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Micronucleated Erythrocytes As an Index of Cytogenetic Damage In [CANCER RESEARCH 50, 5049-5054, August 15. 1990] Micronucleated Erythrocytes as an Index of Cytogenetic Damage in Humans: Demographic and Dietary Factors Associated with Micronucleated Erythrocytes in Splenectomized Subjects1 Daniel F. Smith,2 James T. MacGregor,3 Robert A. Hiatt, N. Kim Hooper, Carol M. Wehr, Beverly Peters, Lynn R. Goldman, Leslie A. Yuan, Philip A. Smith, and Charles E. Becker Environmental Epidemiology and Toxicology Branch, California Department of Health Services, Emeryville, California 94608 [D. F. S., L. R. G., L. A. Y.J; Food Safety Research Unit, Western Regional Research Center, United States Department of Agriculture, Albany, California 94710 [J. T. M., C. M. W., P. A. S.J; The Permanente Medical Group, Division of Research, Oakland, California 94611-5463 [R. A. H., B. P.J; Reproductive and Cancer Hazard Assessment Section, California Department of Health Services, Berkeley, California 94704 [N. K. H.J; Northern California Occupational Health Center, University of California, San Francisco, California 94110 [C. E. B.I ABSTRACT nuclei to be accumulated in culture as binucleate cells which are easily recognized and scored. The ability to determine the Erythrocytes containing micronuclei serve as an indicator of genotoxic cell division being scored is a critical requirement because the exposure in splenectomized individuals. Micronucleated erythrocytes, derived from cytogenetically damaged RBC precursors, are not selectively micronucleus frequency depends on the proportion of cells removed from peripheral blood in individuals who lack splenic function. which have divided following the induction of DNA damage The relationship between micronucleated cell frequencies and demo and the fate of micronuclei in cells which have divided more graphic, environmental, and dietary factors was examined in 44 subjects than once (9). Nevertheless, the assay is still limited by a lack with previous splenectomy due to trauma. Their micronucleated cell in sensitivity to agents which do not produce long-lived lesions counts fit a log-normal distribution, with geometric means of 3.3 micro- in nondividing cells, due to the test cell population not actively nucleus-containing cells/1000 reticulocytes and 2.7/1000 normochromatic dividing in vivo. erythrocytes. A multiple regression analysis showed that drinking five This report suggests using micronucleated peripheral blood cups of coffee or tea/day (relative to none) was associated with an approximately 2-fold higher frequency of micronucleated cells. Weaker erythrocytes in splenectomized subjects as a marker for chro statistical associations were also noted with micronucleus frequency and mosomal damage. The end point measured is the prevalence of the consumption of calcium supplements (associated with a higher fre micronuclei (also known as Howell-Jolly bodies) among periph quency) and vitamins A, C, or E (lower frequency). An apparent trend of eral erythrocytes. Under normal conditions, the human spleen higher micronucleus counts with age was attenuated when other factors removes micronucleus-containing erythrocytes from the periph were considered in the regression. Cigarette smoking and decaffeinated eral blood. But in asplenic individuals, micronucleated cells coffee consumption were among the factors not associated with elevated persist and accumulate with repeated genotoxic exposure (10). micronucleated cell frequencies. Because the occurrence of micronuclei in reticulocytes reflects cytotoxic exposures within the past 3-8 days, it As a consequence, evidence of cytogenetic damage is uniquely may be possible to test directly the relationship of these factors to visible in splenectomized persons as an increased frequency of micronucleus formation through intervention studies. micronucleus-containing RBCs. Although limited to splenec tomized individuals, the advantages of the assay include sim plicity and greater ease of scoring than other currently applied INTRODUCTION assays. With the current concern for the potential genotoxic and The assay can be conducted on two erythrocyte populations: carcinogenic effects of environmental exposures (in air, water, the immature polychromatic erythrocytes which stain positively diet, occupation, etc.), sensitive indicators of DNA damage are for residual RNA (hereafter referred to as reticulocytes) and the needed to determine the extent to which such exposures actually mature normochromatic erythrocytes which do not stain posi contribute to genotoxic damage in humans. tively for RNA (hereafter simply erythrocytes). Newly formed The measurement of micronuclei in human erythrocytes and reticulocytes mature into circulating erythrocytes after only 1- lymphocytes is one such marker of the effect of genotoxic 2 days (11); hence, micronucleated reticulocytes demonstrate exposures (1). The measurement of micronuclei in bone marrow only very recent genotoxic exposures. Erythrocytes have an erythrocytes has become established as a routine screening test average life span of 120 days (11); hence, micronucleated eryth for in vivo cytogenetic damage in experimental animals (2) and rocytes reflect genotoxic exposures within approximately 4 has been applied to humans with exposures to chemotherapeu- months prior to sampling (10). tic drugs or with occupational exposures (3-7). However, the The studies of micronuclei in circulating erythrocytes which invasive nature of bone marrow sampling severely limits the have been reported to date have examined relatively small usefulness of this procedure as a routine assay for cytogenetic numbers of subjects splenectomized due to illness (idiopathic damage in humans. Interest in the peripheral blood lymphocyte thrombocytopenia, Hodgkin's disease, leukemia, etc.) or trau assay (8) has been stimulated recently by the introduction of the cytokinesis-block method (9), which allows first-division matic damage to the spleen. Chemotherapeutic agents known to damage DNA cause > 10-fold increases in micronucleated Received 11/1/89; revised 4/11/90. cells, and the kinetics of the appearance and disappearance of The costs of publication of this article were defrayed in part by the payment micronucleated cells have been shown to be consistent with the of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. kinetics of RBC formation and turnover (10). A marked effect ' Support for this study was provided in part by a grant from the Northern of mild folate deficiency on the frequency of micronucleated California Cancer Program. 2To whom requests for reprints should be addressed, at the Environmental cells has also been reported (12). Epidemiology Section, California Department of Health Services, 5900 Hollis The present investigation sought to examine the frequency Street, Suite E, Emeryville, CA 94608. 'Present address: SRI International, 333 Ravenswood Avenue, Menlo Park. of micronucleated cells in a group of splenectomized (but oth CA 94025. erwise healthy) subjects and to identify common demographic, 5049 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. MICRONUCLEATED ERYTHROCYTES IN HUMANS environmental, and dietary factors that may be correlated with that was approximately 0.05 times the area of every fourth field and is this frequency. based upon at least 100 cells. The percentage of RNA-positive cells in each sample was determined by counting the number of RNA-positive cells in each fourth field and MATERIALS AND METHODS counting the number of RNA-negative cells in a small sector of that field. For a typical proportion of RNA-positives to RNA-negatives, this Enrollment of Study Subjects. Potential study subjects were identified percentage was based upon a total count of approximately 30 RNA- from the records of the Northern California Kaiser Permanente Medical positive cells and approximately 100 RNA-negative cells. Care Program. Computer-stored discharge records for the years 1971- Absence of splenic function was verified by determining the frequency 1984 were searched to obtain a list of individuals who were hospitalized of "pitted" erythrocytes (14) in the glutaraldehyde-fixed blood samples. with trauma/injury (International Classification of Diseases, Rev. 9, 800-999 and E codes) and whose medical records had splenectomy Pitted cells accumulate in the erythrocyte population of subjects who lack a functioning spleen but occur at a very low frequency (<2%) in (surgical procedure code 45.1) as the primary discharge diagnosis. This individuals with normal spleen function. A sample of fixed blood in 1- produced a list of 303 individuals with a trauma-related splenectomy for whom computer-stored information, including date of birth, date 2 drops of fixative solution was placed on a glass slide with coverslip. The specimen was examined under Normarski optics at xlOOO mag of surgical procedure, and address, was available. nification. Pitted cells were defined by the presence of an apparent cell To each person was mailed a recruitment packet that contained a surface depression with a continuous, smooth, and defined edge and letter describing the study and requesting their participation, a human evident shadowing. The size of this apparent depression may range subjects consent form that followed Kaiser Permanente guidelines, and from 0.05-0.2 times the cell diameter. Cells with one or more surface a self-administered questionnaire. The questionnaire included
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