Protocadherin-12 deficiency leads to modifications in the structure and function of arteries in mice.

We studied the role of protocadherin-12 on arterial function. This protein belongs to the cadherin superfamily and is located at the intercellular junctions of endothelial cells where it promotes homotypic cellular adhesion. We previously showed that mice deficient for PCDH12 exhibited developmental growth retardation owing to placenta defects without altering neither survival nor fertility. Here, we investigated the effects of PCDH12 deficiency on the structural, mechanical properties and functionality of arteries from adult mice. Histological studies of the PCDH12(-/-) mouse arteries have shown age-independent modifications such as ramifications of medial elastic lamellae, accompanied by the appearance of radial fibers linking together two successive concentric elastic lamellae. Mechanical studies also revealed some age-independent modifications in the PCDH12(-/-) mice arteries such as an increase in inner-diameter and circumferential mid-wall stress. Moreover, the PCDH12(-/-) mice exhibited a mild reduction of blood pressure, thus maintaining the inner-diameter close to its normal value and a normal circumferential wall stress for vascular cells. This is likely a compensation mechanism enabling normal blood flow in the arteries. The vascular phenotypic differences observed between PCDH12(-/-) and wild type mice arteries did not seem to be age-dependent, except for some results regarding the carotid artery: the reactivity to acetylcholine and the circumferential mid-wall stress decreased with ageing in the PCDH12(-/-) mice, as opposed to the increase observed in the wild types. In conclusion, deficiency in one specific interendothelial junction component leads to significant changes in the structure and function of the vascular wall. Possible explanations for the observed modifications are discussed.

Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of bupropion in rat everted gut sacs.

We have developed a rapid, sensitive and selective LC-MS method for the simultaneous assay of bupropion and its metabolite hydroxybupropion during its intestinal absorption, studied with the rat everted gut sac model. The method was validated in the concentration range of 1-15 microM (0.024-3.58 microg/mL) for bupropion and 0.005-1 microM (0.00127-0.25 microg/mL) for hydroxybupropion with 10 microL injected. Bupropion is used as a probe for the activity of the CYP2B6 isoenzyme of the P450 family of enzymes in man. Its major metabolite hydroxybupropion was found in the serosal media of the gut sac showing that the isoenzyme of the 2B group was active in the intestinal mucosa and metabolized bupropion during its passage across the mucosa. The metabolite was also quantified in the mucosal media indicating its ability to cross the apical membrane of the epithelial cells.

Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of dextromethorphan in rat everted gut sacs.

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous assay of dextromethorphan and its metabolites in tissue culture medium and its intestinal metabolism studied with the rat everted gut sac model. The method was validated in the concentration range of 0.1-2.5 microM (27.1 ng/mL-0.677 microg/mL) for dextromethorphan and 0.005-0.5 microM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL-0.128 microg/mL) and 3-hydroxymorphinan (1.22 ng/mL-0.122 microg/mL). The limits of quantification (LOQ) were 0.0025 microM (12.5 fmoles, 3.4 pg, 5 microL injected) for dextromethorphan; 0.0025 microM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 microL injected. The detection of dextrorphan and 3-methoxymorphinan showed that both the P450 isoforms CYP3A and 2D were active in the intestinal mucosa and metabolised dextromethorphan during its passage across the mucosa.

Liquid chromatographic-mass spectrometric method to assess cytochrome P450-mediated metabolism of testosterone by rat everted gut sacs.

A rapid, sensitive and specific method was developed for the simultaneous assay of testosterone, androstenedione and 6beta-hydroxytestosterone (6beta-OHT) in the TC199 tissue culture medium used in intestinal drug metabolism studies with the rat everted gut sac model. An electrospray LC-MS method was validated in the concentration range of 0.025-9.5 microM (7.2 ng-2.7 microg/mL) for testosterone and androstenedione and 0.01-4 microM (3 ng-1.2 microg/mL) for 6beta-hydroxytestosterone. The limits of quantification (LOQ) with an injection volume of 10 microL were 0.0005 microM (4.9 fmol, 1.4 pg injected), 0.004 microM (0.04 pmol, 11.4 pg injected) and 0.03 microM (0.3 pmol, 91 pg injected), respectively. The method also detected the other testosterone metabolites, the 16alpha-, 16beta-, 2beta- and 2alpha-hydroxytestosterones and was then used to study the metabolism of testosterone during its absorption by rat intestine in vitro, using everted gut sacs.

Development and evaluation of an HPLC urinalysis screening test for occupational exposure to 3,4- and 3,5-dichloroanilines.

OBJECTIVE: In order to develop a screening test to detect human occupational exposure to aromatic amines such as 3,4-dichloroaniline (3,4-DCA) and 3,5-dichloroaniline (3,5-DCA), we first investigated the urinary excretion of these highly toxic compounds in the rat. The study was performed after both oral and dermal application, even though contact with the skin is the major route of contamination in the workplace. The aim of this study was to develop a rapid screening test for risk assessment in the workplace. METHODS: An initial group of 3 rats was treated with 40 microl of 3,4-DCA solution (30% in methanol), applied topically to the shaved dorsal skin. A second group of 3 rats were administered the same dose of the amine orally by gavage before urine sampling. The same procedure was performed with 3,5-DCA (2 other groups of 3 rats). The urine samples were collected for a period of 24 hours after treatment and the excretion of 3,4-DCA, 3,5-DCA was studied using a GC-MS and an HPLC method after urine extraction. The urine of 2 workers potentially exposed to the amines for a period of 161 and 147 days, respectively, was analyzed by the same methods with urine collection before and at the end of the work shift. RESULTS: The study of excretion in the rat showed that unchanged dichloroanilines and some metabolites were excreted 24 hours after administration of the amines. Based on these results, we propose an HPLC method for the screening of risk assessment in the workplace. The presence of 3,4-DCA and 3,5-DCA in the urine of workers showed that they were absorbing amines during the workshift. CONCLUSIONS: These results successfully allowed us to detect contamination due to 3,4-DCA and 3,5-DCA in exposed workers. The HPLC method described provides a satisfactory and sensitive procedure for urine screening in the assessment and monitoring of the occupational exposure to dichloroanilines.

In vivo study of percutaneous absorption of 4-chloroaniline using microdialysis in the rat.

The present work was undertaken to study the percutaneous absorption of 4-chloroaniline (parachloroaniline, PCPA, CAS 106-47-8), a chemical intermediate in pesticide manufacture, using in vivo microdialysis in the rat. The results of the microdialysis study showed that PCPA was able to cross the skin (Tmax = 3 h and area under the curve (AUC) = 332.1 ng.h/ml) and rapidly enter the systemic circulation (Tmax = 3.3 +/- 0.6 h). It could be also shown that PCPA was partly metabolised during its percutaneous absorption. The analysis of cutaneous dialysate samples using gas chromatography/mass spectrometry (GC-MS) showed that the metabolite was 4-chloracetanilide. Taken together, the data obtained showed that contact with the skin is a danger for exposed persons.

The roles of P-glycoprotein and intracellular metabolism in the intestinal absorption of methadone: in vitro studies using the rat everted intestinal sac.

Methadone is used as a treatment for opiate detoxification in methadone maintenance programs. Intra- and inter-patient variations in methadone bioavailability have been observed after oral methadone treatment and this makes it difficult to predict a dosing regimen. Intestinal absorption and metabolism could explain these variations. The in vitro gut sac model was used to study the intestinal absorption of methadone, and it confirmed that methadone is a substrate for P-glycoprotein. The transport of methadone was increased in presence of P-gp inhibitors verapamil and quinidine. The appearance of a major metabolite of methadone, 2-ethylidene-1, 5-dimethyl-3, 3-diphenyl pyrrolidine (EDDP) in the gut sac contents also demonstrated the existence of intestinal metabolism of methadone.

Effect of hydrogen fluoride inhalation on lipid metabolism in guinea pigs.

The action of fluoride in vivo (exposure 96 hrs to 7 mg/m3) on the metabolism of cyclic AMP and relationship between cAMP and lipid metabolism was investigated. The mean values for cAMP, non esterified fatty acids and cholesterol were significantly increased after hydrogen fluoride exposure. cAMP is directly responsible for the increased lipolysis. In animals exposed to HF, theophylline injection causes increases of non esterified fatty acids and not produces modification of cholesterol level.

[Efficacy of PGF2 alpha on milk ejection during the sexual cycle of the cow]. / Etude de l'efficacité de PGF2 alpha sur l'éjection du lait au cours du cycle sexuel de la vache.

After 30 days of lactation, 15 non-pregnant French Friesian cows were divided into groups A and B, balanced by the milk yield and lactation number of the cows. The 7 cows of group A were given a series of intrajugular prostaglandin F2 alpha (Upjohn Dinolytic) injections every morning, administered in increasing logarithmic doses (1, 4, 16, 64, 256, 1 024 micrograms). The aim of the experiment was to determine the minimal dose that would induce an increase in intramammary pressure (IMP) during the sexual cycle and milk let-down through a cannula in the teat cup. The 8 cows of group B were given a single injection of 256 micrograms of PGF2 alpha every morning to determine the changes during the sexual cycle in the response parameters of IMP (latency and amplitude) or of milk let-down (latency and collected volume) through a cannula. The following results were obtained: 1. The minimal dose of prostaglandin inducing milk let-down varied during the cycle; it was lower (1 to 16 micrograms) during the luteal phase (D4 to D12) than during the perioestral phase (256 to greater than 1 024 micrograms from D - 2 to D + 3) (figs. 2, 3). 2. During daily administration of the same dose (256 micrograms) of PGF2 alpha no response was obtained during the perioestral phase, while during the luteal phase; - maximal IMP deflection reached 12 to 14 mmHg (figs. 5, 6); - the volume of milk ejected was on an average higher than 3 liters per quarter (figs. 5, 6); - the latency time between injection and let down response was about 1.8 times shorter between D10 and D12 than between D4 and D5 (fig. 7). 3. The cyclic changes in the milk ejection parameters caused by PGF2 alpha were closely related to the plasma progesterone level (figs. 2, 3, 5, 6). The coefficients of correlation between the progesterone level and the - threshold dose: r = - 0.809** (group A), - volume of ejected milk: r = + 0.872*** (group B), - deflection amplitude: r = + 0.805*** (group B) were always significantly higher than the threshold of 0.001. Various hypotheses concerning the endocrine control of cyclic variations in milk let-down under the effect of PGF2 alpha are discussed.