Modulating effect of Poincianella bracteosa (Tul.) L.P. Queiroz bark (Leguminosae) on DNA damage induced by doxorubicin on the somatic cells of Drosophila melanogaster wings

The aim of this study was to assess the genotoxic and antigenotoxic effects of Poincianella bracteosa bark aqueous extract on DNA damage induced by doxorubicin (DXR) a chemotherapeutic agent using SMART (Somatic Mutation and Recombination Test). The analysis was performed using the somatic mutation and recombination test in Drosophila melanogaster. Larvae from the standard and high-bioactivity crosses were chronically treated with four concentrations of P. bracteosa bark tea, alone and in association with DXR. The results revealed no mutagenic effect of bark extract for any of the concentrations tested. A modulating effect of aqueous extract in reducing the genotoxic action of DXR was observed for all concentrations tested in descendants of both crosses, but inhibition was more effective in those from the high-bioactive cross. The modulating effect observed may be associated with the presence of tannins and reducing sugars, as observed in phytochemical studies, since they are capable of capturing and stabilizing free radicals. Given the widespread use of P. bracteosa bark in folk medicine, further studies to elucidate the mechanism of action of these cellular compounds and with other experimental models would be useful to confirm that P. bracteosa extract is beneficial to human health.


Introduction
Plants have been used for medicinal purposes for thousands of years, ranging from the simplest treatment forms to the industrial manufacture of drugs (Saraiva et al., 2015). According to the World Health Organization (WHO), around 80% of the world's population still depends on medicinal plants for basic health care, with increasing use observed in Western countries (Ouedraogo et al., 2012). In Brazil, around 80% of the population use medicinal plant-based products. However, the lack of suitable information on the safety of these products has hindered more widespread use of these plants (Vargas et al., 2016).
Poincianella bracteosa (Tul.) L.P. Queiroz. (Fabaceae), popularly known as "pau-de-In spite of the therapeutic advantages, the different chemical constituents present in P. bracteosa can be potentially toxic, mutagenic and carcinogenic (Ping, Darah, Yusuf, Yeng, & Sasidharan, 2012). Thus, assessment of the toxic effects of any medicinal plant extract for short and long-term human consumption is extremely important. Furthermore, assessing the toxicity, mutagenicity and genotoxicity of natural products is a crucial step for pharmaceutical companies to consider new therapeutic agents (Sponchiado et al., 2016).
A biological model successfully used in genotoxicity studies is Drosophila melanogaster. It is estimated that nearly 75% of disease-related genes in humans have functional orthologs in the fly. In general, flies and humans share about 80 to 90% identity in functional protein domains (Pandey & Nichols, 2011). As such, these organisms have been used to study the genotoxic and antigenotoxic activities of many medicinal plants (Patenkovic, Stamenkovic-Radak, Nikolic, Markovic, & Andelkovic, 2013;Zafred et al., 2016).
The somatic mutation and recombination test (SMART) in D. melanogaster can detect a wide spectrum of genetic end points, including point mutations, deletions and certain chromosomal abnormalities, as well as mitotic recombination and gene conversion (Graf, Abraham, Rincón, & Würgler, 1998). Considering the sensitivity of the SMART and the use of P. bracteosa in traditional medicine, the present study aimed to assess the phytochemical content, genotoxic and modulatory potential of its stem bark extract, alone or combined with doxorubicin, using this test.

Plant Material
The bark of P. bracteosa was collected from an adult plant in Teresina, Piaui (Northeast of Brazil, geographical coordinates 5º 02´21.36"S and 42º 47´ 22.44"W) in January 2016.
Herbarium specimens containing leaves, flowers and fruits were stored in the Afrânio Fernandes Herbarium at the State University of Piauí (UESPI, Teresina -PI, Brazil; voucher specimen number HAF 03635). The bark was oven dried at 45-50° C and then ground in a blender until a fine powder was obtained. Around four grams of the powder was added to 250 mL of distilled water and boiled for 10 minutes. Next, the aqueous extract of P. bracteosa (AEPb) was filtered and maintained in a refrigerator at 4º C, in a dark jar for 24h. The extract was assessed at four concentrations: 2, 4, 8 and 16 mg/mL. The phytochemical profiles of the Research, Society and Development, v. 9, n. 9, e745997833, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7833 6 extracts were determined by colorimetric reactions that qualitatively detect the primary plant metabolites (Barbosa et al., 2004).

Doxorubicin
Doxorubicin (DXR, Doxolen® lyophilized, Eurofarma Laboratórios Ltda., São Paulo, Brazil, CAS No. 23214-92-8), dissolved in distilled water in the dark at a concentration of 0.125 mg/mL was used as positive control, and distilled water as negative control. DXR is a chemotherapeutic agent that induces single and double-stranded DNA breaks (Rezende et al.

Drosophila Strains and Crosses
Three strains of D. melanogaster were used: 1) multiple wing hairs: y;mwh j (mwh, 3-

Larval Feeding
For the treatments and controls, 72h-old larvae from the ST and HB crosses were transferred to plastic tubes containing 4g of instant mashed potatoes (Yoki®), dissolved in 12 mL of a solution containing AEPb with or without DXR at 0.125 mg/mL. The larvae were fed on the medium until the larval phase was complete (about 2 days). The experiments were performed at 25ºC and 60% relative humidity.

Statistical Analysis
The binomial conditional test was used to evaluate mutagenic potential (Frei & Würgler, 1988). The study compares the number of different classes of spots found between treatments and their negative control. For antimutagenic analysis, the frequencies of each type of spot for each treatment group were submitted to pairwise comparison (DXR vs AEPb + DXR in each class analyzed), using the nonparametric Mann-Whitney U-test and Wilcoxon rank sum test (Frei & Würgler, 1995). The inhibition percentages of stem bark tea were calculated using the frequency of clones per 10 5 cells, corrected by the control, as follows: [(DXR alone -AEPb + DXR/ DXR alone) x 100] (Abraham, 1994).

Results
Both crosses (ST and HB) were supplied with third instar larvae at concentrations ranging from 2 to 16 mg/mL for approximately 48 h. The frequency of positive control spots showed a statistically significant increase in all categories when compared to the negative control (p < 0.05).

Discussion
In the present study, the potential effects of AEPb alone or in combination with the chemotherapeutic agent DXR were tested in chronic treatments on larval descendants of ST and HB crosses, using the somatic mutation and recombination test (SMART) in D.
melanogaster. Similar studies have been carried out to assess medicinal aqueous plant extracts (Fernandes et al., 2013, Jacociunas et al., 2014. The concentrations used in this study were initially based on satisfactory results in genotoxic and cytotoxic tests in meristematic cells from the Allium cepa root, where the extract of P. bracteosa also showed no cytotoxic or genotoxic effects since the number of chromosomal changes did not increase (Souza et al., 2016).
The negative control contained a low number of spontaneous mutations in both crosses, and the positive control exhibited a statistically significant increase in the number of mutations compared to the negative control. This validates the use of the SMART test and demonstrates its good response to the mutagenic agent DXR, which is consistent with data in the literature (Guterres et al., 2013, Vale et al., 2013. In the experimental conditions assessed, in addition to showing no mutagenic/recombinogenic effect, AEPb exhibited a modulatory response in doxorubicin activity. This response was observed in the descendants of both crosses (ST and HB), but inhibition was more effective in those of the HB cross, similar to the results reported in the literature (Fernandes et al., 2014).
The difference between ST and HB crosses is related to the level of CYP450.
Descendants from the former cross contain basal levels of CYP450, while those of the latter showed high CYP450 enzyme expression. This set of enzymes is involved in the metabolism of a wide variety of endogenous and xenobiotic compounds. Some drugs are inactivated through this biotransformation; however, the active properties of certain metabolites generated in this process may increase (Saturnino, Machado, Lopes, & Nepomuceno, 2018), explaining the higher modulating effect of AEPb in HB cross offspring.
The mechanisms by which AEPb reduces the frequency of DXR-induced mutant spots were not directly evaluated in this study. However, it is known that DXR binds strongly to DNA due to its ability to intersperse between pairs of bases, causing ruptures in the molecule and inhibiting DNA and RNA synthesis (Orsolin, Oliveira, & Nepomuceno, 2016).
considered an antimutagenic agent that, when combined with DXR, acts as a DXR-induced free radical scavenger and/or by blocking its interaction with DNA.
The presence of hydrolyzed tannins and reducing sugars may explain the lower number of mutant stains produced by AEPb when associated with DXR. Hydrolyzed tannins consist of gallic acid esters and glycolyzed ellagic acids, formed from shikimate, where the sugar hydroxyl groups are esterified with phenolic acids (Monteiro et al., 2005). Reducing sugars are carbohydrates that contain a free carbonyl group, capable of oxidizing in the presence of oxidative agents in an alkaline solution (Santos et al., 2017). Both can capture and stabilize free radicals, thereby promoting a preventive effect on the carcinogenic process and other degenerative diseases associated with high intercellular concentrations of free radicals (Vale et al., 2013).
The action of AEPb modulation observed in this study may serve as the basis for the development of new coadjuvant drugs in chemotherapy, given that the combined use of extracts with modulatory action can decrease the genotoxic effect of cancer drugs on healthy cells without interfering in the treatment of tumor cells (Felício et al., 2011).

Conclusion
The present study shows that aqueous bark extract did not induce mutation and when associated with DXR, at all concentrations, the extract exhibited a modulating effect on chemotherapy-induced DNA damage in the somatic cells of Drosophila melanogaster wings.
Although promising, further studies the genotoxicity with other experimental models are needed to confirm that P. bracteosa extract is beneficial to human health, since this plant is widely used in folk medicine.