George I. Henderson

Ethanol-induced oxidative stress precedes mitochondrially mediated apoptotic death of cultured fetal cortical neurons.

In utero ethanol exposure elicits apoptotic cell death in the fetal brain, and this may be mediated by oxidative stress. Our studies utilize cultured fetal rat cortical neurons and illustrate that ethanol elicits a rapid onset of oxidative stress, which culminates in mitochondrially mediated apoptotic cell death. Cells exposed to ethanol (2.5 mg/ml) remained attached to their polylysine matrix during a 24‐hr exposure, but they exhibited distinct signs of oxidative stress, decreased viability, and apoptosis. Confocal microscopy of live cortical neurons pretreated with dichlorodihydrofluorescein diacetate demonstrated an increase in reactive oxygen species (ROS) within 5 min of ethanol exposure. The levels of ROS further increased by 58% within 1 hr (P < .05) and by 82% within 2 hr (P < .05), accompanied by increases of mitochondrial 4‐hydroxynonenal (HNE). These early events were followed by decreased trypan blue exclusion of 10% to 32% (P < .05) at the 6‐ to 24‐hr time points, respectively. This culminates in apoptotic death, with increases of Annexin V binding of 43%, 89%, 123%, and 238%, at 2, 6, 12, and 24 hr of ethanol treatment, respectively, as well as DNA fragmentation increases of 50% and 65% by 12 and 24 hr, respectively. Release of cytochrome c by mitochondria increased by 53% at 6 hr of exposure (P < .05), concomitant with activation of caspase 3 (52% at 12 hr, P < .05). Pretreatment with N‐acetylcysteine increased cellular glutathione and prevented apoptosis. These studies provide a time line illustrating that oxidative stress and formation of a proapoptotic lipid peroxidation product, HNE, precede a cascade of mitochondrially mediated events in cultured fetal cortical neurons, culminating in apoptotic death. The prevention of apoptosis by augmentation of glutathione stores also strongly supports a role for oxidative stress in ethanol‐mediated apoptotic death of fetal cortical neurons.

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.

Placental Amino Acid Uptake in Normal and Complicated Pregnancies

Amino acids are the essential substrates for fetal growth and catabolism. The fetus is dependent on the placenta for the provision of amino acids, the first step being concentration of amino acids within the syncytiotrophoblast for subsequent transfer to the fetus. A reliable technique for the isolation of human syncytiotrophoblast plasma membrane has been described, and the suitability of this preparation for the study of amino acid uptake and membrane transport has been well documented. Using this technique, the microvillous vesicle uptake of alpha-aminoisobutyric acid (AIB), a nonmetabolizable amino acid, has been determined over multiple time points for normal (NL) pregnancies and those complicated by pregnancy-induced hypertension (PIH), non-insulin-dependent diabetes mellitus (NIDDM) and those delivering small-for-gestational-age (SGA) neonates. There was no significant difference in AIB uptake between NL pregnancies and those complicated by PIH or NIDDM. Compared to each of the above, AIB uptake by the SGA group was significantly less at each time point. These results suggest that normal placental amino acid transport mechanisms may be altered in SGA pregnancies. If so, such alterations may interfere with the normal provision of nutrients to the fetus and ultimately contribute to impaired growth in utero.

Astrocytes Protect Neurons From Ethanol-Induced Oxidative Stress and Apoptotic Death

Ethanol induces oxidative stress in cultured fetal rat cortical neurons and this is followed by apoptotic death, which can be prevented by normalization of cell content of reduced glutathione (GSH). Because astrocytes can play a central role in maintenance of neuron GSH homeostasis, the following experiments utilized cocultures of neonatal rat cortical astrocytes and fetal cortical neurons to determine if astrocytes could protect neurons from ethanol‐mediated apoptotic death via this mechanism. In cortical neurons cultured in the absence of astrocytes, ethanol (2.5 and 4 mg/ml; 6‐, 12‐, and 24‐hr exposures) decreased trypan blue exclusion and the MTT viability measures by up to 45% (P < 0.05), increased levels of reactive oxygen species (ROS) by up to 81% (P < 0.05), and decreased GSH within 1 hr of treatment by 49 and 51% for 2.5 and 4 mg/ml, respectively (P < 0.05). This was followed by onset of apoptotic cell death as determined by increased Annexin V binding and DNA fragmentation by 12 hr of ethanol exposure. Coculturing neurons with astrocytes prevented GSH depletion by 2.5 mg/ml ethanol, whereas GSH content was increased over controls in neurons exposed to 4 mg/ml ethanol (by up to 341%; P < 0.05). Ethanol generated increases in neuron ROS and apoptosis; decreases in viability were also prevented by coculture. Astrocytes were largely insensitive to ethanol, using the same measures. Only exposure to 4.0 mg/ml ethanol decreased GSH content in astrocytes, concomitant with a 204% increase in GSH efflux (P < 0.05). These studies illustrate that astrocytes can protect neurons from ethanol‐mediated apoptotic death and that this may be related to maintenance of neuron GSH.

The transfer of cocaine and its metabolites across the term human placenta

This study defines human placental transport of cocaine and its two minor, but pharmacologically active, metabolites—norcocaine and cocaethylene. The experimental system was the single, isolated perfused cotyledon of a normal term human placenta, and antipyrine served as a freely diffusible marker. Cocaine was transferred rapidly by the placenta at a rate about 80% that of antipyrine. The transfer had characteristics of passive transport consistent with the high lipid solubility of the drug. We found no evidence of significant placental metabolism of cocaine during its rapid placental transfer. Ethanol did not alter the cocaine transfer rate. Norcocaine and cocaethylene were equally as rapidly transferred. Thus the placenta is no barrier to the transfer of cocaine and its derivatives to the fetus.

Astrocyte control of fetal cortical neuron glutathione homeostasis: up-regulation by ethanol.

Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N‐acetylcysteine or by co‐culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte‐neuron γ‐glutamyl cycle is up‐regulated by Ethanol, enhancing control of neuron GSH in response to the pro‐oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of γ‐glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrosoyte up to 517% (p < 0.05). Ethanol increased both γ‐glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p = 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the γ‐glutamyl cycle that are up‐regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanol‐mediated oxidative stress and apoptotic death.

Antioxidant transport by the human placenta

We investigated the transfer of three antioxidants - melatonin, S-adenosyl methionine (SAM) and various forms of vitamin E - across the term, normal human placenta. The transport technique involved the single, isolated placental cotyledon system in vitro. Melatonin crossed the placental rapidly, equally to the freely diffusible marker, antipyrine. There was no biotransformation of the agent. SAM was transferred slowly, similarly to passively transported L-glucose as a marker. There was a breakdown of SAM to at least one other derivative; the process appeared to be nonenzymatic. Vitamin E was transferred slowly, at a rate only 10% of L-glucose. The natural RRR (nonracemic) form of vitamin E was transported best. Free vitamin, rather than the acetate seems to be transferred best, a finding that will require further study.

Azidothymidine (Zidovudine) Transport by the Human Placenta

The diagnosis of Acquired Immunodeficiency Syndrome (AIDS) is increasingly made in pregnant women, and the disease may be transmitted to the fetus. Azidothymidine (AZT, Zidovudine) is the one therapeutic agent of some promise in this condition. As there is no information on the transport of this drug by the human placenta, such studies were carried out using the single cotyledon placental perfusion system and human placental vesicles. AZT crossed the placenta readily and bidirectionally. The transfer rate was about 70% that of a freely diffusible reference marker, antipyrine, and was comparable in both directions. There was no evidence of active or carrier-mediated transport and no glucuronidated metabolites of the drug were identified in either maternal or fetal compartments. The authors believe that the drug crosses the placenta by diffusion, consistent with its lipophilicity and transport into various blood cells.

Overexpression of Nrf2 Protects Cerebral Cortical Neurons from Ethanol-Induced Apoptotic Death

Ethanol (ETOH) can cause apoptotic death of neurons by depleting GSH with an associated increase in oxidative stress. The current study illustrates a means to overcome this ETOH-induced neurotoxicity by enhancing GSH through boosting Nrf2, a transcription factor that controls GSH homeostasis. ETOH treatment caused a significant increase in Nrf2 protein, transcript expression, Nrf2-DNA binding activity, and expression of its transcriptional target, NQO1, in primary cortical neuron (PCNs). However, this increase in Nrf2 did not maintain GSH levels in response to ETOH, and apoptotic death still occurred. To elucidate this phenomenon, we silenced Nrf2 in neurons and found that ETOH-induced GSH depletion and the increase in superoxide levels were exacerbated. Furthermore, Nrf2 knockdown resulted in significantly increased (P < 0.05) caspase 3 activity and apoptosis. Adenovirus-mediated overexpression of Nrf2 prevented ETOH-induced depletion of GSH from the medium and high GSH subpopulations and prevented ETOH-related apoptotic death. These studies illustrate the importance of Nrf2-dependent maintenance of GSH homeostasis in cerebral cortical neurons in the defense against oxidative stress and apoptotic death elicited by ETOH exposure.

Glutathione content as a potential mediator of the vulnerability of cultured fetal cortical neurons to ethanol-induced apoptosis.

Ethanol ingestion during pregnancy elicits damage to the developing brain, some of which appears to result from enhanced apoptotic death of neurons. A consistent characteristic of this phenomenon is a highly differing sensitivity to ethanol within specific neuron populations. One possible explanation for this “selective vulnerability” could be cellular variations in glutathione (GSH) homeostasis. Prior studies have illustrated that ethanol elicits apoptotic death of neurons in the developing brain, that oxidative stress may be an underlying mechanism, and that GSH can be neuroprotective. In the present study, both multiphoton microscopy and flow cytometry demonstrate a striking heterogeneity in GSH content within cortical neuron populations. Ethanol differentially elicits apoptotic death and oxidative stress in these neurons. When neuron GSH content is reduced by treatment with butathione sulfoxamine, the ethanol‐mediated enhancement of reactive oxygen species is exacerbated. Sorting of cells into high‐ and low‐GSH populations further exemplifies ethanol‐mediated oxidative stress whereby apoptotic indices are preferentially elevated in the low‐GSH population. Western blot analysis of the low‐GSH subpopulations shows higher ethanol‐mediated expression of active caspase 3 and 24‐kDa PARP‐1 fragments compared with the high‐GSH subpopulation. In addition, neuronal content of 4‐hydroxynonenal adducts is higher in low‐GSH neurons in response to ethanol. These studies suggest that GSH content is an important predictor of neuronal sensitivity to ethanol‐mediated oxidative stress and subsequent cell death. The data support the proposition that the differences in proapoptotic responses to ethanol within specific neuron populations reflect a heterogeneity of neuron GSH content.