Vitamin C Dosage For Smokers

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 Shionogi Rats with Osteogenic Disorders

Received:

23 November 2002

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 antioxidative enzyme expression. The osteogenic disorder Shionogi (ODS) rat, which lacks ascorbic acid synthesis enzyme, was administered either minimal amounts (4 mg/day, S4) or high-dose amounts (40 mg/day, S40) of ascorbic acid, and were exposed to cigarette smoke daily for 25 days. The effect on antioxidative 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 antioxidative enzymes, an ascorbic acid recycle enzyme, and xenobiotic metabolizing enzymes.

Cigarette smoking and passive exposure to cigarette smoke are preventable risk factors 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 not to be sufficient to protect the system with those enzymes only. Antioxidative small molecules, such as vitamins C (ascorbic acid) and E, also work as defense systems. Our previous finding showed that 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 a 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 ( Mays et al., 1999), and smokers' recommended intake has increased by 35 mg/day in the U.S. 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 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 disulfide 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, a differential display method has been developed for the analysis of total gene expression patterns (Liang and Pardee, 1992); therefore, differences of gene expression between high-dose 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, and were kept in standard conditions (stainless-steel cages, 18–21°C, 55–60% relative humidity, 12-h–12-h light/dark 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 the stomach once a day. After 7 days, the rats were divided into four groups (n = 6) and were administered either minimal amounts (4 mg/day, S4 and C4) or high amounts (40 mg/day, S40 and C40) of ascorbic acid. The S4 group and S40 group were exposed to cigarette smoke daily, while the C4 and C40 groups were not, as described in our previous reports ( Kurata et al., 1998). Briefly, 6 rats of the S4 or S40 group were placed in a chamber and exposed to side-stream cigarette smoke. Smoke was obtained from the cigarettes, Peace, produced in Japan (contents per cigarette: nicotine 1.9 mg; tarry substances 21.0 mg; carbon monoxide 40–50 mg). The four cigarettes (tobacco and cigarette paper) were burned for 10 min. The rate of airflow through the chamber was about 0.6 m³/h. The cigarette smoke exposure was repeated four times a day with 20-min intervals. The temperature during exposure was about 21°C in a chamber. At the end of the 25-day experiment, the rats were killed under anesthesia. The livers and lungs were removed and immediately frozen in liquid nitrogen and stored at −80°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-treated (Takara Biotech, Tokyo, Japan) total RNA with M-MLV reverse transcriptase (Gibco BRL Products, Gaithersburg, MD) using Random Hexamer (Promega, Madison, WI). The internal standard DNAs for competitive RT-PCR were created by the PCR mutagenesis method described by Ho et al. (1989) with slight modification ( Kono et al., 2001, 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 ( Kono et al., 2001, 2002; Ueta et al., 2001) modified from Inoue et al. (1998). Briefly, to 0.5 μl 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), 10 pmol of forward primer labeled with Cy5 (Amersham Pharmacia Biotech, Uppsala, Sweden), 10 pmol of reverse primer, 1 μl of 10× buffer, and 1 μl 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 β-actin mRNA.

Differential display/RT-PCR (DD/RT-PCR) and sequencing of DD/RT-PCR fragments.

Total RNA was prepared by the guanidine isothiocyanate method as described previously ( Ueta et al., 2001). Differential display (Liang and Pardee, 1998) was performed by the Fluorescence Differential Display kit (Takara, Shiga, Japan) according to manufacturer's instruction. Briefly, one μl of cDNA solution was added 1 μl of 10× LA PCR buffer II (Takara, Shiga, Japan), 0.5 μl of 25 mM MgCl₂, 0.325 μl of 2.5 mM dNTPs, 0.05 μl of 5U/μl TaKaRa LA Taq (Takara, Shiga, Japan), 0.25 μl of 10 pmol/μl rhodamine-labeled downstream primer, and 2.5 μl of 2 pmol/μl upstream primer to make 10 μl. DNA was amplified by heating at 94°C for 2 min, 40°C for 5 min, 72°C for 5 min, followed by 34 cycles of heating at 94°C for 30 s, 40°C for 2 min, and 72°C for 1 min, and final heating at 72°C for 5 min in a thermal cycler. Three μl of product solution was added with 3 μl of the denaturing solution (95% formamide-20 mM EDTA), and was electrophoresed on a 7 M urea-4% polyacrylamide gel at 40 W for 2 h. Bands were analyzed by a FMBIO II Multi-View image analyzer (Hitach software engineering, Tokyo). 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) gel with 1 U/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). 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 Antioxidative Enzyme 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 ). The amount of mRNA in lungs was also measured. The MnSOD mRNA level was significantly lower in the S40 group than the S4 group in liver, but smoke exposure did not show any difference at the same ascorbic acid intake. In lungs, the C40 group showed highest value. CuZnSOD mRNA concentration in liver of S40 and C40 groups showed lower value, but there was 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 the C4 group. The amounts of GPx-1, PhGPx, and GPx-P mRNA in livers and in lungs are shown in Figure 2 . GPx-1 and GPx-P mRNA content in lungs was decreased in the S40 group, but not in livers. PhGPx mRNA content in livers was decreased in the C40 and S40 groups. The catalase mRNA content was decreased in the C40 and S40 groups 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 in mRNA of ascorbic acid synthesis enzyme and recycling enzymes were shown in Figures 3 and 4 . The GLO mRNA was not changed between the groups in liver, but decreased in the 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, we used a differential display method. Representative examples of DD/RT-PCR reactions run on denaturing gels are shown in Figures 5A and 5B . 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 Figure 5B was identified as part of the cytochrome P-450d that is decreased in the samples of high-dose ascorbic acid administration. A total of 23 genes were increased in the high-dose group compared with the low-dose ascorbic acid administration group, and four genes were decreased in the high-dose 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 and six genes to inflammation and drug metabolism; four genes were protease and its inhibitor group. Vcr6 was registered in GenBank 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 was measured by RT-PCR (Fig. 5C ). Figure 5C shows only the genes, the expression of which was different in each group. Increased expression of vcr4 in the case of the S40 group was observed. Vcr5 and vcr9 were also increased in the S40 group. The expression of vcr24 was increased by 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 expressions of vcr4, vcr13, and vcr24 were measured by competitive RT-PCR (Fig. 6 ). The sequence of vcr4 was almost the same as 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 Glutathione S-Transferase (GST) Expression from 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 Figure 7 , those genes were suppressed in the S40 group. Vcr24 (CYP1A2) is a first-phase xenobiotic enzyme and suppressed by high-dose ascorbic acid administration; we also investigated a 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), the same as CYP1A2 mRNA.

DISCUSSION

We fed ODS rats with 4 mg /day or 40 mg/day ascorbic acid. The average body weight of rats was about 200 g at the end of the experiment; therefore this value was 20-mg/day/Kg body weight for the minimal group. This value is very high compared with the human estimated average requirement of ascorbic acid: 75 mg for adult males and 60 mg for females. However, Horio et al. (1985) 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 an ODS rat to prevent scurvy. Therefore, 4 mg/day was chosen for the 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 livers 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% of 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. Among known genes, vcr6 was a known sequence but an unknown function. One gene was an 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); 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 mRNA expression results in liver, most of the antioxidative enzyme expression was suppressed slightly by high-dose ascorbic acid administration. The expression of xenobiotic metabolizing enzymes was upregulated by smoke exposure at low-dose ascorbic acid administration, and this expression was suppressed by high-dose ascorbic acid administration. In the lung, ECSOD, GPx-P, and DHAR mRNAs were increased by smoke exposure at low-dose ascorbic acid administration, and increased expression was decreased by high-dose ascorbic acid administration. Gilks et al. (1998) reported MnSOD mRNA level in lungs was increased more in the smokers group than in the control group by 2 days smoke exposure, but it decreased to normal level by 14 days. In our experiment, we exposed rats to cigarette smoke for 25 days; therefore, the MnSOD mRNA level was not changed by smoke exposure in the low-dose group. Comhair et al. (1999) showed increased extracellular GPx in smokers, the same as our result. It is interesting that ECSOD and GPx-P are extracelluar types. It seems that those enzymes are major defensive enzymes in lungs against cigarette smoke damage.

The effect of ascorbic acid on enzyme expression was investigated mainly on deficiency, but the effect of high-dose ascorbic acid administration on 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 direct effect of high-dose ascorbic acid administration on an antioxidant gene expression. Rohrdanz et al. (2000) showed that the reactive oxygen species-producing agent increased catalase but differently influenced MnSOD. Das et al. (1995) showed reducing agents increased MnSOD. The reason for the difference between the results of those and our data are unknown. CuZnSOD, MnSOD, and catalase expression were decreased by transforming growth factor TGF-β (Kayanoki 1994). The result we obtained here resembled the effect of TGF-β. 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 the inflammatory process ( Blom et al., 1997). Plasma proteinase inhibitor alpha-1-inhibitor III was decreased during the first 24 h of acute phase ( Braciak et al., 1988). It seems that high-dose ascorbic acid administration relieved the cigarette smoke-induced damage, especially inflammatory damage, and returned those gene expressions 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.

TABLE 1

Composition of AIN-76 Purified Diet (%)

Material	Percentage of diet
*Without ascorbic acid.
Casein	20.0
DL-methionine	0.3
α-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

Material	Percentage of diet
*Without ascorbic acid.
Casein	20.0
DL-methionine	0.3
α-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

TABLE 1

Composition of AIN-76 Purified Diet (%)

Material	Percentage of diet
*Without ascorbic acid.
Casein	20.0
DL-methionine	0.3
α-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

Material	Percentage of diet
*Without ascorbic acid.
Casein	20.0
DL-methionine	0.3
α-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

TABLE 2

List of Genes for Which Expression Was Altered by Ascorbic Acid Administration—Analyzed with DD/RT-PCR

Name	Identification	40-mg ascorbic acid group
Note. The genes listed are those whose expression was changed at least twofold by ascorbic acid administration.
Signaling/gene regulation
vcr1	Mouse phosphatidylinositol 3-kinase p85 β subunit homologue	Up
vcr7	Rat regulator of G-protein signaling 5 (Rgs5)	Up
vcr9	Human protein tyrosine phosphatase receptor type f homologue	Up
vcr19	Mouse DEAD box RNA helicase homologue	Up
Inflammation/drug metabolism
vcr8	Rat pre-pro-albumin	Up
vcr16	Rat alpha-1-macroglobulin	Up
vcr22	Mouse major histocompatibility locus class II homologue	Up
vcr23	Rat kininogen	Up
vcr24	Rat cytochrome P-450d	Down
vcr25	Rat preproalbumin	Up
Protease/hydolase
vcr4	Rat pre-alpha-inhibitor, heavy chain 3	Up
vcr10	Rat 26s proteasome subunit p112	Up
vcr13	Rat plasma proteinase inhibitor alpha-1-inhibitor III	Up
vcr20	Rat epoxide hydrolase	Up
EST
vcr14	EST:Rat UI-R-C2-ng-d-11-0-UI.r1 homologue	Down
Others/unknown
vcr2	Mouse 10-day embryo cDNA, RIKEN library,clone: 2610003J06 homologue	Up
vcr3	Rat fetuin-like protein	Up
vcr5	Rat tubulin alpha 4	Up
vcr6	Rat MG87, unknown	Up
vcr11	Mouse 10-day embryo cDNA, RIKEN library, clone: 2610511A05 homologue	Up
vcr12	Rat hepsin	Up
vcr15	Rat mitochondrial genome	Up
vcr17	Rat brain digoxin carrier protein	Up
vcr18	Rat iron-responsive element-binding protein	Down
vcr21	Rat fibronectin 1	Up
vcr26	Rat mitochondrial long-chain enoyl-CoA	Up
vcr27	Mouse RalBP1 associated Eps domain protein homologue	Down

Name	Identification	40-mg ascorbic acid group
Note. The genes listed are those whose expression was changed at least twofold by ascorbic acid administration.
Signaling/gene regulation
vcr1	Mouse phosphatidylinositol 3-kinase p85 β subunit homologue	Up
vcr7	Rat regulator of G-protein signaling 5 (Rgs5)	Up
vcr9	Human protein tyrosine phosphatase receptor type f homologue	Up
vcr19	Mouse DEAD box RNA helicase homologue	Up
Inflammation/drug metabolism
vcr8	Rat pre-pro-albumin	Up
vcr16	Rat alpha-1-macroglobulin	Up
vcr22	Mouse major histocompatibility locus class II homologue	Up
vcr23	Rat kininogen	Up
vcr24	Rat cytochrome P-450d	Down
vcr25	Rat preproalbumin	Up
Protease/hydolase
vcr4	Rat pre-alpha-inhibitor, heavy chain 3	Up
vcr10	Rat 26s proteasome subunit p112	Up
vcr13	Rat plasma proteinase inhibitor alpha-1-inhibitor III	Up
vcr20	Rat epoxide hydrolase	Up
EST
vcr14	EST:Rat UI-R-C2-ng-d-11-0-UI.r1 homologue	Down
Others/unknown
vcr2	Mouse 10-day embryo cDNA, RIKEN library,clone: 2610003J06 homologue	Up
vcr3	Rat fetuin-like protein	Up
vcr5	Rat tubulin alpha 4	Up
vcr6	Rat MG87, unknown	Up
vcr11	Mouse 10-day embryo cDNA, RIKEN library, clone: 2610511A05 homologue	Up
vcr12	Rat hepsin	Up
vcr15	Rat mitochondrial genome	Up
vcr17	Rat brain digoxin carrier protein	Up
vcr18	Rat iron-responsive element-binding protein	Down
vcr21	Rat fibronectin 1	Up
vcr26	Rat mitochondrial long-chain enoyl-CoA	Up
vcr27	Mouse RalBP1 associated Eps domain protein homologue	Down

TABLE 2

List of Genes for Which Expression Was Altered by Ascorbic Acid Administration—Analyzed with DD/RT-PCR

Name	Identification	40-mg ascorbic acid group
Note. The genes listed are those whose expression was changed at least twofold by ascorbic acid administration.
Signaling/gene regulation
vcr1	Mouse phosphatidylinositol 3-kinase p85 β subunit homologue	Up
vcr7	Rat regulator of G-protein signaling 5 (Rgs5)	Up
vcr9	Human protein tyrosine phosphatase receptor type f homologue	Up
vcr19	Mouse DEAD box RNA helicase homologue	Up
Inflammation/drug metabolism
vcr8	Rat pre-pro-albumin	Up
vcr16	Rat alpha-1-macroglobulin	Up
vcr22	Mouse major histocompatibility locus class II homologue	Up
vcr23	Rat kininogen	Up
vcr24	Rat cytochrome P-450d	Down
vcr25	Rat preproalbumin	Up
Protease/hydolase
vcr4	Rat pre-alpha-inhibitor, heavy chain 3	Up
vcr10	Rat 26s proteasome subunit p112	Up
vcr13	Rat plasma proteinase inhibitor alpha-1-inhibitor III	Up
vcr20	Rat epoxide hydrolase	Up
EST
vcr14	EST:Rat UI-R-C2-ng-d-11-0-UI.r1 homologue	Down
Others/unknown
vcr2	Mouse 10-day embryo cDNA, RIKEN library,clone: 2610003J06 homologue	Up
vcr3	Rat fetuin-like protein	Up
vcr5	Rat tubulin alpha 4	Up
vcr6	Rat MG87, unknown	Up
vcr11	Mouse 10-day embryo cDNA, RIKEN library, clone: 2610511A05 homologue	Up
vcr12	Rat hepsin	Up
vcr15	Rat mitochondrial genome	Up
vcr17	Rat brain digoxin carrier protein	Up
vcr18	Rat iron-responsive element-binding protein	Down
vcr21	Rat fibronectin 1	Up
vcr26	Rat mitochondrial long-chain enoyl-CoA	Up
vcr27	Mouse RalBP1 associated Eps domain protein homologue	Down

Name	Identification	40-mg ascorbic acid group
Note. The genes listed are those whose expression was changed at least twofold by ascorbic acid administration.
Signaling/gene regulation
vcr1	Mouse phosphatidylinositol 3-kinase p85 β subunit homologue	Up
vcr7	Rat regulator of G-protein signaling 5 (Rgs5)	Up
vcr9	Human protein tyrosine phosphatase receptor type f homologue	Up
vcr19	Mouse DEAD box RNA helicase homologue	Up
Inflammation/drug metabolism
vcr8	Rat pre-pro-albumin	Up
vcr16	Rat alpha-1-macroglobulin	Up
vcr22	Mouse major histocompatibility locus class II homologue	Up
vcr23	Rat kininogen	Up
vcr24	Rat cytochrome P-450d	Down
vcr25	Rat preproalbumin	Up
Protease/hydolase
vcr4	Rat pre-alpha-inhibitor, heavy chain 3	Up
vcr10	Rat 26s proteasome subunit p112	Up
vcr13	Rat plasma proteinase inhibitor alpha-1-inhibitor III	Up
vcr20	Rat epoxide hydrolase	Up
EST
vcr14	EST:Rat UI-R-C2-ng-d-11-0-UI.r1 homologue	Down
Others/unknown
vcr2	Mouse 10-day embryo cDNA, RIKEN library,clone: 2610003J06 homologue	Up
vcr3	Rat fetuin-like protein	Up
vcr5	Rat tubulin alpha 4	Up
vcr6	Rat MG87, unknown	Up
vcr11	Mouse 10-day embryo cDNA, RIKEN library, clone: 2610511A05 homologue	Up
vcr12	Rat hepsin	Up
vcr15	Rat mitochondrial genome	Up
vcr17	Rat brain digoxin carrier protein	Up
vcr18	Rat iron-responsive element-binding protein	Down
vcr21	Rat fibronectin 1	Up
vcr26	Rat mitochondrial long-chain enoyl-CoA	Up
vcr27	Mouse RalBP1 associated Eps domain protein homologue	Down

FIG. 1.

The effect of cigarette smoke exposure and ascorbic acid dose on SOD mRNA levels. 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. 1.

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. 2.

FIG. 3.

The effect of cigarette smoke exposure and ascorbic acid dose on the antioxidative 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. 3.

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. *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. 4.

FIG. 5.

Identification of genes with altered expression in livers between high-dose and low-dose ascorbic acid administered to rats by differential display and verification by RT-PCR. Rats were fed with high amounts of ascorbic acid (40 mg/day) or minimal amounts 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 7 M 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 a twofold difference between high-dose ascorbic acid and minimal-ascorbic acid administered rats 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°C for 10 min, 16–34 cycle of 95°C for 30 s, with 55–61°C for 30 s and 72°C for 30 s. Numbers in parentheses 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. 5.

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 β-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. 6.

FIG. 7.

The effect of cigarette smoke exposure and ascorbic acid dose on the mRNA level of transcription factors and GST in liver. 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).

FIG. 7.

To whom correspondence should be addressed at Faculty of Medicine, Ochanomizu University, Tokyo 112-8610, Japan. Fax: +81-3-5978-5813. E-mail: yotsuka@cc.ocha.ac.jp.

Present address: Faculty of Applied Life Science, Niigata University of Pharmacy and Applied Life Science, Higashijima, Niitsu, Niigata 956-8603, Japan.

We thank the Gene Research Center, Tottori University, for the facilities and the services. We also thank 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 a Smoking Research Foundation Grant for Biomedical Research.

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Vitamin C Dosage For Smokers

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Vitamin C Dosage For Smokers

Melvin Desember 03, 2021