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 basic depressions meeting these criteria were classified as pitted. At least 500 demographic information, several dietary questions, and questions about exposures to potential genotoxic agents, including X-rays, cells were scored/specimen. Subjects with a frequency of pitted eryth rocytes of >12% were considered to lack splenic function (14). chemotherapy, and potential occupational exposures. Because of the limited life span of erythrocytes and the previous demonstration that Statistical Analysis. Laboratory and questionnaire data were merged and analyzed using the statistical software packages BMDP (15) and micronucleated cell frequencies return to their baseline values within 4 SAS (16, 17). Questionnaire responses were used to define various months after cessation of genotoxic exposures (10), the questionnaire demographic, dietary, or life-style factors that might be associated with asked only about exposures within the last 4 months. Subjects who the frequency of micronucleated cells. All of the codings and groupings agreed to participate were directed to their local health plan facility for of these factors were made without knowledge of any individual's blood drawing. Laboratory Procedures. A blood sample was drawn from each subject micronucleus frequency. into a heparinized Vacutainer tube by Kaiser Permanente laboratory Coffee and tea consumption were combined to create a variable staff. Two drops of blood were transferred to a vial containing 0.5 ml representing daily total caffeine intake from these two sources. The of 3% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The daily consumption of each were weighted proportionately to their sample was gently mixed and held at room temperature at least 24 h. approximate caffeine content (18) and summed as follows: 1 x (cups of caffeinated coffee/day) + 0.4 x (cups of tea/day) = cups of coffee Six or more standard blood smears were prepared using the freshly drawn heparinized blood. Smears were air dried, immersed in absolute equivalents/day. methanol for 2-5 min, and air dried again. All samples were delivered We also asked subjects whether they took regular doses of vitamin to the United States Department of Agriculture laboratory for analysis. or mineral supplements. Because relatively few subjects reported taking Upon receipt at the United States Department of Agriculture labora each specific vitamin or mineral, the responses were combined for those tory, the glutaraldehyde-fixed preparations were refrigerated until taking B vitamins and those taking vitamins C, E, or A. Grouping of scored. vitamins C, E, and A was based on evidence that their antioxidant The methanol-fixed slides were immersed in a solution of acridine properties may defend against genetic and carcinogenic damage (19). orange (0.02 mg/ml in 0.01 M sodium phosphate buffer, pH 7.4) for 4 Occupational title, job duties, and reported exposures to solvents, min and were then rinsed in phosphate buffer for 8 min. Prior to metal fumes, pesticides, or other chemicals were used to group subjects scoring, a coverslip was placed over the wet smear area, and the slide into those with a high potential for genotoxic exposure, those with was blotted to remove excess buffer. The stained slides were scored as relatively less potential, and those with little or no potential. wet mounts. To examine possible effects of X-ray exposures, we considered only Blood samples were scored for the frequency of micronucleus-con- X-rays to major portions of the body (such as trunk, pelvis, limbs) and taining cells in newly formed RNA-positive reticulocytes and mature excluded dental X-rays or X-rays of distal extremities. RNA-negative erythrocytes. Slides were scored as wet mounts at x630 The frequencies of micronucleus-containing cells in the two cell by epifluorescence microscopy using an oil immersion objective and a populations (reticulocytes and erythrocytes) were analyzed separately. Zeiss fluorescein Â¡sothiocyanate filter. The excitation wavelength was To investigate the association between various demographic or life 450-490 nm, the dichromatic beam splitter was set at 510 nm, and the style factors and the frequency of micronucleus-containing cells, we barrier filter was 520 nm. Under these conditions of staining and compared the frequencies between subgroups of categorical variables excitation, RNA-containing bodies fluoresce red-orange, and DNA- (such as gender) or examined scatter plots of micronucleus-containing containing bodies (nuclei and micronuclei) fluoresce bright yellow. cell counts against continuous variables, such as age and coffee con RNA-negative erythrocytes are a dull green color and are easily distin sumption. guished from the red-orange RNA-positive cells. Micronuclei were The frequency distribution of micronucleated cells/1000 cells was defined as intracellular bodies with the characteristic yellow fluores skewed to the right and appeared to follow a log-normal distribution. cence of DNA in addition to the other characteristics of micronuclei Therefore, we derived a log transformation of the frequency of micro- described in the literature (13). The frequencies of micronucleus-con- nucleus-containing cells as In (number of micronucleus-containing taining erythrocytes in each sample were determined using a semiau- cells/number of cells scored). Hence, the means we report for micro- tomated procedure in which the count of total cells scored was based nucleated cell counts/1000 cells are geometric means. upon specified subfields of known area. To assess the independent statistical relationships of several variables The numbers of micronucleus-containing cells among 2,000 RNA- simultaneously, we used a multiple linear regression analysis (20). positive erythrocytes and among at least 10,000 RNA-negative eryth Model goodness of fit was assessed by R2, which is interpreted as the rocytes were determined for each sample. The total number of RNA- proportion of the variance in the log micronuclei frequency accounted positive cells scored was estimated by counting the number of RNA- for by the independent variables in the model (20). The dependent positive cells in every fifth field scored and was based upon a count of variable in each model was the natural log of the number of micronu at least 400 cells. The total number of RNA-negative cells scored was cleus-containing cells/1000 cells, as described above. The regressions estimated by counting the number of RNA-negative cells in an area used as independent variables the demographic and life-style factors 5050

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. MICRONUCLEATED ERYTHROCYTES IN HUMANS that were associated with micronucleus counts in the unadjusted analy sis or that we thought may be associated a priori. For ease of presen tation and interpretation, the independent variables were kept untrans- formed. More complicated representations (e.g., logarithmic, quadratic) n did not significantly improve the fit. Hence, the regression coefficient r> for each independent variable indicates the change in log frequency of CO micronucleated cells associated with a one unit change in the independ ent variable. Alternatively, the antilog of the regression coefficient can be interpreted as the multiplier of the micronucleated cell frequency associated with a unit change in the independent variable. In order to compare the strengths of the associations of various study variables, we used the fitted models to calculate the micronucleus counts 6 10 12 14 predicted by varying each independent variable, holding the others constant (21). Micronucleated Cells per 1000 Reticulocytes

RESULTS Enrollment of Study Subjects. Of the 303 patients identified with a discharge diagnosis of splenectomy, 174 had either terminated their membership in the health plan, had moved without providing the health plan with a forwarding address, or were deceased. Of the remaining 129 persons, 48 (37%) declined to participate, or failed to provide both a blood speci men and a completed questionnaire. This left 81 subjects (63% of those who could be contacted) for whom we received both laboratory and questionnaire data. 0 4 8 10121416 In order to eliminate subjects with evidence of splenosis or Micronucleated Cells per 1000 Erythrocytes regenerated splenic function, we excluded 37 subjects who had Fig. 1. Frequency distribution of micronucleated cells/1000 reticulocytes (top) pitting in <12% of the erythrocytes examined. The statistical and erythrocytes (bottom) from 44 asplenic individuals. analysis below is based on the remaining 44 confirmed asplenic subjects. mean of 2.7/1000. These values are similar to values reported The asplenic subjects contained more males than females (26 for bone marrow erythrocytes (7). and 18, respectively), and the predominant ethnic group was In the unadjusted analysis, micronucleated cell frequencies white, non-Hispanic. The median age of the group was 42 years, appeared directly associated with age and consumption of coffee with the youngest member being 5 years old and the eldest 80 equivalents, while there was no apparent trend with cigarette years old. Nearly half (46%) of the group reported a total consumption (Fig. 2). household income of >$30,000/year. None of these subjects For the multivariate analysis, Table 1 lists the independent reported receiving chemotherapy within the previous 4 months, variables included in the regression, the units for each variable, although eight persons reported receiving major X-ray expo its regression coefficient and P value, and the predicted micro- sure. nucleus frequency multiplier (the exponentiation of the regres The questions about dietary and personal habits asked re sion coefficient) with its 95% confidence interval. These coef spondents to report their average experience during the pre ficients are "adjusted" for all other factors included in the vious 4 months. Sixteen (36%) reported smoking cigarettes, with a median consumption in the category of 0.5-1 pack/day. model, in the sense that they are calculated in the presence of the other factors. Twenty-nine (66%) drank coffee (median consumption, 1-2 For reticulocytes, the regression model has an R- of 0.41, cups/day), 10 (23%) drank decaffeinated coffee (median, <1 indicating that 41% of the variance in log micronucleus fre cup/day), and 12 (27%) drank tea (median, <1 cup/day). quency was accounted for by the 11 variables in the regression. Ten (23%) subjects reported taking daily doses of at least one of the vitamins A, C, or E. Four (9%) took regular doses of B The variables in this model that were statistically significantly vitamins, 10 (23%) took multiple vitamin preparations, and six associated with higher micronucleus frequencies were the con subjects (14%), all of whom were females, reported taking sumption of coffee equivalents and taking calcium supplements. calcium supplements. Taking vitamin A, C, or E supplements was significantly asso ciated with lower frequencies. The four subjects classified as having a job with a high The equivalent analysis for erythrocytes had an R2 of 0.43 potential for genotoxic exposure included three workers (a radiation worker, a pipefitter, and a shipfitter) whose reported (Table 1). Male gender and coffee equivalents consumption exposures included halogenated organic solvents or welding were significantly associated with increased micronuclei. The materials and a hairstylist who worked with hair dyes and coefficients for taking vitamins A, C, or E and calcium supple permanent wave solutions. Subjects in the medium potential ments, while approximately similar in magnitude to those in group included a wallpaper hanger and a rancher who used the reticulocyte analysis, did not reach statistical significance (0.1 < />< 0.2). herbicides. Results of the Micronucleus Assay. The numbers of micro- The regression models given in Table 1 were used to show nucleus-containing cells/1000 reticulocytes in the 44 asplenic the changes in the mean micronucleus-containing cell counts subjects ranged from 0.5-15.9/1000, with a geometric mean of (and 95% confidence intervals) that would be predicted from 3.3/1000 (Fig. 1). The number of micronucleus-containing varying each independent variable (Fig. 3). These predictions cells/1000 erythrocytes was 0.3-8.6/1000, with a geometric were calculated against the predicted micronucleated cell counts 5051

average of 5 cups/day and taking regular doses of calcium S supplements are approximately comparable (an increase of Q fio about 2/1000). An increase of about 1/1000 reticulocytes is predicted/30-year increase in age, but this association was not observed for erythrocytes. Regular daily doses of vitamin A, C, or E would be predicted to lower micronucleus-containing cell 6 399 t 0016X InY . -6.384+ 001IX counts to approximately one-half the baseline value. (p.001) (p.O 09) The results in Fig. 3 are intended to illustrate the relative 20 40 60 20 40 60 strengths of the statistical associations predicted by the factors Age m Years Age in Years investigated in this cross-sectional sample. These are, of course, Â£100- hypothetical predictions derived from a statistical model. In practice, changing the value of one variable (e.g., reducing a person's coffee consumption) might well result in the changing I 10- of another as well (e.g., increasing tea or decaffeinated coffee consumption).

InY . -5936 . 0 IdSX InY . -6119 . 0133X (P.Oon (p.O 03) DISCUSSION

0246 0246 CollÃ©eEqun/aleniCupsper Day Coffee EquivalentCupsper Day This study has demonstrated the feasibility of recruiting splenectomized subjects for an epidemiological study and has shown statistically significant associations between the micronucleus frequency in circulating reticulocytes or erythrocytes and three dietary factors: consumption of caffeinated coffee and/or tea, vitamin A, C, or E, and calcium supplements. Caffeine has long been a suspected carcinogen, primarily for cancers of the pancreas, bladder, kidney, and ovary (22). Caf feine is known to have adverse effects on DNA synthesis and 5 663 O 191X InY . -5 912 â€¢¿002BX(P-091Ã• (p-0 38) mitosis as well as DNA repair processes (23), but, in general, O 05 10 15 0 05 10 '5 human epidemiological studies have not revealed any strong or Packs ol Cigarettes per Day acKSof Cigarettes per Day consistent relationship to any cancer (22). Pancreatic cancer Fig. 2. Micronucleated cells/1000 reticulocytes (A) and erythrocytes (â€¢)from has been most studied because of an initially positive association 44 asplcnic individuals by ago of the individual (lop), caffeine intake measured as cups of coffee equivalents/day (middle), and packs of cigarettes smoked/day with coffee consumption (24), but subsequent studies have not (bottom). Note that the vertical axis is a log scale. The least squares fit line and borne out this relationship (25). Nevertheless, on the basis of the regression equation are given for each plot, as well as the P value for the test the long-term suspicion of caffeine as a carcinogen, the findings that the slope is zero. of the current study are of special interest and deserve further study. We also note that reported intake of decaffeinated coffee for a hypothetical splenectomized subject with "standard" val was not associated with a similar increase in micronuclei. ues for each independent variable: a woman aged 42 years (the Consumption of coffee has recently been reported to be asso mean) who did not drink coffee, decaffeinated coffee, or tea, ciated with increased chromosomal fragility (26) and sister did not take vitamin or calcium supplements, did not work in chromatid exchange frequency (27) in peripheral blood lym a potentially genotoxic job. had not had an X-ray in the last 4 phocytes. months, and did not smoke. The regression models predict that The possible protective role of vitamins A, C, and E in the the mean micronucleus frequencies for such a person would be etiology of cancer has received an increasing amount of support 2.4 micronucleus-containing reticulocytes/1000 and 1.4 mi- from animal as well as human observational and intervention cronucleus-containing erythrocytes/1000. Fig. 3 indicates that studies (28). Vitamin A is important for cell differentiation and the predicted effects of increasing coffee consumption by an proliferation, a deficiency of which is an important factor in

Table 1 Multiple regression analysis of micronucleated cell frequency" among reticulocytes and erythrocytes in 44 splenectomized subjects

ReticulocytesVariableGenderAgeCoffee of of coefficient (95% coefficient (95% coefficient0.2920.1090.135-0.018-0.6480.7370.3780.018-0.1030.021-0.159P"0.220.100.050.870.020.050.360.940.780.940.47Exponentialconfidencelimits)1.34(0.85-21.11 coefficient0.6040.0250.1540.085-0.3590.587-0.6410.020-0.1960.5290.040ErythrocytesP"0.020.720.030.470.190.130.140.940.620.100.86K>confidencelimits)1.83(1.13-2.97)1 vi.female10 y1 (0.98-11.14(1.01-10.98 .03(0.90-1.17(1.02-1.09(0.87-0.70(0.41-1.80(0.86-0.53 equivalentsDecaffeinated cup/day1 coffeeVitamin cup/dayDaily (0.79-112)26)30)22)0.52 ECalciumA. C. or dosevs.Daily (0.32-0.86)2.09(1.04-41.46(0.66-31.02(0.64-10.90(0.44-11.02(0.58-10.85(0.56-1A2 supplementsB dosevi.Daily vitaminsMultiple dosevi.Daily (0.23-1.02(0.62-0.82 vitaminsGenotoxic dosevi.nonononoHigh exposuresX-rayCigarette potentialYesi'i. low (0.38-.17).34).37).19)(.76).22).68).76)1.70(0.92-3.12)1.04(0.66-1.64)0.43 vi.no1 smokingUnitMale pack/dayRegression = 0.4119)22)63)85)81)31)Regression =Exponential 0 Dependent variable expressed as )n(micronucleated cells/1000 cells). * P value for two-tailed test of hypothesis that regression coefficient is 0. 5052

Fig. 3. Micronucleated cells/1000 reticu- loeytes (top) and erythrocytes (bottom) pre dicted by the multiple regression model for changes in the independent variables. The point estimates (*) and 95% confidence inter vals (bars) represent the frequency of micronucleated cells predicted by varying each inde pendent variable separately, as described. The 8 Ã¼ shifts in micronucleus counts are compared to <" o a baseline predicted value ( ) calculated when all independent variables are at their reference levels.

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carcinogenesis. Also, carotene is known to have antioxidant consumption) that were correlated with age. An analysis of our effects which protect against damage to DNA (29). In epide data relating micronucleated cell counts to age only, as, for miolÃ³gica!studies, an association with reduced lung cancer risk example, was done by Fenech and Morley (9), would have has been most strongly supported (28, 30) and enough evidence predicted a 17% increase in micronuclei among reticulocytes, exists to justify randomized double-blind controlled trials of and a 12% increase among erythrocytes, per 10-year increase the effect of ÃŸ-carotenesupplements on cancer outcomes (31). in age. In contrast, the multivariate analysis predicted an in Vitamins C and E are also antioxidants which reduce DNA crease in micronuclei per 10-year increase in age of only 11% damage and mutations and could thereby protect against car for reticulocytes and 3% for erythrocytes. cinogenesis (28). Little evidence currently exists from human Although smoking has been reported to be associated with studies for a protective effect of vitamin C, but a recent study an increased micronucleus frequency in both peripheral blood of serum vitamin E in a large cohort of Finnish men suggests a lymphocytes (37, 39) and in erythropoietic bone marrow cells reduced risk of all cancers among those with high levels com (39), we observed no evidence of an association between smok pared to those with low levels (32). ing and increased micronucleus frequency in the present study. Calcium has, until recently, not been considered of etiological This may have been due in part to the relatively low exposures importance in cancer. However, recent studies from several in this study group. No subject reported smoking as many as 2 sources have suggested that dietary calcium may be associated packs of cigarettes/day, and only three smoked >1 pack/day. with a reduced risk of colon cancer (33, 34). Calcium may bind Serum folate deficiency has recently been shown to increase with fats in the intestine which make these fats less accessible the frequency of micronuclei in erythrocytes (12) but could not to metabolism by bile salts and the subsequent formation of be evaluated in the present study. Recent observations in labo carcinogenic breakdown products (35). Our findings are not ratory animals demonstrated a strong enhancement of caffeine- consistent with the epidemiological literature in that persons induced micronucleus formation by folate deficiency (40). These with high calcium intake in our study had higher levels of findings suggest an interaction between folate status and caf- micronuclei. It may be that taking calcium supplements was a feinated beverage consumption. It will be important to monitor marker for something else not measured in our study, such as folate status in future studies and to assess its possible interac low estrogen levels in women or contaminants in over-the- tion with other factors. counter calcium supplements. The main limitation of this study was its small sample size. Several studies have reported a strong association of micro- Our group of 44 confirmed asplenic subjects was too small to nucleus frequency with age in human peripheral blood lympho adequately assess the effects of factors with low prevalence, cytes (9, 36-38). Simple scatter plots of our erythrocyte data such as specific occupational exposures. Even though 81 sub against age (Fig. 2) showed a trend similar to that reported for jects had completed questionnaire and laboratory work, we lymphocytes. However, the multiple regression analysis sug were required to eliminate 37 subjects (46%) because they gested that the age effect apparent in our unadjusted analysis showed evidence of residual or regenerated splenic function. was partly due to confounding by other factors (such as coffee The proportion of individuals with such regenerated splenic 5053

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. Micronucleated Erythrocytes as an Index of Cytogenetic Damage in Humans: Demographic and Dietary Factors Associated with Micronucleated Erythrocytes in Splenectomized Subjects

Daniel F. Smith, James T. MacGregor, Robert A. Hiatt, et al.

Cancer Res 1990;50:5049-5054.

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