Department of Obstetric and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong1To whom correspondence should be addressed at: Department of Obstetric and Gynaecology, Prince of Wales Hospital,The Chinese University of Hong Kong, Shatin, Hong Kong.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: diclofenac/teratogenicity/whole rat embryo culture
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aspirin and other NSAID act by inhibition of prostaglandin synthesis. Aspirin has been shown to induce a wide range of malformations when given in the first trimester to animals, including central nervous system abnormalities, spina bifida and hindlimb bud abnormalities (Kimmel et al., 1971; Klein et al., 1981
). Teratogenicity of aspirin is gestation dependent. Studies on rat embryos showed that the incidence of abnormalities induced by aspirin treatment on gestation day 9 (the critical period of organogenesis) was higher than on gestation day 11 (Kimmel et al., 1971
). Other malformations, such as cleft palate, have been reported when mice embryos were exposed to NSAID at gestational age of day 13.5 (Montenegro and Palomino, 1990
).
Diclofenac has also been shown to inhibit implantation and embryonic development in rats when given on gestation day 5 (Carp et al., 1988). It was reported that diclofenac at a high concentration of 75 µg/ml was toxic to rat blastocysts. The same study also showed that diclofenac inhibited implantation and caused embryo growth retardation at a concentration of 40 µg/ml. In a recent study, a positive association between use of NSAID during pregnancy and miscarriages was reported (Nielsen et al., 2001
). However, information regarding teratogenicity of NSAID during the critical period of organogenesis is lacking. Because aspirin and other NSAID share a similar mechanism of action, we postulated that NSAID might induce congenital abnormalities when given during the critical period of organogenesis.
The aim of the present study is to investigate the teratogenicity of diclofenac in explanted rat embryos undergoing organogenesis. The whole embryo culture model is a well recognized method of investigation in teratology (Webster et al., 1997). The advantage of this model is that the direct effect of the interested agent on the developing embryo can be studied (Webster et al., 1997
). It is important to study the direct effect of diclofenac because in a previous experiment, we showed that diclofenac crosses the placenta readily in the first trimester of human pregnancy resulting in a fetal diclofenac concentration which is the same as the maternal serum concentration (Siu et al., 2000
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whole embryo culture
The whole embryo culture system was based on the model previously described (New, 1978). Animals were killed by diethyl ether overdose at gestational day 9.5 between 0900 and 1000 h in the morning and embryos were explanted. To minimize variation, only embryos with crownrump length of 1.5 ± 0.3 mm were used for the experiment. Embryos were explanted from four pregnant rats at one time. They were then mixed together and three to five embryos were assigned to a culture bottle belonging to one of the experimental groups. The investigator who assigned the embryos was unaware of which experimental group the embryos were assigned to. Embryos were then cultured for 48 h using a rotating-bottle culture unit, rotating at a constant rate of 60 revolutions per min.
Each culture bottle contained 1 ml of culture medium per embryo. Each ml of culture medium contained: (i) equal volumes of SpragueDawley rat serum and Dulbecco's modified eagle medium (DMEM) (Gibco BRL, USA); (ii) penicillin G (Sigma, UK) and streptomycin sulphate (Sigma) at a final concentration of 60 and 100 µg/ml respectively; and (iii) diclofenac sodium solution (Voltaren, Switzerland) at a final concentration depending on the study group.
During the period of culture, the system was continuously aerated with initially a gas mixture of 5% CO2, 5% O2 and 90% N2 for 24 h, followed by 5% CO2, 20% O2 in 75% N2 for the next 8 h and then 5% CO2, 40% O2 in 55% N2 for the remaining 16 h. The switching of aerating gas was performed automatically by a timer-controlled system. The different types of gas mixtures were premixed and prepared commercially.
Experimental groups
During the first part of the experiment, embryos were randomly assigned to one of the following four study groups. Group 1 was the control group without diclofenac. Embryos in groups 2 to 4 were exposed to diclofenac at a concentration of 1.5, 7.5 and 15 µg/ml respectively. Based on the result of the first part of the experiment, the second part was performed to investigate the lowest teratogenic concentration of diclofenac, and the following diclofenac concentrations were used: 0 (control), 2.5 and 5.0 µg/ml. The concentrations of 1.5 and 2.5 µg/ml were the average peak plasma diclofenac concentrations after a single oral dose of 50 and 150 mg respectively of delayed-release (enteric-coated) diclofenac sodium tablets (American Society of Health System Pharmacist, 2000).
Morphological assessment
Embryos were examined after 48 h of culture at the equivalent of 11.5 days of gestation, by a researcher who was not aware of the study group assignment. Mean yolk sac diameter and crownrump length were measured. Embryonic morphologies were studied according to a standard morphological scoring system (Van Maele-Fabry et al., 1990), which gives a numerical score (05) to 17 morphological features depending on their stages of development. To assess intra-observer error of the scoring process, 10 embryos were set aside and rescored later on the same day. The intraclass correlation coefficient was 0.92. Embryos with total morphological scores of less than two were the most likely embryos to be damaged due to explantation and were therefore excluded from the analysis.
Statistical evaluation
Between-group differences were analysed by the KruskalWallis test; Dunn's test was used as a posteriori test (Dunn., 1964), when a difference was found with the KruskalWallis test. Analyses were performed by the Statistical Package for Social Sciences for Windows version 10.0 (SPSS Inc., Illinois, USA). A P value of <0.05 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have studied rat embryo exposure to diclofenac from gestational days 9.5 to 11.5. The overall embryonic growth, as measured by yolk sac diameter, crownrump length and number of somites were not affected by diclofenac up to 15.0 µg/ml, which is equivalent to 10 times the plasma concentration after a single oral dose. However, significant effects on organogenesis were observed when rat embryos were exposed to 7.5 µg/ml of diclofenac. We also found that the caudal neural tube and hindlimb buds were particularly vulnerable. This pattern of abnormality is similar to those after exposure to aspirin treatment (McGarrity et al., 1981
).
Our results showed that diclofenac demonstrates teratogenic effects in rat embryos at a relatively low concentration (7.5 µg/ml). Although this concentration is still higher than the mean peak plasma concentration achieved after a single oral dose of diclofenac, it is of clinical significance because there is a significant variation in mean peak plasma concentration between different subjects. It has been shown that after a single dose of 100 mg diclofenac sodium tablet (enteric-coated), the peak serum concentration ranged from 2.86.6 µg/ml (El-Sayed et al., 1988). Moreover, diclofenac has been shown to penetrate and accumulate in the synovial cavity; and synovial diclofenac concentrations are increased and sustained for periods up to 12 h following multiple doses, with a ratio of synovial fluid to plasma concentration of ~5 (Davies and Anderson, 1997
). Our previous study also showed that fetal tissue concentration of diclofenac is higher than that in maternal plasma after two oral doses of diclofenac, indicating that the drug may also accumulate in fetal tissue with time (Siu et al., 2000
). In a real clinical situation, diclofenac is usually given in multiple doses rather than a single dose. Therefore, it is possible that fetal tissue concentrations may well reach the teratogenic level in some patients who are taking diclofenac.
Although many mechanisms have been proposed, the exact pathway through which NSAID produce teratogenic effects is still uncertain (Montenegro and Palomino, 1990). It has been postulated that aspirin-induced malformations result from cellular death secondary to disturbed blood supply, which is a consequence of transient vasoconstriction due to the inhibition of synthesis of vasodilatory prostaglandins (Klein et al., 1980
). Given the similar mechanism of pharmacological action and pattern of teratogenic abnormalities produced by aspirin and diclofenac, it is possible that diclofenac induces malformation through a similar pathway. Further studies are required to elucidate the mechanism of teratogenicity of NSAID.
In summary, our study has demonstrated that diclofenac exerts direct teratogenic effects on rat embryos. Since diclofenac can accumulate in fetuses, it is potentially teratogenic in humans. Although results from animal teratogenicity studies may not reflect the circumstances in humans, our findings suggest that adverse effects of diclofenac exposure during early pregnancy warrant further investigation and monitoring. Before more information in humans becomes available, the use of NSAID (especially regular and large therapeutic doses) in women of childbearing age should be treated with a degree of caution.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bitto, A., Gray, R.H. and Simpson, J.L. (1997) Adverse outcomes of planned and unplanned pregnancies among users of natural family planning: a prospective study. Am. J. Pub. Health, 87, 338343.[Abstract]
Carp, H.J.A., Fein, A. and Nebel, L. (1988) Effect of diclofenac on implantation and embryonic development in the rat. Eur. J. Obstet. Gynecol. Reprod. Biol., 28, 273277.
Davies, N.M. and Anderson, K.E. (1997) Clinical pharmacokinetics of diclofenac. Clin. Pharmacokinet., 33, 184213.[ISI][Medline]
Dawood, M.Y. (1993) Nonsteroidal antiinflammatory drugs and reproduction. Am. J. Obstet. Gynecol., 169, 12551265.
Dunn, O.J. (1964) Multiple contrasts using rank sums. Technometrics, 6, 241252.[ISI]
El-Sayed, Y., Suleiman, M.S., Hasan, M. et al. (1988) Comparative bioavailability and in vitro characterization of two brands of diclofenac sodium enteric-coated tablets. In. J. Clin. Pharmacol. Ther. Toxicol., 26, 487491.
Freinkel, N., Lewis, N.J., Akazawa, S. et al. (1984) The honeybee syndrome implications of the teratogenicity of mannose in rat-embryo culture. N. Engl. J. Med., 310, 223230.[Abstract]
Hewitt, M.J., Pratten, M.K., Regan, L. et al. (2000) The use of whole rat embryo culture as a technique for investigating potential serum toxicity in recurrent miscarriage patients. Hum. Reprod., 15, 22002204.
Kimmel, C.A., Wilson, J.G. and Schumacher, H.J. (1971) Studies on the metabolism and identification of the causative agent in aspirin teratogenesis in rats. Teratology, 4, 1524.[ISI][Medline]
Klein, K.L., Scott, W.J. and Clark, K.E.. (1980) A possible mechanism of aspirin teratogenesis. Teratology, 21, 50A.
Klein, K.L., Scott, W.J. and Wilson, J.G. (1981) Aspirin-induced teratogenesis: a unique pattern of cell death and subsequent polydactyly in the rat. J. Exp. Zool., 216, 107112.
McGarrity, C., Samani, N., Beck, F. et al. (1981) The effect of sodium salicylate on the rat embryo in culture: an in vitro model for the morphological assessment of teratogenicity. J. Anat., 133, 257269.[ISI][Medline]
Montenegro, M.A. and Palomino, H. (1990) Induction of cleft palate in mice by inhibitors of prostaglandin synthesis. J. Craniofac. Genet. Dev. Biol., 10, 8394.
New, D.A.T. (1978) Whole embryo culture and study of the mammalian embryo during organogenesis. Biol. Rev., 53, 81112.
Nielsen, G.L., Sorensen, H.T., Larsen, H. et al. (2001) Risk of adverse birth outcome and miscarriage in pregnant users of non-steroidal anti-inflammatory drugs: population based observational study and case-control study. Br. Med. J., 322, 266270.
Siu, S.S.N., Yeung, J.H.K. and Lau, T.K. (2000) A study on placental transfer of diclofenac in first trimester of human pregnancy. Hum. Reprod., 15, 24232425.
Todd, P.A. and Sorkin, E.M. (1988) Diclofenac sodium. A reappraisal of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy. Drugs, 35, 244285.[ISI][Medline]
Van den Veyver, I.B. and Moise, K.J. (1993) Prostaglandin synthetase inhibitors in pregnancy. Obstet. Gynecol. Surv., 48, 493502.
Van Maele-Fabry, G., Delhaise, F. and Picard, J.J. (1990) Morphogenesis and quantification of the development of postimplantation mouse embryos. Toxicol. in Vitro, 4, 149156.[ISI]
Webster, W.S., Brown-Woodman, P.D. and Ritchie, H.E. (1997) A review of the contribution of whole embryo culture to the determination of hazard and risk in teratogenicity testing. Int. J. Develop. Biol., 41, 329335.
Submitted on May 24, 2001; accepted on July 23, 2001.