Circadian Influence on Ethanol-Related Intrauterine Growth Retardation in Mice

Circadian influences on growth and development in response to ethanol were studied in mice. On gestational day 10, pregnant animals received a single intraperitoneal injection of ethanol with the following dose levels: 1.0, 2.5 or 4.0 g/kg at one of four circadian phases (0700, 1300, 1900 or 0100 hr). 48 hrs after injection the embryonic weight and length, protein and DNA content and placental weight and protein were determined. Ethanol-related intrauterine growth retardation were shown to be dose- and circadian phase-dependent, the greatest susceptibility being seen during the dark phase. The variations observed are discussed with regard to changes in drug metabolism and tissue sensitivity.     

Effects of ethanol on the decidual cell reaction in rats.

The effects of ethanol on uterine sensitivity to induction of decidualization and deciduoma growth were determined. Rats were ovariectomized, given an oestrogen-progesterone regimen to optimize induction and growth of deciduoma and randomly assigned to one of three ethanol treatment groups: (i) days 1-4 (pre-induction/period of sensitivity), (ii) days 5-9 (post-induction/period of growth), (iii) days 1-9 (periods of sensitivity and growth); or to a control group not treated with ethanol (pair-fed to treated groups). Ethanol (0, 1, 2, or 4 g kg-1) diluted in water was administered by stomach tube on the days prescribed. Decidualization was induced in one uterine horn by intraluminal injection of sodium phosphate buffer. Uterine sensitivity and decidual growth were assessed as cornu weight. Blood alcohol concentrations were measured by gas chromatography. Alcohol treatment reduced uterine sensitivity, but increased deciduoma growth. Blood alcohol concentrations rose to 133 mg% at 30 min, remained high for 90 min and declined to 82 mg% at 120 min. Thus, blood alcohol concentrations sufficient to induce mild intoxication in humans suppressed uterine sensitivity to decidualization and enhanced deciduoma growth in rats. As all ovarian steroid hormone support was exogenous, the effects of ethanol on deciduoma induction and growth were not due to alterations in the hypothalamic-pituitary-ovarian axis.     

Acute ethanol exposure in pregnancy alters the insulin-like growth factor axis of fetal and maternal sheep

Maternal ethanol intake during pregnancy impairs fetal growth, but mechanisms are not clearly defined. Reduced IGF abundance or bioavailability in the fetus and/or mother may contribute to this growth restriction. We hypothesized that an episode of acute ethanol exposure, mimicking binge drinking would restrict fetal growth and perturb the maternal and fetal IGF axes. Pregnant sheep were infused intravenously with saline or ethanol (1 g/kg maternal wt) over 1 h, on days 116, 117, and 118 of gestation (start of 1st infusion = time 0, term is 147 days). Maternal and fetal plasma IGF and IGF-binding protein (IGFBP) concentrations were measured before and after each infusion. Compared with controls, ethanol exposure reduced fetal weight at day 120 by 19%, transiently reduced maternal plasma IGF-I (-35%) at 30 h, and decreased fetal plasma IGF-II (-28%) from 24 to 54 h after the first infusion. Ethanol exposure did not alter maternal or fetal plasma concentrations of IGFBP-2 and IGFBP-3, measured by Western ligand blotting. We conclude that suppression of maternal and fetal IGF abundance may contribute to fetal growth restriction induced by acute or binge ethanol exposure.     

Rhizomorph Formation in Fungi III. The Effect of Ethanol on the Synthesis of DNA and RNA and Uptake of Asparagine and Phosphate in Armillaria mellea

Growth and rhizomorph formation in Armillaria mellea (Vahl ex Fr.) Quél. can be stimulated by ethanol when grown on a synthetic glucose medium. The content of DNA (deoxy-ribonucleic acid) and RNA (ribonucleic acid) in cultures of A. mellea have been followed during a growth period in relation to growth (dry weight) and ethanol uptake with 3 different initial ethanol concentrations. The continuous increase in dry weight as well as DNA and RNA contents during growth showed similar exponential rates as long as ethanol was present in the medium. The final amounts were proportional to the initial ethanol concentration. A further supply of ethanol caused a similar proportional increase in dry weight and also in the DNA and RNA contents, which indicates that the growth stimulated by ethanol is caused by cell divisions rather than an accumulation of lipids or polysaccharides. Uptake of asparagine and phosphate were also stimulated by ethanol.     

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.     

Effect of maternal diabetes and ethanol interactions on embryo development in the mouse

The aim of this study was to determine the possible fetal effects of interaction between maternal diabetes and acute doses of alcohol. Pregnant TO mice were made diabetic by a single injection of streptozotocin (STZ) on gestation day (GD) 2. Single dose of 0.003 or 0.03 ml/g body weight of fresh ethanol (25% v/v of absolute alcohol in normal saline) was injected into groups of diabetic and nondiabetic animals on GD 7 or 8. One group of diabetic animals had a daily dose of 6-8 IU of insulin subcutaneously. Fetuses were collected on GD 18. There was a significant increase in the incidence of implantation failure in the diabetes plus ethanol groups and insulin control group. Ethanol injection on GD 7 accentuated diabetes-related embryonic resorption and intrauterine growth retardation (IUGR). This effect was less marked in the diabetic group treated with ethanol on GD 8. Diabetes alone produced a greater incidence of IUGR than ethanol alone. Midfacial hypoplasia and minor anomalies were found more frequently in the combination treatment groups. Holoprosencephaly and thymus hypoplasia observed in diabetic groups were found to be reduced in frequency in the diabetes plus ethanol groups, suggesting an antagonistic type of ethanol-diabetes interaction, stage-dependently. Since severely malformed embryos are known to be resorbed/killed in utero in mice, this reduction might reflect the magnitude of early death of severely malformed embryos. These data suggest that the interaction effects are possibly related to alterations in fundamental developmental processes of early embryos.     

In vivo labelling of the proteins in the first leaf of intact oat plants (Avena sativa)

In order to label the primary leaves of intact oat plants ( Avena sativa L. cv. Victory) for studying protein turnover using two-dimensional polyacrylamide gel electrophoresis, the following methods were tested: growth of seedlings on ³⁵S-sulfate-containing Knop medium, labelling with ³⁵S-methionine by vacuum infiltration of the leaf, injection into the leaf base or into the seed near the embryo, or wiping the surface of the leaf with ethanol followed by incubation in the labelled solution. A specific activity of 1.6 kBq per 20 μg of leaf protein applied was minimally necessary to obtain a well-resolved fluorogram of the gels. This level of labelling was reached only upon the treatment with ethanol, which did not require more than 0.55 MBq of ³⁵S-methionine per leaf. The method may be useful locally to apply compounds to intact plants.     

Entrapment of proteins in phosphatidylcholine vesicles.

The trapping efficiency of globular proteins in four different types of phosphatidylcholine vesicles was systematically studied. Vesicles were generated in a mixture of 125I-labeled proteins of various molecular weights. The trapped proteins were separated from untrapped proteins by gel filtration and ultrafiltration and subsequently analyzed by gel electrophoresis and autoradiography. Entrapment of proteins was demonstrated by their resistance to trypsin digestion. The relative amount of each entrapped protein species was then compared to that of the original protein solution. In multilamellar vesicles and large unilamellar vesicles, proteins of molecular weight up to 97 000 had the same trapping efficiency as sucrose. In small unilamellar vesicles generated by either sonication or ethanol injection, however, the relative trapping efficiency of protein decreased progressively as the molecular weight of the protein became greater. For example, the trapping efficiency of alpha-amylase (Mr 97 000) was only half of that for sucrose. The apparent decrease in trapping efficiency with the protein's molecular weight in small unilamellar vesicles canbe accounted for by the combination of the bound water layer at the vesicle's internal surface and the steric hindrance when protein is captured during vesicle formation.     

The response of circulating parameters of bone mineral metabolism to ethanol- and EDTA-induced hypocalcemia in the rat

The mechanism of the acute hypocalcemia that follows acute ethanol administration has not been established. Measurements of parathyroid hormone (PTH) performed during this hypocalcemia reveal conflicting results. We compared the response of ionized calcium (Ca²⁺), immunoreactive PTH and bone Gla protein (BGP) after ethanol- and EDTA-induced hypocalcemia. 103 male Sprague Dawley rats each weighing approximately 300 g received ethanol and 100 rats of similar weight received EDTA. In each of these studies the animals were divided into experimental and control groups. The ethanol-treated rats received ethanol, 2 g/kg body weight, by ip injection and the EDTA-treated rats received 100 mg EDTA/kg body weight by im injection. Controls received normal saline by the corresponding route of administration. Rats were sacrificed at 0, 30, 60, 90, 180 and 360 min for the measurement of the above parameters. In both experimental groups Ca²⁺ levels were significantly reduced to the same degree by 30 min with return to control values by 360 min. There was no significant difference in immunoreactive PTH, and BGP between control and ethanol-treated groups. In the EDTA-treated rats, however, PTH values were significantly increased at 30 (P < 0.005) and BGP at 60 and 90 minutes (P < 0.005) vs. control. Therefore acute ethanol administration appears to blunt the PTH response to hypocalcemia. A direct inhibitory effect of ethanol on osteoblast function ie BGP production cannot be excluded. In addition, PTH may stimulate BGP.     

Effect of maternal diabetes and ethanol interactions on embryo development in the mouse

The aim of this study was to determine the possible fetal effects of interaction between maternal diabetes and acute doses of alcohol. Pregnant TO mice were made diabetic by a single injection of streptozotocin (STZ) on gestation day (GD) 2. Single dose of 0.003 or 0.03 ml/g body weight of fresh ethanol (25% v/v of absolute alcohol in normal saline) was injected into groups of diabetic and nondiabetic animals on GD 7 or 8. One group of diabetic animals had a daily dose of 6–8 IU of insulin subcutaneously. Fetuses were collected on GD 18. There was a significant increase in the incidence of implantation failure in the diabetes plus ethanol groups and insulin control group. Ethanol injection on GD 7 accentuated diabetes-related embryonic resorption and intrauterine growth retardation (IUGR). This effect was less marked in the diabetic group treated with ethanol on GD 8. Diabetes alone produced a greater incidence of IUGR than ethanol alone. Midfacial hypoplasia and minor anomalies were found more frequently in the combination treatment groups. Holoprosencephaly and thymus hypoplasia observed in diabetic groups were found to be reduced in frequency in the diabetes plus ethanol groups, suggesting an antagonistic type of ethanol--diabetes interaction, stage-dependently. Since severely malformed embryos are known to be resorbed/killed in utero in mice, this reduction might reflect the magnitude of early death of severely malformed embryos. These data suggest that the interaction effects are possibly related to alterations in fundamental developmental processes of early embryos. (Mol Cell Biochem 261: 43–56, 2004)     

Changes in nuclear proteins of astrocytes as a result of acute ammonia or ethanol exposure

We have used an in vitro system to monitor the effects of high levels of ammonia and ethanol on glial cells. Nuclei were isolated and the protein profiles examined by two-dimensional gel electrophoresis. Acute exposure of rat astrocyte cell cultures to ammonia or ethanol resulted in changes in cellular morphology and the level of some nuclear proteins. Glial fibrillary acidic protein (GFAP) levels remained constant under both treatments. Several nuclear proteins were increased specifically. Only one protein was visually detected which was unique to treatment with ammonia or ethanol. This protein (p2a) appeared only in the presence of ammonia. There were no changes in previously observed astrocyte-associated proteins (Silverman et al. Neurochem Int. 12, 513–518, 1988). Two proteins appeared de novo upon either treatment with either ammonia or ethanol. These latter proteins had a molecular weight and pI profile similar to the major class of nuclear stress proteins (hsp70). However, results from immunoblot experiments clearly demonstrated that hsp70 was not induced in astroycte cultures following exposure to ammonia or ethanol.     

Influence of vitamin C status on the metabolic rate of a single dose of ethanol-1-(14)C in guinea pigs

The rate of oxidative metabolism after a single i.p. dose of ethanol-1-(14)C was studied in male guinea pigs, previously treated with two different levels of vitamin C (traces or 0.5 g/100 g) in their diet for 5 weeks. While the body weight did not differ between these two groups after 5 weeks of the dietary regimen, the vitamin C concentration in the liver was five times higher in the group with the high vitamin C intake. The cumulative amounts of breathing 14CO2 measured at short time intervals during 24 hours after an ethanol-14C injection (23 mg ethanol and 160 kBq per kg body weight or 2.35 g ethanol and 165 kBq per kg body weight in a parallel experiment) were significantly different. The half-time of ethanol turnover reached a value of 5.1 h versus 6.9 h (9.9 vs 14.4 h in a parallel experiment) in the high and low saturated group respectively. The long-term pretreatment of guinea pigs with large doses of vitamin C accelerated ethanol metabolism. Improvement of the redox state and activation of the cytochrome P450 system in vitamin C-supplemented organism are considered to be the reason for the increased ethanol catabolism.     

Toxic and antigrowth effects of raw and processed field bean (Dolichos lablab) on albino rats

Samples of freeze dried green field bean (Dolichos lablab) and dry mature bean, were subjected to the following processing methods—heat processing, extraction with 80% ethanol, hexane or dilute acid, protein isolation; and these samples were evaluated for growth promoting value and toxicity. Extraction with 80% ethanol or with dilute acid increased survival period of the animals; but these did not promote growth. Heat processing was essential to destroy antinutritional factors and promote growth. Extraction of the beans with 80% ethanol did not however alter the trypsin inhibitor or haemagglutinin activities. The protein isolate and acid-extracted residue which had low trypsin inhibitor and haemagglutinin activities, did not also promote growth. Thus the trypsin inhibtor and haemagglutinin activities did not completely account for the toxicity to albino rats. However, heat processing of ethanol extracted bean flour indicated that the beneficial effect of ethanol extraction was not apparent, once the samples were heat processed. Dry mature bean dhal was more toxic than the whole bean either dry or green. Supplementation of heat processed field bean with methionine and tryptophan promoted good growth of albino rats and significantly increased the protein efficiency ratio. Part of the Ph.D. thesis entitled “Studies on the factors affecting the nutritive value of field beanDolichos lablab”, University of Mysore, 1977.     

Effects of dexamethasone and indomethacin on estrogen-induced uterine growth

Certain aspects of estrogen-induced uterine growth are reminiscent of an inflammatory response. Dexamethasone (DEX) and indomethacin (IND), two anti-inflammatory agents that interfere with arachidonic acid metabolism, were examined with respect to their effects on several growth-associated responses of the uterus to estrogen. Ovariectomized rats were given a s.c. injection of either DEX (2 mg) or IND (8 mg) immediately prior to receiving a s.c. injection of estradiol (10 ωg). At 4 hr, DEX inhibited estrogen-stimulated uterine wet weight and ornithine decarboxylase (ODC) activity by 100% and 48%, respectively. At 24 hr, ³H-leucine incorporation into protein was inhibited 44% and ³H-thymidine incorporation into DNA was depressed 83%. Estrogen-stimulated increases in uterine protein/DNA ratio and epithelial microvilli density at 24 hr were not inhibited by DEX. IND inhibited estrogen-stimulated wet weight by 64% and ³H-thymidine incorporation into DNA by 42%, yet did not inhibit the increases in ODC activity, ³H-leucine incorporation into protein or protein/DNA ratio. These results suggest that the inflammation-like component of estrogen-induced uterine growth is mediated, at least in part, by arachidonic acid metabolites and is directed primarily toward stimulating cell division, and not cell growth.     

Evaluation of allopurinol as a cardioprotectant in ethanol-fed turkey poults

Cardioprotectant effects of allopurinol were investigated in ethanol-fed poults during induction of cardiomyopathy and during a period of ethanol abstinence. In young poults fed ethanol, allopurinol has an additive effect on depression of growth but has little or no effect on caloric consumption. Allopurinol significantly depresses heart weight and heart weight to body weight ratios in young poults. In poults 5 weeks and older, allopurinol depresses significantly body weight and caloric consumption. Although allopurinol depresses heart weight in older poults, it appears to have little or no effect on heart weight to body weight ratios. Cardioprotective effect of allopurinol is more apparent during the inductive process than during the recovery phase.     

Effect of pyrazole on ethanol metabolism in ethanol-tolerant rats.

Adult male rats were pair-fed liquid diets, providing 37% of calories as ethanol or sucrose, for 1 month. Alcohol dehydrogenase (ADH) activity in the cytosol fractions of liver homogenates from the two groups did not differ with respect to total activity per 100 g body weight, Km for ethanol, or Ki for pyrazole. Other rats, fed in the same way, were fasted for 18-24 H, then given an intraperitoneal injection of pyrazole followed 1 h later by an injection of ethanol, 3g/kg. Blood alcohol curves showed an unexplained slower rise to maximum level in the chronic alcohol group. Both groups showed a period of several hours in which the blood alcohol stayed at the respective maximum concentrations, which were higher in the control group. After 7-8h the alcohol concentration began to fall in both groups, significantly more rapidly in the chronic alcohol-fed animals. A kinetic analysis shows that the results are adequately explained by the known effects of pyrazole on the ADH-mitochondrial system. The results are interpreted as evidence against the function of any microsomal ethanol oxidizing system in vivo.     

The effect of long-term, voluntary ethanol consumption on hepatic regeneration in rats

Previous studies have reported conflicting results regarding the effect of ethanol on hepatic regeneration. The purpose of the present study was to determine whether long-term, voluntary consumption of ethanol, within the range reported in humans, has an effect on hepatic regenerative activity in rats following partial hepatectomy. Ninety-four adult male Sprague-Dawley rats (n = 3-9/group) were studied. Based on the amount of 9% ethanol consumed over a 50-day period, low ethanol intake (0.1-1.9 g.kg-1.d-1) and high ethanol intake (2.0-4.0 g.kg-1.d-1) groups were identified. Control groups consisted of rats provided with propylene glycol in equivalent caloric amounts to the ethanol consumed by high ethanol intake rats (isocaloric group) and rats served water only (ad libitum group). An additional two groups from which ethanol was removed 5 days prior to surgery were also studied (low ethanol grace and high ethanol grace). Hepatic regeneration was determined by restitution of liver weight, [3H]thymidine incorporation into DNA, and [14C]leucine incorporation into protein 24, 48, and 72 h following partial (70%) hepatectomy. The results of the study revealed no significant differences in the rate of hepatic regeneration between low and high ethanol consuming rats or between either of these groups and isocaloric or ad-libitum fed control groups. Regeneration in low ethanol grace and high ethanol grace groups were also similar to each other and controls. Moreover, there was no correlation between mean ethanol consumption per rat and restitution of liver weight, [3H]thymidine incorporation into DNA, or [14C]leucine incorporation into protein by the regenerating liver (r = 0.0716, -0.1637, and 0.1395, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of chronic ethanol ingestion on protein metabolism in type-I- and type-II-fibre-rich skeletal muscles of the rat.

1. The effects of chronic ethanol feeding on muscles containing a predominance of either Type I (aerobic, slow-twitch) or Type II (anaerobic, fast-twitch) fibres were studied. Male Wistar rats, weighing approx. 90 g or 280 g, were pair-fed on a nutritionally complete liquid diet containing 36% of total energy as ethanol, or isovolumetric amounts of the same diet in which ethanol was replaced by isoenergetic glucose. After 6 weeks feeding, fractional rates of protein synthesis were measured with a flooding dose of L-[4-(3)H]-phenylalanine and muscles were analysed for protein, RNA and DNA. 2. Ethanol feeding decreased muscle weight, protein, RNA and DNA contents in both small and large rats. Type-II-fibre-rich muscles showed greater changes than did Type-I-fibre-rich muscles. Changes in protein paralleled decreases in DNA. 3. The capacity for protein synthesis (RNA/protein), fractional rates of protein synthesis and absolute rates of protein synthesis were decreased by ethanol feeding in both small and large rats. The amounts of protein synthesized relative to RNA and DNA were also decreased. Changes were less marked in Type-I than in Type-II-fibre-rich muscles. Loss of protein, RNA and DNA was greater in small rats, but protein synthesis was more markedly affected in large rats. 4. It was concluded that chronic ethanol feeding adversely affects protein metabolism in skeletal muscle. Fibre composition and animal size are also important factors in determining the pattern of response.     

Synthesis of poly[(R)-3-hydroxybutyric acid) in the cytoplasm of Pichia pastoris under oxygen limitation

We have constructed a tandem gene expression cassette containing three Ralstonia eutropha poly[(R)-3-hydroxybutyrate] (PHB) synthesis genes under the control of the Pichia pastoris glyceraldehyde-3-phosphate promoter and the green fluorescent protein (Gfp) under the control of the P. pastoris alcohol oxidase promoter. The inducible Gfp reporter protein has been used to rapidly isolate transformed strains with two copies of the entire expression cassette. The isolated strain exhibits Gfp induction kinetics that is twice as fast as that of the strains isolated without cell sorting. In addition, the sorted strains exhibited higher PHB contents in preliminary screening experiments. PHB synthesis was characterized in more detail in the sorted strain and was found to be dependent on culture conditions. It was observed that the specific PHB synthesis rate was dependent on the carbon source utilized and that the conditions of oxygen stress lead to increased fractional PHB content. When this strain is cultivated on glucose under oxygen-limited conditions, the cultures accumulated ethanol during the initial growth phase and then consumed the ethanol for the accumulation of PHB and biomass. While PHB was not synthesized during initial growth on glucose, significant levels of PHB were synthesized when ethanol was subsequently consumed. PHB was also synthesized under aerobic conditions when ethanol was the only carbon source. During growth on ethanol, the specific growth rate of the culture was reduced under oxygen-limited conditions but the specific PHB synthesis rate was relatively unaffected. Thus, the high accumulation of PHB which exceeded 30% of the cell dry weight appears to be the consequence of the decreased biomass growth rate under severe oxygen limitation.     

Changes in dry weight, protein, deoxyribonucleic acid, ribonucleic acid and reserve and structural carbohydrate during aerobic growth cycle of yeast

1. Changes in dry weight, DNA, RNA, protein and reserve and structural carbohydrate were measured during the aerobic growth of yeast on 0.9% glucose in an aerobic synthetic medium. 2. After glucose had been consumed and during the growth of yeast on ethanol and acetate, the rate of formation of DNA remained about the same but the rate of increase of dry weight was greatly diminished. 3. During the second stage of growth the ratios dry weight/DNA, protein/DNA, RNA/DNA and carbohydrate/DNA decreased to about 30% of the corresponding values during the first stage of growth. 4. A higher fraction of the dry weight of the yeast cells could be accounted for by the reserve carbohydrate content of the cells during the second stage of growth. 5. By the end of the first stage of growth an increase in the reserve carbohydrate content of the cells was observed. Part of this reserve carbohydrate was consumed by the cells in the beginning of the second stage of growth. The possibility of adaptation of cells at the expense of their reserves is discussed.