Re: Rajeswari,N., Ahuja,Y.R., Malani,U., Chandrashekar,S., Balakrishna,N., Rao,K.V. and Khar,A. (2000) Risk assessment in the first degree female relatives of breast cancer patients using the alkaline Comet assay. Carcinogenesis, 21, 557–561

Armen K. Nersesyan

Laboratory of Carcinogenesis, Cancer Research Centre, 76 Fanardjian Street, Yerevan 75052, Armenia Email: genetik{at}ysu.am

Dear Sir

I read with great interest the research findings of Dr Rajeswari and co-workers (1). I draw attention to the extremely high background micronucleus (MN) level in exfoliated buccal mucosa cells of untreated cancer patients and their first degree female relatives (3.41 and 1.65%, respectively) compared with controls (0.27%). This means that in buccal cells of cancer patients and their relatives MN frequencies were 12.6- and 6.1-fold higher than in control subjects.

Analysis of some recent publications has shown that after exposure to environmental mutagens, such as pesticides (2), organic solvents and lead-containing pigments (3), radioactive iodine (4) and antineoplastic drugs (5), MN level increased only 2.7–3.7 times (2,3) or did not increase significantly (4,5).

It is extremely unlikely that the difference in MN content in the same healthy population may differ by 6.1 times (female relatives and controls) while after exposure to mutagens the differences between exposed subjects and controls were 2.7–3.7.

My opinion is supported by some recent papers concerning cytogenetic disturbances in lymphocytes of breast cancer patients and their female relatives (6,7). The differences in the levels of chromosomal aberrations in lymphocytes of patients and their relatives were 1.4 (6) and 1.7 (7) and between relatives and controls was 1.5 (6) (data concerning control subjects was not presented in Trivedi et al.; ref. 7). Moreover, a paper by Jyothish et al. (8) showed no differences (neither constitutional nor structural) in lymphocytes of breast cancer patients and their relatives compared with control subjects. However, it is well established that use of MN in exfoliated buccal mucosa cells to detect chromosomal damage induced by various mutagens is less efficient than that of both MN and chromosomal aberrations in lymphocytes (3,5). Thus, if the difference in chromosomal aberration levels in lymphocytes of relatives and control subjects is <2, the difference in MN level in buccal mucosa cells should be at least the same, but no more.

I suggest that the extremely high levels of cells with MN (1) were due to two causes. Rajeswari et al. (1) analyzed only 1000 cells from each subject, although for this purpose at least 3000–5000 (9) and even, in the opinion of Belien et al. (10), 10 000 exfoliated cells should be studied, due to a low baseline MN frequency. The second reason may be the use of Giemsa staining instead of DNA-specific stains (acridine orange, Hoechst 33258 plus pyronin, propidium iodine or DAPI) because the latter can eliminate most of the artefacts associated with use of DNA non-specific stains (9).

In conclusion, I suggest that, along with real MN, Rajeswari et al. (1) registered artefacts due to the few cells scored and the use of Giemsa staining.

References

  1. Rajeswari,N., Ahuja,Y.R., Malani,U., Chandrashekar,S., Balakrishna,N., Rao,K.V. and Khar,A. (2000) Risk assessment in the first degree female relatives of breast cancer patients using the alkaline Comet assay. Carcinogenesis, 21, 557–561.[Abstract/Free Full Text]
  2. Gomez-Arroya,S., Diaz-Sanchez,Y., Meneses-Perez,M.A., Villalobos-Pictrini,R. and De Leon-Rodriguez,J. (2000) Cytogenetic biomonitoring in Mexican floriculture workers exposed to pesticides. Mutat. Res., 466, 117–124.[ISI][Medline]
  3. Pinto,D., Ceballos,J.M., Garcia,G., Guzman,P., Del Razo,L.M., Vera,E., Gomez,H., Garcia,A. and Gonsebatt,E. (2000) Increased cytogenetic damage in outdoor painters. Mutat. Res., 467, 105–111.[ISI][Medline]
  4. Ramirez,M.J., Surralles,J., Galofre,P., Creus,A. and Marcos,R. (1999) FISH analysis of 1cen-1q12 breakage, chromosome 1 numerical abnormalities and centromeric content of micronuclei in buccal cells from thyroid cancer and hyperthyroidism patients treated with radioactive iodine. Mutagenesis, 14, 121–127.[Abstract/Free Full Text]
  5. Burgaz,S., Karahalil,B., Bayrak,P., Taskin,L., Yavuzaslan,F., Bokesoy,I., Anzion,R.B.M., Bos,R.P. and Platin,N. (1999) Urinary cyclophosphamide excretion and micronuclei frequences in peripheral lymphocytes and in exfoliated buccal epithelial cells of nurses handling antineoplastics. Mutat. Res., 439, 97–104.[ISI][Medline]
  6. Roy,S.K., Trivedi,A.H., Bakshi,S.R., Patel,R.K., Shukla,P.H., Patel,S.J., Bhatavdekar,J.M., Patel,D.D. and Shah,P.M. (2000) Spontaneous chromosomal instability in breast cancer families. Cancer Genet. Cytogenet., 118, 52–56.[ISI][Medline]
  7. Trivedi,A.H., Roy,S.K., Bhachech,S.H., Patel,R.K., Dalal,A.A., Bhatavdekar,J.M. and Patel,D.D. (1998) Cytogenetic evaluation of 20 sporadic breast cancer patients and their first degree relatives. Breast Cancer Res. Treat., 48, 187–190.[ISI][Medline]
  8. Jyothish,B., Ankathil,R., Chandini,R., Vinodkumar,B., Nayar,G.S., Roy,D.D., Madhavan,J. and Nair,M.K. (1998) DNA repair proficiency: a potential marker for identification of high risk members in breast cancer families. Cancer Lett., 124, 9–14.[ISI][Medline]
  9. Albertini,R.J., Anderson,D., Douglas,G.R., Hagmar,L., Hemminki,K., Merlo,F., Natarajan,A.T., Norppa,H., Shuker,D.E.G., Tice,R., Waters,M.D. and Aitio,A. (2000) IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutat. Res., 463, 111–172.[ISI][Medline]
  10. Belien,J.A., Copper,M.P., Braakhuis,B.J., Snow,G.B. and Baak,J.P. (1995) Standardization of counting micronuclei: definition of a protocol to measure genotoxic damage in human exfoliated cells. Carcinogenesis, 16, 2395–2400.[Abstract]
Received October 6, 2000; accepted November 7, 2000.