Quantitative study of hepatocytes from minipigs and minipig fetuses exposed to ethanol in vivo

The effect of ethanol on hepatocytes from pregnant minipigs and their half-term fetuses was studied with the aid of morphometric methods. In the pregnant minipigs the hepatocytes of the ethanol-treated animals showed a significant increase in the volume density of mitochondria, autophagic vacuoles, Golgi complexes and smooth endoplasmic reticulum, and a significant decrease of glycogen. In the half-term fetuses the hepatocytes of ethanol-exposed animals showed no significant change in the volume density of mitochondria, peroxisomes, autophagic vacuoles, Golgi complexes, smooth endoplasmic reticulum or glycogen, and no significant change in the surface density of granular endoplasmic cisternae. The present investigation indicates that in the maternal hepatocyte certain cytoplasmic components are quantitatively changed by ethanol, whereas the volume and surface densities of identical components in the fetal hepatocyte are unaffected. The significance of these results is discussed.     

External examination of limb positions in near-term mouse fetuses: an experimental study and review of the literature published in Teratology

The accidental finding of near-term mouse fetuses exhibiting limb positions that could have been interpreted as malformations but that were, in fact, spurious, led us to study the posture of the limbs in the offspring of 100 CD-1 mouse dams. A total of 1,015 fetuses (18 days-old, vaginal plug: day 0), was examined. Fifteen of these fetuses presented positions that deviated markedly from the norm and were therefore considered to be "unusual," although the usual position was recovered in 6 of the 15 by placing the affected limb in a standard position and eventually eliciting locomotion. A broad review of the literature was made in order to evaluate our results fully. This review included 43 reports published in Teratology that recorded abnormal limb positions in near-term mouse and rat fetuses, and another 9 reports where these were not referred to but that included photographs showing such abnormalities. The data obtained from our experimental study and review led us to conclude that for a correct record of abnormal positions in near-term mouse and rat fetuses, authors must follow appropriate, clearly described technical procedures, including maneuvers to rule out spurious displacements; give adequate information in the legends of the photographs; use unequivocal terminology; and carry out further research to determine accurately the range in variation of normal limb positions.     

Influence of maternal dexamethasone treatment on morphometric characteristics of pituitary GH cells and body weight in near-term rat fetuses

Growth hormone (GH) and glucocorticoids have a powerful influence on controlling fetal growth, differentiation and maturation of numerous tissues. In the present study, the effect of maternal dexamethasone (Dx) treatment on GH cells and body weight in 19- and 21-day-old rat fetuses was investigated using immunocytochemical and morphometric methods. Pregnant female rats received daily injections of 1.0-0.5-0.5 mg Dx/kg b.w. on days 16-18 of pregnancy (experimental group), while the control group received an equal volume of saline. Dx treatment of pregnant rats enhanced immunostaining intensity and significantly increased (p<0.05) GH nuclear and cell volume, as well as volume density and number of GH cells per square millimeter in 19-day-old fetuses compared to the controls. In 21-day-old fetuses after maternal Dx administration, immunoreactivity, volume density and number of GH cells remained significantly increased (p<0.05). Dx treatment of pregnant rats resulted in marked body weight reduction of 21-day-old but not 19 days old fetuses in comparison with the corresponding controls. The presented results demonstrate that maternal Dx application has pronounced effect on morphometric parameters of GH cells of 19- and 21-day-old fetuses. Also, in near-term rat fetuses body weight was largely independent of pituitary GH cell activity.     

Cerebrovascular effects of intravenous dopamine infusions in fetal sheep.

Preterm infants are often treated with intravenous dopamine to increase mean arterial blood pressure (MAP). However, there are few data regarding cerebrovascular responses of developing animals to dopamine infusions. We studied eight near-term and eight preterm chronically catheterized unanesthetized fetal sheep. We measured cerebral blood flow and calculated cerebral vascular resistance (CVR) at baseline and during dopamine infusion at 2.5, 7.5, 25, and 75 microg x kg(-1) x min(-1). In preterm fetuses, MAP increased only at 75 microg x kg(-1) x min(-1) (25 +/- 5%), whereas in near-term fetuses MAP increased at 25 microg x kg(-1) x min(-1) (28 +/- 4%) and further at 75 microg x kg(-1) x min(-1) (51 +/- 3%). Dopamine infusion was associated with cerebral vasoconstriction in both groups. At 25 microg x kg(-1) x min(-1), CVR increased 77 +/- 51% in preterm fetuses and 41 +/- 11% in near-term fetuses, and at 75 microg x kg(-1) x min(-1), CVR increased 80 +/- 33% in preterm fetuses and 83 +/- 21% in near-term fetuses. We tested these responses to dopamine in 11 additional near-term fetuses under alpha-adrenergic blockade (phenoxybenzamine, n = 5) and under dopaminergic D(1)-receptor blockade (SCH-23390, n = 6). Phenoxybenzamine completely blocked dopamine's pressor and cerebral vasoconstrictive effects, while D(1)-receptor blockade had no effect. Therefore, in unanesthetized developing fetuses, dopamine infusion is associated with cerebral vasoconstriction, which is likely an autoregulatory, alpha-adrenergic response to an increase in blood pressure.     

Peri- and postnatal development of heterogeneity in the amounts of endoplasmic reticulum in mouse hepatocytes

The surface density and area per cell of the endoplasmic reticulum (ER) in periportal and perihepatic hepatocytes from male ddY mice, "17-, 18-, and 19-day-old fetuses," "newborn and 1-, 5-, 10-, and 20-day-old animals," and "adult animals" were analyzed by quantitative electron microscopy. The surface density of rough ER was not significantly different between periportal and perihepatic cells in all age groups examined, except for 19-day-old fetuses in which the value was greater in periportal cells than perihepatic cells. The surface density of smooth ER and total (rough and smooth) ER did not significantly differ between the periportal and perihepatic cells from 17-day-old fetuses to 5-day-old animals. In 10- and 20-day-old and adult animals, the values of smooth and total ER were greater in perihepatic cells than in periportal cells. When the data were expressed as area per cell, the patterns of subacinar distributions hardly differed, but age-related changes differed considerably from the patterns seen in the surface density data. The differences were generally caused by the increase in hepatocyte volume between 20 days of age and adulthood, especially in perihepatic cells, and by the changes in volume during the perinatal period. The results show that differences in the surface density and area per cell of smooth and total ER between periportal and perihepatic hepatocytes evident in adult animals are not present in fetal and newborn animals but arise during postnatal development.     

Effect of acute, multiple-dose ethanol on maternal and fetal blood gases and acid-base balance in the near-term pregnant ewe

The effect of ethanol on maternal and fetal blood gases and acid-base balance was determined in six conscious instrumented near-term pregnant ewes for maternal intravenous infusion of 3 g ethanol/kg total body weight administered as six doses of 0.5 g ethanol/kg total body weight over 8 h. Maternal and fetal blood ethanol concentrations, determined in two animals, were maximal at 8 h (3.74 and 3.82 mg/mL, respectively) and were virtually identical during the 24-h study. Maternal and fetal blood gases and acid-base balance were not significantly altered during and after ethanol administration compared with preinfusion values. The data demonstrate that, during near-term ovine pregnancy, the equivalent of a binge-type drinking episode does not produce fetal hypoxia or acidosis. Furthermore, these data do not support the postulated involvement of ethanol-induced fetal hypoxia in the mechanism of ethanol teratogenesis.     

Effects of alcohol (ethanol) on the fetus

Alcohol (ethanol) use during pregnancy can produce a wide spectrum of effects in the developing embryo/fetus that are dependent on the maternal drinking pattern. The effects of chronic ethanol exposure on the developing conceptus are reviewed with primary focus on ethanol teratogenesis, manifesting in the human as the fetal alcohol syndrome or fetal alcohol effects. The effects of acute ethanol exposure on the near-term fetus are described, including suppressed fetal breathing movements, electrocorticographic (ECoG) activity and electrooculographic (EOG) activity. The ethanol-induced suppression of fetal breathing movements is a very sensitive index of acute exposure of the near-term fetus to ethanol, and appears to involve a direct mechanism of action rather than an indirect mechanism involving suppression of electrocortical activity. The disposition of ethanol and its pharmacologically active proximate metabolite, acetaldehyde, and the activity of alcohol dehydrogenase and aldehyde dehydrogenase in the near-term maternal-fetal unit are described, and a pharmacokinetic model is proposed. The effects of short-term ethanol exposure on the near-term fetus include the development of tolerance to the ethanol-induced suppression of fetal breathing movements, low-voltage ECoG activity and EOG activity. The development of tolerance occurs more rapidly to the latter two fetal biophysical activities. The mechanism of tolerance development appears to be pharmacodynamic (functional) in nature, as there is no increase in the rate of ethanol elimination from the maternal-fetal unit. The role of prostaglandins (PGs) in the mechanism of the ethanol-induced suppression of fetal breathing movements is described. In the near-term fetus, there is a direct relationship between fetal blood ethanol concentration and fetal plasma PGE2 concentration, and an inverse relationship between the incidence of fetal breathing movements and each of fetal plasma and fetal cerebrospinal fluid (CSF) PGE2 concentrations. Indomethacin, a PG synthetase inhibitor, selectively blocks and reverses the ethanol-induced suppression of fetal breathing movements. These data support the postulates that the ethanol-induced suppression of fetal breathing movements is mediated by increased PGE2 concentration in the near-term fetus and that the ability of indomethacin to antagonize the ethanol-induced suppression of fetal breathing movements is due to its biochemical action to decrease fetal PGE2 concentration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Central angiotensin induction of fetal brain c-fos expression and swallowing activity

The present study examined physiological and cellular responses to central application of ANG II in ovine fetuses and determined the fetal central ANG-mediated dipsogenic sites in utero. Chronically prepared near-term ovine fetuses (130 +/- 2 days) received injection of ANG II (1.5 microg/kg icv). Fetuses were monitored for 3.5 h for swallowing activity, after which animals were killed and fetal brains were perfused for subsequent Fos staining. Intracerebroventricular ANG II significantly increased fetal swallowing in near-term ovine fetuses (1.1 +/- 0.2 to 4.5 +/- 1.0 swallows/min). The initiation of stimulated fetal swallowing activity was similar to the latency of thirst responses (drinking behavior) elicited by central ANG II in adult animals. ANG II evoked increased Fos staining in putative dipsogenic centers, including the subfornical organ, organum vasculosum of the lamina terminalis, and median preoptic nucleus. Intracerebroventricular injection of ANG II also caused c-fos expression in the fetal hindbrain. These results indicate that an ANG II-mediated central dipsogenic mechanism is intact before birth, acting at sites consistent with the dipsogenic neural network. Central ANG II mechanisms likely contribute to fetal body fluid and amniotic fluid regulation.     

Dexamethasone exposure affects paraventricular nucleus and pituitary corticotrophs in female rat fetuses: An unbiased stereological and immunohistochemical study

The hypothalamic paraventricular nucleus (PVN) drives the stress response by activating the hypothalamo-pituitary-adrenal (HPA) axis, particularly vulnerable to glucocorticoid exposure during development. To evaluate the effects of fetal dexamethasone (Dx) exposure on the stereological features of PVN and HPA axis activity in female rat fetuses, pregnant rats received 0.5 mg Dx/kg/b.w./day on days 16, 17 and 18 of pregnancy and 21-day-old fetuses were obtained; controls received the same volume of saline. In an unbiased stereological approach, Cavalieri’s principle and an optical fractionator were used for estimating volume and total cell number of the PVN, respectively. The intensity of corticotropin-releasing hormone (CRH) immunoreactivity in the median eminence (ME) was determined by CRH optical density and the adrenocorticotropic hormone (ACTH) relative fluorescence signal intensity (RIF) in pituitary corticotrophs was measured using Image J. Significant reductions (p < 0.05) in PVN volume and cell number were found in fetuses exposed to Dx. Additionally, CRH optical density in the ME and ACTH RIF (p < 0.05) in the corticotrophs were decreased. The established results suggest that the reduced number of cells in the PVN after maternal Dx administration negatively affects the CRH content in the ME and the ACTH quantity in pituitary corticotrophs in near-term fetuses.     

Development of tolerance to ethanol-induced suppression of breathing movements and brain activity in the near-term fetal sheep during short-term maternal administration of ethanol

The effect of short-term maternal ethanol administration on the ethanol-induced suppression of fetal breathing movements, electrocortical (ECoG) activity, and electroocular (EOG) activity was determined in the near-term fetal sheep. Twelve conscious instrumented pregnant ewes (between 125 and 139 days of gestation; term, 147 days) received 1-h intravenous infusion of 1 g ethanol/kg total body weight daily for six days (n = 6) or an equivalent volume of normal saline daily for six days (n = 6). On the seventh day, the ethanol- and saline-pretreated animals were administered 1 g ethanol/kg total body weight. A further six ewes received 1-h intravenous infusion of 1 g ethanol/kg total body weight (n = 3) or an equivalent volume of normal saline (n = 3) daily for thirteen days with both groups receiving 1 g ethanol/kg total body weight on day fourteen. Fetal ECoG and EOG activities, and fetal breathing movements were monitored continuously over the post- operative and experimental periods. Saline infusion had no significant effect on the parameters studied. Fetal breathing movements were suppressed for 8 h after the first ethanol dose, and were not significantly suppressed after fourteen days of once-daily, maternal ethanol administration. Low-voltage ECoG and EOG activities were suppressed for 3 h after the first ethanol dose, and were not significantly suppressed after seven days of repeated ethanol administration. Maternal and fetal blood gases and acid-base balance were not significantly affected by maternal ethanol administration. These data demonstrate that short-term maternal administration of ethanol results in the development of tolerance to ethanol in the mature fetus.     

Effects of postnatal dexamethasone on blood-brain barrier permeability and brain water content in newborn lambs

We showed that antenatal corticosteroids reduced blood-brain barrier permeability in fetuses at 60 and 80%, but not 90% of gestation, and decreased brain water content in fetuses. Our objective was to examine the effects of postnatal corticosteroids on regional blood-brain barrier permeability and brain water content in newborn lambs. Three dexamethasone treatment groups were studied in 3- to 5-day-old lambs. A 0.01 mg/kg dose was selected to estimate the amount of dexamethasone that might have reached fetuses via antenatal treatment of ewes in our previous studies. The other doses (0.25 and 0.5 mg/kg) were chosen to approximate those used clinically to treat infants with bronchopulmonary dysplasia. Lambs were randomly assigned to receive four intramuscular injections of dexamethasone or placebo given 12 h apart on days 3 and 4 of age. Blood-brain barrier function was measured with the blood-to-brain transfer constant (K(i)) to alpha-aminoisobutyric acid, brain plasma volume was measured with polyethylene glycol for the calculation of K(i,) and brain water was measured by wet-to-dry tissue weights. Postnatal treatment with corticosteroids did not reduce barrier permeability in newborn lambs. Brain blood volume was higher in the 0.25 and 0.5 mg/kg dose dexamethasone groups than in the placebo group. Brain water content did not differ among the groups. We conclude that postnatal treatment with corticosteroids did not reduce regional blood-brain barrier permeability or brain water content but increased the brain plasma volume in newborn lambs. These findings are consistent with our previous work indicating that barrier permeability is responsive to corticosteroids at 60 and 80% of gestation and brain water regulation at 60% of gestation, but not in near-term fetuses or newborn lambs.     

Changes in some chromatin and cytoplasmic enzymes of perinatal rat hepatocytes during culture

Summary Hepatocytes prepared from rats at various perinatal stages were cultured in selective medium that does not allow fibroblastic cell growth. Cell population remained homogeneous during the culture. Hepatocytes undergo divisions for a period, which varies according to the stage of development of the rat. Light and electron microscope observations showed the presence of numerous cytoplasmic organelles; moreover, hydrocortisone-induced structures similar to bile canaliculi. Chromatin protein kinase decreased rapidly during culture except in samples prepared from 17-day fetuses in which it remained unchanged for 2 days and decreased to a lesser extent afterwards. Chromatin nonhistone proteins were incubated with (γ-³²P) ATP and the phosphorylation pattern analyzed on polyacrylamide gels. Many radioactive peaks were observed in chromatin proteins from 17-day fetuses; they were much lower in proteins from 19-day fetuses. The phosphorylation pattern was analyzed in hepatocytes after 2 days of culture. Many radioactive peaks were observed with proteins from heapatocytes taken from 17-day fetuses; no radioactivity was observed in proteins from 19-day fetuses. This is in contrast with the absence of radioactive peaks in chromatin proteins from adult rat hepatocytes. In cytoplasm, aldolase and pyruvate kinase specific activities varied according to the age of the rat. They strongly decreased during culture except in hepatocytes from 15-and 17-day fetuses, in which they remained stable for at least 5 days. The stability of chromatin and cytoplasmic enzymes in hepatocytes from 17-day fetuses could result from their ability to be regulated by hormones that are secreted at this stage of development.     

A COMPARED STUDY ON THE PURIFICATION OF FETAL AND ADULT RAT HEPATOCYTE SUSPENSIONS BY BIOMAGNETIC ISOLATION TECHNIQUES AND FLOW CYTOMETRY ANALYSIS

An analysis of liver cell populations from both adult and 21 day pregnancy rat fetuses (E21) was carried out. The results show that E21 hepatocytes express OX-43, as do endothelial cells but not adult hepatocytes. OX-43 could be used in future as a cell marker for the hepatocyte maturation.     

Isocaloric maternal low-protein diet alters IGF-I,IGFBPs, and hepatocyte proliferation in the fetal rat

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by approximately 30% compared with control hepatocytes (P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma (P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma (P < 0.01) and fetal liver (P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I (P < 0.01) and more 29- to 32-kDa IGFBPs (P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3-4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 +/- 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 +/- 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects.     

Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses

We hypothesized that congenital diaphragmatic hernia (CDH) may decrease distal air space fluid absorption due to immaturity of alveolar epithelial cells from a loss of the normal epithelial Na+ transport, as assessed by amiloride and epithelial Na+ channel (ENaC) and Na-K-ATPase expression, as well as failure to respond to endogenous epinephrine as assessed by propranolol. Timed-pregnant dams were gavage fed 100 mg of nitrofen at 9.5-day gestation to induce CDH in the fetuses, and distal air space fluid absorption experiments were carried out on 22-day gestation (term) fetuses. Controls were nitrofen-exposed fetuses without CDH. Absorption of distal air space fluid was measured from the increase in 131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. In controls, distal air space fluid absorption was rapid and mediated by beta-adrenoceptors as demonstrated by reversal to fluid secretion after propranolol. Normal lung fluid absorption was also partially inhibited by amiloride. In contrast, CDH fetuses continued to show lung fluid secretion, and this secretion was not affected by either propranolol or amiloride. CDH lungs showed a 67% reduction in alpha-ENaC and beta-ENaC expression, but no change in alpha1-Na-K-ATPase expression. These studies demonstrate: 1) CDH delays lung maturation with impaired distal air space fluid absorption secondary to inadequate Na+ uptake by the distal lung epithelium that results in fluid-filled lungs at birth with reduced capacity to establish postnatal breathing, and 2) the main stimulus to lung fluid absorption in near-term control fetuses, elevated endogenous epinephrine levels, is not functional in CDH fetuses.     

No effect of chronic ethanol administration on the activity of alcohol dehydrogenase and aldehyde dehydrogenases in the near-term pregnant guinea pig

The objective of this study was to determine the effect of chronic maternal administration of moderate-dose ethanol on alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the guinea pig at near-term pregnancy. The activity of each enzyme in the maternal liver, fetal liver, and placenta of the guinea pig at 59 days of gestation (term, 66 days) was determined spectrophotometrically following chronic daily oral administration of two doses of 1 g ethanol/kg maternal body weight or isocaloric sucrose solution. There was no experimental evidence of ethanol-induced malnutrition in the mother or growth retardation in the fetus. There was a statistically significant increase (65%) in the microsomal cytochrome P-450 content of the maternal liver for the ethanol treatment compared with the sucrose treatment. The alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the maternal liver, fetal liver, and placenta were not statistically different for the ethanol-treated compared with the sucrose-treated animals. This also was the case for the maternal blood and fetal blood ethanol and acetaldehyde concentrations, determined at 2h after maternal administration of 1 g ethanol/kg maternal body weight. These data demonstrate that the ethanol- and acetaldehyde-oxidizing enzyme activities in the maternal-placental-fetal unit of the guinea pig at near-term pregnancy were not changed by chronic administration of moderate-dose ethanol.     

Effects of ethanol and clofibrate on expression of cytochrome P-450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes

Cultured and cocultured rat hepatocytes were used to study the effects of ethanol and clofibrate on cytochrome P-450 (P-450) enzymes and epoxide hydrolase. We showed that in the presence of ethanol, clofibrate or both compounds, rat hepatocytes were able to express, after 3 days of pure culture, quantitatively and qualitatively reasonable levels of most cytochrome P-450 enzymes and epoxide hydrolase, compared to freshly isolated hepatocytes. However, ethanol induced the P-450IA subfamily, and clofibrate the P-450-IVA subfamily. In cocultures, after 6 days, most P-450 enzymes were still expressed while P-450IIC11 was completely lost. Ethanol and clofibrate had the same effect as in pure culture. These results show, by modifying culture medium conditions and cell-cell interactions, that it is possible to maintain reasonable xenobiotic-metabolizing-enzyme expression; however, these conditions have to be improved in order to preserve better P-450 expression. The mechanism of these effects and the inducibility of these systems remain to be elucidated by a study at molecular level.     

Role of peroxisomal fatty acid beta-oxidation in ethanol metabolism

The contribution of peroxisomal fatty acid beta-oxidation to ethanol metabolism was examined in deermice hepatocytes. Addition of 1 mM oleate to hepatocytes isolated from fasted alcohol dehydrogenase (ADH)-positive deermice in the presence of 4-methylpyrazole or to hepatocytes from fasted or fed ADH-negative deermice produced only a slight and statistically not significant increase in ethanol oxidation. Lactate (10 mM), which is not a peroxisomal substrate, showed a greater effect on ethanol oxidation. There was also a lack of oleate effect on the oxidation of ethanol by hepatocytes of ADH-positive deermice. Furthermore, in ADH-negative deermice, the catalase inhibitor azide (0.1 mM) did not inhibit the increase in ethanol oxidation by oleate and lactate. The rate of oleate oxidation by hepatocytes from fasted ADH-negative deermice was much lower than that of ethanol. These results indicate that in deermice hepatocytes, peroxisomal fatty acid oxidation does not play major role in ethanol metabolism.