Systematic screening of human ABCC3 polymorphisms and their effects on MRP3 expression and function.

The present study was undertaken to identify genetic polymorphisms of multidrug resistance-associated protein 3 (MRP3, gene name ABCC3), an ATP-binding cassette transporter that mediates the transport of substrates across the basolateral membrane into the blood, and to investigate their effects on ABCC3 expression and MRP3 function. We identified genetic polymorphisms of ABCC3 and evaluated the effects by (1) a luciferase reporter gene assay, (2) measuring mRNA levels, and (3) a human pharmacogenomics study with 4-methylumbelliferone glucuronide (4-MUG). Overall, 61 genetic variants were identified in three ethnic populations; of these variants 17 were novel (7 were non-synonymous: 61Arg>Cys, 132Gln>Stop, 221Trp>Stop, 270His>Gln, 548Leu>Gln, 600Lys>Arg, and 1324Arg>His). However, these mutations occurred at very low frequencies (max. 4.7%). The observed allele frequencies showed considerable inter-ethnic differences. The reporter gene assay indicated a significant reduction of transcriptional activity with the -1767G>A allele compared to the wild-type allele; however, a decreased expression of ABCC3 mRNA was not detected in human liver samples. A human pharmacokinetic study showed that the ABCC3 genotype in the promoter region was not associated with changes in the pharmacokinetics of 4-MUG, a substrate of MRP3. This is the first study to assess the effects of ABCC3 polymorphisms on human pharmacokinetics; however, further investigations are needed to complete the picture.

Does the composition of urine change when collected from disposable diapers and other absorbent materials?

The free and conjugated urinary species of non-persistent environmental chemicals or their breakdown products are valid human exposure biomarkers. For convenience, disposable diapers and other absorbent materials are widely used to collect urine specimens from infants and young toddlers. However, the extent to which the different urinary species of the target analytes and other components are recovered after the urine is extracted from these absorbent materials is unknown. In this proof-of-concept study, we investigated the extraction recovery from disposable diapers, cotton pads, and gauzes of the free versus glucuronidated urinary species of three example chemicals: bisphenol A, triclosan, and 4-methylumbelliferone. Although the glucuronides were almost fully recovered, the free species were not. Our results suggest that, in addition to other sampling considerations, the binding affinity and extraction recovery of the target biomarkers to the material used to collect the urine should be considered. Alternative collection approaches that do not require such an extraction (e.g., urine bags routinely used in hospitals) may be worth exploring. Despite its shortcomings, having urinary concentrations for biomonitoring considerably strengthens the exposure assessment, particularly for infants and young toddlers, and the benefits of including biomonitoring data outweigh their potential limitations.

Impaired renal excretion of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) sulfate in breast cancer resistance protein (BCRP1/ABCG2) knockout mice.

Murine breast cancer resistance protein 1 (Bcrp1) is expressed in the brush-border membrane of proximal tubule cells of the kidney. The purpose of the present study is to investigate whether Bcrp1 could be involved in the urinary excretion of the human BCRP substrates, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole sulfate (E3040S) and 4-methylumbelliferone sulfate (4MUS), using Bcrp1(-/-) mice. E3040S and 4MUS were given to the mice by intravenous infusion, and plasma and kidney concentrations and the urinary excretion rate were determined. Knockout of Bcrp1 did not affect the creatinine clearance [7.17 +/- 1.00 and 8.66 +/- 2.02 ml/min/kg for Bcrp1(-/-) and wild-type mice, respectively]. The renal clearance of E3040S was 2.4-fold lower in Bcrp1 (-/-) mice compared with wild-type mice (2.74 +/- 0.41 versus 6.55 +/- 0.52 ml/min/kg). The concentration of E3040S in the kidney was increased in Bcrp1(-/-) mice compared with that in wild-type mice (55.5 +/- 10.5 versus 19.4 +/- 2.7 nmol/g kidney, respectively). In contrast, knockout of Bcrp1 did not affect the pharmacokinetic parameters of 4MUS, although 4MUS was predominantly excreted in the urine. This is to our knowledge the first demonstration of involvement of Bcrp1 in the renal secretion of organic sulfates. However, taking the results of 4MUS into consideration, the renal secretion of organic sulfates cannot be accounted for solely by Bcrp1, and transporters other than Bcrp1 are also involved.

Identification of GM2-gangliosidosis B1 variant carriers.

GM2-gangliosidosis B1 variant, considered a rare disorder with a wide geographical and ethnic distribution, appears to be exceptionally frequent in Portugal. In order to establish a carrier detection method for this disease we have determined the ratio of enzymatic activities against 4MUGS and 4MUG in urine from B1 variant obligate carriers and controls, using the total extract and the Hex A immunobound to a monoclonal antibody. The Hex A immunoassay was applied to the identification of carriers in B1 variant families and the results obtained were compared with those from DNA analysis. The reliability and feasibility of the Hex A immunoassay make it a suitable method for B1 variant carrier screening, which is particularly important for the prevention of this severe neurological disease in the population at risk.