Hydroxyurea embryotoxicity is enhanced in P53-deficient mice

Accepted Manuscript Title: Hydroxyurea embryotoxicity is enhanced in P53-deficient mice Authors: Nazem El Husseini, Barbara F. Hales PII: DOI: Reference:

S0890-6238(18)30117-5 https://doi.org/10.1016/j.reprotox.2018.06.011 RTX 7686

To appear in:

Reproductive Toxicology

Received date: Revised date: Accepted date:

20-3-2018 14-6-2018 19-6-2018

Please cite this article as: Husseini NE, Hales BF, Hydroxyurea embryotoxicity is enhanced in P53-deficient mice, Reproductive Toxicology (2018), https://doi.org/10.1016/j.reprotox.2018.06.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hydroxyurea embryotoxicity is enhanced in P53-deficient mice

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Nazem El Husseini1 and Barbara F. Hales1*

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Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

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To whom correspondence should be addressed:

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Department of Pharmacology and Therapeutics, McGill University,

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3655 Promenade Sir William Osler, Montreal, Quebec, Canada H3G 1Y6

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E-mail: [email protected].

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Highlights In this study we demonstrate that fetal P53 genotype is an important determinant of the embryotoxicity of hydroxyurea during organogenesis. Our findings suggest that P53 is a “hub” for the embryonic response to stress.

ABSTRACT

Hydroxyurea, a ribonucleotide reductase inhibitor, is a potent teratogen in mice, causing

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severe limb and skeletal defects. The exposure of gestation day nine murine embryos to

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hydroxyurea elicits an early embryonic stress response that involves activation of the P53

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transcription factor. The impact of this P53 activation on the embryotoxicity of hydroxyurea- is

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not known. The goal of this study was to test the hypothesis that P53 acts to suppress hydroxyurea embryotoxicity. Trp53+/- timed pregnant mice were treated with saline or

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hydroxyurea (200 or 400 mg/kg) on gestation day nine; fetuses were examined for viability and

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external and skeletal malformations on gestation day eighteen. Neither the deletion of Trp53 nor

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hydroxyurea treatment significantly affected fetal growth although a trend towards a decrease in fetal weights was observed in Trp53-/- fetuses. However, hydroxyurea induced a significantly

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higher incidence of malformations and resorptions in Trp53-/- fetuses compared to their wildtype littermates. Thus, fetal P53 genotype is an important determinant of the effects of hydroxyurea

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on organogenesis-stage embryos.

KEYWORDS: P53; Hydroxyurea; Organogenesis; Teratogen; Developmental Toxicity; Birth Defects 2

ABBREVIATIONS: Trp53: tumor suppressor related protein 53

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GD: gestational day DNA: deoxyribonucleic acid HU: hydroxyurea SAL: saline

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CRL: crown-rump lengths

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1. INTRODUCTION

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P53 is a tumor suppressor protein that acts as a transcription factor in response to various

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stress stimuli, including genotoxic and oxidative stress [1]. P53 concentrations are tightly controlled under normal conditions; in response to stress P53 is post-translationally modified and

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translocated to the nucleus where it upregulates the transcription of several downstream targets

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involved in apoptosis and cell cycle arrest [2, 3]. P53 deletions (e.g. in Trp53-deficient mice) and

[4, 5].

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gene mutations (in mice and humans) are associated with an increase in the incidence of tumors

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Trp53 is expressed in the murine embryo during the critical stage of organogenesis, as early as gestational day (GD) 8 and up to GD 12 [6, 7]. Furthermore, genotoxic stress, such as

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exposure to radiation, can dramatically increase P53 activity in specific regions of the organogenesis-stage embryo [8, 9]. Exposure to genotoxic teratogens may also lead to significant upregulation of P53 protein levels and induce the transcription of many of the P53 downstream targets that are involved in mediating apoptosis [8, 10-12].

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P53 is important for proper embryonic development; a subset of Trp53-null embryos display birth defects, such as exencephaly and limb malformations [4, 13, 14]. Embryos lacking P53 show higher rates of malformations when exposed to several genotoxic teratogens [11, 12,

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15]. However, other teratogens, such as 2-chloro-2’-deoxyadenosine, show a reduced incidence of specific malformations in the absence of P53 [16]. Thus, the role of P53 as a suppressor or inducer of embryotoxicity is not clear.

Hydroxyurea, an anti-neoplastic agent, is used to treat chronic myeloid leukemia and

sickle cell anemia [17, 18]. This drug inhibits ribonucleotide reductase, resulting in the depletion

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of deoxyribonucleotides and DNA replication fork stalling [19]. It induces cell cycle arrest at the

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G1/S phase and leads to the death of proliferating cells [20-22]. Since organogenesis is

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dependent on rapid cell proliferation, embryos at this stage of development are highly susceptible

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to the effects of hydroxyurea [23-25]. The treatment of timed pregnant CD-1 mice with hydroxyurea on GD 9 produces dose-dependent increases in the incidence of resorptions and

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fetuses with severe limb (e.g. syndactyly, oligodactyly), tail (e.g. hypoplastic or curly tails) and

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skeletal defects (e.g. amelia, short ribs) [26, 27]. Exposure to hydroxyurea also elicits an early

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stress response in the embryo that involves the upregulation of P53 and its downstream signaling pathways [2]. The activation of P53 promotes caspase-mediated apoptosis via the cleavage and

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activation of caspase-3 [28]. The impact of this early embryonic stress response related P53 activation in determining the outcome of fate of hydroxyurea exposed embryos is not known.

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Here we have tested the hypothesis that P53 acts as a suppressor of hydroxyurea embryotoxicity.

2. MATERIALS AND METHODS 2.1 Experimental Animals 4

Trp53 transgenic mice (B6.129S2-Trp53tm1Tyj/J, stock number 002101) were purchased from The Jackson Laboratory (Sacramento, CA, USA) and housed in the McIntyre Animal Resource Centre (Montreal, QC, Canada). This strain was originally developed by the Tyler

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Jacks laboratory; a neomycin cassette was inserted in the Trp53 gene to replace exons 2-6 (including the start codon) [29]. The Trp53tm1Tyj/J mutant strain was developed using a 129derived D3 embryonic stem cell line; this mutant strain was then backcrossed to C57BL/6J

inbred mice at least five times by The Jackson Laboratory [30]. To generate test animals in-

house, Trp53 heterozygote females were mated with Trp53 heterozygote males so that only the

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fetal genotypes differed within each litter. One breeding pair per cage was housed overnight and

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the females were separated from the males at 10:00 AM the following day (designated as GD 0).

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All animal treatments and procedures were approved by the McGill University Animal Care

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Committee under protocol #4456 and conducted in accordance with the guidelines outlined in the Canadian Guide to the Care and Use of Experimental Animals. Between 8:00 and 10:00 AM

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on GD 9 pregnant dams were treated with either saline (SAL), 200 mg/kg (HU200) or 400 mg/kg

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(HU400) hydroxyurea (Aldrich Chemical Co., Madison, WI) by intraperitoneal injection.

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Volumes injected were not more than 10 μl/g body weight. No maternal effects were observed

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with any treatment condition.

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2.2 Examination of Pregnancy Outcome On GD 18, all dams were euthanized by CO2 asphyxiation and cervical dislocation. The

uteri were removed and examined for implantation sites and resorptions. Fetuses that did not respond to prodding stimuli were considered dead. All fetuses were numbered according to their site of implantation in ascending order, from left to right. Live fetuses were weighed after 5

explantation and then euthanized by hypothermia. Crown-rump lengths (CRL) were determined using a digital caliper. External malformations were then recorded, including the type, incidence and severity. Internal tissues, as well as tissues from resorption sites, were collected from each

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transgenic fetus for genotyping, after which fetuses were fixed in ethanol in preparation for skeletal staining.

2.3 DNA Extraction

DNA was extracted from the tail tips or internal tissues of Trp53 adult and fetal

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transgenic mice using a modified version of the ethanol precipitation method [31]. Briefly, tissue

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samples were kept overnight at 55° C in 250 μl of DNA extraction buffer (50 mM Tris-HCl, 10

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mM EDTA, 100 mM NaCl, 1% SDS) and 0.4 mg/ml proteinase K (Sigma-Aldrich, St. Louis,

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MO, USA). Samples were then centrifuged at 20,000g for 10 min to remove fur and debris,

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followed by two additions of 100% ethanol to the supernatant. Lastly, the sample was

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centrifuged to precipitate the DNA, the ethanol was discarded and the pellet left to air dry. DNA was then dissolved in distilled water and the concentration was determined using a

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NanoDrop1000 spectrophotometer (Fisher Scientific, Wilmington, DE, USA).

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2.4 Genotyping by High Resolution Melt-Curve Analysis

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The Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems, Foster City, CA)

and the StepOnePlus Real-Time PCR System (Applied Biosystems) were used to carry out high resolution melt-curve analysis to determine the zygosity of adult and fetal samples [32]. Each reaction was composed of 5 μl of 25 ng/μl DNA mixed with 10 μl SYBR Green Master Mix, 0.3 μl of each of the 10 mM forward and reverse primers, completed to a total reaction volume of 20 6

μl with DNAse and RNAse free water. Samples were run in single-plex for each allele. Temperature cycling was determined as per the supplier’s recommended protocol; 95° C for 10 min followed by 40 cycles of 95° C for 15 s, 60° C for 30 s, and 72° C for 30 s. Subsequently,

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the melt-curve stage was as follows: 95° C for 15 s, 60° C for 1 min, followed by incremental increases of 0.3° C up to 95° C. The peak melting temperatures for the mutant and wild type

amplicons matched those of the supplier (~78° C and ~84° C, respectively). The sequences of the forward and reverse primers for the wild type and mutant alleles of Trp53 were provided by the supplier (Wild type forward: AGGCTTAGA GGTGCAAGCTG; mutant forward:

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CAGCCTCTGTTCCACATACACT; common reverse: TGGATGGTGGTATACTCAGAGC)

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and were synthesized by Alpha DNA (Montreal, QC, Canada). The genotypes of all implantation

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sites (resorptions and developed fetuses) were validated by Transnetyx (Cordova, TN), using

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proprietary techniques for DNA extraction and real-time PCR.

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2.5 Alcian Blue and Alizarin Red Staining of Fetal Skeletons Ethanol-fixed fetuses were immersed in a water bath (70°C) for 30 s, skinned,

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eviscerated, and placed in 95% ethanol overnight. The ethanol was decanted and replaced with alcian blue solution (15 mg alcian blue; 80 ml 95% ethanol; 20 ml glacial acetic acid) for 48 h.

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The solution was then replaced with 95% ethanol for 24 h. The ethanol was substituted with

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Alizarin red S solution (25 mg/l Alizarin red S in 1% potassium hydroxide) for 24 h. The dye was replaced with 0.5% potassium hydroxide for 24 h and then the skeletons were placed in a 2:2:1 solution consisting of 2 parts 70% ethanol: 2 parts glycerin: 1 part benzyl alcohol. After 24 h, stained skeletons were placed in 1:1 solution (1 part 70% ethanol: 1 part glycerin) for later evaluation and storage. The evaluator was blinded against the different treatments and genotypes 7

of the fetuses and described all differences in ossification observed during evaluation. Previous reports on normal GD18 fetal skeletal ossification patterns of fore- and hind-limbs were used as

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a reference [33].

2.6 Statistical Analyses

All data were statistically analyzed using the GraphPad Prism Software (version 5, Graph Pad Software Inc., La Jolla, CA.). A chi-square goodness of fit test was used to determine if the offspring genotypes conformed to expected Mendelian ratios. Implantation sites, resorptions and

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malformations were tabulated. Total percentages were calculated based on the total number of

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implantation sites or fetuses examined, of the same genotype, within each treatment group,

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regardless of the litter source. Data were tested by the Fisher’s Exact test or 2-way ANOVA

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followed by a Bonferroni post-hoc multiple comparison correction, as appropriate. The level of

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3. RESULTS

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significance for all statistical tests was set to p < 0.05.

3.1 Trp53-/- fetuses are more susceptible to hydroxyurea-induced

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embryolethality and malformations than their Trp53+/+ littermates

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The numbers of implantation sites, resorptions and fetuses with external malformations

that were observed when GD 9 heterozygote (Trp53+/-) timed pregnancy dams were treated with saline or hydroxyurea are shown in Table 1. Genotyping results showed that Mendelian ratios were produced in the offspring of almost all litters (data not shown). The overall incidence of resorptions among the saline-treated litters was similar to that found in wild type (C57bL/6J) 8

saline-treated animals (data not shown) and can be attributed to the background resorption rate that is inherent to this inbred mouse strain. The resorption rate was significantly elevated after treatment with 400 mg/kg of hydroxyurea, with the highest rate of resorptions in the Trp53-/-

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fetuses (Table 1).

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Table 1: The fetal Trp53 zygosity of implantation sites, resorptions and externally malformed fetuses in transgenic litters treated with saline or hydroxyurea. Treatment

SAL

HU200

HU400

Dams (n) Conceptal Genotype Implantation Sites (n)

5

7

5

Resorptions (n)

+/+

Trp53

+/-

Trp53

-/-

Trp53

+/+

Trp53

8

27

12

12

27

0

2

2

0

3

+/-

Trp53

-/-

Trp53

+/+

Trp53

+/-

Trp53

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Trp53

14

7

20

7

0

3

9

5

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N

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% Resorptions 0 7 17 0 11 0 43 45† 71† Viable Fetuses 8 25 10 12 24 14 4 11 2 (n) Externally Malformed 0 1 3 2 4 9 4 5 2 Fetuses (n) % Externally Malformed 0 4 30 17 17 64* 100† 45† 100 Fetuses Percentages are represented as the resorbed or externally malformed fetuses out of the total implantation

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sites and fetuses examined, respectively, per genotype within each treatment group. *: indicates significant difference compared to Trp53+/+ fetuses within same treatment group. †: indicates significant

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saline, HU = hydroxyurea.

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difference compared to SAL treated fetuses with same genotype. Fischer’s Exact test, p<0.05. SAL =

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-/-

Interestingly, the deletion of Trp53 did not significantly affect fetal growth, as assessed by fetal weights and crown-rump lengths (CRLs) (Fig. 1), although there was a trend towards a decrease in body weights and CRLs in the absence of Trp53. In addition, hydroxyurea treatment

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had no significant effect on fetal weights or CRLs. The lack of a statistical significant effect of

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hydroxyurea on fetal growth may be due to the relatively small sample sizes.

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(1.5 COLUMN)

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Fig. 1: Fetal growth after exposure to embryotoxic doses of hydroxyurea. A: Fetal weights in transgenic fetuses did not differ significantly between the three possible Trp53 genotypes in any

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treatment group. Each bar represents the mean weight in grams of the fetuses examined in each treatment group. B: Fetal crown-rump lengths (CRLs) in transgenic fetuses did not differ

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significantly between the three possible Trp53 genotypes in any treatment group. Each bar represents the mean CRL in millimeters of the fetuses examined in each treatment group. SAL= saline, HU= hydroxyurea.

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3.2 The majority of external and skeletal malformations in Trp53-/hydroxyurea-treated fetuses occurred in the limbs Interestingly, several fetuses among the Trp53+/- and Trp53-/- fetuses in saline-treated

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litters had malformations; these included exencephaly, digit defects and tail defects (Tables 1 and 2). An increased incidence and wider range of malformations were observed among fetuses

exposed to hydroxyurea; these included craniofacial anomalies (e.g. exencephaly), abdominal

defects (e.g. gastroschisis), and a multitude of limb and tail abnormalities (Tables 1 and 2). The

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number of malformed fetuses increased after treatment with 200 mg/kg of hydroxyurea

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(HU200); moreover, the incidence of malformations among the Trp53-/- fetuses was significantly

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higher than that of their Trp53+/+ littermates in this treatment groups (Table 1). The Trp53-/-

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fetuses exposed to 200 mg/kg hydroxyurea showed a trend towards a higher incidence of hypoplastic tails (Table 2, Fig. 2) and a significant increase in skeletal hypoplasia of the forelimb

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phalanges (Table 3, Fig. 2A) compared to their Trp53+/+ littermates. After treatment with 400

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mg/kg hydroxyurea (HU400), all of the Trp53+/+ and Trp53-/- fetuses exhibited external malformations (Table 1). The defects observed among fetuses exposed to 400 mg/kg

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hydroxyurea included deformities in the lumbar vertebrae (Table 3, Fig. 2B).

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(1.5 COLUMN)

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Fig. 2: Hydroxyurea induced skeletal hypoplasia of fetal limbs of Trp53 transgenic fetuses.

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Representative images of fetal skeletons stained for cartilage (Alcian blue) and ossified bone -/-

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(Alizarin Red) after exposure to saline or hydroxyurea. A: A Trp53 fetus from the saline treatment group exhibiting exencephaly (EX), unossified distal phalanges (UDP), unossified

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proximal phalanges (UPP), and curly tail (CT). B: A Trp53+/- fetus from the 400 mg/kg

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hydroxyurea exhibiting incomplete ossification of lumbar vertebrae (IOLV) and UPP.

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Table 2: Types and incidence of external malformations in transgenic litters by Trp53 zygosity, in saline or hydroxyurea treatment groups. SAL Trp53

Trp53

+/-

Trp53

-/-

Trp53

+/+

Trp53

Trp53

+/+

Trp53

+/-

Trp53

10

12

24

14

4

11

2

Exencephaly

-

-

2 (20.0)

-

-

1 (7.1)

-

-

-

Cleft Lip/Palate

-

-

-

-

-

1 (7.1)

-

-

-

Gastroschisis

-

-

-

-

-

2 (14.3)

-

-

-

Edema

-

-

-

-

-

-

-

1 (9.1)

-

Syndactyly

-

-

1 (10.0)

1 (8.3)

-

1 (9.1)

-

Oligodactyly =1

-

-

-

Syndactyly

-

1 (4.0)

-

Oligodactyly =1

-

-

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-

-

-

-

1 (9.1)

-

1 (8.3)

2 (8.3)

2 (21.4)

2 (50.0)

1 (9.1)

-

-

-

-

2 (50.0)

-

1 (50.0)

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-

Curly/ 1 (4.0) 3 (30.0) 1 (8.3) 4 (28.6) 1 (25.0) 3 (27.3) Hypoplastic Percentages (in parentheses) are represented as the malformed fetuses out of the fetuses examined per

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genotype within each treatment group; a single fetus may be represented more than once in listing individual defects. SAL = saline, HU = hydroxyurea. Oligodactyly =1 indicates missing one digit on any

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limb.

-/-

2 (14.3)

2 (8.3)

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Tail

-/-

25

Abdominal

Hindlimb

Trp53

8

Craniofacial

Forelimb

+/-

HU400

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No. of examined fetuses

+/+

HU200

15

1 (50.0)

Table 3: Incidence of skeletal defects in transgenic litters by Trp53 zygosity, in saline or hydroxyurea treatment groups. SAL

HU200

Trp53+/+ Trp53+/- Trp53-/- Trp53+/+ Trp53+/-

HU400 Trp53-/-

Trp53+/+

Trp53+/- Trp53-/-

8

25

10

12

24

14

4

11

2

Vertebrae Forelimb effects

-

-

-

-

1 (4.2)

2 (50.0)

3 (27.3)

-

2 (16.7)

8 (33.3)

2 (50.0)

3 (27.3)

-

-

-

2 (14.3) 11 (78.6)* 2 (14.3)

-

-

-

7 (50.0)

2 (50.0)

3 (27.3)

-

1 (25.0)

1 (9.1)

-

4 (100.0)

9 (81.8)

-

1 (9.1)

Lumbar

2 (25.0)

8 (32.0) 7 (70.0)

-

Proximal Phalanges

2 (25.0)

7 (28.0) 7 (70.0)

2 (16.7)

5 (20.8)

Distal Phalanges

2 (25.0)

5 (20.0) 6 (60.0)

2 (16.7)

8 (33.3)

13 (52.0) 2 (8.0)

7 (70.0)

8 (66.7)

3 (30.0)

-

-

Proximal Phalanges

5 (62.5)

11 (44.0)

7 (70.0)

8 (66.7)

Distal Phalanges

6 (75.0)

12 (48.0)

7 (70.0)

20 (83.3) 7 (29.2)

9 (64.3)* 12 (85.7) 4 (28.6)

16 (66.7)

12 (85.7)

3 (75.0)

7 (63.6)

2 (100.0)

20 (83.3)

11 (78.6)

3 (75.0)

9 (81.8)

2 (100.0)

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Metatarsals

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6 (75.0)

2 (20.0)

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Metacarpals

Hindlimb effects

2 (8.0)

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No. of examined fetuses

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8 (66.7)

Percentages (in parentheses) are represented as the affected fetuses out of the fetuses examined per

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genotype within each treatment group; a single fetus may be represented more than once in listing

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individual findings. *: indicates significant difference compared to Trp53+/+ fetuses within same treatment

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group. Fischer’s Exact test, p<0.05.SAL = saline, HU = hydroxyurea.

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2 (100.0) -

4. DISCUSSION Using a chi-square goodness of fit test, we found that in almost all litters the genotypes of the offspring at GD18 did not differ significantly from the expected Mendelian ratio (1:2:1).

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Earlier studies have reported that non-Mendelian proportions of Trp53-/- offspring were produced from the mating of heterozygote parents (i.e. less than 25%), and suggested that this

phenomenon may be due to a decreased survival of early embryos (i.e. before implantation or in early organogenesis) as a result of P53 deletion [15, 39, 40]. We did not determine the sex of

individual offspring; however, the Jackson Laboratory observed a decreased survival of Trp53-/-

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female offspring before weaning in this mouse strain [30]. This may be due to an increased

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incidence of fatal exencephaly in Trp53-/- females [13, 14].

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The overall impact of hydroxyurea exposure during organogenesis was enhanced in P53

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heterozygote and null fetuses. Specifically, higher overall rates of resorptions and malformations

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were observed in hydroxyurea exposed Trp53-/- fetuses compared to their Trp53+/+ littermates,

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suggesting that the presence P53 plays a role in limiting the embryotoxicity of this drug. The P53 protein has been implicated in the ability of embryos to cope with a teratogenic exposure in

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several studies [34]. Fetuses that were deficient in Trp53 all had increased incidences of

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resorptions and malformations after exposure to ionizing radiation, cyclophosphamide, benzo[α]pyrene or phenytoin compared to their Trp53+/+ counterparts [11, 12, 15, 35, 36]. All

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these agents are well-characterized teratogens that share a common outcome: the induction of apoptosis as a result of oxidative stress and DNA damage [24, 27, 34, 37, 38]. In addition, we observed a higher incidence of malformations in saline-treated Trp53-/fetuses than their Trp53+/+ counterparts. These malformations included limb defects and exencephaly, which are reminiscent of observations from earlier studies of Trp53-/- embryos [14]. 17

During organogenesis, P53 activity is tightly controlled and is mainly restricted to the limb buds, where it helps “sculpt” the digits of the hind- and fore-limbs, and the neural tube, where it regulates the local cell turnover that is essential for proper neural tube closure [41, 42]. These

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processes are driven by P53-regulated cell cycle progression and apoptosis [4, 43]. Thus, an imbalance in P53-mediated regulation of cell cycle progression and apoptosis would disrupt limb development, leading to merged or absent digits, and prevent neural tubes from closing properly. A possible mechanism of P53-mediated suppression of hydroxyurea-induced

embryotoxicity is via apoptosis. P53-mediated apoptosis increases in several tissues after

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exposure to a DNA damaging agent, resulting in the selective disposal of irreparable damaged

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cells to enable preservation of the integrity of the surrounding tissue and allow proper organ

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development [44-46]. Interestingly, hydroxyurea treatment of GD 9 Trp53-/- embryos failed to

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activate caspase-3 and to induce the expression of pro-apoptotic factors (Fas, Trp53inp1, Pmaip1) [28]. Previous studies reported that Trp53+/+ fetuses exposed to ionizing radiation

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displayed a lower rate of malformations than Trp53-/- fetuses, yet showed an increase in

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apoptotic cells that was not observed in Trp53-/- fetuses [12, 35]. Other studies have shown that a

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decrease in apoptosis may lead to unregulated forms of cell death, such as necrosis [47, 48]. Indeed, Trp53-deficient murine limb buds exposed to teratogenic doses of a pre-activated analog

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of cyclophosphamide, 4-hydroperoxycyclophosphamide, displayed higher rates of malformation, compared to wild type, and underwent necrosis, as shown by the rupture of cell membranes and

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lack of the DNA fragmentation characteristic of apoptosis [11]. As such, it is possible that the absence of P53 renders the embryo more susceptible to hydroxyurea embryotoxicity due to its inability to dispose of damaged cells via apoptosis, leading to widespread irrevocable tissue damage and an elevated incidence of malformations and resorptions.

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The ability of P53 to limit hydroxyurea embryotoxicity may be dose-dependent. After exposure to 200 mg/kg hydroxyurea a higher percentage of fetuses were malformed among those that were deficient in P53 (Trp53-/-) than among their littermates that were Trp53+/+; however, in

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the 400 mg/kg treatment group, the rates of malformations and resorptions between Trp53+/+ and Trp53-/- were similar. Likewise, earlier reports showed that when mouse embryos were exposed to a low dose of ionizing radiation (1.2 mGy/min), Trp53-/- fetuses exhibited a higher incidence of malformations compared to their Trp5+/+ littermates; however after exposure to a higher dose of radiation, the incidence of malformations in Trp5+/+ fetuses was markedly elevated [35]. The

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ability of the embryo to counter damaging effects appears to rely on P53 function during

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embryonic development; Zhang et al. demonstrated that mildly-stressed embryonic cells are

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marked for elimination by P53 and are “out-competed” by healthier surrounding cells as an

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adaptive response with the goal of protecting organs from maldevelopment [44]. The differences in embryotoxicity between Trp53 null and wildtype fetuses after exposure to 200 or 400 mg/kg

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hydroxyurea may indicate that the ability of P53 to suppress the embryotoxicity of hydroxyurea

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is dose limited; P53 may be unable to counteract the excessive damage induced by higher doses

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of hydroxyurea, resulting in embryotoxicity in wildtype as well as Trp53 null fetuses. Higher genotoxic stress levels may result in P53 activity reaching a “tipping point”, going from an

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adaptive response to an adverse response, where P53-mediated apoptosis surpasses the adaptive threshold and results in irrevocable tissue death, that is not replaced by healthy tissue, leading to

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malformed organs [49, 50]. Thus, P53 may be able to suppress hydroxyurea-induced birth defects up to a certain “threshold”. While most studies have reported that P53 acts as a suppressor of the embryotoxicity of various teratogens, a few studies have observed the opposite. Wubah et al. demonstrated that

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Trp53-/- fetuses treated with the nucleoside analog 2-chloro-2-deoxyadenosine on GD 8 had lower incidences of eye defects (i.e. microphthalmia) than their wild type counterparts [16]. This effect appears to rely on the ability of P53 to induce apoptosis; after treatment with 2-chloro-2-

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deoxyadenosine Trp53+/+ fetuses displayed more rampant apoptosis in the eye compared to Trp53-/- fetuses [51]. A possible explanation for the discrepancy between studies that indicate P53 is acting as a suppressor of embryotoxicity and others that indicate that it induces

embryotoxicity, is the degree of stress induced by the different teratogens that were examined. It is likely that the DNA lesions caused by 2-chloro-2’-deoxyadenosine initiate a more acute and

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restricted response in the embryo than do hydroxyurea, benzo[α]pyrene, cyclophosphamide or

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phenytoin, all of which may cause widespread oxidative and genotoxic stress, [34, 36].

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Furthermore, the ocular lens seems to be highly sensitive to P53-dependent apoptosis, increasing

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the chance of malformations due to irrevocable tissue death compared to other organs after exposure to P53-activating teratogens [52, 53].

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While the conclusions of our study may be somewhat limited by low sample size, due to

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the poor breeding capability of the B6.129S2-Trp53tm1Tyj/J mouse strain, our findings suggest

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a paradigm in which P53 is capable of acting as a suppressor of embryotoxicity up to a certain threshold. The consequences to the embryo of P53 activation are likely to be dependent on the

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specific teratogen, the level of stress that is induced, and/or the sensitivity of the target organ. While it is clear that P53 is a “hub” for the embryonic response to stress many important

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questions remain about the underlying mechanisms.

5. ACKNOWLEDGMENTS

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We should like to think Han “Aileen” Yan (McGill University, Montreal) for her assistance and instructions with fetal skeletal staining and evaluation.

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6. FUNDING This work was supported by funding from the Canadian Institutes of Health Research

[grant number: MOP-57867]. NELH is the recipient of a student stipend award from the CIHR Training Program in Reproduction, Early Development, and the Impact on Health (CIHR-

REDIH), and the Graduate Excellence Fellowship from McGill University. BFH is a James

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McGill Professor.

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