Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ishii, T.
Right arrow Articles by Yamamoto, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ishii, T.
Right arrow Articles by Yamamoto, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Carcinogenesis, Vol. 21, No. 5, 1013-1016, May 2000
© 2000 Oxford University Press

Carcinogenesis

Induction of murine intestinal and hepatic peroxiredoxin MSP23 by dietary butylated hydroxyanisole

Tetsuro Ishii3, Ken Itoh, Junetsu Akasaka, Toru Yanagawa2, Satoru Takahashi1, Hiroshi Yoshida2, Shiro Bannai and Masayuki Yamamoto1

Department of Biochemistry, Institute of Basic Medical Sciences,
1 Tsukuba Advanced Research Alliance Center and
2 Department of Oral and Maxillofacial Surgery, Institute of Clinical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Feeding mice with 2(3)-t-butyl-4-hydroxyanisole (BHA) induces phase II detoxifying enzymes that inhibit the action of carcinogens. We have found that dietary BHA induces intestinal and hepatic MSP23 (also called peroxiredoxin I), a stress-inducible antioxidant, in a manner similar to the induction of glutathione S-transferases (GSTs). The levels of MSP23 in the proximal intestine and liver, estimated by immunoblotting, increased approximately 1.9- and 1.3-fold, respectively, in mice fed a diet containing 0.7% (w/w) BHA for 7 days. The level of MSP23 mRNA in these tissues also increased more than 2-fold after mice were fed BHA, suggesting that the induction of MSP23 is controlled at the transcription level. Immunostaining of the small intestine shows that MSP23 is expressed mainly in the columnar epithelial cells. The induction of MSP23 may be important to protect the cells and tissues against toxic electrophiles and reactive oxygen species.

Abbreviations: BHA, 2(3)-t-butyl-4-hydroxyanisole; GST, glutathione S-transferase; Prx, peroxiredoxin; TNF-{alpha}, tumor necrosis factor-{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
2(3)-t-Butyl-4-hydroxyanisole (BHA) is a synthetic phenolic antioxidant that is widely used as a food preservative. Dietary administration of BHA protects animals against various carcinogens (1), presumably through the induction of phase II enzymes that detoxify the activated electrophilic metabolites of carcinogens. In the murine liver and intestine, BHA markedly induces the expression of glutathione S-transferases (GSTs) (2), NAD(P)H:quinone reductase (3), UDP-glucuronosyltransferases (4) and epoxide hydrolases (5).

A 23 kDa oxidative stress-inducible protein termed MSP23, which is now classified as murine peroxiredoxin (Prx) I, was first cloned in macrophages by differential screening using diethylmaleate, a sulfhydryl-reactive electrophilic agent, as inducer (6). MSP23 is ubiquitously expressed and its levels are especially high in the liver (6). The peroxiredoxin proteins have conserved cysteine residues that participate in oxidoreductive reactions and protect macromolecules from oxidative damage (7). The mammalian Prx proteins I–III reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-{alpha} (TFN-{alpha}) in the presence of the thioredoxin/thioredoxin reductase system (8,9). Prx I and II regulate the activation of NF-{kappa}B (10) and AP-1 (11) and protect cells from apoptosis by reducing oxidative damage (12).

Since the BHA metabolite t-butylhydroquinone induced MSP23 in murine macrophages in vitro, we examined the effect of dietary BHA on the expression of MSP23 in mouse tissues. In this study we show that dietary BHA leads to induction of MSP23 in the small intestine and liver. The induction of MSP23 may be important in protecting the tissues from oxidative damage.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Treatment of animals
Female ICR mice (Charles River Co.) between 8 and 12 weeks of age were used. Mice of each genotype were randomly assigned to two groups. Both groups were fed either the basal powder diet (MF; Oriental Yeast Co.) or the experimental diet, which was prepared by supplementing the food with BHA at a concentration of 0.7% (w/w) as described previously (13). During the experiment, the mice had free access to food and tap water. After 1–14 days the animals were killed by decapitation. All animal experiments were carried out after receiving permission from the Animal Experiment Committee, University of Tsukuba, and in accordance with the University of Tsukuba's Regulations on Animal Experiments and Japanese Governmental Law No. 105.

Tissue preparation
The tissues were excised immediately after death. The intestine between the stomach and the colon was dissected into 5 cm segments and each segment was cut open and washed with ice-cold phosphate-buffered saline. Aliquots (15 mg wet wt) of washed tissues were homogenized in small glass homogenizers in 1 ml SDS sample buffer (2% w/v SDS, 10% v/v glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 mM Tris–HCl, pH 6.8), boiled for 5 min and stored at –20°C until use. The protein content of tissue homogenates was determined using BCA reagent (Pierce). Samples were mixed with bromophenol blue and 2-mercaptoethanol and boiled for 5 min, then immediately subjected to SDS–PAGE (10% w/v acrylamide). Western blots were visualized using ECL reagent (Amersham). MSP23 was detected with polyclonal rabbit antiserum raised against rat Prx I purified from rat liver (14). Immunostained protein bands were quantified by densitometric analysis using the NIH Image program. Proteins that partially remained in the gels after electrotransfer were stained with Coomassie brilliant blue R 250 (Fluka) to detect the induction of GSTs.

Immunohistological staining
Immunostaining of tissues was performed by the horseradish peroxidase-conjugated streptavidin biotin method (LsAB method; Nichirei) following the manufacturer's protocol. Pieces of proximal intestine were washed in phosphate-buffered saline, then post-fixed with 4% paraformaldehyde for 3 h and embedded in paraffin. The sections were incubated with 1:1000 (v/v) diluted anti-Prx I rabbit antiserum at 4°C overnight. The antigen was stained brownish red. The sections were counterstained with hematoxylin.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
To examine the effects of dietary BHA on the expression levels of MSP23 in tissues, we fed wild-type ICR female mice a diet containing 0.7% BHA for up to 2 weeks and monitored the level of MSP23. Using anti-Prx I antiserum, we detected a single 23 kDa immunostained band corresponding to MSP23 in both intestine and liver from control and BHA-fed mice. The induction efficiency of dietary BHA was monitored by detecting the induced protein bands of {alpha} and µ class GSTs in the intestine (Figure 1Go, upper) and {pi} class GST in the liver, which have apparent masses of ~25 kDa (13). We found that MSP23 was measurably increased in the small intestine after 5 days of continuous BHA treatment and reached a maximum level after 7 days, which lasted for at least 1 week (Figure 1Go, lower). This time course indicated that the induction of intestinal MSP23 by dietary BHA is significantly slower than that of the GSTs. To examine the location of the induced MSP23 in the intestinal duct, we dissected the small intestine (mean length ± SD = 47.5 ± 2.1 cm, n = 5) into nine or 10 5 cm segments and monitored the relative levels of MSP23 in each segment. The inducing effect of BHA was most evident in the proximal to middle part of the small intestine (Figure 2Go, lanes 2–10) and was not significant in either the stomach or the colon (Figure 2Go, lanes 1 and 11). After treatment with BHA for 7 days, the levels of intestinal and hepatic MSP23 were enhanced 1.92 ± 0.39- and 1.25 ± 0.21-fold (mean ± SD, n = 6), respectively (Figure 3Go). The inducing effect was statistically significant (P < 0.001 in intestine and P < 0.05 in liver). The ratio of intestinal to hepatic MSP23 levels in mice fed the normal diet was calculated to be 0.58 (mean of six mice), which increased to 0.89 after treatment with BHA (Figure 3Go).



View larger version (53K):
[in this window]
[in a new window]
  		Fig. 1. Time-dependent induction of MSP23 in the small intestine by dietary BHA. Induction of {alpha} and µ class GSTs is visible as stained protein bands with masses of ~25 kDa (upper). The immunostained MSP23 band (lower) is shown during the time of continuous BHA feeding. Number of days refers to after BHA was added to the diet.

 


View larger version (60K):
[in this window]
[in a new window]
  		Fig. 2. Locus of induction of MSP23 in small intestine. The small intestines of mice fed a diet without (control) or with BHA treatment (+BHA) were cut into 5 cm pieces (lanes 2–10, proximal–middle–distal) from the stomach (lane 1) to the colon (lane 11). The dye stained gel (upper) and the bands immunostained with anti-MSP23 antibodies (lower) are shown.

 


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 3. Densitometric analysis of the effect of dietary BHA on the levels of MSP23 in small intestine and liver. Mice were fed a diet with or without BHA for 7 days. The mean values of the level of MSP23 in the proximal small intestine of control mice were defined as the standard (1.00). Each dot represents the relative value of MSP23 in each mouse.

 
We next examined the effect of dietary BHA on the levels of MSP23 mRNA (1.0 kb) in the proximal intestine and liver by northern blotting, We found that it enhanced the MSP23 mRNA levels 2.30 ± 0.72- and 1.82 ± 0.08-fold (mean ± SD, n = 4 and 6), respectively (Figure 4Go). The inducing effect of dietary BHA was statistically significant (P < 0.001 in both intestine and liver). The effect of dietary BHA on the levels of MSP23 mRNA was roughly parallel to the effect on the levels of MSP23 protein in both tissues (compare Figure 4Go with Figure 2Go). These results suggest that the induction of MSP23 by dietary BHA in these tissues is regulated at the transcriptional level.



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4. Induction of intestinal and hepatic MSP23 mRNA by dietary BHA. Densitometric analysis of MSP23 mRNA levels in the proximal small intestine (left) and liver (right). The mean values of the level of MSP23 mRNA in the small intestine and liver of control mice were defined as the standard (1.00). The relative values were calculated using ß-actin mRNA (2.0 kb) as an internal standard.

 
To examine the localization of MSP23 in the proximal small intestine, a histochemical study was performed using antibody staining with an anti-Prx I antiserum (Figure 5Go). In the intestine, heavy staining was seen in the columnar epithelial cells, especially those located in the middle to lower part of the intestinal villi. In contrast, only slight staining was seen in the Paneth cells, myofibroblasts, goblet cells and muscularis mucosae (Figure 5Go). The location of staining of MSP23 in the intestine was fundamentally the same, but the intensity appeared higher after mice were fed BHA (compare Figure 5a with bGo).



View larger version (130K):
[in this window]
[in a new window]
  		Fig. 5. Localization of MSP23 in proximal intestine. MSP23 (brownish red) was immunostained with antiserum as described in Materials and methods. (a) Control. (b) BHA fed. x370 magnification.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We have shown for the first time that: (i) dietary BHA induces intestinal and hepatic MSP23 in mice (Figures 1–4GoGoGoGo); (ii) MSP23 is mainly expressed in columnar intestinal epithelial cells facing the lumen of the ducts (Figure 5Go). Notably, dietary BHA almost doubled the level of intestinal MSP23 and the enhanced level approached that of the liver, which expressed the highest MSP23 level among the major tissues. No induction of MSP23 by dietary BHA could be detected in the stomach and colon (Figure 2Go) or in brain, heart, lung, spleen and kidney (not shown) by immunoblotting. Thus, the induction of MSP23 by dietary BHA appears specific to intestine and liver.

The administration of BHA or other antioxidants to rodents subsequently induces specific activities of enzymes in the liver and peripheral tissues of these animals (15). These enzymes include GST (2), NAD(P)H:quinone reductase (3), UDP-glucuronosyltransferase (4), microsomal epoxide hydrolase (5), aflatoxin B1 aldehyde reductase (16), glucose 6-phosphate dehydrogenase (17) and {gamma}-glutamylcysteine synthetase (18). The increased levels of these enzymes reduce the susceptibility of cells to higher concentrations of toxic electrophilic compounds and carcinogens. This protective metabolic response provoked by BHA and other agents has been called an `electrophile counterattack' (19). This study indicates that the induction of MSP23, in addition to phase II enzymes, is one of the important adaptive responses induced by dietary BHA. We have no experimental results that explain the reason for the delayed induction of MSP23 compared with that of GST by dietary BHA. One possibility to explain this phenomenon is that dietary BHA gradually causes oxidative damage and that accumulated damage induces MSP23. Another possibility is that MSP23 mRNA is induced early after BHA treatment but that the apparent MSP23 protein level increased later because the protein degradation rate was enhanced in the early stages following treatment. Further precise analysis is necessary to elucidate the delayed induction of MSP23 by dietary BHA.

MSP23 is an oxidative stress-inducible protein belonging to the peroxiredoxin family. Members of this family have the ability to reduce hydrogen peroxide in the presence of the thioredoxin system (10) and to trap thiyl radicals with reactive cysteine residues (8). Transient expression of Prx I or II inhibited activation of NF-{kappa}B by extracellularly added H2O2 or TNF-{alpha} (10), although the level of induced Prx I in NIH 3T3 cells appeared to be less than 2-fold. It has also been shown that overexpression of Prx II in endothelial cells significantly blocks TNF-{alpha}-induced AP-1 activation (11). These results indicate that Prx enzymes play an important role in eliminating peroxides generated during metabolism. Therefore, the enhanced MSP23 level in mouse tissues induced by dietary BHA probably contributes to a reduction in cellular damage by oxidants. Although BHA is known as an antioxidant, it is oxidatively demethylated in mammalian tissues to t-butylhydroquinone. This metabolite is autoxidized to t-butylquinone and may produce reactive oxygen species by redox cycling (20). Using the EPR spin trapping technique, the generation of hydroxy radicals by autoxidation of t-butylhydroquinone to t-butylquinone was detected in human hepatoma HepG2 cells (21). There is a correlation between production of oxygen radicals and the induction of GST Ya gene expression (22). We observed that t-butylquinone supplementation of the culture medium markedly induced MSP23 in murine peritoneal macrophages (unpublished data). BHA, however, only slightly induced MSP23 in these cells. Therefore, BHA has the potential to act as both oxidant and antioxidant, and the generated quinone and/or oxygen radicals may induce phase II enzymes (23) and MSP23.

In conclusion, the present study demonstrates that the induction of MSP23 (Prx I) in intestine and liver is one of the broad defense reactions provoked by dietary BHA. These results suggest that MSP23 plays an important role in protecting these tissues against toxic electrophiles and reactive oxygen species.


   Acknowledgments
 
This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, Research for the Future of the Japanese Society for Promotion of Science (JSPS) and the JSPS. K.I. is a research fellow of the JSPS.


   Notes
 
3 To whom correspondence should be addressed Email: teishii{at}md.tsukuba.ac.jp Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Wattenberg,L.W. (1972) Inhibition of carcinogenic and toxic effects of polycyclic hydrocarbons by phenolic antioxidants and ethoxyquin. J. Natl Cancer Inst., 48, 1425–1430.
  2. Benson,A.M., Batzinger,R.P., Ou,S.-Y,L., Bueding,E., Cha,Y.-N. and Talalay,P. (1978) Elevation of hepatic glutathione S-transferase activities and protection against mutagenic metabolites of benzo(a)pyrene by dietary antioxidants. Cancer Res., 38, 4486–4495.[Abstract/Free Full Text]
  3. Benson,A.M., Hunkeler,M.J. and Talalay,P. (1980) Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc. Natl Acad. Sci. USA, 77, 5216–5220.[Abstract/Free Full Text]
  4. Moldeus,P., Dock,L., Cha,Y.-N., Berggren,M. and Jernstrom,B. (1982) Elevation of conjugation capacity in isolated hepatocytes from BHA-treated mice. Biochem. Pharmacol., 31, 1907–1910.[Web of Science][Medline]
  5. Cha,Y.-N., Martz,F. and Beuding,E. (1978) Enhancement of liver microsome epoxide hydratase activity in rodents by treatment with 2(3)-tert-butyl-4-hydroxyanisole. Cancer Res., 38, 4496–4498.[Abstract/Free Full Text]
  6. Ishii,T., Yamada,M., Sato,H., Matsue,M., Taketani,S., Nakayama,K., Sugita,Y. and Bannai,S. (1993) Cloning and characterization of a 23-kDa stress-induced mouse peritoneal macrophage protein. J. Biol. Chem., 268, 18633–18636.[Abstract/Free Full Text]
  7. Yim.,M.B., Chae,H.Z., Rhee,S.G., Chock,P.B. and Stadtman,E.R. (1994) On the protective mechanism of the thiol-specific antioxidant enzyme against the oxidative damage of biomacromolecules. J. Biol. Chem., 269, 1621–1626.[Abstract/Free Full Text]
  8. Chae,H.Z., Chung,S.J. and Rhee,S.G. (1994) Thioredoxin-dependent peroxide reductase from yeast. J. Biol. Chem., 269, 27670–27678.[Abstract/Free Full Text]
  9. Kang,S.W., Chae,H.Z., Seo,M.S., Kim,K., Baines,I.C. and Rhee,S.G. (1998) Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-{alpha}. J. Biol. Chem., 273, 6297–6302.[Abstract/Free Full Text]
  10. Jin,D.-Y., Chae,H.Z., Rhee,S.G. and Jeang,K.-T. (1997) Regulatory role for a novel human thioredoxin peroxidase in NF-{kappa}B activation. J. Biol. Chem., 272, 30952–30961.[Abstract/Free Full Text]
  11. Shau,H., Huang,A.C., Faris,M., Nazarian,R., de Vellis,J. and Chen,W. (1998) Thioredoxin peroxidase (natural killer enhancing factor) regulation of activator protein-1 function in endothelial cells. Biochem. Biophys. Res. Commun., 249, 682–686.
  12. Zhang,P., Liu,B. Kang,S.W., Seo,M.S., Rhee,S.G. and Obeid,L.M. (1997) Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2. J. Biol. Chem., 272, 30615–30618.[Abstract/Free Full Text]
  13. Itoh,K., Chiba,T., Takahashi,S., Ishii,T., Igarashi,K., Katoh,Y., Oyake,T., Hayashi,N., Satoh,K., Hatayama,I., Yamamoto,M. and Nabeshima,Y. (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun., 236, 313–322.[Web of Science][Medline]
  14. Ishii,T., Kawane,T., Taketani,S. and Bannai,S. (1995) Inhibition of the thiol-specific antioxidant activity of rat liver MSP23 protein by hemin. Biochem. Biophys. Res. Commun., 216, 970–975.[Web of Science][Medline]
  15. Primiano,T., Sutter,T.R. and Kensler,T.W. (1997) Antioxidant-inducible genes. Adv. Pharmacol., 38, 293–328.
  16. Ellis,E.M., Judah,D.J., Neal,G.E. and Hayes,J.D. (1993) An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a sub-family of aldo-keto reductase. Proc. Natl Acad. Sci. USA, 90, 10350–10354.[Abstract/Free Full Text]
  17. Cha,Y.-N. and Bueding,E. (1979) Effect of 2(3)-tert-butyl-4-hydroxyanisole (BHA) administration on the activities of several hepatic microsomal and cytoplasmic enzymes in mice. Biochem. Pharmacol., 28, 1917–1921.[Web of Science][Medline]
  18. Eaton,D.L. and Hamel,D.M. (1994) Increase in {gamma}-glutamylcysteine synthetase activity as a mechanism for butylated hydroxyanisole-mediated elevation of hepatic glutathione. Toxicol. Appl. Pharmacol., 126, 145–149.[Web of Science][Medline]
  19. Prestera,T., Zhang,Y., Spencer,S.R., Wilczak,C.A. and Talalay,P. (1993) The electrophile counterattack response: protection against neoplasia and toxicity. Adv. Enzyme. Regul., 33, 281–296.[Web of Science][Medline]
  20. Kahl,R., Weinke,S. and Kappus,H. (1989) Production of reactive oxygen species due to metabolic activation of butylated hydroxyanisole. Toxicology, 59, 179–194.[Web of Science][Medline]
  21. Pinkus,R., Weiner,M. and Daniel,V. (1996) Role of oxidants and antioxidants in the induction of AP-1, NF-{kappa}B and glutathione S-transferase gene expression. J. Biol. Chem., 271, 13422–13429.[Abstract/Free Full Text]
  22. Pinkus,R., Weiner,L.M. and Daniel,V. (1995) Role of quinone-mediated generation of hydroxyl radicals in the induction of glutathione S-transferase gene expression. Biochemistry, 34, 81–88.[Medline]
  23. Yu,R., Tan,T.-H. and Kong,A.-N.T. (1997) Butylated hydroxyanisole and its metabolite tert-butylhydroquinone differentially regulate mitogen-activated protein kinases: the role of oxidative stress in the activation of mitogen-activated protein kinases by phenolic antioxidants. J. Biol. Chem., 272, 28962–28970.[Abstract/Free Full Text]
Received August 17, 1999; revised December 30, 1999; accepted January 5, 2000.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 International Journal of ToxicologyHome page
Annual Review of Cosmetic Ingredient Safety Assessments--2004/2005
International Journal of Toxicology, March 1, 2006; 25(2_suppl): 1 - 89.
[Full Text] [PDF]

Home page
 J. Histochem. Cytochem.Home page
S. Immenschuh, E. Baumgart-Vogt, M. Tan, S.-i. Iwahara, G. Ramadori, and H. D. Fahimi
Differential Cellular and Subcellular Localization of Heme-Binding Protein 23/Peroxiredoxin I and Heme Oxygenase-1 in Rat Liver
J. Histochem. Cytochem., December 1, 2003; 51(12): 1621 - 1631.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ishii, T.
Right arrow Articles by Yamamoto, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ishii, T.
Right arrow Articles by Yamamoto, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?