Amelioration of Carcinogen-Induced Toxicity in Mice...

Advance Access Publication 19 June 2007 eCAM 20096(1)65ndash75doi101093ecamnem067

Original Article

Amelioration of Carcinogen-Induced Toxicity in Mice byAdministration of a Potentized Homeopathic Drug NatrumSulphuricum 200

Nandini Bhattacharjee Surajit Pathak and Anisur Rahman Khuda-Bukhsh

Cytogenetics and Molecular Biology Laboratory Department of Zoology University of Kalyani Kalyani-741235India

To examine if a potentized homeopathic drug Natrum Sulphuricum 200 (Nat Sulph-200) hasprotective potentials against hepatocarcinogenesis liver tumors were induced in mice throughchronic feeding of P-dimethylaminoazobenzene (p-DAB initiator of hepatocarcinogenesis) andphenobarbital (PB promoter) Mice were divided into five sub-groups fed normal low proteindiet (Gr I normal control) fed normal low protein plus alcohol-200 (vehicle of thehomeopathic remedy) (Gr II) fed diet mixed with 006 p-DAB plus 005 PB (Gr III) feddiet and carcinogens like GrIII plus alcohol 200 (positive control for drug fed mice) (Gr IV)and fed diet and carcinogens like Gr III plus Natrum Sulphuiricum-200 (Gr V drug fed)Mice were sacrificed at day 7 15 30 60 90 and day 120 for study of cytogenetical endpointslike chromosome aberrations (CA) micronuclei (MN) mitotic index (MI) and sperm headanomaly (SHA) and biochemical toxicity parameters like aspartate amino transferase (AST)alanine amino transferase (ALT) acid (AcP) and alkaline (AlkP) phosphatases lipidperoxidation (LPO) and reduced glutathione (GSH) content Less number of liver tumorswere observed in Gr V (drug fed) mice Administration of Nat Sulph 200 reduced genomicdamage activities of AcP AlkP AST ALT LPO and increased GSH content Thereforeindependent replication of the study by others is encouraged

Keywords anti-carcinogenesis ndash azo dye induced liver cancer ndash geno- and cyto-toxicitybiomarkers homeopathic remedy ndash mice

Introduction

Complementary and alternative medicine (CAM) is one

of the growing areas in health care today (1ndash4)

particularly as supportive medicine in treating difficult

to cure diseases like cancer (5ndash7) Homeopathy is

becoming one of the popular alternative modes of

treatment (8ndash10) Because of ultra high dilutions of

most potentized homeopathic drugs their efficacy in

treating diseases is often put to question However our

recent works which showed the efficacy of certainhomeopathic drugs in ameliorating p-dimethylaminoazo-

benzene (p-DAB) induced cancer in mice (11ndash14) have

aroused interest in testing other homeopathic drugs for

their possible anti-cancer effects The azo dye p-DAB

used occasionally as a food additive is known to act as

initiator of liver cancer when used chronically (15ndash19)

and is known to cause hazard to humans and animals

(20ndash21) Phenobarbital (PB) predominantly used as an

anti-epilepticsedative drug is known to have carcino-

genic effect (22) to humans mice and rats when

administrated repeatedly (23ndash26) The effects are more

pronounced when PB is used in combination with the

azo-dye which unfailingly produce liver tumors that

For reprints and all correspondence Anisur Rahman Khuda-BukhshDepartment of Zoology University of Kalyani Kalyani-741235India Tel thorn91-33-25828750 extn 315 Fax thorn91-33-25828282E-mail prof_arkbyahoocoin khudabukhsh_48rediffmailcom

2007 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc20uk) which permits unrestricted non-commercial use distribution and reproduction in any medium provided the original work isproperly cited

ultimately become neoplastic (141527) Therefore this

induced liver carcinoma serves as a good model for the

study of events during carcinogenesis as well as to test

if a drug has any anti-cancerous effect Further

homeopathic mode of treatment does not have any

known side effects and is less expensive than that of

conventional treatments of liver cancer which often needs

surgery and costly medicines in management

Remedy Selection Indication from a Pilot Study

We have for some time been screening some homeopathicremedies which are claimed to have pronounced effectsin protecting liver from toxicity or that are known to helpin recovery of liver dysfunction (eg ChelidoniumLycopodium etc) for their possible anti-hepatotoxicanti-tumorigenic effects since the primary target organ ofthe carcinogens is the liverPotentized form of Natrum Sulphuricum is one such

remedy generally used by homeopathic practitioners forvarious liver disorders having agreeable symptoms (28)As chronic feeding of P-DAB and PB is known to causesymptoms arising out of liver disorders and NatrumSulph being one of the remedies used to alleviate andprotect liver from toxic effects we first conducted a pilotstudy on the efficacy of Nat Sulph-30 with a limitednumber of protocols (data unpublished) On getting apositive result the present investigation was undertakenwith a primary objective of evaluating efficacy of NatSulph-200 (a more diluted higher potency claimed to havestronger and longer lasting effect) in amelioration ofcarcinogen induced toxicity in mice utilizing severalwidely accepted protocols of cytogenetical and biochem-ical studies

Implications In Brief of the Parameters Used

The parameters used in this study are importantgenotoxicity and cyto-toxicity (particularly hepatotoxin)biomarkers either directly or indirectly implicated in thedevelopment of liver tumors The changes observed in thecytogenetical studies led to the studies of LPO which isassociated with tissue damage and necrosis Similarlytissue damage and necrosis can lead to improper liverfunction and thereby causing hepato-toxicity For thisliver function tests that include the assay of variousenzymes like AcP AlkP AST and ALT were selected tothrow light on the extent of liver malfunctioning if anyFurther if hepato-toxicity is generated there will begeneration of free radicals GSH has anti-oxidant activitywhich is accomplished by scavenging free-radicals and thestudy of GSH was therefore of added value Furtherwhile decrease of the other biochemical parametersindicates decreased hepato-toxicity a concomitantincrease in GSH is expected to confirm this

Methods

Feeding the Carcinogens and the Homeopathic Remedy to

Mice

An inbred strain of Swiss albino mice (Mus musculus)

were reared and maintained in the animal house of the

Department of Zoology (with clearance from the

University Ethical Committee and under the supervision

of the Animal Welfare Committee) University of

Kalyani India for the investigation Mice under experi-

ment were provided food and water and kept in a

hygienic condition The general diet was prepared

from wheat gram and powdered milk without any

animal protein supplement A group of 36 healthy

mice weighing between 25ndash30 g were used for

each treatment series namely (i) normal (ii) normalthorn

alcohol 200 (iii) P-dimethylaminoazobenzene (p-DAB)thorn

phenobarbital (PB) (iv) p-DABthornPBthorn alcohol 200

(v) p-DABthornPBthornNat Sulph-200 fed series In our earlier

studies we used to maintain another group only verum

fed control along with normalthorn succussed alcohol fed

control But since the differences in data between these

two series were negligible or insignificant the normalthorn

verum fed group as control was later abandoned for

saving life of mice and costEach group of 36 mice divided into six different sets

consisting of six mice each for six different intervals offixation namely at day seven 15 30 60 90 and 120

The first set of mice (group-I normal negative control)was fed normal low protein diet The second set of mice(group II) was fed diluted alcohol 200 (potentized as per

homeopathic principle of dilution and succussion) withnormal low protein diet The third set of mice (group-III)was fed the same diet but mixed with 006 p-DAB(Sigma D-6760) at a daily dose of 165mgkg body wt

per mouse (152729) and an oral dose of 006ml of005 aqueous solution of PB daily (1430) through aspecially made fine pipette The fourth set of mice(group-IV positive control) was fed 006 p-DAB along

with 005 aqueous solution of PB as above andalso diluted alcohol 200 (alc-200 as the lsquovehiclersquo of thedrug was ethyl alcohol) till they were sacrificed Thefifth set of mice (group-V) was fed p-DAB plus PB as

in group-III but was also fed Nat Sulph-200 once aday till they were sacrificed at day seven 15 30 60 90and 120

Feeding Procedure and Dose

Each mouse was fed one drop (006ml) of stock

solution of Nat Sulph-200 or alc-200 as the case

may be once daily (one dose at 7AM) with the aid of

a fine pipette

66 Anti-carcinogenic potential of homeopathic remedy

Preparation of the Potentized Form of the Drug

The potentized homeopathic drug Nat Sulph-200 wasprocured from lsquoHAPCOrsquo 165 Bipin Behari GangulyStreet Kolkata India The 200 potency of Natrum Sulphwas derived from the mother tincture of sodium sulphateby lsquosuccussion and dilutionrsquo procedure (3132) asrecommended in the Homeopathic Pharmacopoeia ofIndia Vol I (33) The glass vial containing potentizedalc-200 provided by lsquoHAPCOrsquo having been accidentallybroken during the initial days of experiment theavailable lsquoplaceborsquo provided by Boiron Lab LyonFrance had been used However we examined variousphysical properties of both the lsquoplacebosrsquo prepared bylsquoHAPCOrsquo and lsquoBOIRONrsquo through UV FTIR fluores-cence 13 C-NMR and 1H-NMR spectroscopy earlier(34) but failed to observe any significant differencebetween the two lsquoplacebosrsquo

Laboratory Methodology

Cytogenetic Assay

The standard and widely practiced cytogenetic protocolslike assays of chromosome aberration (CA) micronuclei(MN) mitotic index (MI) and sperm head anomaly(SHA) have been adopted in the present study for testinggenotoxicity (1426) A total of 300 bone marrow cellswere observed for CA 3000 cells for MN and 5000 cellsfor MI and 5000 sperm for SHA analysis

Biochemical Assays

After collection of blood by ventricular puncture frometherized mice (approx 15ml from each mouse) theywere sacrificed and their liver and spleen were quicklycollected in an ice trayFor the study of aspartate and alanine amino trans-

ferases (AST and ALT respectively) acid and alkalinephosphatases (AcP and AlkP respectively) and lipidperoxidation (LPO) and reduced glutathione content(GSH) standard methods (14) were adopted

Statistical Analysis

The significance of difference between data of thedifferent series was conducted by Studentrsquos t-test andtwo-way ANOVA (Mini Tab 1301) was performed Thisexperiment was done as per randomized double blindplacebo control method

Results

Tumor Growth

Out of 18 mice sacrificed at three longer fixation intervals(ie at day 60 90 and 120) 15 and 16 mice respectivelyshowed liver tumors in the p-DABthornPB fed andp-DABthornPBthorn alc-200 fed series while altogether eightmice showed liver tumors in the drug fed series (Table 1)The tumors looked pale reddish in color Some of themwere present as solitary or a few individually distinguish-able ones (mostly in drug fed mice) much less inmagnitude than the multiple innumerable nodules(mostly in drug unfed mice) which were considered astrue tumors and the one or two nodules present in a fewwere ignored On this estimate Nat Sulph-200 fed miceshowed an overall protection to the tune of 417(Nat Sulph-30 rendered 311 unpublished data) whendata of all three longer intervals of fixation wereconsidered

Cytogenetical Studies

As compared with normal metaphase plate (Fig1A)there was a palpable increase in the frequencies ofvarious types of CA (Fig 1BndashD) in different treatmentseries during all fixation periods Several types ofchromosome aberrations mdash both of major and minortypes mdash were observed in certain metaphase plates ofp-DABthornPB and p-DABthornPBthorn alc-200 fed mice Thetotal frequencies of aberrations were maximum inp-DABthornPBthorn alc-200 fed mice The frequency of CAwas always greater in p-DABthornPB fed mice than innormal (Gr I) and normalthorn alc-200 fed (Gr II) mice

Table 1 Number of animals showing tumors at different fixation intervals after chronic feeding of p-DAB plus PB for 7 15 30 60 90 and 120 daysin each fixation intervals 6 mice each were used per set

Series No ofspecimensused

Tumorincidence7 days






Normal 36 06 06 06 06 06 06

NormalthornAlc-200 36 06 06 06 06 06 06

p-DABthornPB 36 06 06 06 36 66 66

p-DABthornPBthornAlc-200 36 06 06 06 46 66 66

p-DABthornPBthornNat Sulph-200 36 06 06 06 26 36 36

TOTAL 180 030 030 030 930 1530 1530

eCAM 20096(1) 67

The frequency of CA in p-DABthornPBthornNat Sulph-200fed group was found to be considerably decreased(P5001ndashP50001) at all fixation intervals except atday seven (Fig 2A)

Micronucleated Erythrocytes

The incidence of micronuclei (Fig 1E which represent afragment or broken part or whole arm of a chromo-some) in carcinogen fed as also p-DABthornPBthorn alc-200fed mice was appreciably higher than that of normalcontrol mice at all fixation intervals However there wasa palpable decrease in MN in the drug fed series(P5005ndashP50001) The decrease was a result ofreduction in number of both polychromatic (PCE) andnormochromatic (NCE) erythrocytes (Fig 2B)

Mitotic Index

The mitotic indices represent the rate at which the cellspass through a cell cycle When a cell becomestransformed into a tumor cell the rate becomes increasedfor failure of proper control of cell division and thusmay be used as an indicator of tumorigenicityThe mitotic indices of mice fed either p-DABthornPBor p-DABthornPBthornAlc-200 were higher than that ofthe normal control series In p-DABthornPBthornNat

Sulph-200 fed mice there was a significant decrease(P5005ndashP50001) in MI at all fixation intervals exceptat day seven (Fig 2C)

Sperm Head Anomaly

Genotoxic or carcinogenic or spermatotoxic agents cancause damage to sperm head which may be an indicatorof the risk of transmission of mutagenic trait if thesedeformed sperm by chance fertilize the ovum Greaternumber of sperm with abnormal head morphologies(Fig 1G) was encountered in p-DABthornPB fed andp-DABthornPBthorn alc-200 fed mice at all fixation intervalsThe frequencies of SHA were considerably decreased inp-DABthornPBthornNat Sulph-200 fed group at all fixationintervals (P5005ndashP50001) (Fig 2D) except at day 7

Biochemical Assay

Aspartate Amino Transferase Activity (AST)

The AST activity in liver was increased in bothp-DABthornPB fed and p-DABthornPBthornAlc-200 fed mice atall fixation intervals However the AST activity inp-DABthornPBthornNat Sulph-200 fed series appeared todecrease significantly (P5001ndashP50001) from day15 ndash day 120 (Table 2) in liver

Alanine Amino Transferase Activity (ALT)

Similar trend of reduction in ALT activity (P5001ndashP50001) was noticed in liver of the p-DabthornPBthornNatSulph-200 fed mice when compared to that ofp-DabthornPBthornAlc-200 fed mice at all fixation intervalsexcept at day seven (Table 3)

Acid Phosphatase Activity (AcP)

The activity of AcP in liver showed a trend of steady risefrom day 7 to day 15 in p-DabthornPB fed mice thendecreased at day 60 and 90 only to rise again at day 120The same trend was noticed in the p-DabthornPBthornAlc-200fed series The activity of AcP was decreased significantly(P5001) (Table 4) in the drug fed mice at all fixationintervals except at day 7

Alkaline Phosphatase Activity (AlkP)

The activity of AlkP was enhanced in liver of p-DabthornPBfed mice from day seven through day 120 and the same inp-DabthornPBthornNat Sulph-200 fed mice decreased appreci-ably (P5005 through P50001) at all fixation intervals(Table 5)

Lipid Peroxidation (LPO)

Lipid peroxidation was considerably higher in p-DabthornPBthornAlc-200 fed mice However there was a significant

Figure 1 (AndashF) (A) Photomicrographs of normal metaphase comple-

ment (B) Ring (R) and Centric fusion (CF) (C) Polyploidy

(D) Terminal association (TA) (E) Erythrocyte showing micronucleus

(F) Sperm with abnormal head shape


decrease (P5005 through P50001) in lipid peroxida-

tion in liver of p-DabthornPBthornNat Sulph-200 fed mice at

all fixation intervals (Table 6)

Reduced Glutathione Content (GSH)

The reduced glutathione content decreased in p-Dabthorn

PBthornAlc-200 fed mice at all fixation intervals However

there was an increase in reduced glutathione content of

p-DabthornPBthornNat Sulph-200 fed mice at all fixationintervals (P5005 to P5001) (Table 7) in liver

Results of Statistical Analysis

The significance of differences between data of thedifferent series was determined by Studentrsquos t-test andthe results of two-way ANOVA (Mini Tab 1301) aresummarized in Tables 2ndash7 and Table 8ndash9

Histograms showing of CA of different seriesat different days interval

000

500

1000

1500

2000

2500

3000

7 D 15 D 30 D 60 D 90 D 120 D

7 D 15 D 30 D 60 D 90 D 120 D

Different days intrerval

A B

C D

Different days intrerval7 D 15 D 30 D 60 D 90 D 120 D


7 D 15 D 30 D 60 D 90 D 120 D


o

f ch

rom

oso

me

aber

rati

on

(C

A)

Normal Normal+Alcoholp-DAB+PB p-DAB+PB+Alcohol

Normal+Alcoholp-DAB+PB+Alcohol

p-DAB+PB+Nat Sulph-200

Normalp-DAB+PBp-DAB+PB+Nat Sulph-200

Histograms showing of MNE of differentseries at different days interval

000

020

040

060

080

100

120

140

o

f M

icro

nu

clei

(M

N)

Histograms showing of MI of different seriesat different days interval

000

100

200

300

400

500

600

700

800

900

1000

o

f M

ito

tic

Ind

ex (

MI)

NormalNormal+Alcoholp-DAB+PBp-DAB+PB+Alcoholp-DAB+PB+Nat Sulph-200


Histograms showing of SHA of differnt seriesat different days interval

000

050

100

150

200

250

300

350

400

450

o

f sp

erm

hea

d a

no

mal

y(S

HA

)

b

cc

c

c

ac

cc c

a

c

c

c

c

a

c

c

c

c

Figure 2 (AndashD) Histograms showing the following (A) percentage () of chromosomal aberrations (CA) in different series of mice at different

fixation intervals (B) percentage () of micronucleated erythrocytes (MN) in different series of mice at different fixation intervals (C) percentage

() of mitotic indices in different series of mice at different fixation intervals and (D) percentage () of sperm head anomalies (SHA) in different

series of mice at different fixation intervals Statistical analyses were done between series p-DABthornPBthornAlcohol-200 versus p-DABthornPBthornNat

Sulph-200 The different levels of statistical significances between two given series have been designated by P5005 P5001 P50001

p-DAB p-dimethylaminoazobenzene PB phenobarbital Nat Sulph-200 Natrum Sulphuricum-200

eCAM 20096(1) 69

Table 2 Mean Aspartate amino transferase (AST) activity in (nM 100mg protein Min) in liver of mice treated with p-DABthornPBp-DABthornPBthorn alc-200 p-DABthornPBthornNat Sulph-200 normal and negative controls at different fixation intervals The different levels ofstatistical significances between two given series have been designated by

Fixation intervals in days 7 days 15 days 30 days

Series AST activity SE AST activity SE AST activitySE

Liver Difference in activity Liver Difference in activity Liver Difference in activity

Normal 0017 0002 0012 0001 0021 0001

NormalthornAlcohol 0026 0001 0011 0001 0020 0000

p-DABthornPB 0040 0002 0041 0005 0049 0002

p-DABthornPBthornAlcohol 0043 0003 0045 0001 0054 0002

p-DABthornPBthornNat 0050 0003 0007n 00400001 0005 0036 0001 0018

Sulph-200

60 days 90 days 120 days

AST activity SE AST activity SE AST activitySE

Normal 0024 0004 0010 0001 0054 0002


p-DABthornPB 0047 0006 0056 0004 0075 0007


p-DABthornPBthornNat 0032 0004 0021 0041 0002 0018 0046 0002 0061

Sulph-200

P5005 P5001P50001 nfrac14 non-significantStatistical analyses were done between series p-DABthornPBthornAlcohol versus p-DABthornPBthornNat Sulph-200p-DAB p-dimethylaminoazobenzene PB phenobarbital Nat Sulph-200 Natrum Sulphuricum-200 SE standard error

Table 3 Mean Alanine amino transferase (ALT) activity in (nM 100mg protein Min) in liver of mice treated with p-DABthornPB p-DABthornPBthornalc-200 p-DABthornPBthornNat Sulph-200 normal and negative controls at different fixation intervals The different levels of statistical significancesbetween two given series have been designated by


Series ALT activity SE ALT activity SE ALT activity SE


Normal 0006 0000 0010 0001 0005 0000


p-DABthornPB 0009 0002 0028 0002 0017 0002


p-DABthornPBthornNat 0021 0001 0007n 0026 0001 0010 0014 0002 0008

Sulph-200


ALT activity SE ALT activity SE ALT activity SE

Normal 0006 0000 0006 0001 0004 0000


p-DABthornPB 0015 0001 0015 0002 0019 0001



Sulph-200

P5005 P5001 P50001 nfrac14non-significantStatistical analyses were done between series p-DABthornPBthornAlcohol versus p-DABthornPBthornNat Sulph-200p-DAB p-dimethylaminoazobenzene PB phenobarbital Nat Sulph-200 Natrum Sulphuricum-200 SE standard error


Table 4 Mean Acid phosphatase (AcP) activity in (nM 100mg protein Min) in liver of mice treated with p-DABthornPB p-DABthornPBthorn alc-200p-DABthornPBthornNat Sulph-200 normal and negative controls at different fixation intervals The different levels of statistical significances between twogiven series have been designated by


Series AcP activity SE AcP activity SE AcP activity SE


Normal 0048 0001 0143 0007 0040 0002


p-DABthornPB 0080 0006 0157 0010 0086 0005



Sulph-200


AcP activity SE AcP activity SE AcP activity SE

Normal 0046 0005 0012 0000 0025 0002


p-DABthornPB 0076 0009 0053 0005 0128 0013



Sulph-200


Table 5 Mean Alkaline phosphatase (AlkP) activity in (nM 100mg protein Min) in liver of mice treated with p-DABthornPB p-DABthornPBthornalc-200 p-DABthornPBthornNat Sulph-200 normal and negative controls at different fixation intervals The different levels of statistical significancesbetween two given series have been designated by


Series AlkP activity SE AlkP activity SE AlkP activity SE


Normal 0017 0001 0014 0003 0024 0001


p-DABthornPB 0038 0001 0026 0004 0044 0002



Sulph-200


AlkP activity SE AlkP activity (activitySE) AlkP activity SE

Normal 0021 0001 0019 0001 0012 0001


p-DABthornPB 0034 0001 0045 0004 0139 0006



Sulph-200


eCAM 20096(1) 71

Table 6 Mean Lipid peroxidation (LPO) activity in (nM MDA mg wet tissue) in liver of mice treated with p-DABthornPB p-DABthornPBthornalc-200 p-DABthornPBthornNat Sulph-200 normal and negative controls at different fixation intervals The different levels of statistical significancesbetween two given series have been designated by


Series LPO activitySE LPO activity SE LPO activity SE


Normal 0245 0008 0135 0010 0086 0002


p-DABthornPB 0332 0010 0312 0010 0234 0008



Sulph-200


LPO activitySE LPO activity SE LPO activity SE

Normal 0052 0004 0043 0001 0048 0001


p-DABthornPB 0247 0012 0119 0010 0131 0009


p-DABthornPBthornNat 0141 0009 0113 0090 0004 0034 0118 0004 003 1

Sulph-200


Table 7 Mean Reduced glutathione (GSH) content in (nM mg wet tissue) in liver of mice treated with p-DABthornPB p-DABthornPBthorn alc-200p-DABthornPBthornNat Sulph-200 normal and negative controls at different fixation intervals The different levels of statistical significances betweentwo given series have been designated by


Series GSH content SE GSH contentSE GSH content SE


Normal 0004 0000 0006 0000 0005 0001

NormalthornAl cohol 0004 0001 0006 0000 0004 0001

p-DABthornPB 0001 0000 0001 0000 0001 0000



Sulph-200


GSH content SE GSH contentSE GSH content SE

Normal 0005 0000 0006 0000 0007 0000


p-DABthornPB 0001 0000 0002 0000 0003 0000



Sulph-200



Discussion

In the present study several widely accepted toxicity

markers have been used all of which are known to have

significant role during the carcinogenesis process

(1435ndash44) In brief the significant reduction of biomar-

kers like AST ALT AcP AlkP and LPO and increase of

GSH in the drug fed series speak for the reduction of

hepato-toxicity and injury (1435) Positive modulations

of several other parameters such as blood glucosecholesterol testosterone and estradiol were also noted(data not shown) Similarly there were evidences fromelectron microscopic studies (both scanning Fig 3 and

Table 8 Showing two-way ANOVA analyses for different cytogenetical parameters

Two way ANOVA Mitotic index Chromosome Aberration Micro nucleated Erythrocytes Sperm Head Anomaly

Source of Variation df MS F MS F MS F MS F

Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005

Table 9 Showing two-way ANOVA analyses for different enzymatic parameters

Two way ANOVA AST activity ALT activity AcP activity AlkP activity LPO activity GSH content

Source of Variation df MS F MS F MS F MS F MS F MS F

Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005

Figure 3 Section of liver under SEM showing features of normal

(3a-3b) p-DABthornPBthornAlcohol fed (3c-3d) and p-DABthornPBthornNat

Sulph-200 fed mice (3c-3f)

Figure 4 Liver section under TEM showing features of normal (4a-4b)

p-DABthornPBthornAlcohol fed (4c-4d) and p-DABthornPBthornNat sulph-200

fed mice (4c-4d)

eCAM 20096(1) 73

transmission Fig 4) on liver that the homeopathicremedy played a positive role in protecting ultra-structural integrities (data not shown) Incidentallyreactive oxygen species (ROS) are produced by metabo-lism of various endogenous and exogenous compoundsand are associated in the pathogenesis of different liverdiseases such as cirrhosis and hepatocellular cancer (41ndash44) In cirrhosis and hepatocellular carcinoma theantioxidant system is damaged severely and the GSHlevel falls down the MDA level rises and GSH-dependent enzyme activities are enhanced in cirrhoticand cancer tissues (43) Incidentally positive effects ofhomeopathic preparations on human prostate cancergrowth have recently been studied by Maclaughlin et al(45) and Jonas et al (46) while crude extracts of someplants have also been shown to have anti-cancerousactivities against prostate cancer cell line in human (47)and in skin carcinogenesis (48) and leukemia and aging(49) of miceIn recent years cell death in brain cancer has been

observed by some workers (50) by administering ahomeopathic drug Ruta 6 both on in vivo and in vitrosystems But how the micro doses of the highly dilutedhomeopathic remedy could bring about such positivechanges in diverse parameters of study is not yet properlyunderstood The ability of the homeopathic remedy inbringing down cytogenetical damage along with thepositive biochemical changes can be considered asproofs of their ability towards restoring normalcy in thefunctioning of bodyrsquos important metabolic enzymes andmechanism of repair and retrieval for genome presum-ably by activatingdeactivating the relevant genes respon-sible for regulation of their respective activities (513132)

Conclusions

Finally compared with Nat Sulph-30 Nat Sulph-200appeared to show a little better efficacy in reducing theincidence of tumors in liver and most other commonparameters tested with both potencies particularly atsome longer intervals of fixation Therefore otherresearchers are encouraged to replicate the study inmice andor other mammalian models and see ifpotentized remedies of Natrum Sulphuricum can berecommended as a potential supportive medicine for usein human cancer therapy as well

Acknowledgements

This work was financially supported by an EMR grant ofAYUSH Ministry of Health and Family WelfareNew Delhi to ARKB The authors sincerely thankDr TC Nag Associate Professor Department ofAnatomy All India Institute of Medical Sciences NewDelhi for his kind help in conducting the scanning and

transmission electron microscopic studies The authorsexpress their sincere thanks to Dr Philippe BelonDirector Boiron Lab Lyon France for kindly donatingthe Alc-200 for this work

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medicine in cancer Eur J Cancer 2003392273ndash772 Earnst E Complementary or alternative therapies for osteoarthritis

Nat Clin Pract Rheumatol 2006274ndash803 Ricotti V Delanty N Use of complementary and alternative

medicine in epilepsy Curr Neurol Neurosci Rep 20066347ndash534 Simpson CA Complementary medicine in chronic pain treatment

Phys Med Rehabil Clin N Am 200617451ndash725 Richardson MA Straus SE Complementary and alternative

medicine Opportunities and Challenges for cancer managementand research Seminars in Oncology 200229531ndash45

6 Chrystal K Allan S Forgeson G Issacs R The use ofcomplementary and alternative medicine by cancer patients inNew Zealand and regional cancer treatment center New ZealandMed J 20031161ndash8

7 Molassiotis A Panteli V Patiraki E Ozden G Platin N Madsen Eet al Complementary and alternative medicine use in lung cancerpatients in eight European countries Complement Ther Clin Pract20061234ndash9

8 Molassiotis A Margulies A Fernandez-Ortega P Pud D Panteli VBruyns I et al Complementary and alternative medicine usein patients with hematological malignancies in EuropeComplement Ther Clin Pract 200511105ndash10

9 Bensoussan M Jovenin N Garcia B Vandromme L Jolly DBouche O et al Complementary and alternative medicine use bypatients with inflammatory bowel disease results from a postalsurvey Gastroenterol Clin Biol 20063014ndash23

10 Joos S Rosemann T Szecsenyi J Hahn EG Willich SNBrinkhaus B Use of complementary and alternative medicine inGermany - a survey of patients with inflammatory bowel diseaseBMC Complement Altern Med 2006619 doi 1011861472-6882-6-19 httpwwwbiomedcentralcom1472-6882619

11 Biswas SJ Khuda-Bukhsh AR Effect of a homeopathic drugChelidonium in amelioration of p-Dab induced hepatocarcinogen-esis in mice BMC Complement Altern Med 200221ndash16

12 Biswas SJ Khuda-Bukhsh AR Evaluation of protective potentialsof a potentized homeopathic drug Chelidonium majus during azodye induced hepatocarcinogenesis in mice Indian J Exp Biol200442698ndash714

13 Biswas SJ Pathak S Bhattacharjee N Das JK Khuda-Bukhsh AREfficacy of a potentized homeopathic drug Carcinosin-200 fedalone and in combination with another drug Chelidonium-200in amelioration of p-Dab induced hepatocarcinogenesis in miceJ Altern Complement Med 200511839ndash54

14 Pathak S Das JK Biswas SJ Khuda-Bukhsh AR Protectivepotentials of a potentized homeopathic drug Lycopodium-30in ameliorating azo dye induced hepatocarcinogenesis in miceMol Cell Biochem 2006258121ndash31

15 Daust R Molnar F Cellular populations and mitotic activity in ratliver parenchyma during azo dye carcinogenesis Cancer Res1964241898ndash1909

16 IARC (International Agency for Research on Cancer) On theevaluation of carcinogenic risk of chemicals to man Group-2B19877449

17 Ohnishi S Murata M Degawa M Kawanishi S Oxidative DNAdamage induced by an N-hydroxy metabolite of carcinogenic4-dimethylaminoazobenzene Jpn J Cancer Res 20019223ndash9

18 Merkulova TI Kropachev KY Timofeeva OA Vasiliev GVLevashova ZB Ilnitskaya SI et al Species-specific effects of thehepatocarcinogens 30- methyl-4-dimehylaminoazobenzene andortho-aminoazotoluene in mouse and rat liver Mol Carcinog200544223ndash32

19 Biswas SJ Khuda-Bukhsh AR Cytotoxic and genotoxic effects ofthe azo-dye p-dimethylaminoazobenzene in mice A time-coursestudy Mutat Res 20055871ndash8


20 Siemiatycki J Richardson L Straif K Latreille B Lakhani RCampbell S et al Listing occupational carcinogens researchReview Environ Health Perspect 20041121447ndash59

21 NTP Report on Carcinogens (11th edn) Research Triangle ParkNC National Toxicology Program 2005 (Retrieved from httpntpniehsnihgovindexcfmObjectidfrac1432BA9724-F1F6-975E-7FCE50709CB4C932)

22 IARC (International Agency for Research on Cancer)Phenobarbital and Sodium salt IARC monograph 200179161

23 White SJ McLean AEM Howland C Anticonvulsant drugsand cancer A cohort study in patients with severe epilepsyLancet 1979ii458ndash61

24 Lee GH Paradoxical effects of phenobarbital on mouse carcinogen-esis Toxicol Pathol 200028215ndash25

25 Kinoshita A Wanibuchi H Morimura K Wei M Shen JImaoka S et al Phenobarbital at low dose exerts hormesis in rathepatocarcinogenesis by reducing oxidative DNA damage alteringcell proliferation apoptosis and gene expression Carcinogenesis2003241389ndash99

26 Biswas SJ Pathak S Khuda-Bukhsh AR Assessment of thegenotoxic and cytotoxic potential of an antiepileptic drugPhenobarbital in mice A time course study Mutat Res20045631ndash11

27 Palekar SD Sirsat SM Studies on the hepatocyte in azo dyecarcinogenesis Indian J Exp Boil 1966473ndash8

28 Boericke W Pocket Manual of Homeopathic Materia MedicaIndian edn Calcutta India Sett Dey and Co 1976

29 Kitagawa T Sugano H Enhancing effect of phenobarbital onthe development of enzyme-altered islands and hepatocellularcarcinomas initiated by 30-methyl-4-(dimethylamino) azobenzene ordiethylnitrosamine Gann 197869679ndash87

30 Kitagawa T Sugano H Enhancement of azo-dye hepatocarcino-genesis with dietary phenobarbital in rats Gann 197768255ndash6

31 Khuda-Bukhsh AR Towards understanding molecular mechanismsof action of homeopathic drugs An overview Mol Cell Biochem2003253339ndash45

32 Khuda-Bukhsh AR Laboratory research in homeopathy proIntegr Cancer Ther 20065320ndash32

33 Homeopathic Pharmacopoeia of India Ministry of HealthGovernment of India Press Coimbatore-641019 Controller ofPublications 19711165

34 Pathak S Bhattacharjee N Das JK Chaki Chowdhury S RoyKarmakar S Banerjee P et al Supportive evidences for anti-cancerous potential of an alternative medicine in hepatocarcinogen-esis of mice Res Com Med June issue 2007 14132 (DOI101159000102007)

35 Plaa GL Amdun AM Doull J Klasser CD Toxic responses of theliver 4th edn London UK Pergamon Press 1991

36 Chan KY Wong N Lai PB Squire JA Macgregor PF Behesthi Bet al Transcriptional profiling on chromosome 19p indicated

frequent down regulation of ACP5 expression in hepatocellularcarcinoma Int J cance 2005114902ndash8

37 Reichling JJ Kaplan MM Clinical use of serum enzymes in liverdiseases Dig Dis Sci 1988331601ndash14

38 Wiwanitkit V High serum alkaline phosphatase levels a study in181 Thai adult hospitalized patients BMC Family Practice 2001 22(retrieved from httpwwwbiomedcentralcom1471-229622)

39 Marnett LJ Lipid peroxidation-DNA damage by malondialdehydeMutat Res 199942483ndash95

40 Mishra S Sharma DC Sharma P Studies of biochemicalparameters in breast cancer with and without metastasis Indian JClin Biochem 20041971ndash5

41 Dreher D Junod AF Role of oxygen free radicals in cancerdevelopment Eur J Cancer 199632A30ndash8

42 Jungst C Cheng B Gehrke R Schmitz V Nischalke HDRamakers J et al Oxidative damage is increased in human livertissue adjacent to hepatocellular carcinoma Hepatology2004391663ndash72

43 Balasubramanian S Kowdley KV Effect of alcohol on viralhepatitis and other forms of liver dysfunction Clin Liver Dis2005983ndash101

44 Czeczot H Scibior D Skrzycki M Podsiad M Glutathione andGSH-dependent enzymes in patients with liver cirrhosis andhepatocellular carcinoma Acta Biochem Pol 200653237ndash42

45 Maclaughlin BW Gutsmuths B Pretner E Jonas WB Ives JKulawardane DV et al Effects of homeopathic preparations onhuman prostate cancer growth in cellular and animal models IntegrCancer Ther 20065362ndash72

46 Jonas WB Gaddipati JP Rajeshkumar NV Sharma A et alCan homeopathic treatment slow prostate cancer growthIntegr Cancer Ther 20065343ndash9

47 Adams LS Seeram NP Hardy ML Carpenter C Heber DAnalysis of the Interactions of Botanical Extract CombinationsAgainst the Viability of Prostate Cancer Cell Lines eCAM20063117ndash24

48 Padmavathi B Rath PC Rao AR Singh RP Roots of Withaniasomnifera Inhibit Forestomach and Skin Carcinogenesis in MiceEvid Based Compliment Alernat Med 2005299ndash105

49 Miller SC Echinacea a Miracle Herb against Aging and CancerEvidence In vivo in Mice Evid Based Compliment Alternat Med20052309ndash14

50 Pathak S Multani AS Banerjee P Ruta 6 selectively induced celldeath in brain cancer cells but proliferation in normal peripheralblood lymphocytes A novel treatment for human brain cancerInt J Oncol 200323975ndash82

51 Khuda-Bukhsh AR Potentized homeopathic drugs act throughregulation of gene expression a hypothesis to explain theirmechanism and pathways of action in vivo Com Ther Med1997513ndash46

Received November 27 2006 accepted May 8 2007

eCAM 20096(1) 75

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS


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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

ultimately become neoplastic (141527) Therefore this

induced liver carcinoma serves as a good model for the

study of events during carcinogenesis as well as to test

if a drug has any anti-cancerous effect Further

homeopathic mode of treatment does not have any

known side effects and is less expensive than that of

conventional treatments of liver cancer which often needs

surgery and costly medicines in management

Remedy Selection Indication from a Pilot Study

We have for some time been screening some homeopathicremedies which are claimed to have pronounced effectsin protecting liver from toxicity or that are known to helpin recovery of liver dysfunction (eg ChelidoniumLycopodium etc) for their possible anti-hepatotoxicanti-tumorigenic effects since the primary target organ ofthe carcinogens is the liverPotentized form of Natrum Sulphuricum is one such

remedy generally used by homeopathic practitioners forvarious liver disorders having agreeable symptoms (28)As chronic feeding of P-DAB and PB is known to causesymptoms arising out of liver disorders and NatrumSulph being one of the remedies used to alleviate andprotect liver from toxic effects we first conducted a pilotstudy on the efficacy of Nat Sulph-30 with a limitednumber of protocols (data unpublished) On getting apositive result the present investigation was undertakenwith a primary objective of evaluating efficacy of NatSulph-200 (a more diluted higher potency claimed to havestronger and longer lasting effect) in amelioration ofcarcinogen induced toxicity in mice utilizing severalwidely accepted protocols of cytogenetical and biochem-ical studies

Implications In Brief of the Parameters Used

The parameters used in this study are importantgenotoxicity and cyto-toxicity (particularly hepatotoxin)biomarkers either directly or indirectly implicated in thedevelopment of liver tumors The changes observed in thecytogenetical studies led to the studies of LPO which isassociated with tissue damage and necrosis Similarlytissue damage and necrosis can lead to improper liverfunction and thereby causing hepato-toxicity For thisliver function tests that include the assay of variousenzymes like AcP AlkP AST and ALT were selected tothrow light on the extent of liver malfunctioning if anyFurther if hepato-toxicity is generated there will begeneration of free radicals GSH has anti-oxidant activitywhich is accomplished by scavenging free-radicals and thestudy of GSH was therefore of added value Furtherwhile decrease of the other biochemical parametersindicates decreased hepato-toxicity a concomitantincrease in GSH is expected to confirm this

Methods

Feeding the Carcinogens and the Homeopathic Remedy to

Mice

An inbred strain of Swiss albino mice (Mus musculus)

were reared and maintained in the animal house of the

Department of Zoology (with clearance from the

University Ethical Committee and under the supervision

of the Animal Welfare Committee) University of

Kalyani India for the investigation Mice under experi-

ment were provided food and water and kept in a

hygienic condition The general diet was prepared

from wheat gram and powdered milk without any

animal protein supplement A group of 36 healthy

mice weighing between 25ndash30 g were used for

each treatment series namely (i) normal (ii) normalthorn

alcohol 200 (iii) P-dimethylaminoazobenzene (p-DAB)thorn

phenobarbital (PB) (iv) p-DABthornPBthorn alcohol 200

(v) p-DABthornPBthornNat Sulph-200 fed series In our earlier

studies we used to maintain another group only verum

fed control along with normalthorn succussed alcohol fed

control But since the differences in data between these

two series were negligible or insignificant the normalthorn

verum fed group as control was later abandoned for

saving life of mice and costEach group of 36 mice divided into six different sets

consisting of six mice each for six different intervals offixation namely at day seven 15 30 60 90 and 120

The first set of mice (group-I normal negative control)was fed normal low protein diet The second set of mice(group II) was fed diluted alcohol 200 (potentized as per

homeopathic principle of dilution and succussion) withnormal low protein diet The third set of mice (group-III)was fed the same diet but mixed with 006 p-DAB(Sigma D-6760) at a daily dose of 165mgkg body wt

per mouse (152729) and an oral dose of 006ml of005 aqueous solution of PB daily (1430) through aspecially made fine pipette The fourth set of mice(group-IV positive control) was fed 006 p-DAB along

with 005 aqueous solution of PB as above andalso diluted alcohol 200 (alc-200 as the lsquovehiclersquo of thedrug was ethyl alcohol) till they were sacrificed Thefifth set of mice (group-V) was fed p-DAB plus PB as

in group-III but was also fed Nat Sulph-200 once aday till they were sacrificed at day seven 15 30 60 90and 120

Feeding Procedure and Dose

Each mouse was fed one drop (006ml) of stock

solution of Nat Sulph-200 or alc-200 as the case

may be once daily (one dose at 7AM) with the aid of

a fine pipette





Cytogenetic Assay


Biochemical Assays





Results

Tumor Growth












Normal 36 06 06 06 06 06 06

NormalthornAlc-200 36 06 06 06 06 06 06

p-DABthornPB 36 06 06 06 36 66 66



TOTAL 180 030 030 030 930 1530 1530

eCAM 20096(1) 67




Mitotic Index



Sperm Head Anomaly


Biochemical Assay



























000

500

1000

1500

2000

2500

3000

7 D 15 D 30 D 60 D 90 D 120 D

7 D 15 D 30 D 60 D 90 D 120 D


A B

C D



7 D 15 D 30 D 60 D 90 D 120 D


o

f ch

rom

oso

me

aber

rati

on

(C

A)






000

020

040

060

080

100

120

140

o

f M

icro

nu

clei

(M

N)


000

100

200

300

400

500

600

700

800

900

1000

o

f M

ito

tic

Ind

ex (

MI)




000

050

100

150

200

250

300

350

400

450

o

f sp

erm

hea

d a

no

mal

y(S

HA

)

b

cc

c

c

ac

cc c

a

c

c

c

c

a

c

c

c

c







eCAM 20096(1) 69





Normal 0017 0002 0012 0001 0021 0001


p-DABthornPB 0040 0002 0041 0005 0049 0002



Sulph-200



Normal 0024 0004 0010 0001 0054 0002


p-DABthornPB 0047 0006 0056 0004 0075 0007



Sulph-200






Normal 0006 0000 0010 0001 0005 0000


p-DABthornPB 0009 0002 0028 0002 0017 0002



Sulph-200



Normal 0006 0000 0006 0001 0004 0000


p-DABthornPB 0015 0001 0015 0002 0019 0001



Sulph-200







Normal 0048 0001 0143 0007 0040 0002


p-DABthornPB 0080 0006 0157 0010 0086 0005



Sulph-200



Normal 0046 0005 0012 0000 0025 0002


p-DABthornPB 0076 0009 0053 0005 0128 0013



Sulph-200






Normal 0017 0001 0014 0003 0024 0001


p-DABthornPB 0038 0001 0026 0004 0044 0002



Sulph-200



Normal 0021 0001 0019 0001 0012 0001


p-DABthornPB 0034 0001 0045 0004 0139 0006



Sulph-200


eCAM 20096(1) 71





Normal 0245 0008 0135 0010 0086 0002


p-DABthornPB 0332 0010 0312 0010 0234 0008



Sulph-200



Normal 0052 0004 0043 0001 0048 0001


p-DABthornPB 0247 0012 0119 0010 0131 0009



Sulph-200






Normal 0004 0000 0006 0000 0005 0001


p-DABthornPB 0001 0000 0001 0000 0001 0000



Sulph-200



Normal 0005 0000 0006 0000 0007 0000


p-DABthornPB 0001 0000 0002 0000 0003 0000



Sulph-200



Discussion












Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005




Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005






fed mice (4c-4d)

eCAM 20096(1) 73



Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Cytogenetic Assay


Biochemical Assays





Results

Tumor Growth












Normal 36 06 06 06 06 06 06

NormalthornAlc-200 36 06 06 06 06 06 06

p-DABthornPB 36 06 06 06 36 66 66



TOTAL 180 030 030 030 930 1530 1530

eCAM 20096(1) 67




Mitotic Index



Sperm Head Anomaly


Biochemical Assay



























000

500

1000

1500

2000

2500

3000

7 D 15 D 30 D 60 D 90 D 120 D

7 D 15 D 30 D 60 D 90 D 120 D


A B

C D



7 D 15 D 30 D 60 D 90 D 120 D


o

f ch

rom

oso

me

aber

rati

on

(C

A)






000

020

040

060

080

100

120

140

o

f M

icro

nu

clei

(M

N)


000

100

200

300

400

500

600

700

800

900

1000

o

f M

ito

tic

Ind

ex (

MI)




000

050

100

150

200

250

300

350

400

450

o

f sp

erm

hea

d a

no

mal

y(S

HA

)

b

cc

c

c

ac

cc c

a

c

c

c

c

a

c

c

c

c







eCAM 20096(1) 69





Normal 0017 0002 0012 0001 0021 0001


p-DABthornPB 0040 0002 0041 0005 0049 0002



Sulph-200



Normal 0024 0004 0010 0001 0054 0002


p-DABthornPB 0047 0006 0056 0004 0075 0007



Sulph-200






Normal 0006 0000 0010 0001 0005 0000


p-DABthornPB 0009 0002 0028 0002 0017 0002



Sulph-200



Normal 0006 0000 0006 0001 0004 0000


p-DABthornPB 0015 0001 0015 0002 0019 0001



Sulph-200







Normal 0048 0001 0143 0007 0040 0002


p-DABthornPB 0080 0006 0157 0010 0086 0005



Sulph-200



Normal 0046 0005 0012 0000 0025 0002


p-DABthornPB 0076 0009 0053 0005 0128 0013



Sulph-200






Normal 0017 0001 0014 0003 0024 0001


p-DABthornPB 0038 0001 0026 0004 0044 0002



Sulph-200



Normal 0021 0001 0019 0001 0012 0001


p-DABthornPB 0034 0001 0045 0004 0139 0006



Sulph-200


eCAM 20096(1) 71





Normal 0245 0008 0135 0010 0086 0002


p-DABthornPB 0332 0010 0312 0010 0234 0008



Sulph-200



Normal 0052 0004 0043 0001 0048 0001


p-DABthornPB 0247 0012 0119 0010 0131 0009



Sulph-200






Normal 0004 0000 0006 0000 0005 0001


p-DABthornPB 0001 0000 0001 0000 0001 0000



Sulph-200



Normal 0005 0000 0006 0000 0007 0000


p-DABthornPB 0001 0000 0002 0000 0003 0000



Sulph-200



Discussion












Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005




Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005






fed mice (4c-4d)

eCAM 20096(1) 73



Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Mitotic Index



Sperm Head Anomaly


Biochemical Assay



























000

500

1000

1500

2000

2500

3000

7 D 15 D 30 D 60 D 90 D 120 D

7 D 15 D 30 D 60 D 90 D 120 D


A B

C D



7 D 15 D 30 D 60 D 90 D 120 D


o

f ch

rom

oso

me

aber

rati

on

(C

A)






000

020

040

060

080

100

120

140

o

f M

icro

nu

clei

(M

N)


000

100

200

300

400

500

600

700

800

900

1000

o

f M

ito

tic

Ind

ex (

MI)




000

050

100

150

200

250

300

350

400

450

o

f sp

erm

hea

d a

no

mal

y(S

HA

)

b

cc

c

c

ac

cc c

a

c

c

c

c

a

c

c

c

c







eCAM 20096(1) 69





Normal 0017 0002 0012 0001 0021 0001


p-DABthornPB 0040 0002 0041 0005 0049 0002



Sulph-200



Normal 0024 0004 0010 0001 0054 0002


p-DABthornPB 0047 0006 0056 0004 0075 0007



Sulph-200






Normal 0006 0000 0010 0001 0005 0000


p-DABthornPB 0009 0002 0028 0002 0017 0002



Sulph-200



Normal 0006 0000 0006 0001 0004 0000


p-DABthornPB 0015 0001 0015 0002 0019 0001



Sulph-200







Normal 0048 0001 0143 0007 0040 0002


p-DABthornPB 0080 0006 0157 0010 0086 0005



Sulph-200



Normal 0046 0005 0012 0000 0025 0002


p-DABthornPB 0076 0009 0053 0005 0128 0013



Sulph-200






Normal 0017 0001 0014 0003 0024 0001


p-DABthornPB 0038 0001 0026 0004 0044 0002



Sulph-200



Normal 0021 0001 0019 0001 0012 0001


p-DABthornPB 0034 0001 0045 0004 0139 0006



Sulph-200


eCAM 20096(1) 71





Normal 0245 0008 0135 0010 0086 0002


p-DABthornPB 0332 0010 0312 0010 0234 0008



Sulph-200



Normal 0052 0004 0043 0001 0048 0001


p-DABthornPB 0247 0012 0119 0010 0131 0009



Sulph-200






Normal 0004 0000 0006 0000 0005 0001


p-DABthornPB 0001 0000 0001 0000 0001 0000



Sulph-200



Normal 0005 0000 0006 0000 0007 0000


p-DABthornPB 0001 0000 0002 0000 0003 0000



Sulph-200



Discussion












Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005




Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005






fed mice (4c-4d)

eCAM 20096(1) 73



Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























000

500

1000

1500

2000

2500

3000

7 D 15 D 30 D 60 D 90 D 120 D

7 D 15 D 30 D 60 D 90 D 120 D


A B

C D



7 D 15 D 30 D 60 D 90 D 120 D


o

f ch

rom

oso

me

aber

rati

on

(C

A)






000

020

040

060

080

100

120

140

o

f M

icro

nu

clei

(M

N)


000

100

200

300

400

500

600

700

800

900

1000

o

f M

ito

tic

Ind

ex (

MI)




000

050

100

150

200

250

300

350

400

450

o

f sp

erm

hea

d a

no

mal

y(S

HA

)

b

cc

c

c

ac

cc c

a

c

c

c

c

a

c

c

c

c







eCAM 20096(1) 69





Normal 0017 0002 0012 0001 0021 0001


p-DABthornPB 0040 0002 0041 0005 0049 0002



Sulph-200



Normal 0024 0004 0010 0001 0054 0002


p-DABthornPB 0047 0006 0056 0004 0075 0007



Sulph-200






Normal 0006 0000 0010 0001 0005 0000


p-DABthornPB 0009 0002 0028 0002 0017 0002



Sulph-200



Normal 0006 0000 0006 0001 0004 0000


p-DABthornPB 0015 0001 0015 0002 0019 0001



Sulph-200







Normal 0048 0001 0143 0007 0040 0002


p-DABthornPB 0080 0006 0157 0010 0086 0005



Sulph-200



Normal 0046 0005 0012 0000 0025 0002


p-DABthornPB 0076 0009 0053 0005 0128 0013



Sulph-200






Normal 0017 0001 0014 0003 0024 0001


p-DABthornPB 0038 0001 0026 0004 0044 0002



Sulph-200



Normal 0021 0001 0019 0001 0012 0001


p-DABthornPB 0034 0001 0045 0004 0139 0006



Sulph-200


eCAM 20096(1) 71





Normal 0245 0008 0135 0010 0086 0002


p-DABthornPB 0332 0010 0312 0010 0234 0008



Sulph-200



Normal 0052 0004 0043 0001 0048 0001


p-DABthornPB 0247 0012 0119 0010 0131 0009



Sulph-200






Normal 0004 0000 0006 0000 0005 0001


p-DABthornPB 0001 0000 0001 0000 0001 0000



Sulph-200



Normal 0005 0000 0006 0000 0007 0000


p-DABthornPB 0001 0000 0002 0000 0003 0000



Sulph-200



Discussion












Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005




Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005






fed mice (4c-4d)

eCAM 20096(1) 73



Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Normal 0017 0002 0012 0001 0021 0001


p-DABthornPB 0040 0002 0041 0005 0049 0002



Sulph-200



Normal 0024 0004 0010 0001 0054 0002


p-DABthornPB 0047 0006 0056 0004 0075 0007



Sulph-200






Normal 0006 0000 0010 0001 0005 0000


p-DABthornPB 0009 0002 0028 0002 0017 0002



Sulph-200



Normal 0006 0000 0006 0001 0004 0000


p-DABthornPB 0015 0001 0015 0002 0019 0001



Sulph-200







Normal 0048 0001 0143 0007 0040 0002


p-DABthornPB 0080 0006 0157 0010 0086 0005



Sulph-200



Normal 0046 0005 0012 0000 0025 0002


p-DABthornPB 0076 0009 0053 0005 0128 0013



Sulph-200






Normal 0017 0001 0014 0003 0024 0001


p-DABthornPB 0038 0001 0026 0004 0044 0002



Sulph-200



Normal 0021 0001 0019 0001 0012 0001


p-DABthornPB 0034 0001 0045 0004 0139 0006



Sulph-200


eCAM 20096(1) 71





Normal 0245 0008 0135 0010 0086 0002


p-DABthornPB 0332 0010 0312 0010 0234 0008



Sulph-200



Normal 0052 0004 0043 0001 0048 0001


p-DABthornPB 0247 0012 0119 0010 0131 0009



Sulph-200






Normal 0004 0000 0006 0000 0005 0001


p-DABthornPB 0001 0000 0001 0000 0001 0000



Sulph-200



Normal 0005 0000 0006 0000 0007 0000


p-DABthornPB 0001 0000 0002 0000 0003 0000



Sulph-200



Discussion












Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005




Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005






fed mice (4c-4d)

eCAM 20096(1) 73



Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Normal 0048 0001 0143 0007 0040 0002


p-DABthornPB 0080 0006 0157 0010 0086 0005



Sulph-200



Normal 0046 0005 0012 0000 0025 0002


p-DABthornPB 0076 0009 0053 0005 0128 0013



Sulph-200






Normal 0017 0001 0014 0003 0024 0001


p-DABthornPB 0038 0001 0026 0004 0044 0002



Sulph-200



Normal 0021 0001 0019 0001 0012 0001


p-DABthornPB 0034 0001 0045 0004 0139 0006



Sulph-200


eCAM 20096(1) 71





Normal 0245 0008 0135 0010 0086 0002


p-DABthornPB 0332 0010 0312 0010 0234 0008



Sulph-200



Normal 0052 0004 0043 0001 0048 0001


p-DABthornPB 0247 0012 0119 0010 0131 0009



Sulph-200






Normal 0004 0000 0006 0000 0005 0001


p-DABthornPB 0001 0000 0001 0000 0001 0000



Sulph-200



Normal 0005 0000 0006 0000 0007 0000


p-DABthornPB 0001 0000 0002 0000 0003 0000



Sulph-200



Discussion












Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005




Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005






fed mice (4c-4d)

eCAM 20096(1) 73



Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Normal 0245 0008 0135 0010 0086 0002


p-DABthornPB 0332 0010 0312 0010 0234 0008



Sulph-200



Normal 0052 0004 0043 0001 0048 0001


p-DABthornPB 0247 0012 0119 0010 0131 0009



Sulph-200






Normal 0004 0000 0006 0000 0005 0001


p-DABthornPB 0001 0000 0001 0000 0001 0000



Sulph-200



Normal 0005 0000 0006 0000 0007 0000


p-DABthornPB 0001 0000 0002 0000 0003 0000



Sulph-200



Discussion












Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005




Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005






fed mice (4c-4d)

eCAM 20096(1) 73



Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Discussion












Dues to Series 4 2326 1123 28913 3945 08247 9582 526 1502

Due to Days 5 1167 564 4585 626 003821 444 146 418

P5005




Dues to Series 4 00016054 1790 00002548 1412 0003271 1339 0002043 410 002558 1678 00000218 3303

Due to Days 5 00009453 1054 00000997 552 0006541 2679 000 606 003844 2522 000 752

P5005






fed mice (4c-4d)

eCAM 20096(1) 73



Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Conclusions


Acknowledgements


























































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































eCAM 20096(1) 75






of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Amelioration of Carcinogen-Induced Toxicity in Mice...

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