Antioxidative potential of fluvastatin via the inhibition of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase activity

Article abstract of DOI:10.1248/bpb.26.818

We previously reported that fluvastatin, a potent 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, a strong lipid lowering drug, exerted an anti-atherosclerotic effect at doses insufficient to lower serum lipids in cholesterol fed rabbits. The evidence demonstrated that the superoxide allions from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a critical role in several steps in the development of atherosclerosis. This study was designed to determine the effects of HMG-CoA reductase inhibitors on the production of the superoxide anions of NADPH oxidase in isolated rat peritoneal neutrophils. Fluvastatin (1-10 μM) decreased phorbol 12-myristate 13-acetate (PMA, 10 nM)-dependent reactive oxygen species (ROS) generation in a concentration-dependent manner. It also (10 μM) decreased PMA-dependent O₂ consumption of the rat neutrophils. These effects were reversed by the addition of mevalonate, a metabolite in the HMG-CoA reductase pathway. Treatment with pravastatin did not show any significant changes. Fluvastatin (10 μM) decreased ROS, such as hydroxyl radicals and superoxide anions generated by the Fenton reaction, and by the xanthine-xanthine oxidase system. Rats were treated with either fluvastatin (5 mg/kg per day, p.o.) or pravastatin (5 mg/kg per day, p.o.) for 1 week. Treatment with fluvastatin decreased the PMA-dependent ROS generation. The fluvastatin induced effect on the PMA-dependent ROS generation was reversed by the combined administration with 40 mg/kg mevalonate per day. The antioxidative effect of fluvastatin was thought to have caused not only the scavenging action of the radicals but also to have inhibited ROS generation by inhibiting the NADPH oxidase activity. This antioxidative potential of fluvastatin via the inhibition of NADPH oxidase activity may be profitable in preventing atherosclerosis.

Full text of DOI:10.1248/bpb.26.818

818
Biol. Pharm. Bull. 26(6) 818—822 (2003)
Vol. 26, No. 6
Antioxidative Potential of Fluvastatin via the Inhibition of Nicotinamide
Adenine Dinucleotide Phosphate (NADPH) Oxidase Activity
,
Tsutomu BANDOH, a Eisuke F. SATO,b Hironobu MITANI,c Akinori NAKASHIMA,c
*
b
Katsuji HOSHI,a and Masayasu INOUE
a Department of Clinical Pharmacology, Hokkaido College of Pharmacy; 7–1 Katsuraoka-cho, Otaru, Hokkaido
b
047–0264, Japan: Department of Biochemistry and Molecular Pathology, Osaka City University Medical School; 1–4–3
Asahimachi, Abeno-ku, Osaka 545–8585, Japan: and c Department of Pharmacology, Novartis Tsukuba Research Institute;
8 Ohkubo, Tsukuba 300–2611, Japan. Received November 15, 2002; accepted March 18, 2003
We previously reported that fluvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) re-
ductase inhibitor, a strong lipid lowering drug, exerted an anti-atherosclerotic effect at doses insufficient to lower
serum lipids in cholesterol fed rabbits. The evidence demonstrated that the superoxide anions from nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase plays a critical role in several steps in the development of ath-
erosclerosis. This study was designed to determine the effects of HMG-CoA reductase inhibitors on the produc-
tion of the superoxide anions of NADPH oxidase in isolated rat peritoneal neutrophils. Fluvastatin (1—10 mM) de-
creased phorbol 12-myristate 13-acetate (PMA, 10 nM)-dependent reactive oxygen species (ROS) generation in a
concentration-dependent manner. It also (10 mM) decreased PMA-dependent O2 consumption of the rat neu-
trophils. These effects were reversed by the addition of mevalonate, a metabolite in the HMG-CoA reductase
pathway. Treatment with pravastatin did not show any significant changes. Fluvastatin (10 mM) decreased ROS,
such as hydroxyl radicals and superoxide anions generated by the Fenton reaction, and by the xanthine–xanthine
oxidase system. Rats were treated with either fluvastatin (5 mg/kg per day, p.o.) or pravastatin (5 mg/kg per day,
p.o.) for 1 week. Treatment with fluvastatin decreased the PMA-dependent ROS generation. The fluvastatin in-
duced effect on the PMA-dependent ROS generation was reversed by the combined administration with
40 mg/kg mevalonate per day. The antioxidative effect of fluvastatin was thought to have caused not only the scav-
enging action of the radicals but also to have inhibited ROS generation by inhibiting the NADPH oxidase activity.
This antioxidative potential of fluvastatin via the inhibition of NADPH oxidase activity may be profitable in pre-
venting atherosclerosis.
Key words fluvastatin; nicotinamide adenine dinucleotide phosphate oxidase; antioxidant; 3-hydroxy-3-methylglutaryl coen-
zyme A reductase inhibitor
Oxidative stress plays a critical role in the pathogenesis of fluvastatin on NADPH oxidase activity were also compared
atherosclerosis.1—3) Elevated oxidative stress and superoxide with those of another HMG-CoA reductase inhibitor, pravas-
anion generation could promote the conversion of low den- tatin, which is known as a highly hepatocyte-selective agent
sity lipoprotein (LDL) to atherogenic oxidized LDL (ox- in inhibiting cholesterol synthesis.10)
LDL), contributing to atherosclerosis. There is increasing ev-
idence that the oxidative modification of LDL contributes to MATERIALS AND METHODS
the formation of foam cells in the artery wall.4) Concerning
the modification of LDL, neutrophils rapidly take up ox-LDL
Chemicals PMA was obtained from Sigma (St. Louis,
and generate superoxide anions, which may cause superoxide MO, U.S.A.). L-012 was donated by Wako Pure Chemical
mediated lipid peroxidation in vivo.5) The superoxide anions Co. (Osaka, Japan). The drugs used were fluvastatin, sup-
of human monocytes participate in both the oxidation of plied by Sandoz Research Institute (E. Hanover, NJ, U.S.A),
LDL and its conversion to a cytotoxin.6)
and pravastatin obtained from Sankyo (extracted from
We had reported that fluvastatin, a strong inhibitor of 3-hy- Mevalotin®; Tokyo, Japan). Mevalonate was obtained from
droxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, Tokyo Kasei Kogyo (Tokyo, Japan). Male Wistar rats, 6
the late limiting enzyme of cholesterol biosynthesis, sup- weeks old, were supplied from SLC (Shizuoka, Japan). The
presses atherosclerotic progression, mediated through its in- spin-trapping agent, 5,5-dimethylpyroline-N-oxide (DMPO),
hibitory effect on endothelial dysfunction, lipid peroxidation, was obtained from Sigma (St. Louis, MO, U.S.A.). Ammo-
and macrophage deposition, unrelated to its strong hypocho- nium Iron (II) Sulfate Hexahydrate was obtained from Wako
lesterolemic action.7) Nicotinamide adenine dinucleotide Pure Chemical Co. (Osaka, Japan). Superoxide dismutase
phosphate (NADPH) oxidase is an important and major (SOD) was obtained from Chiron Corporation (CA, U.S.A.).
source of superoxide anions in neutrophils.8)
Preparation of Neutrophils Neutrophils were obtained
Thus, the aim of this study was to determine the direct ef- from 6-week-old male Wistar rats 16 h after an intraperi-
fect of fluvastatin on the NADPH oxidase activity in neu- toneal injection of 2% casein solution at a dosage of 1/10 the
trophils using phorbor 12-myristate 13-acetate (PMA) as a body weight, as described by Morimoto et al.11) After 16 h,
stimulant to clarify the strong anti-atherosclerotic effect migrated neutrophils were collected by an intraperitoneal in-
without serum lipid reduction. PMA activates protein kinase jection of 50 ml calcium-free 0.9% NaCl solution containing
C (PKC) directly, and PKC activity is primarily cytosolic in 10 mM phosphate buffer (pH 7.4), 6 mM KCl, and 1 mM
unstimulated neutrophils, but becomes firmly associated with MgCl2 (Krebs–Ringer phosphate buffer solution, KRP). The
the membrane fraction after PMA treatment.9) The effects of neutrophils were washed 3 times with KRP solution by cen-
* To whom correspondence should be addressed. e-mail: bandoh-t@hokuyakudai.ac.jp
© 2003 Pharmaceutical Society of Japan

June 2003
819
trifugation (1500 rpm, 5 min, 4 °C). The pellet in the bottom
Data and Statistical Analysis The data were expressed
of the tube was collected and then resuspended in KRP as the meanϮS.E. Statistical analyses of the data were per-
(1ϫ108 cells/ml) and kept on ice until used for the experi- formed using one-way analysis of variance, followed by
ments.
Assay for ROS Generation The neutrophils (106 was accepted at pϽ0.05.
cells/ml) were incubated in 0.5 ml of KRP in the presence of
Dunnett multiple comparison test. Statistical significance
400 mM L-012 and fluvastatin or pravastatin of each concen- RESULTS
tration. After incubation for 5 min at 37 °C, the reaction was
started by the addition of 10 nM PMA. During the incubation,
To determine the effect of fluvastatin on the NADPH oxi-
the chemiluminescence (CHL) intensity was recorded contin- dase activity in neutrophils, the effects of fluvastatin on ROS
uously for 20—30 min using a Luminescence Reader BLR- generation and O2 consumption in rat neutrophils were exam-
201 (Aloka, Tokyo, Japan). Cell viability was assessed by ined.
trypan blue exclusion after 30 min incubation with the test
By adding 1 and 10 mM fluvastatin to the neutrophil sus-
compounds, and the compounds did not show cytotoxicity pensions, ROS generation was significantly decreased in a
under these conditions. One hundred mM mevalonate was dis- dose dependent manner, and this effect was reversed by the
solved in ethanol and added to the reaction mixture at a final addition of 100 mM mevalonate. In contrast with fluvastatin,
concentration of 0.1% or less, and mevalonate and ethanol ROS generation was not significantly decreased by treatment
did not show any effect on reactive oxygen species (ROS) with the same concentration of pravastatin, the other HMG-
generation. Values were expressed as a percent change from CoA reductase inhibitor (Fig. 1).
the control mean values.
O2 consumption of rat neutrophils was significantly de-
Hydroxyl Radical Analysis The final concentrations of creased by treatment with 10 mM fluvastatin, and this effect
the mixtures were set at 200 mM H2O2, 100 mM FeSO4 was reversed by the addition of 100 mM mevalonate. On the
(NH4)2SO4·6H2O, 1 mM DMPO, 50 mM potassium phosphate other hand, pravastatin did not show any significant effect on
buffer (pH 7.0), and fluvastatin or pravastatin of each con- O2 consumption up to 10 mM (Fig. 2).
centration. The signal intensities were evaluated as the peak
The direct hydroxyl radical scavenging effect was deter-
height of the second signal of the quartet of DMPO-OH spin mined by ESR studies. In order to evaluate the potency of the
adducts. ESR spectra were measured with a JEOL JES- scavenging effect of fluvastatin on hydroxyl radicals, we
TE200 ESR spectrometer (Tokyo, Japan). Typical ESR set- compared the effect with pravastatin. Ten mM fluvastatin
tings were, field, 3350Ϯ50G; frequency, 9.42 GHz; and mod- showed a significant hydroxyl radical scavenging effect. In
ulation, 100 kHzϫ1G. Values are expressed as a percent contrast, pravastatin did not show any significant effect on
change from the control mean values.
Superoxide and O2 Consumption Analysis Superoxide
the quantity of hydroxyl radicals up to 10 mM (Figs. 3, 4).
The direct superoxide anion scavenging activity of fluvas-
anion was generated by the xanthine–xanthine oxidase sys- tatin was measured by the cytochrome c method. SOD effec-
tem. The concentrations of the mixtures were set at 0.05 mM tively decreased the increasing rate of the absorbance, sug-
xanthine, 50 mM phosphate buffer (pH 7.8), 1 mM cytochrome gesting that the generation of superoxide anion actually oc-
c, 0.01 mM xanthine oxidase, and fluvastatin or pravastatin of curred in the mixture. Ten mM fluvastatin showed significant
each concentration. The direct superoxide anion scavenging scavenging activity of superoxide anions. In contrast, pravas-
activity of fluvastatin or pravastatin was measured by the cy- tatin did not show any significant effect on the quantity of su-
tochrome c method12) at 550 nm using a Shimadzu UV-3100 peroxide anions (Fig. 5).
PC scanning spectro photometer. Values are expressed as a
To confirm whether these in vitro effects actually appear in
percent change from the slope before the addition of each vivo, fluvastatin and pravastatin were administered orally to
drug as a control.
O2 consumption by neutrophils was measured with an oxy-
gen electrode using a Lank Brothers LTD. oxygen monitor
connected to a Pantos unicorder, U-228 recorder. Assays
were conducted at 37 °C with 4ϫ106 cells/ml. The reaction
was started by the injection of 10 nM PMA into the 2 ml
chamber. Values were expressed as a percent change from
the slope of O2 consumption before the addition of each
drug, as a control.
Animal Treatment Male Wistar rats (6 weeks old) were
obtained from SLC (Shizuoka, Japan). The animal room was
maintained at 24Ϯ2 °C, with 55Ϯ10% relative humidity, and
a 12 h light/dark cycle. The rats were given water and com-
mercial laboratory chow (MF; Oriental Yeast Co., Japan) ad
libitum for at least one week before use. Rats were given oral
fluvastatin or pravastatin at a dose of 5 mg/kg once a day in
the evening (17:00—18:00) for one week. Mevalonate was
given orally with fluvastatin at a daily dose of 40 mg/kg. The
control group was given distilled water. Values were ex-
pressed as a percent change from the control mean values.
Fig. 1. Effects of HMG-CoA Reductase Inhibitors on PMA-Dependent
ROS Generation in Isolated Rat Neutrophils
Fluvastatin (FV), pravastatin (PV), and mevalonate (MEV), were added 5 min before
the PMA addition. Values are meanϮS.E. (nϭ6). The basal value of the control group,
after the PMA addition, was 57.7Ϯ9.6 (cpmϫ103). * pϽ0.05, significantly different
from the control group (C, Dunnett multiple comparison test).

820
Vol. 26, No. 6
Fig. 4. Hydroxyl Radical Scavenging Effects of HMG-CoA Reductase In-
hibitors
Fig. 2. Effects of HMG-CoA Reductase Inhibitors on O2 Consumption in
Isolated Rat Neutrophils
The concentration of the DMPO spin adducts of the hydroxyl radicals (DMPO-OH)
formed was recorded after the addition of DMPO. FV; Fluvastatin, PV; Pravastatin, C;
Control. Values are meanϮS.E. (nϭ4). * pϽ0.05, significantly different from the con-
trol group (Dunnett multiple comparison test).
Fluvastatin (FV), pravastatin (PV), and mevalonate (MEV), were added 3.5—4 min
after the PMA addition. Values are meanϮS.E. (nϭ6). Values are expressed as a per-
cent change from the basal slope of O2 consumption before the addition of each drug,
as a control. The basal slope before the addition of each drug was Ϫ4.30Ϯ0.15
(mm/min). * pϽ0.05, significantly different from the control group (C, Dunnett multi-
ple comparison test).
Fig. 5. Superoxide Anion Scavenging Effects of HMG-CoA Reductase In-
hibitors
Superoxide anions were generated by the xanthine–xanthine oxidase system. The di-
rect superoxide radical scavenging activity of HMG-CoA reductase inhibitors was mea-
sured by the cytochrome c method. FV; Fluvastatin, PV; Pravastatin, C; Control, SOD;
33 mg/ml. Values are meanϮS.E. (nϭ4). Values are expressed as a percent change from
the slope before the addition of each drug, as a control. The basal slope of the control
group was 0.027Ϯ0.002 (Dabsorbance at 550 nm/min). * pϽ0.05, significantly different
from the control group (Dunnett multiple comparison test).
Fig. 3. Typical Electron Spin Resornance (ESR) Spectra of DMPO-OH
Generated by the Fenton Reaction
The ESR spectra after the reaction of 1 mM DMPO with 200 mM H2O2 and 100 mM
FeSO4(NH4)2SO4·6H2O in the absence (control), and in the presence of 10 mM fluvas-
tatin (FV) or 10 mM pravastatin (PV).
the rats once a day for 1 week. Treatment with fluvastatin
(5 mg/kg per day, p.o.) for 1 week decreased the PMA-depen-
dent ROS generation. The fluvastatin induced effect on the
PMA-dependent ROS generation was reversed by the com-
bined administration with 40 mg/kg mevalonate per day.
Pravastatin did not show any significant effect (Fig. 6).
DISCUSSION
In the present study, fluvastatin significantly decreased the
PMA-dependent ROS generation. It was thought that this de-
creasing effect of ROS generation with fluvastatin was, at
least in part, attributed to a direct inhibitory action to neu-
trophil NADPH oxidase, because the O2 consumption of neu-
trophils was significantly decreased by treatment with fluvas-
tatin. This inhibitory effect of fluvastatin on ROS generation
and O2 consumption was reversed by the addition of meval-
onate, a metabolite of the HMG-CoA reductase pathway.
This phenomenon indicated that fluvastatin inhibits ROS
Fig. 6. In Vivo Effects of HMG-CoA Reductase Inhibitors on PMA-De-
pendent ROS Generation
Fluvastatin (FV, 5 mg/kg, p.o.) and pravastatin (PV, 5 mg/kg, p.o.) were administered
once a day for 1 week. Mevalonate (MEV) was given orally with fluvastatin for 1 week
at a daily dose of 40 mg/kg. C; Control. Values are meanϮS.E. (nϭ6). The basal value
of the control group was 52.1Ϯ7.1 (cpmϫ103). * pϽ0.05, significantly different from
generation via the inhibition of the cholesterol synthesis the control group (Dunnett multiple comparison test).

June 2003
821
pathway. The neutrophil oxidase consists of 5 major sub- terol synthesis, and it is possible that pravastatin cannot sup-
units: a plasma membrane spanning cytochrome b558 com- press the isoprenylation of the rac2 protein of the neu-
posed of a large subunit gp91phox and a smaller subunit trophils. In addition, different characteristics between pravas-
p22phox, and 3 cytosolic components p40phox, p47phox and tatin and fluvastatin are also found, with respect to a lack of
p67phox.13—15) The low molecular weight G protein rac2 par- the inhibitory effect on the thickening of the intima after bal-
ticipates in the assembly of the active complex and NADPH loon catheterization.23) Thus, although the dose factor cannot
oxidase is activated by isoprenylation of the rac2 protein, be excluded, pravastatin might be less permeable to neu-
which results in a production of superoxide anions.16) HMG- trophils than fluvastatin, as shown by the liver specificity of
CoA reductase generates mevalonate from HMG-CoA, and the former drug action based on its hepatocytes specific car-
mevalonate is further metabolized into a series of iso- rier mediated system and greater hydrophilicity.10) In this in
prenoids. Accordingly, fluvastatin may suppress the isopreny- vivo model, it is considered that fluvastatin did not alter
lation of the rac2 protein, and may finally suppress the pro- serum lipid concentration. Tsujita et al. reported that pravas-
duction of superoxide. The fact that the inhibitory effect of tatin did not reduce serum lipids in rats and mice.24) Further-
fluvastatin on superoxide generation was reversed by the ad- more, Kimura et al. reported that fluvastatin also did not re-
dition of mevalonate is also further evidence to the possibil- duce serum lipids at a dose of 6 mg/kg in rats.25)
ity. Inoue et al. reported that the mRNA levels of p22phox,
Recently, it was demonstrated that the oxidative stress by
and the protein levels of p47phox of NADPH oxidase, were NADPH oxidase in endothelial cells (EC) also plays a role in
decreased by treatment with fluvastatin, and that this effect the early steps of atherosclerosis.26) Meyer et al. confirmed
was reversed by the addition of mevalonate.17) To understand protein expression of NADPH oxidase subunits, such as gp
the detailed mechanism by which fluvastatin inhibits neu- 91phox, p22phox, p47phox, and p67phox in EC, and that
trophil superoxide anion production, additional studies such apocynin, a specific leukocyte NADPH oxidase inhibitor, in-
as an analysis of the translocation of cytosolic subunits hibits the translocation of p47phox to the membrane of stim-
(p40phox, p47phox and p67phox) to the membrane, and their ulated EC. These findings also support the presence of a
assembly with the gp91phox and p22phox subunits, is re- functionally active leukocyte type NADPH oxidase in EC.27)
quired.
From these facts, it is likely that fluvastatin inhibits the pro-
Fluvastatin reduced active oxygen species such as hy- gression of atherosclerosis via the inhibition of NADPH oxi-
droxyl radicals and superoxide anions, generated by the Fen- dase in not only neutrophils but also in EC. Rueckschloss et
ton reaction and by the xanthine–xanthine oxidase system, al. reported that oxidized LDL induces proatherosclerotic
respectively. There are several reports that fluvastatin sup- NADPH oxidase expression and superoxide anion formation
presses lipid peroxidation by scavenging active oxygen in human endothelial cells, and an antioxidative potential of
species such as hydroxyl radicals and the superoxide anions HMG-CoA reductase inhibition via the reduction of vascular
of rat liver microsomes in vivo and in vitro.18,19) Pravastatin NADPH oxidase expression.28) Furthermore, Inoue et al. re-
did not show a chemical scavenging action, such as hydroxyl ported that after the 16-week lipid-lowering therapy, the anti-
radicals and superoxide anions. It is assumed that these phe- ox-LDL titer significantly decreased in the fluvastatin group
nomena originate in its chemical structure.
but did not change in the pravastatin group.29)
Thus, it was confirmed that fluvastatin decreased PMA-de-
Several kinds of molecules such as hydrogen peroxide, hy-
pendent ROS generation in vitro, and we further examined droxyl radical, and singlet oxygen, are known as reactive
the in vivo effects of fluvastatin on PMA-dependent ROS oxygen species which neutrophils produce, beside superox-
generation to confirm whether the observed in vitro effects ide anions. At first, superoxide anions are produced, and then
actually occur in vivo. Treatment with fluvastatin (5 mg/kg other reactive oxygen species are produced from superoxide
per day, p.o.) for 1 week also decreased PMA-dependent anions by the chemical reaction. Accordingly, NADPH oxi-
ROS generation in vivo, and this suppressive effect of fluvas- dase takes part directly only in the production of superoxide
tatin on ROS generation was also reversed by the combined anions.30) Therefore, it was thought that the direct NADPH
administration with mevalonate. Pravastatin with the same oxidase inhibition of fluvastatin is important.
concentration and dose as fluvastatin did not show any signif-
NADPH oxidase is present in neutrophils and EC, and it
icant effect on ROS generation in vitro or in vivo. The most seems to be a source of superoxide anion production in ani-
likely reason for the difference between these HMG-CoA re- mal models of vascular disease, and in human atherosclero-
ductase inhibitors is that greater doses of pravastatin are re- sis.31) Therefore, these effects of fluvastatin may be beneficial
quired to produce these effects in this species of animal and in preventing vascular complications in hyperlipidemia, in
cells. However, pravastatin showed a similar range of lipid addition to its strong hypocholesterolemic action.
lowering effects in humans at 0.3—0.7 mg/kg20): 20—
40 mg/d for pravastatin versus 20—60 mg/d for fluvastatin.21)
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