Effects of chronic prenatal ethanol exposure on mitochondrial glutathione and 8-iso-prostaglandin F2alpha concentrations in the hippocampus of the perinatal guinea pig.

It is hypothesised that oxidative stress is a key mechanism of ethanol neurobehavioural teratogenicity, resulting in altered endogenous antioxidant status and increased membrane lipid peroxidation in the hippocampus of chronic prenatal ethanol exposure (CPEE) offspring. To test this hypothesis, timed pregnant guinea-pigs (term, approximately gestational day (GD) 68) received chronic daily oral administration of (i) 4 g ethanol kg(-1) maternal bodyweight, (ii) isocaloric sucrose with pair feeding, or (iii) water. At GD 65 (term fetus) and postnatal day (PD) 0 (neonate), individual offspring were killed, the brain was excised and the hippocampi were dissected. Glutathione (GSH) concentration was measured in the cytosolic and mitochondrial fractions of hippocampal homogenate. The occurrence of lipid peroxidation was determined by measuring the concentration of 8-iso-prostaglandin F2+/- (8-iso-PGF2+/-). There was CPEE-induced decreased brain weight and hippocampal weight at GD 65 and PD 0, decreased mitochondrial GSH concentration in the hippocampus at PD 0, with no change in mitochondrial GSH concentration at GD 65 or cytosolic GSH concentration at GD 65 or PD 0, and no change in mitochondrial or whole-homogenate 8-iso-PGF2+/- concentration in the hippocampus at GD 65 or PD 0. The data demonstrate that CPEE produces selective mitochondrial dysfunction in the hippocampus of the neonatal guinea-pig, involving GSH depletion.

Role of 4-hydroxynonenal in modification of cytochrome c oxidase in ischemia/reperfused rat heart.

Mitochondrial dysfunction is a characteristic of ischemia/reperfusion (I/R) injury in the heart. While oxidative stress has been implicated in mitochondrial damage in I/R injury, the underlying mechanisms are unclear. 4-Hydroxynonenal (HNE) is a toxic aldehyde generated by lipid peroxidation. The purpose of the present study was to assess the role of HNE in I/R-induced damage of a crucial component of the mitochondrial electron transport chain, cytochrome c oxidase (COX). I/R was induced in male WKY rats by 15 mins of ischemia followed by reperfusion for up to 3 h. COX activity was measured spectrophotometrically at 550 nm. HNE adducts with COX subunits were detected by Western Blot using an HNE-histidine antibody. HNE and reduced glutathione (GSH) contents were measured in mitochondria by HPLC. Following 3 h of reperfusion, COX activity was reduced to 59% of control, accompanied by increases in HNE adducts with COX (P<0.05). Mitochondrial HNE content in reperfused hearts was increased to 165% of control, whereas GSH was decreased to 62% of control (P<0.05). After purified COX was incubated with HNE in vitro, COX activity was decreased progressively with increasing concentrations of HNE, accompanied by concentration-dependent formation of HNE adducts with COX. GSH prevented HNE adduct formation as well as COX inhibition by HNE. These results suggest that HNE, via adduct formation with COX subunits, plays an important role in COX dysfunction caused by reperfusion. The findings also indicate that decreases in mitochondrial GSH stores in reperfused myocardium could potentiate HNE-mediated COX damage.

In utero ethanol exposure causes mitochondrial dysfunction, which can result in apoptotic cell death in fetal brain: a potential role for 4-hydroxynonenal.

BACKGROUND: In utero ethanol exposure causes abnormal fetal brain development that may partly be due to enhanced cell death. The mechanisms underlying this remain to be defined, but ethanol-induced oxidative stress may play a role. The following studies investigated the effects of short-term in utero ethanol exposure on fetal brain mitochondrial events that are known to elicit apoptotic cell death. Evidence is presented suggesting that 4-hydroxynonenal (HNE), a toxic product of lipid oxidation, is a causal factor in the observed mitochondrial damage. METHODS: Mitochondria were isolated from control and ethanol-exposed fetal brains (days 17 and 18 of gestation). Permeability transition was determined spectrophotometrically, and cytochrome c and apoptosis-inducing factor (AIF) release were assessed by Western blotting. Caspase-3 activity and DNA fragmentation were determined both as markers for mitochondrially mediated apoptosis and as consequences of cytochrome c and AIF release. RESULTS: Maternal ethanol intake caused an increase in mitochondrial permeability transition, and this was accompanied by cytochrome c and AIF release from fetal brain mitochondria that exceeded control values by 62 and 25%, respectively (p < 0.05). In utero ethanol exposure resulted in a 30% increase in caspase-3 activity and a 25% increase in DNA fragmentation (p < 0.05) in the fetal brain. HNE levels were increased by 23% (p < 0.05) in mitochondria by in vivo ethanol exposure. In vitro treatment of fetal brain mitochondria with HNE (25-100 microM) also caused increases in mitochondrial permeability transition, as well as dose-dependent releases of cytochrome c and AIF. CONCLUSIONS: These studies illustrate that in utero ethanol exposure can elicit a cascade of events in the fetal brain that are consistent with mitochondrially mediated apoptotic cell death. Additionally, the increase in mitochondrial content of HNE after ethanol intake and the ability of HNE added to fetal brain mitochondria to mimic these effects of in vivo ethanol exposure support a potential role for HNE in the proapoptotic responses to ethanol.

Effect of ethanol on thromboxane and prostacyclin production in the human placenta.

Fetal alcohol syndrome (FAS) is frequently associated with intrauterine growth retardation (IUGR). One cause of ethanol-induced IUGR is thought to be related to increased pressor activity in the human placenta, resulting in decreased oxygenation and nutrient transport to the fetus. Thus, we have investigated the effect of ethanol on paracrine substances, such as thromboxane and prostacyclin, that act as vasoregulators within the intrauterine tissues. In these studies we have utilized the perfused single human cotyledon system to study the effect of ethanol on placental prostanoid production. We assessed the effect of longer (240 min) and more acute (60 min) exposure to ethanol on release of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1 alpha) (6-keto-PGF(1 alpha)) at the maternal and fetal sides of the placenta. Thromboxane was increased by both longer and shorter ethanol exposure, especially on the fetal side of the placenta. Prostacyclin was essentially unchanged with exposure to ethanol. The thromboxane:prostacyclin ratio also tended to increase with both 60- and 240-min ethanol exposure, but a statistically significant increase was seen only at a few time points. In the 60-min ethanol exposure, an increase in thromboxane was observed both during and following exposure to ethanol. The increase in the thromboxane milieu observed with ethanol exposure may lead, at least in part, to the IUGR which is frequently associated with FAS. Prevention of this effect of ethanol on thromboxane production might be a beneficial intervention for FAS.

Formation of malondialdehyde adducts in livers of rats exposed to ethanol: role in ethanol-mediated inhibition of cytochrome c oxidase.

BACKGROUND: Previous studies in our laboratory demonstrated that short-term ethanol consumption by maternal rats increased the hepatic levels of 4-hydroxynonenal (HNE) in both the adult and the fetus. Additionally, HNE inhibited cytochrome c oxidase (COX) by forming adducts with the enzyme subunits. The present study examined modification of COX by another major aldehydic lipid peroxidation product, malondialdehyde (MDA), and its role in COX inhibition by ethanol. METHODS AND RESULTS: It is demonstrated in vitro that MDA inhibits the activity of purified COX while forming adducts with the enzyme. Compared with HNE, MDA is a more potent inhibitor of COX. Overnight incubation at room temperature caused an 80% decrease in COX activity by MDA versus a 67% decrease by HNE. MDA produced marked inhibition of COX activity at physiologically relevant concentrations, e.g., 43% inhibition at 10 microM. Although our previous studies documented that HNE formed adducts primarily with subunit IV of COX via histidine residues, the current report showed that MDA forms adducts with both subunit IV and subunit V via lysine residues. Furthermore, both aldehydes induce carbonyl formation in subunit IV. The in vivo role of MDA in the impairment of COX by ethanol is assessed in both adult and fetal liver after maternal ethanol consumption. CONCLUSIONS: The results showed that: (1) there are significant increases in MDA levels in liver homogenate as well as mitochondria in both adult and fetal livers after ethanol exposure; (2) these MDA levels are in the nanomole/mg protein range, in contrast to picomole/mg protein range of HNE in identical setting; and (3) ethanol-induced production of MDA is accompanied by enhanced formation of MDA adducts with COX. These findings suggest that MDA may play at least as equally an important role as HNE in ethanol-induced inhibition of COX.

Ethanol, oxidative stress, reactive aldehydes, and the fetus.

The fetotoxic effects of maternal ethanol (E) consumption have been documented for over two decades, yet the mechanisms underlying this devastating phenomenon remain uncertain. The wide variety of cellular/biochemical effects of E on fetal tissues is itself a puzzle and strongly suggests that fetotoxic responses to E reflect a multifactorial setting. Many of these responses can be conceptually connected to effects on membrane structure and function. Representative of this, are studies in our laboratory documenting E effects on fetal cell replication, membrane transport systems, membrane fluidity, Na+-K+ pump expression, and EGF receptor expression. Recent studies have provided evidence that oxidative stress may be one mechanism by which E produces these membrane-related events. We initially observed E-induced oxidative stress in cultured fetal rat hepatocytes, the latter exhibiting morphological and biochemical signs of mitochondrial damage. E increased H2O2, O2-, lipid peroxidation products, along with signs of membrane damage. Supplementation with antioxidants or agents that enhance glutathione stores reversed these effects. E was found to inhibit activities of mitochondrial respiratory chain components (a potential source of the enhanced levels of H2O2, and O2-) and this could be reversed by antioxidant treatment. Subsequent studies have documented oxidative stress and membrane lipid peroxidation in fetal brain and liver (gestation day 19) following a two day maternal E consumption and in gestation day 14 and 17 "embryos" immediately following a single dose of E to the pregnant dam. The means by which E can induce oxidative stress in fetal cells is under investigation. We have examined effects of E on activities of key antioxidant enzymes and found no depressant responses. However, the low levels of antioxidants in fetal tissues and an exaggerated response of fetal mitochondria to prooxidant stimulation in vitro, suggest that fetal cells are strongly predisposed to oxidative stress. Additionally, recent studies have suggested that fetal tissues are likewise prone to the formation and subsequent accumulation of at least one toxic lipid peroxidation product, 4-hydroxynonenal. We conclude that maternal E consumption induces oxidative stress in fetal tissues and that this is responsible for some toxic responses to E. Additionally, the low antioxidant defenses in fetal tissues and accumulation of toxic aldehyde products of lipid peroxidation predispose the fetus to oxidative damage.

Formation of 4-hydroxynonenal adducts with cytochrome c oxidase in rats following short-term ethanol intake.

This study addresses the role of the lipid peroxidation product, 4-hydroxynonenal (HNE), in ethanol-related damage of cytochrome c oxidase (COX) in vivo. It utilizes an animal model with acute ethanol exposure in which HNE levels in liver mitochondria are strikingly increased. Pregnant female Sprague-Dawley rats were administered 5 doses of ethanol (4 gm/kg, po at 12-hour intervals) beginning on day 17 of gestation and were sacrificed on day 19. Controls were pair-fed and received dextrose isocaloric to ethanol. Mitochondria were isolated from maternal and fetal livers and COX activities were measured spectrophotometrically. Compared with the pair-fed controls, COX activity was decreased with exposure to ethanol by 25% in maternal rats and 43% in fetal rats (P<.05). Western Blot with an HNE-Histidine antibody showed enhanced formation of HNE adducts with COX from ethanol-exposed rats, which was more pronounced in fetal than in adult livers. The HNE adducts were mainly with subunit IV of COX. The cause and effect relationship between HNE adduct formation and COX inhibition was examined in vitro by incubating purified COX with HNE. COX inhibition was accompanied by concentration-dependent HNE adduct formation that was consistent with those found in in vivo ethanol-exposed samples. These results suggest that the ethanol-related decreases in COX activity found in liver mitochondria could be attributable to HNE adduct formation with the enzyme complex. This could be an important mechanism by which modification of proteins occur in in vivo oxidative stress.

Inhibition of cytochrome c oxidase activity by 4-hydroxynonenal (HNE). Role of HNE adduct formation with the enzyme subunits.

The role of 4-hydroxynonenal (HNE), a major lipid peroxidation product, in oxidative damage to mitochondrial cytochrome c oxidase (COX) was examined. Oxidative stress was induced in mitochondria isolated from livers of male Sprague-Dawley rats by tert-butylhydroperoxide (t-BHP). COX activity was inhibited, with a concomitant increase in endogenous HNE level in mitochondria. COX activity was also inhibited following incubation of mitochondria with 50-450 microM HNE. Blocking HNE degradation intensified COX inhibition by HNE and by t-BHP-induced oxidative stress, the latter accompanied by a simultaneous increase in endogenous HNE production. On the other hand, COX inhibition by HNE was markedly reduced by potentiating HNE degradation via enhancing conjugation of HNE with reduced glutathione (GSH). Incubation of purified COX with 10-400 microM HNE resulted in HNE adduct formation with specific subunits of COX, correlated with inhibition of the enzyme activity. These data suggest that HNE may inhibit mitochondrial COX by forming adducts with the enzyme, and that this could be one mechanism underlying mitochondrial damage caused by oxidative stress. The findings also illustrate a role for GSH in protecting mitochondria from the deleterious effects of HNE.

Histochemical method for localization of hydrogen peroxide and oxygen radicals in the intact neonatal brain.

The generation of reactive oxygen species has been implicated in the pathogenesis of a wide variety of diseases of the central nervous system. Often these pathological conditions involve damage to specific cell types within selected areas of the brain. Thus, there is a marked need for a method which allows microscopic visualization/detection of these oxygen radicals in discrete brain areas. We are reporting a method to histochemically localize, with single cell resolution, hydrogen peroxide (H2O2) and oxygen radicals in the neonatal brain in vivo. This method expands on the technique developed to visualize H2O2 and the superoxide anion radical (O2-) in isolated perfused organs (e.g., lung, heart) (Bobbs, 1994). With our technique, the intact brain is perfused intracardially with warm oxygenated saline to remove blood, followed by perfusion with buffers containing either iron and diethylenetriaminepentaacetate for the detection of H2O2 or manganese for the detection of oxygen radicals. The free radical oxidizes its respective metal, which in turn oxidizes diaminobenzidine (DAB) to form a brown reaction product which can be visualized using light microscopy.

Catalase mediates acetaldehyde formation from ethanol in fetal and neonatal rat brain.

Fetal ethanol (E) exposure has well documented deleterious effects on brain development, yet it is uncertain if the neurotoxicity of maternal E consumption is generated by E itself, by its primary metabolite acetaldehyde (AcHO), or both. The current studies present evidence that homogenates of immature rat brains can generate AcHO via a catalase (CAT)-mediated reaction and that AcHO may be produced in vivo by this system. Homogenates of day 19 fetal rat brain were incubated with E (50 mM). When incubated with CAT inhibitors (sodium azide or 3-aminotriazole), AcHO formation was blocked, whereas neither the alcohol dehydrogenase inhibitor, 4-methylpyrazole, nor P-450 inhibitors decreased AcHO production. Three hours after one oral dose of E (4 g/kg) to a pregnant dam (gestation day 19), AcHO levels in fetal brain increased to 14.28 +/- 1.82 nM/g tissue. Baseline CAT activity in day 19 fetal brains was 4.5 times adult values (p < 0.05). Western blot analysis determined that CAT protein level in the day 19 fetal brain exceeded that in adult brain by 2.5 times. One hour after a single dose of E, CAT activity in day 19 fetal brain increased by 8.2 units/mg protein. In 5-day-old neonatal brains during the "third trimester" brain growth spurt, baseline CAT activity was twice the adult values (p < 0.05) and a 2-day in vivo E regimen increased AcHO levels to four times the control values, with a concomitant 1.7-fold increase in CAT activity. This was prevented by administration of a CAT inhibitor (3-amino-1,2,4-triazole). Immunohistochemical staining of neonatal brains exposed to E illustrated the presence of acetaldehyde-protein adducts. We conclude that AcHO is likely produced in rat fetal and neonatal brain via CAT-mediated oxidation of E. This phenomenon may be an important factor in the neurotoxic effects of in utero E exposure.

4-hydroxynonenal levels are enhanced in fetal liver mitochondria by in utero ethanol exposure.

Lipid peroxidation has been implicated in ethanol-induced liver injury and observed in fetal liver and brain after maternal ethanol consumption with mitochondria being the target organelles. This process generates a highly reactive and toxic product, 4-hydroxynonenal (HNE). In the present study, HNE levels and metabolism were assessed in mitochondria of fetal and maternal liver after in vivo ethanol exposure. Female Sprague-Dawley rats received five doses of ethanol (4 g/kg orally at 12-hour intervals) and were killed on day 19 of gestation. The results showed that HNE levels were enhanced in hepatic mitochondria of fetal rats exposed to ethanol, far in excess of that in adult liver mitochondria. Measurement of HNE metabolism showed that fetal mitochondria had a lower capacity for HNE catabolism than adult mitochondria. In adult mitochondria, HNE could be metabolized by nicotine adenine dinucleotide-dependent oxidation, reduced glutathione conjugation, and reduced nicotine adenine dinucleotide-dependent reduction, whereas in fetal liver only the former two pathways were active, but to a lesser degree than in adult mitochondria. On the other hand, mitochondria from fetal liver showed a higher production of HNE when oxidative stress was induced with t-butyl hydroperoxide. Prior in vivo ethanol exposure further potentiated HNE formation in t-butyl hydroperoxide-stimulated fetal liver mitochondria, but not in adult mitochondria. These findings indicate that increased levels of HNE in fetal liver mitochondria after maternal ethanol consumption reflect a higher susceptibility to HNE formation in addition to a lesser capacity to metabolize it. The enhanced accumulation of this toxic aldehyde may contribute to oxidative damage observed in fetal tissues after in utero ethanol exposure.

Ethanol-induced oxidative stress and enzymatic defenses in cultured fetal rat hepatocytes.

Previously, we have documented an ethanol (E)-induced oxidative stress (OS) in cultured fetal rat hepatocytes (FRH). The cause of this is uncertain, but an inhibition of key antioxidant enzymes could be a/the factor. OS was also observed in fetal liver (FL) during in utero E exposure, but not in maternal liver, a difference that might be related to selectively lower enzymatic defenses in the fetus. Here, we record effects of E on activities of catalase (Cat), superoxide dismutase (Cu, Zn SOD and Mn SOD), glutathione peroxidase (GPX), and glutathione-S-transferase (GST) in FRH isolated from 20-day-old fetuses and exposed to E (2 mg/ml) for up to 24 h and we compare these to adult rat liver data. E treatment decreased fetal liver reduced glutathione (GSH) pools by 23% (p < 0.05) and increased malondialdehyde (MDA) by 14% (p < 0.05) within 24 h of E exposure. E caused an increase in fetal liver Cat by 18%, 32%, and 47% by 3, 6, and 24 h of E, respectively (p < 0.05). A 24-h E exposure increased Cu, Zn SOD by 22% (p < 0.05) and Mn SOD by 21% (p < 0.05). A 24 h E treatment increased GPX by 18% (p < 0.05) and GST by 17% (p < 0.05). Cat in whole FL was 26% of adult (p < 0.05) whereas Cu, Zn SOD and Mn SOD in whole FL were 12% and 11% of adult levels (p < 0.05). GPX and GST in FL were 11% and 28% of adult values (p < 0.05). It is concluded that in FRH, E-induced OS is not caused by impaired activities of these enzymes, but their low basal activities (vs. adult) may predispose the fetus to OS.

Sumatriptan (Imitrex) transport by the human placenta.

Sumatriptan (Imitrex), a selective 5-hydroxytryptamine receptor agonist, has been found to be of therapeutic benefit in the acute management of migraine. There is no information on the transfer of this agent across the human placenta. Accordingly, the current study assessed the transport of this drug across the normal term human placenta, using the isolated perfused single cotyledon technique. We found that only about 15% of a single dose of the agent placed in the maternal reservoir crossed into the fetal compartment over 4 hr. Given the average elimination half-life of 2 hr for sumatriptan, it is evident that only very small amounts of the agent will cross from mother to fetus after single doses of Imitrex. Only the parent drug entered the fetal compartment. Metabolites were not detected in the perfusates, but there was evidence of some metabolism of sumatriptan in the placenta. The nature of the metabolites has not been determined. The mechanism of transfer of the drug across the placenta is passive (i.e., the clearance is similar to L-glucose which is passively transported), the rate of transfer is equal in both directions (maternal to fetal and in the reverse), and the drug does not cross into the fetus against a concentration gradient. This passive transport of sumatriptan across the placenta is consistent with its molecular weight, its water solubility, and its slow penetration across the blood-brain barrier in experimental animals.

Maternal-to-fetal transfer of 5-methyltetrahydrofolate by the perfused human placental cotyledon: evidence for a concentrative role by placental folate receptors in fetal folate delivery.

Folates play a vital role in cellular processes that are essential for fetal growth and viability. Thus the human placenta, which contains high-affinity membrane-associated placental folate receptors (PFRs), maintains a concentrative maternal-to-fetal flux of the vitamin under conditions of minimal dependence on variations of maternal dietary intake. To define transplacental folate transport and the role of PFRs in this mechanism, we utilized the isolated perfused human placental cotyledon. In closed system perfusions with 10 nmol/L 5-methyltetrahydrofolate, placental binding was rapid and extensive (47%), with a gradual maternal-to-fetal transfer of 5-methyltetrahydrofolate. Although hydrophilic PFRs were released into the fetal perfusate, PFR-bound folates constituted only a fraction of net transplacental folate transport. Transfer was bidirectional, not saturable, not inhibited by anion channel blockers, and dependent on perfusate levels. Placental binding far exceeded transfer, and pulsing the maternal circuit with tritiated 5-methyltetrahydrofolate, followed by washout of unbound radiolabel and rechallenge with unlabeled 5-methyltetrahydrofolate or folate, led to release of bound tritiated 5-methyltetrahydrofolate, illustrating reversible binding. Perfusion with the N-hydroxysuccinimide ester of folic acid eliminated essentially all 5-methyltetrahydrofolate binding to PFRs, while increasing net maternal-to-fetal transfer of the vitamin. Finally, because it has been suggested that impaired placental transport of folate may be linked to the fetotoxic effects of ethanol, the effect of this compound on the above processes was examined. An acute 6-hour exposure to ethanol (2.5 to 3.1 mg/ml) had no effect (p > 0.05) on net maternal-to-fetal transfer of 5-methyltetrahydrofolate. These studies suggest that net maternal-to-fetal transfer is a process consisting of two steps. First is the concentrative component in which circulating 5-methyltetrahydrofolate is bound to (captured by) PFRs on the maternally facing chorionic surface. Although kinetics favor binding, there is a dynamic state wherein a gradual release of 5-methyltetrahydrofolate from this pool can add to incoming circulating folates to generate an intervillous blood level approximately 3 times that in the maternal blood. In the second step, folates are passively transferred to the fetal circulation along a downhill concentration gradient. This unique mechanism for transplacental folate transport may be applicable to other small relative molecular mass ligand nutrients that bind to high-affinity placental receptors.

In utero ethanol exposure elicits oxidative stress in the rat fetus.

Prior studies in our laboratory have shown that exposure of cultured fetal rat hepatocytes to ethanol (E) blocks epidermal growth factor-dependent replication and that this is paralleled by cell membrane damage, mitochondrial dysfunction, membrane lipid peroxidation (LP), and enhanced generation of reactive oxygen species. These measures of E-mediated oxidative stress (OS) were mitigated by treatment with antioxidants, and cell replication could be normalized by maintaining cell glutathione (GSH) pools. We have now extended these studies to an in vivo model. Rats were administered E (4 g/kg, po) at 12-hr intervals on days 17 and 18 of gestation and killed on day 19, 1 hr following a final dose of E (a total of 5 doses). Fetal and maternal brain and liver were assayed for signs of OS. The 2-day in utero E exposure increased membrane LP in fetal brain as evidenced by increased malondialdehyde (MDA) levels from 1.76 +/- 0.12 SE (nMol/mg protein) to 2.00 +/- 0.08 (p < 0.05) and conjugated dienes from 0.230 +/- 0.006 SE (OD223/mg lipid) to 0.282 +/- 0.006 (p < 0.05). In fetal liver, MDA levels increased from 2.39 +/- 0.08 SE (nMol/mg protein) to 2.87 +/- 0.08 (p < 0.05), whereas dienes differed significantly only between ad libitum controls and the E and pair-fed control groups (p < 0.05). E decreased GSH levels in fetal brain by 19%, from 19.88 +/- 0.72 to 16.13 +/- 1.06 (nMol/mg protein) (p < 0.05). A 10% decrease in GSH was seen in fetal liver (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of acute ethanol exposure on cultured fetal rat hepatocytes: relation to mitochondrial function.

Studies from our laboratory have shown that short-term ethanol exposure inhibits epidermal growth factor-dependent replication of cultured fetal rat hepatocytes, along with a drop in ATP level, and that these effects could be caused, at least in part, by ethanol-induced oxidative stress. In these prior studies, mitochondrial morphology was abnormal and membrane lipid peroxidation products were increased, along with reduced transmembrane potential and enhanced permeability to sucrose. To define the effects of ethanol on mitochondrial function further, the present study examines the impact of ethanol exposure on mitochondrial electron transport chain components. A 24-hr exposure of cultured fetal rat hepatocytes to ethanol (2.5 mg/ml) reduced mitochondrial complex I activity by 16% (p < 0.05), complex IV by 28% (p < 0.05), and succinate dehydrogenase by 23% (p < 0.05). This reduction was paralleled by lower ADP translocase activity (24%, p < 0.05) and diminished mitochondrial glutathione (GSH) (20%, p < 0.05). Pretreatment with 0.1 mM S-adenosyl methionine, before ethanol exposure, normalized mitochondrial GSH along with activities of complex I, complex IV, and succinate dehydrogenase. A 3-hr exposure of isolated mitochondria (which do not metabolize ethanol) to ethanol (2.5 mg/ml), inhibited the activities of complex I (19%, p < 0.05), complex IV (24%, p < 0.05), and of ATP synthesis (20%, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Transfer of dideoxyinosine across the human isolated placenta.

1. Dideoxyinosine (ddI) has recently been approved for the treatment of patients with HIV infection. As increasing numbers of such patients are pregnant, we wished to define the rate and mechanism(s) of ddI transfer by the placenta to the foetus. Using isolated single perfused human term placental cotyledons, the drug was shown to cross the placenta from mother to foetus at a rate of 25% that of a freely diffusible marker, antipyrine, and at about half the rate of zidovudine (AZT). The transfer of ddI was similar in both directions (maternal to foetal and the reverse), equal to that of L-glucose, a passively transported sugar, and was not inhibited by excess inosine or uric acid (structural analogues of ddI). ddI did not cross to the foetus against a concentration gradient. The transport process appeared to be passive and it was not altered by AZT. 2. ddI was not metabolized in the Krebs Ringer buffer/albumin perfusate, and placental homogenates converted only 4% of ddI to hypoxanthine over the 4 h incubation. However, when maternal term or cord blood was incubated with ddI for 3 h, 50% of the drug was converted to hypoxanthine in maternal blood and to hypoxanthine and uric acid in cord blood. 3. Thus, ddI metabolism in maternal blood should decrease its net transfer to the foetus in vivo. In the foetal circulation, ddI will be further metabolized by erythrocytes to hypoxanthine and possibly to uric acid. Hence, the fraction of administered ddI delivered to foetal tissues should be much lower than that of AZT.

Biotin uptake by basolateral membrane vesicles of human placenta: normal characteristics and role of ethanol.

This study assessed the mechanism of uptake of biotin by the fetal-facing (basolateral) membrane of the term human placenta. Using membrane vesicles, we showed that most of the uptake was attributable to transfer of the vitamin into the vesicle and that the uptake was saturable, Na-dependent, carrier-mediated, and electroneutral. The rate of uptake was less than for biotin uptake by the maternal-facing (apical) membrane of the human placenta. Because ethanol inhibits biotin uptake by the apical membrane, the effect of ethanol on uptake by basolateral vesicles was investigated. With 10-hr exposure at a concentration of 2 and 3 mg/ml, but not 1 mg/ml, ethanol modestly inhibited biotin uptake. The mechanism of inhibition by alcohol is not known.

Ganciclovir transfer by human placenta and its effects on rat fetal cells.

Cytomegalovirus is a common cause of intrauterine infection. Ganciclovir is an accepted therapeutic agent for this infection, but is proscribed in pregnancy, except when there is a life-threatening maternal infection, because of its known teratogenic and embryotoxic effects in experimental animals. There are no such data in humans and the human transplacental transfer of this drug has not been studied. This study defines the rate and mechanism of human-placental ganciclovir transport using maternal-facing syncytiotrophoblast vesicles and the perfused, isolated single-cotyledon system and determines further the effects of ganciclovir on fetal tissue, using cultured rat fetal hepatocytes. Ganciclovir was taken up by the maternal-facing placental membrane by a carrier-dependent, Na-independent system inhibited by adenine, guanine, and acyclovir, but not by cytosine and thymine or thymidine and uridine. By contrast, the overall transfer of the drug by the placenta was passive and without drug metabolism. Therefore, the drug is concentrated initially at the maternal placental surface and then crosses passively into the fetal compartment, with the latter process being rate-limiting. There was little or no toxic effect of high concentrations of ganciclovir on cultured fetal-rat hepatocytes.