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 (23)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Nakamura, Y.
Right arrow Articles by Ohigashi, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakamura, Y.
Right arrow Articles by Ohigashi, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Carcinogenesis, Vol. 21, No. 10, 1899-1907, October 2000
© 2000 Oxford University Press

Carcinogenesis

A simple phenolic antioxidant protocatechuic acid enhances tumor promotion and oxidative stress in female ICR mouse skin: dose- and timing-dependent enhancement and involvement of bioactivation by tyrosinase

Yoshimasa Nakamura, Koji Torikai, Yoshimi Ohto, Akira Murakami1, Takuji Tanaka2 and Hajime Ohigashi3

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502,
1 Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Iwade-Uchita, Wakayama 649-6493 and
2 The First Department of Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The modifying effects of topical application of the phenolic antioxidant protocatechuic acid (PA) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin tumor promotion were investigated. Dimethylbenz[a]anthracene-initiated female ICR mice were treated with TPA (1.6 nmol) twice weekly for 20 weeks to promote papilloma formation. Pre-treatment with 16nmol PA 30 min prior to each TPA treatment significantly inhibited the number of papillomas per mouse by 52% (P < 0.05). On the other hand, PA pre-treatment at a high dose (1600 nmol) significantly enhanced tumor numbers by 38% (P < 0.05). Interestingly, in the group treated with a quite high dose (20000 nmol) of PA 5 min prior to each TPA application, the average number of tumors per mouse was reduced by 38%, whereas the same PA dose 3 h before TPA treatment significantly enhanced tumor numbers by 84% (P < 0.01). These results suggested that topically applied PA was converted to compound(s) lacking antioxidative properties and/or rather possessing the potential to enhance tumor development. A similar tendency was also observed in the short-term experiment of TPA-induced inflammation and oxidative stress; i.e. two groups pre-treated with PA at 20000 nmol, 30min and 3h before TPA treatment, did not show suppression or even significantly enhanced TPA-induced leukocyte infiltration, H2O2 generation, thiobarbituric acid-reacting substances level and proliferating cell nuclear antigen index, while PA treatment together with TPA significantly suppressed these parameters. Treatment with a high dose (20000 nmol) of PA alone for 3h enhanced oxidative stress by reducing glutathione levels in mouse skin, which was counteracted by the tyrosinase inhibitor arbutin. Oxidative stress responses such as leukocyte infiltration and H2O2 generation were also counteracted by arbutin. These results suggested that tyrosinase-dependent oxidative metabolism of PA was at least partially involved in the enhanced effects of PA on TPA-induced inflammatory responses and thus tumor promotion.

Abbreviations: DMBA, dimethylbenz[a]anthracene; GPx, glutathione peroxidase; GSH, glutathione; GST, glutathione S-transferase; HRP, horseradish peroxidase; IE, inhibitory effect; MPO, myeloperoxidase; PA; protocatechuic acid; PCNA, proliferating cell nuclear antigen; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reacting substances; TPA, 12-O-tetradecanoylphorbol-13-acetate.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The frequent consumption of fresh fruit and vegetables is associated with a low incidence of cancer (1). This may be partly due to the presence of several vitamins in plant foodstuffs (2). Such foods also contain certain naturally occurring phenolic compounds that have antioxidative properties (3), such as reactive oxygen species (ROS) scavenging, electrophile scavenging, metal cheletion and inhibition of ROS generation systems. Most naturally occurring phenolic compounds in foods are flavonoids, but others include chlorogenic acid, caffeic acid, ferulic acid, catechins and diallylheptanoids such as curcumin. Besides their antioxidative activities, they have been reported to be antimutagenic and/or anticarcinogenic and to possess several other biological activities. Many antioxidants have been investigated for their potential usefulness as cancer chemopreventive agents. Antioxidants have been thought to mainly suppress carcinogenesis during the initiation phase, since most act as radical scavengers, or inducers or inhibitors of xenobiotics metabolizing enzymes including phase I and II enzymes. On the other hand, several antioxidants that can inhibit the initiation events have been found to be second stage tumor promoters (4). Recently, Ogawa et al. (5) clearly demonstrated stage- and organ-dependent promotional effects of antioxidants in a rat multiorgan carcinogenesis model. Thus, chemopreventive agents that act in the initiation stage do not necessarily exert beneficial effects in the post-initiation phase. Moreover, radical scavengers are known to have pro-oxidative potential because of their conversion to more reactive or stable radicals after they react directly with ROS, which may contribute to the induction of secondary oxidative damage on the target organs.

The simple phenolic protocatechuic acid (PA; Figure 1Go) is one of the major benzoic acid derivatives from edible plants and fruits and shows a strong antioxidative effect, 10-fold higher than that of {alpha}-tocopherol (6). PA even at a 100p.p.m. shows potent chemopreventive effects on colon and oral carcinogenesis in rats (7,8). The present study was initially performed to estimate the effectiveness of PA against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. Interestingly, differences in modifying effects were dependent on the dose (~8.1–20 000 nmol) and timing (~5 min to ~3 h before TPA treatment) of PA. We demonstrated not only the lack of an inhibitory effect but also significant enhancement of mouse skin tumor promotion by pre-treatment with a high dose of PA 3 h before TPA application. The possibility that metabolism by tyrosinase activity of PA to certain compound(s) without antioxidative properties and/or with tumor promotional potency was also suggested.



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 1. Chemical structure of PA.

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals and animals
PA was purchased from Nacalai Tesque Inc. (Kyoto, Japan). TPA was obtained from Research Biochemicals International (Natick, MA). RPMI 1640 medium was purchased from Gibco BRL (Rockville, MD). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Female ICR mice (7 weeks old) were obtained from Japan SLC (Shizuoka, Japan). Mice used in each experiment were supplied with fresh tap water ad libitum and rodent pellets (MF; Oriental Yeast Co., Kyoto, Japan) freshly changed twice a week. Animals were treated in accordance with the Guidelines for Animal Experimentation of Kyoto University. Animals were maintained in a room controlled at 24 ± 2°C with a relative humidity of 60 ± 5% and a 12 h light/dark cycle (06:00 to 18:00). The back of each mouse was shaved with surgical clippers 2 days before each experiment.

Tumor promotion experiment
The modifying effect of PA on TPA-induced tumor promotion was examined by a standard initiation-promotion protocol with dimethylbenz[a]anthracene (DMBA) and TPA as reported previously (9). One group was composed of 15 female ICR mice housed five per cage. The back of each mouse was shaved with a surgical clipper 2 days before initiation. The mice at 7 weeks old were initiated with DMBA (190 or 95 nmol/0.1 ml acetone). One week after initiation, the mice were promoted with TPA (1.6 nmol/0.1 ml acetone) and then twice a week for 20 weeks. In four other groups, the mice were treated with PA (16, 160, 1600 or 20 000 nmol/0.1 ml acetone) 0 min, 40 min or 3 h before each TPA treatment. The modifying effect on TPA-induced tumor promotion was evaluated by both the ratio of tumor-bearing mice and the number of tumors, >1 mm in diameter, per mouse. The data were statistically analyzed using the Student's t-test (two-sided), which assumed unequal variance, for the average number of tumors per mouse, and by the {chi}2-test for the incidence of skin tumors.

Inflammatory biomarker determination
The modifying effect on inflammation induced by single TPA application was determined by two biomarkers, edema formation and myeloperoxidase (MPO) activity, as reported previously (10). Mice were killed by cervical dislocation 18 h after a single application of TPA. Mouse skin punches were obtained with an 8 mm diameter cork borer and weighed in an analytical balance. The inhibitory effects (IE) were expressed by the relative increasing ratio of the weight of a treated punch to that of a control punch; IE (%) = {[(TPA alone) – (test compound + TPA)]/[(TPA) – (vehicle)]}x100. The MPO activity was calculated from the linear portion of the curve and expressed as units of MPO per skin punch. One unit of MPO activity was defined as the activity that degraded 1 µmol of H2O2 per min at 25°C.

Double TPA treatment protocol
One group was composed of five female ICR mice housed at five per cage. PA was topically applied to the shaved area of dorsal skin at various times before application of TPA solution (8.1 nmol/0.1 ml in acetone). This TPA dose (8.1 nmol) was used for the potentiation of oxidative responses compared with the dose for tumor promotion (1.6 nmol). The same doses of PA and TPA or acetone were applied twice at an interval of 24 h for H2O2 determination. Although the timing (24 h apart) of double TPA application was different from tumor promotion protocol, the level of oxidative stress was nearly the same when the time between the two TPA treatments was 24–72 h (data not shown).

Determination of oxidative stress parameters
Mice treated by the double-treatment protocol were killed 1 h after the last TPA treatment. The H2O2 content was determined by the phenol red–horseradish peroxidase (HRP) method (9–11). The final results are expressed as equivalents of nmol of H2O2 per skin punch, on the basis of a standard curve of HRP-mediated oxidation of phenol red by H2O2. Thiobarbituric acid-reacting substances (TBARS) level of mouse epidermis was determined by our previously reported method (10,12). The final results are expressed as equivalents of nmol malondialdehyde per cm2, on the basis of a standard line of TBARS formation using the authentic malondialdehyde.

Histological examination
Mice treated by the double-treatment protocol were killed 1 h after the second TPA treatment. Excised skin was fixed in 10% buffered formalin, and then embedded in paraffin. Skin samples were cut at 3 µm, mounted on silanized slides, dewaxed in xylene, dehydrated through an ethanol series, and stained with hematoxylin and eosin. For each section of skin, the thickness of the epidermis from the basal layer to the stratum corneum was measured at five equidistant interfollicular sites utilizing an image analysis system Leica Q500IW-EX (Leica Co. Ltd, Tokyo, Japan) with a microscope Leica DMRE HC (Leica Co. Ltd). The numbers of infiltrating leukocytes were counted at five different areas of each section using this image analysis system. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed as reported previously (10). Skin sections were treated with 1.2% H2O2 in absolute methanol for 30 min and stained by the indirect avidin–biotin–HRPO method (ABC standard; Vector Laboratories, Burlingame, CA). Color development with diaminobenzidine (Vector Laboratories) was monitored by the appearance of brown PCNA staining in the normal epidermis. The PCNA labeling index was counted at six different areas of each section using the image analysis system, and expressed as (number of positive squamous cell nuclei/total number of squamous cell nuclei)x100.

Glutathione (GSH) assay
Measurement of GSH was performed spectrophotometrically using a commercial kit (BIOXYTECH® GSH-400TM Assay; OXIS International Inc., Portland, OR). Mice were treated with PA at different time intervals and killed by cervical dislocation. The skin samples were homogenized and extracted with 5% metaphosphoric acid solution containing 5 mM EDTA. Then, after centrifugation (10 000 g, 20 min), 50 µl of 12 mM chromogenic reagent in 0.2 M HCl was added to the resulting supernatant (300 µl) and mixed thoroughly. After 50 µl of 7.5 M NaOH was added and mixed, the mixture was incubated at 25°C for 10 min, and then absorbance was determined spectrophotometrically at 400 nm.

Glutathione peroxidase (GPx) and glutathione S-transferase (GST) assays
Measurement of GPx activity was performed spectrophotometrically using a commercial kit (BIOXYTECH® GPx-340TM Assay; OXIS, International, Inc.). The skin samples were minced in 3 ml of 50 mM Tris–HCl buffer (pH 8.0) containing 5 mM sodium azide and 1 mM 2-mercaptoethanol and then homogenized twice at 4°C for 30 s. After centrifugation (10 000 g, 20 min), 700 µl of NADPH reagent containing GSH and GSH reductase were added to the resulting supernatant (70 µl) and mixed thoroughly. After 350 µl of 0.007% (w/w) tert-butyl hydroperoxide solution was added and mixed, increases in absorption at 340 nm were recorded at 0.2 min intervals for 1 min. The GPx activity was calculated from the linear portion of the curve and expressed as units of GPx per skin punch. One unit of GPx activity was defined as the activity that degraded 1 µmol of NADPH per min at 25°C.

GST was measured using 1-chloro-2,4-dinitrobenzene as a substrate according to the method of Habig et al. (14).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Modifying effect of the dose or timing of PA pre-treatment on TPA-induced tumor promotion
The modifying effects of different doses of topically applied PA on TPA-induced tumor promotion were examined in a two-stage mouse skin carcinogenesis model and the results are shown in Table IGo. Tumors began to develop 6 weeks after tumor promotion by TPA. The incidence of tumor-bearing mice and the average number of tumors per mouse in the group given DMBA and TPA were 100% and 14.0, respectively, at the end (20 weeks) of the experiment. In the group treated with 16 nmol of PA 40 min prior to each TPA treatment, the average number of tumors per mouse was reduced by 52% (P < 0.05) and tumor incidence by 42% (P < 0.05). On the other hand, pre-treatment with 160 nmol PA showed slight inhibitory effects on tumor numbers (IE = 8%) and the tumor incidence (IE = 8%). Moreover, 1600 nmol PA significantly enhanced the number of tumors per mouse by 38% (P < 0.05). Mice initiated with DMBA and then treated with acetone or 1600 nmol PA twice weekly for 20 weeks did not develop any tumors.


View this table:
[in this window]
[in a new window]
  		Table I. Modifying effects of pre-treatment (40 min) with different doses of PA on TPA-induced papilloma formation in ICR mouse skin
 
Subsequently, we examined whether the time interval between PA and TPA treatments attenuates its antioxidative properties and thus decreases antitumor promotional activity (Figure 2Go). In this experiment, mice initiated with 95 nmol DMBA and promoted with 1.6 nmol of TPA twice weekly for 20 weeks developed an average of 8.7 ± 6.1 tumors/mouse. In the group treated with 20 000 nmol PA 5 min prior to 1.6 nmol TPA application, the average number of tumors per mouse was reduced by 38%, but this was not statistically significant. On the other hand, pre-treatment with 20 000 nmol PA 3 h before TPA treatment significantly increased the number of tumors per mouse (16.0 ± 7.4) by 84% (P < 0.01).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 2. Modifying effect on TPA-induced tumor promotion of PA in DMBA-initiated ICR mouse skin. One group was composed of 15 female ICR mice. The mice at 7 weeks old were initiated with DMBA (95 nmol). One week after initiation, the mice in group 1 (closed circles) were promoted with TPA (1.6 nmol/0.1 ml in acetone) twice a week for 20 weeks. In the PA-treated experiments, the mice in groups 2 (open triangles) and 3 (open squares) were treated with PA (20 000 nmol/0.1 ml in acetone, respectively) for 5 min and 3 h, respectively, prior to each TPA treatment. The tumor promotion-modifying activity was evaluated by both the ratio of tumor-bearing mice (A) and the number of tumors per mouse (B). Statistical analysis was performed by the {chi}2-test on tumor-bearing mouse and the Student's t-test on the number of tumors per mouse. Significance is expressed as: a, P < 0.005; b, P < 0.05.

 
Modifying effect of PA on inflammatory responses and oxidative stress in ICR mouse skin
The possibility that PA could modify TPA-induced inflammation and oxidative stress was assessed by investigating the effects of different doses and different timing of topical application of PA on single TPA application-induced edema formation and MPO activity enhancement as well as double TPA application-induced H2O2 generation in ICR mouse skin. As shown in Table IIGo, the application of a low dose (81 nmol) of PA 30 min before 8.1 nmol TPA treatment inhibited edema formation, MPO activity and H2O2 generation by 20, 58 (P < 0.05) and 22%, respectively. Whereas, the application of 8100 or 20 000 nmol PA 30 min before TPA treatment showed slight inhibition of expression of these three biomarkers (IEs = –4 to 8%).


View this table:
[in this window]
[in a new window]
  		Table II. Modifying effects of pre-treatment at different times or different doses of PA on TPA-induced enhancement of oxidative stress parameters in ICR mouse skin
 
To determine whether the time interval between PA and TPA treatments modifies its antioxidative properties, the effects of pre-treatment with 20 000 nmol PA 0, 0.5, 1, 3 or 6 h before TPA treatment were evaluated (Table IIGo). The simultaneous application of 20 000 nmol PA together with TPA treatment inhibited expression of all biomarkers by 27, 47 (P < 0.05) and 51% (P < 0.001), respectively. However, as the time intervals between PA and TPA treatment were increased, lesser inhibitory effects on inflammation and oxidative stress elevation were observed. PA application 3 h prior to TPA treatment showed the strongest enhancement of MPO activity and H2O2 generation [166% of control (P < 0.05) and 125% of control (P < 0.05), respectively].

As topically applied PA significantly modified H2O2 generation induced by double TPA application, we assessed whether such PA treatment influences double TPA application-induced TBARS formation, a well-known biomarker of overall oxidative damage to cellular constituents such as membrane lipids. The quantitative data for the levels of TBARS formation in mouse epidermis homogenate are shown in Figure 3Go. The increased level of TBARS caused by the double TPA application was significantly higher than that of the control (0.43 ± 0.09 versus 0.15 ± 0.06 nmol/cm2, P < 0.01). The simultaneous application of PA (20 000 nmol) with TPA treatment significantly inhibited the increase in TBARS level (0.24 ± 0.05 nmol/cm2, P < 0.01 versus double TPA). On the other hand, the application of PA 3 h before TPA treatment significantly enhanced double TPA application-induced TBARS formation (0.63 ± 0.13 nmol/cm2, P < 0.05 versus double TPA).



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3. Modifying effects of PA on TBARS formation in the mouse epidermis. ICR mice (5 mice in each group) were treated by the double-treatment protocol as described in Materials and methods. Mouse skin was treated with PA (20 000 nmol) or acetone 0 or 3 h prior to each TPA treatment. The mice were killed 1 h after the second TPA application, and their epidermis was removed for TBARS assays. Significance determined by the Student's t-test is expressed as: a, versus acetone, P < 0.05; b, versus TPA, P < 0.01; c, versus TPA, P < 0.05.

 
Effects of PA on morphological alterations in mouse skin treated with double TPA application
The finding that PA treatment caused the inverse effects on MPO activity, H2O2 generation and TBARS formation led us to select a double TPA application model of mouse inflammation for histological observation to determine whether PA application enhances leukocyte infiltration and hyperplasia in the cutis. Double application of TPA caused morphological alteration of inflammatory response (Table IIIGo, Figure 4BGo) as compared with the control group (Figure 4AGo), which was well correlated to the results of skin edema formation and MPO activity (Table IIGo). Mouse skin treated with TPA twice within a 24 h interval displayed severe epidermal hyperplasia (Figure 4BGo) and resulted in a marked increase in leukocyte infiltration as compared with treatment with acetone as shown in Table IIGo (8 ± 1 versus 312 ± 41 per mm2). Also, double TPA treatment increased the number of mitoses in epidermal squamous cells (Figure 4BGo). On the other hand, pre-treatment with PA (20 000 nmol) together with TPA application diminished TPA-induced hyperplasia, mitosis (Figure 4CGo) and leukocyte infiltration (Table IIIGo). PCNA immunohistochemistry revealed PCNA-positive nuclei in many epidermal keratinocytes, only at the basal and first suprabasal layer in mice treated with acetone alone (PCNA-labeling index: 36.4 ± 6.7; Table IIIGo). Double application of TPA significantly enhanced the PCNA index (73.4 ± 16.1, P < 0.05) as compared with acetone alone. The PCNA index (36.1 ± 3.2) in mice given double application of TPA and PA at the same time was almost equivalent to that of control mice, and the value was significantly lower than that of mice treated twice with TPA (P < 0.02). While a high proportion of epithelial cell nuclei in hair follicles and glandular appendages was also stained, no significant differences in this pattern were observed between different treatment groups (data not shown). On the other hand, as the interval between PA and TPA treatment was increased, inhibitory effects on TPA-induced increases in numbers of leukocytes and PCNA index were not significant as compared with group 2 treated only with TPA (Table IIIGo).


View this table:
[in this window]
[in a new window]
  		Table III. Modifying effects of PA pre-treatment at different times on TPA-induced morphological changes in mouse skin
 


View larger version (135K):
[in this window]
[in a new window]
  		Fig. 4. Effects of PA on TPA-induced morphological changes in mouse skin. The protocol for animal treatment was as described in Materials and methods. Treatment with: (A) acetone; (B) double doses of TPA; (C) double dose of TPA and PA (20 000 nmol, 0 h prior to TPA); (D) double dose of TPA and PA (20 000 nmol, 3 h prior to TPA). Magnification x20.

 
PA causes tyrosinase-dependent reduction of GSH level and antioxidative enzyme activities
We assessed whether redox alteration is involved in the inverse effects of PA on inflammatory responses in mouse skin. Since the treatment of mouse skin with TPA alone showed little influence on total GSH level (15), the modifying effect of PA on GSH level in mouse skin was examined. As shown in Figure 5Go, the GSH level in mouse skin was reduced by treatment with 20000 nmol PA but not by 81nmol PA. This reduction was completely inhibited by co-administration of the tyrosinase inhibitor arbutin (10 µmol), which showed no modulatory effect on GSH level by itself. The possibility that oxidative metabolism of PA to benzoquinone form, which can readily conjugate to nucleophiles such as GSH, prompted us to determine whether treatment with a high dose of PA inhibits enzyme activity due to protein modification such as conjugation with sulfhydryl residue at the active site. GPx and GST are abundant antioxidative enzymes that can diminish cellular H2O2 level. As shown in Table IVGo, treatment with 20 000 nmol PA significantly inhibited both GPx and GST activities (P < 0.05, respectively), and this effect was also counteracted by arbutin.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5. Effects of PA on total GSH level in ICR mouse skin. (A) Time-dependent reduction of total GSH level induced by PA (20 000 nmol). The mice were treated with PA for different time intervals as indicated in the figure and killed for GSH assay. Significance determined by the Student's t-test is expressed as: *, versus acetone, P < 0.01. (B) Counteracting effects of the tyrosinase inhibitor arbutin on PA-induced reduction of GSH level. The mice (five in each group) were treated with arbutin (10 µM) or acetone 1 h prior to PA (81 or 20 000 nmol) treatment. The mice were killed 3 h after PA application, and skin samples were removed for GSH assays. Significance determined by the Student's t-test is expressed as: a, versus acetone, P < 0.01; b, versus PA 20 000 nmol, P < 0.01.

 

View this table:
[in this window]
[in a new window]
  		Table IV. Effects of PA treatment on GPx and GST activities in mouse skin
 
We also examined the inhibitory effects of arbutin on PA-enhanced inflammatory responses and oxidative stress. As shown in Figure 6Go, the tyrosinase inhibitor arbutin counteracted PA-induced enhancement of edema formation (127% of TPA group versus 107%), MPO activity (256 versus 145%, P < 0.05) and H2O2 level (136 versus 120%).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 6. Counteracting effects of arbutin on PA-induced enhancement of inflammatory biomarkers. ICR mice (five in each group) were treated as described in Materials and methods. The mice were treated with arbutin (10 µM) or acetone 1 h prior to PA (81 or 20 000 nmol) treatment. Mice were treated with PA (20 000 nmol) 3 h prior to each TPA treatment. Significance determined by the Student's t-test is expressed as: *, versus acetone + PA + TPA, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The results of the present study demonstrated that topical application of PA exerts contrasting effects on TPA-induced tumor promotion in mouse skin in a manner that is both dose and timing dependent. A low dose of PA (16 nmol; 10-fold dose of TPA) 40 min prior to TPA application significantly decreased both the incidence and the multiplicity of tumors (Table IGo). The dose-dependent inhibitory effect of PA at doses ranging from 0.1-fold (0.16 nmol) to 10-fold (16 nmol) that of TPA was confirmed (data not shown). This inhibitory effect of PA was prominent as compared with known food phytochemicals (9,16–19). On the other hand, the antitumor promotional effect of PA was inversely correlated with the dose ranging from 10- to 1000-fold that of TPA. As the dose of PA was increased, less antitumor promoting activity was observed within this range (Table IGo). Tseng et al. (20) reported that fairly high doses (5, 10 and 20 µmol) of PA 5 min prior to TPA (15 nmol) treatment twice weekly to mice initiated with benzo[a]pyrene inhibited the incidence of tumors by 19–44%. The effective dose of PA in the present study was ~250-fold lower than those reported previously. Interestingly, experiments using two groups treated with 20 µmol (20 000 nmol) of PA 5 min and 3 h prior to TPA treatment clearly demonstrated completely opposite effects on tumor development: the application of PA 5 min before TPA treatment inhibited tumor formation as described previously by Tseng et al. (20), whereas the application of PA 3 h before TPA treatment lacked a tumor inhibitory effect and even enhanced tumor formation (Figure 2Go). Thus, it is very likely that the excessive amount of PA is converted to compound(s) without antioxidative activity or antitumor promotional properties.

There is increasing evidence indicating important roles of oxidative stress in tumor promotion. Tumor promoters such as TPA enhance the generation of ROS and decrease the ROS detoxification enzymes in both epidermal and inflammatory cells. Kensler et al. (21) proposed the requirement of two applications of TPA for massive ROS generation, and the hypothesis that the first treatment of mouse skin with TPA application causes a chemotactic action, i.e. recruitment of neutrophils responsible for ROS generation with the second TPA treatment, i.e. priming and activation, respectively (22). ROS production by double or multiple TPA treatments is closely associated with the metabolic activation of proximate carcinogens (21–23) and the increased levels of oxidized DNA bases (24–26). We recently demonstrated that a potent inhibitor of leukocyte-derived ROS generation (10) effectively prevented inflammation-related carcinogenesis (27–29). The results of the present study clearly demonstrated that application of PA exerted application dose- and timing-dependent inverse effects on TPA-induced inflammatory oxidative stress, trends which were very closely correlated with modifying effects on tumor development. Even a high dose of PA treatment concurrently with TPA significantly inhibited edema formation, MPO activity and H2O2 generation, whereas PA treatment 3 h prior to TPA significantly enhanced these inflammatory parameters. The present result also indicated that although quite high doses of PA showed strong antioxidative effects in lipid peroxidation in vitro (6), PA when applied 3 h prior to TPA treatment significantly enhanced TBARS formation (Figure 3Go). TBARS is known as an overall oxidative damage biomarker formed downstream of H2O2 generation in the presence of a metal ion as a catalyst. TBARS formation in vivo is considered not to reflect a single particular phenomenon but to indicate widespread oxidative damage including lipid peroxidation, mitochondrial de-energization and degradation of protein or sugar rather than DNA (30). Thus, the opposite effects of PA on oxidative stress consequent on inflammatory responses are likely to be, at least in part, determinants for TPA-induced tumor promotion. Histological studies also demonstrated that PA treatment 3 h prior to TPA diminished the inhibitory effects, which were observed with simultaneous application of PA, on TPA-induced inflammatory responses such as leukocyte infiltration in the cutis and PCNA-labeling index, also supporting our assumption that topically applied PA was converted to the plausible toxic compound(s).

Compounds with a catechol moiety such as caffeic acid derivatives are known to be strong radical scavengers in vitro and to show more potent antioxidative activity than monophenolic compounds (31,32). On the other hand, endogenous and exogenous catechols are oxidants as well as electrophiles. The catechols are enzymatically or spontaneously oxidized to ortho-quinones that undergo redox cycling mediated by cytochrome P450/P450 oxide-reductase or transition metals. In addition, ortho-quinones are Michael reaction acceptors that can readily react with nucleophiles such as thiol groups. Thus, ortho-quinone or semiquinone formation from catechols has been suggested to explain their cytotoxic and/or genotoxic effects at high doses, since their molecular mechanisms of action are attributed to ROS generation by redox cycling and covalent binding with a variety of cellular macromolecules (33,34). The former events by high doses of PA may occur on the basis of the results that the pre-treatment of a high dose PA 3 h prior to TPA induced TBARS formation in larger quantities than did treatment with TPA alone. It is well-known that the reaction of catechol semiquinone with oxygen leads to generation of superoxide anions with subsequent Fe(II)/Fe(III)-catalyzed production of hydroxyl radicals (34). Cu(II) also strongly mediates the oxidation of hydroquinone producing benzoquinone and H2O2 through a Cu(I)/Cu(II) redox mechanism (35). The in vitro abilities of PA to induce Fe(II)/Fe(III) redox-cycle-dependent lipid peroxidation and Cu(I)/Cu(II) redox-cycle-dependent oxidative DNA damage have recently been observed (unpublished data), suggesting that the effects of PA are mediated by ROS generation. The oxidative phenomenon of nucleophilic addition to sulfur groups was supported by the findings that a high dose of PA significantly reduced the total level of GSH, the most abundant thiol in cells, which can protect against the attack of electrophilic toxicants (Figure 5Go). GSH is also known to be an endogenous antioxidant, decomposing H2O2 spontaneously or as a substrate for GPx or GST. The present study clearly demonstrated that the in vitro enzyme activities of GPx and GST were reduced by treatment with PA, although excessive GSH was added in the enzyme activity determination systems. This inhibition may be due to nucleophilic attachment of enzyme proteins, since the metabolism of PA to quinone by tyrosinase was required as discussed below. Thus, the disturbance of ROS detoxification systems such as not only GSH but also GPx and GST by the possible electrophilic metabolite(s) of PA may be partially involved in enhanced oxidative stress induced by PA. On the other hand, it is within the range of possibility that PA-derived electrophilic quinones also play some important roles in anticarcinogenic activity of PA. An appropriate amount of electrophiles such as isothiocyanates are known to stimulate phase II enzyme induction to eliminate chemical carcinogens. Moreover, suppressive effects of PA on tumor development, when its dosage is applied only a short time before the TPA application, could be explained by the cytotoxic properties of ortho-quinones attenuating TPA-induced mitogenic effect, whereas the GSH depletion and consequent oxidative stress potentiate the oxidative and thus tumor-promoting action of TPA when applied 3 h before TPA. Although more extensive studies on action mechanisms should be examined, the present results lead us to hypothesize that PA, in both the protective and enhancing dosage regimes, exerts its action through the electrophiles quinone oxidation products.

Compounds with a carboxylic acid (COOH) moiety including benzoic acid are also known to be immediate- and non-immunological-type irritants in skin (36). In addition, the prior treatment of mouse skin with acetic acid, the pKa (acidity) value of which is quite similar to benzoic acid derivatives, increased the yield of tumors initiated by urethane (37). However, the action mechanism remains unclear. As for disturbance of GSH detoxification systems, the involvement of free COOH group could be ruled out, since some catechol compounds having no COOH group exhibited significant GSH consumption (unpublished data). The permissive role of the COOH moiety of PA should be elucidated.

In the present study, the competitive tyrosinase inhibitor arbutin (38) counteracted not only GSH consumption and enzyme activity inhibition by a high dose of PA, but also PA-induced enhancement of inflammatory oxidative stress (Table IVGo; Figures 5 and 6GoGo). Tyrosinase, which catalyzes the hydroxylation of monophenols and the oxidation of catechols to their quinone compartments (39–41) as well as the conversions of L-tyrosine to L-dopa, and L-dopa to L-dopaquinone, is regarded as the rate-limiting steps of mammalian melanin synthesis (42). Actually, we have recently detected significant tyrosinase activity in ICR mouse skin (manuscript in preparation). Evidence for the generation of a superoxide anion through enzymatic action of tyrosinase has been reported (43). As mentioned above, PA induced redox-cycle-dependent lipid peroxidation and oxidative DNA damage, suggesting that PA itself chemically generates ROS. In addition, several phenolic toxicants such as urushiols (poison ivy) cause immunomodulation such as type IV hypersensitivity through hapten formation in skin keratinocytes and Langerhans cells (44). In this case, the reactive quinone intermediate is believed to bind protein sulfhydryl or lysyl residue and its conjugates are recognized as haptens by macrophages. We recently observed that treatment with high doses of PA induced hypersensitivity in mouse skin (manuscript in preparation). In addition, tyrosinase was a rate-limiting factor of inhibition of GST and GPx by PA in vitro. PA alone did not inhibit enzyme activities whereas addition of tyrosinase resulted in inhibition (data not shown). These results suggested that the formation of PA-quinone intermediate(s) may occur by means of tyrosinase. Actually, the in vitro data regarding the oxidation of catechols by tyrosinase, the reactivity of the corresponding quinones with thiol groups and the cytotoxicity of tyrosinase-generated ortho-quinones have been described (40,41). The occurrence of hapten formation in mouse skin treated with PA and the relationship to immunomodulatory effects such as leukocytic MPO activity enhancement or up-regulation of H2O2 generation ability remains to be elucidated. In any case oxidative conversion of PA to PA-quinonoid(s) by tyrosinase is likely to be the critical step in enhanced oxidative stress in mouse skin. In addition, we more recently observed PA-induced enhancement of inflammatory responses and disturbance of GSH detoxification systems in the tumor promotion-resistant mouse strain B6C3F1 (unpublished data), which indicates reduced responses to TPA, such as hyperplasia, leukocyte infiltration and H2O2 generation (45–50). Thus, the possibility that PA is oxidatively activated to more toxic compounds by TPA-sensitive leukocytic MPO in mouse skin may be excluded, although oxidative conversion of a para-alkyl phenol to toxic quinonoids by MPO was reported (51).

Antioxidants have been considered as a double-edged sword in cancer control. Recently, an interesting study on the evaluation of the sensitivity of transgenic mice with overexpression of GPx or both GPx and superoxide dismutase to skin tumor promotion was reported by Lu et al. (52). Surprisingly, these transgenic mice showed an enhanced tumorigenic response to application of DMBA/TPA. The mechanism responsible for this phenomenon is not yet well understood. They, however, have presumed that the altered ROS-detoxification enzyme levels might influence the process of carcinogenesis by modulating cell growth phenotype, increasing resistance of cells with oxidative damage, or by altering immune function. These results indicated the difficulty in regulation of ROS level and in prevention of cancer by radical-scavenging-type antioxidants.

In conclusion, the dose- and timing-dependent effects of phenolic antioxidant application on skin carcinogenesis and oxidative stress were confirmed. Much attention should be paid to the administration dose of the phenolic antioxidants in chemoprevention studies not only in rodent experiments but also in clinical application in humans.


   Notes
 
3 To whom correspondence should be addressed Email: ohigashi{at}kais.kyoto-u.ac.jp Back


   Acknowledgments
 
We thank Dr M.A.Huffman for his critical reading and helpful comments on earlier versions of this manuscript. This study was supported by grants-in-aid for Scientific Research on Priority Areas (Cancer) (H.O.) and JSPS Research Fellow (Y.N.) from the Ministry of Education, Science, Sports and Culture of Japan and by a subsidy from the Asahi Beer Foundation for the Promotion of Science.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Shibata,A., Paganini-Hill,A., Ross,R.K. and Henderson,B.E. (1992) Intake of vegetables, fruits, ß-carotene, vitamin C and vitamin supplements and cancer incidence among the elderly: a prospective study. Br. J. Cancer, 66, 673–679.[ISI][Medline]
  2. Weisburger,J.H. (1991) Effective human cancer prevention as goal for the regulation of environmental chemicals. Environ. Carcinog. Ecotoxicol. Rev., C8, 339–351.
  3. Ho,C.-T. (1992) Phenolic compounds in food: an overview. In Huang, M.-T., Ho,C.-T. and Lee,C.Y. (eds) Phenolic Compounds in Food and Their Effects on Health II: Antioxidants and Cancer Prevention, American Chemical Society Symposium Series 507. American Chemical Society, Washington, DC, pp. 2–7.
  4. Ito,N. and Hirose,M. (1989) Antioxidants-carcinogenic and chemo- preventive properties. Adv. Cancer Res., 53, 247–302.[ISI][Medline]
  5. Ogawa,K., Futakuchi,M., Hirose,M., Boonyaphiphat,P., Mizoguchi,Y., Miki,T. and Shirai,T. (1998) Stage and organ dependent effects of 1-O-hexyl-2,3,5-trimethylhydroquinone, ascorbic acid derivatives, N-heptadecane-8,10-dione and phenylethyl isothiocyanate in a rat multiorgan carcinogenesis model. Int. J. Cancer, 76, 851–856.[ISI][Medline]
  6. Ueda,J., Saito,N., Shimazu,Y. and Ozawa,T. (1996) A comparison of scavenging abilities of antioxidants against hydroxyl radicals. Arch. Biochem. Biophys., 333, 377–384.[ISI][Medline]
  7. Tanaka,T., Kojima,T., Suzui,M. and Mori,H. (1993) Chemoprevention of colon carcinogenesis by the natural product of a simple phenolic compound protocatechuic acid: suppressing effects on tumor development and biomarkers expression of colon tumorigenesis. Cancer Res., 53, 3908–3913.[Abstract/Free Full Text]
  8. Tanaka,T., Kawamori,T., Ohnishi,M., Okamoto,K., Mori,H. and Hara,A. (1994) Chemoprevention of 4-nitroquinoline 1-oxide-induced oral carcinogenesis by dietary protocatechuic acid during initiation and postinitiation phases. Cancer Res., 54, 2359–2365.[Abstract/Free Full Text]
  9. Nakamura,Y., Kawamoto,N., Ohto,Y., Torikai,K., Murakami,A. and Ohigashi,H. (1999) A diacetylenic spiroketal enol ether epoxide, AL-1, from Artemisia lactiflora inhibits 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion possibly by suppression of oxidative stress. Cancer Lett., 140, 37–45.[ISI][Medline]
  10. Nakamura,Y., Murakami,A., Ohto,Y., Torikai,K., Tanaka,T. and Ohigashi,H. (1998) Suppression of tumor promoter-induced oxidative stress and inflammatory responses in mouse skin by a superoxide generation inhibitor 1'-acetoxychavicol acetate. Cancer Res., 58, 4832–4839.[Abstract/Free Full Text]
  11. Nakamura,Y., Ohto,Y., Murakami,A., Osawa,T. and Ohigashi,H. (1998) Inhibitory effects of curcumin and tetrahydrocurcuminoids on the tumor promoter-induced reactive oxygen species generation in leukocytes in vitro and in vivo. Jpn. J. Cancer Res., 89, 361–370.[ISI][Medline]
  12. Nakamura,Y., Murakami,A., Koshimizu,K. and Ohigashi,H. (1996) Inhibitory effects of pheophorbide a, a chlorophyll-related compound, on skin tumour promotion in ICR mouse skin. Cancer Lett., 108, 247–255.[ISI][Medline]
  13. Wei,H., Wei,L., Frenkel,K., Bowen,R. and Barnes,S. (1993) Inhibition of tumour promoter-induced hydrogen peroxide formation in vitro and in vivo by genistein. Nutr. Cancer, 20, 1–12.[ISI][Medline]
  14. Habig,W.H., Pabst,M.J. and Jakoby,W.B. (1974) Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249, 7130–7139.[Abstract/Free Full Text]
  15. Reiners,J.J.,Jr, Kodari,E., Cappel,R.E. and Gilbert,H.F. (1991) Assessment of the antioxidant/prooxidant status of murine skin following topical treatment with 12-O-tetradecanoylphorbol-13-acetate and throughout the ontogeny of skin cancer. Part II: quantitation of glutathione and glutathione disulfide. Carcinogenesis, 12, 2345–2352.[Abstract/Free Full Text]
  16. Huang,M.-T., Smart,R.C., Wong,C.-Q. and Conney,A.H. (1988) Inhibitory effect of curcumin, chlorogenic acid, caffeic acid and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res., 48, 5941–5946.[Abstract/Free Full Text]
  17. Nishino,H., Iwashima,A., Fujiki,H. and Sugimura,T. (1984) Inhibition by quercetin of the promoting effect of teleocidin on skin papilloma formation in mice initiated with 7,12-dimethylbenz[a]anthracene. Jpn. J. Cancer Res., 75, 113–116.
  18. Murakami,A., Nakamura,Y., Koshimizu,K. and Ohigashi,H. (1995) Glyceroglycolipids from Citrus hystrix, a traditional herb in Thailand, potently inhibits the tumor promoting activity of 12-O-tetra- decanoylphorbol-13-acetate in mouse skin. J. Agric. Food Chem., 43, 2779–2783.
  19. Murakami,A., Ohura,S., Nakamura,Y., Koshimizu,K. and Ohigashi,H. (1996) 1'-Acetoxychavicol acetate, a superoxide anion generation inhibitor, potently inhibits tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in ICR mouse skin. Oncology, 53, 386–391.[ISI][Medline]
  20. Tseng,T.-H., Hsu,J.-D., Lo,M.-H., Chu,C.-Y., Chou,F.-P., Huang,C.-L. and Wang,C.-J. (1998) Inhibitory effect of Hibiscus protocatechuic acid on tumor promotion in mouse skin. Cancer Lett., 126, 199–207.[ISI][Medline]
  21. Kensler,T.W., Egner,P.A., Moore,K.G., Taffe,B.G., Twerdok,L.E. and Trush,M.A. (1987) Role of inflammatory cells in the metabolic activation of polycyclic aromatic hydrocarbons in mouse skin. Toxicol. Appl. Pharmacol., 90, 337–346.[ISI][Medline]
  22. Ji,C. and Marnett,L.J. (1992) Oxygen radical-dependent epoxidation of (7S,8S)-dihydroxy-7,8-dihydrobenzo[a]pyrene in mouse skin in vivo. Stimulation by phorbol esters and inhibition by antiinflammatory steroids. J. Biol. Chem., 267, 17842–17878.[Abstract/Free Full Text]
  23. Kensler,T.W., Egner,P.A., Taffe,B.G. and Trush,M.A. (1989) Role of free radicals in tumour promotion and progression. In Slaga,T.J., Klein-Szanto,A.J.P., Boutwell,R.K., Stevenson,D.E., Spitzer,H.L. and D'Motto,B. (eds) Skin Carcinogenesis, Mechanisms and Human Relevance. Alan R. Liss, Inc., New York, NY, pp. 233–248.
  24. Wei,H. and Frenkel,K. (1991) In vivo formation of oxidized DNA bases in tumor promoter-treated mouse skin. Cancer Res., 51, 4443–4449.[Abstract/Free Full Text]
  25. Wei,H. and Frenkel,K. (1992) Suppression of tumor promoter-induced oxidative events and DNA damage in vivo by sarcophytol A: a possible mechanism of antipromotion. Cancer Res., 52, 2298–2303.[Abstract/Free Full Text]
  26. Wei,H. and Frenkel,K. (1993) Relationship of oxidative events and DNA oxidation in SENCAR mice to in vivo promoting activity of phorbol ester-type tumor promoters. Carcinogenesis, 14, 1195–1201.[Abstract/Free Full Text]
  27. Ohnishi,M., Tanaka,T., Makita,H., Kawamori,T., Mori,H., Satoh,K., Hara,A., Murakami,A., Ohigashi,H. and Koshimizu,K. (1996) Chemopreventive effect of a xanthine oxidase inhihitor, 1'-acetoxychavicol acetate, on rat oral carcinogenesis. Jpn. J. Cancer Res., 87, 349–356.[ISI][Medline]
  28. Tanaka,T., Makita,H., Kawamori,T., Kawabata,K., Mori,H., Murakami,A., Satoh,K., Hara,A., Ohigashi,H. and Koshimizu,K. (1997) A xanthine oxidase inhibitor 1'-acetoxychavicol acetate inhibits azoxymethane-induced colonic aberrant crypt foci in rats. Carcinogenesis, 18, 1113–1118.[Abstract/Free Full Text]
  29. Tanaka,T., Kawabata,K., Kakumoto,M., Makita,H., Matsunaga,K., Mori,H., Satoh,K., Hara,A., Murakami,A., Ohigashi,H. and Koshimizu,K. (1997) Chemoprevention of azoxymethane-induced rat colon carcinogenesis by a xanthine oxidase inhibitor, 1'-acetoxychavicol acetate. Jpn. J. Cancer Res., 88, 821–830.[ISI][Medline]
  30. Nakae,D., Mizumoto,Y., Yoshiji,H., Andoh,N., Horiguchi,K., Shiraiwa,K., Kobayashi,E., Endoh,T., Shimoji,N., Tamura,K. et al. (1994) Different roles of 8-hydroxyguanine formation and 2-thiobarbituric acid-reacting substance generation in the early phase of liver carcinogenesis induced by a choline-deficient, L-amino acid-defined diet in rats. Jpn. J. Cancer Res., 85, 499–505.[ISI][Medline]
  31. Tsuda,T., Shiga,K., Ohshima,K., Kawakishi,S. and Osawa,T. (1996) Inhibition of lipid peroxidation and the active oxygen radical scavenging effect of anthocyanin pigments isolated from Phaseolus vulgaris L. Biochem. Pharm., 52, 1033–1039.[ISI][Medline]
  32. Nakamura,Y., Ohto,Y., Murakami,A. and Ohigashi,H. (1998) Superoxide scavenging activity of rosmarinic acid from Perilla frutescens Britton var. acuta f. viridis. J. Agric. Food Chem., 46, 4545–4550.
  33. Monks,T.J. and Lau,S.S. (1997) Biological reactivity of polyphenolic–glutathione conjugates. Chem. Res. Toxicol., 10, 1296–1313.[ISI][Medline]
  34. Monks,T.J., Hanzlik,R.P., Cohen,G.M., Ross,D. and Graham,D.G. (1992) Comtemporary issues in toxicology. Quinone chemistry and toxicology. Toxicol. Appl. Pharmacol., 112, 2–16.[ISI][Medline]
  35. Li,Y., Trush,M.A. and Yager,J.D. (1994) DNA damage caused by reactive oxygen species originating from a copper-dependent oxidation of the 2-hydroxy catechol of estradiol. Carcinogenesis, 15, 1421–1427.[Abstract/Free Full Text]
  36. Lahti,A., Pylvanen,V. and Hannuksela,M. (1995) Immediate irritant reactions to benzoic acid are enhanced in washed skin areas. Contact Dermatitis, 33, 177–182.[ISI][Medline]
  37. Pound,P.A. (1966) Further observations concerning the influence of preliminary stimulation by croton oil and acetic acid on the intiation of skin tumours in mice by urethan. Br. J. Cancer, 8, 666–671.
  38. Maeda,K. and Fukuda,M. (1996) Arbutin: Mechanism of its depigmenting action in human melanocyte culture. J. Pharmacol. Exp. Ther., 276, 765–769.[Abstract/Free Full Text]
  39. Mayer,A.M. (1987) Polyphenol oxidases in plants: recent progress. Phytochemistry, 26, 11–20.
  40. Cooksey,C.J., Land,E.J., Rushton,F.A.P., Ramsden,C.A. and Riley,P.A. (1996) Tyrosinase-mediated cytotoxicity of 4-substituted phenols: Use of QSAR to forecast reactivities of thiols towards the derived ortho-quinones. Quant. Struct. Act. Relat., 15, 498–503.
  41. Riley,P.A., Cooksey,C.J., Johnson,C.J., Land,E.J., Latter,A.M. and Ramsden,C.A. (1997) Melanogenesis-targeted anti-melanoma pro-drug development: Effect of side-chain variations on the cytotoxicity of tyrosinase-generated ortho-quinones in a model screening system. Eur. J. Cancer, 33, 135–143.
  42. Hearing,V.J. and Jiménez,M. (1987) Mammalian tyrosinase, the critical regulatory control point in melanocyte pigmentation. Int. J. Biochem., 19, 1141–1147.[ISI][Medline]
  43. Koga,S., Nakano,M. and Tero-Kubota,S. (1992) Generation of superoxide during the enzymatic action of tyrosinase. Arch. Biochem. Biophys., 292, 570–575.[ISI][Medline]
  44. Park,B.K., Pirmohamed,M. and Kitteringham,N.R. (1998) Role of drug disposition in drug hypersensitivity: A chemical, molecular and clinical perspective. Chem. Res. Toxicol., 11, 970–988.
  45. Fischer,S.M., Jasheway,D.W., Klann,R.C., Butler,A.P., Patrick,K.E., Baldwin,J.K. and Cameron,G.S. (1989) Correlation of phorbol ester promotion in the resistant C57BL/6J mouse with sustained hyperplasia, but not with ornithine decarboxylase or protein kinase C. Cancer Res., 49, 6693–6699.[Abstract/Free Full Text]
  46. Sisskin,E.E., Gray,T. and Barrett,J.C. (1982) Correlation between sensitivity to tumor promotion and sustained epidermal hyperplasia of mice and rats treated with 12-O-tetradecanoylphorbol-13-acetate. Carcinogenesis, 3, 403–407.[Abstract/Free Full Text]
  47. Mills,K.J. and Smart,R.C. (1989) Comparison of epidermal protein kinase C activity, ornithine decarboxylase induction and DNA synthesis stimulated by TPA or dioctanoylglycerol in mouse strains with different susceptibility to TPA-induced tumor promotion. Carcinogenesis, 10, 833–838.
  48. Fischer,S.M., Baldwin,J.K., Jasheway,D.W., Patrick,K.E. and Cameron, G.S. (1988) Phorbol ester induction of 8-lipoxygenase in inbred SENCAR (SSIN) but not C57BL/6J mice correlated with hyperplasia, edema and oxidant generation but not ornithine decarboxylase induction. Cancer Res., 48, 658–664.[Abstract/Free Full Text]
  49. Yoon,H.L., Marcus,C.B. and Pfeifer,R.W. (1993) Induction of superoxide by 12-O-tetradecanoylphorbol-13-acetate and thapsigargin, a non-phorbol-ester-type tumor promoter, in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: a permissive role for the arachidonic acid cascade in signal transduction. Mol. Carcinog., 7, 116–125.[ISI][Medline]
  50. Wei,L., Wei,H. and Frenkel,K. (1993) Sensitivity to tumor promotion of SENCAR and C57BL/6J mice correlates with oxidative events and DNA damage. Carcinogenesis, 14, 841–847.[Abstract/Free Full Text]
  51. Thompson,D., Constantin-Teodosiu,D., Norbeck,K., Svensson,B. and Moldéus,P. (1989) Metabolic activation of eugenol by myeloperoxidase and polymorphonuclear leukocytes. Chem. Res. Toxicol., 2, 186–192.[ISI][Medline]
  52. Lu,Y.-P., Lou,Y.-R., Yen,P., Newmark,H.L., Mirochnitchenko,O.I., Inouye,M. and Huang,M.-T. (1997) Enhanced skin carcinogenesis in transgenic mice with high expression of glutathione peroxidase or both glutathione peroxidase and superoxide dismutase. Cancer Res., 57, 1468–1474.[Abstract/Free Full Text]
Received April 19, 2000; revised June 15, 2000; accepted June 16, 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
 CarcinogenesisHome page
N. Miyoshi, S. Takabayashi, T. Osawa, and Y. Nakamura
Benzyl isothiocyanate inhibits excessive superoxide generation in inflammatory leukocytes: implication for prevention against inflammation-related carcinogenesis
Carcinogenesis, April 1, 2004; 25(4): 567 - 575.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
J. S. Suzuki, N. Nishimura, B. Zhang, Y. Nakatsuru, S. Kobayashi, M. Satoh, and C. Tohyama
Metallothionein deficiency enhances skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate in metallothionein-null mice
Carcinogenesis, June 1, 2003; 24(6): 1123 - 1132.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
K. R. Martin, C. Trempus, M. Saulnier, F. W. Kari, J.C. Barrett, and J. E. French
Dietary N-acetyl-L-cysteine modulates benzo[a]pyrene-induced skin tumors in cancer-prone p53 haploinsufficient Tg.AC (v-Ha-ras) mice
Carcinogenesis, September 1, 2001; 22(9): 1373 - 1378.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Nakamura, Q. Feng, T. Kumagai, K. Torikai, H. Ohigashi, T. Osawa, N. Noguchi, E. Niki, and K. Uchida
Ebselen, a Glutathione Peroxidase Mimetic Seleno-organic Compound, as a Multifunctional Antioxidant. IMPLICATION FOR INFLAMMATION-ASSOCIATED CARCINOGENESIS
J. Biol. Chem., January 18, 2002; 277(4): 2687 - 2694.
[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