Biomedical subjects

H Nau

Publications and source records attributed to H Nau.

At least 37 records · Page 2Linked to original sources

PubMed2001

Induction of differentiation in F9 cells and activation of peroxisome proliferator-activated receptor delta by valproic acid and its teratogenic derivatives.

The antiepileptic drug valproic acid (VPA) is teratogenic, because it induces birth defects in some children of mothers treated for epilepsy. Cellular and molecular actions associated with teratogenicity were identified by testing differentiation of F9 embryocarcinoma cells. VPA altered cell morphology and delayed proliferation. Specific differentiation markers (e.g., c-fos and keratin 18 mRNA and particularly the activating protein-2 transcription factor protein) were induced. This pattern differs from the pattern induced by other teratogens or F9 cell-differentiating agents. Induction of differentiation correlated with teratogenicity because teratogenic derivatives of VPA, such as (S)-4-yn-VPA, induced differentiation, whereas closely related nonteratogenic compounds, such as (R)-4-yn-VPA, 2-en-VPA, and 4-methyl-VPA, did not. In the cellular signaling network, the peroxisome proliferator-activated receptor delta (PPARdelta) was activated selectively by VPA and teratogenic derivatives. Depletion of PPARdelta by antisense RNA expression precluded the response of F9 cells to VPA. In conclusion, our data show that VPA and its teratogenic derivatives induce a specific type of F9 cell differentiation and that PPARdelta is a limiting factor in the control of differentiation.

Animals↗

PubMed2001

The role of metabolism and toxicokinetics in retinoid teratogenesis.

Retinoids (vitamin A and its analogs) exert profound effects on a wide variety of life processes, including morphogenesis and embryonic development. Several retinoids are also effective drugs for therapy of skin diseases and some types of cancer. However, the applicability of this class of compounds is limited by their teratogenic activity. A major question in retinoid toxicology has been the marked interspecies differences in the lowest teratogenic doses of 13-cis-retinoic acid and retinol. In addition, great attention has been drawn to the risk assessment of embryotoxicity resulting from excessive intake of vitamin A by pregnant women. The present review first gives an overview of the biochemistry, metabolism and mode of action of retinoids as well as their role in embryonic development. It then summarizes the results of recent studies on retinoid metabolism, toxicokinetics, and embryonic exposure and discusses how the available information provides explanation of the aforementioned interspecies variations. Finally, it presents some approaches for risk assessment of high vitamin A intake by humans based on various animal models and epidemiological studies.

Abnormalities, Drug-Induced↗

PubMed2001

Effects of receptor-selective retinoids on CYP26 gene expression and metabolism of all-trans-retinoic acid in intestinal cells.

Retinoids mediate most of their function via interaction with retinoid receptors [retinoic acid receptors (RARs) and retinoid X receptors (RXRs)], which act as ligand-activated transcription factors controlling the expression of a number of target genes. The complex mechanistic pattern of retinoid-induced effects on gene expression of CYP26 and intestinal metabolism of all-trans-retinoic acid (RA) was investigated here by studying the effects of retinoid ligands with relative selectivity for binding and transactivation of the retinoid acid receptors, RARs and RXRs, in human intestinal Caco-2 cells. We show here that CYP26 is expressed in human duodenum and colon. In Caco-2 cells not only all-trans-RA but also synthetic agonists of the RAR induced intestinal CYP26 gene expression and all-trans-RA metabolism as well. The RARalpha ligand Am580 induced the CYP26 gene expression more than the RARbeta ligand CD2019 or the RARgamma ligand CD437 suggesting the highest specificity for RARalpha on intestinal CYP26 gene regulation. RXR ligands alone did not induce CYP26 gene expression or RA metabolism in Caco-2 cells at all. But together with the RARalpha ligand, Am580, there were enhanced effects on the induction of CYP26 gene expression and on the induction of the metabolism of all-trans-RA. We conclude that gene regulation of CYP26 and the metabolism of all-trans-RA in intestinal cells is regulated through RXR and RAR heterodimerization. When coadministered, RAR agonists showed the highest potency for CYP26 gene regulation. Receptor-selective retinoids showed enhanced effects on induction of CYP26 gene expression and all-trans-retinoic acid metabolism.

Caco-2 Cells↗

PubMed2001

Prediction of embryotoxic effects of valproic acid-derivatives with molecular in vitro methods.

Therapy with the antiepileptic drug valproic acid (2-propylpentanoic acid, VPA) during early pregnancy can cause similar teratogenic effects (neural tube defects) in human and mice. In this study a new molecular bioassay is presented using following endpoints: differentiation of F9 teratocarcinoma cells, altered cell morphology, induction of possible targeted genes, and the induction of viral RSV-promoter. The induction of a transiently transfected viral (RSV) promoter driven luciferase gene by VPA was used to screen a set of VPA-derivatives. Structure-activity investigations showed: the longer the aliphatic side chain the more the induction of the RSV-reporter gene. The specific induction was stereoseletive. The teratogenic enantiomer S-4-yn-VPA (2-propyl-4-pentynoic acid) induced the RSV-driven reporter gene while the non teratogenic R-4-yn-VPA does not. Heptyl-4-yn-VPA was the most potent teratogen in vitro and in vivo. Non teratogenic VPA-derivatives like R-4-yn-VPA and 2-en-VPA (2-propyl-2-pentenoic acid) were ineffective in this system. Thus, the teratogenic effect of VPA and VPA-derivatives in the mouse correlated with the specific induction of the viral RSV-promoter controlled reporter in F9-cells. Acid compounds such as fatty acids are known to interact with peroxisome proliferator-activated receptors (PPARs). To test structure-activity relationships by VPA or its derivatives we used CHO cells stably expressing hybrid proteins of the ligand-binding domain of either of the PPARs. The teratogen VPA and the teratogenic derivatives of VPA activated the PPAR-delta construct in a very specific structure- and stereoselective way which correlated well with the activities in the reporter gene assay (bioassay) and those in vivo. No such correlation was found with respect to activation of PPAR-alpha or PPAR-gamma. These structure-activity relationships indicate that PPAR-delta may be a potential mediator of VPA-induced differentiation of F9 cells and may possibly be involved in the mechanism of teratogenicity of VPA in vivo. Furthermore two bioassays were designed with clearly defined endoints, amenable to automation and screening of great number of compounds. The test system allows to replace animal experiments in the preclinical development of new antiepileptics drugs with reduced teratogenic risk. Supported by BgVV-ZEBET (Berlin).

Animals↗

PubMed2000

2,3,7,8-tetrachlorodibenzo-p-dioxin increases serum and kidney retinoic acid levels and kidney retinol esterification in the rat.

Halogenatedorganic environmental contaminants such as dioxins are well-known to affect tissue levels of retinoids. To further investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid homeostasis, adult male Sprague-Dawley rats were killed 1-112 days after a single oral dose of 10 microg TCDD/kg body wt. Additional groups of rats were killed three days after a single oral dose of 0.1, 1, 10, or 100 microg TCDD/kg body wt. Serum and renal retinoic acid levels were measured, as were levels of serum retinol-binding protein (RBP) in liver, kidneys, and serum. Hepatic and renal formation as well as hepatic hydrolysis of retinyl esters were determined, together with hepatic and renal retinoid levels. In addition, one of the retinyl ester hydrolase (REH) activities was investigated in isolated hepatocytes and hepatic stellate cells from rats killed 7 days after a single oral dose of 10 microg TCDD/kg body wt. No increased hepatic REH activity that could explain the decreased hepatic retinyl ester levels following TCDD treatment was found. In the liver, TCDD increased protein levels, but not mRNA levels, of RBP. A causal relationship is suggested for the increased renal lecithin:retinol acyltransferase (LRAT) activity and increased renal retinyl ester levels in TCDD-treated rats. Importantly, TCDD was shown to substantially increase serum and renal levels of retinoic acid. The ability of TCDD to cause increased tissue retinoic acid levels suggests that TCDD may alter the transcription of retinoic acid-responsive genes.

Animals↗

PubMed2000

Selective agonists of retinoic acid receptors: comparative toxicokinetics and embryonic exposure.

Three biologically active synthetic retinoids were investigated that bind selectively to retinoic acid receptors RARs (alpha, beta and gamma). The retinoids were previously demonstrated to have different teratogenic effects in the mouse in terms of potency and regioselectivity. The teratogenic potency rank order (alpha >beta >gamma) was found to be more or less compatible with the receptor binding affinities and transactivation potencies of the retinoid ligands to their respective receptors. The RARalpha agonist (Am580; CD336) induced a wide spectrum of malformations; CD2019 (RARbeta agonist) and especially CD437 (RARgamma agonist) produced more restricted defects. In the current study we tried to address whether the differences in teratogenic effects are solely related to binding affinity and transactivation differences or also due to differences in embryonic exposure. Therefore, transplacental kinetics of the ligands were assessed following administration of a single oral dose of 15 mg/kg of either retinoid given to NMRI mice on day 11 of gestation. Am580 was rapidly transferred to the embryo resulting in the highest embryonic exposure [embryo to maternal plasma area under the time vs concentration curve (AUC)(0-24 h )ratio (E/M) was 1.7], in accordance with its highest teratogenic potency. The low placental transfer of CD2019 (E/M of 0.3) was compatible with its lower teratogenic potential. Of major interest was the finding that the CD437, though being least teratogenic, exhibited considerable embryonic exposure (E/M of 0.6). These findings suggest that both the embryonic exposure and receptor binding transactivation selectivity are crucial determinants of the teratogenicity of these retinoid ligands.

Abnormalities, Drug-Induced↗

PubMed2000

All-trans-retinoic acid and all-trans-retinoyl-beta-D-glucuronide alter the development of axolotl embryos (Ambystoma mexicanum) in vitro.

Retinoids are involved in several physiological processes and are used in the treatment of various skin disorders. Therapy with retinoids during pregnancy may induce severe embryotoxic effects like craniofacial and cardiovascular malformations in the developing embryo. We investigated the effects of all-trans-retinoic acid (ATRA) and all-trans-retinoyl-beta-D-glucuronide (ATRAG) in an amphibian embryotoxicity assay with Ambystoma mexicanum (axolotl) as an alternative in vitro method. Embryos were exposed to various concentrations of ATRA or ATRAG for 48 h beginning with the blastula stage. Kinetic investigations in the embryonic tissue were performed during the exposure period. Both retinoids interfered with the development of the axolotl embryos. Dose-dependent effects observed included growth retardation, craniofacial and cardiovascular malformations, as well as neural tube defects. In the axolotl, ATRA induced slightly more pronounced embryotoxic effects than ATRAG. All-trans-retinal was shown to be a major endogenous retinoid in this species. Endogenous levels of all-trans-retinaldehyde were increased during exposure to both ATRA and ATRAG. The glucuronide, however, was only detected in small amounts after ATRAG exposure. The embryotoxic potential of ATRAG could be explained by deglucuronidation to ATRA.

Abnormalities, Drug-Induced↗

PubMed2000

Biological effects and metabolism of 9-cis-retinoic acid and its metabolite 9,13-di-cis-retinoic acid in HaCaT keratinocytes in vitro: comparison with all-trans-retinoic acid.

9-cis-Retinoic acid (9cRA), a geometric isomer of all-trans-retinoic acid (atRA), is an endogenous high-affinity ligand for retinoid X receptors and retinoic acid receptors activating them with high potency. 9,13-di-cis-Retinoic acid (9,13dcRA) has been described as a major plasma metabolite of 9cRA. In this study, the biological activity and the metabolism of 9cRA and 9,13dcRA were investigated and compared with those of atRA in a retinol-free culture system of HaCaT keratinocytes. 9cRA exhibited a slightly weaker activity overall than atRA in inhibiting cell proliferation, inducing cellular retinoic acid binding protein II (CRABP II) mRNA levels and upregulating cytokeratin 19 expression. 9,13dcRA regulated HaCaT keratinocyte activity only at the highest concentration tested (10(-6) M). In cultures of HaCaT keratinocytes with atRA and 9cRA, rapid intracellular accumulation of atRA was observed within 2 h, and atRA levels were higher with atRA treatment than with 9cRA treatment. 9,13dcRA remained relatively stable in the medium with intracellular 9,13dcRA levels below the level of detection. Taken together, 9cRA seems to be slightly less potent than atRA in regulating the biological activity of HaCaT keratinocytes, while its metabolite 9,13dcRA is effectively inactive at biologically relevant concentrations. Our data suggest a prodrug/drug relationship between 9cRA and atRA in human keratinocytes. 9,13dcRA seems to be a weaker prodrug of atRA or an inactive metabolic derivative.

Alitretinoin↗

PubMed2000

[Substances with pharmacological effects including hormonally active substances in the environment: identification of tetracyclines in soil fertilized with animal slurry].

Many drugs used in human medicine are detectable in surface waters from the low to the very low microgram/L concentration range. In drinking waters only some of these substances were detected, the concentrations are usually an order of magnitude below the concentrations found in surface waters. A risk assessment of long time effects caused by a permanent intake of these low concentrations of drug residues cannot be done at this time. Hormonally active substances in surface waters may present an ecotoxicological risk, there are many investigations currently under way to assess this problem. Our investigations show for the first time that residues of the commonly used veterinary drugs tetracycline and chlortetracycline can be detected in the surface of soil (0-40 cm) fertilized with animal slurry. The maximum concentrations found were 32.3 micrograms/kg and 26.4 micrograms/kg respectively. Leaching of these compounds into seeping water sampled at a depth of 80-140 cm could not be detected with the methods employed. The significance of the detected antibiotic residues in soil samples for the quality of food of animal origin or any ecotoxicological consequences needs further investigations. The knowledge about the concentrations of veterinary drug residues resulting from animal husbandry in the environment is the first step for such a risk assessment.

Animal Husbandry↗

PubMed2000

Metabolism of vitamin A and its active metabolite all-trans-retinoic acid in small intestinal enterocytes.

Retinol and its metabolites (retinoids) are essential for growth and cell differentiation, particularly of epithelial tissue. Retinoids mediate most of their function via interaction with retinoid receptors (retinoic acid receptors and retinoid X receptors), which act as ligand-activated transcription factors controlling the expression of a number of target genes. We have investigated whether retinoid receptor ligands such as all-trans-retinoic acid (RA) are formed in the human intestinal epithelium from dietary vitamin A. We show here that retinol was metabolized to its active metabolite, all-trans-RA, by isolated cytosolic fractions of human small intestinal enterocytes and by human Caco-2 cells. All-trans-RA was metabolized by human small intestinal microsomes to at least two metabolites (all-trans-4-hydroxy-RA and all-trans-4-oxo-RA). When Caco-2 cells were incubated with all-trans-RA, at least three major polar metabolites (all-trans-4-hydroxy-RA, all-trans-4-oxo-RA, and 13-cis-4-hydroxy-RA) were identified by HPLC-UV. The cytochrome P450 (CYP) 1A1 inhibitor alpha-naphthoflavone inhibited the metabolism of all-trans-RA, whereas the CYP1A1 inducer beta-naphthoflavone induced the metabolism of all-trans-RA, suggesting that CYP1A1 is involved. The induction of CYP3A by rifampicin enhanced the metabolism, and the induction of all-trans-RA metabolism was also observed after preincubation of the cells with all-trans-RA. Liarozole almost completely inhibited the RA metabolism. The specific retinoic acid metabolizing CYP26 was induced after RA treatment in Caco-2 cells. It is concluded that in addition to CYP1A1 and CYP3A, CYP26 may be the main CYP enzyme responsible for the metabolism of all-trans-RA in enterocytes. Active ligands such as all-trans-RA are formed in intestinal epithelial cells.

Caco-2 Cells↗

PubMed1999

Human uterine leiomyomata express higher levels of peroxisome proliferator-activated receptor gamma, retinoid X receptor alpha, and all-trans retinoic acid than myometrium.

Uterine leiomyomata are the main indication for a hysterectomy in the United States and occur in 25% of women >35 years. Because uterine leiomyomata can form when ovariectomized guinea pigs are exposed to estradiol and retinoic acids, we tested whether human leiomyomata had high levels of retinoic acids and related nuclear receptors. Compared with normal human myometrium, leiomyomata had 3- to 5-fold higher levels of peroxisome proliferator-activated receptor gamma (PPARgamma), retinoid X receptor alpha proteins, and all-trans retinoic acid, but only during the follicular phase of the menstrual cycle. 9-cis Retinoic acid was undetectable in either leiomyomata or myometrium. PPARgamma mRNA levels were lower in leiomyomata than myometrium, but only during the luteal phase of the cycle. A PPARgamma agonist, troglitazone, was given to guinea pigs along with estradiol and all-trans retinoic acid and produced the largest leiomyomata seen to date in this model. By contrast, no tumors formed when troglitazone was given alone or with estradiol or when troglitazone was given with estradiol and 9-cis retinoic acid. New therapies for human leiomyomata may emerge by combining antagonists for PPARgamma and retinoid X receptor alpha with selective estrogen receptor modulators.

Alitretinoin↗

PubMed1999

New molecular bioassays for the estimation of the teratogenic potency of valproic acid derivatives in vitro: activation of the peroxisomal proliferator-activated receptor (PPARdelta).

Therapy with the antiepileptic drug valproic acid (2-propylpentanoic acid, VPA) during early pregnancy can cause teratogenic effects (neural tube defects) in humans and in mice. VPA and a teratogenic derivative specifically induce differentiation of F9 teratocarcinoma cells and activate PPARdelta. We have now studied structure-activity relationships of 11 VPA-related compounds by quantitatively comparing their teratogenic potency with their effects in the two novel in vitro systems. Based on the induction of a Rous sarcoma virus (RSV) promoter-driven reporter gene, which is associated with the differentiation of F9 cells, a system suitable for high-throughput and quantitative screening was established. Structure-activity investigations showed that only teratogenic derivatives of VPA induced the response in F9 cells as well as activated the PPARdelta-dependent reporter system in Chinese hamster ovary (CHO) cells. Increases in the length of the side chain in the VPA-related 2-alkyl-pentynoic acid generate more potent inducers in the cell-culture-based assays, which also show higher teratogenicity and embryonic lethality rates. Activation of PPARdelta correlated well with the effects in the F9 cell assay and with teratogenic potency in vivo (p < 0.007). Evaluation of the effects of the presented set of compounds allows the conclusion that the in vitro systems faithfully reflect teratogenicity of VPA-related compounds. Whether the activation of PPARdelta is causally related to the disruption of proper embryonic development or whether it reflects other yet unknown VPA-induced events remains to be established.

Animals↗

PubMed1999

Enantioselective synthesis and teratogenicity of propylisopropyl acetamide, a CNS-active chiral amide analogue of valproic acid.

Propylisopropyl acetamide (PID), an amide analogue of the major antiepileptic drug valproic acid (VPA), possesses favorable anticonvulsant and CNS properties. PID contains one chiral carbon atom and therefore exists in two enantiomeric forms. The purpose of this work was to synthesize the two PID enantiomers and evaluate their enantiospecific teratogenicity. Enantioselective synthesis of PID enantiomers was achieved by coupling valeroyl chloride with optically pure (4S)- and (4R)-benzyl-2-oxazolidinone chiral auxiliaries. The two oxazolidinone enolates were alkylated with isopropyl triflate, hydrolyzed, and amidated to yield (2R)- and (2S)-PID. These two PID enantiomers were obtained with excellent enantiomeric purity, exceeding 99.4%. Unlike VPA, both (2R)- and (2S)-PID failed to exert teratogenic effects in NMRI mice following a single 3 mmol/kg subcutaneous injection. From this study we can conclude that individual PID enantiomers do not demonstrate stereoselective teratogenicity in NMRI mice. Due to its better anticonvulsant activity than VPA and lack of teratogenicity, PID (in a stereospecific or racemic form) has the potential to become a new antiepileptic and CNS drug.

Abnormalities, Drug-Induced↗

PubMed1999

Dietary vitamin A and teratogenic risk: European Teratology Society discussion paper.

This review discusses the predictive value of animal models in assessment of possible risk from excess vitamin A consumption during pregnancy and the human evidence concerning risk of congenital malformations from excess vitamin A in the diet, consumed either as a constituent of normal foods or in the form of dietary vitamin supplements. Other sources of exposure to vitamin A include medicines (dermal and oral preparations) and cosmetics, but these are not further considered here. Conservative estimates of the likely safe intake of vitamin A during pregnancy are available, based on results from scientific research to date. However, current uncertainties are such that further research is needed to more clearly define intakes which may be on the borderline between those which are beneficial and those which may pose a risk to the developing embryo and fetus.

Abnormalities, Drug-Induced↗

PubMed1999

Retinoid signaling by all-trans retinoic acid and all-trans retinoyl-beta-D-glucuronide is attenuated by simultaneous exposure of human keratinocytes to retinol.

Retinol and retinyl esters are converted with time to slowly increasing amounts of all-trans retinoic acid (RA) in cultured human keratinocytes. Exogenous RA has been shown to limit retinol oxidation and to increase retinol esterification. Because significant amounts of retinol are present in biologic systems, we examined whether RA and all-trans-retinoyl-beta-D-glucuronide (RAG) interact with retinol in exhibiting their activities on HaCaT keratinocytes maintained in a retinoid-free culture system. RA was more potent than RAG and retinol in inducing ultrastructural changes attributed to retinoids, inhibiting cell proliferation as well as enhancing keratin 19 expression. In addition, retinoids were able to induce cellular retinoic acid-binding protein II mRNA levels in the cultures, whereas early RA and late RAG activity was detected. The described biologic effects of RA and RAG were diminished by simultaneous cell exposure to retinol. HaCaT cells quickly metabolized retinol to retinyl esters and consequently to low amounts of RA. RA treatment led to an early high peak of cellular RA followed by reduction to trace amounts. Treatment with RAG resulted in constantly high cellular RAG and low RA levels. Under the combined RA and retinol treatment retinyl esters were increased and RA was reduced in HaCaT cells, whereas extracellular RA levels were similar to those obtained by RA alone. On the other hand, the combination of RAG and retinol resulted in higher extracellular RAG, similar cellular RAG, and lower cellular RA levels than those obtained by RAG alone without any change in retinyl esters. This study demonstrates that retinoid signaling by RA and RAG is attenuated by simultaneous exposure of HaCaT keratinocytes in vitro to retinol. The presence of retinol in the medium alters the rate of RA or RAG metabolism and thus cellular RA concentrations. The intensity of retinoid signal is probably dependent on cellular RA levels. The resulting "antagonism" among retinoids is consistent with the presence of an auto-regulatory mechanism in human keratinocytes offering protection against excessive accumulation of cellular RA.

Blotting, Northern↗

PubMed1999

Topical 9-cis-retinaldehyde for delivery of 9-cis-retinoic acid in mouse skin.

The 9-cis-retinoic acid (9cRA) is an endogenous ligand of retinoid X nuclear receptors (RXRs). Although the epidermis contains five times more RXRs than RARs, little is known on the activity of topical 9cRA. In order to circumvent surface isomerization of topically applied 9cRA into all-trans-retinoic acid (atRA), we used topical 9-cis-retinaldehyde (9cRAL) as a precursor of 9cRA, hypothesizing that keratinocytes would metabolize 9cRAL into 9-cis-retinoic acid (9cRA). Retinoid content was determined by HPLC analysis of mouse tail skin that had been washed after the application of 9cRAL (0.05% for 14 days) to evaluate the metabolites produced within the epidermis. Biologic activities of 9cRAL and atRAL were analysed by assessing hyperplastic and metaplastic responses, by determining epidermal thickness and the levels of mRNAs encoding for specific keratins. atRAL and derived retinoids were found in skin treated with either atRAL or 9cRAL. The metabolite pattern obtained with 9cRAL was similar to that obtained with atRAL except the presence in 9cRAL samples of an unidentified nonpolar metabolite. However, treatment with 9cRAL yielded higher atRAL and lower retinyl ester concentrations. The biologic activities (hyperplastic and metaplastic responses) resulting from topical application of 9cRAL were lower than those induced by atRAL or atRA at similar concentrations. Taken together, these data show that topical 9cRAL does not deliver significant amounts of 9cRA and exerts less biologic activity than atRAL. Contrary to atRAL, 9cRAL does not appear therefore as a pertinent candidate for topical use in humans.

Administration, Topical↗

PubMed1999

Metabolism of retinaldehyde isomers in pregnant rats: 13-cis- and all-trans-retinaldehyde, but not 9-cis-retinaldehyde, yield very similar patterns of retinoid metabolites.

Retinaldehyde (RAL), a key intermediate in retinoid metabolism, acts as a retinoic acid (RA) precursor, but is also reduced to retinol (ROH), which can subsequently be esterified to retinyl esters, the storage form of vitamin A. Limited information is available on the metabolism of geometric isomers of RAL as well as on the transplacental distribution of their metabolites, including RA isomers. Such information would be very helpful for the assessment of the teratogenic potency of RAL isomers, as teratogenesis represents a major side effect of retinoid use in pharmacotherapy. In the present study we examined concentrations of retinoids in plasma, maternal tissues, and embryos of pregnant rats 2 h after a single oral dose (100 mg/kg body weight) of all-trans-, 13-cis-, or 9-cis-RAL on gestational day 13. The main findings of this study were the very similar patterns of retinoid metabolites (consisting of retinoids with mainly the all-trans-configuration) after administration of all-trans- and 13-cis-RAL, and the high concentrations of 9-cis-RA, 9,13-dicis-RA, and 9-cis-retinoyl-beta-D-glucuronide after dosing with 9-cis-RAL. In addition, all-trans-RA as a RAL metabolite reached the embryos to a much greater extent than any of its cis-isomers. The results are discussed in view of in vitro data on enzymes involved in the biotransformation of RAL isomers.

Animals↗

PubMed1998

Determination of folate patterns in mouse plasma, erythrocytes, and embryos by HPLC coupled with a microbiological assay.

Folates are important cofactors in one-carbon metabolism. Disturbances in folate homeostasis and metabolism may be related to an increased risk of cardiovascular disease and carcinogenesis and may lead to congenital malformations, namely neural tube defects. Determination of these compounds in biological samples is often a problem due to the existence of numerous folate metabolites, their relative instability, and the low contents in serum and most tissues. As existing methods have distinct limitations, we developed a method, which facilitates the separation as well as the sensitive detection of eight folates by coupling HPLC with a microbiological assay. After a simple sample preparation, including deproteinization and enzymatic hydrolysis of folate polyglutamates, extracts were chromatographed, fractions were collected on microtiter plates, and folates were quantitated using the Lactobacillus casei assay. The raw data were processed using a computing system after reconstructing the HPLC chromatogram with the bacterial growth data. Using the described method, the eight physiologically occurring folate monoglutamates could be simultaneously determined. The detection limits were 2-20 fmol per injection. The application of the method was demonstrated with the analysis of the folate pattern in milligram or sub-milligram quantities of plasma, erythrocyte, and embryos of pregnant mice during organogenesis.

Animals↗