Pharmacogenetics and anaesthesia: the value of genetic profiling.

Approximately 50 years ago, pharmacogenetics was described as a new field of medicine that may explain human drug action. Anaesthesia played a key role in the early investigations. An understanding of how a person's DNA influences drug metabolism and effectiveness may allow individually tailored prescriptions, improving outcomes and safety. The ultimate goal of pharmacogenetic research is to offer tailored personalised medicine to improve both the efficacy of medication and patient safety by helping to predict risk of adverse outcomes. In this review, we present a selection of historical landmarks where anaesthesia has been a catalyst for pharmacogenetic development. We examine the level of evidence and cite examples of candidate genes and common polymorphisms known to alter the response to peri-operative medication. Finally, we set forth current views and potential exciting perspectives that may arise from the application of pharmacogenetics to the daily practice of anaesthesia and pain medicine.

In vivo dopamine release and uptake impairments in rats treated with 3-nitropropionic acid.

Recent evidence has suggested that mitochondrial dysfunction may lead to impaired neurotransmitter exocytosis in transgenic Huntington's disease (HD) model mice. To gain insight into the impact of mitochondrial impairment on striatal dopamine release in vivo, we used fast-scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes to measure dopamine release and uptake kinetics in anesthetized Lewis rats continuously treated for 5 days with 3-nitropropionic acid (3NP). Our results indicate that, even though striatal dopamine content was unchanged, remotely stimulated dopamine release evoked per electrical stimulus pulse ([DA](p)) is decreased in 3NP-treated rats (33% of that observed in sham control rats) and that this decrease is uniform throughout all stereotaxic depths tested. Nevertheless, unlike data collected previously from transgenic HD model rodents, the maximum rate of dopamine uptake (V(max)) in 3NP-treated rats is diminished (30% of controls) while K(m) is unchanged. Treatment with 3NP also resulted in a corresponding decrease in locomotor activity, presumably due in part to the impaired dopamine release. These results indicate that dopamine release is degraded in this HD model, as is observed in transgenic HD model rodents; however, the results also imply that there are fundamental differences in dopamine uptake between 3NP-treated animals and transgenic animals.

Xenopus laevis: a model system for the study of embryonic retinoid metabolism. III. Isomerization and metabolism of all-trans-retinoic acid and 9-cis-retinoic acid and their dysmorphogenic effects in embryos during neurulation.

These investigations provide data pertaining to the metabolism and disposition of exogenous 9-cis-retinoic acid and all-trans-retinoic acid during neurulation in Xenopus embryos. Each isomer elicited malformations of the heart, eye, and brain, but approximately 2-fold higher concentrations of all-trans-retinoic acid than 9-cis-retinoic acid were required to produce qualitatively and quantitatively similar dysmorphogenic effects. The dymorphogenic effects of all-trans-retinoic acid could not be attributed to the isomerization of all-trans-retinoic acid to 9-cis-retinoic acid. Evidence is provided that all-trans-retinoic acid and 9-cis-retinoic acid are both direct-acting dysmorphogens. After Xenopus embryos were exposed to all-trans-retinoic acid, elevated levels of 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid, all-trans-retinoyl-beta-glucuronide, and 13-cis-retinoic acid were detected in the embryos, whereas embryonic levels of 9-cis-retinoic acid were actually slightly lower than endogenous levels during early neurulation. After embryos were exposed to 9-cis-retinoic acid during neurulation, elevated levels of 4-oxo metabolites, glucuronides and 9,13-di-cis-retinoic acid were observed in the embryos. At equivalent concentrations, 4-oxo-13-cis-retinoic acid and 13-cis-retinoic acid elicited fewer severe multiple malformations than all-trans isomers 9,13-di-cis isomers, or 9-cis isomers. The dysmorphogenic effect of 9,13-di-cis-retinoic acid may be caused by its isomerization to 9-cis-retinoic acid. All-trans retinoyl-beta-glucuronide was only marginally teratogenic at the highest concentrations tested.

Xenopus laevis: a model system for the study of embryonic retinoid metabolism. I. Embryonic metabolism of 9-cis- and all-trans-retinals and retinols to their corresponding acid forms.

Recently, the temporal and spatial distribution patterns of two established, endogenous retinoid receptor ligands, 9-cis-retinoic acid and all-trans-retinoic acid and various precursor retinoids were described in Xenopus embryos during early development (Creech Kraft et al., Proc. Natl. Acad. Sci. U.S.A. 1994; Biochem. J. 1994). Each of these two receptor ligands is a metabolite of vitamin A (all-trans-retinol), and each is also a potent dysmorphogen in Xenopus embryos as well as in embryos of several other vertebrate species. This study demonstrates early embryonic metabolism of exogenous all-trans-retinol, 9-cis-retinol, all-trans-retinal, and 9-cis-retinal to 9-cis-retinoic acid, all-trans-retinoic acid, and other metabolites in Xenopus embryos during neurulation, a specific stage of development that spans a time period of approximately 8 hr. Our results demonstrate that the Xenopus embryo provides a suitable model system for studying the embryonic bioconversion of retinoids and dysmorphogenic effects within a single time window of development.

Embryogenesis in cultured whole rat embryos after combined exposures to 3,3',5-triiodo-L-thyronine (T3) plus all-trans-retinoic acid and to T3 plus 9-cis-retinoic acid.

Retinoid-induced malformations of the jaw, ears, face, skull, eyes, and heart in humans and rodents are well known. Data on nuclear receptors and developmental toxicity bioassays indicate that thyroid hormones can modulate the biologic activity of retinoids. The present investigation concerned the potential for interactions of all-trans-retinoic acid (RA) with 3,3'5-triiodo-L-thyronine (T3) and of 9-cis-retinoic acid (9-cis-RA) with T3 in the morphogenesis of cultured whole rat embryos. Varying concentrations of retinoids or T3 were microinjected into the amniotic fluid or placed in the culture medium alone or in combinations of T3 with each retinoid. At 200 ng/ml, T3 increased the incidence of branchial arch defects produced by either RA or 9-cis-RA but did not elicit branchial arch defects alone except at concentrations significantly compromising survival (2,000 ng/ml; 32% mortality). Similarly high culture medium concentrations of T3 alone were associated with failure of neural tube closure in the rhombencephalon (rhombencephalic schisis). At this concentration, other dysmorphia were minimal and at 670 ng/ml T3, no dysmorphogenic or embryotoxic effects could be detected. Modulation of T3 effects by the yolk sac placenta was suggested by failure of microinjected T3 to elicit dysmorphia at very high amniotic fluid concentrations. RA (300 ng/ml) or 9-cis-RA (600 ng/ml) alone elicited no or minimal rhombencephalic schisis at the highest concentrations studied. RA plus T3 produced a much greater than additive effect on rhombencephalic schisis, whereas 9-cis-RA plus T3 produced a less than additive effect. Conversely, much greater than additive effects on anterior schisis were observed for 9-cis-RA plus T3 whereas combined effects of RA and T3 were approximately additive. For most other dysmorphia, the combined effects of each retinoid with T3 were greater than additive and were particularly striking for cephalic defects.

The retinoid X receptor ligand, 9-cis-retinoic acid, is a potential regulator of early Xenopus development.

Endogenous retinoids are potential regulators of vertebrate embryogenesis that have been implicated in early anterior-posterior patterning and limb-bud development. We have characterized the temporal and spatial distribution of 9-cis-retinoic acid in the Xenopus embryo and compared it to two other retinoids, all-trans-retinoic acid and all-trans-retinoyl-beta-glucuronide. 9-cis-Retinoic acid is first detected after the midblastula transition and by the end of gastrulation is localized primarily within the anterior and posterior dorsal regions of the embryo. Since 9-cis-retinoic acid is a 6-fold more potent dysmorphogen than trans-retinoic acid, we suggest that it is involved in the early specification of the Xenopus anterior-posterior axis.

v-erbA and citral reduce the teratogenic effects of all-trans retinoic acid and retinol, respectively, in Xenopus embryogenesis.

Treatment of late blastula/early gastrula stage Xenopus embryos with all-trans retinoic acid results in disruption of the primary body axis through effects on both mesoderm and neuroectoderm. This effect of retinoic acid, coupled with the known presence of retinoic acid in Xenopus embryos has led to the proposal that retinoic acid may be an endogenous morphogen providing positional information in early development. To further elucidate the role of retinoic acid in early Xenopus development, we have attempted to interfere with the retinoic acid signalling pathway both at the level of retinoic acid formation, by treatment with citral (3,7-dimethy-2,6-octadienal), and at the level of nuclear retinoic acid receptor function, by microinjection of v-erbA mRNA. The feasibility of this approach was demonstrated by the ability of citral treatment and v-erbA mRNA injection to reduce the teratogenic effects of exogenous retinol and retinoic acid, respectively, in early Xenopus development. Interestingly, v-erbA mRNA injection and citral treatment of gastrula stage embryos resulted in tadpoles with a similar set of developmental defects. The defects were chiefly found in tissues that received a contribution of cells from the neural crest, suggesting that at least a subset of neural crest cells may be sensitive to the endogenous level of retinoic acid. In accord with this proposal, it was found that the expression patterns of two early markers of cranial neural crest cells, Xtwi and XAP-2, were altered in embryos injected with v-erbA mRNA. These results indicate that structures in addition to the primary axis are regulated by retinoic acid signalling during early Xenopus development.

9-cis-retinoic acid: a direct-acting dysmorphogen.

Experiments in vitro with cultured rat conceptuses demonstrated that 9-cis-retinoic acid (9-cis-RA) (300 ng/mL amniotic fluid) produced branchial arch and somite defects similar to those elicited by equal concentrations of all-trans-retinoic acid (all-trans-RA), but with an increase in cephalic defects that included missing optic vesicles. After conceptuses were intraamniotically microinjected with 600 ng 9-cis-RA/mL amniotic fluid on day 10 of gestation, an unusual heart defect was also observed. HPLC analyses indicated that 9-cis-RA readily underwent conversion to the less active metabolite, 13-cis-retinoic acid (13-cis-RA), in cultured conceptuses during the first 4 hr after treatment but only after 6 hr could elevated levels of the potent dysmorphogen all-trans-RA be detected. In separate experiments, conversion of 13-cis-RA or of all-trans-RA to 9-cis-RA could not be detected during a 6-hr embryo culture period. Endogenous levels of 9-cis-RA in whole rat embryos also were below limits of detection but small quantities of this isomer could be detected in neonatal rat eye and human embryonic brain. Our present study strongly suggests that 9-cis-RA is a direct-acting dysmorphogen with probable specific target sites of action.

Tissue levels of retinoids in human embryos/fetuses.

In nonhuman vertebrate embryos, two endogenous retinoids with significant morphogenic activities have been identified thus far: all-trans retinoic acid and 3, 4-didehydroretinoic acid. To date, no information is available concerning endogenous retinoid levels in developing human embryos or fetuses. The purpose of the present study was to provide data relating to normal levels of retinoids in various human embryonic and fetal tissues at various stages of gestation measurable with HPLC techniques. Our investigations show that all-trans-retinoic acid, 13-cis-retinoic acid, retinol, all-trans-retinoyl-beta-glucuronide, and one unidentified metabolite were all present and quantifiable in several human embryos and fetuses investigated. Tissue levels of retinol were consistently much higher than those of the other three detected metabolites; 4-oxo metabolites were below the levels of detection in all samples studied.

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.

Microinjections of cultured rat conceptuses: studies with 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide.

4-Oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships showed that the dysmorphogenic effects produced by these metabolites were qualitatively similar to those of parent all-trans-retinoic acid. Compared with all-trans-retinoic acid (300 ng/ml), the dysmorphogenic effects were elicited by a 2-fold higher concentration of 4-oxo-all-trans-retinoic acid, an approximately 10-fold higher concentration of 4-oxo-13-cis-retinoic acid and a 16-fold higher concentration of all-trans-retinoyl-beta-glucuronide. A surplus of uridine 5'-diphospho-glucuronic acid, microinjected together with 300 ng/ml all-trans-retinoic acid, decreased the observed embryo-toxicity of all-trans-retinoic acid, suggesting the possibility of glucuronidation in tissues of the conceptus per se. The results of the study provide further support for the hypothesis that 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid are, in contrast to the corresponding cis-isomers and glucuronides, ultimate dysmorphogenic retinoids.

Microinjection of cultured rat embryos: studies with retinol, 13-cis- and all-trans-retinoic acid.

Retinol, all-trans-retinoic acid or 13-cis-retinoic acid were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships of the retinoids showed that the dysmorphogenic effects were qualitatively similar for all three, but were elicited by a low concentration of all-trans-retinoic acid (250 ng/ml), a 6- to 7-fold higher concentration of 13-cis-retinoic acid and an approximately 16-fold higher concentration of retinol. After microinjection of 2,000 ng/ml of retinol, no dysmorphogenesis was observed but instead an increase in all growth parameters as compared to the controls.

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)

Transplacental pharmacokinetics of teratogenic doses of etretinate and other aromatic retinoids in mice.

The transplacental pharmacokinetics of single teratogenic doses of etretinate and motretinide were compared with particular emphasis on distribution and concentrations in the exposed embryos of the free acid metabolite, etretin. The three aromatic retinoids were also tested for their direct inhibitory effect on chondrogenesis in the limb bud mesenchymal cell "micromass" culture assay. After a standard dose of 100 mg/kg administered on day 11 of gestation in NMRI mice, all three compounds were teratogenic, but they differed from each other in potency. Etretinate was most active as a teratogen, equalling the potency of our standard all-trans-retinoic acid; every exposed fetus was deformed with severe shortening of all limb bones as well as cleft palate. Etretin was less potent than etretinate, and motretinide was considerably less active as a teratogen than the other two. In the in vitro assay, only etretin suppressed chondrogenesis and this activity was equivalent to that of all-trans-retinoic acid (IC50 of 12 ng/ml). Both etretinate and motretinide (which contain an ethyl ester and ethylamide terminal group, respectively) were essentially inactive in vitro, demonstrating the fact that a free carboxylic group may be a requirement for the in vitro suppression of chondrogenesis. These differences between the results obtained in vivo and in vitro could be resolved by pharmacokinetic investigations using HPLC methods. Both etretinate and motretinide were metabolized in vivo to etretin, their likely common teratogenic metabolite. The high teratogenic potency of etretinate was probably the result of high concentrations as well as AUC values of its metabolite etretin in the embryo. On the other hand, the comparatively low teratogenicity of motretinide could be related to approximately 5 x lower embryonic peak levels as well as AUC values of etretin. A comparison of these results with those previously obtained for all-trans- and 13-cis-retinoic acids confirms the correlation between embryonic exposure and teratogenic potency in the mouse. Our results indicate that pharmacokinetic studies are essential for the interpretation of relative teratogenic potencies of retinoids as well as apparent differences between in vivo and in vitro teratogenesis. A free carboxyl group at the terminal end of the tetraene chain was necessary for high activity of the retinoids studied.

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.