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Keywords:

  • 7,12-dimethylbenz[a]anthracene;
  • beetroot juice;
  • cytochrome P450;
  • phase II enzymes;
  • liver;
  • mammary gland

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgement
  8. Conflict of Interest
  9. REFERENCES

Red beetroot contains a specific class of antioxidants collectively named betalains, which have been shown to have anticarcinogenic and anti-inflamatory potential. We investigated the effect of beetroot juice on the hepatic and mammary gland carcinogen metabolizing enzymes, DNA damage and liver injury, altered by 7,12-dimethylbenz[a]anthracene (DMBA). In the liver, pretreatment with beetroot juice significantly decreased levels and activities of the majority of tested biochemical parameters, elevated by DMBA. Feeding with beetroot juice decreased the activities of CYP1A1 and 1A2 and increased phase II enzymes. The activities of all enzymes tested were enhanced in the animals treated with DMBA alone and in combination with beetroot juice. The most significant changes in the level of the enzymes tested were observed for NAD(P)H:quinone oxidoreductase-1. In mammary gland, beetroot juice induced the level of glutathione S-transferase pi, enzyme involved in active metabolites of DMBA detoxification. The final effects of beetroot juice are tissue specific and depend on the class of carcinogen. Copyright © 2013 John Wiley & Sons, Ltd.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgement
  8. Conflict of Interest
  9. REFERENCES

Red beetroot (Beta vulgaris L.) is a vegetable characteristic of the Eastern and Central European diet. Beetroots are not only consumed as fresh vegetables but are also processed to obtain desiccated or frozen products, juices and their concentrates as well as natural pigments (β-cyans) used as food additives (Kanner et al., 2001). Additionally, drinking beetroot juice provides a more convenient alternative to consuming whole vegetable (Wootton-Beard and Ryan, 2011). The juice or extract of beetroots are used as a popular folk remedy for liver and kidney diseases, for stimulation of the immune and hematopoietic systems and as a special diet in the treatment of cancer. Beetroot juice contains a high level of biologically accessible antioxidants as well as many other health-promoting compounds such as magnesium, folic acid, niacin, biotin, vitamin B6, iron, zinc, calcium, phosphorus, sodium, potassium and soluble fibre (Wootton-Beard and Ryan, 2011). In addition to other active chemicals, beetroots contain a unique class of water-soluble, nitrogen-containing pigments called betalains, which are synthesized from the amino acid tyrosine into two structural groups: the red–violet betacyanins and yellow–orange betaxanthins (Azeredo, 2009). Several lines of evidence suggest that betalains show a number of biological properties, including antioxidant, anti-inflammatory, anticarcinogenic, neuro- and hepatoprotective activities (Khan et al., 2011). The antioxidant effects of betalains have been demonstrated mainly in various in vitro experiments (Kanner et al., 2001). Recently, the proapoptotic action of betanin was reported (Zielińska-Przyjemska et al., 2012). In vivo studies confirmed anticarcinogenic activity of beetroot in mouse lung, skin and liver carcinogenesis models (Kapadia and Rao, 2012). Additionally, Kawano and Umemura (2012) have found that oral intake of beet extract shows potential preventing skin diseases associated with impaired skin barrier function. In our previous studies, we have shown a protective role of beetroot juice against N-nitrosodiethylamine (NDEA)-induced oxidative stress in rats (Kujawska et al., 2009) and in isolated neutrophils (Zielińska-Przyjemska et al., 2009).

Moreover, our previous study has shown that long-term treatment of male Wistar rats with beetroot juice alone or in combination with NDEA can modulate phase I and phase II enzymes and DNA damage in the liver (Krajka-Kuźniak et al., 2012).

In the current study we investigated the effect of beetroot juice using a representative of other class of chemical carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA), in female Sprague–Dawley rats. This strain is susceptible to DMBA-induced mammary gland carcinogenesis. While the metabolic activation of NDEA needs the involvement of CYP2E1 and CYP1A2, polycyclic aromatic hydrocarbon (PAH), DMBA, requires the participation of other forms of CYP450, principally CYP1A1 and CYP1B1. Although the liver is not a target organ of DMBA-induced carcinogenesis, it is very likely that it plays a crucial role in the tumor initiating process, as the most of the activated metabolites of DMBA are formed in hepatocytes, and then reach the mammary gland through the bloodstream (Girolami et al., 2008).

Thus, in this study, we examined the effect of a long-term (28 days) treatment of rats with beetroot juice alone or in combination with DMBA on the hepatic and mammary gland carcinogen metabolizing enzyme expression, DNA damage and liver injury.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgement
  8. Conflict of Interest
  9. REFERENCES
Chemicals

DMBA, ethoxyresorufin, methoxyresorufin, penthoxyresorufin, resorufin, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, glutathione, 1-chloro-2,4-dinitrobenzene (CDNB), 2,6-dichlorophenolindophenol (DPIP), dicoumarol, NADP, NADPH, dithiothreitol, sucrose, bovine serum albumin, low melting point agarose, dimethylsulfoxide (DMSO), propidium iodide and Tris were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Normal melting point agarose was Prona Plus Agarose. Primary and secondary antibodies against CYP1A1/1A2, beta-actin and NAD(P)H:quinone oxidoreductase-1 (NQO1) were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary and secondary antibodies against glutathione S-transferase (GST) alpha, pi and mu, were supplied by Oxford Biomedical Research (Oxford, MI, USA). Primary and secondary antibodies against CYP1B1 and CYP2B1 were obtained from BD Biosciences (Woburn, MA, USA). All the antibodies used in these experiments were specific for their respective proteins, and according to the information provided by suppliers there was no cross-reactivity within the isozymes of the same family. Commercial reagent kits for determination of albumin, bilirubin, cholesterol, creatinine and blood urea nitrogen (BUN) concentrations and alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH) and gamma glutamyl transferase (GGT) activities were provided by Pointe Scientific, Inc. (Canton, MI, USA).

All other chemicals were commercial products of the highest purity available.

Animals and treatments

Female Sprague–Dawley rats (6 weeks of age, 240 ± 10 g), provided by the University of Medical Sciences, Department of Toxicology Breeding Facility (Poznań, Poland), were housed in polycarbonate cages (30×20×25 cm; 4 ≤ rats/cage), containing hardwood chip bedding. Commercial ISO 9001 certified rat food (Labofeed H) and distilled water were available without restriction. The animals were randomly divided into four experimental groups, each of six rats.

The animals were treated by gavage with crude natural beetroot juice (8 ml/kg b.w.) for 28 consecutive days. The chosen juice dose corresponds to approximately 500–600 ml of juice consumed daily by the average-weight adult human. DMBA was administered intraperitoneally in the dose of 10 mg/kg body weight, on the 27th and the 28th days. Control groups of animals received water. Red beetroot var. Chrobry was a gift from Experimental Vegetable Plantation Nochowo (Poland). The juice was prepared fresh daily in a household juice extractor. The content of betaxanthines was 79.3 mg/100 ml and of betacyanins 159.6 mg/100 ml as determined according to the method by Nilson (1970). All procedures were carried out according to the European guidelines for the care and use of laboratory animals and were approved by the Regional Ethics Committee (No. 33/2007).

Preparation of homogenates and cytosolic and microsomal fractions

Twenty four hours after the last treatment, the rats were anesthetized by ketamine, and blood was collected by heart puncture into heparinized tubes and centrifuged (1000 × g for 10 min at 4 °C) to separate plasma for determination of albumin, bilirubin, cholesterol, creatinine, BUN levels and ALT, AST, ALP, SDH, LDH, GGT activities. In a portion of heparinized blood, comet assay was carried out immediately. The livers were removed, rinsed in the ice-cold buffered 0.2 M sucrose (pH 7.5) and homogenized in the same medium. Cytosolic and microsomal fractions were prepared by differential centrifugation as described previously (Krajka-Kuźniak et al., 2004). Mammary tissue was excised, rinsed in the ice-cold buffered 0.2 M sucrose (pH 7.5) and homogenized in the same medium. Homogenates were centrifuged for 20 min, 10 000 × g at 4 °C. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard.

Phase I and phase II enzymes activity assays

The activities of ethoxyresorufin-O-deethylase (EROD), methoxyresorufin-O-demethylase (MROD) and penthoxyresorufin-O-depentylase (PROD) were measured as described previously (Burke et al., 1994). Cytosolic NQO1 activity was assayed as described by Ernster (1967) and modified by Benson et al. (1986) with NADPH as the electron donor and DPIP as the electron acceptor. The activity of GST was measured by the method of Habig et al. (1974), using CDNB as a substrate.

Protein immunoblotting

Cytosolic and microsomal proteins (20–100 µg) were separated on 10% or 12% SDS-PAGE slab gels, and the proteins were transferred to nitrocellulose membranes (Laemmli, 1970; Towbin et al., 1979). After blocking with 5% or 10% skimmed milk, the proteins were probed with mouse anti-rat CYP1A1/1A2, rabbit anti-rat CYP1B1, goat anti-rat CYP2B1, rabbit anti-human GST alpha, goat anti-rat GST mu, rabbit anti-human GST pi, goat anti-human NQO1 and rabbit anti-mouse beta-actin antibodies. The beta-actin protein was used as an internal standard. As the secondary antibodies in the staining reaction, the alkaline phosphatase-labeled anti-goat IgG, anti-mouse IgG or anti-rabbit IgG were used. The amount of immunoreactive product in each lane was determined by densitometric scanning using BioRad GS 710 Image Densitometer (BioRad Laboratories, Hercules, CA, USA). Values were calculated as relative absorbance units (RQ) per mg protein.

Alkaline single cell gel electrophoresis (comet) assay

Alkaline comet assay in whole blood leukocytes was performed according to the method of Hartmann et al. (2003). The cell lysis buffer was enriched with 10% DMSO. DNA unwinding and electrophoresis were carried out at pH > 13. After neutralization, the slides were dehydrated in absolute ethanol, dried, stored at room temperature and protected from light. Immediately before evaluation (Zeiss fluorescence microscope, magnification 400×), the slides were rehydrated and stained with ethidium bromide. Images of comets were recorded with a digital camera. For quantification, 100 comets were evaluated for each slide. The comets were classified according to the degree of DNA damage and divided into five groups (Collins, 2004). Multiplication of number of cells assigned to each grade of damage by the numeric value of the grade and summing over all grades resulted in the total damage score for the slide.

Statistical analysis

The statistical analysis was performed by one-way ANOVA. Statistical significance between the experimental groups and their respective controls was assessed by Tukey's post hoc test, with p <0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgement
  8. Conflict of Interest
  9. REFERENCES

Effect of beetroot juice and DMBA on selected biochemical parameters in blood

The effects of beetroot juice and DMBA on selected biochemical parameters (ALT, AST, ALP, SDH, LDH, GGT activities and the concentrations of albumin, bilirubin, cholesterol, creatinine, BUN) are presented in Tables 1 and 2, respectively. Treatment of rats with DMBA alone resulted in a statistically significant increase in all tested enzyme activities in blood plasma in comparison to the values from control group (by 28–127%). The concentrations of bilirubin, cholesterol, creatinine and BUN were also increased by 46%, 23%, 55% and 49%, respectively. With the exception of albumin and AST, the pretreatment with beetroot juice significantly decreased the levels and activities of all tested biochemical parameters tested elevated by DMBA.

Table 1. Effect of beetroot juice and DMBA on the selected plasma enzymatic markers of rat liver function. (Values in parentheses represent percent of control)
ParameterControlBeetroot juiceDMBABeetroot juice + DMBA
  • a

    Values are means ± SEM from 6 animals. Each assay was run in triplicate.

  • b

    ALT, AST, ALP, SDH, LDH, GGT are expressed in IU/l.

  • *

    Significantly different from control, p < 0.05.

  • **

    Significantly different from DMBA-treated rats, p < 0.05.

ALTa,b44.2 ± 1.0353.2 ± 0.6 (120)79.5 ± 0.76 (180)*57.8 ± 0.73 (131)**
ASTb88.7 ± 1.49102.0 ± 0.86 (115)113.5 ± 2.18 (128)*107.6 ± 0.78 (121)
ALPb144.9 ± 4.05163.6 ± 4.01 (112)205.3 ± 3.76 (142)*140.4 ± 1.78 (97)**
SDHb4.4 ± 0.144.4 ± 0.05 (100)10.0 ± 0.29 (227)*4.0 ± 0.13 (91)**
LDHb159.1 ± 3.48120.8 ± 4.09 (76)226.3 ± 8.54 (142)*135.0 ± 1.89 (85)**
GGTb6.6 ± 0.105.2 ± 0.80 (78)9.9 ± 0.08 (150)*5.2 ± 0.25 (79)**
Table 2. Effect of beetroot juice and DMBA on the selected plasma biochemical markers of rat liver functions. (Values in parentheses represent percent of control)
ParameterControlBeetroot juiceDMBABeetroot juice + DMBA
  • a

    Values are means ± SEM from 6 animals. Each assay was run in triplicate.

  • b

    Albumin is expressed in g/dl.

  • c

    Bilirubin, cholesterol, creatinine and BUN are expressed in mg/dl.

  • *

    Significantly different from control, p < 0.05.

  • **

    Significantly different from DMBA-treated rats, p < 0.05.

Albumina,b4.37 ± 0.014.37 ± 0.01 (100)4.14 ± 0.01 (95)4.22 ± 0.02 (97)
Bilirubinc1.15 ± 0.021.20 ± 0.02 (104)1.68 ± 0.03 (146)*1.27 ± 0.02 (110)**
Cholesterolc98.89 ± 0.99102.42 ± 0.75 (104)121.51 ± 1.05 (123)*108.43 ± 0.73 (110)**
Creatininec0.47 ± 0.010.51 ± 0.01 (109)0.73 ± 0.01 (155)*0.54 ± 0.01 (115)**
BUNc20.00 ± 0.2420.42 ± 0.18 (102)29.72 ± 0.42 (149)*20.64 ± 0.10 (103)**

Effect of beetroot juice and DMBA on phase I and II enzymes activities in the liver

The effects of beetroot juice and DMBA on the activities of cytochrome P450-dependent enzymes, GST and NQO1 in rat liver are summarized in Figs. 1A and 2A. Twenty eight days of forced feeding with beetroot juice alone decreased the activities of EROD (the marker of CYP1A1) and MROD (the marker of CYP1A2) by 30% and 29%, respectively, in comparison with the results from the control group of animals receiving water only.

image

Figure 1. Effect of beetroot juice and DMBA on the CYP isozymes activity (A) and protein level (B) in the rat liver. (A) Data (mean ± SEM, n = 6) are expressed as percentage of value obtained in respective control group. EROD: 28.06 ± 1.62; MROD: 23.13 ± 1.36; PROD: 16.34 ± 0.43 pmol resorufin formed/min/mg of protein. Each assay was run in triplicate. Asterisks above bars denote statistically significant differences from * control group, p < 0.05. (B) Western blot analysis – representative blot is shown: C-Control, BJ-Beetroot juice, DMBA-7,12-dimethylbenz[a]anthracene, BJ + DMBA – Beetroot juice + 7,12-dimethylbenz[a]anthracene. Data below the blot (CYP2B) present the expression of tested isozyme as percentage of control groups, from two separate experiments run in triplicate. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.

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image

Figure 2. Effect of beetroot juice and DMBA on the GST and NQO1 activities (A) and protein level (B) in the rat liver. (A) Data (mean ± SEM, n = 6) are expressed as percentage of value obtained in respective control group. GST: 536.40 ± 25.94 nmol 1-chloro-2,4-dinitrobenzene conjugated formed/min/mg of protein; NQO1: 160.44 ±19.22 nmol 2,6-dichloroindophenol reduced/min/mg of protein. Each assay was run in triplicate. Asterisk above bars denote statistically significant differences from * control group, p < 0.05. (B) Western blot analysis – representative blot is shown: C-Control, BJ-Beetroot juice, DMBA-7,12-dimethylbenz[a]anthracene, BJ + DMBA – Beetroot juice + 7,12-dimethylbenz[a]anthracene. Data below the blot present the expression of tested isozymes as percentage of control groups, from two separate experiments run in triplicate.

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DMBA induced EROD most extensively (~16-fold). The activity of MROD was enhanced by ~4.6-fold and of PROD by ~1.4-fold in comparison to effects measured in the control group of animals.

Beetroot juice increased the activities of GST and NQO1 by 103% and 41%, respectively. About 40–45% induction of these enzymes was also observed in the DMBA-treated animals. The combined treatment with beetroot juice and DMBA did not change phase I and II enzymes activities in comparison to the results obtained in the DMBA-treated rats.

Effect of beetroot juice and DMBA on phase I and II enzyme expressions in the liver

Western blot analysis with CYP1A1/1A2 and CYP1B1 specific antibodies (Fig. 1B) did not show constitutive expression of the hepatic CYP1A1/1A2 and CYP1B1 in controls and beetroot juice alone treated rats, whereas a significant increase in the levels of these enzymes in the animals treated with DMBA alone or in combination with beetroot juice was observed. The level of CYP2B was slightly increased in all treated groups of animals as compared to the results from controls.

Fig. 2B presents the immunoblots of GST isozymes and NQO1 and their quantitative analysis. The levels of GST alpha and mu isozymes were enhanced (by about 25–28%) in animals exposed to DMBA alone, but the pretreatment with beetroot juice prior to DMBA administration did not change the level of these isozymes. Increased activity of NQO1 was accompanied by an elevated level of the enzyme protein as a result of beetroot juice treatment.

Effect of beetroot juice and DMBA on phase I and II enzyme expressions in the mammary gland

Mammary gland CYPs and phase II enzyme activities were not measured because of the limited amounts of the available tissue. Fig. 3 shows an increase in the CYP1A1/1A2 levels in DMBA treated animals in comparison with the effects obtained in the control group. Similar results were observed in the animals treated with beetroot juice and DMBA. Among the GST isozymes tested, the most significant changes were observed in GST pi. The treatment with beetroot juice increased the level of GST pi by 20%. A similar effect was also observed in DMBA-treated animals and in those subjected to the combined treatment with beetroot juice and DMBA.

image

Figure 3. Effect of beetroot juice and DMBA on the expression of CYP and GST isozymes in the rat mammary gland. A representative immunoblots from two independent experiments are shown. Data below the blot present the expression of tested isozymes as percentage of control groups, from two separate experiments run in triplicate.

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Analysis of DNA damage in comet assay

The effects of beetroot juice and DMBA treatments on DNA damage are presented in Fig. 4. DMBA significantly augmented the scale of DNA damage in the whole blood leukocytes (by ~ 100%) in comparison with the results from untreated controls.

image

Figure 4. Effect of beetroot juice and DMBA on the extent of DNA damage in blood leukocytes of rats. Data (mean ± SEM) are expressed as percentage of value obtained in control group (103.00 ± 3.144 arbitrary points). Asterisk above bar denote statistically significant differences from * control group, p < 0.05

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The pretreatment with beetroot juice prior to the DMBA challenge did not change the extent of nuclear DNA damage in blood leukocytes.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgement
  8. Conflict of Interest
  9. REFERENCES

Beetroot is a common ingredient of Central European diet and folk medicine component, but so far it has not been a subject of intensive studies. However, as indicated by the data available, beetroot products may inhibit carcinogenesis in in vivo and in vitro models. Anti-proliferative effects of betanin, a principle betacyanin pigment isolated from the fruits of Opuntia ficus-indica on human chronic myeloid leukemia cell line-K562, were demonstrated by Sreekanth et al. (2007). Oral administration of betanin inhibited also the DMBA initiated and UV-B promoted skin tumorigenesis. (Kapadia et al., 2003). DMBA is one of the most potent initiators of skin and mammary gland tumorigenesis. As other PAHs, DMBA in order to exert its carcinogenic activity requires a metabolic activation by cytochrome P450-dependent enzymes, principally CYP1A1 and CYP1B1, leading to formation of DNA adducts (Lambard et al., 1991). Major part of DMBA metabolism occurs in the liver upon CYP1A1 action, while CYP1B1 enzymes exert their activity predominantly in extra-hepatic tissues, such as the mammary gland (Girolami et al., 2008). Their expression is mediated by aryl hydrocarbon receptor (AhR). Upon activation, the ligand-bound AhR translocates to the nucleus and dimerizes with the AhR nuclear translocator. This protein dimer interacts with a specific gene promoter sequence (xenobiotic response element) and initiates transcription of CYP1 enzyme family (Brauze et al., 2006). Besides these P450 isozymes, also CYP2B1 and 2C6 are involved in metabolic activation of DMBA (Lambard et al., 1991). Thus, the modulation of CYP induction and activity may contribute to anticarcinogenic action of beetroot.

In this study, we evaluated the effect of long-term feeding of rats with beetroot juice on DMBA-induced expression and the activity of CYPs 1A1, 1A2 and 2B in the rat liver and mammary gland. As expected, the treatment with DMBA alone induced the activities of EROD, MROD and PROD (markers of CYP1A1, CYP1A2 and CYP2B, respectively) in the liver. Although the combined treatment with beetroot juice and the carcinogen did not change the phase I enzymes in comparison to the effects found in the DMBA-treated group, beetroot juice alone reduced the activities of EROD and MROD. Inhibition of MROD, a marker of constitutive expression of CYP1A2, seems more important for chemoprevention as CYP1A1 is not constitutively expressed in the liver.

More importantly, the beetroot juice treatment increased the activities of hepatic phase II enzymes, GST and NQO1. Consistent with the observations of other authors (Nijhoff et al., 1993) and our previous studies (Krajka-Kuźniak et al., 2008), the GST pi protein was not detected in the liver. The enhanced activity of GST after the treatment with DMBA correlated with higher levels of GST alpha and mu. These GST isoforms are involved in the detoxification of reactive metabolites of PAHs, including DMBA (Coles and Ketterer, 1999). Increased activity of NQO1 observed after beetroot juice treatment was also accompanied by an elevated level of the enzyme protein. In our previous study on male Wistar rats (Krajka-Kuźniak et al., 2012), we observed a similar effect, which suggest that the animal strain or gender do not have an important effect on NQO1 activity and expression. Lee et al. (2005) have also found the induction of NOQ1 in the murine hepatoma cells upon exposure to beetroot extract, both in vitro and in vivo. NQO1 is a key enzyme responsible for detoxification of DMBA. This enzyme is a cytosolic flavoprotein that catalyzes metabolic detoxification of quinones, including benzo[a]pyrene quinones, by converting them into hydroquinones via a single-step two-electron reduction, thus avoiding the formation of deleterious reactive semiquinones (Keum et al., 2006). NQO1 was reported to protect cells against DMBA action as the NQO1-deficient mice were found more susceptible to DMBA-induced skin cancer than the wild-type animals (Long et al., 2001).

In the mammary gland our present results showed DMBA induction of CYP1A1/1A2 and GST. Among the GST isozymes tested, the most elevated was the protein level of GST pi and to lesser extent GST mu. However, the levels of these enzymes were not affected by the combined treatment with beetroot juice and DMBA. The constitutive expression of GST mu and GST pi was moderately increased as a result of the beetroot treatment. As GST pi represents the main class of GST which is involved in conversion of DMBA to inactive metabolites, the induction of GST pi may represent one of the potential mechanisms explaining the anticarcinogenic effects of beetroot on DMBA-induced carcinogenesis in animal models (Han et al., 2007). The role of the GST pi isoform in DMBA detoxification was indicated in the study in which GSTp-null mice were treated with DMBA to induce skin tumor formation. GSTp-null mice had increased DMBA-induced tumor formation relative to the control animals (Henderson et al., 1998), indicating a potential role for GSTp-mediated GSH conjugation to DMBA as a mechanism of detoxification in the ovary (Bhattacharya and Keating, 2012).

Feeding with beetroot juice had no effect on the DMBA-induced DNA damage in rat blood leukocytes, indicating that beetroot did not alter the blood antioxidant status. The same effect was observed in our previous study on chokeberry juice (Szaefer et al., 2011). Thus, the results of our current and previous studies may indicate that DMBA is capable of inducing large DNA lesions in peripheral blood cells, which cannot be prevented by diverse antioxidant-containing diets. On the other hand, Klewicka et al. (2012) noticed the increased serum antioxidant capacity as a result of combined treatment of rat with heterocyclic amine PhIP (2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine) and fermented red beetroot juice (FBJ). However, the results of this study, which showed also the reduction of genotoxicity of fecal water upon rat treatment with FBJ, cannot be compared with the results from our experiment since the carcinogen type and treatment protocols were different.

These findings do not exclude the damaging effects in carcinogen-specific target tissues. In this regard, Jung et al. (2006) found a significant inhibition of DMBA-DNA adduct formation in rats fed with grape juice and moderate 8-oxo-dG adducts level in mammary gland.

In our previous studies, we showed a protective role of beetroot juice against NDEA-induced oxidative stress and the liver injury in male rats (Kujawska et al., 2009; Krajka-Kuźniak et al., 2012). The results of our present study indicate that beetroot juice is able to protect female rat also against the DMBA-induced liver damage. With the exception of albumin and AST, the pretreatment with beetroot juice before the administration of DMBA reduced the markers of hepatic damage elevated as a result of DMBA treatment. The most significant effect was observed for SDH, which is a key enzyme in the polyol pathway converting sorbitol to fructose, whose level is significantly enhanced in diabetes. The reduction of SDH activity may suggest that beetroot phytochemicals have not only hepatoprotective activity, but may also prevent diabetic complications, by improving the polyol pathway as was shown for ursolic acid (Jang et al., 2010).

In summary, the results of our current study and our earlier observations indicate that metabolic alterations induced by beetroot may change the activation of chemical carcinogens. The final effects, however, are tissue specific and depend on the class of carcinogen. In addition, consumption of beetroot juice may protect against liver damage and modulate hepatic polyol pathway.

Acknowledgement

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgement
  8. Conflict of Interest
  9. REFERENCES

This study was supported by the Ministry of Science and Higher Education of Poland, grant 094/P06/2003/19.

Conflict of Interest

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgement
  8. Conflict of Interest
  9. REFERENCES

The authors have declared that there are no conflicts of interest.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgement
  8. Conflict of Interest
  9. REFERENCES
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