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A note on the utility of Fischer-344 rat in the micronucleus test
Toxicology Letters, 17 (1983) 201-204 Elsevier
201
Short Communication A NOTE ON THE UTILITY OF FISCHER-344 RAT IN THE MICRONUCLEUS TEST (Fischer-344; micronucleus;
B. BHASKAR GOLLAPUDI,
mast cells; basophilic granules)
V. ANN LINSCOMBE and ANIL K. SINHA
Health and Environmental Sciences-Texas, Lake Jackson Research Center, Dow Chemical U.S.A., Freeport, TX 77541 (U.S.A.) (Received December 13th, 1982) (Accepted January 7th, 1983)
SUMMARY Bone marrow smears from Fischer-344 rats possessed such a high incidence of basophilic granules that accurate detection of micronucleated erythrocytes was not possible. In contrast, preparations from Sprague-Dawley rats had far fewer such granules, so that the micronucleated marrow cells were clearly visualized and counted. The difference in the incidence of basophilic granules was attributed to strain differences in mast cell population size.
INTRODUCTION
The micronucleus test is a simple and rapid in vivo assay for detecting agents capable of breaking chromosomes and interfering with spindle function [l]. Acentric chromosomal fragments, or even whole chromosomes, that are not included in the daughter nuclei following cell division are expected to form one or more micronuclei independent of the major nuclei. Micronuclei are routinely scored from the bone marrow erythrocytes. An increase in the frequency of micronucleated erythrocytes at an appropriate interval following the administration of a test substance is indicative of the genotoxic potential of the agent under investigation PI. The laboratory rat has been used as an animal model for a variety of toxicological investigations. Utilization of the rat in genetic toxicology testing would facilitate correlation of genotoxicity data with other toxicological data. Only a few rat 0378-4274/83/S 03.00 0 1983 Elsevier Science Publishers B.V.
Fig. 1. Photographs showing rat bone marrow smears: (A) Sprague-Dawley; arrow indicates micronucleated erythrocyte; (B) Fischer-344: note numerous overlying positively stained basophilic granules.
203
micronucleus studies have appeared in the literature [3-51, and no strain differences in micronucleus detectability have thus far been reported. This communication relates to the observation of such a difference. MATERIALS
AND METHODS
Bone marrow smears were prepared according to Schmid [l] from Fischer-344 (Charles River, Portage, MI and Timco, Inc., Houston, TX) and Sprague-Dawley (Timco, Inc.) rats. The animals were killed by cervical dislocation. Femoral bone marrow was aspirated into a lo-ml syringe containing 3 ml of fetal calf serum (GIBCO, Grand Island, NY). The samples were transferred into siliconized centrifuge tubes and spun at 700 rev./min for about 10 min. The pellet was resuspended in a drop of serum with a siliconized Pasteur pipet and then smeared on a microscope slide. The slides were stained with Giemsa [6]. RESULTS
AND DISCUSSION
The Fischer rat smears had so many basophilic granules that accurate detection of micronucleated erythrocytes was not possible. This was the case whether the strain was obtained from Charles River or Timco, Inc. On the other hand, use of Sprague-Dawley rats resulted in preparations with far fewer basophilic granules so that the typical micronucleated marrow cells could be clearly visualized and counted (Fig. 1). Mast cells are normally found in stromal connective tissue throughout the body. The cytoplasmic granules of these cells contain several biologically active amines, and stain basophilic and blue with ordinary histological stains [7]. Tissue manipulation readily causes cytoplasmic rupture and scattering of granules in the vicinity of the disrupted cells. The incidence of mast cells in the marrows of Fischer-344 and Sprague-Dawley rats was evaluated in order to understand the remarkable difference in the distribution of basophilic granules among the two strains of the rats. TABLE
I
FREQUENCIES MARROW
OF MAST
SMEARS
CELLS
OF FISCHER
Fischer-344
(PER
1000 NUCLEATED
CELLS
344 AND SPRAGUE-DAWLEY
SCORED)
IN THE
RATS
Sprague-Dawley
Animal
Number
Animal
Number
No.
mast cells
No.
mast cells
1 2
25 24
1 2
5 2
of
3
17
3
2
4
14
4
2
5
28
5
0
of
BONE
204
Fischer rats exhibited much higher numbers of mast celis in comparison to those in the Sprague-Dawley (Table I). It appears that the rupturing of such a large population of mast cells in Fischer rats was responsible for releasing the basophilic granules over various marrow cell types making the detection of micronuclei quite difficult. Thus, we have abandoned the use of Fischer-344 rats in micronucleus testing. ACKNOWLEDGEMENT
The authors are grateful to Dr. Wayne Taylor for his advice on bone marrow cytology. REFERENCES
1 W. Schmid,
Mutation
2 W. Schmid, Chemical York, 1976, p. 31. 3 P. Goetz, 4 D.R. 5 R.J.
R.J.
G.L.
6 B.B. Gollapudi 7 S.L. Robbins
Principles
Sram and J. Dohnalova,
Goodman, Trzos,
Res., 31 (1975) 9. Mutagens:
P.J.
Hakkinen,
Petzold, and O.P.
J.H.
M.N.
Mutation Nemenzo
Brunden
Kamra,
and R.S. Cotran,
and Methods
Mutation
Pathologic
for Their Detection,
Vol. 4, Plenum,
New
Res., 31 (1975) 247. and M. Vore, Mutation
and J.A.
Swenberg,
Mutation
Res., 48 (1977) 295. Res.,
58 (1978) 79.
Res., 64 (1979) 45. Basis of Disease,
Saunders,
Philadelphia,
PA, 1979, p. 77.
201
Short Communication A NOTE ON THE UTILITY OF FISCHER-344 RAT IN THE MICRONUCLEUS TEST (Fischer-344; micronucleus;
B. BHASKAR GOLLAPUDI,
mast cells; basophilic granules)
V. ANN LINSCOMBE and ANIL K. SINHA
Health and Environmental Sciences-Texas, Lake Jackson Research Center, Dow Chemical U.S.A., Freeport, TX 77541 (U.S.A.) (Received December 13th, 1982) (Accepted January 7th, 1983)
SUMMARY Bone marrow smears from Fischer-344 rats possessed such a high incidence of basophilic granules that accurate detection of micronucleated erythrocytes was not possible. In contrast, preparations from Sprague-Dawley rats had far fewer such granules, so that the micronucleated marrow cells were clearly visualized and counted. The difference in the incidence of basophilic granules was attributed to strain differences in mast cell population size.
INTRODUCTION
The micronucleus test is a simple and rapid in vivo assay for detecting agents capable of breaking chromosomes and interfering with spindle function [l]. Acentric chromosomal fragments, or even whole chromosomes, that are not included in the daughter nuclei following cell division are expected to form one or more micronuclei independent of the major nuclei. Micronuclei are routinely scored from the bone marrow erythrocytes. An increase in the frequency of micronucleated erythrocytes at an appropriate interval following the administration of a test substance is indicative of the genotoxic potential of the agent under investigation PI. The laboratory rat has been used as an animal model for a variety of toxicological investigations. Utilization of the rat in genetic toxicology testing would facilitate correlation of genotoxicity data with other toxicological data. Only a few rat 0378-4274/83/S 03.00 0 1983 Elsevier Science Publishers B.V.
Fig. 1. Photographs showing rat bone marrow smears: (A) Sprague-Dawley; arrow indicates micronucleated erythrocyte; (B) Fischer-344: note numerous overlying positively stained basophilic granules.
203
micronucleus studies have appeared in the literature [3-51, and no strain differences in micronucleus detectability have thus far been reported. This communication relates to the observation of such a difference. MATERIALS
AND METHODS
Bone marrow smears were prepared according to Schmid [l] from Fischer-344 (Charles River, Portage, MI and Timco, Inc., Houston, TX) and Sprague-Dawley (Timco, Inc.) rats. The animals were killed by cervical dislocation. Femoral bone marrow was aspirated into a lo-ml syringe containing 3 ml of fetal calf serum (GIBCO, Grand Island, NY). The samples were transferred into siliconized centrifuge tubes and spun at 700 rev./min for about 10 min. The pellet was resuspended in a drop of serum with a siliconized Pasteur pipet and then smeared on a microscope slide. The slides were stained with Giemsa [6]. RESULTS
AND DISCUSSION
The Fischer rat smears had so many basophilic granules that accurate detection of micronucleated erythrocytes was not possible. This was the case whether the strain was obtained from Charles River or Timco, Inc. On the other hand, use of Sprague-Dawley rats resulted in preparations with far fewer basophilic granules so that the typical micronucleated marrow cells could be clearly visualized and counted (Fig. 1). Mast cells are normally found in stromal connective tissue throughout the body. The cytoplasmic granules of these cells contain several biologically active amines, and stain basophilic and blue with ordinary histological stains [7]. Tissue manipulation readily causes cytoplasmic rupture and scattering of granules in the vicinity of the disrupted cells. The incidence of mast cells in the marrows of Fischer-344 and Sprague-Dawley rats was evaluated in order to understand the remarkable difference in the distribution of basophilic granules among the two strains of the rats. TABLE
I
FREQUENCIES MARROW
OF MAST
SMEARS
CELLS
OF FISCHER
Fischer-344
(PER
1000 NUCLEATED
CELLS
344 AND SPRAGUE-DAWLEY
SCORED)
IN THE
RATS
Sprague-Dawley
Animal
Number
Animal
Number
No.
mast cells
No.
mast cells
1 2
25 24
1 2
5 2
of
3
17
3
2
4
14
4
2
5
28
5
0
of
BONE
204
Fischer rats exhibited much higher numbers of mast celis in comparison to those in the Sprague-Dawley (Table I). It appears that the rupturing of such a large population of mast cells in Fischer rats was responsible for releasing the basophilic granules over various marrow cell types making the detection of micronuclei quite difficult. Thus, we have abandoned the use of Fischer-344 rats in micronucleus testing. ACKNOWLEDGEMENT
The authors are grateful to Dr. Wayne Taylor for his advice on bone marrow cytology. REFERENCES
1 W. Schmid,
Mutation
2 W. Schmid, Chemical York, 1976, p. 31. 3 P. Goetz, 4 D.R. 5 R.J.
R.J.
G.L.
6 B.B. Gollapudi 7 S.L. Robbins
Principles
Sram and J. Dohnalova,
Goodman, Trzos,
Res., 31 (1975) 9. Mutagens:
P.J.
Hakkinen,
Petzold, and O.P.
J.H.
M.N.
Mutation Nemenzo
Brunden
Kamra,
and R.S. Cotran,
and Methods
Mutation
Pathologic
for Their Detection,
Vol. 4, Plenum,
New
Res., 31 (1975) 247. and M. Vore, Mutation
and J.A.
Swenberg,
Mutation
Res., 48 (1977) 295. Res.,
58 (1978) 79.
Res., 64 (1979) 45. Basis of Disease,
Saunders,
Philadelphia,
PA, 1979, p. 77.