Assessment of Labrasol/Labrafil/Transcutol (4/4/2, v/v/v) as a non-clinical vehicle for poorly water-soluble compounds after 4-week oral toxicity study in Wistar rats.

Drug safety research is frequently faced with the challenge of the selection of appropriate vehicles for use in in vivo non-clinical safety assessment studies. Reported here are the results of blend Labrasol, Labrafil and Transcutol, [L/L/T, (4/4/2, v/v/v)], excipients used as bioavailability enhancer and solubilizer for poorly water-soluble compounds and tested daily for 4 weeks by oral route in Wistar rats (10/sex/group) at dose volumes of 5, 10 or 20 mL/kg/day and compared to controls given 20 mL/kg/day of 1% (w/v) hydroxyethylcellulose in purified water. L/L/T was broadly well tolerated at 5 mL/kg/day and lethal at 20 mL/kg/day in 1 of 20 rats treated at this level. Changes in appearance and behaviour were observed from 10 mL/kg/day with volume-related incidence, severity and duration. Reduced feed intake observed from 5 (females) or 10 mL/kg/day (males) resulted in low bodyweights for high volume males only (-11% of controls). There was a volume-related induction of hepatic CYP 1A1/2, 2B1/2 and/or 2E1 subfamilies from 5 mL/kg/day, with high liver weight, centrilobular hepatocellular hypertrophy and high ALT, triglyceride and cholesterol serum values at 20 mL/kg/day. Renal tubular dilation in medulla, cortical cell degeneration/necrosis with granular material in adjacent glomerular spaces, crystal deposits in the inner medulla, papilla and/or renal pelvis, and tubular mineralization, associated with proteinuria and calcium oxalate crystalluria, were observed at 20 mL/kg/day as well as vacuolation in the adrenal cortex, with a sex-dependant localization. According to these results, 5 mL/kg/day was considered as an acceptable volume for further use of L/L/T (4/4/2, v/v/v) blend as a vehicle for poorly water soluble drugs in Wistar rat toxicity studies.

Comparative effects of drugs on P-glycoprotein expression and activity using rat and human trophoblast models.

The drug efflux transporter P-glycoprotein (P-gp) is an active component of the placental barrier which protects the fetus against maternal xenobiotics. The goal of the study was to compare species difference between human and rat in terms of susceptibility to drugs at the level of the placental barrier using in vitro models in order to improve translation from rat to human. Effects of selected drugs (Aspirin, Methadone, a Cardiovascular Proprietary Compound, Thalidomide) on cytotoxicity and P-gp expression and activity were compared using human and rat trophoblast cultures. No direct cytotoxicity of drugs on trophoblasts was noted in both invitro models, but for Thalidomide a proliferative effect on human trophoblast primocultures was observed. All tested drugs induced changes towards P-gp; for each drug the same profile was noted in both human and rat trophoblast models except for Thalidomide. Observation of this similar response between these two in vitro trophoblast models is promising for assessment between P-gp expression and activity of drugs towards placental function.

Development and characterisation of a new model of rat trophoblasts.

The placenta plays a key role during pregnancy. In vitro models have proven to assess the role of placental transporters in the exchange of nutrients, waste products and the distribution of drugs between the maternal and fetal compartments. Therefore, a primoculture of Wistar rat trophoblasts from the labyrinth zone was developed and characterised. Expression of placental transporters including P-glycoprotein (P-gp) and bcrp was evaluated by western blot and their activity using different inhibitors. A time-dependent increase in P-gp expression was noted from primocultures Day 2 to Day 4 followed by a plateau thereafter, whereas bcrp expression was stable throughout the culture period. P-gp and bcrp expression was maintained after seven passages in primocultures and in cryopreserved trophoblasts (up to 3 freezings and 10 passages). Activity of efflux transporters was confirmed in both placental primocultures and cryopreserved trophoblasts by an approximately 60% inhibition with cyclosporin A and valspodar for P-gp and 55% with elacridar for bcrp. In sum, this new in vitro model seems promising for a better understanding of the role of P-gp and bcrp in the toxicity of drugs during pregnancy and could be considered as an additional step towards the minimization of animal testing during drug development.

Cataractogenesis in rats induced by in utero exposure to RG 12915, a 5-HT3 antagonist.

RG 12915, a selective 5-HT3 antagonist developed for the treatment of emesis and nausea associated with cancer chemotherapy, was administered by gavage to four groups of pregnant rats from Gestation Day 6 to 17 at doses of 0, 1, 10, and 100 mg/kg/day, as part of a Segment II (developmental toxicity) study. The 100 mg/kg/day dose was maternally toxic as indicated by decreased body weight gain and food consumption during the treatment period. A portion of the rats were allowed to deliver and rear their litters and three pups from two litters in the 100 mg/kg/day group were observed to have lens opacities (visible to the naked eye) at weaning. At a later examination, when the offspring were approximately 4 months old, four additional animals from the same two litters had cataracts. A slight growth retardation was also observed postweaning in the offspring of the 100 mg/kg/day group. To confirm the lens findings and more precisely define the no-effect dose, another study was conducted in which pregnant rats were administered daily RG 12915 doses of 0, 10, 30, 60, or 100 mg/kg/day from Gestation Day 6 to 17. There was a dose-related decrement in maternal body weight gain during the treatment period in the 30, 60, and 100 mg/kg/day groups (12, 28, and 47%, respectively) compared to the control group. A treatment-related incidence of nuclear cataract was observed in the offspring of the 60 and 100 mg/kg/day groups (litter incidence 6 and 45%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Anionic sites of the basement membrane of rat seminiferous tubules during ontogenesis.

The anionic sites of the basement membrane of rat seminiferous tubules were demonstrated ultrastructurally in the lamina densa by using cationic polyethyleneimine (PEI). The sites were largely digested out after incubation with heparitinase, indicating a large proportion of heparan sulfates. The anionic sites were present as early as day 16 of gestation on the interstitial side of the lamina densa, and after gestation day 20 they were symmetrically organized on both sides of the lamina densa. The number of sites is not modified postnatally. They appear more irregular in density with advancing age. Experimental conditions as cryptorchidism, fetal irradiation, and ligation of the ductuli efferents lead to unspecific alterations in the distribution of the anionic sites that are parallel to the modifications in the basement membrane.

Anionic sites in the basement membrane of the rat epididymal epithelium during ontogenesis and after testicular and gonadal tract lesions.

Anionic binding sites in the lamina densa of the basement membrane of the rat epididymal epithelium were demonstrated ultrastructurally with the use of cationized polyethyleneimine (PEI). Enzyme digestion with heparitinase removed the anionic sites, indicating that they consist largely of heparan sulfates. The anionic sites are present as early as the 16th day of gestation on the interstitial face of the lamina densa; later during gestation they are localized on both faces of the lamina densa without further modification after birth. The distribution of the anionic sites was identical all along the epididymal duct. After castration and ligation of efferent ducts or in the state of cryptorchidism the sites were more numerous and located inside the thicker portion of the lamina densa. These alterations were more prominent in the initial segment compared to the distal segments, suggesting a differential androgen dependence of the reactive sites and their patterns of distribution.

[Postnatal development of zinc levels in the epididymis and testis in rats under normal and experimental conditions]. / Evolution postnatale de la teneur en zinc de l'épididyme et du testicule chez le rat dans les conditions normales et expérimentales.

Postnatal testicular and epididymal zinc concentration in the rat was investigated by means of differential pulse polarography. The zinc concentration increased gradually from birth to day 90 in the testis and up to day 60-90 in the epididymis with an abrupt increase on day 21. No marked variation in the zinc content was observed all along the epididymal duct. Experimental castration and efferent duct ligation were carried out in order to assess the influence of blood-borne and luminal androgens on epididymal zinc content. In prepubertal rats, unilateral castration and efferent duct ligation did not affect the zinc content of the epididymis. Moreover, zinc concentration was not affected by bilateral castration which induced very low plasma testosterone levels. These results suggested that epididymal zinc content did not depend upon endocrine testicular secretions, especially androgens. On the other hand, in adult rats efferent duct ligation and cryptorchidism resulted in about 50 and 70% reduction, respectively of the testicular and epididymal zinc content. A correlation was found between the absence of testicular fluid and spermatozoa or the alteration of germ cells and the decrease in epididymal and testicular zinc content.

Influence of testicular secretions on differentiation in the rat epididymis: ultrastructural studies after castration, efferent duct ligation and cryptorchidism.

The differentiation of the rat epididymis was studied in prepubertal castrated, ligated or cryptorchid rats, in order to assess the influences of blood-borne and luminal androgens. The principal cells showed partial differentiation: decrease in cell height, decreased numbers of cytoplasmic organelles implicated in the elaboration phenomena (Golgi apparatus, smooth endoplasmic reticulum), whereas the organelles implicated in the absorptive function remained relatively intact. The lamina densa of the basement membrane underlying the epithelium was irregular, thicker than normal and followed the irregular outline of the basal parts of the epithelial cells. These changes were evident in castrated rats, to a lesser degree in ligated and cryptorchid rats, and were more prominent in the initial part of the duct. On the other hand, the narrow cells and the clear cells followed a normal differentiation pattern in the experimental rats, suggesting that a differential androgen dependence exists among the various type of epididymal cells.

Ultrastructural study of epithelial cells and basement membrane. Differentiation of the rat epididymis after prenatal irradiation.

The effects of prenatal irradiation on the testis are well documented, but less is known about its effects on epididymal differentiation. Pregnant rats were irradiated on the 18th day of gestation. The increase in microfilaments and lipid inclusions in the epithelial cells, in favor of a direct radiation effect, is maximal at birth and disappears thereafter. Narrow cells and clear cells show a normal differentiation pattern. On the other hand, the principal cell maturation is largely altered. The synthesis capacities are decreased based on a reduction in the size of the Golgi apparatus and the smooth endoplasmic reticulum. The aspects of invaginations of the apical plasmalemma, coated vesicles and multivesicular bodies are not modified, suggesting normal absorption functions. The epithelial basement membranes become irregular and thicker than normal, enfolding the basal part of the epithelial cells. The basement membrane proteoglycans, demonstrated by the cationic marker polyethyleneimine, are irregularly distributed in contrast to the normal pattern. These modifications of the principal cells and the basement membrane are more prominent in the proximal epididymis. This suggests a differential maturation dependence of the epithelial cells on the luminal factors, normally secreted by the testis, and likely disturbed by prenatal irradiation which leads to germ cell degeneration, and then to a new balance in the seminiferous epithelium.

[Ultrastructural analysis of anionic sites of the basal membrane of the seminiferous tubules of subjects with severe oligospermia]. / Analyse ultrastructurale des sites anioniques de la lame basale du tube séminifère de sujets présentant une oligospermie sévère.

Several morphological alterations of the basement membrane of the seminiferous tubules have been reported in the patients presenting with a testicular sterility. The proteoglycans are one of the major components of the basal membrane and may be ultrastructurally defined by the use of the cationic marker polyethyleneimine (PEI). In comparaison to the normal distribution of the anionic sites labelled by PEI which is characterized by a regular pattern of distribution along both the epithelial and the interstitial faces of the lamina densa, the pattern of distribution of the sites is largely altered in 6 patients, 27 to 40 years of age, with oligo- or azoospermia. Several types of alterations are reported: parallel thickening of the lamina densa with an abnormal distribution of the anionic sites inside the lamina densa, stratifications of the basal membrane depressing the seminiferous epithelium with anionic sites labelled on both faces of the lamina densa-like layers, massive expansions of the lamina densa with either a regularly circumferential labelling of the sites or a random type of distribution. The digestion of the sites by the enzyme heparitinase is highly suggestive of the presence of proteo-heparan-sulfate. The analysis of the distribution of the anionic sites may represent a usefull tool for studying the pathogenesis of testicular sterility.

Influence of testicular secretions on epididymis oxidative metabolism in the rat.

The oxidative metabolism of the epididymis has been investigated in 40-day-old rats under normal and experimental conditions. No difference in the oxidative metabolism was observed between the initial, middle and terminal segments of the epididymal duct of normal rats. After bilateral or unilateral castration or efferent duct ligation, the oxidative metabolism was found to be exclusively dependent upon plasmatic androgens in the terminal segment in contrast to the proximal segment oxidative metabolism which is dependent of both normal luminal secretions and normal plasmatic androgens.

Differentiation of the rat epididymis after withdrawal of androgen.

The differentiation capacity of the rat epididymis after depletion of androgen was studied in organ culture and in castrated rats. The differentiation of "narrow cells' in 5- and 10-day-old explants and in 10-day-old castrated rats suggests that: (i) the testicular androgens are not essential for their differentiation, (ii) a differential androgen dependence exists among the epididymal cell types, (iii) the undifferentiated epithelial cells are the precursors of the narrow cells.

Experimental synovitis induced by aluminium phosphate in rabbits. Comparison of the changes produced in synovial tissue and in articular cartilage by aluminium phosphate, carrageenin, calcium hydrogen phosphate dihydrate, and natural diamond powder.

The goal of this experimental study was to examine the effect on articular tissue of tribasic aluminium phosphate (crystalline and amorphous forms) after intraarticular injection in rabbit and to compare it with that of various phlogistic compounds such as carrageenin, calcium hydrogen phosphate dihydrate and diamond powder, as a control. Synovium and cartilage were studied with light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy dispersive micro-analysis (EDM). Crystalline and amorphous aluminium phosphate could induce a synovitis with articular effusion in rabbits. With TEM, lysosomal inclusions of phagocytosed material were observed. Through SEM coupled with EDM, aluminium associated with phosphate was found in cellular elements.

In vitro development of seminiferous tubules from immature rat testis: ultrastructural and morphometric studies.

Seminiferous tubules from 5 day-old rats maintained in a semi-solid culture system were examined for 4, 8 or 12 days. Morphometric and ultrastructural studies show a significant increase in the seminiferous tubule diameter and normal Sertoli cell differentiation. All the germinal cells degenerate except the spermatogonia.

[Postnatal development of oxidative metabolism of the epididymis in rats]. / Evolution postnatale du métabolisme oxydatif de l'épididyme de rat.

The oxidative metabolism of the epididymis decreases significantly from birth to 12 days and increases gradually after 20 days. Its evolution is contemporary with the development of the interstitial gland and follows the variations of plasmatic and tissular testosterone levels.

[Toxicity and pharmacokinetics of zirconium oxychloride in mice and rats]. / Toxicité et pharmacocinétique de l'oxychlorure de zirconium chez la souris et chez le rat.

The experimental toxicity of zirconium compounds is examined in the Mouse (acute toxicity) and in the Rat (short time toxicity). The absorption, the distribution and the elimination of zirconium are evaluated by zirconium cation assay in some biological fluids and tissues. After a single oral dose, zirconium oxyd is not toxic, zirconium oxychlorure slightly toxic and zirconium chlorure moderately toxic. At certain concentrations, cerebral and pulmonary disorders are observed, particularly with zirconium chlorure. In considering molar toxicity, the studied zirconium compounds are more toxic than certain aluminium salts mentioned in the literature. The zirconium oxychlorure doesn't influence the growth curve after iterative administrations (0.23 g zirconium/kg/day). Only a weak fraction of administered zirconium is absorbed and is electively fixed in the ovaries, in a lesser degree in the lung and the bone. In the ovary the zirconium induces vascular variation (hypervascularization) which appear one month after the end of the treatment. The absorbed zirconium is eliminated by the urinary tract. The fecal elimination can be essentially explained by an important quantity of non absorbed zirconium.

Inflammatory effect of aluminium phosphate on rat paws.

The aim of this experiment was to determine the inflammatory effect of aluminium phosphate on the rat paw and to compare it with those of carrageenan, calcium hydrogen phosphate dihydrate and natural diamond powder. At the probability threshold of the various tests used there was a significant increase in volume of the treated paw relative to the control paw for all the substances at all times, except for the two concentrations of diamond (effect no longer significant from 30 min on), of calcium phosphate (not significant from 2 h), and of aluminium phosphate (not significant from 24 h). The effects were not significantly different between diamond and calcium phosphate (both concentrations). The effects of aluminium phosphate were significantly different from those of the two until at least 2 h after the injection ; there was no significant difference between the two doses of this substance. The kinetics of the effects produced by carrageenan differed from the kinetics of the other materials, its effects were not significantly different from those produced by aluminium phosphate until 2 h and later. The inflammatory effect that we have demonstrated in this study is added information suggesting that in addition to apatite, calcium pyrophosphate dihydrate, and sodium monourate, aluminium compounds can play a role in the unexpected appearance of inflammatory manifestations in haemodialysed patients with chronic renal insufficiency treated with aluminium gels.