Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction Emmanuel Mfotie Njoya a,b,n, Paul F. Moundipa a, Helga Stopper b a b
Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 11 March 2014 Received in revised form 27 May 2014 Accepted 15 June 2014
Ethnopharmacological relevance: Codiaeum variegatum, grown in many varieties, has been widely used as a houseplant based on its brightly decorative foliage. In addition, a variety of this plant has been used for a long time against bloody diarrhea by the local population in Cameroon. Aim of the study: In our previous study, the aqueous extract of this plant and an isolated sub-fraction exhibited significant anti-amoebic activity on axenic culture of Entamoeba histolytica. Due to the medicinal value of these extracts, we promptly initiated to investigate their genotoxic and mutagenic potential in order to assure their safe and rationale usage in traditional healthcare system. Material and methods: Both extracts were incubated with L5178Y mouse lymphoma cells, primary hepatic cells and HepG2 human hepatocellular carcinoma cells and their genotoxicity and mutagenicity were evaluated by quantifying DNA damage and chromosomal aberrations through comet assay, micronucleus assay and mouse lymphoma mutation assay. Results: The aqueous extract of Codiaeum variegatum is not cytotoxic up to 2000 mg/mL while the amoebicidal fraction is significantly cytotoxic (r 40–55%) on L5178Y mouse lymphoma and HepG2 cells at concentrations higher than 500 mg/mL. Besides, no significant DNA damage and induction of micronucleus formation were identified at concentrations up to 2000 mg/mL. Moreover, the mutagenic potential of these extracts after short (4 h) and long term (24 h) treatment, revealed no significant gene mutation induction. Conclusion: The aqueous extract of Codiaeum variegatum and the amoebicidal fraction SF9B are neither genotoxic on non-competent or metabolic competent cell lines, nor mutagenic in mouse lymphoma mutation assay and therefore they could be safely used at lower doses for medicinal purpose. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Codiaeum variegatum (Euphorbiaceae) Aqueous extract Amoebicidal sub-fraction SF9B Cytotoxicity Genotoxicity Mutagenicity Safety
1. Introduction Medicinal plants are important because they produce a large variety of metabolites which are known to possess therapeutic importance for maintaining human health and improving the well-being, thus justifying their use in traditional medicine system. However, many adverse effects such as genotoxicity, mutagenicity and carcinogenicity of medicinal plants have been published in various medical reviews (Soliman, 2001; Konan et al., 2007; Sowemimo et al., 2007; Akaneme and Amaefule, 2012). Codiaeum variegatum, also referred to as “garden croton”, belongs to the family of Euphorbiaceae and is a tropical-origin ornamental shrub with diverse beautiful and attractive foliage. In addition to its decorative usage as an indoor plant, Codiaeum variegatum is n Corresponding author at: Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812 Yaoundé, Cameroon. Tel.: þ 237 75526943. E-mail addresses: [email protected], [email protected] (E. Mfotie Njoya).
also widely used for its medicinal properties. Freeze-dried leaf decoction of Codiaeum variegatum is taken as tea by Filipinos and drinking crushed leaves cures diarrhea (Saffoon et al., 2010). The root and bark are used against syphilis, constipation, stomachache, loss of appetite and dysuria. From the fresh latex, a cyanoglucoside was isolated and displayed virucidal activity against influenza A virus without impairment of haemagglutination properties (Forero et al., 2008). An evaluation of tumor promoter contents by high-performance liquid chromatography coupled with tumorpromoting activity assessment showed that cultivars of Codiaeum variegatum were devoid of tumor-promoting compounds (Vogg et al., 1999). Despite the beneficial effects of this plant, it has been reported that the latex or sap produced from the bark, root and leaves is toxic and can cause skin eczema in humans after repeated exposure (Hausen and Shulz, 1977; Ogunwenmo et al., 2007). There are more than 300 cultivars known as mutants or hybrids and grouped into nine species based on their leaf morphology (Deng et al., 2010). The cultivar used in Cameroon belongs to the royal-like species with green oak leaves and white spots.
http://dx.doi.org/10.1016/j.jep.2014.06.038 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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This plant is widely used by the local population against intestinal disorders such as bloody and watery diarrhea (Moundipa et al., 2005). The aqueous extract from the leaves of this plant and an isolated sub-fraction were identified with relevant antiamoebic efficacy against the intestinal parasite Entamoeba histolytica (Mfotie et al., 2014). The rational and safe usage of Codiaeum variegatum is absolutely important to gain benefit from its medicinal value. Until now, no study has been carried out to evaluate the genotoxicity and the mutagenicity of this medicinal plant. Nowadays, detection of chromosome or DNA damage represents an important tool used for prioritizing compounds early in the drug development process. Therefore, the in vitro toxicity of the aqueous extract from the leaves of Codiaeum variegatum and its amoebicidal sub-fraction was assessed by evaluating the cytotoxicity and quantifying DNA damage and chromosomal alterations respectively through comet and micronucleus assays. Moreover, the evaluation of the mutagenic potential of these substances was carried out by the mouse lymphoma (TK) mutation assay after short and long term treatment.
2. Material and methods 2.1. Preparation of the aqueous extract and sub-fraction isolation Freshly leaves of Codiaeum variegatum var. mollucanum were collected in Yaoundé (Cameroon) and were thoroughly washed, rinsed with distilled water, dried at room temperature and grounded. The powder obtained (100 g) was prepared with 1 L of distilled water for 1 h. After cooling, the mixture was filtered with Whatman No.1 filter paper and the filtrate was dried by lyophilisation to obtain a residue which constitutes the aqueous extract. From the aqueous extract, the amoebicidal sub-fraction named SF9B was obtained as earlier described (Mfotie et al., 2014). 2.2. Chemical profile of the tested substances Qualitative phytochemical screening was done as described by (Trease and Evans, 1989) to obtain the chemical composition of the aqueous extract of Codiaeum variegatum. Some major groups of compounds such as alkaloids, anthranoids, sugar, leuco-anthocyanins, coumarins, tannins, saponins, polyphenols, flavonoids, sterols and triterpenes were analyzed. It resulted from this analysis that the aqueous extract of Codiaeum variegatum contains polyphenols, tannins, coumarins and traces of sugar. The fingerprint of the sub-fraction SF9B was defined in this study using analytical high-performance liquid chromatography (HPLC) by injecting 20 ml of the sub-fraction dissolved in methanol through a silica gel column under an inverse stationary phase of KROMASILs C18. The solvent system used is composed of H2O (35%)/CH3CN (65%) flowing at a debit of 0.8 mL/min. The detection was done under ultra-violet light at λ ¼254 nm using a GILSON 170 Diode Array Detectors. The HPLC-UV fingerprint of the subfraction SF9B is shown in Fig. 1.
Fig. 1. HPLC-UV fingerprint analysis of the sub-fraction SF9B. Elution under isocratic condition with the solvent system H2O (35%)/CH3CN (65%) using an inverse stationary phase of KROMASILs C18; debit: 0.8 mL/min; detection: GILSON 170 Diode Array Detectors at λ ¼ 254 nm.
10% (v/v) fetal calf serum, 1% (v/v) L-glutamine, 1% (v/v) of sodiumpyruvate (100 mM) and 0.4% (v/v) antibiotics (50 U/mL penicillin and 50 mg/mL streptomycin). Primary cells were isolated from freshly prepared rat liver (ZUC-Leprfa) previously washed with ice-cold phosphate buffered saline (PBS). The liver was transferred into 3 mL of an ice-cold HepG2 culture medium and rapidly cut on ice into small pieces. The primary cells were collected by filtration through a filter mesh pore size of 100 mm (Heidelberg, Germany), centrifuged at 100g for 5 min at 4 1C and finally suspended in 5 mL of HepG2 culture medium. The viability and the number of cells were determined using trypan blue exclusion technique on the Neubauer chamber. 2.4. Treatment of cells Primary rat hepatic cells, HepG2 and L5178Y cell lines were inoculated at a density of 6 105 cells in 3 mL culture medium on 6-well microtiter plates. After seeding, cells were treated with increasing concentrations of the aqueous extract and the subfraction SF9B dissolved either in phosphate buffered saline (PBS) or in dimethylsulfoxide (DMSO). The maximum concentrations of the aqueous extract and the sub-fraction SF9B used in these experiments were determined based on their solubility in different solvents (sterile PBS and DMSO) with the highest tested concentration showing insolubility. The aqueous extract and the sub-fraction were used in culture medium up to a concentration of 2000 and 1000 mg/mL (w/v) respectively. Methyl methanesulfonate and cyclophosphamide (Sigma-Aldrich, Steinheim, Germany) all dissolved in DMSO were used as positive controls respectively on L5178Y cells and HepG2 or primary cells. During each experiment, the maximal concentration of DMSO in the medium did not exceed 1%. After incubation for 4 or 24 h at 37 1C with 5% CO2, cells were collected by centrifugation in fresh culture medium for the following assays. 2.5. Cytotoxicity assay
2.3. Cells and culture media The L5178Y mouse lymphoma cells (obtained from Dr. W. J. Caspary, NIEHS/USA) were cultured in suspension at 37 1C with 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) heatinactivated horse serum, 1% (v/v) L-glutamine (200 mM), 1% (v/v) sodium-pyruvate (100 mM) and 0.4% (v/v) antibiotics (50 U/mL penicillin and 50 mg/mL streptomycin). The HepG2 human hepatocellular carcinoma cells (ATCC HB8065) are adherent cells which multiply in monolayer at 37 1C with 5% CO2 in the DMEM low glucose (1 g/l) supplemented with
Cytotoxicity is considered as the potential of a compound to induce cell injury or death. In this assay, the cell viability was assessed by using the fluorescein diacetate/gel red technique. The staining solution contained 30 mg/mL of fluorescein diacetate and gel red (1:1000) dissolved in PBS. Treated cells in suspension (70 ml) were mixed with 30 ml of the staining solution and 20 ml of this mixture was applied on the slide and covered with a cover slip (21 26 mm2). The ratio of green and red cells in a total of 3 100 cells was counted at a 200-fold magnification under a microscope using the FITC filter. Viable cells stain green because their active
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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esterases cleave the acetate groups from fluorescein diacetate, which then starts to fluoresce. Dead cells appear red because gel red can cross their defective membrane and intercalates in the DNA. 2.6. Comet assay The endpoint of this assay is to detect single and double strand breaks as well as alkali-labile lesions. In this assay, L5178Y, HepG2 and primary cells were treated as described previously. Treated cells were harvested and 20 ml of this suspension were mixed with 180 ml of 0.5% low-melting point agarose and 45 ml of this mixture was deposited on slides covered previously with 1.5% high melting point agarose. The slides were incubated in lysis solution (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, and 10 g/l Sodium N-lauroylsarcosine adjusted to pH 10 with NaOH 5 M) containing 1% Triton X-100 and 10% DMSO for at least 1 h at 4 1C. Thereafter, the slides were washed with distilled water and then placed in the electrophoresis chamber and covered with the electrophoresis solution (5 M NaOH and 0.2 M EDTA [pH413.0]) for 20 min. The electrophoresis was conducted for 20 min at 25 V and 300 mA adjusted with the electrophoresis solution. The slides were neutralized in 0.4 M Tris buffer (pH 7.5), followed by cell fixation in cold methanol for 5 min at 20 1C. The slides were then left at 37 1C in an incubator to dry for 10 min and stored at room temperature afterward. Before evaluation, 20 ml of gel red (1:100)/DABCO (1/4) solution were added to each slide and covered with cover slips (21 26 mm2). Images of 100 randomly selected cells at the middle of the slide (50 per replicate slide) for each sample were analyzed with a fluorescence microscope (Labophot 2; Nikon GmbH, Germany) at 200-fold magnification using image analysis software (Komet 5; BFI Optilas, Germany). The percentage of DNA in the tail was used to quantify DNA damage and the average determined within three independent experiments. 2.7. Micronucleus frequency assay In this experiment, L5178Y and HepG2 cells were treated as previously described. On the one hand, treated L5178Y cells were collected and incubated in fresh medium for 20 h at 37 1C with 5% CO2 with addition of the cytokinesis-inhibitor cytochalasin B (final concentration: 3 mg/mL). Micronucleus analysis was limited to binucleated cells which have divided once since substance exposure, and a proliferation index was calculated for assessment of cytostatic effects (see the formula below). On the other hand, treated HepG2 cells were incubated in fresh medium without the addition of cytochalasin B for 40 h. HepG2 cells did not tolerate cytochalasin B treatment well. Next, cells (both cell lines) were collected, washed with PBS, brought onto slides by cytospin centrifugation and fixed by incubation in methanol at 20 1C for at least 2 h. Cells were stained in the dark for 7 min with a gel green (1:100) solution and slides were washed with PBS, and mounted with diazabicyclo-octan (DABCO) for microscopy. Micronucleus count was performed at 400-fold magnification under a microscope using a FITC filter. The number of mononucleated (MN), binucleated (BN) and multinucleated (MuN) cells was enumerated as well as the frequency of binucleated cells or mononucleated cells (for HepG2 cells) containing micronuclei in 1000 cells per slide. For each experiment, two slides were analyzed and the average of micronucleus formation was calculated within three independent experiments. CBPI
¼
ð1 MNÞ þ ð2 BNÞ þ ð3 MuNÞ MNþ BN þ MuN
CBPI: Cytochalasin B Proliferation Index; MN: mononucleated cells; BN: binucleated cells and MuN: multinucleated cells.
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2.8. Mammalian mutation assay in L5178Y (TK þ / ) mouse lymphoma cells In this assay, mouse lymphoma L5178Y cells were exposed to a short and a long term treatment as recommended by the International Conference on Harmonization (ICH) and the Organization for Economic Co-operation and Development (OECD) (Clive and Spector, 1975; Cole et al., 1999; Clements, 2000). Before any experiment, an exponentially growing culture of L5178Y mouse lymphoma cells was first cleansed from spontaneous mutations by incubation at 37 1C with 5% CO2 for 24 h in culture medium containing thymidine (9 mg/mL), hypoxanthine (15 mg/mL), methotrexate (0.3 mg/mL) and glycine (22.5 mg/mL). These cells were further incubated for 24 h in the same medium without methotrexate. This treatment thereby permits to reduce the background level of spontaneous TK / cells. After collection of cells by centrifugation at 80g for 10 min, 106 cells were transferred in culture flasks containing 5 mL of fresh culture medium. These cells were submitted to short (4 h) and long (24 h) term treatment with at least four concentrations of the aqueous extract (dissolved in PBS) and the sub-fraction SF9B (dissolved in DMSO). Methyl methanesulfonate was used as the positive control at the concentrations of 100 and 50 mM respectively after 4 and 24 h treatment. Treated cells were washed with fresh medium and 0.5 106 cells were added in 43 mL of culture medium containing 4.5 mL of sterilized granulated agar 3.5% (Gibcon BRL, Life Technologies, Scotland). After homogenization, the mixture was plated onto 2 culture dishes (100 20 mm) and these culture dishes were allowed to solidify at room temperature. After 40 h of phenotypic expression, selection of mutant cells was carried out by addition of an overlayer of a semisolid medium containing trifluorothymidine (final concentration: 3 mg/mL). Cloning efficiency and viability were determined by addition of 600 cells in 90 mL of culture medium containing 9 mL of sterilized granulated agar 3.5% and plated onto 3 culture dishes. All culture dishes were incubated at 37 1C with 5% CO2 for colony growth. Colonies were counted manually both for the evaluation of the cloning efficiency and viability 9 days after platting and for the mutation frequency (TFTresistant colonies) 10 days after TFT addition. Based on the colony count, the viability and the cloning efficiency are evaluated by comparing the total growth of surviving cells out of 600 cells seeded in the test against the negative control. The mutation frequency is obtained from the number of TFT-resistant cells divided by the cloning efficiency per million cells used in the experiment. 2.9. Statistical analysis All assays were performed in three independent experiments and the results are presented as mean7SD (standard deviation) values. Statistical analysis was carried out with the GraphPad Instat 3.0 software and student's t-test was used to determine P-values for the differences observed between test substances and negative control. Results were considered significantly different when Pr 0.05.
3. Results 3.1. Viability of treated cells with fluorescein diacetate/gel red The cytotoxic effect of the aqueous extract and the sub-fraction were evaluated in mouse lymphoma L5178Y and HepG2 cells after 4 and 24 h of treatment. Results showed that the aqueous extract did not cause significant cytotoxicity in both cell lines up to 2000 mg/mL, the concentration at which this extract become insoluble in the culture medium. The sub-fraction SF9B did not
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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cause reduction of cell viability up to the concentration of 250 mg/mL. When tested above this concentration, the subfraction SF9B was significantly cytotoxic with 10–55% reduction in cell viability (Fig. 2).
positive controls methyl methanesulfonate and cyclophosphamide induced significant elevations of DNA damage in all cases.
3.3. Induction of micronucleus formation 3.2. DNA damage in treated cells The comet assay was used to evaluate DNA damage in treated cells. Results obtained from comet assay reveal usually an early or immediate response which occurs in DNA of a cell when treated with a genotoxic agent. In fact, this DNA damage may be transitory and susceptible to repair. Thus, the effect obtained during each of our experiment represents the interaction between the generation of DNA damage and repair mechanisms. Quantification of the percentage DNA in tail showed no significant DNA damage in mouse lymphoma L5178Y cells and HepG2 cells after 4 and 24 h treatment with concentrations up to 2000 mg/mL for the aqueous extract and 1000 mg/mL for the sub-fraction SF9B. On primary cells isolated from fresh rat liver, the treatment with the aqueous extract and the amoebicidal sub-fraction SF9B was done for 2 h and also no significant DNA damage was observed. The respective
Micronuclei are biomarkers of genotoxic events and chromosomal instability which originate from chromosome breakage or chromosome loss during anaphase of the cell division. The formation of a micronucleus following exposure to a genotoxic compound reflects a serious and irreversible damage of the DNA of treated cells. The genotoxic effect is thereby identified by a significant increase in micronuclei or micronucleus containing cells in the treated cell population. In this assay, the aqueous extract and the sub-fraction SF9B showed no significant induction of micronucleus formation in mouse lymphoma L5178Y and HepG2 cells after 4 and 24 h of incubation compared to the negative control. Moreover, the treatment of L5178Y cells with high concentrations of the aqueous extract (2000 mg/mL) and the sub-fraction SF9B (1000 mg/mL) significantly reduced the cell proliferation as shown by the cytochalasin B proliferation index (CBPI) (Fig. 3). In addition, methyl methanesulfonate, an alkylating agent which forms DNA adducts and as result of
Fig. 2. Cell viability with fluorescein diacetate/gel red of mouse lymphoma L5178Y cells (A) and HepG2 cells (B) after 4 and 24 h treatment with the sub-fraction SF9B; methyl methanesulfonate (MMS) was used at the concentration of 200 mM and cyclophosphamide (CP) at 2.5 mM. (n means significant difference between the assay and the negative control, p r0.05).
Fig. 3. Micronucleus formation and proliferation index in mouse lymphoma L5178Y cells treated with the aqueous extract (A) and the sub-fraction SF9B (B) for 4 and 24 h; methyl methanesulfonate (MMS) was used at the concentration of 200 mM. CBPI1 and CBPI2 represent respectively the proliferation index of cells treated with each substance for 4 and 24 h before addition of cytochalasin B. (n means significant difference between the test and the negative control, p r0.05).
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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repair of those also causes DNA single and double-strand breaks, showed a significant induction of micronucleus formation which increased significantly over the treatment period. For cyclophosphamide, a prodrug which needs metabolic activation from mixed-function oxidase (cytochrome P450), also caused significant induction of micronucleus formation.
3.4. Mouse lymphoma mutation assay The mutation induction by the test substance is identified by the resistance of cells to trifluorothymidine thereby leading to the formation of colonies. Table 1 Total colony growth and cloning efficiency of L5178Y mouse lymphoma cells after 4 h treatment with the aqueous extract Surviving colonies Mutant colonies Cloning efficiency (%) Control 250 mg/mL 500 mg/mL 1000 mg/mL 2000 mg/mL 100 mM MMS
530 7 40 520 7 44 5067 52 4717 84 463 7 62 3137 52n
707 4 717 6 757 5 907 20 66 7 19 320 7 80nnn
88.0 7 6.2 86.6 7 7.3 84.17 8.4 78.4 7 13.9 77.17 10.4 52.2 7 8.6n
Each value represents the mean7 standard deviation within three independent experiments (Student-Newman-Keuls t- test. nmeans significant difference between the assay and the control, p r0.05). MMS represents methyl methanesulfonate.
Table 2 Total colony growth and cloning efficiency of L5178Y mouse lymphoma cells after 4 h treatment with the amoebicide sub-fraction SF9B. Surviving colonies Mutant colonies Cloning efficiency (%) Control 125 mg/mL 250 mg/mL 500 mg/mL 1000 mg/mL 100 mM MMS
451 727 407 711 369 740 239 727n 115 710n 258 716
1127 29 1137 42 987 23 1257 25 64 7 20n 365 7 69nnn
75.2 7 4.5 67.9 7 1.8 61.6 7 6.7 39.9 7 4.5n 19.2 7 1.6nnn 43.0 7 2.7n
Each value represents the mean7 standard deviation within three independent experiments.(Student-Newman-Keuls t- test: nmeans significant difference between the assay and the control, p r0.05). MMS represents methyl methanesulfonate.
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3.4.1. Short term treatment The number of colonies enumerated after short term (4 h) treatment with the aqueous extract and the sub-fraction SF9B are represented respectively in Tables 1 and 2. There was no significant variation in the number of surviving and mutant colonies between the negative control and the tested concentrations of the aqueous extract. Also, the cloning efficiency did not show any significant difference. On the contrary, treatment of cells with the sub-fraction SF9B causes a significant decrease in surviving colonies from 250 mg/mL and in mutant colonies at 1000 mg/mL. This effect on the surviving colonies is directly correlated to a significant decrease in the cloning efficiency. Treatment with methyl methanesulfonate used as positive control, resulted in a significant decrease in surviving colonies and a significant increase in mutant colonies which is three times higher than the negative control. The growth of surviving colonies and TFT-resistant colonies on the semi-solid agar medium containing nutrients was used for the evaluation of cytotoxicity and the mutation frequency of each substance. This result confirmed the non-cytotoxic effect of the aqueous extract on mouse lymphoma L5178Y cells according to the viability which remains above 90% up to the concentration of 2000 mg/mL. Moreover, the aqueous extract did not affect the mutation frequency whose difference is not significant compared to the negative control (Fig. 4A). However, there was a significant increase in the mutation frequency from 500 mg/mL of sub-fraction SF9B (Fig. 4B). This was not due to an increase in the absolute number of mutant colonies which remained non-significant up to 500 mg/mL, and subsequently decreases significantly at 1000 mg/ mL as compared to the negative control (see Table 2). The increase in the mutation frequency (number of mutants/million viable cells) is due to the cytotoxicity of the sub-fraction SF9B which left less than 50% of viable cells able to form colonies at 500 mg/mL and less than 20% at 1000 mg/mL. The positive control methyl methanesulfonate induced a significant increase of the mutation frequency correlated with significant increase of mutant colonies.
3.4.2. Long term treatment The count of surviving and mutant colonies after long term (24 h) of treatment of mouse lymphoma cells with the aqueous extract and sub-fraction SF9B is indicated respectively in Tables 3 and 4. It is shown from these results that the aqueous extract did
Fig. 4. Mutation frequency (MF) and viability against the concentration of the aqueous extract (A) and the sub-fraction SF9B (B) during a short-term treatment (4 h) of mouse lymphoma L5178Y cells; methyl methanesulfonate (MMS) was used at the concentration of 100 mM. (n significant difference between the test and the negative control, p r 0.05).
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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not cause significant variation in surviving and mutant colonies while the sub-fraction SF9B caused significant decrease in surviving and mutant colonies from 500 mg/mL. Mutation frequency and viability were determined from the number of surviving and mutant colonies and these results are resumed in Fig. 5. The aqueous extract did not lead to a significant difference in the mutation frequency and viability (Fig. 5A). The sub-fraction SF9B was significantly more cytotoxic from 500 mg/ mL with a viability of less than 20% of the control concerning surviving colonies (Fig. 5B). Due to high cytotoxic effect of the subfraction SF9B at 1000 mg/mL, very little or no cell growth was observed either with or without TFT preventing the calculation of the mutation frequency.
Table 3 Total colony growth and cloning efficiency of L5178Y mouse lymphoma cells after 24 h treatment with the aqueous extract
Control 250 mg/mL 500 mg/mL 1000 mg/mL 2000 mg/mL 50 mM MMS
Surviving colonies
Mutant colonies
Cloning efficiency (%)
5377 61 520 7 59 5747 20 4797 55 548 7 100 3367 35n
927 16 967 15 1077 13 917 28 1027 4 3007 13n
89.5 7 9.9 86.6 7 9.7 95.6 7 3.2 79.8 7 9.1 91.3 7 16.7 55.8 7 5.8n
Each value represents the mean7 standard deviation within three independent experiments. (Student-Newman-Keuls t- test: nmeans significant difference between the assay and the control, p r0.05). MMS represents methyl methanesulfonate.
Table 4 Total colony growth and cloning efficiency of L5178Y mouse lymphoma cells after 24 h treatment with the amoebicide sub-fraction SF9B.
Control 125 mg/mL 250 mg/mL 500 mg/mL 1000 mg/mL 50 mM MMS
Surviving colonies
Mutant colonies
Cloning efficiency (%)
4737 118 4187 139 3757 80 777 19nn 17 1nnn 287 7 42n
63 718 71 718 79 716 24 77nn 1 72nnn 277 731n
78.8 7 19.7 69.7 7 23.2 62.5 7 13.5 12.7 7 3.3nn 0.17 0.2nnn 47.8 7 6.9n
Each value represents the mean7 standard deviation within three independent experiments. (Student-Newman-Keuls t- test: nmeans significant difference between the assay and the control, p r0.05). MMS represents methyl methanesulfonate.
4. Discussion Selective toxicity is the key to all chemotherapy and the goal of any chemotherapeutic treatment is to selectively attenuate or destroy pathogenic micro-organisms or cells with minimal side effects to the host (Wink, 2012). In fact, cell exposure to chemical agents or drugs can lead directly or indirectly to DNA damage. Generally, such damage may result in mutagenesis and initiation of carcinogenesis, thus justifying a potential impact on human genome. This is a real concern in the process of acceptance and validation of substances for pharmaceutical use. In our experiment which aims to assess the cytotoxicity and the genotoxicity of the aqueous extract and the sub-fraction SF9B, mouse lymphoma L5178Y cells were used to evaluate the direct toxicity of these substances while human hepatocellular carcinoma HepG2 cells and primary hepatic cells implied in testing their indirect toxicity after metabolic activation. Actually, mouse lymphoma L5178Y cells are often used in routine genotoxicity tests (Stopper et al., 2000; Schmitt et al., 2003). Moreover, HepG2 cells and primary hepatic cells are used in the assessment of the toxicity of xenobiotics due to the presence of cytochrome P450 and Phase II enzymes with metabolic activity (Westerink and Schoonen, 2007,Guo et al., 2011). The genotoxicity testing was performed through the comet and the micronucleus assays. The comet assay reflects immediate and transitory response of DNA damage which may be repaired (Collins, 2004; Shaposhnikov et al., 2008). It is often used in combination with the micronucleus assay which represents an irreversible damage not subjected to any repair mechanism (Kirsch-Volders et al., 2003). Micronuclei represent chromosomal fragments or whole chromosomes similar to the main nucleus and easily observable in cells during interphase (Fenech et al., 2011). The combination of these two markers of genotoxicity is justified because they reflect different levels of genotoxicity with different consequences. We noted no significant cytotoxicity of the aqueous extract while the sub-fraction SF9B was significantly cytotoxic at high concentrations (500 and 1000 mg/mL). In addition, no immediate or permanent DNA damage was observed even at concentrations of the sub-fraction SF9B which showed significant cytotoxicity to different cell lines. The amoebicidal sub-fraction (SF9B) at concentrations up to 1000 mg/mL caused a high cytotoxicity and a slight reduction in micronucleus formation was observed because micronuclei are usually more expressed in cells which complete
Fig. 5. Mutation frequency (MF) and viability against the concentration of the aqueous extract (A) and the sub-fraction SF9B (B) during a long-term treatment (24 h) of mouse lymphoma L5178Y cells; methyl methanesulfonate (MMS) was used at the concentration of 50 mM. (n significant difference between the test and the negative control, p r0.05).
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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nuclear division. In any case, these high concentrations of the subfraction SF9B would not be used in therapy since in our previous report this sub-fraction exhibited significant anti-amoebic activity with an efficient concentration (EC50) of 2.75 mg/mL (Mfotie et al., 2014). In order to further investigate the in vitro toxicity of the aqueous extract and the sub-fraction SF9B, we assessed gene mutation induction in L5178Y mouse lymphoma cells which detects mutagenic effect at the thymidine kinase locus by measuring TFTresistant colonies (Lloyd and Kidd, 2012). This assay involves the exposure of cells to a short term treatment with or without a metabolic activation system and a long term treatment (Cole et al., 1999). Since the metabolically competent primary cells and HepG2 cells did not show any difference in their reaction in the comet assay and the micronucleus assay compared to L5178Y cells, the mutation assay was only performed in the absence of an exogenous metabolic activation system. The aqueous extract did not cause any gene mutation. With the sub-fraction SF9B, under conditions of marked toxicity at the two highest concentrations (500 and 1000 mg/mL), an elevation of the mutation frequency occurred. However, the absolute number of mutant colonies was not elevated, and it must be considered that the calculation of the mutation frequency, which integrates the cloning efficiency may be misleading under conditions of high toxicity. Therefore, although the induction of gene mutation at concentrations of 500 mg/mL of the sub-fraction SF9B cannot be excluded at this point, the data can also not be considered proof for a true mutagenic property of this sub-fraction. As discussed above, this concentration is almost 200-fold higher than the EC50 for antiamoebic activity and therefore highly unlikely to be of human therapeutic relevance. 5. Conclusion In summary, the aqueous extract of Codiaeum variegatum is not cytotoxic and the amoebicidal sub-fraction SF9B is significantly cytotoxic when tested at concentrations higher than 500 mg/mL. Both extracts are neither genotoxic on non-competent or metabolic competent cell lines, nor mutagenic in mouse lymphoma mutation assay at non-toxic or moderately toxic concentrations. Thus, they could be safely used at lower doses for medicinal purpose. Besides, these results support the safe usage of this cultivar of Codiaeum variegatum in the traditional health-care system and will be further investigated through in vivo studies in order to promote its application in drug development. Acknowledgments Authors want to thank Christin Thiel for her technical assistance during experimental design. We are also grateful to acknowledge the German Academic Exchange Service (DAAD) for financial assistance to Emmanuel Mfotie Njoya through the PhD sandwich program. References Akaneme, F.I., Amaefule, C.C., 2012. Evaluation of the cytotoxicity and genotoxicity of aqueous leaf extracts of Azadirachta indica A. Juss using the Allium test. Journal of Medicinal Plants Research 6, 3898–3907.
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Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction Emmanuel Mfotie Njoya a,b,n, Paul F. Moundipa a, Helga Stopper b a b
Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 11 March 2014 Received in revised form 27 May 2014 Accepted 15 June 2014
Ethnopharmacological relevance: Codiaeum variegatum, grown in many varieties, has been widely used as a houseplant based on its brightly decorative foliage. In addition, a variety of this plant has been used for a long time against bloody diarrhea by the local population in Cameroon. Aim of the study: In our previous study, the aqueous extract of this plant and an isolated sub-fraction exhibited significant anti-amoebic activity on axenic culture of Entamoeba histolytica. Due to the medicinal value of these extracts, we promptly initiated to investigate their genotoxic and mutagenic potential in order to assure their safe and rationale usage in traditional healthcare system. Material and methods: Both extracts were incubated with L5178Y mouse lymphoma cells, primary hepatic cells and HepG2 human hepatocellular carcinoma cells and their genotoxicity and mutagenicity were evaluated by quantifying DNA damage and chromosomal aberrations through comet assay, micronucleus assay and mouse lymphoma mutation assay. Results: The aqueous extract of Codiaeum variegatum is not cytotoxic up to 2000 mg/mL while the amoebicidal fraction is significantly cytotoxic (r 40–55%) on L5178Y mouse lymphoma and HepG2 cells at concentrations higher than 500 mg/mL. Besides, no significant DNA damage and induction of micronucleus formation were identified at concentrations up to 2000 mg/mL. Moreover, the mutagenic potential of these extracts after short (4 h) and long term (24 h) treatment, revealed no significant gene mutation induction. Conclusion: The aqueous extract of Codiaeum variegatum and the amoebicidal fraction SF9B are neither genotoxic on non-competent or metabolic competent cell lines, nor mutagenic in mouse lymphoma mutation assay and therefore they could be safely used at lower doses for medicinal purpose. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Codiaeum variegatum (Euphorbiaceae) Aqueous extract Amoebicidal sub-fraction SF9B Cytotoxicity Genotoxicity Mutagenicity Safety
1. Introduction Medicinal plants are important because they produce a large variety of metabolites which are known to possess therapeutic importance for maintaining human health and improving the well-being, thus justifying their use in traditional medicine system. However, many adverse effects such as genotoxicity, mutagenicity and carcinogenicity of medicinal plants have been published in various medical reviews (Soliman, 2001; Konan et al., 2007; Sowemimo et al., 2007; Akaneme and Amaefule, 2012). Codiaeum variegatum, also referred to as “garden croton”, belongs to the family of Euphorbiaceae and is a tropical-origin ornamental shrub with diverse beautiful and attractive foliage. In addition to its decorative usage as an indoor plant, Codiaeum variegatum is n Corresponding author at: Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812 Yaoundé, Cameroon. Tel.: þ 237 75526943. E-mail addresses: [email protected], [email protected] (E. Mfotie Njoya).
also widely used for its medicinal properties. Freeze-dried leaf decoction of Codiaeum variegatum is taken as tea by Filipinos and drinking crushed leaves cures diarrhea (Saffoon et al., 2010). The root and bark are used against syphilis, constipation, stomachache, loss of appetite and dysuria. From the fresh latex, a cyanoglucoside was isolated and displayed virucidal activity against influenza A virus without impairment of haemagglutination properties (Forero et al., 2008). An evaluation of tumor promoter contents by high-performance liquid chromatography coupled with tumorpromoting activity assessment showed that cultivars of Codiaeum variegatum were devoid of tumor-promoting compounds (Vogg et al., 1999). Despite the beneficial effects of this plant, it has been reported that the latex or sap produced from the bark, root and leaves is toxic and can cause skin eczema in humans after repeated exposure (Hausen and Shulz, 1977; Ogunwenmo et al., 2007). There are more than 300 cultivars known as mutants or hybrids and grouped into nine species based on their leaf morphology (Deng et al., 2010). The cultivar used in Cameroon belongs to the royal-like species with green oak leaves and white spots.
http://dx.doi.org/10.1016/j.jep.2014.06.038 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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This plant is widely used by the local population against intestinal disorders such as bloody and watery diarrhea (Moundipa et al., 2005). The aqueous extract from the leaves of this plant and an isolated sub-fraction were identified with relevant antiamoebic efficacy against the intestinal parasite Entamoeba histolytica (Mfotie et al., 2014). The rational and safe usage of Codiaeum variegatum is absolutely important to gain benefit from its medicinal value. Until now, no study has been carried out to evaluate the genotoxicity and the mutagenicity of this medicinal plant. Nowadays, detection of chromosome or DNA damage represents an important tool used for prioritizing compounds early in the drug development process. Therefore, the in vitro toxicity of the aqueous extract from the leaves of Codiaeum variegatum and its amoebicidal sub-fraction was assessed by evaluating the cytotoxicity and quantifying DNA damage and chromosomal alterations respectively through comet and micronucleus assays. Moreover, the evaluation of the mutagenic potential of these substances was carried out by the mouse lymphoma (TK) mutation assay after short and long term treatment.
2. Material and methods 2.1. Preparation of the aqueous extract and sub-fraction isolation Freshly leaves of Codiaeum variegatum var. mollucanum were collected in Yaoundé (Cameroon) and were thoroughly washed, rinsed with distilled water, dried at room temperature and grounded. The powder obtained (100 g) was prepared with 1 L of distilled water for 1 h. After cooling, the mixture was filtered with Whatman No.1 filter paper and the filtrate was dried by lyophilisation to obtain a residue which constitutes the aqueous extract. From the aqueous extract, the amoebicidal sub-fraction named SF9B was obtained as earlier described (Mfotie et al., 2014). 2.2. Chemical profile of the tested substances Qualitative phytochemical screening was done as described by (Trease and Evans, 1989) to obtain the chemical composition of the aqueous extract of Codiaeum variegatum. Some major groups of compounds such as alkaloids, anthranoids, sugar, leuco-anthocyanins, coumarins, tannins, saponins, polyphenols, flavonoids, sterols and triterpenes were analyzed. It resulted from this analysis that the aqueous extract of Codiaeum variegatum contains polyphenols, tannins, coumarins and traces of sugar. The fingerprint of the sub-fraction SF9B was defined in this study using analytical high-performance liquid chromatography (HPLC) by injecting 20 ml of the sub-fraction dissolved in methanol through a silica gel column under an inverse stationary phase of KROMASILs C18. The solvent system used is composed of H2O (35%)/CH3CN (65%) flowing at a debit of 0.8 mL/min. The detection was done under ultra-violet light at λ ¼254 nm using a GILSON 170 Diode Array Detectors. The HPLC-UV fingerprint of the subfraction SF9B is shown in Fig. 1.
Fig. 1. HPLC-UV fingerprint analysis of the sub-fraction SF9B. Elution under isocratic condition with the solvent system H2O (35%)/CH3CN (65%) using an inverse stationary phase of KROMASILs C18; debit: 0.8 mL/min; detection: GILSON 170 Diode Array Detectors at λ ¼ 254 nm.
10% (v/v) fetal calf serum, 1% (v/v) L-glutamine, 1% (v/v) of sodiumpyruvate (100 mM) and 0.4% (v/v) antibiotics (50 U/mL penicillin and 50 mg/mL streptomycin). Primary cells were isolated from freshly prepared rat liver (ZUC-Leprfa) previously washed with ice-cold phosphate buffered saline (PBS). The liver was transferred into 3 mL of an ice-cold HepG2 culture medium and rapidly cut on ice into small pieces. The primary cells were collected by filtration through a filter mesh pore size of 100 mm (Heidelberg, Germany), centrifuged at 100g for 5 min at 4 1C and finally suspended in 5 mL of HepG2 culture medium. The viability and the number of cells were determined using trypan blue exclusion technique on the Neubauer chamber. 2.4. Treatment of cells Primary rat hepatic cells, HepG2 and L5178Y cell lines were inoculated at a density of 6 105 cells in 3 mL culture medium on 6-well microtiter plates. After seeding, cells were treated with increasing concentrations of the aqueous extract and the subfraction SF9B dissolved either in phosphate buffered saline (PBS) or in dimethylsulfoxide (DMSO). The maximum concentrations of the aqueous extract and the sub-fraction SF9B used in these experiments were determined based on their solubility in different solvents (sterile PBS and DMSO) with the highest tested concentration showing insolubility. The aqueous extract and the sub-fraction were used in culture medium up to a concentration of 2000 and 1000 mg/mL (w/v) respectively. Methyl methanesulfonate and cyclophosphamide (Sigma-Aldrich, Steinheim, Germany) all dissolved in DMSO were used as positive controls respectively on L5178Y cells and HepG2 or primary cells. During each experiment, the maximal concentration of DMSO in the medium did not exceed 1%. After incubation for 4 or 24 h at 37 1C with 5% CO2, cells were collected by centrifugation in fresh culture medium for the following assays. 2.5. Cytotoxicity assay
2.3. Cells and culture media The L5178Y mouse lymphoma cells (obtained from Dr. W. J. Caspary, NIEHS/USA) were cultured in suspension at 37 1C with 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) heatinactivated horse serum, 1% (v/v) L-glutamine (200 mM), 1% (v/v) sodium-pyruvate (100 mM) and 0.4% (v/v) antibiotics (50 U/mL penicillin and 50 mg/mL streptomycin). The HepG2 human hepatocellular carcinoma cells (ATCC HB8065) are adherent cells which multiply in monolayer at 37 1C with 5% CO2 in the DMEM low glucose (1 g/l) supplemented with
Cytotoxicity is considered as the potential of a compound to induce cell injury or death. In this assay, the cell viability was assessed by using the fluorescein diacetate/gel red technique. The staining solution contained 30 mg/mL of fluorescein diacetate and gel red (1:1000) dissolved in PBS. Treated cells in suspension (70 ml) were mixed with 30 ml of the staining solution and 20 ml of this mixture was applied on the slide and covered with a cover slip (21 26 mm2). The ratio of green and red cells in a total of 3 100 cells was counted at a 200-fold magnification under a microscope using the FITC filter. Viable cells stain green because their active
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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esterases cleave the acetate groups from fluorescein diacetate, which then starts to fluoresce. Dead cells appear red because gel red can cross their defective membrane and intercalates in the DNA. 2.6. Comet assay The endpoint of this assay is to detect single and double strand breaks as well as alkali-labile lesions. In this assay, L5178Y, HepG2 and primary cells were treated as described previously. Treated cells were harvested and 20 ml of this suspension were mixed with 180 ml of 0.5% low-melting point agarose and 45 ml of this mixture was deposited on slides covered previously with 1.5% high melting point agarose. The slides were incubated in lysis solution (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, and 10 g/l Sodium N-lauroylsarcosine adjusted to pH 10 with NaOH 5 M) containing 1% Triton X-100 and 10% DMSO for at least 1 h at 4 1C. Thereafter, the slides were washed with distilled water and then placed in the electrophoresis chamber and covered with the electrophoresis solution (5 M NaOH and 0.2 M EDTA [pH413.0]) for 20 min. The electrophoresis was conducted for 20 min at 25 V and 300 mA adjusted with the electrophoresis solution. The slides were neutralized in 0.4 M Tris buffer (pH 7.5), followed by cell fixation in cold methanol for 5 min at 20 1C. The slides were then left at 37 1C in an incubator to dry for 10 min and stored at room temperature afterward. Before evaluation, 20 ml of gel red (1:100)/DABCO (1/4) solution were added to each slide and covered with cover slips (21 26 mm2). Images of 100 randomly selected cells at the middle of the slide (50 per replicate slide) for each sample were analyzed with a fluorescence microscope (Labophot 2; Nikon GmbH, Germany) at 200-fold magnification using image analysis software (Komet 5; BFI Optilas, Germany). The percentage of DNA in the tail was used to quantify DNA damage and the average determined within three independent experiments. 2.7. Micronucleus frequency assay In this experiment, L5178Y and HepG2 cells were treated as previously described. On the one hand, treated L5178Y cells were collected and incubated in fresh medium for 20 h at 37 1C with 5% CO2 with addition of the cytokinesis-inhibitor cytochalasin B (final concentration: 3 mg/mL). Micronucleus analysis was limited to binucleated cells which have divided once since substance exposure, and a proliferation index was calculated for assessment of cytostatic effects (see the formula below). On the other hand, treated HepG2 cells were incubated in fresh medium without the addition of cytochalasin B for 40 h. HepG2 cells did not tolerate cytochalasin B treatment well. Next, cells (both cell lines) were collected, washed with PBS, brought onto slides by cytospin centrifugation and fixed by incubation in methanol at 20 1C for at least 2 h. Cells were stained in the dark for 7 min with a gel green (1:100) solution and slides were washed with PBS, and mounted with diazabicyclo-octan (DABCO) for microscopy. Micronucleus count was performed at 400-fold magnification under a microscope using a FITC filter. The number of mononucleated (MN), binucleated (BN) and multinucleated (MuN) cells was enumerated as well as the frequency of binucleated cells or mononucleated cells (for HepG2 cells) containing micronuclei in 1000 cells per slide. For each experiment, two slides were analyzed and the average of micronucleus formation was calculated within three independent experiments. CBPI
¼
ð1 MNÞ þ ð2 BNÞ þ ð3 MuNÞ MNþ BN þ MuN
CBPI: Cytochalasin B Proliferation Index; MN: mononucleated cells; BN: binucleated cells and MuN: multinucleated cells.
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2.8. Mammalian mutation assay in L5178Y (TK þ / ) mouse lymphoma cells In this assay, mouse lymphoma L5178Y cells were exposed to a short and a long term treatment as recommended by the International Conference on Harmonization (ICH) and the Organization for Economic Co-operation and Development (OECD) (Clive and Spector, 1975; Cole et al., 1999; Clements, 2000). Before any experiment, an exponentially growing culture of L5178Y mouse lymphoma cells was first cleansed from spontaneous mutations by incubation at 37 1C with 5% CO2 for 24 h in culture medium containing thymidine (9 mg/mL), hypoxanthine (15 mg/mL), methotrexate (0.3 mg/mL) and glycine (22.5 mg/mL). These cells were further incubated for 24 h in the same medium without methotrexate. This treatment thereby permits to reduce the background level of spontaneous TK / cells. After collection of cells by centrifugation at 80g for 10 min, 106 cells were transferred in culture flasks containing 5 mL of fresh culture medium. These cells were submitted to short (4 h) and long (24 h) term treatment with at least four concentrations of the aqueous extract (dissolved in PBS) and the sub-fraction SF9B (dissolved in DMSO). Methyl methanesulfonate was used as the positive control at the concentrations of 100 and 50 mM respectively after 4 and 24 h treatment. Treated cells were washed with fresh medium and 0.5 106 cells were added in 43 mL of culture medium containing 4.5 mL of sterilized granulated agar 3.5% (Gibcon BRL, Life Technologies, Scotland). After homogenization, the mixture was plated onto 2 culture dishes (100 20 mm) and these culture dishes were allowed to solidify at room temperature. After 40 h of phenotypic expression, selection of mutant cells was carried out by addition of an overlayer of a semisolid medium containing trifluorothymidine (final concentration: 3 mg/mL). Cloning efficiency and viability were determined by addition of 600 cells in 90 mL of culture medium containing 9 mL of sterilized granulated agar 3.5% and plated onto 3 culture dishes. All culture dishes were incubated at 37 1C with 5% CO2 for colony growth. Colonies were counted manually both for the evaluation of the cloning efficiency and viability 9 days after platting and for the mutation frequency (TFTresistant colonies) 10 days after TFT addition. Based on the colony count, the viability and the cloning efficiency are evaluated by comparing the total growth of surviving cells out of 600 cells seeded in the test against the negative control. The mutation frequency is obtained from the number of TFT-resistant cells divided by the cloning efficiency per million cells used in the experiment. 2.9. Statistical analysis All assays were performed in three independent experiments and the results are presented as mean7SD (standard deviation) values. Statistical analysis was carried out with the GraphPad Instat 3.0 software and student's t-test was used to determine P-values for the differences observed between test substances and negative control. Results were considered significantly different when Pr 0.05.
3. Results 3.1. Viability of treated cells with fluorescein diacetate/gel red The cytotoxic effect of the aqueous extract and the sub-fraction were evaluated in mouse lymphoma L5178Y and HepG2 cells after 4 and 24 h of treatment. Results showed that the aqueous extract did not cause significant cytotoxicity in both cell lines up to 2000 mg/mL, the concentration at which this extract become insoluble in the culture medium. The sub-fraction SF9B did not
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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cause reduction of cell viability up to the concentration of 250 mg/mL. When tested above this concentration, the subfraction SF9B was significantly cytotoxic with 10–55% reduction in cell viability (Fig. 2).
positive controls methyl methanesulfonate and cyclophosphamide induced significant elevations of DNA damage in all cases.
3.3. Induction of micronucleus formation 3.2. DNA damage in treated cells The comet assay was used to evaluate DNA damage in treated cells. Results obtained from comet assay reveal usually an early or immediate response which occurs in DNA of a cell when treated with a genotoxic agent. In fact, this DNA damage may be transitory and susceptible to repair. Thus, the effect obtained during each of our experiment represents the interaction between the generation of DNA damage and repair mechanisms. Quantification of the percentage DNA in tail showed no significant DNA damage in mouse lymphoma L5178Y cells and HepG2 cells after 4 and 24 h treatment with concentrations up to 2000 mg/mL for the aqueous extract and 1000 mg/mL for the sub-fraction SF9B. On primary cells isolated from fresh rat liver, the treatment with the aqueous extract and the amoebicidal sub-fraction SF9B was done for 2 h and also no significant DNA damage was observed. The respective
Micronuclei are biomarkers of genotoxic events and chromosomal instability which originate from chromosome breakage or chromosome loss during anaphase of the cell division. The formation of a micronucleus following exposure to a genotoxic compound reflects a serious and irreversible damage of the DNA of treated cells. The genotoxic effect is thereby identified by a significant increase in micronuclei or micronucleus containing cells in the treated cell population. In this assay, the aqueous extract and the sub-fraction SF9B showed no significant induction of micronucleus formation in mouse lymphoma L5178Y and HepG2 cells after 4 and 24 h of incubation compared to the negative control. Moreover, the treatment of L5178Y cells with high concentrations of the aqueous extract (2000 mg/mL) and the sub-fraction SF9B (1000 mg/mL) significantly reduced the cell proliferation as shown by the cytochalasin B proliferation index (CBPI) (Fig. 3). In addition, methyl methanesulfonate, an alkylating agent which forms DNA adducts and as result of
Fig. 2. Cell viability with fluorescein diacetate/gel red of mouse lymphoma L5178Y cells (A) and HepG2 cells (B) after 4 and 24 h treatment with the sub-fraction SF9B; methyl methanesulfonate (MMS) was used at the concentration of 200 mM and cyclophosphamide (CP) at 2.5 mM. (n means significant difference between the assay and the negative control, p r0.05).
Fig. 3. Micronucleus formation and proliferation index in mouse lymphoma L5178Y cells treated with the aqueous extract (A) and the sub-fraction SF9B (B) for 4 and 24 h; methyl methanesulfonate (MMS) was used at the concentration of 200 mM. CBPI1 and CBPI2 represent respectively the proliferation index of cells treated with each substance for 4 and 24 h before addition of cytochalasin B. (n means significant difference between the test and the negative control, p r0.05).
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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repair of those also causes DNA single and double-strand breaks, showed a significant induction of micronucleus formation which increased significantly over the treatment period. For cyclophosphamide, a prodrug which needs metabolic activation from mixed-function oxidase (cytochrome P450), also caused significant induction of micronucleus formation.
3.4. Mouse lymphoma mutation assay The mutation induction by the test substance is identified by the resistance of cells to trifluorothymidine thereby leading to the formation of colonies. Table 1 Total colony growth and cloning efficiency of L5178Y mouse lymphoma cells after 4 h treatment with the aqueous extract Surviving colonies Mutant colonies Cloning efficiency (%) Control 250 mg/mL 500 mg/mL 1000 mg/mL 2000 mg/mL 100 mM MMS
530 7 40 520 7 44 5067 52 4717 84 463 7 62 3137 52n
707 4 717 6 757 5 907 20 66 7 19 320 7 80nnn
88.0 7 6.2 86.6 7 7.3 84.17 8.4 78.4 7 13.9 77.17 10.4 52.2 7 8.6n
Each value represents the mean7 standard deviation within three independent experiments (Student-Newman-Keuls t- test. nmeans significant difference between the assay and the control, p r0.05). MMS represents methyl methanesulfonate.
Table 2 Total colony growth and cloning efficiency of L5178Y mouse lymphoma cells after 4 h treatment with the amoebicide sub-fraction SF9B. Surviving colonies Mutant colonies Cloning efficiency (%) Control 125 mg/mL 250 mg/mL 500 mg/mL 1000 mg/mL 100 mM MMS
451 727 407 711 369 740 239 727n 115 710n 258 716
1127 29 1137 42 987 23 1257 25 64 7 20n 365 7 69nnn
75.2 7 4.5 67.9 7 1.8 61.6 7 6.7 39.9 7 4.5n 19.2 7 1.6nnn 43.0 7 2.7n
Each value represents the mean7 standard deviation within three independent experiments.(Student-Newman-Keuls t- test: nmeans significant difference between the assay and the control, p r0.05). MMS represents methyl methanesulfonate.
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3.4.1. Short term treatment The number of colonies enumerated after short term (4 h) treatment with the aqueous extract and the sub-fraction SF9B are represented respectively in Tables 1 and 2. There was no significant variation in the number of surviving and mutant colonies between the negative control and the tested concentrations of the aqueous extract. Also, the cloning efficiency did not show any significant difference. On the contrary, treatment of cells with the sub-fraction SF9B causes a significant decrease in surviving colonies from 250 mg/mL and in mutant colonies at 1000 mg/mL. This effect on the surviving colonies is directly correlated to a significant decrease in the cloning efficiency. Treatment with methyl methanesulfonate used as positive control, resulted in a significant decrease in surviving colonies and a significant increase in mutant colonies which is three times higher than the negative control. The growth of surviving colonies and TFT-resistant colonies on the semi-solid agar medium containing nutrients was used for the evaluation of cytotoxicity and the mutation frequency of each substance. This result confirmed the non-cytotoxic effect of the aqueous extract on mouse lymphoma L5178Y cells according to the viability which remains above 90% up to the concentration of 2000 mg/mL. Moreover, the aqueous extract did not affect the mutation frequency whose difference is not significant compared to the negative control (Fig. 4A). However, there was a significant increase in the mutation frequency from 500 mg/mL of sub-fraction SF9B (Fig. 4B). This was not due to an increase in the absolute number of mutant colonies which remained non-significant up to 500 mg/mL, and subsequently decreases significantly at 1000 mg/ mL as compared to the negative control (see Table 2). The increase in the mutation frequency (number of mutants/million viable cells) is due to the cytotoxicity of the sub-fraction SF9B which left less than 50% of viable cells able to form colonies at 500 mg/mL and less than 20% at 1000 mg/mL. The positive control methyl methanesulfonate induced a significant increase of the mutation frequency correlated with significant increase of mutant colonies.
3.4.2. Long term treatment The count of surviving and mutant colonies after long term (24 h) of treatment of mouse lymphoma cells with the aqueous extract and sub-fraction SF9B is indicated respectively in Tables 3 and 4. It is shown from these results that the aqueous extract did
Fig. 4. Mutation frequency (MF) and viability against the concentration of the aqueous extract (A) and the sub-fraction SF9B (B) during a short-term treatment (4 h) of mouse lymphoma L5178Y cells; methyl methanesulfonate (MMS) was used at the concentration of 100 mM. (n significant difference between the test and the negative control, p r 0.05).
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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not cause significant variation in surviving and mutant colonies while the sub-fraction SF9B caused significant decrease in surviving and mutant colonies from 500 mg/mL. Mutation frequency and viability were determined from the number of surviving and mutant colonies and these results are resumed in Fig. 5. The aqueous extract did not lead to a significant difference in the mutation frequency and viability (Fig. 5A). The sub-fraction SF9B was significantly more cytotoxic from 500 mg/ mL with a viability of less than 20% of the control concerning surviving colonies (Fig. 5B). Due to high cytotoxic effect of the subfraction SF9B at 1000 mg/mL, very little or no cell growth was observed either with or without TFT preventing the calculation of the mutation frequency.
Table 3 Total colony growth and cloning efficiency of L5178Y mouse lymphoma cells after 24 h treatment with the aqueous extract
Control 250 mg/mL 500 mg/mL 1000 mg/mL 2000 mg/mL 50 mM MMS
Surviving colonies
Mutant colonies
Cloning efficiency (%)
5377 61 520 7 59 5747 20 4797 55 548 7 100 3367 35n
927 16 967 15 1077 13 917 28 1027 4 3007 13n
89.5 7 9.9 86.6 7 9.7 95.6 7 3.2 79.8 7 9.1 91.3 7 16.7 55.8 7 5.8n
Each value represents the mean7 standard deviation within three independent experiments. (Student-Newman-Keuls t- test: nmeans significant difference between the assay and the control, p r0.05). MMS represents methyl methanesulfonate.
Table 4 Total colony growth and cloning efficiency of L5178Y mouse lymphoma cells after 24 h treatment with the amoebicide sub-fraction SF9B.
Control 125 mg/mL 250 mg/mL 500 mg/mL 1000 mg/mL 50 mM MMS
Surviving colonies
Mutant colonies
Cloning efficiency (%)
4737 118 4187 139 3757 80 777 19nn 17 1nnn 287 7 42n
63 718 71 718 79 716 24 77nn 1 72nnn 277 731n
78.8 7 19.7 69.7 7 23.2 62.5 7 13.5 12.7 7 3.3nn 0.17 0.2nnn 47.8 7 6.9n
Each value represents the mean7 standard deviation within three independent experiments. (Student-Newman-Keuls t- test: nmeans significant difference between the assay and the control, p r0.05). MMS represents methyl methanesulfonate.
4. Discussion Selective toxicity is the key to all chemotherapy and the goal of any chemotherapeutic treatment is to selectively attenuate or destroy pathogenic micro-organisms or cells with minimal side effects to the host (Wink, 2012). In fact, cell exposure to chemical agents or drugs can lead directly or indirectly to DNA damage. Generally, such damage may result in mutagenesis and initiation of carcinogenesis, thus justifying a potential impact on human genome. This is a real concern in the process of acceptance and validation of substances for pharmaceutical use. In our experiment which aims to assess the cytotoxicity and the genotoxicity of the aqueous extract and the sub-fraction SF9B, mouse lymphoma L5178Y cells were used to evaluate the direct toxicity of these substances while human hepatocellular carcinoma HepG2 cells and primary hepatic cells implied in testing their indirect toxicity after metabolic activation. Actually, mouse lymphoma L5178Y cells are often used in routine genotoxicity tests (Stopper et al., 2000; Schmitt et al., 2003). Moreover, HepG2 cells and primary hepatic cells are used in the assessment of the toxicity of xenobiotics due to the presence of cytochrome P450 and Phase II enzymes with metabolic activity (Westerink and Schoonen, 2007,Guo et al., 2011). The genotoxicity testing was performed through the comet and the micronucleus assays. The comet assay reflects immediate and transitory response of DNA damage which may be repaired (Collins, 2004; Shaposhnikov et al., 2008). It is often used in combination with the micronucleus assay which represents an irreversible damage not subjected to any repair mechanism (Kirsch-Volders et al., 2003). Micronuclei represent chromosomal fragments or whole chromosomes similar to the main nucleus and easily observable in cells during interphase (Fenech et al., 2011). The combination of these two markers of genotoxicity is justified because they reflect different levels of genotoxicity with different consequences. We noted no significant cytotoxicity of the aqueous extract while the sub-fraction SF9B was significantly cytotoxic at high concentrations (500 and 1000 mg/mL). In addition, no immediate or permanent DNA damage was observed even at concentrations of the sub-fraction SF9B which showed significant cytotoxicity to different cell lines. The amoebicidal sub-fraction (SF9B) at concentrations up to 1000 mg/mL caused a high cytotoxicity and a slight reduction in micronucleus formation was observed because micronuclei are usually more expressed in cells which complete
Fig. 5. Mutation frequency (MF) and viability against the concentration of the aqueous extract (A) and the sub-fraction SF9B (B) during a long-term treatment (24 h) of mouse lymphoma L5178Y cells; methyl methanesulfonate (MMS) was used at the concentration of 50 mM. (n significant difference between the test and the negative control, p r0.05).
Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
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nuclear division. In any case, these high concentrations of the subfraction SF9B would not be used in therapy since in our previous report this sub-fraction exhibited significant anti-amoebic activity with an efficient concentration (EC50) of 2.75 mg/mL (Mfotie et al., 2014). In order to further investigate the in vitro toxicity of the aqueous extract and the sub-fraction SF9B, we assessed gene mutation induction in L5178Y mouse lymphoma cells which detects mutagenic effect at the thymidine kinase locus by measuring TFTresistant colonies (Lloyd and Kidd, 2012). This assay involves the exposure of cells to a short term treatment with or without a metabolic activation system and a long term treatment (Cole et al., 1999). Since the metabolically competent primary cells and HepG2 cells did not show any difference in their reaction in the comet assay and the micronucleus assay compared to L5178Y cells, the mutation assay was only performed in the absence of an exogenous metabolic activation system. The aqueous extract did not cause any gene mutation. With the sub-fraction SF9B, under conditions of marked toxicity at the two highest concentrations (500 and 1000 mg/mL), an elevation of the mutation frequency occurred. However, the absolute number of mutant colonies was not elevated, and it must be considered that the calculation of the mutation frequency, which integrates the cloning efficiency may be misleading under conditions of high toxicity. Therefore, although the induction of gene mutation at concentrations of 500 mg/mL of the sub-fraction SF9B cannot be excluded at this point, the data can also not be considered proof for a true mutagenic property of this sub-fraction. As discussed above, this concentration is almost 200-fold higher than the EC50 for antiamoebic activity and therefore highly unlikely to be of human therapeutic relevance. 5. Conclusion In summary, the aqueous extract of Codiaeum variegatum is not cytotoxic and the amoebicidal sub-fraction SF9B is significantly cytotoxic when tested at concentrations higher than 500 mg/mL. Both extracts are neither genotoxic on non-competent or metabolic competent cell lines, nor mutagenic in mouse lymphoma mutation assay at non-toxic or moderately toxic concentrations. Thus, they could be safely used at lower doses for medicinal purpose. Besides, these results support the safe usage of this cultivar of Codiaeum variegatum in the traditional health-care system and will be further investigated through in vivo studies in order to promote its application in drug development. Acknowledgments Authors want to thank Christin Thiel for her technical assistance during experimental design. We are also grateful to acknowledge the German Academic Exchange Service (DAAD) for financial assistance to Emmanuel Mfotie Njoya through the PhD sandwich program. References Akaneme, F.I., Amaefule, C.C., 2012. Evaluation of the cytotoxicity and genotoxicity of aqueous leaf extracts of Azadirachta indica A. Juss using the Allium test. Journal of Medicinal Plants Research 6, 3898–3907.
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Please cite this article as: Mfotie Njoya, E., et al., in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.038i
