Perfluorooctanesulfonic acid (PFOS) and its derivatives have been used in a range of industrial and commercial applications, including the manufacture of surfactants, adhesives, anticorrosion agents, and insecticides. PFOS is found at detectable concentrations in human and wildlife tissues and in the global environment. N-Substituted perfluorooctanesulfonamides are believed to be degraded to PFOS and, therefore, contribute to the accumulation of PFOS in the environment. N-Ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide (N-EtFOSE) is converted to PFOS in experimental animals. The objective of this study was to elucidate the pathways for the biotransformation of N-EtFOSE, which is a major precursor and component of PFOS-based compounds. N-EtFOSE and several putative metabolites were incubated with liver microsomes and cytosol and with liver slices from male Sprague-Dawley rats. Microsomal fractions fortified with NADPH catalyzed the N-deethylation of N-EtFOSE to give N-(2-hydroxyethyl)perfluorooctanesulfonamide (FOSE alcohol) and of FOSE alcohol to give perfluorooctanesulfonamide (FOSA). These N-dealkylation reactions were catalyzed mainly by male rat P450 2C11 and P450 3A2 and by human P450 2C19 and 3A4/5. Rat liver microsomal fractions incubated with UDP-glucuronic acid catalyzed the O-glucuronidation of N-EtFOSE and FOSE alcohol and the N-glucuronidation of FOSA. Cytosolic fractions incubated with NAD(+) catalyzed the oxidation of FOSE alcohol to perfluooctanesulfonamidoacetate (FOSAA). The oxidation of N-EtFOSE to N-ethylperfluorooctanesulfonamidoacetate (N-EtFOSAA) was observed in liver slices but not in cytosolic fractions. FOSA was biotransformed in liver slices to PFOS, albeit at a low rate. These results show that the major pathway for the biotransformation of N-EtFOSE is N-dealkylation to give FOSA. The biotransformation of FOSA to PFOS explains the observation that PFOS is found in animals given N-EtFOSE.

ABSTRACT:N-Alkylperfluorooctanesulfonamides have been used in a range of industrial and commercial applications. Perfluorooctanesulfonamide (FOSA) is a major metabolite of N-alkylperfluorooctanesulfonamides and has a long half-life in animals and in the environment and is biotransformed to FOSA N-glucuronide. The objective of this study was to identify and characterize the human and experimental animal liver UDP-glucuronosyltransferases (UGTs) that catalyze the N-glucuronidation of FOSA. The results showed that pooled human liver and rat liver microsomes had high Nglucuronidation activities. Expressed rat UGT1.1, UGT2B1, and UGT2B12 in HK293 cells catalyzed the N-glucuronidation of FOSA but at rates that were lower than those observed in rat liver microsomes. Of the 10 expressed human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, and 2B17) studied, only hUGT2B4 and hUGT2B7 catalyzed the N-glucuronidation of FOSA. The kinetics of N-glucuronidation of FOSA by rat liver microsomes and by hUGT2B4/7 was consistent with a single-enzyme Michaelis-Menten model, whereas human liver microsomes showed sigmoidal kinetics. These data show that rat liver UGT1.1, UGT2B1, and UGT2B12 catalyze the N-glucuronidation of FOSA, albeit at low rates, and that hUGT2B4 and hUGT2B7 catalyze the N-glucuronidation of FOSA.

ABSTRACT:Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of ␣-haloacids, including dichloroacetic acid (DCA), and the penultimate step in the tyrosine degradation pathway. DCA is a rodent carcinogen and a common drinking water contaminant. DCA also causes multiorgan toxicity in rodents and dogs. The objective of this study was to determine the expression and activities of GSTZ1-1 in rat tissues with maleylacetone and chlorofluoroacetic acid as substrates. GSTZ1-1 protein was detected in most tissues by immunoblot analysis after immunoprecipitation of GSTZ1-1 and by immunohistochemical analysis; intense staining was observed in the liver, testis, and prostate; moderate staining was observed in the brain, heart, pancreatic islets, adrenal medulla, and the epithelial lining of the gastrointestinal tract, airways, and bladder; and sparse staining was observed in the renal juxtaglomerular regions, skeletal muscle, and peripheral nerve tissue. These patterns of expression corresponded to GSTZ1-1 activities in the different tissues with maleylacetone and chlorofluoroacetic acid as substrates. Specific activities ranged from 258 ؎ 17 (liver) to 1.1 ؎ 0.4 (muscle) nmol/min/mg of protein with maleylacetone as substrate and from 4.6 ؎ 0.89 (liver) to 0.09 ؎ 0.01 (kidney) nmol/min/mg of protein with chlorofluoroacetic acid as substrate. Rats given DCA had reduced amounts of immunoreactive GSTZ1-1 protein and activities of GSTZ1-1 in most tissues, especially in the liver. These findings indicate that the DCA-induced inactivation of GSTZ1-1 in different tissues may result in multiorgan disorders that may be associated with perturbed tyrosine metabolism.Glutathione transferases (GST 1 ) are a multigene family of phase II drug-metabolizing enzymes that catalyze the conjugation of glutathione with a range of endogenous and exogenous substrates (Armstrong, 1997;Salinas and Wong, 1999). Soluble and membrane-bound GSTs are expressed in many tissues, and soluble GSTs constitute 1 to 5% of total cytosolic protein (Morgenstern et al., 1984;Sundberg et al., 1993;Rowe et al., 1997). Activities with model substrates and the subcellular localization of GSTA, GSTM, GSTP, GSTT, and GSTO class GSTs and of microsomal GST1 have been previously characterized (Meyer et al., 1991;Hiratsuka et al., 1994;Mannervik and Widersten, 1995;Armstrong, 1997;Otieno et al., 1997;Yin et al., 2001).The expression of GSTs is regulated pre-and post-translationally in a tissue-specific manner resulting in protein products that differ quantitatively in different tissues (Tu et al., 1983;Rozell et al., 1993;Rowe et al., 1997). Testes express the highest amount of total GST protein per milligram cytosolic protein followed by the liver, brain, pancreas, adrenals, heart, and lung (DePierre and Morgenstern, 1983;Listowsky et al., 1998). Major GST-expressing tissues are the principal sites for drug and chemical metabolism indicative of the role of GSTs in the biotransformation of xenobiotics (Pabst et al., 1973;Armstrong, 1997). Studies...

A previously identified human herpesvirus 6B (HHV-6B) origin of DNA replication contains two binding sites for the origin-binding protein (OBPH6B). We have investigated the functional significance of these sites by determining the replication efficiencies of mutated origin sequences, using a transient replication assay. The results indicate that both sites are required for DNA replication. In addition, we have tested the functional consequences of linear sequence amplifications in the origin. The data show that tandemized origin elements are more efficiently replicated than single-copy origins. Finally, we have determined the extent of interstrain origin sequence variation that exists among HHV-6 isolates by cloning, sequencing, and analyzing origins from a number of virus isolates, including examples of both HHV-6A and HHV-6B.

The lytic-phase origin of DNA replication from human herpesvirus 6B (HHV-6B oriLyt) contains two binding sites for the origin-binding protein (OBP H6B ), both of which are required for DNA replication and which are separated by an AT-rich spacer. We have tested the functional significance of the structural, spatial and sequence characteristics of this spacer element by constructing a series of mutated origin sequences and analysing their replication efficiency. Changes in the sequence composition or length of the spacer resulted in dramatic decreases in replication efficiency. Furthermore, in contrast to what has been observed for herpes simplex virus type 1 (HSV-1) ori S , insertion of a complete helical turn of DNA into the spacer also resulted in abrogation of origin function. These data suggest that the arrangement of OBP sites in HHV-6B oriLyt is stringently constrained in terms of spacing and intervening sequence.