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In Vivo Assessment of Genotoxic, Antigenotoxic and Anticarcinogenic Activities of Solanum lycocarpum Fruits Glycoalkaloidic Extract

In Vivo Assessment of Genotoxic, Antigenotoxic and Anticarcinogenic Activities of Solanum lycocarpum Fruits Glycoalkaloidic Extract

  • Carla Carolina Munari, 
  • Pollyanna Francielli de Oliveira, 
  • Luis Fernando Leandro, 
  • Leandra Mara Pimenta, 
  • Natália Helen Ferreira, 
  • Juliana de Carvalho da Costa, 
  • Jairo Kenupp Bastos, 
  • Denise Crispim Tavares
PLOS
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Abstract

The fruits of Solanum lycocarpum, known as wolf-fruit, are used in folk medicine, and because of that we have evaluated both the genotoxic potential of its glycoalkaloidic extract (SL) and its influence on the genotoxicity induced by methyl methanesulfonate. Furthermore, the potential blocking effect of SL intake in the initial stage of colon carcinogenesis in Wistar rats was investigated in a short-term (4-week) bioassay using aberrant crypt foci (ACF) as biomarker. The genotoxic potential was evaluated using the Swiss mice peripheral blood micronucleus test. The animals were treated with different doses of SL (15, 30 and 60 mg/kg b.w.) for 14 days, and the peripheral blood samples were collected at 48 h, 7 days and 14 days after starting the treatment. For antigenotoxicity assessment, MMS was administered on the 14th day, and after 24 h the harvesting of bone marrow and liver cells was performed, for the micronucleus and comet assays, respectively. In the ACF assay, male Wistar rats were given four subcutaneous injections of the carcinogen 1,2-dimethylhydrazine (DMH, 40 mg/kg b.w.), twice a week, during two weeks to induce ACF. The treatment with SL (15, 30 and 60 mg/kg b.w.) was given for four weeks during and after carcinogen treatment to investigate the potential beneficial effects of SL on DMH-induced ACF. The results demonstrated that SL was not genotoxic in the mouse micronucleus test. In animals treated with SL and MMS, the frequencies of micronucleus and extensions of DNA damage were significantly reduced in comparison with the animals receiving only MMS. Regarding the ACF assay, SL significantly reduced the frequency of ACF induced by DMH.

Introduction

Genomic instabilities are induced by the usual metabolic processes, as well as by exogenous factors comprising diet, life style, and environmental stresses, such as solar radiation [1]. There is increasing evidence that cancer and other mutation-related diseases can be prevented not only by avoiding exposures to recognized risk factors, but also by favoring the intake of chemoprophylactic agents, as well as by modulating the defense mechanisms of the host organisms. This strategy, referred to as chemoprevention, can be pursued either by means of suitable pharmacological agents and/or by dietary factors [2]. Plants are considered among the main sources of biologically active chemicals, and a wide number of new compounds originated from natural resources have been developed into commercial drugs [3].

Plants of Solanum genus display anti-inflammatory [4], hypotensive [5], hepatoprotective [6], antiviral [7], antiurolithiasic [8] and antiallergic activities [9]. Solanum species are known for their high content of alkaloids, mainly for the two major glycoalkaloids, solamargine and solasonine, which are found in at least 100 species. Studies on the activities of these glycoalkaloids showed that it inhibit the growth of different cancer cell types, e.g. human colon (HT-29, HCT-15), human prostate (LnCaP, PC-3), human breast (T47D, MDA-MB-231, MCF-7), human hepatoma (HepG2, SMMC-7721), human cervical cancer (JTC-26, HeLa), human leukemia (K562), human glioblastoma (M059J, U343, U251), human osteosarcoma (U2OS), and murine melanoma (B16F10) [10]; [11]; [12].

Solanum lycocarpum A. St.-Hil. (Solanaceae) is a native plant found in the Southeast and West-Central Brazilian savanna called “cerrado”. The fruits of this plant, known as wolf-fruit, are used in folk medicine as antiepileptic, antispasmodic, hypoglycemic and hypocholesterolemic agent, as well as for controlling obesity [13].

Considering the frequent use of S. lycocarpum fruit in folk medicine and its medicinal properties, we have undertaken the genotoxic, antigenotoxic and anticarcinogenic activities of S. lycocarpum fruits glycoalkaloidic extract in rodents.

Materials and Methods

1. Plant material, attainment and chromatographic analysis of alkaloidic extract

Fruits of S. Lycocarpum A. St.-Hil. Solanaceae were collected in the Campus of the University of São Paulo in Ribeirão Preto, São Paulo State, Brazil. The plant material was identified by Prof. Dr Milton Groppo Junior and a voucher specimen (code: SPFR: 11638) is deposited in the herbarium of the Faculty of Philosophy Sciences and Letters, University of São Paulo, Ribeirão Preto (SP), Brazil.

Fresh fruits were chopped and dried using an air circulating oven at 40°C. The alkaloidic extract was obtained by acid-base selective extraction [14]. The powdered dried fruits (1 kg) of S. lycocarpum were submitted to hydrochloric acid (0.5 M) extraction, three times sequentially, overnight by maceration, followed by percolation and centrifugation. Next, the aqueous acid extracts were alkalinized by adding NaOH (3.0 M) to achieve pH 12.0. Enough time was allowed for complete precipitation of the alkaloids. After precipitation, the supernatants were decanted and the wet precipitated biomass was centrifuged, combined and dried in an oven at 40°C. The dried rich alkaloidic extract was powdered in a knife mill, suspended in distilled ethanol and was submitted to sonication in an ultrasonic bath at room temperature (25°C). The ethanol soluble fraction was filtered, concentrated under reduced pressure and lyophilized to furnish the alkaloidic extract (9.2 g).

2. HPLC-DAD analysis

Chromatographic analyses were performed in an HPLC equipped with a diode array detector (HPLC-DAD) (Shimadzu) with Shimadzu Class-VP version 5.02 software, using a Zorbax SB-C18, 250×4.6 mm, colunm (Agilent). The glycoalkaloid analysis was undertaken employing an isocratic method of elution using acetonitrile/phosphate buffer (0.01 M) at pH 7.2 (36.5∶63.5). Standards of solasonine and solamargine were previously isolated from the alkaloidic extract. The flow rate was 1 mL min−1 for 20 min, and detection was set at 200 nm [14].

For the experiments, S. lycocarpum fruits glycoalkaloidic extract (SL) was dissolved in sterile distilled water.

3. Animals

Male Swiss mice (Mus musculus), 6–8 weeks old and weighing approximately 25 g, and male Wistar rats aged five weeks and weighing approximately 120 g were supplied by the Animal House of the School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto (São Paulo, Brazil). The animals were kept in plastic boxes in an experimental room under controlled conditions of temperature (232°C) and humidity (5010%), under a 12-h light-dark cycle, with free access to regular laboratory diet chow and tap water. The study protocol was approved by the Ethics Committee for Animal Use of the University of Franca (process no. 033/009).

4. DNA damage-inducing agents

Methyl methanesulfonate (MMS, CAS: 66-27-3, Sigma-Aldrich, St. Louis, USA) was dissolved in distilled water and used as the positive control for the genotoxicity and antigenotoxicity tests. The MMS dose (40 mg/kgb.w.) was selected based on its effectiveness in inducing DNA damage.

The well-known colon carcinogen DMH (Sigma-Aldrich) was dissolved immediately before use in 1 mmol/L EDTA (ethylenediaminetetraacetic acid). A total dose of 160 mg/kg b.w. was divided into four subcutaneous (sc) injections of 40 mg/kg b.w., which was administered twice a week for two weeks (weeks 2 and 3).

5. Genotoxic and antigenotoxic assessment

The genotoxic potential was evaluated using the Swiss mice peripheral blood micronucleus test. The doses of SL were selected based on literature data [15] and preliminary experiments using the doses of 15, 30, 60, 120, 250, 500 and 1000 mg SL/kg b.w. The doses above 60 mg/kg b.w. showed to be toxic (data not shown). Therefore, the animals were treated by gavage (0.5 mL/animal) with 15, 30 and 60 mg/kg b.w. of SL or water (negative control) for 14 days. Peripheral blood samples were collected at 48 h, 7 days and 14 days after treatment for genotoxic evaluation. For antigenotoxicity experiments, on the 14th day, it was administered MMS (i.p.; 0.3 mL/animal), and after 24 h it was performed the harvesting of bone marrow and liver cells, for the micronucleus and comet assays, respectively. Body weight and water consumption were measured throughout the experimental period.

6. Micronucleus assay

The in vivo peripheral blood micronucleus assay in bone marrow was performed according to the protocols described by MacGregor et al. [16]; [17]. For the determination of the frequency of micronucleated polychromatic erythrocytes (MNPCEs), 2000 polychromatic erythrocytes (PCEs) per animal were analyzed by light microscopy with a 100x immersion objective. A total of 400 erythrocytes per animal were scored to calculate the PCE/PCE+NCE (normochromatic erythrocytes) ratio in order determine the cytotoxicity of the treatments [18].

7. Comet assay

The alkaline comet assay was performed according to Burlisson et al. [19] under dim indirect light. Briefly, 20 µL of the liver cells suspension were mixed with 120 µL of molten 0.5% low-melting-point agarose and layered on a slide precoated with a thin layer of normal-melting-point agarose. The slides were placed into a lysis solution (2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris, 1% sodium laurylsarcosine, pH 10; with 1% Triton X-100 and 10% DMSO added just before use) for 24 h. Then, the slides were washed in PBS and placed into a horizontal electrophoresis unit filled with freshly made alkaline buffer (1 mmol/L EDTA and 300 mmol/L NaOH, pH 13). After 20-min of DNA unwinding period, electrophoresis was carried out in the same buffer at 25 V and 300 mA for 20 min. Afterwards, the slides were neutralized (0.4 mol/L Tris, pH 7.5) and fixed with 100% ethanol for 10 min.

All the slides of the experiment were coded before analysis. The slides were stained with 40 µL ethidium bromide solution (20 µg/mL in H2O) and covered with a coverslip. The comet DNA damage was visualized an AXIO Imager fluorescence microscope (Carl Zeiss, Germany) under 40× objective, and the images were captured with image analysis software (Comet imager V.2.0.0). DNA damage was quantified using a total of 100 nucleoids per repetition, resulting in a total of 600 nucleoids per treatment. The DNA damage was measured using DNA percentage in comet tail. As reported in an earlier paper [20], DNA percentage in tail is considered the best parameter for representation of DNA damage using comet assay.

For evaluating the viability, it was used the Trypan blue staining. Briefly, a fresh prepared solution of 20 µL Trypan blue (0.4%) in distilled water was mixed with 20 µL of each liver cell suspension obtained from comet assay, spread onto a microscope slide and covered with a coverslip. Non-viable cells appeared blue. At least 200 liver cells were counted per animal. Liver cells viability was calculated as a percentage of the respective control values.

8. Calculation of DNA damage reduction percentage

For antigenotoxicity experiments, the percentage reduction in MMS-induced damage by SL was calculated according to Waters et al. [21] using the following formula:where A corresponds to the DNA damage observed for the treatment with MMS (positive control), B is the group treated with SL plus MMS, and C is the negative control.

9. Anticarcinogenic assessment

The anticarcinogenic potential of SL was performed by aberrant foci crypt (ACF) assay in Wistar rats. The preliminary experiments using the doses of 15, 30, 60 and 120 mg SL/kg b.w. in rats were evaluated. The doses above 60 mg/kg b.w. showed to be toxic, leading to weight loss and animal deaths. Each treatment group consisted of six animals fed with a regular chow throughout the experiment (five weeks). After one week of acclimation, animals were divided into six treatment groups, as following: SL (60 mg/kg b.w.); DMH (160 mg/kg b.w.); SL I (15 mg/kg b.w.) and DMH; SL II (30 mg/kg b.w.) and DMH; SL III (60 mg/kg b.w.) and DMH; and negative EDTA controls. Negative and positive control groups received EDTA (0.05 mL/10 g b.w.) and DMH (40 mg/kg b.w.), respectively, twice a week for two weeks (weeks 2 and 3). SL was administered to rats five times a week for four weeks by gavage (1.0 mL/animal), during and after DMH treatment. DMH and EDTA were administered by subcutaneous injections. All animals were anesthetized with sodium pentobarbital (45 mg/kg b.w., i.p.) and euthanized five weeks after starting the experiment (i.e., four weeks after the first DMH treatment). Body weight and water consumption were measured five times a week throughout the experimental period.

After laparotomy, the colons were excised, flushed with 0.9% saline, cut open along the longitudinal axis, and fixed in 10% phosphate-buffered formalin (pH 6.9–7.1) for 24 h. Immediately before analysis, the colon was stained with 0.02% methylene blue for five minutes, mounted on microscope slides with the mucosal side facing upward, and observed under a light microscope at 100× magnification. Fifty sequential fields of the distal colon were screened for ACF, which were characterized by elongated, slit-shaped lumens surrounded by thickened epithelium that stained more intensely than the surrounding normal crypts [22] (Figure 1). The number of ACF and crypt multiplicity (number of crypts in each focus) was recorded. The multiplicity of ACF was expressed as aberrant crypts/focus. Each colon specimen was examined by at least three observers in a blinded manner.

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Figure 1. Photomicrograph of methylene blue-stained, whole-mount preparations of colon from Wistar rats treated with DMH with ACF contaning one (A) and five (B) aberrant crypts/focus.

https://doi.org/10.1371/journal.pone.0111999.g001

10. Statistical analysis

The data were analyzed statistically by analysis of variance for completely randomized experiments (ANOVA), and a significant difference among treatment groups was evaluated by Tukey test. The results were considered statistically significant at p<0.05. All statistical analyses were made using GraphPad Prism 5.0.

Results

1. Glycoalkaloids present in the S. lycocarpum alkaloidic extract

HPLC analysis of S. lycocarpum fruit’s glicoalkaloidic extract used in the present study, and obtained by selective extraction, allowed the quantification of two major glycolakaloids: solasonine (45%) and solamargine (44%) (Figure 2).

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Figure 2. HPLC chromatogram of the alkaloidic extract of Solanum lycocarpum fruits.

Zorbax-SB (5 µm), 250×4.6 mm column; mobile phase of acetonitrile and phosphate buffer 0.01 M (36.5∶63.5); flow rate of 1.0 mL/min and detection at 200 nm. SLN = Solasonine; SLM = Solamargine.

https://doi.org/10.1371/journal.pone.0111999.g002

2. Genotoxic and antigenotoxic assessment

No significant difference in the frequencies of MNPCEs in peripheral blood was observed between animals treated with SL, for the three different period of sample collection (48 h, 7 days and 14 days), in comparison with the negative control (P<0.05). The frequencies of MNPCEs in bone marrow and DNA damage in liver cells demonstrated that the dose of 60 mg/kg b.w. was not statistically different from the negative control (P<0.05). These findings indicated the absence of genotoxic effects of SL, in the experimental conditions used in this study (Tables 1, 2 and 3).

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Table 1. Frequencies of micronucleated polychromatic erytrocytes (MNPCEs) and polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratio obtained from peripheral blood of Swiss mice after treatment with SL and/or MMS, and their respective controls.

https://doi.org/10.1371/journal.pone.0111999.t001

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Table 2. Frequencies of micronucleated polychromatic erytrocytes (MNPCEs) and polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) obtained from bone marrow of Swiss mice after treatment with SL and/or MMS, and their respective controls.

https://doi.org/10.1371/journal.pone.0111999.t002

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Table 3. Mean percentage of DNA damage by the comet assay and cell viability by Trypan blue exclusion dye method in the liver cells of Swiss mice after treatment with SL and/or MMS, and their respectives controls.

https://doi.org/10.1371/journal.pone.0111999.t003

The animals treated with different doses of SL associated with MMS showed significant reduction in both MNPCEs frequencies in bone marrow and DNA damage extensions in liver cells in comparison with the negative control. The reductions ranged from 40.96 to 44.18% in bone marrow cells and 45.45 to 54.98% in liver cells of Swiss mice. The gradual increase in the dose of SL did not result in a proportional increase in reducing the MMS-induced genotoxicity, indicating absence of a dose-response (Tables 2 and 3).

No significant reduction of PCEs in relation to the total number of erythrocytes was observed in any of the treatment groups in comparison with the negative control, evidencing the absence of cytotoxicity of the different treatments (Tables 1 and 1). The viability of liver cells by Trypan blue exclusion method was >93% for all experimental and control groups (Table 3).

No statistically significant differences in the body weight gain and water consumption, during the experimental period were observed between groups (Table 4).

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Table 4. Initial body weight, final body weight, body weight gain and water consumption of Swiss mice after treatment for 14 days with SL and/or MMS, and their respective controls.

https://doi.org/10.1371/journal.pone.0111999.t004

3. Anticarcinogenic evaluation

Mean values and standard deviation of ACF and aberrant crypts observed in the distal colon of male Wistar rats are shown in Table 5. The groups of animals treated with EDTA (negative control) and SL (60 mg/kg b.w.) did not show ACF (data not shown). All DMH-treated groups developed ACF in the colon. In the 4-week ACF assay, the number of ACF and aberrant crypts were significantly reduced in the treated animals by 47%, 33% and 36%, respectively, for the doses of 15, 30 and 60 mg/kg b.w. of SL in comparison with the untreated group. Regarding the number of aberrant crypt/ACF, there was a prevalence of one aberrant crypt/ACF.

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Table 5. Mean number (± SD) of aberrant crypt foci (ACF) and aberrant crypt in the distal colon of rats in response to SL during and after DMH treatment.

https://doi.org/10.1371/journal.pone.0111999.t005

Treatment with SL resulted in a lower weight gain of the animals, although these differences were not statistically significant for the animals treated with the different doses of SL in comparison with the control group (Table 6).

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Table 6. Initial body weight, final body weight, body weight gain and water consumption of rats after treatment for four weeks with SL and/or DMH, and their respective controls.

https://doi.org/10.1371/journal.pone.0111999.t006

Discussion

S. lycocarpum dried fruits bear approximately 1% of glycoalkaloids, and the use of standardized selective acid extraction allows to concentrate the glycoalkaloids in a precipitated fraction using only hydrochloric acid and water. To enrich the alkaloid content, it is necessary to submit the crude alkaloid fraction to digestion with ethanol. Then, it furnishes extracts with the content of glycoalkaloids ranging from 80 to 90% in a very reproducible way. Therefore, the developed extractions procedure is simple, reproducible and of low cost, which would allow the production of these alkaloids in an industrial scale.

The Swiss mice treated for 15 days with doses ≤60 mg/kg b.w. of SL showed no sign of toxicity, in the used protocols. However, SL led to lower weight gain to Wistar rats treated for four weeks. Previous studies showed that the administration of S. lycocarpum fruit did not produce body weight variations in male Wistar rats and Swiss mice, although a significant weight change was observed in some organs. Significant weight loss was observed only in the ventral prostate of mice receiving the high dose treatment [23]. Its administration to pregnant rats during the periods of blastocyst preimplantation and implantation [24] did not interfere with these processes. However, significant changes in the placenta, fetal, lung and kidneys weights were observed when administered to pregnant rats [25].

Maruo et al. [26] showed that long-term consumption of S. lycocarpum fruit can produce sight toxic effects in exposed adult rats. In this study, male (60 days treatment) and female (37 days treatment) adult rats received a diet containing 3% S. lycocarpum fruit. It was observed that while significant reductions in uterus and liver weight occurred in treated females, no alterations were reported in males. In addition, no significant differences were observed in other organ weights, blood enzymes or proteins.

Soares-Mota et al. [27] evaluated the toxic effects on male and female rats exposure to 10% S. lycocarpum fruit from weaning (21 days old) until adult age (8 weeks treatment). It was observed weight change in some organs of treated males, while in females, no differences were observed in organ weights. The histopathologic study showed no alterations between groups. Serum biochemical parameters showed triglyceride reductions in treated animals of both sexes. In females, an increase in both albumin and alanine aminotransferase levels, and a reduction in total protein levels were noted.

The present study demonstrated absence of genotoxicity of SL in Swiss mice micronucleus assay. The protective effect of SL against chromosomal and DNA damage induced by MMS were observed in Swiss mice bone marrow and liver cells, respectively. Furthermore, the SL significantly reduced the number of preneoplastic lesions induced by DMH, demonstrating anticarcinogenic activity. The chemopreventive effect can be attributed to the presence of the glicoalkaloids, solamargine and solasonine, major compounds in the glycolacaloidic extract of S. lycocarpum.

MMS has been used as an experimental model compound for several decades to elucidate the genotoxic mechanisms of alkylating agents. It is a direct monofunctional alkylating agent which monoadducts in the DNA, as well as crosslink causing mutations that involve different base substitutions [28]. There are reports of alkylating agents that rapidly replete gluthatione-S-transferase (GSH) in cells, thereby generating oxidative stress [29]. It has been postulated that the loss of GSH may compromise cellular oxidant defenses, with consequence accumulation of reactive oxygen species (ROS) generated as byproducts of normal cellular function. The generation of ROS may play a role in MMS-induced genotoxicity.

The DMH used to induce preneoplastic lesions is a carcinogen that requires metabolism within the organism to become active. The metabolic activation includes oxidation of DMH in azometane, azoximetane, N-hydroxylated and methylazoximetane. The methylazoximetane chemical structure is unstable at body temperature. During the process, it forms an oxidant agent, methyldiazonium, which generates a dioxide ion able to methylate the DNA, RNA and proteins [30]. DMH has been reported to induce carcinogenesis in rats and mice due to its high production of free radicals, which can result in cellular damage [31].

Although the mechanisms of action involved in chemoprevention of SL are not clear, some hypothesis can be drawn. The first is related to the reduction of the damage effects of ROS, due to possible indirect antioxidant action of natural product tested. S. lycocarpum fruits glycoalkaloid extract showed no genotoxic activity and revealed protective effect against both genomic and chromosomal damages induced by methy methanesulfonate [32]. Tavares et al. [15] demonstrated that the S. lycocarpum fruits hydroalcoholic extract not only exerted no genotoxic effect, but also it significantly reduced the frequency of chromosomal aberrations induced by DXR in both in vitro and in vivo mammalian cells. The authors attributed the chemopreventive effect observed to sum of interactions between different compounds of this complex mixture which contains glicoalkaloids and phenolic compounds, as major secondary metabolites. Both the retention times and UV spectra of the major peaks obtained in HPLC analysis confirmed the presence of solamargine (4.60%), solasonine (6.57%) and polyphenols (3.60%). It should be pointed out that the content of both alkaloids in the assayed alkaloidic extract was approximately 90%. Therefore, they might be mostly responsible for the reported activities.

Other species of Solanum have been investigated considering the mutagenic/antimutagenic potential. Chacon et al. [33] observed that the S. melogena hydroalcoholic extract had no mutagenic effect on Wistar rat bone marrow and peripheral blood cells using the micronucleus and chromosome aberrations assays. No mutagenicity was found in the immature fruit extract of S. nigrum in Ames test using TA 98 and TA100 strains, with or without the S9 mixture. However, S. nigrum extract showed antimutagenic effect for the same strains [34]. The presence of S. nigrum hydroalcoholic extract protected DNA against oxidative damage to its deoxyribose sugar moiety in the reaction mixture containing calf thymus DNA and free radicals generation system [35].

In plants of the genus Solanum, solasodine is a steroidal alkaloid and it occurs mainly as the glycoalkaloids solamargine and solasonine, which contain three sugar moieties, also called steroidal saponins. Hanausek et al. [36] observed 62% and 74% reduction by avicins, saponin (Acacia victoriae), in H-ras mutation at codon 61 during the initiation and promotion of skin carcinogenesis, respectively, as well as significant inhibition of the modified DNA base formation (8-OH-dG), inhibition of hydrogen peroxide generation, nuclear fator-κB (NF-κB) activation and nitric oxide synthase induction.

The second hypothesis is the modulating action of SL on the protein p53 and DNA repair genes. The protein p53, encoded by TP53 gene, has been identified as a tumor suppressor protein to protect cells from DNA damage. Following cellular stresses, p53 is stabilized and binds to DNA as a tetramer, in a sequence-specific manner that results in the transcription regulation of genes that are involved in mediating key cellular processes, such as cell-cycle arrest, senescence, apoptosis and DNA repair [37]. There are evidences to suggest that the base excision repair (BER) induced by the base-damaging agent MMS is partially deficient in cells lacking functional p53. Base damage is frequently generated by ROS including superoxide, hydroxyl radical and hydrogen peroxide, and this DNA damage is corrected by BER. Moreover, base alkylation damages from endogenous alkylating agents (e.g., S-adenosylmethionine) and from monofunctional alkylating agents (e.g., MMS and vinyl chloride) are also repaired by BER [1]. According to Weissenberg [38] solasodine potential value as a precursor raw material for the synthesis of steroidal drugs is effective in the repair of DNA fragments.

In conclusion, under the present experimental conditions, SL did not shown genotoxic effects and revealed chemopreventive action. Additional studies involving the analysis of gene expression should be conducted to further elucidate the action mechanisms.

Acknowledgments

We are thankful to Dr. Milton Groppo Junior for the plant identification.

Author Contributions

Conceived and designed the experiments: CCM PFO LFL LMP NHF JCC JKB DCT. Performed the experiments: CCM PFO LFL LMP NHF JCC. Analyzed the data: CCM PFO LFL LMP NHF JCC. Contributed reagents/materials/analysis tools: JKB DCT. Wrote the paper: CCM JKB DCT.

References

  1. 1. Seo YR, Jung HJ (2004) The potential roles of p53 tumor suppressor in nucleotide excision repair (NER) and base excision repair (BER). Experim Mol Med 36: 505–509.
  2. 2. Ferguson LR, Bronzetti G, De Flora S (2005) Mechanistic approaches to chemoprevention of mutation and cancer. Mutat Res 591: 3–7.
  3. 3. Khazir J, Mir BA, Mir SA, Cowan D (2013) Natural products as lead compounds in drug discovery. J Asian Nat Prod Res 15: 764–788.
  4. 4. Anwikar S, Bhitre M (2012) Study of synergistic anti-inflammatory activity of Solanum xanthocarpum Schrad and Wendl and Cassia fistula Linn. Int J Ayurveda Res 3: 167–171.
  5. 5. Bahgat A, Abdel-Aziz H, Raafat M, Mahidy A, El-Kihatib AS, et al. (2008) Solanum indicum ssp. distichum extract is effective against L-NAME-induced hypertension in rats. Fundam Clin Pharmacol 6: 693–699.
  6. 6. Gupta RK, Hussain T, Panigrahi G, Das A, Singh GN, et al. (2011) Hepatoprotective effect of Solanum xanthocarpum fruit extract against CCl4 induced acute liver toxicity in experimental animals. Asian Pac J Trop Med 12: 964–968.
  7. 7. Javed T, Ashfag UA, Riaz S, Rehman S, Reazuddin S (2011) In-vitro antiviral activity of Solanum nigrum against Hepatitis C Virus. Virol J 19: 8–26.
  8. 8. Patel P, Patel M, Saralai M, Gandhi T (2012) Antiurolithiatic effects of Solanum xanthocarpum fruit extract on ethylene-glycol-induced nephrolithiasis in rats. J Young Pharm 3: 164–170.
  9. 9. Nirmal SA, Patel AP, Bhawar SB, Pattan SR (2012) Antihistaminic and antiallergic actions of extracts of Solanum nigrum berries: possible role in the treatment of asthma. J Ethnopharmacol 142: 91–97.
  10. 10. Li X, Zhao Y, Wu WKK, Liu S, Cui M, et al. (2011) Solamargine induces apoptose associated with p53 transcription-dependent and transcription-independent pathways in human osteosarcoma UO2S cells. Life Scinces 88: 314–321.
  11. 11. Ding X, Zhu FS, Li M, Gao SG (2012) Induction of apoptosis in human hepatoma SMMC-7721 cells by solamargine from Solanum nigrum L. J Ethnopharmacol. 139: 599–604.
  12. 12. Munari CC, Oliveira PF, Campos JCL, Martins SPL, Costa JC, et al. (2013) Antiproliferative activity of Solanum lycocarpum alkaloidic extract and their constituents, solamargine and solasonine in tumor cell lines. J Nad Med 68: 231–41.
  13. 13. Farina F, Piassi FG, Moysés MR, Bazzolli DM, Bissoli NS (2010) Glycemic and urinary volume responses in diabetic mellitus rats treated with Solanum lycocarpum. Appl Physiol Nutr Metab 35: 40–44.
  14. 14. Tiossi RFJ, Miranda MA, Sousa JPB, Praça FSG, Bentley MVLB, et al. (2012) A Validated reverse phase HPLC analytical method for quantitation of glycoalkaloids in Solanum lycocarpum and its extracts. J Anal Methods Chem 2012, ID947836.
  15. 15. Tavares DC, Munari CC, Araújo MGF, Beltrame MC, Furtado MA, et al. (2011) Antimutagenic potential of Solanum lycocarpum A. St.-Hil. against induction of chromosomal aberrations in V79 cells and micronuclei in mice by doxorubicin. Planta Med 13: 1489–1494.
  16. 16. MacGregor JT, Wehr CM, Gould DH (1980) Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test. Environ Mutagen 2: 509–514.
  17. 17. MacGregor JT, Heddle JA, Hite M, Margolin BH, Ramel C, et al. (1987) Guidelines for the conduct of micronuclei assay in mammalian bone morrow erythrocyte. Mutat Res 189: 103–112.
  18. 18. Krishna G, Hayashi M (2000) In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat Res 455: 155–166.
  19. 19. Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, et al. (2007) Fourth International Workgroup on Genotoxicity testing: Results of the in vivo Comet assay workgroup. Mutat Res 627: 31–5.
  20. 20. Collins AR, Oscoz AA, Brunborg G, Gaivao I, Giovannelli L, et al. (2008) The comet assay: topical issues. Mutagenesis 23: 143–151.
  21. 21. Waters MD, Brady AL, Stack HF, Brockman HE (1990) Antimutagenicity profiles for some model compounds. Mutat Res 238: 57–85.
  22. 22. Bird RP (1987) Observation and qualification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett 37: 147–51.
  23. 23. Sá RCS, Vireque AP, Reis JEP, Guerra MO (2000) Evaluation of the toxicity of Solanum lycocarpum in the reproductive system of male mice and rats. J Ethnopharmacol 73: 283–287.
  24. 24. Peters VM, Pinheiro NL, Reis JEP, Guerra MO (2001) Absence of interceptive effect in rats treated with Solanum lycocarpum (St. Hill). Contraception 63: 53–55.
  25. 25. Maruo VM, Soares MR, Bernardi MM, Spinosa HS (2003) Embryotoxic effects of Solanum lycocarpum St. Hill fruits consumption during preimplanttion and organogenesis in rats. Neurotoxicol Teratol 25: 627–631.
  26. 26. Maruo VM, Bernardi MM, Spinosa HS (2003) Toxicological evaluations of long term consumption of Solanum lycocarpum St. Hill fruits in male and female adult rats. Phytomedicine 10: 48–52.
  27. 27. Soares-Mota MR, Scwarz A, Bernardi MM, Maiorka PC, Spinosa HS (2010) Toxicological evaluation of 10% Solanum lycocarpum St. Hill fruit consumption in the diet of growing rats: Hematological, biochemical and histopathological effects. Exp Toxicol Pathol 62: 549–553.
  28. 28. Wyatt MD, Pittman DL (2006) Methylating agents and DNA repair responses: methylated bases and sources of strand breaks. Chem Res Toxicol 19: 1580–1594.
  29. 29. Van der Walters B, Zoeteweij JP, Nagelkerke JF (1996) Alkylation-induced oxidative cell injury of renal proximal tubular cells: involvement of glutathione redox-cycle inhibition. Arch Biochem Biophys 327: 71–80.
  30. 30. Gangadhar C (1998) Human health perspective on environmental exposure to hydrazines: a review. J Nat Prod 37: 801–843.
  31. 31. Tomasi A, Albano E, Botti B, Vannini V (1987) Detection of free radical intermediates in the oxidative metabolism of carcinogenic hydrazine derivates. Toxicol Pathol 15: 178–183.
  32. 32. Munari CC, Oliveira PF, Lima IMS, Martins SPL, Costa JC, et al. (2012) Evaluation of cytotoxic, genotoxic and antigenotoxic potential of Solanum lycocarpum fruits glicoalkaloid extract in V79 cells. Food Chem Toxicol 50: 3693–3701.
  33. 33. Chacon DR, Libera AND, Cintra DEC, Carvalho JCT, Oliveira GA, et al. (2002) Absence of genotoxic and antigenotoxic effects of a standardized extract of the medicinal plant Solanun melongena on peripheral blood and bone marrow cells of Wistar rats. Cytologia 67: 417–422.
  34. 34. Yen GC, Chen HY, Peng HH (2001) Evaluation of the cytotoxicity, mutagenicity and antimutagenicity of emerging edible plants. Food Chem Toxicol 39: 1045–1053.
  35. 35. Sultana S, Perwaiz S, Igbal M, Athar M (1995) Crude extracts of hepatoprotective plants, Solanum nigrum and Cichorium intybus inhibit free radical-mediated DNA damage. J Ethnopharmacol 45: 189–192.
  36. 36. Hanausek M, Ganesh P, Walaszek Z, Arntzen CJ, Slaga TJ, et al. (2001) Avicins, a family of triterpenoid saponins from Acacia victoreae (Bentham), suppress H-ras mutations and aneuploidy in a murine skin carcinogenesis model. Proc Nat Acad Sci USA 98: 11551–11556.
  37. 37. Reinhardt HC, Schumacher B (2012) The p53 network: cellular and systemic DNA damage responses in aging and cancer. Trends in Genet 3: 128–136.
  38. 38. Weissenberg M (2001) Isolation of solasodine and other steroidal alkaloids and sapogenins by direct hydrolysis-extraction of Solanum plants or glycosides therefrom. Phytochemistry 58: 501–508.