Reduction of doxorubicin-induced genotoxicity by Handroanthus impetiginosus in mouse bone marrow revealed by micronucleus assay

Handroanthus impetiginosus has long been used in traditional medicine and various studies have determined the presence of bioactive chemical compounds and potential phytotherapeutics. In this study, the genotoxicity of the lyophilized tincture of H. impetiginosus bark (THI) was evaluated in mouse bone marrow using micronucleus assays. The interaction between THI and genotoxic effects induced by the chemotherapeutic agent, doxorubicin (DXR), was also analyzed. Experimental groups were evaluated 24 to 48 h after treatment with N-nitroso-N-ethylurea (NEU; 50 mg/kg), DXR (5 mg/kg), sodium chloride (NaCl; 150 mM), and THI (0.5-2 g/kg). Antigenotoxic assays were carried out using THI (0.5 g/kg) in combination with NEU or DXR. Analysis of the micronucleated polychromatic erythrocytes (MNPCEs) indicated no significant differences between treatment doses of THI (0.5-2 g/kg) and NaCl. Polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) ratios did not indicate any statistical differences between DXR and THI or NaCl, but there were differences between THI and NaCl. A significant reduction in MNPCEs and PCE/NCE ratios was observed when THI was administered in combination with DXR. This study suggested the absence of THI genotoxicity that was dose-, time-, and gender-independent and the presence of moderate systemic toxicity that was dose-independent, but timeand gender-dependent. The combination of THI and DXR also suggested antigenotoxic effects, indicating that THI reduced genotoxic effects induced by chemotherapeutic agents.

Although numerous studies have supported the effectiveness of different forms of the crude extract of H. impetiginosus or its phytochemical compounds alone, few recent studies aimed to understand their genotoxic and mutagenic effects (Vanni et al., 1998;De Sousa et al., 2009;Lemos et al., 2012;Zabka et al., 2013).To contribute to information about the genotoxic potential of herbal and natural products, the present study evaluated the genotoxic effects (clastogenicity and aneugenicity) of the lyophilized tincture of H. impetiginosus bark in the bone marrow of mice using a micronucleus assay.The action of this tincture on genotoxic effects induced by the chemotherapeutic agent, DXR, was also studied (antigenotoxic evaluation).

System -test in vivo
Healthy, heterogeneous, young adult male and female Swiss albinus (Unib: SW) mice (between 7 and 12 weeks -pubescent period), with a body weight between 30 g and 40 g (i.e., the variation weight between the animals, for each sex, should not exceed the ±20% of medium mass) were provided by CEMIB (Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório -UNICAMP; http://www.cemib.unicamp.br),and erythrocytes from the bone marrow of these mice were used in the micronucleus assay (OECD, 1997;Boriollo et al., 2014a, b).The animals were kept in groups of the same sex, in polypropylene boxes, in an air-conditioned environment to 22 °C ±3 °C, with relative air humidity of 50% ±20%, and with 12-hour day-night cycles (i.e., 12 h light and 12 h dark).These were fed with Purina  Labina commercial rations (Nestlé Purina Pet Care Company) and water ad libitum, and acclimated to laboratory conditions for 7 days (a trial period) before the execution of the experiment.At the end of the trial period, each animal was weighed and, according to the weight, received 2 ml/100g body weight of the indicated liquid (negative control, positive control, chemotherapeutic and phytotherapeutic).After the experimental treatment, the animals were euthanized by CO 2 asphyxiation in adapted acrylic chambers (Boriollo et al., 2014a, b).This research was approved by Committee of Ethics in Research Involving Animals of UNIFENAS (CEPEAU Protocol No. 08A/2014).
• Genotoxicity test (phytotherapeutics): 500, 1,000, 1,500 and 2,000 mg/kg of THI lyophilized and diluted in water type 1.The maximum tolerated dose (MTD) was defined as (i) the highest dose that can be administered without inducing lethality or excessive toxicity during the study causing moribund euthanasia, or (ii) a dose that produces some indication of toxicity of the bone marrow (e.g. a reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow), or (iii) 2,000 mg/kg (OECD, 1997).

Processing the bone marrow and cell analysis
Shortly after euthanasia, the femora were surgically and aseptically removed, and the animals appropriately discarded.Each femur was sectioned at the proximal end and the contents of the spinal canal were washed with 1.5 ml of 150 mM NaCl solution and transferred to a 15 ml centrifuge tube (OECD, 1997;Boriollo et al., 2014a, b).This material was resuspended with a Pasteur pipette to ensure a random distribution of bone marrow cells.The suspension was then centrifuged at 1,000 rpm (Centrífuga de Bancada Microprocessada, Mod.NT 810, Nova Técnica Ind. e Com. de Equip.para Laboratório Ltda., Piracicaba, SP, Brazil) for 5 minutes.The supernatant was discarded and the resulting sediment was resuspended in 500 μL of 150 mM NaCI solution added 4% formaldehyde.The slides were prepared by smearing (2 slides per animal), dried at room temperature for 24 h and stained with Leishman's eosin methylene blue dye [pure dye for 3 min, followed by diluted dye in water type 1 (1:6) for 15 min] to differentiate polychromatic erythrocyte (PCE) from normochromatic erythrocyte (NCE).
Polychromatic erythrocytes (PCEs) were observed at a magnification of 1,000× using optical microscopy (Nikon Eclipse E-200), counted (at least 2,000 polychromatic erythrocytes anucleated per animal were scored for the incidence of micronucleated polychromatic erythrocytes) with the aid of a digital cell counter (Contador Diferencial CCS02, Kacil Indústria e Comércio Ltda., PE, Brazil) and photographed using an 8.1 Megapixel Digital Camera (DC FWL 150).The number of PCEs and NCEs, the number and frequency of micronucleated polychromatic erythrocytes (MNPCEs) were reported.In order to evaluate bone-marrow toxicity, the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) was also observed (OECD, 1997;Boriollo et al., 2014a, b).This PCE/NCE ratio is an indicator of the acceleration or inhibition of erythropoiesis and it has been reported to vary with scoring time.A continuous decline in the PCE/NCE ratio may be due to the inhibition of cell division, the killing of erythroblasts, the removal of damaged cells, or dilution of the existing cell pool with newly formed cells (OECD, 1997;Boriollo et al., 2014a, b).

Statistical analysis
The data obtained in the micronucleus assay were submitted to one-way analysis of variance (ANOVA), using a factorial scheme of 9 × 2 × 2 (treatment × sex × euthanasia time), and medium comparison with Tukey's test (α = 0.05) using SAS  version 9.3 computer software.
The acute toxicity and antiedematogenic and antinociceptive effects of an aqueous extract of H. impetiginosus inner bark were previously studied in various animal models (Swiss mice and Wistar rats, male and female) (De Miranda et al., 2001).In that study, H. impetiginosus presented antinociceptive and antiedematogenic activities, in which the antinociceptive effect was associated with the adenosine system.The mortality index was observed over 48 h and there was no LD 50 when mice received the aqueous extract at concentrations of 1, 3, and 5 g/kg (p.o.).However, the authors indicated that the plant had a low toxicity profile (De Miranda et al., 2001).The genotoxic potential of H. impetiginosus bark and stem and its association with the reference mutagen DXR (antigenotoxic effects) were evaluated using a wing spot test in Drosophila melanogaster (Somatic Mutation and Recombination Test -SMART) using the standard (ST) version to evaluate direct action and the high bioactivation (HB) version to evaluate indirect action (De Sousa et al., 2009).The bark and stem of H. impetiginosus were toxic at a higher concentration (40% w/w), but did not induce Means with the same letter (A, B, C or D) are not significantly different (p < 0.05).Shown are data from the controls (NaCl, NEU and DXR), an evaluation of the genotoxicity of THI (0.5-2 g/kg), and an evaluation of the antigenotoxicity of THI (0.5 g/kg) (THI + DXR and THI + NEU).♀ and ♂ correspond to gender of female and male Swiss albinus mice, respectively.MNPCEs: micronucleated polychromatic erythrocytes.PCE / (PCE + NCE): ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE).somatic mutation and recombination in D. melanogaster from ST and HB crossbreeding.That is, H. impetiginosus alone neither acted as a genotoxin nor exerted any antigenotoxic effects on spontaneous DNA lesions.However, H. impetiginosus displayed a considerable potentiating effect on DXR genotoxicity (0.125 mg/ml) that was inversely proportional to the concentration applied (10%, 20%, and 40% w/w), indicating a dose-response relationship with toxicity (De Sousa et al., 2009).In other studies, the genotoxic activities of the hydroalcoholic extract of H. impetiginosus inflorescences were assessed using a comet assay [Single Cell-Gel Electrophoresis (SCGE)] in order to detect the extent of DNA damage in blood and liver cells from male Wistar rats (Lemos et al., 2012).The analysis of liver and blood cells after 24 h of treatment (100, 300, and 500 mg/kg of body weight) revealed extensive DNA damage when compared to negative control (drinking-water), with the exception of the treatment at 100 mg/kg when considering only blood cells.These data suggested a dose-dependent genotoxicity of H. impetiginosus inflorescence in Wistar rats and, therefore, inferences about its use in alternative medicine requires caution (Lemos et al., 2012).
Among the phytochemical compounds identified in H. impetiginosus, lapachol and β-lapachone stand out for their pharmacological activity (Silva et al., 2003;Gómez Castellanos et al., 2009).Characterization of the genotoxic mechanisms of β-lapachone (commercial source) was performed in cultured Chinese hamster ovary (CHO) cells (Vanni et al., 1998).In that study, β-lapachone was cytotoxic (on S phase cells) and genotoxic (induced DNA strand breaks) in logarithmically growing CHO cells at concentrations around 10 μM.β-lapachone inhibited the activity of poly (ADP-ribose) polymerase (PARP) and increased the activity of topoisomerase I.In addition, these data taken together suggested that β-lapachone did not directly damage DNA.It is possible that the strong genotoxic potential observed in this study reflected the interference of β-lapachone with the delicate DNA metabolizing machinery, in which both PARP and topoisomerase I play a central role (Vanni et al., 1998).However, it is also possible that β-lapachone induced a small number of DNA strand breaks owing to its ability to produce activated oxygen species (Docampo et al., 1979;Molina Portela and Stoppani, 1996;Molina Portela et al., 1996).
β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] pyran-5,6-dione, ARQ 501), a natural o-naphthoquinone and a major component in an ethanol extract of H. impetiginosus bark, displayed promising antitumor activity in various tumor cells (Pardee et al., 2002;Tagliarino et al., 2003;Terai et al., 2009;Tan et al., 2012) and has been tested as an antitumor drug in phase I, II, and III clinical trials in combination with other chemotherapeutic agents (Pardee et al., 2002;Bentle et al., 2007).The anticancer activity of β-lapachone may involve its catalysis by NAD(P) H:quinone oxidoreductase (NQO1, DT-diaphorase), which used NAD(P)H or NADH as an electron source to yield the two-electron reduction of β-lapachone (Pardee et al., 2002;Reinicke et al., 2005).In the presence of NQO1, β-lapachone underwent reduction to an unstable hydroquinone, resulting in the generation of ROS, including superoxides (Pink et al., 2000;De Witte et al., 2004;Choi et al., 2007).In turn, these reactive oxygen species oxidized thiol groups of the mitochondrial potential transition pore complex, leading to increased inner membrane permeability, reduced membrane depolarization, release of cytochrome c, and cell death (Lemasters et al., 1998;Smaili et al., 2000).The expression of NQO1 in solid cancers is higher than in normal tissues (Belinsky and Jaiswal., 1993), including in carcinoma of the liver (Schlager and Powis, 1990), colon (Smitskamp-Wilms et al., 1995), breast (Schlager and Powis, 1990;Smitskamp-Wilms et al., 1995), brain (Berger et al., 1985), and lung (Schlager and Powis, 1990).In addition, NQO1 has been shown to be an important factor in β-lapachone-induced cell death in many kinds of cancer cells (Pink et al., 2000;Reinicke et al., 2005), including breast cancer (Tagliarino et al., 2003), glioma (Park et al., 2011), and prostate cancer (Pink et al., 2000).In this context, other methods have been examined to increase NQO1 expression or activity in cancer cells (Terai et al., 2009;Tan et al., 2012;Satsu et al., 2012) in order to increase the clinical efficacy of β-lapachone.More recently, the genotoxic impact and genotoxic activities of the combination of β-lapachone and hydroxyurea (another anticancer drug and an inhibitor of ribonucleotide reductase) were reported in Allium cepa root meristem cells (Zabka et al., 2013).Treatment with β-lapachone (100 mM) and hydroxyurea (0.75 mM) generated hydrogen peroxide (H 2 O 2 ) and induced DNA double strand breaks, which was correlated with γ-phosphorylation of H2AX histones by using an immunoassay.However, the extent of H2AX phosphorylation was considerably reduced in root meristem cells treated jointly with β-lapachone and hydroxyurea.In addition, treatment with β-lapachone (100 mM) alone resulted in a lower number of M-phase cells, an increase in the occurrence of mitotic abnormalities (over-condensation and enhanced stickiness of chromosomes, formation of anaphase bridges, and micronucleation), a reduction in mitotic spindles, and induction of apoptosis-like programmed cell death.In turn, hydroxyurea treatment alone led to adaptation of cells to replication stress and promotion of abnormal nuclear divisions with biphasic interphase/mitotic states of chromatin condensation (Zabka et al., 2013).
The presence of multiple molecules in plants may be advantageous because some may counteract the toxicity of others, and as a result, the net effect may be beneficial for therapeutic purposes.For example, the effect of various concentrations of A. marmelos (Venkatesh et al., 2007), C. langsdorffii (Alves et al., 2013), H. annuus (Boriollo et al., 2014a), and Z. joazeiro (Boriollo et al., 2014b) on doxorubicin (DXR)-induced genotoxic effects in mice bone marrow was studied.The treatment of mice with A. marmelos, for consecutive days before DXR treatment, significantly reduced the frequency of DXR-induced micronuclei and increased the PCE/NCE ratio at all scoring times compared with DXR treatment alone.This chemoprotective effect may be assigned to the sum total of interactions between different ingredients of this complex mixture.However, the degree of protection may depend on the interaction of components individually or collectively with the genotoxic agent.The plausible mechanisms of action of A. marmelos in protecting against DXR-induced genomic insult were scavenging of O 2 •-and • OH and other free radicals, increase in antioxidant status, restoration of topoisomerase II activity, and inhibition of the formation of DXR-iron complex (Venkatesh et al., 2007).The results of other studies demonstrated that C. langsdorffii was not itself genotoxic and that, in animals treated with the combination of C. langsdorffii Desf.and DXR, the number of micronuclei significantly decreased compared to animals receiving DXR alone, according to the Swiss mouse peripheral blood micronucleus test.
The putative antioxidant activity of one or more of the active compounds of C. langsdorffii, including two major flavonoid heterosides (quercitrin and afzelin) may explain the effect of this plant on DXR-induced genotoxicity (Alves et al., 2013).More recently, the absence of genotoxicity of the tincture and two sources of oils of H. annuus was observed in the bone marrow of Swiss albinus mice using micronucleus assay, but indications of antigenotoxic effects were related to combination treatment with the tincture and DXR, suggesting a partial protective mechanism against DXR-induced genotoxic effects (Boriollo et al., 2014a).Finally, the nongenotoxicity of Z. joazeiro and its antigenotoxic effects when administered together with DXR were also related as shown by micronucleus assay in the bone marrow of Swiss albinus mice (Boriollo et al., 2014b).
In conclusion, this study reports information with an emphasis on mutagenesis, especially on the possible genotoxic and antigenotoxic effects of a lyophilized tincture of H. impetiginosus bark using in vivo micronucleus assay in mouse bone marrow, which has recently been the subject of little research.The results suggested the absence of genotoxicity (clastogenicity and aneugenicity) of H. impetiginosus [dose (0.5-2 g/kg), time-(24-48 h), and gender-independent (male and female)].Relatively little systemic toxicity (acute and chronic in bone marrow) was observed in the micronucleus test conditions by the proportion of PCE/NCE data.However, combination treatment with H. impetiginosus (0.5 g/kg) and DXR (5 mg/kg) showed antigenotoxic effects (anticlastogenicity and antianeugenicity) and a significant reduction in toxicity, which suggested that the extract of H. impetiginosus bark provided protection against toxic and genotoxic effects induced by the chemotherapeutic agent, DXR, in the bone marrow of mice (time-and gender-independent antigenotoxic effect, time-and gender-dependent antitoxic effect).
More studies involving the evaluation of the genotoxicity of extracts and phytochemical compounds found in H. impetiginosus (Mart.ex DC.) Mattos should be conducted, including mutagenicity assays with Salmonella typhimurium test (Ames test) as an indicator of potential carcinogenicity to mammals, gene mutation test in mammalian cells (mouse lymphoma assay), cytogenetic and aneuploidy tests in vitro, micronucleus tests in cultured cells in vitro, fluorescent in situ hybridization (FISH) tests for mutagenesis, comet tests to detect DNA damage and repair in individual cells, and functional genomic and proteomic tests for mutagenesis (cDNA microarrays and other array analyses) to characterize their potential genotoxic and antigenotoxic effects and the molecular mechanisms involved.In addition, studies should be conducted to establish limits for human consumption, delineate potential risks to human health, and provide rational strategies for implementing chemo-preventive measures.

Figure 1 .
Figure 1.Box-plots showing the incidence of micronucleated polychromatic erythrocytes (MNPCEs) and ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) in bone marrow of male (♂) and female (♀) Swiss albinus mice after testing for 24h and 48h.Shown are data from the controls (150 mM NaCl, NEU 50 mg/kg and DXR 5 mg/kg), an evaluation of the genotoxicity of the H. impetiginosus bark (THI: 0.5-2 g/kg), and an evaluation of the antigenotoxicity of THI bark (THI 0.5 g/kg + DXR 5 mg/kg; THI 0.5 g/kg + NEU 50 mg/kg).Means with the same letter (A, B, C or D) are not significantly different (p < 0.05).

Table 1 .
The mean incidence of MNPCEs and PCE/NCE ratio in bone marrow of male and female Swiss albinus mice after testing for 24h and 48h.Shown are data from the controls (NaCl, NEU and DXR), an evaluation of the genotoxicity of the H. impetiginosus bark (THI), and an evaluation of the antigenotoxicity of THI bark (THI + DXR and THI + NEU).