An inbred strain of Swiss albino mice (Mus musculus), reared and maintained in the animal house of the Department of Zoology, (under the supervision of the Animal Welfare Committee), Kalyani University, served as materials for the present study. Mice were provided food and water ad libitum. The food was made of wheat, gram and powdered milk without any animal protein supplement. With due permission from the Animal Welfare Committee, Kalyani University, which also oversees ethical issues of animal experimentation, the present investigation was undertaken. A group of 25 healthy mice weighing between 25–30 grams were used for each of the three long term fixation intervals viz. 60, 90 and 120 days. Each group was further divided into five different sets consisting of five mice each. The first set of mice were allowed normal low protein diet mixed with 0.06% p-DAB (Sigma, D-6760), a known "initiator" of hepatocarcinoma, and water ad libitum, till 30 days after which the water was replaced with 0.05% aqueous solution of PB, a known "promoter", till they were sacrificed. The second set of mice were provided with low protein diet without p-DAB and 0.05% aqueous solution of PB instead of pure water after one month as in group (i) till they were sacrificed. For the third set of mice the low protein diet was neither mixed with p-DAB nor water was replaced with PB. The third set served as negative control. The fourth set of mice were given p-DAB and PB in the same way as that of the first group but were also fed 0.06 ml of stock solution of the drug-Ch-30 thrice a day (6 A.M, 12 Noon, 6 P.M) from first day onward of p-DAB feeding, for seven days, and then twice a day (6 AM, 6 PM) till they were sacrificed. In the fifth set of mice the feeding of p-DAB, PB and Ch-200 followed the same manner as that of the fourth set of mice, except that the drug was fed twice a day (6AM, 6PM) all along till they were sacrificed.
Preparation of the potentized homeopathic drug
The two potencies of Chelidonium, procured from "HAPCO", 165, Bipin Behari Ganguli Street, Kolkata, were prepared as per the standard procedure of homoeopathic drug preparation. The dry drug material of Chelidonium majus (whole plant) was extracted in 44% ethyl alcohol (i.e. the "mother tincture"). 1 ml of the mother tincture was subsequently diluted with 99 ml HPI approved solvent (IP 96/HPI grade ethyl alcohol) and "succussed" 10 times to make the potency 1. The potency 2 was similarly made by diluting 1 ml of potency 1 with 99 ml of ethyl alcohol and giving 10 jerks, and the procedure was repeated to get the microdoses of Ch-30 and Ch-200.
Preparation of stock solution of the drug
1 ml each of Ch-30 and Ch-200 was finally diluted separately with 20 ml of double distilled water to make the stock solution of Ch-30 and Ch-200, respectively.
Feeding procedure and dose
Each mouse was fed 1 drop (0.06 ml) of either Ch-30 or Ch-200 from the stock solutions at a time with the aid of a fine pipette.
Mice were intra-peritonially injected with 0.03% colchicine @ 1 ml/100 gm body weight 1 hr and 30 min before sacrifice. Marrow of the femur was flushed in 1% sodium citrate solution at 37°C and fixed in acetic acid/ethanol (1:3). Slides were prepared by the conventional flame drying technique followed by Giemsa staining for scoring bone marrow chromosome aberrations. A total of 500 bone marrow cells were observed, 100 from each of 5 mice of a set.
For micronucleus (MN) preparation, a part of the suspension of bone marrow cells in 1% sodium citrate was smeared on clean grease free slides, briefly fixed in methanol and subsequently stained with May-Grunwald followed by Giemsa. Approximately 5000 bone marrow cells, comprising both polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were scored and the ratios between PCE and NCE were calculated.
The mitotic index (MI) was determined from the same slide which was scanned for MN. The non-dividing and dividing cells were recorded and their ratios ascertained.
For sperm head anomaly (SHA), the epididymis of each side of mouse of both (control and treated) sets was dissected out and its inner content squeezed out into 10 ml of 0.87% normal saline separately. It was made free of fats, vas deferens and other tissues. The content was thoroughly shaken, filtered through a silken cloth and dropped on grease free clean slides. The slides were allowed to air dry and then stained by dilute Giemsa (1 ml Giemsa in 10 ml distilled water).
Mice were sacrificed and their liver, spleen and kidney were quickly isolated. The tissues were homogenized with cold 0.87% normal saline, followed by centrifugation at 3000 g for 20 minutes in cooling centrifuge (C24, Remi Instruments). Before carrying out the enzymatic estimations the quantitative estimation of total protein was made by the method of Lowry et al. To 0.1 ml of the sample, 0.9 ml of 0.1 (N) NaOH was added. Then 5 ml of alkaline solution was added to the sample solution followed by 0.5 ml of Folin-Phenol reagent and after 30 minutes the extinction was read at 750 nm against appropriate blank in spectrophotometer (Shimadzu, Double beam Spectrophotometer UV-180, Japan).
Estimation of Lipid Peroxidase
The lipid peroxidation was estimated from the supernatant by the method of Buege and Aust . 1 ml of sample (homogenate containing 0.1–0.2 mg of protein) was mixed thoroughly with 2 ml of TCA-TBA-HCl (15% w/v TCA and 0.375% w/v TBA in 0.25 N HCl,). The absorbance of the sample was determined at 535 nm in a double beam spectrophotometer against a suitable blank The malonaldehyde concentration of the sample was calculated by using extinction coefficient of 1.56 × 105 M-1cm-1.
Estimation of Acid and Alkaline Phosphatases
For the study of acid and alkaline phosphatases method of Walter and Schutt  was followed. For acid phosphatase, to 0.2 ml tissue homogenate 1 ml of acid buffer was added to make the volume 1.2 ml. It was mixed and incubated at 37°C for 30 minutes. Then 2 ml of 0.1 (N) NaOH was added. The absorbance was measured at 405 nm against a blank. Then the activity of acid phosphatase was calculated as mM phenol liberated per mg of protein after 30 min of incubation at 37°C using suitable standard curve.
For alkaline phosphatase activity the 0.05 ml homogenate was mixed with 2 ml alkaline buffer so that the volume always stood at 2.05 ml. It was incubated at 37°C for 30 minutes, then 10 ml of 0.05 N NaOH was added and the absorbance was measured at 405 nm against a blank. The activity of alkaline phosphatase was calculated as mM phenol liberated per mg of protein after 30 min of incubation at 37°C using suitable standard curve.
Statistical analysis and scoring of data
The significance test between different series of data was conducted by student's t-test. During preparation of slides for cytogenetical observation and biochemical estimation of the different enzymes, the "observer" was kept "blinded" in order to remove any "bias" in observation and to keep uniformity in scoring data of both treated and control sets of mice.