Effects of cocaine and cocaine metabolites on mouse developmentin vitro

Pergamon

Toxicology in Vitro 10 (1996) 407-414

Effects of Cocaine and Cocaine Metabolites on Mouse Development In Vz’tro E. S. HUNTER, tDepartment University

III*t and

T.

W.

SADLERt$

of Cell Biology and Anatomy, Laboratories of Developmental Biology and Toxicology, of North Carolina at Chapel Hill. Chapel Hill, NC 27599 and IBirth Defects Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

(Accepted 28 February 1996) Abstract-The use of cocaine use has been associated with adverse developmental effects in humans, and cocaine administration produces developmental toxicity in animal models. However, whether the adverse effects produced during organogenesis are due directly to the effects of cocaine or its metabolites remains to be established. This study was therefore undertaken to compare the morphological effects of cocaine and its metabolites, ecgonine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in whole embryo culture (WEC) using early somite stage ICR mice. Cocaine produced a concentration-dependent induction of defects including effects on craniofacial development such as neural tube closure defects (NTDs). Concentrations of cocaine of 51.4 pM or more produced dysmorphogenesis and 100% of the embryos exhibited NTDs at 441 ELM.EME also induced defects at concentrations of 400 pM or above. Neither ecgonine nor BE altered embryogenesis at concentrations of 2000 pM or less. The incidence of cocaine-induced NTDs was dependent on the length of exposure to cocaine. At 294 PM, exposures of 3 hr or more were required to alter development when evaluated at the end of a 24-hr culture period. Lower cocaine concentrations required longer exposure periods (6 or I2 hr) to produce dysmorphogenesis. The incidence of NTDs appears to follow the area under the concentration time curve and is not solely dependent on the peak cocaine concentration m the medium. Exposure of conceptuses to a combination of cocaine and EME produced a high incidence of NTDs. These results suggest that the concentration of cocaine or EME required to induce NTDs in c,irro is higher than the teratogenic concentration in cit,o. Additionally. the time required for high concentrations of cocaine to induce NTDs is longer than the serum half-life of cocaine reported in t,iro following a single administration. Thus, NTDs produced by cocaine administration appear not to be due solely to the effect of cocaine or its metabolites on the conceptus but may involve effects on extraembryonic and/or maternal tissues as well. Published by Elsevier Science Ltd.

INTRODUCTION

Cocaine abuse during pregnancy has been associated with a variety of adverse developmental effects. For example, high rates of spontaneous abortions, foetal deaths and stillbirths occur in women who use cocaine during pregnancy (Bingo1 rf ul., 1987; Chasnoff et al., 1985; Ryan et al., 1987). Many children (2040%) born to cocaine-using mothers are growth retarded (i.e. birth weights less than 2500 g) (Chasnoff et al., 1989; MacGregor, 1987). Similarly, 616% of infants of cocaine-using mothers exhibit congenital malformations compared with l-2% in non-drug-exposed children (Bingo1 et al., 1987; Chasnoff et al.. 1988 and 1989; Lipshultz et al., 1991;

*Author for correspondence

at: US Environmental Reproductive Toxicology tion Agency, National Health and Environmental Research tory, MD No. 67, TRP, NC 2771 I. USA. Abbreviations: AUC = area under the time and tration curve; BE = benzoylecgonine; EME = methyl ester: NTDs = neural tube closure VYS = visceral yolk sac; WEC = whole embryo 0887-2333/96/$15.00 + 0.00 Published PII SO887-2333(96)00022-7

ProtecDivision, Laboraconcenecgonine defects: culture.

by Elsevier Science

Little ct u/.. 1988 and 1989: MacGregor, 1987: Zuckerman r/ a/.. 1989). A wide variety of structures appear to be affected by maternal cocaine use including the cranium (anencephaly), heart (septal defects) and urogenital system (hydronephrosis). In mice, administration of cocaine during organogenesis induces developmental toxicity including maldevelopment of the cardiovascular system, urogenital system (hydronephrosis), neural tube (exencephaly), eye (anophthalmia), limb, and decreased foetal weight (Fantel and MacPhail, 1982; Finnel et al., 1990; Fisher et al., 1994; Hunter et al., 1995; Mahalik et al., 1980; Mehanny et a/., 1991). To date it is not clear whether cocaine, its metabolites or a combination of these xenobiotics produce the developmental toxicity. Previous studies have documented that cocaine has direct effects on embryogenesis. In chick embryos, cocaine inhibited neural tube closure at a concentration of 500 PM in culture medium (Burin C?al., 1991). At 662 PM, a 2-hr exposure to cocaine produced a 61% incidence of neurulation defects. When metabolites of cocaine were evaluated, neither benzoylecgonine (BE) (I 175 Ltd. All rights

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in Great Britain

E. S. Hunter

408

and T. W. Sadler

J1M) nor ecgonine (1533 pM) affected chick development. Similarly, Sandstrom and Pennington (1993) reported that cocaine suppressed embryonic growth when administered in ow. Cocaine toxicity has also been reported in cultured mammalian embryos. In day-10 rat embryos exposed to 100 PM cocaine. a decrease in protein content, crown-rump length and viability were induced (Fantel rf rrl.. 1990). These effects were exacerbated by exposure to cocaine under hypoxic conditions. In addition. an axial asymmetry. similar to that produced by nitro-heterocycles. was induced in some embryos by cocaine (Fantel ct d.. 1990). Cocaine produced a concentration-dependent decrease in embryonic protein content and induced lethality (300 FM) in IO- 12 somitr rat embryos cultured for 48 hr (El-Bizri (‘t ul.. I9Y 1). In moust embryos. the effects of a 4%hr exposure to IO 66 l,tgjml (29.4-194 pM) cocaine have been reported (Fisher et al., 1994). Concentrations as IOH as IO pg/ml produced a reduction in a composite measure of embryonic development and 20 pg:‘ml produced growth retardation and increased oedema. open neural tubes and other neural defects. The present study was designed to investigate the time course for cocaine-induced defects. the potential toxicity of cocaine metabolites and their interaction with cocaine.

Warner (1984). Protein content was measured by the technique of Lowry (Lowry et ul., 1951) or the BCA technique (Pierce Biochemical, Rockford, IL, USA). The concentration-dependent effects of cocaine, ecgonine. EME and BE were determined using a 24-26-hr exposure period. All solutions were prepared immediately before use in filter-sterilized saline and protected from light. Cocaine concentration ranged from 2.94 to 441 pM. The effects of EME were evaluated at 200-- 1200 pM and ecgonine and BE concentrations ranged from 200 to 2000 pM. The time-dependency of cocaine-induced effects was also determined using concentrations of 73.5, 147 and 294 11~ and 3. 6 and I?-hr exposure periods. At the end of the exposure period, embryos were rinsed and transferred to control medium to complete the 26-hr culture period. The interaction of cocaine and EME ~‘as evaluated by exposing conceptuses to cocaine (0. 2Y.4 and 51.4 pM) plus EME (0.200,300 and 400 PM) for 24~26 hr and comparing the incidence of defects to embryos exposed to the agents separately. .Stuti.stic’.\ The incidence of neural tube defects produced by exposure to agents was compared with control using Fisher‘s exact t-test (Dowdy and Weardon, 1984). RESCILTS

MATERIALS

A’SD

METHODS

Cocaine hydrochloride. BE. ecgomne hydrochloride and ecgonine methyl ester hydrochloride (EM E) were purchased from Sigma Chemical Co. (St L.nui$. MO, USA). Animals HSD:(ICR) BR mice (from Harlan Sprague Dawley, Inc.. IN. USA) were housed under ;I 14-IO-hr light/dark cycle within a temperature and humidity controlled vivarium. Mice were giLen unlimited access to Purina rodent laborator) cho\+ and filtered tap water and housed at least 2 wk priotto breeding. Males were housed individually and females were housed three per cage. A male mouse was placed into the cage of the females overnight (17.00 to 9.00). Females were examined the following morning for the presence of a vaginal plug that indicated that mating had occurred and this day was designated day I. Whole embryo cultuw Whole embryo culture (WEC) was performed as previously described (Sadler, 1979). Briefly. day-9 embryos with 3-6 pairs of somites were prepared for culture by removal of maternal tissues, parietal yolk sac and Reichert’s membrane. At the end of the morphological development was culture period, assessed using criteria described by Sadler and

Mouse conceptuses placed into culture at the early somite stage (3 6 pairs of somites) exhibited normal growth and development during a 2426-hr culture period (Sadler. 1979). Craniofacial development, including closure of the cranial neural tube. establishment of the optic and otic anlagen and formation of the pharyngeal arches. occurred in normal fashion. The heart tube completed its initial the embryo rotated and attained a loopmg. ventroflexed curvature. and there were 20-23 pairs of somites (Plate 1). Embryos exposed to cocaine exhibited abnormal development at concentrations of 51.4 pM or above (Plate I ). The cocaine-induced defects included incomplete embryonic rotation as well as effects on craniofacial and heart development. Non-closure of the crantal neural tube was produced and in the most severely malformed embryos occurred throughout the brain region. Hypoplasia and growth retardation of the prosencephalon occurred as an isolated defect or in combination with neural tube non-closure. Eye development appeared to be affected by cocaine exposure, but this defect was only seen in association with prosencephalic hypoplasia and may be secondary to the effect on the neural tube. Hypoplasia of the pharyngeal arches was also noted in some embryos. Effects on heart development appeared as a reduction in the size of the heart tube and a lack of looping which led to a ‘V-shaped heart tube. In addition to the effects of cocaine on the embryo, at the highest concentrations tested cocaine exposure

Plate. I, Photomicrographs of ICR mouse embryos after a 24-hr culture period. Panel A shows an embryo grown in control medium. Embryonic growth and development during this 24-hr period is the same as that in r~it~ over the same time period. Panels B and C show embryos grown in the presence of cocaine. The embryo in Panel B exhibits marked hypoplasia of the prosencephalon and first arch as well as the non-closure in the mesencephalon. Panel C shows an embryo having a reduction of the prosencephalon. maldevelopment of the first arch. incomplete rotation and incomplete looping of the heart tube.

409

411

Cocaine-induced developmental effects

80 -

0

Contro12.9429.4

51.4 73.5 147 220 294 441

j.rMcocaine Fig. 1. Incidence of NTDs produced by cocaine following a 24-hr culture period. The number of embryos evaluated in each group is shown on the figure.

affected development of the vascular system in the viscera1 yolk sac (VYS). In the most severe form of this maldevelopment, yolk sac vascnlature was not apparent and only ‘blood islands’ were present. However, in less severely affected conceptuses, ramification of the large-calibre vessels appeared to be reduced, but no quantitative assessments of vessel diameter or ramification were performed. Cocaine produced a concentration-dependent increase in NTDs (Fig. 1). No increase in NTDs was produced at concentrations less than 29.4 FM, but all embryos exhibited this defect at 441 pM. The time-dependency of cocaine-induced defects was determined using concentrations of 13.5, 147 and 294 pM (Fig. 2). Conceptuses were exposed to cocaine for 3, 6, or 12 hr and then transferred to control medium to complete the 24-hr culture period. Embryos exposed to 294 pM exhibited an increased incidence of NTDs when exposed for 3 hr or more. The incidence of NTDs produced by a 12-hr cocaine exposure was similar to the incidence of NTDs when conceptuses were exposed for the entire 24-hr period. Neither 73.5 PM nor 147 PM increased NTDs following a 3-hr exposure period. Using these data, the area under the time and concentration curves (AUCs) were calculated and plotted against the incidence of NTDs (Fig. 3) and suggested that the induction of defects by cocaine followed the AUC

3 hr

6 hr 12hr Hr of exposure

0

10002000300040005000600070008000 AUC (pM/hrcocaine)

Fig. 3. The incidence of NTDs compared with the area under the time and cocaine concentration curve.

and was not solely dependent on the peak serum concentration. EME produced no increase in NTDs at a concentration of 200 pM (Fig. 4), but produced this defect at concentrations of 300 pM or above. Exposure of conceptuses to ecgonine or BE did not significantly increase the incidence of NTDs compared with controls (Figs 5 and 6, respectively) at concentrations ranging from 200 to 2000 pM and 400 to 2000 PM, respectively. To determine whether there was an interaction between cocaine and EME, conceptuses were exposed to a combination of these agents (Table 1). At a concentration of 29.4 PM cocaine, that did not produce NTDs alone, the incidence of NTDs produced by the combination of cocaine and EME were similar to those induced by EME alone. However, when 51.4 FM cocaine was employed, the incidence of NTDs produced by the combination of cocaine and EME showed trends to be different than that produced by cocaine or EME alone. For example, the combination of 51.4 pM cocaine and 400 pM EME produced 53% NTDs, while cocaine and EME produced 15 and 35%, respectively. Although this value is different from that produced by cocaine alone (P < 0.05) there is no statistical difference compared with 400 PM EME. When 51.4 PM cocaine was combined with 200 FM EME, 33% of embryos exhibited NTDs. This rate was greater (P < 0.05) than that produced by EME alone (0%; O/16 embryos), but not statistically greater than the

24 hr

Fig. 2. Incidence of neural tube defects produced by short-term exposure to cocaine. The number of embryos evaluated in each group is shown on the figure. The incidence of neural tube defects in control embryos was 10.5% (4/38).

Control 200

300

400 600 uM EME

800 1200

Fig. 4. Incidence of NTDs produced by EME in oitro. The number of embryos evaluated in each group is shown on the figure.

E. S. Hunter and T. W. Sadler

IX

4

X

II

800

I200

I -5

r---t

400

Control

2000

pM ecgonine

Fig. 5. Incidence

of NTDs

produced

by ecgonme

1~ r//ro.

The number of embryos evaluated is shown on the figure for each group,

rate of NTDs 4’27 embryos).

produced

by cocaine

alone

(14.8%:

DISCUSSION

This study, involving an evaluation of neurulation staged mouse embryo grown in whole embryo culture system. confirmed the direct embryotoxicity of cocaine (Burin l’t u/.. 1991: El-Bizri (‘t (I/.. 1991: Fantel ef (II.. 1990; Fisher et d., 1994) and describes the concentration-dependent induction of neural tube defects. Additionally. cocaine exposure affected development of the VYS. Since the VYS is important in histiotrophic nutrition, the embryotoxicity produced by cocaine may be mediated through a direct effect on the embryo, through a direct effect on the VYS, or a combination of these targets. The lowest peak cocaine concentration required to produce NTDs was 51.4 PM. However, cocaine both in rim and in rim undergoes hydrolysis to BE in aqueous media and is hydrolysed by plasma esterases to EME (Stewart et u/.. 1977 and 1979). El-Bizri 1’1al. (1991) reported that at the end of a 48-hr embryo culture period, 18% of the initial concentration of cocaine was present in the culture medium. How this change in medium cocaine concentration compares with the change in cocaine in riw remains to be established. The peak concentration of cocaine m the culture medium required to induce abnormalities is approximately 20-30 times greater than the

,r 29

10

8

Control

200

500

8

750 FM BE

8

I-t

1200

2000

I

Fig. 6. Incidence of NTDs produced by BE 01 ci/ro. The number of embryos evaluated is indicated above each group.

peak serum concentrations in vivo. In rats, a 50 PM/kg ip dose produced a 1682 k 260 pmol/ml (1.68 FM) concentration of cocaine (El-Bizri ef a/., 1991). Peak venous blood levels in humans after “typical cocaine doses” average 200-600 ng/ml (0.6-l .7 JAM) (Benowitz. 1993). The mean concentration of cocaine that IS lethal to adults is 20.4 pM with a range of 0.33-68.9 pM (Mittleman ef al., 1989). The differences in the concentration of cocaine required to alter development in vitro and the peak concentrations of cocaine achieved in z~iw suggest that factors other than cocaine alone, namely metabolites or vascular effects (e.g. vasoconstriction), contribute to the embryotoxicity observed following cocaine exposure. The time course for cocaine-induced NTDs was also evaluated by exposing conceptuses for 3, 6 or I? hr and transferring the embryo to medium that did not contain cocaine. The effects of cocaine were dependent on both the initial concentration of cocaine as well as the length of exposure. At the highest concentration employed (294 PM). a 3-hr exposure period produced NTDs. However, at lower concentrations (147 and 73.5 PM), exposures between 6 and I2 hr were required to alter development. This suggests that the direct toxicity of cocaine on the conceptus is dependent on the AUC and not solely dependent on the peak serum concentration. The importance of comparing peak serum concentrations and AUC in understanding the teratogenic response has been demonstrated for a number of xenobiotics (Nau. 1986). The exposure periods required to produce dysmorphogenesis in vitro are longer than the half-life of cocaine in rVw (0.5-1.5 hr after iv administration (Ambre LJ~[II.. 1988; Jeffcoat et ul., 1989) but may be as long as 5 hr after intranasal exposure (Benowitz, 1993)). This further suggests that neural tube defects produced by cocaine administration are not due solely to the effects of cocaine on the conceptus. As previously reported for chick embryos in vitro, BE and ecgonine did not alter development (Burin r/ f/l., 1991). Our study confirmed these results in rodent embryos and showed that even higher concentrations than those previously reported were non-toxic. Since BE is produced by non-enzymatic hydrolysis of cocaine and ecgonine produced from the degradation of BE and EME, degradation of cocaine to these products separately results in detoxification. In contrast, EME produced malformations similar to those produced by cocaine. However, the lowest concentration of EME to induce NTDs was 300 pM, which is about sixfold higher than the concentration of cocaine that induced defects. The dose-response curve for EME-induced NTDs was not steep so that 80% of embryos exhibited NTDs at a concentration of 1200 PM. Thus, in comparison to the direct effect of cocaine on the conceptus, EME is a less potent developmental toxicant since much higher concentrations of EME

Cocaine-induced developmental effects

413

Table I. Incidence of neural tube defects produced by combinations of cocaine and ecgonine methyl ester (EME) in mouse embryos in vitro Concentration Cocaine 0 PM 29.4 PM 51.4 LLM

of EME

0 PM

200 PM

300 FM

3.6 (l/28) 0 (O/17) 14.8 (4127)

0 (O/16) 0 (O/16) 33*t, (8/24)

17.6 (3/17) 0 (O/14) 37.51 (6116)

400 phi 35.3* 27.8’t 53.3*t

(6117) (5/18) (8/15)

The percent of embryos exhibiting NTDs and (the number of embryos with NTDs/total number cultured) are shown for each combination of cocaine and EME.*P < 0.05 compared with control; tP < 0.05 compared with the same concentration of cocaine; $P < 0.05 compared with the same concentration of EME.

are required to directly affect development. Although the in vivo concentrations of EME following administration of teratogenic doses have not been fully described, EME has a much longer [l/2 [3.56 hr (Jatlow, 1987)] than cocaine (0.5-1.5 hr). Thus, the conceptus would be exposed to EME for longer periods than exposure to cocaine in vivo. Although it is generally held that the cocaine metabolites are without biological effect, a recent report documented that cocaine, as well as its metabolites, were vasoactive (Madden and Powers, 1990). Unlike cocaine, which is vasoconstrictive, EME produced vasodilatation at a concentration of 10 PM. The relationship between the vascular effects of EME and developmental toxicity is currently unknown. The adverse effects of the combination of cocaine and EME indicated that there was no synergistic interaction between these xenobiotics, but may suggest additivity at the concentrations used. In only one group (200 PM EME + 51.4 PM cocaine) was the incidence of NTDs greater than the sum of that produced by cocaine and EME alone. The most compelling suggestion of additivity is shown by the combination of 51.4 PM cocaine and 400 PM EME, which separately produced malformations in 14.8 and 35.3% of embryos, respectively. In combination there is a 53.3% incidence of dysmorphology. However, the incidence of defects produced by the cocaine EME combination is not statistically different than that produced by EME alone. The lack of a demonstrable synergistic interaction between these very high concentrations of cocaine and EME further suggests that the embryonic effects produced by cocaine administration are not produced solely by the effects of cocaine and its metabolites on the conceptus. On the basis of the high concentrations of xenobiotics needed to alter development, the lack of a greater than additive interaction between agents and the relatively long exposure periods required to induce dysmorphogenesis, this study suggests that the toxicity produced by maternal administration of cocaine is not due simply to the effects of cocaine and its metabolites on the mouse embryo. This assessment further supports previous studies (Fantel et al., 1990; Fisher et al., 1994; Zimmerman et al., 1994) that provide evidence of a combination of toxic effects on the conceptus and maternal system leading to developmental defects. The ability of the whole embryo culture technique to support normal embry-

onic growth and development during this critical stage of development allows this type of interaction to be assessed. Thus, the vasoconstrictive effect of cocaine on the maternal vasculature in combination with its direct effect, and that of its metabolites, on the conceptus appear to contribute to the adverse developmental effects of cocaine administration. Disclaimer: This manuscript has been reviewed by the Health Effects Research Laboratory, US EPA and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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