Screening of cytoprotectors against methotrexate-induced cytogenotoxicity from bioactive phytochemicals

Introduction

Methotrexate (MTX) is one of the most intensively investigated and effective chemotherapeutic agents for inhibiting dihydrofolate reductase, resulting in depletion of tetrahydrofolate. However, the genotoxic effects of MTX have been reported in the somatic cells employing chromosome aberration and micronucleus test as the end points of evaluation (Choudhury & Palo, 2004). A present study has confirmed that MTX induced cytotoxic and genotoxic effects in the germ cells (Padmanabhan et al., 2008). Furthermore, MTX-treated RA patients have an increasing incidence of melanoma, non-Hodgkin’s lymphoma, and lung cancer (Buchbinder et al., 2008). In addition, MTX chemotherapy for gestational trophoblastic tumors and acute lymphoblastic leukemia greatly increases the risk of second tumors (Schmiegelow et al., 2009). Being a structural analogue of folic acid (FA), MTX competes with the normal substrate FA for the binding site on dihydrofolate reductase (DHFR), which is the critical enzyme involved in the synthesis of essential DNA precursors such as thymidylates and purines. Suppression of DHFR leads to depletion of tetrahydrofolates which are required for the synthesis of purines and thymidilate and thereby perturb the DNA synthesis (Aggarwal et al., 2006). On the other hand, the deficiency of folates induced by MTX interfering in the folate metabolism is often accompanied by genotoxic damage including strand breaks, chromosomal abnormalities, extensive incorporation of uracil in place of thymine into the DNA, defective DNA repair, anomalous DNA methylation patterns and increased somatic mutation rates (Vinson & Hales, 2002; Branda et al., 2001; Knock et al., 2008; Kapiszewska et al., 2005; Branda et al., 2007). In addition, Coleshowers’s work indicated that MTX causes oxidative stress by reducing the activities of superoxide dismutase, catalase and glutathione reductase (Coleshowers et al., 2010). Gressier et al. (1994) demonstrated that MTX increases the amount of hydrogen peroxide and other free radicals which may lead to toxicity thus accelerating the rate of cellular damage. Several studies suggested that oxidative stress in the pathogenesis of MTX-induced damage play an important role in the various organs (Vardi, Parlakpinar & Ates, 2012). Thus, the development of efficient protective agents against methotrexate-induced cytogenotoxicity has attracted more and more attention.

In recent years, a number of natural plant products have been investigated as cytoprotectors to defend normal cells from the damage induced by MTX. Horie et al. (2006) reported that aged garlic extract could protect IEC-6 cells from the MTX-induced intestinal damage. Verschaeve & Van Staden (2008) found that apricot and β-carotene treatment could alleviate the impairment of oxidative stress and ameliorate MTX-induced intestine damage. Chang et al. (2013) showed that MTX-induced apoptosis of IEC-6 cells could be repressed by the pre-treatment of lutein. Daggulli et al. (2014) observed that carvacrol significantly reduced deleterious effects of MTX on testicular tissue. Vardi, Parlakpinar & Ates (2012) demonstrated that chlorogenic acid treatment may protect against the impairment of oxidative stress and ameliorate MTX-induced cerebellar damage in rats. It appears that proanthocyanidin from Vitis vinifera can protect the small intestine of rats and inhibits methotrexate-induced oxidative stress (Gulguna et al., 2010). There is evidence indicating that grape seed extract could ameliorate the MTX-induced oxidative injury in the rat liver (Cetin et al., 2008). Contrasting with garlic, apricot, Origanum onites L. panax ginseng, chlorogenic acid, proanthocyanidin and American ginseng, the cytoprotective effect of those bioactive phytochemicals against methotrexate-induced cytogenotoxicity has been rarely reported, such as eleutherosides (Siberian ginseng root), gingerols (ginger root), ginkgo flavone (ginkgo leaf), ginsenosides (ginseng root), polyphenols (green tea), polysaccharides (reishi mushroom), and isoflavones (soybean). Actually, ginger phenolic compounds exhibited potential antioxidant properties, moderate activity against xanthine oxidase, monoamine oxidase-A and α-glucosidase, and protection of PC-12 and primary rat liver cells against H₂O₂-induced damage (Peng et al., 2012). Ginkgo flavonoids might protect against apoptosis of hippocampal neurons through inhibiting death receptor pathway or mitochondrial pathway underTNF-α background (Guo etal., 2015). Ginsenoside can increase the activities of antioxidant enzymes such as T-SOD and GSH-Px and decrease the level of lipid peroxidation such as TBARS and protein carbonyl to block oxidative pathways (Zhao, Li & Li, 2011). Green tea polyphenols can protect against oxidative stress/damage and bladder cell death (Coyle et al., 2008). The antimutagenic and antioxidant properties of mushrooms polysaccharides have been also reported in literature (Delmanto et al., 2001; Kozarski et al., 2011; Wang et al., 2015). The antioxidant ability of soybean isoflavone is well known. However, most of the previous studies are focused on the cytoprotection of individual plant extracts or combined with other agents, such as -carotene and quercetin. Studies on the protective potentials of various bioactive phytochemicals against MTX-induced damage, such as Siberian ginseng eleutherosides, chrysanthemum chlorogenic acid, ginger gingerols, grape seed proanthocyanidins, green tea polyphenols and so on, especially plant extract combinations have received less attention. To develop a more efficient protective agent against MTX-induced cytogenotoxicity, cytoprotective activity of ten different extracts and some extracts combinations were evaluated in vivo and in vitro tests. The combination of grape seed proanthocyanidins and eleutherosides from Siberian ginseng as well as green tea polyphenols and eleutherosides owned potentials to be used as new cytoprotectors.

Materials and Methods

Statistical analysis

Values are presented as means ± standard deviation (SD).The data were analyzed for statistical significance using t-test (SPSS 13.0 for Windows). A p value of less than 0.05 was deemed as significant.

Results

Evaluation of the antimutagenic potentials of cytoprotector candidates by micronucleus test

The micronucleus assay is internationally recognized as the standard method to detect the mutagenicity of chemicals. In order to assess the protective effects of the candidate cytoprotectors against the genotoxicity caused by MTX, micronucleus assay was employed in the next trial. With administration of the candidate plant extract combinations, the data of the bone marrow polychromatic erythrocyte micronucleus test in mice exposed to MTX were presented in Fig. 4. Whether male or female, there were statistically significant increases (p < 0.01) in the frequency of micronucleated polychromatic erythrocytes and ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) in model group and control group. However, the treatment of the candidate cytoprotectors markedly restrained the incidence of mice bone marrow micronucleus, and improved the ratio of PEC and NEC. Furthermore, there were significant differences between treatment groups and model group (p < 0.01). In terms of the inhibition of micronucleus formation, the combination of grape seed proanthocyanidins and eleutherosides from Siberian ginseng was superior to the combination of green tea polyphenols and eleutherosides. Meanwhile, the two treatment groups did not cause differences between male and female mice. It could be considered that the two combinations had promising effects on suppression of MTX-induced micronuclei in mice bone marrow cells.

Influence of cytoprotector candidates on reproductive toxicity induced by MTX

To investigate whether the combinations of bioactive phytochemicals treatment could strengthen or weaken the reproductive toxicity induced by MTX, sperm tests had been adopted. The method is one reliable and easy way to detect reproductive toxicity. In this study, the kinds of abnormal sperm in any group were mainly no hooks, amorphous, bananas and triangular head heads. As could be seen from Fig. 5, the incidence of mouse sperm head deformity of treatment groups had no significant difference from that of the control (p > 0.05). Significant differences could be observed between the treatment groups and model groups (p < 0.01). The summary of no hooks and amorphism heads in the model accounted up to 90% of the total sperm head morphology, significantly higher than that of control group and treatment group. The results illustrated that the two combinations of bioactive phytochemicals may alleviate the reproductive toxicity caused by MTX. Compared with the model group, the percentage of different types of abnormal sperm showed significant difference. The percent of Lack hook, Amorphous, Banana-like and triangular in Treatment I were 39.9%, 47.2%, 9.8% and 3.1%, respectively. In the case of sperm aberration, the result of the Treatment II was closes to the data of control group. It seemed that cytoprotective activities of proanthocyanidins and eleutherosides combination were superior to that of polyphenols and eleutherosides.

Discussion

The effective estimating for antimutagenic properties of bioactive phytochemicals includes the bacterial gene mutation assay Ames test (Verschaeve & Van Staden, 2008; Horn & Vargas, 2003), mammalian cell culture benzo(a)pyrene metabolism assay (Cassady et al., 1988), the mammalian cell gene mutation assay (Mersch-Sundermann et al., 2004), and the in vitro micronucleus assay (Serpeloni et al., 2008). Compared with animal-cell-based systems, microbe-based assays for screening cytoprotectors present several advantages, such as the simplicity of the procedures, the relatively short time needed to obtain results, and cost-effective. Nevertheless, except for the Ames test, there are few reports in the literature describing the studies of evaluation for antimutagenic properties of bioactive phytochemicals by umu test so far. In this study, we selected a short-term bacterial test system, the umu test, to evaluate the antimutagenic potential of plant extracts. Moreover, based on comparison of the umu test results (486 chemicals) with the Ames test (274 compounds) as well as rodent carcinogenicity data (179 compounds), Reifferscheid & Heil (1996) found good agreement between the umu test and the Ames test results. Thus umu test could be developed an effective high-throughput screening assay to evaluate antimutagenic potential of phytochemicals same as Ames test and comet assay.

Natural plant medicine is an important resource to develop new cytoprotectors. Cytoprotective effects of many bioactive phytochemicals have been proved in the past several decades. Isoflavones from soybean seeds have showed antimutagenic activity in S. typhimurium TA1535/pSK1002 and TA100 (Miyazawa et al., 1999). Garlic extract has been proved that it could reduce apoptotic cell injury induced by MTX. It should be noted that garlic was most popular supplement in US households (Amagase et al., 2001). Meanwhile, polyphenols from green tea (Chinese Gunpowder and Japanese Sencha) exhibited high antimutagenic activity in the Ames test as well as in S. cerevisiae D7 test. Moreover, in the peripheral blood lymphocytes method reduced number of abberant cells as well as decreased number of chromosome breaks was observed using both green tea extracts (Bunkova, Marova & Nemec, 2005). What’s more, grape seed extract played a role in attenuating the genotoxicity induced by cisplatin (Attia et al., 2008). Contrasting with allicin, proanthocyanidins, polyphenols and isoflavones, chlorogenic acid, proanthocyanidin and American ginseng, cytoprotective effect of those bioactive phytochemicals against methotrexate-induced cytogenotoxicity has been reported rarely, such as Siberian ginseng eleutherosides, ginger gingerols, ginkgo flavone, ginseng ginsenosides, mushroom polysaccharides, and soybeam isoflavones. Our results suggested that allicin, proanthocyanidins, polyphenols, eleutherosides and isoflavones showed stronger antimutagenic activities than other five kinds of plant extracts. Moreover, it was demonstrated results single eleutherosides and its combinations with proanthocyanidins, polyphenols had strong antimutagenic effect. It is well known that the detrimental effects of MTX was partly due to its direct toxic action by increasing reactive oxygen species production, although the exact mechanisms of methotrexate-induced toxicity had not yet been elucidated to date (Oktem, 2006). Coleshowers’s work indicated that MTX causes oxidative stress by reducing the activities of superoxide dismutase, catalase and glutathione reductase (Coleshowers et al., 2010). Gressier et al. (1994) demonstrated that MTX increases the amount of hydrogen peroxide and other free radicals which may lead to toxicity thus accelerating the rate of cellular damage. Several studies have well confirmed the contribution of oxidative stress in the pathogenesis of MTX-induced damage in the various organs (Vardi, Parlakpinar & Ates, 2012). It is supposed that the oxidative stress induced by MTX was reduced and the cell damage caused by free radical accumulation was alleviated owing to potential antioxidant activity of the grape seed proanthocyanidins, green tea polyphenols, soybean isoflavone and Siberian ginseng eleutherosides. Gulguna et al. (2010) demonstrated that proanthocyanidin from Vitis vinifera can inhibits methotrexate-induced oxidative stress. Yu et al. (2016) found that a polyphenol-rich Herb (Scutellariae radix) ingestion increased the systemic exposure and mean residence time of MTX via modulation on efflux transporters multidrug resistance–associated protein 2 and breast cancer resistance protein. Chiang et al. (2005) observed that the coadministration of Pueraria lobata root decoction (an isoflavone-rich herb) significantly decreased the elimination and resulted in markedly increased exposure of MTX in rats. Recently, it was proved that eleutherosides (as one kind of polyphenols) were promising chemical substances with antioxidant properties. Substantially, grape seed proanthocyanidins, green tea polyphenols, soybean isoflavone and Siberian ginseng eleutherosides belong to the members of polyphenols which have strong antioxidant capacity. In this radical scavenging mechanism, polyphenols sacrificially reduce ROS/RNS, such as ^∙OH, ${\text{O}}_2^{\bullet -}$, NO^∙, or OONO⁻ after generation, preventing damage to biomolecules or formation of more reactive ROS (Perron & Brumaghim, 2009). Previous research found that water extract of Siberian ginseng showed significant antioxidant activity and protective effect against oxidative DNA damage induced by H₂O₂ (Park et al., 2006). Moreover, the methanol extract of root, stem, and leaf of Eleutherococcous senticosus showed inhibitory effects on the mutagenicity induced by 2-AF or Trp-P-1 in S. typhimurium TA98 (Park et al., 2002). Therefore, we inferred that the cytoprotector candidates may play a key role in attenuating the methotrexate-induced cytogenotoxicity due to their antimutagenic and antioxidant activity.

In addition, the previous study had proved that MTX could bring out the reproductive toxicity (Padmanabhan et al., 2008). MTX treatment significantly reduced the sperm count and increased the occurrence of sperm head abnormalities. However, the administration of phytochemicals combinations to Kunming specific pathogen-free mice appeared clearly decreases in sperm abnormality rate in the case of MTX exposure. Akram et al. (2012) ever reported that American ginseng extract treatment exhibited therapeutic effects on sperm parameters in rats treated with Cyclophosphamide (CP), which is an antineoplastic agent and immunosuppressive medicine in the treatment of various types of tumors, and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis (Tripathi & Jena, 2009). Moreover, ginseng has been demonstrated to have a cytoprotective effects against these toxins, in which administration of Panax ginseng extract was reported to markedly decrease the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced pathological and genotoxical damages in rat testes (Lee et al., 2007). Recent studies had confirmed that green tea and soybean extracts showed protective effects against reproductive toxicity induced by CP. Fahmy et al. (2014) found a significant decrease in the percentage of sperm abnormalities in orally administrated soybean extracts. Zanchi et al. (2015) demonstrated that polyphenols improved CP-induced damage on reproductive system, and its effect is probably due to high concentrations of catechins and antioxidant activity.

Although some evidence suggested that Siberian ginseng may not stimulate immune function (Wang et al., 2003), it has been indicated that polyphenols from green tea and grape seed proanthocyanidin have apparent immunomodulatory effects. Sheikhzadeh found that decaffeinated green tea in lower doses of administration could enhance the immunity of rainbow trout (Sheikhzadeh et al., 2011). The dietary administration of green tea supplementation positively raises the cellular immune responses and disease resistance of kelp grouper Epinephelus bruneus to Vibrio carchariae (Harikrishnan, Balasundaram & Heo, 2011). Tong et al. (2011) demonstrated that grape seed proanthocyanidins (GSPs) could improve functional activation of the immune system, and the antitumor effects of GSPs were achieved by immunostimulating properties. Our findings implied that the two combinations of phytochemical compounds not only could affect the immune organ indices of experimental animal Kunming mice, but also could effectively relieve the immune toxicity caused by MTX. According to the above results, it can be deduced that the combination of grape seed proanthocyanidins and Siberian ginseng eleutherosides as well as green tea polyphenols and eleutherosides was able to weaken the reproductive toxicity induced by MTX.

Conclusions

In this study, we developed an effective screening assay to evaluate the antimutagenic potential of bioactive phytochemicals based on an umu assay coupled with a micronucleus test. Allicin, proanthocyanidins, polyphenols, eleutherosides and isoflavones showed higher antimutagenic activity than other phytochemicals. Moreover, of all the bioactive phytochemicals combinations, the combination of proanthocyanidins and eleutherosides as well as polyphenols and eleutherosides obtained higher cytoprotective effects. In the case of MTX exposure, the administration of the two combinations of bioactive phytochemicals to Kunming mice clearly presented decreases in micronucleus induction and in sperm abnormality rate. Moreover, obvious increases in thymus and spleen indices were observed in the cytoprotector candidates treated groups. The results implied that phytochemicals combination of proanthocyanidins, eleutherosides and polyphenols could be used as new cytoprotectors.

Supplemental Information