In VitroCell.Dev.Biol.--Animal39:98-105,January.

and February"2003
9 2003 Societyfor In VitroBiology
1071-2690/03 $18.00+0.00

HOMOCYSTEINE OXIDATION A N D APOPTOSIS: A POTENTIAL

CAUSE OF CLEFT PALATE
LYNDA KNOTT,l TOM HARTRIDGE,NATHANL. BROWN, JASON P. MANSELL,ANDJONATHON R. SANDY
Division of Child Dental Health, Dental School, Universityof Bristol, United Kingdom BS1 2LY
(Received 29 January 2003; accepted 11 March 2003)

SUMMARY
Cleft palate is the most common craniofacial anomaly. Affected individuals require extensive medical and psychosocial
support. Although cleft palate has a complex and poorly understood etiology, low maternal folate is known to be a risk
/actor for craniofacial anomalies. Folate deficiency results in elevated homocysteine levels, which may disturb palatogenests by several mechanisms, including oxidative stress and perturbation of matrix metabolism. We examined the effect
of homocysteine-induced oxidative stress on human embryonic palatal mesenchyme (HEPM) ceils and demonstrated that
biologically relevant levels of homocysteine (20-100 I~M) with copper (10 ~M) resulted in dose-dependant apoptosis,
which was prevented by addition of catalase but not superoxide dismutase. Incubation of murine palates in organ culture
with homocysteine (100 IxM) and CuSO4 (10 trM) resulted in a decrease in palate fusion, which was not significant.
Gelatin gel zymograms of HEPM cell-conditioned media and extracts of cultured murine palates, however, showed no
change in the expression or activation of pro-matrix metalloproteinase-2 with homocysteine (20 I~M-1 mM) with or
without CuSO4 (10 I~M). We have demonstrated that biologically relevant levels of homocysteine in combination with
copper can result in apoptosis as a result of oxidative stress; therefore, homocysteine has the potential to disrupt normal
palate development.
Key words: folic acid; oxidative stress; craniofacial development.
INTRODUCTION

for neural tube defects, independent of folate status (Steegers-Theunissen et at., 1994). Furthermore, hyperhomocysteinemia due to vitamin B6 or B12 deficiency or due to genetic defects of methylenetetrahydrofolate reductase and cystathione [3-synthase is associated with an increased incidence of nonsyndromic orofacial clefts
(Mills et al., 1999; Wong et al., 1999).
Homncysteine is currently the subject of much research and discussion because of its potential link with cardiovascular disease
(Refsum et al., 1998). Most of the information on the potential
mechanisms of homocysteine action on cellular function has been
obtained irom studies using vascular cells and tissues. It is of relevance that congenital malformations involving heart septation, neural tube closure, and oral clefting often occur together (e.g., "Catch
22"; Sergi, 1999), the common factor being the origin of the ceils
involved. The neural crest ceils that uhimately form these structures
derive from adjacent areas of the neural ectoderm and appear to be
particularly sensitive to a variety of environmental and genetic factors (Rosenquist et al., 1996; Burgoon et al., 2002). There is little
direct research on the effects of homocysteine on embryological
events or processes. However, Rosenquist et al. (1996, 2001) and
Limpach et al. (2000) have reported homocysteine-induced malformations in the developing chick embryo, including abnormal palate
formation. However, development of the avian palate is very different from that of the mammalian palate, and further work is evidently
required in this area to investigate the role of homocysteine in the
development of cleft palate.
Many mechanisms of action have been proposed for homocysteine

Cleft palate represents the most common craniofacial anomaly in

man, with an incidence of approximately 1 in 700 live births. Although cleft lip and palate have a complex, muhifactorial etiology,
it is well documented that low maternal folate status is an important
risk factor for neural tube defects and craniofacial anomalies (Finnell et al., 1998). Both folic acid requirement and its metabolism
are elevated during pregnancy (Christensen and Rosenblatt, 1995).
Reduced serum folate is also associated with maternal smoking and
anticonvulsant medication for epilepsy, and both groups have a
higher incidence of oral clefts (for review, see Hartridge et al.,
1999).
The cellular mechanism for the role of folate in palate development is not clear. Folate deficiency results in elevated levels of
set~lm homocysteine, a sulfur-containing amino acid generated during the cellular remethylation and transulfuration pathways for
which folate is required. Homocysteine can enter the amniotie fluid
of the developing fetus, at levels correlating with maternal plasma
concentrations, and elevated levels of homoeysteine in amniotie fluid have been found in women bearing fetuses with neural tube defects (Steegers-Theunissen et al., 1995; Wang et al., 2000). Homoeysteine has been implicated epidemiologieally as a risk factor
To whom correspondence should be addressed at Matrix Biology Group,
Department of Clinical Veterinary Science, University of Bristol, Churchill
Building, Langford, Bristol, United Kingdom BS40 5DT. E-mail: 1.knon@
bris.ac.uk
98

99

HOMOCYSTEINE AND CLEFT PALATE

including hypomethylation, protein homocysteinylation, interaction
with receptors, and oxidative stress, one or more of which may be
involved in the disruption of palate development. We have concentrated our initial investigations on the role of homocysteine-induced
oxidative stress because neural crest cells have a reduced capacity
to deal with reactive Oxygen species (ROS; Davis et al., 1990; Sivan
et al., 1997). The free radicals generated by ethanol, for example,
are thought to contribute to the development of cleft palate and
other abnormalities involving cranial neural crest cells in fetal alcohol syndrome (Chen et al., 1996).
Homoeysteine is readily oxidized to the disulphide honmcystine;
oxidation is a process augmented by transition metals such as copper that may be elevated in homoeysteinaemie patients (Dudman
and Wilcken, 1983). The oxidation of homoeysteine results in the
liberation of highly reactive hydrogen peroxide (H202) and superoxide anions ( 0 2 ) , both of which are directly damaging to cells,
altering their function, inducing apoptosis, or both (Starkebamn and
Harlan, 1986; Hultberg et al., 1997; Davies, 1999). Neural c r e s t derived vascular endothelial cells (VEC) in contrast to non-nem'al
crest-derived vascular cells also have an abnornml oxidative stress
response (Outinen et al., 1998). Homocysteine precipitates a decrease in intracellular glutathione peroxidase messenger ribonueleic
acid (mRNA) and glutathione peroxidase activity, thereby impairing
the ability of VEC to detoxify H2Oz (Upehurch et al., 1997). Data
from differential display and complementary deoxyribonucleic acid
micromTay analysis further suggest that VEC exposure to homoeysteine results in reductive stress, although the levels of homoeysteine
required to elicit these responses are very high (Austin et al., 1998;
Outinen et al., 1999).
Homocysteine and ROS can also disturb the extracellular matrix
and its metabolism. Homocysteine has been variously described to
activate matrix metalloproteinase-2 (MMP-2; Bescond et al., 1999;
Mujumdar et al., 2001), induce tissue inhibitor of metalloproteinases-1 (Torres et al., 1999), inhibit and promote AP-1 (an MMP
promotor) activity (Torres et al., 1999; Suzuki et al., 2000), increase
collagen synthesis (Majors et al., 1997), and alter collagen crosslinking (Jackson, 1973). Oxidative stress in general has been shown
to increase MMP-1 mRNA (Brenneisen et al., 1997) and regulate
both MMP activity and collagen synthesis (Rajagopalan et al., 1996;
Siwik et al., 2001). The extracellular matrix, principally collagen,
and its metabolism (Morris-Wiman et al., 1999; Mansell et al.,
2000), undergo temporal changes during secondary palate development. Normal development of the palate requires a complex series
of events involving mesenehymal proliferation in the maxillary processes, elevation of the palatal shelves, and breakdown of the contacting medial edge epithelium (MEE) (Brinkley, 1980; Ferguson,
1988). Many of these processes require metabolism of the matrix,
and we and others have previously demonstrated that disruption of
MMP function can result in nonfusion of palates in vitro (Blavier
et al., 2001; Brown et al., 2002).
We therefore hypothesize that elevated levels of homocysteine
during embryogenesis can result in the development of cleft palate.
To test this hypothesis, we first examined the effect of homocysteineinduced oxidative stress on both human embryonic palatal mesenchyme (HEPM) cells and on murine palate organ culture. Second,
we investigated the effects of homocysteine and oxidative stress on
matrix turnover, specifically, MMP-2 production.

MATERIALS AND METHODS

All materials were purchased from Sigma Chemical Co. (Poole, U.K.) unless otherwise stated.
Human embryonic palatal mesenchyme cell culture. Human embryonic palatal mesenchyme cells Were purchased from the European Collection Of Cell
Culture (Salisbury, U.K.). All cell cultures were carried out at 37 ~ C in a
humidified atmosphere containing 5% CO2. Before any experimentation, cells
were maintained in modified Eagle medium supplemented with 5% (v/v) fetal
calf serum (FCS; GIBCO, Paisley, U.K.), 4 mM L-glutamine, penicillin-streptomycin (100 U/ml and 0.1 mg/ml, respectively), and 1% v/v nonessential
amino acids.
Human embryonic palatal mesenchyme cells were seeded into 12-well
plates (Helena Biosciences, Sunderland, U.K.) and left for 24 h until 5060% confluent. The medium was then removed and replaced with medium
supplemented with 1% FCS, and the cells were left for a further 24 h to
reduce the basal level of activity. Both CuSO4 and DL-homocysteine were
prepared as stocks (10 nrM) in 1 urM hydrochloric acid on the d of use.
Concentrations of homocysteine between 10 and 100 p~M were used with 10
IxM CuSO4 and 100 IxM homocysteine with 0.2-10 ~M CuSQ. Control experiments using the disulphides homocystine and cystine and the thiol cysteine together with 10 p.M CuSO4 were also conducted. The concentrations
of homocysteine and copper are within the ranges observed clinically (Cartwright and Wintrobe, 1964; Finkelstein and Martin, 2000). To investigate the
role of superoxide dismutase (SOD) or catalase (or both), stock solutions were
prepared in media and added to wells at a final concentration of 200 and
1000 U/ml.
Each experiment was repeated three times (n = 6 for each condition per
experiment). Cell number was quantified using a Coulter counter after disassociation with trypsin-ethylenediamine-tetraacetic acid (GIBCO). The use
of colorimetric methods such as the 3-(4,5-dimethyhhiazole-2-yl)-2,5-biphenyl tetrazolimn bromide assay could not be considered because of thiol interference.
Determining the mechanism of cell death. Cell death can occur by either
apoptosis or necrosis, and exposure of cells to oxidative stress can result in
cell death by either mechanism, depending on stress level, cell type, and
cell environment (Davies, 1999). Morphological and histochemical observations were made after 3 h of incubation with 100 txM homocysteine and 10
txM CuSO4, when the initial effects can be observed by phase contrast microscopy (cell shrinkage), before the dead cells detach and finally lyse.
A fluorescein isothiocyanate (FITC)-labeled cell-permeable caspase inhibitor, valyl-alanyl-[o-methyl]-fluoromethylketone (VAD-FMK), was used to label activated caspases to allow identification of apoptosing cells with a fluorescent microscope. Cells were grown on three-well slides until 50% comquent and challenged with 100 ~M homocysteine per 10 ta3//CuSO4 in media
containing 1% FCS for 3 h. The CaspACE ~ FITC-VAD-FMK in situ marker
(Promega, Southampton, U.K.) was then added to the media at a final concentration of 20 txM, and incubation at 37 ~ C continued for 90 min. The
cells were then fixed in 10% buffered formalin in the dark at room temperature and subsequently washed in phosphate-buttered saline. A slide mountant supplemented with 4',6-diamidine-2-phenylindole (DAN) (Vector Laboratories, Peterborough, U.K.) enabled nuclear localization, when cells were
viewed under the fluorescence microscope.
Gelatin gel zymography of cell-conditioned media. Human embryonic palatal mesenchyme ceils wei~ cultured as described above and were challenged
with homocysteine and copper levels that did not result in significant cell
death (20 tzM homocysteine and 10 txM CuSO4). In addition, HEPM cells
were cultured with 100 txM homocysteine alone.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography
was performed as previously described (Mansell et al., 1998). Briefly, cellconditioned media were diluted in nonreducing sample buffer (2 as described by Laemmli (1970), but containing twice the concentration of SDS
(2% w/v). Samples and MMP-2 standards (Calbiochem, Nottingham, U.K.)
were eleetrophoresed using 10% polyacrylamide (Bio-Rad, Hertfordshire,
U.K.) gels eopolymerized with 0.5 mghul d gelatin (bovine skin; Sigma). Gels
were washed in 2.5% Triton X-100 to displace SDS and then incubated
overnight at 37 ~ C in proteolysis buffer (500 mM NaC1, 50 mM CaC12, 50
nrM Tris-HC1, pH 7.8) containing 200 txM p-aminophenylmercuric acetate.
The gels were then stained with Coomassie brilliant blue and the proteolytically clarified zones quantified using an Agfa Studiostar scanner and Scion
Image (Scion Corporation, MD) software.
Palate organ culture. CD1 mice were subjected to a reverse day-night

100

KNOTT ET AL.

FIG. i. The effect of homocysteine (lacy) and copper (CuSO~) on human embryonic palatal mesenchymc cell nmnber. Human embryonic
palatal mesenchyme cells were cultured under standard conditions, challenged for 24 h, and cell number quantified using an automated
Coulter counter. Cell number was not significantly affected with 100 p~M homocysteine or 10 p~M CuSQ alone; however, there was a
homocysteine concentration-related decrease in cell number when combined with 10 taM CuSO4. Data are presented as percentages of
controls and standard deviations. **, P < 0.01 compared with control; n = 6.

cycle and the males introduced to females for a period of 2 h during the
night phase. Females with vaginal plugs were isolated and if pregnant sacrificed by cervical dislocation 13 d postcoitum _+ 2 h. This method ensured
accuracy and consistency in obtaining embryonic day 13 mice. The palates
were then prepared for organ culture, as described previously (A1-Obdaidi et
at., 1995).
Palates were cultured in sialinized roller tubes (Supelco, Sigma, Poole,
U.K.) containing BGJ-B medium (GIBCO-BRL) supplemented with 850 t~M
ascorbic acid, 10 taM CuSO4, and 0.6% (w/v) bovine serum albumin and
were purged with 95% 02 and 5% COs before roller culturing at 37 ~ C for
72 h. The tubes were purged for 3 min at 24-h intervals and the media
changed after 48 h. Palates were cultured in the presence of 100 IxM homocysteine or vehicle. In addition, palates were also cultured with 5 taM
retinoic acid as a control for clefting.
Palate histology. Palates were fixed in formal saline for 24 h and prepared
for histology on a Shandon 2LE processor with graded methylated spirits,
xylene, and paraffin wax baths. Wax was removed with 100% xylene, and
the palates were air-dried overnight. Palates were photographed using a microscope and an external light source. Palates were then embedded in paraffin wax, sectioned on a microtome (2 ~m), and stained with hematoxylin
and eosin.
Gelatin gel zymography of cultured palate extracts. A second series of palates, cultured as described above, were homogenized in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid buffer at pH 7.4 to a concentration of 50 mg/ml. Homogenates were centrifuged at 5000 rpm for 10 rain at
4~ C, and the supernatant was used for zymography. A protein assay (BioRad) was used to measure the protein concentration for each sample, and
the equivalent of 5 t~g protein was subjected to electrophoresis as described
earlier.
Statistics. The data from the cell culture experiments were analyzed using
a one-way analysis of variance after testing the assumptions of a normal
population and equal standard deviations by the methods of Kohnogorov and
Smimov, and Bartlett, respectively. A Fisher exact test was used to analyze
a contingency table of the organ culture data. The tests were carried out
using Instat | (Grapbpad Software Inc., San Diego, CA).
RESULTS

The effect of homocysteine and copper on HEPM cell number. Incubation of HEPM cells with either 100 v,M homocysteine or lO
p,M CuSO4 had no effect on cell number. However, when cells were

incubated with homocysteine together with C u S Q , there was a doserelated decrease in cell number (P < 0.01; Fig. 1). Cysteine, but
not the disulphide homocystine, also resulted in cell death when
incubated with CuSO4 (P < 0.01; Fig. 2), suggesting that coppercatalyzed oxidation of thiols resulted in cell death.
The decrease in cell number was abrogated by the addition of
catalase but not SOD or boiled catalase (P < 0.01; Fig. 3), indicating that the cell death is triggered by the generation of n202
rather than by O2 . Furthermore, the addition of catalase to the cells
significantly increased cell number above controls (P < 0.01) because of the reduction in the background levels of oxidative s t r e s s related cell death in standard media.
CaspACE labeling. Observed under a standard phase contrast
microscope, the ceils do not show any morphological changes until
4 - 5 h of incubation with bomocysteine and CuSO4. The cells initially shrink and become rounded with condensed chromatin characteristic of apoptotic cells. After 8 h, the cells swell and become
detached from the surface, followed by extensive lysis after 24 h of
incubation.
The use of the FITC-labeled caspase inhibitor, VAD-FMK, enabled identification of apoptotic ceils after only 3 h of incubation
with homocysteine and copper. The majority of cells were positively
stained for active caspase, with bright DAPI-fluorescing condensed
nuclei and some blebbing of nuclei (Fig. 4).
Organ culture. The addition of CuSO4 alone was found to have
no effect on palate fusion, and therefore 10 p A / C u S Q was routinely
added to the standard media to reduce the number of experimental
animals required. The results of the organ culture are summarized
in Table 1. Seventy-six percentage (13 of 17) of palates cultured in
control standard medium (containing 10 ~M CuSO~) had evidence
of fusion, whereas only 13% (2 of 16, P < 0.001 compared with
control) fused when cultured in the presence of 5 p.M retinoic acid
(clefting control). In the experimental gToup, addition of 100 p,M
homocysteine to the standard medium resulted in a reduction in

HOMOCYSTEINE AND CLEFT PALATE

101

FIG. 2. The effect of thiols and disulphides on human embryonic palatal mesenchyme cell number. Human embryonic palatal mesenchyme cells were cultured under standard conditions, challenged for 24 h, and cell number quantified using an automated Coulter
counter. Cell number was not significantly affected with 100 IxM homocystine or cysteine with or without 10 txM CuSO,. However 100
I-tM cysteine with 10 IxM CuSO4 resulted in a decrease in cell number similar to that produced by 100 p.M homocystine (Fig. 1). Data
are presented as percentages of controls and standard deviations. **, P < 0.01 compared with control; n = 6.

palatal fusion, with 52% (11 of 21, not significant compared with
control) of palates fused.

Gelatin gel zymography. Pro-metalloproteinase-2 was the major

gelatinase produced by HEPM cells in cuhure, with no evidence of
MMP-9 and small, but unquantifiable, levels of activated MMP-2.
There was no significant change in the production or activation of
MMP-2 in HEPM ceils incubated with 20 IxM homocysteine and
10 p~M CuSO4 for up to 48 h (Fig. 5a). Similarly, there was no
change in expression of pro-MMP-2 or in its activation in cells
challenged with 100 p~M homocysteine alone (data not shown).

Both pro- and active MMP-2 were present in extracts from cultured murine palates. Ahhough there was a decrease in the levels
of MMP-2 with 1 mM homocysteine, there were no significant
changes in the levels of pro- or active MMP-2 with addition of any
of the concentrations of homocysteine compared with controls (Fig.
5b).
DISCUSSION
Given the link between cleft palate and folate deficiency and the
reported susceptibility of neural crest-derived cells to the effects

FIG. 3. The effect of catalase and superoxide dismutase (SOD) on homocysteine- and copper-induced cell death. Human embryonic
palatal mesenchyme cells were cultured under standard conditions, challenged for 24 h, and cell number quantified using an automated
Coulter counter. Catalase prevented the cell death induced by homocysteine and CuSO4, whereas SOD had no effect. Inactivation of the
catalase by boiling abolished its protective effect. Data are presented as percentages of controls and standard deviations. **, P < 0.01
compared with control; n = 6.

102

KNOTT ET AL.

FIG. 4. Activated easpase labeling in homoeysteine- and copper-treated human embryonic palatal mesenehyme cells. Hmnan embryonic palatal mesenchyme ceils were cultured on chamber slides, challenged, and after 3 h, labeled with fluorescein isothiocyanate (FITC)labeled CaspACE, counterstained with 4',6-diamidine-2-phenylindole (DAPI), and then examined using a fluorescence microscope to
determine extent of apoptosis. (a, b) Control cells, treated with vehicle only, FITC and DAPI fluorescence, respectively. No CaspACElabeled, apoptotic cells are present, and all nuclei have normal morphology. (c, d) Cells treated with 100 ~M homocysteine and 10 txM
CuSO~, FITC and DAPI fluorescence, respectively, showing five apoptosing cells with strong CaspACE labeling with condensed, brightly
fluorescing shrunken nuclei under DAPI fluorescence (large arrow), one normal cell with no labeling and normal nucleus (small arrow).
Magnification: X400.

TABLE 1
FREQUENCY OF PALATALFUSION OF CULTUREDMUR1NE PALATE
EXPLANTS. PALATESWERE CULTUREDIN STANDARDMEDIA
(CONTAINING 10 IxM CuSO~), SUPPLEMENTEDWITH 5 txM
RETINOIC ACID (CLEFTINGCONTROL), 100 ~M HOMOCYSTEINE,
OR VEHICLE
Fused
Nonfused
Total
Vehicle
Retinoic acid
Homocysteine
Total

13
2
11
26

4
14"**
10
28

17
16
21
54

***P < 0.001 compared with vehicle, as determined using the Fisher
exact test.

of homoeysteine and associated oxidative stress, there has been little investigation into the role of elevated homocysteine and the development of this common eraniofaeial anomaly or the potential
cellular mechanisms involved. This study has demonstrated that the
oxidation of homoeysteine can result in palatal mesenchyme cell
death in vitro and the possible disruption of normal murine palatogenesis in organ culture. The mechanism of action that we investigated was the potential for oxidative stress, induced by oxidation
of homoeysteine, to disrupt HEPM cell function because neural
crest derived-cells such as palatal mesenchyme cells are known to
be susceptible to changes in redox status (Outinen et al., 1998).
Starkebaum and Harlan (1986) first demonstrated that oxidative

stress as a consequence of homocystine oxidation by copper (free

or bound as ceruloplasmin) could result in endothelial cell death.
However, it is important to note that in the study by Starkebaum
and Harlan (1986) and in several other studies investigating the
pathological effect of homocystine, supraphysiologieal levels of homocysteine (100-500 ixM) were used. A study by Hultberg et al.
(1997) demonstrated that oxidative stress induced by low levels of
homocysteine and copper resulted in cell damage, and in our study,
we have demonstrated a dose-related increase in cell death with
elevated, but clinically relevant, levels of both homocysteine (10100 /xM) and copper (1-10 t~M) (Cartwright and Wintrobe, 1964;
Finkelstein and Martin, 2000).
Transition metal-catalyzed autooxidation of free thiols produces
H~02 and O~ ROS. Reactive oxygen species can induce a range
of cellular events depending on the level of oxidative stress, inducing proliferation, growth arrest, apoptosis, and necrosis under
both pathologic and nonpathologie conditions (Davies, 1999; Simon et al., 2000). In this study, the oxidative stress resulting from
incubation of HEPM cells with homocysteine (20-100 ~M) and
copper (0.1-10 IxM) led to apoptosis rather than necrosis. Reactive oxygen species can induce apoptosis through several different
mechanisms, including the triggering of death receptors, mitoehondrial disruption, lysosomal rupture, oxidative damage to key
proteins, and disruption of the cellular redox status (Kehrer, 2000;
Simon et al., 2000; Antunes et al., 2001). Although not all apoptoses involve easpases, these intraeellular eysteine proteinases are
integral to many of the apoptotie pathways, and as such, the pres-

HOMOCYSTEINE AND CLEFT PALATE

103

FIG. 5. Gelatin gel zylnographyof (a) human embryonic palatal mesenchyme (HEPM)-conditioned media and (b) cultured murine
palate extracts. (a) Media taken from HEPM cells cultured under standard conditions and challenged for 48 h with either vehicle (v) Or
20 tiM homocysteineand 10 tAMCuSO4 (h). (b) Extracts were taken from routine palates roller cultured for 72 h in media containing 10
tAM CuSO4,with 5, 1, or 0.1 mM homocysteine or controts with vehicle only. There were no significant differences in the levels of proor active MMP-2 in either cell or organ culture experiments.
ence of activated caspases is often used to identify apoptosing
ceils. Caspase 3 has been previously identified in VEC in which
apoptosis was induced by a relatively high level of homocysteine
(500 IxM) and 10 IxM copper (Bessede et al., 2001). In this study,
the use of the CaspACE in situ marker enabled the identification
of early apoptosing cells before (3 h) the morphological changes
were observed under phase contrast microscopy after 4-5 h of
incubation. After 8 h, few ceils were caspase positive, as the majority of apoptosing cells had progressed to secondary necrosis or
disintegrated. Caspases are regulated by ROS; however, as cysteine proteases, they are themselves sensitive to the redox status
of the cell, and at higher levels of oxidative stress, caspases may
become inactivated, thereby blocking some apoptotic pathways
(Hampton et al., 1998). Apoptosis has been proposed along with
several other mechanisms for the loss of the MEE during palatal
shelf fusion, in addition to being an important process in developmental processes in general. Recently published data suggest
that programmed cell death is required for successful palate shelf
fusion (Cuervo et al., 2002), and any disruption in the balance of
apoptosis and cell survival would result in disruption of palate
formation.
Human embryonic palatal mesenchyme apoptosis in response
to homocysteine and copper was abrogated with the addition of
catalase but not SOD, indicating that the cell death was triggered
by the generation of H202 rather than O2 . Homocysteine also
affects other aspects of the cell's ability to respond to oxidative
stress therefore potentially exacerbating the effects of the ROS
generated by homocysteine autooxidation. Elevated levels of homocysteine are known to inhibit production and activity of glutathione peroxidase, which is responsible for the removal of n202
(Nishio and Watanabe, 1997; Outinen et al., 1998). High levels
of homocysteine (0.5-5 raM) were used in several of these studies,
and it is possible that different mechanisms may be involved at
these pharmacological levels; however, it is likely that the mechanism whereby homocysteine damages cells is multifactorial. Indeed, several cellular mechanisms of action have been proposed

for homocysteine-induced responses, including hypomethylation,

protein homoeysteinylation, and interaction with N-mehyl-D-aspartate receptors.
We, and others, have previously described the importance of gelatinases during palate development (Mansell et al., 2000) and also
that disruption of MMP function results in cleft palate (Blavier et
al., 2001; Brown et al., 2002). Both homocysteine and ROS have
been reported to disrupt MMPs, from transcription to activation;
however, in this study, we were unable to demonstrate any significant changes in MMP-2 levels or activation. Homocysteine and oxidative stress have been reported to have opposing effects on MMPs,
and ROS have been shown to increase MMP transcription and activity. Homocysteine can inhibit the activity of the transcription factor AP-1, thereby decreasing MMP-1, -3, and -9 production (Suzuki
et al., 2000; Siwik et al., 2001), although MMP-2 transcription is
controlled by AP-2. The overall expectation for MMP involvement
is therefore not clear or predictable, and this aspect of palatal development may not be affected by oxidative stress in this situation.
Normal human palate development requires a complex and coordinated sequence of events including palatal shelf elevation with
subsequent tissue reorganization and differentiation into definitive
structures. In addition to our study of palatal mesenchyme ceils, we
have used a murine organ culture method to test whether homocysteine can disrupt palate development at the tissue level. Palatogenesis differs among species, but human and mouse palate development is comparable. Both species have individual shelves of the
secondary palate that grow vertically and need to reorientate into a
position above the tongue, contact, and exclude the MEE to complete fusion. This makes mouse a suilable model to study pal,ate
development, and murine palatal organ culture is an established
technique for examining the potential of teratogenic agents to induce cleft palate. Our palatal culture results suggest that 100 IxM
homocysteine with 10 IAM CuSQ might be teratogenic, although the
findings indicate a reduction in the number of fused palates under
these conditions, and the data obtained did not reach stalistical
significance. Further studies of the effect of homocysteine on palate

104

KNOTT ET AL.

culture would be valuable to clarify these data and also to investigate the possible changes in the levels of apoptosis in the palate
in light of the data from the cell culture experiments. The organ
culture data may suggest, however, that the main effect of homocysteine toxicity demonstrated in cell culture may not be in the
later stages of palate elevation and fusion examined in this study
but during earlier stages of palate formation such as during neural
crest cell migration within the developing palate.
It is apparent from this and other studies that homocysteine does
have the potential to disrupt normal palate development. We have
demonstrated that biologically relevant levels of homocysteine and
copper can result in apoptosis induced by oxidative stress. Clearly,
the role of homocysteine in the development of cleft palate requires
further investigation, and such studies would not only provide insights into the etiology of cleft palate in general but also the complex process of palatogenesis.

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