Complementary Action of Antioxidant Enzymes in
the Protection of Bioengineered Insulin-Producing
RINm5F Cells Against the Toxicity of Reactive
Oxygen Species
Markus Tiedge, Stephan Lortz, Rex Munday, and Sigurd Lenzen
To determine the importance of different antioxidative
enzymes for the defense status of insulin-producing
cells, the effects of stable overexpression of glutathione
peroxidase (Gpx), catalase (Cat), or Cu/Zn superoxide
dismutase (SOD) in insulin-producing RINm5F cells on
the cytotoxicity of hydrogen peroxide (H2O2), hypoxanthine/xanthine oxidase (H/XO), and menadione have
been investigated. Single overexpression of Cat or Gpx
provided less protection than the combined expression
of Cat plus SOD or Cat plus Gpx, while single overexpression of SOD either had no effect on the toxicity of
the test compounds or increased it. RINm5F cells were
also susceptible to butylalloxan, a lipophilic alloxan
derivative that is selectively toxic to pancreatic -cells.
Overexpression of enzymes, both alone and in combination, did not protect against butylalloxan-induced toxicity while SOD overexpression increased it, as evident
from a half maximally effective concentration (EC50)
value. The addition of Cat to the culture medium completely prevented the toxic effects of H2O2 and H/XO but
had no significant effect on the toxicity of menadione or
butylalloxan. Extracellular SOD had no effect on the
toxicity of any of the test compounds. The results of
this study show the importance of a combination of
antioxidant enzymes in protecting against the toxicity of
reactive oxygen species. Thus, overexpression of Cat
and Gpx, alone or in combination with SOD, by use of
molecular biology techniques can protect insulin-producing cells against oxidative damage. This may represent a strategy to protect pancreatic -cells against
destruction during the development of autoimmune diabetes and emphasizes the importance of optimal antioxidative enzyme equipment for protection against free
radical–mediated diseases. Diabetes 47:1578–1585, 1998
From the Institute of Clinical Biochemistry (M.T., S.Lo., S.Le.), Hannover
Medical School, Hannover, Germany; and AgResearch (R.M.), Ruakura
Agricultural Research Centre, Hamilton, New Zealand.
Address correspondence and reprint requests to S. Lenzen, Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany.
Received for publication 6 April 1998 and accepted in revised form 1 July
1998.
ANOVA, analysis of variance; Cat, catalase; DIG, digoxigenin; EC 50, half
maximally effective concentration; ECL, enhanced chemiluminescence;
Gpx, glutathione peroxidase; H/XO, hypoxanthine/xanthine oxidase; H2 O2,
hydrogen peroxide; KRB, Krebs-Ringer bicarbonate; MTT, 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; NBT, nitro-bluetetrazolium; PVDF, polyvinylidene fluoride; SOD, superoxide dismutase.
1578
R
eactive oxygen species participate in the pathogenesis of inflammatory and autoimmune diseases, reperfusion injury, and cancer (1). The
destruction of pancreatic -cells in autoimmune
diabetes is a multifactorial process involving islet antigen
autoantibodies, toxic T-cells, and cytokines as humoral mediators (2–10). It is generally accepted that reactive oxygen
species and nitric oxide participate in the toxic actions that
lead to necrosis or apoptosis of the insulin-producing cells
(11–14). During autoimmune attack, reactive oxygen species
are generated by macrophages and -cells in response to
humoral factors by a mechanism that is presently unknown.
An exceptional susceptibility toward oxidative damage is,
irrespective of the specific pathways of autoimmune destruction, a central characteristic of the pancreatic -cell (15,16).
The low expression level of antioxidant enzymes, in particular glutathione peroxidase (Gpx) and catalase (Cat), which
destroy hydrogen peroxide (H2O2), might provide an explanation for this high degree of sensitivity (15,17–19). Since the
adaptive properties of antioxidant enzyme gene expression
are very limited in insulin-producing cells (19), stable overexpression of antioxidant enzymes is an attractive option
for strengthening cellular resistance against oxidative stress.
In the experiments described in this study, we bioengineered
RINm5F cells with high expression levels of Gpx, Cat, and
Cu/Zn superoxide dismutase (SOD). To test the susceptibility of these cells toward reactive oxygen species, we exposed
them to H2O2, hypoxanthine/xanthine oxidase (H/XO), and
menadione. In addition, we investigated the effect of overexpressed antioxidant enzymes on the toxicity of butylalloxan, a compound that is selectively toxic to -cells (20), and
which, unlike alloxan itself, is also toxic to RINm5F cells. Our
results show that the best protection of insulin-secreting
cells against reactive oxygen species is provided by a combination of antioxidant enzymes rather than any single
enzyme in isolation.
RESEARCH DESIGN AND METHODS
Materials. Restriction enzymes, the SP6/T7 Transcription Kit, bovine catalase, and
the Digoxigenin (DIG) Nucleic Acid Detection Kit were obtained from Boehringer
Mannheim (Mannheim, Germany). Hybond N nylon membranes, the enhanced
chemiluminescence (ECL) detection system, and autoradiography films were
from Amersham (Braunschweig, Germany) and Immobilon-P polyvinylidene fluoride (PVDF) membranes were from Millipore (Bedford, MA). Cu/Zn SOD (from
bovine liver), Gpx (from bovine erythrocytes), hypoxanthine/xanthine oxidase,
DIABETES, VOL. 47, OCTOBER 1998
M. TIEDGE AND ASSOCIATES
menadione, nitro-blue-tetrazolium (NBT), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide (MTT), and peroxidase-labeled antirabbit-IgG antibody were
purchased from Sigma (St. Louis, MO). Menadione was recrystallized from
methanol before use. Guanidine thiocyanate was from Fluka (Neu-Ulm, Germany). All other reagents of analytical grade were from Merck (Darmstadt, Germany). Butylalloxan (N-butylalloxan) was synthesized as described previously (21).
The pcDNA3 expression vector and Zeocin were from Invitrogen (Leek, Netherlands). The cDNAs coding for rat cytoplasmic Cu/Zn SOD and rat Gpx were kindly
provided by Y.-S. Ho (Detroit, MI). Human Cat cDNA was obtained from the American Tissue Culture Collection (Rockville, MD). The pcDNA3Zeo expression vector was kindly provided by M. Marget (Kiel, Germany). Antibodies against rat
Cu/Zn SOD and Gpx were kindly provided by K. Asayama (Yamanashi, Japan). The
antibody against bovine Cat was purchased from Rockland (Gilbertsville, PA). All
tissue culture equipment was from GIBCO Life Technologies (Gaithersburg, MD).
Tissue culture. RINm5F tissue culture cells (passage 55–70) were cultured as
described (19) in RPMI-1640 medium, supplemented with 10 mmol/l glucose, 10%
(vol/vol) fetal calf serum, penicillin, and streptomycin in a humidified atmosphere
at 37°C and 5% CO2. Control and transfected RINm5F cells were seeded at a concentration of 5 104 cells/well in 100 µl culture medium in 96-well microplates
and incubated at 37°C with the test compounds. The cells were exposed to serial concentrations of H2O2 for 2 h in Hepes-supplemented (20 mmol/l) Krebs-Ringer
bicarbonate (KRB) medium with 5 mmol/l glucose and, after removal of H2O2, for
a further 18 h at in RPMI-1640 medium. Cells were incubated with a mixture of
hypoxanthine and xanthine oxidase or with menadione in RPMI-1640 medium for
18 h. Butylalloxan was dissolved in 10 mmol/l HCl immediately before the experiment and added to Hepes-supplemented (20 mmol/l) KRB medium without glucose for 2 h. The buffer was then removed, and the cells were incubated for a further 18 h in RPMI-1640 medium. After the incubation period, the viability of the
cells was determined using a microtiter plate–based MTT assay (22). Viability was
expressed as the percentage of the untreated samples. In experiments investigating
the effects of exogenous addition of antioxidant enzymes, Cu/Zn SOD (60 U/ml)
and/or Cat (6 U/ml) were added to the incubation medium immediately before addition of H2O2 (100 µmol/l), hypoxanthine and xanthine oxidase (0.2 mmol/l and 4
mU/ml, respectively), menadione (20 mmol/l), or butylalloxan (5 mmol/l). Control experiments using boiled preparations of SOD and Cat were conducted in parallel. The effectiveness of protein inactivation by boiling was verified by measurement of enzyme activities.
Overexpression of antioxidant enzymes. The cDNAs of rat cytoplasmic Gpx
(23), human Cat (24), and rat cytoplasmic Cu/Zn SOD (25) were subcloned into
the pcDNA3 or pcDNA3-Zeo expression vector by standard molecular biology techniques (26,27). RINm5F cells were transfected with the vector DNA by the use of
lipofectamine (GIBCO). Positive clones were selected through resistance against
G 418 (250 µg/ml) (GIBCO) and verified by Northern blot analysis, Western blot
analysis, and measurement of enzyme activity. The combined overexpression of
Cat and Gpx and of Cat and Cu/Zn SOD was achieved by a second transfection
with the pcDNA3-Zeo vector and selection through resistance against Zeocin
(100 µg/ml). Of the clones, 15–20 were obtained in each transfection. Clones
with expression levels comparable to those in the liver were selected for the present study. Selenium (10 nmol/l) as an essential cofactor for Gpx was added to the
tissue culture medium of Gpx-transfected cells (28). Selenium did not affect
enzyme expression or protection against reactive oxygen radicals. Transfection
with the pcDNA3 or pcDNA3-Zeo vectors without insert did not affect the expression of the cytoprotective enzymes in control experiments.
Northern blot analyses. RINm5F cells were homogenized in buffered 4 mol/l
guanidine thiocyanate solution. Total RNA was isolated according to Chomczynski and Sacchi (29). The 20 µg total RNA per lane was separated through electrophoresis on denaturing formamide/formaldehyde 1% agarose gels and
hybridized using DIG-labeled antisense cRNA probes coding for rat cytoplasmic
Cu/Zn SOD (25), rat Gpx (23), or human Cat (24). The DIG-labeled hybrids were
detected by an enzyme-linked immunoassay followed by chemiluminescence
detection. The intensity of the bands was quantified through densitometry with
the National Institutes of Health (NIH) Image 1.52 program (Bethesda, MD).
Western blot analyses. RINm5F cells and liver were homogenized in ice-cold
medium (20 mmol/l Hepes, 210 mmol/l mannitol, 70 mmol/l sucrose; pH 7.4). Tissue homogenates were centrifuged at 1,000g and 4°C for 10 min. The supernatant
was used for Western blot analyses. Protein was determined by the BCA assay
(Pierce, Rockford, IL). The 10 µg protein was fractionated by reducing 10% SDS
polyacrylamide gel electrophoresis and was electroblotted to PVDF membranes.
Immunodetection was performed using specific primary antibodies against Gpx
(30) or Cu/Zn SOD (31).The hybrids were visualized through chemiluminescence
using the ECL detection system.
Antioxidant enzyme activities. Cells were homogenized in 50 mmol/l potassium
phosphate buffer (pH 7.8) through sonication on ice for 1 min in 15-s bursts at 90
W with a Braun-Sonic 125 sonifier (Braun, Melsungen, Germany). The homogenates
were then centrifuged at 35,000g and 4°C for 40 min and the supernatant stored at
–20°C until assayed. SOD activities were measured spectrophotometrically by the
DIABETES, VOL. 47, OCTOBER 1998
method of Oberley and Spitz (32) using NBT as the indicator reagent. One unit of
activity was defined as the amount of SOD protein, which gave a half maximal inhibition of NBT reduction. The activities of the RINm5F cell clones were plotted
against a standard curve of purified SOD from bovine liver. The addition of 5 mmol/l
NaCN to the samples specifically inhibited Cu/Zn SOD. Subtraction of SOD activity after NaCN treatment from total SOD activity gave the Mn SOD activity of the
sample. Cat activity was measured by ultraviolet spectroscopy, monitoring the
decomposition of H2O 2 at 240 nm (33). One unit of Cat activity was defined as that
which destroyed 1 µmol of H2O2 per minute at 25°C. Gpx activity was measured
in a photometric assay at 37°C using glutathione reductase and NADPH in a coupled reaction (34). The decrease of NADPH absorbance was monitored at 340 nm.
Activities were calculated against a Gpx standard according to Cornelius et al. (35)
and expressed as units of measurement per milligram protein.
Statistical analyses. Data are expressed as mean values ± SE. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons. EC50 values were calculated from nonlinear
regression analyses using least-square algorithms of the Prism analysis program
(Graphpad, San Diego, CA).
RESULTS
Overexpression of antioxidant enzymes in RINm5F cells.
Successful overexpression of Gpx, Cat, and Cu/Zn SOD at the
mRNA and protein level was verified in Northern and Western
blots, respectively (Figs. 1 and 2). The lengths of the Gpx
mRNA and of the Cu/Zn SOD mRNA were slightly higher from
the transfected gene because of the effective polyadenylation
from the expression vector (Figs. 1 and 2). In single-transfected clones, we achieved enzyme activities of Gpx and Cat
that were higher than those in the liver, while in cells overexpressing Cu/Zn SOD, the expression level was equal to that of
the liver (Table 1). The expression of both enzymes was much
higher than that in islets (Table 1). In RINm5F clones with
combined overexpression of Cat and Gpx or Cat and Cu/Zn
SOD, the expression levels of Cat and SOD were slightly lower
than in the liver but significantly higher than in nontransfected
controls and islets (Table 1). The procedure of stable transfection did not generally affect the pattern of the other antioxidant enzymes, the only exception being a doubling of Mn
SOD activity in cells overexpressing Gpx and a decrease of Mn
SOD activity in cells overexpressing Cu/Zn SOD (Table 1).
Transfection with the various antioxidant enzyme cDNAs did
not affect insulin content, nor did it modify basal and KClinduced insulin secretion (data not shown).
Effects of overexpression of antioxidant enzymes on
the toxicity of H2O2. The EC50 value of H2O2 in nontransfected RINm5F cells was 46 µmol/l (Fig. 3A, Table 2). Overexpression of Cat greatly increased cellular resistance
against H2O2, giving an EC50 value of 592 µmol/l (Fig. 3A,
Table 2). Gpx overexpression resulted in a small increase in
the EC50 value for H2O2 toxicity, which did not reach the level
of significance, while overexpression of SOD did not alter the
resistance of the cells (Fig. 3A, Table 2). The combined overexpression of Cat and Gpx was no more effective than Cat
alone, but a combined overexpression of Cat and Cu/Zn SOD
gave very effective protection, with an EC50 value of 973
µmol/l (Fig. 4A, Table 2).
Effects of overexpression of antioxidant enzymes on
the toxicity of H/XO. Superoxide radicals and H2O2 were
generated through the H/XO system according to Fridovich
(36). In the presence of H/XO, a concentration-dependent
decrease in viability of the control RINm5F cells was
recorded (Fig. 3, Table 2). Overexpression of Cat gave some
protection against the toxic effects of H/XO (Fig. 3, Table 2),
but overexpression of Gpx or Cu/Zn SOD was without effect
(Fig. 3, Table 2). However, the combined overexpression of
1579
ANTIOXIDANT ENZYMES IN BIOENGINEERED RINm5F CELLS
FIG. 1. Stable overexpression of Gpx, Cat, or Cat and Gpx in RINm5F
cells: (1) liver, (2) nontransfected controls, (3) Gpx-transfected cells,
(4) Cat-transfected cells, and (5) Cat/Gpx-transfected cells. A representative blot of three independent experiments is shown.
FIG. 2. Stable overexpression of Cu/Zn SOD or Cat and Cu/Zn SOD in
RINm5F cells: (1) liver, (2) nontransfected controls, (3) Cu/Zn SODtransfected cells, and (4) Cat and SOD-transfected cells. A representative blot of three independent experiments is shown.
Cat and Gpx or of Cat and Cu/Zn SOD provided excellent protection against H/XO toxicity (Fig. 4, Table 2). The viability of
cells with the combined overexpression decreased by only
20% at concentrations of H/XO, which caused complete cell
death in nontransfected controls (Fig. 4). Because of the
extraordinary resistance of these cells, it was not possible to
calculate EC50 values for the clones overexpressing Cat and
Gpx or Cat and Cu/Zn SOD (Fig. 4).
Effects of overexpression of antioxidant enzymes on
the toxicity of menadione. The EC50 value of menadione
in control cells was 14.3 µmol/l (Fig. 3, Table 2). Cells overexpressing Gpx were much more resistant to menadioneinduced toxicity than controls, with an EC50 value of 25.5
µmol/l, although overexpression of Cat gave no protection
(Fig. 3, Table 2). Overexpression of Cu/Zn SOD increased
the toxicity of menadione, with an EC50 value of 10.2 µmol/l
(Fig. 3, Table 2). The combined overexpression of Cat and Gpx
gave no significant protection and was thus less efficient
than Gpx alone (Fig. 4, Table 2), but excellent protection
was recorded in cells overexpressing Cat and Cu/Zn SOD,
with an EC50 value of 30.2 µmol/l (Fig. 4, Table 2).
Effects of overexpression of antioxidant enzymes on
the toxicity of butylalloxan. Alloxan was not toxic to nontransfected RINm5F cells at a concentration of 10 mmol/l (data
not shown). In contrast, the N-substituted derivative of this substance, butylalloxan, induced a concentration-dependent cellular damage to these cells (Fig. 5), with an EC50 value of 4
mmol/l (Table 2). Overexpression of Cat or Gpx alone or in combination had no effect on the toxicity of butylalloxan, but overexpression of Cu/Zn SOD increased the toxicity of this substance (Table 2). Combined overexpression of Cu/Zn SOD with
Cat prevented this increase of butylalloxan toxicity (Table 2).
Effects of exogenous antioxidant enzymes on the toxicity of H2O2, H/XO, menadione, and butylalloxan. The
addition of Cat or a mixture of Cat and Cu/Zn SOD to the incubation medium protected completely against the toxic action
of H2O2 and H/XO, although Cu/Zn SOD alone was without
significant effect (Table 3). Neither Cat nor Cu/Zn SOD, either
alone or in combination, influenced the toxicity of menadione or butylalloxan (Table 3).The activity of the enzymes
was completely destroyed by boiling, and none of the effects
observed with the native enzymes was seen with the denatured material (data not shown).
1580
DISCUSSION
All aerobic cells contain a battery of defenses against reactive
oxygen species. The primary enzymatic defenses are SOD,
which catalyses the conversion of superoxide radicals into
H2O2 plus Cat and Gpx, both of which destroy H2O2. It would
be anticipated that cells containing low levels of one or more
of these enzymes would be particularly vulnerable to oxidative damage. This appears to be the situation in pancreatic cells, which are readily destroyed by oxidants, and in which
there is unusually low expression of antioxidant enzymes, particularly of Cat and Gpx (15,17–19). This study focused on two
aspects of pancreatic -cell damage, namely, the evaluation
of the toxic potential of reactive oxygen species and the elucidation of the relative importance of components of the cellular antioxidant defenses. For this purpose, various antioxidant enzymes, alone and in combination, were overexpressed in RINm5F insulin-producing tissue culture cells
resulting in cell clones with different patterns of antioxidant
enzyme activity. Through overexpression of Gpx, Cat, and/or
Cu/Zn SOD, we generated insulin-producing cells with
DIABETES, VOL. 47, OCTOBER 1998
M. TIEDGE AND ASSOCIATES
TABLE 1
Cu/Zn SOD, Mn SOD, Cat, and Gpx activities in RINm5F cells overexpressing Cu/Zn SOD, Cat, and/or Gpx
Overexpression
Cu/Zn SOD
(U/mg protein)
None
Gpx
Cat
Cat/Gpx
SOD
Cat/SOD
Islets
Liver
70 ± 7 (4)
62 ± 4 (4)
99 ± 1 (4)
78 ± 3 (4)
154 ± 26 (4)†
120 ± 3 (4)*
50 ± 5 (5)
161 ± 12 (6)
Mn SOD
(U/mg protein)
13 ± 2 (4)
26 ± 4 (4)*
11 ± 5 (4)
12 ± 1 (4)
5 ± 1 (4)
17 ± 4 (4)
5 ± 1 (4)
21 ± 3 (6)
Cat
(U/mg protein)
Gpx
(U/mg protein)
6 ± 1 (4)
8 ± 1 (4)
564 ± 18 (4)†
212 ± 2 (4)†
9 ± 1 (4)
326 ± 4 (4)†
4 ± 1 (4)
324 ± 30 (6)
0.17 ± 0.03 (4)
3.90 ± 0.35 (4)†
0.12 ± 0.01 (4)
4.40 ± 0.29 (4)†
0.09 ± 0.01 (4)
0.21 ± 0.02 (4)
0.19 ± 0.01 (6)
0.76 ± 0.06 (6)
Data are means ± SE (numbers of experiments). Enzyme activities were determined in 35,000 g supernatants from sonicated tissue
homogenates by spectrophotometric assays. Data for islets and liver are given for the purpose of comparison and were taken from
the study by Tiedge et al. (19). *P < 0.05, †P < 0.01 compared with control cells overexpressing none of the antioxidant enzymes.
antioxidant enzyme expression levels comparable to those
found in the liver, an organ with particularly high levels of
such enzymes (17–19).
Overexpression of Cat in RINm5F cells gave excellent protection against the toxicity of extracellular H2O2, in accord
with the results of earlier studies of Cat overexpression in
other cells types (37,38). In contrast, Gpx, the other enzyme
that destroys H2O2, showed only a modest protective effect,
and a combination of Cat and Gpx was no better than Cat
alone. This difference may reflect the different kinetic behavior of the two enzymes with a low affinity of Cat and a high
affinity of Gpx for the substrate H2O2. (39). Cat is thus more
effective than Gpx in protecting against high concentrations
of H2O2, while Gpx gives more efficient protection than Cat
when H2O2 concentrations are low (40). Therefore, in the
situation in which H2O2 is presented to cells at high concentration, Cat will be largely responsible for its detoxification.
It has been suggested that the species responsible for the
cytotoxicity of H2O2 is the hydroxyl radical, formed through
the superoxide-driven Haber-Weiss reaction (41). There is
good evidence for this process in cells exposed to an extracellular source of both iron and H2O2, and such cells were protected by addition of either SOD or Cat to the medium (42).
However, RIN5mF cells transfected with SOD were no more
resistant to H2O2 than control cells, suggesting that in this situation the intracellular production of OH• via the superoxidedriven Haber-Weiss reaction plays little part in the mechanism
of H2O2 cytotoxicity. Although cells transfected with SOD
alone were not protected from H 2O2, a combination of SOD
and Cat gave much better protection than Cat alone, reflecting the ability of SOD to maintain Cat in its active form
(43,44). Thus, in the presence of SOD the antioxidant defense
can be maintained and protection is thereby improved.
As expected, the addition of Cat to the medium gave complete protection against the toxicity of H2O2. Cat also gave
complete protection against H/XO, but SOD had no significant
effect. Although H/XO generates both H2O2 and superoxide
(45), the effect of extracellular Cat indicates that the cytotoxic
effects of H/XO that were observed in the present experiments
were solely attributable to H2O2. Similar results have been
obtained in other cell systems (46,47). This is to be expected,
since superoxide does not readily traverse membranes (48),
and because of its rapid spontaneous dismutation (49), most
will decay extracellularly to H2O2. Although H2O2 was the
DIABETES, VOL. 47, OCTOBER 1998
toxic agent both after addition of this substance to the cells
and in the H/XO system, the pattern of delivery in the two situations was quite different. After the addition of H2O2 as a single bolus, high levels of H2O2 would initially be present extracellularly, and the peroxide would rapidly enter the cell
across the concentration gradient. Largely through the action
of CAT, as discussed above, the intracellular level of H2O2
would rapidly decrease. In the case of enzymatically generated peroxide through H/XO, the toxin will be produced over
a prolonged period, albeit at a relatively low concentration.
H2O2 will therefore enter the cell continuously, and the oxidative challenge will continue throughout the period of exposure
to the H/XO system. In the present experiments, Cat was
again a more effective protective agent than Gpx, suggesting
that H2O2 levels were sufficiently high to favor destruction by
Cat rather than Gpx. However, in contrast to the situation
involving the addition of H2O2, the combination of Cat and
Gpx was better than either enzyme alone. This observation
can be explained by an inhibition of Cat during prolonged
exposure to low substrate concentrations of H2O2 (50),
which can be counteracted by overexpression of Gpx. Alternatively, the prolonged exposure to H2O2 could lead to lipid
peroxidation, which is preferentially prevented by Gpx,
thereby improving cell survival (51). A combination of SOD
and Cat also gave very good protection. Again, this could
reflect maintenance of Cat activity, as discussed above, or it
could be due to inhibition of superoxide-mediated propagation reactions of lipid peroxidation (52).
No significant protection was given against menadione toxicity by extracellular SOD or Cat, consistent with an intracellular
site of free radical production from this substance (53,54). The
activation of menadione (Q) involves one-electron reduction by
flavoenzymes to the corresponding semiquinone (reaction 1)
(53,55–57). The latter undergoes autooxidation (reaction 2),
reforming the quinone with concomitant generation of superoxide. By dismutation, either spontaneous or mediated by
SOD, the latter forms H2O2 (reaction 3):
Q + e– → Q• –
Q• – + O2 ↔ Q + O2• –
2O2• – + 2H+ → H2O2 + O2
(1)
(2)
(3)
Transfection of the RIN5mF cells with SOD alone
increased the toxicity of menadione, but double transfection
1581
ANTIOXIDANT ENZYMES IN BIOENGINEERED RINm5F CELLS
A
A
B
B
C
C
FIG. 3. Effects of overexpression of antioxidant enzymes in RINm5F
cells on the toxicity of H 2O 2 (A), H/XO (B), and menadione (C). Nontransfected cells ( ), cells overexpressing Gpx ( ), cells overexpressing Cat ( ), and cells overexpressing Cu/Zn SOD ( ) are shown.
Viability was measured by the MTT assay and expressed as percentage of the untreated cells. Data are means ± SE from four to six individual experiments.
FIG. 4. Effects of combined overexpression of Cat and Gpx or Cat and
SOD in RINm5F cells on the toxicity of H2O2 (A), H/XO (B), and menadione (C). Nontransfected cells ( ), cells overexpressing Cat and
Gpx ( ), and cells overexpressing Cat and SOD ( ) are shown. Viability was measured by the MTT assay and expressed as a percentage
of the untreated cells. Data are means ± SE from four to six individual experiments.
with both SOD and Cat gave good protection. These results
may be explained in terms of the processes described in
reactions 1–3. The reaction of the semiquinone with molecular oxygen (reaction 2) is reversible (58), so that an equilibrium will be established involving the quinone and superoxide radical. This reaction is fast (59), and will therefore compete with the spontaneous dismutation of superoxide, so
that the rate of production of H2O2 will be low. In the presence
of SOD, however, the equilibrium will be driven to the right
(60), decreasing the concentration of semiquinone but
increasing that of H2O2 . In low-Cat control cells, therefore,
SOD will increase the toxicity of menadione, while in the
presence of SOD and Cat, the excess peroxide will be
destroyed and protection given. Transfection of RIN5mF
cells with Cat had no effect on the toxicity of menadione, but
protection was given by Gpx, consistent with earlier findings
1582
DIABETES, VOL. 47, OCTOBER 1998
M. TIEDGE AND ASSOCIATES
TABLE 2
Half-maximal concentrations (EC50) for toxicity in the MTT assay of H2O2, H/XO, menadione, and butylalloxan in RINm5F cells after
overexpression of Cu/Zn SOD, Cat, and/or Gpx
Overexpression
H2O2 (µmol/l)
H/XO (mU/ml)
None
Gpx
Cat
Cat and Gpx
SOD
Cat and SOD
46 ± 1 (6)
71 ± 3 (6)
592 ± 37 (6)†
556 ± 54 (6)†
52 ± 2 (6)
973 ± 42 (6)†
2.5 ± 0.1 (5)
3.2 ± 0.3 (5)
4.4 ± 0.3 (5)†
>6 (5)†
2.3 ± 0.1 (5)
>6 (5)†
Menadione (µmol/l)
14.3 ± 0.4 (5)
25.5 ± 2.3 (7)†
14.8 ± 0.9 (7)
19.9 ± 0.5 (5)
10.2 ± 0.3 (5)
30.2 ± 1.8 (5)†
Butylalloxan (mmol/l)
4.0 ± 0.1 (4)
3.9 ± 0.1 (4)
3.6 ± 0.3 (4)
3.1 ± 0.2 (4)
2.3 ± 0.3 (4)†
3.2 ± 0.2 (4)
Data are means ± SE (numbers of experiments). H/XO (expressed in milliunits per milliliter) is a mixture containing a fixed ratio
of 20 parts xanthine oxidase (mU/ml) and 1 part hypoxanthine (mmol/l). Cells were exposed to serial concentrations of the test compounds as used in the concentration dependencies shown in Figs. 3 and 4. Viability of the cells was determined by the MTT assay.
The EC50 values were calculated by nonlinear regression analyses from the curves in Figs. 3 and 4. *P < 0.05, †P < 0.01 compared
with control cells overexpressing none of the antioxidant enzymes.
in T47D cells overexpressing the latter enzyme (61). Menadione semiquinone, being a lipophilic free radical, may initiate lipid peroxidation. The organic hydroperoxides, so
formed, would be destroyed by Gpx, but not by Cat, so that
the former, but not the latter, would give protection. It should
be noted that the cells transfected with Gpx contained higher
levels of Mn SOD than control cells. This could have contributed to the protection afforded to these cells, since, as discussed above, the higher level of SOD would lead to a lower
concentration of semiquinone.
There is evidence that the diabetogenic agent, alloxan,
exerts its toxic effects on -cells through production of reactive oxygen species (2) involving a chain of redox cycling reactions (21,62–64). Alloxan itself was not toxic to RINm5F cells
at 10 mmol/l, in accord with similar observations in other cell
systems in vitro (65,66). This may be due to the low expression level of GLUT2 glucose transporter in the RINm5F cell
clone used for antioxidant enzyme transfection in the present study (67), which has been shown to be responsible for
the lack of susceptibility of insulin-secreting cells toward
alloxan toxicity (68,69). However, butylalloxan (the more
lipophilic and selectively -cell toxic alloxan derivative
[20,21]), was toxic to RINm5F cells, with an EC50 value of 4
mmol/l. The autooxidation of dialuric acid and its derivatives is strongly inhibited by SOD (21,62). Thus one might
expect protection of SOD as observed recently in Cu/Zn SOD
transgenic mice (70,71). However, in RINm5F cells, no protection was recorded in the present study against butylalloxan
through both overexpression of SOD and the addition of
exogenous SOD; rather, transfection with SOD increased the
toxicity of this substance. Furthermore, no protection was
given by Cat or Gpx, either alone or in combination. Thus, as
considered for alloxan (16,68,69,72), mechanisms other than
free oxygen radical toxicity, such as interactions with specific
proteins in the plasma membrane or the expression of protective heat shock proteins, may also be involved in the toxicity of butylalloxan.
The results of the present study emphasize the cooperative
nature of antioxidant enzymes in providing protection
against various oxidizing species. It is clear from the results
of the present experiments that all three enzymes are
involved in maintaining protection against oxidative stress
according to their specific characteristics, as evident from the
participation of Gpx and Cat in destroying different concentrations of H2O2, the unique ability of Gpx to reduce lipid peroxides, and the prevention of superoxide-driven radical
FIG. 5. Effects of overexpression of antioxidant enzymes in RINm5F cells on the toxicity of butylalloxan. A: Single overexpression: nontransfected
cells ( ), cells overexpressing Gpx ( ), cells overexpressing Cat ( ), and cells overexpressing Cu/Zn SOD ( ) are shown. B: Combined overexpression: nontransfected cells ( ), cells overexpressing Cat and Gpx ( ), and cells overexpressing Cat and SOD ( ) are shown. Viability
was measured by the MTT assay and expressed in percentage of the untreated cells. Data are means ± SE from four individual experiments.
DIABETES, VOL. 47, OCTOBER 1998
1583
ANTIOXIDANT ENZYMES IN BIOENGINEERED RINm5F CELLS
TABLE 3
Effects of Cat, Cu/Zn SOD or Cat, and Cu/Zn SOD added to the incubation medium of nontransfected RINm5F cells on the toxicity
of H2O2, H/XO, menadione, and butylalloxan
Enzyme addition
None
Cat
SOD
Cat/SOD
H2O2 (100 µmol/l) H/XO [(0.2 mmol · 1–1 · (4 mU)–1 )]
22 ± 7 (4)
98 ± 6 (4)†
28 ± 11 (4)
102 ± 5 (4)†
18 ± 6 (4)
103 ± 21 (4)*
47 ± 28 (4)
106 ± 20 (4)*
Menadione (20 µmol/l)
5 ± 5 (5)
13 ± 7 (5)
2 ± 6 (5)
7 ± 4 (5)
Butylalloxan (5 mmol/l)
39 ± 7 (4)
18 ± 3 (4)
31 ± 7 (4)
24 ± 5 (4)
Data are means ± SE (number of experiments). Nontransfected RINm5F cells were exposed to the various test compounds at concentrations given in parentheses in the presence of exogenously added Cu/Zn SOD (60 U/ml) and/or Cat (6 U/ml). Viability of the cells
was determined by the MTT assay and expressed in percentage of the control cells without addition of an antioxidant enzyme to the
incubation medium. *P < 0.05; †P < 0.01 compared with cells without addition of an antioxidant enzyme to the incubation medium.
chain reactions by SOD (73). The complementary behavior of
the enzymes is also shown in the ability of one to protect
against inhibition by the substrates of others preventing an
imbalance in the antioxidant enzyme complement of the
cell. As shown in this study, the overexpression of antioxidant enzymes by molecular biology techniques is a very efficient method of increasing the levels of antioxidant enzymes
in insulin-producing cells to a range that can provide significant protection against the deleterious effects of reactive
oxygen species. Since reactive oxygen species play a central
role in the development of autoimmune diabetes, formed
extracellularly by macrophages and intracellularly following
cytokine action (3,11,12,74,75), these genetically modified
RINm5F cells can contribute to the understanding of the
mechanisms of free radical–mediated toxicity in the development of IDDM. Furthermore, overexpression of antioxidant enzymes may represent an approach to bioengineer
surrogate -cells that are resistant to autoimmune destruction after implantation into a diabetic organism.
ACKNOWLEDGMENTS
This work has been supported by grants from the Juvenile Diabetes Foundation International (JDFI), New York, and the
Waikato Medical Research Foundation, Hamilton, New Zealand.
The authors are grateful to Y.S. Ho (Detroit, MI) for kindly
providing the cDNAs for Gpx and Cu/Zn SOD, to M. Marget
(Kiel, Germany) for the pcDNA3Zeo expression vector, and
to K. Asayama (Yamanashi, Japan) for the antibodies against
rat Gpx and rat Cu/Zn SOD. Some of the results were
obtained during thesis work by S. Lortz.
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