Intraluminal Medium Research Articles

Sex differences in hepatic and intestinal contributions to nevirapine biotransformation in rats

The understanding of the intestine contribution to drug biotransformation improved significantly in recent years. However, the sources of inter-individual variability in intestinal drug biotransformation, namely sex-differences, are still elusive. Nevirapine (NVP) is an orally taken anti-HIV drug associated with severe idiosyncratic reactions elicited by toxic metabolites, with women at increased risk. As such, NVP is a good model to assess sex-dimorphic metabolism. The aim of this study was to perform a comparative profiling of NVP biotransformation in rat intestine and liver and evaluate whether or not it is organ- and sex-dependent. Therefore, nevirapine-containing solutions were perfused through the intestine, in a specially designed chamber, or incubated with liver slices, from male and female Wistar rats. The levels of NVP and its Phase I metabolites were quantified by HPLC-UV. Liver incubation experiments yielded the metabolites 2-, 3-, 8-, and 12-OH-NVP, being 12-OH-NVP and 2-OH-NVP the major metabolites in males and females, respectively. Inter-sex differences in the metabolic profile were also detected in the intestine perfusion experiments. Herein, the metabolites 3- and 12-OH-NVP were only found in male rats, whereas 2-OH-NVP levels were higher in females, both in extraluminal (p<0.01) and intraluminal media. The metabolite 8-OH-NVP was not detected in the intraluminal media from either males or females. In this study, important inter-sex differences were detected in both organs, providing further clues to the sex-dimorphic profile of NVP toxicity. Moreover, an extra-hepatic contribution to NVP biotransformation was observed, strengthening the relevance of the intestinal contribution in the biotransformation of orally taken-drugs.

Understanding the preclinical toxicity profile of standard of care oncology agent carboplatin: Effect of a 7-cycle treatment on bone marrow and peripheral cell depletion and recovery in the Han Wistar rat

s / Toxicology Letters 229S (2014) S40–S252 S241 formed to evaluatewhether sex-dependent differences exist in this context. Nevirapine-containing solutions were perfused through the intestine, in a specially designed chamber, or incubated with liver slices frommale and female wistar rats. The levels of NVP and its Phase I metabolites were quantified by HPLC-UV. Liver incubationexperimentsyielded themetabolites2-, 3-, 8-, and12-OH-NVP. 12-OH-NVP and 2-OH-NVP were the major metabolites in males and females, respectively. Inter-sex differences in the metabolic profile were also detected in the intestine. Whilst 3and 12-OHNVP were only found in male rats, 2-OH-NVP levels were higher in females, both inextraluminal (p<0.01) and intraluminalmedia. The metabolite 8-OH-NVP was not detected in the intraluminal media, nor in the extraluminal media, from either males or females. An extra-hepatic contribution toNVP biotransformationwas observed herein. In particular, NVP biotransformation in the intestine generated a metabolite profile different from the liver. Moreover, important inter-sex differences were detected in both organs, providing further clues to the sex-dimorphic profile of NVP toxicity. http://dx.doi.org/10.1016/j.toxlet.2014.06.803

Basic mechanism of in vitro pulsed-dye laser-induced vasodilation.

Vasodilation of rabbit carotid arteries induced by a pulsed-eye laser was studied in vitro to clarify the underlying mechanism. Artery segments were double cannulated in a pressure-perfusion apparatus which, under physiological conditions, allows for differential application of various solutions, pharmacological agents, and pulsed-dye laser light. Vasoconstriction was activated using both pharmacological and nonpharmacological agonists. Laser energy at a wavelength of either 480 or 575 nm was applied intraluminally in 1-microseconds pulses, which caused dilation of the arteries if hemoglobin was present in the lumen at sufficient concentration. Induced vasodilation did not specifically require the presence of hemoglobin; the same phenomenon could be repeated using an inert dye such as Evans blue as an optical absorber of laser energy. The optical density of the absorber, the number of applied laser pulses, and total amount of applied energy directly influenced the vasodilatory response. Laser-induced vasodilation was possible in both normal vessels and vessels denuded of endothelium. Pulsed-dye laser-induced vasodilation is therefore not a phenomenon mediated through chemical processes, but is rather a purely physical process initiated by the optical absorption of laser energy by the intraluminal medium, which probably induces cavitation bubble formation and collapse, resulting in the vasodilatory response of the vessel.

Facial nerve regeneration through semipermeable chambers in the rabbit.

Peripheral neural regeneration, over a 10-mm transectional gap, was determined in 70 rabbit buccal divisions of the facial nerve using two entubational systems (semipermeable and impermeable silicone chambers) prefilled with three natural occurring media (serum, blood, and saline) during a 5-week period. The number of myelinated axonal regenerates at the midchamber and at 2 mm in the distal transected neural stump were counted in each group and compared to pooled myelinated axonal counts in 9 normal rabbit buccal divisions of the facial nerve. Semipermeable porous chambers had an overall greater regeneration success rate (75% vs. 42.8%) and regained, on the average, a higher number of myelinated axons (51.4% vs. 26.1%) than silicone chamber regenerates. Semipermeable chambers prefilled with serum or blood had significantly higher regeneration success rates, myelinated axonal counts, and percentages of neural innervation of the distal transected neural stump. Both entubational systems produced similar axonal counts with intraluminal saline. The highest overall success rate (93.7%) and average number of myelinated axons per chamber (3072) were achieved in semipermeable chambers prefilled with serum. The greatest variability in myelinated axonal counts (0 to 3266 axons) and percentage of distal stump innervation (5.5% to 98.1%) was seen in silicone chambers filled with saline. The percentage of myelinated axons from the midchamber that innervated the distal stump was greater in semipermeable chambers with blood (73%) and serum (54%) than in silicone saline chambers (43%). On the average, the distal stumps from semipermeable chambers filled with serum (47%) and blood (33.5%) regained a higher percentage of normal myelinated axonal counts than silicone-saline chambers (12.5%). These results suggest that both the construction of entubational chamber and the intraluminal medium can have significant influence on neurite regeneration. Semipermeable chambers prefilled with serum have a strong neurite-promoting potential in peripheral neural regeneration of rabbit facial nerves.

Effets du chrome hexavalent sur les fonctions de la bordure en brosse de l'intestin de la truite arc en ciel ( Salmo gairdneri R.)

A new procedure has been developed to study the effects of hexavalent chromium (potassium dichromate) on brush border membrane functions of the intestines of living trout (alkaline phosphatase and maltase activities, glycine absorption). Manifold recylced perfusions were carried out with saline solutions containing the specific substrate, the hydrolysis of which liberates glucose (Bussiere et al., 1987). At the end of each period, glucose concentrations were determined using the glucose oxydase method (Hugget and Nixon, 1957). For the determination of alkaline phosphatase activity, glucose-1-phosphate (20 mM) was used as substrate rather than classic para-nitrophenyl phosphate because the product of its hydroloysis; para-nitrophenol, is rapidly absorbed by the intestine (Bussiere et al., 1987). Phloridzin (10 −4 M) and KCl (150 mM), were added to the perfusates to prevent the glucose absorption, pH was adjusted to 10 with CHES (20 mM). For the determination of maltase activity, maltose (28 mM) was used as substrate in KCl (150 mM) solutions containing phloridzin (10 −4 M). pH was adjusted to 6.5 with MES (20 mM). The absorption of glycine by the intestine of trout was also determined in vivo using a perfusion technique (Pérès et al., 1973). The intestinal lumen was perfused with a saline solution containing glycine 14C (0.5 mM) and buffered at pH 7.4 with HEPES 20 mM. The perfusate was recycled in the intestine during 30 min periods and non absorbed glycine was collected and measured. Effects of hexavalent chromium on enzyme activities On each trout, a perfusion was first carried out in the absence of toxicant. Chromium was then added to the solutions and succeeding 30 min perfusions were carried out. After the chromium perfusions, the intestines were rinsed and perfused with a medium devoid of chromium. At the end of each period, alkaline phosphatase and maltase activities were measured. Two different dichromate concentrations were used, 1.36 and 0.136 mM. The highest concentration is generally lethal for trout after 24 h exposure in the aquatic environment. Chromium leads to a severe decrease of the alkaline phosphatase activity which increases with the chromium concentration and with time. An approximative 50% inhibition of this activity was observed after 30 min with 1.36 mM dichromate. Such an inhibition is obtained after 90 min with the lowest chromium concentration. During the early perfusion with 1.36 mM dichromate, a transient stimulation of the alkaline phosphatase activity has been observed which is progressively replaced by a characteristic inhibition. After chromium removal from the perfusion medium, the enzyme activity remains at a lower level than in the first control experience. This means that no recovery of the initial activity exists. No effect of chromium on maltase activity has been observed. Effect of hexavalent chromium on glycine absorption The intestinal absorption of amino acids in fish requires, as in mammals, the presence of sodium in the intraluminal medium, and can be highly depressed when sodium is replaced by potassium (Boge et al., 1980a). To avoid prejudicial effects of potassium on amino acid absorption, potassium dichromate was replaced by sodium dichromate and sodium was present in the perfusate at a final concentration of 272 mM. Chromium effects on each trout were studied after 60 min perfusions following control determinations. The results obtained revealed that chromium is a potent inhibitor of glycine absorption in trout but only when the concentration is very high: a 90% inhibition was obtained at 136 mM dichromate. This effect disappears when this concentration is ten times lower. These results point out the particular interest in ecotoxicology of the alkaline phosphatase activity which is a sensible target for chromium in trout intestine. A new method was presented which permits the measurement of this activity on living fishes, in relation to the presence of a toxic molecule.

Metabolism of exogenous noradrenaline in constricted and in distended rabbit ear arteries.

(1) The metabolism of extraluminal and of intraluminal 3H-noradrenaline (3H-NA; 0.18 mumol/l) was examined in arteries from reserpine-pretreated rabbits. The arteries were perfused in Ca medium (a) in the absence of prazosin to permit constriction to occur, (b) in the presence of prazosin, and (c) at a high intraluminal pressure (110 mm Hg; prazosin present) in order to distend the vessel. (2) The constrictor activity of the extraluminal NA was extremely weak, and antagonism by prazosin was not associated with a change in metabolite formation. The only change accompanying distension was a small (24%) increase in 3,4-dihydroxyphenylethylene glycol (DOPEG) formation. (3) The constrictor activity of intraluminal NA was much stronger than that of extraluminal NA. Antagonism of constriction by prazosin was accompanied by a 5-fold increase in DOPEG formation and a 2-fold increase in normetanephrine (NMN) formation. Distension resulted in a further 3-fold increase in DOPEG formation, but had little effect on NMN formation. (4) Since DOPEG is neuronal in origin, it is suggested that the changes in DOPEG formation in point 3 above reflect changes in the rate at which the intraluminal NA diffuses into the region of the sympathetic nerve terminals. In support of this suggestion, (a) distension was without effect when DOPEG formation was related to the vascular dimensions which determine rate of diffusion of NA, instead of to vascular mass, and (b) effects of constriction and distension on the rate of diffusion of intraluminal 14C-sorbitol across the artery wall paralleled their effects on DOPEG formation. (5) The effects of constriction on NMN formation in point 3 are attributed to diminished uptake2 (on which NMN formation depends) in the contracted smooth muscle, in association with tendencies for both NMN formation, and contractile activity, to be localized to the region of the vessel wall closest to the surface of entry of the NA. (6) Diffusion coefficients of 14C-sorbitol were estimated. The estimates indicate that (a) the changes in vascular dimensions are the prime determinants of rates of diffusion, (b) diffusivity is decreased when the vessel constricts, and (c) there may be bulk flow of the intraluminal medium into the vessel wall when it is distended by a high intraluminal pressure.