Correlations between conceptal concentrations of all-trans-retinoic acid and dysmorphogenesis after microinjections of all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-retinoyl-beta-glucuronide, or retinol in cultured whole rat embryos.

Retinol (4,000 ng/ml), all-trans-retinoyl-beta-glucuronide (4,000 ng/ml), and 13-cis-retinoic acid (1,500 ng/ml) each produced dysmorphogenic effects qualitatively similar to those elicited by 250 ng/ml of all-trans-retinoic acid after microinjections of the respective individual retinoids into the amniotic cavities of cultured whole rat embryos. Subsequent HPLC analyses of the cultured whole conceptuses, embryos proper, yolk sacs, and culture media (24 hr after microinjections) indicated that conceptal biotransformation of each of the retinoids had occurred during the culture period. All-trans-retinoic acid was present in the embryos proper at quantitatively similar concentrations (20-100 nM) after microinjections of the selected quantities of each of the microinjected retinoids: retinol, all-trans-retinoyl-beta-glucuronide, 13-cis-retinoic acid, or all-trans-retinoic acid. The results suggested that all-trans-retinoic acid acted as an ultimate dysmorphogen for the retinoids tested with respect to the anomalies monitored in the embryo culture system.     

Steady state pharmacokinetics of oral treatment with 13-cis-retinoic acid or all-trans-retinoic acid in male and female adult rats

Male and female Sprague-Dawley rats were orally gavaged with 13-cis-retinoic acid (7.5 or 15 mg/kg) or all-trans-retinoic acid (10 or 15 mg/kg) for 7 consecutive days. Blood was collected out to 8 hr after the last gavage on day 7. HPLC serum concentrations of 13-cis-retinoic acid, all-trans-retinoic acid, and 13-cis-4-oxo-retinoic acid were subjected to model independent pharmacokinetic analyses. Peak serum levels of 563 to 1640 ng/ml were observed for rats treated with 13-cis-retinoic acid at 1.5-2 hr after gavage. Peak serum levels of 183 to 267 ng/ml at 1.5 hr after gavage were observed for all-trans-retinoic acids. The elimination half-life of 13-cis-retinoic acid was about 1.5 hr while the elimination half-life of all-trans-retinoic acid was slightly longer. There were no sex differences for any parameter. Serum levels resulting from the 7.5 mg/kg 13-cis-retinoic acid were similar to those of human Accutane users.     

Plasma pharmacokinetics and metabolism of 13-cis- and all-trans-retinoic acid in the cynomolgus monkey and the identification of 13-cis- and all-trans-retinoyl-beta-glucuronides. A comparison to one human case study with isotretinoin.

In order to compare the disposition and metabolism of 13-cis-retinoic acid (13-cis-RA) and all-trans-retinoic acid (all-trans-RA) in the nonpregnant female cynomolgus monkey, the plasma concentrations of the parent compound, the oxidized metabolites 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-retinoic acid, and the conjugate metabolites 13-cis-retinoyl-beta-glucuronide (13-cis-RAG) and all trans-retinoyl-beta-glucuronide (all-trans-RAG), were determined on day 1 and day 10 after oral dosing of 2 and 10 mg 13-cis- and all-trans-RA/kg/day. Both 13-cis-RAG and all-trans-RAG have been identified as major plasma metabolites in these studies using thermospray/HPLC/mass-spectrometry of the intact conjugates. AUC comparisons from 0-24 hr after administration indicated that 13-cis-RA treatment resulted in primarily cis metabolites and all-trans-RA treatment resulted in primarily trans metabolites, although low levels of isomerization products were observed. Comparison of the two doses (2 and 10 mg/kg, po) revealed that the AUCs were proportional to the dose administered. Although qualitatively similar, elimination of 13-cis-RA in the monkey was more rapid than in the human, and approximately a 10-fold greater dose of 13-cis-RA was required in the monkey to produce the AUC values comparable to the human. The elimination of all-trans-RA in monkey was faster than that of 13-cis-RA and tended to increase with repeated dosing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Teratogenicity and placental transfer of all-trans-, 13-cis-, 4-oxo-all-trans-, and 4-oxo-13-cis-retinoic acid after administration of a low oral dose during organogenesis in mice

13-cis-Retinoic acid (isotretinoin) is teratogenic in humans at therapeutic doses (0.5-1.5 mg/kg) but only marginally teratogenic in the mouse at a high dose of 100 mg/kg. Previous results explained why the cis isomer of retinoic acid was much less teratogenic than the trans isomer in mice. It was found that the placental transfer of all-trans retinoic acid to the mouse embryo was far greater than that of the 13-cis isomer. Since our previous study had been performed with exceedingly high doses (100 mg/kg) of 13-cis-retinoic acid and all-trans-retinoic acid, we have now performed additional experiments with 10-fold lower doses. Studies were also done with the main metabolites of the two retinoids (the 4-oxo-derivatives) to elucidate the metabolism, pharmacokinetics, and teratogenicity of each single compound. It was shown that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid were extremely teratogenic, whereas their corresponding cis isomers caused only 2% cleft palate. Embryonic exposure to the trans isomers was likewise higher than that to the cis isomers, as shown by the far higher embryonic peak concentrations and by the 30-fold higher areas under the concentration-time curve values reached for the trans isomers compared with the cis isomers. At 8 hr, embryo/maternal plasma ratios were higher than 1 after administration of the all-trans compounds. Concentrations found in the placenta and yolk sac were higher for the trans forms than for the cis forms. We propose a model for a facilitated transport of the all-trans forms to the developing embryo and suggest that the conversion to the trans isomer and trans metabolite could play a major role in the teratogenicity of 13-cis-retinoic acid in humans.     

Embryonic subcellular distribution of 13-cis- and all-trans-retinoic acid indicates differential cytosolic/nuclear localization.

Isotretinoin (13-cis-retinoic acid [13CRA], Accutane) is used for the treatment of dermatological diseases. Isotretinoin is, however, teratogenic in animals and humans. The mechanism of action of its teratogenicity is still not clearly identified. It has little or no binding properties to cytosolic retinoid-binding proteins or nuclear retinoid receptors (RAR, RXR). One hypothesis is that the teratogenicity of 2 approximately equipotent teratogenic doses of 13CRA and all-trans-retinoic acids (ATRA) could mainly be correlated to ATRA in the nuclei, where the retinoic acid receptors (RARs) are located. To test this hypothesis, female mice at gestational day 11 were treated with approximately equipotent teratogenic doses of 13-cis-retinoic acid (100 mg/kg orally) or all-trans-retinoic acid (10 mg/kg orally) and sacrificed 1 h and 4 h after administration. Embryos were homogenized and centrifuged into 4 fractions, and the purity of the fractions was tested by quantification of marker constituents for various cell compartments. We analyzed, by RP-HPLC, nuclear, mitochondrial, microsomal, and cytosolic fractions, as well as embryo homogenate and maternal plasma. After treatment with 13-cis-retinoic acid, this substance was mainly located in the nuclear fraction of the embryo (approximately 82%), whereas all-trans-retinoic acid, after ATRA treatment, was mainly located in the cytosolic supernatant (approximately 64%). The binding to cellular retinoid-binding protein (CRABP) may limit the access of ATRA to the nucleus, in contrast to 13CRA, which does not bind to CRABP. The concentration of ATRA in the nuclear fraction was similar after administration of either 13CRA or ATRA. The teratogenic activity of 13-cis-retinoic acid could therefore be explained by its access to the nucleus and its possible conversion to all-trans-retinoic acids, which will interact with the nuclear retinoid receptors.     

Low teratogenicity of 13-cis-retinoic acid (isotretinoin) in the mouse corresponds to low embryo concentrations during organogenesis: comparison to the all-trans isomer

13-cis-Retinoic acid (isotretinoin) is teratogenic in man at therapeutic doses (0.5-1.5 mg/kg body wt), but only marginally teratogenic in the mouse at exceedingly high doses (greater than 100 mg/kg). On the other hand, the isomer all-trans-retinoic acid (tretinoin) is teratogenic in the mouse at dose levels which are 10 times lower than those for the 13-cis isomer. We have therefore studied whether the greatly different teratogenic potencies of these two compounds in the mouse are the result of differences in their transplacental kinetics. Following a single oral dose of 100 mg all-trans- or 13-cis-retinoic acid per kg body wt, concentrations of the parent drugs, of the C-13 isomerization products, as well as of their 4-oxo metabolites were determined in maternal plasma and embryo at two sensitive stages of organogenesis, i.e., Days 9 or 11 of gestation. All-trans-retinoic acid and its 4-oxo metabolite were transferred to the embryo to a much greater extent (embryo/maternal plasma concentration ratios, approximately 0.4) than the 13-cis-retinoic acid and its 4-oxo metabolite (embryo/maternal plasma concentration ratios, approximately 0.02). Embryo concentrations of all-trans-retinoic acid on Day 9 of gestation exceeded those on Day 11, whereas the embryo levels of 13-cis-retinoic acid were minimal at both gestational stages. The concentration of the 4-oxo metabolite of all-trans-retinoic acid was generally lower than that of the parent drug, whereas the level of the 4-oxo metabolite of the 13-cis-retinoic acid was comparable with or even higher than that of the parent compound. Concentrations of the C-13 isomerization products in maternal plasma were less than 20% of corresponding parent drug levels. However, due to the different extent of transfer of the two isomers, the concentration of all-trans-retinoic acid in the embryo exceeded that of the cis isomer even after administration of 13-cis-retinoic acid. Our results indicate that the low teratogenicity of 13-cis-retinoic acid in the mouse is the result of minimal placental transfer of this compound and of its 4-oxo metabolite, which contrast sharply with extensive placental transfer and high teratogenicity of the corresponding isomers with the all-trans configuration.     

Identification of 4-oxo-13-cis-retinoic acid as the major metabolite of 13-cis-retinoic acid in human blood

The metabolites of 13-cis-retinoic acid (Accutane) were investigated in blood samples from human volunteers on chronic treatment for dermatological disorders. The major metabolite was isolated by reverse-phase high-pressure liquid chromatography and identified as 4-oxo-13-cis-retinoic acid by comparison of its mass and NMR spectra to the spectra of the reference compound. 4-Oxo-all-trans-retinoic acid was also identified, but the extent to which this compound was a metabolite of 13-cis-retinoic acid or an artifactual isomerization product of the major metabolite is unknown. Chromatographic data suggested that small amounts of 13-cis-retinoic acid, 4-hydroxy-13-cis-retinoic acid, and dioxygenated metabolites of 13-cis-retinoic acid may also be present in the blood. This study indicates that a major metabolic pathway of 13-cis-retinoic acid in humans is oxidation at C4 of the cyclohexenyl group.     

Disposition of all-trans- and 13-cis-retinoic acids and n-hydroxyethylretinamide in mice after intravenous administration

The disposition of 13-cis- and all- trans-retinoic acids and N-hydroxyethylretinamide has been studied in mice after an intravenous dose of 10 mg/kg. As determined by high-ressure liquid chromatography, serum levels of 13-cis-retinoic acid, in a distribution phase, decreased rapidly over a 30-min period. This was followed by an apparently exponential phase of elimination. N-Hydroxyethylretinamide had a distribution phase lasting for 1 hr followed by an exponential phase. All-trans-retinoic acid had a similar distribution phase, but this was followed by a nonexponential phase. Tissue levels of 13-cis-retinoic acid, although generally not as high as those in serum, decreased in a manner similar to that for serum. Levels of all-trans-retinoic acid in liver, kidney, lung, brain, and small intestine were generally higher than those of serum throughout the period of observation. At 8 hr after injection of the mice, relatively high levels of all-trans-retinoic acid remained in the brains, and detectable levels of 13-cis-retinoic acid persisted in the lungs. N-Hydroxyethylretinamide also persisted in tissues for long periods of time, with tissue levels generally higher than those in serum. At 18 hr after injection of mice, relatively high levels of this compound were found in the liver, kidneys, and testes.     

Chronic oral treatment with 13-cis-retinoic acid (isotretinoin) or all-trans-retinoic acid does not alter depression-like behaviors in rats.

Oral treatment with the anti-acne drug Accutane (isotretinoin, 13-cis-retinoic acid) has been associated with suicide ideation and depression. Here, depression-like behaviors (i.e., behavioral despair and anhedonia) were quantified in adult Sprague-Dawley rats gavaged daily beginning at postnatal day (PND) 82 with 13-cis-RA (7.5 or 22.5 mg/kg) or all-trans-retinoic acid (10 or 15 mg/kg ). Tested at PND 130-131 in the Forced Swim Test, 7.5 mg/kg 13-cis-RA marginally decreased immobility and slightly increased climb/struggle durations whereas neither all-trans-retinoic acid group differed from controls. Voluntary saccharin solution (0.03%) intake at PND 102-104 and PND 151-153 was not different from controls in any treated group, although all RA-treated groups had lower intakes. Swim speed in a water maze at PND 180 was similar across groups, indicating no RA-induced differences in physical ability. Open field activity was mildly decreased at PND 91 in 7.5 mg/kg-treated males only, but it was within the control range at PND 119, 147, and 175. Thus, at serum levels similar to those in humans receiving the drug, chronic 13-cis-RA treatment did not severely affect depression-like behaviors in rats. These data do not substantiate the hypothesis of 13-cis-RA-induced depression.     

Metabolism of 13-cis-retinoic acid by a rat liver 9000g supernatant preparation

The in vitro metabolites formed on incubation of 13-cis-retinoic acid (13-cis-RA, isotretinoin) with a 9000g rat liver supernatant system were isolated by HPLC and identified by their mass and NMR spectra. The major metabolic pathway was hydroxylation at C4 to give 4-hydroxy-13-cis-RA, which was rapidly oxidized to 4-oxo-13-cis-RA, the major isolated metabolite. Further metabolism of this 4-oxo metabolite led to two novel compounds, 2-hydroxy-4-oxo-13-cis-RA and 3-hydroxy-4-oxo-13-cis-RA. In addition, small amounts of 13-cis-RA and 4-oxo-13-cis-RA were enzymatically converted to their all-trans isomers. Support for these pathways was obtained by the metabolism of reference samples of 4-hydroxy-13-cis-RA, 4-oxo-13-cis-RA, all-trans-RA, and 4-oxo-all-trans-RA. The predominant formation of 4-oxo metabolites of 13-cis-RA in this in vitro rat system and the results from previously reported in vivo metabolism studies suggest that oxidation at C4 is a major metabolic pathway of 13-cis-RA in both rats and humans.     

Pharmacokinetics and metabolism of all-trans- and 13-cis-retinoic acid in pulmonary emphysema patients

Retinoids promote lung alveolarization in animal models and were administered to patients as part of the Feasibility of Retinoid Therapy for Emphysema (FORTE) study. This FORTE substudy investigated the pharmacokinetic profiles of 2 retinoic acid isomers-all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid (13-cRA)-in subjects with emphysema, evaluated strategies to overcome self-induced ATRA catabolism, and identified pharmacodynamic relationships. Comprehensive and limited pharmacokinetics were obtained at multiple visits in emphysema subjects treated with placebo (n = 30), intermittent dosing (4 days/week) with low-dose ATRA (1 mg/kg/day, n = 21), or high-dose ATRA (2 mg/kg/day, n = 25) or daily administration of 13-cRA (1 mg/kg/day, n = 40). High-dose ATRA produced the highest peak plasma ATRA Cmax. However, at follow-up, plasma ATRA C(max) was significantly decreased from baseline in subjects whose day 1 levels exceeded 100 ng/mL (P < .0001). In contrast, administration of 13-cRA produced lower plasma ATRA C(max) (<100 ng/mL), but the levels were significantly higher at follow-up than those on day 1 (P < .001). Plasma ATRA levels as determined on day 1 correlated with changes in pulmonary diffusing capacity at 6 months, consistent with concentration-dependent biologic effects (r2 = -0.25). The authors conclude that intermittent therapy with high-dose ATRA produced the greatest ATRA exposure, but alternative approaches for limiting self-induced ATRA catabolism should be sought.     

Quantitation of 4-oxo-4-(3-pyridyl)butanoic acid and enantiomers of 4-hydroxy-4-(3-pyridyl)butanoic acid in human urine: A substantial pathway of nicotine metabolism

A liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC-APCI-MS/MS) method was developed to analyze human urine for 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and the enantiomers of 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid) to test our hypothesis that (S)-hydroxy acid could be a biomarker of metabolic activation of the tobacco-specific carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) while (R)-hydroxy acid would be formed predominantly from nicotine, as indicated by studies with rats. Urine was collected from smokers, and from the same individuals after they had stopped smoking and used a nicotine transdermal system (nicotine patch) for 3 weeks. If (S)-hydroxy acid were a biomarker of NNK and NNN metabolic activation, its levels should be higher in the urine of smokers than in nicotine patch users because tobacco smoke, but not the nicotine patch, contains NNK and NNN. Internal standard, [2,2,3,3,4-D5]hydroxy acid, was added to an aliquot of urine, which was then subjected to solid phase extraction. The eluant containing hydroxy acid was esterified with acidic methanol, followed by treatment with (S)-(-)-alpha-methylbenzyl isocyanate, producing methyl-4(S)- or methyl-4(R)-[(S)-alpha-methylbenzylcarbamoyl]-4-(3-pyridyl)buta noate [(S,S)- or (R,S)-MMPB, respectively]. After HPLC purification, the MMPB diastereomers were separated and quantified by LC-APCI-MS/MS. Mean levels of (S)- and (R)-hydroxy acid were 14.1 +/- 8.0 and 1120 +/- 600 ng/mL, respectively, in smokers during ad lib smoking (n = 18), while the corresponding levels during nicotine patch use (n = 18) were 4.1 +/- 3.3 and 363 +/- 228 ng/mL. The amounts of (S)-hydroxy acid were far higher than could be formed from NNK and NNN, and the total amount of hydroxy acid indicated that it was a substantial urinary metabolite of nicotine, in contrast to results with rats. Therefore, the study was extended to quantify keto acid. This was accomplished by NaBH4 treatment of urine, which converted keto acid to hydroxy acid quantitatively, which was in turn analyzed as described above. Levels of keto acid while subjects were smoking and using the nicotine patch were 228 +/- 129 (n = 8) and 97.5 +/- 80.6 ng/mL (n = 8), respectively. These results indicate that conversion of nicotine to keto acid and hydroxy acid is a substantial metabolic pathway in humans, accounting for an estimated 14% of the nicotine dose. Apparently, keto acid is extensively converted to hydroxy acid in humans, in contrast to the results with rats. (S)-Hydroxy acid in human urine cannot be used as a biomarker of NNK and NNN metabolic activation because it is overwhelmed by the (S)-hydroxy acid formed from nicotine, despite the fact that >98% of the urinary hydroxy acid has the (R)-configuration. These results provide new insights about nicotine metabolism in humans.     

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 beta-glucuronides in the cynomolgus monkey

Retinoid concentrations were analyzed in plasma of nonpregnant female cynomolgus monkeys after oral administration of retinol or retinyl acetate at doses of 2, 10, or 50 mg (as retinol) per kilogram body weight dissolved in acetone/soybean oil (1/9) or acetone/Tween 20/water (1/5/4). All-trans-retinoic acid, 13-cis-retinoic acid, all-trans-4-oxoretinoic acid, and 13-cis-4-oxoretinoic acid as well as the conjugates of retinol and all-trans-retinoic acid with beta-D-glucuronic acid represented major polar plasma retinoids after high doses of vitamin A. The relative bioavailability of vitamin A as well as the biotransformation to more polar retinoids was independent of the molecular form of vitamin A (retinol or retinyl acetate) used for dosing. After administration of 2 mg/kg and, in particular, after a 10 mg/kg dose, the metabolic formation of polar retinoids in plasma was much more extensive with the detergent-based vehicle compared to the oil-based vehicle. At 50 mg/kg, comparable metabolism was observed for both forms. Polar metabolites of retinol were increased in a more than linear fashion with the detergent-based vitamin A preparations between 2 and 10 mg/kg and with the oil-based preparations between 10 and 50 mg/kg. Since retinoic acid compounds have previously been shown to be potent teratogens in various animal species and humans, their metabolic formation may be of significance for the teratogenic activity of high doses of vitamin A. In addition to the dose, the pharmaceutical preparation of vitamin A could therefore be a major determinant of the developmental toxicity of vitamin A.     

抗坏血酸对成骨细胞生长作用的研究

目的观察不同浓度的抗坏血酸对鼠成骨细胞生长的作用。方法在体外培养小鼠成骨细胞的过程中,分别加入不同浓度的抗坏血酸,通过检测碱性磷酸酶的含量观察其对成骨细胞的促进作用。结果抗坏血酸10ng/ml组、15ng/ml组对成骨细胞的生长均有较明显的促进作用,其中又以10ng/ml组最明显,25ng/ml组对成骨细胞的生长有抑制作用。结论抗坏血酸对成骨细胞的生长是剂量依赖性的,在10ng/ml这个浓度中对成骨细胞生长的促进作用是最显著的。     

Simultaneous determination of unlabeled and carbon-13-labeled etoricoxib, a new cyclooxygenase-2 inhibitor, in human plasma using HPLC-MS/MS

A method for the simultaneous determination of etoricoxib and its carbon-13 analog ((13)C(6)-etoricoxib) from human plasma has been developed and used to support bioavailability studies. Plasma samples (0.5 mL) were extracted by using a 3M Empore 96-well plate (C(8)) and the resulting extracts were analyzed by using a PE-Sciex API-3000 HPLC-MS/MS with a heated nebulizer interface (500 degrees C). The method was validated with two different calibration curve ranges, one for etoricoxib (5 to 2500 ng/mL) determined in the presence of lower concentrations of (13)C(6)-etoricoxib (0.5 to 250 ng/mL), and a second curve for the quantitation of similar concentrations of both etoricoxib and (13)C(6)-etoricoxib (0.5 to 250 ng/mL). Extraction recoveries of etoricoxib, (13)C(6)-etoricoxib, and a methylated internal standard were >70% over the range of concentrations included in both calibration curves. Intraday precision and accuracy for the quantitation of etoricoxib were 7.8% relative standard deviation (RSD) or less and within 3.4% respectively over the range of 5 to 2500 ng/mL, and 10.8% RSD or less and within 4 % respectively over the range of 0.5 to 250 ng/mL. Within-batch precision and accuracy for the quantitation of (13)C(6)-etoricoxib over the range of 0.5 to 250 ng/mL were 8.3% RSD or less and within 2.3%, respectively. The validated assay was used in support of human clinical trials.     

Transplacental pharmacokinetics and teratogenicity of a single dose of retinol (vitamin A) during organogenesis in the mouse

Pregnant mice received 10 or 100 mg retinol/kg body wt. by gavage on day 11 of gestation (plug day = day 0). One group of animals was used for a pharmacokinetic study. At various times after dosing, plasma and tissue samples were collected and analyzed by HPLC for retinyl esters, retinol, 13-cis- and all-trans-retinoic acid and 13-cis-4-oxo and all-trans-4-oxoretinoic acid. In the other group the fetuses were removed on day 18 and examined for malformations. After 10 mg/kg retinol, no teratogenic effect was observed. The pharmacokinetic investigation revealed a moderate increase of retinyl esters, retinol and all-trans-retinoic acid in plasma, embryonic tissue, placenta, yolk sac membranes and extraembryonic fluid. A high incidence of severe fetal malformations occurred after 100 mg/kg retinol. These malformations included limb defects (81% of fetuses) and cleft palate (55% of fetuses) which are characteristically found after administration of a single teratogenic dose of an active retinoid on day 11 of gestation. The concentration-time profile of retinoids after 100 mg/kg on day 11 showed a pronounced increase of retinyl esters and retinol in all compartments including the embryo and a massive generation of the polar metabolites all-trans-retinoic acid and all-trans-4-oxoretinoic acid. These polar metabolites were found in the embryo with peak concentrations of 327 +/- 115 and 143 +/- 20.7 ng/g (mean +/- SE) wet tissue, respectively. It is likely that all-trans-retinoic acid and all-trans-4-oxoretinoic acid, both well-known teratogens, largely contributed to the teratogenic outcome. The in-vivo oxidation of retinol may be an important factor in the teratogenic activity of high doses of vitamin A.     

Identification of human cytochrome P450 isoforms that contribute to all-trans-retinoic acid 4-hydroxylation

The role of specific human cytochrome P450 (CYP) isoforms in the oxidative metabolism of all-trans-retinoic acid was investigated by studies in human liver microsomes using isoform-specific chemical inhibitors and inhibitory antibodies. Studies using individual isoforms expressed in lymphoblastoid cells and correlation analysis using different microsome preparations were also performed. With expressed isoforms, evidence for a role for CYP2C8, CYP3A4, CYP2C9, and CYP1A1 in 4-hydroxylation was obtained, with the highest catalytic efficiency being observed for CYP2C8. Using inhibition studies and correlation analysis, we also concluded that CYP2C8 was the major all-trans-retinoic acid 4-hydroxylating cytochrome P450 in human liver microsomes, though CYP3A4 and, to a lesser extent CYP2C9, also made a contribution. In addition, we compared the rate of retinoic acid degredation in HepG2 cells when cultured in the absence and presence of 3-methylcholanthrene or all-trans-retinoic acid. Culture in the presence of all-trans-retinoic acid decreased the half-life twofold and resulted in an increased sensitivity of retinoic acid degredation to ketoconazole. Since no induction of either CYP1A1, CYP2C8, CYP2C9, or CYP3A4 was detected using immunoblotting and as mRNA encoding another cytochrome P450 enzyme, CYP26, has been previously demonstrated to be induced by retinoic acid treatment of HepG2 cells and to be highly sensitive to ketoconazole, this enzyme in addition to CYP2C8, CYP2C9 and CYP3A4 likely plays a role in all-trans-retinoic acid oxidation in the liver at high retinoic acid levels.     

Catalysis of the 4-hydroxylation of retinoic acids by cyp3a7 in human fetal hepatic tissues.

Cytochrome P4503A7 (CYP3A7) is the primary CYP isoform expressed in human fetal hepatic microsomes, and its potential role in human embryotoxicity has attracted considerable investigative attention. In this study, we investigated the 4-hydroxylation of highly embryotoxic and teratogenic retinoic acids (RA) as catalyzed by human fetal liver microsomes (HFLM) and demonstrated that CYP3A7 is an efficient RA hydroxylase. When all-trans-retinoic acid (tRA), 9-cis-retinoic acid (9cRA), or 13-cis-retinoic acid (13cRA) were incubated with HFLM (54-109 gestational days) plus NADPH, each of these three retinoic acids was rapidly converted to its corresponding 4-hydroxy and 4-oxo metabolites. The reactions were strongly inhibited by CO (CO:O(2), 80:20) and were NADPH-dependent, indicating that the reactions were catalyzed by P450 isoenzymes. At 54 to 89 gestational days, 4-hydroxylase activities were relatively low. However, at gestational days 96 to 109, activities were much higher. Selective inhibitors were employed for elucidation of the roles of individual CYP isoenzymes in HFLM. alpha-Naphthoflavone, paclitaxel, and diethyldithiocarbamate showed little or no effects on HFLM-catalyzed reactions, indicating that CYP1A1, CYP1A2, CYP1B1, CYP2C8, and CYP2E1 did not play significant roles in the catalysis. By contrast, troleandomycin strongly inhibited the reaction (70-75% inhibition), suggesting that CYP3A7 was primarily responsible for the observed catalysis. It was also discovered that CYP3A7 SUPERSOMES efficiently catalyzed the 4-hydroxylations of tRA, 9cRA, and 13cRA. Because 4-hydroxylated metabolites of RA are much less potent embryotoxins and teratogens, the results indicated that the 4-hydroxylation of RA, catalyzed prenatally by CYP3A7, might play an important role in protecting the human fetus against RA-induced embryotoxicity.