homogenized in 400  l of ice - cold 50  mm hepes [ 4-(2-hydroxyethyl)-piperazineethanesulfonic acid ] buffer ( ph 7.4 ) , 1  mm dtt ( dithiothreitol ) , 0.1  mm edta ( disodium ethylenediaminetetraacetic acid ) , and 0.1% chaps ( 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate ) , and nonsoluble material was removed by centrifugation at 10,000  g ( 10  min at 4c ) .
aliquots from each supernatant were assayed for total protein   and caspase-3 activity [ 5 , 22 ] .
as the fluorogenic acetylated - aspartate - glutamate - valine - aspartate-7-amino-4-methylcoumarin ( acdevd - amc ) substrate was cleaved by activated caspase-3 , the liberated amc ( 7-amino-4-methylcoumarin ) was detected by fluorescence at an excitation wavelength of 360  nm and an emission wavelength of 460  nm .
caspase-3 activities are reported as units of caspase-3/mg protein , and one unit of caspase-3 is defined as the release of 1  pmol of amc liberated per minute from the 0.3  mm acdevd - amc substrate at 30c . in order to study the effects of exogenous etoh and/or taurine on brain hocys levels ,
the isolation and quantification of hocys was achieved by modifying the technique of yi et al . .
embryonic brains were homogenized in 500  l of ice - cold 50  mm hepes buffer ( ph 7.4 ) , 1  mm dtt , and 0.1  mm edta , and nonsoluble material was removed by centrifugation at 10,000  g ( 5  min at 4c ) .
supernatants were deproteinated by the addition of 400  l of 0.4  m perchloric acid ( pca ) .
protein - containing precipitates were removed by centrifugation at 10,000  g ( 5  min at 4c ) and the ph was readjusted to approximately 6.5 to 7.5 by adding 15  l of 6  m naoh to each supernatant .
hocys and methionine derivatives were prepared as previously described [ 1 , 22 , 23 ] .
the separation and quantification of hocys derivative was achieved by hplc using a 15  cm  4.6  mm discovery bio - wide pore c 18 hplc column ( 5  m beads ) using a high - pressure liquid chromatography system ( hplc ) equipped with a fluorescence detector .
the mobile phase was 50  mm sodium monophosphate , 1  mm octane sulfonic acid , and 2% acetonitrile and adjusted to ph 2.7 with 85% phosphoric acid .
the isocratic flow rate was 1  ml / min ( 1800 to 2100 psi ) and the excitation wavelength was 230  nm and the emission wavelength was 418  nm . using these hplc conditions ,
hocys concentrations within biological samples were determined from a standard curve prepared from known concentrations of d / l - homocysteine ( 0 to 10  mol hocys ) .
effects of exogenous etoh and/or taurine on reduced glutathione ( gsh ) levels within embryonic chick brains utilized the protocols of anderson [ 24 , 25 ] .
embryonic brains were homogenized in 500  l of ice - cold 50  mm sodium phosphate buffer ( ph 7.4 ) and 0.1  mm edta , and nonsoluble material was removed by centrifugation at 10,000  g ( 5  min at 4c ) .
an additional aliquot of 50  l was removed from each supernatant and deproteinated by adding 190  l of 4% 5-sulfosalicylic acid and the protein - containing precipitate removed by centrifugation at 15,000  g ( 5  min at 4c ) .
an aliquot of 200  l was then removed and 1  ml of ellman 's reagent ( 0.1  mm 5,5-dithio - bis(2-nitrobenzoic acid ) in 0.1  m sodium phosphate buffer , ph 8.0 ) was added to each aliquot and incubated at 37.5c for 30  min . as reduced gsh was converted into oxidized gssg ( oxidized glutathione disulfide ) , 5,5-dithiobis(2-nitrobenzoic acid ) was converted into 2-nitro-5-thiobenzoic acid , and the absorbance was measured at 412  nm .
serum aliquots of 50  l were deproteinized by adding 50  l of cold 0.2  m 5-sulfosalicylic acid to each sample , and nonsoluble material removed by centrifugation at 11,500  g ( 2  min at 4c ) .
columns containing 1  ml of dowex 1  4 resin over 3  ml of dowex-50w-8 resin in 12  ml reservoirs fitted with 1.5  cm frits were prewashed with 12  ml of 1  m hcl .
after 40  l of 100  m homoserine was added to each supernatant as an internal standard , supernatants were layered onto a dual - bed column and taurine and homoserine were eluted with 4  ml of h2o .
opa ( ortho - phthalaldehyde ) derivatives were synthesized according to waterfield 's approach   and filtered through 0.2  m millex - fg13 ptfe filters .
aliquots of 20  l were injected onto a 15  cm  4.6  mm discovery bio - wide pore c 18 hplc column ( 5  m beads ) on a scm 1000 high - pressure liquid chromatography system ( hplc ) equipped with dual detectors including a fluorescence detector and a uv - vis detector .
the uv - vis detector was set at a wavelength of 250  nm and the excitation wavelength of the fluorescence detector was 350  nm with an emission wavelength of 476  nm [ 2 , 3 ] .
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 this protocol , 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 .   because reactive oxygen species ( oxygen radicals ( o2 ) and hydroxyl radicals ( ho ) ) can attack any unsaturated neutral lipid or any unsaturated phospholipid , it became imperative to determine if exogenous etoh and/or taurine altered brain membrane fatty acid composition .
embryonic brains were homogenized in 400  l of ice - cold 50  mm hepes buffer ( ph 7.4 ) , 1  mm dtt , and 0.1  mm edta , and total lipids were extracted by the folch technique .
samples were concentrated by the evaporation of solvent under n2 and stored overnight at 40c .
neutral lipids were separated from membrane lipids on silicic acid columns [ 46 , 29 ] .
liquid chromatography columns ( 12  ml reservoirs ) were packed with anhydrous 100200 mesh sil - r to a bed height of 3.5  cm .
after columns were washed several times with chloroform , total lipids were dissolved in 0.5  ml of 2  :  1 chloroform - methanol ( v / v ) and applied to a column .
neutral lipids were eluted with 4  ml of chloroform , and membrane lipids , including phospholipids and some glycolipids , were eluted with 4  ml of methanol .
after fractionated samples were dried under n2 , membrane lipid samples were saponified and liberated fatty acids were methylated according to metcalfe et al . .
fatty acid methyl esters ( fames ) were separated on an omega - wax capillary gas chromatography ( gc ) column ( 30  m  0.53  mm  0.25  m film thickness ) in a gc .
injector temperature was 225c and the flame ionization detector was set at 250c with a he carrier gas rate of 2025 ml / min .
after a 5  min delay , the column temperature was increased to 205c at a rate of 2c / min .
after methyl pentadecanoate was added as an internal standard ( 48.75  nmol / sample ) , individual fames within each biological sample were identified by their retention times , quantified as compared to the internal standard , and data expressed as % mol .
lipid hydroperoxides ( lpos ) are peroxidation intermediates   and were extracted by homogenizing brains in 500  l of ice - cold 50  mm hepes buffer ( ph 7.4 ) , 1  mm dtt , and 0.1  mm edta .
samples were deproteinated by adding 500  l of meta - phosphoric acid ( 20  mg / ml in methanol ) to each sample , and protein - containing precipitates were removed by centrifugation ( 5,000  g for 5  min ) .
nonpolar lipid hydroperoxides were extracted from each supernatant by adding 3  ml of chloroform , followed by centrifugation for 5  min at 2,000  g .
an aliquot of 1  ml was removed from each organic layer and spectrophotometrically assayed for lpos by using lipid hydroperoxide assay kits .
lpos are highly unstable and readily promote the conversion of fe to fe and were detected by using thiocyanate ions as the chromogen .
membrane fatty acid composition is reported as % mol . because percentages may form binomial distributions rather than normal distributions , all % mol data were subjected to arcsine transformations prior to analysis of variance ( anova ) followed by post hoc tukey tests ( honestly significant difference tests ) .
other data sets were not subjected to arcsine transformation before anova analyses were followed by post hoc tukey tests ( honestly significant difference tests ) . the significance level in post hoc tukey tests was p  0.05 .
early embryonic exposure to etoh ( 3  mmol / kg egg ) and taurine ( 4  mol / kg egg ) caused approximately a 1.8-fold and a 1.6-fold decrease in brain masses at 11 days of development , respectively ( table 1 ) . while the combination of exogenous etoh and taurine caused reduced brain masses , the reductions were insignificant .
brain caspase-3 activities increased by approximately 1.8-fold when exposed to either 1.5  mmol etoh / kg egg or 3  mmol etoh / kg egg as compared to controls .
meanwhile , exposure to 4  mol of exogenous taurine / kg egg caused approximately a 1.7-fold increase in brain caspase-3 activities as compared to controls .
the combined treatments of etoh and taurine ( 1.5  mmol etoh and 4  mol taurine / kg egg or 3  mmol etoh and 4  mol taurine / kg egg ) also caused increased brain caspase-3 activities as compared to controls ( table 1 ) . hence , etoh and/or taurine caused increased brain apoptosis rates .
early embryonic etoh ( 3  mmol / kg egg ) exposure ( e02 ) caused increased brain and hepatic caspase-3 activities and reduced brain masses at 11 days of development ( midembryogenesis ) [ 13 , 68 , 32 ] .
however , the observations that exogenous taurine also caused reduced brain masses and promoted increased brain caspase-3 activities are novel and indicate that exogenous etoh and/or taurine are embryopathic . in a preliminary study in this laboratory , a 50  mg of exogenous taurine / kg egg dosage ( 400  mol taurine / kg egg ) during early embryogenesis ( e02 ) caused reduced percentage of living chick embryos at 11 days of development ( theoretical stage 37 ) to 31.9  10.2% as compared to 86.7  3.1% within controls ( t = 10.80 ; df = 6 ; p  0.0001 ) ( unpublished data ) . in this study ,
living embryos were defined as possessing a beating heart , and there were 4 rounds of injections ( n = 4 ) with 18 to 21 eggs within each injection group . in this study , a correlation coefficient ( r ) of 0.52 ( f = ( 1 , 22 ) 8.20 ; p = 0.009 ) was observed when serum taurine levels were correlated to brain caspase-3 activities across all groups .
this last observation indicates that exogenous taurine is more toxic in chick embryos at 11 days of development as compared to chick embryos at 18 days of development .
the exogenous dosage of 4  mol taurine / kg egg during the first three days of development previously failed to impact brain caspase-3 activities within 18-day chick embryos ( late embryogenesis ) and was , therefore , thought to be  semi - safe 
( unpublished data ) . in this study , an exogenous dosage of 4  mol taurine / kg egg during the first three days of development caused increased brain caspase-3 activities within 11 day chick embryos ( midembryogenesis ) ( table 1 ) .
taurine - induced toxicity within chick embryos was first reported by van gelder and belanger   who reported that a single injection of 100  mol taurine/50  g egg ( 2  mmol taurine / kg egg ) caused increased incorporation of taurine into developing hearts and brains at 15 days of development and was associated with ataxia , reduced muscle strength , a loss of motor coordination , and reduced embryo viability .
consequently , we have still not found a safe exogenous taurine dosage for chick embryos at 11 days of development ( tables 1 , 2 , and 3 ) .
exogenous etoh , taurine , and the combination of etoh and taurine all caused increased brain hocys levels ( table 1 ) .
exposure to 3  mmol etoh / kg caused a 1.9-fold increase in brain hocys levels and exposure to 4  mol exogenous taurine / kg egg caused a 2.3-fold increase in brain hocys levels as compared to controls .
the combined treatments of etoh and taurine ( 1.5  mmol etoh and 4  mol taurine / kg egg and 3  mmol etoh and 4  mol taurine / kg egg ) both caused elevated brain hocys levels ( table 1 ) .
when brain caspase-3 activities were correlated to brain hocys levels across all groups , pearson 's product moment ( r ) was 0.35 ( f = ( 1 , 47 ) 6.48 ; p = 0.0004 ) .
thus , etoh- and taurine - induced increased brain caspase-3 activities correlated to etoh- and taurine - induced increased brain hocys levels ( table 1 ) .
etoh - induced and taurine - induced increases in brain hocys levels can promote the synthesis of reactive oxygen species ( ros ) .
two hocys molecules can undergo autooxidation and form a dimer ( homocysteine : oxidized disulfide ) by liberating two hydrogen ions and two electrons . in doing so ,
hydrogen peroxide and hydroxyl radicals can be generated and thus produce reactive oxygen species , that is , hydroxyl radicals ( ho ) and oxygen radicals ( o2 ) .
early embryonic exposure to etoh ( 1.5  mmol etoh / kg egg and 3  mmol / kg egg ) and taurine ( 4  mol / kg egg ) caused approximately a 1.2-fold to 1.3-fold decrease in brain gsh levels at 11 days of development , respectively ( table 1 ) .
the combined treatments of etoh and taurine ( 1.5  mmol etoh and 4  mol taurine / kg egg or 3  mmol etoh and 4  mol taurine / kg egg ) both caused approximately a 1.2-fold to 1.0-fold decrease in brain gsh levels as compared to controls
. total brain thiol ( gsh and gssg ) levels were largely unaffected ( table 1 ) .
when brain caspase-3 activities were correlated to brain gsh levels within all groups , pearson 's product moment ( r ) was 0.29 ( f = ( 1 , 48 ) 4.40 ; p = 0.004 ) .
thus , as apoptosis rates increased ( brain caspase-3 activities ) , brain gsh levels decreased .
recently , we demonstrated that etoh ( 3  mmol / kg egg ) exposure during the first three days of development ( e02 ) caused decreased brain selenium - dependent glutathione peroxidase ( gpx ; ec .
1.11.1.9 ) activities and non - selenium - dependent gpx activities at 11 days of development ( midembryogenesis ) ( unpublished data ) ( p < 0.05 ) .
glutathione peroxidases ( gpx ) , both selenium - dependent and non - selenium - dependent , are well - known first - line defense antioxidant enzymes because they catalyze the reduction and ultimate removal of lipid hydroperoxides ( lpos ) , and other peroxides , as follows [ 35 , 36 ] : 

 consequently , etoh - induced reduction in brain gsh - dependent gpx activities could certainly contribute to oxidative stress and slow the catabolism of etoh - induced increased lpo levels ( table 1 ) . increased endogenous serum taurine levels were caused by exogenous etoh ( 1.5  mmol etoh / kg egg and 3  mmol / kg egg ) , taurine ( 4  mol / kg egg ) , and etoh and taurine ( 1.5  mmol etoh and 4  mol taurine / kg egg and 3  mmol etoh and 4  mol taurine / kg egg ) ( table 1 ) .
early embryonic exposure to 1.5  mmol exogenous etoh / kg egg and 3  mmol exogenous etoh / kg egg caused approximately a 2.2-fold increase and a 3.1-fold increase in serum taurine levels , respectively .
meanwhile , exposure to 4  mol of exogenous taurine / kg egg caused approximately a 2.4-fold increase in serum taurine levels .
the combined treatments of etoh and taurine ( 1.5  mmol etoh and 4  mol taurine / kg egg and 3  mmol etoh and 4  mol taurine / kg egg ) both caused elevated serum taurine levels as compared to controls ( table 1 ) . when serum taurine levels were correlated to brain caspase-3 activities within all groups , pearson 's product moment ( r ) was 0.61 ( f = ( 1 , 24 ) 11.54 ; p = 0.003 ) and when serum taurine levels were correlated to brain hocys levels within all groups , pearson 's product moment ( r ) was 0.43 ( f = ( 1 , 24 ) 6.05 ; p = 0.003 ) . therefore , etoh - induced and taurine - induced increases in serum taurine levels correlated to increased apoptosis rates and increased hocys levels within embryonic chick brains . presumably , exogenous taurine caused increased endogenous serum taurine levels ( table 1 ) by diffusing into the yolk and then entering the embryo during vascularization .
previously , it was demonstrated that exogenous taurine exposure during the first three days of embryonic development ( e02 ) caused increased endogenous chick serum taurine levels during late embryogenesis ( 18th day of development ) ( unpublished data ) .
this last observation has now been extended to midembryogenesis ( 11 days of incubation ; table 1 ) . while it was not surprising that exogenous taurine caused increased serum taurine levels ( table 1 ) , it was surprising to find that early exogenous etoh exposure caused increased levels of serum taurine ( table 1 ) .
one hypothesis in explaining this phenomenon is etoh - induced increases de novo taurine synthesis rates .
4.1.1.29 ) then catalyzes the decarboxylation of cysteine sulfinic acid to hypotaurine and taurine ( figure 1 ) .
chick embryos can synthesize taurine from cysteine , and very high cysteine lyase ( ec .
4.4.1.10 ) activities are found in the area vasculosa within 10-somite ( approximately 33  hr ) chick embryos .
cysteine lyase catalyzes the synthesis of cysteine sulfinic acid through the following reaction : 
  chick neurons and glia readily incorporate cysteine sulfinic acid , and incorporated cysteine sulfinic acid is quickly converted into cysteic acid and is decarboxylated , via cysteine sulfinic acid decarboxylase ( ec .
besides synthesizing taurine from cysteine , adult rats , adult chickens , and chick embryos also have the ability to synthesize taurine from serine [ 3942 ] . in this very old evolutionary pathway , inorganic sulfate
is added to atp and converted to 3-phosphoadenosine-5-phosphosulfate ( paps ) followed by the transfer of the sulfate from paps to a hydroxylated organic compound , such as serine , to form taurine and is catalyzed by the pyridoxine - dependent enzyme , paps transferase ( ec .
this pathway is illustrated as follows : 
 ( 3)atp+so42  atp - sulfurylaseadenosine-5-phosphosulfate  ( aps)(4)aps+atp  aps  kinaseadp  + 3-phosphoadenosine-5-phosphate  ( paps)(5)paps+serinepaps  transferasetaurine we suspect that the hypothesis concerning exogenous etoh - induced increased de novo taurine synthesis rates within chick embryos is false .
elevated d / l - hocys levels are a potent inhibitor of na - dependent cysteine sulfanilic acid uptake into chick astrocytes and neurons   and should slow de novo brain taurine synthesis [ 11 , 13 ] .
etoh - induced and taurine - induced increases in brain hocys levels have been observed in this study ( table 1 ) , and previous reports have demonstrated that exogenous etoh ( 3  mmol / kg egg ) during the first three days of development ( e02 ) caused reduced brain and hepatic taurine levels at 11 days of development [ 2 , 3 ] .
thus , an alternative hypothesis predicts etoh - induced increases in membrane fluidity and permeability may stimulate an efflux of taurine , and possibly k , from neural cells into the circulatory system .
etoh - induced increases in membrane fluidity are documented [ 7 , 27 ] and etoh - induced and hocys - induced decreased chick brain membrane phosphatidylcholine ( pc ) levels [ 5 , 44 ] , increased chick brain membrane phosphatidylethanolamine ( pe ) levels [ 5 , 44 ] , and decreased chick brain membrane pufa levels [ 4 , 68 , 22 , 27 , 44 ] have been reported .
etoh - induced increases in taurine and pe levels within chick allantoic fluid have been reported and allantoic taurine and pe levels exceeded serum levels .
as cells lose cylindrically shaped phospholipids , such as pc , and gain triangularly shaped phospholipids , such as pe , membrane fluidity and permeability increase [ 7 , 27 , 46 ] .
an etoh- or hocys - induced taurine efflux from brain and hepatic cells into the circulatory system could create hypertonic environments and promote cell shrinkage that is typically observed during apoptosis [ 11 , 17 ] . in biopsies of adult human gliomas ,
histology revealed significant correlations between apoptotic cell density and taurine efflux in nonnecrotic ( r = 0.73 , p < 0.003 ) and necrotic ( r = 0.63 , p  0.0005 ) biopsies and , thus , the authors argued that taurine efflux is a potential marker of apoptosis . increased extracellular taurine levels have been implicated as the mechanism in apoptosis - related cell shrinkage in several cell types [ 4850 ] and p38 mitogen - activated protein kinase activation before taurine and k efflux have been associated with caspase-3 activation . early embryonic etoh exposure altered brain membrane fatty acid composition during midembryogenesis ( table 2 ) .
etoh ( 3  mmol / kg egg ) caused a 2.2-fold reduction in brain membrane docosahexaenoic acid ( 22  :  6 , n-3 ) levels ; a 1.7-fold reduction in docosapentaenoic acid ( 22  :  5 , n-6 ) levels ; a 1.5-fold reduction in arachidonic acid ( 20  :  4 , n-6 ) levels ; a 2.2-fold reduction in linolenic acid ( 18  :  3 , n-6 ) levels ; and a 2.3-fold reduction in palmitolenic acid ( 16  :  3 , n-3 ) levels as compared to controls ( table 2 ) . meanwhile ,
etoh caused a 1.5-fold increase in brain membrane palmitic acid ( c16  :  0 ) levels and a 3.5-fold increase in myristic acid ( 14  :  0 ) levels when compared to controls ( table 2 ) .
early embryonic taurine ( 4  mol / kg egg ) exposure also altered brain membrane fatty acid composition during midembryogenesis ( table 2 ) .
taurine caused a 1.4-fold reduction in brain membrane docosahexaenoic acid ( 22  :  6 , n-3 ) levels and a 1.7-fold reduction in adrenic acid ( 22  :  4 , n-6 ) levels as compared to controls .
meanwhile , taurine - induced increases in brain membrane linoleic acid ( 18  :  2 , n-6 ) levels were observed . the combination of etoh and taurine ( 3  mmol etoh and 4  mol taurine / kg egg ) caused a 3.0-fold reduction in brain membrane docosahexaenoic acid ( 22  :  6 , n-3 ) levels ; a 1.6-fold reduction in docosapentaenoic acid ( 22  :  5 , n-6 ) levels ; a 2.2-fold reduction in adrenic acid ( 22  :  4 , n-6 ) levels ; a 1.4-fold reduction in arachidonic acid ( 20  :  4 , n-6 ) levels ; and a 1.2-fold decrease in palmitoleic acid ( 16  :  1 , n-7 ) levels as compared to controls . meanwhile , a 5.6-fold increase in brain membrane linoleic acid ( 18  :  2 , n-6 ) levels was observed in etoh- and- taurine - treated embryos as compared to controls ( table 2 ) . in order to look at etoh - induced and taurine - induced changes in brain membrane fatty acid composition in a simplified and holistic manner , membrane fatty acid levels ( table 2 ) were converted into two ratios ( table 3 ) .
the first ratio was the sum of unsaturated / saturated membrane fatty acids (  unsaturated/ saturated fatty acids ) and the second ratio was the sum of long - chain polyunsaturated fatty acids / short - chain membrane fatty acids (  long - chain pufas/ short - chain membrane fatty acids ) ( table 3 ) . in this study , long - chain pufas are defined as having hydrocarbon lengths 20 carbons , and short - chain fatty acids had hydrocarbon chain length 20 carbons . when  unsaturated/ saturated fatty acids ratios were correlated to  long - chain pufas/ short - chain membrane fatty acids in all groups , a pearson 's product moment ( r ) value of 0.37 ( f = ( 1 , 31 ) 4.99 ;
this correlation coefficient indicates that long - chain pufas are relatively more unsaturated as compared to short - chain fatty acids .
etoh - treated embryos had a 1.5-fold decrease in  unsaturated/ saturated membrane fatty acid levels and a 1.9-fold decrease in  long - chain pufas/ short - chain membrane fatty acid levels as compared to controls ( table 3 ) .
taurine - treated embryos and etoh- and- taurine - treated embryos demonstrated insignificant differences in  unsaturated/ saturated membrane fatty acid levels as compared to controls .
however , taurine - treated embryos had a 1.3-fold decrease in  long - chain pufas/ short - chain membrane fatty acid levels and etoh- and- taurine - treated embryos had a 2.0-fold decrease in  long - chain pufas/ short - chain membrane fatty acid levels as compared to controls .
thus , etoh - treated , taurine - treated- , and etoh- and- taurine - treated embryos all demonstrated decreased levels of membrane long - chain pufas as compared to controls ( table 3 ) . because reduced pufa levels are observed during lipid peroxidation [ 7 , 10 , 27 ] , brain lipid hydroperoxides ( lpos ) levels
lpos are lipid peroxidation intermediates   and etoh - treated embryos had a 5.0-fold increase in lpo levels , taurine - treated embryos had a 3.6-fold increase in lpo levels , and etoh- and- taurine - treated embryos had a 4.8-fold increase in lpo levels as compared to controls ( p < 0.05 ) .
when  long - chain pufas/ short - chain membrane fatty acid values were correlated to brain lpo levels within all groups , a pearson 's product moment ( r ) of 0.78 ( f = ( 1 , 28 ) 42.86 ; p < 0.0001 ) was observed .
taurine , and especially hypotaurine , has membrane - protective and antioxidant properties in a variety of systems undergoing oxidative - stress [ 11 , 14 , 15 ] .
taurine supplementation ameliorated etoh - ameliorated lipid peroxidation , alcohol - induced liver damage , and etoh - induced increased cytochrome p450 - 2e1 ( cyp 2e1 ) activities within rats .
consequently , etoh - induced and/or hocys - induced taurine efflux from the brain into the circulatory system [ 2 , 3 ] may remove a powerful antioxidant from the neurons and/or astrocytes that are undergoing etoh - induced oxidative stress ( tables 2 and 3 ) .
etoh - induced formation of reactive oxygen species via the oxidation of fe , increased xanthine oxidase activities , and increased cytochrome p450 - 2e1 ( cyp 2e1 ; ec 1.14.13n7 ) activities have been discussed [ 7 , 27 ] .
etoh- and taurine - induced increased reactive oxygen species levels can attack long - chain pufas and momentarily create lpo intermediates which cleave long - chain pufas into less saturated membrane fatty acids and produce a number of cytotoxic reactive aldehydes ( tables 2 and 3 ) [ 27 , 31 ] .
while the sources of reactive oxygen species during membrane lipid peroxidation in taurine - treated chick embryos are unclear ( tables 2 and 3 ) , the commonality between etoh - induced membrane lipid peroxidation and taurine - induced lipid peroxidation ( tables 2 and 3 ) elevates brain hocys levels as seen in both exogenous treatments ( table 1 ) .
this paper has failed to demonstrate that an exogenous dosage of 4  mol taurine / kg egg can ameliorate etoh - induced lipid peroxidation , oxidative - stress , and apoptosis within embryonic chick brains during midembryogenesis .
both exogenous etoh and exogenous taurine treatments during early embryogenesis ( e02 ) caused increased apoptosis rates ( increased caspase-3 activities ) , increased oxidative - stress ( decreased gsh levels ) , and increased membrane lipid peroxidation ( reduced long - chain pufas and increased lpo levels ) within embryonic chick brains ( tables 1 , 2 , 3 ) .
etoh - induced and taurine - induced increases in brain hocys levels were associated with decreased brain gsh levels and brain taurine levels ( tables 1 and 2 ) and may indicate etoh - induced and taurine - induced decreased metabolism via the transsulfuration pathway ( figure 1 ) that may contribute to elevated hocys levels . because increased brain hocys levels can be a source of reactive oxygen species , elevated hocys levels appear to be associated with increased rates of apoptosis , oxidative - stress , and lipid peroxidation within embryonic chick brains and possibly cause a taurine efflux from brain and hepatic cells into the blood and may contribute to apoptosis - related osmotic stress and cell shrinkage ( table 1 ) [ 2 , 3 ] . while the exogenous dosage of 4  mol / kg egg failed to impact brain masses , brain caspase-3 activities , or brain lpo levels within chick embryos at 18 days of development ( late embryogenesis )
, this same dosage clearly promotes increased rates of membrane lipid peroxidation and apoptosis within embryonic chick brains at 11 days of development ( midembryogenesis ) ( tables 1 , 2 , 3 ) .
increased exogenous taurine - induced toxicity , as seen in embryonic chick brains at midembryogenesis as compared to late - embryogenesis , may not be surprising because chick brain and hepatic glutathione - dependent glutathione peroxidases ( gpx ; ec .
1.11.1.9 ) activities increase by 1.4-fold to 2.8-fold when comparing embryos at midembryogenesis ( 11 days of development ) to late - embryogenesis ( 18 days of development ) .
that is , the protective capabilities of antioxidant enzymes are greater during later stages of development as compared to midembryogenesis . while a lower exogenous taurine dosage may have antioxidant capabilities within chick brains during midembryogenesis as compared to the prooxidant capabilities of the 4  mol / kg egg dosage used in this study , this paper calls for caution and more study .
energy drinks  has become popular and a concern within european and north american communities [ 52 , 53 ] .
taurine levels in  energy drinks  can range from 10  mg ( 79.9  mol ) to 1000  mg ( 7990  mol ) .
thus , a pregnant woman , weighing from 140 pounds ( 63.5  kg ) to 180 pounds ( 81.6  kg ) , who consumes a single drink of alcohol mixed with an energy drink that contains 1000  mg taurine ( 7990  mol ) , consumes etoh mixed with an exogenous taurine dosage ranging from 97.9  mol / kg to 125.8  mol / kg .
these dosages are from 25.5-fold to 31.5-fold higher than the 4  mol exogenous taurine / kg egg dosage used in this study which caused apoptosis and membrane lipid peroxidation within chick brains at midembryogenesis .
this is a concern and warrants caution because a truly safe exogenous taurine dosage within a human fetus is currently unknown .
 because taurine alleviates ethanol- ( etoh- ) induced lipid peroxidation and liver damage in rats , we asked whether exogenous taurine could alleviate etoh - induced oxidative stress in chick embryos . 
 exogenous etoh ( 1.5  mmol / kg egg or 3  mmol / kg egg ) , taurine ( 4  mol / kg egg ) , or etoh and taurine ( 1.5  mmol etoh and 4  mol taurine / kg egg or 3  mmol etoh and 4  mol taurine / kg egg ) were injected into fertile chicken eggs during the first three days of embryonic development ( e02 ) . at 11 days of development ( midembryogenesis ) , serum taurine levels and brain caspase-3 activities , homocysteine ( hocys ) levels , reduced glutathione ( gsh ) levels , membrane fatty acid composition , and lipid hydroperoxide ( lpo ) levels were measured . early embryonic etoh exposure caused increased brain apoptosis rates ( caspase-3 activities ) ; increased brain hocys levels ; increased oxidative - stress , as measured by decreased brain gsh levels ; decreased brain long - chain polyunsaturated levels ; and increased brain lpo levels . 
 although taurine is reported to be an antioxidant , exogenous taurine was embryopathic and caused increased apoptosis rates ( caspase-3 activities ) ; increased brain hocys levels ; increased oxidative - stress ( decreased brain gsh levels ) ; decreased brain long - chain polyunsaturated levels ; and increased brain lpo levels . 
 combined etoh and taurine treatments also caused increased apoptosis rates and oxidative stress . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions