The present studies have been executed to explore the protective mechanism of carnosic acid (CA) against NaAsO2-induced hepatic injury. CA exhibited a concentration dependent (1–4
Arsenic (As) is a toxic metalloid, which raises much disquiet in the health standpoints for human and animals [
Carnosic acid (CA), (4aR,10aS)-5,6-dihydroxy-1,1-dimethyl-7-propan-2-yl-2,3,4,9,10,10a-hexahydrophenanthrene-4a-carboxylic acid, is a naturally occurring phenolic diterpene (Figure
Structure of carnosic acid.
CA, bovine serum albumin (BSA), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Bradford reagent, and collagenase type I were procured from Sigma-Aldrich Chemical Company, MO, USA. Antibodies for immunoblotting were bought from Novus Biologicals, CO, USA. The solvents (HPLC grade) were obtained from Merck, Mumbai, India. Kits for the measurement of different biochemical parameters were procured from Span Diagnostics Ltd., India. 1-Chloro-2,4-dinitrobenzene, (NH4)2SO4, NaAsO2, 2,4-dinitrophenylhydrazine, ethylenediaminetetraacetic acid, 5,5-dithiobis(2-nitrobenzoic acid), N-Ethylmaleimide, nitro blue tetrazolium, NADH, KH2PO4, phenazine methosulphate, Na4P2O7, GSH, NaN3, thiobarbituric acid, 5-thio-2-nitrobenzoic acid, and CCl3COOH were obtained from Sisco Research Laboratory, Mumbai, India.
Healthy Swiss albino mice (♂, ∼2 month old, 25 ± 5 g) were procured from Chakraborty Enterprise, Kolkata, India and were housed in standard polypropylene cages (29 × 22 × 14 cm) in the animal house of the Department of Pharmaceutical Technology, Jadavpur University, India. The mice were maintained with temperature (22 ± 2°C), humidity (40 ± 10%), and 12 h light–dark cycle [
Hepatocytes were isolated from the liver of immediately sacrificed albino mice by two-step in situ collagenase perfusion as described by Dua et al. [
To determine the effect of CA on hepatocytes, hepatocytes (~2 × 106 cells/set) were exposed to CA (1, 2, 4, 6, and 10
To determine the cytoprotective effect of CA, hepatocytes (~2 × 106 cells/set) were exposed to NaAsO2 (12
Hoechst 33258 nuclear staining has been executed to study the cytotoxic events [
The flow cytometric study has been performed to accomplish the nature of cell death. Briefly, hepatocytes were exposed to NaAsO2 (12
Different sets of hepatocytes, each containing 1 ml of suspension (~2 × 106 cells/ml), were used in experiments. The prophylactic role of CA against NaAsO2 intoxication was analyzed by incubating hepatocytes with CA (4
The protein samples of hepatocytes for specific cellular components namely whole cell, cytosolic, and mitochondrial fractions were separated following standard sequential fractionation procedure as described by Baghirova and coworkers [
The
Gr I: normal control, mice received only 1% tween 80 in distilled water (1 ml,
Gr II: toxic control, mice were treated with NaAsO2 (10 mg/kg body weight,
Gr III: animals were treated with CA (10 mg/kg body weight,
Gr IV: animals were treated with CA (20 mg/kg body weight,
The doses of CA were selected on the basis of
The food intake and water intake were monitored on a daily basis. After 15 days, the mice were fasted overnight and were sacrificed by cervical dislocation under CO2 anesthesia. Before sacrificing the mice, body weight was recorded. For measurements of haematological parameters and biochemical markers in the sera, blood samples were collected from retro-orbital venous plexus after applying tetracaine (0.5%, one drop) ophthalmic anesthetic drop to the eyes. The livers were excised and cleaned with PBS. The weight of liver was recorded. The organs were homogenized immediately in Tris–HCl (0.01 M) + EDTA (0.001 M) buffers of pH 7.4 and centrifuged (12,000
A schematic outline of
Total erythrocyte count was measured using a haemocytometer and haemoglobin content was estimated using a haemoglobinometer. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) in the sera were estimated by commercially available kits (Span Diagnostics Limited, India) following manufacturer’s protocol.
The As contents in liver and urine of experimental animals were analyzed following the method of Das et al. [
Cytosolic fractions from livers of experimental mice were isolated following subcellular fractionation protocol employing centrifugation methods mentioned by Abcam, Cambridge, MA, USA. Cytosolic ATP level in the hepatic tissue was estimated by the commercially available assay kit (Abcam, Cambridge, MA, USA) following manufacturer’s protocol. The extent of DNA fragmentation was measured by the diphenylamine reaction as described by Lin et al. [
The protein samples of liver for specific cellular components, namely cytosolic, mitochondrial, and nuclear fractions, were separated, employing sequential fractionation process [
The livers from the experimental mice were immediately fixed in 10% buffered formalin and were processed for paraffin sectioning. Sections (~5
The experimental data were denoted as mean ± SD. The data were statistically examined by one-way ANOVA followed by Dunnett’s
In silico ADME properties of CA were predicted using QikProp module of Maestro Schrödinger software [
The high-resolution X-ray crystallographic structures of proteins were retrieved from Protein Data Bank (PDB) accessed on June 2017 [
Following molecular docking, the protein-ligand complex was subjected to molecular dynamics (MD) simulation study using the Desmond program (Desmond Molecular Dynamics System and Maestro-Desmond Interoperability Tools, Schrödinger) [
Reduction of cell viability is the index of cytotoxicity. In search of the cytotoxic effect of NaAsO2, hepatocytes were incubated with NaAsO2 at different concentrations for 2 h. The cell viability was reduced by NaAsO2 in a concentration-dependent manner (Figure
The cell viability, image, and flow cytometric assays in the absence (NaAsO2) and presence of CA (NaAsO2 + CA)
The hepatocytes incubated with CA (1–6
NaAsO2 (12
The cytoprotective effect of CA has been estimated by Hoechst staining following visualization through fluorescence microscope (Figure
To investigate the nature of cell death, hepatocytes under different treatments were assessed by flow cytometric analysis. Flow cytometric data (Figure
The prophylactic effects of CA on NaAsO2-induced oxidative stress in isolated murine hepatocyte have been depicted in Figure
The effect on ROS accumulation, lipid peroxidation, protein carbonylation, and endogenous redox systems in the absence (NaAsO2) and presence of CA (NaAsO2 + CA) in isolated murine hepatocytes. Values are represented as mean ± SD (
The prophylactic effects of CA on NaAsO2-induced intrinsic and extrinsic apoptotic signaling have been shown in Figure
The effect on intrinsic and extrinsic apoptotic signaling in the absence (NaAsO2) and presence of CA (NaAsO2 + CA)
To investigate the proapoptotic effect of NF-
The effect on I
During the tenure of experiment, no significant change in food and water intake was recorded in the animals of either of experimental group. NaAsO2 (10 mg/kg) treated mice exhibited significant (
Effects on body weight, liver weight, hepatic As content, and urinary As content in the absence (NaAsO2) and presence of CA (CA + NaAsO2) in mice.
Parameters | Gr I | Gr II | Gr III | Gr IV |
---|---|---|---|---|
Body weight (g) | 28.16 ± 3.11 | 21.77 ± 2.08# | 26.08 ± 3.01 |
26.92 ± 3.33 |
Liver weight (g) | 1.24 ± 0.21 | 1.15 ± 7.29 | 1.17 ± 0.13 | 1.22 ± 0.25 |
Liver As ( |
0.06 ± .0001 | 0.51 ± 0.04# | 0.35 ± 0.04 |
0.32 ± 0.02 |
Urinary As ( |
2.34 ± 0.31 | 19.65 ± 2.17# | 23.67 ± 2.98 |
26.01 ± 3.22 |
Values are expressed as mean ± SD (
Blood parameters give crucial impression of pathological state within the system. The effects of CA on blood parameters of experimental mice were reported in Table
Effects on haematological and serum biochemical parameters in the absence (NaAsO2) and presence of CA (CA + NaAsO2) in mice.
Parameters | Gr I | Gr II | Gr III | Gr IV |
---|---|---|---|---|
Total erythrocyte count (×106/mm3) | 6.28 ± 0.42 | 3.22 ± 0.34# | 4.04 ± 0.25 |
5.19 ± 0.67 |
Haemoglobin (g/dl) | 8.76 ± 0.72 | 4.89 ± 0.43# | 6.12 ± 0.48 |
6.88 ± 0.87 |
ALT (IU/I) | 68.54 ± 5.81 | 112.98 ± 9.63# | 89.85 ± 7.62 |
82.17 ± 6.15 |
AST (IU/I) | 58.12 ± 4.63 | 82.91 ± 7.29# | 73.62 ± 5.82 |
64.57 ± 5.51 |
CK (IU/mg protein) | 10.87 ± 1.05 | 18.65 ± 1.24# | 14.22 ± 0.98 |
13.78 ± 1.33 |
LDH (U/l) | 171.34 ± 13.42 | 252.67 ± 21.50# | 198.33 ± 20.12 |
187.54 ± 17.72 |
Values are expressed as mean ± SD (
In
The effect on ROS accumulation, lipid peroxidation, protein carbonylation, and endogenous redox systems in the absence (NaAsO2) and presence of CA (NaAsO2 + CA)
In this study, the expressions of different apoptotic proteins in the liver of experimental mice were assessed by Western blotting (Figure
The effect on intrinsic and extrinsic apoptotic signaling in the absence (NaAsO2) and presence of CA (NaAsO2 + CA)
To investigate the effect on NF-
The effect on I
In this study, NaAsO2 (10 mg/kg) treatment caused significant (
The effect on ATP, DNA fragmentation and DNA oxidation in the absence (NaAsO2) and presence of CA (NaAsO2 + CA)
The histological sections of livers of experimental mice were depicted in Figures
Histological sections 100x (a) and 400x (b) of the livers of experimental mice in the absence (NaAsO2) and presence of CA (NaAsO2 + CA). The liver sections of normal mice revealed normal portal vein and hepatocytes. NaAsO2-exposed liver section exhibited dilated portal vein (red arrow), vacuolated cytoplasm (blue arrows), apoptosis (yellow arrows), and leucocytes infiltration (green arrows) when compared with the section of normal control liver. CA treatment could reinstate NaAsO2 mediated the aforementioned pathological changed. Gr I: normal control; Gr II: toxic control; Gr III: CA (10 mg/kg) + NaAsO2 (10 mg/kg), Gr IV: CA (20 mg/kg) + NaAsO2 (10 mg/kg).
The drug-likeness and pharmacokinetic properties of CA was evaluated using QikProp module of Schrödinger. Drug-likeness and physicochemical properties were found to be as molecular weight = 332.4, H-bond donor = 3, H-bond acceptor = 3.5, predicted water partition coefficient = 3.7, aqueous solubility = −4.4, and total solvent accessible surface area = 575.8. The pharmacokinetic profiles, such as predicted brain/blood partition coefficient = −0.7, predicted apparent Caco-2 cell permeability = 182.7, number of likely metabolic reactions = 4, and human oral absorption = 89.3%, are significantly compliant with recommended values of drug-likeness. In Table
Prediction of drug-likeness and ADME profiles of CA.
Sl. number | Descriptors | Predicted values for CA | Recommended values (based on properties of 95% of known drugs) |
---|---|---|---|
1. | Molecular weight | 332.4 | 130.0 to 725.0 |
2. | SASA | 575.8 | 300.0 to 1000.0 |
3. | WPSA | 0.0 | 0.0 to 175.0 |
4. | H-bond donor | 3.0 | 0.0 to 6.0 |
5. | H-bond acceptor | 3.5 | 2.0 to 20.0 |
6. | glob | 0.8 | 0.75 to 0.95 |
7. | QPlogPo/w | 3.7 | −2.0 to 6.5 |
8. | QPlogS | −4.4 | −6.5 to 0.5 |
9. | QPlogHERG | −1.9 | Concern below −5 |
10. | QPPCaco | 182.7 | 25 poor; >500 great |
11. | QPlogBB | −0.7 | −3.0 to 1.2 |
12. | QPPMDCK | 100.2 | <25 poor, >500 great |
13. | QPlogKp | −3.2 | −8.0 to −1.0 |
14. | #metab | 4.0 | 1.0 to 8.0 |
15. | QPlogKhsa | 0.3 | −1.5 to 1.5 |
16. | % of human oral absorption | 89.3 | >80% is high; <25% is poor |
SASA: total solvent accessible surface area; WPSA: weakly polar component of the SASA; glob: globularity descriptor; QPlogPo/w: predicted octanol/water partition coefficient; QPlogS: predicted aqueous solubility; QPlogHERG: predicted IC50 value for blockage of HERG K+ channels; QPPCaco: predicted apparent Caco-2 cell permeability; QPlogBB: predicted brain/blood partition coefficient; QPPMDCK: predicted apparent MDCK cell permeability in nm/sec; QPlogKp: predicted skin permeability; #metab: number of likely metabolic reactions; QPlogKhsa: prediction of binding to human serum albumin.
Molecular docking analysis was attempted to dig into the possible binding patterns and interactions of CA with the selective signal proteins. Generated docking poses were ranked on the basis of Glide score. The lowest scored conformer was considered to be the best docking pose. Glide score represents the most capable best fit for a ligand in the active site of the target protein. In this study, Apaf-1 (PDB: 1Z6T), cytochrome C (PDB: 3ZCF), Bid (PDB: 4QVE), Fas (PDB: 3EZQ), and p53 (PDB: 2XWR) did not demonstrate significant docked pose after docking with CA; therefore, these proteins were discarded for further in silico analysis. Hydrogen bond (H-bond) interactions with the catalytic amino acid residues along with Glide score and Emodel values of other proteins were presented in Table
Dock score, Emodel value, and interacting residues in molecular docking analysis.
Sl. number | Proteins | Glide scores (Kcal/mol) | Glide Emodel values | Interacting residues in H-bond interaction | Other interactions |
---|---|---|---|---|---|
1. | Bax | −3.115 | −15.341 | Arg 37, Leu 122 | Cation- |
2. | Bcl-2 | −4.986 | −29.586 | — | H-bond with H2O |
3. | Caspase 9 | −3.596 | −23.725 | Arg 355 | π-π stacking, salt bridge with Arg 355 |
4. | Caspase 3 | −4.263 | −30.352 | Arg 207, Thr 62 | — |
5. | Caspase 8 | −4.060 | −19.905 | Glu 396, Thr 467 | — |
6. | I |
−6.524 | −33.382 | Asp 103, Leu 21 | — |
7. | NF- |
−6.042 | −36.121 | Arg 416, Leu 472 | π-π stacking with Arg 408 |
8. | JNK | −4.642 | −17.079 | Asn 114 | — |
9. | p38 | −5.608 | −30.359 | — | — |
Docking interactions of CA with Bax (a), Bcl-2 (b), caspase 9 (c), caspase 3 (d), and caspase 8 (e) proteins.
The binding interactions of CA with I
Docking interactions of CA with I
The MD simulation was carried out for further refinement and stabilization of the docked complexes in dynamic environment to evaluate the most energetically stable binding conformation. The simulation time range of 20–40 ns was used to allow and permit reorganization of the interaction configuration of protein-ligand complex. In the simulation study, the compactness of each simulated complex has been analyzed through root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration, and the secondary structure elements (SSE) of CA ligand interactions with the selective protein along with stable H-bond and hydrophobic interactions. The RMSD plot of Bax-CA complex showed that there is significant deviation till 30 ns of run. However, the complex was thereafter found to be appropriately stable and consistent until reaching to the end of the run (Figure
MD-simulated RMSD plot of Bax (a), Bcl-2 (b), caspase 9 (c), caspase 3 (d), and caspase 8 (e) backbones and CA ligand complex.
MD-simulated RMSD plot of I
Arsenites-contaminated drinking water has been regarded as a major hazard over 70 countries in the world [
Present studies have been executed to evaluate the probable prophylactic role of CA against As intoxication in the liver of experimental mice employing established
Haematological and serum biochemical status give primary indications of toxicological proceedings within the body and/or in the precise organs. In this study, NaAsO2 exposure significantly reduced erythrocyte count and thereby exhibited a significant reduction in haemoglobin level in mice. As-induced oxidative haematotoxicity has been reported in earlier literatures [
The earlier explanations revealed that NaAsO2 exposure significantly promoted intracellular ROS generation in both
Cellular redox defense systems play an imperative role to neutralize oxidative stress [
ROS has been reported to endorse apoptosis via alteration in the expressions of different signal proteins from their normal transcriptional levels [
Utilizing chemometric analysis, valuable information on molecular basis has been elucidated for different protein-ligand complex structures in silico. In this study, Bax, Bcl-2, caspase 9, caspase 3, caspase 8, I
NaAsO2-mediated production and accumulation of oxidative free radicals play significant role in the As-induced hepatotoxicity. The present study demonstrated that NaAsO2 can elicit hepatocellular apoptosis by triggering NF-
Schematic presentation probable protective mechanism of CA against NaAsO2-mediated hepatic injury. The red lightning bolts indicated the pathological events involved within NaAsO2-exposed hepatic cells. The green lines denoted the activity restricted by CA.
The authors declare that there is no conflict of interests.
Saikat Dewanjee, Prasenjit Manna, Supratim Ray, and Vincenzo De Feo designed the experiments. Saikat Dewanjee and Prasenjit Manna supervised the work. Swarnalata Joardar, Prasenjit Manna, and Jatin Kalita performed
The authors gratefully acknowledge the University Grants Commission (UGC), New Delhi, India, for providing the Rajiv Gandhi National Fellowship (Ref. no. F1-17.1/2013-14/RJNF-2013-14-SC-ASS-52029 (SA-III/Website) dated 06.02.3014) to Mister Sonjit Das. The authors are thankful to the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, for providing the Ramalingaswami Re-Entry Fellowship (Grant no. BT/RLF/Re-Entry/34/2013) to Dr. Prasenjit Manna. The authors are thankful to Jadavpur University, Kolkata, India, and the Director, CSIR-NEIST for providing necessary facilities for this study. The authors are thankful to Ms. Cassandra Warden, Vanderbilt University Medical Center, Department of Ophthalmology, USA, for excellent editing of this manuscript.
Supplementary Figure 1: effect of NaAsO2 at different concentrations on cell viability in isolated murine hepatocytes. Values are expressed as mean ± SD (