Characterization of functional polymorphisms and glucocorticoid-responsive elements in the promoter of TDO2, a candidate gene for ethanol-induced behavioural disorders.

AIMS: In response to acute ethanol consumption, tryptophan 2,3-dioxygenase (TDO) induces the kynurenine pathway (KP) through a glucocorticoid-mediated mechanism, which could lead to a dramatic accumulation of neurotoxic metabolites in association with serotonin depletion. As a result, interindividual variability in ethanol-induced behavioural disorders, such as black-outs and violent impulsive behaviours (BOVIBs) following binge drinking, could be partly explained by genetic polymorphisms affecting the KP. The aim of this study was to identify polymorphisms on the promoter of the TDO2 gene that could affect expression and/or activity of TDO through glucocorticoid induction. METHODS: Polymorphisms were screened using a PCR-sequencing strategy applied to 31 alcohol-dependent patients and 49 unrelated healthy volunteers, and functionally analysed with bioinformatic prediction tools and gene reporter assays in HepG2 and A549 cell lines. RESULTS: We identified 12 polymorphisms in the human TDO2 promoter region, 2 of them corresponding to previously unknown single-nucleotide polymorphisms (SNPs) and 3 of them located in putative glucocorticoid-responsive elements (GREs). Gene reporter assays using HepG2 and A549 cell lines confirmed the presence of several functional GREs in the promoter region of TDO2 and revealed that some of the identified polymorphisms affect the promoter activity under glucocorticoid receptor over-expression and dexamethasone exposure conditions. CONCLUSIONS: Correlational studies in larger samples could help to determine whether these polymorphisms are responsible for variations of expression and/or activity of TDO, in particular under conditions where release of glucocorticoids is increased, such as acute ethanol intake. If confirmed, such results would be of major interest in explaining part of the interindividual variability observed in behavioural responses to acute ethanol consumption.

Metabolites of tryptophan catabolism are elevated in sera of patients with myelodysplastic syndromes and inhibit hematopoietic progenitor amplification.

Tryptophan catabolism, which is mediated by the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), produces kynurenine. Kynurenine itself is converted by downstream enzymes into secondary catabolites. We evaluated the serum levels of primary and secondary tryptophan catabolites in a cohort of patients with myelodysplastic syndromes (MDS). The MDS patients showed significantly higher levels tryptophan catabolites which correlated with cytopenia. The tryptophan catabolites inhibited progenitor expansion during the in vitro culture of hematopoietic cells. Thus, MDS patients are characterized by high tryptophan catabolism resulting in elevated primary and secondary metabolites, which both have inhibitory effects on hematopoiesis.

Genetic polymorphism of CYP4A11 and CYP4A22 genes and in silico insights from comparative 3D modelling in a French population.

The CYP4A subfamily is known to ω-hydroxylate the endogenous arachidonic acid into 20-hydroxyeicosatetranoic acid, which has renovascular and tubular functions. The aim of this work was to report a comprehensive investigation of the CYP4A11 and CYP4A22 genetic polymorphisms in a French population. Using PCR-SSCP and sequencing strategies, a total of 26 sequence variations were identified comprising 3 missense mutations for CYP4A11 (Ser404Phe, Phe434Ser and Arg505His) and 7 missense mutations for CYP4A22 (Arg126Trp, Gly130Ser, Asn152Tyr, Val185Phe, Cys231Arg, Leu428Pro and Leu509Phe). In comparison with SNPs reported in the database (dbSNP) of the National Center for Biotechnology information (NCBI), 6 and 3 novel polymorphisms were identified in CYP4A11 and CYP4A22, respectively. The potential impact of the amino acid substitutions on the structure and/or catalytic activity of the enzymes has been estimated by the construction and validation of the CYP4A 3D models. These results could be helpful for further investigations of the potential role of CYP4A variants in the genetic susceptibility to cardiovascular diseases in humans such as arterial hypertension.

Acute arsenic poisoning: clinical, toxicological, histopathological, and forensic features.

This report describes a suicide case by acute arsenic intoxication via intravenous injection. A 30-year-old woman injected arsenic As (V) (sodium arseniate disodique: Disodium Hydrogena Arsenik RP) in a successful suicide attempt. Three hours following administration, the woman developed severe digestive symptoms. She was admitted to a hospital and transferred to the intensive care unit within 12 h of the massive administration of arsenic. Despite therapeutic efforts, over the next 2 h she developed multiorgan failure and died. A postmortem examination was performed. Pulmonary edema and congestion of liver were apparent. As (V) and As (III) were determined by high performance liquid chromatography and inductively coupled plasma mass spectrometry after mineralization of samples by concentrated nitric acid. Toxicological analysis revealed high concentrations of arsenic in biological fluids as well as in organs. Histopathological examination showed a typical indication of myocarditis. These findings were in agreement with acute arsenic poisoning. The symptoms developed by this young woman (intoxication by intravenous administration) were comparable to oral intoxication. The clinical signs, survival time, and administration type are discussed in light of the literature on acute and chronic arsenic poisoning.

Effect of prenatal stress on alcohol preference and sensitivity to chronic alcohol exposure in male rats.

RATIONALE: In rats, prenatal restraint stress (PRS) induces persistent behavioral and neurobiological alterations leading to a greater consumption of psychostimulants during adulthood. However, little is known about alcohol vulnerability in this animal model. OBJECTIVES: We examined in adolescent and adult male Sprague Dawley rats the long-lasting impact of PRS exposure on alcohol consumption. METHODS: PRS rats were subjected to a prenatal stress (three daily 45-min sessions of restraint stress to the mothers during the last 10 days of pregnancy). Alcohol preference was assessed in a two-bottle choice paradigm (alcohol 2.5%, 5%, or 10% versus water), in both naïve adolescent rats and adult rats previously exposed to a chronic alcohol treatment. Behavioral indices associated with incentive motivation for alcohol were investigated. Finally, plasma levels of transaminases (marker of hepatic damages) and ΔFosB levels in the nucleus accumbens (a potential molecular switch for addiction) were evaluated following the chronic alcohol exposure. RESULTS: Alcohol preference was not affected by PRS. Contrary to our expectations, stressed and unstressed rats did not display signs of compulsive alcohol consumption. The consequences of the alcohol exposure on locomotor reactivity and on transaminase levels were more prominent in PRS group. Similarly, PRS potentiated alcohol-induced ΔFosB levels in the nucleus accumbens. CONCLUSION: Our data suggest that negative events occurring in utero do not modulate alcohol preference in male rats but potentiate chronic alcohol-induced molecular neuroadaptation in the brain reward circuitry. Further studies are needed to determine whether the exacerbated ΔFosB upregulation in PRS rats could be extended to other reinforcing stimuli.

Screening of xenobiotics by ultra-performance liquid chromatography-mass spectrometry using in-source fragmentation at increasing cone voltages: library constitution and an evaluation of spectral stability.

In clinical and forensic toxicology, general unknown screening is used to detect and identify exogenous compounds. In this study, we aimed to develop a fast (15 min) comprehensive screening method for 500 toxicologically relevant analytes based on ultra-performance liquid chromatography (UPLC) coupled with a quadrupole mass spectrometer (MS) system operated in full scan mode. Data were acquired using both positive and negative electrospray ionization by scanning across the range m/z 80-650. For each ionization mode, data were also collected under multiple fragmentation conditions (i.e., at six different cone voltages). Consequently, each molecule could be characterized by a combination of retention time and up to a maximum of 12 individual spectra. Investigation of the 500 analytes resulted in the compilation of a library containing 2975 spectra. An assessment of the stability of these spectra was evaluated under various conditions, that is, the impact of increasing drug concentration and the presence of biological matrix. In addition, the transferability of the spectral library was assessed by comparison with data acquired using several other instruments of same model and from the same manufacturer. These data are presented in addition to the utility of the method for the analysis of routine clinical and forensic samples. Following extraction, identified compounds were compared to those found with two other techniques, one based on immunoassay and the other, on high-performance liquid chromatography-photodiode-array.

Profiling gene expression of whole cytochrome P450 superfamily in human bronchial and peripheral lung tissues: Differential expression in non-small cell lung cancers.

Susceptibility to lung diseases, such as lung cancer and chronic obstructive pulmonary disease, is largely influenced by the metabolic capacity of lung tissues. This capacity is partly determined by the expression profile of the cytochromes P450 (CYPs), a superfamily of enzymes that have relevant catalytic properties toward exogenous and endogenous compounds. Using quantitative real-time RT-PCR, we conducted a comprehensive analysis of the expression profile of the 57 human CYP genes in non-tumoral (bronchial mucosa and pulmonary parenchyma) and tumoral lung tissues of 18 patients with non-small cell lung cancer. This study highlights (i) inter-individual variations in lung expression for some CYPs, (ii) different CYP expression patterns between bronchial mucosa and pulmonary parenchyma, that indicate distinctive susceptibility of these tissues toward the deleterious effects of inhaled chemical toxicants and carcinogens, (iii) high intertumoral variability, that could have major implications on lung tumor response to anti-cancer drugs.

Impact of tryptophan metabolism on the vulnerability to alcohol-related blackouts and violent impulsive behaviours.

AIMS: We examined (1) the association of SLC6A4 genotypes and alcohol dependence (AD) in a sample of alcoholics; (2) the validity of lifetime occurrence of blacked-out violent impulsive behaviour (BOVIB) during binge drinking bouts as a criterion for subtyping AD patients and (3) a mechanistic hypothesis for BOVIB involving tryptophan-2,3-dioxygenase (TDO) activity. METHODS: Three common polymorphisms of the SLC6A4 gene (5-HTTLPR, A/G SNP of LPR region and VNTR in intron 2) were genotyped. An oral tryptophan (Trp) load (OTL) was administered to a sample of patients seeking help for AD. BOVIB history and psychological status were screened by BOVIB-Q, depression (BDI), anxiety (BAI, STAI) and personality (TCI) questionnaires. During the 7 h following Trp load, serum kynurenine (Kyn) and Trp were monitored. RESULTS: BOVIB+ patients showed significantly higher scores on depression, anxiety and character scales but no significant association was found between SLC6A4 polymorphisms and BOVIB. Patients with a history of BOVIB (BOVIB+ subgroup) differed from those exempt from such episodes (BOVIB- subgroup) for TDO activity response to OTL assessed by the Kyn:Trp ratio (P = 0.043) and the slope of concentration increase ratio (SCIR) of serum Kyn (P = 0.043). CONCLUSIONS: Put together, these findings support the validity of the BOVIB criterion to differentiate a sub-group of vulnerable AD subjects and suggest that OTL may help to concurrently define a specific endophenotype.

Ethanol attenuates spatial memory deficits and increases mGlu1a receptor expression in the hippocampus of rats exposed to prenatal stress.

BACKGROUND: Although it is generally believed that chronic ethanol consumption impairs learning and memory, results obtained in experimental animals are not univocal, and there are conditions in which ethanol paradoxically improves cognitive functions. In the present work, we investigated the effects of prenatal stress and of chronic ethanol exposure during adulthood on spatial memory in rats. METHODS: Rats were subjected to a prenatal stress delivered as 3 daily 45-minute sections of restraint stress to the mothers during the last 10 days of pregnancy (PRS rats). After 7 months of ethanol exposure (ethanol 10%, oral intake), memory performances were evaluated in a spatial discrimination test in control and PRS male rats. Then, the oxidative damages and the expression of metabotropic glutamate (mGlu) receptors were assessed in their hippocampus. RESULTS: Chronic ethanol exposure resulted in a reduced performance in a spatial recognition task in control animals. Unexpectedly, however, the same treatment attenuated spatial memory deficits in rats that had been subjected to prenatal stress. This paradigm of ethanol administration did not produce detectable signs of oxidative damage in the hippocampus in either unstressed or PRS rats. Interestingly, ethanol intake resulted in differential effects in the expression of mGlu receptor subtypes implicated in mechanisms of learning and memory. In control rats, ethanol intake reduced mGlu2/3 and mGlu5 receptor levels in the hippocampus; in PRS rats, which exhibited a constitutive reduction in the levels of these mGlu receptor subtypes, ethanol increased the expression of mGlu1a receptors but did not change the expression of mGlu2/3 or mGlu5 receptors. CONCLUSION: Our findings support the idea that stress-related events occurring before birth have long-lasting effects on brain function and behavior, and suggest that the impact of ethanol on cognition is not only dose- and duration-dependent, but also critically influenced by early life experiences.

Indoleamine 2,3-dioxygenase activity of acute myeloid leukemia cells can be measured from patients' sera by HPLC and is inducible by IFN-gamma.

The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan to kynurenine, blocking T-cell activation and inducing immunosuppression. In patients with acute myeloid leukemia (AML), the serum kynurenine/tryptophan ratio (Kyn/Trp) was raised, suggesting a higher IDO activity than in healthy people. Patients with higher Kyn/Trp ratios showed lower survival. IDO activity was also detected in AML cells after exposure to IFN-gammain vitro, suggesting that the higher Kyn/Trp ratio in serum of AML patients might have resulted from stimulated leukemic blast cells. Thus, in AML, the activity of IDO can be easily monitored, providing a tool for future clinical testing of IDO-blocking drugs.

Quantitative determination of glyphosate in human serum by 1H NMR spectroscopy.

The determination and quantification of glyphosate in serum using (1)H NMR spectroscopy is reported. This method permitted serum samples to be analysed without derivatization or any other sample pre-treatment, using 3-trimethylsilyl 2,2',3,3'-tetradeuteropropionic acid (TSP-d(4)) as a qualitative and quantitative standard. Characterization of the herbicide N-(phosphonomethyl)glycine was performed by analysing chemical shifts and coupling constant patterns. Quantification was performed by relative integration of CH(2)-P protons to the TSP-d(4) resonance peak. The method was tested for repeatability (n=5) and yielded coefficients of variation of 1% and 3%, respectively: detection and quantification limits were also determined and were 0.03 and 0.1mmol/L, respectively. The method was applied to the quantification of glyphosate in a case of acute poisoning.

Prevalence of psychoactive substances in truck drivers in the Nord-Pas-de-Calais region (France).

A previous study conducted in 1995 showed that psychoactive drug use by workers was higher in safety/security workstations than in the rest of the labour force. In order to verify this finding, we conducted a new study in 2003-2004 in the Nord-Pas-de-Calais region, restricted to truck drivers. The aim of this study was to allow harmonizing the professional practice of the occupational physicians, proposing drug prevention and drug testing policies, validating the analytical methods and the guidelines in case of positive testing results. One thousand truck drivers were studied. Urines were tested for amphetamines, cannabinoids, cocaine, opiates, benzodiazepines, buprenorphine and methadone by immunoassay. Urine ethanol determinations were performed by an ADH method. Positive urines for drugs of abuse, methadone or buprenorphine were then tested by gas chromatography or liquid chromatography coupled to mass spectrometry. Out of the 1000 drivers, cannabinoids were detected in 85 cases, opiates in 41 cases, amphetamines in 3 cases and cocaine in only one case. Buprenorphine was detected in 18 cases, methadone in 5 cases and benzodiazepines in 4 cases. Urine ethanol was positive in 50 cases. We found only one case with 6-monoacetylmorphine. Other positive opiates were metabolites of antitussives. The relatively low number of benzodiazepine positive urines could be explained by the lack of sensitivity of the test we used. All these results confirm those of the previous study for cannabinoids and ethanol in safety/security workstations. Positive results for methadone and buprenorphine are eight times higher than in the general population. In conclusion, the authors think that it will be of a great interest to test urine of truck drivers for other classes of psychoactive drugs, using a liquid chromatography-mass spectrometry method.

Impact of an acute exposure to ethanol on the oxidative stress status in the hippocampus of prenatal restraint stress adolescent male rats.

Prenatal restraint stress (PRS) in rats is associated with hippocampal dysfunctions and several behavioural and endocrine disorders related to this brain area. Recently, we have reported that the PRS modifies the hypothalamic-pituitary-adrenal (HPA) response to an ethanol challenge in adolescent animals. Since hippocampus is particularly sensitive to the deleterious effects of ethanol during adolescence, we investigated in this study the combined effects of PRS and ethanol administration on the oxidative status in the hippocampus of 28-day-old male rats. Thirty minutes after an intraperitoneal (i.p.) injection of ethanol (1.5 g/kg), the activities of several antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) but also non-enzymatic antioxidant (reduced glutathione) were assayed. Thiobarbituric acid reactive substances (TBARS) levels were also measured as a marker of lipid peroxidation. Ethanol enhanced superoxide dismutase activity in control rats but not in PRS rats. At basal level, catalase activity was lower in PRS rats than in control rats, indicating a potentially higher sensitivity to oxidative damages after this early stress. However, the hippocampal TBARS levels were not significantly affected by the ethanol administration, showing that an acute ethanol exposure does not induce oxidative damage in adolescent male rats. In conclusion, our data suggest that PRS affects both basal antioxidant status in the hippocampus and antioxidant response after an acute ethanol exposure. These findings extend previous works showing that PRS leads to hippocampal dysfunctions and raise the question of the potential increase of the hippocampal oxidative damage in PRS rats after repeated exposure to ethanol.

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS. Application to the determination of MDMA after low ecstasy intake.

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C(18) 5 microm, 2.1 mm x 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 degrees C in NaOH 1M before liquid-liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1-50 ng/mL in blood and urine; in the range 5-500 pg/mg for MA, MDMA, MDEA and MBDB, and 20-500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T+12h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D+8) and scalp hair at day 60 (D+60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.

Molecular analysis of the CYP2F1 gene: identification of a frequent non-functional allelic variant.

The CYP2F1 is a human cytochrome P450 that is selectively expressed in lung tissue and involved in the metabolism of various pneumotoxicants with potential carcinogenic effects. In the present study, we report the first systematic investigation of the genetic polymorphism of this enzyme. We analyzed the nucleotidic sequence of the CYP2F1 gene in DNA samples from 90 French Caucasians consisting in 44 patients with lung cancer and 46 control individuals, using single-strand conformation polymorphism analysis of PCR products (PCR-SSCP). We identified 24 novel mutations distributed in the promoter region of the gene, as well as in the coding regions and their flanking intronic sequences. In addition to the wild-type CYP2F1*1 allele, seven allelic variant, CYP2F1*2A, *2B, *3, *4, *5A, *5B and *6, were characterized. The most frequent allelic variant, CYP2F1*2A (25.6%), harbors a combination of 9 mutations, including 2 missense mutations (Asp218Asn and Gln266His) and a 1-bp insertion (c.14_15insC) that creates a premature stop codon in exon 2, probably leading to the synthesis of a severely truncated protein with no catalytic activity. The identification of around 7% of homozygotes for the frameshift mutation in our Caucasian population suggests the existence of an interindividual variation of the CYP2F1 activity and, consequently, the possibility of interindividual differences in the toxic response to some pneumotoxicants and in the susceptibility to certain chemically induced diseases. However, our preliminary results did not show any evidence that the CYP2F1 genetic polymorphism has implications in the pathogenesis of lung cancer.

Lipid-lowering drugs in the MPTP mouse model of Parkinson's disease: fenofibrate has a neuroprotective effect, whereas bezafibrate and HMG-CoA reductase inhibitors do not.

We tested the ability of simvastatin, atorvastatin, fenofibrate and bezafibrate (two synthetic peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonists) to prevent dopaminergic cell death in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Tyrosine hydroxylase (TH) immunochemistry was performed 8 days after acute MPTP intoxication. When orally administered for the week prior to intoxication and a week thereafter, fenofibrate prevented the MPTP-induced dopaminergic cell loss in the substantia nigra pars compacta (SNpc) and attenuated the loss of tyrosine hydroxylase immunoreactivity in the striatum. The dosage of 1-methyl-4-phenyl pyridinium (MPP+) in the striatum by high-performance liquid chromatography indicated that fenofibrate did not affect MPTP metabolism. Bezafibrate had no effect and, strikingly, simvastatin and atorvastatin had a negative effect. We also demonstrated the presence of PPAR-alpha in the dopaminergic neurons of the murine substantia nigra. Our data suggest that PPAR-alpha activation by fenofibrate could have a neuroprotective effect in PD through inhibition of inflammation, oxidative stress and/or apoptosis.

Major metabolites of zolpidem: expeditious synthesis and mass spectra.

An expeditious route to the two major metabolites of Zolpidem-and readily applicable to the synthesis of the drug-was established via a cyclization reaction between a 2-aminopyridine and a suitable alpha-bromoacetophenone. The structures of the target compounds were confirmed from a 2D (1)H-(15)N NMR correlation. Their mass spectra contribute to a reliable toxicological identification of the drug in the case of drug-facilitated crimes.

[Xenobiotic-metabolizing polymorphic enzymes. An opportunity for individualized drug treatment]. / Le polymorphisme génétique des enzymes du métabolisme des médicaments. Une opportunité pour un traitement individualisé.

Interindividual variability in drug responses can complicate patient management. This variability is partly due to genetic factors that affect pharmacokinetic and pharmacodynamic behavior Pharmacogenetics is a discipline focusing on the molecular mechanisms underlying drug responses. Its overriding goal is to optimize drug treatments, in terms of both their efficacy and their safety. Polymorphisms of genes that encode drug-metabolizing enzymes, transporter molecules and receptors have a well-documented impact on the distribution and effects of many medications. This review examines the scope of pharmacogenetics, the molecular bases of interindividual variations in drug responses, and the methods used to assess the individual risk of drug failure or toxicity. Pharmacogenetic approaches have already entered the clinical arena, resulting in significant improvements in patient management. Clinical validation of new pharmacogenetic tests and the development of new efficient genotyping technologies should rapidly lead to patient-tailored therapy.

Hypo-response of the hypothalamic-pituitary-adrenocortical axis after an ethanol challenge in prenatally stressed adolescent male rats.

The period of adolescence and environmental factors, such as stress, are important in determining ethanol vulnerability in both humans and rats. Ethanol is a powerful activator of the hypothalamic-pituitary-adrenal (HPA) axis but attenuated responses of the HPA axis to ethanol have been described in populations with a high risk of ethanol abuse. In rats, prenatal stress leads to prolonged stress-induced corticosterone secretion and increases the vulnerability to drugs of abuse, such as amphetamine and nicotine in adulthood and 3,4-methylenedioxymethamphetamine in adolescent rats. The aim of the present study was to assess the impact of a prenatal stress on HPA axis responsiveness to a moderate dose of ethanol (1.5 g/kg i.p.) in adolescent male rats (28 days old). The parameters evaluated were plasma adrenocorticotropic hormone, plasma corticosterone and mRNA expression of HPA axis central markers (mineralocorticoid receptor, glucocorticoid receptor, corticotropin-releasing hormone and pro-opiomelanocortin). Contrary to prior expectations, our results demonstrate that prenatal stress blunts the HPA axis responsiveness to a moderate dose of ethanol in adolescent rats in spite of similar blood ethanol levels. These data suggest that prenatal stress may have the opposite effect on the response to stress depending on the attributes of the stressor stimulus. They thus raise questions about the possible impact of prenatal stress on the further development of ethanol vulnerability.

Evidence for a functional genetic polymorphism of the human retinoic acid-metabolizing enzyme CYP26A1, an enzyme that may be involved in spina bifida.

BACKGROUND: CYP26A1, together with CYP26B1 and CYP26C1, are key enzymes of all-trans retinoic acid (RA) inactivation and their specific and restricted expression in developing embryos participate in the fine tuning RA levels. As RA is a critical regulator of gene expression during embryonic development, the imbalance between the synthesis and degradation of RA during embryogenesis could contribute to malformations and developmental defects. METHODS: A PCR-single strand conformation polymorphism (PCR-SSCP) strategy was developed to screen for CYP26A1 sequence variations that could affect the enzyme expression and/or activity and applied to DNA samples from 80 unrelated Caucasians, comprising 40 French healthy volunteers and 40 Italian patients with spina bifida. The consequence of the 1-bp deletion identified in the coding sequence was investigated by an in vitro functional assay using COS-7 cells. RESULTS: A total of 7 polymorphisms were identified, comprising 1 nucleotide deletion in the coding sequence (g.3116delT) that results in a frameshift and consequently in the creation of a premature stop codon. The g.3116delT mutation is of particular interest because it was identified in a patient with spina bifida and likely encodes a truncated protein with no enzymatic activity, as demonstrated by our preliminary in vitro data. CONCLUSIONS: Despite the fact that our findings could not show any evidence that the CYP26A1 genetic polymorphism has implications in the pathogenesis of spina bifida, this work represents the first description of a functional genetic polymorphism affecting the coding sequence of the human CYP26A1 gene.