TERATOLOGY 4555-63 (1992)
Inhibition of lsotretinoin Teratogenicity by Acetylsalicylic Acid Pretreatment in Mice STAN KUBOW School ofDietetics and Human Nutrition, Macdonald Campus of McGill University, Ste. Anne de Belleuue, PQ, Canada H9X ZCO
ABSTRACT Although isotretinoin (ITR) has been suggested to cause malformations via cytopathic effects on embryonic cells, the molecular mechanisms of ITR cytotoxicity in teratogenesis are not clear. Since ITR undergoes metabolism by prostaglandin synthase to a potentially cytotoxic peroxyl free radical, the possible role of prostaglandin synthase metabolism as a modulator of ITR teratogenicity was evaluated. Craniofacial and limb abnormalities were noted in fetuses on day 18.5 of gestation following administration of ITR to pregnant CD-1 mice in a three dose regime of 100 mglkg a t 4 hr intervals on day 10.5 of gestation (plug day = day 0.5 of gestation). Mice were also treated with acetylsalicylic acid (ASA), an irreversible inhibitor of the cyclooxygenase component of prostaglandin synthase, a t doses of 20 and 60 mglkg body weight 2 hr prior to each ITR dose. ASA pretreatment of mice receiving ITR treatment showed a dose-dependent decrease in the overall incidence of malformations, number of defects per fetus, and the incidence of specific craniofacial and limb defects. Equivalent doses of ASA given to control mice did not cause malformations or alter the incidence of resorptions. These results demonstrate that ASA is able t o ameliorate the teratogenic effects of ITR observed in fetal mice near term and indicate that prostaglandin metabolism could play a mechanistic role in ITR teratogenicity. Although a great deal is known concerning the teratological outcome of excessive retinoid intake, very little is known concerning its mechanism of teratogenicity. Specific influences of retinoids on cell death, cell proliferation, and cell membrane stability have been proposed as mechanisms to account the broad spectrum of retinoidinduced congenital defects encompassing the craniofacial, skeletal, cardiovascular, and nervous system regions. Recent theories on retinoid teratogenicity have focused on retinoid induction of excessive cellular necrosis in areas associated with programmed cell death (PCD) (Alles and Sulik, '89) and on specific cytotoxic effects of retinoids on cells of the chondrogenic cell lineage (Kochhar et al., '87). The molecular mechanisms by which retinoids may be cytotoxic are unclear but have been related to the action of retinoids to labilize lysosomal membranes, modify genetic information or induce glycosylation of proteins (Alles and Sulik, '89). Some workers have suggested 0 1992 WILEY-LISS,
INC.
that a recently discovered nuclear retinoic acid receptor may play a role in morphogenetic activities of retinoic acid and that retinoic acid may induce excessive cell death in zones of PCD in a similar fashion to glucocorticoids by modulating gene expression (Petkovich et al., '87; Alles and Sulik, '89). In exploring the molecular basis of the teratogenic activity of retinoids it is important to determine whether such compounds could also be further metabolized to more teratologically potent forms. These are several interconvertible forms of retinoids which undergo a number of biotransformations which may be toxicologically important including conjugation, esterification, decarboxylation, glucoronic acid conjugation, and side-chain cleavage (Lotan, '80; Satre et al., '89). Involvement of mixed func-
Received April 24, 1991; accepted September 10, 1991.
56
S. KUBOW
I Arachidonatel Fatty Acid Cyclooxyqenase ..
1
Hydroperoxidase
!
NADPH
I
Cytotoxicity Teratogenesis
O2
1
Fig. 1. Postulated co-oxidation of ITR to a teratogenic free radical by prostaglandin synthase and the metabolism of ITR by cytochromes P-450. Fatty acid cyclooxygenase and hydroperoxidase are the two catalytic components of prostaglandin synthase. PGGS, prostaglandin G2; PGH2, prostaglandin H2.
tion oxidase cytochromes P-450 (Roberts et al., '79) and prostaglandin synthase (Samokvszyn et al., '84) has been suggested in the enzymatic formation of retinoid metabolites (Fig. 1).Under physiological stimuli, the release of arachidonic acid from membrane phospholipids initiates prostaglandin synthesis. The cyclooxygenase component of prostaglandin synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2). PGG2 is reduced by the hydroperoxidase component of prostaglandin synthase to form prostaglandin PGH2 which is subsequently metabolized to a variety of prostaglandins and thromboxanes. The latter reaction requires reducing equivalents and a variety of drugs have been shown to serve as electron donors to the peroxidase leading to the formation of electrondeficient drug metabolites (Eling et al., '83). Co-oxidative metabolism of ITR by the hydroperoxidase component of prostaglandin synthase has been demonstrated to form a ITR peroxyl radical as well as 4hyroxyl-ITR and tretinoin (Samokvszyn et al., '84). Additionally, enzymatic formation of 4-hydroxy-ITR is also suggested via cytochromes P-450-mediated metabolism of ITR which would require NADPH and O2 (Frolik, '81; Hill and Struck, '83). Free radical formation may cause oxidant stress, initiate lipid peroxidation, andlor bind covalently to essential cellular macromolecules, thereby causing cytotoxic effects and
fetal abnormalities. Since embryos demonstrate high prostaglandin synthase activity (Mitchell et al., '85), embryonic prostaglandin synthase metabolism of ITR could contribute to teratogenicity by production of ITR-peroxyl free radicals and other potentially toxic ITR metabolites. Recent evidence has indicated that embryonic prostaglandin synthase-mediated bioactivation of phenytoin (Kubow and Wells, '89; Wells et al., '89a) and trimethadione (Wells et al., '89b) to reactive free radicals intermediates may be a mechanism of teratogenicity of these drugs. The potential role of prostaglandin synthase as a modulator of ITR teratogenicity was evaluated in the present study by pretreating pregnant CD-1 mice with acetylsalicylic acid (ASA, aspirin), a potent and irreversible inhibitor of the cyclooxygenase component of prostaglandin synthase (Flower et al., '85). Malformations were induced in mice by the administration of ITR in a three dose regime of 100 mg/kg at 4 hr intervals on day 10.5 of gestation and detected on day 18.5 of gestation. Craniofacia1 and limb malformations were noted as previously observed in mice exposed on day 10.5 to a similar regimen of ITR (Kochhar and Penner, '87). It was observed that mice receiving an ASA pretreatment showed a ASA dose-dependent reduction of ITRinduced fetal craniofacial and limb malformations in terms of overall incidence and mean number of defects per fetus. MATERIALS AND METHODS
ITR (isotretinoin, 13-cis retinoic acid) (Sigma Chemical Co., St. Louis, MO) was stored a t -20°C in the dark in a sealed, lightproof container under nitrogen. The drug was suspended in safflower oil and dosing solutions were sonicated and vortexed to obtain a uniform suspension. ASA (acetylsalicylic acid, aspirin) (Sigma Chemical Co., St. Louis, MO) was vortexed in a 0.9% saline under warm tap water until completely dissolved. Pregnant CD-1 (ICR) mice (Charles River Canada, Inc., St. Constant, Quebec) were housed in a environmentally controlled room under a 12-hr light-dark cycle (light cycle 7 A.M. to 7 P.M.) and were maintained on Purina Mouse Chow and tap water ad libitum throughout the whole experiment. A group of three virgin females were caged overnight with one male. Mating was confirmed
ASPIRIN REDUCES ISOTRETINOIN MALFORMATIONS
by the presence of a vaginal plug the next morning (8:30 A.M.) which was designated day 0.5 of gestation. Experiments were begun on the morning of gestational day 10.5 when pregnant females were randomly assigned to control and experimental groups. A previous study found that oral doses of 100 mg/kg ITR given three times to the same dam a t either 3 or 8 hr intervals on day 11 of pregnancy induced malformations in a majority of ICR mouse fetuses (Kochhar and Penner, '87). This dose regime was selected in the present investigation except that a 4 hr interval was selected between ITR doses. Control treatment groups included positive controls which received an intubation of 100 mg/kg ITR intragastrically on the morning (11 A.M.), afternoon (3 P.M.), and evening (7 P.M.) on day gestational 10.5. Experimental groups received ASA doses of 20 and 60 mgl kg i.p. 2 hours prior to each ITR dose. At the same time points as the ASA pretreatments in the experimental groups, the saline controls received i.p. injections of sterile physiological saline and the ASA controls received 0.3-0.5 ml safflower oil intragastrically and either 20 or 60 mg/kg ASA i.p. All animals were killed by cervical dislocation on gestational day 18.5. After laparotomy, each fetus was dissected free of the placenta and fetal membranes, weighed, sexed, and examined for external malformations. The number and weight of the resorptions, the number of implantation sites, the pup location, and the viability of the pups were recorded. Placentas were matched to the pups and weighed. Each litter was fixed in Carnoy's solution and examined for anomalies for the craniofacial region by free hand sectioning. Statistical analyses were performed on both an individual fetus and a litter basis. The incidence of specific malformation(s) was calculated as the number of fetuses with the malformation(s) divided by the number of fetuses. Analyses for comparison of incidence of malformed fetuses, types of anomalies and total resorptions were performed by chi-square test. For the purposes of calculating overall incidence of malformations, embryos with complex defects were counted once; for the mean number of defects per fetus, the number of abnormalities per fetus were counted separately. Therefore, the sum of abnormalities per fe-
57
tus may exceed one. A two-way analysis of variance was performed for the statistical comparison of litter size, fetal body weight, implantation sites, placental weights, resorptions per litter, and mean of defects per fetus using Statistical Analysis System for personal computers (SAS, '86). The significance of observed differences among the groups was evaluated by Tukey's test. A probability of P 5 0.05 was accepted as the minimal level of significance. RESULTS
ITR treatment under the chosen experimental conditions was characterized by a spectrum of severe craniofacial deformities and limb reduction abnormalities. The most prominent malformations in the present experiment were cleft palate (72%), micrognathia (16%), forelimb (8%) and hindlimb defects (5%),and kinky and stub tail (6%). The majority of surviving fetuses exposed to ITR treatment had a t least one of the above malformations (79%). Cleft palate and reduction defects of the anterior and posterior limbs are typically observed in mouse fetuses exposed to retinoids on gestational days 10.5 or 11(Kochhar, '73). The high incidence of cleft palate obtained in mice treated with the three dose regimen of 100 mg/kg ITR over a 8 hr time period was similar to that observed with a similar dosing treatment by Kochhar and Penner ('87) in ITR-exposed mice (88%). There were, however, relatively fewer fetuses with a reduction of the limbs in the present experiment than was observed in the Kochhar and Penner ('87) study (> 50%). Since the timing of the ITR treatments and the mouse strain used were identical in the two studies, this difference is likely due to the shorter time interval of 3 hr between doses used by Kochhar and Penner ('87) as compared to the 4 hr interval used in the present study. Kochhar and Penner ('87) showed a much lower frequency of limb defects in mice when longer time intervals were used between the three 100 mg/kg doses of ITR as compared to a 3 hr time interval. Table 1 shows the effects of the dosing agents on gestational and developmental data as observed on day 18 of pregnancy. No effect of ITR, ASA, or ITR plus ASA was observed on implantation sites or number of resorptions on a per litter basis (Table 1). Treatment with 60 mg/kg ASA in the pres-
58
S. KUBOW
TABLE I ,Effects on pregnancy outcome after administration of ITR, ASA, and vehicle doses given to CD-1 mice three times at 4 hr intervals on gestational day 10.5l ITR (100 mg/kg) Safflower oil control ASA (mgikg) Vehicle 20 60 Vehicle 20 60 Litters (n) 19 23 12 9 6 7 Irnplantati~n~,~ sitesilitter 12.10 ? 0.554 11.26 ? 0.68 13.92 ? 0.40 13.44 ? 0.80 12.17 ? 1.68 12.43 ? 0.57 Live fetusesilitter 11.16 ? 0.47ab 10.04 ? 0.65" 12.67 f 0.38b 12.11 ? 0.74"b 10.83 f 1.7OUb12.29 f 0.68"' Fetal weight (g) 1.42 f 0.01" 1.32 f O.Olb 1.37 2 0.01' 1.36 ? 0.01' 1.47 ? O.Old 1.37 2 0.01' Placental weight ( g ) 0.11 t 0.002" 0.11 ? 0.021" 0.11 ? 0.019" 0.10 f 0.017' 0.10 5 0.017b 0.09 ? 0.016b Resorptions/litter' 0.07 f 0.02 0.10 f 0.02 0.9 ? 0.02 0.10 t 0.03 0.12 ? 0.03 0.01 f 0.03 Total resorptions' 7.83" 10.81" 8.98" 9.92" 10.96" 1.15' 'ITR, isotretinoin; ASA, acetylsalicylic acid. 'Plug day = 0.5 of gestation. Fetuses examined on day 18.5 of gestation. 3Means not sharing a common superscript are significantly different at P 4Mean -t S.E.M. 'Values based on implantations
ence of ITR resulted in an increase in the number of live fetuses per litter when compared to 20 mglkg ASA plus ITR. Treatment with 20 mglkg ASA resulted in an increase in fetal weight as compared to saline-control but ASA treatments caused a decrease in fetal weight in the presence of ITR. Administration of ITR, however, was associated with an increased fetal weight relative to saline controls (Table 1).ITR treatment also caused an increase in placental weight relative to the ASA controls. The data for the saline-treated mice (Table 1) show a low background incidence of resorptions with a normal number of implantation sites, fetal and placental weights, and live fetuses per litter consistent with other published reports for mice (Mehanny et al., '91; Lofberg et al., '90; Kochhar and Penner, '87). ASA pretreatment alone did not cause any observable fetal toxicity as indicated by the low incidence of resorptions, no decrease in mean fetal body weight compared to salinetreated controls, and only two malformed fetuses observed with the 20 and 60 mg/kg dose treatments of ASA alone (Table 1).The single malformed fetus observed following 60 mglkg ASA treatment exhibited micrognathia, while a hindlimb defect was observed in the sole malformed fetus in the 20 mgkg ASA group. In fact, percent total resorptions actually decreased in the 60 mgl kg ASA-exposed fetuses as compared to saline controls. The lack of teratogenic effect of ASA is likely to be dose-related since approximately 400-500 mglkg ASA is needed to cause teratogenic effects in the mouse (Szabo, '89; Larsson et al., '63). Pretreatment with ASA produced a reduction in the incidence of ITR-induced
5
0.05.
craniofacial defects (cleft palate and micrognathia) and limb defects (hindlimb and tail) that was dose dependent (Figs. 2,3). In contrast, no significant effect of ASA was observed on ITR-induced forelimb defects (Fig. 3). The incidences of malformed fetuses (Fig. 4) and the average number of abnormalities per fetus (see Fig. 5) induced by ITR were also significantly reduced by ASA pretreatment. In terms of a percentage reduction of ITR-induced abnormalities, pretreatment with ASA caused a 75% reduction of posterior defects (hindlimb and tail) and a 75%reduction in the mean number of abnormalities per fetus in the posterior region (Figs. 4, 5). The anterior region was less sensitive to the protective action of ASA although a 26% reduction in total anterior defects (craniofacial and forelimb) and a 28% reduction in the number of defects per fetus in the anterior region was observed (Figs. 4, 5). DISCUSSION
These findings demonstrate that ASA pretreatment, when administered 2 hr prior to ITR, reduces the incidence of developmental malformations of ITR in a dose-dependent fashion. Pretreatment with the 60 mg/kg dose of ASA resulted in decreases in overall incidences of ITR-induced craniofacia1 (cleft palate and micrognathia) and limb (forelimb, hindlimb, and tail) defects. Moreover, the presence of specific abnormalities in ASA-pretreated fetuses exposed to ITR were accompanied by a diminished severity so that fewer abnormalities per fetus were observed than in ITR fetuses. As ASA irreversibly inhibits the activity of prostaglandin synthase (Flower et al.,
59
ASPIRIN REDUCES ISOTRETINOIN MALFORMATIONS (1131
(nbfetuses ITR
P e r
60
[7
ITR.20
r;Xl
ITR+BO mg/Kg A S A
mg/Kg ASA
C
e n t
40
20
* 521
n Cleft Palate
Mi crag nat hia
Fig. 2. Effects of acetylsalicylic acid (ASA) pretreatment on isotretinoin (1TR)-induced craniofacial defects in the CD-1 mouse. ITR, 100 mg/kg, was given intragastrically in three consecutive doses at 4 hr intervals to pregnant CD-1 mice on gestational day 10.5. ASA was administered i.p. 2 hr prior to each ITR dose. Fetuses were examined on day 18.5 of gestation. *Significantly different from ITR control, P c 0.05.
(nbfetuses ITR
P e r
0
8
I T R * 2 0 mp/Kg ASA
I T R * 8 0 m p l K g ASA
C
e
7
6
F
r e
4
q
*
U
e n
c
(152)
2
Y n "
Forelimb Defects
Hindlirnb Defects
Tail Defects
Fig. 3. Effects of acetylsalicylic acid (ASA) pretreatment on isotretinoin (1TR)-induced limb defects in the CD-1 mouse. ITR, 100 mgkg, was given intragastrically in three consecutive doses at 4 h r intervals to pregnant CD-1 mice on gestational day 10.5. ASA was administered i.p. 2 hr prior to each ITR dose. Fetuses were examined on day 18.5 of gestation. *Significantly different from ITR control, P 5 0.05.
1985), these results indicate that prostaglandin synthase metabolism plays a mechanistic role in ITR teratogenicity. The halflife of ASA is 15 min and the salicylate metabolite has a half-life of 2-3 hr (Flower et al., '85). The 2 hr time interval between the ASA and ITR treatments used in the present study would thus permit the major-
ity of the ASA and salicylate metabolite to be eliminated prior to ITR administration. ASA pretreatment in the mouse has previously been demonstrated to decrease the teratogenic effects of a number of drugs including alcohol (Randall and Anton, '841, phenytoin (Wells et al., '89a), and trimethadione (Wells et al., '89b). The mecha-
60
S. KUBOW Malformed Fetuses 100
(nbfetuses ITR
P e r
0
80
I T R * 2 0 rnglKg ASA
ITR*dO rnglKg ASA
C
e n t
60
F r e
40
9 U
e n C
20
Y
0
Overall Malformed
Anterior Defects
Posterior Defects
Fig 4 Effects of acetylsalicylic acid (ASA) pretreatment on the incidence of isotretinoin (1TR)-induced malformed fetuses in the CD-1 mouse ITR, 100 mgkg, was given intragastrically in three consecutive doses at 4 hr intervals to pregnant CD-1 mice on gestational day 10 5 ASA was administered i p 2 hr prior to each ITR dose. Fetuses were examined on day 18.5 of gestation *Significantly different from ITR control, P c 0 05 (nkfetuses
ITR.20 mglKg ASA I T R * 6 0 mglKg ASA
Overal I
Anterior
Posterior
Defects
Defects
Defects
Fig 5 Effects of acetylsalicylic acid (ASA) pretreatment on the number of defects per fetus induced by isotretinoin (ITR) in the CD-1 mouse ITR, 100 mg/kg, was given intragastrically in three consecutive doses at 4 hr intervals to pregnant CD-1 mice on gestational day 10.5 ASA was administered 1 p 2 hr prior to each ITR dose. Fetuses were examined on day 18 5 of gestation Values are expressed as means + SE *Significantly different from ITR control, P c 005
nism of the ASA inhibition of drug-induced teratogenicity has been proposed to be due to either inhibition of prostaglandin synthase metabolism of these drugs to free radicals or to modulation of embryonic and maternal prostaglandin levels. In the present study, the anterior regions
of mouse fetuses demonstrated enhanced susceptibility to ITR-induced defects as compared to malformations induced by ITR in the posterior regions (Figs. 4, 5). A similar differential sensitivity of forelimb and hindlimb regions to malformations induced by retinoic acid has been previously noted in
ASPIRIN REDUCES ISOTRETINOIN MALFORMATIONS
DBA/2J mouse fetuses (Kochhar, '73). Forelimbs were observed t o be more sensitive to retinoic acid on day 12 than on day 13 while the reverse was true for hindlimbs. The more dramatic reduction in the incidence and severity of ITR-induced posterior (hindlimb and tail) defects by ASA pretreatment as compared to ITR-induced anterior (craniofacial and forelimb) defects could be explained by the developmental stage of these embryonic tissues with respect to the time of dosing of ITR and ASA. The posterior regions of the mouse embryo which contain the tail and hindlimb buds are developmentally delayed by approximately 24 hr at gestational day 10 as compared to the anterior regions which include the forelimb buds and palatal shelves (Rugh, '68). The delay in hindlimb and tail development relative to the anterior region at the time when the mice received the ITR and ASA treatments is likely to have decreased the sensitivity of the posterior regions to ITR. This developmental delay may have also permitted ASA to have more time to exert greater protective effects and allowed normal development of the posterior regions to be completed. Alternatively or in addition, it is possible that the differential protective effects of ASA could be accounted for by variations in the mechanisms of ITR teratogenesis with developmental stage, region, or tissue site. The limited protective effects of ASA against ITR teratogenicity may involve a number of mechanisms. Enzymatic bioactivation of ITR to a reactive oxygen-centered free radical has been demonstrated (Samokvszyn et al., '84) and is a possible means by which prostaglandin synthase metabolism could contribute to ITR teratogenicity. The toxicity of oxygen radicals t o embryonic tissues has been noted in embryo culture studies. For example, rat embryos exposed to xanthine oxidase-generated oxygen free radicals in culture showed severe malformations that were attributed to excessive cellular death and cell cycle delay (Jenkinson et al., '86). Since excessive cell death has been proposed as a mechanism of teratogenesis for ITR administration (Sulik et al., "9,the possibility exists that cytotoxic effects exerted by oxygen free radicals produced from embryonic prostaglandin synthase metabolism of ITR could be a mechanism of ITR teratogenesis. Support for the formation of ITR-derived cytotoxic
61
free radicals has been obtained from recent work which has indicated a relationship between ITR-induced generation of toxic oxygen radical species and depressed neural crest cell viability and proliferation observed upon exposure of isolated chick neural crest cells to ITR (Davis et al., '90). The addition of free radical scavenging enzymes, superoxide dismutase, and catalase to the culture medium increased cell viability and decreased concentrations of oxygen radicals. Additionally, ASA could decrease ITR teratogenicity by inhibiting maternal generation of 4-oxo-ITR, a potent teratogenic metabolite that may arise from prostaglandin synthase metabolism of ITR (Samokvszyn et al., '84); 4-oxo-ITR is about four-fold more effective as a teratogen in mice than is the ITR parent compound (Kochhar and Penner, '87). An inhibition of prostaglandin synthase biotransformation of ITR to 4-0x0ITR by ASA in maternal tissues could be of teratological significance since the enhanced potency of 4-oxo-ITR has been indicated to be due t o more efficient placental transfer of 4-oxo-ITR relative to ITR (Kochhar and Penner, '87; Creech Kraft et al., '87). One further potential mechanism of ASA protection could involve the pathway proposed by Sporn and Roberts ('83) in which retinoids control gene expression via interactions with protein kinases. Control of gene expression via protein kinases has been proposed to initiate a receptor cascade system which integrates phospholipid degradation, prostaglandins, cyclic AMP, cyclic GMP, calmodulin, and calcium (Nishizuka, '84). Since a regulatory role for prostaglandin E, has been suggested with the early events of chondrogenesis (Biddulph et al., '84) and palatal development (Jones and Greene, '861, a toxic imbalance of prostaglandins induced by ITR may be in part responsible for the causation of limb defects and cleft palate. Inhibition of prostaglandin metabolism by ASA could thus be of importance in terms of its protective action against ITR malformations. It may also be possible that ASA was protective via other mechanisms aside from its action on prostaglandin synthase. Nevertheless, research prompted by initial studies demonstrating protective effects of ASA pretreatment against particular drug-induced malformations have supported a role of prostaglandin metabolism in these toxic-
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S. KUBOW
ities. For example, the demonstration of ASA protection against ethanol teratogenicity (Randall and Anton, '84)has been a catalyst for a wide body of research supporting a role of prostaglandins in ethanol teratogenicity (Smith et al., '91). Additionally, a role of prostaglandin synthase in phenytoin metabolism and teratogenicity, suggested by the protective action of ASA pretreatment (Wells et al., '891, has been supported by more in-depth mechanistic studies (Kubow and Wells, '89). It should also be noted that the therapeutic benefits produced by ASA and its salicylate metabolites have thus far been attributed t o inhibition of prostaglandin synthase metabolism (Vane and Botting, '87). Moreover, as opposed to other non-steroidal anti-inflammatory drugs, ASA does not inhibit lipoxygenase activity (Vane and Botting, '87) and thus is more specific in its inhibitory action on prostaglandin synthase. While the mechanism for the protective action of ASA on ITR teratogenicity requires further exploration, the data warrants consideration of the hypothesis that ITR induces malformations by undergoing metabolic transformation by prostaglandin synthase to more teratogenic metabolites and by causing alterations of prostaglandin metabolism. ACKNOWLEDGMENTS
The author would like t o express his grateful appreciation to Susan Smith for her expert technical assistance. REFERENCES Alles, A.J., and K.K. Sulik (1989) Retinoic-acid-induced limb-reduction defects: Perturbation of zones of programmed cell death as a pathogenic mechanism. Teratology, 40:163-171. Biddulph, D.M., L.M. Sawyer, and W.P. Smales (1984) Chondrogenesis of chick limb mesenchyme in vitro. Exp. Cell Res., 153:270-274. Creech Kraft, J., D.M. Kochhar, W.J. Scott, and H. Nau (1987) Low teratogenicity of 13-cis retinoid acid (isotretinoin) in the mouse corresponds to low embryo concentrations during organogenesis: Comparison to all-trans isomer. Toxicol. Appl. Pharmacol., 87.474482. Davis, W.L., L.A. Crawford, O.J. Cooper, G.R. Farmer, D. Thomas, and B.L. Freeman (1990) Generation of radical oxygen species by neural crest cell treated in vitro with isotretinoin and 4-0x0-isotretinoin.J . Craniofac. Genet. Dev. Biol., 10:295-310. Eling, T.E., J.A. Boyd, G.A. Reed, R.P. Mason, and K. Sivarajah (1983) Xenobiotic metabolism by prostaglandin endoperoxide synthetase. Drug Metab. Rev., 14t1023-1053. Flower, R.J., S. Moneada, and J.R. Vane (1985) Anal-
gesic-antipyretics and anti-inflammatory agents: Drugs employed in the treatment of gout. In: The Pharmacological Basis of Therapeutics. A.G. Gilman, L.S. Goodman, T.W. Rall and F. Murad, eds. Macmillan, New York, 7th ed., pp. 674-689. Frolik, C.A. (1981) In vitro and in vivo metabolism of all-trans and 13-cisretinoic acid in the hamster. Ann. N.Y. Acad. Sci., 359:37-44. Hill, D.L., and R.F. Struck (1983) Pharmacolaogic disposition of chemopreventive retinoids. Anticancer Res., 3.171-180. Jenkinson, P.C., D. Anderson, and S.D. Gangoli (1986) Malformations induced in cultured rat embryos by enzymatically generated active oxygen species. Teratogen. Carcin. Mutagen., 6.547-554. Jones, J., and R. Greene (1986) Identification of prostaglandin E, receptor sites in the embryonic murine palate. Prostaglandins, Leukotrienes Med., 22.139-141. Kochhar, D.M. (1973) Limb development in mouse embryos. I. Analysis of teratogenic effects of retinoic acid. Teratology, 7:289-298. Kochhar, D.M., and J.D. Penner (19871Developmental effects of isotretinoin and 4-0x0-isotretinoin:The role of metabolism in teratogenicity. Teratology, 36:67-75. Kochhar, D.M., J . Kraft, and H. Nau (1987) Teratogenicity and disposition of various retinoids in vivo and in vitro. In: Pharmacokinetics in Teratogenesis. H. Nau and W.J. Scott, Jr., eds. CRC Press, Boca Raton, Florida, pp. 173-186. Kubow, S., and P.G. Wells (1989) In vitro bioactivation of phenytoin to a reactive free radical intermediate by prostaglandin synthase, horseradish peroxidase and thyroid peroxidase. Mol. Pharmacol., 35504-511. Larsson, K.S., H. Bostrom, and B. Ericson, B. (1963) Salicylate-induced malformations in mouse embryos. Acta Paediatr. Scand., 52:36-40. Lofberg, B., I. Chahoud, G. Bochert, and H. Nau (1990) Teratogenicity of the 13-cis and all-trans-isomers of the aromatic retinoid etretin: Correlation to transplacental pharmacokinetics in mice during organogenesis after a single oral dose. Teratology, 41:707-716. Lotan, R. (1980) Effects of vitamin A and its analogs (retinoids) on normal and neoplastic cells. Biochem. Biophys. Acta, 605:33-91. Mehanny, S.Z., M.S. Abdel-Rahrman, and Y.Y. Ahmed (1991) Teratogenic effects of cocaine and diazepam in CF1 mice. Teratology, 43:ll-17. Mitchell, M.D., S.P. Brennecke, S.A. Saeed, and D.M. Strickland (1985) Arachidonic acid metabolism in the fetus and neonate. In: Biological protection with prostaglandins. .M.M. Cohen, ed. CRC Press, Boca Raton, Florida, pp. 27-44. Nishizuka, Y. (1984) Turnover of inositol phospholipids and signal transduction. Science, 225:1365-1369. Petkovich, M., N.J. Brand, A. Krust, and P. Chambon (1987) A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature, 330.444450. Randall, C.L., and Anton, R.F. (1984) Aspirin reduces alcohol-induced perinatal mortality and malformations in mice. Alcohol. Clin. Exp. Res., 8513-515. Roberts, A.B., M.D. Nichols, D.L. Newton, and M.B. Sporn (1979) In vitro metabolism of retinoic acid in hamster intestine and liver. J. Biol. Chem., 254: 6296-6302. Rugh, R. (1968) The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, Minn. pp. 209-212. Samokvszyn, V.M., B.F. Sloane, K.V. Honn, and L.J. Marnett (1984) Cooxidation of 13-cis-retinoic acid by
ASPIRIN REDUCES ISOTRETINOIN MALFORMATIONS prostaglandin H synthase. Biochem. Biophys. Res. Commun., 124:430-436. Smith, G.N., J. Patrick, K.R. Sinervo, and J.F. Brien (1991) Effects of ethanol exposure on the embryo-fetus: Experimental considerations, mechanisms, and the role of prostaglandins. Can. J. Physiol. Pharmacol., 69.550-569. Szabo, K.T. (1989) Congenital Malformations in Laboratory and Farm Animals. Academic Press, New York. D. 36. SAS (686)SAS/STAT Guide for Personal Computers. SAS Institute, Cary, NC. Satre M.A., J.D. Penner, and D.M. Kochhar (1989) Pharmacokinetic assessment of teratologically effective concentrations of a n endogenous retinoic acid metabolite. Teratology, 39:341-348. Sporn, M.B., and Roberts, A.B. (1983) Role of retinoids in differentiation and carcinogenesis. Cancer Res., 43: 3034-3040.
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Sulik, K.K., C.S. Cook, and W.S. Webster (1988) Teratogens and craniofacial malformations: Relationships to cell death. Development, 103r213-232. Vane, J., and R. Botting (1987) Inflammation and the mechanism of action of anti-inflammatory drugs. FASEB J., 1:89-96. Wells, P.G., Zubovits, J.T., Wong, S.T., Molinari, L.M., and Ali, S. (1989a) Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicylic acid, caffeic acid, and alpha-pheny1-Nt-butylnitrone: Implications for bioactivation by prostaglandin synthase. Toxicol. Appl. Pharmacol., 97: 192-202. Wells, P.G., M.K. Nagai, and G. Spano Greco (1989b) Inhibition of trimethadione and dimethadione teratogenicity by cyclooxygenase inhibitor acetylsalicylic acid: A unifying hypothesis for the teratologic effects of hydantoin anticonvulsants and structurally related compounds. Toxicol. Appl. Pharmacol., 97:406-414.
Inhibition of lsotretinoin Teratogenicity by Acetylsalicylic Acid Pretreatment in Mice STAN KUBOW School ofDietetics and Human Nutrition, Macdonald Campus of McGill University, Ste. Anne de Belleuue, PQ, Canada H9X ZCO
ABSTRACT Although isotretinoin (ITR) has been suggested to cause malformations via cytopathic effects on embryonic cells, the molecular mechanisms of ITR cytotoxicity in teratogenesis are not clear. Since ITR undergoes metabolism by prostaglandin synthase to a potentially cytotoxic peroxyl free radical, the possible role of prostaglandin synthase metabolism as a modulator of ITR teratogenicity was evaluated. Craniofacial and limb abnormalities were noted in fetuses on day 18.5 of gestation following administration of ITR to pregnant CD-1 mice in a three dose regime of 100 mglkg a t 4 hr intervals on day 10.5 of gestation (plug day = day 0.5 of gestation). Mice were also treated with acetylsalicylic acid (ASA), an irreversible inhibitor of the cyclooxygenase component of prostaglandin synthase, a t doses of 20 and 60 mglkg body weight 2 hr prior to each ITR dose. ASA pretreatment of mice receiving ITR treatment showed a dose-dependent decrease in the overall incidence of malformations, number of defects per fetus, and the incidence of specific craniofacial and limb defects. Equivalent doses of ASA given to control mice did not cause malformations or alter the incidence of resorptions. These results demonstrate that ASA is able t o ameliorate the teratogenic effects of ITR observed in fetal mice near term and indicate that prostaglandin metabolism could play a mechanistic role in ITR teratogenicity. Although a great deal is known concerning the teratological outcome of excessive retinoid intake, very little is known concerning its mechanism of teratogenicity. Specific influences of retinoids on cell death, cell proliferation, and cell membrane stability have been proposed as mechanisms to account the broad spectrum of retinoidinduced congenital defects encompassing the craniofacial, skeletal, cardiovascular, and nervous system regions. Recent theories on retinoid teratogenicity have focused on retinoid induction of excessive cellular necrosis in areas associated with programmed cell death (PCD) (Alles and Sulik, '89) and on specific cytotoxic effects of retinoids on cells of the chondrogenic cell lineage (Kochhar et al., '87). The molecular mechanisms by which retinoids may be cytotoxic are unclear but have been related to the action of retinoids to labilize lysosomal membranes, modify genetic information or induce glycosylation of proteins (Alles and Sulik, '89). Some workers have suggested 0 1992 WILEY-LISS,
INC.
that a recently discovered nuclear retinoic acid receptor may play a role in morphogenetic activities of retinoic acid and that retinoic acid may induce excessive cell death in zones of PCD in a similar fashion to glucocorticoids by modulating gene expression (Petkovich et al., '87; Alles and Sulik, '89). In exploring the molecular basis of the teratogenic activity of retinoids it is important to determine whether such compounds could also be further metabolized to more teratologically potent forms. These are several interconvertible forms of retinoids which undergo a number of biotransformations which may be toxicologically important including conjugation, esterification, decarboxylation, glucoronic acid conjugation, and side-chain cleavage (Lotan, '80; Satre et al., '89). Involvement of mixed func-
Received April 24, 1991; accepted September 10, 1991.
56
S. KUBOW
I Arachidonatel Fatty Acid Cyclooxyqenase ..
1
Hydroperoxidase
!
NADPH
I
Cytotoxicity Teratogenesis
O2
1
Fig. 1. Postulated co-oxidation of ITR to a teratogenic free radical by prostaglandin synthase and the metabolism of ITR by cytochromes P-450. Fatty acid cyclooxygenase and hydroperoxidase are the two catalytic components of prostaglandin synthase. PGGS, prostaglandin G2; PGH2, prostaglandin H2.
tion oxidase cytochromes P-450 (Roberts et al., '79) and prostaglandin synthase (Samokvszyn et al., '84) has been suggested in the enzymatic formation of retinoid metabolites (Fig. 1).Under physiological stimuli, the release of arachidonic acid from membrane phospholipids initiates prostaglandin synthesis. The cyclooxygenase component of prostaglandin synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2). PGG2 is reduced by the hydroperoxidase component of prostaglandin synthase to form prostaglandin PGH2 which is subsequently metabolized to a variety of prostaglandins and thromboxanes. The latter reaction requires reducing equivalents and a variety of drugs have been shown to serve as electron donors to the peroxidase leading to the formation of electrondeficient drug metabolites (Eling et al., '83). Co-oxidative metabolism of ITR by the hydroperoxidase component of prostaglandin synthase has been demonstrated to form a ITR peroxyl radical as well as 4hyroxyl-ITR and tretinoin (Samokvszyn et al., '84). Additionally, enzymatic formation of 4-hydroxy-ITR is also suggested via cytochromes P-450-mediated metabolism of ITR which would require NADPH and O2 (Frolik, '81; Hill and Struck, '83). Free radical formation may cause oxidant stress, initiate lipid peroxidation, andlor bind covalently to essential cellular macromolecules, thereby causing cytotoxic effects and
fetal abnormalities. Since embryos demonstrate high prostaglandin synthase activity (Mitchell et al., '85), embryonic prostaglandin synthase metabolism of ITR could contribute to teratogenicity by production of ITR-peroxyl free radicals and other potentially toxic ITR metabolites. Recent evidence has indicated that embryonic prostaglandin synthase-mediated bioactivation of phenytoin (Kubow and Wells, '89; Wells et al., '89a) and trimethadione (Wells et al., '89b) to reactive free radicals intermediates may be a mechanism of teratogenicity of these drugs. The potential role of prostaglandin synthase as a modulator of ITR teratogenicity was evaluated in the present study by pretreating pregnant CD-1 mice with acetylsalicylic acid (ASA, aspirin), a potent and irreversible inhibitor of the cyclooxygenase component of prostaglandin synthase (Flower et al., '85). Malformations were induced in mice by the administration of ITR in a three dose regime of 100 mg/kg at 4 hr intervals on day 10.5 of gestation and detected on day 18.5 of gestation. Craniofacia1 and limb malformations were noted as previously observed in mice exposed on day 10.5 to a similar regimen of ITR (Kochhar and Penner, '87). It was observed that mice receiving an ASA pretreatment showed a ASA dose-dependent reduction of ITRinduced fetal craniofacial and limb malformations in terms of overall incidence and mean number of defects per fetus. MATERIALS AND METHODS
ITR (isotretinoin, 13-cis retinoic acid) (Sigma Chemical Co., St. Louis, MO) was stored a t -20°C in the dark in a sealed, lightproof container under nitrogen. The drug was suspended in safflower oil and dosing solutions were sonicated and vortexed to obtain a uniform suspension. ASA (acetylsalicylic acid, aspirin) (Sigma Chemical Co., St. Louis, MO) was vortexed in a 0.9% saline under warm tap water until completely dissolved. Pregnant CD-1 (ICR) mice (Charles River Canada, Inc., St. Constant, Quebec) were housed in a environmentally controlled room under a 12-hr light-dark cycle (light cycle 7 A.M. to 7 P.M.) and were maintained on Purina Mouse Chow and tap water ad libitum throughout the whole experiment. A group of three virgin females were caged overnight with one male. Mating was confirmed
ASPIRIN REDUCES ISOTRETINOIN MALFORMATIONS
by the presence of a vaginal plug the next morning (8:30 A.M.) which was designated day 0.5 of gestation. Experiments were begun on the morning of gestational day 10.5 when pregnant females were randomly assigned to control and experimental groups. A previous study found that oral doses of 100 mg/kg ITR given three times to the same dam a t either 3 or 8 hr intervals on day 11 of pregnancy induced malformations in a majority of ICR mouse fetuses (Kochhar and Penner, '87). This dose regime was selected in the present investigation except that a 4 hr interval was selected between ITR doses. Control treatment groups included positive controls which received an intubation of 100 mg/kg ITR intragastrically on the morning (11 A.M.), afternoon (3 P.M.), and evening (7 P.M.) on day gestational 10.5. Experimental groups received ASA doses of 20 and 60 mgl kg i.p. 2 hours prior to each ITR dose. At the same time points as the ASA pretreatments in the experimental groups, the saline controls received i.p. injections of sterile physiological saline and the ASA controls received 0.3-0.5 ml safflower oil intragastrically and either 20 or 60 mg/kg ASA i.p. All animals were killed by cervical dislocation on gestational day 18.5. After laparotomy, each fetus was dissected free of the placenta and fetal membranes, weighed, sexed, and examined for external malformations. The number and weight of the resorptions, the number of implantation sites, the pup location, and the viability of the pups were recorded. Placentas were matched to the pups and weighed. Each litter was fixed in Carnoy's solution and examined for anomalies for the craniofacial region by free hand sectioning. Statistical analyses were performed on both an individual fetus and a litter basis. The incidence of specific malformation(s) was calculated as the number of fetuses with the malformation(s) divided by the number of fetuses. Analyses for comparison of incidence of malformed fetuses, types of anomalies and total resorptions were performed by chi-square test. For the purposes of calculating overall incidence of malformations, embryos with complex defects were counted once; for the mean number of defects per fetus, the number of abnormalities per fetus were counted separately. Therefore, the sum of abnormalities per fe-
57
tus may exceed one. A two-way analysis of variance was performed for the statistical comparison of litter size, fetal body weight, implantation sites, placental weights, resorptions per litter, and mean of defects per fetus using Statistical Analysis System for personal computers (SAS, '86). The significance of observed differences among the groups was evaluated by Tukey's test. A probability of P 5 0.05 was accepted as the minimal level of significance. RESULTS
ITR treatment under the chosen experimental conditions was characterized by a spectrum of severe craniofacial deformities and limb reduction abnormalities. The most prominent malformations in the present experiment were cleft palate (72%), micrognathia (16%), forelimb (8%) and hindlimb defects (5%),and kinky and stub tail (6%). The majority of surviving fetuses exposed to ITR treatment had a t least one of the above malformations (79%). Cleft palate and reduction defects of the anterior and posterior limbs are typically observed in mouse fetuses exposed to retinoids on gestational days 10.5 or 11(Kochhar, '73). The high incidence of cleft palate obtained in mice treated with the three dose regimen of 100 mg/kg ITR over a 8 hr time period was similar to that observed with a similar dosing treatment by Kochhar and Penner ('87) in ITR-exposed mice (88%). There were, however, relatively fewer fetuses with a reduction of the limbs in the present experiment than was observed in the Kochhar and Penner ('87) study (> 50%). Since the timing of the ITR treatments and the mouse strain used were identical in the two studies, this difference is likely due to the shorter time interval of 3 hr between doses used by Kochhar and Penner ('87) as compared to the 4 hr interval used in the present study. Kochhar and Penner ('87) showed a much lower frequency of limb defects in mice when longer time intervals were used between the three 100 mg/kg doses of ITR as compared to a 3 hr time interval. Table 1 shows the effects of the dosing agents on gestational and developmental data as observed on day 18 of pregnancy. No effect of ITR, ASA, or ITR plus ASA was observed on implantation sites or number of resorptions on a per litter basis (Table 1). Treatment with 60 mg/kg ASA in the pres-
58
S. KUBOW
TABLE I ,Effects on pregnancy outcome after administration of ITR, ASA, and vehicle doses given to CD-1 mice three times at 4 hr intervals on gestational day 10.5l ITR (100 mg/kg) Safflower oil control ASA (mgikg) Vehicle 20 60 Vehicle 20 60 Litters (n) 19 23 12 9 6 7 Irnplantati~n~,~ sitesilitter 12.10 ? 0.554 11.26 ? 0.68 13.92 ? 0.40 13.44 ? 0.80 12.17 ? 1.68 12.43 ? 0.57 Live fetusesilitter 11.16 ? 0.47ab 10.04 ? 0.65" 12.67 f 0.38b 12.11 ? 0.74"b 10.83 f 1.7OUb12.29 f 0.68"' Fetal weight (g) 1.42 f 0.01" 1.32 f O.Olb 1.37 2 0.01' 1.36 ? 0.01' 1.47 ? O.Old 1.37 2 0.01' Placental weight ( g ) 0.11 t 0.002" 0.11 ? 0.021" 0.11 ? 0.019" 0.10 f 0.017' 0.10 5 0.017b 0.09 ? 0.016b Resorptions/litter' 0.07 f 0.02 0.10 f 0.02 0.9 ? 0.02 0.10 t 0.03 0.12 ? 0.03 0.01 f 0.03 Total resorptions' 7.83" 10.81" 8.98" 9.92" 10.96" 1.15' 'ITR, isotretinoin; ASA, acetylsalicylic acid. 'Plug day = 0.5 of gestation. Fetuses examined on day 18.5 of gestation. 3Means not sharing a common superscript are significantly different at P 4Mean -t S.E.M. 'Values based on implantations
ence of ITR resulted in an increase in the number of live fetuses per litter when compared to 20 mglkg ASA plus ITR. Treatment with 20 mglkg ASA resulted in an increase in fetal weight as compared to saline-control but ASA treatments caused a decrease in fetal weight in the presence of ITR. Administration of ITR, however, was associated with an increased fetal weight relative to saline controls (Table 1).ITR treatment also caused an increase in placental weight relative to the ASA controls. The data for the saline-treated mice (Table 1) show a low background incidence of resorptions with a normal number of implantation sites, fetal and placental weights, and live fetuses per litter consistent with other published reports for mice (Mehanny et al., '91; Lofberg et al., '90; Kochhar and Penner, '87). ASA pretreatment alone did not cause any observable fetal toxicity as indicated by the low incidence of resorptions, no decrease in mean fetal body weight compared to salinetreated controls, and only two malformed fetuses observed with the 20 and 60 mg/kg dose treatments of ASA alone (Table 1).The single malformed fetus observed following 60 mglkg ASA treatment exhibited micrognathia, while a hindlimb defect was observed in the sole malformed fetus in the 20 mgkg ASA group. In fact, percent total resorptions actually decreased in the 60 mgl kg ASA-exposed fetuses as compared to saline controls. The lack of teratogenic effect of ASA is likely to be dose-related since approximately 400-500 mglkg ASA is needed to cause teratogenic effects in the mouse (Szabo, '89; Larsson et al., '63). Pretreatment with ASA produced a reduction in the incidence of ITR-induced
5
0.05.
craniofacial defects (cleft palate and micrognathia) and limb defects (hindlimb and tail) that was dose dependent (Figs. 2,3). In contrast, no significant effect of ASA was observed on ITR-induced forelimb defects (Fig. 3). The incidences of malformed fetuses (Fig. 4) and the average number of abnormalities per fetus (see Fig. 5) induced by ITR were also significantly reduced by ASA pretreatment. In terms of a percentage reduction of ITR-induced abnormalities, pretreatment with ASA caused a 75% reduction of posterior defects (hindlimb and tail) and a 75%reduction in the mean number of abnormalities per fetus in the posterior region (Figs. 4, 5). The anterior region was less sensitive to the protective action of ASA although a 26% reduction in total anterior defects (craniofacial and forelimb) and a 28% reduction in the number of defects per fetus in the anterior region was observed (Figs. 4, 5). DISCUSSION
These findings demonstrate that ASA pretreatment, when administered 2 hr prior to ITR, reduces the incidence of developmental malformations of ITR in a dose-dependent fashion. Pretreatment with the 60 mg/kg dose of ASA resulted in decreases in overall incidences of ITR-induced craniofacia1 (cleft palate and micrognathia) and limb (forelimb, hindlimb, and tail) defects. Moreover, the presence of specific abnormalities in ASA-pretreated fetuses exposed to ITR were accompanied by a diminished severity so that fewer abnormalities per fetus were observed than in ITR fetuses. As ASA irreversibly inhibits the activity of prostaglandin synthase (Flower et al.,
59
ASPIRIN REDUCES ISOTRETINOIN MALFORMATIONS (1131
(nbfetuses ITR
P e r
60
[7
ITR.20
r;Xl
ITR+BO mg/Kg A S A
mg/Kg ASA
C
e n t
40
20
* 521
n Cleft Palate
Mi crag nat hia
Fig. 2. Effects of acetylsalicylic acid (ASA) pretreatment on isotretinoin (1TR)-induced craniofacial defects in the CD-1 mouse. ITR, 100 mg/kg, was given intragastrically in three consecutive doses at 4 hr intervals to pregnant CD-1 mice on gestational day 10.5. ASA was administered i.p. 2 hr prior to each ITR dose. Fetuses were examined on day 18.5 of gestation. *Significantly different from ITR control, P c 0.05.
(nbfetuses ITR
P e r
0
8
I T R * 2 0 mp/Kg ASA
I T R * 8 0 m p l K g ASA
C
e
7
6
F
r e
4
q
*
U
e n
c
(152)
2
Y n "
Forelimb Defects
Hindlirnb Defects
Tail Defects
Fig. 3. Effects of acetylsalicylic acid (ASA) pretreatment on isotretinoin (1TR)-induced limb defects in the CD-1 mouse. ITR, 100 mgkg, was given intragastrically in three consecutive doses at 4 h r intervals to pregnant CD-1 mice on gestational day 10.5. ASA was administered i.p. 2 hr prior to each ITR dose. Fetuses were examined on day 18.5 of gestation. *Significantly different from ITR control, P 5 0.05.
1985), these results indicate that prostaglandin synthase metabolism plays a mechanistic role in ITR teratogenicity. The halflife of ASA is 15 min and the salicylate metabolite has a half-life of 2-3 hr (Flower et al., '85). The 2 hr time interval between the ASA and ITR treatments used in the present study would thus permit the major-
ity of the ASA and salicylate metabolite to be eliminated prior to ITR administration. ASA pretreatment in the mouse has previously been demonstrated to decrease the teratogenic effects of a number of drugs including alcohol (Randall and Anton, '841, phenytoin (Wells et al., '89a), and trimethadione (Wells et al., '89b). The mecha-
60
S. KUBOW Malformed Fetuses 100
(nbfetuses ITR
P e r
0
80
I T R * 2 0 rnglKg ASA
ITR*dO rnglKg ASA
C
e n t
60
F r e
40
9 U
e n C
20
Y
0
Overall Malformed
Anterior Defects
Posterior Defects
Fig 4 Effects of acetylsalicylic acid (ASA) pretreatment on the incidence of isotretinoin (1TR)-induced malformed fetuses in the CD-1 mouse ITR, 100 mgkg, was given intragastrically in three consecutive doses at 4 hr intervals to pregnant CD-1 mice on gestational day 10 5 ASA was administered i p 2 hr prior to each ITR dose. Fetuses were examined on day 18.5 of gestation *Significantly different from ITR control, P c 0 05 (nkfetuses
ITR.20 mglKg ASA I T R * 6 0 mglKg ASA
Overal I
Anterior
Posterior
Defects
Defects
Defects
Fig 5 Effects of acetylsalicylic acid (ASA) pretreatment on the number of defects per fetus induced by isotretinoin (ITR) in the CD-1 mouse ITR, 100 mg/kg, was given intragastrically in three consecutive doses at 4 hr intervals to pregnant CD-1 mice on gestational day 10.5 ASA was administered 1 p 2 hr prior to each ITR dose. Fetuses were examined on day 18 5 of gestation Values are expressed as means + SE *Significantly different from ITR control, P c 005
nism of the ASA inhibition of drug-induced teratogenicity has been proposed to be due to either inhibition of prostaglandin synthase metabolism of these drugs to free radicals or to modulation of embryonic and maternal prostaglandin levels. In the present study, the anterior regions
of mouse fetuses demonstrated enhanced susceptibility to ITR-induced defects as compared to malformations induced by ITR in the posterior regions (Figs. 4, 5). A similar differential sensitivity of forelimb and hindlimb regions to malformations induced by retinoic acid has been previously noted in
ASPIRIN REDUCES ISOTRETINOIN MALFORMATIONS
DBA/2J mouse fetuses (Kochhar, '73). Forelimbs were observed t o be more sensitive to retinoic acid on day 12 than on day 13 while the reverse was true for hindlimbs. The more dramatic reduction in the incidence and severity of ITR-induced posterior (hindlimb and tail) defects by ASA pretreatment as compared to ITR-induced anterior (craniofacial and forelimb) defects could be explained by the developmental stage of these embryonic tissues with respect to the time of dosing of ITR and ASA. The posterior regions of the mouse embryo which contain the tail and hindlimb buds are developmentally delayed by approximately 24 hr at gestational day 10 as compared to the anterior regions which include the forelimb buds and palatal shelves (Rugh, '68). The delay in hindlimb and tail development relative to the anterior region at the time when the mice received the ITR and ASA treatments is likely to have decreased the sensitivity of the posterior regions to ITR. This developmental delay may have also permitted ASA to have more time to exert greater protective effects and allowed normal development of the posterior regions to be completed. Alternatively or in addition, it is possible that the differential protective effects of ASA could be accounted for by variations in the mechanisms of ITR teratogenesis with developmental stage, region, or tissue site. The limited protective effects of ASA against ITR teratogenicity may involve a number of mechanisms. Enzymatic bioactivation of ITR to a reactive oxygen-centered free radical has been demonstrated (Samokvszyn et al., '84) and is a possible means by which prostaglandin synthase metabolism could contribute to ITR teratogenicity. The toxicity of oxygen radicals t o embryonic tissues has been noted in embryo culture studies. For example, rat embryos exposed to xanthine oxidase-generated oxygen free radicals in culture showed severe malformations that were attributed to excessive cellular death and cell cycle delay (Jenkinson et al., '86). Since excessive cell death has been proposed as a mechanism of teratogenesis for ITR administration (Sulik et al., "9,the possibility exists that cytotoxic effects exerted by oxygen free radicals produced from embryonic prostaglandin synthase metabolism of ITR could be a mechanism of ITR teratogenesis. Support for the formation of ITR-derived cytotoxic
61
free radicals has been obtained from recent work which has indicated a relationship between ITR-induced generation of toxic oxygen radical species and depressed neural crest cell viability and proliferation observed upon exposure of isolated chick neural crest cells to ITR (Davis et al., '90). The addition of free radical scavenging enzymes, superoxide dismutase, and catalase to the culture medium increased cell viability and decreased concentrations of oxygen radicals. Additionally, ASA could decrease ITR teratogenicity by inhibiting maternal generation of 4-oxo-ITR, a potent teratogenic metabolite that may arise from prostaglandin synthase metabolism of ITR (Samokvszyn et al., '84); 4-oxo-ITR is about four-fold more effective as a teratogen in mice than is the ITR parent compound (Kochhar and Penner, '87). An inhibition of prostaglandin synthase biotransformation of ITR to 4-0x0ITR by ASA in maternal tissues could be of teratological significance since the enhanced potency of 4-oxo-ITR has been indicated to be due t o more efficient placental transfer of 4-oxo-ITR relative to ITR (Kochhar and Penner, '87; Creech Kraft et al., '87). One further potential mechanism of ASA protection could involve the pathway proposed by Sporn and Roberts ('83) in which retinoids control gene expression via interactions with protein kinases. Control of gene expression via protein kinases has been proposed to initiate a receptor cascade system which integrates phospholipid degradation, prostaglandins, cyclic AMP, cyclic GMP, calmodulin, and calcium (Nishizuka, '84). Since a regulatory role for prostaglandin E, has been suggested with the early events of chondrogenesis (Biddulph et al., '84) and palatal development (Jones and Greene, '861, a toxic imbalance of prostaglandins induced by ITR may be in part responsible for the causation of limb defects and cleft palate. Inhibition of prostaglandin metabolism by ASA could thus be of importance in terms of its protective action against ITR malformations. It may also be possible that ASA was protective via other mechanisms aside from its action on prostaglandin synthase. Nevertheless, research prompted by initial studies demonstrating protective effects of ASA pretreatment against particular drug-induced malformations have supported a role of prostaglandin metabolism in these toxic-
62
S. KUBOW
ities. For example, the demonstration of ASA protection against ethanol teratogenicity (Randall and Anton, '84)has been a catalyst for a wide body of research supporting a role of prostaglandins in ethanol teratogenicity (Smith et al., '91). Additionally, a role of prostaglandin synthase in phenytoin metabolism and teratogenicity, suggested by the protective action of ASA pretreatment (Wells et al., '891, has been supported by more in-depth mechanistic studies (Kubow and Wells, '89). It should also be noted that the therapeutic benefits produced by ASA and its salicylate metabolites have thus far been attributed t o inhibition of prostaglandin synthase metabolism (Vane and Botting, '87). Moreover, as opposed to other non-steroidal anti-inflammatory drugs, ASA does not inhibit lipoxygenase activity (Vane and Botting, '87) and thus is more specific in its inhibitory action on prostaglandin synthase. While the mechanism for the protective action of ASA on ITR teratogenicity requires further exploration, the data warrants consideration of the hypothesis that ITR induces malformations by undergoing metabolic transformation by prostaglandin synthase to more teratogenic metabolites and by causing alterations of prostaglandin metabolism. ACKNOWLEDGMENTS
The author would like t o express his grateful appreciation to Susan Smith for her expert technical assistance. REFERENCES Alles, A.J., and K.K. Sulik (1989) Retinoic-acid-induced limb-reduction defects: Perturbation of zones of programmed cell death as a pathogenic mechanism. Teratology, 40:163-171. Biddulph, D.M., L.M. Sawyer, and W.P. Smales (1984) Chondrogenesis of chick limb mesenchyme in vitro. Exp. Cell Res., 153:270-274. Creech Kraft, J., D.M. Kochhar, W.J. Scott, and H. Nau (1987) Low teratogenicity of 13-cis retinoid acid (isotretinoin) in the mouse corresponds to low embryo concentrations during organogenesis: Comparison to all-trans isomer. Toxicol. Appl. Pharmacol., 87.474482. Davis, W.L., L.A. Crawford, O.J. Cooper, G.R. Farmer, D. Thomas, and B.L. Freeman (1990) Generation of radical oxygen species by neural crest cell treated in vitro with isotretinoin and 4-0x0-isotretinoin.J . Craniofac. Genet. Dev. Biol., 10:295-310. Eling, T.E., J.A. Boyd, G.A. Reed, R.P. Mason, and K. Sivarajah (1983) Xenobiotic metabolism by prostaglandin endoperoxide synthetase. Drug Metab. Rev., 14t1023-1053. Flower, R.J., S. Moneada, and J.R. Vane (1985) Anal-
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