Metabolic Hydrolysis of Aromatic Amides in Selected Rat, Minipig, and Human In Vitro Systems.

The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5-10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.

Previous PICC placement may be associated with catheter-related infections in hemodialysis patients.

BACKGROUND: Catheter-related infections (CRIs) are a significant source of morbidity and mortality in hemodialysis patients. The identification of novel, modifiable risk factors for CRIs may lead to improved outcomes in this population. Peripherally inserted central catheters (PICCs) have been hypothesized to compromise vascular access due to vascular damage and venous thrombosis, whereas venous thrombosis has been linked to the development of CRIs. Here we examine the association between PICC placement and CRIs. METHODS: A retrospective review was performed of all chronic hemodialysis catheter placements and exchanges performed at a large university hospital from September 2003 to September 2008. History of PICC line use was determined by examining hospital radiologic records from December 1993 to September 2008. Catheter-related complications were assessed and correlated with PICC line history. RESULTS: One hundred eighty-five patients with 713 chronic tunneled hemodialysis catheter placements were identified. Thirty-eight of those patients (20.5%) had a history of PICC placement; these patients were more likely to have CRIs (odds ratio = 2.46, 95% confidence interval = 1.71-3.53, p < .001) compared with patients without a history of PICC placement. There was no difference between the two groups in age or number of catheters placed. CONCLUSION: Previous PICC placement may be associated with catheter-related infections in hemodialysis patients.

The metabolism and toxicity of furosemide in the Wistar rat and CD-1 mouse: a chemical and biochemical definition of the toxicophore.

Furosemide, a loop diuretic, causes hepatic necrosis in mice. Previous evidence suggested hepatotoxicity arises from metabolic bioactivation to a chemically reactive metabolite that binds to hepatic proteins. To define the nature of the toxic metabolite, we examined the relationship between furosemide metabolism in CD-1 mice and Wistar rats. Furosemide (1.21 mmol/kg) was shown to cause toxicity in mice, but not rats, at 24 h, without resulting in glutathione depletion. In vivo covalent binding to hepatic protein was 6-fold higher in the mouse (1.57 +/- 0.98 nmol equivalent bound/mg protein) than rat (0.26 +/- 0.13 nmol equivalent bound/mg protein). In vivo covalent binding to mouse hepatic protein was reduced 14-fold by a predose of the cytochrome P450 (P450) inhibitor, 1-aminobenzotriazole (ABT; 0.11 +/- 0.04 nmol equivalent bound/mg protein), which also reduced hepatotoxicity. Administration of [(14)C]furosemide to bile duct-cannulated rats demonstrated turnover to glutathione conjugate (8.8 +/- 2.8%), gamma-ketocarboxylic acid metabolite (22.1 +/- 3.3%), N-dealkylated metabolite (21.1 +/- 2.9%), and furosemide glucuronide (12.8 +/- 1.8%). Furosemide-glutathione conjugate was not observed in bile from mice dosed with [(14)C]furosemide. The novel gamma-ketocarboxylic acid, identified by nuclear magnetic resonance spectroscopy, indicates bioactivation of the furan ring. Formation of gamma-ketocarboxylic acid was P450-dependent. In mouse liver microsomes, a gamma-ketoenal furosemide metabolite was trapped, forming an N-acetylcysteine/N-acetyl lysine furosemide adduct. Furosemide (1 mM, 6 h) became irreversibly bound to primary mouse and rat hepatocytes, 0.73 +/- 0.1 and 2.44 +/- 0.3 nmol equivalent bound/mg protein, respectively, which was significantly reduced in the presence of ABT, 0.11 +/- 0.03 and 0.21 +/- 0.1 nmol equivalent bound/mg protein, respectively. Furan rings are part of new chemical entities, and mechanisms underlying species differences in toxicity are important to understand to decrease the drug attrition rate.

Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes.

Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and on exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug-metabolizing enzymes, vary widely between species, among individual humans, and on exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. Here we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a second-generation doxycycline-inducible gene expression system and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely phase I (cytochrome P-450 2E1; CYP2E1) and phase II (glutathione S-transferase P1; GSTP1) drug metabolism, and three transcription factors that respond to chemical stress [nuclear factor erythroid 2 p45-related factors (NRF)1 and 2 and NFKB1 subunit of NF-kappaB]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.