Abstract
Momordica charantia, popularly known as bitter melon, is a plant widely used in ethnobotanical medicine. It has antibacterial, antifungal, anthelmintic, antidiabetic, antiviral, and antimalarial activities, among others. The goal of this study was to evaluate the genotoxic and/or antigenotoxic activity of the aqueous extracts obtained from the aerial parts and fruit of this plant by means of the Drosophila melanogaster wing spot test. Third-stage larvae that obtained standard (ST) cross and high bioactivation (HB) cross were treated with aqueous extracts of the aerial parts (IQA) and fruit (IQF) of M. charantia, following two protocols (genotoxicity and antigenotoxicity). The aqueous extracts are not genotoxic in lower concentrations. The frequencies of mutant spots observed in the descendants of the ST and HB crosses treated with doxorubicin (DXR) alone were 8.65 and 9.25, respectively, whereas in those cotreated with IQA and DXR, the frequencies ranged from 15.90 to 29 in the ST cross and from 15.05 to 24.78 in the HB cross. In cotreatment with IQF, the frequencies ranged from 30.10 to 30.65 in the ST cross and from 13.60 to 14.50 in the HB cross, whereas the frequencies obtained with DXR were 32.50 in the ST cross and 26.00 in the HB cross. In conclusion, the IQA has a synergistic effect, enhancing the genotoxicity of DXR in the ST cross and the HB cross, whereas the IQF has antigenotoxic effects in the HB cross.
Introduction
M
Previous investigation has shown that crude extracts of fruits, stem, and cucurbitane triterpenoids of M. charantia L. possess antidiabetic activity, and many cucurbitane-type triterpenoids have been isolated from the fruits, seeds, leaves, and stems of M. charantia. 5 –7
Kubola and Siriamornpun, 8 in their study, show that aqueous extracts of the leaf and fruit of M. charantia L. exhibited a high value of antioxidant activity and demonstrated that bitter melon fractions are rich in phenolics and have a strong antioxidant activity. Antioxidants have diverse biological effects, such as anti-inflammatory, antiatherosclerotic, and anticarcinogenic effects. 9
The efficacy of the extract of M. charantia L. was evaluated as an anticancer agent 10 in human cells of breast cancer, MCF-7 line, 11 and in glandular breast tissue, MDAMB-231 line, as an in vitro model. 12 The authors reported a significant inhibition of breast cancer cell growth; the extract of this plant can be used as a dietary supplement for the prevention of breast cancer.
Currently, the alternative strategy used to prevent or reverse cancer is the consumption of anticarcinogenic or antigenotoxic agents in vegetables and fruits. 13 Thus, studies on genotoxicity and antigenotoxicity can help assess the safety of many plants used in folk medicine.
Among several genotoxicity assays, the somatic mutation and recombination test (SMART) developed by Graf et al. 14 allows detecting the loss of heterozygosity of genetic markers identified in the phenotypes expressed in wing trichomes of Drosophila melanogaster. 15 This assay can detect agents that cause point mutations, chromosomal abnormalities, recombinations, deletions, rearrangements, and aneuploidy. For this purpose, two crosses are typically used: the standard (ST) cross 16 and the high bioactivation (HB) cross. 17 The ST cross present basal levels of the metabolizing cytochrome P450 enzyme (Cyp6A2) and is used to detect direct-acting genotoxins. The HB cross uses strains with high levels of Cyp6A2 and is used to detect indirect acting genotoxins that exert their genotoxic activity only when metabolized. 17–18
The compounds to be analyzed by SMART can be pure substances, 19 plant extracts, 20 beverages, and various other substances. 21
Considering the wide use of M. charantia L. as food and in folk medicine to treat various diseases, this study aimed to evaluate the genotoxic and/or antigenotoxic activity of the aqueous extracts prepared with the fruit and aerial parts of M. charantia L. through the SMART trial in somatic cells of D. melanogaster.
Materials and Methods
Collection and obtaining of extracts
The aerial parts (leaves and stems) and fruit of M. charantia L. were collected in the Chácara dos Poderes in Campo Grande, state of Mato Grosso do Sul, and identified by Prof. Dr. Arnildo Pott (Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, MS, Brazil).
The aqueous extracts of the aerial parts of M. charantia L. (IQA) were prepared with 4 g of dried aerial parts dissolved in 200 mL of distilled boiling water, with a prolonged extraction of 20 min.
The aqueous extracts of fruit (IQF) were prepared with 4 g of fresh ripe fruit dissolved in 200 mL of distilled water. The aqueous extracts (IQA and IQF) that were diluted with distilled water and filtered through Millipore filters (0.22 μm) were used in the experiments at room temperature.
Somatic mutation and recombination test
For testing with SMART, mutant strains of D. melanogaster were provided by Dr. Mário Antônio Spanó, Instituto de Genética e Bioquímica–Universidade Federal de Uberlândia, Minas Gerais, Brazil.
The SMART was performed through experimental crosses between strains (mwh, flr3 , and ORR/flr3 ) of D. melanogaster. The two different crosses were as follows: (1) ST cross—standard cross between mwh males and flr3 virgin females 16 and (2) HB cross—high bioactivation cross between mwh males and ORR/flr3 virgin females. 17
Approximately 400 virgin females of strains flr3 and ORR were crossed with ∼200 mwh males for a 48-h period; thereafter, couples were transferred for a period of 8 h to vials containing a solid agar base, covered by a layer of yeast (Saccharomyces cerevisiae) supplemented with sugar, for egg collection. After 72±4 h, the third instar larvae were washed with tap water and collected with the aid of a fine mesh sieve. Groups of ∼100 larvae were transferred to glass tubes containing 1.5 g of alternative culture medium (Yoki® instant mashed potatoes) and were treated according to the two protocols (genotoxicity and antigenotoxicity).
In the genotoxicity protocol, the aqueous extracts of the fruit (IQF) and aerial parts (IQA) of M. charantia L. were evaluated at different concentrations (2.5, 5.0, 10.0, and 20.0 mg/mL). For the antigenotoxicity assessment, the IQF was evaluated at 2.5 and 5.0 mg/mL concentrations and IQA at 1.25, 2.5, and 5.0 mg/mL concentrations. These were associated with doxorubicin (DXR) at 0.125 mg/mL. The concentration (0.125 mg/mL) of DXR used in SMART was established in previous experiment. 22 The filtered water was used as a negative control and DXR as a positive control.
Emerging adults with both types of genotypes, mwh +/+ flr3 (marked trans-heterozygous—MH) and mwh +/+ TM3, BdS (balanced heterozygous—BH), were collected and fixed in 70% ethanol. Wings were detached and mounted between slides and coverslips in the Faure solution (30 g gum arabic, 50 g chloral hydrate, 100 mL water, and 20 mL glycerol) and analyzed for the occurrence of different types of mutant spots in an optical microscope with 400×magnification.
Statistical analysis
To evaluate the statistical significance of the results, we used the procedure proposed by Frei and Würgler, 23 an analysis of multiple decisions that generate four different diagnoses: positive, weak positive, negative, or inconclusive (significance level P<.05). The frequency of each type of mutant spot per individual of a series treated was compared with its corresponding negative control test using the binomial conditional test of Kastenbaum and Bowman. 24 To assess the negative results, multiplication factors (m) were introduced in the test. These include m=2 for single small spots and total spots due to their high spontaneous frequency, and m=5 for single large spots and twin spots, which rarely arise spontaneously. 14,23,25 In this way, criteria are established for a positive diagnosis, which requires a frequency of mutations to be treated in the m times greater than obtained in the negative control frequency. 26
Results
The genotoxic potential of the aqueous extracts of the aerial parts (IQA) and fruit (IQF) of M. charantia L. was investigated in independent experiments. Table 1 shows the results of analysis of mutant spots' frequency in the offspring of the ST cross treated with different concentrations of IQA. The frequency of mutations in the negative control was 0.55, whereas in the groups treated with IQA varied from 0.45 to 0.55. The frequencies of mutations in the groups treated with IQA are closer to the value obtained in the negative control.
Statistical diagnosis according to Frei and Würgler. 23 U-test, two sided; probability levels: −, negative; +, positive; i, inconclusive.
Including rare single spots flr3 .
Considering mwh clones from mwh single and twin spots.
Frequency of clone formation: clones/fly/48,800 cells (without size correction).
Toxic.
DXR, doxorubicin; ST, standard; IQA, aqueous infusion of aerial parts of Momordica charantia L; IQF, aqueous infusion of fruit of Momordica charantia L.
The descendants of the ST cross treated with different concentrations of IQF presented frequencies of mutations from 0.45 to 0.95, while the one obtained in the negative control was 0.40. The values observed in the groups treated with 2.5 and 5.0 mg/mL concentrations were not statistically different from that observed in the negative control.
Table 2 presents the results of the HB cross, whose descendants were treated with different concentrations of IQA and IQF. The frequency of mutant spots in the negative control was 0.90, whereas in the groups treated with IQA ranged from 0.55 to 0.70, and with IQF from 0.80 to 1.40. It is noted that the frequencies of mutations found in the groups treated with IQA at all concentrations and IQF at 2.5 and 5.0 mg/mL were not statistically different from that obtained in the negative control.
Statistical diagnosis according to Frei and Würgler. 23 U-test, two sided; probability levels: −, negative; +, positive; i, inconclusive.
Including rare single spots flr3 .
Considering mwh clones from mwh single and twin spots.
Frequency of clone formation: clones/fly/48,800 cells (without size correction).
Toxic.
HB, high bioactivation.
However, in the descendants of the ST and HB crosses treated with 10.0 mg/mL of IQF, the frequencies of mutations were statistically different from that observed in their negative controls, indicating genotoxic effects, while concentrations of 20.0 mg/mL was toxic.
The antigenotoxicity assessment was performed with the nongenotoxic concentrations. The results of cotreatment of IQA and DXR in the offspring of the ST cross are shown in Table 3. It is found that the mutation frequency obtained in the positive control (DXR) was 8.65, whereas the cotreated groups showed frequencies ranging from 15.90 to 29.10 distributed in all classes (single small spot, single large spot, and twin spots). The frequencies of mutations of the groups cotreated with IQA and DXR differ statistically from the positive control (DXR).
Statistical diagnosis according to Frei and Würgler. 23 U-test, two sided; probability levels: −, negative; +, positive; i, inconclusive.
Including rare single spots flr3 .
Considering mwh clones from mwh single and twin spots.
Frequency of clone formation: clones/fly/48,800 cells (without size correction).
w+, weak positive.
Similar results were observed in the offspring of the ST cross cotreated with IQF and DXR, and the mutation frequency in the positive control was 32.55, whereas in the groups cotreated with IQF, the frequencies ranged from 30.10 to 30.65, distributed in all categories (Table 3). The frequencies of mutations of the groups cotreated with IQF and DXR did not differ statistically from the positive control (DXR).
Table 4 lists the frequencies of mutant spots in descendants of the HB cross cotreated with IQA and DXR. The frequency of mutations obtained in the positive control was 9.25, whereas the groups cotreated with IQA exhibited frequencies varying between 15.05 and 24.78. The frequencies of mutations of the groups cotreated with IQA and DXR are above the value obtained in the positive control, with statistical difference.
Statistical diagnosis according to Frei and Würgler. 23 U-test, two sided; probability levels: −, negative; +, positive; i, inconclusive.
Including rare single spots flr3 .
Considering mwh clones from mwh single and twin spots.
Frequency of clone formation: clones/fly/48,800 cells (without size correction).
w+, weak positive.
Nevertheless, in descendants of the HB cross cotreated with IQF and DXR, the frequencies of mutations ranged from 13.60 to 14.50, while in the positive control was 26.00. The values found in the groups cotreated with IQF are lower than that obtained with DXR, differing significantly from the positive control (Table 4).
Discussion
By comparing the frequencies of mutations caused by IQA and IQF on descendants of the standard (ST) cross and high metabolic bioactivation (HB) cross, which contain high levels of the cytochrome P-450 enzyme for xenobiotics metabolization, it is observed that the results are similar because both played their roles independent of the metabolizing enzymes activity. 17 However, 10.0 and 20.0 mg/mL concentrations of IQF were genotoxic and toxic, respectively.
Our results are consistent with those of Hanusch et al. 27 who evaluated the mutagenic activity of ethanol extracts prepared with all parts of M. charantia L. (leaf, stem, root, fruit pulp, seed, and fruit peel) at concentrations of 10, 25, 50, and 70 mg/L using the Allium cepa bioassay. The highest rates of chromosomal alterations were registered in the roots of A. cepa bulbs exposed to extracts of root, fruit pulp, seed, and fruit peel of M. charantia L. and are therefore considered genotoxic in this test system. According to Grover and Yadav, 3 the fruit and seeds of M. charantia L. are more toxic than the leaves and aerial parts.
Varella et al. 28 evaluated the mutagenicity and antimutagenicity of ethanol extract of M. charantia L. at different concentrations (0.64, 1.27, 2.55, and 3.84 mg/plate) by means of the AMES test, using strains TA100, TA98, and TA102 of Salmonella typhimurium with microsomal activation. In the concentrations tested, the extract had no mutagenic activity but acted as an antimutagenic agent.
The antigenotoxic activity of the aqueous extracts of the fruit of M. charantia L. was evaluated by Sumanth and Chowdary 29 using the micronucleus assay and chromosome aberration test in mouse bone marrow. Animals were treated orally with 900 mg/kg for 24, 48, and 72 h. The mice treated with the extract showed a significant decrease in the frequency of micronuclei or chromosomal aberrations, statistically different from that observed in the positive control (cyclophosphamide, 100 mg/kg), and this result indicates that the extract at this concentration is antigenotoxic.
The studies related to genotoxicity and antigenotoxicity of extracts from M. charantia L. using test systems (A. cepa, AMES, Micronuclei, and Chromosomal Aberration) indicate that at lower concentrations the extract has no genotoxic activity, but rather antigenotoxic effect.
Meanwhile, in this study, using the SMART, the highest concentrations of IQF presented genotoxic and toxic effects. The toxic activity can be attributed to the compounds, cucurbitacines and oxygenated tetracyclic triterpenoids, found in this plant. These substances have a wide spectrum of biological activities, including antioxidant, 30 cytotoxic, antitumor, anti-inflammatory, and antimicrobial activities. 2
The results obtained with the cotreatment of IQA and DXR indicate that under the experimental conditions, they exert synergistic activity because the frequencies of mutations observed in treated groups are significantly different compared with the positive control (DXR), enhancing the genotoxic effect of this chemotherapeutic agent. The results obtained with IQF in descendants of the standard (ST) cross indicate that it did not protect DNA from genotoxic damages caused by DXR.
However, in the HB cross, the cotreatment with IQF and DXR reduced the genotoxic effect of this chemotherapeutic agent, which can be due to the activation of cytochrome oxidase 17 and glutathione peroxidase, 31 because according to Semiz and Sen, 32 rats treated with 200 mg/mL of the fruit extract of M. charantia showed increased activity of antioxidant enzymes, including SOD, catalase, and glutathione peroxidase.
When comparing the obtained results with studies described in the literature where extracts of M. charantia L. had antigenotoxic activity in various test systems, the results obtained can be ascribed to secondary metabolites found, in the IQA and IQF, which in the presence of DXR potentiated its genotoxic effect in the SMART. Nonetheless, the results are similar to those described with other substances associated with DXR in the SMART assay.
Rezende et al. 33 investigated the mutagenicity and recombinogenicity of (-)-cubebin in the SMART assay. When evaluated alone, cubebin did not induce mutation and recombination, and when combined with DXR at low concentrations, it reduced the frequency of spots induced by DXR, but at high concentrations, it potentiated the mutagenic effect of DXR. The authors suggest that (-)-cubebin can act as a scavenger of free radicals (antioxidants) at low concentrations and as a prooxidant at higher concentrations when it interacts with the enzyme system, which catalyzes the metabolic detoxification of DXR.
Souza et al. 34 evaluated the genotoxicity and antigenotoxicity of a commercial preparation of powdered bark and stem of Tabebuia impetiginosa also using the SMART assay. The authors verified that the powder did not alter the frequency of spontaneous mutant spots in both crosses compared with the negative control. Mean time, when combined with DXR, T. impetiginosa was toxic in both crosses, at the highest concentration, and in the HB cross, it potentiated the genotoxic effect of DXR. The authors suggested that this is due to the constituents of T. impetiginosa that can interact with enzyme systems that catalyze the metabolic detoxification of DXR or the constituents that produce superoxide radicals and stimulate the microsomal oxidation of NAD(P)H.
The aqueous extracts of the aerial parts and fruit of M. charantia L. present substances with Janus behavior, a term used to describe substances that behave as genotoxic and antigenotoxic agents depending on cell types or doses used. 35 Tavares et al. 36 observed the phenomenon Janus in the ethanol extract of green propolis: once, this extract at lower concentrations inhibited the damage caused by DXR, whereas at larger concentrations, it increased the genetic damage.
In short, the results with IQA and IQF obtained from M. charantia L., added to those found in the literature, allows us to conclude that IQA and IQF have no genotoxic activity when evaluated pure, but at high concentrations, they present toxic activity. Nevertheless, nongenotoxic concentrations combined with the chemical agent DXR potentiate the genotoxicity thereof. However, the cotreatment of IQF and DXR, after metabolization, has antigenotoxic effect.
The results obtained with the SMART assay can be extrapolated to the vertebrates, including humans, whereas the sequencing of the genome of D. melanogaster revealed a high evolutionary conservation when compared with the human genome, not only at the level of DNA sequence but also mainly in relation to gene functions, showing high genetic homology. 37
The data obtained from the analysis of the proteome, since 60% of the 289 genes related to human diseases have counterparts in Drosophila, of which 75% sheep like protein sequences similar in these two bodies, thus there is a high conservation between biochemical pathways and regulatory functions between the two species. 38
However, further studies should be performed to confirm the synergistic, genotoxic, and toxic effects verified with the extracts of this plant.
Footnotes
Acknowledgments
The authors thank the State University of Mato Grosso do Sul (UEMS) and Brazilian agencies CAPES, FUNDECTMS, and CNPq.
Author Disclosure Statement
No competing financial interests exist.