This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants--cisplatin, gentamicin, and puromycin--to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg). Groups of rats were sacrificed at various times after administration of these compounds for standard clinical chemistry, urine analysis, and histological evaluation of the kidney. RNA was extracted from the kidney for microarray analysis. Principal component analysis and gene expression-based clustering of compound effects confirmed sample separation based on dose, time, and degree of renal toxicity. In addition, analysis of the profile components revealed some novel changes in the expression of genes that appeared to be associated with injury in specific portions of the nephron and reflected the mechanism of action of these various nephrotoxicants. For example, although puromycin is thought to specifically promote injury of the podocytes in the glomerulus, the changes in gene expression after chronic exposure of this compound suggested a pattern similar to the known proximal tubular nephrotoxicants cisplatin and gentamicin; this prediction was confirmed histologically. We conclude that renal gene expression profiling coupled with analysis of classical end points affords promising opportunities to reveal potential new mechanistic markers of renal toxicity.
This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants-cisplatin, gentamicin, and puromycin-to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg). Groups of rats were sacrificed at various times after administration of these compounds for standard clinical chemistry, urine analysis, and histological evaluation of the kidney. RNA was extracted from the kidney for microarray analysis. Principal component analysis and gene expression-based clustering of compound effects confirmed sample separation based on dose, time, and degree of renal toxicity. In addition, analysis of the profile components revealed some novel changes in the expression of genes that appeared to be associated with injury in specific portions of the nephron and reflected the mechanism of action of these various nephrotoxicants. For example, although puromycin is thought to specifically promote injury of the podocytes in the glomerulus, the changes in gene expression after chronic exposure of this compound suggested a pattern similar to the known proximal tubular nephrotoxicants cisplatin and gentamicin; this prediction was confirmed histologically. We conclude that renal gene expression profiling coupled with analysis of classical end points affords promising opportunities to reveal potential new mechanistic markers of renal toxicity.
Monocrotaline and trichodesmine are structurally closely related pyrrolizidine alkaloids (PAs) exhibiting different extrahepatic toxicities, trichodesmine being neurotoxic (LD(50) 57 mu mol/kg) and monocrotaline pneumotoxic (LD(50) 335 mu mol/kg). We have compared certain physicochemical properties and metabolic activities of these two PAs in order to understand the quantitative and qualitative differences in toxicity. Both PAs were metabolized in the isolated, perfused rat liver to highly reactive pyrrolic dehydroalkaloids that appear to be responsible for the toxicity of PAs. More dehydrotrichodesmine (468 nmol/g liver) than dehydromonocrotaline (116 nmol/g liver) was released from liver into perfusate on perfusion for 1 hr with 0.5 mM of the parent PA. Dehydrotrichodesmine had a significantly longer aqueous half-life (5.4 sec) than that of dehydromonocrotaline (3.4 sec). In vivo, significantly higher levels of bound pyrroles were found in the brain 18 hr after injection of trichodesmine (25 mg/kg; i.p.) than were seen following either an equal dose (25 mg/kg; i.p.) or an equitoxic dose (90 mg/kg; i.p.) of monocrotaline. Trichodesmine had a higher partition coefficient than monocrotaline for both chloroform and heptane, indicating its greater lipophilicity. The pK(a) of trichodesmine (7.07) was only slightly higher than that of monocrotaline (pK(a¿ 6.83), suggesting that a difference in degree of ionization was not a major factor affecting the relative ability of the dehydroalkaloids to cross the blood-brain barrier. We conclude that the greater lethality and neurotoxicity of trichodesmine compared to monocrotaline is due to two structural characteristics: (i) steric hindrance at position 14 of dehydrotrichodesmine results in greater resistance to hydrolysis, allowing more to be released from the liver and to be delivered to the brain; (ii) the larger isopropyl substituent at position 14 of dehydrotrichodesmine renders the molecule more lipophilic, leading to greater penetration of the brain.
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