Oxidative Status and Lipofuscin Accumulation in Urothelial Cells of Bladder in Aging Mice

<div><p>Age-related changes in various tissues have been associated with the onset of a number of age-related diseases, including inflammation and cancer. Bladder cancer, for instance, is a disease that mainly afflicts middle-aged or elderly people and is mostly of urothelial origin. Although research on age-related changes of long-lived post-mitotic cells such as neurons is rapidly progressing, nothing is known about age-related changes in the urothelium of the urinary bladder, despite all the evidence confirming the important role of oxidative stress in urinary bladder pathology. The purpose of this study was thus to investigate the oxidative status and age-related changes in urothelial cells of the urinary bladder of young (2 months) and aging (20 months) mice by means of various methods. Our results demonstrated that healthy young urothelium possesses a powerful antioxidant defence system that functions as a strong defence barrier against reactive species. In contrast, urothelial cells of aging bladder show significantly decreased total antioxidant capacity and significantly increased levels of lipid peroxides (MDA) and iNOS, markers of oxidative stress. Our study demonstrates for the first time that ultrastructural alterations in mitochondria and accumulation of lipofuscin, known to be one of the aging pigments, can clearly be found in superficial urothelial cells of the urinary bladder in aging mice. Since the presence of lipofuscin in the urothelium has not yet been reported, we applied various methods to confirm our finding. Our results reveal changes in the oxidative status and structural alterations to superficial urothelial cells similar to those of other long-lived post-mitotic cells.</p> </div

Ultrastructural appearance of mitochondria in superficial cells of aging mice urothelium.

<p>(<b>a</b>) Enlarged and dilated mitochondria with no visible cristae inside. (<b>b</b>) Mitochondria of normal size and shape but with electron-dense inclusions (arrows). (<b>c</b>) Mitochondria of normal size and shape but with partial or complete destruction of cristae (star frames). L-lumen of the urinary bladder.</p

Age-related differences in the levels of antioxidants, lactate dehydrogenase and malondialdehyde in the urothelium and serum of young and aging C57BL/6JOlaHsd female mice.

<p>Significant differences between groups of young and aging mice were evaluated by a test of independence (t test) and when variances of the two samples were different, we used the Mann-Whitney (Wilcoxon) W test. The results were considered statistically significant at P<0.05.</p><p>Values are mean ± SEM.</p><p><u>Legend</u>: n.a., not analysed; ns, non-significant; CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulphide; LDH, lactate dehydrogenase; MDA, malondialdehyde; SOD, superoxide dismutase; TAS, total antioxidant status; TP, total protein concentration.</p

Immunolabeling of Ki-67, active caspase 3, uroplakins and cytokeratin 20 in aging mice urothelium.

<p>(<b>a</b>) Individual Ki-67 positive cells with red fluorescent signal (asterisk) are present in the urothelium of aging mice. Nuclei are stained with DAPI (blue). Arrows show lipofuscin granules. (<b>b</b>) No apoptotic cells (red fluorescence) were detected in the urothelium. Apoptotic fibroblast in connective tissue is marked with asterisk. Nuclei are stained with DAPI (blue). Arrows show lipofuscin granules. (<b>c</b>) Immunoreaction against uroplakins shows typical labeling (red fluorescence) of the apical plasma membrane and the membranes of specific vesicles - fusiform vesicles in the cytoplasm of superficial and intermediate cells. Nuclei are stained with DAPI (blue). (<b>d</b>) Immunoreaction against cytokeratin 20 (green fluorescence) reveals typical network of this cytokeratin in the apical cytoplasm of superficial cells. Nuclei are stained with DAPI (blue). Arrows show lipofuscin granules. SC-superficial cells, IC-intermediate cells, BC-basal cells, L-lumen of the urinary bladder.</p

Electron micrographs of lipofuscin in superficial cells of aging mice urothelium.

<p>(a) Ultrastructural appearance of lipofuscin granules (arrowheads), heterogeneous in their size and structure. (<b>b</b>) Lipofuscin surrounded by double membrane (arrowheads), indicating that the structure is an early autophagic vacuole (autophagosome). (<b>c</b>) Lipofuscin in late autophagic vacuole (autophagolysosome), limited by single membrane (arrow). (<b>d</b>) The lipofuscin granule (arrow) is filled with electron-dense precipitate as the reaction product of enzyme histochemistry for detection of acid phosphatase activity as the main lysosomal enzyme. Precipitate at the sites of acid phosphatase activity proves that lipofuscin-loaded autophagolysosome contains active lysosomal enzymes trying to degrade the undegradable lipofuscin. FV-fusiform vesicles, typical structures of superficial urothelial cells, L-lumen of the urinary bladder.</p

Structure of aging and young mice urothelium.

<p>(<b>a</b>) Semi-thin section of urothelium in aging mice showing normal structure of three-layered epithelium with numerous lipofuscin granules in the cytoplasm of superficial cells (arrows). (<b>b</b>) Semi-thin section of urothelium in young mice showing normal structure of three-layered epithelium with no lipofuscin granules in the superficial cells. (<b>c</b>) Ultrathin section of aging urothelium. Ultrastructure of superficial cell fulfiled with specific fusiform vesicles and numerous osmiophilic lipofuscin granules. (<b>d</b>) Ultrathin section of young urothelium. Typical ultrastructural appearance of superficial cell with large amounts of fusiform vesicles in the cytoplasm but no lipofuscin granules. SC-superficial cells, IC-intermediate cells, BC-basal cells, N-nucleus, L-lumen of the urinary bladder.</p

Cardiac autonomic modulation induced by doxorubicin in a rodent model of colorectal cancer and the influence of fullerenol pretreatment

<div><p>The very effective anticancer drug doxorubicin (DOX) is known to have cardiotoxic side effects, which could be accompanied by autonomic modulation. Autonomic disbalance might even be an initiating mechanism underlying DOX-induced cardiotoxicity and can be studied noninvasively by the analysis of heart rate variability (HRV). A number of strategies have been assessed to predict chemotherapy-induced cardiac dysfunction while HRV, a potential detecting tool, has not yet been tested. Thus, we aimed to determine the effect of DOX treatment on HRV in a rat model of colorectal cancer. While pretreatment with fullerenol (Frl) acts protectively on DOX-induced cardiotoxicity, we aimed to test the effect of Frl pretreatment on DOX-induced HRV alterations. After the induction of colorectal cancer, adult male Wistar rats were treated with saline (n = 7), DOX (1.5 mg/kg per week, n = 7) or DOX after pretreatment with Frl (25 mg/kg per week, n = 7) for three weeks (cumulative DOX dose 4.5 mg/kg). One week after treatment rats were anaesthetized, standard ECG was measured and HRV was analyzed in time and frequency domain. During autopsy the intestines and hearts were gathered for biochemical analysis and histopathological examination. DOX treatment significantly decreased parasympathetically mediated high-frequency component (p<0.05) and increased the low-frequency component of HRV (p<0.05), resulting in an increased LF/HF ratio (p<0.05) in cancerous rats. When pretreated with Frl, DOX-induced HRV alterations were prevented: the high-frequency component of HRV increased (p<0.01), the low-frequency decreased (p<0.01), LF/HF ratio decreased consequently (p<0.01) compared to DOX only treatment. In all DOX-treated animals, disbalance of oxidative status in heart tissue and early myocardial lesions were found and were significantly reduced in rats receiving Frl pretreatment. Autonomic modulation accompanied the development of DOX-induced cardiotoxicity in rat model of colorectal cancer and was prevented by Frl pretreatment. Our results demonstrated the positive prognostic power of HRV for the early detection of DOX-induced cardiotoxicity.</p></div

Representative histologic pictures of the heart sections.

<p>(a) DMH-treated Wistar rats, control group; (b) after DOX therapy; (c) DOX therapy in combination with Frl pretreatment, haematoxylin-eosin staining, magnification 400x.</p

Frequency domain indices in control, DOX and Frl/DOX groups of animals.

<p>Statistically significant differences of control and Frl/DOX group were found in nLF, nHF and LF/HF compared to DOX. Control: group of rats with colorectal carcinoma without anticancer treatment, DOX: group of rats with colon cancer, treated with doxorubicin (DOX), Frl/DOX: group of rats with colon cancer, treated with doxorubicin, pretreated with Fullerenol. *—statistically significant difference of control group compared to DOX (p<0.05), #—statistically significant difference of Frl/DOX group compared to DOX (p<0.01). nLF: integral under the low-frequency (LF) part of the power spectral density function expressed in normalized units (NU), nHF: integral under the high frequency (HF) part of the power spectral density function expressed in NU; LF/HF: the ratio between power spectrum density in HF part and LF part of the total power spectrum.</p

Representative histologic pictures of the colon lesions.

<p>All DMH-treated Wistar rats developed small microscopically visible colon lesions such as hyperplastic (a) or dysplastic crypts (b), adenomas (c) and adenocarcinomas (d). Kreyberg staining, magnification 400x (a,b), 200x (c,d).</p