The Teratogenic Effects of 6-Mercaptopurine on Chick Embryos in Ovo1 RANDALL B GRUBB AND ETHEL C MONTIEGEL D e p c i r t m e n t oj Broloqy, W e s t Vtrginici U n i u e r & z t y , Morgrintown, West V i r g t n m 26506
1
ABSTRACT Fertilized eggs of single-combed White Leghorn hens were each injected through a shell window directly beneath the embryo at 70 or 96 h of incubation with 2 mg of the sodium salt of 6-MP dissolved in 0.1 M phosphate buffer, and at 11 days of incubation the embryos were examined for gross morphological abnormalities. Various gross malformations and growth retardation were found, the most frequently and severely affected structures being limbs, beak, and eyes. Treatment at 70 h caused more severe abnormalities than at 96 h, but the spectrum of defects was not very different.
6-Mercaptopurine (6-MP), first synthesized by Elion et al. ('52), became the subject of many diverse investigations soon after its development. Early studies established it as a potential chemotherapeutic agent (Clarke et al., '53, '54; Sugiura, '55) and revealed its detrimental effect on frog (Bieber et al., '52) and sand-dollar (Karnofsky, '60) development. Chick embryos exhibited only slight weight inhibition when 6-MP was injected into eggs at 4 days of incubation (Karnofsky, '55) but later under similar conditions inconsisten tly developed facial deformities and short lower beak (Karnofsky, '60). The drug caused no adverse effects when added to the culture medium of chick embryos explanted at 6-7 h of incubation (Nicholas, '63). It inhibited rabbit blastocyst implantation (Adams et al., '61) and caused stunting and resorption of rat fetuses (Thiersch, '54) but did not produce gross abnormalities in either species. In subsequent studies with rats, however, 6-MP produced syndac tyly, hypomorphic pelvic bones, absent tibiae and tails, and irregular ossification (Murphy, '60; Bragonier et al., '64); and numerous other abnormalities including cleft lip, jaw, and palate (Schmitz et al., '72). There have been a myriad of biochemically oriented studies involving 6-MP(e.g., Hitchings and Rhoads, '54; Elion and Hitchings, '57; Hitchings and Elion, '67) which have revealed a number of loci of action for 6-MP (Hitchings and Elion, '72) but its precise mechanism of action TEFIATOLOGY. 1 1 : 17S186.
has not yet been elucidated (Zimmerman et al., '74). In view of the facts that (1) 6-MP adversely influences development of echinoderm, amphibian, and mammalian embryos, (2) Karnofsky got inconsistent results when administering 6-MP to chick embryos, ( 3 ) independent studies involving administration of 6-MP to rats resulted in divergent findings, and (4) many reports showed that 6-MP alters basic biochemical functions in a variety of biological systems, it seemed logical to reinvestigate the effect of 6-MP on development of chick embryos. The present paper reports the results of such a study. MATERIALS AND METHODS
Eggs of single-combed White Leghorn hens, maintained a t the West Virginia University Poultry Farm, were incubated at 37 f 0.1"C. in a mild-breathing-airmovement incubator (Petersime Incubator Company, Gettysburg, Ohio) with relative humidity controlled at 49 t 5 % . Eggs were rotated three times a day until shell windows were made but were not rotated thereafter. Eight hours before the eggs were injected they were candled and winReceived July 1 , '74. Accepted Nov. 4 , ' 7 4 . 1 The material in this paper is taken from a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, West Virginia University, Morgantown, West Virginia 26506. 2 Present address: Department of Anatomical Sciences, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190. 3 Present address: Department of Biology, West Virginia University, Morgantown, West Virginia 26506.
179
180
RANDALL B . GRUBB AND ETHEL C . MONTIEGEL
dows cut in their shells by Hamburger's method ('60), modified by using a dental drill with inverted-cone bit for cutting the shell and by placing a drop of sterile Locke's solution on the shell membrane before its removal. Egg nests (Betz, '63) were used to position eggs during window preparation and for holding the eggs during injections. Water soluble 6-MP in the form of the sodium salt, in 0.5-g sterile vials,4 was solubilized under sterile conditions just prior to use by adding 25 ml sodium phosphate solution to each vial. This solution was prepared by mixing 0.1 M dibasic sodium phosphate and 0.1 M tribasic sodium phosphate in a ratio of 1:15 and sterilized by filtration through a Millipore filter of 0.45-p pore size. The control solution was sodium phosphate, which had the same pH (10.5 +- 0.1) as the 6-MP solution. The volume for experimental and control injections was 0.1 ml and the treatment dose was 2 mg/egg 6-MP. In preliminary studies this was determined to be the LDjo for embryos injected at 96 h of incubation and incubated for 11 days. Eggs were injected using 2.5-ml sterile disposable hypodermic syringes with 22gauge, 1.5-in, 12"-beveled needles. Injections were made through the shell windows directly beneath the embryo on the right side. For eggs injected at 96 h of incubation needles were inserted through the area vasculosa making sure no vessels were punctured in the process; for injections at 70 h needles were inserted through the area vitellina interna (Hamilton, '52) just outside the sinus terminalis. After injection shell windows were closed by sealing no. 2, 18-mm, round cover slips into position with melted paraffin, and the eggs were returned to the incubator for further development. During the subsequent incubation period eggs were candled every other day and dead embryos were removed and examined. After 11 days of incubation living embryos were killed by opening the eggs and their wet weights determined. All embryos were fixed in Bouin's fluid, staged (Hamburger and Hamilton, '51), and examined for gross abnormalities. Three independent series were injected at 96 h of incubation and one at 70 h. Data were analyzed statistically by the chi-square test of independence and
analysis of variance for unequal subclasses (Fryer, '66). RESULTS
One hundred and fifty-seven eggs (106 experimentals and 51 controls) were injected at 96 h in the three series. Fiftythree (50%) experimentals and nine (17.6% ) controls died during the 7-day incubation period following treatment, seven experimentals and five controls during the first 24 h and seven experimentals within the first 60 h. These 19 dead embryos were too small to analyze for gross abnormalities but the others had developed to stage 30 or over and were large enough for gross examination. Fifty-two eggs (35 experimentals and 17 controls) were treated at 70 h. Only eight (22.9% ) experimentals were alive by the end of day 11 of incubation, whereas 16 (94.1%) controls survived through this period. Seventeen of the 27 dead experimentals died within 60 h of treatment and could not be examined for gross abnormalities but the remaining ten, having developed to stage 30 or over, were inspected for malformations. In all three series injections at 96 h produced embryos with significantly reduced weight (P < .005), (fig. 1) and with gross morphological abnormalities (figs. 3-5; table l), involving beak, long bones and digits of wings and legs, eyes, and tail. Beak abnormalities included those of the upper and lower beak. Both parts were shortened, curved, or bent to one side; in addition upper beaks had serrated ventrolateral borders and lower beaks possessed short ventrolateral projections from their posterior portions. Wing and leg long bones were short or curved or both. Digit abnormalities included short or totally absent wing and leg digits and fusion of various leg digits. Eye abnormalities ranged from slight bulges over the pupil to large sacs covering the entire eye from upper to lower eyelid. In some cases eyes bulged anteroventrally, had irregular eyelids, or were smaller than normal. Tail abnormalities simply involved shortening of the tail. It should be stressed that various degrees of each type of abnormality 4 Supplied by Dr. George H. Hitchings of Burroughs Wellcome and Company, Inc., Research Triangle Park, North Carolina 27709.
181
6-MP EFFECTS ON CHICK DEVELOPMENT
P
1
ABSTRACT Fertilized eggs of single-combed White Leghorn hens were each injected through a shell window directly beneath the embryo at 70 or 96 h of incubation with 2 mg of the sodium salt of 6-MP dissolved in 0.1 M phosphate buffer, and at 11 days of incubation the embryos were examined for gross morphological abnormalities. Various gross malformations and growth retardation were found, the most frequently and severely affected structures being limbs, beak, and eyes. Treatment at 70 h caused more severe abnormalities than at 96 h, but the spectrum of defects was not very different.
6-Mercaptopurine (6-MP), first synthesized by Elion et al. ('52), became the subject of many diverse investigations soon after its development. Early studies established it as a potential chemotherapeutic agent (Clarke et al., '53, '54; Sugiura, '55) and revealed its detrimental effect on frog (Bieber et al., '52) and sand-dollar (Karnofsky, '60) development. Chick embryos exhibited only slight weight inhibition when 6-MP was injected into eggs at 4 days of incubation (Karnofsky, '55) but later under similar conditions inconsisten tly developed facial deformities and short lower beak (Karnofsky, '60). The drug caused no adverse effects when added to the culture medium of chick embryos explanted at 6-7 h of incubation (Nicholas, '63). It inhibited rabbit blastocyst implantation (Adams et al., '61) and caused stunting and resorption of rat fetuses (Thiersch, '54) but did not produce gross abnormalities in either species. In subsequent studies with rats, however, 6-MP produced syndac tyly, hypomorphic pelvic bones, absent tibiae and tails, and irregular ossification (Murphy, '60; Bragonier et al., '64); and numerous other abnormalities including cleft lip, jaw, and palate (Schmitz et al., '72). There have been a myriad of biochemically oriented studies involving 6-MP(e.g., Hitchings and Rhoads, '54; Elion and Hitchings, '57; Hitchings and Elion, '67) which have revealed a number of loci of action for 6-MP (Hitchings and Elion, '72) but its precise mechanism of action TEFIATOLOGY. 1 1 : 17S186.
has not yet been elucidated (Zimmerman et al., '74). In view of the facts that (1) 6-MP adversely influences development of echinoderm, amphibian, and mammalian embryos, (2) Karnofsky got inconsistent results when administering 6-MP to chick embryos, ( 3 ) independent studies involving administration of 6-MP to rats resulted in divergent findings, and (4) many reports showed that 6-MP alters basic biochemical functions in a variety of biological systems, it seemed logical to reinvestigate the effect of 6-MP on development of chick embryos. The present paper reports the results of such a study. MATERIALS AND METHODS
Eggs of single-combed White Leghorn hens, maintained a t the West Virginia University Poultry Farm, were incubated at 37 f 0.1"C. in a mild-breathing-airmovement incubator (Petersime Incubator Company, Gettysburg, Ohio) with relative humidity controlled at 49 t 5 % . Eggs were rotated three times a day until shell windows were made but were not rotated thereafter. Eight hours before the eggs were injected they were candled and winReceived July 1 , '74. Accepted Nov. 4 , ' 7 4 . 1 The material in this paper is taken from a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, West Virginia University, Morgantown, West Virginia 26506. 2 Present address: Department of Anatomical Sciences, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190. 3 Present address: Department of Biology, West Virginia University, Morgantown, West Virginia 26506.
179
180
RANDALL B . GRUBB AND ETHEL C . MONTIEGEL
dows cut in their shells by Hamburger's method ('60), modified by using a dental drill with inverted-cone bit for cutting the shell and by placing a drop of sterile Locke's solution on the shell membrane before its removal. Egg nests (Betz, '63) were used to position eggs during window preparation and for holding the eggs during injections. Water soluble 6-MP in the form of the sodium salt, in 0.5-g sterile vials,4 was solubilized under sterile conditions just prior to use by adding 25 ml sodium phosphate solution to each vial. This solution was prepared by mixing 0.1 M dibasic sodium phosphate and 0.1 M tribasic sodium phosphate in a ratio of 1:15 and sterilized by filtration through a Millipore filter of 0.45-p pore size. The control solution was sodium phosphate, which had the same pH (10.5 +- 0.1) as the 6-MP solution. The volume for experimental and control injections was 0.1 ml and the treatment dose was 2 mg/egg 6-MP. In preliminary studies this was determined to be the LDjo for embryos injected at 96 h of incubation and incubated for 11 days. Eggs were injected using 2.5-ml sterile disposable hypodermic syringes with 22gauge, 1.5-in, 12"-beveled needles. Injections were made through the shell windows directly beneath the embryo on the right side. For eggs injected at 96 h of incubation needles were inserted through the area vasculosa making sure no vessels were punctured in the process; for injections at 70 h needles were inserted through the area vitellina interna (Hamilton, '52) just outside the sinus terminalis. After injection shell windows were closed by sealing no. 2, 18-mm, round cover slips into position with melted paraffin, and the eggs were returned to the incubator for further development. During the subsequent incubation period eggs were candled every other day and dead embryos were removed and examined. After 11 days of incubation living embryos were killed by opening the eggs and their wet weights determined. All embryos were fixed in Bouin's fluid, staged (Hamburger and Hamilton, '51), and examined for gross abnormalities. Three independent series were injected at 96 h of incubation and one at 70 h. Data were analyzed statistically by the chi-square test of independence and
analysis of variance for unequal subclasses (Fryer, '66). RESULTS
One hundred and fifty-seven eggs (106 experimentals and 51 controls) were injected at 96 h in the three series. Fiftythree (50%) experimentals and nine (17.6% ) controls died during the 7-day incubation period following treatment, seven experimentals and five controls during the first 24 h and seven experimentals within the first 60 h. These 19 dead embryos were too small to analyze for gross abnormalities but the others had developed to stage 30 or over and were large enough for gross examination. Fifty-two eggs (35 experimentals and 17 controls) were treated at 70 h. Only eight (22.9% ) experimentals were alive by the end of day 11 of incubation, whereas 16 (94.1%) controls survived through this period. Seventeen of the 27 dead experimentals died within 60 h of treatment and could not be examined for gross abnormalities but the remaining ten, having developed to stage 30 or over, were inspected for malformations. In all three series injections at 96 h produced embryos with significantly reduced weight (P < .005), (fig. 1) and with gross morphological abnormalities (figs. 3-5; table l), involving beak, long bones and digits of wings and legs, eyes, and tail. Beak abnormalities included those of the upper and lower beak. Both parts were shortened, curved, or bent to one side; in addition upper beaks had serrated ventrolateral borders and lower beaks possessed short ventrolateral projections from their posterior portions. Wing and leg long bones were short or curved or both. Digit abnormalities included short or totally absent wing and leg digits and fusion of various leg digits. Eye abnormalities ranged from slight bulges over the pupil to large sacs covering the entire eye from upper to lower eyelid. In some cases eyes bulged anteroventrally, had irregular eyelids, or were smaller than normal. Tail abnormalities simply involved shortening of the tail. It should be stressed that various degrees of each type of abnormality 4 Supplied by Dr. George H. Hitchings of Burroughs Wellcome and Company, Inc., Research Triangle Park, North Carolina 27709.
181
6-MP EFFECTS ON CHICK DEVELOPMENT
P
