Developmental patterns in human blood-brain barrier and blood-cerebrospinal fluid barrier ABC drug transporter expression.

When drugs exert their effects in the brain, linear extrapolation of doses from adults could be harmful for children as the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) function is still immature. More specifically, age-related variation in membrane transporters may impact brain disposition. As human data on brain transporter expression is scarce, age dependent [gestational age (GA), postnatal age (PNA), and postmenstrual age (PMA)] variation in immunohistochemical localization and staining intensity of the ABC transporters P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins 1, 2, 4, and 5 (MRP1/2/4/5) was investigated. Post mortem brain cortical and ventricular tissue was derived from 23 fetuses (GA range 12.9-39 weeks), 17 neonates (GA range 24.6-41.3 weeks, PNA range 0.004-3.5 weeks), 8 children (PNA range 0.1-3 years), and 4 adults who died from a wide variety of underlying conditions. In brain cortical BBB, immunostaining increased with age for Pgp and BCRP, while in contrast, MRP1 and MRP2 staining intensity appeared higher in fetuses, neonates, and children, as compared to adults. BCSFB was positively stained for Pgp, MRP1, and MRP2 and appeared stable across age, while BCRP was not detected. MRP4 and MRP5 were not detected in BBB or BCSFB. In conclusion, human BBB and BCSFB ABC membrane transporters show brain location and transporter-specific maturation.

Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

Four QTLs determine crown rust (Puccinia coronata f. sp. lolii) resistance in a perennial ryegrass (Lolium perenne) population.

Crown rust resistance is an important selection criterion in ryegrass breeding. The disease, caused by the biotrophic fungus Puccinia coronata, causes yield losses and reduced quality. In this study, we used linkage mapping and QTL analysis to unravel the genomic organization of crown rust resistance in a Lolium perenne population. The progeny of a pair cross between a susceptible and a resistant plant were analysed for crown rust resistance. A linkage map, consisting of 227 loci (AFLP, SSR, RFLP and STS) and spanning 744 cM, was generated using the two-way pseudo-testcross approach from 252 individuals. QTL analysis revealed four genomic regions involved in crown rust resistance. Two QTLs were located on LG1 (LpPc4 and LpPc2) and two on LG2 (LpPc3 and LpPc1). They explain 12.5, 24.9, 5.5 and 2.6% of phenotypic variance, respectively. An STS marker, showing homology to R genes, maps in the proximity of LpPc2. Further research is, however, necessary to check the presence of functional R genes in this region. Synteny at the QTL level between homologous groups of chromosomes within the Gramineae was observed. LG1 and LG2 show homology with group A and B chromosomes of oat on which crown rust-resistance genes have been identified, and with the group 1 chromosomes of the Triticeae, on which leaf rust-resistance genes have been mapped. These results are of major importance for understanding the molecular background of crown rust resistance in ryegrasses. The identified markers linked to crown rust resistance have the potential for use in marker-assisted breeding.

Exposure related mutagens in urine of rubber workers associated with inhalable particulate and dermal exposure.

AIMS: To determine the relation of the inhalation and dermal exposure routes and mutagenic activity in the urine of rubber workers (n = 105). METHODS: Mutagenic activity of ambient total suspended particulate matter (TSPM), surface contamination wipes, and Sunday and weekday urine samples was assessed with S typhimurium YG1041 in the presence of a metabolic activation system. Each subject was grouped into one of two exposure categories for dermal exposure (high (>/=25 revertants/cm(2)), low (<25 revertants/cm(2))) based on the mutagenic activity detected on likely skin contact surfaces and into two airborne mutagenic exposure categories (high (>/=210 revertants/m(3)), low (<210 revertants/m(3))). The potential influence of skin aberrations and acetylation status (NAT2) on urinary mutagenicity levels was also evaluated. RESULTS: A non-significant increase of +1605 revertants/g creatinine in urinary mutagenicity during the workweek relative to levels observed on Sunday was observed for the total population. Subsequent multivariate regression analyses, with the subjects' weekday urinary mutagenicity levels as the dependent variable, revealed associations with environmental and mainstream tobacco smoke exposure, with the level of mutagenic contamination on surfaces with which the subjects had likely contact, with the subjects' inhalable particulate exposure level, with observed mild skin aberrations, and when the subjects had a slow acetylation phenotype. Similar associations, although weaker were observed with Sunday urinary mutagenicity levels as well, except for the association with slow acetylation phenotype. Based on measured exposure levels it could be estimated that a high potential for exposure to surface contamination with mutagenic activity increased weekday urinary mutagenicity by about 62% when compared to low exposed workers, while high inhalable particulate exposure levels increased weekday urinary mutagenicity levels by about 21%. Subjects with mild skin aberrations had an additional, non-significant, increase in weekday urinary mutagenic activity compared to subjects without any skin aberrations. DISCUSSION: Results suggest that the dermal exposure route may contribute more to the level of genotoxic compounds in urine of rubber workers than the inhalation route. Although the study was limited in size, the results warrant further investigation in the importance of and ways to effectively control the dermal exposure route in the rubber industry.

Further localization of the gene for hereditary paragangliomas and evidence for linkage in unrelated families.

Paragangliomas of the head and neck are slow-growing tumors that rarely show malignant progression. Familial transmission has been described, consistent with an autosomal dominant gene that is maternally imprinted. Clinical manifestations of hereditary paraganglioma are determined by the sex of the transmitting parent. All affected individuals have inherited the disease gene from their father, expression of the phenotype is not observed in the offspring of an affected female or female gene carrier until subsequent transmittance of the gene through a male gene carrier. Recently, we assigned the gene responsible for paragangliomas (PGL) to chromosome 11q23-qter by linkage in a single large Dutch kindred. We now report confirmation of this localization in five unrelated Dutch families with hereditary paragangliomas. On the basis of segregation of haplotypes in the available family material, we localize the PGL locus between markers STMY and CD3D on chromosome 11q22.3-q23.

Stereospecific in vitro embryotoxicity of l-homocysteine in pre- and post-implantation rodent embryos.

Recently a derangement of homocysteine metabolism has been suggested as a possible risk factor for neural tube defects and recurrent spontaneous abortion. To investigate a possible role of homocysteine in the aetiology of neural tube defects we tested the in vitro embryotoxicity of l-homocysteine by culturing day 10 post coitum post-implantation rat embryos in whole embryo culture (WEC) for 24 hr and day 2 post coitum pre-implantation mouse embryos for 48 hr. With an area under curve (AUC) of 6.3 mm/hr, l-homocysteine significantly reduced the percentage of mouse embryos that developed into blastocysts. In rat WEC, an AUC for l-homocysteine of 3.6 mm/hr reduced the mitotic index of the neural epithelium of the rhombencephalon and the cell density of the mesenchyme adjacent to it, while at an AUC of 7.2 mm/hr l-homocysteine reduced the total morphological score and the number of malformations was increased. Malformations most often seen were transparent rhombencephalon, no or delayed formation of forelimb buds, dysmorphogenesis of the somites, and blister formation dorso-laterally of the place of forelimb bud formation. The embryotoxicity of l-homocysteine was stereospecific since d-homocysteine caused no embryotoxic effects. Also the oxidation product l-homocystine (AUC, 72 mm/hr) and the metabolite l-methionine (AUC, 144 mm/hr) were not embryotoxic. Both stereoisomers of homocysteinethiolactone were embryotoxic at an AUC of 72 mm/hr. The results are discussed in relation to the metabolism of homocysteine and methionine and their possible role in the neurulation process.

Sex difference in Aroclor 1254 induction of rat hepatocytes: Consequences for in vitro embryotoxicity and mutagenicity of cyclophosphamide.

The sequential culture of rat hepatocytes and post-implantation rat embryos has been proposed as a model for the in vitro testing of pro-teratogens. Comparing this model with a model in which embryos and hepatocytes are cultured simultaneously a striking difference in sensitivity was noted. To address the question of whether this difference could be explained by different sex and/or Aroclor 1254 pretreatment of the rats providing the hepatocytes, an experiment was designed with four groups: male Aroclor 1254 pretreated (M(1)), male untreated, pregnant female Aroclor 1254 pretreated (F(1)) and pregnant female untreated rats. Hepatocytes were incubated in the presence of cyclophosphamide (CP) and rat embryos were cultured in the media derived from the hepatocyte culture (i.e. the sequential culture model). Additionally, the CP concentrations of the media were analysed and subsequently the media were tested in a bacterial mutagenicity test (Salmonella typhimurium TA1535). With a CP concentration of 300 mum, M(1) produced maximum embryotoxicity and mutagenicity after 4 hr of hepatocytes incubation. All other groups showed no or only a slight increase in embryotoxicity and mutagenicity for all hepatocyte incubations. M(1) was also quickest to eliminate CP from the medium. These results indicate that despite a strong increase in total cytochrome P-450 in both sexes as a result of Aroclor 1254 pretreatment, and in the absence of a significant difference in total cytochrome P-450 between M(1) and F(1), Aroclor 1254 pretreatment has a much more pronounced effect in male rats than in pregnant female rats with regard to the production of embryotoxic and mutagenic metabolites of CP.

Growth and liver morphology after long-term ethanol consumption of rats.

Ethanol was administered to female and male Wistar rats by mixing it with their drinking water. Ethanol concentrations were gradually increased up to either 8% or 15%. Female rats receiving 8% ethanol in their drinking water consumed 5-13 g, males 4-10 g daily. The ethanol/total food caloric intake percentages were 13 to 20% and 9 to 15% for female and male rats, respectively. There was no difference in body weight and relative liver weight between treated rats and their controls. Female and male rats receiving 15% of ethanol in their drinking water consumed 8-14 g ethanol per kg body weight per day. The percentages of ethanol/total food caloric intake were stabilized at about 25% for both sexes. Growth of the rats differed only slightly from controls; a tendency for a higher increase of body weight of the control rats was found. No difference in relative liver weight between ethanol-treated and control rats was observed. Microscopic examinations revealed that the ethanol treatment resulted in fat accumulation in the liver cells. A proliferation of the Smooth Endoplasmic Reticulum (SER) was more marked in the 15% dosed rats than in the 8% dosed rats and more distinct in female rats than in male rats in both dosage groups.