Lycopene counteracts the hepatic response to 7,12-dimethylbenz[a]anthracene by altering the expression of Bax, Bcl-2, caspases, and oxidative stress biomarkers

Abstract

Context: Lycopene is a carotenoid found in tomato, watermelon, pink grapefruit, and guava in high concentration. Dietary intake of lycopene has been proposed to inversely correlate with the risk of cancer. It has also been reported to provide protection against cellular damage caused by reactive oxygen species, which makes it worthwhile to study the effect of lycopene on liver damage in rat model.

Objective: In this study, we report the effect of lycopene on 7,12-dimethylbenz[a]-anthracene (DMBA)-induced expression of Bax, Bcl-2, caspases, and oxidative stres biomarkers in the liver.

Materials and methods: Lycopene was administered orally at 20 mg/kg body weight for 20 weeks followed by the intraperitoneal injection of DMBA (50 mg/kg body weight) on day 1 and day 30 of the experiment. Control rats received vehicle (olive oil) or DMBA alone. Rats were sacrificed after completion of the treatment.

Results: We observed that the levels of Bax, caspase-3, and caspase-9 decreased to 44, 67, and 43%, respectively, and Bcl-2 increased by 80% in DMBA-treated rats. Lycopene reversed the changes in the respective groups, and decreased the level of Bcl-2 to 25%, while increasing the Bax to 42% when compared to DMBA control. Lycopene increased the expression of caspase-3 (82.09%) and caspase-9 (58.96%), and attenuated the level of hepatic malondialdehyde (41%) and 8-isoprostane (40%) when compared to the respective controls. Glutathione (GSH) decreased significantly in DMBA group (15.89%), but reached the normal level in lycopene-treated animals. Hepatic lycopene concentration in treated rats was 8.2 nmol/g tissue.

Conclusion: The study reports that lycopene counteracts the hepatic response to DMBA by altering the expression of Bax, Bcl-2, caspases, and oxidative stress biomarkers in animal model.

Keywords::

• Animal models
• cancer prevention
• apoptosis
• liver

Introduction

Polycyclic aromatic hydrocarbons are ubiquitous environmental contaminants produced by the partial combustion of organic substances. These compounds bind to and activate the cytosolic transcription factor, aryl hydrocarbon receptor (AhR), and ultimately stimulate the xenobiotic response elements in a variety of genes including cytochrome P450 family 1 (CYP1) members (Shimada & Fujii-Kuriyama, 2004). The AhR-induced CYP1 enzymes are of particular interest for their ability to metabolize polycyclic aromatic hydrocarbons into reactive metabolites which may cause toxicity and generate DNA adducts (Ma & Lu, 2007; Tompkins & Wallace, 2007). The genomic profiling of hepatic AhR in rodents suggests an alteration of genes involved in cellular proliferation, endocrine disruption, immunomodulation, metabolism, transport, and neoplastic transformation (Vezina et al., 2004; Yoon et al., 2006; Singhal et al., 2008), suggesting its role in multiple toxicological endpoints. In contrast to the effects of polycyclic aromatic hydrocarbonss, nutrients with antioxidant capacity such as lycopene have been reported to have beneficial effects in several chronic disease conditions, and reduce the risk of several types of cancer.

Lycopene is a nonprovitamin A carotenoid that imparts red color to tomatoes, guava, watermelon, and pink grapefruit. It is a major carotenoid and highly potent antioxidant that provides protection against cellular damage caused by reactive oxygen species (Giovannucci, 1999; Rao & Agarwal, 1999). Consumption of tomato and/or its products has been reported to increase the level of lycopene in blood and reduce oxidative damage to biomolecules including lipid, protein, and DNA (Agarwal & Rao, 1998). Epidemiological studies have shown that high intake of lycopene-containing vegetables is inversely associated with the incidence of certain types of cancer including digestive tract, prostate, and cervix cancer (Giovannucci, 1999; Seren et al., 2008a,b; Amin et al., 2009). However, little is known about the effect of lycopene on the expression of Bax, Bcl-2, caspases and the levels of 8-isoprostane and glutathione (GSH) in the liver in response to 7,12-dimethylbenz[a] anthracene (DMBA), a polycyclic aromatic hydrocarbon. DMBA has been widely used as a model compound in the studies on cancer. In this study, lycopene is reported to partially attenuate the effect of DMBA related to apoptosis and oxidative stress in the liver.

Materials and methods

Animals and experimental procedure

Thirty male Wistar rats (8-weeks-old, weighing 180–200 g) obtained from Firat University Research Center (Elazig, Turkey) were kept as per the protocol approved by the Firat University Veterinary Faculty for the care of experimental animals. All procedures were conducted in strict compliance with the guidelines established by the Institutional Animal Care and Use Committee. Animals were housed in standard cages, and kept in a room maintained at 23 ± 2°C with a 12 h/12 h light/dark cycle, and had free access to commercial standard laboratory diet and water ad libitum. Rats were divided into three groups. Group 1, normal control rats, did not receive any treatment and was fed with standard rat chow. Group 2 was administered intraperitoneally DMBA (50 mg/kg body weight) on day 1 and day 30 of the experiment. Rats in group 3 received DMBA and lycopene in olive oil. Lycopene (DSM, Istanbul, Turkey) was suspended in olive oil and administered intragastrically three times per week to each rat at a dose level of 20 mg/kg body weight. At this dose level, hepatic lycopene concentration is 8 nmol/g tissue, which has been found to be pharmacologically active (Sahin et al., 2011). All animals were kept for 20 weeks. First dose of lycopene was administered on day 2 following the exposure to carcinogen. The dose of lycopene was selected on the basis published data (Wei et al., 2010). After 5 h of administering the final dose of lycopene, rats were anesthetized using diethyl ether and sacrificed. The liver was isolated, blotted, weighed, frozen in liquid nitrogen, and stored at −80°C until futher use.

Laboratory analyses

For analyses, the frozen liver was thawed and homogenized gently for about 45 s in 9 volume of ice-cold 10 mM phosphate-buffered saline (PBS, pH 7.4) containing 1.15% KCl, and centrifuged at 800g to remove cell debris and nuclei. Supernatant was further centrifuged at 10,000g for 10 min and kept for analysis.

Concentrations of lycopene in the rat liver supernatant was determined by high-performance liquid chromatography (HPLC), as described by Wang et al., (2010). Briefly, 100 mg liver or plasma (1 mL) was homogenized in 3 mL solution containing saline and ethanol (1:2, vol/vol). Lycopene was extracted from the biological samples in 5 mL solution of hexane and ether (1:1, vol/vol) by vortexing for 3 min. The mixture was centrifuged at 2,000g for 10 min at 4°C, and the upper layer was collected. The sample was extracted three times and evaporated under nitrogen gas, and reconstituted with 100 mL of ethanol and ether (2:1, vol/vol). The extract (50 μl) was injected into the HPLC system to measure lycopene. Malondialdehyde concentration of liver tissue was measured as described previously (Karatepe, 2004) with minor modifications by HPLC (Shimadzu, Tokyo, Japan) using Shimadzu UV-vis SPD–10 AVP detector and C_18- ODS-3, 5 μm, 4.6 × 250 mm column. The mobile phase was 30 mM KH₂PO₄-methanol (82.5 ± 17.5, vol/vol %, pH 3.6) and the flow rate was 1.2 mL min⁻¹. Chromatograms were monitored at 250 nm and injection volume was 20 µL (Karatepe, 2004). The homogenized tissue (in PBS) was purified in the presence of 0.01% butylated hydroxytoluene and then processed for analysis of 8-isoprostane as previously described (Wong et al., 2006) and determined using rat specific enzyme-linked immunosorbent assay (ELISA) kits (Cayman Chemical, Ann Arbor, MI) according to the manufacturers’ protocols. Tissue GSH was determined using GSH-400 kit (Oxis Int, Portland, OR).

Western blot analysis

For western blot, liver was homogenized in PBS with protease inhibitor cocktail (Calbiochem, San Diego, CA) and the protein concentration was quantitated. The sample (20 mg of protein per lane) was mixed with sample buffer, boiled for 5 min, separated by SDS-polyacrylamide (12%) gel electrophoresis under denaturing conditions, and electroblotted onto nitrocellulose membrane. Nitrocellulose blots were washed in PBS and blocked with 1% bovine serum albumin in PBS for 1 h before application of the primary antibody. Primary antibody was diluted (1:1,000) in the same buffer containing 0.05% Tween-20. The nitrocellulose membrane was incubated overnight at 4°C with protein antibody. Antibodies against Bcl-2, Bax, cleaved caspase-3 (19-kDa), and β-actin were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. The next day, the immunoreaction was continued with the secondary goat antirabbit horseradish-peroxidase-conjugated antibody after washing for 2 h at room temperature. Specific binding was detected using diaminobenzidine and H₂O₂ as substrates. Protein levels were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD).

Statistical analyses

Sample size was calculated based on a power of 85% and a p value of 0.05. Given that assumption, a sample size of 15 per treatment was calculated. The data were analyzed using the GLM procedure of SAS (2002). Results were compared using analysis of variance; p < 0.05 was considered to be statistically significant. Results were analyzed by the analysis of variance for repeated measurements followed by Fisher’s post hoc test for all groups.

Results

Western blot analysis of Bax, Bcl-2, and caspases in the liver of DMBA- and lycopene-treated rats

The antiapoptotic effect of lycopene was studied in DMBA-treated rats on the expression level of Bax, Bcl-2, and caspase-3 and -9. Bax expression was down by 44.41% (Figure 1A), and Bcl-2 increased by 80.06% (Figure 1B) in DMBA group compared to the control group. Further, cleaved caspase-3 and caspase-9 expression was downregulated by 66.86 and 42.68%, respectively, when compared to the control group. These changes were found to reverse in lycopene-treated rats, where the Bcl-2 expression decreased to 24.77% and Bax expression increased by 42.35% in comparison to the DMBA control. Lycopene also significantly increased the expression of cleaved caspase-3 (82.09%; Figure 1C) and caspase-9 (58.96%; Figure 1D) in rat liver (Figure 2).

Figure 1.  Effect of lycopene on (A) hepatic Bax, (B) Bcl-2, (C) caspase-3, and (D) caspase-9 expression in rats with DMBA-induced liver injury. The band intensity was quantified by densitometric analysis. Each bar represents the SEM (n = 3). β-Actin was used to ensure equal protein loading. Data points with superscripts a, b, c indicate significant difference at the level of p < 0.05 (Fisher’s multiple comparison test). DMBA, 7,12-dimethylbenz[a]anthracene.

Figure 2.  Effect of lycopene supplementation on (A) hepatic malondialdehyde, (B) 8-isoprostane, and (C) GSH. Each bar represents the SEM. Data points with different superscripts a, b, c indicate significant difference at the level of p < 0.05 (Fisher’s multiple comparison test). DMBA, 7,12-dimethylbenz[a]anthracene; GSH, glutathione.

Oxidative stress biomarkers in DMBA- and lycopene-treated rats

The levels of hepatic malondialdehyde (Figure 2A) and 8-isoprostane (Figure 2B) increased in DMBA-treated rats, and were attenuated by lycopene. Malondialdehyde (MDA) and 8-isoprostane increased up to 79.26 and 59.77%, respectively, when compared to the normal (untreated) control group. The protection by lycopene was 41.29 and 40.23%, respectively, when compared to the group II. The tripeptide, GSH decreased significantly in DMBA group (15.89%) when compared to the control group (Figure 2C). Lycopene could replenish the depleted levels of GSH.

Hepatic lycopene concentration

The concentration of lycopene in the lycopene-treated group of rats, which were orally administered lycopene (20 mg/kg body weight) was found to be 8.20 nmol/g. Lycopene was not detected in control rats.

Discussion

This study reports that lycopene partially attenuates the effects of DMBA related to apoptosis and oxidative stress in rat liver. Lycopene supplementation attenutaed the hepatic response to DMBA by altering the expression of Bax, Bcl-2, caspases, and oxidative stress biomarkers (Figures 1 and 2). Earlier, in vitro animal and clinical studies have reported that lycopene attenuates the liver injury and possibly prevent the development of hepatocellular carcinoma (Seren et al., 2008b). Patients with hepatocellular damage contain lower serum and hepatic retinol, tocopherols, lutein, lycopene, α-carotene, β-carotene, and higher serum malodialdehyde (Yadav et al., 2002). Lycopene has singlet-oxygen-quenching ability twice that of β-carotene and 10 times higher than that of α-tocopherol. Its oxygen-quenching ability is suggested to protect against oxidative DNA damage both in vitro and in vivo, thereby preventing potential mutations that might induce cancer initiation (DiMascio et al., 1989; Conn et al., 1991). Metabolism of DMBA leads to free radical formation, which results in pathological changes in the liver (Prasad et al., 2007; Choi & Kim, 2009). DMBA is metabolized by cytochrome P4501A1 and cytochrome P4501B1 in liver microsomes to form diol epoxides and other toxic reactive oxygen species such as peroxides, hydroxyl, and superoxide anion radicals (Shou et al., 1996; Wakui et al., 2006; Prasad et al., 2007). Lycopene might protect the liver against DMBA-induced changes by shifting the oxidant-antioxidant balance in favor of the later. DMBA treatment resulted in a significant increase in MDA and 8-isoprostane production, and lycopene supplementation attenuated the changes (Figure 2). Lycopene was detected in the blood of rats supplemented with lycopene, correlating the increased level of lycopene with the tissue antioxidant status.

The mechanism by which lycopene might affect free radical and peroxide production has been proposed to be due to its singlet-oxygen and free radical scavenging capability (Seren et al., 2008b). We propose that the antioxidant activity of lycopene might be a potential mechanism to counteract the hepatic response to DMBA. Our data are in agreement with the studies reported by others investigating antioxidant supplementation. In a recent study, Wang et al., (2010) have reported that lycopene supplementation reduced MDA, cytochrome P450 2E1, inflammatory foci and mRNA expression of proinflammatory cytokines (tumor necrosis factor-α, IL-1β, and IL-12) of the livers in rats receiving diethylnitrosamine and high-fat diet. Lycopene has also been reported to significantly inhibit oxidative damage (Choi & Kim, 2009), and protect the hepatocyte from the effects of the hepatocarcinogen aflatoxin at cellular and molecular level (Reddy et al., 2006). Of particular interest in this study is the ability of lycopene to protect cellular GSH, which is proposed to be the result of lycopene on lipid peroxidation and its free radical scavenging activity (Leal et al., 1999).

The Bcl-2 family of proteins in association with caspases is known for regulating apoptosis, either as activator (Bax) or as inhibitor (Bcl-2). The gene product of Bcl-2 and Bax function at the mitochondrial level to prevent or promote the release of apoptogenic factors such as cytochrome c. Bcl-2, a major antiapoptotic protein located primarily in the outer mitochondrial membrane, inhibits apoptosis by preventing cytochrome c release from the mitochondria and inhibit caspase activation (Oltvai et al., 1993; Kirkin et al., 2004). Bcl-2 overexpression, a key event in malignant transformation is associated with the down-regulation of Bax, a proapoptotic member of the Bcl-2 family (Marone et al., 1998). A direct role of Bax on tumor suppression has been demonstrated in a mouse epithelial tumor model (Yin et al., 1997). In this study, the expression of Bax decreased and Bcl-2 increased in DMBA rats, suggesting survival of the mutated cells. On the other hand, lycopene supplemented rats showed an increased Bax and decreased Bcl-2. Like Bax, the expression of caspases also decreased in DMBA rats and increased in rats supplemented with lycopene (Figure 1A and 1B). These data indicate that lycopene may modulate the Bax/Bcl-2 level and activate apoptosis in DMBA rats.

Apoptosis involves an orchestrated series of biochemical events that lead to a variety of morphological changes, including blebbing, changes in the cell membrane (e.g., loss of membrane asymmetry and attachment), cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. A number of factors have been reported to induce or inhibit cell death. In a study by Choi and Kim (2009), DMBA significantly decreased Bcl-2 expression and increased the level of Bax as well as inhibited caspase-3 expression in mice. However, data in this study do not agree with this finding. We found over-expression of Bcl-2, and inhibition of Bax and caspases in the liver of DMBA-treated rats (Figure 1). The effect was reversed in lycopene supplemented rats.

Conclusion

The lycopene supplementaion is reported to increase the expression levels of Bax, a proapoptotic protein, as well as caspases, and inhibit the level of survival protein Bcl-2 in DMBA-treated rats, suggesting protective effect on cancer.

Acknowledgement

The authors thank the DSM for providing lycopene. S.A. acknowledges Firat University and Turkish and Indian Academy of Sciences for providing the opportunity to work together.

Declaration of interest

This study was supported by Firat University (FUBAP–1686). The authors declared no conflicts of interest.