Purple carrot extract protects against cadmium intoxication in multiple organs of rats: Genotoxicity, oxidative stress and tissue morphology analyses

Accepted Manuscript Title: Purple carrot extract protects against cadmium intoxication in multiple organs of rats: Genotoxicity, oxidative stress and tissue morphology analyses Author: Samuel Rangel Claudio Andrea Pittelli Boiago Gollucke Hirochi Yamamura Damila Rodrigues Morais Giovana Anceski Bataglion Marcos Nogueira Eberlin Rogerio Correa Peres Celina Tizuko Fujiyama Oshima Daniel Araki Ribeiro PII: DOI: Reference:

S0946-672X(15)30030-4 http://dx.doi.org/doi:10.1016/j.jtemb.2015.08.006 JTEMB 25713

To appear in: Received date: Revised date: Accepted date:

24-7-2015 23-8-2015 31-8-2015

Please cite this article as: Claudio Samuel Rangel, Gollucke Andrea Pittelli Boiago, Yamamura Hirochi, Morais Damila Rodrigues, Bataglion Giovana Anceski, Eberlin Marcos Nogueira, Peres Rogerio Correa, Oshima Celina Tizuko Fujiyama, Ribeiro Daniel Araki.Purple carrot extract protects against cadmium intoxication in multiple organs of rats: Genotoxicity, oxidative stress and tissue morphology analyses.Journal of Trace Elements in Medicine and Biology http://dx.doi.org/10.1016/j.jtemb.2015.08.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Purple carrot extract protects against cadmium intoxication in multiple organs of rats: genotoxicity, oxidative stress and tissue morphology analyses Running title: Purple carrot and cadmium exposure Samuel Rangel Claudio1, Andrea Pittelli Boiago Gollucke1, Hirochi Yamamura1, Damila Rodrigues Morais2, Giovana Anceski Bataglion2, Marcos Nogueira Eberlin2, Rogerio Correa Peres1, Celina Tizuko Fujiyama Oshima3, Daniel Araki Ribeiro1*  [email protected] 1

Department of Biosciences, Federal University of Sao Paulo, UNIFESP, SP, Brazil

2

ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of

Campinas (UNICAMP), Campinas, SP, Brazil 3

Department of Pathology, Paulista Medical School, Federal University of Sao Paulo,

UNIFESP, SP, Brazil *

Corresponding author at: Departamento de Biociências, Universidade Federal de São

Paulo - UNIFESP, Av. Ana Costa 95, Zip code: 11060-001, Santos – SP, Brazil. Tel./Fax +55 13 38783823.  

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Abstract  The aim of this study was to investigate if purple carrot extract is able to protect against the noxious activities induced by cadmium exposure in multiple organs of rats. For this purpose, histopathological analysis, genotoxicity and oxidative status were investigated in this setting. A total of twenty Wistar rats weighing 250g on the average, and 8 weeks age were distributed into four groups (n=5), as follows: Control group (non-treated group, CTRL); Cadmium group (Cd) and Purple carrot extract groups at 400 mg/L or 800 mg/L. Histopathological analysis revealed that liver from animals treated with purple carrot extract improved tissue degeneration induced by cadmium intoxication. Genetic damage was reduced in blood and hepatocytes as depicted by comet and micronucleus assays in animals treated with purple carrot extract. SOD-CuZn and cytocrome C gene expression increased in groups treated with purple carrot extract. Purple carrot extract also reduced the 8OHdG levels in liver cells when compared to cadmium group. Taken together, our results demonstrate that purple carrot extract is able to protect against cadmium intoxication by means of reducing tissue regeneration, genotoxicity and oxidative stress in multiple organs of Wistar rats.  Keywords: purple carrot; cadmium; rat; genotoxicity; oxidative stress.  

 

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Introduction  Nowadays, carrots (Daucus carota L.) are considered the 10 most economically important vegetable crops grown worldwide [1]. Carrot cultivars appear in five taproot color types: purple, orange, yellow, red, and white. In general, orange carrots contain high amounts of α-and β-carotene; yellow carrots contain lutein, the red color of carrots is due to lycopene, while polyphenol substances, mostly anthocyanins are typical for purple roots [2]. In the last years, purple carrots (D. carotas sp.sativus var.atrorubens Alef.) are increasing popularity, because they contain high amounts of anthocyanin in their flesh taproots [3]. Anthocyanins from purple carrots are commonly used as natural food colorants in candies, ice cream, and beverages. This is because they remain stable when exposed to heat and light, and have increased pH values [4]. Purple carrots also contain some trace amounts of peonidin- or pelargonidin-based anthocyanins in their taproots [5].  Cadmium is a toxic heavy metal extremely harmful to humans and other mammalian species. It is present in air, soil, sediments, water and smoking. After intake, cadmium accumulates in multiple organs and tissues, particularly in liver and kidneys [6]. Long-term or even short term exposure leads to a wide range of noxious health effects, including renal dysfunction, cardiovascular disease, hypertension, osteoporosis, hepatotoxicity, pancreatic activity changes and cancers in many organs [7,8]. For this reason, International Agency for Research on Cancer has categorized cadmium as carcinogenic to humans and animals [9]. Unfortunately, human exposure to cadmium is increasing in the next decades, mainly in developing countries due to rapidly growing industries with increasing consumption and subsequent release into the environment.

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Several underlying mechanisms at cellular or even molecular levels have been purposed for cadmium toxicity. Cadmium is involved in the disruption of many genomic processes, the mechanisms of which are being gradually understood so far. It has been established that oxidative stress plays a crucial role for cadmium toxicity. Following the intracellular contact of cadmium, some enzymes are inactivated through direct displacement from their binding site [10]. The cellular pro-oxidative stress induced by cadmium is most likely mediated by disruption of redox homeostasis associated with mishandling of redox-active transition metals result in lipid peroxidation, membrane protein damage, DNA damage and alter gene expression [11]. Nevertheless, cadmium is not able to induce reactive oxygen species (ROS) directly because it is not effective of accepting or donating electrons under physiological conditions [12]. Even so, cadmium induces genetic damage, mutagenesis and inhibits DNA repair system [13].  A growing interest in the scientific community has been focused on plausible manners of protection from adverse effects induced by cadmium exposure. Because numerous effects to cadmium toxicity result from its genotoxic as far as pro-oxidative properties, it seems reasonable that special attention should be directed to agents that can prevent or reduce this metal-induced oxidative stress and genetic damage in several tissues, organs and systems such as liver, and peripheral blood cells [14,15]. Many authors assume that free radical scavengers and antioxidants are feasible for protecting against cadmium toxicity. Among them, fruits and vegetables have recognized nutritional valuable for human health mainly due to neutralization of reactive oxygen species. Of particular interest, carrot has high nutritional value as a result of bioactive constituents present in the vegetable [16].

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The aim of this study was to investigate if purple carrot extract is able to protect against the noxious activities induced by cadmium exposure in multiple organs of rats. For this purpose, histopathological analysis, genotoxicity and oxidative status were investigated in this setting.   

Materials and Methods  Animals and experimental design  All experimental protocols involving animals are conformed to procedures described in the Principles for the Use of Laboratory Animals Guidelines. The study was approved by the Animal Ethics Committee of Federal University of Sao Paulo, UNIFESP, SP, Brazil (Protocol CEUA n. 4698240214).  A total of twenty Wistar rats weighing 250g on the average, and 8 weeks age were distributed into four groups (n=5), as follows: Control group (non-treated group, CTRL); Cadmium group (Cd) and Purple carrot extract groups at 400 mg/L or 800 mg/L. All animals were provided from Development Center of Experimental Models for Medicine and Biology (CEDEME) of Federal University of São Paulo, SP, Brazil, and they were maintained under controlled conditions of temperature (23 ± 1ºC), lightdark periods of 12h and free access to water and diet. The experimental design was established in previous studies conducted by our research group [17,18].  Animals from control group received a single intraperitoneal (ip) water injection while those from the groups Cd and purple carrot extract received a single ip injection of cadmium chloride (1.2 mg/kg body weight) as established elsewhere [17]. After 15 days, purple carrot groups received purple carrot extract (Hansen, Campinas Brasil) for

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400mg/L or 800mg/L in drinking water ad libitum. The daily dose was calculated in order to provide the equivalent in humans of 2g polyphenols/day taking into consideration the rat metabolism (twice faster than humans). The amount was reported by the American Dietetic Association (ADA) as sufficient to promote beneficial health effects (ADA, 2004). This value corresponds to 400 mg/L of purple carrot extract [19]. Control and cadmium groups received drinking water during the same experimental period ad libitum. All animals were checked daily for behavior and general health conditions and body mass was recorded weekly. At the end of the experimental period, all animals were anesthetized with inhalational anesthetic halothane (Tanohalo™, Cristália™, SP, Brazil) and euthanatized for tissue collection.

Quantification of Total Phenols, Determination of Radical Scavenging Activity and Anthocyanins Total phenols were measured by the Folin-Ciocalteu assay using gallic acid (Sigma-Aldrich®, St Louis, MO, USA) for the standard curve and the results being expressed in mg gallic acid equivalents (GAE)/kg. The readings (in triplicates) were taken at 740 nm using a Genesis 2 spectrometer. For the evaluation of the antioxidant activity in vitro, the DPPH (1,1-diphenyl-2-picrylhydrazil) (Sigma-Aldrich®’, Steinheim, BW, Germany) assay was used based on the methods of Brand-Williams [20]. The absorbance was measured with a Beckman spectrometer at 517 nm before addition of samples and after 30 minutes; the difference was plotted on a vitamin C (ascorbic acid) (Merck®, USA) standard curve. Analyses were carried out in triplicates and the results expressed in mg Vitamin C equivalents (VCEAC)/kg. The presence of

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anthocyanins was measured in duplicate by UV-visible spectroscopy as described elsewhere [21]. 

Characterisation of purple carrot extract by electrospray ionization mass spectrometer (ESI-MS) The sample (100 mg) was extracted with 1000 µL of methanol. The extract solution was thoroughly mixed for 1 h with magnetic bar and filtrated. Then 80µL of this extract was diluted in 1000 µL of methanol. Formic acid (0,1%) was added to evaluate samples in positive mode and ammonium hydroxide (0,1%) was added to evaluate samples in negative mode. Samples were direct infused and analyzed by a 7.2T LTQ FT (FT-ICR) Ultra mass spectrometer (ThermoScientific, Bremen, Germany). The full scan spectra were acquired in the m/z range of 100-1200 Da. General ESI conditions were as follow: gas pressure of 0.3 psi, capillary voltage of ± 3.0 kV, tube lens of + 115 and - 125 V, capilar temperature of 280 °C and a flow rate of 5 µl min-1. Mass spectra were processed via the Xcalibur 2.0 software (ThermoScientific, Bremen, Germany).

Histopathological analysis Histopathological changes in liver, including steatosis and inflammation, were analyzed by the semi-quantitative method (Table 1) according to Aguiar et al [22].

Single cell gel (Comet) Assay 

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The protocol used for peripheral blood and liver cells followed the guidelines outlined by Tice et al.[23]. Peripheral blood was collected by cardiac puncture and liver cells were obtained by liver tissue maceration with PBS. Cells were transferred to individual plastic tubes, containing 1 mL of cold phosphate buffer solution (PBS, Ca+2, Mg+2 free, pH 7.3), and centrifuged for 5 min, 1000 rpm, at room temperature. The supernatant was removed and the cell suspensions (~10 µL) were used for single cell gel (Comet) assay. A volume of 10 µL of cellular suspension was added to 120 µL of 0.5% low-melting point agarose at 37oC, layered onto a pre-coated slide with 1.5% regular agarose, and covered with a coverslip. After brief agarose solidification in refrigerator, the coverslip was removed and the slides immersed in lysis solution (2.5 M NaCl, 100 mM EDTA– Merck™, Darmstadt, Germany; 10 mMTris–HCl buffer, pH 10 – Sigma Aldrich™, St Louis, MO, EUA; 1% sodium sarcosinate – Sigma™, St Louis, MO, EUA; with1% Triton X-100 – Sigma™, St Louis, MO, EUA; 10% dimethyl sulphoxide –Merck™, Darmstadt, Germany) for about 1 h. Afterwards, the slides were washed in ice-cold PBS (Ca+2, Mg+2 free, pH 7.3) for 5 min, left in electrophoresis buffer (0.3 mM NaOH and 1 mM EDTA – Merck™, Darmstadt, Germany, pH > 13) for DNA unwinding during 20 min, and electrophoresed in the same buffer for 20 min at 25 V (0.400 V/cm) and 300 mA. Following electrophoresis, slides were neutralized in 0.4 M Tris–HCl (pH 7.5, Sigma Aldrich™, St Louis, MO, EUA), fixed in absolute ethanol and stored at room temperature until analysis in a fluorescence microscope at 400x magnification. All steps were performed under reduced light.  A total of 50 randomly captured comets per animal (25 cells from each slide) were examined blindly by one expert observer at 400x magnification using a fluorescent microscope (Olympus™, Orangeburg, NY, USA). The microscope was connected through a black and white camera to an image analysis system (Comet Assay II,

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Perceptive Instruments™, Suffolk, Haverhill, UK) calibrated previously according to the manufacturer’s instructions. To measure DNA damage, we used the tail moment defined as the product of the tail length and the fraction of DNA in the Comet tail [23].    

Micronucleus Test  After completing the experimental period, the micronucleus test was performed in bone marrow and liver tissues. The bone marrow micronucleus test was performed according to Ribeiro et al. [24]. For this purpose, femoral bones were collected and stored in sodium chloride 0.9%. The proximal epiphyses of the bones were removed and 1mL of fetal bovine serum (FBS; Cultilab™, Campinas, São Paulo, Brazil) was injected into the medullar canal. A smear on glass slides was performed with the suspension formed by the bone marrow and fetal bone serum. After drying the slides, they were stained with Giemsa (Merck™, Darmstadt, Germany). For liver micronucleus test, paraffin sections (3µm) were stained by Feulgen and counterstained with Fast Green (Sigma Aldrich™, USA). A total of one thousand polychromatic erythrocytes or hepatocytes were analyzed per animal. Slides were scored blindly using a light microscope with a 100x immersion objective.  

Real Time PCR  Liver tissue at –80oC was homogenized and total RNA was isolated using cold Trizol Reagent (Invitrogen™, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Total

RNA

was

determined

using

a

NanoDrop™ND-1000

spectrophotometer (NanoDrop Technologies™, Wilmington, DE). RNA samples were

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treated with DNAse (DNase Amplification Grade™, Applied Biosystems™, Foster City, CA, USA) to avoid contamination with genomic DNA. cDNA synthesis was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Foster City, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed in the 7500 Fast Real-Time PCR System (Applied Biosystems™, Foster City, CA, USA) using the Power SYBR™ Green Kit (PCR Master Mix 2x, Applied Biosystems™, Foster City, CA, USA). Primers for the specific amplification of each cDNA were designed using the Primer Express software (Applied Biosystems™,Foster City, CA, USA), considering established criteria, such as product length, optimal PCR annealing temperature and the likelihood of primer self-annealing. The primers sequence are: GAPDH: sense 5’ – CAA CTC CCT CAA GAT TGT CAG CAA – 3’ and anti-sense 5’ – GGCATG GAC TGT GGT CAT GA – 3’; CuZn-Superoxide Dismutase: sense 5’ – CCAGTG CAG GAC CTC ATT TT – 3’ and anti-sense 5´– CCT TTC CAG CAG TCA CAT TG-3´; Manganese-SOD: sense 5’ – AAC ATT AAC GCG CAG ATC A – 3’ and anti-sense 5´ – AAT ATG TCC CCC ACC ATT GA – 3´;Catalase: sense 5’ – AGC GGA TTC CTG AGA GAG TG – 3’ and anti-sense 5’ – GAG AAT CGA ACG GCA ATA GG – 3’; Cytocrome C: forward 5′-CCAGTGCAGGACCTCATTTT-3′ and reverse 5′CCTTTCCAGCAGTCACATTG-3′. PCR reactions were performed in duplicate containing 20µL final volume using 2.0 µL of a 1:5 (v/v) dilution of cDNA, 2.0 µL of primer mix (forward and reverse),10.0 µl of Power SYBR™ Green (PCR Master Mix 2x,Applied Biosystems™, Foster City, CA, USA) and DPEC water (Ultrapure™ DEPC Treated Water, Invitrogen™, Carlsbad, CA, USA). The reactions were performed in Micro Amp™ 96-well plates (Applied Biosystems™, Foster City, CA, USA) covered with optical adhesive (Applied

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Biosystems™, Foster City, CA, USA). Samples were submitted to forty cycles of 95°C for 10 min, 95°C for 15 sec and 60°C for 1 min. An amplification efficiency curve using different cDNA dilutions was also performed for each gene tested. To normalize the data for the control and experimental groups, arbitrary units were calculated as: arbitrary unit = 2-ΔΔCT, and ΔΔCT = sample ΔCT – control ΔCT, where CT is the threshold cycle.

OHdG Immunohistochemistry  Liver serial sections of 4 m were desparafinized in xylene and rehydrated in graded ethanol (99.5%), then pretreated in a microwave with 10mM citric acid buffer (pH 6, 0.1M citric acid – Synth™, São Paulo, Brazil; 0.1M sodium citrate – Synth™, São Paulo, Brazil) for 3 cycles of 5min each for antigen retrieval. They were preincubated with 0.3% hydrogen peroxide for inactivation of endogenous peroxidase and then blocked with 5% normal goat serum for 30 min. The specimens were then incubated

with

anti-8-hydroxy-20-deoxyguanosine

(8-OHdG,

Santa

Cruz

Biotechnologies Inc™, MO, USA) at 1:100 dilution, overnight, at 4oC. This was followed by two washes in PBS and further incubation with a biotinylated secondary antibody, diluted 1:100 in PBS for 1 h. The sections were washed twice with PBS followed by the application of preformed avidin biotin complex (Vector Technologies™, USA) for 45 minutes. The bound complexes were visualized by the application of a 0.05% solution of 3,3-diaminobenzidine (Sigma™, St Louis, MO, EUA) and counterstained with hematoxylin (Sigma™, St Louis, MO, EUA). Sections stained using immunohistochemistry were analyzed for the percentages of

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immunopositive cells in liver. A total of 1000 hepatocytes were evaluated in 3 to 5 fields at 400x magnification. These values were used as labeling indices.    

Statistical Analysis  All the data are expressed as mean ± standard deviation (SD). For micronucleus test, immunohistochemistry and Real Time PCR data was used one-way analysis of variance (one way – ANOVA) followed Tukey's multiple comparisons post-hoc. For Comet Assay, two-way analysis of variance (two way – ANOVA) was performed followed by Tukey’s multiple comparisons test. Statistical analysis was performed using Graph Pad Prism™ 6.0 program. p<0.05 was considered to be significant.   

Results  Chemical analyses The results of the total phenol content of purple carrot extract showed a concentration of 101.4±1.1 g gallic acid equivalents/kg with an antioxidant activity of 14.6±0.2 g vitamin C equivalents/kg. The total amount of monomeric anthocyanins present in the purple carrot extract was 20.3 g/kg. Figure 1 and 2 show the ESI-MS in the positive and negative ion mode for the qualitative evaluation of the carrot extract. A total of 8 constituents were identified on the measurements of their accurate masses and comparisons with fragmentation profiles (Table 2). Several phenolic compounds were identified as their deprotonated molecules such

as

cyanidin-3-(2”-xylosyl-galactoside),

cyanidin-3-(2”xylopiranose-6”-(6”’-

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feruloylglucopyranose)-galactopyranose),

cyanidin-3-(2”xylosyl-6”-(6”’-

sinapoylglucoside)-galactoside), caffeoyl N- tryptophan, caffeoyl N- tryptophan hexoside, caffeoylquinic acid and citric acid.

Clinical findings Neither complications nor behavioral changes were observed. No statistically significant differences (p>0·05) were observed to the final body weight and the weight gain for all groups evaluated. No animals died during the experiment. Such findings are showed to Table 3.

Histopathological analysis Table 4 shows the positive effect of purple carrot extract on the degree of histopathological changes in liver tissues in normal and cadmium-intoxicated rats. Under microscopic evaluation, liver tissues from the control group presented ordinary architecture based on hepatocytes, sinusoidal vessels and portal field preserved (Figure 3a). In the group intoxicated with cadmium, all animals presented extensive hepatic degeneration after 30 days of intoxication (Figure 3b) when compared to the negative control group (Figure 3a). Degenerative vacuoles in hepatic tissues, increased steatosis in lobular central bands, spotty necrosis and focal necrosis were observed in the majority of animals exposed to cadmium. Such changes were categorized as score number 3 for all animals. In specimens exposed to cadmium and purple carrot extract, apparent normal architecture of liver tissues containing mild changes was found for both doses tested when compared to cadmium exposed group. (Figure 3c). A total of

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four animals were categorized as score 1 and one animal as score 2 in these groups. No remarkable histopathological differences were noticed between the two doses tested.

Comet Assay   The single cell (comet) assay was used to detect DNA strand breaks. Peripheral blood data showed a significant increase for DNA damaging cadmium group when compared to the control group. In groups exposed to purple carrot extract and cadmium, a reduction was detected and significant statistically differences (p<0.05) were noticed when compared to cadmium group for both doses used in this study. Such data are demonstrated in Figure 4. Liver was also evaluated for genetic damage in this experimental design by comet assay. Cadmium was very genotoxic as depicted by tail moment results when compared to negative control. Purple carrot extract was able to reduce DNA damage in liver cells in rats exposed to cadmium. This occurred to similar manner for both doses used in this study.  

Micronucleus Test  Micronucleus test in bone marrow and liver data showed that cadmium was able to stimulate micronuclei formation in both tissues indistinctly as a result of chromosomal breakage or loss. Nevertheless, a significant decrease in the number of micronucleated erythrocytes was observed to animals exposed to cadmium and treated with purple carrot extract when compared to animals only exposed to cadmium. Such anti-mutagenic findings were observed for both doses tested. In liver, the same picture

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did not occur. Although cadmium intoxication be mutagenic in liver cells, purple carrot extract decreased the micronucleus frequency at the highest dose tested in this study. Control animals presented lower frequency of micronucleated cells when compared to other groups. Such findings are summarized in Figure 5.  

Immunohistochemistry  8OHdG immunomarker was detected in the nucleus or cytoplasm of liver cells. In the negative control group, weak immunoreactivity for 8OHdG was found (Figure 6a). In the group exposed to cadmium, the majority of liver cells were positive for 8OHdG immunomarker (Figure 6b). Purple carrot extract was able to reduce the 8OHdG levels when compared to cadmium group, being significant statistically differences (p<0.05) between groups (Figure 6c). These numerical results can be better visualized in Figure 7.   

Real Time PCR  Antioxidant enzymes Copper-Zinc Superoxide Dismutase (CuZn-SOD), Manganese Superoxide Dismutase (Mn-SOD) and Catalase (CAT) were analyzed by Real Time Polymerase Chain Reaction (qPCR). CuZn-SOD increased after treatment with purple carrot extract for both doses used in this study. However, Mn-SOD expression did not show statistically significant difference between groups (p>0.05). Regarding catalase expression, the same picture occurred, i.e. no significant statistically differences (p>0.05) were noticed to purple carrot extract group at 400mg/L. The group

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treated with purple carrot extract at 800mg/L decreased catalase levels (p<0.05) when compared to cadmium group. Such findings are summarized in Figure 8. Cytocrome C gene expression increased in groups treated with purple carrot extract in a dose-dependent manner (Figure 9).

Discussion  In the last decades, discussion regarding the relationship on development of diseases induced by environmental pollutants is increasing in the scientific literature. Among the numerous toxic substances to which, human and other mammalian species are exposed is cadmium, a heavy metal whose half-life range between 10 and 30 years. It promotes extensive damage to several tissues such as liver, kidney, lungs and blood [25]. The aim of this study was to evaluate if purple carrot extract is able to protect against the noxious activities induced by cadmium in rats. For this purpose, histopathological analysis, genotoxicity and oxidative status were evaluated in this study. To the best of our knowledge, the approach has not been addressed so far.  Histopathological analysis from liver revealed that animals intoxicated with cadmium presented extensive necrosis of parenchyma, presence of hemorrhagic areas, congested vessels and tissue degeneration. However, animals treated with purple carrot extract improved the microscopic changes induced by cadmium exposure as depicted by reduction of necrosis and hemorrhagic areas. Therefore, we assume that purple carrot extract protects liver against cadmium induced tissue damage. By comparison, some studies have demonstrated that black, red, and white tea exerted a varied impact on the microscopic architecture and innervations of the small intestine wall as well as on the absorptive function of small intestine mucosa in rats poisoned with cadmium [26].

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Physalis (family Solanaceae) treatment was also able to reverse the histopathological changes in liver and kidney tissues after cadmium intoxication using experimental models [27]. Cadmium is able to induce DNA lesions by interactions with the cellular DNA damage response system [28].  The micronucleus test and comet assay showed that cadmium greatly increased the number of micronucleus and DNA strand breaks, respectively, in blood and liver cells. These tests are widely employed to detect the genotoxicity of xenobiotics as a result of genetic damage that could be induced by oxidative stress [29]. According to Tapisso et al. [30], the presence of cadmium is related with increased frequencies of micronucleus and, even at low concentrations for inducing DNA damage.  Protection against genetic damage and modulation of DNA repair system play an important role on prevention of mutations. Several fruits and vegetables, as well as its bioactive compounds, have been investigated for its antioxidant effects [31]. In this study, it was observed that purple carrot extract was able to reduce mutagenic and genotoxic effects of this metal in erythrocytes and hepatocytes. Probably, it was due to its antioxidant activity. In fact, some authors have assumed that bioactive phenols present in lamiaceae plants may prevent carcinogenesis through scavenging free radicals and inhibiting DNA damage induced by cadmium exposure [32]. A previous study conducted by our research group has demonstrated that pretreatment with higher doses of the purified anthocyanin (10 and 20mg/kg b.w.) led to a statistically significant reduction (p<0.05) in the frequency of micronuclei in polychromatic erythrocytes induced by cyclophosphamide. The pattern of reduction ranged from 48% to 57% independent of concentration used. No apparent genotoxicity and mutagenicity was found for either the anthocyanin or delphinidin extracts [33].

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Accumulating evidence suggests that cadmium induces oxidative damage by disturbing the antioxidant defense systems, and the enhancement of ROS production is responsible to suppress free-radical scavenger enzymes, such as superoxide dismutase and catalase [34]. Higher cadmium concentration in tissues cause oxidative stress by increasing malondialdehyde as a mean of altering antioxidant defense system and deterioration of bioelements in rat liver, kidney, and heart [35]. CuZn-SOD and MnSOD are powerful superoxide scavengers able to catalyze the conversion of O2− radicals into O2 and H2O2 [36]. Some published studies reveal different results regarding the activity of antioxidant enzymes in animals exposed to cadmium. Ognjanović et al. [37] administered cadmium chloride (15 mg kg−1−1for 4 weeks) to rats, and their results showed a decrease in liver SOD activity. Jihen et al. [38] offered cadmium chloride in tap water (200 mg Cd/L) during 35 days to rats and observed an increase on CuZn-SOD activity, whereas Mn-SOD activity was not affected by cadmium. Our results demonstrated that CuZn-SOD expression increased after treatment with purple carrot extract. MnSOD did not show remarkable differences between groups. Catalase decreased after treatment with purple carrot extract at the highest dose (800mg/L). This result is new and could be explained by the fact that the mechanism of action of polyphenols and anthocyanins present in purple carrot extract is related to metals removal as well as scavenging free radicals [39]. Low levels of the enzyme catalase activity in this study may be due to the action of bioactive compounds present in the purple carrot extract and should have acted in scavenging free radicals without the need to activate the antioxidant properties present in mammalian cells. Further studies are welcomed to elucidate the issue. The interaction of reactive oxygen species with the bases of the DNA strand, as guanine, leads to the formation of 8-hydroxyguanine (8-OHGua) or its nucleoside form

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deoxyguanosine (8-hydroxy-2’-deoxyguanosine). This induces the generation of radical adducts and synthesis of 8-hydroxy-2’-deoxyguanosine (8-OHdG), one of the most important marker for measuring endogenous oxidative damage to DNA and cancer [40]. However, the increased exposure of mitochondrial DNA to ROS also causes elevated levels of 8OHdG and may result in an increased formation of DNA-protein crosslinks [41]. According to Kitada and colleagues [42], 8-OHdG expression can be widely observed in various forms of chronic liver disease, especially because 8-OHdG hepatic localization is not always similar due to the differences in the source and target of oxidative stress among the liver diseases. Furthermore, Nomoto et al. [43] showed that in nonalcoholic steato-hepatitis and steatosis, the 8-OHdG cytoplasmatic expression reflects oxidative damage to the mitochondrial DNA of hepatocytes. This confirms our results once cadmium exposed group showed a substantial

8-OHdG immunoexpression either to nucleus or cytoplasm of liver cells. Treatment with purple carrot extract decreased the immunoexpression of 8-OHdG in liver cells. Carrots contain many active phenolic compounds which can eliminate free radicals and prevent aggressive metabolites that act as mutagenic agents. Taken a whole, we assume that purple carrot extract possess antioxidant activity in liver cells protecting against oxidative stress induced by cadmium as a result of decreasing 8-OHdG levels. Mitochondria have been reported to play a critical role in the regulation of apoptosis [44]. The loss of apoptogenic factors, such as cytocrome C from mitochondria into the cytosol is associated with apoptosis. Some authors have revealed that overexpression of cytosolic copper-zinc SOD attenuates cytochrome c release after some pathological conditions such as cerebral ischemia, whereas there is an exacerbation after loss of the mitochondrial antioxidant MnSOD, or CuZnSOD [45]. These data give evidence that increases in cellular superoxide, cytosolic or mitochondrial, can serve as the signaling event for cytochrome c release after tissue injury. The release of cytochrome  c  may result in further ROS production by inhibition

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of the respiratory chain [46]. In the present study, the release of cytocrome c was observed in the animals exposed to cadmium and treated with purple carrot extract in a dose-dependent manner. Some authors have postulated that anthocyanins attenuated the dysregulation of Ca(2+), ROS accumulation, activation of AMPK, and increase in percentage of apoptotic cells [47]. Others have mentioned that anthocyanins act against human breast cancer cells in vitro and in vivo by inducing apoptosis and suppressing angiogenesis [48]. Therefore, it seems that purple carrot extract is able to trigger apoptosis process, by means of intrinsic signaling pathway as a result of cytocrome c activity. In summary, our results demonstrate that purple carrot extract is able to prevent tissue degeneration, genotoxicity and oxidative stress induced by cadmium exposure in multiple organs of Wistar rats. 

Financial Support This study was supported by CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de NIvel Superior).

Conflict of Interest  None declared.

Acknowledgements  The authors are grateful to Hansen Company (Campinas, SP, Brazil) for providing us the purple carrot extract.  

 

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23. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay, guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35(3): 206-221. 24. Ribeiro DA, Grilli DG, Salvadori DM. Genomic instability in blood cells is able to predict the oral cancer risk, an experimental study in rats. J Mol Histol. 2008; 39(5):481-486. 25. Nair PM, Park SY, Chung JW, Choi J. Transcriptional regulation of glutathione biosynthesis genes, γ-glutamyl-cysteine ligase and glutathione synthetase in response to cadmium and nonylphenol in Chironomus riparius. Environ Toxicol Pharmacol. 2013; 36(2):265-273. 26. Tomaszewska E, Winiarska-Mieczan A, Dobrowolski P. Hematological and serum

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32. Thirugnanasampandan R, Jayakumar R. Protection of cadmium chloride induced DNA damage by Lamiaceae plants. Asian Pac J Trop Biomed. 2011; 1(5):391394. 33. Azevedo L, Alves de Lima PL, Gomes JC, Stringheta PC, Ribeiro DA, Salvadori DM. Differential response related to genotoxicity between eggplant (Solanum melanogena) skin aqueous xtract and its main purified anthocyanin (delphinidin) in vivo. Food Chem Toxicol. 2007; 45(5):852-858. 34. Cichoż-Lach H, Michalak A. Oxidative stress as a crucial factor in liver diseases. World J Gastroenterol. 2014; 20(25): 8082-8091. 35. Erdem O, Yazihan N, Kocak MK, Sayal A, Akcil E. Influence of chronic cadmium exposure on the tissue distribution of copper and zinc and oxidative stress parameters in rats. Toxicol Ind Health. 2015; in press. 36. Klaunig JE, Kamendulis LM, Hocevar BA. Oxidative stress and oxidative damage in carcinogenesis. Toxicol Pathol. 2010; 38(1): 96-109. 37. Ognjanović BI, Marković SD, Pavlović SZ, Zikić RV, Stajn AS, Saicić ZS. Effect of chronic cadmium exposure on antioxidant defense system in some tissues of rats, protective effect of selenium. Physiol Res. 2008;57(3):403-411. 38. Jihen el H, Sonia S, Fatima H, Mohamed Tahar S, Abdelhamid K. Interrelationships between cadmium, zinc and antioxidants in the liver of the rat exposed orally to relatively high doses of cadmium and zinc. Ecotoxicol Environ Saf. 2011; 74(7): 2099-2104. 39. Flora SJ, Shrivastava R, Mittal M. Chemistry and pharmacological properties of some natural and synthetic antioxidants for heavy metal toxicity. Curr Med Chem. 2013;20(36):4540-4574. 40. Valavanidis A, Vlachogianni T, Fiotakis C. 8-hydroxy-2' -deoxyguanosine (8OHdG), A critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2009; 27(2): 120-39. 41. Cahill A, Wang X, Hoek JB. Increased oxidative damage to mitochondrial DNAfollowing chronic ethanol consumption. BiochemBiophys Res Commun. 1997;235(2):286-290. 42. Kitada T, Seki S, Iwai S, Yamada T, Sakaguchi H, Wakasa K. In situ detectionof oxidative DNA damage, 8-hydroxydeoxyguanosine, in chronic human liver disease.J Hepatol. 2001 ;35(5):613-618. 43. Nomoto K, Tsuneyama K, Takahashi H, Murai Y, Takano Y. Cytoplasmic finegranular expression of 8-hydroxydeoxyguanosine reflects early mitochondrialoxidative DNA

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26 

Figure Captions Figure 1. Chemical characterization of purple carrot extract by mass spectrometry. Figure 2. Characterization of the carrot extract by ESI-MS in the negative ion mode. Figure 3. Photomicrographies from rat liver exposed to cadmium and treated with purple carrot extract. G1 – Control group; G2- Cadmium group; G3 – Cadmium and purple carrot extract at 400 mg/L; G4 – Cadmium and purple carrot extract at 800 mg/L. Figure 4. Mean ± S.D. of DNA damage in peripheral blood cells (A) and liver cells (B) following cadmium exposure and treatment with purple carrot extract. G1 – Control group; G2- Cadmium group; G3 – Cadmium and purple carrot extract at 400 mg/L; G4 – Cadmium and purple carrot extract at 800 mg/L. *p<0.05 when compared to G2; **p<0.05 when compared to control (G1). Figure 5. Total number of micronucleated cells in bone marrow cells (A) and liver cells (B) induced by cadmium exposure and treated with purple carrot extract. G1 – Control group; G2- Cadmium group; G3 – Cadmium and purple carrot extract at 400 mg/L; G4 – Cadmium and purple carrot extract at 800 mg/L. *p<0.05 when compared to G2; **p<0.05 when compared to control. Results are expressed as Mean ± S.D. Figure 6. Mean ± S.D. of 8-OHdG immunoreactivity in liver cells of rats exposed to cadmium and treated with purple carrot extract. G1 – Control group; G2- Cadmium group; G3 – Cadmium and purple carrot extract at 400 mg/L; G4 – Cadmium and purple carrot extract at 800 mg/L. Figure 7. Semi-quantitative analysis of 8-OHdG positive cells in liver following cadmium exposure and treatment with purple carrot extract. G1 – Control group; G2-

27 

Cadmium group; G3 – Cadmium and purple carrot extract at 400 mg/L; G4 – Cadmium and purple carrot extract at 800 mg/L. *p<0.05 when compared to G2; **p<0.05 when compared to control (G1). Results are expressed as Mean ± S.D. Figure 8. Gene expression of CuZn-SOD (A); Mn-SOD (B) and catalase (C) in liver cells after cadmium exposure and treatment with purple carrot extract. G1 – Control group; G2- Cadmium group; G3 – Cadmium and purple carrot extract at 400 mg/L; G4 – Cadmium and purple carrot extract at 800 mg/L. *p<0.05 when compared to G2. Results are expressed as Mean ± S.D Figure 9. Gene expression of cytocrome C in liver cells after cadmium exposure and treatment with purple carrot extract. G1 – Control group; G2- Cadmium group; G3 – Cadmium and purple carrot extract at 400 mg/L/; G4 – Cadmium and purple carrot extract at 800 mg/L. *p<0.05 when compared to G2; **p<0.05 when compared to G3. Results are expressed as Mean ± S.D.

28  Figure 1  9 1 9 .2 4 9 4 1

100 95 90 85 80 75 70 65

Relative Abundance

60 4 5 5 .1 1 6 0 7

3 7 7 .0 8 4 4 0

55 50

5 2 7 .1 5 8 3 9

45 40 35

3 5 5 .1 0 2 7 5 C 16 H 19 O 9

30

1 .0 8 9 0 6 p p m

7 3 1 .1 7 9 8 8 C 34 H 35 O 18

25

-2 .6 1 8 2 7 p p m

20

8 6 9 .2 7 5 2 8

15

6 8 9 .2 1 1 7 2

5 8 1 .1 5 0 6 6 C 26 H 29 O 15

10

0 .9 7 4 0 2 p p m

4 3 3 .1 3 4 5 2 C 18 H 25 O 12

3 0 9 .2 0 3 9 1

7 6 1 .2 3 3 6 6 7 9 8 .3 2 5 2 2 C 45 H 50 O 13

1 .0 7 9 3 6 p p m

5

9 4 9 .2 6 1 6 9

8 5 1 .2 6 4 8 4

9 9 1 .3 3 7 0 3

0 .7 8 7 6 8 p p m

2 7 6 .1 4 4 4 6 0 200

250

300

350

400

450

500

550

600 m /z

650

700

750

800

850

900

950

1000

 

  Figure 2  545.11464 C 22 H 25 O 16 1.71053 ppm

100 95 90 85 80 75 70 65

Relative Abundance

60 55 353.08790

50 45 40 35 30 25 20

601.12920 C 39 H 21 O 7 1.69665 ppm

15 515.12550 C 18 H 27 O 17

191.01984 10

399.09345

5

225.06174 174.04090

0 100

200

2.36964 ppm

925.23445 949.26151

308.09887 300

400

500

600 m/z

   

707.18283 C 32 H 35 O 18 1.47396 ppm

 

700

800

900

1000

 

29    Figure 3 

    Figure 4 

   

 

30  Figure 5  Mean number of micronucleated cells Mean number of micronucleated cells

A

B

*

15

* 10

** 5

0 G1

G2

G3

G4

1 G1

2 G2

3 G3 Groups

Groups

4 G4

 

      Figure 6 

     

 

31  Figure 7  Mean number of 8-oHDG positive cells

* 120 100 80

**

**

60 40 20 0 1 G1

2 G2

3 G3

4 G4

Groups

 

            Figure 8 B

A

C

2-

Ct

*

G1

Groups

   

G2

 

G3

G4

d

G4

d

G3 Groups

O

G2

G

G1

G1

G2

G3 Groups

G4

 

32  Figure 9 

 

33 

Tables Table 1. Score criteria for histopathological changes in liver adopted in this study (21). Score

Necrosis area

Structural changes

0

0

Tissue structure preserved

1

<30

Watering degeneration and small necrosis.

2

>30

Increased of eosinophilie, presence of congested vessels, vacuolization.

3

>50

Severe degeneration, necrosis and loss of structure.

34 

Table 2. Compounds tentatively identified in purple carrot extract using ESI-MS in the negative and positive ion mode. Compound ESI(+)-MS Unknown Unknown Caffeoyl N- tryptophan Vinylgualacol aglicon Unknown Caffeoyl N- tryptophan hexoside Cyanidin-3-(2”-xylosyl-galactoside) Unknown Unknown Unknown Cyanidin-3-(2”xylopiranose-6”-(6”’feruloylglucopyranose)galactopyranose) Cyanidin-3-(2”xylosyl-6”-(6”’sinapoylglucoside)-galactoside) ESI(-)-MS Citric Acid Caffeoylquinic Acid Unknown Unknown Unknown Unknown

Precursor Ion m/z

MS/MS Fragment Ion m/z

309.20391 355.10275 365 433.13452 455.11607 527.15839 581.15066 689.21172 731.17988 869.27528

291, 263 314, 163 203 127, 145 395, 329, 149 365 287

919.24941

287, 355, 554, 625

949.26169

191.01984 353.08790 399.09345 482.13053 545.11464 707.18283

377 527

287, 365, 589

87, 111 191, 135 191, 353 353, 191 191, 111 191, 353

35 

Table 3. Mean final body weight and weight gain (±S.D.) of rats intoxicated with cadmium and treated with purple carrot extract CTRL

Final body weight (g) Weight (g)

gain

Cd

Cd+ Purple carrot extract (400mg/

Cd+ Purple carrot extract (400mg/

L)

L)

399.5 ± 1.9

363.4 ± 46.7

407.6 ± 12.7

373.4 ± 27.3

111.3 ± 11.6

91.2 ± 46

195 ± 36.8

166 ± 4.5

p>.0.05. CTRL: control group; Cd: cadmium group; n=5 per group.

36 

Table 4. Total number of rats in all groups according to degree of liver histological changes. Groups

0

1

2

3

Concentrations

Number of animals

Control

5

5

0

0

0

Cadmium

5

0

0

0

5

L)*

5

0

4

1

0

Cadmium + Purple carrot extract (800mg/L)*

5

0

4

1

0

Cadmium + Purple carrot extract (400mg/

Semi quantitative scoring evaluation in liver histology (According to Aguiar Jr et al. 2011).* Significantly different when compared with its respective control group (p<0.05)