Effect of magnetic resonance imaging on Xenopus laevis embryogenesis

Mognerrc Resonance Prmted in the USA.

l

Imogmng. Vol. 6, pp. 501-506, All rights reserved.

1988 Copyright

0730-725X/88 $3.00 + .OO C 1988 Pergamon Press pk

Original Contribution

EFFECT OF MAGNETIC RESONANCE IMAGING ON XENOPUS LAE VIS EMBRYOGENESIS H.H. KAY,*

R.J. HERFKENS,~

AND B.K.

KAY$

Departments of *Obstetrics and Gynecology and tRadiology, Duke University Medical Center, Durham, N.C. and IDepartment of Biology, University of North Carolina, Chapel Hill, North Carolina, USA Xenopus luevis embryos, exposed to various lengths of magnetic resonance imaging (MRI), demonstrated

no abnormal morphology, function, or developmental delays. The overall protein profiles and nucleic acid ratios were similar compared to controls. Results suggest there are no adverse effects of MRI components on the development of this vertebrate.

Keywords: Xenopus laevis; Magnetic resonance imaging; Development;

of MRI that may lead to fetal abnormalities should presumably be detected in the early stages of development when such processes as cellular division, determination and migration, and tissue differentiation occur. All of these events can be easily monitored during Xenopus laevis development. This animal model has been successfully utilized to study the toxic and teratogenic effects of aromatic amines, ethanol,12 semicarbazide,” and various environmental chemicals such as di(2-ethylhexyl) phthalate (DEHP), methylmercury chloride, and the thalidomide analog, EM12.4 Significant developmental defects (abnormal limb formation, abnormal tail development causing immobility, and craniofacial malformations) were noted in 80 to 100% of embryos when exposed to these reagents. We, therefore, expect that any significant, adverse developmental effects from the combined components of MRI would be observed in a similar fashion during Xenopus embryogenesis.

INTRODUCTION Magnetic resonance imaging (MRI) is a tool that is fast becoming the accepted mode of imaging in many instances. It offers certain features that computerized tomography scanning (CT) does not offer. Nevertheless, before MRI can be routinely performed upon patients, particularly human fetuses in utero, it is necessary to demonstrate its safety in biologic systems. Although there is no ionizing radiation, the components of MRI, which include static magnetic fields, induced electric currents and changing fields of magnetic gradients and radiofrequency fields, may have potential harm. Studies of the independent effects of these components upon biologic systems have not demonstrated any significant adverse effects.‘~10.14.1x However, there have been no studies looking directly at the teratogenic effects of these combined MRI components utilized in imaging systems. The purpose of this study is to evaluate the potential effects of MRI upon early vertebrate development by studying the effects on Xenopus laevis embryogenesis. We chose to study Xenopus Zaevis, the South African frog, because (a) it is a vertebrate; (b) its embryology has been well studied; (c) the early development of the embryos can be easily monitored; and (d) the development from a fertilized ovum to a tadpole occurs rapidly within 3 days. Any adverse effects

RECEIVGD 10/16/87;

Ackndwledgment-We

ACCEPTED

MATERIALS

Burn, R.T. for his technical Robert

AND METHODS

Xenopus laevis females were induced to ovulate with human chorionic gonadotropin injections. The eggs were then simultaneously fertilized with testis explants and the embryos were placed in 100 mm plastic petri dishes containing dechlorinated tap water.

2/8/88.

would like to thank

Embryogenesis.

Address

A. 501

correspondence

assistance in this project. to H.H. Kay, M.D.

Magnetic Resonance Imaging l Volume 6, Number 5, 1988

502

MRI was performed in a General Electric prototype imaging system operating at 1.5 T (64 MHz). Spin echo technique employed included a multislice (5 slice) routine with a TR of 2000 and a TE value of 20 and 80 msec. The slice thickness was 1 cm with a maximum gradient of approximately 1 Gauss per centimeter. The radiofrequency pulses were adjusted to produce an appropriate 90 and 180 degree pulse for the small imaging volume provided. Two experiments were performed, Fig. 1. One experiment (I) evaluated 1 hour exposures 24 hours apart and the other experiment (II) evaluated prolonged exposure covering the stages of blastula, gastrula and neurula. In each experiment, one petri dish was placed within the magnet and a second dish, the control, was kept on a bench in an adjacent room. The control embryos were exposed to less than 20 Gauss and virtually no significant RF exposure during the experiments. They experienced identical handling and transporting conditions as the experimental embryos, but only similar lighting and temperature conditions, since they were not in the magnet. After exposure, the embryos were maintained

I

at room temperature far removed from the magnet and examined daily for their morphologic and functional development into tadpoles. Undeveloped eggs due to unsuccessful fertilization were removed on day 1. Embryos were staged according to Nieuwkoop and Faber.13 Embryos were examined with a Wild dissecting microscope and the anatomic morphology and function were evaluated. In addition, embryos from experiment II were examined biochemically. Embryos, at stages 24 and 41, representing two different developmental stages, were chosen from the control and MRI groups and homogenized in lysis buffer (65 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol). The boiled protein mixtures were then resolved by electrophoresis in a 10% polyacrylamide/SDS gel. &’ The proteins, along with molecular weight standards, were stained in the gel with Coomassie Blue (BioRad, Richmond, Ca.). This analysis allowed gross evaluation of general protein stability and accumulation. To evaluate the potential presence of aneuploidy in

I

r

Magnetic

t

Resonance Exposure

24 HOUfS

72

(Fertilization) t;: : fZT$ 9

Embryo Stages

48

19

&~~e;i.,

Geurula)

(jailbud)

(tadpole)

~blbs&la) (gasirula)

MoJor Events In Development

Cell

Cell

Tlrrur

:F””

Detcrmlnotbn,

Diffrrenllollon

Cdl

Estobllshmwt

d Dorsal/

Migration Old Morphogrnrrlr

Ventrot PolarMy

Fig. 1. Details of experiments I and II. Simultaneous fertilization occurred at time zero and magnetic resonance exposure within the imaging unit occurred at the times indicated by the black bars. The duration of exposure is indicated by the width of the bars. Exposure occurred at different stages of embryogenesis which includes various developmental events.

Effect of MRI on Embryogenesis 0 H. H.

the exposed embryos, ten stage 43 embryos from both control and MRI groups of experiment II were homogenized and the RNA and DNA fractions were separated by the Schmidt-Tannhauser method.16 Nucleic acids were quantitated spectrophotometrically with a Beckman DU spectrophotometer. RESULTS

In experiment I, a total of 171 embryos were examined in the control group and 180 embryos in the MRI group. There was no delay in development between the two groups, nor were there morphologic or functional differences between them (Fig. 2). All the tadpoles swam without any abnormalities. Each group had two tadpoles with curved tails and each had one embryo with slower development. In experiment II, which had prolonged exposure, there were a total of 264 embryos in the control and 235 embryos in the MRI group. Again, there was no significant difference between the rate of development, nor in the morphology or behavior of the embryos (Fig. 3). Among the control embryos as a whole there were 6 hydroceles, 1 blunt tail, 1 crooked tail, 1 split tail, and 1 lumpy belly. In the MRI group

Fig. 2. Representative opment.

The difference

KAY ET AL.

503

there was 1 curved tail, 1 short embryo and 1 with a side bulge. These isolated findings are consistent with normal development where these variations are occasionally found (unpublished observation). The temperature of the water was checked before and after exposure and averaged 22 +- 2°C. At no time did it go above 25°C which is the lethal temperature for the embryos.13 The overall profiles of extracted proteins were identical between the experimental and control embryos, at the level of one dimensional polyacrylamide gel electrophoresis (Fig. 4). This finding demonstrates that protein synthesis, accumulation and stability are normal in MRI exposed embryos. The total amounts of nucleic acids were similar between the MRI and the control groups and are similar to published values for DNA3 and RNA” (Table 1). The RNA/DNA ratio were the same, with values of 1.61 for the control embryos and 1.63 for the experimental embryos. This suggests that there is no polyploidy in the majority of cells within the embryo nor is there significant aneuploidy resulting from exposure to MRI. Any significant aneuploidy would not have been compatible with the normal development observed in this study.

stage 43 embryos from the control (C) and NMR exposed (M) groups in experiment 1 at day 13 of develin pigmentation patterns is within the range seen for these outbred animals. Bar = 1.0 mm.

504

Magnetic Resonance Imaging 0 Volume 6, Number 5, 1988

Fig. 3. Representative embryos from control (C) and NMR exposed (M) groups in experiment II, prolonged exposure, after being allowed to develop to day 1, 2, 3 and 10 corresponding to stages 18 (A), 24 (B), 41 (C) and 43 (D) respectively. Bar = 1.O mm.

DISCUSSION

Xenopus laevis is a good model for studying developmental malformations due to environmental effects. Embryos can be simultaneously fertilized and abnormal rates of development can be easily observed. Development from a fertilized ovum to a tadpole occurs rapidly in three days and allows detailed studies of its embryology. They are large embryos and monitoring abnormal morphology can be easily performed by observing with simple magnification. Moreover, their swimming behavior can be observed by the naked eye.. Lastly, it is a vertebrate which allows us to extrapolate the likely effects of MRI upon human development. Although the Xenopus oocyte is larger (1.3 mm)13 than the human oocyte (0.13 mm),” the Xenopus tadpole is comparable in size to the human embryo. A four week human embryo with complete closure of the neural tube, a primitive heart, and arm and leg buds is approximately 6 mm in length. l l This is comparable to the tadpole between stages 24 (3 mm) and 41 (8 mm) as shown in Figure 3, B and C. These size similarities minimize any effects of potential induced

currents in tissues and allow us to extrapolate any effects seen in the Xenopus embryo to the human embryo. Our study examined the question of whether or not developmental defects may result from magnetic resonance imaging of embryos. We did not observe any delay in development, nor any morphologic or behavioral abnormalities in our exposed embryos. Each stage of development was monitored by visual inspection. Thus, we conclude that there does not appear to be any adverse outcome of MRI on early developmental stages of Xenopus. Temperature effect has been a concern in MRI. Xenopus embryos are particularly sensitive to rises in temperature which may increase their rate of development. There were no abnormalities noted over the range of temperatures at which the experiments were conducted. In our experiments, MRI occurred at all the early major developmental stages (Fig. 1). It also occurred over a prolonged period of time at energy levels higher than encountered in routine imaging. Because of the many demands for imaging time, we were not able to image the embryos for more than 12 hours at

Effect of MRI on Embryogenesis 0 H. H. KAY H-AL.

505

;

Fig. 4. One dimensional polyacrylamide gels demonstrate protein profiles of single embryos from experiment II at stages 24 and 41 for the control (Cl, C2) and NMR exposed (Ml, M2) embryos. (S) represents molecular weight standards. kD = kilodaltons. Gel was stained with Coomassie Blue.

Table

1. Nucleic acid analysis

of embryos

exposed

to prolonged

magnetic

resonance

imaging. RNA/DNA

RNA

DNA

Control

72.3 pg/lO embryos 7.23 pg/embryo

44.9 pg/lO embryos 4.49 pg/embryo

1.61

MRI

81.6 pg/lO embryos 8.16 pg/embryo

49.9 c(g/lO embryos 4.99 pg/embryo

1.63

Ten stage 43 embryos from experiment II of exposed and control groups were homogenized in lysis buffer and whole cell RNA and DNA fractions were separated by the Schmidt-Tannhauser method. The fractionated nucleic acids were then quantitated by spectrophotometry at 260 nanometers. One optical density unit was assumed to correspond to 50 microgram (pg) or 40 pg/milliliter for DNA or RNA, respectively.

a time, and thus we cannot rule out the possibility that longer exposures could be detrimental. However, clinical imaging is never pursued for such a prolonged period of time, and we feel that our experiments have encompassed an adequate interval of exposure. We were also unable to follow these embryos to full development and reproduction since that would have taken a year’s time. However, we feel that with all growth, motility and anatomy being normal it would

be unlikely that any later developmental problems would occur. Effects during pregnancy have been investigated by several investigators. No adverse effects were noted in several reports of MRI in pregnancy.8,‘9s20 A recent study by Heinrichs et al’ also detected no embryotoxicity or postnatal developmental disturbances in outbred rats exposed to magnetic resonance spectroscopy in utero. Another report by McRobbie et al9 studied

506

Magnetic Resonance Imaging 0 Volume 6, Number 5, 1988

pulsed magnetic field exposure in pregnant mice and also found no adverse effects on development. Since frog embryos develop without extraembryonic tissues, we cannot rule out any potential adverse effects on human fetuses due to alterations of surrounding tissues such as the placenta, amniotic fluid and membranes. Our biochemical analysis of protein groups shows no difference between the exposed and the control embryos. Although it is known that many embryonic proteins are derived from the oocyte, our results demonstrate that, for the proteins in the size range analyzed, there is at least no major alteration of their stability and replacement. To refine this analysis further, one might either study the de nova synthesis of proteins by the incorporation of radioisotope-labelled amino acids, or follow the presence of new protein species by 2-dimensional gel electrophoresis. The observations that the RNA content, DNA content and the RNA/DNA ratios are similar between control and MRI exposed embryos strongly suggests that there is no significant degree of aneuploidy due to exposure to MRI. The slight difference in the actual quantities most likely reflects a better recovery of the extracted nucleic acids in the MRI versus the control embryos. Cell division and replication appear to continue without alterations in nuclear content. Without karyotyping of embryonic cells, we cannot, however, exclude that some slight degree of aneuploidy did occur. In conclusion, we do not find any adverse effect upon Xenopus embryogenesis from exposure to MRI either at intermittent intervals or for a prolonged period of time. There also do not appear to be any alterations of protein or nucleic acid content. Although these are negative results, they have not been previously reported and we feel that they represent an important step in establishing that MRI most likely has no adverse effects upon vertebrate development because we studied critical early embryonic stages. In summary, we extrapolate from the results presented in this paper that the components of MRI most likely will cause no adverse effects on the development of human fetuses.

REFERENCES 1. Budinger, T.F.; Cullander, C. Biophysical phenomena and health hazards of in vivo magnetic resonance. A.R. Margulis, C.B. Higgins, L. Kaufman, L.E. Crooks, eds. Clinical Magnetic Resonance Imaging. St. Louis: Mosby Company; 1983: 303-320. 2. Davis, K.R.; Schultz, T.W.; Dumont, J.N. Toxic and

3. 4.

5.

6.

7. 8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

teratogenic effects of selected aromatic amines on embryos of the amphibian Xenopus laevis. Arch. Environm. Contam. Toxicol. 10:371-391; 1981. Dawid, I.B. Deoxyribonucleic acid in amphibian eggs. J. Mol. Biol. 12:581-599; 1965. Dumpert, K.; Zietz, E. Platanna (Xenopus laevis) as a test organism for determining the embryotoxic effects of environmental chemicals. Ecotoxicol. Environ. Safety 8:55-74; 1984. Heinrichs, L.; Heinrichs, S.; Wiener, S.; Dalesandro, J.; Stanton, M.; Moseley, M. Influence of prenatal exposure to magnetic resonance spectroscopy (MRS) on postnatal development in rats. Book of Abstracts, Society for Gynecologic Investigation, Atlanta, 1987, p. 183, number 301. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 277:680-688; 1970. Maizel, J.V., Jr. Polyacrylamide gel electrophoresis of viral proteins. Methods Virol. 5: 180-190; 1971. McCarthy, S.M.; Filly, R.A.; Stark, D.D.; Hricak, H.; Brant-Zawadzki, M.N.; Callen, P.W.; Higgins, C.B. Obstetrical magnetic resonance imaging: fetal anatomy. Radiology 154:427-432; 1985. McRobbie, D.; Foster, M.A. Pulsed magnetic field exposure during pregnancy and implications for NMR fetal imaging: A study with mice. Magn. Reson. fmaging 3:231-234; 1985. Mild, K.H.; Sandstrom, M.; Loutrop, S. Development of Xenopus laevis embryos in a static magnetic field. Bioelectromagnetics 2: 199-20 1; 198 1. Moore, K.L. Before we are born: Basic embryology and birth defects. Saunders, W.B., Philadelphia. 2nd edition. 1983: 56. Nakatsuji, N. Craniofacial malformation in Xenopus laevis tadpoles caused by the exposure of early embryos to ethanol. Teratology 28:299-305; 1983. Nieuwkoop, P.; Faber, J. Normal tables of Xenopus laevis (Daudin). Amsterdam: North-Holland. 2nd edition. 1967. Prasad, N.; Wright, D.A.; Forster, J.D. Effect of nuclear magnetic resonance on early stages of amphibian development. Magn. Reson. Imaging 1:35-38; 1982. Sargent, T.D.; Dawid, LB. Differential gene expression in the gastrula of Xenopus laevis. Science 222: 135-l 39; 1983. Schmidt, G.; Tannhauser, S.J. A method for the determination of desoxyribonucleic acid, ribonucleic acid and phosphoproteins in animal tissues. J. Biol. Chem. 161:83-89; 1945. Schultz, T.W.; Dumont, J.N.; Epler, R.G. The embryotoxic and osteolothyrogenic effects of semicarbazide. Toxicology 36:183-198; 1985. Schwartz, J.L.; Crooks, L.E. NMR imaging produces no observable mutations or cytotoxicity in mammalian cells. AJR 139:583-585; 1982. Smith, F.W.; MacLennan, F. NMR imaging in human pregnancy: A preliminary study. Magn. Reson. Imaging 2:57-64; 1984. Weinreb, J.C.; Lowe, T.W.; Santos-Ramos, R.; Cummingham, F.G.; Parkey, R. Magnetic resonance imaging in obstetric diagnosis. Radiology 154:157-161; 1985.