Magnetic Resonancr Imaging. Vol. I, pp 35-38. 1982 0730 725X/X2/OlOO35 44S3 00/O Printed in the USA. All rights reserved. Copyright F 19X2 Pergsmon Press Ltd.

l Research Article

EFFECT OF NUCLEAR MAGNETIC RESONANCE ON EARLY STAGES OF AMPHIBIAN DEVELOPMENT

NARESH PRASAD, Ph.D.

Department of Radiology, Baylor College of Medicine, Houston, Texas 77030, USA

DAVID A. WRIGHT, Ph.D.

Department of Genetics, University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas

JOSEPH D. FORSTER, M.S.E.E.

Department of Physiology, Baylor College of Medicine, Houston, Texas

Frog smtozoa, fertilized eggs (during second meiotic division), and embryos (during cleavage) were subjected separately to 30 MHz continuous wave NMR exposure in a static magnetic tield of 7.05 kC for 20 min and were compared to unexposed groups, at similar stages, with respect to damage ia g-tic material, interferemx with meiotic cell divisii and impairment in the development of embryos. No significant differences were observed following treatment suggesting tbat NMR exposure, at the dose used, does not cause detectable adverse effects in this amphibian.

Keywords: Nuclear magnetic resonance, Bioeffect, Meiotic division, Spermatozoa, Embryos.

INTRODUCTION

Nuclear magnetic resonance (NMR) imaging is a noninvasive new technique that does not use ionizing radiation in diagnostic procedures. NMR imaging shows great promise for advanced medical imag- ing5.7.8.18; it also provides information about the chemi- cal and physiological status of the test organ and its anatomy. 3.10 The imaging procedure is based on mea- suring the response of the protons within the specific tissue to a known radio-frequency (RF) stimulus in the presence of a large magnetic field.’ The potential health hazards of the NMR imaging procedure may lie either in the radio-frequency electromagnetic field, the static magnetic field, the time-dependent magnetic field, or a combination of some or all of these. The individual field utilized in the NMR imaging does not exceed the recommended safe level,” but there is lack of information on potential health hazard if these fields are utilized together. A great interest is being demon- strated in medical research for the use of NMR in clinical diagnosis.3.‘0 However, before NMR can be accepted as a routine diagnostic procedure, the

absence of deleterious bioeffects must be demon- strated.

The present study was designed to investigate the effects of NMR exposure on three phases of early amphibian development to answer some of the above questions. Frog spermatozoa, fertilized eggs during second meiotic division and cleavage stage embryos were utilized in this investigation. The frog spermato- zoon prior to fertilization is sensitive to radiation’2*1*15 and chemically-induced genetic damage.* Typically, this damage is manifested as developmental abnormal- ities, chromosome damage or haploidy in embryos resulting from fertilization of eggs with treated sperm. Cytotoxic effects reducing the viability or mobility of the spermatozoa are seen in reduced frequency of fertilization. Before fertilization, the mature egg is arrested in metaphase of the second meiotic division. After activation by the spermatozoon, the ovum com- pletes meiosis, forms the second polar body and the haploid female pronucleus. Temperature shock and hydrostatic pressure have been shown to interfere with this process so that the polar body does not form and

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35

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36 Magnetic Resonance Imaging 0 Volume 1, Number I, I982

Table 1. Nuclear magnetic resonance exposure of sperm: Effects on fertilization and development

Experiment Sperm

Treatment No. Eggs (No.1

Cleaving

(%) (x1)

Normal Tail Bud

(No.1 (%l (x?

I Exposed 92 82 89 0.07 64 78 0.02 Control 82 69 84 - 52 75 -

II Exposed 69 53 77 0.06 46 87 0.02 Control 91 66 73 - 55 83 -

III Exposed 80 69 86 0.05 56 81 0.02 Control 65 57 88 - 48 84 -

the female pronucleus becomes diploid.‘V6V9 The early

cleavage stages of the frog egg show the most rapid mitotic activity in the life cycle of the animal. The time between ceil divisions in early cleavage is approxi- mately 60 min at 18 “C in Rana pipiens.” Any damage during these stages would be expected to have pro- found effects at later stages of development.

METHODS

Rana pipiens (leopard frogs) were obtained from J. M. Hazen (Alburg, UT). Procedures for artificial induction of ovulation and fertilization in Ranapipiens were essentially those of Rugh.16 The medium for growth of embryos and preparation of sperm suspen- sions contained 0.35 g NaCl, 0.01 g CaCl, - 2Hz0, 0.01 g MgC12 - 6Hz0, 0.007 g KHCO, per liter in 0.01 M tris-HCl buffer at pH 7.6.

Exposure was performed using a Bruker SXP NMR Spectrometer (Bruker Instruments, Inc., Billerica, MA). The magnet bore size was 5 cm and the static magnetic field utilized in this study was 7.05 kG. Sperms or eggs were placed in a 20 mm NMR tube, which was inserted into the radio-frequency coil. The coil was 2 cm in diameter and 4 cm long. The RF exposure was at a 30 MHz continuous wave with a total power input of 1 mW and a resulting power per unit volume of 8.66 x lo-’ W/cm3.

Sperm suspensions, prepared by mincing both testes of a single male in 10 ml of medium, were treated for 20 min and used to fertilize eggs immediately follow- ing exposure. Eggs were kept in 100 mm diameter plastic petri dishes. Eggs undergoing second meiosis were treated for 20 min after being exposed to an untreated sperm suspension for 12 min. Cleavage stage embryos were also treated for 20 min. Treatment was administered between the 2- and 32-cell stages. Con- trol eggs and sperm were placed in the same kind of tube as experimentals for 20 min without exposure to NMR and left on the bench. After treatment of eggs, they were removed from the test tube used for exposure and placed in 100 mm diameter petri dishes.

The eggs from all experimental treatments were examined under a stereomicroscope 4 to 5 h after fertilization and the number of cleaving embryos counted. The embryos were allowed to develop at 18 “C. After 4 days, the number of normal and ab- normal tail bud embryos in stage 18 were counted.” Contingency x2 tests were performed comparing experimental and control for the number of cleaving eggs in the total eggs used or the number of normal tail bud embryos of the total cleaving in each experimental treatment. Some of the embryos were treated for 4 h with colchicine (20 mg/ml), the cells were dissociated with 25 mM EDTA, 75 mM NaCl, 10 IYtM Tris, pH 8.0

Table 2. Exposure of fertilized eggs to nuclear magnetic resonance during the second meiotic division

Experiment Treatment No. Eggs (No.1

Cleaving

(%I (x2)

Normal Tail Bud

(No.1 (%I (x2)

I Exposed 28 15 53 1.59 12 80 0.02 Control 82 69 84 - 52 75 -

II Exposed 25 21 84 0.09 19 90 0.007 Control 23 17 74 - 16 94 -

111 Exposed 35 27 77 0.07 22 81 0.02 Control 33 28 85 - 24 86 -

Elkct of NMR on amphibian development 0 Naresh Prasad et al. 37

Table 3. Exposure of embryos to nuclear magnetic resonance during cleavage

Experiment Stage of

Treatment

Cleaving Embryos

(No.) (No.)

Normal Tail Bud

(%) (x2)

I 8 cell Control

II 2 cell 4 cell Control

II1 16 cell 32 cell Control

33 25 16 0.006 38 28 14 -

20 19 95 0.0004 11 11 100 0.01 17 16 94 -

23 21 91 0.02 16 14 87 0.002 28 24 86 -

solution for 45 min followed by distilled water for 20 min and fixed in 3:l methanol acetic acid. Slides were prepared and chromosomes stained with Giemsa.

RESULTS AND DISCUSSION

No significant differences were seen between treated and control groups in the development of embryos on the basis of x2 tests. Table 1 summarizes the data from three different experiments where sperm suspensions were treated. Tables 2 and 3 show the data from fertilized eggs treated in meiosis and during cleavage stages, respectively. In experiment I (Table 2), treatment during the second meiotic division, the low number of surviving embryos which showed nor- mal cleavage, was probably due to physical damage to the eggs in removing them with a spatula from the treatment tube. Since the egg’s jelly coat is still very sticky at this stage, it was necessary to use a fine wire to dislodge the eggs without damage after treatment in experiments II and III.

If the sperm treatment had caused any damage to the genetic material, developmental abnormalities similar to those seen with ultraviolet light or X-rays would have been obtained.13 The results suggest that NMR at the dose level used does not induce detectable genetic damage, embryonic abnormalities or chromo- some loss. NMR treatment of fertilized eggs during the period of the second meiotic division might have inhibited this process in a manner similar to tempera- ture shock or hydrostatic pressure.‘.6 In that event, triploid animals identified by their larger cell size and chromosome number might have been seen. The nor- mal cell size and normal chromosome number of

treated samples indicates that NMR does not interfere with meiotic cell division.

The lack of an increased number of abnormal embryos among the embryos treated during cleavage stages compared to controls (Table 3) also suggests that NMR at the dose used does not interfere in any detectable way with the cleavage process during that critical stage in development.

Recent reports on in vitro4*‘9*20 and in vivo14 studies have shown no adverse effects of NMR imaging either on chromosome aberrations, sister-chromatid ex- changes, DNA synthesis, or mutation rate. It is appar- ent from these studies that the NMR imaging is an excellent procedure without known risk. Our present data also support the safety of NMR at the dose utilized in these experiments. However, the results are very limited and additional data are needed for an adequate analysis of biohazard before definitive con- clusions may be reached. Various radio frequencies and gradient pulses are being investigated in our ongoing experiments.

SUMMARY

Continuous wave NMR at 30 MHz in a static magnetic field of 7.05 kG was used to treat frog spermatozoa, fertilized eggs during meiotic division, and cleavage stage embryos; for 20 min. The resulting embryos were allowed to develop to late tail bud stage and examined. No significant differences between control and experimental embryos were seen. These results suggest that NMR exposure at the dose used causes no adverse effects.

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