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A description of ultrasonically-induced chromosomal anomalies in Vicia faba
Radia&~z B&t!),,
1973. Vol. 13, Pp. 2 11 to 2 13. Pergamon
A DESCRIPTION CHROMOSOMAL FREDERICK L. CATALDO, Department of Radiation
Press. Printed in Great Britain.
OF ULTRASONICALLY-INDUCED ANOMALIES IN TrlcIA FABA* MORTON W. MILLERand WINBORN D. GREGORY Biology and Biophysics, School of Medicine and Dentistry and
Department
of Electrical
Engineering,
EDWIN L. CARSTENSEN The University of Rochester, Rochester, New York 14627, U.S.A. (Received
CATALDO F. sonically-induced
16 February
1973 ; revised)
L., MILLER
M. W., GREGORY W. D. and CARSTENSEN E. L. A description of ultraanomalies in Vicia faba. RADIATION BOTANY 13,2 11-2 13, 1973.-An investigation of the effect of2.0 MHz ultrasound at 8.0 W/cm% on chromosomes of Viciafaba root me&tern cells. No “classical” chromosome aberrations werefound, but chromosome damage in the form of bridged prophases and metaphases and agglomerated mitotics was observed during the first mitosis after sonication. chromosomal
INTRODUCTION THE APPLICATION of ultrasound at medically relevant powers and intensities has been shown to produce pronounced effects upon the growth and cytology ofroot meristem cells of Viciufaf~a. These effects include reduction ofgrowth rate (BLEANEY and OLI~ER!~)), and depression of mitotic index (GREGORY (5)). MACINTOSH and DAVEY (*) also reported an induction of deletion and exchange types of chromosome aberrations with ultrasonicated lymphocytes although four other investigators(2*3*7t11) have failed to confirm their work. HILL(~) has described the biological and physical-biochemical mechanism by which ultrasound may exert effects. We have observed a type of chromosomal anomaly, previously unreported, which we believe to be at least partially responsible for the observed growth and cytological effects of ultrasonication on Vi& fuba roots. We have classified
these aberrations into three categories, based upon their general appearances: Bridged Prophases (BP), Bridged Metaphases (BM), and Agglomerated Mitotics (AM). That other researchers (with the possible exception of CONGER,(~) who observed some globule-like nuclei in Trudescuntiu fialudosu after X-rays and sonication at 9.1 kHz) have not observed these particular types of aberrations may be attributable to scoring technique. A “standard” technique for scoring metaphases for chromosome aberrations consists of scanning the slide with a high dry objective (450x), locating a scoreable metaphase, and then switching to an oil immersion objective (1000 X) for analysis. This “standard” technique automatically selects against mitotic figures other than those which include well-spread, easily recognizable metaphase chromosomes; one would simply not “see” bridged and agglomerated mitotic figures.
*This paper is based upon work performed under contract with the U.S. Atomic Energy Commission at The University of Rochester Atomic Energy Project and has been assigned Report No. UR 3490-275. This work was also partially supported by Grant USPHS GM 0933. 211
212
FREDERICK MATERIALS
AND
L. CATALDO
METHODS
Seedsof Vi& fuba obtained from the Kellogg Seed Company (Ventura, California 93001) were usedfor the experiment. Seedswere selected for intact seedcoats, soaked for 5 min in Clorox, and germinated overnight in running tap water. Seed coats were removed and the seedsplanted in moist vermiculite, where they were allowed to grow for five days at 19°C (vi& READ(~)). The seedlings, with plumules removed, were then transferred to ZO-liter stainless steel tanks containing a modified VOTH’S(~~) No. 5 nutrient solution. The tank was aerated and kept at 19°C for two days before exposing the roots to ultrasound. Ultrasound was delivered by means of a ceramic transducer, 2.5 cm dia, powered by an oscillator and amplifier. The frequency employed was 2 MHz and the intensity 8 W/cm2 as measured by a radiation pressure balance. The roots were exposedin a lucite chamber filled with degassedwater; the bottom waslined with sound absorbing natural rubber. Roots were placed horizontally in the tank, secured on a platform, and the transducer, immersed in the water and mounted on a motorized traverse, moved back and forth over the row of root tips at one sweep per min for 10 min, each root receiving an effective sonication doseof 0.1 min per sweep, or a total effective sonication of 1 min. After exposure, roots were returned to the culture tank. Preparation for microscopic examination included excision of root tips into 0.04 per cent aerated colchicine solution for 3 hr, fixation overnight in 3 : 1. ethanol:acetic acid, 10 min hydrolysis in 1N HCI at 60°C and staining for 2 hr by the Feulgen reaction. Squash preparations were made from the stained meristemsand were observed under oil immersion (1250 x ) . Photomicrographs were taken with a Carl Zeiss Photomicroscope, using Kodak high contrast copy film. OBSERVATIONS
Figures 1A and 1B (2 170 x ) show a normal prophase and normal metaphase, respectively, for the purpose of comparison. Figure 2 (2 100 x ) shows an assortment of examples of the type of anomaly which we have called Bridged Prophase. The chromosomes appear to be of the same
el al.
average thickness as normal prophase chromosomes,but they are clumped together in places and appear to be “welded” or fused together in many other places. Figure 3 (2100 X) shows several examples of Bridged Metaphases. It is conceivable that these represent later stages of the same type of anomaly shown in Fig. 2, with the increasing degree of chromosomecontraction occurring in the transition from prophase to metaphase causing this rather striking appearance. Figure 4 (2 100x ) showsthe third category of damage, the Agglomerated Mitotic, which consistsof clumped massesof nuclear material, with no discernible chromosomal morphology. We have observed none of these aberrations in cells obtained from control roots. At present, additional experiments are under way in an attempt to elucidate the mechanism of this cellular damage, and to quantitate its frequency of occurrence into a usable form for possiblecorrelation with the other effects induced by ultrasound.
REFERENCES 1. BLEANEY B. I. and OLIVER R. (1972) The effect of irradiation of Viciu faba roots with 1.5 MHz ultrasound. Brit. 3. Radiol. 45, 358-361. 2. BUCKTON K. E. and BAKER N. V. (1972) An investigation into possible chromosome damaging effects of ultrasound on human blood cells. Brit. 3. Radiol. 45,340-342. 3. COAKLEY W.T., SLADE J.S., BRAEMAN J. M.and MOORE J. L. (1972) Examination of lymphocytes for chromosome aberrations after ultrasonic irradiation. Brit. 3. Radiol. 45, 328-332. 4. CONGER A. D. (1948) The cytogenetic effect of sonic energy applied simultaneously with X-rays. Proc. Nat1 Acad. Sci. 34,470-474. 5. GREGORY W. D. (1972) The effects of ultrasound and low frequency magnetic fields on the growth and cytology of Vicia fuba. MS. Thesis, The
University of Rochester,Rochester,New York. 6. HILL C. R. (1972) Ultrasonic exposure thresholds for changes in cells and tissues. 3. Acoust. Sot. Am. 52,667-672. 7. HILL C.R., JOSHIG. P.and RJWELL S. H.(1972) A search for chromosome damage following exposure of Chinese hamster cells to high intensity pulsed ultrasound. Brit. 3. Radiol. 45,333-334. 8. MACINTOSH I. J. C. and DAVEY D. A. (1972) Relationshipbetween intensity of ultrasound and
FIG.
1A.
Normal
prophase.
FIG.
1B. Normal
metaphase.
R.B.
Ep. 212
.
. ,::.
FIG. 2. Bridged 8 W/cm2.
Arrows
prophases: chromosomal anomalies induced mark some of the more easily seen bridged focal plane.
by 2 MHz or “welded”
ultrasound at areas in this
FIG. 3. Bridged metaphases: chromosomal anomalies induced 8 W/cm2. Arrows mark some of the more easily seen bridged focal plane.
by 2 MHz or “welded”
ultrasound at areas in this
FIG. 4. Agglomerated
mitotics:
chromosomal anomalies at 8 W/cm2.
induced
by 2 MHz
ultrasound
ULTRASONICALLY-INDUCED induction
of chromosome
aberrations.
Radiol. 45, 320-327. 9. READ J. (1959) Radiation Biology of Vicia Relation to the General Problem. Charles C.
CHROMOSOMAL Brit.
3.
faba in Thomas,
Springfield, Ill., 270 pp. 10. VOTH P. D. and HOER K. C. (1940) Responses
ANOMALIES
IN
VZCZA
FABA
213
of Marchantia polymorpha to nutrient supply and photoperiod. Botan. Car. 102, 169-205. 11. WATTS P. L., HALL A. J. and FLEMING J. E. E. (1972) Ultrasound and chromosome damage. Brit.
3.
Radiol.
45,335-339.
1973. Vol. 13, Pp. 2 11 to 2 13. Pergamon
A DESCRIPTION CHROMOSOMAL FREDERICK L. CATALDO, Department of Radiation
Press. Printed in Great Britain.
OF ULTRASONICALLY-INDUCED ANOMALIES IN TrlcIA FABA* MORTON W. MILLERand WINBORN D. GREGORY Biology and Biophysics, School of Medicine and Dentistry and
Department
of Electrical
Engineering,
EDWIN L. CARSTENSEN The University of Rochester, Rochester, New York 14627, U.S.A. (Received
CATALDO F. sonically-induced
16 February
1973 ; revised)
L., MILLER
M. W., GREGORY W. D. and CARSTENSEN E. L. A description of ultraanomalies in Vicia faba. RADIATION BOTANY 13,2 11-2 13, 1973.-An investigation of the effect of2.0 MHz ultrasound at 8.0 W/cm% on chromosomes of Viciafaba root me&tern cells. No “classical” chromosome aberrations werefound, but chromosome damage in the form of bridged prophases and metaphases and agglomerated mitotics was observed during the first mitosis after sonication. chromosomal
INTRODUCTION THE APPLICATION of ultrasound at medically relevant powers and intensities has been shown to produce pronounced effects upon the growth and cytology ofroot meristem cells of Viciufaf~a. These effects include reduction ofgrowth rate (BLEANEY and OLI~ER!~)), and depression of mitotic index (GREGORY (5)). MACINTOSH and DAVEY (*) also reported an induction of deletion and exchange types of chromosome aberrations with ultrasonicated lymphocytes although four other investigators(2*3*7t11) have failed to confirm their work. HILL(~) has described the biological and physical-biochemical mechanism by which ultrasound may exert effects. We have observed a type of chromosomal anomaly, previously unreported, which we believe to be at least partially responsible for the observed growth and cytological effects of ultrasonication on Vi& fuba roots. We have classified
these aberrations into three categories, based upon their general appearances: Bridged Prophases (BP), Bridged Metaphases (BM), and Agglomerated Mitotics (AM). That other researchers (with the possible exception of CONGER,(~) who observed some globule-like nuclei in Trudescuntiu fialudosu after X-rays and sonication at 9.1 kHz) have not observed these particular types of aberrations may be attributable to scoring technique. A “standard” technique for scoring metaphases for chromosome aberrations consists of scanning the slide with a high dry objective (450x), locating a scoreable metaphase, and then switching to an oil immersion objective (1000 X) for analysis. This “standard” technique automatically selects against mitotic figures other than those which include well-spread, easily recognizable metaphase chromosomes; one would simply not “see” bridged and agglomerated mitotic figures.
*This paper is based upon work performed under contract with the U.S. Atomic Energy Commission at The University of Rochester Atomic Energy Project and has been assigned Report No. UR 3490-275. This work was also partially supported by Grant USPHS GM 0933. 211
212
FREDERICK MATERIALS
AND
L. CATALDO
METHODS
Seedsof Vi& fuba obtained from the Kellogg Seed Company (Ventura, California 93001) were usedfor the experiment. Seedswere selected for intact seedcoats, soaked for 5 min in Clorox, and germinated overnight in running tap water. Seed coats were removed and the seedsplanted in moist vermiculite, where they were allowed to grow for five days at 19°C (vi& READ(~)). The seedlings, with plumules removed, were then transferred to ZO-liter stainless steel tanks containing a modified VOTH’S(~~) No. 5 nutrient solution. The tank was aerated and kept at 19°C for two days before exposing the roots to ultrasound. Ultrasound was delivered by means of a ceramic transducer, 2.5 cm dia, powered by an oscillator and amplifier. The frequency employed was 2 MHz and the intensity 8 W/cm2 as measured by a radiation pressure balance. The roots were exposedin a lucite chamber filled with degassedwater; the bottom waslined with sound absorbing natural rubber. Roots were placed horizontally in the tank, secured on a platform, and the transducer, immersed in the water and mounted on a motorized traverse, moved back and forth over the row of root tips at one sweep per min for 10 min, each root receiving an effective sonication doseof 0.1 min per sweep, or a total effective sonication of 1 min. After exposure, roots were returned to the culture tank. Preparation for microscopic examination included excision of root tips into 0.04 per cent aerated colchicine solution for 3 hr, fixation overnight in 3 : 1. ethanol:acetic acid, 10 min hydrolysis in 1N HCI at 60°C and staining for 2 hr by the Feulgen reaction. Squash preparations were made from the stained meristemsand were observed under oil immersion (1250 x ) . Photomicrographs were taken with a Carl Zeiss Photomicroscope, using Kodak high contrast copy film. OBSERVATIONS
Figures 1A and 1B (2 170 x ) show a normal prophase and normal metaphase, respectively, for the purpose of comparison. Figure 2 (2 100 x ) shows an assortment of examples of the type of anomaly which we have called Bridged Prophase. The chromosomes appear to be of the same
el al.
average thickness as normal prophase chromosomes,but they are clumped together in places and appear to be “welded” or fused together in many other places. Figure 3 (2100 X) shows several examples of Bridged Metaphases. It is conceivable that these represent later stages of the same type of anomaly shown in Fig. 2, with the increasing degree of chromosomecontraction occurring in the transition from prophase to metaphase causing this rather striking appearance. Figure 4 (2 100x ) showsthe third category of damage, the Agglomerated Mitotic, which consistsof clumped massesof nuclear material, with no discernible chromosomal morphology. We have observed none of these aberrations in cells obtained from control roots. At present, additional experiments are under way in an attempt to elucidate the mechanism of this cellular damage, and to quantitate its frequency of occurrence into a usable form for possiblecorrelation with the other effects induced by ultrasound.
REFERENCES 1. BLEANEY B. I. and OLIVER R. (1972) The effect of irradiation of Viciu faba roots with 1.5 MHz ultrasound. Brit. 3. Radiol. 45, 358-361. 2. BUCKTON K. E. and BAKER N. V. (1972) An investigation into possible chromosome damaging effects of ultrasound on human blood cells. Brit. 3. Radiol. 45,340-342. 3. COAKLEY W.T., SLADE J.S., BRAEMAN J. M.and MOORE J. L. (1972) Examination of lymphocytes for chromosome aberrations after ultrasonic irradiation. Brit. 3. Radiol. 45, 328-332. 4. CONGER A. D. (1948) The cytogenetic effect of sonic energy applied simultaneously with X-rays. Proc. Nat1 Acad. Sci. 34,470-474. 5. GREGORY W. D. (1972) The effects of ultrasound and low frequency magnetic fields on the growth and cytology of Vicia fuba. MS. Thesis, The
University of Rochester,Rochester,New York. 6. HILL C. R. (1972) Ultrasonic exposure thresholds for changes in cells and tissues. 3. Acoust. Sot. Am. 52,667-672. 7. HILL C.R., JOSHIG. P.and RJWELL S. H.(1972) A search for chromosome damage following exposure of Chinese hamster cells to high intensity pulsed ultrasound. Brit. 3. Radiol. 45,333-334. 8. MACINTOSH I. J. C. and DAVEY D. A. (1972) Relationshipbetween intensity of ultrasound and
FIG.
1A.
Normal
prophase.
FIG.
1B. Normal
metaphase.
R.B.
Ep. 212
.
. ,::.
FIG. 2. Bridged 8 W/cm2.
Arrows
prophases: chromosomal anomalies induced mark some of the more easily seen bridged focal plane.
by 2 MHz or “welded”
ultrasound at areas in this
FIG. 3. Bridged metaphases: chromosomal anomalies induced 8 W/cm2. Arrows mark some of the more easily seen bridged focal plane.
by 2 MHz or “welded”
ultrasound at areas in this
FIG. 4. Agglomerated
mitotics:
chromosomal anomalies at 8 W/cm2.
induced
by 2 MHz
ultrasound
ULTRASONICALLY-INDUCED induction
of chromosome
aberrations.
Radiol. 45, 320-327. 9. READ J. (1959) Radiation Biology of Vicia Relation to the General Problem. Charles C.
CHROMOSOMAL Brit.
3.
faba in Thomas,
Springfield, Ill., 270 pp. 10. VOTH P. D. and HOER K. C. (1940) Responses
ANOMALIES
IN
VZCZA
FABA
213
of Marchantia polymorpha to nutrient supply and photoperiod. Botan. Car. 102, 169-205. 11. WATTS P. L., HALL A. J. and FLEMING J. E. E. (1972) Ultrasound and chromosome damage. Brit.
3.
Radiol.
45,335-339.