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Vitamin A teratogenicity and risk assessment in the macaque retinoid model
Reproductive Toxicology 15 (2001) 445– 447
www.elsevier.com/locate/reprotox
Letter to the editor Vitamin A teratogenicity and risk assessment in the macaque retinoid model To the Editor: We read with great interest the article by Hendrickx et al., recently published in your journal [1]. While this paper provides valuable information on the teratogenicity of vitamin A in the macaque retinoid model, we think that the human risk assessment approach is inadequate and therefore has led the authors to misleading conclusions, including their statement (last sentence in the abstract) about “. . . safe levels of vitamin A during human pregnancy in the range of ⬃25,000 to 37,000 IU/day.” The available data on the metabolism of vitamin A in humans and cynomolgus monkeys from previous studies, which were not cited in the paper, do not fully justify the choice of the macaque model as the most suitable one for human risk assessment. Thus, the -glucuronides of retinol and all-trans-RA, two major plasma metabolites of vitamin A in monkeys [2,3] but not in humans [4,5] were not determined by Hendrickx et al. Also, neither 9,13-di-cisRA, nor 9-cis-RA, nor 14-hydroxy-4,14-retro-retinol, recently identified in human plasma following liver consumption [5], was searched for in the study by Hendrickx et al., probably due to analytical limitations. Furthermore, Hendrickx et al. suggested that the teratogenic potency of vitamin A is less than that of 13-cis-RA in the monkey based on comparative data for maternal exposure to 13-cis-RA following 13-cis-RA or vitamin A administration. This appears very problematic to us, mainly due to the following reasons: (1) The hypothesis that the teratogenicity of 13-cis-RA and vitamin A result from the action of the same proximate teratogen (13-cis-RA) is totally arbitrary. (2) The authors have apparently overlooked maternal exposure to all-trans-RA and 13-cis-4-oxo-RA in their study. Nevertheless, even in the presence of relatively low plasma concentrations of all-trans-RA following vitamin A dosing, embryonic exposure to all-trans-RA may be pronounced, as was recently observed in vitamin A dosed rabbits and was ascribed to efficient transplacental passage of all-trans-RA to the embryo and/or metabolic generation of all-trans-RA from retinol taking place in the embryo [6]. (3) The identification of the ultimately teratogenic metabolite of vitamin A in the monkey is virtually impossible in the absence of embryonic retinoid exposure data. Unfortu-
nately, embryonic retinoid pharmacokinetics following dosing with vitamin A were not examined in the monkey study. In light of the aforementioned criticism, we consider the validity of the risk assessment for the human, as presented by Hendrickx et al. [1], questionable. The authors attempted to estimate safe levels of vitamin A intake by humans on the basis of Recommended Dietary Allowances (RDA) in humans and monkeys and the NOAEL in the monkey study, or, alternatively, based on the NOAEL/safety factor concept. We are seriously concerned about the soundness of these concepts, because (1) the experimentally derived NOAEL is only an estimate of the true threshold dose of vitamin A for teratogenicity; (2) interspecies differences in pharmacokinetics and metabolism were not considered; therefore, the safety factor (10) used for extrapolation to the human is far too low; and (3) there is no evidence to conclude that the NOAEL/RDA ratio is the same for humans and monkeys. Finally, the vitamin A intakes proposed by the authors as safe (25,000 to 37,000 IU/day) may actually endanger the health of the mother and the developing conceptus. It has previously been shown that a daily consumption of 25,000 IU can lead to liver cirrhosis [7] and intakes greater than 5,000 IU/day can lead to reduced bone density and a doubling of the risk of hip fracture [8]. For all the aforementioned reasons, we disagree with the proposal by Hendrickx et al. [1] about “safe dose levels of vitamin A during pregnancy in the range of 25,000 to 37,000 IU/day.” In contrast, we see no reason to alter the opinion of both the Teratology Society [9] and the European Teratology Society [10] that overall vitamin A intake by women should not exceed 8,000 to 10,000 IU/day during pregnancy in order to avoid any risk for developmental toxicity.
0890-6238/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 0 - 6 2 3 8 ( 0 1 ) 0 0 1 4 6 - 0
Georg Tzimas Tzimas-Dimolios, Co., Edessis 3, GR-546 25 Thessaloniki, Greece Heinz Nau Department of Food Toxicology, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173, Hannover, Germany E-mail address: [email protected]
446
Letters to the Editor / Reproductive Toxicology 15 (2001) 445– 447
References [1] Hendrickx AG, Peterson P, Hartmann D, Hummler H. Vitamin A teratogenicity, and risk assessment in the macaque retinoid model. Reprod Toxicol 2000;14:311–23. [2] Eckhoff C, Wittfoht W, Nau H, Slikker W Jr. Characterization of oxidized and glucuronidated metabolites of retinol in monkey plasma by thermospray liquid chromatography/mass spectrometry. Biomed Environ Mass Spectrom 1990;19:428 –33. [3] Eckhoff C, Bailey JR, Collins MD, Slikker W Jr, Nau H. 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 -glucuronides in the cynomolgus monkey. Toxicol Appl Pharmacol 1991;111:116 –127. [4] Eckhoff C, Collins MD, Nau H. Human plasma all-trans-, 13-cis- and 13-cis-4-oxoretinoic acid profiles during subchronic vitamin A supplementation: Comparison to retinol and retinyl ester plasma levels. J Nutr 1991;121:1016 –25. [5] Arnhold T, Tzimas G, Wittfoht W, Plonait S, Nau H. Identification of 9-cis-retinoid acid, 9,13-di-cis-retinoid acid, and 14-hydroxy-4,14retro-retinol in human plasma after liver consumption. Life Sci 1996; 59PL:169 –77. [6] Tzimas G, Collins MD, Bu¨rgin H, Hummler H, Nau H. Embryotoxic doses of vitamin A to rabbits result in low plasma but high embryonic concentrations of all-trans-retinoic acid: Risk of vitamin A exposure in humans. J Nutr 1996;126:2159 –71. [7] Geubel AP, De Galocsy C, Alves N, Rahier J, Dive C. Liver damage caused by therapeutic vitamin A administration: Estimate of doserelated toxicity in 41 cases. Gastroenterology 1991;100:1701–9. [8] Melhus H, Michaelsson K, Kindmark A, Bergstrom R, Holmberg L, Mallmin H, Wolk A, Ljunghall S. Excessive dietary vitamin A intake is associated with reduced bone mineral density and increased risk for hip fracture. Ann Intern Med 1998;129:770 –78. [9] Public Affairs Committee of the Teratology Society. Recommendations for vitamin A use during pregnancy. Teratology 1987;35:269 – 75. [10] Dolk H, Nau H, Hummler H, Barlow SM. Dietary vitamin A and teratogenic risk: European Teratology Society Discussion Paper. Eur J Obst Gynecol Reprod Biol 1999;83:31– 6.
In reply: The comments by Heinz Nau and Georg Tzimas are surprising to the authors because of our past collaborations, and joint publications on retinoid teratogenicity, and toxicokinetics in the macaque model. First, it is generally accepted that safety assessment is not done following a well-defined formula, rather it is amenable to variable approaches depending on numerous factors. It is also generally accepted that there is no single animal species that is identical to the human regarding dosage, time of exposure, metabolism, and placental transfer. However, for any given experimental situation or safety assessment, it is desirable to utilize the animal that most closely models the human. In our investigation [1], the cynomolgus macaque was selected because of its very close similarity to the human in normal embryonic development and the considerable experience with 13-cis-retinoic acid (13-cis-RA), a known human teratogen. Several statements in the letter of Nau and Tzimas regarding the pathway of glucuronidation need to be clarified. The glucuronides were not determined in our studies, because previous research has documented that after admin-
istration of 13-cis-RA, the -glucuronides show much lower plasma concentrations than the parent compounds in monkeys [2,3] as well as in humans [4]. In contrast, -glucuronidation predominates in the rabbit [5]. Moreover, there is limited placental passage of the -glucuronides in all species examined to date, including the mouse, rat, rabbit, and monkey [6]. The metabolites 9,13-di-cis-RA, 9-cis-RA, and 14-hydroxy-4,14-retroretinol in monkeys were not measured since the role of these compounds in teratogenicity has not been established. Following administration of vitamin A, 13-cis-RA and 13-cis-4-oxo-RA are the major metabolites with regard to plasma concentrations (not necessarily amounts) in the human and in the cynomolgus monkey. In contrast, the major plasma metabolite in the rabbit is 9,13di-cis-RA, which only crosses the placenta to a limited extent and is not biologically active [7]. In our human studies, 9,13-di-cis-RA in plasma was below the limit of quantification of the assay in most subjects. We feel that not all compounds present in plasma must be analyzed, unless there is a suspected relationship to the effect being investigated. Regarding the proximate teratogen issue, the transplacental transfer of both all-trans-RA, and 13-cis-RA has been measured in the primate model [2,6]. Although it would be of considerable interest to similarly determine these values for the parent compound (vitamin A), it was not feasible to undertake this aspect in the study under discussion [1]. However, as noted in our paper, further monkey and human work is forthcoming and many, but not all of the issues raised by Nau and Tzimas will be addressed. Collectively, the studies with cynomolgus monkeys have shown that the teratogenic effect induced by 13-cis-RA in this species cannot solely result from the action of all-trans-RA, but may include 13-cis-RA and 13-cis-4-oxo-RA [6]. The results of transplacental transfer studies in the rabbit appear more conflicting. There was no difference in embryonic exposure to all-trans-RA between nonteratogenic and teratogenic doses of 13-cis-RA [8]. The transplacental transfer of 13-cis-RA and 13-cis-4-oxo-RA was low, but the teratogenic effect was attributed to prolonged exposure to these retinoids [5]. Following embryonic doses of vitamin A, no 13-cis-RA concentrations were measurable in the embryo, and the concentrations of all-trans-RA were only slightly elevated above endogenous levels. Therefore, alltrans-RA was claimed to be the proximate teratogen in the rabbit under those experimental conditions [7]. It is noteworthy that the embryonic exposure to endogenous alltrans-RA is substantial (embryo/maternal plasma ratio ⫽ 15). These data indicate that the issue on the proximate teratogen is not yet clear. Results in the embryo should not be overemphasized, particularly since measurements do not allow differentiation of free and bound retinoids. Furthermore, embryo data are not commonly used for risk assessment, but rather provides information on cellular/molecular mechanisms.
www.elsevier.com/locate/reprotox
Letter to the editor Vitamin A teratogenicity and risk assessment in the macaque retinoid model To the Editor: We read with great interest the article by Hendrickx et al., recently published in your journal [1]. While this paper provides valuable information on the teratogenicity of vitamin A in the macaque retinoid model, we think that the human risk assessment approach is inadequate and therefore has led the authors to misleading conclusions, including their statement (last sentence in the abstract) about “. . . safe levels of vitamin A during human pregnancy in the range of ⬃25,000 to 37,000 IU/day.” The available data on the metabolism of vitamin A in humans and cynomolgus monkeys from previous studies, which were not cited in the paper, do not fully justify the choice of the macaque model as the most suitable one for human risk assessment. Thus, the -glucuronides of retinol and all-trans-RA, two major plasma metabolites of vitamin A in monkeys [2,3] but not in humans [4,5] were not determined by Hendrickx et al. Also, neither 9,13-di-cisRA, nor 9-cis-RA, nor 14-hydroxy-4,14-retro-retinol, recently identified in human plasma following liver consumption [5], was searched for in the study by Hendrickx et al., probably due to analytical limitations. Furthermore, Hendrickx et al. suggested that the teratogenic potency of vitamin A is less than that of 13-cis-RA in the monkey based on comparative data for maternal exposure to 13-cis-RA following 13-cis-RA or vitamin A administration. This appears very problematic to us, mainly due to the following reasons: (1) The hypothesis that the teratogenicity of 13-cis-RA and vitamin A result from the action of the same proximate teratogen (13-cis-RA) is totally arbitrary. (2) The authors have apparently overlooked maternal exposure to all-trans-RA and 13-cis-4-oxo-RA in their study. Nevertheless, even in the presence of relatively low plasma concentrations of all-trans-RA following vitamin A dosing, embryonic exposure to all-trans-RA may be pronounced, as was recently observed in vitamin A dosed rabbits and was ascribed to efficient transplacental passage of all-trans-RA to the embryo and/or metabolic generation of all-trans-RA from retinol taking place in the embryo [6]. (3) The identification of the ultimately teratogenic metabolite of vitamin A in the monkey is virtually impossible in the absence of embryonic retinoid exposure data. Unfortu-
nately, embryonic retinoid pharmacokinetics following dosing with vitamin A were not examined in the monkey study. In light of the aforementioned criticism, we consider the validity of the risk assessment for the human, as presented by Hendrickx et al. [1], questionable. The authors attempted to estimate safe levels of vitamin A intake by humans on the basis of Recommended Dietary Allowances (RDA) in humans and monkeys and the NOAEL in the monkey study, or, alternatively, based on the NOAEL/safety factor concept. We are seriously concerned about the soundness of these concepts, because (1) the experimentally derived NOAEL is only an estimate of the true threshold dose of vitamin A for teratogenicity; (2) interspecies differences in pharmacokinetics and metabolism were not considered; therefore, the safety factor (10) used for extrapolation to the human is far too low; and (3) there is no evidence to conclude that the NOAEL/RDA ratio is the same for humans and monkeys. Finally, the vitamin A intakes proposed by the authors as safe (25,000 to 37,000 IU/day) may actually endanger the health of the mother and the developing conceptus. It has previously been shown that a daily consumption of 25,000 IU can lead to liver cirrhosis [7] and intakes greater than 5,000 IU/day can lead to reduced bone density and a doubling of the risk of hip fracture [8]. For all the aforementioned reasons, we disagree with the proposal by Hendrickx et al. [1] about “safe dose levels of vitamin A during pregnancy in the range of 25,000 to 37,000 IU/day.” In contrast, we see no reason to alter the opinion of both the Teratology Society [9] and the European Teratology Society [10] that overall vitamin A intake by women should not exceed 8,000 to 10,000 IU/day during pregnancy in order to avoid any risk for developmental toxicity.
0890-6238/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 0 - 6 2 3 8 ( 0 1 ) 0 0 1 4 6 - 0
Georg Tzimas Tzimas-Dimolios, Co., Edessis 3, GR-546 25 Thessaloniki, Greece Heinz Nau Department of Food Toxicology, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173, Hannover, Germany E-mail address: [email protected]
446
Letters to the Editor / Reproductive Toxicology 15 (2001) 445– 447
References [1] Hendrickx AG, Peterson P, Hartmann D, Hummler H. Vitamin A teratogenicity, and risk assessment in the macaque retinoid model. Reprod Toxicol 2000;14:311–23. [2] Eckhoff C, Wittfoht W, Nau H, Slikker W Jr. Characterization of oxidized and glucuronidated metabolites of retinol in monkey plasma by thermospray liquid chromatography/mass spectrometry. Biomed Environ Mass Spectrom 1990;19:428 –33. [3] Eckhoff C, Bailey JR, Collins MD, Slikker W Jr, Nau H. 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 -glucuronides in the cynomolgus monkey. Toxicol Appl Pharmacol 1991;111:116 –127. [4] Eckhoff C, Collins MD, Nau H. Human plasma all-trans-, 13-cis- and 13-cis-4-oxoretinoic acid profiles during subchronic vitamin A supplementation: Comparison to retinol and retinyl ester plasma levels. J Nutr 1991;121:1016 –25. [5] Arnhold T, Tzimas G, Wittfoht W, Plonait S, Nau H. Identification of 9-cis-retinoid acid, 9,13-di-cis-retinoid acid, and 14-hydroxy-4,14retro-retinol in human plasma after liver consumption. Life Sci 1996; 59PL:169 –77. [6] Tzimas G, Collins MD, Bu¨rgin H, Hummler H, Nau H. Embryotoxic doses of vitamin A to rabbits result in low plasma but high embryonic concentrations of all-trans-retinoic acid: Risk of vitamin A exposure in humans. J Nutr 1996;126:2159 –71. [7] Geubel AP, De Galocsy C, Alves N, Rahier J, Dive C. Liver damage caused by therapeutic vitamin A administration: Estimate of doserelated toxicity in 41 cases. Gastroenterology 1991;100:1701–9. [8] Melhus H, Michaelsson K, Kindmark A, Bergstrom R, Holmberg L, Mallmin H, Wolk A, Ljunghall S. Excessive dietary vitamin A intake is associated with reduced bone mineral density and increased risk for hip fracture. Ann Intern Med 1998;129:770 –78. [9] Public Affairs Committee of the Teratology Society. Recommendations for vitamin A use during pregnancy. Teratology 1987;35:269 – 75. [10] Dolk H, Nau H, Hummler H, Barlow SM. Dietary vitamin A and teratogenic risk: European Teratology Society Discussion Paper. Eur J Obst Gynecol Reprod Biol 1999;83:31– 6.
In reply: The comments by Heinz Nau and Georg Tzimas are surprising to the authors because of our past collaborations, and joint publications on retinoid teratogenicity, and toxicokinetics in the macaque model. First, it is generally accepted that safety assessment is not done following a well-defined formula, rather it is amenable to variable approaches depending on numerous factors. It is also generally accepted that there is no single animal species that is identical to the human regarding dosage, time of exposure, metabolism, and placental transfer. However, for any given experimental situation or safety assessment, it is desirable to utilize the animal that most closely models the human. In our investigation [1], the cynomolgus macaque was selected because of its very close similarity to the human in normal embryonic development and the considerable experience with 13-cis-retinoic acid (13-cis-RA), a known human teratogen. Several statements in the letter of Nau and Tzimas regarding the pathway of glucuronidation need to be clarified. The glucuronides were not determined in our studies, because previous research has documented that after admin-
istration of 13-cis-RA, the -glucuronides show much lower plasma concentrations than the parent compounds in monkeys [2,3] as well as in humans [4]. In contrast, -glucuronidation predominates in the rabbit [5]. Moreover, there is limited placental passage of the -glucuronides in all species examined to date, including the mouse, rat, rabbit, and monkey [6]. The metabolites 9,13-di-cis-RA, 9-cis-RA, and 14-hydroxy-4,14-retroretinol in monkeys were not measured since the role of these compounds in teratogenicity has not been established. Following administration of vitamin A, 13-cis-RA and 13-cis-4-oxo-RA are the major metabolites with regard to plasma concentrations (not necessarily amounts) in the human and in the cynomolgus monkey. In contrast, the major plasma metabolite in the rabbit is 9,13di-cis-RA, which only crosses the placenta to a limited extent and is not biologically active [7]. In our human studies, 9,13-di-cis-RA in plasma was below the limit of quantification of the assay in most subjects. We feel that not all compounds present in plasma must be analyzed, unless there is a suspected relationship to the effect being investigated. Regarding the proximate teratogen issue, the transplacental transfer of both all-trans-RA, and 13-cis-RA has been measured in the primate model [2,6]. Although it would be of considerable interest to similarly determine these values for the parent compound (vitamin A), it was not feasible to undertake this aspect in the study under discussion [1]. However, as noted in our paper, further monkey and human work is forthcoming and many, but not all of the issues raised by Nau and Tzimas will be addressed. Collectively, the studies with cynomolgus monkeys have shown that the teratogenic effect induced by 13-cis-RA in this species cannot solely result from the action of all-trans-RA, but may include 13-cis-RA and 13-cis-4-oxo-RA [6]. The results of transplacental transfer studies in the rabbit appear more conflicting. There was no difference in embryonic exposure to all-trans-RA between nonteratogenic and teratogenic doses of 13-cis-RA [8]. The transplacental transfer of 13-cis-RA and 13-cis-4-oxo-RA was low, but the teratogenic effect was attributed to prolonged exposure to these retinoids [5]. Following embryonic doses of vitamin A, no 13-cis-RA concentrations were measurable in the embryo, and the concentrations of all-trans-RA were only slightly elevated above endogenous levels. Therefore, alltrans-RA was claimed to be the proximate teratogen in the rabbit under those experimental conditions [7]. It is noteworthy that the embryonic exposure to endogenous alltrans-RA is substantial (embryo/maternal plasma ratio ⫽ 15). These data indicate that the issue on the proximate teratogen is not yet clear. Results in the embryo should not be overemphasized, particularly since measurements do not allow differentiation of free and bound retinoids. Furthermore, embryo data are not commonly used for risk assessment, but rather provides information on cellular/molecular mechanisms.