Inhibitory effects of astaxanthin on azoxymethane-induced colonic preneoplastic lesions in C57/BL/KsJ-db/db mice

Background Obesity and related metabolic abnormalities, including excess oxidative stress and chronic inflammation, are associated with colorectal carcinogenesis. Astaxanthin, a xanthophyll carotenoid found in aquatic animals, is known to possess antioxidant, anti-inflammatory, and antineoplastic properties. The present study examined the effects of astaxanthin on the development of azoxymethane (AOM)-induced colonic premalignant lesions in C57BL/KsJ-db/db (db/db) obese mice. Method Male db/db mice were administered 4 weekly subcutaneous injections of AOM (15 mg/kg body weight) from 5 weeks of age and subsequently, from 1 week after the last injection of AOM, were fed a diet containing 200 ppm astaxanthin throughout the experiment (8 weeks). Result The development of colonic premalignant lesions, i.e., aberrant crypt foci and β-catenin accumulated crypts, was significantly inhibited in mice treated with astaxanthin than in mice fed the basal diet. Astaxanthin administration markedly reduced urinary levels of 8-OHdG and serum levels of d-ROMs, which are oxidative stress markers, while increasing the expression of mRNA for the antioxidant enzymes GPx1, SOD1, and CAT in the colonic mucosa of AOM-treated db/db mice. The expression levels of IL-1β, IL-6, F4/80, CCL2, and CXCL2 mRNA in the colonic mucosa of AOM-treated mice were significantly decreased by astaxanthin. Dietary feeding with astaxanthin also resulted in a reduction in the numbers of NF-κB- and PCNA-positive cells that were increased by AOM exposure, in the colonic epithelium. Conclusion These findings suggest that astaxanthin inhibits the development of colonic premalignant lesions in an obesity-related colorectal carcinogenesis model by reducing oxidative stress, attenuating chronic inflammation, and inhibiting NF-κB activation and cell proliferation in the colonic mucosa. Astaxanthin, therefore, may be a potential candidate as a chemoprevention agent against colorectal carcinogenesis in obese individuals.

of adipokines, induction of oxidative stress, and a state of chronic inflammation [3][4][5]. Obese and diabetic mice are susceptible to chemically induced colon tumorigenesis [9]. Diet-induced obesity significantly promotes colon tumor development in mice [10]. On the other hand, recent studies have demonstrated that certain types of phytochemicals such as curcumin and (−)-epigallocatechin gallate inhibit the development of obesity-related colorectal carcinogenesis in mice by attenuating chronic inflammation [11,12]. Administration of angiotensin-converting enzyme inhibitor also suppresses the early phase of colorectal carcinogenesis in diabetic and hypertensive rats by attenuating inflammation and oxidative stress [13]. These reports suggest that targeting obesity-related metabolic abnormalities including chronic inflammation and oxidative stress using phytochemicals and specific agents is an effective strategy for preventing CRC development in obese individuals [3].
Astaxanthin, a conventional red-colored xanthophyll, is an oxygenated carotenoid derivative occurring naturally in a wide variety of living organisms including microalgae, fungi, salmon, trout, shrimp, and some birds [14,15]. Astaxanthin has been shown to exert numerous pharmacological effects due to its antioxidant, anti-inflammatory, antidiabetic, and antineoplastic properties [8,14,15]. Supplementation with astaxanthin actually decreased oxidative stress and inflammation in a clinical trial [16]. In preclinical animal studies, dietary astaxanthin was found to significantly inhibit chemically induced colorectal [17,18], urinary bladder [19], and oral carcinogenesis [20]. In particular, anti-inflammatory activity is one of the key mechanisms by which astaxanthin prevents colitis-related CRC development [17,18].
C57BL/KsJ-db/db (db/db) mice, which lack the long form of the leptin receptor, develop hyperphagic obesity and diabetes [21]. Interestingly, the development of colonic premalignant lesions induced by azoxymethane (AOM), a colonic carcinogen widely used to produce preneoplastic and neoplastic colonic lesions that mimic those observed human colon, is significantly enhanced in db/db mice [22]. A preclinical animal model using AOM and db/db mice [22] has proved useful in investigating specific agents for their ability to prevent inflammation-related colorectal carcinogenesis caused by obesity [11,12,[23][24][25]. In the present study, we investigated the effects of astaxanthin on the development of colonic premalignant lesions, i.e., aberrant crypt foci (ACF) and β-catenin accumulated crypts (BCAC) [26][27][28], using this obesity-related colorectal carcinogenesis model, with special focus on the reduction of oxidative stress and the attenuation of inflammation.

Animals and chemicals
Four-week-old male db/db mice were purchased from Japan SLC, Inc. (Shizuoka, Japan) and were maintained humanely at the Gifu University Life Science Research Center in accordance with the Institutional Animal Care Guidelines. AOM was purchased from Sigma-Aldrich (St. Louis, MO, USA). Astaxanthin from Haematococcus pluvialiswas was supplied by Fuji Chemical Industry (Toyama, Japan).

Experimental procedure
All experimental protocols involving animals were approved by the Animal Research Committee, Gifu University Graduate School of Medicine. Forty male db/db mice were divided into the following 4 groups: untreated control (n = 10); 200 ppm astaxanthin alone (n = 10); AOM alone (n = 10); and, AOM and 200 ppm astaxanthin (n = 10). At 5 weeks of age, mice in AOM alone group and AOM + astaxanthin group were injected with AOM (15 mg/kg body weight) subcutaneously once a week for 4 weeks. None treatment group and AOM alone group were fed the basal diet CRF-1 (the formula of Charles River, Oriental Yeast, Tokyo, Japan) throughout the experiment. The composition of the CRF-1 diet was as follows: 8.1 g/100 g water, 22.6 g/100 g protein, 5.6 g/100 g fat, 6.6 g/100 g minerals, 3.3 g/100 g fiber, and 53.8 g/100 g carbohydrates. Astaxanthin alone group and AOM + astaxanthin group were fed the basal diet supplemented with 200 ppm astaxanthin for 8 weeks, starting 1 week after the last injection of AOM. The dosage of astaxanthin was determined according to a previous report [18]. Food intakes in all groups were measured daily, while body weights were recorded once a week during the study. At the termination of the study (17 weeks of age), all mice were killed, and the development of ACF and BCAC was analyzed.

Identification and quantification of ACF and BCAC
The numbers of ACF and BCAC were determined according to standard procedures [12,23,24,29]. After fixing flat in 10% buffered formalin for 24 hours, we stained the colons with methylene blue (0.5% in distilled water) to count ACF. The number of ACF was recorded along with the number of aberrant crypts (ACs) in each focus. The data are expressed per colon. The distal parts of the colon (1 cm from anus; mean area, 0.7 cm 2 /colon) were then resected and embedded in paraffin, and a total of 20 serial sections (each 4 μm thick) per mouse were cut by an en face preparation to identify BCAC intramucosal lesions [12,23,24].

Clinical chemistry
Blood samples were collected from the inferior vena cava at sacrifice after 6 hours of fasting for chemical analyses. The serum concentrations of insulin (Shibayagi, Gunma, Japan), glucose (BioVision Research Products, Mountain View, CA, USA), adiponectin (R&D Systems, Minneapolis, MN, USA), leptin (R&D Systems), triglyceride (Wako, Osaka, Japan), and TNF-α (R&D Systems) were determined using an enzyme immunoassay, according to the manufacturer's protocol.

Statistical analyses
The measures are presented as mean ± SD and were statistically analyzed using the GraphPad InStat software program, Version 3.05 (GraphPad Software, San Diego, CA, USA) for Macintosh. One-way analysis of variance (ANOVA) was used to compare groups. If the ANOVA analysis indicated significant differences, the Tukey-Kramer multiple comparisons test was performed to compare the mean values among the groups. The differences were considered significant when the two-sided P value was less than 0.05.

General observations
During the experiment, dietary feeding with astaxanthin did not cause any clinical symptoms. As listed in Table 2, the mean body weight of the AOM alone group was lower than that of AOM-untreated control group (group 1, P < 0.05) at the termination of the experiment. This might be due to the toxicity of AOM, as observed in our previous studies [12,23,24]. The mean relative liver weight (g/100 g body weight) of AOM alone group was also significantly lower than that of none treatment group (P < 0.05). No significant differences were observed in the mean relative weights of adipose tissue among the groups. Histopathological examination of the liver, kidney, and spleen confirmed the absence of toxicity of dietary astaxanthin (data not shown).

Effects of astaxanthin on AOM-induced ACF and BCAC formation in experimental mice
ACF ( Figure 1A) developed only in the colons of mice that received AOM. The total mean numbers of ACF/ colon, aberrant crypts/colon, and large ACFs/colon were significantly reduced by astaxanthin administration (Table 3; P < 0.05). Moreover, the number of BCAC ( Figure 1B), which also developed only in the colons of AOM-treated mice, markedly decreased when these mice were fed astaxanthin-containing diets (Table 3; P < 0.05), indicating that two types of precursor lesions for CRC were suppressed by astaxanthin administration. These findings suggest that astaxanthin prevented the early phase of obesity-related colorectal carcinogenesis.
Effects of astaxanthin on systemic oxidative stress and expression levels of GPx1, SOD1, and CAT mRNA in the colonic mucosa of experimental mice Oxidative stress is implicated in obesity-related colorectal tumorigenesis [4]. Therefore, we determined the levels of oxidative stress and antioxidant biomarkers in the experimental mice. AOM injection significantly increased the levels of urinary 8-OHdG (Figure 2A; P < 0.05), which reflects DNA damage induced by oxidative stress, and serum d-ROMs ( Figure 2B; P < 0.01), marker for hydroperoxide levels of serum, as compared to those observed in the control group. However, the level of 8-OHdG significantly decreased with astaxanthin administration (Figure 2A; P < 0.05). As shown in Figure 2C, the expression of GPx1 mRNA, which encodes an antioxidant enzyme, was reduced by AOM injection (P < 0.05), but astaxanthin significantly increased the expression of GPx1, as well as of SOD1 and CAT, both of which encode antioxidant enzymes, in the colonic mucosa of db/db mice treated with AOM (P < 0.05).
Effects of astaxanthin on serum levels of TNF-α and expression levels of TNF-α, IL-1β, IL-6, F4/80, CCL2, and CXCL2 mRNA in the colonic mucosa of experimental mice Chronic inflammation plays a critical role in the pathogenesis of obesity and CRC development [4,31]. Therefore, the effect of astaxanthin on the levels of inflammatory mediators including TNF-α, IL-1β, IL-6, F4/80, CCL2, and CXCL2 in experimental mice was examined. Serum levels of TNF-α were significantly higher in the AOM-injected mice ( Figure 3A; P < 0.05), but not in the astaxanthintreated mice, than those in the control mice. As shown in Figure 3B, there was a marked increase in the expression levels of TNF-α, IL-1β, IL-6, and F4/80 (P < 0.05 compared to the untreated control group) in the colonic mucosa of group treated with AOM alone. However, astaxanthin administration significantly decreased the expression of IL-1β, IL-6, and F4/80 mRNA in the colonic mucosa of the AOM-treated db/db mice (P < 0.05). The expression of CCL2 and CXCL2, which are associated with colorectal carcinogenesis [32][33][34], in the colonic mucosa of the AOM-treated db/db mice was also significantly decreased by astaxanthin treatment (P < 0.05).  Effects of astaxanthin on NF-κB activity and cell proliferative activity in the colonic mucosa of experimental mice NF-κB activation is critically involved in the progression of inflammation and the activation of cell proliferation in colonic mucosa [35]. Therefore, the effects of astaxanthin on NF-κB activity and cell proliferative activity were examined in the colonic mucosa of experimental mice. The indices of phospho-NF-κB p65-positive cells, which were increased by AOM injection in the colonic epithelium, were significantly reduced by astaxanthin treatment ( Figure 4A; P < 0.001 for each comparison). As shown in Figure 4B, astaxanthin treatment also significantly decreased the PCNA-labeling indices of nonlesional crypts (P < 0.01), which had been increased by  AOM injection (P < 0.001). These findings indicate that astaxanthin significantly inhibits NF-κB activity and cell proliferation in the colonic mucosa of AOM-treated db/db mice.

Effects of astaxanthin on serum parameters in experimental mice
Diabetes, dyslipidemia, and adipokine imbalance, which are complications often associated with obesity, are involved in colorectal tumorigenesis [3][4][5]. Therefore, serum parameters associated with these metabolic disorders were assessed. AOM exposure did not affect either serum levels of adiponectin, triglycerides, glucose, and insulin or the value of QUICKI, a useful index of insulin sensitivity [36], in experimental mice ( Table 4). The serum level of leptin was significantly elevated by AOM irrespective of astaxanthin administration (P < 0.05). None of these metabolic parameters had been altered by astaxanthin administration at the end of this study.

Discussion
Obesity, which is one of the most serious healthcare problems worldwide, is a significant risk factor for the development of CRC [4,5]. Oxidative stress and chronic inflammation are key mechanisms linking obesity and colorectal carcinogenesis [37,38]. In particular, increased adipose tissue creates an oxidative environment that can upregulate the expression of various pro-inflammatory cytokines including TNF-α and IL-6, and this is critically associated with CRC development because these cytokines stimulate tumor growth and progression [31,39]. The results of the present study show that astaxanthin exerts preventive effects on the development of the AOMinduced colonic premalignant lesions ACF [26,27] and BCAC [28] in db/db obese mice, mainly through the reduction of oxidative stress and attenuation of inflammation. Saturated fatty acids from obesity-induced lipolysis are capable of activating macrophages and thereby activating  NF-κB signaling, which in turn leads to transcriptional activation of genes encoding pro-inflammatory factors including IL-1β and IL-6 [39]. IL-1β plays a key role in obesity-induced inflammation [39] and inflammationrelated carcinogenesis by modulating the gene expression involved in proliferation, survival, and angiogenesis [40]. The expression of CCL2, which is associated with infiltration and migration of tumor-related macrophages, has been demonstrated in several tumor tissues including CRC [32,33]. In addition, CXCL2, an inflammatory cytokine, has been shown to participate in the early phase of colorectal carcinogenesis [34]. In the present study, the expression levels of IL-1β, IL-6, F4/80, CCL2, and CXCL2 mRNA were decreased by astaxanthin in the experimental mice. These findings indicate that overexpression of these inflammatory mediators, which connect obesity and carcinogenesis, may be one of the critical targets of astaxanthin in preventing the development of obesity-related CRC. These findings are also consistent with those from a previous study showing that feeding with astaxanthin significantly suppressed colitis and colitis-related colorectal carcinogenesis by inhibiting NF-κB activation, decreasing the expression of IL-1β and IL-6, and suppressing cell proliferation in mice [18]. In particular, NF-κB signaling pathway is regarded as one of the key targets of astaxanthin to exert chemopreventive effects [41]. Obesity and chronic inflammation are often accompanied with increased generation of reactive oxygen species (ROS), which are derivatives of molecular oxygen such as superoxide and hydrogen peroxide. These derivatives can induce mutagenic changes and may damage DNA repair proteins, resulting in cancer development [40,42]. Cancer cells are also known to cause oxidative stress by generating ROS and modulating antioxidant enzymes [43]. In the present study, oxidative stress markers, including urinary levels of 8-OHdG and serum levels of d-ROMs, were significantly suppressed, while the expression levels of GPx1, SOD1, and CAT mRNA, which encode antioxidant enzymes, in the colonic mucosa were increased by astaxanthin intake in AOMinjected db/db mice. These results strongly indicate that attenuation of oxidative stress and recuperation from high oxidation state via antioxidative effects are critical mechanisms by which astaxanthin suppressed the occurrence of premalignant lesions, ACF and BCAC, in obese mice. It should be mentioned that astaxanthin has even been called a super-antioxidant because it is a superior antioxidant and scavenger of free radicals as compared with other carotenoids such as β-carotene [44].
Epidemiologically, it is still unclear whether the intake of carotenoids such as astaxanthin is associated with a reduced risk of CRC development. The results of randomized trials using β-carotene supplementation provided no evidence to support an effect of carotenoids on CRC chemoprevention [45,46]. Rather, intervention trials using high-dose β-carotene supplements showed an increase in the incidence of lung cancer in high-risk patients, like smokers and/or workers exposed to asbestos [47,48]. On the other hand, astaxanthin has been demonstrated to be safe in several human clinical trials [16,49,50]. Moreover, astaxanthin supplementation has positive effects on lipid profiles and oxidative stress in overweight and obese subjects, at least in part by activating the antioxidant defense system [49,50]. Taken together, these observations suggest that obese individuals, who are at high-risk of developing CRC and colorectal adenomas [5], may be appropriate subjects for interventional trials using astaxanthin for the prevention of colorectal tumorigenesis.
Previous reports using metabolic syndrome animal models have shown that astaxanthin reduces insulin resistance, recovers insulin sensitivity, and increases serum levels of adiponectin [51,52]. A double-blind randomized controlled trial also reported that astaxanthin consumption significantly increases blood adiponectin levels [53]. Furthermore, targeting insulin resistance and adipokine imbalance are suggested to be effective methods of preventing obesity-related colorectal tumorigenesis [3]. Therefore, we initially expected that astaxanthin would inhibit the development of ACF and BCAC in the AOMtreated db/db mice by ameliorating insulin resistance and improving adipokine imbalance. However, abnormalities in serum levels of glucose, insulin, adiponectin, and leptin were not improved by astaxanthin administration in this study. We suggest that this was likely due to the duration of the experiment (8 weeks) and the particular animal model studied, because previous studies demonstrating effects of astaxanthin on insulin sensitivity were long-term studies (22 weeks) [51] and the animals were not genetically obese [51,52]. Our results were consistent with another study investigating the effect of astaxanthin on the apoptosis of retinal ganglion cells using db/db mice, in which insulin resistance was not improved by astaxanthin, but apoptosis of retinal ganglion cells was attenuated via the suppression of oxidative stress [54]. Future long-term studies should be conducted to confirm that astaxanthin inhibits the early phase of obesity-related colon tumorigenesis by improving insulin resistance and the imbalance of adipokines in several animal models. In our experimental model, astaxanthin suppressed the development of obesity-related colorectal tumorigenesis by targeting oxidative stress, inflammation, and cell proliferation.

Conclusion
In summary, the results from this study showed that reduction of oxidative stress and attenuation of inflammation in the colonic mucosa are crucial mechanisms by which astaxanthin acts to prevent the early phase of obesity-related colorectal carcinogenesis. Since the risk of CRC increases with obesity and obesity-related metabolic abnormalities, which are impending health crises worldwide, targeting obesity-related metabolic abnormalities, including chronic inflammation and induction of oxidative stress, may be an attractive and effective strategy for preventing the development of CRC in obese individuals. Astaxanthin seems to be a potentially effective and viable candidate for this purpose because this agent attenuates chronic inflammation while reducing oxidative stress without causing side effects.