AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 5, 1992 Mary Ann Fiebert, Inc., Publishers

The Effects of AZT and DDI on Pre- and Postimplantation Mammalian Embryos: An In Vivo and In Vitro Study EMMANUEL SIEH,1 M. LUISA COLUZZI,1 M. GABRIELLA CUSELLA DE ANGELES.1'2 ANTONIO MEZZOGIORNO,1 MARCO FLORIDIA,3 RITA CANIPARI,1 GIULIO COSSU,1 and STEFANO VELLA3

ABSTRACT the effects of the nucleoside analogs dideoxyinosine (DDI) and 3'-azido-3'deoxythymidine mammalian (AZT) embryonic development. When administered to pregnant mice (at concentrations 300 from 10 to mg/kg/day), through all or part of gestation, AZT and DDI did not result in any visible ranging effect on mouse embryos nor did they cause any obvious malformation or defect at birth or during postnatal growth. Similarly, when embryonic or fetal mouse or human cells (from brain, limb buds, or different organ rudiments) were exposed to AZT or DDI in vitro, cytotoxicity was observed only in the mM range, with AZT showing slightly higher cytotoxicity and brain cells appearing slightly more sensitive to both nucleosides. However, even in cultures treated with very high concentrations of AZT or DDI, the reduction in the number of terminally differentiated skeletal myotubes, cardiocytes, neurons, and chondrocytes was similar to the reduction in the total number of cells, indicating that AZT and DDI did not selectively inhibit differentiation of any of the above-mentioned cell types. Finally, preimplantation mouse embryos (at the 2-ceIl or 4-cell stage), treated in vitro with micromolar concentrations of AZT, were arrested at the 4-cell stage. DDI or other nucleoside analogs tested did not have this effect.

This

study reports on

INTRODUCTION

Nucleoside therapeutic

been utilized in recent years as of acquired immunodetreatment in the agents and related (AIDS) syndromes.1 3'-Azidosyndrome ficiency 3'-deoxythymidine (AZT), the most widely used until now, has shown considerable proficiency in protecting lymphocytes from HIV infection,2 however, it also shows a significant cytotoxicity on bone marrow progenitor cells, so that its prolonged use at high doses frequently causes severe anemia. ' Other nucleoside analogs such as 2',3'-dideoxyinosine (DDI) and 2'.3'-dideoxyadenosine, are being considered as potential therapeutic agents on the basis of their ability to interfere with viral reverse transcriptase at concentrations significantly below the threshold of cytotoxicity.4 Even though their mechanism of action has not yet been fully elucidated, agreement exists on their inhibitory analogs have

action being due to selective inhibition of viral polymerase or chain termination upon incorporation into DNA orto both these mechanisms. An imbalance of the nucleotide pool, at high concentrations, might also contribute to possible cytotoxic

effects.7

AZT and DDI have entered practical use and are being considered for treatment during pregnancy. Recently it has been reported that AZT and dideoxycytidine (DDC) treatment of pregnant mice results in increased embryotoxicity. but only at very high doses.8,9 No teratogenic effects were detected. On the other hand, it is becoming increasingly clear that retroviruses do infect the fetus through the placenta. Since this has serious ">~12 animal models have consequences for the infected infant, been developed to study such phenomena in detail.I4_'"'' We have therefore compared possible embryotoxic and/or teratogenic effects of AZT and DDI by exposing mouse embryos

'Institute of Histology and General Embryology. Medical School. University of Rome "La Sapienza." Via A. Scarpa 14, institute of Normal and Pathological Citomorfology, C.N.R.. Via dei Vestini, 66100 Chieti, Italy. 'Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299. 00161 Rome. Italy. 639

00161 Rome,

Italy,

640

sif:h et al.

and murine and human embryonic cells in vitro to different concentrations of these nucleoside analogs.

MATERIALS AND METHODS

Dideoxyinosine (DDI) was a generous gift of Bristol-Mayer Squibb Company. 3'-Azido-3'deoxythymidine (AZT). 8'-azaguanine (AZG). and 5'-azido-3'deoxycytidine (AZC) were purchased from Sigma. Tissue culture reagents were from GIBCO. CD1 outbreed mice

were

obtained from Charles River.

In vivo studies CD I outbreed 2-month-old female mice

were

used in this

study. Pregnancy was determined by inspection of vaginal plugs in the morning (0.5 days postcoitum, dpc). Pregnant mice received daily administration of AZT (orally in 5 ml of drinking water) or of DDI (by subcutaneous injection). Doses and periods of treatment were as specified in Table 1. On day 11, one mouse from each group was sacrificed (ex-

cept from groups H and I, where one mouse was sacrificed at day 15), the number of embryos alive and the number of

résorptions were scored and the embryos were examined under a dissecting microscope for external malformations. The embryos were fixed for histology, scanning electron microscopy, or immunofluorescence (see below). When the offspring of the remaining mice were born, the number born alive was recorded and the mice were examined daily for increase in weight, opening of the eye lids, appearance of hair, and beginning of deambulation. Upon reaching sexual maturity, at least four siblings of each group were mated and their offspring similarly examined.

In vitro studies

Postimplantation embryos. Pregnant CD I outbreed female were sacrificed on days 11 and 16 of gestation. The embryos were dissected into different organ anlagens such as brain, heart, limb buds, etc. Cells were prepared from these mice

Table 1. Protocol

Experimental group

A B

C D E F G H I J K L M

of

Treatment

of

anlagens by gentle pipetting in a calcium-magnesium-free phosphate-buffered saline (PBS) (CMF) using blue and yellow tips of Gilson pipettes. This treatment increases cell yield and reduces cell death caused by proteolytic treatments. In the case of 16 dpc fetuses however, a brief (5 min) treatment with a low concentration of trypsin (0.05%) was necessary to obtain single cell suspensions from various organs.I5 17 Human embryos were obtained from legal abortions, staged according to crown-rump length as described before,'8 and similarly dissected into different organ rudiments. All cells were cultured on collagen-coated dishes in Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), l%glutamine, and 1% penicillin-streptomycin solution. Two hours after plating, cells received AZT or DDI or AZG (used as a known cytotoxic control) at concentrations ranging from 1 p.M to 25 mM. The medium was changed daily and after 3 or 7 days the cultures were either analyzed for protein or DNA content or fixed for immunofluorescence, Alcian blue or hematoxylin-eosin staining. AZG was dissolved in dimethyl sulfoxide (DMSO) while AZT and DDI were dissolved directly into the culture medium. Preimplantation embryos. CD1 outbreed female mice (about 2 months old) were primed with 10 IU of pregnant mare serum gonadotropin (PMSG) and 48 h later injected with human chorionic gonadotropin (hCG). They were mated overnight with their male counterparts and the following morning they were examined for the presence of a vaginal plug. Two-cell and four-cell embryos were collected approximately 1.5 and 2 days after mating, respectively. The oviducts were isolated and the embryos were flushed with Medium 2 containing 4 mg/ml bovine serum albumin (BSA) (M2 + BSA).1'' The embryos

cultured in CZB medium,20 in 24-multiwell culture dishes with a daily change of medium. Incubations were carried out at 37CC in an atmosphere of 5% CO,. After varying numbers of days in culture, the embryos were examined for compaction and blastocyst formation. The number of blastomeres was determined under epifluorescence microscopy, after staining with 0.1 mM 33258 Hoechst (Fluka) in PBS for 10 min at room were

temperature.

Pregnant Mice

with

Nucleoside Analogs

Number of mice! group

Average offspring born/ experimental group

mg/kg/day

15 5 5 15 15 5 5 5 5 5 5 15 15

12.66 12.00 13.22 11.33 11.61 11.10 12.00 13.12 12.85 13.75 13.00 12.60 11.53

AZT 10 AZT 30 AZT 100 AZT 300 AZT 100 AZT 300 AZT 100 AZT 300 DDI 10 DDI 30 DDI 100 DDI 300

Period

Mice received AZT or DDI as specified in Methods. No external malformation birth in any of the offspring examined.

of

treatment

1 1 1 1 1 1

dpc- birth dpc- birth dpc- birth dpc birth dpc- lOdpc dpc- lOdpc 8dpc- birth 8 dpc birth 1 dpc- birth 1 dpc birth 1 dpc- birth 1 dpc- birth was

observed at

EFFECT OF AZT AND DDI ON MOUSE DEVELOPMENT

641

FIG. 2. Sagittal section of one of the 15 dpc mouse embryos treated with the highest dose of AZT from day 8 of development. The morphology of the embryo appears normal.

against myosin heavy chain, tropomyosin, neurofilafibronectin, and laminin. Embryos to be processed for

directed ments,

scanning electron microscopy (SEM) were fixed overnight in 2% glutaraldehdye. stained with osmium tetroxide, and examined with an Hitachi X-200 scanning microscope.

Immunofluorescence

M FIG. 1. (A) Scanning electron micrograph of one of the 11 dpc embryos treated with the highest dose of AZT from day I of development. (B) Higher magnification shows a dorsal view of the trunk at the level of the forelimb bud. Note the normal development of external structures.

scanning electron microscopy Embryos and fetuses were fixed overnight in freshly prepared 4% paraformaldehyde, then washed in PBS, and dehydrated through a graded series of ethanol solutions (50, 70, 80, 90, 95,

Histology,

and

100%) followed by two washes with toluene. The embryos were then infiltrated and embedded in paraffin. Sample sections, 6 p.m thick sagittal, parasagittal, and transverse sections were cut, rehydrated. and stained with emallume. Alcian blue staining was performed as described.21 Other embryos were similarly fixed, dehydrated through serial wash in 8. 18, and 30% sucrose in PBS, embedded in OCT. and quickly frozen in liquid N2-cooled isopentane. Seven micron sagittal or transverse sections of these embryos were cut in a cryostat. The sections were placed on gelatin-coated slides and processed (as described below) for immunofluorescence with a variety of antibodies

For immunofluorescence, cells (or tissue sections) were fixed for 10 min at 4°C with freshly prepared 4% paraformaldehyde in PBS. They were permeabilized by 0.1% Triton X-100 in PBS (10 min at 4°C), then washed with PBS containing 0.1% bovine serum albumin (BSA) for 15 min at 25°C and incubated with one of the following antibodies: MF20 (monoclonal antibody directed against sarcomeric myosin heavy chains, donated by D. Fischman, Cornell University, NY), anti-K68 neurofilament (Sigma), anti-a skeletal tropomyosin (donated by M. Fiszam, Institut Pasteur, Paris), antifibronectin and antilaminin (donated by F. Walsh, University Hospital, London). All of the first antibodies were used at 1:50 (except MF20. 1:10) dilution for 1 h at 25°C. The cells were then washed twice in PBS-BSA 0.1% for 30 min each and incubated with second antibodies (fluoresceine-conjugated goat anti-rabbit or rodamine-conjugated goat anti-mouse IgG, Cappel) at 1:30 dilution for 1 h at 25CC. After an additional two PBS-BSA washes, the cells were mounted with buffered glycerol and examined using a Zeiss epifluorescence

microscope.22

ELISA and Alcian blue extraction The amount of skeletal myosin in control and treated cultures measured as detailed in Cossu et al.23 The amount of K68 neurofilament was determined similarly by adsorbing known aliquots of total homogenate to ELISA plates which were then was

SIEH ET AL.

642 120 -,

100

H

60

H

o

ï 8 C

S

20

1 I I ~I 1mM 5m M control 0.04mM 0.2mM DDI concentration

25 m M

Lunbbuds

1mM control 0.04mM 0.2mM 5mM AZT concentration

25mM

[)yj Heart J Brain

The effect of (a) DDI or (b) AZT on protein content (expressed as a percentage of the control) in cultures of limb buds (solid bars), heart (hatched bars), or brain cells, (dotted bars) from 11 dpc mouse embryos. Cells received (a) DDI or (b) AZT 2 h

FIG. 3.

after plating and with successive daily medium changes. Cells were harvested after 3 days of culture. Each point is the average of at least five separate experiments, each performed in duplicate, with errors ranging within 10% of the reported values.

incubated with anti-K68 neurofilament monoclonal antibody (Sigma, at 1:50 dilution). The amount of glycosaminoglycans (GAG) in both control and treated cultures was determined by Alcian blue extraction as described.2'

Protein

synthesis and electrophoresis

To evaluate protein synthesis after 2 days in culture, control and treated embryos were incubated for 5 h with 2 mCi/ml, 20 p.M [35S]methionine (Amersham. spec. act. 10 Ci/mmol), washed in PBS, placed in 15 u.1 of electrophoresis lysis buffer and subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli. Gels were then fixed in 7% acetic acid, dried, and exposed on Kodak X-Omat film at —70°C for autoradiography.

day 8 pc), one animal per experimental group was sacrificed and the embryos were examined. The number of résorptions were not significantly increased in treated embryos (95% correlation exists between the teratogenic effect of a drug and its ability to inhibit terminal differentiation of neurons and chondrocytes.27 We then exposed, to AZT and DDI, murineand human embryonic cells, and could not detect inhibition of proliferation or differentiation of neurons, chondrocytes. cardi-

-

647

EFFECT OF AZT AND DDI ON MOUSE DEVELOPMENT

Table 3. Blastocyst Formation in 4-Cell Embryos Treated with Nucleoside Analogs

Experimental conditions

Control DDI 0.5 mM AZC 0.5 mM AZT 0.5 p.M AZT 5 p.M AZT 0.05 mM AZT 0.5 mM AZT 0.5 mM Reverted

No. bla.stocy.sts/ total embryos

Percent of maturation

53/64 49/61 20/26 26/35 12/32 5/28 12/63 7/30

83% 80% 76% 75% 39% 18% 6% 24%

Four-cell embryos were cultured for 48 h in control medium in the presence of the nucleoside analogs, at the concentrations indicated. In one case, embryos were cultured for 24 h in the presence of AZT (0.5 mM). then shifted to control medium and cultured for an additional 24 h (AZT Reverted).

J5

or

pa

1)

S

3

synthesis in the blastomeres. This would imply that the sensitivity of early cells to AZT, but not to DDI, is much higher than that exhibited by postimplantation cells. However, when pregnant high doses of AZT (corresponding to 300 mg/kg/day). embryonic development continued on a normal mice were treated with

This therefore suggests that after oral administration the real concentration of AZT in the maternal blood and/or the microenvironment of the embryo probably is too low to elicit any effect. Recent data on the pharmacokinetics of AZT in near-term pregnant macaques.2'' revealed that the nucleoside course.

24

Time in hours

FIG. 9. Time-course of cell division in 2-cell (upper panel) and 4-cell (lower panel) embryos, continuously treated with 0.5 mM AZT (v). 0.5 mM AZC ( ), 0.5 mM DDI (•). Control embryos (O). Each point is the average ± SE of three separate experiments each carried out on pools of at least 25 embryos per experimental group.

ocytes, or skeletal myotubes. However, a notable exception to the correlation between teratogenicity and inhibition of differ-

entiation was represented by thalidomide.27 and this should impose caution on our in vitro results. Perhaps, in vitro culture of organ rudiments which complete part of their morphogenesis in vitro, might offer a more appropriate method in the future. At the present time however, reproducible correct morphogenesis occurs only in a percentage of organs cultured which is too low to permit the testing of possible teratogens.28 One result of this study merits consideration on its own: The cytostatic effect of AZT, but not of DDI or AZC, on the early cleavage of mouse embryos. Toltzis et al.8 showed a similar effect of AZT, but they did not use DDI or other nucleoside analogs. We also observed that the cytostatic effect of AZT was reproducible at concentrations which did not interfere with ongoing protein synthesis (at least during the first 2 days of treatment). It therefore appeared to be caused by a block of DNA

BCD Autoradiogram of newly synthesized proteins of embryos, labeled with [ ,5S]methionine and analyzed by

FIG. 10. mouse

SDS-PAGE as described in Materials and Methods. (Lane A) Control, (Lane B) 0.5 mM AZT. (Lane C) 0.5 mM DDI, and (Lane D) 0.5 mM AZC. The homogenate from the same number of embryos (20) was loaded on each lane.

648

SIEH ET AL.

freely crosses the placenta3" and probably does accumulate in the late fetus. Data on the possible accumulation of AZT or DDI on earlier embryos are not available and the size of mouse embryos suggests that this might be measured only by radioactive methods. Also no similar data are available as yet for primates or humans. Even so, extrapolation of the data on preimplantation mouse embryos to humans would suggest that an effect of AZT similar to that reported in mice, should cause death of the conceptus and not malformations. In conclusion, the studies reported above demonstrate a lack of major teratogenic effects of DDI and AZT on rodents in vivo and in vitro and on human cells in vitro. The possible cytotoxic effect of AZT on mouse preimplantation embryos might cause abortions, but is less likely to cause malformations (since cell division is arrested in embryos at a very early stage). Taken together, these data allow a prudent but optimistic attitude toward clinical experimentation during pregnancy.

7.

8.

9.

10.

11.

12.

13.

ACKNOWLEDGMENTS This work was supported by grants from the Italian Ministry of Health-Istituto Superiore di Sanità-AIDS projects 1990 and 1991 n. 6203-009 to GC and from CNR, Progetto Finalizzato FATMA, n. 91.00264.41 toSV. ES was supported by a fellowship from The Third World Academy of Science. We thank Mr. Q. Giustiniani for his help with the scanning electron microscope, Mr. S. Greci for excellent technical help, and Dr. Paula Murphy for critical reading of the manuscript. We thank Bristol-Myers Squibb for generously donating

dideoxyinosine.

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