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Year : 2004  |  Volume : 41  |  Issue : 2  |  Page : 72--80

Protective action of an anti-oxidant (L-Ascorbic acid) against genotoxicity and cytotoxicity in mice during p-DAB-induced hepatocarcinogenesis

B Surjyo, KB Anisur Rahman 
 Cytogenetics Laboratory, Department of Zoology, University of Kalyani, Kalyani - 741235, West Bengal, India

Correspondence Address:
K B Anisur Rahman
Cytogenetics Laboratory, Department of Zoology, University of Kalyani, Kalyani - 741235, West Bengal
India

Abstract

BACKGROUND : DNA damage from micronutrient deficiencies has been suggested as one major cause of cancer. Therefore studies involving vitamin supplementaion , particularly with those with anti-oxidant activity, in combating cancer have routinely been carried out in both in vivo and in vitro systems, but relatively much less in mice. AIMS : The present study examines if L-Ascorbic acid (AA; vitamin C) administration has any protective abilities in combating p-DAB induced hepatocarcinogenesis in mice at cytogenetical, biochemical, histological and ultra-structural levels. SETTINGS AND DESIGN : To test if AA had a protective action against genotoxicity, cytotoxicity and tissue damage in liver during p-dimethylaminoazobenezene (p-DAB) induced hepatocarcinogenesis in mice, a group of mice were chronically fed 0.06% p-DAB and 0.05% phenobarbital (PB) for a varying period of time (7, 15, 30, 60, 90 and 120 days). A sub-group of the p-DAB plus PB fed mice were also fed 1% L-ascorbic acid. Several assays were periodically conducted (at the six intervals of fixation) for determination of genotoxic (based on chromosomal, nuclear and sperm head anomalies), cytotoxic (based on the marker enzymes aspartate transaminase; AST, alanine aminotransferase; ALT; acid phosphatase; ACP; alkaline phosphatase; ALKP; lipid peroxidation; LPO); and tissue damaging (based on optical and electron microscopic studies of liver at day 60 only) effects in these different groups of mice as compared to normal healthy control. METHODS AND MATERIAL: Adult healthy mice of Swiss Albino strain, reared and maintained in the animal house of the Department of Zoology,. Kalyani University, under supervision of Animal Welfare Committee (which oversees ethical issues), served as materials for the present study. Widely practiced standard technique has been followed for each protocol. STATISTICAL ANALYSIS USED: The significance test between different series of data was conducted by student¢s t-test. RESULTS AND CONCLUSIONS : The results of all these studies indicated that AA had protective action against p-DAB induced hepatocarcinogenesis in mice.



How to cite this article:
Surjyo B, Anisur Rahman K B. Protective action of an anti-oxidant (L-Ascorbic acid) against genotoxicity and cytotoxicity in mice during p-DAB-induced hepatocarcinogenesis .Indian J Cancer 2004;41:72-80


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Surjyo B, Anisur Rahman K B. Protective action of an anti-oxidant (L-Ascorbic acid) against genotoxicity and cytotoxicity in mice during p-DAB-induced hepatocarcinogenesis . Indian J Cancer [serial online] 2004 [cited 2021 Jun 22 ];41:72-80
Available from: https://www.indianjcancer.com/text.asp?2004/41/2/72/12349


Full Text

 Introduction



Many epidemiological and experimental studies indicate that diets rich in micronutrients may reduce the risk of cancer and mutation.[1],[2],[3],[4] Vitamins are very important as micronutrients in protecting macromolecules. Ascorbic acid (AA) is one such important micronutrient, which functions as a factor in several metabolic reactions.[4],[5] Further, DNA damage from micronutrient deficiencies has also been attributed as a major cause of cancer.[6] Therefore, various studies have been conducted during recent years to investigate the genotoxic and antigenotoxic properties of AA[7],[8],[9] in both in vitro and in vivo systems, though at a much lesser scale on in vivo system, particularly in mice.[10],[11],[12]

However some conflicting role of ascorbic acid (AA) has been suggested in either inhibiting or enhancing carcinogenesis. While AA has been reported to have tumor enhancing effect in some mammalian models like guinea pig, rats[13],[14] and mice[15] its anti-carcinogenic role has also been advocated by others.[16],[17],[18],[19] The growth of solid form of Ehrlich ascites tumor has been reported to be significantly slower in mice maintained on distilled water supplemented with 1% AA.[20] Therefore we felt inclined to examine if this dose of 1% AA could also have a similar inhibitory effect in reducing p-DAB induced tumor induction/growth in liver and produce visible and quantifiable changes with regard to some cytogenetical and biochemical parameters. Further, visible changes or modulations brought forth by AA in mice, if any, were also carefully analyzed at histological and ultrastructural levels, which had not been done before in mice.

 Methods



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 (which oversees ethical issues) , University of Kalyani, served as materials for the present study. Mice provided with food and water ad libitum served as normal controls. The food was made up of wheat, gram and powdered milk without any animal protein supplement. The chronic dietary method used by several workers[21],[22] in producing hepatic liver nodules and subsequent hepatocarcinoma was adopted. In the treated group, mice (45 samples) were allowed to take 0.06% p-dimethylaminoa-zobenzene (p-DAB) (Sigma, USA) mixed with food and 0.05% phenobarbital (PB) (Sigma, USA) instead of pure water. A group (45 samples) of p-DAB+PB fed mice were also administered orally with 0.06 ml of 1% L-ascorbic acid [SRL, India], i.e. 0.6 mg approx per mouse every day with the aid of a fine specially made pipette until they were sacrificed at day 7, 15, 30, 60, 90 or 120 of feeding.

Slides for chromosomal assay were prepared by the conventional flame drying technique followed by Giemsa staining. Chromosome aberrations of various nature have been pooled into two categories: the “major” type comprising aberrations like break, fragments, ring, polyploidy, etc. and the “other” types comprising less significant aberrations like gaps, erosions, precocious centromeric separation, pycnosis, stretching, etc. A total of 500 bone marrow cells were observed, either 100 from each of 5 mice or 50 from each of 10 mice (longer intervals) of a set.

For micronucleus (MN) preparation, clean grease free slides with smeared bone marrow cells were briefly fixed in methanol and subsequently stained with May-Grunwald (Sigma, USA) followed by Giemsa (Gurr, Germany). Approximately 5000 bone marrow cells, comprising both polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were scored and the ratios between PCE and NCE ascertained.

The mitotic index (MI) was assessed from the same slide that was scanned for MN. The non-dividing and dividing cells were recorded and their ratios calculated.

For sperm head anomaly (SHA) study, the technique of Wyrobek[23] followed by Giemsa staining was adopted.

The lipid peroxidation was estimated from the supernatant by the method of Buege and Aust.[24] 1 ml of sample (homogenate containing 0.1-0.2 mg of protein) was mixed thoroughly with 2 ml of TCA-TBA-HCL mixture. The absorbance of the sample was determined at 535 nm in a double beam spectrophotometer (Schimadzu, UV 180 model) against a suitable blank. The malonaldehyde concentration of the sample was then calculated. Similarly, the methods of Bergmeyer and Brent[25] was adopted for estimation of AST and ALT. For the study of acid and alkaline phosphatases, the method of Walter and Schutt[26] was followed.

For preparation of histological slides of liver at day 60, the standard methodology using Bouin's fixative and microtome sectioning has been followed.

For electron microscopy of liver at day 60, the standard gold coating technique using critical point-drier (CPD-Biorad, Microscience Division, Warford England), sputter-coater (Agar Sputter Coater, Model 198, Stansted, United Kingdom) etc was adopted in case of scanning electron microscopy (LEO, 435VP, United Kingdom). For transmission electron microscopy, (TEM CM-10, Philips Microscope) the ultrathin sections (60-90 nm) were stained with uranyl acetate and lead citrate (Sigma, USA).

The significance test between different series of data was conducted by student¢s t-test.

 Results



The number of animals showing tumor nodules in their liver on autopsy has been shown in [Table:1]. Tumors started appearing at day 60 onwards, in all 10 out of 10 mice at each interval of fixation in the p-DAB+PB fed mice. However, in the p-DAB+PB+AA fed mice, 7 out of 10 mice at day 60, 8 out of 10 at day 90 and 7 out 10 mice at day 120 showed the formation of tumor nodules, indicating thereby positive protective effect of AA in combating tumorigenesis in mice.

As compared to typical diploid metaphase spreads in normal control, there were various types of aberrations [Figure:1] in the p-DAB+PB fed mice. The frequencies of chromosome aberration steadily increased in the p-DAB+PB fed series at all fixation intervals (pet al[36] demonstrated that such spermatotoxic effect might be due to alteration of testicular DNA and sperm chromatin structure. The feeding of AA could combat the spermatotoxic effects to some extent. As ascorbic acid has marked nucleophilic properties it might intercept the reactive metabolites thereby preventing their attack on nucleophilic sites on DNA, hence blocking adduct formation.[37],[38]

Earlier, Levine[39] implicated the role of AA in the physiology of testis in regard to protein metabolism. Many enzymatic functions of (AA) are believed to be essential for the normal integrity and function of testis i.e. synthesis, development and maintenance of normal sperm.[40] Therefore, the protective role of AA on sperm head observed in the present study could also be due to its regulatory effect on protein metabolism and repair activities in the germinal cells.

Quite appreciable changes also took place in their enzymatic activities. Some of these enzymes like ACP and ALKP have even been directly implicated to the extent of cellular damage and toxicity,[33],[41],[42],[43] as also in lipid peroxidation. Similarly, changes in AST and ALT have also been implicated to the altered transaminase activities necessitated in connection with combating/repairing protein damage and necrosis.[44] Several noticeable changes in histological patterns of liver were also noted. Similar destructive changes were also reported to occur in liver sections of guinea-pigs and rats studied through electron microscopy after these animals had been treated with carcinogens like nitrosamine and dimethylaminoazobenzene.[45] TEM also revealed significant destructive changes in sub-cellular organelles and ultra-structural organization of liver in the carcinogen fed mice, but signs of recovery were noticeable in the mice also fed AA.

Along with the above genotoxic and cytotoxic changes in several tissue and histological changes observed in liver, visible external changes were also noticeable in form of tumor nodules in the liver of most mice treated with p-DAB + PB at day 60 and afterwards.

Thus the results of the present study clearly demonstrated protective ability of AA against the tumorigenic and clastogenic potentials of p-DAB and PB in mice, and were in support of several other works that suggested the anti-cancerous effects of AA[16],[17],[18],[19] in other mammals. The mechanisms through which AA played a protective role could also be attributed to its free radical scavenging, anti-oxidant, apoptosis-inducing and nucleophilic properties as suggested by other workers.[19],[38],[44],[46] However, although the scavenging capacity of antioxidant vitamins has mainly been believed to prevent oxidative damage by neutralizing the free radicals,[12],[47] sometimes under a different condition these can also have co-genotoxic activity instead of normal anti-genotoxic action as reported in cultured mammalian cells[48] and in fish in vivo.[49],[50]

 Acknowledgements



The authors are highly indebted to: Dr. T.C. Nag, Asst. Professor of Electron Microscope Facility at All India Institute of Medical Sciences, New Delhi, for kindly providing SEM and TEM facilities and reading the histo-pathological changes, and to the University of Kalyani, for financial support of the work.

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