In this study, Euphorbia grisophylla aerial part water extract (EAE) was investigated for genotoxic, cytotoxic, and oxidative potential. Micronucleus (MN) findings, one of the endpoint assays, correlate with the clastogenic and or aneugenic effect. In this test findings, the statistically insignificant increase in the number of MN was due to the increase in the extract concentration. In addition, MN formations induced by EAE are significantly lower than the positive control (mitomycin C, MMC). The similarities between the MN increases detected at high concentrations of the test substance and the DNA damage findings in the comet test draw attention. The fact that the outputs of these two experiments seem to support each other may be related to the possible clastogenic activity of the test substance. However, insignificant increases in the number of revertant colonies observed in the Ames test indicate that EAE did not induce the point mutation. The clastogenic and cytotoxic effect of EAE may be related to disruption of the DNA synthesis/repair function, oxidative stress, or the inadequacy of oxidative energy production.
Although the oxidative stress index (OSI) measured in the cell culture medium showed partial fluctuations, it was not statistically significant. The cause for this result is either the TOC did not significantly increase, or TAR did not respond to the current situation. The fact that Euphorbia extract possibly reduces the amount of dissolved oxygen in the liquid medium may help to explain the present results. According to previous studies supporting this view, Euphorbia extract significantly reduces the amount of dissolved oxygen depending on the dose (Akpa et al. 2009; Idowu et al. 2019). Decreased dissolved oxygen probably suppresses oxidative stress parameters and oxidative ATP production in cells. This approach also supports the argument that the test substance is possible genotoxic and apparent cytotoxic.
Previous studies have data that supports some of our findings and contradicts others. When it comes to the clastogenic potential, including DNA breaks, with results of a few comprehensive studies conducted recently generally show similarities with the DNA damage findings we have detected. But a small number of them have reported that euphorbia products do not damage DNA and even have DNA protective activity, contrary to our findings. In one study, the whole extract and fractions of Euphorbia rigida significantly stimulated DNA strand breaks (Basaran et al. 1996). Similarly, in the micronucleus test performed with Euphorbia hyssopifolia ethanolic extract in the hepatocarcinoma cell line (HepG2), some concentrations induced severe DNA damage that led to death (de Sousa Araújo et al. 2015). Euphorbia triaculeata extract exhibited a genotoxic effect by increasing the mean DNA damage percentage in a time-dependent manner against MCF-7 and PC 3 cancer lines, but this did not occur in the HEPG2 cancer cell line (Al-Faifi et al. 2017). In the comet experiment with the MCF-7 cell line, Euphorbia hirta extract exhibited a genotoxic effect by increasing the average DNA damage rate in a time-dependent manner (Ping et al. 2013). In the Allium cepa test performed with the extract of the same euphorbia species, an increase in chromosome aberrations and a decrease in the mitotic index were observed in response to the high extract concentration (Mohamed and Aly 2018). Contrary to the above, both the aqueous extract and the latex of the Euphorbia tirucalli are not genotoxic in human leukocyte cells in vitro (de Oliveira et al. 2015). Some euphorbia species have DNA protective activity; the methanolic extract of Euphorbia dracunculoides showed a healing effect against oxidative stress and DNA damage caused by CCI4 in rat liver (Batool et al. 2017).
2.3-butanediol, one of the most abundant components in the test substance extract, did not show embryotoxic effects in 10 days pregnant albino Wistar rats (Priscott 1985). In a study with human blood, the 30% concentration of 2.3-butanediol showed relatively low toxicity for erythrocytes. However, the time-dependent hemolytic effect draws attention. The hemolysis rate detected 5 hours after treatment of the agent reached 2%, 6% after 21 hours, and 60% after 46 hours (Pubchem).
Hexanoic acid, another component of euphorbia extract, has shown a growth-arresting effect in early childhood malignant tumors (Atasever-Arslan et al. 2015). This result is consistent with our cytotoxicity findings.
According to the Ames test results in our study, the test substance did not cause a significant increase in colony numbers of Salmonella typhimurium mutant auxotrophic strains (TA 98 and TA 100). Here the extract did not induce frameshift and base shift mutations in the auxotrophic strains. Similarly, in different studies, complete extracts and fractions of some medicinal plants, including euphorbia, did not produce a positive mutagenic effect in Salmonella typimurium mutant strains (TA98 and TA100), regardless of whether there was metabolic activation or not (Basaran et al. 1996). The natural or lyophilized Euphorbia splendens latex did not have an acute toxic effect on bioluminescent Photobacterium phosphoreum. In addition, it did not cause mutagenic activity in Salmonella typhimurium TA98 and TA100 strains in the presence or absence of S9 (Schall et al. 1991). According to these results, Euphorbia extract does not induce nucleotide substitution and frameshift mutations.
In the cell culture used in our study, the aqueous extract of spurge did not induce any oxidative stress parameters (TOC and TAR) and thus oxidative stress index. However, in a different study, the antioxidant effect of Euphorbia hirta and the galactoside and glucoside derivatives isolated from Euphorbia kansui were discovered to have antioxidative and anti-fatigue properties in mice (Yu et al. 2006; Karim et al. 2015). An extract combination (KIOM-79) obtained from a group of medicinal plants, including Euphorbia pekinensis, was studied in rat pancreatic beta (RINm5F) cells. Against oxidative stresses induced by streptozotocin (STZ) in RINm5F (rat insulinoma) cells, KIOM-79 showed a potential cytoprotective effect by inhibiting the ERK pathway (Kang et al. 2007). In contrast to the above study, euphorbia factor L1 (EFL1), a lathyran-type diterpenoid obtained from the Euphorbia lathyris plant, decreased human gastric mucosal epithelium cells (GES-1) survival. Moreover, this agent increased LDH leakage and induced abnormal ROS, MDA, and SOD production in GES-1 cells. Some parameters such as reduced mitochondria membrane potential (MMP), pyknotic nucleus, fragmented DNA, and increased apoptosis rate are reporters of mitochondria-mediated apoptosis. EFL1 has reduced the survival of GES-1 cells by several mechanisms, including oxidative stress, mitochondria-mediated apoptosis activation, and inhibition of the PI3K/AKT/mTOR pathway(Zhu et al. 2019). The above result was confirmed by genotoxicity data, which damages DNA and increases oxidative damage at 1% and 10% concentrations of Euphorbia tirucalli (Aveloz) extract. Since aveloz contains phorbol ester, it can act as a genotoxic agent (Machado et al. 2016). Contrary to the previous finding, Euphorbia dracunculoides methanolic extract showed an ameliorative effect against CCI4-induced oxidative stress and DNA damage (hepatic toxicity) in rat liver (Batool et al. 2017). Euphorbia bicolor latex extract showed antioxidant activity by scavenging free radicals in vitro analyses. (Basu et al. 2019). In a different study, diterpenoids isolated from ethyl acetate extract obtained from the aerial parts of Euphorbia antiquorum significantly suppressed the production of the lipopolysaccharide-induced oxidation marker nitric oxide (NO) in murine macrophage J774.A1 cells (Choodej et al. 2020).
The difference between our findings and the results of some studies may be due to differences in plant species, habitat, harvest time, extraction method, and test protocol. Our experiments were carried out directly on a single type of healthy cell culture medium in vitro. Possibly, the lack of homeostatic reflexes of cells to an exogenous effect may be one of the reasons for the discrepancy between study findings.
In our study, the applied Euphorbia grisophylla plant extract decreased the nuclear division index (NDI) value non-randomly compared to the control. In another study, extracts from Euphorbia platyphyllos showed cytotoxic effects on human breast metastatic carcinoma (MCF-7) cells in a dose- and time-dependent manner in vitro. It has been evaluated that this cytotoxicity is due to the genotoxic effect (Aslantürk and Çelik 2010). Contrary, no significant cytotoxic effect of Euphorbia splendens latex was detected in Chinese hamster ovary (CHO) cells (Schall et al. 1991). The ethanolic extract isolated from Euphorbia hyssopifolia species did not show significant cytotoxic activity in the MTT test in HepG2 cell culture. However, the 1.0 mg/ml concentration of the extract induced severe cell damage and cytotoxic effect leading to death in the micronucleus test (de Sousa Araújo et al. 2015). There is significant toxicity in the MTT test performed with a methanolic extract of a different species of spurge (Euphorbia triaculeata) (Al-Faifi et al. 2017). The increase in methanolic extract concentrations of Euphorbia hirta significantly decreased the mitotic index in Allium cepa cells. In the current situation, the mitodepressive nature of the methanolic extract is remarkable. (Mohamed and Aly 2018). In a study including in silico approaches to examine the apoptogenic potential of Euphorbia peplus methanolic extract (EPME) in rats, EPME induced strong renal and cardiac p53 expression and moderate cardiac TNF-a expression, which are markers of apoptosis in rats. Moreover, according to molecular docking simulation data, di-(2-ethylhexyl) phthalate (DEHP), one of the components of the extract, was predicted to have a higher docking affinity to the unique apoptosis dimer interface. Here DEHP docked and inhibited to apoptosis dimer interface that the mouse double minute 2 (MDM2) homologous-p53, compared to the standard reference compound. Long-term exposure to EPME adversely affects heart and kidney tissues, possibly due to stimulating inflammatory and apoptotic activities. According to in silico data, EPME inhibits MDM2-mediated p53 degradation, suggesting possible anticancer potential in kidney and heart tissues (Abd-Elhakim et al. 2019). One of the different diterpenoids isolated from Euphorbia neriifolia exhibited moderate cytotoxicity on HepG2 and multi-drug resistant HepG2/Adr cells. While another component of the same plant exhibited significant cytotoxicity in the HepG2 cell line, it did not show any cytotoxic activity against the HepG2/Adr cell line (Li et al. 2019). The semi-synthetic ingenol derivative (ingenol-3-dodecanoate, IngC) from Euphorbia tirucalli caused dose-dependent cytotoxicity and effective colony reduction. Exposure to IngC regulated a broad spectrum of signaling effectors relevant to survival and cell cycle management by inducing S-phase arrest associated with overexpression of the p21CIP/WAF1 pathway. In addition, an IngC derivative directed glioma cells to autophagy through the accumulation of LC3B-II. When combined with a specific autophagic inhibitor, bafilomycin A1, cells exhibited increased cytotoxic sensitivity (Silva et al. 2019). In a study related to the induction of apoptosis, the hydroalcoholic extract of Euphorbia microsciadia increased the cytotoxic effect on the metastatic mammary epithelium (MDA-MB-231) cell line in a dose-dependent manner. According to qRT-PCR data, the same extract increased the expression levels of Let-7, miR-15, miR-16, miR-29, and miR-34a molecules, which caused increased apoptosis (Mahmoudian-Sani and Asadi-Samani 2020). Zhou et al. (2021) observed that 80% ethanol extract obtained from the aerial parts of Euphorbia helioscopia exhibited cytotoxic activities against six different kidney cancer cell lines. (Zhou et al. 2021). Another study has reported that seven compounds, three of them novel, isolated from Euphorbia peplus, did not show cytotoxic activities against five human tumor cell lines at 40 µM concentration (Chen et al. 2021). One of the eight compounds, ingol-3.7.12-triacetate-8-benzoate, isolated from Euphorbia royleana has shown a modulatory effect on the P-glycoprotein-mediated multidrug-resistant cancer cell (HepG2/DOX) line without apparent cytotoxic effect. This effect increased the effectiveness of the anticancer drug doxorubicin (DOX) and proved to be approximately 105 times stronger than the positive drug verapamil (Shaker et al. 2020). As a result of the phytochemical analysis of Euphorbia kopetdaghi aerial part extract, which grows wild in the northeastern regions of Iran, two new different cyclomyrcinol macrocyclic diterpenes with apoptotic effects were discovered. The apoptotic potential of these compounds has been associated with excessive ROS production and loss of mitochondrial membrane potential (ΔΨm). One of the compounds abovementioned, copetdaghinane A, inhibits the growth of MCF-7 breast cancer cells through the mitochondrial apoptotic pathway activation (Riahi et al. 2020). On the other hand, none of the compounds in Euphorbia antiquorum root extracts showed cytotoxic activity (Tran et al. 2022). Fraction compounds of Euphorbia umbellata latex hexane extracts have caused morphological changes compatible with apoptosis induction against leukemic cells, such as cell cycle alteration, depolarized cell population increase, and caspase 3/7 activation. The bio-guided fractionation study suggested that latex samples of Euphorbia umbellata synergistically enhance the activity of phytocomplexes used against leukemia cells and that this cytotoxicity may be due to apoptosis (Cruz et al. 2020). The chloroform fraction (Em-C) of Euphorbia milii methanol extract showed significant cytotoxicity against HepG2 cells compared with the standard compound 5-fluorouracil (5-FU). However, Em-C did not show similar significant effects on the human cervical cancer cell (HeLa) line (Chohan et al. 2020). GRC-2, a prostratin analog isolated from Euphorbia grandicornis, is ten times more potent than prostratin used to inhibit the growth of human non-small cell lung cancer (NSCLC) (A549) cells. According to flow cytometry analysis, GRC-2 and prostratin induce apoptosis by inhibiting cell cycle progression in the G2/M phase. There is evidence that the cytotoxic effect of GRC-2 and prostratin accompany Protein kinase C-δ (PKC-δ) and Protein kinase D (PKD) activation and nuclear translocation as well as ERK hyperactivation. Here, prostratin and its more potent analog, GRC-2, has reduced cell viability in A549 cells, partly through activation of the PKC-δ/PKD/ERK pathway (Tsai et al. 2020). In a similar study, two new daphnane-type diterpenoids (fischerianin A and fischerianin B) and two known diterpenoids (langduin A and langduin A6) were isolated from the extract of the dry roots of Euphorbia fischeriana Steud. These four compounds have shown moderate inhibitory activity against human cancer cell lines (A375, HepG2, HL-60, K562, and HeLa) (Xie et al. 2021).
Some of the research on Euphorbia plant extract and its components are in vivo studies. However, cancer cell lines have been used in most in vitro studies. The originality of our experiments is the application of Euphorbia grisophylla plant extract to healthy cells in vitro under short-term treatment. According to our findings, Euphorbia grisophylla extract showed a clear and consistent cytotoxic effect. In most previous studies, Euphorbia sp plant extracts have cytotoxic and antioxidant potential in cancer cell lines.
As mentioned above, according to our findings, spurge extract had a partial cytogenotoxic effect on lymphocytes. The cause for this effect has not been clearly explained. Presumably, this situation may have resulted from the retention of the dissolved oxygen present in the culture medium by the extract. The oxygen binding prediction has not yet been experimentally confirmed. However, the practice of using euphorbia in fishing with the conventional method among the people forms the basis of this idea. Presumably, the fact that the extract does not stimulate oxidative stress indicators may be related to the oxygen binding/retention characteristic in the environment.
In summary, in this study, moderate genotoxic and cytotoxic effects of Euphorbia grisophylla extract were observed at subtoxic concentrations in cultured healthy human peripheral lymphocyte cultures. However, the agent has not caused significant changes in parameters related to oxidative stress. The fact that a small number of similar studies have been carried out before increases the importance of this project. Original scientific research to be carried out by impartial laboratories with euphorbia extract or components in different cell systems and with innovative methods, alone or in combination, will be groundbreaking in that area. In summary, it is a strong possibility that the findings obtained from these studies will donate new paradigms to the pharmaceutical industry.