1 Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
2 Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA
3 Department of Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA
4 To whom correspondence should be addressed Email: skatiyar{at}uab.edu
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Abstract |
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Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; DXA, dual-energy X-ray absorptiometry; GSP, grape seed polyphenols; LPO, lipid peroxidation; TPA, 12-O-tetradecanoylphorbol-13-acetate; TBMD, total bone mineral density; TBMC, total bone mineral content; UV, ultraviolet
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Introduction |
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Chemoprevention refers to the prevention of diseases through dietary manipulation or pharmacological intervention. It is important to mention that worldwide interest is considerably increasing on the use of naturally occurring dietary supplements, which can be used as chemopreventive agents and/or as complementary and alternative medicine. Ideally, such chemopreventive agents should be non-toxic in nature. In our continuing efforts to identify newer and effective dietary botanicals, which can inhibit solar UV radiation-induced skin cancers, studies were performed to assess the anti-photocarcinogenic efficacy of proanthocyanidins isolated from grape seeds (hereafter referred to as GSP) using animal skin tumor model. Grapes are widely consumed all over the world and are rich in polyphenolic compounds also called procyanidins or proanthocyanidins (7). Preliminary studies have shown that proanthocyanidins from grape seeds have anti-inflammatory (8), and anti-oxidant properties (911). Arii et al. (12) have shown that oral administration of 1% grape seed extract in the diet to Min mice inhibits APC mutation-associated intestinal adenoma formation. Topical treatment of GSP has been shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated CD-1 mice skin (13). Inhibition of tumor promotion by GSP treatment was also associated with the inhibition of TPA-induced ornithine decarboxylase and myeloperoxidase activities (13), which indicates the antitumor promoting properties of GSP. Zhao et al. (14) have shown that topical treatment of GSP to SENCAR mice skin significantly inhibited tumor incidence, tumor multiplicity and tumor size in TPA-promoted and DMBA-initiated mouse skin model.
Since the incidence of solar UV radiation-induced skin cancer (photocarcinogenesis) is higher among American people, particularly in Caucasians, compared with chemical carcinogen-initiated skin cancers, we tested the chemopreventive efficacy of GSP on photocarcinogenesis in vivo animal model. Moreover, in recent years, the human population is constantly and increasingly using complementary and alternative medicine prepared or developed from naturally occurring dietary botanicals. Therefore, the present study was designed and conducted to determine whether dietary feeding of GSP would prevent UVB radiation induced (i) photocarcinogenesis in terms of tumor incidence, tumor multiplicity and tumor size and (ii) malignant conversion of benign papillomas to carcinomas in SKH-1 hairless mouse model. To accomplish this study, GSP (0.2 and 0.5%, w/w) was given in an AIN76 control diet to mice. In these experiments, the effect of GSP was determined on different stages of photocarcinogenesis, such as, (i) UVB-induced tumor initiation stage, (ii) UVB-induced tumor promotion stage and (iii) UVB-induced complete carcinogenesis stage (included both UVB-induced tumor initiation and promotion protocols). The rationale for the selection of these three different protocols was to dissect out the protective effects of GSP at the different stages of photocarcinogenesis. Additionally, we were also interested in determining whether (iv) the feeding of GSP to animals prevents UV radiation-induced oxidative damage (e.g. lipid peroxidation) and (v) long-term intake of GSP in the diet has any apparent toxic effect in animals. For this purpose, the effect of dietary feeding of GSP was determined on the body weight and in vivo body composition like bone free soft-lean tissue mass (LTM), fat mass (FM), total bone mineral content (TBMC) and total bone mineral density (TBMD) in experimental mice and compared with that of non-GSP-fed animals. These parameters were determined by using dual-energy X-ray absorptiometry (DXA) (GE-Lunar PIXImus, software version 1.44, Madison, WI).
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Materials and methods |
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Chemicals and GSP
DMBA and TPA were purchased from Sigma Chemical Co. (St Louis, MO). All chemicals employed in this study were of analytical grade and purchased from Sigma Chemical Co. Purified GSP was provided as a gift by Kikkoman Corporation, Japan, for this study. Chemical composition of GSP was analyzed by Kikkoman Corporation and given in Table I.
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UVB irradiation of mice and UVB light source
UVB irradiation of mice was performed as described previously (15). Briefly, the dorsal skin area of the mice was exposed to UVB radiation from a band of four FS-20 fluorescent lamps from which short wavelengths of UVB (280290 nm) and UVC normally not present in natural solar light were filtered out using Kodacel cellulose film (Eastman Kodak Co., Rochester, NY). After filtration with a Kodacel film, the majority of the resulting wavelengths of UV radiation were in UVB (290320 nm) and UVA range with peak emission at 314 nm as monitored. The UVB emission was monitored with an IL1700 phototherapy radiometer equipped with an IL SED 240 detector fitted with a W side angle quartz diffuser and a SC5 280 filter (all from International Light, Newburyport, MA).
Photocarcinogenesis experiment and protocol
One week after their arrival in the animal facility, the mice were divided into different treatment groups with 20 mice in each group. Three long-term tumorigenesis protocols were employed to determine the photoprotective effect of GSP: (i) UVB-induced complete carcinogenesis protocol (included both UVB-induced tumor initiation and promotion stages), (ii) UVB-induced tumor initiation and (iii) UVB-induced tumor promotion as detailed previously (15,16). Briefly, these protocols are summarized below.
Anti-complete photocarcinogenesis protocol
For the studies to determine whether dietary feeding of GSP will inhibit UVB-induced complete carcinogenesis, mice in group 1 were given AIN76 control diet while groups 2 and 3 were given GSP at the dose of 0.2 and 0.5% (w/w), respectively, in the AIN76 diet throughout the experimental protocol to determine the dose-dependent effect of GSP on complete photocarcinogenesis protocol. Mice in groups 2 and 3 were given GSP for 14 days, thereafter on day 15 the mice belonging to groups 1, 2 and 3 were irradiated every day with UVB (180 mJ/cm2) and continued for a total of 10 days. Thus, the total UVB-irradiation dose during tumor initiation was 1800 mJ/cm2 fractionated equally in 10 days. One week after the last UVB exposure of initiation, the mice were again UVB irradiated with the same dose (180 mJ/cm2) three times a week for total 30 weeks from the last UVB exposure, which served as the tumor promoter. Tumor yield and size stabilized at 24 weeks, but to determine the effect of GSP on malignant conversion of papillomas to carcinoma, this experiment was continued until week 30. The fourth group of mice received 1% GSP in diet but did not receive any other treatment to determine whether dietary feeding of GSP alone has any effect on tumor formation.
Anti-initiation protocol
For studies to determine the effect of GSP on UVB-induced initiation stage of photocarcinogenesis, one group of mice was given the AIN76 control diet and a second group was given GSP (0.5%, w/w) in the AIN76 diet. As 0.5% GSP in diet was more chemopreventive than 0.2% GSP to prevent skin tumorigenesis, in further experiments 0.5% dietary feeding of GSP was used. Fourteen days after GSP feeding regimen, on day 15, mice in both groups were UVB irradiated (180 mJ/cm2) everyday for a total of 10 days. Thus, the total UVB dose of 1800 mJ/cm2 was delivered on the dorsal skin of each mouse in 10 days. One week after the last UVB exposure, animals in both groups were treated topically with 10 nmol of TPA as a tumor promoter in 100 µl acetone per mouse per application (15). The TPA treatment was given thrice a week up to the end of 24 weeks of the experiment following the last UVB exposure.
Anti-promotion protocol
For studies to determine the effect of dietary feeding of GSP on UVB-induced tumor promoting stage of photocarcinogenesis, one group of mice was given the AIN76 control diet, and the second group of mice was given GSP (0.5%, w/w) in the AIN76 diet only during tumor promotion stage of photocarcinogenesis. Before tumor promotion, the mice in the second group (GSP + AIN76) received only the AIN76 diet as in group 1. The mice in this protocol were topically treated with a single application of 200 nmol (51.2 µg) of DMBA (Aldrich Chemical Co., Milwaukee, WI) in 100 µl of acetone per mouse (15). One week after tumor initiation with DMBA, the mice in both groups were UVB irradiated (180 mJ/cm2) three times a week to achieve tumor promotion phase until 24 weeks from the start of the UVB irradiation.
During the experimental protocols, mice were regularly monitored for food and water consumption and any apparent signs of toxicity, such as weight loss or mortality. The body weight of mice in each group was recorded fortnightly throughout the experimental protocol and their dietary intake was also recorded weekly. Skin papillomas or suspected carcinomas were recorded weekly. The tumors >1 mm in diameter that persisted for 2 weeks or more were recorded. Tumor data were recorded until 24 weeks when their yield and size were stabilized. At this time point, the dimensions of all of the tumors on each mouse were recorded. The tumor volumes were calculated by the hemiellipsoid model formula: tumor volume = 1/2 (4/3) x (l/2) x (w/2) x h, where l = length, w = width and h = height, as followed earlier (15). The carcinoma incidence and multiplicity were recorded until 30 weeks of the experimental protocol. At this time, carcinoma incidence and multiplicity were stable. The diagnosis of carcinoma was confirmed histologically either at the time when carcinoma bearing mice died or at the termination of the experiment at 30 weeks. Moreover, because of ulcerations and larger size of carcinomas, the Institutional Animal Care and Use Committee (IACUC) at University of Alabama at Birmingham, Birmingham, did not allow these experiments for a longer time, therefore, experiments were stopped at this stage.
Assay of lipid peroxidation
To determine whether anti-carcinogenic activity of GSP is mediated through the inhibition of UVB-induced oxidative damage of the skin, lipid peroxidation (LPO) was estimated in the epidermal microsomal fraction of the skin as described previously (17,18). Briefly, LPO was measured in terms of the formation of thiobarbituric acid, and the color intensity was measured at 530 nm on a spectrophotometer. In in vitro LPO estimation with GSP and other antioxidants, these agents were dissolved in equal amount of acetone and added to the incubation mixture prior to the start of the incubation. Data were calculated and presented as percent inhibition of lipid peroxidation by the treatment of GSP or other antioxidants.
Evaluation of non-toxicity of dietary feeding of GSP
Physico-chemical observations were recorded to determine the non-toxic nature of dietary feeding of GSP to the animals. To evaluate the non-toxic nature of GSP in the diet, we determined weekly consumption of diet and fortnightly body weight per mouse and compared with the control, non-GSP-fed group of mice. At the termination of the photocarcinogenesis experiment at 24 or 30 weeks, mice were killed, internal body organs like spleen, liver and kidney were dissected out and observations were recorded to test the apparent toxicity of GSP, if any, in terms of organ weights and/or lengths.
Determination of in vivo body composition of mice by DXA
To assess the non-toxicity of long-term dietary feeding of GSP in mice, we also determined the effect of dietary feeding of GSP on the bone free soft LTM, FM, TBMC and TBMD in experimental mice and compared with non-GSP-fed animals. At the end of photocarcinogenesis experiment at week 30, mice were killed and kept at -80°C until they were analyzed for these parameters. These parameters were determined by using DXA as described previously (19). The head of the mouse was not included in any of these measures, and the DXA analysis was done on thawed animals.
Statistical analysis
In tumorigenesis experiments, the statistical significance of difference between UVB alone and GSP + UVB treatment groups in terms of tumor incidence and tumor multiplicity was evaluated by the 2 analysis and Wilcoxon rank sum test, respectively. An advantage of Wilcoxon rank sum test is that its validity does not depend on any assumption about the shape of the distribution of tumor multiplicities. Kinetics of tumor multiplicity was analyzed by using FisherIrwin exact test. Student's t-test was used to determine the statistical significance of difference in lipid peroxidation, tumor volume and fat content data analysis between UVB alone irradiated and GSP + UVB-irradiated groups of animals.
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Results |
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Dietary feeding of GSP prevents photocarcinogenesis
The chemopreventive effect of dietary feeding of GSP was evaluated in three different protocols of photocarcinogenesis, as described below.
Prevention against complete photocarcinogenesis
Dietary feeding of GSP (0.2 and 0.5%, w/w) during complete photocarcinogenesis protocol resulted in a dose-dependent reduction in photocarcinogenesis when expressed in terms of percent of mice with tumors and tumor multiplicity compared with that of non-GSP-fed but UVB-irradiated control animals, as shown in Figure 1 (top row). Feeding of GSP at the dose of 0.2 and 0.5% resulted in 20 (not significant) and 35% (P< 0.05) inhibition of tumor incidence (percent of mice with tumors), respectively, at the termination of the experiment at 24 weeks as compared with non-GSP-fed animals (Figure 1, top row, left panel). Non-GSP-fed animals achieved 100% tumor incidence at the 16th week of tumor promotion while GSP-fed animals could not achieve 100% tumor incidence up to the end of the 24th week when tumor incidence was stabilized. Further, feeding of GSP increased the latency period of tumors by 2 weeks during a 24 week long tumor protocol. In complete photocarcinogenesis protocol, a total of 360 tumors (18 ± 2.5 tumors/tumor bearing animal) were recorded in the non-GSP-fed group of 20 animals while 192 (12 ± 2 tumors/tumor bearing animal) and 126 tumors (9.7 ± 2 tumors/tumor bearing animal) were recorded with dietary feeding of 0.2 and 0.5% of GSP, respectively, in the animals. Thus, feeding of 0.2 and 0.5% GSP significantly inhibited tumor multiplicity by 46 (P<0.05) and 65% (P<0.005), respectively, as compared with non-GSP-fed animals, as shown in Figure 1 (top row, right panel) and Table II. Further, the kinetics of tumor multiplicity in GSP-fed (0.5%) animals was significantly prevented (P<0.05, Fisher-Irwin exact test) throughout the experimental protocol compared with the non-GSP-fed control group. As shown in Table II, dietary feeding of 0.2 and 0.5% GSP significantly inhibited tumor size when expressed in terms of total tumor volume per group (6678%, P<0.001) or tumor volume per tumor bearing mouse by 5766% (P<0.001). When data were compared in terms of average tumor volume per tumor between GSP-fed and non-GSP-fed groups, a significant reduction was observed by 0.2 and 0.5% GSP (35%, P<0.05) as shown in Table II. Further, a group of animals was subjected to dietary feeding of GSP (1.0%, w/w) alone for the whole experimental protocol to determine whether feeding of GSP had any effect on tumor incidence. At the termination of the experiment at 24 weeks, we were unable to record any tumor appearance on these animals. Thus, these observations indicated that long-term feeding of GSP alone did not induce a carcinogenic effect in laboratory animals.
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Prevention against UVB-induced tumor promotion
Tumor promotion stage is a reversible stage of multistage carcinogenesis, therefore, is most suitable for anticarcinogenic agents to prevent, reverse or slow down the process of carcinogenesis. Following DMBA-initiation and UVB-induced tumor promotion protocol, latency period of tumors was reduced by 3 weeks compared with anti-complete and anti-initiating protocol as shown in Figure 1. The animals in the control group (non-GSP-fed) achieved 100% tumor incidence at 15th week while GSP-fed animals achieved only 60% tumor incidence at this time, and 70% tumor incidence was recorded at the end of this protocol at 24 weeks, as shown in Figure 1 (bottom row, left panel). Thus, dietary feeding of GSP prevented tumor incidence by 30%. When tumor data were analyzed in terms of tumor multiplicity, a total of 178 tumors (8.9 ± 2 tumors/tumor bearing animal) were recorded in the non-GSP-fed group of animals compared with 71 tumors (5.1 ± 2 tumors/tumor bearing animal) in the GSP-fed group of animals. Thus, dietary feeding of GSP resulted in a 60% reduction (P<0.005) in the total number of tumors per group. Additionally, tumor data were also analyzed in terms of tumor size in both treatment groups. As shown in Table II, dietary feeding of GSP resulted in 74% reduction (P<0.001) in terms of total tumor volume per group or 63% reduction (P<0.001) in terms of tumor volume per tumor bearing mouse compared with the non-GSP-fed group of animals. Further feeding of GSP to UVB-irradiated mice also resulted in a 29% reduction (P<0.05) in terms of average tumor volume per tumor compared with non-GSP-fed and UVB-irradiated group of control mice.
Prevention of malignant conversion of papillomas to carcinomas
After observing that dietary feeding of GSP significantly prevented UVB radiation-induced skin tumorigenesis in different photocarcinogenesis protocols employed, we were interested to look at the chemopreventive efficacy of GSP on malignant conversion of papillomas into carcinomas. Again, because dietary feeding of 0.5% GSP was more highly chemopreventive than that of 0.2% GSP, we continued these experiments with only 0.5% GSP treatment, and only against complete photocarcinogenesis protocol. This experiment was continued up to 30 weeks of tumor promotion. As shown in Figure 2, histological observations indicated that papillomas had started transforming into carcinomas at 21 weeks of tumor promotion. As evidenced by Figure 2, 70% mice developed carcinoma in non-GSP-fed control mice compared with only 25% in GSP-fed group of mice. Thus, 45% (P<0.05) prevention in terms of carcinoma incidence was observed by dietary feeding of GSP, as shown in Figure 2 (top panel). The weekly progression of carcinoma multiplicity in terms of total number of carcinomas per group and number of carcinomas per carcinoma bearing mouse is shown in the middle and bottom panels, respectively, of Figure 2. When the data were analyzed in terms of carcinoma multiplicity, we found that a total 18 papillomas were converted into carcinomas in non-GSP-fed but UVB-irradiated group of mice in comparison to only seven in GSP + UVB-irradiated group of mice. Histologically, these carcinomas were distinguished as keratoacanthomas and squamous cell carcinomas (data not shown). The data indicated that dietary feeding of GSP resulted in prevention of UVB-induced transformation of benign papillomas to carcinomas by 61% (P<0.005), as shown in Table III. But when the data were analyzed in terms of number of carcinomas per carcinoma bearing mouse, the protective effect of GSP was not evident (Figure 2, bottom panel). Further, dietary feeding of GSP inhibited the growing size of carcinoma. As evidenced by the analysis of physical characteristics of the carcinomas shown in Table III, feeding of GSP to UVB-irradiated mice resulted in a significant reduction in total carcinoma volume per group and average carcinoma volume per carcinoma by 75 (P<0.001) and 36% (P<0.05), respectively, compared with the UVB alone-irradiated group of mice.
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Dietary feeding of GSP is non-toxic to the animals
Further, we were interested to determine whether long-term feeding of GSP has any apparent toxic effect in animals. For this assessment, we determined and compared physico-chemical characteristics of animals between GSP-fed and non-GSP-fed groups of mice. To determine any adverse effect caused by dietary feeding of GSP treatment on body weight loss or extra weight gain as a marker of apparent toxicity, animals in each group, with and without GSP treatment, were weighed fortnightly. We observed that there was no apparent loss in body weights or extra weight gain in those animals, which were given dietary feeding of GSP compared with non-GSP-fed animals over the whole period of the experiment (data not shown). These observations demonstrated that mice treated with GSP (both 0.2 and 0.5%, w/w) did not produce toxicity in them as it would have been manifested by loss in their weight or gain in body weight. Additionally, there was no significant difference in water or total diet consumption per mouse per day among the animals of different treatment groups (data not shown), further suggesting that dietary feeding of GSP has no adverse effect on diet consumption. The data also reflected that experimental animals did not dislike the feeding of GSP in the diet and therefore it did not affect their appetite. Further, we evaluated the adverse physical effect of dietary feeding of GSP, if any, on internal body organs of the animals. At the termination of the experiment at week 30, animals were killed and internal body organs like spleen, liver and kidney were dissected out and their weights or lengths were measured. There was no significant difference in weights or lengths of these internal body organs among GSP-fed and non-GSP-fed groups of animals (data not shown). However, UVB irradiation alone to animals increased spleen length and weight when compared with non-UVB-irradiated animals. It appeared that UVB irradiation did induce a kind of toxicity in the spleens of experimental animals, which resulted in larger size and weight of spleen. Dietary feeding of GSP alone to animals did not affect the size of spleen but at the same time feeding of GSP to UVB-irradiated animals resulted in reduction of UVB-induced increase in weight of the spleens (2431%, P<0.05) but did not significantly reduce the length of the spleen when compared with non-GSP-fed but UVB-irradiated animals. These observations suggested that long-term feeding of GSP to the animals has no apparent toxic effect on the internal body organs. Similar observations of dietary feeding of GSP (0.5%, w/w) on body weight, and weight and length of internal body organs were also found in mice under anti-initiating and anti-promoting protocols of photocarcinogenesis (data not shown).
Dietary feeding of GSP has no apparent toxicity to bone mineral density and bone mineral content but significantly reduced fat content in the body
To further confirm that long-term feeding of GSP has no apparent toxic effect in animals, animals were subjected to assess whether dietary feeding of GSP (0.5%, w/w) has any apparent adverse effect on lean (bone-free tissue mass), FM, TBMD and TBMC compared with non-GSP-fed mice. These parameters were assessed at the end of photocarcinogenesis protocols at week 24 or 30 by using DXA. The data obtained from DXA demonstrated that there was no significant change in TBMD, TBMC, lean and total body mass (mass of lean + fat) in GSP-fed mice compared with non-GSP-fed mice (data not shown), but at the same time feeding of GSP to UVB-irradiated animals resulted in a significant reduction of tissue fat content (27%, P<0.05) which tempted to suggest its possible role, at least in part, in prevention of photocarcinogenesis in animals by GSP treatment. Together, cumulative observations on non-toxicity of GSP in diet to animals indicated that long-term dietary feeding of GSP has no apparent toxic effect in the animals. Similar observations of dietary feeding of GSP (0.5%) on TBMD, TBMC, lean and fat were also noted in mice under anti-initiating and anti-promoting protocols of photocarcinogenesis (data not shown).
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Discussion |
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The present study provides additional information on the effectiveness of GSP as an inhibitor of malignant conversion of benign skin papillomas to carcinomas in SKH-1 hairless mice (Figure 2 and Table III). It is well known that transformation of benign papillomas to carcinomas further requires genetic and epigenetic changes in the tumor cells and this can be achieved by using free radical-generating agents (26,27) or genotoxic substances (28). In this photocarcinogenesis protocol, it appeared that chronic exposure of UVB radiation performed these alterations/functions. Although, the exact mechanism of prevention afforded by GSP is not clearly understood by the present study but it appeared that the inhibition of UVB-induced oxidative damage of lipids (Figure 3), and global DNA hypomethylation in the skin by dietary feeding of GSP, at least in part, are the possible determinant factors (29). It has been shown that the increase in the rate of malignant transformation by free radical-generating compounds may be related to free radical-mediated enhancement of genetic instability, ultimately increasing the progression rate of non-malignant lesions (30). Therefore, the results of this study suggest that feeding of GSP might prevent UVB-induced free radical-mediated enhancement of genetic instability by decreasing the total number of carcinomas per group as well as the percentage of mice with carcinomas when compared with that of non-GSP-fed and UVB-irradiated group of mice. Earlier studies suggest that the transformation of benign papillomas to carcinomas requires further genetic changes in papillomas, and it can be achieved by tumor initiating agents (28,31). In the present photocarcinogenesis protocols (complete and tumor promoting), repeated exposure of UVB radiation to papillomas would provide further genetic changes in tumor cells (32), which enhanced the transformation of benign papillomas to carcinomas. Therefore, it can be suggested that dietary feeding of GSP affords protection against genetic and epigenetic alterations caused by UVB radiation. Similar observations were also found when topical treatment of green tea polyphenols inhibited benzoyl peroxide (free radical-generating agent)- and 4-nitroquinoline-N-oxide (initiating agent)-induced malignant conversion of chemically induced papillomas into carcinomas (33).
Proanthocyanidins rich extract from grape seeds contains several types of polyphenolic components such as dimers, trimers, tetramers, oligomers and monomeric flavanols like catechins and epicatechin derivatives (Table I) (7). Zhao et al. (14) suggested that dimerization and trimerization of monomers (catechins and epicatechins) could lead to anticarcinogenic effects in DMBA-initiated and TPA-promoted skin tumorigenesis possibly due to increase in their antioxidant activity. It has also been shown that dimeric procyanidins are absorbed into the blood stream (34,35), and some of the products of hydrolytes of the higher oligomers and polymers were presumed to be absorbed through the intestinal membrane and then the absorbed procyanidins and/or hydrolytes of procyanidins might display antioxidative activity in vivo (36,37). Thus, antioxidative activity of GSP might be responsible for the protection against photocarcinogenesis. Similar to GSP, resveratrol is another antioxidant found mainly in the skin of grapes in small amounts and has been shown to prevent UVB-induced markers of tumor promotion, such as edema, COX-2 expression and LPO, when treated topically on SKH-1 hairless mouse skin (38). In comparison with resveratrol, the availability of GSP in grape seeds is quite high and the molecular structures are complex because GSP are mainly dimers, trimers, tetramers and oligomers in nature (39).
In efforts to determine the photoprotective mechanism of GSP, we found that treatment of GSP in vivo as well as in vitro systems significantly inhibited UVB-induced or Fe3+-induced LPO (Figure 3). Lipid peroxidation in biological membranes is a free radical-mediated event and is regulated by the availability of substrates in the form of polyunsaturated fatty acids, pro-oxidants which promote peroxidation and antioxidant defenses such as -tocopherol, glutathione, ß-carotene and superoxide dismutase (4042). LPO is highly detrimental to cell membrane structure and function. Elevated level of LPO has been linked to injurious effects such as loss of fluidity, inactivation of membrane enzymes and receptors, increased permeability of ions and, eventually rupture of cell membranes leading to release of cell organelles (40,43). Peroxidation products can also result in damage to crucial biomolecules, including DNA (44,45). Thus, inhibition of UV-induced elevated level of LPO by dietary feeding of GSP would result in reduction of the risk factors associated with UV-induced ROS-mediated tumor promoting effects of UV radiation in cutaneous inflammatory responses and malignancies.
Further, there has not been a systematic report, which defines the toxicity or non-toxicity of proanthocyanidins in vivo in animals, especially when GSP is given in diet for a longer time. In the present study, mice were given GSP in diet for 30 weeks and it was observed that during this time period animals neither lose weight nor gain weight (data not shown), which may have distorted their physical or metabolic activity. Additionally, long-term feeding of GSP did not adversely affect the internal body organs of animals, which is evidenced by determining the physical characteristics, such as weights and lengths of the internal body organs like liver, spleen and kidney (data not shown). Most importantly, long-term dietary feeding of GSP did not affect the overall body mass (lean + fat), bone mineral density and bone mineral content when analyzed by DXA (data not shown). These observations provided ample evidence for the first time that dietary feeding of GSP has no apparent toxic effects in animals.
The most significant observation was that dietary feeding of GSP inhibited UVB-induced tumor incidence and multiplicity concomitant with significant reduction in tissue fat content in mice compared with that of non-GSP-fed but UVB-irradiated mice. Although several dietary modifications are known to inhibit carcinogenesis, and some may decrease body fat levels in rodents (e.g. caloric restriction) (4649), this is the first study with proanthocyanidins from grape seeds to show a close relationship between the inhibition of photocarcinogenesis and reduction in tissue fat levels. Similar observations were also noted with green tea and black tea on the inhibition of UVB-induced skin carcinogenesis concomitant with reduction in tissue fat (50). The results of the present study suggest that the inhibitory effect of dietary feeding of GSP on photocarcinogenesis may be relatedat least in partto their effects on the reduction in tissue fat levels including the level of fat content in skin layers. This may be attributed to administration of GSP increased lipolysis or decreased the absorption or synthesis of fat without changing body weight. It may be of interest to determine the effects of dietary feeding of GSP on the profile of fatty acids in epidermal phospholipids and in the neutral fat of the parametrial fat pads.
In summary, as we are aware, this is the first study which demonstrates that dietary feeding of proanthocyanidins from grape seeds has the potential to prevent photocarcinogenesis and malignant conversion of papillomas to carcinomas in mice, and this prevention is closely associated with the inhibition of UVB-induced LPO or photo-oxidative damage of lipids, and reduction in tissue fat levels. Additional studies are needed to determine whether chemopreventive effects of GSP on photocarcinogenesis are mediated through: (i) antioxidant mechanism of GSP and/or (ii) their effects on skin fat content, and also whether treatment of GSP decreases the arachidonic acid content or modifies the levels of other fatty acids in dermal or epidermal phospholipids. Decreased levels of arachidonic acid in the epidermis could result in decreased levels of prostaglandins that have been implicated in carcinogenesis.
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Acknowledgments |
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References |
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