Curcumin and enalapril ameliorate renal failure by antagonizing inflammation in 5/6 nephrectomized rats: role of phospholipase and cyclooxygenase.

Previously, we showed that curcumin prevents chronic kidney disease (CKD) development in ⅚ nephrectomized (Nx) rats when given within 1 wk after Nx (Ghosh SS, Massey HD, Krieg R, Fazelbhoy ZA, Ghosh S, Sica DA, Fakhry I, Gehr TW. Am J Physiol Renal Physiol 296: F1146-F1157, 2009). To better mimic the scenario for renal disease in humans, we began curcumin and enalapril therapy when proteinuria was already established. We hypothesized that curcumin, by blocking the inflammatory mediators TNF-α and IL-1ß, could also reduce cyclooxygenase (COX) and phospholipase expression in the kidney. Nx animals were divided into untreated Nx, curcumin-treated, and enalapril-treated groups. Curcumin (75 mg/kg) and enalapril (10 mg/kg) were administered for 10 wk. Renal dysfunction in the Nx group, as evidenced by elevated blood urea nitrogen, plasma creatinine, proteinuria, segmental sclerosis, and tubular dilatation, was comparably reduced by curcumin and enalapril, with only enalapril significantly lowering blood pressure. Compared with controls, Nx animals had higher plasma/kidney TNF-α and IL-1ß, which were reduced by curcumin and enalapril treatment. Nx animals had significantly elevated kidney levels of cytosolic PLA(2), calcium-independent intracellular PLA(2), COX 1, and COX 2, which were comparably reduced by curcumin and enalapril. Studies in mesangial cells and macrophages were carried out to establish that the in vivo increase in PLA(2) and COX were mediated by TNF-α and IL-1ß and that curcumin, by antagonizing the cytokines, could significantly reduce both PLA(2) and COX. We conclude that curcumin ameliorates CKD by blocking inflammatory signals even if it is given at a later stage of the disease.

Evaluation of xenobiotic impact on urban receiving waters by means of statistical methods.

Xenobiotics in urban receiving waters are an emerging problem. A sound knowledge of xenobiotic input, distribution and fate in the aquatic environment is a prerequisite for risk assessments. Methods to assess the impact of xenobiotics on urban receiving waters should address the diverse characteristics of the target compounds and the spatiotemporal variability of concentrations. Here, we present results from a one-year-monitoring program concerning concentrations of pharmaceuticals, additives from personal care products and industrial chemicals in an urban drainage catchment in untreated and treated wastewater, surface water and groundwater. Univariate and multivariate statistical methods were applied to characterize the xenobiotic concentrations. Correlation and principal component analysis revealed a pronounced pattern of xenobiotics in the surface water samples. The concentrations of several xenobiotics were characterized by a negative proportionality to the water temperature. Therefore, seasonal attenuation is assumed to be a major process influencing the measured concentrations. Moreover, dilution of xenobiotics the surface water was found to significantly influence the concentrations. These two processes control more the xenobiotic occurrence in the surface water than the less pronounced concentration pattern in the wastewater sources. For the groundwater samples, we assume that foremost attenuation processes lead to the found differentiation of xenobiotics.

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males.

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n=16) or frozen-thawed semen (Group B, n=9) at a concentration of 100x10(6) spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.

Curcumin ameliorates renal failure in 5/6 nephrectomized rats: role of inflammation.

TNF-alpha and NF-kappaB play important roles in the development of inflammation in chronic renal failure (CRF). In hepatic cells, curcumin is shown to antagonize TNF-alpha-elicited NF-kappaB activation. In this study, we hypothesized that if inflammation plays a key role in renal failure then curcumin should be effective in improving CRF. The effectiveness of curcumin was compared with enalapril, a compound known to ameliorate human and experimental CRF. Investigation was conducted in Sprague-Dawley rats where CRF was induced by 5/6 nephrectomy (Nx). The Nx animals were divided into untreated (Nx), curcumin-treated (curcumin), and enalapril-treated (enalapril) groups. Sham-operated animals served as a control. Renal dysfunction in the Nx group, as evidenced by elevated blood urea nitrogen, plasma creatinine, proteinuria, segmental sclerosis, and tubular dilatation, was significantly reduced by curcumin and enalapril treatment. However, only enalapril significantly improved blood pressure. Compared with the control, the Nx animals had significantly higher plasma and kidney TNF-alpha, which was associated with NF-kappaB activation and macrophage infiltration in the kidney. These changes were effectively antagonized by curcumin and enalapril treatment. The decline in the anti-inflammatory peroxisome proliferator-activated receptor gamma (PPARgamma) seen in Nx animals was also counteracted by curcumin and enalapril. Studies in mesangial cells were carried out to further establish that the anti-inflammatory effect of curcumin in vivo was mediated essentially by antagonizing TNF-alpha. Curcumin dose dependently antagonized the TNF-alpha-mediated decrease in PPARgamma and blocked transactivation of NF-kappaB and repression of PPARgamma, indicating that the anti-inflamatory property of curcumin may be responsible for alleviating CRF in Nx animals.

Protein profiling of non-malignant and malignant ascites by SELDI-TOF MS: proof of principle.

Ascites is a common clinical symptom in liver cirrhosis, inflammatory disorders of the abdomen and a major late manifestation of metastatic malignancies. Standard cytopathological techniques and immunocytochemistry have specificities and sensitivities of approximately 95 and 60%, respectively for the presence of tumor cells. Development of faster and more accurate screening methods would be of great clinical utility. In this work we examined differential analysis of the unbound proteins in the supernatant of ascites fluid by Protein-Chip SELDI mass spectrometry. There were 21 tumor cell-positive and 34 tumor cell-negative samples. We used principal component analysis coupled with linear regression applied to the mass spectra of the samples to distinguish between the sample groups. Two sample sets for statistical analysis were created after randomization, a training set with 37 samples and a validation set with 18 samples resulting in a specificity of 93% and a sensitivity of 83% on the training set. The validation set yielded a specificity and sensitivity of 75%. This study suggests that SELDI-TOF mass spectrometry appears to have great potential as a surrogate diagnostic tool to evaluate effusion specimens.

N,N-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry.

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol-ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.

Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity.

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as "reactive-tracers" for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.

Analyzing effects of photodynamic therapy with 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) in urothelial cells using reverse phase protein arrays.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) is clinically established approach for a number of defined applications. However, in order to optimize the therapeutic benefits of PDT, the specific mode of cell destruction should be better defined. Apoptosis is favored over necrosis for clinical practice as the latter causes more side-effects. In the present study, we analyse PDT-induced cell death and its correlation to various PDT parameters (different doses applied, time after PDT treatment) in vitro using reverse phase protein arrays. Human urothelial cell lines with varying degrees of differentiation (UROtsa, RT4, RT112, J82) were subjected to in vitro-PDT using increasing doses of irradiation. In addition, positive controls for apoptosis, necrosis and un-/specific cellular damage were included. Cells were harvested over a specified time course, lysed and arrayed onto nitrocellulose-covered glass slides. The arrays were analyzed for expression of apoptosis-related proteins by immunohistochemistry. Analysis of caspase-3 and -9 expression, the activation of HIF-1alpha, Bcl2, Cox2 and the phosphorylation of AKT reveals signal activation due to a PDT-stimulus in correlation with the positive controls. Data were analyzed by unsupervised hierarchical clustering and depicted as a heat map revealing cell-specific patterns of pathway stimulation. Higher differentiated phenotypes showed a more distinct signal response in general and a higher apoptotic response in detail. Lower differentiated cell lines lost pathway regulation capabilities according to their state of dedifferentiation. Reverse phase protein arrays are a promising technique for signal pathway profiling: they exceed the range of traditional western blots by sensitivity, high-throughput capability, minimal sample consumption and easy quantification of results obtained.

A modified Ce/Mg-BCIP-NBT formazan/indigoblue technique for demonstration of non-specific alkaline phosphatase activity.

The wide ranged structurally variability of formazans and their accessibility for auxiliary additives as redoxmediators or metals provide an easy tunable chromogenic visualization technique. We present here an improved nitro blue tetrazolium (NBT) 5-bromo-4-chloro-3-indolyl phosphate (BCIP) method which is superior to the classical McGadey's procedure regarding proper precipitation and localization as well as sensitivity. Different metal additives as well as the overall reaction course modifying additives (redox mediators, chelating additives, buffer) were optimized.

New fluorescent method for the histochemical detection of dipeptidyl peptidase IV using glycyl-l-prolyl-2-anthraquinonyl hydrazide as substrate.

Glycyl-4-prolyl-2-anthraquinonylhydrazide (Gly-Pro-2-AH) was synthesized and used as a new fluorogenic substrate for the histochemical detection of dipeptidyl peptidase IV activity (DPP IV). The enzymatic hydrolysis liberates 2-anthraquinonyl hydrazine (2-AH). Further on, the primary reaction product reacts with an aromatic aldehyde to give an insoluble hydrazone. The final reaction product fluoresces orange-red when exited by green light (lambda(exc)=520-580 nm) and marks sites of enzymatic activity by an intensive fluorescence. This fluorescent method permits highly sensitive enzyme detection and causes only very low background tissue fluorescence. Thus enzyme locations in the capillary bed endothelium properly and sensitively stained, which has not been achieved by now. The new method is used successfully to demonstrate the enzyme in cryotome tissue sections from several rat organs.

New fluorescent method for the histochemical detection of tripeptidyl peptidase I using glycyl-l-prolyl-l-met-2-anthraquinonyl hydrazide as substrate.

A new substrate for tripeptidyl peptidase I (TPP I; E.C.3.4.14.9)-Gly-L-Pro-L-Met-2-anthraquinonyl hydrazide (Gly-Pro-Met-2-AH) is synthesized and used for the fluorescent histochemical detection of the enzyme. The enzyme liberates low soluble 2-anthraquinonylhydrazine, which-couples quickly with 3-nitrobenzaldehyde (3-NBA) yielding a highly fluorescent water-insoluble hydrazone--3-nitrobenzylidene-2-anthraquinonylhydrazone. The latter compound is localized precisely at sites of enzymatic activity and marks them with a very bright and stable orange-red fluorescence after excitation with conventional monochromatic andlaser green light (lambda(exc)=520-580 nm). The new technique is used successfully for the visualization of the enzyme in tissue sections of different rat organs - and represent the first fluorescent histochemical method for that peptidase.

[Precancerous conditions in the urothelium. Early detection and molecular understanding through endoscopic fluorescence diagnosis]. / Präkanzerosen des Urothels. Früherkennung und molekulares Verständnis durch endoskopische Fluoreszenzdiagnostik.

Fluorescence diagnosis after application of 5-aminolevulinic acid (ALA) detects red-fluorescing preneoplastic and neoplastic lesions using light excitation. The principle of the method is the relative tumor-selective accumulation of the metabolite protoporphyrin IX (PPIX), which is built intracellularly out of exogenously applied ALA. The early detection of tumors and especially preneoplasias is an ideal prerequisite for genetic analysis of these lesions. With this approach, methods such as fluorescence in situ hybridization and loss of heterozygosity analysis for deletion mapping as well as gene sequencing data could be compared. New data are presented on deletions, numeric chromosomal aberrations, and oligoclonality of tumors found in about 30% of cases. The phenomenon of tumor-selective fluorescence was further investigated by parallel biochemical analysis, which showed marked differences in heme metabolism. The analysis of gene and protein expression may aid in identifying tumor-specific molecules associated with heme metabolism.

Recent advances in catalytic peroxidase histochemistry.

Immunoassays have developed to become an important analytical tool in life sciences for detection of endogenous and exogenous targets. Among the most important enzyme labels horseradish peroxidase (HRP), alkaline phosphatase (AP), and beta-D-galactosidase (GAL) is HRP the smallest enzyme and plays nowadays an outstanding role. The oldest substrates are chromogens widely applied for localization of sites of peroxidase (PO) activity in histochemistry as well as for colorimetric applications. They are represented by a diversity of aromatic amines and phenols. Encouraged by development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels represent the most sensitive and worthwhile detection tools to date. In contrast to chromogens fluorescent labels for detection PO activity are confined only to a few substrates developed more recently. These substrates are mostly applied in histochemistry at a short time scale due to their frequently high solubility. At the long time scale sole exception is so far the tyramine based fluorochome deposition technique (more general: catalytic reporter deposition, CARD). Despite quite different staining behavior both fluorometric and product deposition related principles are based on 4-hydroxy phenylalkyl substrates. The following article reviews basic principles of peroxidatic substrate degradation processes including chromogenic and fluorescent approaches with emphasis on recent advances in development of chromogens and fluorogens for application in histology. As a result of systematic efforts towards the design of substrates, the range of classical precipitating chromogens as well as fluorescent techniques could be complimented by novel highly sensitive substrates with superior staining capabilities: a) Metal chelating 2-hydroxy benzylamines are derived from classical aniline substrates (two steps) and utilize metal catalytic effects in an efficient intramolecular way. The enzymatically yielded dark colored polycondensation products are applicable in histochemistry, in colorimetry and especially as precipitating electron opaque labels with enhanced osmiophilic properties for light and electron microscopy. b) Fluorescent 4-hydroxy-styryl derivatives are capable of oxidative selfanchoring reactions at the cellular level close to sites of PO activity. In contrast to deposition of tyramine conjugated fluorochromes an altered fluorochrome with improved fluorescence properties is furnished during oxidative crosslinking of the substrate. This results in a highly specific and photostable fluorescence response and an outstanding low background staining. Histochemical and immunohistochemical applications are presented.

Improving the use of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) for the gastrointestinal tract by esterification--an in vitro study.

Possible approaches to improve the diagnostic and therapeutic effects of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) are the esterification of ALA for enhanced uptake and the choice of wavelength for irradiation. The human colonic cell lines HT29 [G2] and CCD18 (fibroblasts) were incubated with 0.6 mM ALA, ALA-hexylester or -benzylester respectively, and for further assays with hypotaurine, in addition. PPIX-accumulation was analyzed by flow cytometry and fluorescence spectroscopy. PPIX formation kinetics were continuously recorded. Incubated cells were irradiated with an incoherent light source lambda = 400-700 nm or lambda = 590-700 nm, respectively. After PDT treatment, clonogenicity assays were performed to determine cell viability. Esterification leads to increased PPIX-accumulation, decreased time for production of detectable amounts of PPIX as well as increased response to PDT. Tumor specificity is always maintained or exceeds values for ALA alone. ALA enters the cells via beta transporter whereas esters by passive diffusion. Altering irradiation wavelengths showed the independence of wavelength rather than light dose. Results emphasize the role of heme metabolism for generating tumor specificity rather than the process of ALA-uptake, an important detail for future clinical application.

The effects of 5-aminolevulinic acid esters on protoporphyrin IX production in human adenocarcinoma cell lines.

Photodynamic diagnosis (PDD) and photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) is an interesting approach to detect and treat dysplasia and early cancers in the gastrointestinal tract. Because of low lipophilicity resulting in poor penetration across cell membranes, high doses of ALA should be administered in order to reach clinically relevant levels of PPIX. One way of increasing PPIX accumulation is derivatization of ALA into a more lipophilic molecule. In our in vitro study, different esterifications of ALA were investigated to analyze the effects on PPIX accumulation in human adenocarcinoma cell lines. For systematic analysis of cell type-specific PPIX accumulation, three human adenocarcinoma cell lines (SW480, HT29 and CaCo2) and a fibroblast cell line (CCD18) were tested. 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) assays were performed to ensure that the ALA esters showed no cellular dark toxicity. Different concentrations (ranging from 0.012 to 0.6 mmol/L, 3 h) and incubation times (5, 10, 30, 180 min; 0.12 mmol/L) were examined. PPIX accumulation was measured using flow cytometry. ALA esters, especially ALA-hexylester and ALA-benzylester, induced significant higher PPIX levels in adenocarcinoma cell lines when compared with ALA and may be promising candidates for PDT and PDD.

Protoporphyrin IX distribution following local application of 5-aminolevulinic acid and its esterified derivatives in the tissue layers of the normal rat colon.

Photodynamic diagnosis and especially therapy after sensitization with 5-aminolevulinic acid (ALA) is hampered by limitations of uptake and distribution of ALA due to its hydrophilic nature. Chemical modification of ALA into its more lipophilic esters seems to be promising to overcome these problems. The aim of the present study was to investigate the comparative kinetics of protoporphyrin IX (PPIX) fluorescence in rat colonic tissue after topical application of ALA and its esterified derivatives, ALA-hexylester (h-ALA), ALA-methylester (m-ALA) and ALA-benzylester (b-ALA). Fluorescence intensity induced by PPIX in normal colonic tissue was quantified using fluorescence microscopy at 1, 2, 4, 6 and 8 h after sensitization. Mucosa exhibited higher fluorescence levels compared to the underlying submucosa or smooth muscle. Peak fluorescence intensities were seen 4 h after local sensitization with 86.0 mol ml(-1) ALA (513 +/- 0.57 counts per pixel), 6.6 mol ml(-1) m-ALA (508 +/- 35.50) and 4.8 mol ml(-1) h-ALA (532 +/- 128.80), and 6 h after sensitization with 4.6 mol ml(-1) b-ALA (468 +/- 190.27). A 13-18 times lower concentration of ALA esters was required for fluorescence intensities reached with ALA alone. A similar degree of the fluorescence ratio between mucosa and muscularis (5-6:1) was detected for ALA and its derivatives. The time point of the maximum value of this ratio was consistent with peak fluorescence levels for ALA and each ALA-ester. The clinical feasibility and the advantages of topical ALA ester-based fluorescence for detection of malignant and premalignant lesions need further investigations.

Rapid detection of antibodies to immunoglobulin A molecules by using the particle gel immunoassay.

BACKGROUND AND OBJECTIVES: Antibodies to immunoglobulin A (IgA) molecules are thought to be frequently responsible for anaphylactic reactions in transfusion medicine, but practical tests for the detection of antibodies to IgA are not yet available. MATERIAL AND METHODS: Red, high-density polystyrene beads were coated with purified IgA molecules and then used to test serum samples collected from unselected healthy blood donors (n = 105) and patients with common variable immunodeficiency and/or IgA deficiency (n = 44). For testing, the standard gel-agglutination technique (ID-Micro Typing System) was employed. RESULTS: None of the normal serum samples were reactive with IgA-coated beads and samples from only 10 patients were positive (titre range 1 : 2 to 1 : 256). Only one out of all patients studied had a history of an anaphylactic reaction and this was related to the administration of Rh(D) prophylaxis (anti-D immunoglobulin). The beads did not show non-specific agglutination and could be used repeatedly for longer than 6 months. The results were reproducible in all patients tested. CONCLUSION: The new test allows a specific and rapid detection of antibodies to IgA molecules. In order to evaluate the clinical relevance of the test, analysis is required of a wider range of antibodies that produce anaphylactic reactions.

Optimization of differential photodynamic effectiveness between normal and tumor urothelial cells using 5-aminolevulinic acid-induced protoporphyrin IX as sensitizer.

Photodynamic therapy using 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX is a promising tool in bladder-cancer therapy. However, little is known about the cellular mechanisms of phototoxicity. Our aim was to characterize the cellular damage and to optimize differential photodynamic effectiveness between tumor and normal urothelial cells. RT4 tumor and UROtsa normal urothelial cells were used to simulate a papillary bladder tumor in contrast to normal urothelium. Photodynamically induced damage in plasma membrane and mitochondria was monitored by flow cytometry with propidium iodide exclusion and analysis of aggregate formation of the dye JC-1. Cell morphology was investigated by phase-contrast and fluorescence microscopy following acridine orange staining. Long incubation times (3 hr) led to complete RT4 tumor cell kill accompanied by a marked fraction of damaged normal UROtsa cells. Shorter incubation intervals (1 hr) also resulted in complete RT4 tumor cell kill; however, most UROtsa cells retained their cell properties, including intact plasma membrane and active mitochondria as well as intact cellular morphology. Phototoxicity depends not only on cellular sensitizer accumulation but also on intracellular localization. Analysis of phototoxic mechanisms is an important step for planning combination therapy regimens with, e.g., DNA-damaging agents. Further, data indicate that differential phototoxicity in normal and tumorous urothelium can be enhanced using differences in cellular protoporphyrin IX distribution following short 5-ALA incubation times. These data are encouraging for the in vivo situation since short incubation times are a more practical approach for local photodynamic therapy of early tumor stages not only in the bladder but also, e.g., in the gastro-intestinal tract or bronchial mucosa.

Progression after release of obstructive nephropathy.

Progressive glomerular and tubulointerstitial fibrosis develop in 1-year-old rats even after relief (R) of unilateral ureteral obstruction (UUO) at 5 days of age. The present study investigated whether a progressive renal injury model of UUO could be achieved after reversal of UUO (RUUO) in adult instead of neonatal rats. The potential for alpha-tocopherol modulation of mRNA for the fibrogenic cytokine, transforming growth factor-beta1 (TGFbeta1), apoptosis (TUNEL assay), and the presence of the stress protein, heat shock protein-70 (HSP-70), was also studied in this post-obstructive model. Male Sprague-Dawley rats weighing 125-150 g were randomly assigned to groups of 4 animals each for durations of 7 or 14 days of alpha-tocopherol supplementation after RUUO. The groups included: (i) sham, regular chow; (ii) RUUO, regular chow; (iii) RUUO, contralateral nephrectomy (NX); and (iv) RUUO, NX plus alpha-tocopherol supplementation. We found a significant increase in the ratio of kidney weight/body weight in the RUUO+NX group at 14 days compared with the sham and RUUO groups. This rise in the RUUO+NX group was significantly reduced after 14 days of alpha-tocopherol administration. The elevated level of kidney TGFbeta1 mRNA in the RUUO+NX group was only partially reduced at 7 days. But at 14 days this became significantly reduced with the continued alpha-tocopherol treatment. The HSP-70 staining and the apoptosis of the kidney showed results parallel to those of TGFbeta mRNA at 14 days. To separate the effects of hypertrophy after unilateral NX from the RUUO studies, we carried out a second experiment in control animals subjected to NX, with and without alpha-tocopherol supplementation. Fourteen days after NX, the apoptosis and TGFbeta1 mRNA showed no significant differences from the control animals. Our data suggest that a model of progressive renal injury in RUUO can be established in adult rats. After contralateral NX, the progressive injury is evidenced by the increase in the ratio of kidney weight/total body weight, the apoptotic counts, as well as fibrogenic cytokine TGFbeta1 mRNA in the post-obstructed kidney. Finally, our data also support the concept that alpha-tocopherol is renal protective, as judged by TGFbeta1 mRNA, apoptosis, and HSP-70 staining, even in the progressive disease process of the post-obstructed model.