TERATOLOGY 54150-156 (1996) All-Trans-Retinoyl-P-Glucuronide Is a Potent Teratogen in the Mouse Because of Extensive Metabolism to All-Trans-RetinoicAcid H. NAU, M.M.A. ELMAZAR, R. ROHL, R. THIEL, AND J.O. SASS Znstitut fur Toxikologie und Embryopharmakologie, Fachbereich Humunmedizin, Freie Universitiit Berlin, 0-14195 Berlin, Germany (H.N., R.R., R.T.); King Suud University, College of Pharmacy, Riyudh 1145,Saudi Arabia M.M.A.E.); Universitatsklinik fur Kinder- und Jugendheilkunde, A-9020 Innsbruck, Austria (J.O.S.); Department of Food Toxicology, Veterinary Medical University of Hannover, 0-30173 Hannover, Germany (H.N.) Al I - trans- retinoyl- p - D- g IucuABSTRACT ronide (all-trans-RAG) is a water-soluble derivative of all-frans-retinoic acid (all-frans-RA) and has been characterized as an endogenous metabolite of vitamin A in rat bile and kidney. All-frans-RAG was previously demonstrated to be a maior metabolite after application of all-trans-RA in several species (mouse, rat, rabbit, monkey); all-transRAG was described in these experiments to exhibit a very low placental transfer to the embryo. Because retinoid-like activity has been found after application of all-frans-RAG in vivo as well as in several in vitro systems, and because of its low placental transfer, this glycoconjugate appeared to be an interesting retinoid with possible therapeutic activity, but reduced teratogenicity. Here we investigated the teratogenic activity of all-transRAG in comparison to all-frans-RA in mice, and performed accompanying pharmacokinetic studies. Surprisingly,all-frans-RAGwas moreteratogenic than equimolar doses of all-frans-RA following subcutaneous application on day 11 of gestation in the mouse (20 prnol/kg body weight). Pharmacokinetic studies revealed that all-trans-RAG was extensively hydrolyzed to all-trans-RA and that the plasma area under the concentration-time curve (AUC) of all-trans-RA following all-trans-RAG application exceeded the plasma AUC value of allfrans-RA following application of all-frans-RA. Extensive hydrolysis of all-trans-RAG was also observed after intravenous application of this glycoconjugate. Transfer of all-frans-RAG to the embryo was low, but transfer was high to maternal organs such as the liver and kidney. These in vivo studies suggest that all-trans-RAG serves as a precursor of all-trans-RA by the intravenous and subcutaneous routes, and application of all-transRAG results in high and teratogenic in vivo exposure to all-frans-RA. o 1996 WiIey-Liss, Inc. tabolite in rat bile, small intestine, and liver (Dunagin et al., '65; Zile e t al., '82; McCormick et al., '83). This glycoconjugate of all-truns-retinoic acid (all-truns-RA) was also purported to occur as a n endogenous component in human plasma (Barua and Olson, '86). Subsequently, all-truns-RAG was identified as a major metabolite after application of pharmacologic or toxic doses of retinol and all-truns-RA to mice (Creech Kraft et al., '91a), rats (Collins e t al., '95), rabbits (Tzimas et al., '94a), and monkeys (Eckhoff et al., '90, '91; Creech Kraft et al., '91b; for reviews see Nau, '93, '94). p-Glucuronides of retinol, 13-cis-retinoic acid and 9-cis-retinoic acid (Eckhoff et al., '91; Creech &aft et al., '91a,b; Sass e t al., '94, '95; Tzimas e t al., '94b), were also identified and quantitated following administration of the respective aglycones. All-truns-RAG attracted attention because it exerted retinoid-like biological activity in a variety of in vitro systems such a s the cultured human promyelocytic leukemia cell line HL-60 (Zile et al., '87; Janick-Buckner et al., '91) and mammary gland in organ culture (Mehta et al., '91). In these systems, all-truns-RAG acted potently in regard to inhibition of proliferation and induction of differentiation. Administration of alltrans-RAG promoted the growth of vitamin A deficient rats. However, from those experiments it was not clear if all-truns-RAG acted directly or following hydrolysis to all-truns-RA. When analytical measurements were performed it became clear that a considerable portion of all-truns-RAG had been hydrolyzed in vitro and the resulting all-truns-RA was found in the medium as well as in the cultured tissue (Tzimas et al., '94c; Ruhl et al., '94; Foerster et al., '96). Considerable hydrolysis was also observed after intraperitoneal (i.p.) application of a tracer dose of 3H-all-truns-RAG in the rat Received April 22, 1996; accepted July 29, 1996. All-truns-retinoyl-p-D-glucuronide(all-truns-RAG) was first identified as an endogenous vitamin A me- 0 1996 WILEY-LISS, INC. Address reprint requests to Dr. Heinz Nau, Dept. of Food Toxicology, Veterinary Medical University of Hannover, Biinteweg 15/115, D-30173 Hannover, Germany. ALL-!l"S-RETINOYL-P-GLUCURONIDE (Barua and Olson, '89). Retinoid glucuronides do not interact with cytosolic retinoid binding proteins (Mehta et al., '92) or retinoid receptors (Sani et al., '92), which could be taken as an argument against a direct action of these glycoconjugates. Also further retinoid-glycoconjugates were shown to exert biological activity including all-truns-retinoyl-pglucose (Barua and Olson, '91, '92), all-truns-retinyl-pglucuronide (Barua and Olson, '871, all-truns-retinyl-pglucose (Barua and Olson, ,921, the p-glucuronide of N-(4-hydroxypenyl)-retinamide(Bhatnagar et al., '91) and derivatives (Doepner et al., '92; Kaleagasioglu et al., '93; Panigot et al., '94; Robarge et al., '94). Teratogenicity is an extremely serious side effect of essentially all active retinoids synthesized and tested up to now (Kochhar, 1967; for a review, cf. Nau et al., 1994). It is likely that the teratogenic activity is mediated by nuclear retinoid receptors which may also be crucially involved in the pharmacological effect of retinoid drugs in dermatology or oncology (Chambon, '93). A number of retinoid receptors have been shown to be expressed in embryonic tissue in a very specific spatial-temporal fashion, and it appears to be extremely difficult to develop novel retinoids which interact with the retinoid receptors in the desired target tissue of pharmacologic action, but do not interact with embryonic receptors. We have been following another lead to retinoids with low teratogenic activity: retinoids exhibiting limited transfer to the embryo. We have demonstrated that all-truns-RAG, produced as metabolite of alltruns-RA, was transferred to the embryo to a very limited degree, and only a few percent of maternal plasma all-truns-RAG concentrations were found in the embryo of the mouse (Creech Kraft et al., '91a), rat (Collins et al., '94, '951, rabbit (Tzimas et al., '94a; Collins et al., '95), and monkey (Hummler et al., '94). Very low embryo/maternal plasma concentration ratios were also found for the p-glucuronides of 13-cis-retinoic acid (Creech-Kraft et al., '91a; Tzimas et al., '94a), 9 4 s retinoic acid (Sass et al., '94; Tzimas et al., '94b) and retinol (Collins et al., '94). The low placental transfer of retinoid-p-glucuronides, together with the biological activity of these glycoconjugates in vitro discussed above, prompted us to study the teratogenic activity of all-trans-RAG in vivo in mice. Gestational day (GD) 11 represents a suitable stage for treatment of mice, in order to assess teratogenic potencies of retinoids by morphologic evaluation of near-term fetuses; resorption, growth retardation, cleft palate and limb reduction defects are reliable end-points (Kochhar and Satre, '93). Parallel pharmacokinetic studies were performed for the rational interpretation of the results from our teratology study. Area under the concentration-time curve (AUC) values were used as exposure parameters because they have been previously shown to correlate well with teratogenic activity of retinoids (Nau, '90, '94). 151 MATERIALS AND METHODS Laboratory precautions All work with retinoids was performed under dim amber light to avoid photodegradation. Retinoids and their solutions as well as biological samples for retinoid analysis were stored at -20°C. Chemicals Retinoic acid isomers were provided by Hoffmann-La Roche (Basel, Switzerland). All-truns-RAGwas synthesized following a modification of the method of Barua and Olson ('87, '89) which has been reported recently (Foerster et al., '96). All chemicals for the synthesis were generously provided by BASF AG (Ludwigshafen, Germany). Organic solvents of highest purity and trifluoracetic acid were purchased from Merck (Darmstadt, Germany). Water was purified with a Milli-Q water purification system (Millipore, Eschborn, Germany). Animals NMRI mice (Han: NMRI; Zentralinstitut fiir Versuchstierzucht, Hannover, Germany, or Harlan-Winkelmann, Borchen, Germany) were kept under specific pathogen free (spf) conditions and a 12-hr standard light-dark cycle. They received a standard pellet diet (Altromin 1324; Altromin, Lage, Germany) and tap water ad libitum. The animals were mated during 2-hr period in the morning. The following 24 hr interval was considered gestational day (GD)0. Teratologic study On GD 11,mice received a single subcutaneous (s.c.) dose of 20 pmol all-truns-RAG or all-truns-RAkg body weight (b.wt.). The dosing volume was 1 mlkg; the vehicle was dimethyl sulfoxide (DMSO). A control group was administered the pure solvent. On GD 18, the dams were killed by cervical dislocation. Implantation sites, resorptions, and live fetuses were counted. Live fetuses were weighed individually and examined for external malformations. The fetuses were then preserved in 5% formalin solution until examined for internal malformations. Skeletal abnormalities were examined after staining with alizarin red S. Results are compared with those of the vehicle control group using Student's t-test (fetal weight) or chi-square test (others). Toxicokinetic study Pregnant mice were treated with 20 pmol alltruns-RA or all-trans-RAGlkg b.wt. on GD 11 as described for the teratologic study. To examine retinoid pharmacokinetics in the plasma of one group of mice, serial blood samples were collected under brief ether anesthesia from the retroorbital sinus into heparinized capillary tubes 0.25,0.5,1,2,4,8,16,24,32, and 48 hr after treatment. Plasma was prepared by centrifuga- 152 H. NAU ET AL. TABLE 1. Prenatal effects induced by all-trans-RA or all-trans-RAG (both 20 pmolkg, s.c.) in NMRI mice on GD 11 No. of litters Implantations Resorptions Live fetuses Fetal weight ( g k SD) Exencephaly Kinked tail Full-length cleft palate' Dislocated hind limb Extra ribs' Dislocated sternum2 Radius (bent)' Radius (short)' Ulna (bent) Control' (%) 9 110 13 (11.8) 97 (88.2) 1.30? 0.11 0 (0) 0 (0) 0 (0) 3 (3.1) 10 (10.3) 9 (9.28) (0) (0) (0) (0) all-trans-RA(%) 9 108 6 (5.6) 102 (94.4) 1.24 f 0.13** (0) l(0.98) 4 (3.9)* 6 (5.9) 15 (14.7) 14 (13.7) (0) (0) (0) all-trans-RAG (%) 12 142 16 (11.3) 126 (88.7) 1.23 f 0.11** 2 (1.6) 0 (0) 27 (21.4)** 4 (3.2) 62 (49.2)** 40 (31.7)** 25 (19.8)** 12 (9.5)** 4 (3.2) 3 (2.4) Ulna (short) (0) 'One ml DMSOkg b.wt. S.C. 'Significantly higher in the all-trans-RAG-treatedgroup than in the all-tram-RA-treated group. *P < 0.05;**P< 0.01;compared with the control group using the Student's t-test (fetal weight) and chi-squaretest (others). tion of the blood for 10 min at 1,500g and 4°C. Two other groups of mice were also administered all-transRAG as described. These animals were killed by cervical dislocation 2 and 6 hr after treatment, respectively, after blood had been collected. Plasma was prepared from this blood as described, and embryos, yolk sacs, placentas, and maternal livers, kidneys, and spleens were collected. To reduce their blood content, liver samples were rinsed with ice-cold 0.9% (w/v) aqueous NaCl solution. All tissues were weighed and immediately frozen. For examination of plasma retinoid concentrations at different time points after intravenous 6v.) administration, non-pregnant (female) mice were administered the all-trans-RAG solution or an equimolar solution of all-trans-RA in DMSO. They received a single i.v. dose (injected into the tail vein) of 5 kmol/kg (corresponding to 10 p1 dosing solution per mouse). (eluent A: water + 0.2% trifluoracetic acid; eluent B: acetonitrile + 0.2% trifluoracetic acid) which has been developed especially for separation of isomers of retinoyl-f3-D-glucuronide and retinoic acid (Sassand Nau, '94). Calibration was performed using solutions of bovine serum albumin spiked with defined concentrations of reference compounds. Plasma AUC values were calculated using the trapezoidal rule. RESULTS Teratology Both all- trans-RA and all-truns-RAG were administered on GD 11 in the NMRI mouse with a dose of 20 kmol/kg b.wt. As expected, this administration regimen was clearly teratogenic with all-trans-RA and a number of retinoid-specific malformations were observed such as limb defects, other skeletal defects, and cleft palate (Table 1). The fetal weight was slightly reduced by both drugs, and the embryolethality was Retinoid analysis not affected. Plasma, embryo, and yolk sac samples were exSurprisingly, all-trans-RAG exhibited a higher tertracted with a threefold volume of isopropanol, folatogenic potency compared to all-trans-RA: retinoidlowed by solid phase extraction according to the specific malformations such as limb reduction defects method described by Collins et al. ('92). Analytes were and cleft palate were more pronounced after applicaenriched on AASP C2 solid phase extraction cartridges tion of all-trans-RAG than after all-trans-RA (Table I). (silica modified with ethyl groups; ICT, Bad Homburg, Germany). Sample preparation of maternal tissues was Toxicokinetics based on this method. However, prior to extraction I.v. administration. All-trans-RA as well as allwith isopropanol, the tissues-except livers-were homogenized in an equal volume of water. If necessary, trans-RAG, dissolved in DMSO, were administered to tissues were minced with scissors before homogeniza- non-pregnant mice via the i.v. route. The concentration. Livers were homogenized with 9 volumes of ice- tions observed in plasma are shown in Figure 1. cold 0.9% (w/v) NaCl solution in a teflon-glass potter. Both drugs reached high initial concentrations. AllFollowing solid phase extraction, retinoid concentra- trans-RA was rapidly cleared, while the elimination of tions were determined with a reversed phase high per- all-trans-RAG from mouse plasma proceeded much formance liquid chromatographic (HPLC) method more slowly. Most importantly, considerable concen- ALL-TRANS-RETINOYL-P-GLUCURONIDE 153 A B all-trans-RAG i.v. all-trans-RA i . v . 104, all-trans-RAG 103 102, 100 I 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Time [hours] Time [hours] Fig. 1. Plasma concentrations of retinoids following i.v. administration of 5 pmoykg b.wt. all-transRAG (A) or all-trans-RA (B) in NMRI mice. 1 o3 ,RAG lo3 1 o2 a I I - trans-RA 10' I 0 . I ' I ' I * I 2 4 6 8 Time [ h o u r s ] 10' 0 2 4 6 8 Time [ h o u r s ] F5g. 2. Plasma concentrations of retinoids (mean ? SD, n = 3-6) following 8.c. administrationof 20 pmol/kg b.wt. all-trans-RAG(A) or all-trans-RA (B) in NMRI mice; samples from retro-orbital sinuses. Kinetic analysis yields elimination half-lives for all-trans-RA and all-trans-RAG of 2.6 and 3.0 hr, respectively. 154 H.NAU ET AL. present in the embryo in minute concentrations only in spite of high maternal plasma levels (Table 3). In contrast, high concentrations of both all-trans-RA and alltrans-RAG were found in kidney, which even exceeded AUC,-,h, (FMhr) maternal plasma levels. Considerable levels of both Administration of All-trans-RA All-trans-RAG all-trans-RA and all-trans-RAG were observed in liver, All-trans-RA (n = 6) 0.44 f 0.11 Trace placenta, yolk sac, and spleen. Other isomers of RA and 2.04 f 1.07* 8.84 f 1.48 All-trans-RAG (n = 3) RAG were present in plasma and tissues in much lower *Significantlyhigher than the AUC of all-trans-RAfollowing concentrations. administrationof all-trans-RA(P < 0.01; Student's two-tailed t-test). DISCUSSION The most important finding of the present study was trations of all-trans-RA were found in plasma after administration of all-trans-RAG. Some hemolysis of the the high teratogenicity observed in mice following adblood was observed after i.v. application of the drug ministration of all-trans-RAG. The teratogenic potency of all-trans-RAG exceeded that of all-trans-RA in the preparations as well as the pure vehicle (DMSO). S.C. application. Plasma retinoid levels following experimental protocol of the present study. These reS.C. application of all-trans-RA and all-trans-RAG to sults are readily explained by our toxicokinetic study: mice are shown in Figure 2. Again, high initial con- all-trans-RA levels and AUC values following admincentrations of all-trans-RAG (Fig. 2A) as well as all- istration of all-trans-RAG were high because of contintrans-RA (Fig. 2B) were observed after application of uous hydrolysis of persistent all-trans-RAG levels. In the respective drugs. All-trans-RA was cleared rapidly contrast, all-trans-RA administration resulted in rapid after all-trans-RA administration, while all-trans-RAG clearance of this compound. Similar findings were obwas cleared more slowly. Again, considerable all- served after S.C. as well as i.v. administration regitrans-RA levels were observed after all-trans-RAG ap- mens. It is thus clear that all-trans-RAG acts as a preplication (Fig. 2A). The plasma concentrations of all- cursor for all-trans-RA, which is formed via hydrolysis trans-RAG following all-trans-RA application were in the maternal organism and can transfer to the emvery low. Little hemolysis was observed after S.C.ap- bryonic compartment and act as a teratogen. Previous studies showed that all-trans-RAG and its plication of the DMSO preparations. The plasma and tissue concentrations of retinoids isomers-formed as metabolites from the respective found 2 and 6 h r after administration of all-trans-RAG retinoic acid-transfer to the embryo in small concento pregnant mice are shown in Tables 2 and 3. The trations only: in several species (mouse, rat, rabbit, plasma AUC values after administration of the drug monkey) embryonic/maternal plasma ratios of allconjugate (Table 2) were 8.84 p M h r all-trans-RAG and trans-RAG were well below 0.1. It was therefore hoped 2.04 pM/hr all-tram-RA. The elimination half-life of that all-tram-RAG would be a n interesting retinoid all-trans-RAG in maternal plasma, determined from with low teratogenic activity. Our present results demthe data displayed in Figure 2A, was 3.0 h r (r = onstrate, however, that all-trans-RAG is readily hydro0.9999 for 2-8 hr). As expected, all-trans-RA exhibits lyzed to the teratogenic all-trans-RA after S.C. as well good transfer to the embryo, while all-trans-RAG was as i.v. administration. All-trans-RAG is therefore a TABLE 2. Plasma AUC values following administration of all-trans-RAor all-trans-RAG(both 20 pmolkg, s.c.) in NMRI mice on GD 11 TABLE 3. Plasma and tissue concentrations (nM) following S.C.administration of 20 pmoykg b.wt. all-trans-RAG to pregnant mice on GD 11 Plasma/ tissue Plasma Embryo Yolk sac Placenta Liver Kidney Spleen Time after administration (hr)' All-trans-RAG 2 6 2 6 2 6 2 6 2 6 2 6 2 6 1,703 f 674 854 193 18.5 4.2 37.2 f 15.2 465 2 199 482 f 227 634 t 256 883 -t 224 1,017 f 536 1,023 2 69.5 3,254 f 249 3,372 f 762 385 t 344 193 f 82.0 * * 13-cis-RAG Concentrations (nM) 9-cis-RAG All-trans-RA 46.4 25.5 64.9 17.6 26.6 f22.7 * 30.5 * 15.9 -2 * - -2 -2 -2 <20 20.9 L25.4 <20 21.1 k23.9 - - 115 f 19.5 195 62.4 291 41.6 8.90 7.0 7.60 2 3.3 111 2 16.8 106 f 29.2 132 f 17.2 20.5 7.2 11.7 3.9 'N = 5 mice (2 hr); N = 4 mice (6 hr). 'Small peaks and/or overlapped by interfering fractions. * * * * * * * 555 91.2 14.8 2.8 424 f 226 23.9 t 4.9 378 f 208 21.2 2 9.6 468 f 144 - 4,070 f 1,051 729 2 89.3 2,003 t 71 123 f 24.0 307 f 191 - 13-cis-RA 23.9 6.5 9-cis-RA 9.73 7.3 23.6 +. 10.9 27.5 f 14.9 27.9 f 17.7 18.3 k24.7 4.02 2 2.7 * - - -2 * - - 18.2 ? 7.3 - -2 -2 307 +. 220 258 58.1 2 15.4 34.1 ? 9.9 21.1 k23.4 - - 2 - - 161 ALL-TRANS-RETINOYL-P-GLUCURONIDE 155 precursor for all-trans-RA after systemic application, and the hoped-for advantage of all-trans-RAG over alltrans-RA in regard to an improved safety ratio is not seen. The recent report that all-trans-RAG is of low teratogenicity after oral application in rats (Gunning et al., '93) must be interpreted with great caution: alltrans-RAG was only poorly absorbed under the experimental protocol used in that study (oral application). Our present study (s.c. application) clearly shows that all-trans-RAG, if absorbed, is a potent teratogen in vivo. Further experiments need to be undertaken to show if all-trans-RAG has a n advantage over all-trans-RA in other administration regimens. All-trans-RAG has been described to be less irritant to skin than alltrans-RA following topical application; however, much higher dosages of topical all-trans-RAG than topical all-trans-RA had to be used to exert a pharmacological effect (Gunning et al., '94). The possible systemic exposure to all-trans-RA following topical all-trans-RAG application has not yet been studied. The present results suggest that all-trans-RAG will be teratogenic if available systemically and that this retinoid should not be used in women of childbearing age until a formulation can be developed which exerts the desired pharmacological activity, but does not result in systemic retinoid exposure. ACKNOWLEDGMENTS This work was supported by the European Commission (BIOTECH program BI02-CT93-0471). The excellent technical assistance of C. Plum (HPLC) and I. Dillmann (teratology) is gratefully acknowledged. U. Schwikowski prepared the glossy prints of the figures. LITERATURE CITED Barua, A.B., and J.A. Olson (1986)Retinoyl p-glucuronide: An endogenous compound of human blood. Am. J. Clin. Nutr., 43:481-485. Barua, A.B., and J.A. Olson (1987) Chemical synthesis and growthpromoting activity of all-trans-retinyl p-D-glucuronide. Biochem. J., 244:231-234. Barua, A.B., and J.A. Olson (1989) Chemical synthesis of all-trans[ll-3Hlretinoyl p-glucuronide and its metabolism in rats in vivo. Biochem. J .,263:403 -409. Barua, A.B., and J.A. Olson (1991) All-trans retinoyl p-glucose: Chemical synthesis, growth-promoting activity, and metabolism in the rat. Int. J . Vit. Nutr. Res., 61 :258-263. Barua, A.B., and J.A. Olson (1992) Chemical synthesis, growth-promoting activity, and metabolism of all-trans retinyl p-glucose in the rat. Int. J. Vit. Nutr. Res., 62:298-302. Bhatnagar, R., H. Abou-Issa, R.W. Curley, Jr., A. Koolemans-Beynen, M.L. Moeschberger, and T.E. Webb (1991) Growth suppression of human breast carcinoma cells in culture by N-(4-hydroxyphenyD retinamide and its glucuronide and through synergism with glucarate. Biochem. Pharmaml., 41:1471-1477. Chambon, P. (1993) The molecular and genetic dissection of the retinoid signalling pathway. Gene, 135:223-228. Collins, M.D., C. Eckhoff, I. Chahoud, G. Bochert, and H. Nau (1992) 4-Methylpyrazol partially ameliorated the teratogenicity of retinol and reduced the formation of all-trans-retinoic acid in the mouse. Arch. Toxicol., 66:652-659. Collins, M.D., G. Tzimas, H. Hummler, H. Burgin, and H. Nau (1994) Comparative teratology and transplacental pharmamkinetics of all-trans-retinoic acid, 13-cis-retinoic acid, and retinyl palmitate following daily administrations in rats. Toxicol. Appl. Pharmacol., 127:132-144. Collins, M.D., G. Tzimas, H. Burgin, H. Hummler, and H. Nau (1995) Single versus multiple dose administration of all-trans-retinoic acid during organogenesis: Differential metabolism and transplacental kinetics in rat and rabbit. Toxicol. Appl. Pharmacol., 130:9-18. Creech Kraft, J., C. Eckhoff, D.M. Kochhar, G. Bochert, I. Chahoud, and H. Nau (1991a) Isotretinoin (13-cis-retinoic acid) metabolism, cis-trans isomerization, glucuronidation and transfer to the mouse embryo: Consequences for teratogenicity. Teratogen. Carcinogen. Mutagen., 11:21-30. Creech Kraft, J., W. Slikker, Jr., J.R. Bailey, L.G. Roberts, B. Fischer, W. Wittfoht, and H. Nau (1991b) Plasma pharmacokinetics and metabolism of 13-cis- and all-trans-retinoic acid in the cynomolgus monkey and the identification of 13-cis- and all-tmns-retinoyl-pglucuronides. A comparison to one human case study with isotretinoin. Drug Metab. Dispos. 19:317-324. Doepner, G., C.-D. Gerharz, U. v.Deessen, K. Kaiser, and H.K. Biesalski (1992) Effects of novel retinoids on growth and differentiation of a rhabdomyosareoma cell line. Arzneim. Forsch./Drug Res., 42fW: 1036-1040. Dunagin, P.E., Jr., E.H. Meadows, Jr., and J.A. Olson (1965) Retinoyl beta-glucuronic acid A major metabolite of vitamin A in rat bile. Science, 148:86-87. Eckhoff, C., W.Wittfoht, H. Nau, and W. Slikker, Jr. (1990) Characterization of oxidized and glucuronidated metabolites of retinol in monkey plasma by thermospray liquid chromatography/mass spectrometry. Biomed. Environ. Mass Spectrom., 19:428-433. Eckhoff, C., J.R. Bailey, M.D. Collins, W. Slikker, Jr., and H. Nau (1991)Influence of dose and pharmaceutical formulation of vitamin A on plasma levels of retinyl esters and retinol and metabolic generation of retinoic acid compounds and p-glucuronides in the cynomolgus monkey. Toxicol. Appl. Pharmacol., 111:116-127. Foerster, M., J.O. Sass, R. Ruhl, and H. Nau (1996) Comparative studies on effects of all-trans-retinoic acid and all-trans-retinoy1-Pglucuronide on the development of fetal mouse thymus in a n organ culture system. Toxicol. In Vitro, 10:7-15. Gunning, D.B., A.B. Barua, and J.A. Olson (1993) Comparative teratogenicity and metabolism of all-trans-retinoic acid, all-transretinoyl p-glucose, and all-tram-retinoyl p-glucuronide in pregnant Sprague-Dawley rats. Teratology, 47:29-36. Gunning, D.B., A.B. Barua, R.A. Lloyd, and J.A. Olson (1994) Retinoyl P-glucuronide: A nontoxic retinoid for the topical treatment of acne. J. Dermatol. Treat., 5:181-185. Hummler, H., A.G. Hendrickx, and H.Nau (1994) Maternal toxicokinetics, metabolism and embryo exposure following a teratogenic dosing regimen with 13-cis-retinoic acid (isotretinoin) in the cynomolgus monkey. Teratology, 50:184-193. Janick-Buckner, D., A.B. Barua, and J.A. Olson (1991) Induction of HL60 cell differentiation by water-soluble and nitrogen-containing conjugates of retinoic acid and retinol. FASEB J., 5:320-325. Kaleagasioglu, F., G. Doepner, H.K.Biesalski, and M.R. Berger (1993) Antiproliferative activity of retinoic acid and some novel retinoid derivatives in breast and colorectal cancer cell lines of human origin. Anneim. ForschJDrug Res., 43fZ):487-490. Kochhar, D.M. (1967) Teratogenic activity of retinoic acid. Acta Pathol. Microbiol. Sand., 70:398-404. Kochhar, D.M., and M.A. Satre (1993) Retinoids and fetal malformations. In: Dietary Factors and Birth Defects. R.P. Sharma, ed. San Francisco: Pacific Division AAAS, pp. 134-229. McCormick, A.M., K.D. Kroll, and J. Napoli (1983) 13-cis-retinoic acid metabolism in vivo. Biochemistry, 22:3933-3940. Mehta, R.G., A.B. Barua, J.A. Olson, and R.C. Moon (1991) Effects of retinoid glucuronides on mammary gland development in organ culture. Oncology, 48:505-509. Mehta, R.G., A.B. B a a , J.A. Olson, and R.C. Moon (1992) Retinoid glucuronides do not interact with retinoid binding proteins. Int. J. Vit. Nutr. Res., 62:143-147. 156 H. NAU ET AL. Nau, H. (1990) Correlation of transplacental and maternal pharmacokinetics of retinoids during organogenesis with teratogenicity. Methods Enzymol., 190:437-448. Nau, H. (1993) Teratogenesis, transplacental pharmacokinetics, and metabolism of some retinoids in the mouse, monkey, and human. In: Retinoids: Progress in Research and Clinical Applications. M.A. Livrea and L. Packer, eds. New York: Marcel Dekker, pp. 599615. Nau, H. (1994) Toxicokinetics and structure-activity relationships in retinoid teratogenesis. Ann. Oncol., 5(Suppl. 9):S39443. Nau, H., I. Chahoud, L. Dencker, E. Lammer, and W.J. Scott (1994) Teratogenicity of vitamin A and retinoids. In: Vitamin A in Health and Disease. R. Blomhoff, ed. New York: Marcel Dekker, pp. 615664. Panigot, M.J., K.A. Humphries, and R.W. Curley, Jr. (1994) Preparation of 4-retinamidophenyl- and 4-retinamidobenzyl-C-glycosyl and C-glucuronosyl analogues of the glucuronide of 4-hydroxyphenyl-retinamide as potential stable cancer chemopreventive agents. J. Carbohydr. Chem., 13:303-321. Fbbarge, M.J., J.J. Repa, K.K. Hanson, S. Seth, M. Clagett-Dame, H. Abou-Issa, and R.W. Curley (1994) N-linked analogs of retinoid 0-glucuronides: Potential cancer chemopreventivelchemotherapeutic agents. Bioorg. Med. Chem. Lett., 4:2117-2122. Ruhl, R., D. Hofmann, H. Nau, and S. Klug (1994) Influence of alltrans-retinoyl-p-D-glucuronide on growth and differentiation in two in vitro systems of different biological complexity. Arch. Pharm a d , 349(SupplJ:R108. Sani, B.P., A.B. Barua, D.L. Hill, T.-W. Shih, and J.A. Olson (1992) Retinoyl p-glucuronides: Lack of binding to receptor proteins of retinoic acid as related to biological activity. Biochem. Pharmacol., 43:919-922. Sass, J.O., and H. Nau (1994) Single-run analysis of isomers of retinoyl-p-D-glucuronideand retinoic acid by reversed-phase high- performance liquid chromatography. J. Chromatogr., 685:182188. Sass, J.O., G. Tzimas, and H. Nau (1994) 9-cis-retinoyl-p-D-glucuronide is a major metabolite of 9-cis-retinoic acid. Life Sci., 54:PL 69-74. Sass, J.O.,E. Masgrau, J.-H. Saurat, and H. Nau (1995) Metabolism of oral 9-cis-retinoic acid in the human: Identification of 9-cis-retinoyl-P-glucuronide and 9-cis-4-oxo-retinoyl-~-glucuronide as urinary metabolites. Drug Metab. Dispos., 23:887-891. Tzimas, G., H. Biirgin, M.D. Collins, H. Hummler, and H. Nau (1994a) The high sensitivity of the rabbit to the teratogenic effects of 13-cis-retinoic acid (isotretinoin) is a consequence of prolonged exposure of the embryo to 13-cis-retinoic acid and 13-cis-4-0x0-retinoic acid, and not of isomerization to all-tmns-retinoic acid. Arch. Toxicol., 68: 119-128. Tzimas, G., J.O. Sass, W. Wittfoht, M.M.A. Elmazar, K. Ehlers, and H. Nau (1994b) Identification of 9,13-dicis-retinoic acid as a major plasma metabolite of 9-cis-retinoic acid and limited transfer of 9-cis-retinoic acid and 9,13-dicis-retinoic acid to the mouse and rat embryos. Durg Metab. Dispos., 22:928-936. Tzimas, G., J.O. Sass, R. Ruhl, S. Klug, M.D. Collins, and H. Nau (1994~)Proximate retinoid teratogens. In: From Basic Science to Clinical Applications. M.A. Livrea and G. Vidali, eds. Base1 Birkhauser Verlag, pp. 179-195. Zile, M.G., R.C. Inhorn, and H.F. DeLuca (1982) Metabolism in vivo of all-trans-retinoic acid. Biosynthesis of 13-cis-retinoic acid and alltmns- and 13-cis-retinoyl glucuronides in the intestinal rnucosa of the rat. J . Biol. Chem., 257:3544-3550. Zile, M.H., M.E. Cullum, R.U. Simpson, A.B. Barua, and D.A. Swartz (1987) Induction of differentiation of human promyelocytic leukemia cell line HL-60 by retinoyl glucuronide, a biologically active metabolite of vitamin A. Roc. Natl. Acad. Sci. U.S.A., 84:22082212.