Two Homeopathic Remedies Used Intermittently Provide Additional Protective Effects Against Hepatotoxicity Induced by Carcinogens in Mice

J Acupunct Meridian Stud 2012;5(4):166e175

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Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com

- RESEARCH

ARTICLE

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Two Homeopathic Remedies Used Intermittently Provide Additional Protective Effects Against Hepatotoxicity Induced by Carcinogens in Mice Nandini Bhattacharjee, Anisur Rahman Khuda-Bukhsh* Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani, India Available online Jun 8, 2012 Received: May 27, 2011 Revised: Dec 9, 2011 Accepted: Dec 14, 2011 KEYWORDS biomarkers; combined therapy; hepatocarcinogenesis; homeopathy; oxidative stress

Abstract The purpose of the study was to evaluate whether potentized cholesterinum (Chol) intermittently used with another homeopathic remedy, Natrum Sulphuricum (Nat Sulph) can provide additional benefits in combating hepatotoxicity generated by chronic feeding of carcinogens, p-dimethylaminoazobenzene (p-DAB), and phenobarbital (PB). Mice were categorized into subgroups: normal untreated (Gr-1); normal þ alcohol “vehicle” (Alc) (Gr-2), 0.06% p-DAB þ0.05% PB (Gr-3), p-DABþPBþAlc (Gr-4), p-DABþPBþNat Sulph-30 (Gr-5), p-DABþPBþChol-200 (Gr-6), p-DABþPBþNat Sulph30þChol-200 (Gr-7), p-DABþPBþNat Sulph-200 (Gr-8), and DABþPBþNat Sulph200þChol-200 (Gr-9). Hepatotoxicity was assessed through biomarkers like aspartate and alanine aminotransferases (AST and ALT), acid and alkaline phosphatases (AcP and AlkP), reduced glutathione content (GSH), glucose 6-phosphate dehydrogenase (G6PD), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), and analysis of lipid peroxidation (LPO) at 30, 60, 90, and 120 days and antioxidant biomarkers like superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) were assayed. Electron microscopic studies (scanning and transmission) and gelatin zymography for matrix metalloproteinases were conducted in liver. The feeding of the homeopathic drugs showed intervention in regard to the increased activities of AST, ALT, AcP, AlkP, GGT, LDH, and LPO and decreased activities of G6PD, SOD, CAT, GR, and GSH noted in the intoxicated mice, more appreciable in Groups 7 and 9. Thus, combined therapy provided additional antihepatotoxic and anticancer effects.

* Corresponding author. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India. E-mail: [email protected] Copyright ª 2012, International Pharmacopuncture Institute doi:10.1016/j.jams.2012.05.004

Added benefit of combined homeopathy

1. Introduction Azo dye induced carcinogenesis in mice has been extensively used as a tool in various toxicological studies earlier [1e3] including in complementary and alternative medicine (CAM) practices [4]. P-dimethylaminoazobenzene (p-DAB) is a potent carcinogen (initiator) [5] and phenobarbital (PB) is a carcinogen [6] that acts as a promoter of hepatic tumors [7]. When these two chemicals are chronically fed to mice or rats, liver tumors develop at 60 days onward, some of which subsequently transform into neoplastic growth. Since the primary site of metabolism of the carcinogen is liver, various toxicity biomarkers in liver have often been studied to follow the periodical/sequential changes in hepatotoxicity after chronic administration of the carcinogens in mice [8,9], which could be correlated with phases of tumorigenesis. Earlier, the homeopathic remedy, Natrum Sulphuricum 200c (Nat Sulph-200) was reported by us to have ameliorating potentials against azo dye induced toxicity in mice [10], but neither efficacy of Natrum Sulphuricum 30c (Nat Sulph-30) nor the efficacy of cholesterinum 200c (Chol-200, which is also recommended for use against stubborn cases of liver ailment) had been studied alone or in combination with Nat Sulph earlier in mice. Cholesterinum 200 used intermittently with Lycopodium 30 and Lycopodium 200 has been reported to reduce genotoxicity and cytotoxicity induced by carcinogens in mice more than when the drugs were used alone [11] Different potencies of Nat Sulph, lycopodium, and cholesterinum are routinely used in treatment of various liver disorders in humans, with apparently good effect [12,13]. Therefore, in the present study, the hypothesis to be tested was that whether Chol-200 could provide protective effects against hepatotoxicity as well as against liver carcinogenesis if administered alone or could provide additional protective effects if used intermittently with either of the two potencies of the homeopathic remedy, Nat Sulph-30, or Nat Sulph-200.

2. Methods

167 for each sex, weighing between 25e30 g, were initially randomized from the colony. All animals were acclimatized seven days prior to the commencement of the treatment and allowed free access to food and water. Experiments on animals were performed with clearance from the Animal Ethics Committee, University of Kalyani, and under the guidelines and supervision of Animal Welfare Committee, University of Kalyani.

2.2. Source and preparation of stock solution of drug Alcoholic preparations (90%) of Nat Sulph-30 and 200, Chol200, and 90% alcohol (used as placebo) prepared by following the principle of homeopathic dilution and potentization procedure as recommended in the Homeopathic Pharmacopoeia of India [14] were procured from HAPCO in Kolkata. Cholesterinum was originally derived from the epithelium lining of gall bladder of humans [13].

2.3. Feeding procedure and dose Each mouse was fed with the aid of a fine pipette 0.06 ml of stock solution [1 ml of each drug or alcohol (vehicle) was diluted separately with 20 ml of double distilled water] of either Nat Sulph-30, or Nat Sulph-200, or Chol-200, as the case may be, that conformed a single dose. As per the set protocol followed in the present study, Nat Sulph-30 was fed twice a day and Nat Sulph-200 was fed once daily. Cholesterinum in higher potency (200 c) reportedly gives better result [11] for which this potency was selected. Chol-200 was fed once a week as an intermittent remedy to mice receiving either Nat Sulph-30 and 200 fed mice. For a better comparison of results, Chol-200 was also fed alone once a week in another group of experimental mice. The doses selected for chronic feeding were derived on the basis of our earlier work [8e10].

2.4. Experimental groups 2.1. Animals Healthy inbred adult (from the same source and batch) albino Swiss strain mice (Mus musculus), in equal number Table 1

A total of 54 healthy mice of both sexes, weighing between 25e30 g were used for each of four fixation intervals, namely, 30, 60, 90, and 120 days, making a total

Number of mice with tumor incidence at different fixation intervals and in different groups.

Groups

Normal Normal þ Alc p-DABþPB p-DABþPBþAlc p-DABþPBþNat Sulph-30 p-DABþPBþChol-200 p-DABþPBþNat Sulph-30þChol-200 p-DABþPBþNat Sulph-200 p-DABþPBþNat Sulph-200þChol-200

Number of Specimens

24 24 24 24 24 24 24 24 24

Tumor Incidence and Intensity 30 Days

60 Days

90 Days

120 Days

0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

0/6 0/6 6/6 6/6 2/6 3/6 2/6 2/6 2/6

0/6 0/6 6/6 6/6 3/6 4/6 2/6 3/6 1/6

0/6 0/6 6/6 6/6 3/6 3/6 1/6 3/6 1/6

(3þþþ, 3þþ) (4þþþ, 2þþ) (1þþ, 1þ) (2þþ, 1þ) (2þ) (1þþ, 1þ) (2þ)

Six mice were used in each group for fixation intervals at 30, 60, 90, and 120 days. (þ) Z low intensity tumor; (þþ) Z moderate intensity tumor; (þþþ) Z high intensity tumor.

(5þþþ, 1þþ) (5þþþ, 1þþ) (2þþ,1þ) (2þþ,2þ) (1þþ,1þ) (2þþ,1þ) (1þþ)

(6þþþ) (6þþþ) (2þþ, 1þ) (3þþ) (1þ) (1þþ, 2þ) (1þ)

168 Table 2

N. Bhattacharjee, A.R. Khuda-Bukhsh Tests of between-subjects effects dependant variable: tumor.

Source

Type III Sum of Squares

df

Mean Square

F

Significance

Model Days Series Error

276.222* 46.972 89.222 32.778

12 3 8 24

23.019 15.657 11.153 1.366

16.854 11.464 8.166

0.001 0.001 0.001

* r2 Z 0.894 (adjusted r2 Z 0.841). df Z degree of freedom.

of 216 animals for the entire study. For every fixation interval, six mice each were fed nine different diets. Animals were randomly divided into the following nine groups: (a) The first group of six healthy untreated mice reared under normal laboratory conditions with normal low protein diet (control-1; Group-1); these data can be taken as baseline data for Day 0, (b) the second group of mice fed alcohol with normal low protein diet (control-2; Group-2), c) the third group of mice fed diet mixed with 0.06% p-dimethylaminoazobenzene (p-DAB; initiator; Sigma, D-6760, Sigma-Aldrich Corporation, Bangalore, India) and provided 0.05% aqueous solution of phenobarbital (PB; promoter) instead of water (carcinogen treated; Group-3), (d) another group of mice chronically fed 0.06% p-DAB along with 0.05% aqueous solution of PB plus succussed alcohol (as the “vehicle” of the drug was ethyl alcohol (Alc), carcinogen treated positive control-3; Group-4; as in earlier experiments in our laboratory, succussed alcohol 30 and 200 did not yield significant differences in results, only succussed alcohol 30 was uniformly used as ethyl alcohol control), (e) the fifth group of mice chronically fed p-DABþPB plus Nat Sulph-30 (treated drugfed-1; Group-5), (f) a group of mice fed p-DABþPB plus Chol-200 (treated drug-fed-2; Group-6), (g) the seventh group of mice fed p-DABþPB plus Nat Sulph-30 plus Chol200 (treated drug-fed-3; Group-7), (h) the eighth group of mice fed p-DABþPB plus Nat Sulph-200 (treated drug-fed4; Group-8), and (i) another group of mice fed p-DABþPB plus Nat Sulph-200 plus Chol-200 (treated drug-fed-5;

Table 3 test.

2.00

3.00

4.00

2.5. Laboratory methodology 2.5.1. Biochemical assays The detailed procedures for preparation of tissue homogenates, blood collection, and different biochemical assays etc. have been provided in our earlier publications [4,8,9,15].

Multiple comparisons of tumor incidence dependant variable: tumor least significant difference according to Tukey Days

1.00

Group-9). Mice were sacrificed at four different intervals of fixation, namely at 30, 60, 90, and 120 days for inspection of tumor incidence and intensity and for analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase (AcP), alkaline phosphatase (AlkP), lipid peroxidation (LPO), reduced glutathione content (GSH), glucose 6-phosphate dehydrogenase (G6PD), gamma glutamyl transferase (GGT), and lactate dehydrogenase (LDH) and at two intervals, viz. 90 and 120 days, for superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), scanning and transmission electron microscopic studies (SEM and TEM, respectively), and gelatin zymography for matrix metalloproteinases (MMP), along with inspection of tumor incidence and intensity. However, as results of 60, 90 and 120 days were not significantly different in terms of most of the parameters used, we put our focus restricted to one of the three longer fixation intervals, namely, at 120 days along with the one of the shortest, namely, 30 days, to avoid a cumbersome presentation of a voluminous data.

2.00 3.00 4.00 1.00 3.00 4.00 1.00 2.00 4.00 1.00 2.00 3.00

Mean Difference 2.5556* 2.7778* 2.5556* 2.5556* 0.2222 0.0000 2.7778* 0.2222 0.2222 2.5556* 0.0000 0.2222

Standard Error

0.5509 0.5509 0.5509 0.5509 0.5509 0.5509 0.5509 0.5509 0.5509 0.5509 0.5509 0.5509

Based on observed means. * The mean difference is significant at the 0.05 level.

Significance

0.001 0.001 0.001 0.001 0.690 1.000 0.001 0.690 0.690 0.001 1.000 0.690

95% Confidence Interval Lower Bound

Upper Bound

3.6926 3.9148 3.6926 1.4185 1.3592 1.1370 1.6408 0.9148 0.9148 1.4185 1.1370 1.3592

1.4185 1.6408 1.4185 3.6926 0.9148 1.1370 3.9148 1.3592 1.3592 3.6926 1.1370 0.9148

Added benefit of combined homeopathy 2.5.2. Statistical analysis and scoring of data The significance test between different series of the data was conducted first by student’s t-test. Since we were more concerned with the differences between the drug-fed series and p-DABþPBþAlc fed control (positive), these were highlighted. Inter-group statistical comparisons were assessed by a two-way analysis of variance using the SPSS software (ver. 11.0 for Windows; SPSS, Inc., Chicago, IL, USA) package for Windows. Post-hoc testing was performed by Tukey test. A value of p < 0.05 was considered to indicate a significant difference between groups.

169 The experimental animals were kept in suitable cages with “coded” labels of the type of treatment they received. The correspondent author fed the groups according to the study protocol. The first author, who did all the other experimental works was ‘blinded’ during biochemical estimation of enzymes, and pathological studies, and also for studies of the expression of MMP, and after all observations were completed, the codes were deciphered. Thus, any bias in observation was avoided and uniformity was maintained in sorting data of both treated and control mice.

2.6. Blinding 2.7. Electron microscopic studies The observers were “blinded” during observation as to whether the sample originated from the drug or placebo treated groups.

The procedures for SEM and TEM electron microscopic studies have been described elsewhere [4].

Figure 1 (A,B) Representative photomicrographs of liver sections under SEM showing features of normal; (B) p-DABþPBþAlc fed; (C,D) and p-DABþPBþNat Sulph-30þChol-200 fed; (E) and p-DABþPBþNat Sulph-200þChol-200; and (F) fed mice. Arrows show (A) normal hepatocyte; (C) damaged hepatocyte; and (E) hepatocyte where damage is reduced.

170

2.8. Level of matrix metalloproteinase expression For the study of matrix metalloproteinase activity, liver tissue homogenates were prepared as per the procedure described in preparation of tissue homogenates. The supernatant was collected and subjected to routine gelatin zymography [7,9,16]. Liver extracts were thawed on ice and mixed 3:1 with substrate gel sample buffer (10% SDS, 4% sucrose, 0.25 M Tris-HCl, pH 6.8, 0.1% bromophenol blue). Each sample (20 mg) was loaded under non-reducing conditions on to electrophoretic mini-gels (SDS-PAGE) containing 1 mg/mL of type-1 gelatin (Sigma, USA). The gels were run at a running buffer temperature of 4 C. After SDS-PAGE, the gels were washed twice in 2.5% Triton X-100 for 30 minutes each, rinsed in water and incubated overnight in a substrate buffer at 37 C (Tris-HCl 50 mM, CaCl2 5 mM, NaN3 0.02%, pH 8) The gels were stained with Coomassie brilliant blue R250, and gelatinolytic activity of matrix metalloproteinases was detected as clear bands on a blue background [7,9,16].

3. Results 3.1. Tumor growth On autopsy, Groups 1 and 2 showed healthy liver while liver tumors were encountered in other groups of mice

N. Bhattacharjee, A.R. Khuda-Bukhsh receiving the carcinogens for 60 days or more. As compared to all carcinogen fed mice showing distinct sign of tumor formation in the form of pale reddish multiple nodules, the incidence and intensity of tumor was found to be greater in the carcinogen fed mice (more pronounced in the p-DABþPB and p-DABþPBþAlc fed series) at 60, 90, and 120 days (Tables 1e3), the incidence and growth of tumors found in the drug fed series was less, both numerically and qualitatively. Further, in the conjoint drug fed series, the number of tumors was also lesser than in the single drug fed series.

3.2. Effect on biochemical parameters In mice fed p-DABþPB and p-DABþPBþAlc, the activities of AST (Histogram 1A), ALT (Histogram 1B) AcP (Histogram 2A), AlkP (Histogram 2B) and LPO (Histogram 3) were greater in carcinogen fed series of mice than that of the drug treated ones. The differences were statistically significant for all or most parameters. In Nat Sulph-30 plus Chol-200 fed mice, the positive intervention in activities was more pronounced, particularly at day 90 and day 120 in liver. Nat Sulph-200 alone showed considerable ameliorative effect in the liver. However, Nat Sulph-200 along with Chol-200 exhibited greater combative effects in liver particularly conspicuous at longer fixation intervals.

Figure 2 (A) Representative photomicrographs of liver sections under TEM showing features of normal; (B) p-DABþPBþAlc fed; (C) p-DABþPBþNat Sulph-30þChol-200 fed and (D) p-DABþPBþNat Sulph-200þChol-200 fed mice.

Added benefit of combined homeopathy

171 There was generally a decline in activity of G6PD (Histogram 6) in p-DABþPB and p-DABþPBþAlc fed series. Compared to carcinogen fed series, the levels of G6PD activity in all the drug-fed series were close to the normal control, denoting thereby that the drugs did intervene in the deleterious changes to take place in the level of this enzyme in the carcinogen fed mice. In p-DABþPBþNat Sulph-200 fed mice, a significant ameliorative effect was noted as compared to controls. The effect appeared to be greater than that observed in the p-DABþPBþNat Sulph-30 and p-DABþPBþChol-200 fed mice at longer fixation intervals. Further, in p-DABþPBþNat Sulph-200þChol-200 fed mice, the positive modulating effect was more discernible than in the p-DABþPBþNat Sulph-30þChol-200 fed mice. The GGT (Histogram 7A) and LDH (Histogram 7B) activities were significantly more in mice chronically fed p-DABþPB and p-DABþPBþAlc. Administration of Nat Sulph30 or Nat Sulph-200 separately along with p-DABþPB brought about considerable positive intervention in the levels of serum GGT and serum LDH. Administration of Chol200 alone brought about considerable positive intervention

A

A critical analysis of the data of different series of mice would reveal the following general trends except for minor deviations observed here and there: Chronic feeding of p-DABþPB and p-DABþPBþAlc decreased content of GSH (Histogram 4) and activities of SOD (Histogram 5A), CAT (Histogram 5B), and GR (Histogram 5C). Administration of homeopathic remedies appeared to show these data to be significantly close towards normal. As compared to controls, administration of p-DABþPBþNat Sulph-30 generally showed greater modulating abilities in respect of all biochemical assays, excepting at some fixation intervals. Administration of Chol-200 alone did not allow the remarkable changes to take place to the extent observed in the GSH content and activities of SOD, CAT and GR in liver of the untreated carcinogen intoxicated mice. Administration of Chol-200 intermittently with Nat Sulph-30 showed better abilities to keep the activities of these biomarkers under control than that of Nat Sulph-30 fed alone. Similarly, administration of p-DABþPBþNat Sulph200 appeared to show a little more ability to keep the toxic changes under control in the carcinogen intoxicated mice as compared to that shown by Nat Sulph-30 fed mice, more noticeable at longer fixation intervals, but the combined series of Nat Sulph-200 plus Chol-200 manifested apparently better ability to restrict the toxicity level as compared to that found in mice fed Nat Sulph-200 alone.

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

0.14

nmol/mg protein/min

0.12 0.1 0.08 0.06 0.04 0.02 0 30D

120 D Fixation intervals (in days)

B

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200 0.025

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

0.02 nmol/mg protein/min

Figure 3 Gelatin zymogram of liver samples showing the expression of MMP in experimental mice sacrificed at (A) Day 90; and (B) Day 120. Lane 1, normal; lane 2, p-DABþPB; lane 3, p-DABþPBþAlc; lane 4, p-DABþPBþNat Sulph-30; lane 5, p-DABþPBþChol-200; lane 6, p-DABþPBþNat Sulph-30þChol200; lane 7, p-DABþPBþNat Sulph-200; lane 8, p-DABþPBþNat Sulph-200þChol-200; M, molecular weight marker.

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

0.015

0.01

0.005

0 30D

120 D Fixation intervals (in days)

Histogram 1 Activities (nmol/mg protein/min) of AST (1A) and ALT (1B) in different series of mice at 30 and 120 days fixation intervals [Data presented as mean  S.E.]

172

N. Bhattacharjee, A.R. Khuda-Bukhsh

A

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

0.018

0.16

0.016

0.14

nmol/mg tissue

nmol/100 mg protein/min

0.014

0.12 0.1 0.08 0.06

0.006

0.002 0 30D

0 120D

Histogram 4 Reduced glutathione (nmol/mg tissue) content in different series of mice at 30 and 120 days fixation intervals [Data presented as mean  S.E.]

Fixation intervals (in days)

B

120 D Fixation intervals (in days)

30D

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

0.18 0.16 0.14 nmol/100 mg prptein/min

0.01 0.008

0.004

0.04 0.02

0.12

of serum GGT and LDH levels. But p-DABþPBþNat Sulph30þChol-200 produced slightly better intervention in comparison to Nat Sulph-30 or Chol-200 fed mice. Similarly, in p-DABþPBþNat Sulph-200þChol-200 fed mice, positive modulating values were greater when these were compared with those observed in the p-DABþPBþNat Sulph-200 fed mice.

0.1 0.08

3.3. Electron microscopic studies

0.06 0.04 0.02 0 30D

120D Fixation intervals (in days)

Histogram 2 Activities (nmol/100 mg protein/min) of AcP (2A) and AlkP (Fig. 2B) in different series of mice at 30 and 120 days fixation intervals [Data presented as mean  S.E.]

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

0.3 0.25 nmol MDA/mg tissue

0.012

0.2 0.15

3.3.1. SEM As compared with the normal hepatocytes (Fig. 1A and B) found in untreated controls, damaged hepatocytic cells were found in the carcinogen fed series (Fig. 1C and D). In the combined drug fed mice [Nat Sulph-30þChol-200 (Fig. 1E) and Nat Sulph-200þChol-200 (Fig. 1F)], the damage to hepatocytes was clearly less severe. 3.3.2. TEM Compared with normal euchromatinized nuclei of normal cells (Fig. 2A), heterochromatinized nuclei with abnormal liver cells were found in carcinogen fed mice (Fig. 2B). In contrast to the intact nuclear membrane observed in liver cells of normal (Fig. 2A), the nuclear membrane of liver cells of carcinogen treated mice (Fig. 2B) seemed to be broken. Further, there was indication of deposition of black lipid droplets in some areas in the p-DABþPB and p-DABþPBþAlc fed mice. Destructive changes were prominent in the cristae of carcinogen fed mice along with an increased number of mitochondria. These features were less conspicuous in mice that were fed Nat Sulph-30þChol200 (Fig. 2C) and Nat Sulph-200þChol-200 (Fig. 2D).

0.1 0.05 0 30D

120 D Fixation intervals (in days)

Histogram 3 Lipid peroxidation (nmol MDA/mg tissue) in different series of mice at 30 and 120 days fixation intervals [Data presented as mean  S.E.]

3.3.3. Level of MMP expression At Day 90 (Fig. 3A), in p-DABþPB and p-DABþPBþalcohol fed mice there were two bands, of which the one near 97 kDa possibly represented MMP-9 appeared to be overexpressed. In the drug fed series, only a single band representing possibly MMP-9 was expressed, but to a lesser extent. Similar results were obtained at Day 120 also (Fig. 3B).

Added benefit of combined homeopathy

A

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

173 Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

4

0.08

3.5

0.07

3 G6PD activity (U/g Hb)

0.06 unit enzyme/mg protein

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

0.05 0.04 0.03 0.02

2.5 2 1.5 1 0.5

0.01

0 0

30D 90 D

120 D

120 D Fixation intervals (in days)

Fixation intervals (in days)

B

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

Histogram 6 G6PD activity (U/g Hb) in different series of mice at 30 and 120 days fixation intervals [Data presented as mean  S.E.]

10 9 unit enzyme/mg protein

8 7 6

A

5 4 3 2

80

0 120 D Fixation intervals (in days)

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

30

70 GGT activity (IU/L)

90 D

C

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

90

1

60 50 40 30 20 10

25 µmol NADPH/mg protein

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

0 30D

20

120 D Fixation intervals (in days)

15

B

10

Normal p-DAB+PB p-DAB+PB+Nat Sulph-30 p-DAB+PB+Nat Sulph-30+Chol-200 p-DAB+PB+Nat Sulph-200+Chol-200

5

Normal+Alc p-DAB+PB+Alc p-DAB+PB+Chol-200 p-DAB+PB+Nat Sulph-200

800

0 90 D

120 D

700

Fixation intervals (in days)

4. Discussion From the above findings, several aspects become clear: (a) the chronic feeding of the carcinogens generate hepatotoxicity and oxidative stress during the course of carcinogenesis, as revealed from the elevated activities of AST, ALT, AcP, AlkP, LDH, GGT, and LPO and decline in activities of SOD, CAT, GR, G6PD and GSH, (b) the administration of

LDH activity (IU/L)

Histogram 5 Activities (Unit enzyme/mg protein) of SOD (5A), CAT (5B) and GR (5C) in different series of mice at 90 and 120 days fixation intervals [Data presented as mean  S.E.]

600 500 400 300 200 100 0 30D

120 D Fixation intervals (in days)

Histogram 7 Activities of serum g-GGT (IU/L) (A) and serum LDH (IU/L) (B) in different series of mice at 30 and 120 days fixation intervals [Data presented as mean  S.E.]

174 the homeopathic remedies individually demonstrated ability of intervention showing less remarkable changes in toxicity levels of these toxicity biomarkers which are also reflected in the histological features of liver, and (c) that the degree of intervention was greater in the combined drug treatment group of mice as compared to the single drug treatment group, particularly discernible in the Nat Sulph-200þChol-200 fed group (Tables 2 and 3). These would provide evidence that the homeopathic drugs, when treated alone or in combination, were capable of producing a combative effect in the form of antihepatotoxic action that possibly was also responsible for ameliorating the oxidative stress effects presumably generated by the reactive oxygen species (ROS) in mice. The development of a lesser number and size of the tumor nodules in the drug fed mice would also suggest that the drugs had an overall anti-cancer effect as well. There are numerous studies to suggest the roles played by the individual toxicity biomarkers used in the present study, linking up each of these to the process of carcinogenesis [17e20]. A critical appraisal of these would reveal that hepatotoxicity also possibly generated ROS and oxidative stress over the lapse of time, because excessive generation of ROS is known to hamper antioxidant defenses [21]. Reduced glutathione (GSH), a prominent cellular reductant, is associated with defence against free radicals, peroxides and other toxic compounds [22]. Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) [23]. The ability of homeopathic remedies to regulate activities of SOD, CAT, GR, and G6PD should be considered as a further positive effect in rendering internal environment of the cell relatively free of toxic elements. The reduced activities of the enzymes GGT and LDH noted in the drug fed intoxicated mice would further support the ability of the homeopathic drugs to intervene in the generation of toxicity induced by the carcinogens in mice. Further, the lack of over-expression of MMPs in drug-fed group renders supportive evidence of their antitumorigenic effects at the oncoprotein expression level. That the homeopathic remedies actually played a combative role in restricting the cellular and sub-cellular damage, was also supported by the histologic features studied through TEM and SEM. Incidentally, in our earlier studies [10,15] histologic changes in target organ suggestive of protective role played by some other homeopathic remedies had also been reported for Carcinosin, when administered singly or conjointly with Nat Sulph. The azo-dye p-DAB is known to metabolize to monoamino azobenzene (MAB) by N-demethylation and subsequently to amino azobenzene (AAB) after its entry inside living organism; AAB is considered to be the carcinogenic factor [3]. Therefore, the homeopathic drugs could either block at the metabolism level of p-DAB, or else, could act at the genic level where the carcinogenic factor(s) actually transformed the proto-oncogenes to oncogenes for triggering the carcinogenesis process. Although the exact mechanism of action of potentized homeopathic remedies, particularly those diluted beyond Avogadro’s limit, has been a subject of controversy for long, Khuda-Bukhsh [24e27] proposed a hypothesis that the potentized homeopathic drugs might have the ability to act

N. Bhattacharjee, A.R. Khuda-Bukhsh as a “molecular trigger” for switching “on” and “off” certain relevant genes, and through a cascade of subsequent gene action/interaction, a series of biochemical changes could follow, that in turn could bring about the alterations observed in the parameters of the present study. The role of some nanoparticles has also been demonstrated in altering physico-chemical property of the drug and its biological action during the process of homeopathic dynamization [28]. As CAM therapy [29] and homeopathy in particular, is becoming popular day by day, more such studies are should be performed independently by other researchers to verify and confirm (or refute) these interesting findings.

5. Conclusion The results of this study, supported with statistical analysis of individual data, would indicate that both Nat Sulph and Chol, even when administered singly had anti-hepatotoxic and anti-cancer effects in carcinogen treated mice, and when Chol-200 was intermittently administered with Nat Sulph, there was a clear additive effect. Therefore, these data could be extrapolated to arrive at a conclusion that Cholesterinum-200 could be more fruitfully and beneficially used along with the homeopathic remedy Nat Sulp in treating stubborn cases of human liver disorders, suspected to have neoplastic growth.

Acknowledgments This work was financially supported by an Extra-Mural Research Grant of AYUSH, Ministry of Health and Family Welfare, Government of India, New Delhi, sanctioned to Professor A.R. Khuda-Bukhsh, Department of Zoology, University of Kalyani, Kalyani, West Bengal. The authors are thankful to Dr. P. Belon and Dr. N. Boujedaini, Lyon, France, for encouragement.

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