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The persistence of micronuclei in peripheral blood erythrocytes: detection of chronic chromosome breakage in mice
Mutation Research, 104 (1982)367-369
367
Elsevier BiomedicalPress
The persistence of micronuclei in peripheral blood erythrocytes: detection of chronic chromosome breakage in mice R o b e r t Schlegel a n d J a m e s T. M a c G r e g o r University of California, School of Public Health, Berkeley, CA, and USDA, Western Regional Research Center, Berkeley, CA (U.S.A.)
(Accepted 12 February 1982)
Micronuclei in bone-marrow erythrocytes have been recognized for some time as an indicator of acute cytogenetic damage (e.g., Schmid, 1976; Heddle, 1973). Until recently, it was believed that bone-marrow clastogens did not increase the frequency of micronucleated erythrocytes in peripheral blood and that the spleen was involved in the removal of these abnormal cells (Von Ledebur and Schmid, 1973). It has been recently demonstrated in mice that single acute treatments with bone-marrow clastogens do, in fact, increase the frequency of micronuclei in newly formed ( p o l y c h r o m a t i c ) erythrocytes of the peripheral blood and that the peak incidences are approximately equal to those in bone marrow (MacGregor et al., 1980). We now present evidence that chromosomal breakage in bone-marrow erythroblasts produces an accumulation o f micronuclei in the older ( n o r m o c h r o m a t i c ) erythrocytes o f the peripheral blood in mice, and that there is little, if any, selective removal of these micronucleated cells. Male, Swiss-Webster mice weighing approximately 20 g (Simonsen Laboratories, Gilroy, CA) received 8 intraperitoneal (i.p.) injections of 0.2 mg/kg triethylenemelamine (TEM, Polysciences Inc., Lot No. LK-16-84, Warrington, PA) in a volume of 5 ml/kg isotonic saline over a period of 17 days. Controls received only isotonic saline. Peripheral blood samples were taken from the ventral tail artery. The initial control samples were taken on day 1, immediately preceding the first injection. The artery was penetrated with a 25 gauge needle, approximately 10/~1 of blood was collected in a capillary tube, and a blood smear was made after quickly mixing it with a drop o f newborn calf serum on a microscope slide. Slight pressure was applied to the penetration site after sampling to prevent blood loss. Blood smears were air-dried, fixed in absolute methanol within an hour of sampling, and stained with filtered Wright-Giemsa stain. The staining procedure of Address reprint requests to Robert Schlegelor James T. MacGregor, U.S. Department of Agriculture, Western Regional Research Center, 800 Buchanan Street, Berkeley, CA 94710, U.S.A. 0165-7992/82/0000-0000/$02.75 © Elsevier Biomedical Press
368
Schalm et al. (1975a) was used with the following modifications: the prepared stain was aged for at least 1 week; phosphate buffer, pH 6.8, was used; and the exposure to concentrated stain was extended to 5 min. The incidence of micronuclei was scored at 1000 x under oil immersion by a person who was unaware of the identity of the randomized and coded slides. 2000 normochromatic erythrocytes from each animal were scored at each time point, except day 8, when 1000 cells were scored. The ratio of polychromatic to normochromatic erythrocytes in peripheral blood was determined by counting the number of polychromatic cells present in the fields containing the first 1000 normochromatic cells. The incidence of micronucleated cells was also scored in 500 polychromatic erythrocytes. Selected slides were stained with Feulgen stain and fast green counterstain (Preece, 1972) to assure that similar frequencies of micronuclei were obtained with a DNA-specific stain. The incidence of micronuclei in normochromatic peripheral blood erythrocytes increased steadily during the period of TEM treatment (Fig. 1A). This elevated level
A
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I 20
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1 50
60
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B
0
0
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DAYS Fig. 1. A: Incidence of micronucleated cells among normochromatic erythrocytes (NCE) in the peripheral blood of mice given 0.2 mg/kg TEM i.p. on days 1, 3, 6, 8, 10, 15, and 17. A, control animals; [], treated. Each point is the mean of the determinations on 7 treated animals and 6 controls. The error bars represent the standard error of the mean, and the duration of TEM treatment is given by the bar in the upper left corner. The Wilcoxon rank sum test was used for statistical analysis, comparing the treated group to controls at each time point. *P < 0.02; **P < 0.005. B: Percent polychromatic erythrocytes (PCE) among peripheral blood erythrocytes of mice. Animal treatment, statistical analysis, and symbols are identical to A. Error bars are not shown when the error was smaller than the figure symbols.
369
of micronucleated cells did not return to control values until approximately 30 days after the treatment was dicontinued. The incidence of micronuclei in polychromatic peripheral blood erythrocytes was also examined during and after TEM dosing to establish that micronuclei were being produced during treatment and that production ceased when treatment was stopped. Significantly elevated frequencies of micronucleated polychromatic cells were observed at sampling points during TEM administration (days 8 and 15), and these frequencies returned to control levels on day 20, 3 days after the final injection. The ratio of polychromatic to normochromatic peripheral blood erythrocytes was not affected by TEM treatment (Fig. 1B), indicating that the dose administered did not alter the rate of red blood cell formation. The mean life span of normal red blood cells in mice has been estimated by a variety of methods, with reported values ranging from 20 to 45 days (Schalm et al., 1975b). Since the elevated levels of micronucleated normochromatic erythrocytes remained higher than control values for approximately 30 days after the last TEM injection, the life span of these micronucleated ceils must be very close to that estimated for normal red blood cells. These results provide strong evidence that micronucleated erythrocytes induced by a bone-marrow clastogen accumulate in the peripheral blood of mice, and that there is very little, or no, selective removal of these aberrant cells from the circulation. Since micronucleated erythrocytes accumulate in peripheral blood and are easily measured in a simple blood smear, samples obtained from animals in ongoing chronic or subchronic toxicity studies may permit an assessment of in vivo chromosomal breakage at very little additional effort or expense.
References Heddle, J.A. (1973) A rapid in vivo test for chromosome damage, Mutation Res., 18, 187-190. MacGregor, J.T., C.M. Wehr and D.H. Gould (1980) Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test, Environ. Mutagen., 2, 509-514. Preece, A. (1972) A Manual for Histologic Technicians, Little, Brown, Boston, MA, pp. 325-327, 181. Schalm, O.W., N.C. Jain and E.J. Carrol (1975a) Veterinary Hematology, 3rd edn., Lea and Febiger, Philadelphia, PA, p. 29. Schalm, O.W., N.C. Jain and E.J. Carrol (1975b) Veterinary Hematology, 3rd edn., Lea and Febiger, Philadelphia, PA, p. 390. Schmid, W. (1976) The micronucleus test for cytogenetic analysis, in: A. Hollaender (Ed.), Chemical Mutagens, Vol. 4, Plenum, New York, pp. 31-53. Von Ledebur, M., and W. Schmid (1973) The micronucleus test, Methodological aspects, Mutation Res., 19, 109-117.
367
Elsevier BiomedicalPress
The persistence of micronuclei in peripheral blood erythrocytes: detection of chronic chromosome breakage in mice R o b e r t Schlegel a n d J a m e s T. M a c G r e g o r University of California, School of Public Health, Berkeley, CA, and USDA, Western Regional Research Center, Berkeley, CA (U.S.A.)
(Accepted 12 February 1982)
Micronuclei in bone-marrow erythrocytes have been recognized for some time as an indicator of acute cytogenetic damage (e.g., Schmid, 1976; Heddle, 1973). Until recently, it was believed that bone-marrow clastogens did not increase the frequency of micronucleated erythrocytes in peripheral blood and that the spleen was involved in the removal of these abnormal cells (Von Ledebur and Schmid, 1973). It has been recently demonstrated in mice that single acute treatments with bone-marrow clastogens do, in fact, increase the frequency of micronuclei in newly formed ( p o l y c h r o m a t i c ) erythrocytes of the peripheral blood and that the peak incidences are approximately equal to those in bone marrow (MacGregor et al., 1980). We now present evidence that chromosomal breakage in bone-marrow erythroblasts produces an accumulation o f micronuclei in the older ( n o r m o c h r o m a t i c ) erythrocytes o f the peripheral blood in mice, and that there is little, if any, selective removal of these micronucleated cells. Male, Swiss-Webster mice weighing approximately 20 g (Simonsen Laboratories, Gilroy, CA) received 8 intraperitoneal (i.p.) injections of 0.2 mg/kg triethylenemelamine (TEM, Polysciences Inc., Lot No. LK-16-84, Warrington, PA) in a volume of 5 ml/kg isotonic saline over a period of 17 days. Controls received only isotonic saline. Peripheral blood samples were taken from the ventral tail artery. The initial control samples were taken on day 1, immediately preceding the first injection. The artery was penetrated with a 25 gauge needle, approximately 10/~1 of blood was collected in a capillary tube, and a blood smear was made after quickly mixing it with a drop o f newborn calf serum on a microscope slide. Slight pressure was applied to the penetration site after sampling to prevent blood loss. Blood smears were air-dried, fixed in absolute methanol within an hour of sampling, and stained with filtered Wright-Giemsa stain. The staining procedure of Address reprint requests to Robert Schlegelor James T. MacGregor, U.S. Department of Agriculture, Western Regional Research Center, 800 Buchanan Street, Berkeley, CA 94710, U.S.A. 0165-7992/82/0000-0000/$02.75 © Elsevier Biomedical Press
368
Schalm et al. (1975a) was used with the following modifications: the prepared stain was aged for at least 1 week; phosphate buffer, pH 6.8, was used; and the exposure to concentrated stain was extended to 5 min. The incidence of micronuclei was scored at 1000 x under oil immersion by a person who was unaware of the identity of the randomized and coded slides. 2000 normochromatic erythrocytes from each animal were scored at each time point, except day 8, when 1000 cells were scored. The ratio of polychromatic to normochromatic erythrocytes in peripheral blood was determined by counting the number of polychromatic cells present in the fields containing the first 1000 normochromatic cells. The incidence of micronucleated cells was also scored in 500 polychromatic erythrocytes. Selected slides were stained with Feulgen stain and fast green counterstain (Preece, 1972) to assure that similar frequencies of micronuclei were obtained with a DNA-specific stain. The incidence of micronuclei in normochromatic peripheral blood erythrocytes increased steadily during the period of TEM treatment (Fig. 1A). This elevated level
A
1144=,,4 I=',.4
z_ o 2
UU
0
I 10
I 20
I 30
I 40
1 50
60
10
B
0
0
I
I
I
I
I
I0
20
30
40
50
60
DAYS Fig. 1. A: Incidence of micronucleated cells among normochromatic erythrocytes (NCE) in the peripheral blood of mice given 0.2 mg/kg TEM i.p. on days 1, 3, 6, 8, 10, 15, and 17. A, control animals; [], treated. Each point is the mean of the determinations on 7 treated animals and 6 controls. The error bars represent the standard error of the mean, and the duration of TEM treatment is given by the bar in the upper left corner. The Wilcoxon rank sum test was used for statistical analysis, comparing the treated group to controls at each time point. *P < 0.02; **P < 0.005. B: Percent polychromatic erythrocytes (PCE) among peripheral blood erythrocytes of mice. Animal treatment, statistical analysis, and symbols are identical to A. Error bars are not shown when the error was smaller than the figure symbols.
369
of micronucleated cells did not return to control values until approximately 30 days after the treatment was dicontinued. The incidence of micronuclei in polychromatic peripheral blood erythrocytes was also examined during and after TEM dosing to establish that micronuclei were being produced during treatment and that production ceased when treatment was stopped. Significantly elevated frequencies of micronucleated polychromatic cells were observed at sampling points during TEM administration (days 8 and 15), and these frequencies returned to control levels on day 20, 3 days after the final injection. The ratio of polychromatic to normochromatic peripheral blood erythrocytes was not affected by TEM treatment (Fig. 1B), indicating that the dose administered did not alter the rate of red blood cell formation. The mean life span of normal red blood cells in mice has been estimated by a variety of methods, with reported values ranging from 20 to 45 days (Schalm et al., 1975b). Since the elevated levels of micronucleated normochromatic erythrocytes remained higher than control values for approximately 30 days after the last TEM injection, the life span of these micronucleated ceils must be very close to that estimated for normal red blood cells. These results provide strong evidence that micronucleated erythrocytes induced by a bone-marrow clastogen accumulate in the peripheral blood of mice, and that there is very little, or no, selective removal of these aberrant cells from the circulation. Since micronucleated erythrocytes accumulate in peripheral blood and are easily measured in a simple blood smear, samples obtained from animals in ongoing chronic or subchronic toxicity studies may permit an assessment of in vivo chromosomal breakage at very little additional effort or expense.
References Heddle, J.A. (1973) A rapid in vivo test for chromosome damage, Mutation Res., 18, 187-190. MacGregor, J.T., C.M. Wehr and D.H. Gould (1980) Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test, Environ. Mutagen., 2, 509-514. Preece, A. (1972) A Manual for Histologic Technicians, Little, Brown, Boston, MA, pp. 325-327, 181. Schalm, O.W., N.C. Jain and E.J. Carrol (1975a) Veterinary Hematology, 3rd edn., Lea and Febiger, Philadelphia, PA, p. 29. Schalm, O.W., N.C. Jain and E.J. Carrol (1975b) Veterinary Hematology, 3rd edn., Lea and Febiger, Philadelphia, PA, p. 390. Schmid, W. (1976) The micronucleus test for cytogenetic analysis, in: A. Hollaender (Ed.), Chemical Mutagens, Vol. 4, Plenum, New York, pp. 31-53. Von Ledebur, M., and W. Schmid (1973) The micronucleus test, Methodological aspects, Mutation Res., 19, 109-117.