By combining the results of the melting curve analysis of

By combining the results of the melting curve analysis of exon 5 and exon 6, the allelic setup of our 250 subjects was determined; the four allelic variants are shown in Table 1. However, GSTP1-1 *A/*C and GSTP1-1 *B/*D could not be distinguished from one another because both genotypes are characterized by heterozygous patterns on both exons. Therefore, for the 20 subjects with this kind of pattern, we performed an ARMS assay [15] able to distinguish between the two genotypes. The restriction pattern obtained in these samples led to a GSTP1-1 *A/*C genotype as shown in Fig. 2. The genotype distribution for GSTP1-1 “H”-site polymorphism in our 250 subjects representative of an Italian population is summarized in Table 2. The allelic frequencies observed are the following: f(A)=0.710, f(B)=0.236, f(C)=0.054 and f(D)=0. The observed genotypes are in Hardy–Weinberg equilibrium (χ2=0.71, df=4, P=0.95). The increasing request for the evaluation of GSTP1-1 genotype in several clinical settings warrants the development of fast, accurate and automated methodologies. The recently introduced allelic discrimination method based on FRET has allowed the determination of GSTP1-1 exon 5 genotype [14]. We have extended this application to exon 6, thus improving the method for GSTP1-1 genotyping for both exon 5 and exon 6, based on a hybridization probe-formatted PCR performed with the Light-Cycler Instrument. This method provides the ability to genotype 30 samples in 2 h, and it represents a fast, reliable and automated methodology to determine GSTP1-1 polymorphism in order to perform population-based molecular epidemiological studies. This method, coupled with ARMS when appropriate, has allowed us to determine the complete genotype distribution for GSTP1-1 “H”-site polymorphism in a large sample of an adult Italian population coming from the area of Rome.
Acknowledgements
Introduction The diuretic drug ethacrynic acid, an α,β-unsaturated ketone, is known to be conjugated to glutathione (GSH), chemically as well as catalyzed by glutathione S-transferases (GST) [1]. Both the parent compound ethacrynic porcine and the ethacrynic acid-glutathione conjugate (EASG) are potent reversible inhibitors of GSTs with I50 values in the range of <0.1–11 μM, and ethacrynic acid inhibits GST of the pi class by covalent binding [2]. Because of these inhibiting properties ethacrynic acid has been studied as an agent to overcome multidrug resistance against alkylating drugs, since GSTs may play a role in that phenomenon [3]. Additionally, it has recently been shown that the glutathione conjugate of EA is a very good substrate and an inhibitor for the multidrug resistance associated protein (MRP) or GS-X pump [4]. This pump plays a role in drug resistance as well. MRP was first detected in a multidrug resistant cell line [5]and transports glutathione conjugates of both endogenous and exogenous molecules 6, 7. Glutathione conjugation of α,β-unsaturated carbonyl compounds, such as ethacrynic acid, can lead to the formation of two possible diastereoisomers. Catalysis by GSTs often results in preferential formation of one of the two diastereomers 8, 9, 10, 11. For example the GST isoenzyme M2-2 was stereoselective for the formation of one of the diastereomers of 4-phenyl-3-buten-2-one (PBO) [12]. In view of the multiple effects of ethacrynic acid and especially of its EASG conjugate, taking into account the possible different biological effects of the diastereoisomers, in this study we investigated the stereoselectivity of the GST catalyzed conjugation of ethacrynic acid, using 1H NMR spectroscopy, both using purified enzymes and in human melanoma cells.
Materials and methods
Results The most relevant section of a typical NMR spectrum of the EASG diastereoisomeric mixture is shown in Fig. 1A. Fig. 2 presents the structures of ethacrynic acid, an intermediate glutathione conjugate and the relevant parts of the two diastereoisomers of EASG. The addition of the sulfur of glutathione to the β-carbon (C10), followed by protonation of the negatively charged Cα center (C9) thus formed, results in generation of a chiral α-carbon C9. Because the proton most likely originates from the solvent or from a pool of protons that readily exchanges with the solvent 9, 10, protonation can occur on either site of the carbon.