No abstract

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypotheticallya pplicable in preclinical screens within the drug development process. A toxicological study was set up in rats orally treated up to 11 days with 300 mg kg(–1) per day(–1) amiodarone (AMD). Light and transmission electron microscopy investigations were performed to confirm the presence of lamellar bodies indicative of phospholipid accumulation. The effects of AMD upon the transcriptome of these tissues were estimated using DNA microarray technology. Microarray data analysis showed that a total of 545 and 8218 genes were modulated by AMD treatment in heart and blood, respectively. Some genes implicated in the phospholipid accumulation in cells, such as phospholipase A2, showed similar alterations of gene expression. After transcriptome criteria of analysis and target selection, including also the involvement in the onset of PLD, 7 genes (Pla2g2a, Pla2g7, Gal, Il1b, Cebpb, Fcgr2b, Acer 2) were selected as candidate biomarkers of PLD in heart and blood tissues, and their potential usefulness as a sensitive screening test was screened and confirmed by quantitative Real-Time PCR analysis. Collectively, these data underscore the importance of transcriptional profiling in drug discovery and development, and suggest blood as a surrogate tissue for possible phospholipid accumulation in cardiomyocytes.

The atrial natriuretic peptide (ANP) and its precursor (N-terminal fragment of atrial natriuretic peptide, NT-proANP) are natriuretic peptides released into the circulation as a consequence of an acute atrial stretch. As for the brain natriuretic peptide and its N-terminal fragment, the biological significance of ANP and NT-proANP has been widely studied in humans, but the literature is lacking information about the determination of these biomarkers in veterinary medicine and, in particular, in the toxicological species used in preclinical pharmaceutical drug development. This paper describes the evaluation of ANP and NT-proANP levels in a healthy population of Han Wistar and Sprague-Dawley rats, as well as in a rodent model of hypertension (Spontaneously Hypertensive rats). Both biomarkers were measured by mean of two commercially available enzyme immunoassays and serum levels were correlated with heart weight and histopathological findings in the heart, with the aim of building an integrated assessment of the significance of these biomarkers. Results obtained demonstrated that NT-proANP and ANP can be accurately measured in the different rat strains, with NT-proANP concentrations higher than those of ANP, as expected because of its longer half-life. In addition, both correlated well with cardiac hypertrophy evaluated by means of heart weight and histopathological examination. NT-proANP and ANP represent reliable markers of cardiac hypertrophy in the rat.

Little is known concerning the sensitivity of aged rats to xenobiotics inducing kidney damage. To increase this knowledge, the age-dependent response of the kidney to hexachloro-1 : 3-butadiene (HCBD) or potassium dichromate (chromate) was investigated. Rats were treated at different ages with a single dose of segment-specific nephrotoxicants of the proximal tubule, chosen on the basis of their specificity for S(3) and for S(1)-S(2) segments, respectively. The toxicological impact of these xenobiotics has been evaluated through biochemical and genomic markers, and histopathological investigation of kidney samples. HCBD treatment induced tubular necrosis of the S(3) segment of the proximal tubule associated with changes of toxicological markers unrelated to the age. In contrast, chromate treatment induced an increased kidney damage related to the rat age. In fact, histopathological investigation revealed that at 1 month of age tubular vacuolar degeneration was seen affecting S(1)-S(2) segments of the proximal tubule, whereas at 3 months of age tubular necrosis occurred in the same segments associated with tubular dilation of the distal portions. Consistently, biochemical analysis confirmed a direct correlation among genomic and biochemical marker variability and animal age. Altogether, the results show that during aging there is an increased sensitivity of kidney to chromate but not to HCBD-induced damage and evidence differential age-related selectivity of rats for nephrotoxic compounds. Significance for human risk assessment is discussed.

CE is a good alternative to the colorimetric methods currently used for the determination of bilirubin in rat serum.