Kidney Injury Monitoring in Tobramycin-Treated Rhesus Monkeys: Supplementing Urinary Kidney Biomarkers With Kidney Biopsy Gene Expression Profiling.

Kidney biopsies are used sparingly to diagnose kidney injury in the clinic. Here we have conducted a small exploratory study to directly compare the low-grade kidney injury monitoring performance of serum safety biomarkers, novel urine safety biomarkers, microscopic histopathology and targeted gene expression alterations in kidney biopsy specimens in rhesus monkeys treated with tobramycin. Targeted gene expression increases were observed in the kidney biopsy samples and whole kidney sections for kidney injury molecule 1 (KIM-1), clusterin (CLU), osteopontin (OPN) messenger RNA transcripts. In addition, increases of the urinary kidney safety protein biomarkers including KIM-1, CLU, OPN were also observed. These increases in gene expression and urinary protein end point were in concordance with the eventual low-grade kidney lesions seen in terminal tissue sections. In contrast, conventional serum biomarkers blood urea nitrogen and serum creatinine were not as sensitive in monitoring kidney injury. Although these data do not support routinely adding kidney biopsies to regular toxicology studies, they provide evidence on the value and limitations of incorporating gene expression profiling on kidney biopsy specimens, further underscore the value of urinary kidney safety biomarkers for improved low-grade kidney injury monitoring, and open the door for future definitive studies.

Gamma-glutamyl transpeptidase null mice fail to develop tolerance to coumarin-induced Clara cell toxicity.

Coumarin was used as a model Clara cell toxicant to test the hypothesis that tolerance to injury requires increased gamma-glutamyl transpeptidase (GGT) activity. Wildtype (GGT(+/+)) and GGT-deficient (GGT(-/-)) mice on a C57BL/6/129SvEv hybrid background were dosed orally with corn oil (vehicle) or coumarin (200 mg/kg). In vehicle-treated mice, Clara cell secretory protein (CC10) expression was distributed throughout the bronchiolar epithelium. After one dose of coumarin, CC10 expression was dramatically reduced and the bronchiolar epithelium was devoid of Clara cells in GGT(+/+) and GGT(-/-) mice. In wildtype mice, 9 doses of coumarin produced tolerance, characterized as a renewed bronchiolar epithelium with Clara cells expressing CC10 along with a 40% increase in total glutathione (GSH) and a 7-fold increase in GGT activity in the lung. In contrast, tolerance was not observed in GGT(-/-) mice. To assess whether changes in whole lung levels of GSH and GGT activity reflect Clara cell specific changes an enriched population of cells was isolated from female wildtype B6C3F1 mice made tolerant to coumarin. Compared to Clara cells from control mice, GSH and GGT activity increased 3- and 13-fold, respectively. Collectively, these data suggest Clara cell tolerance to coumarin toxicity requires increased GGT activity favoring enhanced GSH synthesis.

Roles for epoxidation and detoxification of coumarin in determining species differences in clara cell toxicity.

Coumarin-induced mouse Clara cell toxicity is thought to result from the local formation of coumarin 3,4-epoxide (CE). However, this toxicity is not observed in the rat, indicating species differences in coumarin metabolism. The purpose of the present work was to characterize the in vitro kinetics of coumarin metabolism in mouse, rat, and human whole lung microsomes, and to determine whether species differences in coumarin-induced Clara cell toxicity correlate with coumarin epoxidation or detoxification. In B6C3F1 mouse lung microsomes, coumarin was metabolized to CE, which in the absence of glutathione spontaneously rearranges to o-hydroxyphenylacetaldehyde (o-HPA). The K(m) and V(max) for o-HPA formation were 155 microM and 7.3 nmol/min/mg protein, respectively. In contrast, the K(m) and V(max) were 2573 microM and 1.75 nmol/min/mg protein, respectively, in F344 rat lung microsomes. Since the intrinsic clearance through the epoxidation pathway was 69 times higher in the mouse, the epoxidation rate was shown to correlate with species sensitivity to toxicity. To determine whether detoxification reactions contribute to species differences in toxicity, the fate of CE and o-HPA were examined. Detoxification of CE via conjugation with glutathione was evaluated in lung cytosol from mice and rats, and the K(m) of this reaction was approximately 800 microM in both species, whereas the V(max) was 3.5 and 6 nmol/min/mg protein, respectively, indicating that conjugation is faster in the rat. Oxidation of o-HPA to o-hydroxyphenylacetic acid (o-HPAA) was examined in lung cytosol from mice and rats. The K(m) of this reaction was approximately 1.5 microM in both species, whereas the V(max) was 0.08 and 0.33 nmol/min/mg protein in mice and rats, respectively, indicating that oxidation is faster in the rat. While the rate of epoxidation correlates with species sensitivity to coumarin, it is likely that Clara cell toxicity is modulated by CE and o-HPA detoxification. In contrast to rodent lung microsomes, bioactivation of coumarin to o-HPA did not occur in 16 different human lung microsomes, which suggests metabolism-dependent toxicity in the human lung is unlikely following low level coumarin exposure.

Identification of the cytochromes P450 that catalyze coumarin 3,4-epoxidation and 3-hydroxylation.

Coumarin, a widely used fragrance ingredient, is a rat liver and mouse lung toxicant. Species differences in toxicity are metabolism-dependent, with injury resulting from the cytochrome P450-mediated formation of coumarin 3,4-epoxide (CE). In this study, the enzymes responsible for coumarin activation in liver and lung were determined. Recombinant human and rat CYP1A forms and recombinant human CYP2E1 readily catalyzed CE production. Coinhibition with CYP1A1/2 and CYP2E1 antibodies blocked CE formation by 38, 84, and 67 to 92% (n = 3 individual samples) in mouse, rat, and human hepatic microsomes, respectively. Although CYP1A and 2E forms seem to be the most active catalysts of CE formation in liver, studies conducted with the mechanism-based inhibitor 5-phenyl-pentyne demonstrated that CYP2F2 is responsible for up to 67% of CE formation in whole mouse lung microsomes. In contrast to the CE pathway, coumarin 3-hydroxylation is a minor product of coumarin in liver microsomes from mice, rats, and humans and is catalyzed predominately by CYP3A and CYP1A forms, confirming that CE and 3-hydroxycoumarin are formed via distinct metabolic pathways.