TERATOLOGY 41:699-706 (1990)

Marked Accumulation of Valproic Acid in Embryonic Neuroepithelium of the Mouse During Early Organogenesis LENNART DENCKER, HEINZ NAU, AND ROLAND DARGY Department of Toxicology, University of Uppsala, S-75124 Uppsala, Sweden (L.D., R.d'A.); Institute of Toxicology and Embryopharmacology, Free University ofBerlin, 0 - 1 0 0 0 Berlin 33, Federal Republic of Germany

(H.N.)

ABSTRACT Valproic acid, an antiepileptic drug, causes neural tube defects in mice and man. 14C-labeled valproic acid (sodium-salt) was administered to pregnant mice on days 8 and 9 of gestation (period of high sensitivity in regard to formation of neural tube defects in this species). Two dose levels of valproic acid (1 and 400 mglkg) were used; in each case the total radioactivity administered was the same: 400 FCilkg or 14.7 MBqIkg. Autoradiography combined with computerized densitometry revealed that in low-dose animals most of the radioactivity was confined to maternal liver and kidney, while at high doses more activity was observed in soft tissues and fluids, including amniotic fluid. In the embryo, the neuroepithelium showed the highest concentration, irrespective of dose and survival interval (30 min, 3 h, and 6 h). Upon administration of the high dose, up to five times more radioactivity (approximately 2,000 times more valproic acid) was recovered in embryonic tissues than after the low dose. It is concluded that high doses of VPA saturate the capacities of metabolism, excretion, and protein binding in the maternal organism, resulting in a higher proportion of the dose reaching the embryo, allowing more of the drug to be accumulated by the target organ, the neuroepithelium. Valproic acid (VPA; 2-propyl-pentanoic acid) has been used as an antiepileptic drug in Europe since the mid sixties and in the U.S.A. since 1978 (Wilder and Karas, '82). In the eighties, it has been implicated as a human teratogen causing neural tube defects, especially spina bifida aperta, in retrospective (Robert and Rosa, '83; Lindhout and Meinardi, '84) as well as in prospective (Jager-Roman et al., '86; Nau et al., '81a; Lindhout and Schmidt, '87) studies. Neural tube defects (exencephaly) are also the most apparent external malformations observed when VPA is administered to pregnant mice in high doses (Nau et al., '81c; Kao et al., '81). Rat embryos cultured in vitro in the presence of VPA also develop neural tube defects (Kao et al., '81; Bruckner et al., '83; Lewandowski et al., '86). The mechanism of VPA-induced teratogenicity is not known; interaction of the drug with embryonic lipid metabolism (Clarke and Brown, '87) or embryonic folate metabolism (Trotz et al., '87) or with an alteration of the relatively high embryonic pH, (Nau and Scott, '86) may 0 1990 WILEY-LISS, INC.

play a significant role (for overview see Nau and Hendrickx, '87; Coakley et al., '86). Because of the apparently high sensitivity of processes involved in the development of the neural tube area, we decided to use radio-labeled VPA to study whether the developing central nervous system is a special target of the compound and whether a longer retention of VPA in the embryo occurs in this structure. Therefore 14C-labeled VPA was administered to pregnant mice during the sensitive phase of gestation, and the label was studied by autoradiography at various time intervals. We found a strong labeling of the embryonic neuroepithelium during the sensitive stage of early organogenesis.

Received February 24, 1989; accepted October 31, 1989.

700

L. DENCKER ET AL.

Brain Site of injection

Kidney

Liver

Blood

Intestines

Kidney

Brain Site of injection

Blood

Bone

Liver

Bone

Intestines

Fig. 1. Whole-body autoradiograms showing the distribution of radioactivity after the S.C.administration of 14C-VPA. The animals were killed 30 min after a 1 mgikg dose (a),or a 400 mgikg dose (b),and 3 h after a 1 mgikg dose (c). The amount of radioactivity administered was the same in each experiment (400 FCi or 14.7 MBqikg bd. wt.). Note that the low dose resulted in asubstantial accumulation of radioactivity (white ar-

eas) in the liver and kidney (a), while the high dose resulted in increased distribution into soft tissue (b). In c, a marked accumulation in the olfactory bulb of the brain can be observed. The lateral nasal gland is mainly a serous gland which is known to metabolize xenobiotics. Magnification x 1.5 for a and b, x 4 for c. Figure continued on next page.

MATERIALS AND METHODS

Chemicals Custom-labeled 14C-valproic acid (14CVPA) with a specific activity of 59 mCii mmol (2165 MBq/mmol) was prepared by Amersham International plc, Buckinghamshire, England. Non-labeled valproic acid (VPA) was a gift from Dr. Schafer, DesitinWerke Carl Klinke, F.R.G. The internal standard, 2-Methyl-2-ethylcaproic acid, was obtained from Fluka (Neu-Ulm, F.R.G.). All

Animals Mice of the NMRI strain (from Anticimex, Sollentuna, Sweden) 3 months old were used in this study. They were fed a pellet diet and were allowed free access to water. Females were caged with males overnight, and the presence of a vaginal plug the next morning was taken as a sign of pregnancy ( = day 0).

VALPROIC ACID IN NEUROEPITHELIUM DURING ORGANOGENESIS

Olfactory bulb

701

Brain

Lateral nasal gland Fig. l c .

chemicals, reagents, and solvents were obtained in the highest purity available. Two different preparations of Na-VPA were made for injection into the mice, one yielding 1 mg/kg body weight of VPA, the other 400 mg/kg, by the addition of NaOH in amounts equimolar to VPA in the two different concentrations. For both doses, the dose of radioactivity was 400 pCi/kg (14.7 MBq/kg); the rest of the dose was made up by non-labeled drug. The injection volume was 10 m l kg. Experiments On days 8 and 9 of gestation mice were injected subcutaneously, five with the low dose and five with the high dose, on each of the gestational days. They were killed in a carbon dioxide atmosphere at 30 min i 2 mice), 3 h (2 mice), and 6 h (1mouse), respectively. Plasma was collected by heart puncture. The abdomen was opened, the

uterine vessels were ligated, and the whole genital tract was removed. The abdominal wall was sutured. The animal and its genital tract were each embedded in a n aqueous gel of carboxymethyl cellulose and then immediately frozen in n-hexane cooled with dry ice (-78°C). Thereafter, thin (20 p,M) sections were cut while adherent to tape (3M) (Ullberg, 54,'77), through the whole animal and through the long axis of the uterus. The sections were allowed to dry a t a temperature of -2O"C, and were then apposed together with some [14C]Micro-scales (Amersham International plc) against Xray film (Industrex, Kodak) for 90 days. The film was then separated from the sections and was developed. Selected sections were stained in hematoxylin-eosin. Plasma levels of radioactivity were measured by liquid scintillation counting, and the level of VPA was measured by a GCMS-computer procedure (Nau et al., '81b).

702

L. DENCKER ET AL.

Quantification by computerized densitometry The concentration of radioactivity in maternal blood, decidua, amniotic fluid, and in the embryonic neuroepithelium and mesenchymal structures was determined by computerized densitometry. The optical density (OD) of the autoradiograms was measured with a solid-state black-and-white videocamera (Hitachi KP-230/231), which was mounted on a light microscope (Leitz Dialux). The camera was connected to a computer (Cromemco S2HD) through a video interface (Cromemco SDD), which digitized the light into 256 gray levels. The image is digitized in a format that is determined by the choice of objective, and consists of 384 x 241 pixels. The “unit area” (pixel) in this investigation was selected to be about 30 pm. The digitized picture was displayed on a color monitor, and regions for calculating of mean OD were selected interactively by using a digitizer pad (d’Argy et al., ’90). For each region, the average OD of three slices was used, which minimized the effect of any slight variations in slice thickness. The densitometric readings were further converted from OD to radioactivity (nCiig) by using the set of [14C] Micro-scales mentioned above.

administration of the low dose (Fig. lc). This was observed also with the high dose at the 6 h time interval. The high-dose animals showed an accumulation in calcified tissues. A comparison of the results obtained by densitometry with GC-MS measurements showed that in the plasma of the low dose mice, only 10-1576 of the radioactivity was represented by unchanged VPA. This may indicate a high rate of biotransformation at these low concentrations (below 1 kg/ml). In the high-dose mice, on the other hand, 7080% of the radioactivity was unchanged VPA at 30 min (600 pg/ml) and 3 h (277 pg/ml), and 30-50% was unchanged at 6 h (28 pg/ml). This corresponds well with previous results in the pregnant mouse which indicate that, with teratogenic doses, the plasma levels of VPA exceeded those of the sum of the metabolites measured around the time period of maximal plasma concentrations.

Fetoplacental unit The autoradiograms illustrating the genital tract also showed a clear difference between the two dose levels. At 30 min, the radioactivity of all tissues as well as amniotic fluid was, when the low dose (1mg/kg) was given, far below that of the maternal blood, both a t day 8 and at day 9 (Figs. 3a,c). RESULTS The highest concentration was found in the Maternal neuroepithelium, and was above that of the The distribution of radioactivity in the embryonic mesenchyme and amniotic fluid. maternal body varied considerably between This tendency was even more pronounced at the low dose (1 mg/kg) and the high dose 3 h (Figs. 2b, 3a,c), when the radioactivity of (400 mgikg) of valproate. Thus, after the ad- the neuroepithelium was a t the level of that ministration of the low dose, the liver and of maternal blood and two to three times as the kidney cortex dominated the distribu- high as that of mesenchyme and amniotic tion pattern at all survival intervals (30 fluid. This was true also a t 6 h, although at min; Fig. l a , 3 h and 6 h), showing concen- a lower concentration. trations above that of the blood. After a high When the high dose (400 mg/kg) was addose, on the other hand, the blood concen- ministered, proportionally more of the dose tration exceeded that of all organs, both at reached the embryo. Clearly, at 30 min the 30 min (Fig. lb) and at 3 h. Another differ- amniotic fluid and all embryonic structures ence was that soft tissues such as muscle were “flooded”by radioactivity, resulting in and connective tissues including the brain higher concentrations there than in the mahad a much higher concentration of radio- ternal blood (Figs. 3b,d). The neuroepitheactivity after the high dose. Such a more lium in particular reached a high concentrablurred distribution pattern may indicate tion. At 3 h, all tissues and amniotic fluid that at the high dose an increased portion of had decreased to levels below that of the the drug-related radioactivity was unbound maternal blood except the neuroepithelium to plasma proteins. (Figs. 2a,c,d, 3b,d), which retained radioacInterestingly, the olfactory bulb of the tivity approximately at the same level as brain showed a marked accumulation of ra- found at 30 min. At 6 h, the concentration in dioactivity at all survival intervals after the most tissues had decreased considerably,

VALPROIC ACID IN NEUROEPITHELIUM DURING ORCANOGENESIS

Neural folds

Brain vesicle

Neural Amniotic tube fluid Fig. 2.

703

Brain vesicle

Amniotic fluid

Neural fold

Amniotic fluid

704

L. DENCKER ET AL.

while the radioactivity in neuroepithelium was still above that in the surrounding tissues and maternal blood. With a low dose, the decidua was always below maternal blood but above amniotic fluid, while after a high dose the radioactivity in the decidua was approximately the same as in the amniotic fluid.

the mother from 1 to 400 mgikg increased the concentration in the neuroepithelium (at 30 min) not 400-fold but approximately 2,000-fold. Considering the fact that higher doses apparently result in lower rates of biotransformation, i.e., in increased VPAI metabolite ratios in plasma, and that the measurement in the embryonic tissues is based entirely on radioactivity, it is likely DISCUSSION that the above-mentioned effect would be The main finding in this study was that even more pronounced if VPA specifically the target organ for the teratogenic action of and not total radioactivity were measured. VPA, the neuroepithelium, showed a higher We are presently studying whether GC-MS uptake of VPA or metabolites than the ma- measurements can be accomplished in the ternal blood, amniotic fluid, and also other neuroepithelium and other relevant sites of embryonic tissues. The retention time in the the embryo during early organogenesis. neuroepithelium was also longer than in the Our present data may well help explain other embryonic structures. Various other the previous finding that it is not the total drugs, such as salicylic acid and hexachlo- dose, or the area under the concentrationrophene, accumulate in the neuroepithe- time curve value in maternal plasma or lium relatively non-specifically and with embryo, which determines the degree of terhitherto unknown mechanisms (for review, atogenicity of VPA, but rather the peak conDencker and Danielsson, '87). Retinoic acid, centration, which may only last for a short on the other hand, accumulates in specific period of time (Nau, '85). Thus, even though areas along the neural tube (Dencker et al., plasma concentrations of VPA measured '87), apparently bound to the cellular retin- with GC-MS decreased by half from 30 min oic acid binding protein. to 3 h, the radioactivity in embryonic neuWe have used two widely spaced dose lev- roepithelium stayed relatively constant. els in this study to determine the dose-de- This means that the concentrations in the pendency of the uptake of VPA-related ra- target organ for the teratogenic action probdioactivity in the embryo. The higher dose ably do not fluctuate as much as the conwas 10-20 times the human therapeutic centrations in the maternal serum when dose and produced peak drug concentrations bolus doses are administered. What the in mouse plasma which were four to six high-bolus dose accomplishes, which the times above the peak levels usually ob- same dose administered as an infusion does served during human therapy (Nau, '85, not, is the production of high concentrations '86). The lower dose level was chosen be- of free VPA that can enter the embryonic cause it corresponded to the dose made up compartment, where it can accumulate in from labeled drug without dilution with sufficient concentrations in the neuroepinon-radioactive VPA. The two dose levels thelium and be retained there for a considwere so widely spaced because we wanted to erable time interval. An interesting observation was made that test whether the transfer of VPA to the neuroepithelium was saturable, which would radioactivity accumulated in the olfactory imply an active or facilitated transport. bulb of the maternal brain, especially when Such a saturable uptake was not observed; low doses were administered. This accumuquite t o the contrary, increasing the dose to lation may be of interest in relation to the antiepileptic effect of VPA (Loscher and Nau, '83). Both experimental and clinical studies suggest that the anticonvulsant efFig. 2. Details of autoradiograms showing part of fect of VPA takes considerably longer to uteri with embryos from dams being on day 8 (a) and day 9 (b-d) of gestation. It is 3 h after the administra- build up during the initial drug treatment than expected from plasma kinetics; followtion of 1mg/kg (b) and 400 mgikg (a, c , d) of 14C-VPA to the mothers. The amount of radioactivity administered ing discontinuation of drug treatment, the was the same in each experiment (400 FCi or 14.7 MBq/ anticonvulsant effect persisted longer than kg bd. wt.). Note the relatively higher concentration of drug concentrations in plasma ("carryradioactivity (white areas) in amniotic fluid and embryover"). Both effects may well be explained onic structures (especially neuroepithelium of neural folds and neural tube) after the high dose. x 25. by accumulation and persistence of VPA in

705

VALPROIC ACID IN NEUROEPITHELIUM DURING ORGANOGENESIS nC$

ncvg

DAY 8 L O W DOSE DAY 9 LOW DOSE

300

300.

200

200

ELL.... 100

d.., '...

'.._ %..

100

C

a

nC@

300

!

0.5

HOURS

0.5

3

HOURS

nCvg DAY 8 HIGH DOSE

DAY 9 HIGH DOSE

300.

200.

100.

d

~~

0.5

i-

HOURS

i,

Fig. 3. Radioactivity concentration-time curves, determined by computerized densitometry, in maternal blood and embryonic structures after the administration of 14C-UPA to pregnant mice on day 8 (a,b)and

day 9 (c,d).Means i SE of four embryos from two dams (30 min, 3 h) or two embryos from one dam (6 h). SE not indicated if too low to be visible in figure, and not at 6 h since data are from only two embryos.

specific brain areas such as the olfactory bulb (Fig. lc), which has been considered to be of special significance in regard to antiepileptic therapy. A similar accumulation has previously been observed for other weak acids and has been interpreted a s a transport from the nasal mucosa, possibly by axonal flow, into the brain (Ghantous et al., '90).

ACKNOWLEDGMENTS

This study was supported by grants from the Swedish Medical Research Council (nr B89-04X-07899) and the Deutsche Forschungsgemeinschaft (SFB 174, C-06). LITERATURE CITED Bruckner, A,, Y.L. Lee, K.S. O'Shea, and R.C. Henneberry (1983) Teratogenic effects of valproic acid and

706

L. DENCKER ET AL.

diphenylhydantoin on mouse embryos in culture. Teratology, 27:29-42. Clarke, D.O., and N.A. Brown (1987) Valproic acid teratogenesis and embryonic lipid metabolism. Arch. Toxicol. [Suppl .1 11:143-1 47. Coakley, M.E., S.J. Rawlings, and N.A. Brown (1986) Short-chain carboxylic acids, a new class of teratogens: Studies of potential biochemical mechanisms. Environ. Health Perspect., 70: 105-1 11. dArgy, R., G.O. Sperber, B.S. Larsson, and S. Ullberg (1990) Computer-assisted quantification and image processing of whole- body autoradiograms. J. of Pharmacol. Methods (in press). Dencker, L., R. dArgy, B.R.G. Danielsson, H. Ghantous, and G.O. Sperber (1987) Saturable accumulation of retinoic acid in neural crest and neural crest derived cells in early embryonic development. Dev. Pharmacol. Ther., 10r212-223. Dencker, L., and B.R.G. Danielsson (1987) Transfer of drugs to the embryo and fetus after placentation. In: Pharmacokinetics in Teratogenesis. Vol. I: Interspecies Comparison and MaternaliEmbryonic-Fetal Drug Transfer. H. Nau, and W.J. Scott, eds. CRC Press, Boca Raton, Florida, pp. 56-69. Ghantous, H., L. Dencker, J. Gabrielsson, B.R.G. Danielsson, and K. Bergman (1990) Accumulation and turnover of metabolites of toluene and xylene in nasal mucosa and olfactory bulb. Toxicol. Pharmacol. 66:87-92. Jager-Roman, E., A. Deichel, S. Jacob, A. Hartmann, S. Koch, D. Rating, R. Steldinger, H. Nau, and H. Helge (1986) Fetal growth, major malformations and minor anomalies in infants born to women receiving valproic acid. J . Pediatr., 108:997-1004. Kao, J., N.A. Brown, B. Schmid, E.H. Goulding, and S. Fabro (1981) Teratogenicity of valproic acid In vivo and in vitro investigations. Teratogenesis Carcinog. Mutagen., 1 :367-382. ,ewandowski, C., S. Klua, H. Nau, and D. Neubert (1986) Pharmacokinetic- aspects of drug effects in vitro: Effects of serum protein binding on concentration and teratogenicity of valproic acid and 2-en-valproic acid in whole embryos in culture. Arch. Toxicol., 58:239-242. (indhout,D., and H. Meinardi (1984) Spina bifida and in-utero exposure to valproate. Lancet, iit396. Lindhout, D., and D. Schmidt (1986) In-utero exposure to valproate and neural tube defects. Lancet, i:13921393. Loscher, W., and H. Nau (1983) Distribution of valproic acid and its metabolites in various brain areas of dogs

and rats after acute and prolonged treatment. J. Pharmacol. Exp. Ther., 226:845-854. Nau, H. (1985) Teratogenic valproic acid concentrations: Infusion by implanted minipumps vs conventional injection regimen in the mouse. Toxicol. Appl. Pharmacol., 80t243-250. Nau, H. (1986) Transfer of valproic acid and its main active unsaturated metabolite to the gestational tissue: Correlation with neural tube defect formation in the mouse. Teratology, 33:21-27. Nau, H. and W.J. Scott, Jr (1986) Weak acids may act as teratogens because they accumulate in the basic milieu of the early mammalian embryo. Nature, 323: 276-278. Nau, H., and A.G. Hendrickx (1987) Valproic acid teratogenesis. IS1 Atlas Sci. Pharmacol., 1 :52-56. Nau, H., D. Rating, S. Koch, I. Hauser, and H. Helge (1981a) Valproic acid and its metabolites: Placental transfer, neonatal pharmacokinetics, transfer via mother’s milk and clinical status in neonates of epileptic mothers. J . Pharmacol. Exp. Ther., 219:768777. Nau, H., R. Zierer, H. Spielmann, D. Neubert, and C. Gansau (1981b) A new model for embryotoxicity testing: Teratogenicity and pharmacokinetics of valproic acid following constant-rate administration in the mouse using human therapeutic drug and metabolite concentrations. Life Sci., 29:2803-2814. Nau, H., W. Wittfoht, H. Schafer, C. Jakobs, D. Rating, and H. Helge (1981~)Valproic acid and several metabolites: Quantitative determination in serum, urine, breast milk and tissues by gas chromatography-mass spectrometry using selected ion monitoring. J . Chromatogr., 226r69-78. Robert, E., and F. Rosa (1983) Valproate and birth defects. Lancet, ii:1142. Trotz, M., C. Wegner, and H. Nau (1987) Valproic acidinduced neural tube defects: Reduction by folinic acid in the mouse. Life Sci., 41:103-110. Ullberg, S. (1954) Studies on the distribution and fate of S35-labeled benzylpenicillin in the body. Acta Radiol. [Suppl.], 118:l-110. Ullberg, S. (1977) The technique of whole body autoradiography. Cryosectioning of large specimens. Sci. Tools (LKB Instru. J.) Special issue on Whole-Body Autoradiography, pp. 2-29. Wilder, B.J., and B.J. Karas (1982) Valproate: Relation of plasma concentration to seizure control. In: Antiepileptic Drugs, 2nd ed. D.M. Woodbury, J.K. Penry, and C.E. Pippenger, eds. Raven Press, New York, pp. 591599.

Marked accumulation of valproic acid in embryonic neuroepithelium of the mouse during early organogenesis.

Valproic acid, an antiepileptic drug, causes neural tube defects in mice and man. 14C-labeled valproic acid (sodium-salt) was administered to pregnant...
764KB Sizes 0 Downloads 0 Views