Amelioration of Carcinogen-Induced Toxicity in Mice by Administration of a Potentized Homeopathic Drug, Natrum Sulphuricum 200

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1 Advane Aess Publiation 19 June 2007 ecam 2009;6(1)65 75 doi: /eam/nem067 Original Artile Amelioration of Carinogen-Indued Toxiity in Mie by Administration of a Potentized Homeopathi Drug, Natrum Sulphurium 200 Nandini Bhattaharjee, Surajit Pathak and Anisur Rahman Khuda-Bukhsh Cytogenetis and Moleular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani , India To examine if a potentized homeopathi drug, Natrum Sulphurium 200 (Nat ) has protetive potentials against hepatoarinogenesis, liver tumors were indued in mie through hroni feeding of P-dimethylaminoazobenzene (p-dab; initiator of hepatoarinogenesis) and phenobarbital (PB; promoter). Mie were divided into five sub-groups: fed normal low protein diet (Gr. I, normal ontrol); fed normal low protein plus alohol-200 (vehile of the homeopathi remedy) (Gr. II); fed diet mixed with 0.06% p-dab plus 0.05% PB (Gr. III); fed diet and arinogens like Gr.III, plus alohol 200 (positive ontrol for drug fed mie) (Gr. IV) and fed diet and arinogens like Gr. III, plus Natrum Sulphuirium-200 (Gr. V; drug fed). Mie were sarified at day 7, 15, 30, 60, 90 and day 120 for study of ytogenetial endpoints like hromosome aberrations (CA), mironulei (MN), mitoti index (MI) and sperm head anomaly (SHA) and biohemial toxiity parameters like aspartate amino transferase (AST), alanine amino transferase (ALT), aid (AP) and alkaline (AlkP) phosphatases, lipid peroxidation (LPO) and redued glutathione (GSH) ontent. Less number of liver tumors were observed in Gr. V (drug fed) mie. Administration of Nat Sulph 200 redued genomi damage, ativities of AP, AlkP, AST, ALT, LPO and inreased GSH ontent. Therefore, independent repliation of the study by others is enouraged. Keywords: anti-arinogenesis azo dye indued liver aner geno- and yto-toxiity biomarkers homeopathi remedy mie Introdution Complementary and alternative mediine (CAM) is one of the growing areas in health are today (1 4), partiularly as supportive mediine in treating diffiult to ure diseases like aner (5 7). Homeopathy is beoming one of the popular alternative modes of treatment (8 10). Beause of ultra high dilutions of most potentized homeopathi drugs, their effiay in treating diseases is often put to question. However, our For reprints and all orrespondene: Anisur Rahman Khuda-Bukhsh, Department of Zoology, University of Kalyani, Kalyani , India. Tel: þ extn. 315; Fax: þ ; prof_arkb@yahoo.o.in; khudabukhsh_48@rediffmail.om reent works whih showed the effiay of ertain homeopathi drugs in ameliorating p-dimethylaminoazobenzene (p-dab) indued aner in mie (11 14) have aroused interest in testing other homeopathi drugs for their possible anti-aner effets. The azo dye p-dab, used oasionally as a food additive, is known to at as initiator of liver aner when used hronially (15 19) and is known to ause hazard to humans and animals (20 21). Phenobarbital (PB), predominantly used as an anti-epilepti/sedative drug, is known to have arinogeni effet (22) to humans, mie and rats when administrated repeatedly (23 26). The effets are more pronouned when PB is used in ombination with the azo-dye, whih unfailingly produe liver tumors that ß 2007 The Author(s). This is an Open Aess artile distributed under the terms of the Creative Commons Attribution Non-Commerial Liense ( lienses/by-n/2.0/uk/) whih permits unrestrited non-ommerial use, distribution, and reprodution in any medium, provided the original work is properly ited.

2 66 Anti-arinogeni potential of homeopathi remedy ultimately beome neoplasti (14,15,27). Therefore this indued liver arinoma serves as a good model for the study of events during arinogenesis as well as to test if a drug has any anti-anerous effet. Further, homeopathi mode of treatment does not have any known side effets and is less expensive than that of onventional treatments of liver aner whih often needs surgery and ostly mediines in management. Remedy Seletion: Indiation from a Pilot Study We have for some time been sreening some homeopathi remedies, whih are laimed to have pronouned effets in proteting liver from toxiity or that are known to help in reovery of liver dysfuntion (e.g. Chelidonium, Lyopodium, et.), for their possible anti-hepatotoxi/ anti-tumorigeni effets sine the primary target organ of the arinogens is the liver. Potentized form of Natrum Sulphurium is one suh remedy generally used by homeopathi pratitioners for various liver disorders having agreeable symptoms (28). As hroni feeding of P-DAB and PB is known to ause symptoms arising out of liver disorders and Natrum Sulph being one of the remedies used to alleviate and protet liver from toxi effets, we first onduted a pilot study on the effiay of Nat Sulph-30 with a limited number of protools (data unpublished). On getting a positive result the present investigation was undertaken with a primary objetive of evaluating effiay of Nat (a more diluted higher poteny laimed to have stronger and longer lasting effet) in amelioration of arinogen indued toxiity in mie utilizing several widely aepted protools of ytogenetial and biohemial studies. Impliations In Brief of the Parameters Used The parameters used in this study are important genotoxiity and yto-toxiity (partiularly hepatotoxin) biomarkers, either diretly or indiretly impliated in the development of liver tumors. The hanges observed in the ytogenetial studies led to the studies of LPO, whih is assoiated with tissue damage and nerosis. Similarly, tissue damage and nerosis an lead to improper liver funtion and thereby ausing hepato-toxiity. For this, liver funtion tests, that inlude the assay of various enzymes like AP, AlkP, AST and ALT were seleted to throw light on the extent of liver malfuntioning, if any. Further, if hepato-toxiity is generated, there will be generation of free radials. GSH has anti-oxidant ativity whih is aomplished by savenging free-radials and the study of GSH was therefore of added value. Further, while derease of the other biohemial parameters indiates dereased hepato-toxiity, a onomitant inrease in GSH is expeted to onfirm this. Methods Feeding the Carinogens and the Homeopathi Remedy to Mie An inbred strain of Swiss albino mie (Mus musulus) were reared and maintained in the animal house of the Department of Zoology (with learane from the University Ethial Committee and under the supervision of the Animal Welfare Committee), University of Kalyani, India, for the investigation. Mie under experiment were provided food and water and kept in a hygieni ondition. The general diet was prepared from wheat, gram and powdered milk without any animal protein supplement. A group of 36 healthy mie weighing between g were used for eah treatment series, namely, (i) normal, (ii) normal þ alohol 200 (iii) P-dimethylaminoazobenzene (p-dab) þ phenobarbital (PB), (iv) p-dab þ PB þ alohol 200, (v) p-dab þ PB þ Nat fed series. In our earlier studies, we used to maintain another group, only verum fed ontrol along with normal þ suussed alohol fed ontrol. But sine the differenes in data between these two series were negligible or insignifiant, the normal þ verum fed group as ontrol was later abandoned for saving life of mie and ost. Eah group of 36 mie divided into six different sets onsisting of six mie eah for six different intervals of fixation, namely, at day seven, 15, 30, 60, 90 and 120. The first set of mie (group-i, normal, negative ontrol) was fed normal low protein diet. The seond set of mie (group II) was fed diluted alohol 200 (potentized as per homeopathi priniple of dilution and suussion) with normal low protein diet. The third set of mie (group-iii) was fed the same diet but mixed with 0.06% p-dab (Sigma, D-6760) at a daily dose of 165 mg/kg body wt. per mouse (15,27,29) and an oral dose of 0.06 ml of 0.05% aqueous solution of PB daily (14,30) through a speially made fine pipette. The fourth set of mie (group-iv, positive ontrol) was fed 0.06% p-dab along with 0.05% aqueous solution of PB as above and also diluted alohol 200 (al-200: as the vehile of the drug was ethyl alohol), till they were sarified. The fifth set of mie (group-v) was fed p-dab plus PB as in group-iii, but was also fed Nat one a day till they were sarified at day seven, 15, 30, 60, 90 and 120. Feeding Proedure and Dose Eah mouse was fed one drop (0.06 ml) of stok solution of Nat or al-200, as the ase may be, one daily (one dose at 7 A.M) with the aid of a fine pipette.

3 ecam 2009;6(1) 67 Preparation of the Potentized Form of the Drug The potentized homeopathi drug, Nat, was proured from HAPCO, 165, Bipin Behari Ganguly Street, Kolkata, India. The 200 poteny of Natrum Sulph was derived from the mother tinture of sodium sulphate by suussion and dilution proedure (31,32) as reommended in the Homeopathi Pharmaopoeia of India, Vol. I (33). The glass vial ontaining potentized al-200 provided by HAPCO having been aidentally broken during the initial days of experiment, the available plaebo provided by Boiron Lab, Lyon, Frane, had been used. However, we examined various physial properties of both the plaebos prepared by HAPCO and BOIRON through UV, FTIR, fluoresene, 13 C-NMR and 1H-NMR spetrosopy earlier (34) but failed to observe any signifiant differene between the two plaebos. Laboratory Methodology Cytogeneti Assay The standard and widely pratied ytogeneti protools like assays of hromosome aberration (CA), mironulei (MN), mitoti index (MI) and sperm head anomaly (SHA) have been adopted in the present study for testing genotoxiity (14,26). A total of 300 bone marrow ells were observed for CA, 3000 ells for MN and 5000 ells for MI and 5000 sperm for SHA analysis. Biohemial Assays After olletion of blood by ventriular punture from etherized mie (approx 1.5 ml from eah mouse), they were sarified and their liver and spleen were quikly olleted in an ie tray. For the study of aspartate and alanine amino transferases (AST and ALT, respetively), aid and alkaline phosphatases (AP and AlkP, respetively) and lipid peroxidation (LPO) and redued glutathione ontent (GSH) standard methods (14) were adopted. Statistial Analysis The signifiane of differene between data of the different series was onduted by Student s t-test and two-way ANOVA (Mini Tab 13.01) was performed. This experiment was done as per randomized double blind plaebo ontrol method. Results Tumor Growth Out of 18 mie sarified at three longer fixation intervals (i.e. at day 60, 90 and 120), 15 and 16 mie, respetively, showed liver tumors in the p-dab þ PB fed and p-dab þ PB þ al-200 fed series, while altogether eight mie showed liver tumors in the drug fed series (Table 1). The tumors looked pale reddish in olor. Some of them were present as solitary or a few individually distinguishable ones (mostly in drug fed mie) muh less in magnitude than the multiple innumerable nodules (mostly in drug unfed mie), whih were onsidered as true tumors and the one or two nodules present in a few were ignored. On this estimate, Nat fed mie showed an overall protetion to the tune of 41.7% (Nat Sulph-30 rendered 31.1%, unpublished data) when data of all three longer intervals of fixation were onsidered. Cytogenetial Studies As ompared with normal metaphase plate (Fig.1A), there was a palpable inrease in the frequenies of various types of CA (Fig. 1B D) in different treatment series during all fixation periods. Several types of hromosome aberrations both of major and minor types were observed in ertain metaphase plates of p-dab þ PB and p-dab þ PB þ al-200 fed mie. The total frequenies of aberrations were maximum in p-dab þ PB þ al-200 fed mie. The frequeny of CA was always greater in p-dab þ PB fed mie than in normal (Gr. I) and normal þ al-200 fed (Gr. II) mie. Table 1. Number of animals showing tumors at different fixation intervals after hroni feeding of p-dab plus PB for 7, 15, 30, 60, 90 and 120 days, in eah fixation intervals 6 mie eah were used per set Series No. of speimens used Tumor inidene 7 days Tumor inidene 15 days Tumor inidene 30 days Tumor inidene 60 days Tumor inidene 90 days Normal 36 0/6 0/6 0/6 0/6 0/6 0/6 Normal þ Al /6 0/6 0/6 0/6 0/6 0/6 p-dab þ PB 36 0/6 0/6 0/6 3/6 6/6 6/6 p-dab þ PB þ Al /6 0/6 0/6 4/6 6/6 6/6 p-dab þ PB þ Nat 36 0/6 0/6 0/6 2/6 3/6 3/6 TOTAL 180 0/30 0/30 0/30 9/30 15/30 15/30 Tumor inidene 120 days

4 68 Anti-arinogeni potential of homeopathi remedy fed mie, there was a signifiant derease (P50.05 P50.001) in MI at all fixation intervals exept at day seven (Fig. 2C). Sperm Head Anomaly Genotoxi or arinogeni or spermatotoxi agents an ause damage to sperm head, whih may be an indiator of the risk of transmission of mutageni trait, if these deformed sperm by hane fertilize the ovum. Greater number of sperm with abnormal head morphologies (Fig. 1G) was enountered in p-dab þ PB fed and p-dab þ PB þ al-200 fed mie at all fixation intervals. The frequenies of SHA were onsiderably dereased in p-dab þ PB þ Nat fed group at all fixation intervals (P50.05 P50.001) (Fig. 2D) exept at day 7. Biohemial Assay Figure 1. (A F). (A) Photomirographs of normal metaphase omplement, (B): Ring (R) and Centri fusion (CF), (C): Polyploidy, (D): Terminal assoiation (TA), (E): Erythroyte showing mironuleus; (F): Sperm with abnormal head shape. The frequeny of CA in p-dab þ PB þ Nat fed group was found to be onsiderably dereased (P50.01 P50.001) at all fixation intervals exept at day seven (Fig. 2A). Mironuleated Erythroytes The inidene of mironulei (Fig. 1E; whih represent a fragment or broken part or whole arm of a hromosome), in arinogen fed as also p-dab þ PB þ al-200 fed mie was appreiably higher than that of normal ontrol mie at all fixation intervals. However, there was a palpable derease in MN in the drug fed series (P50.05 P50.001). The derease was a result of redution in number of both polyhromati (PCE) and normohromati (NCE) erythroytes (Fig. 2B). Mitoti Index The mitoti indies represent the rate at whih the ells pass through a ell yle. When a ell beomes transformed into a tumor ell, the rate beomes inreased for failure of proper ontrol of ell division and thus may be used as an indiator of tumorigeniity. The mitoti indies of mie fed either p-dab þ PB or p-dab þ PB þ Al-200 were higher than that of the normal ontrol series. In p-dab þ PB þ Nat Aspartate Amino Transferase Ativity (AST) The AST ativity in liver was inreased in both p-dab þ PB fed and p-dab þ PB þ Al-200 fed mie at all fixation intervals. However, the AST ativity in p-dab þ PB þ Nat fed series appeared to derease signifiantly (P50.01 P50.001) from day 15 day 120 (Table 2) in liver. Alanine Amino Transferase Ativity (ALT) Similar trend of redution in ALT ativity (P50.01 P50.001) was notied in liver of the p-dab þ PB þ Nat fed mie when ompared to that of p-dab þ PB þ Al-200 fed mie at all fixation intervals exept at day seven (Table 3). Aid Phosphatase Ativity (AP) The ativity of AP in liver showed a trend of steady rise from day 7 to day 15 in p-dab þ PB fed mie, then dereased at day 60 and 90, only to rise again at day 120. The same trend was notied in the p-dab þ PB þ Al-200 fed series. The ativity of AP was dereased signifiantly (P50.01) (Table 4) in the drug fed mie at all fixation intervals exept at day 7. Alkaline Phosphatase Ativity (AlkP) The ativity of AlkP was enhaned in liver of p-dab þ PB fed mie from day seven through day 120 and the same in p-dab þ PB þ Nat fed mie dereased appreiably (P50.05 through P50.001) at all fixation intervals (Table 5). Lipid Peroxidation (LPO) Lipid peroxidation was onsiderably higher in p-dab þ PB þ Al-200 fed mie. However, there was a signifiant

5 ecam 2009;6(1) 69 Histograms showing % of CA of different series at different days interval Histograms showing % of MNE of different series at different days interval % of hromosome aberration (CA) Normal p-dab+pb p-dab+pb+nat b Normal+Alohol p-dab+pb+alohol % of Mironulei (MN) Normal p-dab+pb p-dab+pb+nat a Normal+Alohol p-dab+pb+alohol D 15 D 30 D 60 D 90 D 120 D D 15 D 30 D 60 D 90 D 120 D Different days intrerval Different days intrerval A B Histograms showing % of MI of different series at different days interval Histograms showing % of SHA of differnt series at different days interval % of Mitoti Index (MI) Normal Normal+Alohol p-dab+pb p-dab+pb+\alohol p-dab+pb+nat a 7 D 15 D 30 D 60 D 90 D 120 D Different days intrerval % of sperm head anomaly (SHA) Normal Normal+Alohol p-dab+pb p-dab+pb+\alohol p-dab+pb+nat a 7 D 15 D 30 D 60 D 90 D 120 D Different days intrerval C D Figure 2. (A D). Histograms showing the following: (A) perentage (%) of hromosomal aberrations (CA) in different series of mie at different fixation intervals; (B) perentage (%) of mironuleated erythroytes (MN) in different series of mie at different fixation intervals; (C) perentage (%) of mitoti indies in different series of mie at different fixation intervals; and (D) perentage (%) of sperm head anomalies (SHA) in different series of mie at different fixation intervals. Statistial analyses were done between series p-dab þ PB þ Alohol-200 versus p-dab þ PB þ Nat. The different levels of statistial signifianes between two given series have been designated by *P50.05, **P50.01, ***P p-dab, p-dimethylaminoazobenzene; PB, phenobarbital; Nat, Natrum Sulphurium-200. derease (P50.05 through P50.001) in lipid peroxidation in liver of p-dab þ PB þ Nat fed mie at all fixation intervals (Table 6). Redued Glutathione Content (GSH) The redued glutathione ontent dereased in p-dab þ PB þ Al-200 fed mie at all fixation intervals. However, there was an inrease in redued glutathione ontent of p-dab þ PB þ Nat fed mie at all fixation intervals (P50.05 to P50.01) (Table 7) in liver. Results of Statistial Analysis The signifiane of differenes between data of the different series was determined by Student s t-test and the results of two-way ANOVA (Mini Tab 13.01) are summarized in Tables 2 7 and Table 8 9.

6 70 Anti-arinogeni potential of homeopathi remedy Table 2. Mean Aspartate amino transferase (AST) ativity in (nm/ 100 mg protein/ Min) in liver of mie treated with p-dab þ PB, p-dab þ PB þ al-200, p-dab þ PB þ Nat, normal and negative ontrols at different fixation intervals. The different levels of statistial signifianes between two given series have been designated by *, **, *** Fixation intervals in days 7 days 15 days 30 days Series AST ativity SE AST ativity SE AST ativity SE Liver Differene in ativity Liver Differene in ativity Liver Differene in ativity Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat n ** *** 60 days 90 days 120 days AST ativity SE AST ativity SE AST ativity SE Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat ** *** *** *P50.05, **P50.01,***P50.001, n ¼ non-signifiant. Statistial analyses were done between series p-dab þ PB þ Alohol versus p-dab þ PB þ Nat. p-dab, p-dimethylaminoazobenzene; PB, phenobarbital; Nat, Natrum Sulphurium-200; SE, standard error. Table 3. Mean Alanine amino transferase (ALT) ativity in (nm/ 100 mg protein/ Min) in liver of mie treated with p-dab þ PB, p-dab þ PB þ al-200, p-dab þ PB þ Nat, normal and negative ontrols at different fixation intervals. The different levels of statistial signifianes between two given series have been designated by *, **, *** Fixation intervals in days 7 days 15 days 30 days Series ALT ativity SE ALT ativity SE ALT ativity SE Liver Differene in ativity Liver Differene in ativity Liver Differene in ativity Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat n ** ** 60 days 90 days 120 days ALT ativity SE ALT ativity SE ALT ativity SE Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat ** *** ** *P50.05, **P50.01, ***P50.001, n ¼ non-signifiant. Statistial analyses were done between series p-dab þ PB þ Alohol versus p-dab þ PB þ Nat. p-dab, p-dimethylaminoazobenzene; PB, phenobarbital; Nat, Natrum Sulphurium-200; SE, standard error.

7 ecam 2009;6(1) 71 Table 4. Mean Aid phosphatase (AP) ativity in (nm/ 100 mg protein/ Min) in liver of mie treated with p-dab þ PB, p-dab þ PB þ al-200, p-dab þ PB þ Nat, normal and negative ontrols at different fixation intervals. The different levels of statistial signifianes between two given series have been designated by *, **, *** Fixation intervals in days 7 days 15 days 30 days Series AP ativity SE AP ativity SE AP ativity SE Liver Differene in ativity Liver Differene in ativity Liver Differene in ativity Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat n ** ** 60 days 90 days 120 days AP ativity SE AP ativity SE AP ativity SE Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat ** ** ** *P50.05, **P50.01, ***P50.001, n ¼ non-signifiant. Statistial analyses were done between series p-dab þ PB þ Alohol versus p-dab þ PB þ Nat. p-dab, p-dimethylaminoazobenzene; PB, phenobarbital; Nat, Natrum Sulphurium-200; SE, standard error. Table 5. Mean Alkaline phosphatase (AlkP) ativity in (nm/ 100 mg protein/ Min) in liver of mie treated with p-dab þ PB, p-dab þ PB þ al-200, p-dab þ PB þ Nat, normal and negative ontrols at different fixation intervals. The different levels of statistial signifianes between two given series have been designated by *, **, *** Fixation intervals in days 7 days 15 days 30 days Series AlkP ativity SE AlkP ativity SE AlkP ativity SE Liver Differene in ativity Liver Differene in ativity Liver Differene in ativity Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat n * * 60 days 90 days 120 days AlkP ativity SE AlkP ativity (ativity SE) AlkP ativity SE Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat ** * *** *P50.05, **P50.01, ***P50.001, n ¼ non-signifiant. Statistial analyses were done between series p-dab þ PB þ Alohol versus p-dab þ PB þ Nat. p-dab, p-dimethylaminoazobenzene; PB, phenobarbital; Nat, Natrum Sulphurium-200; SE, standard error.

8 72 Anti-arinogeni potential of homeopathi remedy Table 6. Mean Lipid peroxidation (LPO) ativity in (nm/ MDA/ mg wet tissue) in liver of mie treated with p-dab þ PB, p-dab þ PB þ al-200, p-dab þ PB þ Nat, normal and negative ontrols at different fixation intervals. The different levels of statistial signifianes between two given series have been designated by *, **, *** Fixation intervals in days 7 days 15 days 30 days Series LPO ativity SE LPO ativity SE LPO ativity SE Liver Differene in ativity Liver Differene in ativity Liver Differene in ativity Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat n ** * 60 days 90 days 120 days LPO ativity SE LPO ativity SE LPO ativity SE Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat *** *** ** *P50.05, **P50.01, ***P50.001, n ¼ non-signifiant. Statistial analyses were done between series p-dab þ PB þ Alohol versus p-dab þ PB þ Nat. p-dab, p-dimethylaminoazobenzene; PB, phenobarbital; Nat, Natrum Sulphurium-200; SE, standard error. Table 7. Mean Redued glutathione (GSH) ontent in (nm/ mg wet tissue) in liver of mie treated with p-dab þ PB, p-dab þ PB þ al-200, p-dab þ PB þ Nat, normal and negative ontrols at different fixation intervals. The different levels of statistial signifianes between two given series have been designated by *, **, *** Fixation intervals in days 7 days 15 days 30 days Series GSH ontent SE GSH ontent SE GSH ontent SE Liver Differene in ativity Liver Differene in ativity Liver Differene in ativity Normal Normal þ Al ohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat * 60 days 90 days 120 days GSH ontent SE GSH ontent SE GSH ontent SE Normal Normal þ Alohol p-dab þ PB p-dab þ PB þ Alohol p-dab þ PB þ Nat ** * * *P50.05, **P50.01, ***P50.001, n ¼ non-signifiant. Statistial analyses were done between series p-dab þ PB þ Alohol versus p-dab þ PB þ Nat. p-dab, p-dimethylaminoazobenzene; PB, phenobarbital; Nat, Natrum Sulphurium-200; SE, standard error.

9 ecam 2009;6(1) 73 Table 8. Showing two-way ANOVA analyses for different ytogenetial parameters Two way ANOVA Mitoti index Chromosome Aberration Miro nuleated Erythroytes Sperm Head Anomaly Soure of Variation df MS F MS F MS F MS F Dues to Series * * * * Due to Days * * * * *P50.05 Table 9. Showing two-way ANOVA analyses for different enzymati parameters Two way ANOVA AST ativity ALT ativity AP ativity AlkP ativity LPO ativity GSH ontent Soure of Variation df MS F MS F MS F MS F MS F MS F Dues to Series * * Due to Days * *P50.05 Figure 3. Setion of liver under SEM: showing features of normal (3a-3b), p-dab þ PB þ Alohol fed (3-3d) and p-dab þ PB þ Nat fed mie (3-3f). Disussion In the present study, several widely aepted toxiity markers have been used, all of whih are known to have signifiant role during the arinogenesis proess (14,35 44). In brief, the signifiant redution of biomarkers like AST, ALT, AP, AlkP and LPO and inrease of GSH in the drug fed series speak for the redution of hepato-toxiity and injury (14,35). Positive modulations Figure 4. Liver setion under TEM: showing features of normal (4a-4b). p-dab þ PB þ Alohol fed (4-4d) and p-dab þ PB þ Nat sulph-200 fed mie (4-4d). of several other parameters, suh as, blood gluose, holesterol, testosterone and estradiol were also noted (data not shown). Similarly, there were evidenes from eletron mirosopi studies (both sanning, Fig. 3; and

10 74 Anti-arinogeni potential of homeopathi remedy transmission, Fig. 4) on liver that the homeopathi remedy played a positive role in proteting ultrastrutural integrities (data not shown). Inidentally, reative oxygen speies (ROS) are produed by metabolism of various endogenous and exogenous ompounds and are assoiated in the pathogenesis of different liver diseases, suh as irrhosis and hepatoellular aner (41 44). In irrhosis and hepatoellular arinoma, the antioxidant system is damaged severely and the GSH level falls down, the MDA level rises and GSHdependent enzyme ativities are enhaned in irrhoti and aner tissues (43). Inidentally, positive effets of homeopathi preparations on human prostate aner growth have reently been studied by Malaughlin et al. (45) and Jonas et al., (46) while rude extrats of some plants have also been shown to have anti-anerous ativities against prostate aner ell line in human (47) and in skin arinogenesis (48) and leukemia and aging (49) of mie. In reent years, ell death in brain aner has been observed by some workers (50) by administering a homeopathi drug, Ruta 6, both on in vivo and in vitro systems. But how the miro doses of the highly diluted homeopathi remedy ould bring about suh positive hanges in diverse parameters of study is not yet properly understood. The ability of the homeopathi remedy in bringing down ytogenetial damage along with the positive biohemial hanges an be onsidered as proofs of their ability towards restoring normaly in the funtioning of body s important metaboli enzymes and mehanism of repair and retrieval for genome, presumably by ativating/deativating the relevant genes responsible for regulation of their respetive ativities (51,31,32). Conlusions Finally, ompared with Nat Sulph-30, Nat appeared to show a little better effiay in reduing the inidene of tumors in liver, and most other ommon parameters tested with both potenies, partiularly at some longer intervals of fixation. 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