TERATOLOGY 46~293-300 (1992)
Studies of the Role of Ischemia/Reperfusion and Superoxide Anion Radical Production in the Teratogenicity of Cocaine ALAN G. FANTEL, CHARLES V. BARBER, MARY B. CARDA, RUSLAN W. TUMBIC, AND B. MACKLER Central Laboratory for Human Embryology, Child Development and Mental Retardation Center, Department of Pediatrics, University of Washington, Seattle, Washington 98195
ABSTRACT
The administration of multiple doses of cocaine on a single day during late gestation is teratogenic in rats in which hind limb ectrodactyly is a major finding (Webster and Brown-Woodman, '90). We have previously hypothesized that these limb malformations result from the generation of reactive oxygen species during the process of ischemiaireperfusion in vivo. In order to study the direct effects of cocaine versus the aberrant oxygenation it may induce, we have developed a system for culturing rat embryos between days 14 and 15 of gestation. Growth and development of cultured embryos are comparable to that of in vivo controls. Exposure to normoxia (95% 0,) with or without cocaine failed to induce limb malformations and exposure to a single long period of hypoxia (20% 0,) only reduced limb growth in the anterior-posterior axis. By contrast, embryos receiving multiple brief exposures to hypoxia developed a significant incidence of hind limb ectrodactyly that appeared indistinguishable from that induced by cocaine in vivo. By incubating day 14 embryos in a nitroblue tetrazolium derivative, l-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT),it was shown that superoxide anion radical appears in the digital rays following two episodes of reperfusion. Little reaction product was seen under the other conditions. Finally, mitochondria1 electron transport particles prepared from teratogenically sensitive limb buds spontaneously "leak' electrons to form superoxide anion radical whereas those from insensitive heart fail to do so. We propose that cocaine and other exposures that can transiently reduce conceptal oxygenation during late gestation are teratogenic by virtue of their capacity to induce ischemia/reperfusion. This process generates toxic oxygen radicals that can overwhelm the immature antioxidant defenses. b 1992 Wiley-Liss, Inc.
It has been proposed recently that hypoxiaireperfusion plays a major role in the late gestational teratogenicity of cocaine (Fantel et al., '92). Because common dysmorphogenic features have been found to result from several experimental teratogenic exposures including cocaine (Webster and Brown-Woodman, 'go), uterine vascular clamping (Webster et al., '87; Leist and Grauweiler, '74), and fetal intraperitoneal epinephrine (Jost et al., '691, a common teratogenic pathway involving vascular disruption and conceptal hypoxia has been proposed (Webster and Brown-Woodman, '90; Brent, '90; Plessinger and Woods, '91). Malformations of the distal portions of the limbs 0 1992 WILEY-LISS. INC.
have been a most frequent finding. The term "acroblapsie" has been used to describe distal limb necrosis secondary to hemorrhagic lesions (Jost, '53). A vascular disruptive sequence involving gut and urogenital tissue has also been proposed as the basis of malformations seen in association with prenatal cocaine exposure in humans (Chavez et al., '89; Hoyme et al., '90). Although each of these exposures can induce conceptal hy-
Received March 5, 1992; accepted May 19. 1992. Address reprint requests to Alan G. Fantel, Department of Pediatrics, RD-20, University of Washington, Seattle, WA 98195.
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poxia, we have proposed a n alternative mechanistic model for their developmental toxicity. The proposed model of embryotoxicity states that reactive oxygen species (ROS), most likely generated during periods of hypoxiaireperfusion, are the common ultimate toxicants (Fantel et al., '92). It is well recognized that cocaine has potent vasoconstrictive activity t h a t can reduce uterine perfusion in gravid ewes (Moore, '86) resulting in decreased fetal oxygenation (Woods e t al., '87; Burchfield e t al., '91). Key features of late embryos or fetuses that could predispose them to the generation of toxic ROS include their increasing hemoglobin content (Thomas and Yoffey, '62) and increasing oxygenation. Evidence of changing oxygenation comes from demonstrations that oxygen concentrations that are toxic to early postimplantation r a t embryos in vitro are required at later stages (New, '78; Miki et al., '88). Additionally, prenatal antioxidant activities tend to be extremely low prior to the perinatal period and may in fact be developmentally regulated (Allen and Balin, '89; Frank, '91). These factors could place late gestational conceptuses a t heightened risk for oxidative attack on proteins, nucleic acids, or lipid membranes. Hypoxialreperfusion, first defined by Hearse et al. ('73), involves the generation of ROS shortly after the resumption of circulation in transiently ischemic tissues. This generation has been detected in most organs studied in vivo as well as in tissues and cells subject to hypoxiaireoxygenation in vitro. It has been associated with numerous disease states including myocardial arhythmias, intestinal ischemia, neonatal necrotizing enterocolitis, pancreatitis, hepatitis, regional stroke, and others. In order to test the role of hypoxiaireperfusion in the teratogenicity of cocaine, we developed a system for culturing rat embryos between day 14 and 15 of gestation. During that time, embryos were exposed twice to cocaine, hypoxia, or the combination of the two. Controls were cultured under normoxic conditions while other embryos were exposed to a single long period of hypoxia. Because malformations comparable to those caused by cocaine in vivo were observed only in embryos exposed to repeated hypoxia and reoxygenation, we assessed the capacity of embryonic tissues to generate ROS (superoxide anion radical)
under comparable conditions. Finally, we compared the superoxide-generating capacities of electron transport particles (ETP) prepared from the mitochondria of tissues sensitive (limb bud) and insensitive (heart) to hypoxialreoxygenation embryotoxicity. MATERIALS AND METHODS
Embryo cultures Primigravid Sprague-Dawley rats were obtained from a commercial vendor (Tyler Laboratories, Bellevue, WA). Laparotomy was performed on the morning of gestational day 14 (gd-14) under halothane, nitrous oxide, and oxygen. Embryos were explanted in Hanks' balanced salt solution (HBSS) where they were exteriorized from the yolk sac and amniotic membranes which remained attached. Immediately after explantation, each was placed in prewarmed medium that was continuously flushed with 95% o,, 5% co,. Embryos were grown in flat, 650-ml tissue culture flasks in a Dubnoff metabolic incubator a t 37°C with gentle shaking. The cultures were continuously flushed with a water-saturated 0, plus CO, mixture (955). This gas mixture was designated "normoxia." The medium, consisted of 120 ml of Earle's balanced salt solution (EBSS) plus heat-inactivated, immediately centrifuged r a t serum (3:1), penicillin (12,000 U), and streptomycin (12,000 pg). When all embryos were explanted, 10 were randomly placed in each of the culture flasks and cultured under the control conditions described above for 2 hr. At the end of this time, each bottle was subject to one of the experimental or control conditions outlined below. 1. Two 30-min exposures to cocaine HC1 (25 pM) in normoxia separated by 30 min in control medium (EBSS). 2. Two 30-minute hypoxic exposures (02, CO,, N,, 20:5:75) separated by 30 min in normoxic control medium. 3. Two simultaneous 30-min exposures to cocaine (as group 1)and hypoxia (as group 2) separated by 30 min in normoxic control medium. 4. A single 90-min hypoxic exposure. Control embryos were subject to all manipulations in normoxia and control medium including flask changes. All media were replaced after the first 8 h r of culture. Following exposures, embryos were cultured for a n additional 18 hr.
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Whenever embryos were moved between flasks or when medium was replaced, embryos were checked for viability as determined by the presence of a n active heart beat and nonviable ones were removed and examined. At the end of the culture period, viability was again assessed and although abnormalities were recorded for all embryos, growth and protein content were not assessed in nonviable ones. Greatest length of viable embryos was measured with a n optical micrometer and protein was determined by the method of Lowry e t al. ('51). After morphology and growth were determined, hind limbs were removed, traced with the aid of a projecting microscope, and photographed. In order to determine the adequacy of the culture procedures, several dams were sacrificed on day 15 in order to obtain normal embryonic growth parameters for comparative purposes.
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from embryos and photographed in transmitted light.
Superoxide generation by mitochondria1 electron transport particles ETP Electron transport particles were prepared from limb buds, and embryonic and adult hearts. After the tissues were washed free of blood in HBSS, they were suspended in cold 5% sucrose solution. All procedures were performed a t 5°C. The tissues were homogenized at medium speed for 3 min with a motor driven Teflon pestle in a glass homogenizer immersed in ice. The tissue suspensions were then centrifuged for 30 rnin at 3,OOOg in a Sorvall centrifuge. The pellet was discarded and the supernatant suspension recentrifuged at 140,OOOgfor 45 rnin in a Spinco ultracentrafuge. The resulting pellet was resuspended in 3 ml of 5% sucrose solution. One milliliter of the suspension was retained on ice for enzymatic assays Embryonic superoxide generation and the remaining 2 ml were recentrifuged Experiments were conducted to confirm for 45 min at 140,OOOg. The pellet was susthe presence and location of superoxide an- pended in HBSS and the suspension was ion radical under experimental conditions. again centrifuged for 45 min a t 140,OOOg to Embryo incubations were performed under remove fetal hemoglobin. The pellet was reconditions identical to those described above suspended in HBSS and centrifuged a s with the exceptions that rat serum was re- above once more. The final pellet was susplaced in the culture medium with 0.7% bo- pended to the original volume of 2 ml with a vine serum albumin and exposures to co- solution of sucrose (0.25 M), Tris (0.02 M), caine and cocaine plus hypoxia were and EDTA ( 5 mM), pH 7.5 (STY solution). omitted. These two exposures were excluded The STV suspensions of ETP were free of because the former failed to induce malfor- hemoglobin and were used for determinamations comparable to those that develop tion of cytochrome b concentrations in the in vivo and no embryos survived the latter. preparations from difference spectra (dithioDay 14 rat embryos were explanted and nite reduced minus oxidized). Determinaincubated in medium containing MTT (1- tions were performed and recorded in a n [4,5-dimethylthiazol-2-yll-2,5-diphenyltetraAminco DW-2 recording spectrophotometer zolium bromide, Sigma, 125 kM) under nor- at 562-572 nm (Chance and Williams, '55). moxic conditions for 30 min. MTT, which Assays of superoxide anion radical generwas added to culture medium in 50 p1 ation were performed in two ways. The first DMSO is a dimethyl derivative of nitroblue measured the maximum capacity of ETP to tetrazolium, a n indicator of superoxide an- generate superoxide from the oxidation of ion radical (Seidler, '91). It easily crosses NADH. Superoxide production was detercell membranes, reacting avidly with super- mined by calculating the difference in the oxide anion radical to form a n insoluble rate of cytochrome c reduction before and blue precipitate, formazan. Following the after the addition of superoxide dismutase initial 30-min incubation, embryos were ex- (SOD) (Mackler et al., '85). The complete asposed to one of the hypoxia or control proto- say contained 0.2 ml of 0.2 M potassium cols described above. After 150 min, em- phosphate buffer (pH 7.5), 0.1 ml of a 1% bryos were removed from the culture flasks solution of cytochrome c, 10 mM sodium and placed in petri dishes on ice. They were azide, 0.01 ml of a 1% solution of NADH, examined under a stereoscopic dissecting and sufficient ETP and water to a final volmicroscope where the presence and location ume of 1 ml. Sodium axide was used to inof blue color was recorded and embryos pho- hibit cytochrome oxidase activity. Determitographed. Finally, the limbs were removed nations were made spectrophotometrically
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TABLE 1. Growth mrameters of live ernbrvos Crown rump (mm) L SEM Controls 1 Cocaine x 2 2 Hypoxia x 2 3Coc;hyp x 2 4 Hypoxia x 1 In viva day 14 In vivo day 15
12.9 13.6
13.0
-
TABLE 2. Survival and dysrnorphology
Protein (mg) SEM
*
0.6 0.4 0.7
6.74 6.17 6.09 -
-
0.8
6.50 5.22 6.07
0.57 0.36 0.65
-
14.0 11.4
0.1
13.8
0.6
0.65 0.44 0.47
Survival Controls 1Cocaine (2 x ) 2 Hypoxia (2 x ) 3 Coc+hyp ( 2 x ) 4 Hypoxia (1X )
N
(7%)
74 21 37 15 74
73
Defects Tail Limbs (%) 5
(%a)
52 27
38
0 0
22
8l
0
53
40
4
0 112
P values by Fisher's exact test. 'Ectrodactyly (P = 0.042). 'Reduced growth (I' = 0.0049).
a t 550 nm and 37°C in a recording Gilford spectrophotometer. The second assay was performed in an oxygen polarograph maintained at 37°C (Estabrook, '67). The assay contained 0.35 ml of 0.2 M potassium phosphate buffer (pH 7.5), 0.10 ml of a 1%NADH solution, ETP, and sufficient water to make a final volume of 1.8 ml. Superoxide generation by uninhibited ETP, calculated from the change in the oxygen disappearance rate after the addition of SOD, represented the amount of superoxide normally produced by the preparation. After the addition of SOD, the rate of oxygen disappearance would be expected to be reduced by 1 pmol of oxygen for every 2 pmol of superoxide produced due t o the release of oxygen by the dismutation of superoxide.
GROUP IV
RESULTS
The effects of the various exposures on embryonic growth and development are presented in Table 1 along with comparable data from in vivo and in vitro controls. There were no significant differences in the average greatest length or protein content in any group and these features were comparable to those of in vivo controls. As can be seen in Table 2, viability was decreased in all experimental groups, dropping from 73% in controls to 52% in group 1, 40% in group 4,27%in group 2, and 0% in group 3. Defects of the distal tail, consisting of kinks, bulbs, or blunt ends were observed in all cultured embryos and were highest in those exposure to cocaine. Tracings of representative hind limbs are presented in Figure 1. Growth reduction in the anterior-posterior plane of the hind limb was seen in 29% of group 4 embryos exposed to a single 90-min episode of hypoxia. Ectrodactyly of the hind limb was observed only in group 2 embryos, exposed t o two 30-min episodes of hypoxia (8%, P = 0.04). The right side was affected
GROUP I I
Fig. 1. Tracings of hind limbs of rat embryos cultured between gestational days 14 and 15. Approximate magnification 110 X . Top row: Embryo cultured under normoxic conditions (95%OJ. Middle row: Embryos exposed to a single 90-min period of hypoxia (20% 02). Remainder of culture period was in normoxic conditions. Bottom row: Embryos exposed to two 30-min periods of hypoxia separated by lhr of normoxia. Remainder of culture period was in normoxic conditions.
in all instances with one case of bilateral ectrodactyly. Photographs of representative limbs are presented in Figure 2. Experiments employing MTT permitted visual localization of superoxide anion radical generation. Because of its extreme localization, it was not possible to quantify superoxide production and relate it to a given volume of tissue or protein. Blue staining from formazan was apparent in the
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Fig. 2. Photographs of hind limbs of embryos exposed to two 30-min periods of hypoxia separated by 1 hr of normoxia.
yolk sacs of all groups. In the embryos per se, it could be visualized only in the limb buds of embryos subject to repeated reoxygenation. Photographs of representative limbs are presented in Figure 3. The highest concentration appeared to occurred in hind limbs, with the earliest appearance on the right side.
Table 3 presents the results of studies of superoxide anion radical generation by ETP prepared from embryonic limb bud, and embryonic and adult heart. Although protein content of the preparations is presented, it could not be used to calculate enzyme activities since variable amounts of conceptal hemoglobin were present in all preparations of
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TABLE 3 . Superoxide generation by mitochondria1 ETP (average i- SEMI h i d e inhibited Uninhibited (pmoUmini (pmollminl Preparation nmol cyt b ) nmol cyt 6 ) Adult heart 0.332 ? 0.058 0 Heart (GD16) 0.528 5 0.144 O1 Heart (GD14) 0.628 -t 0.202 Limb bud (GD16) 0.754 i- 0.300 0.092 2 0.034 [35% of total 0,1 Limb bud (GD14) 0.506 5 0.114 0.0952 [22% of total O,] ‘Not done. ‘One assay
significant (P = 0.1). No superoxide could be detected in uninhibited (azide-free) preparations of adult or embryonic heart ETP. In contrast, ETP from gd-14 or -16 limb buds readily produced superoxide at rates of approximately 0.1 ~mol/min/nmol of cytochrome b. This represented 22 and 35%, respectively, of the total oxygen utilization by the preparations. DISCUSSION
Exposure of gd-14 r a t embryos to two 30min periods of hypoxia in vitro induced a significant incidence of limb abnormalities that appeared to closely model those resulting from 2 exposures to cocaine in vivo and other inducers of “acroblapsie” (secondary hind limb ectrodactyly). In contrast to the effect of episodic hypoxia, a single hypoxic exposure of longer duration or direct exposures to cocaine in vitro failed to elicit comparable limb effects. The combination of cocaine and hypoxia was embryolethal in all instances and embryos appear to have died prior to manifesting malformations. These initial findings led us to discount a primary Fig. 3. Photographs of limb buds of day 14 embryos cultured in the presence of 1-[4,5-dimethylthiazo1-2-y1]- role for isolated hypoxia in the genesis of 2,5-diphenyltetrazolium bromide (MTT). Fore limbs are these malformations. I t was proposed inpresented at the top and hind limbs at the bottom of the stead that developing limbs were insulted photographs. Note the presence of reaction product in the reoxygenated embryos indicating generation of su- by reactive oxygen species generated by hypoxialreoxygenation in vitro that modeled peroxide anion radical in the digital rays. ischemialreperfusion in vivo. Support for this hypothesis included pubembryonic tissues. Rates of production of su- lished evidence, discussed previously, that peroxide were, therefore, expressed relative coneceptal oxygenation and hemoglobin to the cytochrome b content of the prepara- content increase with proceeding gestation. tions since cytochrome b is a stoichiometric Molecular oxygen is required for the generconstituent and specific component of ETP. ation of ROS; and iron (or another transiThe maximal capacity to generate superox- tion metal such as copper) is required for the ide was greater in azide-inhibited ETP pre- reduction of hydrogen peroxide to the prepared from embryonic tissues than from sumed ultimate toxicant, hydroxyl radical. adult heart, although the difference was not Significantly, studies of the teratogenicity
COCAINE TERATOGENICITY AND ISCHEMINREPERFUSION
of cocaine during early organogenesis, when iron and oxygen concentrations are presumably lower in the rat have been largely negative (Fantel and MacPhail, ’82; Church et al., ’88; Hutchings et al., ’89). The apparent nonteratogenicity of single exposures to cocaine (Webster and Brown-Woodman, ’90) was also considered supportive of this hypothesis since Clark and Gewertz (’91) have shown that repeated hypoxiaireperfusion results in generation of greater amounts of ROS than do single episodes. In order to strengthen the proposed role of conceptal ischemiaireperfusion in the genesis of the limb malformations induced by cocaine and the other exposures, it was necessary to demonstrate that (1)the conditions that result in these defects are associated with increased generation of ROS and (2) sensitive tissues generate greater amounts of ROS than insensitive ones under experimental conditions. Alternatively, the antioxidant activities of sensitive tissues could be relatively low, a possibility not necessarily mutually exclusive with (2) and one not yet investigated. The results of experiments designed to examine these issues are consistent with the proposed hypothesis. Studies with MTT clearly indicated that increased amounts of superoxide anion radical are generated when embryos are exposed to repeated cycles of hypoxia and reoxygenation. Furthermore, the greatest amounts of superoxide appeared in precisely those tissues that display maximal teratogenic sensitivity, i.e., the distal limb buds. Reaction product appears to be concentrated in the digital rays, and it is the digits that have been shown to be most affected by the in vivo exposures. Although both mitochondria and cytosol have been proposed as sites of superoxide generation during ischemiaireperfusion in adults, studies of embryonic tissue have not been performed. As demonstrated above, electron transport particles prepared from gd-14 or gd-16 limb buds show an unusually large capacity to spontaneously reduce molecular oxygen to superoxide. By contrast, no superoxide was produced by heart ETP unless the NADH oxidase activity of the preparations was inhibited by the addition of azide. Azide acts by inhibiting electron transport at cytochrome oxidase, thus maintaining the electron transport chain in a reduced state upstream and favoring electron “leakage” to molecular oxygen.
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Turrens et al. (’91) have suggested that cytoplasmic Ca2 levels increase during periods of hypoxia. Upon reoxygenation, Ca2 levels in the mitochondria activate phospholipase A,, releasing free fatty acids that uncouple and inhibit electron transport between complexes I and I11 of the electron transport chain. NADH dehydrogenase becomes highly reduced, favoring the leakage of electrons directly t o moleculal‘ oxygen and forming superoxide anion radical. Embryonic cocaine concentrations might also help t o maintain the electron transport chain in the reduced state since Faritel et al. (’91) have shown that it is an effective inhibitor of antimycin-sensitive oxygen utilization in mitochondria1 ETP prepared from adult steer heart. A major role for cytosolic xanthine oxidase is commonly cited in the generation of ROS on reperfusion. It has been shown that during hypoxic periods, continued catabolism of ATP results in the accumulation of hypoxanthine and xanthine. Under normal conditions, these purines are oxdized to uric acid by xanthine dehydrogenase which utilizes NAD+ as its oxidant. During hypoxia, however, xanthine dehydrogenase is converted to xanthine oxidase by calcium-dependent proteases. As it result, when 0, is reintroduced, the purines are rapidly oxidized, but xanthine oxidase is now reoxidized by molecular oxygen, generating superoxide anion radical (Roy and McCord, ’83). It seems likely, however, that the excess superoxide that is generated under conditions of embryonic hypoxiai reoxygenation is generated at the mitochondria but it may represent cytosolic production superimposed on that from the immature mitochondria of the sensitive tissues. In conclusion, we have developed an in vitro model for the late embryonic toxicity of cocaine. Malformations induced by repeated hypoxiaireoxygenation of rat embryos in vitro closely resemble those resulting from multiple exposures to cocaine at comparable gestational stages. These malformations also model those resulting from other protocols that can cause transient reductions in uterine perfusion at these gestational stages. Although there is apparently sufficient antioxidant activity in embryonic tissues under normal conditions, prenatal and antioxidant activities may be inadequate to prevent toxicity from the in+
+
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creased amounts of oxygen radicals generated during hypoxiaheoxygenation. ACKNOWLEDGMENTS
This research was funded by grants from the NIH (ES 05623), the Alcohol and Drug Abuse Institute of the University of Washington, and the March of Dimes Summer Science Research Program for Medical Students (MBC). LITERATURE CITED Allen, R.G., and A.K. Balin (1989) Oxidative influence on development and differentiation: An overview of a free radical theory of development. Free Rad. Biol. Med., 6t631-661. Brent, R.L. (1990) Editorial comment: Relationship between uterine vascular clamping, vascular disruption syndrome and cocaine teratogenicity. Teratology, 41: 757-760. Burchfield, D.J., R.M. Abrams, R. Miller, and C.L. DeVane (1991) Disposition of cocaine in pregnant sheep. 11. Physiological responses. Dev. Pharmacol. Ther., 16:130-138. Chance, B., and G.R. Williams (1955) Respiratory enzymes in oxidative phosphorylation. J . Biol. Chem., 217:383-451. Chavez, G.F., J. Mulinare, and J.F. Corder0 (1989) Maternal cocaine use during early pregnancy a s a risk factor for congenital urogenital anomalies. J. Am. Med. Assoc., 262:795-798. Church, M.W., B.A. Dintcheff, and P.K. Gessner (1988) Dose-dependent consequences of cocaine on pregnancy outcome in the Long-Evans rat. Neurotoxicol. Teratol., 1 0 5 - 5 8 . Clark, E.T., and B.L. Gewertz (1991) Intermittent ischemia potentiates intestinal reperfusion injury. J. Vasc. Surg., 13:601-606. Estabrook, R.W. (1967) Mitochondrial respiratory control and the polarographic measurement of ADPO ratios. Methods Enzymol., 10t41-47. Fantel, A.G., and B.J. Macphail(1982) The teratogenicity of cocaine. Teratology, 16:17-19. Fantel, A.G., C.V. Barber, and B. Mackler (1992) Ischemiakeperfusion: A new hypothesis for the developmental toxicity of cocaine. Teratology, 46.285-292. Fantel, A.G., C.J. Burroughs-Gleim, R.E. Person, and B. Mackler (1991) The direct embryotoxicity of cocaine in rats: Effects on mitochondria1 activity, cardiac function and growth and development in vitro. Teratology, 4235-43. Frank. L. (1991) Developmental aspects of experimental pulmonary oxygen toxicity. Free Rad. Biol. Med., 11 :463-494. Hearse, D.J., S.M. Humphrey, and E.B. Chain (1973) Abrupt reoxygenation of the anoxic potassiumarrested rat heart: A study of myocardial enzyme release. J. Mol. Cell Cardiol., 5:395-407. Hoyme, H.E., K.L. Jones, S.D. Dixon, T. Jewett, J.W. Hanson, L.K. Robinson, M.E. Msall, and J.E. Allanson (1990) Prenatal cocaine exposure and fetal vascular disruption. Pediatrics, 85:743-747.
Hutchings, D.E., T.A. Fico, and D.L. Dow-Edwards (1989) Prenatal cocaine: Maternal toxicity, fetal effects and locomotor activity in rat offspring. Neurotoxicol. Teratol., lI:65-69. Jost, A. (1953) La degenerescence des extremites du foetus de rat sous des actions hormonales (acroblapsie experimentale) et la theorie des bulles myelencephaliques de Bonnevie. Arch. Franc. Pediatr., 10r865870. Jost, A., J . Roffi, and M. Cowitat (1969) Congenital amputations determined by the br gene and those induced by adrenalin injection in the rabbit fetus. In: Limb Development and Deformity: Problems of Evaluation and Rehabilitation. C.A. Swinyard, ed. Springfield, CC Thomas, pp. 187-199. Leist, K.H., and J. Grauwiler (1974) Fetal pathology in rats following uterine-vessel clamping on day 14 of gestation. Teratology, 10.55-68. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall (1951) Protein measurement with the Folinphenol reagent. J. Biol. Chem., 193:265-275. Mackler, B., R. Person, and K.A. Davis (1985) Stumes of pyridine nucleotide oxidizing enzymes from human neutrophils. Biochem. Int., 11:319-325. Miki, A,, E. Fujimoto, T. Obsaki, and H. Mizoguti (1988) Effects of oxygen concentration on embryonic development in rats: A light and electron microscopic study using whole-embryo culture techniques. Anat. Embryol., 178t337-343. Moore, T.R., S. Sorg, L. Miller, T.C. Key, and R. Resnik (1986) Hemodynamic effects of intravenous cocaine on the pregnant ewe and fetus. Am. J. Obstet. Gynecol., 155:883-888. New, D.A.T. (1978) Whole-embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev., 5331-122. Plessinger, M.A., and J.R. Woods (1991) The cardiovascular effects of cocaine use in pregnancy. Reprod. Toxicol., 5t99-113. Roy, R.S., and J.M. McCord (1983) Superoxide and ischemia: Conversion of xanthine dehydrogenase t o xanthine oxidase. Oxy Radicals and Their Scavenger Systems, Vol. 2, Cellular and Medical Aspects, R.A. Greenwood and G. Cohen, eds., New York, Elsevier, pp. 145-153. Seidler, E. (1991) The tetrazolium-formazan system: design and histocheniistry. Prog. Histochem. Cytochem., 24:l-86. Thomas, D.B., and J.M. Yoffey (1962) Human foetal hemopoesis. The cellular composition of foetal blood. Br. J . Hematol., 8:290-295. Turrens, J.F., M. Beconi, J . Barilla, U.B. Chavez, and J.M. McCord (1991) Mitochondrial generation of oxygen radicals during reoxygenation of ischemic tissues. Free Rad. Res. Commun., 12-13:681-689. Webster, W.S., and P.D.C. Brown-Woodman (1990) Cocaine as a cause of congenital malformations of vascular origin: Experimental evidence in the rat. Teratology, 41:689-697. Webster. W.S.. A.H. Lioson. and P.D.C. Brown-Woodman (1987) Uterine trauma and limb defects. Teratology, 35:253-260. Woods, J.R., M.A. Plessinaer. and K.E. Clark (1987) Effect of cocaine on uterike blood flow and fetal oxygenation. J. Am. Med. Assoc., 257:957-961.
Studies of the Role of Ischemia/Reperfusion and Superoxide Anion Radical Production in the Teratogenicity of Cocaine ALAN G. FANTEL, CHARLES V. BARBER, MARY B. CARDA, RUSLAN W. TUMBIC, AND B. MACKLER Central Laboratory for Human Embryology, Child Development and Mental Retardation Center, Department of Pediatrics, University of Washington, Seattle, Washington 98195
ABSTRACT
The administration of multiple doses of cocaine on a single day during late gestation is teratogenic in rats in which hind limb ectrodactyly is a major finding (Webster and Brown-Woodman, '90). We have previously hypothesized that these limb malformations result from the generation of reactive oxygen species during the process of ischemiaireperfusion in vivo. In order to study the direct effects of cocaine versus the aberrant oxygenation it may induce, we have developed a system for culturing rat embryos between days 14 and 15 of gestation. Growth and development of cultured embryos are comparable to that of in vivo controls. Exposure to normoxia (95% 0,) with or without cocaine failed to induce limb malformations and exposure to a single long period of hypoxia (20% 0,) only reduced limb growth in the anterior-posterior axis. By contrast, embryos receiving multiple brief exposures to hypoxia developed a significant incidence of hind limb ectrodactyly that appeared indistinguishable from that induced by cocaine in vivo. By incubating day 14 embryos in a nitroblue tetrazolium derivative, l-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT),it was shown that superoxide anion radical appears in the digital rays following two episodes of reperfusion. Little reaction product was seen under the other conditions. Finally, mitochondria1 electron transport particles prepared from teratogenically sensitive limb buds spontaneously "leak' electrons to form superoxide anion radical whereas those from insensitive heart fail to do so. We propose that cocaine and other exposures that can transiently reduce conceptal oxygenation during late gestation are teratogenic by virtue of their capacity to induce ischemia/reperfusion. This process generates toxic oxygen radicals that can overwhelm the immature antioxidant defenses. b 1992 Wiley-Liss, Inc.
It has been proposed recently that hypoxiaireperfusion plays a major role in the late gestational teratogenicity of cocaine (Fantel et al., '92). Because common dysmorphogenic features have been found to result from several experimental teratogenic exposures including cocaine (Webster and Brown-Woodman, 'go), uterine vascular clamping (Webster et al., '87; Leist and Grauweiler, '74), and fetal intraperitoneal epinephrine (Jost et al., '691, a common teratogenic pathway involving vascular disruption and conceptal hypoxia has been proposed (Webster and Brown-Woodman, '90; Brent, '90; Plessinger and Woods, '91). Malformations of the distal portions of the limbs 0 1992 WILEY-LISS. INC.
have been a most frequent finding. The term "acroblapsie" has been used to describe distal limb necrosis secondary to hemorrhagic lesions (Jost, '53). A vascular disruptive sequence involving gut and urogenital tissue has also been proposed as the basis of malformations seen in association with prenatal cocaine exposure in humans (Chavez et al., '89; Hoyme et al., '90). Although each of these exposures can induce conceptal hy-
Received March 5, 1992; accepted May 19. 1992. Address reprint requests to Alan G. Fantel, Department of Pediatrics, RD-20, University of Washington, Seattle, WA 98195.
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poxia, we have proposed a n alternative mechanistic model for their developmental toxicity. The proposed model of embryotoxicity states that reactive oxygen species (ROS), most likely generated during periods of hypoxiaireperfusion, are the common ultimate toxicants (Fantel et al., '92). It is well recognized that cocaine has potent vasoconstrictive activity t h a t can reduce uterine perfusion in gravid ewes (Moore, '86) resulting in decreased fetal oxygenation (Woods e t al., '87; Burchfield e t al., '91). Key features of late embryos or fetuses that could predispose them to the generation of toxic ROS include their increasing hemoglobin content (Thomas and Yoffey, '62) and increasing oxygenation. Evidence of changing oxygenation comes from demonstrations that oxygen concentrations that are toxic to early postimplantation r a t embryos in vitro are required at later stages (New, '78; Miki et al., '88). Additionally, prenatal antioxidant activities tend to be extremely low prior to the perinatal period and may in fact be developmentally regulated (Allen and Balin, '89; Frank, '91). These factors could place late gestational conceptuses a t heightened risk for oxidative attack on proteins, nucleic acids, or lipid membranes. Hypoxialreperfusion, first defined by Hearse et al. ('73), involves the generation of ROS shortly after the resumption of circulation in transiently ischemic tissues. This generation has been detected in most organs studied in vivo as well as in tissues and cells subject to hypoxiaireoxygenation in vitro. It has been associated with numerous disease states including myocardial arhythmias, intestinal ischemia, neonatal necrotizing enterocolitis, pancreatitis, hepatitis, regional stroke, and others. In order to test the role of hypoxiaireperfusion in the teratogenicity of cocaine, we developed a system for culturing rat embryos between day 14 and 15 of gestation. During that time, embryos were exposed twice to cocaine, hypoxia, or the combination of the two. Controls were cultured under normoxic conditions while other embryos were exposed to a single long period of hypoxia. Because malformations comparable to those caused by cocaine in vivo were observed only in embryos exposed to repeated hypoxia and reoxygenation, we assessed the capacity of embryonic tissues to generate ROS (superoxide anion radical)
under comparable conditions. Finally, we compared the superoxide-generating capacities of electron transport particles (ETP) prepared from the mitochondria of tissues sensitive (limb bud) and insensitive (heart) to hypoxialreoxygenation embryotoxicity. MATERIALS AND METHODS
Embryo cultures Primigravid Sprague-Dawley rats were obtained from a commercial vendor (Tyler Laboratories, Bellevue, WA). Laparotomy was performed on the morning of gestational day 14 (gd-14) under halothane, nitrous oxide, and oxygen. Embryos were explanted in Hanks' balanced salt solution (HBSS) where they were exteriorized from the yolk sac and amniotic membranes which remained attached. Immediately after explantation, each was placed in prewarmed medium that was continuously flushed with 95% o,, 5% co,. Embryos were grown in flat, 650-ml tissue culture flasks in a Dubnoff metabolic incubator a t 37°C with gentle shaking. The cultures were continuously flushed with a water-saturated 0, plus CO, mixture (955). This gas mixture was designated "normoxia." The medium, consisted of 120 ml of Earle's balanced salt solution (EBSS) plus heat-inactivated, immediately centrifuged r a t serum (3:1), penicillin (12,000 U), and streptomycin (12,000 pg). When all embryos were explanted, 10 were randomly placed in each of the culture flasks and cultured under the control conditions described above for 2 hr. At the end of this time, each bottle was subject to one of the experimental or control conditions outlined below. 1. Two 30-min exposures to cocaine HC1 (25 pM) in normoxia separated by 30 min in control medium (EBSS). 2. Two 30-minute hypoxic exposures (02, CO,, N,, 20:5:75) separated by 30 min in normoxic control medium. 3. Two simultaneous 30-min exposures to cocaine (as group 1)and hypoxia (as group 2) separated by 30 min in normoxic control medium. 4. A single 90-min hypoxic exposure. Control embryos were subject to all manipulations in normoxia and control medium including flask changes. All media were replaced after the first 8 h r of culture. Following exposures, embryos were cultured for a n additional 18 hr.
COCAINE TERATOCENICITY AND ISCHEMINREPERFUSION
Whenever embryos were moved between flasks or when medium was replaced, embryos were checked for viability as determined by the presence of a n active heart beat and nonviable ones were removed and examined. At the end of the culture period, viability was again assessed and although abnormalities were recorded for all embryos, growth and protein content were not assessed in nonviable ones. Greatest length of viable embryos was measured with a n optical micrometer and protein was determined by the method of Lowry e t al. ('51). After morphology and growth were determined, hind limbs were removed, traced with the aid of a projecting microscope, and photographed. In order to determine the adequacy of the culture procedures, several dams were sacrificed on day 15 in order to obtain normal embryonic growth parameters for comparative purposes.
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from embryos and photographed in transmitted light.
Superoxide generation by mitochondria1 electron transport particles ETP Electron transport particles were prepared from limb buds, and embryonic and adult hearts. After the tissues were washed free of blood in HBSS, they were suspended in cold 5% sucrose solution. All procedures were performed a t 5°C. The tissues were homogenized at medium speed for 3 min with a motor driven Teflon pestle in a glass homogenizer immersed in ice. The tissue suspensions were then centrifuged for 30 rnin at 3,OOOg in a Sorvall centrifuge. The pellet was discarded and the supernatant suspension recentrifuged at 140,OOOgfor 45 rnin in a Spinco ultracentrafuge. The resulting pellet was resuspended in 3 ml of 5% sucrose solution. One milliliter of the suspension was retained on ice for enzymatic assays Embryonic superoxide generation and the remaining 2 ml were recentrifuged Experiments were conducted to confirm for 45 min at 140,OOOg. The pellet was susthe presence and location of superoxide an- pended in HBSS and the suspension was ion radical under experimental conditions. again centrifuged for 45 min a t 140,OOOg to Embryo incubations were performed under remove fetal hemoglobin. The pellet was reconditions identical to those described above suspended in HBSS and centrifuged a s with the exceptions that rat serum was re- above once more. The final pellet was susplaced in the culture medium with 0.7% bo- pended to the original volume of 2 ml with a vine serum albumin and exposures to co- solution of sucrose (0.25 M), Tris (0.02 M), caine and cocaine plus hypoxia were and EDTA ( 5 mM), pH 7.5 (STY solution). omitted. These two exposures were excluded The STV suspensions of ETP were free of because the former failed to induce malfor- hemoglobin and were used for determinamations comparable to those that develop tion of cytochrome b concentrations in the in vivo and no embryos survived the latter. preparations from difference spectra (dithioDay 14 rat embryos were explanted and nite reduced minus oxidized). Determinaincubated in medium containing MTT (1- tions were performed and recorded in a n [4,5-dimethylthiazol-2-yll-2,5-diphenyltetraAminco DW-2 recording spectrophotometer zolium bromide, Sigma, 125 kM) under nor- at 562-572 nm (Chance and Williams, '55). moxic conditions for 30 min. MTT, which Assays of superoxide anion radical generwas added to culture medium in 50 p1 ation were performed in two ways. The first DMSO is a dimethyl derivative of nitroblue measured the maximum capacity of ETP to tetrazolium, a n indicator of superoxide an- generate superoxide from the oxidation of ion radical (Seidler, '91). It easily crosses NADH. Superoxide production was detercell membranes, reacting avidly with super- mined by calculating the difference in the oxide anion radical to form a n insoluble rate of cytochrome c reduction before and blue precipitate, formazan. Following the after the addition of superoxide dismutase initial 30-min incubation, embryos were ex- (SOD) (Mackler et al., '85). The complete asposed to one of the hypoxia or control proto- say contained 0.2 ml of 0.2 M potassium cols described above. After 150 min, em- phosphate buffer (pH 7.5), 0.1 ml of a 1% bryos were removed from the culture flasks solution of cytochrome c, 10 mM sodium and placed in petri dishes on ice. They were azide, 0.01 ml of a 1% solution of NADH, examined under a stereoscopic dissecting and sufficient ETP and water to a final volmicroscope where the presence and location ume of 1 ml. Sodium axide was used to inof blue color was recorded and embryos pho- hibit cytochrome oxidase activity. Determitographed. Finally, the limbs were removed nations were made spectrophotometrically
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TABLE 1. Growth mrameters of live ernbrvos Crown rump (mm) L SEM Controls 1 Cocaine x 2 2 Hypoxia x 2 3Coc;hyp x 2 4 Hypoxia x 1 In viva day 14 In vivo day 15
12.9 13.6
13.0
-
TABLE 2. Survival and dysrnorphology
Protein (mg) SEM
*
0.6 0.4 0.7
6.74 6.17 6.09 -
-
0.8
6.50 5.22 6.07
0.57 0.36 0.65
-
14.0 11.4
0.1
13.8
0.6
0.65 0.44 0.47
Survival Controls 1Cocaine (2 x ) 2 Hypoxia (2 x ) 3 Coc+hyp ( 2 x ) 4 Hypoxia (1X )
N
(7%)
74 21 37 15 74
73
Defects Tail Limbs (%) 5
(%a)
52 27
38
0 0
22
8l
0
53
40
4
0 112
P values by Fisher's exact test. 'Ectrodactyly (P = 0.042). 'Reduced growth (I' = 0.0049).
a t 550 nm and 37°C in a recording Gilford spectrophotometer. The second assay was performed in an oxygen polarograph maintained at 37°C (Estabrook, '67). The assay contained 0.35 ml of 0.2 M potassium phosphate buffer (pH 7.5), 0.10 ml of a 1%NADH solution, ETP, and sufficient water to make a final volume of 1.8 ml. Superoxide generation by uninhibited ETP, calculated from the change in the oxygen disappearance rate after the addition of SOD, represented the amount of superoxide normally produced by the preparation. After the addition of SOD, the rate of oxygen disappearance would be expected to be reduced by 1 pmol of oxygen for every 2 pmol of superoxide produced due t o the release of oxygen by the dismutation of superoxide.
GROUP IV
RESULTS
The effects of the various exposures on embryonic growth and development are presented in Table 1 along with comparable data from in vivo and in vitro controls. There were no significant differences in the average greatest length or protein content in any group and these features were comparable to those of in vivo controls. As can be seen in Table 2, viability was decreased in all experimental groups, dropping from 73% in controls to 52% in group 1, 40% in group 4,27%in group 2, and 0% in group 3. Defects of the distal tail, consisting of kinks, bulbs, or blunt ends were observed in all cultured embryos and were highest in those exposure to cocaine. Tracings of representative hind limbs are presented in Figure 1. Growth reduction in the anterior-posterior plane of the hind limb was seen in 29% of group 4 embryos exposed to a single 90-min episode of hypoxia. Ectrodactyly of the hind limb was observed only in group 2 embryos, exposed t o two 30-min episodes of hypoxia (8%, P = 0.04). The right side was affected
GROUP I I
Fig. 1. Tracings of hind limbs of rat embryos cultured between gestational days 14 and 15. Approximate magnification 110 X . Top row: Embryo cultured under normoxic conditions (95%OJ. Middle row: Embryos exposed to a single 90-min period of hypoxia (20% 02). Remainder of culture period was in normoxic conditions. Bottom row: Embryos exposed to two 30-min periods of hypoxia separated by lhr of normoxia. Remainder of culture period was in normoxic conditions.
in all instances with one case of bilateral ectrodactyly. Photographs of representative limbs are presented in Figure 2. Experiments employing MTT permitted visual localization of superoxide anion radical generation. Because of its extreme localization, it was not possible to quantify superoxide production and relate it to a given volume of tissue or protein. Blue staining from formazan was apparent in the
COCAINE TERATOGENICITY AND ISCHEMINREPERFUSION
297
Fig. 2. Photographs of hind limbs of embryos exposed to two 30-min periods of hypoxia separated by 1 hr of normoxia.
yolk sacs of all groups. In the embryos per se, it could be visualized only in the limb buds of embryos subject to repeated reoxygenation. Photographs of representative limbs are presented in Figure 3. The highest concentration appeared to occurred in hind limbs, with the earliest appearance on the right side.
Table 3 presents the results of studies of superoxide anion radical generation by ETP prepared from embryonic limb bud, and embryonic and adult heart. Although protein content of the preparations is presented, it could not be used to calculate enzyme activities since variable amounts of conceptal hemoglobin were present in all preparations of
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TABLE 3 . Superoxide generation by mitochondria1 ETP (average i- SEMI h i d e inhibited Uninhibited (pmoUmini (pmollminl Preparation nmol cyt b ) nmol cyt 6 ) Adult heart 0.332 ? 0.058 0 Heart (GD16) 0.528 5 0.144 O1 Heart (GD14) 0.628 -t 0.202 Limb bud (GD16) 0.754 i- 0.300 0.092 2 0.034 [35% of total 0,1 Limb bud (GD14) 0.506 5 0.114 0.0952 [22% of total O,] ‘Not done. ‘One assay
significant (P = 0.1). No superoxide could be detected in uninhibited (azide-free) preparations of adult or embryonic heart ETP. In contrast, ETP from gd-14 or -16 limb buds readily produced superoxide at rates of approximately 0.1 ~mol/min/nmol of cytochrome b. This represented 22 and 35%, respectively, of the total oxygen utilization by the preparations. DISCUSSION
Exposure of gd-14 r a t embryos to two 30min periods of hypoxia in vitro induced a significant incidence of limb abnormalities that appeared to closely model those resulting from 2 exposures to cocaine in vivo and other inducers of “acroblapsie” (secondary hind limb ectrodactyly). In contrast to the effect of episodic hypoxia, a single hypoxic exposure of longer duration or direct exposures to cocaine in vitro failed to elicit comparable limb effects. The combination of cocaine and hypoxia was embryolethal in all instances and embryos appear to have died prior to manifesting malformations. These initial findings led us to discount a primary Fig. 3. Photographs of limb buds of day 14 embryos cultured in the presence of 1-[4,5-dimethylthiazo1-2-y1]- role for isolated hypoxia in the genesis of 2,5-diphenyltetrazolium bromide (MTT). Fore limbs are these malformations. I t was proposed inpresented at the top and hind limbs at the bottom of the stead that developing limbs were insulted photographs. Note the presence of reaction product in the reoxygenated embryos indicating generation of su- by reactive oxygen species generated by hypoxialreoxygenation in vitro that modeled peroxide anion radical in the digital rays. ischemialreperfusion in vivo. Support for this hypothesis included pubembryonic tissues. Rates of production of su- lished evidence, discussed previously, that peroxide were, therefore, expressed relative coneceptal oxygenation and hemoglobin to the cytochrome b content of the prepara- content increase with proceeding gestation. tions since cytochrome b is a stoichiometric Molecular oxygen is required for the generconstituent and specific component of ETP. ation of ROS; and iron (or another transiThe maximal capacity to generate superox- tion metal such as copper) is required for the ide was greater in azide-inhibited ETP pre- reduction of hydrogen peroxide to the prepared from embryonic tissues than from sumed ultimate toxicant, hydroxyl radical. adult heart, although the difference was not Significantly, studies of the teratogenicity
COCAINE TERATOGENICITY AND ISCHEMINREPERFUSION
of cocaine during early organogenesis, when iron and oxygen concentrations are presumably lower in the rat have been largely negative (Fantel and MacPhail, ’82; Church et al., ’88; Hutchings et al., ’89). The apparent nonteratogenicity of single exposures to cocaine (Webster and Brown-Woodman, ’90) was also considered supportive of this hypothesis since Clark and Gewertz (’91) have shown that repeated hypoxiaireperfusion results in generation of greater amounts of ROS than do single episodes. In order to strengthen the proposed role of conceptal ischemiaireperfusion in the genesis of the limb malformations induced by cocaine and the other exposures, it was necessary to demonstrate that (1)the conditions that result in these defects are associated with increased generation of ROS and (2) sensitive tissues generate greater amounts of ROS than insensitive ones under experimental conditions. Alternatively, the antioxidant activities of sensitive tissues could be relatively low, a possibility not necessarily mutually exclusive with (2) and one not yet investigated. The results of experiments designed to examine these issues are consistent with the proposed hypothesis. Studies with MTT clearly indicated that increased amounts of superoxide anion radical are generated when embryos are exposed to repeated cycles of hypoxia and reoxygenation. Furthermore, the greatest amounts of superoxide appeared in precisely those tissues that display maximal teratogenic sensitivity, i.e., the distal limb buds. Reaction product appears to be concentrated in the digital rays, and it is the digits that have been shown to be most affected by the in vivo exposures. Although both mitochondria and cytosol have been proposed as sites of superoxide generation during ischemiaireperfusion in adults, studies of embryonic tissue have not been performed. As demonstrated above, electron transport particles prepared from gd-14 or gd-16 limb buds show an unusually large capacity to spontaneously reduce molecular oxygen to superoxide. By contrast, no superoxide was produced by heart ETP unless the NADH oxidase activity of the preparations was inhibited by the addition of azide. Azide acts by inhibiting electron transport at cytochrome oxidase, thus maintaining the electron transport chain in a reduced state upstream and favoring electron “leakage” to molecular oxygen.
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Turrens et al. (’91) have suggested that cytoplasmic Ca2 levels increase during periods of hypoxia. Upon reoxygenation, Ca2 levels in the mitochondria activate phospholipase A,, releasing free fatty acids that uncouple and inhibit electron transport between complexes I and I11 of the electron transport chain. NADH dehydrogenase becomes highly reduced, favoring the leakage of electrons directly t o moleculal‘ oxygen and forming superoxide anion radical. Embryonic cocaine concentrations might also help t o maintain the electron transport chain in the reduced state since Faritel et al. (’91) have shown that it is an effective inhibitor of antimycin-sensitive oxygen utilization in mitochondria1 ETP prepared from adult steer heart. A major role for cytosolic xanthine oxidase is commonly cited in the generation of ROS on reperfusion. It has been shown that during hypoxic periods, continued catabolism of ATP results in the accumulation of hypoxanthine and xanthine. Under normal conditions, these purines are oxdized to uric acid by xanthine dehydrogenase which utilizes NAD+ as its oxidant. During hypoxia, however, xanthine dehydrogenase is converted to xanthine oxidase by calcium-dependent proteases. As it result, when 0, is reintroduced, the purines are rapidly oxidized, but xanthine oxidase is now reoxidized by molecular oxygen, generating superoxide anion radical (Roy and McCord, ’83). It seems likely, however, that the excess superoxide that is generated under conditions of embryonic hypoxiai reoxygenation is generated at the mitochondria but it may represent cytosolic production superimposed on that from the immature mitochondria of the sensitive tissues. In conclusion, we have developed an in vitro model for the late embryonic toxicity of cocaine. Malformations induced by repeated hypoxiaireoxygenation of rat embryos in vitro closely resemble those resulting from multiple exposures to cocaine at comparable gestational stages. These malformations also model those resulting from other protocols that can cause transient reductions in uterine perfusion at these gestational stages. Although there is apparently sufficient antioxidant activity in embryonic tissues under normal conditions, prenatal and antioxidant activities may be inadequate to prevent toxicity from the in+
+
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creased amounts of oxygen radicals generated during hypoxiaheoxygenation. ACKNOWLEDGMENTS
This research was funded by grants from the NIH (ES 05623), the Alcohol and Drug Abuse Institute of the University of Washington, and the March of Dimes Summer Science Research Program for Medical Students (MBC). LITERATURE CITED Allen, R.G., and A.K. Balin (1989) Oxidative influence on development and differentiation: An overview of a free radical theory of development. Free Rad. Biol. Med., 6t631-661. Brent, R.L. (1990) Editorial comment: Relationship between uterine vascular clamping, vascular disruption syndrome and cocaine teratogenicity. Teratology, 41: 757-760. Burchfield, D.J., R.M. Abrams, R. Miller, and C.L. DeVane (1991) Disposition of cocaine in pregnant sheep. 11. Physiological responses. Dev. Pharmacol. Ther., 16:130-138. Chance, B., and G.R. Williams (1955) Respiratory enzymes in oxidative phosphorylation. J . Biol. Chem., 217:383-451. Chavez, G.F., J. Mulinare, and J.F. Corder0 (1989) Maternal cocaine use during early pregnancy a s a risk factor for congenital urogenital anomalies. J. Am. Med. Assoc., 262:795-798. Church, M.W., B.A. Dintcheff, and P.K. Gessner (1988) Dose-dependent consequences of cocaine on pregnancy outcome in the Long-Evans rat. Neurotoxicol. Teratol., 1 0 5 - 5 8 . Clark, E.T., and B.L. Gewertz (1991) Intermittent ischemia potentiates intestinal reperfusion injury. J. Vasc. Surg., 13:601-606. Estabrook, R.W. (1967) Mitochondrial respiratory control and the polarographic measurement of ADPO ratios. Methods Enzymol., 10t41-47. Fantel, A.G., and B.J. Macphail(1982) The teratogenicity of cocaine. Teratology, 16:17-19. Fantel, A.G., C.V. Barber, and B. Mackler (1992) Ischemiakeperfusion: A new hypothesis for the developmental toxicity of cocaine. Teratology, 46.285-292. Fantel, A.G., C.J. Burroughs-Gleim, R.E. Person, and B. Mackler (1991) The direct embryotoxicity of cocaine in rats: Effects on mitochondria1 activity, cardiac function and growth and development in vitro. Teratology, 4235-43. Frank. L. (1991) Developmental aspects of experimental pulmonary oxygen toxicity. Free Rad. Biol. Med., 11 :463-494. Hearse, D.J., S.M. Humphrey, and E.B. Chain (1973) Abrupt reoxygenation of the anoxic potassiumarrested rat heart: A study of myocardial enzyme release. J. Mol. Cell Cardiol., 5:395-407. Hoyme, H.E., K.L. Jones, S.D. Dixon, T. Jewett, J.W. Hanson, L.K. Robinson, M.E. Msall, and J.E. Allanson (1990) Prenatal cocaine exposure and fetal vascular disruption. Pediatrics, 85:743-747.
Hutchings, D.E., T.A. Fico, and D.L. Dow-Edwards (1989) Prenatal cocaine: Maternal toxicity, fetal effects and locomotor activity in rat offspring. Neurotoxicol. Teratol., lI:65-69. Jost, A. (1953) La degenerescence des extremites du foetus de rat sous des actions hormonales (acroblapsie experimentale) et la theorie des bulles myelencephaliques de Bonnevie. Arch. Franc. Pediatr., 10r865870. Jost, A., J . Roffi, and M. Cowitat (1969) Congenital amputations determined by the br gene and those induced by adrenalin injection in the rabbit fetus. In: Limb Development and Deformity: Problems of Evaluation and Rehabilitation. C.A. Swinyard, ed. Springfield, CC Thomas, pp. 187-199. Leist, K.H., and J. Grauwiler (1974) Fetal pathology in rats following uterine-vessel clamping on day 14 of gestation. Teratology, 10.55-68. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall (1951) Protein measurement with the Folinphenol reagent. J. Biol. Chem., 193:265-275. Mackler, B., R. Person, and K.A. Davis (1985) Stumes of pyridine nucleotide oxidizing enzymes from human neutrophils. Biochem. Int., 11:319-325. Miki, A,, E. Fujimoto, T. Obsaki, and H. Mizoguti (1988) Effects of oxygen concentration on embryonic development in rats: A light and electron microscopic study using whole-embryo culture techniques. Anat. Embryol., 178t337-343. Moore, T.R., S. Sorg, L. Miller, T.C. Key, and R. Resnik (1986) Hemodynamic effects of intravenous cocaine on the pregnant ewe and fetus. Am. J. Obstet. Gynecol., 155:883-888. New, D.A.T. (1978) Whole-embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev., 5331-122. Plessinger, M.A., and J.R. Woods (1991) The cardiovascular effects of cocaine use in pregnancy. Reprod. Toxicol., 5t99-113. Roy, R.S., and J.M. McCord (1983) Superoxide and ischemia: Conversion of xanthine dehydrogenase t o xanthine oxidase. Oxy Radicals and Their Scavenger Systems, Vol. 2, Cellular and Medical Aspects, R.A. Greenwood and G. Cohen, eds., New York, Elsevier, pp. 145-153. Seidler, E. (1991) The tetrazolium-formazan system: design and histocheniistry. Prog. Histochem. Cytochem., 24:l-86. Thomas, D.B., and J.M. Yoffey (1962) Human foetal hemopoesis. The cellular composition of foetal blood. Br. J . Hematol., 8:290-295. Turrens, J.F., M. Beconi, J . Barilla, U.B. Chavez, and J.M. McCord (1991) Mitochondrial generation of oxygen radicals during reoxygenation of ischemic tissues. Free Rad. Res. Commun., 12-13:681-689. Webster, W.S., and P.D.C. Brown-Woodman (1990) Cocaine as a cause of congenital malformations of vascular origin: Experimental evidence in the rat. Teratology, 41:689-697. Webster. W.S.. A.H. Lioson. and P.D.C. Brown-Woodman (1987) Uterine trauma and limb defects. Teratology, 35:253-260. Woods, J.R., M.A. Plessinaer. and K.E. Clark (1987) Effect of cocaine on uterike blood flow and fetal oxygenation. J. Am. Med. Assoc., 257:957-961.
