Integration of clinical chemistry, expression, and metabolite data leads to better toxicological class separation.

A large number of databases are currently being implemented within toxicology aiming to integrate diverse biological data, such as clinical chemistry, expression, and other types of data. However, for these endeavors to be successful, tools for integration, visualization, and interpretation are needed. This paper presents a method for data integration using a hierarchical model based on either principal component analysis or partial least squares discriminant analysis of clinical chemistry, expression, and nuclear magnetic resonance data using a toxicological study as case. The study includes the three toxicants alpha-naphthyl-isothiocyanate, dimethylnitrosamine, and N-methylformamide administered to rats. Improved predictive ability of the different classes is seen, suggesting that this approach is a suitable method for data integration and visualization of biological data. Furthermore, the method allows for correlation of biological parameters between the different data types, which could lead to an improvement in biological interpretation.

Pharmacokinetics of alpha-naphthyl isothiocyanate in rats.

Many naturally occurring and synthetic isothiocyanates can inhibit chemical carcinogenesis in animal models. Recently, we found that alpha-naphthyl isothiocyanate (1-NITC) inhibited P-glycoprotein- and multidrug resistance associated protein 1-mediated efflux, indicating the potential application of 1-NITC as a chemosensitizing agent for cancer chemotherapy. The objective of this study was to explore the pharmacokinetic characteristics of 1-NITC in rats. A single dose of 10, 25, 50, or 75 mg/kg of 1-NITC was administered intravenously or orally to female Sprague-Dawley rats (n = 4 for each group). Dose-normalized concentration-time profiles were not superimposable following intravenous or oral dosing, indicating that the disposition of 1-NITC in rats was nonlinear. As doses increased from 10 to 75 mg/kg following iv administration, the total clearance decreased from 2.2 +/- 0.9 to 0.8 +/- 0.3 L/h/kg; oral availability averaged 0.46 for oral doses of 10-75 mg/kg. A nonlinear two-compartment open model with capacity-limited absorption and capacity-limited elimination from the central compartment best fit the data, based on goodness-of-fit criteria. The mechanism underlying the nonlinear elimination of 1-NITC in rats is most likely due to the capacity-limited metabolism of 1-NITC. This study represents the first report of the pharmacokinetics of 1-NITC.

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.

Role of MRP2 and GSH in intrahepatic cycling of toxins.

MRP2 is a canalicular transporter in hepatocytes mediating the transport of a wide spectrum of amphipathic compounds. This includes organic anions but also compounds complexed with GSH as, e.g. alpha-naphthylisothiocyanate (ANIT) and arsenite. These reversible complexes may fall apart in bile after MRP2-mediated transport, which induces high concentrations of the toxic compound in the biliary tree. To further investigate the role of MRP2 in transport and toxicity of both compounds, we conducted experiments in transduced polarized epithelial cells and in vivo, using the Mrp2-deficient TR(-) rat as a model. Our results show, that in MRP2-transduced MDCK II cells both compounds induce disproportionally strong apical GSH secretion. This induction of GSH secretion was not observed in the parent cells lacking MRP2 expression. This indicated that after transport via MRP2 both complexes released GSH upon which the compound could re-enter the cells. The resulting cycling of both toxins led to concentration dependent GSH depletion of the cells. To further test our hypothesis we administered arsenite (12.5 micromol absolute i.v.) to Wistar and Mrp2-deficient TR(-) rats and collected bile. While both arsenite and GSH secretion were absent in TR(-) rats, the total secretion of arsenite into Wistar bile (2.91 micromol) was accompanied by a excess secretion of 24 micromol GSH, indicating that arsenite undergoes multiple cycles of GSH complexation. We also administered ANIT to both animal models and could show that TR(-) rats are protected from ANIT induced cholestasis. This indicates that Mrp2-mediated biliary secretion of GS-ANIT is a prerequisite for development of cholestasis in rats. We hypothesize that the toxic parent compound ANIT is regenerated in the biliary tree where it can exert its toxic properties on bile duct epithelial cells.

1-Naphthyl isocyanate and 1-naphthylamine as metabolites of 1-naphthylisothiocyanate.

The importance of the bioactivation of 1-naphthylisothiocyanate was studied. Forty minutes after 1-naphthylisothiocyanate administration to rats, bile was collected over a 2.5-h period; the liver was then excised and homogenized. 1-naphthylisothiocyanate and its metabolites in bile and liver of rats were identified and quantified using coupled gas chromatography-mass spectrometry. Three main compounds were found in all 1-naphthylisothiocyanate-treated animals. They were identified as 1-naphthyl isocyanate, 1-naphthylamine and the parent compound, 1-naphthylisothiocyanate. When rats were given cycloheximide, which attenuates 1-naphthylisothiocyanate toxicity, 30 min before 1-naphthylisothiocyanate (300 mg/kg), 1-naphthyl isocyanate concentration was significantly lower than in rats receiving only 1-naphthylisothiocyanate. The appearance of 1-naphthylamine was also inhibited by cycloheximide, although not to the same extent as 1-naphthyl isocyanate. On the other hand, phenobarbital, which potentiates 1-naphthylisothiocyanate hepatotoxicity, enhanced 1-naphthyl isocyanate and 1-naphthylamine formation. It is suggested that 1-naphthyl isocyanate, 1-naphthylamine and the highly reactive sulfur released from 1-naphthylisothiocyanate might be involved in the hepatotoxic effect of 1-naphthylisothiocyanate.

Naphthylisothiocyanate disposition in bile and its relationship to liver glutathione and toxicity.

1-Naphthylisothiocyanate (ANIT), but not 2-naphthylisothiocyanate (BNIT), produces cholangiolitic hepatitis in rats after a single, oral administration. The mechanisms responsible for the disparate toxic outcomes for these closely related structural isomers are not fully understood. Recent reports suggest that ANIT-induced hepatotoxicity is dependent upon the formation and biliary excretion of a reversible glutathione-ANIT conjugate. To understand better the relationship between hepatic glutathione, secretion into bile and hepatotoxicity, the bile concentrations and hepatotoxicities of ANIT and BNIT were examined in rats with and without pretreatment with buthionine sulfoximine (BSO). ANIT (100 mg/kg, p.o.) caused a 3-fold elevation of plasma alanine aminotransferase activity (ALT), a 6-fold elevation of total plasma bilirubin, and a > 90% reduction in bile flow 24 hr after administration. BNIT, at this same dose and route of administration, did not alter significantly these markers of liver injury. Accumulation of ANIT and BNIT in bile occurred with the same temporal characteristics; however, BNIT accumulated to markedly larger concentrations (292 +/- 83 and 235 +/- 100 microM BNIT and 78 +/- 19 and 29 +/- 13 microM ANIT at 1 and 4 hr, respectively). The accumulation of ANIT and BNIT in bile was coincident with a > 2-fold elevation of reduced glutathione in bile. Pretreatment of rats with BSO decreased hepatic glutathione concentration and reduced the concentration of naphthylisothiocyanates in bile by 85%. Associated with this reduction was an attenuation of ANIT hepatotoxicity. Altogether, these findings indicate that both ANIT and BNIT accumulate in bile in a glutathione-dependent manner, yet they yield different hepatotoxic outcomes. Therefore, the disparity in hepatotoxicities observed with these isomers is not related to a difference in ability to enter bile. Other differences, such as in metabolism, chemical reactivity, conjugate stability and/or cytotoxic potential to bile duct epithelial cells may be more important determinants of hepatotoxicity.