The cytotoxicities of three aconitum alkaloids- aconitine, hypaconitine and mesaconitine, and their abilities to bind DNA have been explored. Rat myocardial cells H9c2 were treated with aconitum alkaloids and assessed the cytotoxicities by using MTT assay and flow cytometry. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay. Aconitine was found to be the most toxic in rat myocardial cells H9c2 in three aconitum alkaloids. At the same time, DNA adducts were isolated and then analyzed by UV-Vis spectroscopy after exposure to alkaloids, which indicated that three alkaloids could bind to DNA in rat myocardial cells H9c2. Furthermore, their binding modes were investigated by UV-Visible, fluorescence, DNA melting studies and ionic strength effect. Results indicated that the interaction between three alkaloids and DNA were intercalation coupled with electrostatic effect. The estimated binding constants were between 4.83 × 105 M−1 to 9.85 × 105 M−1 for three alkaloids at 298 K.
The Aconitum species (Ranumculaceae) including Aconitum carmichaeli Debx. and Aconitum Kusnezoffii Reichb. are widespread throughout Asia, Europe and North America. Aconite is usually applied for various diseases, such as painful joints, gastroenteritis, rheumatic fever, and various tumors[
So far as we know, different Aconitum sources contain varies alkaloid: MA is the main compound of Aconitum kusnezoffii, AC is the main alkaloid in Aconitum napellus, and the main constituents of Aconitum carmichaeli are HA and MA. Aconitine, which has a narrow therapeutic index, is considered to be the foremost highly toxic DDA in aconitum alkaloids[
The interaction of drug and DNA has long been the focus of research in chemistry and life science, especially important for the designing of new drug and DNA damage mechanism study. So far as we know, drug-DNA interaction is considered to be the reason for the DNA damage of some drugs and pollutants, such as ochratoxin A (OTA) and aristolochic acids (AAs)[
In recent years, there were still a few clinical cases of aconite intoxication reported from many countries[
The cytotoxicity of three alkaloids in rat myocardial cells H9c2 was determined using MTT assay. The cell viability results following incubation with various concentrations of AC, MA and HA for 24, 48 and 72 h were shown in Fig. 2 and IC
A morphological change observed in rat myocardial cells H9c2 treated with DDAs was shown in Fig. 3. Cells without any treatment and cells treated with 0.5% DMSO (group of control) were egg and shuttle shaped; while cells treated with DDAs were shrinkage, irregularities, and cell rounding in contour and size. Rat myocardial cells H9c2 exposed to AC (6.9 × 10
PI is used to distinguish necrotic cells from apoptotic and living cells by supravital staining without prior permeabilization. Annexin V, which has a high affinity for phosphatidylserine (PS), labeled with a fluorophore can identify apoptotic H9c2 by binding to PS. Early apoptotic cells tested positive for annexin V and negative for PI staining, whereas late apoptotic cells undergoing secondary necrosis were positive for both annexin V and PI staining. As shown in Fig. 4, all of the three alkaloids stimulated apoptosis in H9c2 cells without causing any obvious necrosis. In the control group, the percentage of apoptotic H9c2 cells was 2.3%; however, the percentage of apoptotic H9c2 increased up to 28.1%, 24.9% and 17.3% after treatment with aconitine, hypaconitine and mesaconitine, respectively.(A) Apoptosis induced by AC, HA and MA in H9c2 cells for 24 h. (B) The summary (**p < 0.05 vs. control group).
UV-Vis spectroscopic data collected from DNA, which was isolated from rat myocardial cells H9c2, is displayed in Fig. 5A (line a: cells treated with AC; line b: cells treated with HA; line c: cells treated with MA; line d: control). The absorption spectrum of DNA isolated from the cells in control group had a peak at 258 nm (line d), which was generated from the strong absorption of purine and pyrimidine bases in DNA. The ratio of the absorbance at 260 nm and 280 nm was greater than 1.8, indicating that the DNA was sufficiently free from protein[
In general, if a small molecule of drug interacts with DNA, changes in absorbance and in the position of the band should occur. Hypochromic effects and a bathochromic shift could be observed especially when drug interacts with DNA by either the electrostatic effect or the intercalation binding. As we know, neutral red (NR) is reported as a mutagenic intercalating dye for DNA[
In this work, none of the DDAs (AC, MA and HA) or DDA-DNA complex exhibited fluorescence in the buffer solution (Tris-HCl, pH = 7.4). Thus, molecule probe must be employed to explore the characteristics of fluorescence spectra of DDA-DNA interaction. As we know that neutral red is a kind of planar phenazine dye, which is structurally alike with other planar dyes, e.g., thiazine, acridine and xanthenes. Besides, upon addition of DDAs to a NR solution, neither significant change on fluorescence intensity for NR has been observed nor new fluorescence peaks developed. Based on this, NR was selected as the probe in this work, because of its lower toxicity and higher stability. The emission spectrum of the DNA-NR complex in the absence and presence of AC, MA and HA were shown in Fig. 5C, respectively. There was an obvious maximum emission at 612 nm when the DNA-NR system was excited at 546 nm. With an increasing sequentially adding of each DDA, the fluorescence intensity of the complex decreased without notable changes in the wavelength of maximum emission. Moreover, three of the alkaloids had the ability for fluorescence quenching of DNA-NR system. Figure 5C also showed a significant difference in quenching degree for three of the DDAs, which revealed the difference binding ability and binding constant (K
In this work, DDAs could not induce obvious change on the fluorescence intensity of NR, which means DDA and NR could not bind together. Considering UV-vis spectroscopic studies above, it seems that the decrease in the emission intensity on the addition of DDAs reflected the binding of AC, MA and HA to DNA by intercalation. The observation revealed that DDA competed against NR in binding with DNA and NR molecules intercalated in DNA double helix were excluded by alkaloids. Consequently, a decrease in the emission was observed. Thus, it can be deduced that the AC, MA and HA intercalated into DNA and formed the DDA-DNA complex.
In order to elucidate the fluorescence quenching mechanism, the classical Sterne-Volmer equation was utilized for data analysis.F0/F=1+Kqτ0[D]=1+Ksv[D]
F
In general, the mechanisms of fluorescence quenching are classified as either dynamic or static quenching, which can be distinguished by their difference manifestations dependence on temperatures. Figure 5D showed the curves of F
Moreover, the binding stoichiometry (n) of the system and the apparent binding constant (K
K
A DNA melting analysis is another strong evidence for drug intercalates into the base pairs of DNA. Interaction of small molecules with DNA can influence melting temperature (Tm) of DNA significantly. The intercalation binding of drug into double helix could stabilize the structure of DNA and Tm increases by about 5-8 °C, meanwhile, the other binding modes causes no obvious increase in Tm[
Experiments were carried out by controlling various temperatures ever 5 °C from 20 to 100 °C in the absence and the presence of DDAs while monitoring the absorbance of DNA at 260 nm. Figure 5E showed the Tm value of CT-DNA was 74.1 °C under our experimental conditions, however it was increased to 79.2 °C, 80.1 °C and 81.7 °C in the presence of AC, HA and MA respectively. The results suggested that DDAs molecules intercalated into the base pairs and stabilized DNA helix. It is in a good agreement with the absorption spectra and fluorescence quenching study above.
The effects of sodium chloride on the fluorescence quenching of AC, MA and HA were studied respectively, and the results were shown as Fig. 5F. Generally, there are three predominant non-covalent modes for small molecules binding to DNA, including intercalation, electrostatic interaction and groove binding. If the electrostatic binding is one of the interaction modes, with the adding of NaCl, small molecules of the alkaloids will release[
As shown in Fig. 5F, with the addition of increasing concentrations of NaCl (0.1 M for line a, 0.2 M for line b, 0.3 M for line c), the fluorescence quenching by DDA for DNA-NR complex was decrease. The results indicated that besides intercalation, electrostatic binding was another reasonable binding mode between DDAs and DNA.
Many studies have demonstrated that diterpenoid alkaloids often had damage to tumor cells and normal cells with their cytotoxicity that interfere the DNA synthesis during the cell divisions. Several C19-norditerpenoids like neoline, aconitine, pubescenine, 14-deacetylajadine, lycoctonine, dehydrotakaosamine and ajadelphinine had irreversible effects on SkMel25, SW480, HeLa and PC12 cell lines[
Among the C19-diterpenoid alkaloids in our study, aconitine exhibited the strongest cytotoxic activity against the cells. Some case reports of Aconitum poisoning also illustrated that aconitine was a possible reason for fatal cardiac poisoning[
It is known that drug-DNA interaction is considered to be the reason for the DNA damage of some drugs[
Rat myocardial cells H9c2 were obtained from Shanghai Institutes for Biological Sciences (Shanghai, China). AC, MA and HA were provided by Chengdu Must Bio-Technology Co., LTD (Chengdu, China). Trypsin/ethylene diaminetetraacetic acid (EDTA), fetal bovine serum (FBS) and Dimethyl sulfoxide (DMSO) were obtained from Gibco (NY, USA). Glutamine Minimum essential medium (DMEM) (99.9%), NR and Calf thymus DNA (CT-DNA) were obtained from Sigma (MO, USA).
CT-DNA was dissolved in 50 mM Tris-HCl buffer (pH 7.4) (including 150 mM NaCl). The concentration of stock solution for DNA was calculated to be 2 mM by absorption at 260 nm, following Beer-Lambert Law, using the molar absorption coefficient ε
The stock solutions were stored at 4 °C for more than 24 h to obtain homogeneity.
Rat myocardial cells H9c2 were maintained in DMEM supplemented with 1% glutamine, 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin in an atmosphere at 37 °C humidified with 5% CO
Rat myocardial cells H9c2 were seeded at 10
The morphologies of rat myocardial cells H9c2 after exposure to 6.9 × 10
After treated with AC (2.5 × 10
After exposure to AC (12.0 × 10
0.4 mL of 2 mM CT-DNA, 1 mL of 0.1 mM NR and 4 milliliter of Tris-HCl buffer, were mixed into a 10 mL test tube. Different concentrations of DDAs (AC: 4.0~16.0 × 10
In order to determine the optimal molar ratio of probe and CT-DNA, the fluorescence intensity of 0.1 μM NR with varying the concentrations of CT-DNA from 0 μM to 2 μM were measured. Then, the solution, containing a certain concentration of NR-DNA complex ([DNA]/[NR] = 8, the fluorescence intensity of NR dye could not increase with sequentially adding DNA into NR solution), was titrated by successive addition of DDAs (AC: 4.0~16.0 × 10
Thermal denaturation experiments were carried out by monitoring the absorbance intensities of the CT-DNA with or without DDAs at different temperatures. The absorbance at 259 nm were plotted as a function of the temperature ranging from 20 °C to 100 °C. The melting temperature (Tm) of DNA and DNA adduct were estimated using the equation f
Sodium chloride was used to study the influence of ionic strength on the fluorescence intensity of the DDA-NR-DNA systems. DNA-NR and DDA-NR-DNA solutions fixed with NaCl at concentrations of 0.1 M, 0.2 M and 0.3 M were prepared respectively, and fluorescence intensities were measured.
SPSS 16.0 statistical software package was carried out for statistical analysis. Multiple comparisons were performed by one-way ANOVA, following by the SNK test for differences between groups. The significance was tested as p < 0.05.
A correction to this article is available online at https://doi.org/10.1038/s41598-018-30986-6.
This work was supported by Hebei Education Department, China (Grant number Z2014030); Hebei Food and Drug Administration, China (Grant number PT2014054); Doctoral Fund of Hebei University of Chinese Medicine (Grant number BSZ2017009).
The experiment was designed by W.K., X.T., X.H. and X.L. performed the experiment and analyzed the data. N.L. collected the samples and supervised all data analysis. F.L. wrote the manuscript.
The authors declare that they have no competing interests.
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By Fei Liu; Xiaoxin Tan; Xu Han; Xiang Li; Nan Li and Weijun Kang