Abstract
Tiglium seed is a seed of mature Croton Tiglium Linne containing croton oils, which have been traditionally used as laxative or purgative. As it contains phorbol derivatives, we investigated the mutagenicity and tumor-promoting activity of Tiglium seed. Tiglium seed extract produced the mutagenic responses in five Salmonella typhimurium strains in Ames assay, whereas it did not alter the frequencies of chromosomal aberrations or micronuclei, indicating that it exerted the mutagenic potential, not clastogenicity. Accompanied with phosphorylation of connexin43 (Cx43) and extracellular signal-regulated kinases 1/2 (ERK1/2), Tiglium seed extract inhibited gap junctional intercellular communication (GJIC) associated with tumor-promoting potential. Importantly, these effects were blocked by a protein kinase C (PKC) inhibitor or mitogen-activated protein kinase (MAPKs) inhibitors, suggesting that Tiglium seed-induced GJIC inhibition was regulated by phosphorylation of Cx43 via PKC and MAPKs signaling. In conclusion, Tiglium seed has mutagenicity, possibly linking to tumor-promoting potential through the dysfunction of GJIC.
Graphical Abstract
Schematic representation on the inhibition of GJIC by the Tiglium seed extract in WB-F344 cells.
Many medicinal plants have been widely used as dietary adjuncts and in the treatment of a range of diseases. Although herbal remedies result in an important contribution to public health care in many countries, the medicinal plants have been known to possess various adverse effects which are related to their inherent toxicity or interactions with other herbs and drugs.Citation1−5) Thus, the evaluations for safety and toxicity, including mutagenic and clastogenic activities, are necessary for the medicinal plants to be developed as therapeutic agents.
Croton, a large genus of Euphorbiaceae, is distributed in tropical regions of South-East Asia and China. Many researchers previously reported the pharmacological effects of Croton species on several diseases, such as diabetes, gastritis, and digestive disorders.Citation4,6–9) Tiglium seed is a seed of mature Croton Tiglium Linne, which contains large amounts of croton oils. The croton oil is a source of phorbol derivatives,Citation10) in particular, 12-O-tetradecanocylphorbol-13-acetate (TPA), one of the components from the Tiglium seed, is known as a potent tumor promoter.Citation4) Thus, it is expected that the Tiglium seed extract may act like TPA with carcinogenic properties.
Gap junction is a plaque-like protein structure which forms cell-to-cell communication channel. Each channel is made up of two connexons, which are themselves each constructed out of six connexin (Cx).Citation11) They are directly connected to neighboring cells and provide a pathway for diffusion of small molecules (<1 kDa) such as ions, amino acids, nucleotides, and second messengers (e.g. Ca2+, cAMP, cGMP, and inositol triphosphate).Citation12,13) Particularly, gap junctional intercellular communication (GJIC) plays a pivotal role in regulation of cell growth, embryonic development, and maintenance of cellular homeostasis. Therefore, the dysfunction of GJIC causes loss of homeostasis and is known to be associated with carcinogenesis.Citation14−16)
The present study was designed to evaluate the mutagenic and tumor-promoting potentials of the Tiglium seed using GJIC analysis, a rapid and simple protocol to detect tumor promoters,Citation17,18) and standard assay battery for the genotoxicity, such as an Ames test, a chromosome aberration test, and a micronucleus test.
Materials and methods
Preparation of Tiglium seed extract
The extraction procedures of the Tiglium seed were performed at S&D Co., Ltd (Chuncheon, Korea). In brief, the Tiglium seeds obtained from a traditional herbal market (Seoul, Korea) were peeled from testae and ground into powder. After removing oil from the Tiglium seeds, hexane was added to the Tiglium seed and kept for 24 h at room temperature. They were filtered through filter paper and dried in the dark. After adding ethyl acetate, extracts were stored for 24 h, filtered, and dried. Distilled water was added to the dried extracts and stirred for 72 h at 4 °C. Then, they were filtered with membrane paper (0.24 μm) and concentrated to half by evaporation (Eyela Tokyo Rikakikai Co., Ltd, Tokyo, Japan). Finally, the Tiglium seed extract was frozen, dried for 24 h, and dissolved in distilled water.
Ames test
Mutagenicity was determined with five Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537, which were provided from Ministry of Food and Drug Safety (Osong, Korea), according to the Organization for Economic Cooperation and Development (OECD) guideline 471.Citation19) The bacterial strains were incubated with the Tiglium seed extract with or without S9 mix in the dark at 37 °C for 48 h. 2-nitrofluorene, sodium azide, mitomycin C, 9-aminoacridine, and 2-aminoanthracene (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. The extract was considered positive only when the number of histidine-independent (His+) revertants was double the spontaneous yield.
Chromosome aberration assay
The test was carried out in accordance with the OECD guideline 472.Citation20) Chinese hamster lung (CHL) fibroblast cells were seeded at a density of 1 × 105 cells/mL in minimum essential medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO) in 25 cm2 flask and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. After the treatment with the Tiglium seed extract in the presence or absence of S9 mix for 6 h or 24 h, the cells were washed and incubated in complete medium for 18 h. For this assay, mitomycin C and cyclophosphoamide (Sigma-Aldrich) were used as positive controls. After colcemid (0.2 μg/mL, GIBCO) was added for 2 h, the cells were treated with hypotonic solution, fixed in 3:1 methanol/glacial acetic acid, and stained with 4% Giemsa.
In vivo bone marrow micronucleus assay
The assay was conducted according to the OECD guideline 474.Citation21) Eight-weeks-old male ICR mice (Orient bio, Seongnam, Korea) were orally gavaged with the Tiglium seed extract dissolved in 1% methylcellulose daily for 4 d. Mitomycin C was used as a positive control and was administered via a single intraperitoneal injection at a dose of 2 mg/kg of body weight. After the mice were sacrificed at 24 h after the last dose of the Tiglium seed extract, femoral bone marrow cells were isolated from each mouse. The slides were stained with 5% Giemsa and evaluated for at least 1000 polychromatic erythrocytes (PCE) for the presence of micronuclei per animal. The ratio of polychromatic to all erythrocytes (normochromatic erythrocytes (NCE) and PCE) was also determined.
Cytotoxicity assay
Cytotoxicity was measured by MTT assay as described previously.Citation22) WB-F344 rat liver epithelial cells (WB-F344 cells), a non-tumorigenic diploid cell line derived from a Fischer 344 rat,Citation23) were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO), supplemented with 5% FBS and 0.5% PSN antibiotic mixture (GIBCO) at 37 °C in a 5% CO2 humidified incubator. After the cells were cultured in 96 well plates (1 × 104 cells/well) for 24 h, the Tiglium seed extract was treated up to another 24 h. MTT solution (2 mg/mL, Sigma-Aldrich) was added and incubated for 4 h. After removing the medium and adding DMSO, absorbance was measured at 540 nm using an ELISA microplate reader (Molecular devices, Sunnyvale, CA, USA).
Scrape-loading/dye transfer for GJIC
GJIC was analyzed using scrape-loading/dye transfer (SL/DT) technique as described previously.Citation24) WB-F344 cells have been known as a good model to study the effects of chemical on GJIC because they have large gap junctions and are highly coupled.Citation25) Therefore, we used WB-F344 cells to identify the effects of Tiglium seed extract on GJIC. When WB-F344 cells reached about 80–90% confluency, they were exposed to the Tiglium seed extract. The cells were washed three times with D-PBS (PBS without Ca2+ and Mg2+). Then, 0.05% lucifer yellow (Sigma-Aldrich) was added and at least six scrapes per dish were made with a steel surgical blade. After the incubation for 3 min, the cells were carefully washed with D-PBS and then fixed in 4% paraformaldehyde solution. For protein kinase C (PKC) or mitogen-activated protein kinase (MAPKs) inhibition study, the cells were pretreated with PKC inhibitor (10 μM bisindolymaleimide I (BIM I)) (Calbiochem, San Diego, CA, USA) or MAPKs inhibitors (10 μM MAPK kinase (MEK) inhibitor U0126 and 50 μM extracellular signal regulated kinases (ERK) inhibitor PD98059) (Sigma-Aldrich) for 30 min prior to the treatment with the Tiglium seed extract at 312.5 μg/mL for 1 h. Dye-transferred cell images were captured with an inverted fluorescence microscope IX71 (Olympus, Okaya, Japan). The number of communicating cells was counted in five areas per scrape line.
Immunofluorescence staining of Cx43
Immunofluorescence staining was performed as previously described.Citation26,27) After the treatment of cells with the Tiglium seed extract for 1 h, WB-F344 cells were washed with D-PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. The cells were further washed and blocked with 3% bovine serum albumin (Amresco, Solon, OH, USA) in D-PBS containing 0.05% Tween 20 for 1 h. The cells were incubated with rabbit polyclonal anti-Cx43 antibody (Sigma-Aldrich) at 4 °C for overnight and then incubated with an Alexa fluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. After washing, the slides were mounted in vectashield with DAPI (Vector Laboratory, Burlingame, CA, USA) and observed using an inverted fluorescence microscope.
Western blot analysis of Cx43 and p-ERK1/2
WB-F344 cells were harvested in RIPA buffer (Millipore, Bedford, MA, USA) containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor (Roche, Indianapolis, IN, USA). The protein contents were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein lysates were subjected to 10% SDS-polyacrylamide gel and electrophoretically transferred to a PVDF membrane (Bio-Rad, Richmond, CA, USA). Membranes were blocked with 5% skim milk, subsequently probed with anti-Cx43 (Zymed, San Francisco, CA, USA), anti-pERK1/2, or anti-ERK1/2 antibodies (Cell signaling Technology, Beverly, MA, USA) at 4 °C for overnight, and incubated with secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. Image detection was performed using an enhanced chemiluminescence kit (GE Healthcare, Buckinghamshire, UK). The relative band intensity was quantified using Image J program from the National Institutes of Health (Bethesda, MD, USA).
Statistical analysis
The data were expressed as means ± SD. All data were analyzed by a one-way ANOVA or Student’s t-test using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). p values ≤ 0.05 were considered statistically significant.
Results
Bacterial reverse mutation assay (Ames test)
Positive results should show a twofold or more increase above the negative control in the number of His+ mutants with dose-dependent relationship. Expected positive responses were also observed in all the positive control groups for respective S. typhimurium strains, indicating that the test conditions and metabolic activation system were adequate. The Tiglium seed extract significantly produced His+ mutants at the highest concentration 16.8 mg/plate in S. typhimurium TA98 with or without S9 mix (Table ). And, it also produced His+ mutants dose dependently in TA100, 102, 1535, and 1537 in the presence or absence of S9 mix, although the production of His+ mutants in TA1537 without S9 fraction did not reach statistical significance.
Table 1. S. typhimurium reversion assay with the Tiglium seed extract.
Chromosomal aberration assay
The different chromosomal aberrations, including breaks, fragments, exchanges, and other multiple damages, were analyzed. Under the conditions of this study, no significant increase in the incidence of chromosomal aberrations was observed in CHL cells treated with the Tiglium seed extract with or without S9 mix at all concentrations tested (Table ). In contrast, significant higher chromosome aberrations in the positive control groups were found.
Table 2. Chromosomal aberration induced by the Tiglium seed extract.
Table 3. MNPCEs in mice bone marrow following the treatment with the Tiglium seed extract.
In vivo bone marrow micronucleus assay
The results of in vivo micronucleus test are presented in Table
Micronucleated polychromatic erythrocytes (MNPCE) and PCE/(PCE + NCE) ratio in the positive control group were found to be significantly different from those in the negative control group. In contrast, the Tiglium seed extract did not exhibit any statistically significant decrease in PCE/(PCE + NCE) ratio at concentrations of 0.1, 0.3, and 0.5 g/kg of body weight. And, there was no increase in MNPCE observed at any doses of the Tiglium seed extract, suggesting that the Tiglium seed extract showed no clastogenic potential after in vivo exposure.
Cytotoxicity
Based on our findings on the mutagenicity of the Tiglium seed extract, we postulated that the Tiglium seed extract also has tumor-promoting potential. A cell viability test was carried out prior to GJIC analysis. Dose-dependent cytotoxicity was observed at different concentrations (0–2500 μg/mL) of the Tiglium seed extract for 24 h in WB-F344 cells (Fig. ). On the basis of our result that the Tiglium seed extract exhibited approximately 80% cell viability at concentrations less than 312.5 μg/mL, the maximum dose of the Tiglium seed extract for further experiments was set to 312.5 μg/mL.
Fig. 1. Cytotoxicity of the Tiglium seed extract in WB-F344 cells by MTT assay.
Notes: Data expressed as means ± SD (*p < 0.05 and **p < 0.01).
Effects of Tiglium seed extract on the GJIC and expression levels of Cx43
The effect of the Tiglium seed extract on GJIC was assessed using SL/DT method. As a result, GJIC was dramatically diminished by nearly 90% at 1 h after the treatment with 312.5 μg/mL Tiglium seed extract, followed by a partial restoration thereafter (Fig. (A) and (B)). Based on this time-course result, we selected 1 h as an optimal time point for dose-response analysis. After exposing the cells to TPA as a positive control for 1 h, apparent inhibition of GJIC was observed. Importantly, the Tiglium seed extract exhibited a dose-dependent inhibitory effect for 3.125 (35% of inhibition), 31.25 (55% of inhibition), or 312.5 μg/mL (92% of inhibition) (Fig. (A) and (B)).
Fig. 2. Time course study of the Tiglium seed extract on GJIC in WB-F344 cells.
Notes: (A,B) Representative SL/DT assay (A) for GJIC analysis and quantitative analysis (B) after the treatment with 312.5 μg/mL Tiglium seed extract (original magnification ×200). Data expressed as means ± SD (*p < 0.05 and **p < 0.01). (a) The treatment for 0 min; (b) 30 min; (c) 1 h; (d) 3 h; (e) 12 h; (f) 24 h.
Fig. 3. Dose-dependent inhibition of GJIC and expression levels of Cx43 in WB-F344 cells after the treatment with the Tiglium seed extract.
Notes: (A,B) Representative SL/DT assay (A) for GJIC analysis and quantitative analysis (B) after the treatment with different concentrations of the Tiglium seed extract for 1 h (original magnification ×200). Data expressed as means ± SD (*p < 0.05 and **p < 0.01). (C) Distribution of Cx43 levels (green) was detected by immunofluorescence staining (original magnification ×400). Nuclei were stained with DAPI (blue). (a) Control; (b) 10 ng/mL TPA; (c) 3.125 μg/mL Tiglium seed extract; (d) 31.25 μg/mL Tiglium seed extract; (e) 312.5 μg/mL Tiglium seed extract.
WB-F344 cells have been known to express Cx43 predominantly as their major gap junction protein.Citation28,29) We also found that Cx43 proteins were functionally localized at gap junction plaques between adjacent cells. TPA, which had GJIC-inhibiting capacity, caused a decreased immunostaining of Cx43 on the cell membranes of WB-F344 cells, as evidenced by immunofluorescence staining (Fig. (C)). In line with the results from SL/DT assay, the expression levels of Cx43 at cell membrane were decreased in a dose-dependent manner at 1 h after the exposure with the Tiglium seed extract at 3.125, 31.25, and 312.5 μg/mL.
Recovery effects of PKC or MAPKs inhibitors on the inhibition of GJIC and expression levels of Cx43
PKC, known as an intercellular receptor of TPA and can activate MAPKs signaling pathway, plays a critical role in the regulation of cell growth and differentiation.Citation30,31) Therefore, to elucidate the involvement of PKC and MAPKs pathways in GJIC inhibited by the Tiglium seed extract, we pretreated the cells with PKC inhibitor (BIM I), MEK inhibitor (U0126), or ERK inhibitor (PD98059). It was interesting to note that the Tiglium seed extract-induced inhibition of GJIC was significantly attenuated by the pretreatment with these inhibitors (Fig. (A) and (B)). Consistently, the effects of the Tiglium seed extract on the internalization of Cx43 and decreased levels of Cx43 at cell membrane were also clearly prevented by the pretreatment with BIM I, U0126, or PD98059 (Fig. (C)), suggesting that PKC and MAPKs pathways might be closely involved in the Tiglium seed extract-induced GJIC inhibition associated with the Cx43 internalization and degradation.
Fig. 4. Recovery effects of MAPKs and PKC inhibitors on the Tiglium seed extract-induced inhibition of GJIC in WB-F344 cells.
Notes: The cells were pretreated with 10 μM MEK inhibitor U0126, 50 μM ERK inhibitor PD98059 and 10 μM PKC inhibitor BIM I for 30 min prior to the treatment with 312.5 μg/mL Tiglium seed extract for 1 h. (A,B) Representative SL/DT assay (A) for GJIC analysis and quantitative analysis (B) (original magnification ×200). Data expressed as means ± SD (*p < 0.05 and **p < 0.01). (C) Distribution of Cx43 levels (green) was detected by immunofluorescence staining (original magnification ×400). Nuclei were stained with DAPI (blue). (a) Control; (b) 10 ng/mL TPA; (c) 312.5 μg/mL Tiglium seed extract (T.S.); (d) 10 μM U0126 + 312.5 μg/mL Tiglium seed extract; (e) 50 μM PD98059 + 312.5 μg/mL Tiglium seed extract; (f) 10 μM BIM I + 312.5 μg/mL Tiglium seed extract.
Effects of PKC or MAPKs inhibitors on the phosphorylation of Cx43 and ERK1/2
One unphosphorylated form (P0) and two phosphorylated forms (P1 and P2) of Cx43 were detectable in the untreated cells, as indicated by western blot analysis (Fig. ). When the cells were exposed to TPA, the hyperphosphorylation of Cx43 was observed. In the cells treated with the Tiglium seed extract for 1 h, disappearance of P0 isoform and marked induction of P1/P2 isoforms were clearly observed. In contrast, the pretreatment of BIM I, U0126, or PD98059 for 30 min reversed these phosphorylated shifts of Cx43 induced by the Tiglium seed extract. Noteworthy, the Tiglium seed extract also induced the phosphorylation of ERK1/2 in a dose-dependent manner. And, this was markedly attenuated by the pretreatment with BIM I, U0126, or PD98059.
Fig. 5. Effects of the Tiglium seed extract on phosphorylation of Cx43 and ERK1/2 in WB-F344 cells.
Notes: The cells were pretreated with 10 μM MEK inhibitor U0126, 50 μM ERK inhibitor PD98059 and 10 μM PKC inhibitor BIM I for 30 min prior to the treatment with 312.5 μg/mL Tiglium seed extract for 1 h. Representative Western blot and quantitative analysis. (a) Control; (b) 10 ng/mL TPA; (c) 3.125 μg/mL Tiglium seed extract (T.S.); (d) 31.25 μg/mL Tiglium seed extract; (e) 312.5 μg/mL Tiglium seed extracts; (f) 10 μM U0126 + 312.5 μg/mL Tiglium seed extract; (g) 50 μM PD98059 + 312.5 μg/mL Tiglium seed extract; (h) 10 μM BIM I + 312.5 μg/mL Tiglium seed extract.
Discussion
Natural plants, herbs, are widely used in the treatment of many diseases over the years.Citation5) Although many people believe that they are safe, the safety of medicinal plants is not well established and many adverse effects are reported.Citation32) The Tiglium seed is a seed of Croton Tiglium Linne and contains amounts of croton oil. The croton oil is a complex mixture of several volatiles, fatty acids (i.e. crotonoleic acid and tiglic acid), and toxic proteins (i.e. croton globulin and croton albumin). Moreover, the major constituents of croton oil are many different types of phorbol derivatives including TPA.Citation4,33) TPA is a well-known cocarcinogen and used as a tumor promoter in two-stage (initiator–promoter) mouse skin carcinogenesis model.Citation34) Despite their pharmacological effects of Croton species reported, it was also known to be associated with promotion phase of tumors.Citation35,36) The first aim of this study was to determine the genotoxic potential of the Tiglium seed using the Ames test, the chromosome aberration test, and the micronucleus test for gene mutation, chromosome aberration, and cytogenetic damage, respectively, in different test systems, including bacteria and mammalian cells.
In the Ames test, we used five different S. typhimurium tester strains, indicating frame shift (TA98 and TA1537), base-pair substitution (TA100 and TA1535), or oxidative and cross-linking (TA102) mutations.Citation37) The Tiglium seed extract showed a clear mutagenic potential in S. typhimurium TA100, TA102, and TA1535 strains cultured with and without S9 fraction. And, there was obviously higher mutagenicity in TA98 strain, both in the presence and absence of metabolic activation, at the highest concentration (16.8 mg/plate) of the Tiglium seed extract. The Tiglium seed also produced His+ mutants dose dependently in TA1537, although the production of His+ mutants in TA1537 without S9 fraction did not reach statistical significance. These results suggest that the Tiglium seed can be classified as a base pair substitution, frameshift, or cross-linking and oxidizing mutagens, although it remains to be determined which components of the extract can affect this mutagenicity of the Tiglium seed.
To further assess the clastogenic effect, the in vitro chromosomal aberration assay and in vivo micronucleus assay were also performed using CHL cells and male ICR mice, respectively. Chromosomal aberrations are the classical genotoxic response to tumor initiation and development processes.Citation38) The data of chromosome aberration assay demonstrated that the Tiglium seed extract did not affect the chromosomes of CHL cells, suggesting that it is non-clastogenic as evidenced by the lack of significant structural and numerical aberrations. Micronuclei are indirect indicators of numerical and structural chromosomal abberrations.Citation39) In the current study, the observations from the in vivo micronucleus assay in mice that is considered as more reliable method also did not show any genotoxic potential of the Tiglium seed extract, suggesting that the Tiglium seed extract was considered to be non-clastogenic at up to the highest feasible concentration that could be evaluated.
Genotoxicity is one of important risk factors for long-term effects, including carcinogenicity.Citation40) Especially, the mutagenic potential may be only the first step in some pathways that lead to cancer.Citation41) Since we observed the mutagenicity of the Tiglium seed extract in the current study, it is hypothesized that the Tiglium seed extract also has tumor-promoting potential. For this reason, we additionally performed SL/DT assay in WB-F344 cells treated with the Tiglium seed extract to evaluate GJIC associated with tumor promotion stage of carcinogenesisCitation42) in the current study. We observed that the Tiglium seed extract found to be strongly inhibited GJIC in a dose-dependent manner from 3.125 to 312.5 μg/mL for 1 h following the treatment, indicating that the Tiglium seed extract results in the loss of intercellular communication associated with the tumor promotion.
PKC is a phospholipid-dependent serine–threonine kinase which plays a critical role in the regulation of cell growth and differentiation. In addition, PKC is known as an intercellular receptor of TPA and can activate MAPKs signaling pathway through activation of Raf kinase.Citation30,31) MAPKs signaling pathways, which includes ERK1/2, are also important enzymes involved in cell growth, differentiation, and stress responses.Citation43) Therefore, PKC and MAPKs pathways activated by TPA might be closely related to the loss of cell–cell communication in variety of cell lines, including WB-F344 cells, through conformational changes of Cx plaques, such as phosphorylation of Cx. This led us to hypothesize that the inhibition of GJIC by the Tiglium seed extract is correlated with the activation of PKC and MAPKs pathway similar to TPA (Fig. ). In this study, we demonstrated that inhibition of GJIC by the Tiglium seed extract was significantly prevented by inhibition of PKC, MEK, and ERK. The internalization/phosphorylation of Cx43 and the phosphorylation of ERK1/2 pathways were also attenuated by the Tiglium seed extract, supporting our hypothesis on the involvement of PKC and MAPKs pathways in the tumor-promoting potential of the Tiglium seed. Leithe and RivedalCitation44) demonstrated that the ubiquitination of Cx43 in response to TPA occurred concomitantly with the phosphorylation and degradation of Cx43. Furthermore, Cx43 degradation was blocked by proteasomal inhibitors, indicating that proteasome was associated with Cx43 degradation. Therefore, proteasome-related ubiquitination can also be involved in Tiglium seed extract-induced Cx43 phosphorylation and degradation.
Fig. 6. Schematic representation on the inhibition of GJIC by the Tiglium seed extract in WB-F344 cells.
In the present study, the Tiglium seed extract newly showed the mutagenic activity in Ames assay, although previous reports demonstrated that both croton oil and TPA induced no mutagenic activity.Citation7,35) Thus, it is possible that either croton resin or other complex mixtures of unknown components in the Tiglium seed extracts can be responsible for this mutagenic activity. In addition to 11 known phorbol diesters (nine phorbol diesters and two 4-deoxy-4α-phorbol diesters), Zhang et al.Citation45) newly isolated and characterized eight phorbol diesters (three phorbol diesters and five 4-deoxy-4α-phorbol diesters) from Croton Tiglium. Particularly, 13-O-acetylphorbol-20-oleate was found to exhibit the most cytotoxic activity using the SNU387 hepatic tumor cell line. Further studies with possible candidates are needed to demonstrate the properties of Tiglium seed extract. The Tiglium seed extract also markedly inhibited GJIC by the phosphorylation of Cx43 and ERK1/2 through the activation of PKC and MAPKs pathways. Based on the current studies, it can be concluded that the Tiglium seed has the potential to be mutagenic. Furthermore, this mutagenicity of the Tiglium seed could be linked, directly or indirectly, to the tumor-promoting potential and carcinogenicity involved in the dysfunction of GJIC.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
We thank Ji-Hyun Pak and Ji-Sook Kim for their excellent technical supports.
Additional information
Funding
Notes
Abbreviations: GJIC, gap junctional intercellular communication; Cx43, connexin 43; ERK, extracellular signal regulated kinases; PKC, protein kinase C; MAPKs, mitogen-activated protein kinases; TPA, 12-O-tetradecanocylphorbol-13-acetate; OECD, Organization for Economic Cooperation and Development; CHL, Chinese hamster lung; FBS, fetal bovine serum; MN, micronucleus; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; WB-F344 cells, WB-F344 rat liver epithelial cells; DMEM, Dulbecco’s modified Eagle’s medium; SL/DT, scrape loading and dye transfer; BIM I, bisindolymaleimide I; MNPCE, micronucleated polychromatic erythrocytes.
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