Department of Cancer Biology
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo New York USA 14263
Tel: 716-845-8296
Fax: 716-845-1349

Education
BSc, Chemistry, Gauhati University, Assam, India, 1960
MS, Chemistry, Tufts University, Medford, Massachusetts, 1965
PhD, Chemistry, University of Southampton, United Kingdom, 1970

General Research Interest
My research activities involve the development of sensitive techniques for the detection of macromolecular modifications without using radioisotopes and the application of the techniques to study Chemoprevention of genotoxicity induced by cancer chemotherapy. The ongoing studies are targeted to understand the molecular mechanisms involved in DNA damage induced by anti-breast cancer agent Tamoxifen and the role of antioxidants to prevent such damages in exposed cells.

Program: Antioxidants & Chemoprevention Involving Tamoxifen

Progress
1) To design and develop highly sensitive techniques to assay macromolecular modifications without handling and disposal of radioisotopes.

A novel assay for DNA damage has been developed. This assay combines enzymatic digestion of the DNA with fluorescence postlabeling. When coupled with the laser-induced fluorescence (LIF) detector, the technique offers detection sensitivity comparable to radiolabeling without handling and disposing issues of radiolabeling. The test results showed the general applicability of the technique to assay different types of DNA modifications. DNA modifications induced by commonly used chemotherapeutic agents, such as fluoropyrimidines, platinated drugs and AZT were detected by this technique using human carcinoma cells exposed in culture to these agents. Since administration of radiolabeled drugs is not required, this method has potential for in vivo analysis of DNA damage induced by drugs.

Fluorescence postlabeling assay for DNA damage has been extended for protein modification analysis. All aliphatic amino acid analyses are potential targets of modification by radiolysis, but aromatic acid residues of proteins are particularly sensitive to oxidation by ozone, singlet oxygen, radiolysis and metal-ion catalyzed oxidation (MCO). Two oxidized amino acids of tyrosine, namely DOPA and DT, have been detected in the laboratory using peptides and proteins exposed to ionizing radiation.

2) To evaluate the use of anti-oxidants to reduce the toxicity of oxidative metabolites of tamoxifen.

Tamoxifen (Tam), an antiestrogenic compound, is used in the treatment of breast cancer. In addition to its established chemotherapeutic use, it is being currently evaluated as a prophylactic agent in women at high risk for breast cancer. However, chronic administration of Tam to breast cancer patients is reported to be associated with increased risk for endometrial cancer.

A major microsomal metabolite of Tam (4-OHTam) has been observed in the uterus of both humans and rodents. Since high specific activities of peroxidase are present in endometrial tissues, peroxidase-mediated oxidation of 4-OH Tam can generate reactive electrophile. Expression of CYPs in human uterine endometrium suggests that endometrial tissue have potential to generate genotoxic Tam metabolites. Cellular DNA is susceptible for modification by electrophiles. Whether Tam undergoes oxidative metabolism in endometrial tissue or its metabolism occurs in liver with subsequent accumulation of metabolites in susceptible tissues, inhibition of further oxidation to reactive electrophile by antioxidants will inhibit subsequent damage to macromolecules in the endometrium. Preliminary results using commercial DNA, shows that both ascorbic acid and N-acetylcysteine inhibit the formation of 4-OHTam induced DNA damage in a dose dependent manner. Similar observations were made later using cell culture model. Also, as an ongoing effort to design a more improved methodology for DNA modifications, another novel technique has been developed to detect tamoxifen-DNA adduct by HPLC using post-column, on-line photochemical irradiation without relying on fluorescence postlabeling.

Future Plans

Ascorbic acid and NAC are inexpensive and remarkably nontoxic. The goal is to develop a program to evaluate whether Tam in combination with these dietary antioxidants will offer better prophylactic action for breast cancer risk than Tam or any other SERM alone, as is being used currently. A good understanding of cellular mechanisms behind the anticipated biological effects of antioxidant nutrient on Tam toxicity using the proposed model and the innovative technique developed in the laboratory to assay DNA damage will help to achieve this goal.

Key Publications

• Sharma M, Freund HG. Development of laser-induced fluorescence detection to assay DNA damage. In: Optical Methods for Ultrasensitive Detection and Analysis: Techniques and Applications, B. L. Fearey, ed., Proc. SPIE 1435:280-291, 1991.
• Sharma M, Jain R, Ionescu E, Slocum HK. Capillary electrophoretic separation and laser-induced fluorescence detection of the major DNA adduct of cisplatin and carboplatin. Anal. Biochem. 228:307-311, 1995.
• Sharma M, Jain R. Isolation and analysis of dityrosine from enzyme-catalyzed oxidation of tyrosine and X-irradiated peptide and proteins. Chem.-Biol. Interact. 108:171-185, 1998.
• Sharma M, Slocum HK. Prevention of quinone-mediated DNA arylation by antioxidants. Biochem. Biophys. Res. Commun. 262:769-774, 1999.
• Sharma M. Analysis of tamoxifen-DNA adducts by HPLC using post-column on-line photochemical activation. Biochem. Biophys. Res. Commun. 273:40-44, 2000.