Mutation Research, 283 (1992) 87-89 87 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

MUTLET 0706

Inf luence of white b lood cell count on S C E frequency in per ipheral lymphocytes *

M.A Bender a R.J. Preston b, R.C. Leonard c, B.E. Pyatt a and P.C. Gooch d a Medical Department, Brookhaven National Laboratory * *, Upton, NYl1973, USA, h Chemical Industry Institute of Toxicology, Research Triangle Park, NC 27709, USA,

c Department of Environmental Health and Safety, ARCO Chemical, Newtown Square, PA 19073, USA and a Environmental Safety and Health Compliance, Oak Ridge National Laboratory * * *, Oak Ridge, TN 37831, USA

(Received 8 May 1992) (Accepted 22 May 1992)

Keywords." Sister-chromatid exchange; Lymphocytes; Human peripheral blood; 5-Bromodeoxyuridine

We have earlier reported that in addition to cigarette smoking status and sex, the white blood cell count (WBC) of the subject has a statistically significant influence on the mean frequency of sister-chromatid exchanges (SCE) observed in pe- ripheral lymphocytes in short-term whole blood cultures (Bender et al., 1988, 1989). We have now had the opportunity to further investigate this phenomenon in light of more detailed haemato- logical data obtained for the blood samples ob- tained for culture (i.e., data not yet available at the time of culture or initial data analysis). We present here the results of these analyses.

Correspondence: Michael A Bender, Medical Department, Brookhaven National Laboratory, Upton, NY 11793, USA.

* Research Supported by Interagency Agreements Y01- ES-20099 and Y01-ES-20101 between the National Toxi- cology Program and the U.S. Department of Energy.

** Operated by Associated Universities, Inc. for the U.S. Department of Energy under Contract No. DE-AC02- 76CH00016; accordingly, by acceptance of this article, the publisher and /o r recipient acknowledges the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

*** Operated by Martin Marietta Energy Systems Inc. for the U.S. Department of Energy under Contract No. DE-AC85-85OR-21400.

Materials and methods

We reported details on the nature of our study, population, culture and preparation protocols in our initial publication (Bender et al., 1988). Briefly, we obtained blood samples from a strati- fied random sample of the Brookhaven National Laboratory workforce and, to extend the age range represented, samples of Brookhaven re- tirees and of healthy children seen at a local hospital. Conventional phytohaemagglutinin- stimulated cultures were made in RPMI 1640 medium with 15% foetal calf serum and 25 /zM 5-bromodeoxyuridine (BrdU). These were fixed at 56 h in culture after 2-h treatment with col- cemid (0.2 ~g /ml ) . At least two samples of 25 second in vitro metaphases were scored for SCE on conventionally prepared differentially stained slides. We were concerned about the influence of cell inoculum on BdrU-induced SCE that had been discussed by Carrano and Moore (1982). Their point was that because of the known influ- ence of the level of BrdU incorporation on SCE frequency (higher levels giving higher SCE fre- quency), larger inocular, with more cells compet- ing for a limited amount of BrdU, would give lower SCE frequencies. While it was impractical to get lymphocyte counts on our blood samples

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promptly, we did do quick WBCs on the blood samples and inoculated the 10-ml cultures with enough whole blood to give a constant 5 × 10 6

leukocytes/culture. However, an aliquot of each blood sample was also sent to the haematology laboratory for a differential, haemoglobin and haematocrit. We have now entered these labora- tory findings into our data base, so that they could be used for further analysis of the as yet unexplained influence of WBC on SCE fre- quency.

Results and discussion

Limiting our analysis, as before (Bender et al., 1989, 1992), to the first complete 50 metaphase sample scored for SCE from each subject and to those for which there was a complete hematology report on the same sample, we were able to study data from 362 cases. The old 'quick' WBC values agree quite well with the corresponding values from the haematology laboratory. A correlation analysis gives an R of 0.91, which is significantly different from zero.

The mean SCE frequency, 8.21 ± 0.08 S C E / cell, agrees very well with that reported earlier for this population sample (8.12 _+ 0.08 SCE/cel l ) . The earlier-observed influences of sex and of cigarette-smoking status are again evident. For males ( N = 199) the mean is 8.02 +_ 0.10 while for females ( N = 163) it is 8.45 + 0.12, a statistically significant difference of about 5% (p = 0.006). For male cigarette smokers (N = 30) the mean is 9.02+_0.31, compared to that for male non- smokers ( N = 169) of 7 .8+0.01. For female cigarette smokers (N = 40) the mean is 9.0 + 0.25, while that for female non-smokers ( N = 123) is 8.25 _+ 0.13. The difference, about 14%, is statisti- cally highly significant (p < 0.001). The WBC in- fluence is also still observed, whether the old or the new WBC values are used (R = 0.23, p < 0.001 and R = 0.21, p < 0.001, respectively, by linear regression analysis). It should be noted that this is a positive effect, with SCE mean frequency tending to increase with increasing WBC, superficially the opposite of the influence discussed by Carrano and Moore (1982).

Though all were tested, no other significant correlations with any of the haematological pa-

rameters were observed (i.e., of SCE frequency with monocyte or lymphocyte differential, or with haemoglobin or haematocrit).

However, it will be recalled that the blood volumes inoculated were adjusted to give a con- stant leukocyte inoculum; this needs to be taken into account.

It has been reported that WBC values tend to be elevated among cigarette smokers (Frick, 1976; Kasezawa and Sakurai, 1982; Petitti and Kipp, 1986; Roger et al., 1987). Though there is a numerical difference in the expected direction, such a correlation is not, however, established by our data (by the Welch equality of means test, F = 2.07; p = 0.15 tha t the subpopulations are the same). It is also well-known that haemoglobin values tend to be elevated among cigarette smok- ers, presumably because elevated blood CO levels tie up some haemoglobin as non-functional methaemoglobin. To test the possibility that the WBC effect on SCE frequency might be a mani- festation of such a smoking effect, we calculated total haemoglobin added to each culture and tested for a correlation with smoking status. Again, however, no such correlation could be demonstrated (F = 2.19, p = 0.09).

However, still prompted by possible influence of blood inoculum volume, we also calculated the absolute numbers of each of the white blood cell types in each inoculum and again tested for their influence on mean SCE frequency. By step-wise multiple linear regression, in addition to smoking status and sex, two factors, and no others, were identified as contributing to the overall variance. These were total lymphocyte inoculum and total haemoglobin added. The contributions of these four factors to reduction in variance are, respec- tively: smoking status, 0.096; total lymphocyte in- oculum, 0.033; sex, 0.013; total haemoglobin, 0.001. The relationship of total lymphocytes is an inverse one. Fig. 1 shows a scatter plot of the regression of SCE frequency on total lymphocytes inoculated. The slope of the best-fit linear regres- sion line is -5 .96 × 10-4; R = -0.20.

Total haemoglobin and total lymphocyte in- oculum are, of course, highly correlated through the size of the whole blood inoculum used. We conclude, therefore, that the earlier-observed in- fluence of WBC on SCE frequency is, in fact, just

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Fig. 1. Scatter plot of the relationship between total lympho- cyte inoculum (in thousands) and mean SCE frequency. Dashed line is best-fit linear regression. Numerals indicate

numbers of cases with the same values.

a manifestation of the effect of competition of the lymphocytes inoculated for the available BrdU at relatively low culture medium BrdU concentra- tions, the effect which was earlier described by Carrano and Moore (1982). We had attempted, perhaps naively, to avoid this complication by adjusting our inoculum to give constant leukocyte numbers, since the results of the differential counts that would allow adjustment to give con- stant lymphocyte inoculae could not be obtained promptly. We had deliberately rejected the stratagem suggested by Carrano and Moore (1982) of using very high culture medium BrdU levels to minimize the competition effect, because we

89

wished to avoid artificially elevating the 'sponta- neous' SCE frequencies we wanted to measure. Whether we would have done better to simply use a single whole blood inoculum size instead is a question that must await availability of compa- rable constant-inoculum data, so that the vari- ances associated with the two strategies can be compared.

References

Bender, M.A, R.J. Preston, R.C. Leonard, B.E. Pyatt, P.C. Gooch and M.D. Shelby (1988) Chromosomal aberration and sister chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample, Mutation Res., 204, 421-433.

Bender, M.A, R.J. Preston, R.C. Leonard, B.E. Pyatt and P.C. Gooch (1989) Chromosomal aberration and sister- chromatid exchange frequencies in peripheral blood lym- phocytes of a large human population sample, II. Exten- sion of age range, Mutation Res., 212, 149-154.

Bender, M.A, R.J. Preston, R.C. Leonard, B.E. Pyatt and P.C. Gooch (1992) On the distribution of spontaneous SCE in human peripheral blood lymphocytes, Mutation Res., 281, 227-232.

Carrano, A.V., and D.H. Moore (1982) The rationale and methodology for quantifying sister chromatid exchange in humans, in: J.A. Heddle (Ed.), Mutagenicity: New Hori- zons in Genetic Toxicology, Academic Press, New York, pp. 267-304.

Frick, R. (1976) Rauchen und Ver~inderung der Hepari- nozytenzahl, Z. Ges. Inn. Med., 31, 946-948.

Kasezawa, N., and N. Sakurai (1982)The influence of cigarette smoking on blood constituents - variations of white blood cell counts and hemogram in data of healthy subjects, Jpn. J. Hyg., 37, 272.

Petitti, D.B., and H. Kipp (1986) The leukocyte count: associ- ations with intensity of smoking and persistence of effect after quitting, Am. J. Epidemiol., 123, 89-95.

Roger, R.S.C., K. Fletcher, B.J. Fail, H. Rahman, L. Sviland and P.J. Hamilton (1987) Factors influencing haematologi- cal measurements in healthy adults, J. Chron. Dis., 40, 943-947.

Communicated by F.H. Sobels