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Purpose: To assess different degree of hepatic fibrosis using gadoxetic acid-enhanced MRI in patients with chronic liver disease. Materials and Methods: 31 patients with chronic liver disease and 7 healthy subjects were studied using a 1.5 Tesla MRI. Unenhanced and dynamically enhanced images were acquired using gadoxetic-acid contrast (0.025 mmol/kg). Hepatobiliary phase was obtained with a 20 minute time delay. Liver relative enhancement (LRE) values were calculated sampling 1 cm Regions Of Interest (ROI) per segment. According to Metavir score, patients were divided into different groups: Group A = F0-F1 (absent/mild fibrosis), Group B = F2 (moderate fibrosis) and Group C = F3-F4 (advanced fibrosis). Mann-Whitney test with Bonferroni correction ad hoc was adopted to compare LRE values between classes. Receiver Operating Characteristics (ROC) curves were created for prediction of different degree of fibrosis. Results: When comparing LRE mean values a statistical difference between F0-F1 and F3-F4 groups (p=0.008) was observed. A significant report was also observed between moderate and advanced fibrosis (p=0.02). In the comparison between group A and group B no significant difference for LRE mean values (p=0.93) was observed. For prediction of advanced fibrosis, ROC curve showed an Area Under Curve (AUC) of 0.793, with sensitivity of 94.12% and specificity of 57.89% using a threshold LRE value 1.0031. For prediction of 1.0031. Conclusion: LRE values showed significant difference only between moderate and advanced fibrosis. In patients with chronic liver disease, the assessment of early stages of hepatic fibrosis (mild or moderate fibrosis) is not statistically reliable using gadoxetic acid-enhanced MRI.

1,1-Dichloroethylene (DCE) exposure to mice elicits lung toxicity that selectively targets bronchiolar Clara cells. The toxicity is mediated by DCE metabolites formed via cytochrome P450 metabolism. The primary metabolites formed are DCE epoxide, 2,2-dichloroacetaldehyde, and 2-chloroacetyl chloride. The major metabolite detected is 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. In this investigation, studies were undertaken to test the hypothesis that CYP2E1 and CYP2F2 are involved in bioactivation of DCE to the epoxide in murine lung. We have developed a method using liquid chromatography/mass spectrometry (LC/MS) to evaluate the kinetics of the rates of production of conjugate [C] by recombinant CYP2E1 and CYP2F enzymes and lung microsomes. Concentration-dependent formation of conjugate [C] was found in incubations of DCE with recombinant CYP2E1 and CYP2F enzymes and lung microsomes from CD-1, wild-type (mixed 129/Sv and C57BL), and CYP2E1-null mice. Recombinant rat CYP2E1 exhibited greater affinity and catalytic efficiency for DCE metabolism than did recombinant human CYP2E1, mouse CYP2F2, goat CYP2F3 or rat CYP2F4. In the lung microsomal incubations, the rates of conjugate [C] production were higher in CD-1 mice than in either wild-type or CYP2E1-null mice; the level of [C] in CYP2E1-null mice was about 66% of that in wild-type mice. These results demonstrated that LC/MS analysis is a suitable method for detection and quantitation of conjugate [C], and that CYP2E1 and CYP2F2 catalyze the bioactivation of DCE to the epoxide in murine lung. The results also demonstrated that CYP2E1 is the high-affinity enzyme involved in DCE bioactivation.

3-Methylindole (3 MI) is a selective pulmonary toxicant, and cytochrome P450 (P450) bioactivation of 3 MI, through hydroxylation, epoxidation, or dehydrogenation pathways, is a prerequisite for toxicity. CYP2F1 and CYP2F3 exclusively catalyze the dehydrogenation of 3 MI to 3-methyleneindolenine, without detectable formation of the hydroxylation or epoxidation products. It was not known whether 3 MI is simply an excellent dehydrogenation substrate for all P450 enzymes, or whether certain cytochrome P450s responsible for 3 MI bioactivation have unique active sites that only catalyze the dehydrogenation of the molecule, while other P450s would catalyze only the oxygenation of 3 MI. Therefore, the kinetics of product formation by the CYP2F1 and CYP2F3 enzymes were compared with other cytochrome P450 enzymes. The enzymes tested were CYP1A1, CYP1A2, CYP1B1, and CYP2E1. The CYP1A1 and CYP1A2 enzymes produced all three 3 MI metabolites: the dehydrogenation product, 3-methyleneindolenine (V(max)/K(m) = 4 and 22, respectively); the hydroxylation product, indole-3-carbinol (V(max)/K(m) = 42 and 100, respectively); and the epoxidation product, 3-methyloxindole (V(max)/K(m) = 4 and 72, respectively). These CYP1A enzymes catalyzed oxygenation of 3 MI at much faster rates than dehydrogenation. CYP1B1 produced indole-3-carbinol (V(max)/K(m) = 85) and 3-methyloxindole (V(max)/K(m) = 7), and CYP2E1 only produced 3-methyloxindole (V(max)/K(m) = 98), but neither enzyme catalyzed the formation of the dehydrogenated product. Six additional P450 enzymes that were tested formed none of the dehydrogenation product. The ability of the various CYP1 family enzymes to catalyze the formation of all three major 3 MI metabolites, along with the specific oxygenation by CYP2E1, illustrates that dehydrogenation of 3 MI is not a substrate-directed process, but that the members of the CYP2F family possess unique active sites that specifically catalyze only the dehydrogenation mechanism.

3-Methylindole (3MI) is a naturally occurring pulmonary toxin that requires metabolic activation. Previous studies have shown that 3MI-induced pneumotoxicity resulted from cytochrome P-450-catalyzed dehydrogenation of 3MI to an electrophilic methylene imine (3-methyleneindolenine), which covalently bound to cellular macromolecules. Multiple cytochrome P-450s are capable of metabolizing 3MI to several different metabolites, including oxygenated products. In the present study, the role of human CYP2F1 in the metabolism of 3MI was examined to determine whether it catalyzes dehydrogenation rather than hydroxylation or ring oxidation. Metabolism was examined using microsomal fractions from human lymphoblastoid cells that expressed the recombinant human CYP2F1 P-450 enzyme. Expression of CYP2F1 in the lymphoblastoid cells proved to be an appropriate expression system for this enzyme. Products were analyzed using HPLC and the mercapturate, 3-[(N-acetylcystein-S-yl)methyl]indole, of the reactive intermediate was identified and quantified. Product analysis showed that human CYP2F1 efficiently catalyzed the dehydrogenation of 3MI to the methylene imine without detectable formation of indole-3-carbinol or 3-methyloxindole. High substrate concentrations of 3MI strongly inhibited production of the dehydrogenated product, a result that may indicate the existence of mechanism-based inhibition of CYP2F1 by 3MI. Recombinant CYP2F1 demonstrated remarkable selectivity for the bioactivation of 3MI to the putative dehydrogenated reactive electrophile. Bioactivation of naphthalene to its pneumotoxic epoxide by CYP2F1 was also demonstrated.