Abstract
Previous studies using cell and whole embryo cultures have shown that free radicals play an important role in the ethanol-induced death of mouse neural crest cells (NCCs; a significant cell type with respect to the genesis of alcohol-related birth defects). This investigation was spurred by reports of increased iron in ethanol-exposed fetuses and the knowledge that iron can initiate the production of reactive oxygen species. Initially, the ameliorative potential of two iron chelators, deferoxamine and phenanthroline, relative to ethanol-induced cell death was examined. Cotreatment of cultured NCCs with 100 mM ethanol and either 1 or 10 μM deferoxamine or 10, 50, or 250 μM phenanthroline significantly increased the percentage of viable cells as compared with exposure to 100 mM ethanol alone. These data indicate that iron is involved in the ethanol-induced cytotoxicity. To support this premise, the direct toxicity of iron to NCCs was also examined. As expected, loading the cells with Fe(II)/Fe(III) using 8-hydroxyquinoline as a carrier had an adverse effect on their viability as did treatment with a neurotoxin, 6-hydroxydopamine, that releases iron from ferritin storage. Cotreatment with an antioxidant,N-acetylcysteine, significantly diminished the toxicity of ethanol alone, that resulting from iron loading, as well as from the combination of ethanol exposure and iron loading. These results confirm the role of free radical-mediated damage in ethanol-induced cytotoxicity and highlight the potential role of iron relative to the genesis of alcohol-related birth defects.
A number of in vitro (including whole embryo culture) studies have illustrated that excessive cell death in selected cell populations and subsequent malformations induced by teratogenic concentrations of ethanol can be ameliorated using antioxidants (Kotch et al., 1995; Chen and Sulik, 1996: Smith et al., 1999). However, the sources of the free radicals that initiate the damage are yet to be identified. This study was designed to investigate iron as a mediator of cytotoxicity/cell death in cultured, ethanol-exposed mouse neural crest cells (NCCs). Iron was chosen as a focus because ethanol exposure can lead to an increase in the concentration of free iron (Sanchez et al., 1988;Mendelson and Jiner, 1994), which catalyzes the production of free radicals (Halliwell and Gutteridge, 1984). Cultured NCCs were selected as a model system due to the relevance of this cell population to craniofacial abnormalities as occur in fetal alcohol syndrome (FAS) (Kotch and Sulik, 1992; Cartwright and Smith, 1995).
Duane et al. (1992) have indicated that iron absorption is enhanced by chronic alcohol consumption, whereas others have shown that alcohol consumption increases the available iron in the liver (Mazzanti et al., 1987; Sanchez et al., 1988). It is also noteworthy that ethanol exposure during pregnancy increases fetal iron (Dreosti, 1984;Mendelson and Jiner, 1994).
It is clear that cellular oxidant damage is markedly potentiated by the presence of iron (Halliwell and Gutteridge, 1984; Balla et al., 1990;Hata et al., 1997). In normal tissue, iron rarely exists as a free ion but rather is bound to a variety of active proteins including hemoglobin and myoglobin, transport proteins such as transferrin, and storage proteins such as ferritin. Ethanol metabolism affects the liberation of iron from bound intracellular reserves (Cedarbaum, 1989;Shaw, 1989; Rouach et al., 1990). Specifically, the oxidation of ethanol by alcohol dehydrogenase enhances the cellular production of the reducing agent NADH, which mobilizes iron stored as ferritin (Shaw et al., 1988). In addition, the metabolism of acetaldehyde by aldehyde oxidase or xanthine oxidase may generate free radicals, or superoxide, which in turn also mobilizes iron from ferritin (Shaw, 1989; Shaw and Jayatilleke, 1990). Iron overloading can participate in the production of lethal hydroxyl radicals and the induction of cell death (Kawabata et al., 1997; Double et al., 1998). It has been suggested that the release of free iron may be the primary mechanism for ethanol-induced lipid peroxidation in vivo (Ferrali et al., 1990; Shaw and Jayatilleke, 1990) and ethanol-induced cytotoxicity (Nordmann et al., 1992).
For this investigation, the ameliorative potential of two iron chelators, deferoxamine (DFX) and phenanthroline (PHE) and of the antioxidant, N-acetylcysteine (Nac), relative to ethanol-induced cell death, as well as the direct toxicity of iron to NCCs, were examined. Our results confirm the role of free radical-mediated damage in ethanol-induced cytotoxicity and highlight the potential role of iron relative to the genesis of alcohol-related birth defects.
Materials and Methods
Animal Care.
C57BL/6J (C57) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). They were maintained on a 12-h light/dark cycle and had access to commercially formulated rodent chow and water ad libitum. Mice were mated for 1 h early in the light cycle. The plug detection day was designated gestational day 0.
Establishment of Primary NCC Cultures.
Primary NCC cultures were established as described previously (Chen and Sulik, 1996; Chen et al., 2000). Briefly, on gestational day 8, mouse embryos having 6 to 16 somite pairs were freed of extraembryonic tissues using fine forceps under a dissecting microscope. The cranial neural folds were excised and the explants were placed, separately, in individual wells of 24-well culture plates coated with human fibronectin (50 μg/ml). Explants were cultured in 1.0 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% (v/v) fetal calf serum and 5% (v/v) horse serum at 37°C in a humidified atmosphere of 5% CO2. NCCs were allowed to grow out from the neural fold explants for 60 h, after which the primary explant was removed and experiments were initiated.
Experimental Conditions.
After removal of the explant from the culture dish, the remaining NCCs were washed three times with serum-free DMEM. They were then cultured for an additional 16 h under control conditions or in the presence of 100 mM ethanol; in 1 or 10 μM DFX or 10, 50, or 250 μM PHE, both of which are iron chelators; in 50, 100, or 200 μM concentrations of the neurotoxin 6-hydroxydopamine (6-OHDA); or in 0.01, 0.1, or 1 mM Nac, an antioxidant. In addition, combinations of the ethanol exposure with each of the above were made. The above concentrations were selected based on brief pilot studies and literature reports (Balla et al., 1990; Gillissen et al., 1997; Double et al., 1998).
For the iron-loading experiments, culture plates were washed free of medium using two washes with Hanks' balanced salt solution at 37°C. FeCl2 was mixed with ferric ammonium citrate in Hanks' balanced salt solution and was added along with 8-hydroxyquinoline (8HQ) to culture plates for 30 min at 37°C. At the end of the incubation, the iron-loading buffer was removed, then the cells were washed and cultured in control medium or in the presence of ethanol alone or in combination with the antioxidant for 16 h.
For all of the experiments involving ethanol exposure, evaporation of the ethanol was diminished by tightly wrapping culture plates with parafilm. Parallel cultures of control cells were treated in the same manner as the treated cells with respect to wrapping of plates. At the end of the culture period, ethanol concentration in the culture medium was measured spectrophotometrically using a Sigma kit (cat. no. 333B).
Viability Assessments and Statistical Analyses.
At the end of the culture period, cell viability was determined by trypan blue exclusion (Morgan and Darling, 1993). Cell suspensions were prepared by incubation with 0.25% trypsin for 2 to 3 min. Equal portions of the cell suspension and 0.05% trypan blue were combined and mixed, then transferred to a hemocytometer for counting under a Nikon microscope. Cells from four to six cultures were examined for each culture condition. Data are expressed as mean values ± S.E. Differences between control and treated groups or between treated groups were evaluated using Student's t test.
Results
As previously reported and as reaffirmed in this study, after a 16-h exposure to 100 mM ethanol, there is a significant decrease in the percentage of viable NCCs (54% viable) as compared with that for the control cultures (84% viable) (Fig. 1). The ethanol-induced loss of viability is diminished both by membrane-impermeable (DFX) and membrane-permeable (PHE) chelators of iron (Figs. 1 and 2). Both 1 and 10 μM concentrations of DFX provided comparable levels of protection from ethanol-induced cell death. Although the lowest concentration of PHE tested (10 μM) did not provide a statistically significant result, concentrations of 50 and 250 μM PHE did provide a statistically significant degree of protection from the ethanol treatment. For these experiments, loss of ethanol due to evaporation did not present a major problem, as ethanol concentrations in the culture media even after 24 h were determined to be 91.8 ± 1.5% of that added.
Using the chelating agent, 8HQ, as a carrier to transfer iron across the intact plasma membrane of the cultured NCCs, the deleterious effect of iron by itself and in combination with ethanol exposure was shown (Fig. 3). Others have shown that 8HQ transfers iron into the cell membrane or the cytoplasm by virtue of forming charge-neutral and hydrophobic iron complexes (Balla et al., 1990). Although brief exposure (30-min) to 10 μM 8HQ by itself was somewhat toxic, reducing viability from 83 to 73%, in combination with 10 μM Fe(II)/Fe(III) the NCC viability dropped to 49%. Iron loading in combination with ethanol decreased the viability from 46% with ethanol and the carrier alone to 20% when the NCCs were iron-loaded.
The toxicity of iron was also examined using the neurotoxin 6-OHDA. This agent is highly effective in vitro in releasing iron from ferritin storage (Monteiro and Winterbourn, 1989; Double et al., 1998). As shown in Fig. 4, the addition of 6-OHDA to the culture medium reduced the percentage of viable NCCs in a concentration-dependent manner. At 50 μM 6-OHDA, 67% of the cells remained viable; at 100 μM, 44% were viable; and at 200 μM, only 20% were viable. Similarly, these concentrations of 6-OHDA in combination with 100 mM ethanol resulted in a concentration-dependent reduction in NCC viability relative to the cultures using ethanol exposure alone. The combined effect of ethanol exposure with the highest concentration of 6-OHDA resulted in the survival of only 3% of the cells.
Based on the results of the study involving iron chelators in combination with ethanol exposure and knowledge of iron-mediated oxidant damage, antioxidant exposure was expected to provide protection under the experimental conditions described above. For this investigation, the effectiveness of the antioxidant, Nac, was studied. Concentrations of 0.01, 0.1, and 1.0 mM Nac alone did not result in increased incidences of cell death (Fig.5). Exposure of the NCCs to these concentrations of Nac during the 16 h of ethanol challenge significantly reduced the amount of ethanol-induced cell death, with the 1.0 mM concentration of Nac affording the greatest degree of protection. Similarly, as shown in Fig. 6in which experimental data is expressed relative to percentage of control values, Nac cotreatment diminished the cytotoxicity/cell killing caused by iron loading and iron release. Specifically, in contrast to a survival rate (relative to control) of 47% for the iron-loaded NCCs, in the presence of 1.0 mM Nac this rate was 90% of the control value. Coculture with 6-OHDA and Nac resulted in a survival rate that was 88% of the control values as compared with a rate of 50% for the cultures that did not contain the antioxidant. As shown in Fig. 7, Nac also provided protection for NCCs exposed to excess iron in combination with ethanol, with survival values increasing from 27 to 86% for the ethanol plus iron-loaded cells and from 24 to 68% in the OHDA plus ethanol-exposed cultures.
Discussion
Ethanol abuse during pregnancy results in a wide variety of malformations, including craniofacial anomalies. The typical craniofacial features of individuals with full-blown FAS include microcephaly, short palpebral fissures, deficiencies of the philtral region, and a long upper lip (Hanson et al., 1976). Research using rodent and avian animal models has shown that ethanol has a major affect on cranial NCCs. This affect appears to contribute heavily to the subsequent abnormalities (Sulik et al., 1981; Kotch and Sulik, 1992; Cartwright and Smith, 1995).
Because alcohol affects the metabolism of various trace elements, the possibility exists that the ethanol-induced NCC death and abnormalities described in FAS may, in part, be related to alterations in the metabolism of these trace elements. Reports from various laboratories have indicated that alcohol treatment increased iron levels in the fetal carcass and fetal liver in rats (Mendelson and Huber, 1980;Dreosti, 1984; Mendelson and Jiner, 1994). It is well known that elevated intracellular reduced NADH, as results from alcohol metabolism, can release iron from ferritin by donating an electron to convert ferric to ferrous iron (Tophan et al., 1989). In addition, ethanol exposure has been shown to result in increased xanthine oxidase O2- production, and that superoxide radicals are able to release iron from ferritin (Aust et al., 1985; Nordmann et al., 1987). Alcohol may also impair proper intracellular processing of iron into nontoxic ferritin, thus making available catalytic ferrous iron.
DFX is a naturally occurring iron chelator derived fromStreptomyces pilosis. It has very high binding affinity for iron (Kd = 1021) as compared with transferrin and ferritin. (Halliwell and Gutteridge, 1986). However, DFX has very low membrane permeability (Lloy et al., 1991). PHE is a chelator of divalent Fe2+ and is membrane-permeable. Although it can also bind to other metal ions, PHE has been widely used as an iron chelator in iron overloading studies due to its high binding affinity for Fe2+ (Jones and Johnson, 1967; Hiraishi et al., 1994). The results of this study show that both DFX and PHE can prevent ethanol-induced death of cultured NCCs. Although this suggests that some of the iron-mediated toxicity takes place in the extracellular space, the possibility that the DFX may enter the cell by pinocytosis during the culture period must be taken into account (Lloyd et al., 1991). As expected, artificially elevating cellular free iron either via 8HQ-mediated iron loading or 6-OHDA-mediated intracellular iron release diminishes NCC viability. The percentage of viable NCCs is further diminished by cotreatment with 100 mM ethanol after iron overload. These results strongly suggest that iron overloading is an important factor in ethanol-induced NCC death.
It is well known that iron can cause injury to cells by catalyzing free radical generation. In this investigation, the effectiveness of the antioxidant, Nac, was studied. This agent reduces disulfide bonds, and rapidly deacetylates and supplies cysteine for cellular GSH synthesis. It also directly scavenges reactive oxygen species (Vanderbist et al., 1996; Gillissen et al., 1997). The effectiveness of Nac in reducing NCC injury induced by iron overloading, by ethanol, as well as by the combination of ethanol exposure and iron loading, supports this free radical mechanism.
In conclusion, although the mechanism of the cytotoxicity of ethanol is most likely not entirely iron-mediated, the results of our study suggest that a major factor underlying ethanol-induced NCC death is iron overloading, which initiates the formation of free radicals. The lethal effect of the free radicals is expected to entail lipid peroxidation as well as signal transduction cascades that trigger apoptosis (Anderson et al., 1999). Interestingly, iron-mediated damage to another NCC population, those comprising dorsal root ganglia, has been linked to another disorder, Friedreich's ataxia (Jitpimolmard et al., 1993; Waldvogel et al., 1999). Our work supports a role for iron-mediated cytotoxicity as a factor underlying damage to NCCs and alcohol-related birth defects, and indicates that administration of antioxidants such as Nac may provide a potential therapeutic approach.
Acknowledgment
We thank Deborah B. Dehart for excellent assistance.
Footnotes
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Send reprint requests to: Kathleen K. Sulik, Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090. E-mail: mouse{at}med.unc.edu
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↵1 This work was supported by National Institutes of Health Grant AA11605 from the National Institute of Alcohol Abuse and Alcoholism.
- Abbreviations:
- NCCs
- neural crest cells
- DFX
- deferoxamine
- PHE
- phenanthroline
- 8HQ
- 8-hydroxyquinoline
- 6-OHDA
- 6-hydroxydopamine
- Nac
- N-acetylcysteine
- FAS
- fetal alcohol syndrome
- DMEM
- Dulbecco's modified Eagle's medium
- Received December 16, 1999.
- Accepted March 27, 2000.
- The American Society for Pharmacology and Experimental Therapeutics