Department of Cell Biology and Anatomy and Bowles Center for
Alcohol Studies, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina
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.
 |
Introduction |
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.
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Fig. 1.
DFX, a membrane-impermeable chelator of iron,
prevents ethanol-induced NCC death. Data are expressed as mean
values ± S.E. (*P < .05, **P < .01 versus the ETOH-treated group).
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Fig. 2.
PHE, a membrane-permeable chelator of iron, can
protect NCCs against the cytotoxicity induced by ethanol. Data are
expressed as mean values ± S.E. (*P < .05, **P < .01 versus the ETOH-treated group).
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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.
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Fig. 3.
Iron loading induces cytotoxicity in NCCs and
sensitizes NCCs to ethanol-induced cytotoxicity. Data are expressed as
mean values ± S.E. (*P < .05, **P < .01 versus the control group.
§P < .05, §§P < .01 versus the ETOH-treated
group).
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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.
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Fig. 4.
Ferritin-bound iron released by 6-OHDA induces
cytotoxicity in NCCs and increases ethanol-induced cell death. Data are
expressed as mean values ± S.E. (*P < .05, **P < .01 versus the control group.
§P < .05, §§P < .01 versus the ETOH-treated
group).
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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. 6
in 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.
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Fig. 5.
Nac significantly reduces ethanol-induced
cytotoxicity in NCCs. Data are expressed as mean values ± S.E.
(*P < .05, **P < .01 versus
the ETOH-treated group).
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Fig. 6.
Cytotoxicity induced by iron loading (Fe + 8HQ) or
6-OHDA-released iron can be ameliorated by Nac. Data are expressed as
mean values ± S.E. (*P < .05, **P < .01 iron-loading group versus iron loading + Nac group. §P < .05, §§P < .01, 6-OHDA-treated group
versus 6-OHDA + Nac group).
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Fig. 7.
Nac ameliorates the combined effect of iron and
ethanol on NCC cytotoxicity. Data are expressed as mean values ± S.E. (*P < .05, **P < .01 iron-loading + ETOH group versus iron-loading + ETOH + Nac group.
§P < .05, §§P < .01, 6-OHDA+ ETOH versus
6-OHDA+ ETOH + Nac group).
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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 from
Streptomyces 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.
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.
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Abstract
Introduction
