ToxSci Advance Access published April 15, 2003
The Effect of Cigarette Smoke Exposure and Ascorbic Acid Intake on Gene Expression of Antioxidant Enzymes and Other Related
Enzymes in the Livers and Lungs of Osteogenic Disorder Shionogi Rats.
Etsuko Ueta*1,Yuko Tadokoro*, Tomoko Yamamoto$, Chiyuki Yamane $, Emiko Suzuki#, Eiji Nanba!, Yuzuru Otsuka*, and Tadao
Kurata*2.
*
Institute of Environmental Science for Human Life, Ochanomizu University, Tokyo, 112-8610, Japan; $Faculty of Education and
Regional Sciences, Tottori University, Tottori, 680-8551, Japan, #Faculty of Life Science, Ochanomizu University, Tokyo, 112-8610,
Japan; and !Gene Research Center, Tottori University, Yonago, 683-0826, Japan
Address for correspondence to :Dr. Yuzuru Otsuka, Institute of Environmental Science for Human Life, Ochanomizu University,
Tokyo, 112-8610, Japan; Tel: +81-3-5978-5808; Fax: +81-3-5978-5813; E-mail: [email protected].
Running title: Effect of cigarette smoke and ascorbate
Present Address 1 Faculty of Medicine, Tottori University, Yonago, Tottori 683-0826, Japan.
2
Faculty of Applied Life Science, Niigata University of Pharmacy and Applied Life Science, Higashijima, Niitsu, Niigata 956-8603,
Japan.
1
Copyright (c) 2003 Society of Toxicology
Abstract
Cigarette smoking causes many chronic diseases but is a preventable risk factor in developing countries. However, it may be
possible to relieve the smoke induced damage by increasing the protective defense system. As vitamin C intake reduces smoking risk,
it is recommended that smokers should take more vitamin C. However, the molecular mechanism of vitamin C intake on smokers has
not been thoroughly investigated. We have found there to be suppression of smoke induced cytochrome P-450 1A1 (CYP1A1)
mRNA expression by high dose ascorbic acid administration. Therefore, we surveyed other genes the expressions of which were
altered by the administration of high dose ascorbic acid. As cigarette smoking increases oxidative stress, we investigated the effect on
anti-oxidative enzymes expression. The Osteogenic Disorder Shionogi (ODS) rat which lacks ascorbic acid synthesis enzyme, were
administered either minimal amounts (4mg/day, S4) or high dose amounts (40mg/day, S40) of ascorbic acid, and were exposed to
cigarette smoke daily for 25 days. The effect on anti-oxidative enzymes mRNA expression in the liver was measured by competitive
reverse transcription - polymerase chain reaction method (competitive RT-PCR). CuZn-superoxide dismutase (SOD), MnSOD,
catalase and protein disulfide isomerase (PDI) were significantly decreased by high dose ascorbic acid administration, and plasma
glutathione peroxidase was also decreased but not significantly. Cigarette smoke exposure slightly increased gene expression of PDI
and catalase, but not significantly. The differently expressed 27 genes in the liver were found by differential display methods. From 27
genes, altered expression of plasma proteinase inhibitor alpha-1-inhibitor III and CYP1A2 were confirmed by competitive RT-PCR.
These results show that ascorbic acid intake influences gene expression of anti-oxidative enzymes, an ascorbic acid recycle enzyme
and xenobiotic metabolizing enzymes.
Key words - Ascorbic acid, Cigarette smoke, Gene expression, Differential display, Competitive RT-PCR, CYP1A2, Plasma
proteinase inhibitor alpha-1-inhibitor III.
.
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INTRODUCTION
Cigarette smoking and passive exposure to cigarette smoke are a preventable risk factor, and introduce many chronic diseases to
increase morbidity and mortality. However, it may be possible to relieve the smoke induced damage by increasing the protective
defense system. Cigarette smoke contains various chemically reactive molecular species including reactive oxygen species and
radicals (Church and Pryor, 1991). Because of these oxidants and chemicals, the cigarette smoke exposure increased antioxidant
enzymes (Gilks et al., 1998) and drug metabolizing enzymes (Willy et al., 1997). However it seems to be not sufficient to protect the
system with those enzymes only. Anti-oxidative small molecules, such as vitamin C (ascorbic acid) and E, also work as defense
systems. Our previous finding showed cigarette smoke exposure decreased plasma ascorbic acid levels (Kurata et al., 1998), and loss
of ascorbic acid recycling by cigarette smoking was also reported (Maranzana and Mehlhorn, 1998). The lower vitamin C status of
smokers is most likely due to the result of increased oxidative stress (Kallner et al., 1981). Therefore, it is suggested that supplemental
intake of ascorbic acid might be useful means of preventing the oxidative damage induced by cigarette smoke (Lykkesfeldt et al.,
2000). Therefore, vitamin C supplementation may decrease the potential hazard of smoking (Mayer et al., 1999), and smokers’
recommended intake is increased by 35 mg/day in the United States and Canada recently. However, optimal level of intake is not
certain (Cross and Halliwell, 1993), because high dose ascorbic acid administration may act as an oxidant (Podmore et al., 1998,
Rehman et al., 1998). As high dose ascorbic acid intake is controversial (Hemila, 1997), basic biochemical research on ascorbic acid is
needed to evaluate the acceptable amount of ascorbic acid administration for smokers and smoke exposed persons. However, the
molecular mechanism of ascorbic acid intake for smokers has not been sufficiently investigated as yet.
Cigarette smoking increased many enzyme expressions. Gilks et al. (1998) reported increased mRNA of manganese superoxide
dismutase (MnSOD) and glutathione peroxidase (GPx) by cigarette smoke, however, Mukherjee et al (1993) showed increased SOD
but decreased GPx. Hilbert and Mohsenin (1996) have also shown that smokers increased catalase and GPx but decreased SOD and
ascorbic acid supplementation increased catalase activity. On the other hand, the effect of ascorbic acid intake on the enzyme
expression is poorly understood. CYP1A1 and CYP1A2 mRNA were increased by ascorbic acid deficiency (Mori et al., 1997). Clarke
et al. (1996) reported that ascorbic acid – treatment selectively reduced the expression of CYP2E proteins. However, the effect of high
dose ascorbic acid administration had not been investigated before. The inhibition of arylhydrocarbon hydroxylase activity by
3
phenobarbital and the reduction of biphenyl-4-hydroxylase activity by high dose ascorbic acid administration have only been reported
by Khanduja et al. (1990), and by Sutton et al. (1982), but there are no reports at the mRNA level experiment. We have found that
induced CYP1A1 gene expression by cigarette smoke exposure was decreased by high dose ascorbic acid administration (Ueta et al.,
2001). In this study, we developed a competitive reverse transcription – polymerase chain reaction (RT-PCR) method to measure the
amount of mRNA of antioxidative enzymes of SODs, GPxs, ascorbic acid recycling enzymes of glutathione-dependent
dehydroascorbate reductase (DHAR), glutaredoxine (GRX) and protein disulphide isomerase (PDI), an ascorbic acid synthesis
enzyme of L-gulono-gamma-lactone oxidase (GLO), and a drug metabolizing enzyme of cytochrome P4502B1 (CYP2B1). With this
system we evaluated the effect of ascorbic acid intake on mRNA level in cigarette smoke exposed rats. Osteogenic Disorder Shionogi
(ODS) rats that lack an ascorbic acid synthesis enzyme were used. Unlike the human estimated average daily requirement of ascorbic
acid, 75 mg for adult males and 60 mg for females, the ODS rats required 3 mg/day ascorbic acid to prevent scurvy (Horio et al., 1985).
Recently, differential display method has been developed for the analysis of total gene expression patterns (Liang and Pardee, 1992),
therefore, difference of genes expression between high dose ascorbic acid administration and low dose administration in smoke
exposed rat liver was also analyzed by this method.
MATERIALS AND METHODS
Animals. Twenty four male ODS rats weighing about 130 g at 7 or 8 weeks of age were purchased from Nihon Clea Co.,
Tokyo Japan, and were kept in standard conditions (stainless-steel cages, 18 –21 [degree] C, 55 % - 60 % relative humidity, 12 h – 12 h
day night cycle).All experiments were carried out under the guidance of “Standards Relating to the Care and Management, etc. of
Experimental Animals, Notification of Japanese Prime Minister's Office, 1980”. On the first and second days, the rats were fed with
AIN76 purified diet (Table 1, Nihon Clea Co., Tokyo Japan) without ascorbic acid. Then the rats were given 4 mg ascorbic acid
directly into stomach once a day. After 7 days, the rats were divided into four groups (n=6) and were administered either minimal
amount (4 mg/day, S4 and C4) or high amount (40 mg/day, S40 and C40) of ascorbic acid. The S4 group and S40 group were exposed
to cigarette smoke daily, while the C4 group and C40 group were not as described in our previous reports (Kurata et al., 1998). Briefly,
6 rats of S4 or S40 group were placed in a chamber and exposed to side stream cigarette smoke. Smoke was obtained from the
cigarettes, Peace tobacco, produced in Japan (contents per cigarette: nicotine 1.9 mg; tarry substances 21.0 mg; carbon monoxide 40 –
4
50 mg). The four cigarettes (tobacco and cigarette paper) were burned ten min. The rate of airflow through the chamber was about 0.6
m[superscript]3/h. The cigarette smoke exposure was repeated four times a day with 20 min intervals. The temperature during
exposure was about 21 [degree] C in a chamber. At the end of the 25 days experiment, the rats were killed under anesthesia. The livers
and lungs were removed and immediately frozen in liquid nitrogen, and were stored at –80 [degree] C.
Preparation of RNA and internal standard DNA. Total RNA was prepared with the guanidine isothiocyanate method followed
by ultracentrifugation described in a previous report (Ueta et al., 2001). The cDNA was synthesized from DNase I (Takara Biotech,
Tokyo, Japan) treated total RNA with M-MLV Reverse Transcriptase (Gibco BRL Products, Gaithersburg, MD, USA) using Random
Hexamer (Promega, Madison, WI, USA). The internal standard DNAs for competitive RT-PCR were created by PCR mutagenesis
method described by Ho et al. (1989) with slight modification (Kono et al., 2001, Kono et al., 2002). Primer sequence not listed in
previous reports will be available on request.
RT-PCR and Competitive RT-PCR. Competitive RT-PCR with a DNA competitor was performed as described previously
(Ueta et al., 2001, Kono et al., 2001, Kono et al., 2002) modified from Inoue et al. (1998). Briefly, 0.5 [mu]l of cDNA (equivalent to 40
ng of starting total RNA) was added 0.5 units of Taq polymerase (Gene Amp Taq Gold, Perkin-Elmer, Wellesley, MA, USA), 10 pmol
of forward primer labeled with Cy5 (Amersham Pharmacia Biotech, Uppsala, Sweden), 10 pmol of reverse primer and 1 [mu]l of
10xbuffer and 1 [mu]l of diluted competitor plasmid solution. After PCR, the product was electrophoresed on a 6% polyacrylamide
and 6 M urea gel on an ALFred DNA Sequencer system (Amersham Pharmacia Biotech). The peaks were analyzed by Allele Links
software (Amersham Pharmacia Biotech). The amount of target mRNA was expressed as the ratio to [beta]-actin mRNA.
Differential Display/RT-PCR (DD/RT-PCR) and sequencing of DD/RT-PCR fragments. Total RNA was prepared by
guanidine isothiocyanate method as described previously (Ueta et al., 2001). Differential display (Liang and Pardee, 1998) was
performed by Fluorescence Differential Display kit (Takara, Shiga, Japan) according to manufacturer's instruction. Briefly, one [mu]l
of cDNA solution was added 1 [mu]l of 10x LA PCR buffer II (Takara, Shiga, Japan), 0.5 [mu]l of 25 mM MgCl2, 0.325 [mu]l of
2.5mM dNTPs, 0.05 [mu]l of 5U/[mu]l TaKaRa LA Taq (Takara, Shiga, Japan), 0.25 [mu]l of 10 pmol/[mu]l Rhodamine labeled
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downstream primer, and 2.5 [mu]l of 2 pmol/[mu]l upstream primer to make 10 [mu]l. DNA was amplified by heating at 94 [degree]
C for 2 min, 40 [degree] C for 5 min, 72 [degree] C for 5 min, followed by 34 cycle of heating at 94 [degree] C for 30 sec, 40 [degree]
C for 2 min and 72 [degree] C for 1 min, and final heating at 72 [degree] C for 5 min in a thermal cycler. Three [mu]l of product
solution was added with 3 [mu]l of the denaturing solution (95% formamide 20 mM EDTA), and was electrophoresed on a 7M
urea-4% polyacryl amide gel at 40W for 2 hr. Bands were analyzed by a FMBIO II Multi-View image analyzer (Hitach software
engineering, Tokyo Japan). The differently expressed bands (more than 2 times difference between the groups) were removed, and
DNA was re-amplified, and electrophoresed on a 2.5 % Nusieve (BMA-Takara, Shiga, Japan) – 0.5 % agarose S (Wako, Tokyo,
Japan) gel with 1U/ml H.A.-Yellow dye (Hanse Analytik-Takara, Japan). We used 84 selections from the combination of 24 upstream
and 9 downstream primers. The band extracted from the agarose gel was sequenced directly with BigDye Primer Cycle Sequencing
FS Ready Kit (Applied Biosystems Japan, Tokyo Japan) and direct sequencing primer 1 or 2 (Takara, Shiga Japan) using an
ABIPRISM 310 Genetic analyzer (Applied Biosystems Japan, Tokyo Japan). The sequencing data were analyzed by BLAST in the
Entrez Home Page. (http:// www.ncbi.nlm.nih.gov/entrez/query.fcgi )
Statistical analysis. Statistical analysis was performed with the ANOVA method to compare the means between the groups using
STAT View (SAS Inc.). The level of significance for all analysis was P<0.05.
RESULTS
Effect of smoke exposure and dose of ascorbic acid on the antioxidative enzymes expression. Amounts of mRNA of
CuZnSOD, MnSOD and ECSOD against the amount of glyceroaldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in the liver
of four groups of ODS rats were measured by a competitive RT-PCR with DNA competitor (Fig 1). Amount of mRNA in lungs was
also measured. The MnSOD mRNA level was significantly lower in S40 group than S4 group in liver. But smoke exposure did not
show any difference at the same ascorbic acid intake. In lungs, C40 group showed highest value. CuZnSOD mRNA concentration in
liver of S40 and C40 groups showed lower value, but there is no significant difference in lungs. There is no difference in the ECSOD
mRNA content between the groups in livers, but in lungs, the mRNA content in C40 group was about 4 times higher than that in C4
group. The amounts of GPx-1, PhGPx and GPx-P mRNA in livers and in lungs are shown in Fig 2. GPx-1 and GPx-P mRNA content
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in lungs was decreased in S40 group, but not in livers. PhGPx mRNA content in livers was decreased in the C40 and the S40 groups.
The catalase mRNA content was decreased in the C40 and the S40 group in livers but increased in the C40 group in lungs (Fig.3). The
G6PD mRNA content was decreased in lungs of the S40 group. Contents of mRNA of ascorbic acid synthesis enzyme and recycling
enzymes were shown in Fig.3 and 4. The GLO mRNA was not changed between the groups in liver, but decreased in smoke exposed
group in lungs. DHAR mRNA content was decreased in the S40 group in liver and in lungs. PDI mRNA was also decreased in the
S40 group in liver. The amount of CYP2B1 mRNA in the livers of the S40 group was three times higher than that of the C4 group.
Differential Display/RT-PCR analysis on ODS rat liver in high and low dose ascorbic acid administration.
To investigate other genes the expression of which was altered by high dose ascorbic acid administration, a differential display
method was used. Representative examples of DD/RT-PCR reactions run on denaturing gels are shown in Fig. 5(A,B). Finally, these
sequences were analyzed by means of BLAST search to identify known genes with established functions (Table 2). The nucleotide
sequence of the band indicated as 4-11-1 in Fig.5 (B) was identified as part of cytochrome P-450d that is decreased in the samples of
high dose ascorbic acid administration. A total of 23 genes were increased in high dose ascorbic acid administration group to compare
with the low dose ascorbic acid administration group, and 4 genes were decreased in high dose ascorbic acid administration group.
These genes will be referred to as vitamin C responsible genes (vcr). The sequence of vcr4 was almost completely matched to the
sequence of pre-alpha-inhibitor heavy chain 3 (Blom et al., 1997). The sequence of vcr24 was very similar to cytochrome P-450d
(Kawajiri et al., 1984). Four genes were related to signaling and gene regulation, 6 genes were related to inflammation and drug
metabolism, and 4 genes were protease and its inhibitor group. Vcr6 was registered in Gene Bank as MG87, but the function was
unknown. The vcr14 is represented in the expressed sequence tag (EST) database.
Verification and determination of the extent of induction/repression of mRNAs by RT-PCR, corresponding with the partial cDNAs
isolated by DD/RT-PCR.
To confirm the differential expression pattern observed by DD/RT-PCR, the expression of those isolated genes were measured by
RT-PCR (Fig.5C). Fig.5 (C) shows only the genes the expression of which was different in each group. Increased expression of vcr4 in
the case of S40 group was observed. Vcr5 and vcr9 were also increased in S40 group. The expression of vcr24 was increased by
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cigarette smoke exposure at low ascorbic acid intake (S4) and decreased at 40 mg ascorbic acid administration (S40). Other genes
were not changed by those conditions (data were not shown.).
Verification and determination of the extent of induction/repression of mRNAs by competitive RT-PCR, corresponding with the partial
cDNAs isolated by DD/RT-PCR.
To confirm the result obtained above, the expression of vcr4, vcr13 and vcr24 were measured by competitive RT-PCR (Fig.6). The
sequence of vcr4 was almost the same to pre-alpha-inhibitor, heavy chain 3 (Blom et al., 1997). The sequence of vcr13 was almost the
same as rat plasma proteinase inhibitor alpha-1-inhibitor III (Braciak et al., 1988), and vcr24 was the same as cytochrome P-450d
(Kawajiri et al., 1984). The vcr4 was increased by high dose ascorbic acid administration measured by competitive RT-PCR, and the
vcr24 was decreased by high dose ascorbic acid administration. The results of competitive RT-PCR of these three genes were
consistent with the results of DD/RT-PCR.
Changes in transcription factors and glutathion-S-transferase (GST) expression by smoke exposure and high dose ascorbic acid
administration.
To investigate the mechanism of gene regulation of vcr24 by smoke exposure and ascorbic acid intake, redox sensitive transcription
factors of octamer-binding protein (Oct-1) and CCAAT/enhancer binding protein (C/EBP) mRNA contents were measured. As shown
in Fig.7, those genes were suppressed in S40 group. Vcr24 (CYP1A2) is first phase xenobiotic enzyme and suppressed by high dose
ascorbic acid administration, we also investigated second phase enzyme of GST-alpha (Fig.7). The mRNA content of GST alpha was
also increased by smoke exposure at low dose ascorbic acid administered group, and suppressed by high dose ascorbic acid
administration (S40) as same as CYP1A2 mRNA.
DISCUSSION
We fed ODS rats with 4 mg /day ascorbic acid or 40 mg/day. The average body weight of rats was about 200 g at the end of
experiment, therefore this value was 20 mg/day/Kg body weight for the minimal group. This value is very high to compare with
human estimated average requirement of ascorbic acid, 75 mg for adult males and 60 mg for females. However, Horio et al. (1985)
8
showed that 150 mg/Kg diet ascorbic acid concentration was of minimal value to prevent scurvy. Assuming 20 g of diet was
consumed by a rat per day, this value is 3 mg/day for a ODS rat to prevent scurvy. Therefore, 4 mg/day was chosen for minimal level
of ascorbic acid administration with the risk of adequacy, and 40 mg/day was chosen for high dose administration. The tissue
concentrations of ascorbic acid in liver of those rats were 2.26, 3.33, 2.41 and 6.33 mg/100 g tissue for C4, S4, C40 and S40 groups
respectively.
We performed extensive DD/RT-PCR analysis on ODS rat liver exposed to cigarette smoke with different amounts of ascorbic
acid administration as a means to identify genes involved in regulation by high dose ascorbic acid administration. We applied 84
different primer combinations in our DD/RT-PCR analysis that should represent 39% the entire repertoire of mRNAs. We identified 27
genes with modulated expression in high dose ascorbic acid administered ODS rat liver. Among 27 genes, 26 genes were known gene.
Among known genes, vcr6 was a known sequence but an unknown function. One gene was EST of unknown function. Among 27
known genes, the expressions of 10 genes were confirmed by RT-PCR, and pre-alpha-inhibitor, heavy chain 3 (vcr4), plasma
proteinase inhibitor alpha-1-inhibitor III (vcr13) and cytochrome P450d (vcr24) mRNA expressions were further confirmed by
competitive RT-PCR analysis, and the results were consistent with the result of DD/RT-PCR analysis.
To summarize the mRNA expression results; in liver, most of anti-oxidative enzyme expression was suppressed slightly by
high dose ascorbic acid administration. The expression of xenobiotic metabolizing enzymes were upregulated by smoke exposure at
low dose ascorbic acid administration, and this expression was suppressed by high dose ascorbic acid administration. In lung, ECSOD,
GPx-P and DHAR mRNAs were increased by smoke exposure at low dose ascorbic acid administration, and increased expression
were decreased by high dose ascorbic acid administration. Gilks et al. (1998) reported MnSOD mRNA level in lungs was increased in
smokers group than control group by 2 days smoke exposure but decreased to normal level by 14 days. In our experiment, we exposed
rat to cigarette smoke 25 days, therefore, the MnSOD mRNA level was not changed by smoke exposure in low dose group. Comhair
et al. (1999) showed increased extracellular GPx in smokers as same as our result. It is interesting that ECSOD and GPx-P are
extracelluar type. It seems that those enzymes are major defensive enzymes in lungs against cigarette smoke damage.
The effect of ascorbic acid dose on the enzyme expression was investigated mainly on deficiency, but effect of high dose
ascorbic acid administration on the enzyme expression has not been investigated at the mRNA level. In this study, we showed that
MnSOD and catalase mRNA in liver were decreased significantly by high dose ascorbic acid administration. This is the first report of a
9
direct effect of high dose ascorbic acid administration on an antioxidant gene expression. Rohrdanz et al. (2000) showed that reactive
oxygen species producing agent increased catalase but differently influenced on MnSOD. Das et al. (1995) showed reducing agents
increased MnSOD. The reason of difference between the results of those and our data are unknown. CuZnSOD, MnSOD and catalase
expression were decreased by Transforming Growth Factor (TGF)-beta (Kayanoki 1994). The result we obtained here was resembled
to the effect of TGF-Beta. Therefore, we measured the expression of TGF mRNA, but no obvious change was observed. On the other
hand, Oct-1 and C/EBP transcription factor mRNAs were decreased.
The pre-alpha-inhibitor, heavy chain 3 (vcr4), plasma proteinase inhibitor alpha-1-inhibitor III (vcr13) and major acute phase
alpha-1-protein (vcr23) were inflammation related genes. Pre-alpha-inhibitor heavy chain 3 bound to bikunin that is synthesized with
alpha-1-macroglobulin (vcr16) and is cleaved at inflammatory process (Blom et al., 1997). Plasma proteinase inhibitor
alpha-1-inhibitor III was decreased during first 24 hours of acute phase (Braciak et al., 1988). It seems to that high dose ascorbic acid
administration relieve the cigarette smoke induced damage, especially inflammatory damage, and returned those genes expression to
normal. This is the same as the CYP1A1 gene that we have found (Ueta et al., 2001). It is interesting to know the mechanism of gene
regulation by high dose ascorbic acid administration.
Acknowledgements- We thank the Gene Research Center, Tottori University, for the facilities and the services. We also thank to
Professor M. Oshimura, Tottori University, Faculty of Medicine, for his support. This work was supported in part by a Grant-in-Aid for
Scientific Research (Project No.10558006) from the Ministry of Education and Culture of Japan, and also by Smoking Research
Foundation Grant for Biomedical Research.
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Nature 392, 559.
Rehman, A., Collis, C. S., Yang, M., Kelly, M., Diplock, A. T., Halliwell, B., and Rice-Evans, C. (1998). The effect of iron and vitamin
C co-supplementation on oxidative damage to DNA in healthy volunteers. Biochem. Biophys. Res. Comm. 246, 293-298.
Sutton, J. L., Basu, T. K., and Dickerson, J. W. T. (1982). Effect of high dose doses of ascorbic acid on the mixed function oxidase
system in guinea pig liver. Biochem. Pharmacol. 31, 1591 – 1594.
Ueta, E., Suzuki, E., Nanba, E., Tadokoro, Y., Otsuka, Y., and Kurata, T. (2001). Regulation of cigarette smoke-induced cytochrome
P4501A1 gene expression in osteogenic disorder shionogi rat liver and in lung by high dose ascorbic acid dose. Biosci. Biotechnol.
Biochem. 65, 2548-2551.
Willy, J. C., Coy, E. L., Frampton, M. W., Torres, A., Apostolakos, M. J., Hoehn, G., Schuermann, W. H., Thilly, W. G., Olsen, E.,
Hammersley, J. R., Cespi, C. L., and Utell, M. J. (1997). Quantitative RT-PCR measurement of cytochromes p450 1A1, 1B1, and 2B7,
microsomal epoxide hydrolase, and NADPH oxidoreductase expression in lung cells of smokers and nonsmokers. Am. J. Respir. Cell
Mol. Biol. 17,114-124.
13
Figure legends
Fig. 1. The effect of cigarette smoke exposure and ascorbic acid dose on the SOD mRNA level.
The contents of SOD mRNA in livers and in lungs were measured by competitive RT-PCR. Values are means+SD of the ratio to
GAPDH mRNA. C4: The control group was administered 4 mg ascorbic acid per day; C40: The control group was administered 40
mg ascorbic acid per day; S4: The cigarette smoke exposed group was administered 4 mg ascorbic acid per day; S40: The cigarette
smoke exposed group was administered 40 mg ascorbic acid per day. MnSOD: Mn-superoxide dismutase; CuZnSOD:
CuZn-superoxide dismutase; ECSOD: extracellular superoxide dismutase;
*:Significantly different from C4 (P<0.05). #:Significantly different from S4 (P<0.05). (A): The effect of ascorbic acid dose is
significant (P<0.05).
Fig 2. The effect of cigarette smoke exposure and ascorbic acid dose on the GPxs mRNA level.
GPx-P: Plasma glutathione peroxidase; GPx-1: Cellular glutathione peroxidase; PhGPx: Phospholipid hydroperoxide glutathione
peroxidase.
*:Significantly different from C4 (P<0.05). #:Significantly different from S4 (P<0.05). (A): The effect of ascorbic acid dose is
significant (P<0.05).
Fig 3.The effect of cigarette smoke exposure and ascorbic acid dose on the anitoxidative enzymes mRNA level.
G6PD: Glucose-6-phosphate dehydrogenase; GLO: L-gulono-gamma-lactone oxidase.
*:Significantly different from C4 (P<0.05). #:Significantly different from S4 (P<0.05). (A): The effect of ascorbic acid dose is
significant (P<0.05).
Fig 4. The effect of cigarette smoke exposure and ascorbic acid dose on the anitoxidative enzymes mRNA level.
DHAR: Glutathione dependent dehydroascorbate reductase; GRX: Glutaredoxine; PDI: Protein disulfide isomerase; CYP2B1:
Cytochrome P-450 2B1.
14
*:Significantly different from C4 (P<0.05). #:Significantly different from S4 (P<0.05). (A): The effect of ascorbic acid dose is
significant (P<0.05). (S): The effect of cigarette smoke exposure is significant (P<0.05).
Fig. 5. Identification of genes with altered expression in livers between high dose ascorbic acid administered rats and low dose ascorbic
acid administered rats by differential display and verification by RT-PCR.
Rats were fed with high amount of ascorbic acid (40 mg/day) or minimal amount of ascorbic acid (4 mg/day), and exposed to
cigarette smoke daily. After 25 days, total RNA was isolated from liver and differential display was performed. (A) The gel
electrophoresis pattern on 7M Urea-4% polyacrylamide gel with No. 4 down primer and upper primers from 7 to 12. 4:4 mg/day
ascorbic acid administered and cigarette smoke exposed rat liver. 40:40 mg/day ascorbic acid administered and cigarette smoke
exposed rat liver. (B) The band (circled in A) that shows at least two fold difference between high dose ascorbic acid administered rat
and minimal ascorbic acid administered rat was re-amplified by PCR and analyzed by agarose gel electrophoresis with 1U/ml
H.A.-Yellow, 2.5%NuSieve agarose and 0.5 % agarose S gel. A single band was separated by this electrophoresis to two bands (4-11-1
and 4-11-2). (C) The expression of the genes shown in Table 2 was confirmed by RT-PCR. The PCR conditions were: denaturation at
95 [degree] C for 10 min, 16 – 34 cycle of 95 [degree] C for 30 sec with 55 - 61 [degree] C for 30 sec and 72 [degree] C for 30 sec.
Numbers in parenthesis are annealing temperature (left) and cycling time (right). Vcr shows tentatively named gene number. C4: The
control group was administered 4 mg ascorbic acid per day; C40: The control group was administered 40 mg ascorbic acid per day;
S4: The cigarette smoke exposed group was administered 4 mg ascorbic acid per day; S40: The cigarette smoke exposed group was
administered 40 mg ascorbic acid per day.
Fig. 6. Competitive RT-PCR confirmation of altered expressed genes by ascorbic acid dose.
The concentration of mRNA was measured by the competitive RT-PCR method. Values are relative amount of mRNA
concentration against [beta]-actin mRNA. *:Significantly different from C4 (P<0.05). #:Significantly different from S4 (P<0.05). (A):
The effect of ascorbic acid dose is significant (P<0.05). (S): The effect of cigarette smoke exposure is significant (P<0.05).
Fig 7 . The effect of cigarette smoke exposure and ascorbic acid dose on the mRNA level of transcription factors and GST in liver.
15
Oct-1: Octamer binding protein; C/EBP: CCAAT/enhancer binding protein; GST: glutathione-S-transferase alpha.
*:Significantly different from C4 (P<0.05). #:Significantly different from S4 (P<0.05). (A): The effect of ascorbic acid dose is
significant (P<0.05). (S): The effect of cigarette smoke exposure is significant (P<0.05).
ABBREVIATIONS
DD/RT-PCR: differential display with reverstranscription – polymerase chain reaction method; CYP1A1: cytochrome P-450 1A1;
ODS: Osteogenic Disorder Shionogi; MnSOD: Mn-superoxide dismutase; CuZnSOD: CuZn-superoxide dismutase; ECSOD:
extracellular superoxide dismutase; GPx-P: plasma glutathione peroxidase; GPx-1: cellular glutathione peroxidase; PhGPx:
phospholipid hydroperoxide glutathione peroxidase; G6PD: glucose-6-phosphate dehydrogenase; GLO: L-gulono-gamma-lactone
oxidase; DHAR: glutathione dependent dehydroascorbate reductase; GRX: glutaredoxine; PDI: protein disulfide isomerase; TGF:
transforming growth factor; CYP2B1: cytochrome P-450 2B1; Oct-1: octamer binding protein; C/EBP: CCAAT/enhancer binding
protein; GST: glutathione-S-transferase; EST: expressed sequence tag; vcr: vitamin C responsible gene; C4: The control group was
administered 4 mg ascorbic acid per day; C40: The control group was administered 40 mg ascorbic acid per day; S4: The cigarette
smoke exposed group was administered 4 mg ascorbic acid per day; S40: The cigarette smoke exposed group was administered 40 mg
ascorbic acid per day.
16
Table1. Composition of AIN-76 Purified Diet (%)
----------------------------------------------------------------------------------------Casein
20.0
DL-Methionine
0.3
-Cornstarch
65.0
Fiber
5.0
Corn Oil
5.0
AIN-76 Mineral Mixture
3.5
AIN-76 Vitamin Mixture*
1.0
Choline Bitartrate
0.2
---------------------------------------------------------------------------------------* Without ascorbic acid.
17
Table 2. List of altered expression genes by ascorbic acid administration analyzed with DD/RT-PCR. The genes that expression were
changed at least 2 fold by ascorbic acid administration were listed.
Name
Identification
Signaling / gene regulation
vcr1
Mouse phosphatidylinositol 3- kinase p85 beta subunit homologue
vcr7
Rat regulator of G- protein signaling 5 (Rgs5)
vcr9
Human protein tyrosine phosphatase receptor type f homologue
vcr19
Mouse DEAD box RNA helicase homologue
Inflamation / drug metabolism
vcr8
Rat preproalbumin
vcr16
Rat alpha- 1- macroglobulin
vcr22
Mouse major histocompatibility locus class II homologue
vcr23
Rat kininogen
vcr24
Rat cytochrome P- 450d
vcr25
Rat preproalbumin
Protease / hydolase
vcr4
Rat pre- alpha- inhibitor, heavy chain 3
vcr10
Rat 26s proteasome subunit p112
vcr13
Rat plasma proteinase inhibitor alpha- 1- inhibitor III
vcr20
Rat epoxide hydrolase
EST
vcr14
EST:Rat UI- R- C2- ng- d- 11- 0- UI.r1 homologue
Others / unknown
vcr2
Mouse 10 days embryo cDNA, RIKEN library, clone:2610003J06 homologue
vcr3
Rat fetuin- like protein
vcr5
Rat tubulin alpha 4
vcr6
Rat MG87, unknown
vcr11
Mouse 10 days embryo cDNA, RIKEN library, clone:2610511A05 homologue
vcr12
Rat Hepsin
vcr15
Rat mitochondrial genome
vcr17
Rat brain digoxin carrier protein
vcr18
Rat iron- responsive element- binding protein
vcr21
Rat fibronectin 1
vcr26
Rat mitochondrial long- chain enoyl- CoA
vcr27
Mouse RalBP1 associated Eps domein protein homologue
18
40mg Ascorbic
acid group
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Down
Fig. 1
ECSOD Lung
0.015
0.01
0.005
C40 S40
(A)
0.4
0.3
0.2
0.1
0
C4 S4
C40 S40
0.08
0.06
0.04
0.02
C4 S4
C40 S40
MnSOD Lung
0.2
0.08
#
0.06
0.04
0.02
C4 S4
C40 S40
MnSOD/GAPDH
MnSOD/GAPDH
C4 S4
0.1
(A)
0.1
0
#
0.1
0
C40 S40
MnSOD Liver
*
CuZnSOD Lung
CuZnSOD/GAPDH
CuZnSOD/GAPDH
CuZnSOD Liver
0.2
0
0
C4 S4
0.3
(A)
*
0.4
0.02
ECSOD/GAPDH
ECSOD/GAPDH
ECSOD Liver
Yuzuru Otsuka
0.16
0.12
0.08
0.04
0
C4 S4
C40 S40
Fig. 2
GPx-P Liver
GPx-P Lung
10
8
GPx-P/GAPDH
GPx-P/GAPDH
0.04
0.03
0.02
0.01
6
C4 S4
2
0
C40 S40
C4 S4
GPx-1 Liver
GPx-1/GAPDH
GPx-1/GAPDH
0.2
0.15
0.1
0.05
0.08
0.04
C4 S4
0.08
0.04
C40 S40
C4 S4
C40 S40
PhGPx Lung
PhGpx/GAPDH
PhGPｘ/GAPDH
0.12
(A)
#
0.12
(A)
0.16
0
0.16
0
C40 S40
PhGPx Liver
C40 S40
GPx-1 Lung
0.25
C4 S4
#
4
0
0
Yuzuru Otsuka
0.2
0.15
0.1
0.05
0
C4 S4
C40 S40
Yuzuru Otsuka
Fig. 3
Catalase Liver
0.8
0.4
0
Catalase Lung
0.2
Catalase/GAPDH
Catalase/GAPDH
1.2
(A)
C4 S4
0
GLO/GAPDH
0.25
C4 S4
C40 S40
G6PD/GAPDH
0.02
*
0.016
#
0.012
0.008
0.004
0
C4 S4
C4 S4
C40 S40
GLO Liver
0.2
0.004
C40 S40
GLO Lung
0.003
0.15
0.002
0.1
0.001
0.05
0
0.04
GLO/GAPDH
G6PD/GAPDH
4×10-6
0.08
G6PD Lung
G6PD Liver
8×10-6
0.12
0
C40 S40
1.2×10-5
0.16
C4 S4
C40 S40
0
C4 S4
C40 S40
Fig. 4
0.3
DHAR/GAPDH
DHAR/GAPDH
*
1
0.4
0.2
0.1
C4 S4
0.8
0.6
0.2
0
C40 S40
GRX/GAPDH
GRX/GAPDH
C4 S4
C40 S40
GRX Lung
0.03
0.02
#
0.4
GRX Liver
*
0.01
0
(A)
DHAR Lung
DHAR Liver
0
Yuzuru Otsuka
0.08
0.06
0.04
0.02
C4 S4
0
C40 S40
PDI Liver
(A)
C4 S4
C40 S40
PDI Lung
0.6
0.15
#
0.1
PDI/GAPDH
PDI/GAPDH
0.2
0.2
0.05
0
0
0.012
C40 S40
CYP2B1 Liver
*
(A)(S)
*
0.008
0.004
0
C4 S4
C40 S40
C4 S4
C40 S40
CYP2B1 Lung
CYP2B1/GAPDH
C4 S4
CYP2B1/GAPDH
0.4
(A)
*
2
#
1.5
1
0.5
0
C4 S4
C40 S40
Fig. 5
Up primer
No.
Sample
7
8
9
10
Yuzuru Otsuka
11
vcr4
12
(55,20)
4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40
C4 C40 S4 S40
vcr5
(55,28)
(A)
C4 C40 S4 S40
vcr9
(57,32)
C4 C40 S4 S40
vcr13
(55,18)
S4 C40 C4 S40
vcr20
(61,22)
Band ID No.
3-1 3-2 4-1 4-2 6
11
S4 C40 C4 S40
vcr24
(B)
(55,26)
4-11-1
C4 C40 S4 S40
beta-actin
(C)
(55,22)
C4 C40 S4 S40
Fig. 6
(A)
#
0.6
0.4
0.2
0
C40 S40
#*
1.0
vcr13 /beta-actin
0.03
(A)(S)
*
0.02
#
0.01
0
C4 S4
vcr 13 Liver
0.8
0.6
0.4
0.2
0
vcr 24 Liver
vcr24 /beta-actin
vcr4 /beta-actin
vcr 4 Liver
Yuzuru Otsuka
C4 S4
C40 S40
C4 S4
C40 S40
Fig. 7
Yuzuru Otsuka
Oct-1/beta-actin
0.05
0.0004
0.0002
0
0.06
cEBP/beta-actin
(A)
Oct-1 Liver
0.04
#
0.03
0.02
0.01
C4 S4
C/EBP
(S)
0.04
0.02
0
0
C40 S40
C4 S4
C40 S40
C4 S4
C40 S40
GST Liver
1.2×10-9
GST/beta-actin
TGF-beta/beta-actin
TGF-beta Liver
0.0006
(A)(S)
*
0.8×10-9
#
0.4×10-9
0
C4 S4
C40 S40