1. health and fitness
2. disease
3. cholesterol

Toxicology
Letters
ELSEVIER
Toxicology Letters 91 (1997) 169-178
Pro-oxidant
effects of 6 -aminolevulinic acid (6 -ALA) on
Chinese hamster ovary (CHO) cells
Rachel Neal a, Ping Yang a, James Fiechtl b, Deniz Yildiz a, Hande Gurer a,
Nuran Ercal a,*
a Chemistry Department, University of Missouri, 142 Schrenk Hall, Rolla, A40 6.5409, USA
b Life Sciences Department, University of Missouri, Rolla, MO, USA
Receive’d24 October 1996; received in revised form 17 February 1997; accepted 17 February 1997
Abstract
6-Aminolevulinic
Acid (a-ALA) is a heme precursor accumulated in lead poisoning and acute intermittent
porphyria. Although no single mechanism for lead toxicity has yet been defined, recent studies suggest at least some
of the lead-induced damage may originate from d-ALA-induced oxidative stress. The present study was designed to
test the hypothesis that a-ALA accumulation in Chinese hamster ovary (CHO) cells contributes to the cumulative
oxidative challenge of lead poisoning as indicated by the oxidative stress parameters glutathione (GSH), glutathione
disulfide (GSSG), malondialdehyde equivalents (MDA), and catalase (CAT). It will also examine the possibility that
this oxidative challenge can be reversed by treatment with an antioxidant such as N-acetylcysteine (NAC). First in
vitro administration of B-ALA to CHO cells was found to have a concentration-dependent
inhibitory effect on colony
formation and cell survival. NAC administration
was shown to alleviate this inhibition in CHO survival. The
oxidative status of CIHO cell cultures exposed to increasing concentrations of B-ALA was then examined. Decreases
in GSH levels (P < 0.05) were observed in the a-ALA-treated cultures as compared to the controls, while GSSG and
MDA levels were significantly increased in d-ALA-treated cells (P < 0.05). CAT activity was not significantly
affected. NAC administration concurrent with B-ALA exposure resulted in GSH and GSSG levels similar to the
control levels, while :no significant improvement in MDA was observed. These results indicate a state of oxidative
stress and suggest that the a-ALA- induced inhibitory effect on CHO colony formation may be due to its pro-oxidant
effect. To assess whether this oxidative challenge would induce antioxidant increases during extended exposure to
S-ALA, CHO cells were exposed to 5 mM d-ALA for increasing time periods. The GSH and GSSG levels were
measured and a rebound effect was observed after 12 h of B-ALA exposure. 0 1997 Elsevier Science Ireland Ltd.
Keywords:
6Aminolevulinic
acid; Lead-induced
damage; Oxidative stress; Chinese hamster ovary cells
* Corresponding author. Tel.: + 1 573 3416950; fax: + 1 573 3416033; e-mail: nercal@umr.edu.
0378-4274/97/$17.000 11997Elsevier Science Ireland Ltd. All rights reserved.
PIZSO378-4274(97)03887-3
170
R. Neal et al. /Toxicology
1. Introduction
6 -Aminolevulinic acid (6 -ALA), a component
of the heme biosynthesis pathway, is accumulated
in lead poisoning and in acute intermittent porphyria [l]. Lead’s inhibitory effect on critical enzymes such as S-ALA dehydratase (6-ALAD), an
enzyme in the heme biosynthesis pathway which
catalyzes the condensation of two molecules of
S-ALA to porphobilinogen
[2], has been well
documented (Fig. 1). Lead has been shown to
decrease the activity of 6-ALAD, leading to an
accumulation of J-ALA [3,4]. Although no single
mechanism for lead toxicity has been discovered,
recent studies suggest the lead-induced damage
may originate in part from 6 -ALA-induced oxidative stress. d-ALA has been shown to have a
proclivity for enhancing reactive oxygen intermediates (ROI) and lipid peroxidation [5,6]. The
promotion of ROI formation by d-ALA was examined by Monteiro et al. [7]. Spin-trapping experiments were used to support the contention
that 6 -ALA-induced oxyhemoglobin autooxidation occurs in conjunction with d-ALA autooxidation.
Additionally,
Oteiza
and
Bechara
determined a role for d-ALA-induced lipid peroxidation and membrane leakage by using phosphatidyl choline:cardiolipin liposomes as a model
system [S]. &-ALA induced the formation of thiobarbituric acid reactive substances (TBARS) in
the liposomes as well as stimulating leakage of
carboxyfluorescein
from the liposome into the
media. The formation of TBARS and membrane
leakage was also found to be inhibited by the free
radical scavenger, a-tocopherol.
Recent studies have implicated the mechanism
of d-ALA-induced ROI formation in the pathophysiology of acute intermittent porphyria and
plumbism [5]. In situ generation of ROI due to
the accumulation of d-ALA in bone marrow and
nerve cells has been proposed to trigger the neurological abnormalities typical of porphyrias and
plumbism [6].
The present study was designed to investigate
the hypothesis that d-ALA accumulation during
lead poisoning contributes to the cumulative oxidative challenge, with CHO cells used as an in
vitro model system. Colony formation assays were
Letters 91 (1997) 169-l 78
performed to determine d-ALA’s inhibitory effect
on cell proliferation. The observed inhibition was
investigated by measuring indicators of oxidative
stress in the presence and absence of d-ALA and
without the addition of 1 mM NAC. NAC, a
well-known antioxidant and GSH precursor, was
included to evaluate the possible therapeutic role
of antioxidants
on 6 -ALA-induced
oxidative
stress. A time course study of GSH and GSSG
levels in response to b-ALA challenge was included to evaluate a possible induction of an
antioxidant response.
2. Materials and methods
2.1. Materials
The N-( 1-pyrenyl)-maleimide,
1,1,3,3-tetramethoxypropane,
and 2-vinyl pyridine were purGlycine+ Succinyl-CoA
ALA-synthetase
&
b-AminolevulinicAcid (GALA)
AL.A-dehydratase(ALAD)
J
Porphobiiogm
4
Uroporphyrinogen
4
Coproporphyrinogen
i
ProtoporphyrinIx
&
Heme
Fig. 1. The formation
of B-ALA from glycine
Co-A and the condensation
reaction of h-ALA
phobilinogen.
and succinyl
to form por-
R. Neal et al. /Toxicology
chased from Aldrich
other chemicals were
Louis, MO, USA).
used in GSH, GSSG
(Milwaukee,
WI, USA). All
purchased from Sigma (St.
HPLC grade reagents were
and MDA analysis.
Letters 91 (1997) 169-I 78
the S-ALA
exposed
scale vs. the d-ALA
scale.
2.5. Oxidative
2.2. Colony formation
and counting
of colonies
The cells were fixed by decanting
the media
and
Carnoy’s
fixative
adding
(3:1,
methanol:acetic
acid) for 5 min. After the cells
were washed, crystal violet was added for 5 min
to stain the colonies.
The plates were washed
with distilled water, allowed to air dry, and the
number of colonies were then counted. The plating efficiency (PE) was calculated as follows:
PE = Colonies counted/cells
seeded x IO0
Results
reported
from colony formation
assays represent at lf:ast three separate experiments
performed each time in triplicate.
2.4. Construction
A cell
ting the
counted,
times the
groups
on
concentration
a logarithmic
on a linear
stress studies
assays
CHO cells, an established
tumor
cell line,
were propagated
in Ham’s F-12 culture media
supplemented
with 10% fetal calf serum (FCS)
and maintained
at 37°C in 5% CO,/95%
air.
For
colony
formation
assays,
exponentially
growing cells were collected after trypsinization
and centrifuged
at 1000 x g for 5 min. The resulting cell pellets were resuspended
in fresh media and counted on a hemocytometer.
Between
100-2000
cells were plated into small (60 mm)
petri dishes and incubated
for 4 h to allow cell
attachment
to the surface. The respective concentration
of B-ALA, either with or without
1
mM NAC, was then added to the petri dishes.
After 3-4 h of d-ALA or d-ALA plus 1 mM
NAC exposure,
the media was replaced
with
fresh media. The cells were incubated
for 7-10
days, then fixed and stained (see below).
2.3. Staining
171
of cell survival curve
survival curve was constructed
by plotsurvival
fraction
(number
of colonies
divided by the number of cells seeded,
plating efficiency of the control) from
Separate
cultures
of exponentially
growing
- 5 x lo6 cells/ml were esCHO cells containing
tablished.
After overnight
incubation,
the media
was replaced with fresh media containing
varying concentrations
of d-ALA with or without 1
mM NAC. The b-ALA-exposed
groups were incubated for 3 h with the &-ALA while the control was incubated
in media alone during this
time. At the end of the incubation,
the media
was removed and lactate dehydrogenase
(LDH)
activity was immediately
assayed in the media.
The cells were washed, trypsinized,
and homogenized for the determination
of GSH, GSSG,
MDA, and CAT activity. Results for oxidative
stress studies are from a minimum
of three separate experiments.
2.6. Glutathione and glutathione
determinations by HPLC
disuljide
2.6.1. GSH determination
A new method of GSH determination
was developed in this laboratory
to analyze y-glutamyl
cycle intermediates
[9]. Cell pellets were resuspended in serine-borate
buffer (100 mM TrisHCl, 10 mM borate,
5 mM serine,
1 mM
diethylenetriaminepentacetic
acid (DETAPAC),
pH 7.0). The cell suspension
was homogenized
on ice for 2 min with 5-s intervals of homogenization
and rest, and derivatized
with N-(lpyrenyl)-maleimide
(NPM).
This
compound
reacts
with free sulfhydryl
groups
to form
fluorescent
derivatives (Fig. 2). Each sample was
first diluted with distilled water to make a volume of 250 ~1 NPM (750 ~1, 1 mM in acetonitrile) was then added; the resulting solution was
mixed and incubated
at room temperature
for 5
min. One ~1 of 2 N HCl was added to stop the
reaction.
After filtration
through
a 0.2 pm
acrodisc,
the derivatized
samples were injected
in a reverse phase
onto a 3 pm C,, column
HPLC system.
R. Neal et al. /Toxicology
172
Letters 91 (1997) 169-178
SR
-_15
0
0
/
i>
/
I +Ii-SR
0
A
l
NPM
THIOL
Fig. 2. Reaction of thiol with N-(I-pyrenyl)-maleimide
2.62. GSSG determination
The determination
of GSSG was accomplished
by adding 44 ~1 of water to 40 ~1 of the sample.
To the diluted
sample,
16 ~1 of 6.25% 2vinylpyridine
in absolute ethanol was added, and
the mixture
was allowed to incubate
at room
temperature
for 60 min. After 95 ,ul of a 2 mg/ml
solution of NADPH and 5 ~1 of a 2 U/ml solution of glutathione
reductase
were added, the
solution was subsequently
mixed and an aliquot
of 100 ~1 was immediately
withdrawn.
To this
aliquot, 150 ~1 of HPLC grade water and 750 ~1
of 1.0 mM NPM were immediately
added to
perform the GSH derivatization,
as mentioned
above.
2.6.3. HPLC system
The HPLC system (Shimadzu)
comprised
a
model LC-1OA pump, a Rheodyne injection valve
with a 20-~1 injection filling loop, and a model
RF535 fluorescence
spectrophotometer
operating
at an excitation
wavelength
of 330 nm and an
emission
wavelength
of 375 nm. The HPLC
column was 100 x 4.6 mm and packed with 3 ,um
particles
of C,, packing
material.
The mobile
phase was 35% water and 65% acetonitrile
containing 1 ml/l acetic acid and 1 ml/l o-phosphoric
acid. The NPM derivatives were eluted from the
column isocratically
at a flow rate of 0.5 ml/min.
Quantitation
of the peaks from the HPLC system
was performed
with a Chromatopac,
model CR601 (Shimadzu).
THIOL-NPM
DERIVATIVE
to produce fluorescent thiol-NPM derivative.
2.7. Lipid peroxidation determinations by HPLC
The cell pellets were homogenized
in serine
borate buffer. To 0.250 ml of homogenate,
0.650
ml of 5% trichloroacetic
acid (TCA) and 0.100 ml
of 500 ppm butylated
hydroxytoluene
(BHT) in
methanol
were added.
The sample
was then
heated in a boiling water bath for 30 min. After
cooling on ice, the sample was centrifuged.
The
supernatant
was mixed 1: 1 with saturated thiobarbituric acid (TBA) [lO,l 11. The sample was once
again heated in a boiling water bath for 30 min.
After cooling on ice, 0.50 ml of the sample was
extracted
with 1.00 ml of n-butanol
and centrifuged to facilitate the separation
of the two
phases.
The resulting
organic
layer was first
filtered through
a 0.45 pm acrodisc and then
injected onto a reverse phase 250 x 4.6 mm 3 pm
C,, column. The mobile phase for this system was
composed of 30% acetonitrile
and 0.6% tetrahydrofuran in 5 mM phosphate buffer. The reaction
complexes were eluted from the column isocratitally at a flow rate of 0.75 ml/min.
2.8. Catalase activity assays
CAT activity was determined
spectrophotometrically by the method of Beers and Sizer, and was
expressed in U/mg protein as described by Aebi
[12,13]. This method
measures
the exponential
disappearance
of H,O, (10 mM) at 240 nm in the
presence of cellular homogenates.
The equation
which was used to fit the exponential
decay of
H,O, is as follows:
R. Neal et al. /Toxicology
Letters 91 (1997) 169-l 78
113
A,, = Aini e - kt
3.2.
where k is the rate constant, which is dependent
on the catalase activity.
Table 1 displays the results of the GSH and
GSSG measurements after culture exposure to
various concentrations of S-ALA. GSH levels in
CHO cells treated with 3 mM and 5 mM d-ALA
were significantly lower than those levels observed
in control cultures (P < 0.05). GSSG levels were
found to be significantly elevated in CHO cultures
treated with &-ALA when compared to those
levels in control cultures (P < 0.05). Additionally,
the ratios of GSH/GSSG were significantly decreased in the CHO cultures exposed to J-ALA
when compared to those ratios observed in the
control cultures (P < 0.05). Simultaneous exposure to 1 mM NAC and S-ALA returned the
GSH levels to control levels and significantly decreased the levels of GSSG.
2.9. Lactate dehydrogenase activity
The lactate dehydrogenase (LDH) activity assay was performed according to the method of
Hassoun et al. [14] with a minor modification.
The activity of LDH in 100 ~1 of media was
determined by direct calculation based on the
decrease in absorbance [15].
2. IO. Protein determination
The Bradford method was used to determine
the protein content of the cell samples using concentrated Coomassie Blue (Bio-Rad) and optical
density determinaf.ons at 595 nm [16]. A standard
curve using bovine serum albumin was constructed. The homogenized cell pellets were subjected to appropriate dilutions before protein
determination was performed.
2. Il. Statistical analysis
Tabulated values represent means f SD. of at
least three separate experiments. One-way analysis
of variance (ANOVA) and the Student-NewmanKeuls multiple comparison test were used to analyze data from experimental and control groups.
P-values < 0.05 were considered significant.
3.3.
GSH and GSSG
GSHIGSSG
time course
Fig. 4 shows the GSH/GSSG ratio in CHO
cells exposed to 5 mM d-ALA over a 12-h period
compared to the GSH/GSSG levels in control
CHO cells (media alone). The GSH/GSSG ratio
of 6-ALA-exposed CHO cells decreases between 3
and 6 h of exposure. By 12 h, the GSH/GSSG
level of J-ALA-exposed cells had begun to increase.
3. Results
3.1.
Survival curve
Fig. 3 represents a survival curve, generated by
plotting the survival fractions of d-ALA-treated
cultures against increasing 6 -ALA concentrations.
J-ALA inhibited CHO colony formation in a
concentration-dependent
manner. As shown in
Fig, 3, 1 mM NAC incubated concurrent with 1,
3 and 5 mM concentrations of S-ALA results in
significantly increased cell survival.
I
0.1
0
-AlA+NAC
.-..-- Am
I
1
I
I
,
1
2
3
4
5
delta-ALA concentrations (IIIM)
Fig. 3. Survival curve of CHO
and without 1 mM NAC.
cells exposed
to d-ALA,
with
174
R. Neal et al. /Toxicology
Table 1
Effect of 1, 3 and 5 mM J-ALA,
Control
1 mM
3 mM
5 mM
1 mM
I mM
3 mM
5 mM
a-ALA
a-ALA
h-ALA
NAC
B-ALA+NAC
6-ALA+NAC
S-ALA+NAC
with or without
GSH
(nmol/mg
72.98
76.47
65.35
60.49
85.71
90.50
77.69
69.64
k
k
k
+
k
*
k
f
Letters
91 (1997) 169-178
1 mM NAC supplementation,
protein)
0.95
1.24
1.27*
4.84*
3.22
1.15**
4.03**
0.29**
All values represent mean k S.D. for three separate experiments.
*P<O.O5 compared to control.
**P<O.O5 NAC treated groups compared to corresponding
d-ALA
3.4. Ca talase
Table 2 contains the results from CAT activity
assays. CAT activity had not significantly decreased in CHO cultures exposed to S-ALA when
compared to the control cultures. NAC (1 mM)
treatment during exposure to S-ALA resulted in
no significant improvement in CAT activity.
3.5. Malondialdehyde equivalents (MDA)
Table 2 shows the results of MDA levels for
control and S-ALA- exposed CHO cells. MDA
levels of &-ALA-exposed CHO cells were significantly higher than those observed for the control
CHO cells (P < 0.05). NAC had no observed
beneficial role.
3.6. Lactate dehydrogenase (LDH) activity
Table 2 also shows the results of the LDH
activity assay. As shown, no significant differences
were seen between control, d-ALA, and NACtreated cells.
4. Discussion
Lead has wide-ranging effects on a number of
human organ systems. It has been implicated in
cases of anemia and immunosuppression [17- 191.
The mechanism by which lead causes its deleteri-
GSSG
5.33
7.64
13.85
15.45
7.50
8.82
8.81
9.83
(nmol/mg
*
+
+
k
k
*
+
f
1.38
2.10
1.27*
3.82*
1.84
1.45
1.97**
1.23**
on CHO
protein)
cell GSH
and GSSG
levels
GSH/GSSG
13.70
10.01
4.72
3.92
11.43
10.26
8.82
7.08
+ 0.69
f 0.59*
k 1.OO*
k 1.27*
* 1.75
& 0.79
k 2.05**
_+0.24**
groups.
ous effects has yet to be elucidated; however, part
of lead’s effect may be due to the accumulation of
d-ALA. The accumulation of d-ALA originates
from lead’s inhibition of ALAD, an enzyme in the
heme biosynthesis pathway which catalyzes the
condensation of two molecules of b-ALA to porphobilinogen [2-41. ALAD is sensitive to oxygen,
and requires a high exogenous thiol concentration
for full catalytic activity due to the oxidative
susceptibility of the enzyme’s 32 reactive thiol
groups composing many possible active sites.
Lead is known to bind to ALAD through one of
these active sites causing dramatically lowered
enzyme activity resulting in J-ALA accumulation
[4]. At a pH range of 7.0-8.0, J-ALA enolizes
and this enol undergoes autooxidation resulting in
the formation of the superoxide and the hydroxyl
radical. d-ALA has also been shown to undergo
iron-catalyzed oxidation with ROI generation and
to induce Ca2+ release from mitochondria
through oxidative damage to the inner mitochondrial membrane [8].
To investigate b-ALA’s role in lead-induced
oxidative stress, first colony formation assays
were performed in the presence of increasing concentrations of d-ALA. The results of these assays,
as shown in Fig. 3, indicated that J-ALA acts in
a concentration-dependent
manner to inhibit
CHO cell colony formation. This was evidenced
by a decrease in survival fraction as d-ALA concentration increases. The administration of 1 mM
NAC results in increased survival fraction at all
R. Neal et al. /Toxicology
Letters 91 (1997) 169-l 78
175
-CONTROL
20-
--+- 5 mM G-ALA
/--4
_H-----
time (hours)
Fig. 4. The effect of 5 mM J-ALA on the GSH/GSSG ratio of CHO cells exposed to d-ALA for 12 h
S-ALA concentrations
studied. Since d-ALA has
been shown to have a proclivity
for enhancing
ROI, the observed inhibition
is believed to be the
result of 6-ALA-induced
oxidative stress [5]. The
degree of oxidative challenge can be quantified by
measuring
GSH,
GSSG,
lipid
peroxidation
byproducts
(MDA), and the activities of antioxidant enzymes such. as catalase, superoxide dismutase, and glutathione
peroxidase.
Together these
proteins combine to mediate the intracellular
response to oxidative
stress [20]. GSH functions
both as a direct scavenger
of ROI and as a
cofactor in their metabolic detoxification
[21-231.
As a result of oxidative stress, GSH is oxidized
rapidly to GSSG; consequently,
a decrease in
GSH and an increase in GSSG (i.e. a decreasing
ratio of GSH to GSSG) is suggestive of oxidative
stress [24,25]. Results from the study illustrated a
decrease in the GSH/GSSG
ratio after d-ALA
indicating
that d-ALA
was inducing
exposure,
oxidative stress in CHO cells. A time course experiment was performed to evaluate the impact of
prolonged
d-ALA exposure on the GSH/GSSG
ratio in CHO cells. In Fig. 4, even at 3 h, d-ALA
is shown to induce oxidative stress as the GSH/
GSSG ratio falls. This trend continues until 12 h
when an increase in the GSH/GSSG
ratio of the
d-ALA-treated
cells was observed,
indicating
a
possible initiation
of antioxidant
defense.
Catalase is a heme-containing
antioxidant
enzyme that converts H,O, to 0, and water [26]. Its
activity has been shown to change upon oxidative
challenge in vitro. The activities of antioxidant
enzymes are dependent
upon the nature of the
oxidant
and the duration
of challenge.
In the
present study, no increases in CAT activity were
observed in the d-ALA concentrations
studied. It
is possible that these concentrations
were simply
not significant
to cause an observed change in
CAT activity.
Oxidative stress is also shown by an increase in
lipid peroxidation
byproducts,
such as MDA
[27,28]. MDA is a degradation
product
of the
highly unstable lipid peroxides that are generated
from the interaction
of pro-oxidants
such as the
hydroxyl radical with membrane
lipids. It is routinely measured to show the degree of lipid peroxidation due to oxidative stress [29,30]. Lead does
not directly undergo an oxidation-reduction
cycle
that produces ROI. Therefore,
the effect of lead
on lipid peroxidation
in our previous study may
have been due to an indirect mechanism involving
the inhibition
of enzymes such as ALAD [31].
Such inhibition
could result in increased
substrates capable of undergoing
a redox cycle that
would produce ROI [25,32]. To assess this hypothesis, CHO cells were exposed to increasing
concentrations
of d-ALA. We have shown that
MDA levels increased in CHO cell cultures ex-
R. Neal et al. /Toxicology
176
Table 2
Effect of 1, 3 and 5 mM d-ALA,
hamster ovary (CHO) cells
Group
Control
1 mM
1 mM
3 mM
5 mM
1 mM
3 mM
5 mM
both
with and without
CAT activity
NAC
b-ALA
b-ALA
b-ALA
B-ALAfNAC
O-ALA+NAC
6-ALA+NAC
All values represent
*PiO.O5 compared
0.041
0.039
0.028
0.034
0.036
0.030
0.032
0.029
(Ujmg
Letters 91 (1997) 169-l 78
I mM NAC supplementation,
protein)
* 0.007
* 0.008
+ 0.006
f 0.004
f 0.009
f 0.006
& 0.008
f 0.002
MDA
(nmol/lOO
12.3 f 0.7
11.9+2.3
29.2 + 2.2*
25.5 + 5.5*
26.1 + 2.0*
33.2 k 3.0
25.1 & 3.3
28.1 * 1.5
on CAT,
mg protein)
MDA,
and
LDH
in Chinese
LDH
(U/ml)
40.25
40.01
39.36
38.60
37.78
40.60
39.12
37.78
+
*
k
+
*
+
f
&
2.62
3.14
2.72
2.31
1.81
2.07
3.56
6.66
mean + S.D.
to control.
posed to increasing
concentrations
of d-ALA,
once again indicating
that S-ALA can induce
oxidative stress in CHO cells.
Lipid peroxidation
is known to change membrane fragility, motility and permeability
[33-351.
LDH is a cytoplasmic
enzyme and its leakage
from injured cells into the culture medium has
been shown to be useful as an indicator of cellular
membrane damage. In the present study, we measured LDH activity in a culture medium in order
to assess the membrane
damage induced by 6ALA-derived
ROI. No differences in LDH activity were noted regardless of whether incubation
time or d-ALA concentration
was the independent variable. These findings suggest two possibilities. The concentrations
in this study of d-ALAderived ROI does not change the native membrane permeability
of the CHO cells as HermesLima et al. proposed
for mitochondrial
inner
membrane
[5]. Another possibility is that B-ALA
may simply inhibit LDH activity.
When the results of the GSH, GSSG, and
MDA in 6-ALA-exposed
CHO cells are examined, the hypothesis that d-ALA is at least partially responsible
for the ROI-induced
oxidative
stress present in lead poisoning is supported. One
possible remedy for this oxidative stress would be
replenishment
of the cellular GSH pool. However,
because GSH does not readily cross the outer cell
membrane,
direct GSH supplementation
is not
insuccessful [36]. To circumvent
this limitation,
tracellular
L-cysteine pools may be elevated by
NAC administration.
NAC is readily deacetylated
to yield L-cysteine, which is the rate limiting component of GSH synthesis. NAC is also a wellknown antioxidant
with a proven high toxicity
threshold in vivo and a well-documented
history
of clinical application
[37]. The antioxidant
effects
of NAC supplementation
were evidenced by the
increase in GSH/GSSG
ratios (Table 1) and increased cell survival (Fig. 3). These findings support a protective role of NAC in J-ALA exposed
cultures
and suggest that NAC might have a
therapeutic effect on diseases such as lead poisoning where d-ALA accumulation
occurs.
Our previous study showed that lead-exposed
CHO cells undergo increased oxidative stress [31].
The present study was undertaken
to examine
d-ALA’s contribution
to lead-induced
oxidative
stress. Study results based on data obtained
through the measurement
of the various oxidative
stress parameters indicate that d-ALA does play a
role in the induction
of oxidative stress in CHO
cells. This study, in conjunction
with our previous
study [31], suggests that oxidative stress in lead
poisoning
is caused by lead both directly and
indirectly
by the accumulation
of S-ALA. This
leads to the conclusion
that antioxidants
such as
NAC should be employed in cases of lead poisoning to help restore oxidative balance.
R. Neal et al. /Toxicology
Acknowledgements
The authors are thankful to Dr. D. Spitz and
Dr.
P. Lutz
for
their
comments
on
the
manuscript.
Special thanks also to A. Gambill,
J.T. Cochran and Dr. Serdar Oztezcan for their
technical
help. Dr. Ercal was supported
by
lR15ES08016-01
from the NIEHS, NIH and the
contents of this paper are solely the responsibility
of the authors and do not necessarily
represent
the official views 01‘the NIEHS or NIH. H. Gurer
was supported by the Turkish Scientific and Technical Research Council.
References
[l] Hindmarsh,
J.T. (1986)The porphyrias:
recent advances.
Clin. Chem. 32, 1;!55-1263.
[2] Haeger-Aronsen,
B., Abdulla,
M. and Fristedt,
B.I.
(1971) Effect of lead on delta aminolevulinic
acid dehydratase activity in red blood cells. Arch. Environ. Health
23, 440-445.
[3] Ribarov, S.R. and Bochev, P.G. (1982) Lead-hemoglobin
interaction
as a possible source of reactive oxygen species
a chemiluminescent
study. Arch. Biochem. Biophys.
213, 288-292.
[4] Gibbs, P.N.B., Gore, M.G. and Jordan,
P.M. (1991)
Investigation
of the effect of metal ions on the reactivity
of thiol groups in human 5-aminolevulinic
dehydratase.
Biochem. J. 225, 573-580.
[5] Hermes-Lima,
M.. Valle, G.R.V..
Vercesi. A.E. and
Bechara, E.J.H. (1991) Damage to rat liver mitochondria
promoted
by delta-aminolevulinic
acid-generated
reactive
oxygen species: connections
with acute intermittent
porphyria and lead poisoning. Biochim. Biophys. Acta. 1056,
57-63.
A. and
161Monteiro, H.P., Abdalla, D.S.P., Faljoni-Alario,
Bechara,
E.J.H. (1989) Free radical generation
during
delta-aminolevulinic
acid autoxidation:
induction
of
hemoglobin
and ccnnections
with porphyropathies.
Arch.
Biochem. Biophys. 271, 206-216.
H.P.,
Abdalla,
D.S.P.,
Augusto,
0. and
[71Montiero,
Bechera, E.J.H. (1086) Generation
of active oxygen species during coupled autoxidation
of oxyhemoglobin
and
delta-aminolevulinic
acid. Biochim. Biophys. Acta 881,
100-106.
PI Oteiza, P.I. and BI:chara, E.J.H. (1993) 5-aminolevulinic
acid induces lipid peroxidation
in cardiolipin-rich
liposomes. Arch. Biochem. Biophys. 305, 282-287.
J., Ercal, N., Matthews,
R.H.
191Winters, R.A., Zukowski,
and Spitz, D.R. (1995) Analysis
of glutathione,
glutathione disulfide, ssysteine, homocysteine,
and other biological thiols by high performance
liquid chromatography
Letters
91 (1997) 169-l 78
171
following
derivatization
with N-(1-pyrenyl)maleimide.
Anal. Biochem. 227, 14-21.
H., Wu, J.,
[lOI Draper, H.H., Squires, E.J., Mahmoodi,
Agarwal, S. and Hadley, M.A. (1993) Comparative
evaluation of thiobarbituric
acid methods for the determination of malondialdehyde
in biological
materials.
Free
Rad. Biol. Med. 15, 353-363.
H., Schaur,
R.J. and Zollner,
H. (1991)
[Ill Esterbauer,
Chemistry and biochemistry
of 4-hydroxynonenal,
malonaldehyde and related aldehydes. Free Rad. Biol. Med. 11,
81-128.
u21 Beers, R.F. and Sizer, I.W. (1952) A spectrophotometric
method for measuring the breakdown
of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140.
1131Aebi, H. (1984) Catalase in vitro. In: L. Packer (Ed.),
Methods in Enzymology,
Vol. 105, Academic Press, New
York, pp. 121-126.
E.A., Roche, V.F. and Stohs, J.S. (1993) Reu41 Hassoun,
lease of enzymes by ricin from macrophages
and Chinese
hamster ovary cells in culture. Toxicol. Methods 3, 119129.
A.R. and Kochmar, J.R. (1986)
u51 Moss, D.W., Henderson,
Enzymes.
In: N.W. Tietz (Ed.), Textbook
of Clinical
Chemistry,
WB Saunders, Philadelphia,
pp. 619-663.
M.A. (1976) Rapid and sensitive method for
1161Bradford,
the quantitation
of microgram
quantities of protein utilizing the principle of protein-dye
binding. Anal. Biochem.
72, 248-256.
produced
by
P71 Koller, L.D. (1973) Immunosuppression
lead, cadmium, and mercury. Am. J. Vet. Res. 34, 14571458.
F., Bingham, E. and Stemmer, K.L. (1963)
[181Springman,
The acute effects of lead alkyls. Arch. Environ. Health 6,
469-412.
H.P., Abdalla,
D.S.P., Arcuri,
A.S.A. and
u91 Monteiro,
Bechara, E.J.H. (1985) Oxygen toxicity related to exposure to lead. Clin. Chem. 31, 1673-1676.
G.. Larsson, R.L., MuehlematPO1 Cerruti, P.A., Krupitza,
ter, D., Crawford,
D. and Amstad, P. (1988) Physiological and pathological
effects
of oxidants
in mouse
epidermal cells. Ann. NY Acad. Sci. 551, 75-82.
Pll Hsu, J.M. (1981) Lead toxicity as related to glutathione
metabolism.
J. Nutr. 1 I I, 26-33.
PI Ketterer, B., Coles, B. and Meyer, D.J. (1983) The role of
glutathione
in detoxification.
Environ.
Health Perspect.
49, 49-69.
1231Bray, T.M. and Taylor, C.G. (1993) Tissue glutathione,
nutrition,
and oxidative stress. Can. J. Physiol. Pharmacol. 71, 746-175.
~241Nemeth, I. and Boda, D. (1989) The ratio of oxidized/reduced glutathione
as an index of oxidative stress in various experimental
modes of shock syndrome.
Biomed.
Biochim. Acta 48, 53-57.
1251Sandhir, R., Julka, D. and Gill, K.D. (1994) Lipoperoxidative damage on lead exposure
in rat brain and its
implications
on membrane
bound enzymes. Pharmacol.
Toxicol. 74, 66-71.
R. Neal et al. /Toxicology
178
WI Spitz, D., Adama,
[271
[281
[291
1301
1311
~321
D., Sherman,
C. and Roberts,
R.
(1992) Mechanism of cellular resistance to hydrogen peroxide, hyperoxia,
and 4-hydroxy-2-nonenal
toxicity: the
significance
of increased catalase activity in H,O,-resistant fibroblasts.
Arch. Biochem. Biophys. 292, 221-227.
Koizumi, T. and Li, Z.G. (1992) Role of oxidative stress
in single-dose, cadmium-induced
testicular cancer. J. Toxicol. Environ. Health 37, 25-36.
Muller, L. (1986) Consequences
of cadmium toxicity in
rat hepdtocytes:
mitochondrial
dysfunction
and lipid peroxidation, Toxicology 40, 2855292.
Belghmi, K., Nicolas, J.C. and Crastes de Paul&, A.
(1988) Chemiluminescent
assay of lipid hydroperoxides.
J.
Biolumin. Chemilumin.
2, 113-l 19.
Janero, D.R. (1990) Malondialdehyde
and thiobarbituric
acid reactivity as diagnostic
indices of lipid peroxidation
and peroxidative
tissue injury. Free Rad. Biol. Med. 9,
515-540.
Ercal, N., Treeratphan,
P., Lutz, P., Hammond,
T.C. and
Matthews, R.H. (1996) N-acetylcysteine
protects Chinese
Hamster Ovary (CHO) cells from lead-induced
oxidative
stress. Toxicology
108, 57764.
Monteiro,
H., Bechara, E. and Abdalla, D. (1991) Free
Letters 91 (1997) 169-178
radicals
involvement
in neurological
porphyrias
and lead
poisoning.
Mol. Cell. Biochem. 103, 73383.
[33] Barros, S.B.M., Videla, L.A., Simizu, K., Halsema,
and Junqueira,
V.B.C.
stress. II. Time course
status.
Xenobiotica
(1988) Lindane-induced
of changes in hepatic
L.V.
oxidative
glutathione
18, 1305- 1310.
[34] Junqueira,
V.B.C., Simizu, K., Van Halsema, L.V., Koch,
O.R., Barros, S.B.M. and Videla, L.A. (1988) Lindane-induced oxidative
stress. 1. Time course of changes
in
hepatic
microsomal
parameters,
antioxidant
enzymes,
lipid peroxidative
indices and morphological
chardcteristics. Xenobiotica
[35] Barros, S.B.M.,
Liver
lipid
18, 1297- 1304.
Simizu, K. and Junqueira,
peroxidation-related
term administration
of hexachlorocyclohexane
rats. Toxicol. Lett. 56, 1377144.
[36] Moldeus, P. (1994) N-Acetylcysteine.
Methods
Diego,
V.B.C. (1991)
parameters
in Enzymology,
after
In: L. Packer
Vol. 234, Academic
short-
isomers
to
(Ed.),
Press, San
pp. 482-492.
[37] Janes, J. and Routledge,
P. (1992) Recent developments
in the management
of paracetamol
(acetaminophen)
poisoning.
Drug
Safety
7, 170- 177.