DOI 10.1515/jbcpp-2013-0134 J Basic Clin Physiol Pharmacol 2014; 25(4): 329–339
Sabbir Khan and Gopabandhu Jena*
Sodium valproate, a histone deacetylase inhibitor ameliorates cyclophosphamide-induced genotoxicity and cytotoxicity in the colon of mice Abstract Background: Cyclophosphamide (CP) is an alkylating anticancer drug used for the treatment of various cancer and noncancer disorders. Toxicity of CP is well characterized using different test systems. However, its intestinal genotoxicity and cytotoxicity are the least explored and the mechanism is not fully investigated. Valproic acid (VPA) has been reported as a histone deacetylase (HDAC) inhibitor, which modulates the cytotoxicity of anticancer drugs. The present study aimed to investigate the influence of VPA on CPinduced genotoxicity and cytotoxicity in the colon of mice. Methods: In the 16-day experiment, animals were treated with VPA alone (500 mg/kg/day), CP alone (50 mg/kg, on the 4th, 8th, 12th, and 16th days), and the combination of CP and VPA, while in the 28-day experiment, animals were treated with VPA alone (300 mg/kg/day, 5 days/week), CP alone (100 mg/kg/week), and the combination of low and high dose of VPA (VPA150+CP and VPA300+CP). Animals were sacrificed 24 h after the administration of the last dose. The influence of VPA treatment on CP-induced genotoxicity and cytotoxicity was assessed by the evaluation of oxidative stress, DNA damage, histology, and the expression of 8-hydroxy-guanosine and phosphorylated histone H2AX by immunohistochemistry. Results and conclusions: The present study’s results demonstrated that VPA treatment significantly decreased the CP-induced DNA damage, cytotoxicity, and expression of γH2AX in the colon as revealed by the comet assay and histological as well as immunohistochemical evaluation. VPA treatment significantly ameliorated the CP-induced DNA damage and cytotoxicity in the colon of mice. Keywords: colon; cyclophosphamide; DNA damage; HDAC inhibitor; sodium valproate. *Corresponding author: Dr. Gopabandhu Jena, Facility for Risk Assessment and Intervention Studies, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab-160062, India, Phone: +91-172-2214683 ext. 2152, Fax: +91-172-2214692, E-mail: [email protected], [email protected]
Sabbir Khan: Facility for Risk Assessment and Intervention Studies, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab, India
Introduction Cyclophosphamide (CP), a cytotoxic bifunctional alkylating agent that belongs to the nitrogen mustard class, is used for the treatment of cancer, rheumatoid arthritis, and systemic lupus erythematosus and as an immune suppressant [1, 2]. Further, CP induces gene mutations, chromosome aberrations, micronuclei, sister chromatid exchanges, and gonadal as well as intestinal toxicity [1, 3–5]. However, the mechanism of intestinal genotoxicity and cytotoxicity of CP is not fully understood and explored [5–7]. Sodium valproate [valproic acid (VPA)] has been reported as a histone deacetylase (HDAC) inhibitor, and it modulates several molecular pathways of the cell as well as the cytotoxicity of anticancer drugs [8–12]. Many clinical trials are in progress for the evaluation of the anticancer efficacy of the combination treatment of HDAC inhibitors and cytotoxic drugs [9, 13]. VPA induces chromatin decondensation by HDAC inhibition, which is a dose- and time-dependent phenomenon, and thereby increases the accessibility of DNA for transcription factors, macromolecules, and xenobiotics [14, 15]. Moreover, HDAC inhibitors induce apoptosis in the tumor as well as normal cells, but they respond differently to the normal and tumor cells [8, 16, 17]. The dynamic nature of the epigenome and its responsivity to multiple cellular signaling pathways suggest that epigenetic variations could potentially be established at distinct stages of life in specific tissues, which has subsequent implications on the drug action and toxic effects. Further, cell-specific epigenetic variation could result in the differential pharmacodynamics, pharmacokinetics, and toxicity of drugs [18]. VPA and butyrate are short-chain fatty acid (SCFA) HDAC inhibitors, which regulate colonic epithelial cell
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330 Khan and Jena: Valproate ameliorates cyclophosphamide toxicity maturation, cell cycle arrest, lineage-specific differentiation, and apoptosis through several mechanisms [19, 20]. Further, microbe-derived SCFAs regulate the host gene expression, which is involved in intestinal homeostasis as well as in carcinogenesis by the modulation of micro-RNAs [21]. HDAC inhibitors such as SAHA and trichostatin A suppress cisplatin-induced p53 activation and apoptosis as well as cytotoxicity in the renal tubular cells [17, 22]. Moreover, HDAC inhibitors act as a radioprotectant, which suppresses cutaneous mucositis as well as DNA damage in vivo [23–25]. VPA is the most promising pleiotropic agent and exerts several viable clinical therapeutic opportunities for the treatment of different cancerous as well as noncancerous diseases [10, 26–28]. Additionally, VPA has exhibited a protective role in colitis, burns, and inflammation in experimental models, which indicates the multiple mechanisms of VPA [26, 29, 30]. Together, it is appropriate to investigate the influence of VPA (HDAC inhibitor) on CP-induced toxicity in proliferating cells such as the colon. Therefore, we hypothesized that VPA treatment may ameliorate CP-induced genotoxicity and cytotoxicity in the colon of mice.
Materials and methods Animals All the animal experiment protocols were approved by the Institutional Animal Ethics Committee (IAEC), and experiments were performed on male albino Swiss mice (25–30 g and approximately 8 weeks of age) in accordance with the Committee for the Purpose of Control and Supervision of Experimentation on Animals guidelines. The IAEC approval number for the present study was IAEC/10/36. All the animals were kept under controlled environmental conditions at room temperature (22 ± 2°C) with 50% ± 10% humidity and an automatically controlled 12-h light and dark cycle. Standard laboratory animal feed and water were provided ad libitum.
Chemicals Sodium valproate (CAS no. 1069-66-5), CP (CAS no. 6055-19-2), bovine serum albumin (CAS no. 9048-46-8), hematoxylin and eosin (H&E), Trizma (CAS no. 77-86-1), 3,3′diaminobenzidine tetrachloride (CAS no. 868272-85-9), and SYBR Green I (CAS no. 163795-75-3) were purchased from Sigma-Aldrich Chemicals (Saint Louis, MO, USA). Dimethylsulfoxide (DMSO), normal melting point agarose (NMPA), low melting point agarose (LMPA), Triton X-100, ethylenediaminetetraacetic acid (EDTA), and Hank’s balanced salt solution (HBSS)
were obtained from HiMedia Laboratories Ltd. (Mumbai, India). Mouse anti-8-hydroxy-guanosine (8-oxy-dG) was purchased from Abnova Corporation (Taipei city, Taiwan), and rabbit anti-phosphorylated histone H2AX (γH2AX), goat antimouse, and goat antirabbit secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Dose selection, experimental design, and animal treatment The initial dose of CP (50 mg/kg) for the 16-day experiment was selected on the basis of studies conducted by Tripathi and Jena [31], while the dose of VPA (500 mg/kg) was selected on the basis of previous reports [15, 32]. The doses of CP (100 mg/kg) and VPA (150 and 300 mg/kg) for the 28-day experiment were selected on the basis of the results of the 16-day experiment. Both VPA and CP were dissolved in distilled water and administrated by the intraperitoneal route according to the body weights of animals. In the present work, two separate 16- and 28-day experiments were performed. For the 16-day experiment, all the animals were randomized into four groups consisting of 10 animals per group to assess the influence of VPA on CP-induced genotoxicity and cytotoxicity in the colon. The experimental design consists of different treatment groups as follows: group 1, control (untreated animals); group 2, CP alone; group 3, VPA alone; and group 4, combination of CP and VPA. VPA was given at the dose of 500 mg/kg/day for 16 days, while CP was given at the dose of 50 mg/kg on the 4th, 8th, 12th, and 16th days. For the 28-day experiment, all the animals were randomized into five groups consisting of 10 animals in each group: group 1, control (untreated animals); group 2, CP alone; group 3, VPA alone (VPA 300 mg/kg); groups 4 and 5, combination of low and high dose of VPA with CP (CP+VPA150 and CP+VPA300). VPA was given at the dose of 150 and 300 mg/kg for 5 days/week for 4 weeks and CP was given at the dose of 100 mg/kg/week for 4 weeks. To ensure that amelioration of CP-induced toxicity is really due to VPA treatment, we designed the 28-day experiment at two doses of VPA to differentiate the effect of VPA and/or CP, which was unclear in the 16-day experiment. All the animals were sacrificed by cervical dislocation 24 h after the administration of the last dose. The colon was isolated immediately and cleaned in cold normal saline, then placed in different buffers/mediums for respective assays as mentioned in the method section.
Determination of malondialdehyde level The lipid peroxidation [malondialdehyde (MDA)] level in the colon was measured according to a method previously described [33], with some modifications. The colon tissue was collected and homogenized in ice-cold phosphate buffer (pH7.4) containing EDTA for the determination of lipid peroxidation levels. After homogenization and centrifugation, the supernatant was collected and the MDA level was estimated spectrophotometrically as an end product of lipid peroxidation using the thiobarbituric acid reactive substance method. Lipid peroxidation was calculated from the standard curve generated using 1,1,3,3-tetramethoxy propane (97%) and expressed as nM MDA/mg of protein.
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Khan and Jena: Valproate ameliorates cyclophosphamide toxicity 331
Determination of reduced glutathione level
extent of structural damage in the mucosal epithelium as follows: 0 = normal morphology, 1 = loss of goblet cells, 2 = loss of goblet cells in large areas, 3 = loss of crypts, and 4 = loss of crypts in large areas.
An equal volume of 5% sulfosalicylic acid was added to the colon supernatant from above and was vortexed. The mixture was kept for 30 min in ice bath then centrifuged at 10,000 rpm for 10 min, and the supernatant was collected carefully without disturbing the pellet. Glutathione (GSH) content was measured using Ellmann’s reagent [5,5′-dithiobis(2-nitro-benzoic acid)] solution according to a method described previously [34]. GSH level was calculated by a standard reference curve using reduced GSH as a standard. Results were expressed as μM GSH/mg protein for each group.
Alkaline comet assay in the colon A small piece of the colon was placed in 1 mL cold HBSS containing 20 mM EDTA and 10% DMSO and was minced to make a single cell suspension. In brief, 10 μL of cell suspension was mixed with 90 μL LMPA and layered over the surface of a frosted slide (precoated with 1% NMPA) to form a microgel and was allowed to solidify at 4°C for 5 min. A second layer of 1% LMPA was added and allowed to solidify at 4°C for 5–10 min. The slides were immersed in a lysis solution at 4°C for 24 h. After, the 24-h slides were placed in a specifically designed horizontal electrophoresis tank and DNA was allowed to unwind for 20 min in alkaline running buffer containing 300 mM NaOH and 1 mM EDTA (pH > 13.0) and electrophoresis was carried out at 300 mA and 30 V for 30 min, then stained with SYBR Green I (1:10,000 dilution). The fluorescent labeled DNA was visualized (200 × ) using an AXIO Imager M1 fluorescence microscope (Carl Zeiss, Germany) and the resulting images were captured on a computer and processed with image analysis software (Comet Imager V.2.0.0). The parameters for the DNA damage analysis include tail length (in μM), tail moment (TM), olive TM, and percentage tail DNA (%DNA).
Determination of protein content Protein concentration in the colon homogenate was determined as described by Khan et al. [34], with bovine serum albumin as the standard protein. The principle of this method is based on the reaction of Cu+, produced by the oxidation of peptide bonds with FolinCiocalteu reagent and which produced a blue complex, which is proportional to protein concentration.
Histological evaluation and quantification Histological slides were prepared as per standardized protocol followed in our laboratory and all histological quantifications were performed as described [34]. In brief, the colon was fixed in 10% neutral buffer formalin, dehydrated gradually in ethanol and xylene, then embedded in paraffin and sections were deparaffinized with xylene and rehydrated with alcohol and water. The rehydrated sections were stained using H&E and mounted with DPX mounting media, then examined under a microscope (Olympus BX51 microscope; Tokyo, Japan). Further, histological alterations were quantified in the colon as described by Yoshihara et al. [35]. For histological score, randomly, 10–15 fields were selected from each animal and assigned the score depending upon the
A
32
Control
CP
VPA
VPA+CP
B
28 26
*a
24
*b
@a @b
22
Body weight, g
Body weight, g
After deparaffinization and rehydration, sections were incubated in the citrate buffer (0.01 M, pH6.0) at 95°C for 20–30 min for antigen retrieval and endogenous peroxidase was blocked by 3% H2O2 for 10 min. Nonspecific binding was blocked by incubating with bovine serum albumin milk, then incubation with mouse anti-8-oxo-dG and rabbit anti-γH2AX primary antibody (dilution, 1:50) at 4°C for overnight in a humidified chamber followed by incubation with HRP
34 32
30
20
Immunohistochemistry of γH2AX and 8-oxo-dG in the colon sections
30 28 26 24 Control CP VPA300
22 0
8 Days
16
20
0
1
VPA150+CP VPA300+CP
2 Weeks
3
4
Figure 1 (A and B) Effect of VPA and CP alone as well as the combination treatment on the body weight of mice in the 16- and 28-day experiments. Values are expressed as mean ± SEM, (n = 10). @p
Sabbir Khan and Gopabandhu Jena*
Sodium valproate, a histone deacetylase inhibitor ameliorates cyclophosphamide-induced genotoxicity and cytotoxicity in the colon of mice Abstract Background: Cyclophosphamide (CP) is an alkylating anticancer drug used for the treatment of various cancer and noncancer disorders. Toxicity of CP is well characterized using different test systems. However, its intestinal genotoxicity and cytotoxicity are the least explored and the mechanism is not fully investigated. Valproic acid (VPA) has been reported as a histone deacetylase (HDAC) inhibitor, which modulates the cytotoxicity of anticancer drugs. The present study aimed to investigate the influence of VPA on CPinduced genotoxicity and cytotoxicity in the colon of mice. Methods: In the 16-day experiment, animals were treated with VPA alone (500 mg/kg/day), CP alone (50 mg/kg, on the 4th, 8th, 12th, and 16th days), and the combination of CP and VPA, while in the 28-day experiment, animals were treated with VPA alone (300 mg/kg/day, 5 days/week), CP alone (100 mg/kg/week), and the combination of low and high dose of VPA (VPA150+CP and VPA300+CP). Animals were sacrificed 24 h after the administration of the last dose. The influence of VPA treatment on CP-induced genotoxicity and cytotoxicity was assessed by the evaluation of oxidative stress, DNA damage, histology, and the expression of 8-hydroxy-guanosine and phosphorylated histone H2AX by immunohistochemistry. Results and conclusions: The present study’s results demonstrated that VPA treatment significantly decreased the CP-induced DNA damage, cytotoxicity, and expression of γH2AX in the colon as revealed by the comet assay and histological as well as immunohistochemical evaluation. VPA treatment significantly ameliorated the CP-induced DNA damage and cytotoxicity in the colon of mice. Keywords: colon; cyclophosphamide; DNA damage; HDAC inhibitor; sodium valproate. *Corresponding author: Dr. Gopabandhu Jena, Facility for Risk Assessment and Intervention Studies, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab-160062, India, Phone: +91-172-2214683 ext. 2152, Fax: +91-172-2214692, E-mail: [email protected], [email protected]
Sabbir Khan: Facility for Risk Assessment and Intervention Studies, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab, India
Introduction Cyclophosphamide (CP), a cytotoxic bifunctional alkylating agent that belongs to the nitrogen mustard class, is used for the treatment of cancer, rheumatoid arthritis, and systemic lupus erythematosus and as an immune suppressant [1, 2]. Further, CP induces gene mutations, chromosome aberrations, micronuclei, sister chromatid exchanges, and gonadal as well as intestinal toxicity [1, 3–5]. However, the mechanism of intestinal genotoxicity and cytotoxicity of CP is not fully understood and explored [5–7]. Sodium valproate [valproic acid (VPA)] has been reported as a histone deacetylase (HDAC) inhibitor, and it modulates several molecular pathways of the cell as well as the cytotoxicity of anticancer drugs [8–12]. Many clinical trials are in progress for the evaluation of the anticancer efficacy of the combination treatment of HDAC inhibitors and cytotoxic drugs [9, 13]. VPA induces chromatin decondensation by HDAC inhibition, which is a dose- and time-dependent phenomenon, and thereby increases the accessibility of DNA for transcription factors, macromolecules, and xenobiotics [14, 15]. Moreover, HDAC inhibitors induce apoptosis in the tumor as well as normal cells, but they respond differently to the normal and tumor cells [8, 16, 17]. The dynamic nature of the epigenome and its responsivity to multiple cellular signaling pathways suggest that epigenetic variations could potentially be established at distinct stages of life in specific tissues, which has subsequent implications on the drug action and toxic effects. Further, cell-specific epigenetic variation could result in the differential pharmacodynamics, pharmacokinetics, and toxicity of drugs [18]. VPA and butyrate are short-chain fatty acid (SCFA) HDAC inhibitors, which regulate colonic epithelial cell
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330 Khan and Jena: Valproate ameliorates cyclophosphamide toxicity maturation, cell cycle arrest, lineage-specific differentiation, and apoptosis through several mechanisms [19, 20]. Further, microbe-derived SCFAs regulate the host gene expression, which is involved in intestinal homeostasis as well as in carcinogenesis by the modulation of micro-RNAs [21]. HDAC inhibitors such as SAHA and trichostatin A suppress cisplatin-induced p53 activation and apoptosis as well as cytotoxicity in the renal tubular cells [17, 22]. Moreover, HDAC inhibitors act as a radioprotectant, which suppresses cutaneous mucositis as well as DNA damage in vivo [23–25]. VPA is the most promising pleiotropic agent and exerts several viable clinical therapeutic opportunities for the treatment of different cancerous as well as noncancerous diseases [10, 26–28]. Additionally, VPA has exhibited a protective role in colitis, burns, and inflammation in experimental models, which indicates the multiple mechanisms of VPA [26, 29, 30]. Together, it is appropriate to investigate the influence of VPA (HDAC inhibitor) on CP-induced toxicity in proliferating cells such as the colon. Therefore, we hypothesized that VPA treatment may ameliorate CP-induced genotoxicity and cytotoxicity in the colon of mice.
Materials and methods Animals All the animal experiment protocols were approved by the Institutional Animal Ethics Committee (IAEC), and experiments were performed on male albino Swiss mice (25–30 g and approximately 8 weeks of age) in accordance with the Committee for the Purpose of Control and Supervision of Experimentation on Animals guidelines. The IAEC approval number for the present study was IAEC/10/36. All the animals were kept under controlled environmental conditions at room temperature (22 ± 2°C) with 50% ± 10% humidity and an automatically controlled 12-h light and dark cycle. Standard laboratory animal feed and water were provided ad libitum.
Chemicals Sodium valproate (CAS no. 1069-66-5), CP (CAS no. 6055-19-2), bovine serum albumin (CAS no. 9048-46-8), hematoxylin and eosin (H&E), Trizma (CAS no. 77-86-1), 3,3′diaminobenzidine tetrachloride (CAS no. 868272-85-9), and SYBR Green I (CAS no. 163795-75-3) were purchased from Sigma-Aldrich Chemicals (Saint Louis, MO, USA). Dimethylsulfoxide (DMSO), normal melting point agarose (NMPA), low melting point agarose (LMPA), Triton X-100, ethylenediaminetetraacetic acid (EDTA), and Hank’s balanced salt solution (HBSS)
were obtained from HiMedia Laboratories Ltd. (Mumbai, India). Mouse anti-8-hydroxy-guanosine (8-oxy-dG) was purchased from Abnova Corporation (Taipei city, Taiwan), and rabbit anti-phosphorylated histone H2AX (γH2AX), goat antimouse, and goat antirabbit secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Dose selection, experimental design, and animal treatment The initial dose of CP (50 mg/kg) for the 16-day experiment was selected on the basis of studies conducted by Tripathi and Jena [31], while the dose of VPA (500 mg/kg) was selected on the basis of previous reports [15, 32]. The doses of CP (100 mg/kg) and VPA (150 and 300 mg/kg) for the 28-day experiment were selected on the basis of the results of the 16-day experiment. Both VPA and CP were dissolved in distilled water and administrated by the intraperitoneal route according to the body weights of animals. In the present work, two separate 16- and 28-day experiments were performed. For the 16-day experiment, all the animals were randomized into four groups consisting of 10 animals per group to assess the influence of VPA on CP-induced genotoxicity and cytotoxicity in the colon. The experimental design consists of different treatment groups as follows: group 1, control (untreated animals); group 2, CP alone; group 3, VPA alone; and group 4, combination of CP and VPA. VPA was given at the dose of 500 mg/kg/day for 16 days, while CP was given at the dose of 50 mg/kg on the 4th, 8th, 12th, and 16th days. For the 28-day experiment, all the animals were randomized into five groups consisting of 10 animals in each group: group 1, control (untreated animals); group 2, CP alone; group 3, VPA alone (VPA 300 mg/kg); groups 4 and 5, combination of low and high dose of VPA with CP (CP+VPA150 and CP+VPA300). VPA was given at the dose of 150 and 300 mg/kg for 5 days/week for 4 weeks and CP was given at the dose of 100 mg/kg/week for 4 weeks. To ensure that amelioration of CP-induced toxicity is really due to VPA treatment, we designed the 28-day experiment at two doses of VPA to differentiate the effect of VPA and/or CP, which was unclear in the 16-day experiment. All the animals were sacrificed by cervical dislocation 24 h after the administration of the last dose. The colon was isolated immediately and cleaned in cold normal saline, then placed in different buffers/mediums for respective assays as mentioned in the method section.
Determination of malondialdehyde level The lipid peroxidation [malondialdehyde (MDA)] level in the colon was measured according to a method previously described [33], with some modifications. The colon tissue was collected and homogenized in ice-cold phosphate buffer (pH7.4) containing EDTA for the determination of lipid peroxidation levels. After homogenization and centrifugation, the supernatant was collected and the MDA level was estimated spectrophotometrically as an end product of lipid peroxidation using the thiobarbituric acid reactive substance method. Lipid peroxidation was calculated from the standard curve generated using 1,1,3,3-tetramethoxy propane (97%) and expressed as nM MDA/mg of protein.
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Khan and Jena: Valproate ameliorates cyclophosphamide toxicity 331
Determination of reduced glutathione level
extent of structural damage in the mucosal epithelium as follows: 0 = normal morphology, 1 = loss of goblet cells, 2 = loss of goblet cells in large areas, 3 = loss of crypts, and 4 = loss of crypts in large areas.
An equal volume of 5% sulfosalicylic acid was added to the colon supernatant from above and was vortexed. The mixture was kept for 30 min in ice bath then centrifuged at 10,000 rpm for 10 min, and the supernatant was collected carefully without disturbing the pellet. Glutathione (GSH) content was measured using Ellmann’s reagent [5,5′-dithiobis(2-nitro-benzoic acid)] solution according to a method described previously [34]. GSH level was calculated by a standard reference curve using reduced GSH as a standard. Results were expressed as μM GSH/mg protein for each group.
Alkaline comet assay in the colon A small piece of the colon was placed in 1 mL cold HBSS containing 20 mM EDTA and 10% DMSO and was minced to make a single cell suspension. In brief, 10 μL of cell suspension was mixed with 90 μL LMPA and layered over the surface of a frosted slide (precoated with 1% NMPA) to form a microgel and was allowed to solidify at 4°C for 5 min. A second layer of 1% LMPA was added and allowed to solidify at 4°C for 5–10 min. The slides were immersed in a lysis solution at 4°C for 24 h. After, the 24-h slides were placed in a specifically designed horizontal electrophoresis tank and DNA was allowed to unwind for 20 min in alkaline running buffer containing 300 mM NaOH and 1 mM EDTA (pH > 13.0) and electrophoresis was carried out at 300 mA and 30 V for 30 min, then stained with SYBR Green I (1:10,000 dilution). The fluorescent labeled DNA was visualized (200 × ) using an AXIO Imager M1 fluorescence microscope (Carl Zeiss, Germany) and the resulting images were captured on a computer and processed with image analysis software (Comet Imager V.2.0.0). The parameters for the DNA damage analysis include tail length (in μM), tail moment (TM), olive TM, and percentage tail DNA (%DNA).
Determination of protein content Protein concentration in the colon homogenate was determined as described by Khan et al. [34], with bovine serum albumin as the standard protein. The principle of this method is based on the reaction of Cu+, produced by the oxidation of peptide bonds with FolinCiocalteu reagent and which produced a blue complex, which is proportional to protein concentration.
Histological evaluation and quantification Histological slides were prepared as per standardized protocol followed in our laboratory and all histological quantifications were performed as described [34]. In brief, the colon was fixed in 10% neutral buffer formalin, dehydrated gradually in ethanol and xylene, then embedded in paraffin and sections were deparaffinized with xylene and rehydrated with alcohol and water. The rehydrated sections were stained using H&E and mounted with DPX mounting media, then examined under a microscope (Olympus BX51 microscope; Tokyo, Japan). Further, histological alterations were quantified in the colon as described by Yoshihara et al. [35]. For histological score, randomly, 10–15 fields were selected from each animal and assigned the score depending upon the
A
32
Control
CP
VPA
VPA+CP
B
28 26
*a
24
*b
@a @b
22
Body weight, g
Body weight, g
After deparaffinization and rehydration, sections were incubated in the citrate buffer (0.01 M, pH6.0) at 95°C for 20–30 min for antigen retrieval and endogenous peroxidase was blocked by 3% H2O2 for 10 min. Nonspecific binding was blocked by incubating with bovine serum albumin milk, then incubation with mouse anti-8-oxo-dG and rabbit anti-γH2AX primary antibody (dilution, 1:50) at 4°C for overnight in a humidified chamber followed by incubation with HRP
34 32
30
20
Immunohistochemistry of γH2AX and 8-oxo-dG in the colon sections
30 28 26 24 Control CP VPA300
22 0
8 Days
16
20
0
1
VPA150+CP VPA300+CP
2 Weeks
3
4
Figure 1 (A and B) Effect of VPA and CP alone as well as the combination treatment on the body weight of mice in the 16- and 28-day experiments. Values are expressed as mean ± SEM, (n = 10). @p
