HPLC analysis of benzo[a]pyrene-albumin adducts in benzo[a]pyrene exposed rats. Detection of cis-tetrols arising from hydrolysis of adducts of anti- and syn-BPDE-III with proteins

Quantitation of protein-benzo[a]pyrene adducts represent a more sensitive analysis method than quantitation of benzo[a]pyrene-DNA adducts. By accurate analysis of benzo[a]pyrene-protein adducts several different molecular adduct forms can be studied. Male Wistar rats were injected i.p. with benzo[a]pyrene, and serum albumin was isolated and subjected to acid hydrolysis at 90 degrees C for 3 h. The hydrolysate was analyzed by HPLC with fluorescence detection. The HPLC profiles obtained after albumin hydrolysis from benzo[a]pyrene exposed animals were compared to similar HPLC profiles from in vitro adducted bovine serum albumin (BSA) and direct hydrolysis of both r-10,t-9-dihydrodiol-c-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE-III) and r-10,t-9-t-dihydrodiol-t-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-III). After acid hydrolysis of albumin from benzo[a]pyrene exposed rats, 6 fluorescent peaks were separated. Four of the peaks were isomers of benzo[a]pyrene-tetrahydrotetrols, (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol. In addition we found two fluorescent peaks, named X1 and X2 with retention times similar to the benzo[a]pyrene-tetrols. The unknown fluorescent peaks reacted similar to the four known tetrols in both dose response experiments and time course experiments. Fluorescent material with retention times equal to X1 and X2 were found after acid hydrolysis of syn-BPDE-III and anti-BPDE-III in acid and in hydrolysates from BSA treated in vitro with syn-BPDE-III and anti-BPDE-III. The ratio X1/X2 was relatively constant indicating epimerization equilibrium between these to species. Synchronous fluorescence analysis of fractions containing X1 or X2 from both in vivo and in vitro experiments showed fluorescence spectra characteristic of benzo[a]pyrene tetrols using a wavelength difference of 34 nm.     

Removal and transformation of high molecular weight polycyclic aromatic hydrocarbons in water by live and dead microalgae

The removal and transformation of seven high molecular weight polycyclic aromatic hydrocarbons (PAHs), namely benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, indeno[1,2,3-c,d]pyrene and benzo[g,h,i]perylene, by a freshwater microalga Selenastrum capricornutum under gold and white light irradiation was studied. The two light sources did not result in significant differences in the biodegradation of the selected PAHs in live algal cells, but white light was more effective in promoting photodegradation than was gold light in dead cells. The removal efficiency of seven PAHs, as well as the difference between live and dead microalgal cells, was PAH compound-dependent. Benz[a]anthracene and benzo[a]pyrene were highly transformed in live and dead algal cells, and dead cells displayed greater transformation levels than live cells. Further investigation comparing the transformation of single PAH compound, benzo[a]pyrene, by S. capricornutum and another green microalgal species, Chlorella sp., demonstrated that the transformation in dead cells was similar, indicating the process was algal-species independent. Dead algal cells most likely acted as a photosensitizer and accelerated the photodegradation of PAHs.     

Application of the method of measurement of fluorescence intensity in vivo in biological tissues for pharmacokinetic studies of different chlorin-based photosensitizers

A precise and reproducible method of quantitative determination of the photosensitizer (PS) Photolon in liver samples of laboratory animals by means of a spectrophotometric assay with preliminary extraction has been developed. Conditions of the PS extraction have been optimized and validated for the quantitative determination of the PS Photolon by the spectrophotometric assay and the main analytical characteristics have been investigated. Using the method of quantitative determination of the PS Photolon in liver tissue samples a quantitative estimation of in vivo fluorescence of the liver tissue was performed after PS administration to animals. There was a high correlation (R = 0.99) between results obtained by spectrophotometry ex vivo and spectrofluorimetry in vivo. The method of fluorescence detection in vivo is applicable for studies of the pharmacokinetics of different photosensitizers.     

Phenotypic expression of genetically-controlled natural resistance to Mycobacterium bovis (BCG)

Mycobacterium bovis (BCG), when maintained in vitro, readily incorporates [3H]uracil, the RNA precursor. The rate of [3H]uracil incorporation into bacilli is sharply reduced when the BCG is phagocytized by murine adherent resident peritoneal macrophages and subsequently released by the lysis of monolayers. Macrophages derived from mouse strains that are innately resistant to BCG infection in vivo (Bcgr) are able to inhibit the [3H]uracil incorporation into the bacilli in a significantly more effective way than macrophages from BCG-susceptible (Bcgs) strains. This difference is best demonstrated with a low rate of infection (BCG: macrophage ratio between 1:1 and 2:1), and is most pronounced at 4 to 5 days after in vitro infection of macrophage monolayers. In vivo interaction of BCG with peritoneal macrophages in situ results in the same pattern of enhanced inhibition of [3H]uracil incorporation by Bcgr macrophages. The use of Bcg-congenic mouse strains has confirmed that the Chromosome 1 Bcg (Ity, Lsh) locus is regulating the antimycobacterial activity of macrophages. We conclude that the resident macrophage is the cell population that expresses the phenotype of genetically determined resistance to BCG infection.     

The light-dependent cytotoxicity of benzo[a]pyrene: effect on human erythrocytes, Escherichia coli cells, and Haemophilus influenzae transforming DNA

In vitro, the photodynamic compound benzo[a]pyrene (BAP) generates singlet oxygen efficiently when irradiated in organic solvents. It also photogenerates superoxide anion radical in water and can act as a photoreducing agent in the absence of oxygen. In vivo, the hemolysis of human erythrocytes, the inactivation of Escherichia coli cells representing a series of strains differing in excision repair and catalase proficiency, and the inactivation of Haemophilus influenzae transforming DNA activity were used to characterize the phototoxicity of BAP in the presence of near-UV light (290-400 nm). The results are consistent with BAP behaving as a photosensitizer that generates both superoxide and singlet oxygen, and that damages chiefly membranes. DNA does not seem to be a major target in the phototoxic reactions investigated.     

Properties and applications of photodynamic therapy

Photodynamic therapy (PDT) is the treatment of malignant lesions with visible light following the systemic administration of a tumor-localizing photosensitizer. Pharmacological and photochemical properties of the photosensitizer are combined with precise delivery of laser-generated light to produce a treatment which can offer selective tumoricidal action. Hematoporphyrin derivative (HD) and a purified component called Photofrin II are currently being used in clinical PDT. Initial patient results have been encouraging, and considerable interest has developed in the synthesis and evaluation of new photosensitizers with improved photochemical and pharmacological characteristics. In addition, there has been a gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT. This report provides an overview of the properties and applications of PDT. Information and data related to drug development, photochemistry, subcellular targets, in vivo responses, and clinical trials of PDT are presented.     

Photodynamic therapy for cutaneous leishmaniasis: the effectiveness of topical phenothiaziniums in parasite eradication and Th1 immune response stimulation.

Photodynamic therapy (PDT) is emerging as a therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). The efficacy of PDT against CL has been demonstrated previously with aminolevulinic acid, although the prolonged terms of therapy were less than ideal, and the search for new photosensitizers (PS) is ongoing. However, phenothiaziniums have demonstrated high parasiticidal effects in vitro. The subject of our investigation is the in vivo activity of two PS, 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride (EtNBSe) and (3,7-Bis(N,N-dibutylamino) phenothiazinium bromide (PPA904). The results of our comparative analysis of the efficacy of these two phenothiazinium analogues demonstrated a high antiparasitic activity of EtNBSe in vitro, and the higher efficacy of PPA904 in a mouse model of CL. The kinetics of photodestruction are different in parasite and mammalian cells, and with both dyes, the macrophages are more susceptible to photodynamic effects than L. major parasites. As the number of parasites in the lesions undergoes a biphasic change, temporarily increasing on days 2-4 and decreasing on days 5-7, more than one treatment is required within an interval of 5 to 7 days. We have also shown that PPA904-PDT can provide an immunomodulating, dose-dependent efflux on IL-12p70 production. This mechanism could be responsible for promoting a more rapid healing in PPA904-PDT treated mice. Our initial data indicate that phenothiaziniums exhibit a high parasiticidal effect in vivo against CL; this finding may be of use in establishing curative PDT regimens for future clinical trials.     

Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids

We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[125I]iodonaphthyl-1-azide ([125I]INA). [125I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently with 125I. In this report we demonstrate that fluorescent phospholipids and sphingolipids containing N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-6-aminocaproic acid serve as appropriate photosensitizers for [125I]INA. Using Chinese hamster ovary fibroblasts, we optimized the labeling conditions with respect to lipid concentration and time of irradiation and then examined the profiles of cellular proteins that were labeled when fluorescent analogs of ceramide, sphingomyelin, and phosphatidic acid were used as photosensitizers in living cells. The use of different fluorescent lipids, which label different subcellular compartments of cells as determined by fluorescence microscopy, derivatized different sets of cellular proteins with 125I. The labeled proteins were subsets of the total set of proteins available for derivatization as determined by direct activation of [125I]INA. Most proteins labeled by this procedure were pelleted by centrifugation of cell lysates at high speed (260,000 x g), but several soluble proteins were also labeled under these conditions. The implications of using this technique for identification of compartment-specific proteins and proteins involved in lipid metabolism and transport are discussed.     

Photodynamic therapy and some clinical applications in oncology

Photodynamic therapy (PDT), a new treatment modality for localized cancers involving the selective interaction of visible light with photosensitizers, such as hematoporphyrin derivatives (HpD) or dihematoporphyrin ether/ester (DHE) (Photofrin II). Photodynamic therapy of malignant tumors includes biological, photochemical and photophysical processes. These processes involve: (i) absorption of photosensitizing agent; (ii) selective retention of photosensitizer in tumors and (iii) irradiation of sensitized tumor by laser irradiation. This paper provides a review of photosensitizers, photochemistry, subcellular targets, side effects and lasers involved in photodynamic therapy. In addition, gradual increase in knowledge related to in vivo and in vitro mechanisms of action of PDT, as well as some clinical applications of photodynamic therapy are presented.     

7-aryl 1,5-dihydro-benzo[e][1,4]oxazepin-2-ones and analogs as non-steroidal progesterone receptor antagonists

Novel 7-aryl benzo[1,4]oxazepin-2-ones were synthesized and evaluated as non-steroidal progesterone receptor (PR) modulators. The structure activity relationship of 7-aryl benzo[1,4]oxazepinones was examined using the T47D cell alkaline phosphatase assay. A number of 7-aryl benzo[1,4]oxazepinones such as 10j and 10v demonstrated good in vitro potency (IC(50) of 10-30 nM) and selectivity (over 100-fold) at PR over other steroidal receptors such as glucocorticoid and androgen receptors (GR and AR). Several 7-aryl benzo[1,4]oxazepinones were active in the rat uterine decidualization model. In this in vivo model, compounds 10j and 10u were active at 3 mg/kg when dosed orally.     

Photothermal studies of modulating effect of photoactivated chlorin on interaction of blood cells with bacteria.

BACKGROUND: Bactericidal application of photosensitizers (PS) is a new and promising area. Up to now the action of PS against bacteria was studied without regard to immunocompetent cells, doses of drugs and radiation being used usually are not safe for such cells and therefore there is still no efficient model for PS application. METHODS: Action of chlorin, a plant derived photosensitizer, on a system of interacting cells and microbes was studied. Cell monitoring was done with a photothermal microscopy method. The two bacteria used were gram-positive (S. aureus) and gram-negative (E. coli) in a mixture of basic blood cells and neutrophils. The latter were used to model phagocytosis as fast cell response reaction. RESULTS: The strongest bactericidal effect was found when the cells and photoactivated chlorin act together. Selective bactericidal regime of PS application which does not affect cell viability was demonstrated. CONCLUSIONS: The results obtained suggest that this photosensitizer may enhance cell natural defense capabilities.     

Interest of RGD-containing linear or cyclic peptide targeted tetraphenylchlorin as novel photosensitizers for selective photodynamic activity

Destruction of the neovasculature is essential for tumor eradication by photodynamic therapy. Since the over-expression of integrins is correlated with tumor angiogenesis, we conjugated a photosensitizer (5-(4-carboxyphenyl)-10,15,20-triphenylchlorin or porphyrin) to the alpha(v)beta(3) integrin specific peptide RGD (H-Arg-Gly-Asp-OH) motif as a common sequence. We reported an efficient solid-phase synthesis of a new family of peptidic photosensitizers with linear or cyclic[RGDfK] RGD motif and compared conjugates in vitro selectivity and photodynamic activity. The conjugates were characterized by (1)H NMR, MALDI, UV-visible spectroscopy and singlet oxygen formation was performed. Chlorins containing linear and constrained RGD motif were incorporated up to 98- and 80-fold more, respectively, than the unconjugated photosensitizer over a 24-h exposure in human umbilical vein endothelial cells (HUVEC) over-expressing alpha(v)beta(3) integrin. Peptidic moiety also led to a non-specific increased cellular uptake by murine mammary carcinoma cells (EMT-6), lacking RGD binding receptors. Survival measurements demonstrated that HUVEC were greatly sensitive to conjugates-mediated photodynamic therapy.     

Studies on preparation and photodynamic mechanism of chlorin P6-13,15-N-(cyclohexyl)cycloimide (Chlorin-H) and its antitumor effect for photodynamic therapy in vitro and in vivo

Photodynamic therapy (PDT) represents a promising method for treatment of cancerous tumors. The chemical and physical properties of used photosensitizer play key roles in the treatment efficacy. In this study, a novel photosensitizer, Chlorin-H [-13,15-N-(cyclohexyl)cycloimide] which displayed a characteristic long wavelength absorption peak at 698 nm was synthesized. Following flash photolysis with 355 nm laser, Chlorin-H is potent to react with O₂ and then produce ¹O₂. This finding indicates that Chlorin-H takes its effects through type II mechanism in PDT. Generally, Chlorin-H is localized in mitochondria and nucleus of cell. After light irradiation with 698 nm laser, it can kill many types of cell, inhibit cell proliferation and colony formation, suppress cancer cell invasiveness and trigger apoptosis via the mitochondrial pathway in A549 cells in vitro. In addition, Chlorin-H–PDT can destroy A549 tumor in nude mice and a necrotic scab was formed eventually. The expression levels of many genes which regulated cell growth and apoptosis were determined by RT-PCR following Chlorin-H–PDT. The results showed that it either increased or decrease. Among which, the expression level of TNFSF13, a member of tumor necrosis factor superfamily, increased significantly. Silencing of TNFSF13 caused by RNA interference decreased the susceptibility of A549 cells to Chlorin-H–PDT. In general, Chlorin-H is an effective antitumor photosensitizer in vitro and in vivo and is worthy of further study as a new drug candidate. TNFSF13 will be an important molecular target for the discovery of new photosensitizers.     

Photodynamic therapy: an update

Photodynamic therapy (PDT) is a promising local treatment modality based on the selective accumulation of a photosensitizer in malignant tissues and the subsequent irradiation with laser light. Photodynamic therapy of malignant tumors includes biological, photochemical and photophysical processes. These processes involve: (a) absorption of photosensitizing agent; (b) selective retention of the photosensitizer in tumors and (c) irradiation of sensitized tumor by laser radiation. This report provides a review of photosensitizers, photochemistry, subcellular targets, side effects and laser involved in photodynamic therapy. In addition, gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT, as well as some clinical applications of photodynamic therapy are presented.     

Effect of vitamins A, C and glutathione on the mutagenicity of benzo[a]pyrene mediated by S9 from vitamin A-deficient rats

Vitamin A deficiency has been shown to enhance the mutagenicity of benzo[a]pyrene (Narbonne et al., 1985). Here we report that this is not a result of increased benzo[a]pyrene metabolism but might be a consequence of either a lack of vitamin A or a decreased level of scavengers (ascorbic acid and glutathione) in the liver. However, the addition of vitamin A in vitro in the form of retinyl palmitate strongly inhibits the benzo[a]pyrene mutagenicity. An enhancing effect on the mutagenicity of benzo[a]pyrene is observed with addition of ascorbic acid when incubated with high amounts of the precarcinogen. In vivo addition of high levels of glutathione also reduces the mutagenicity of benzo[a]pyrene.     

Improved protection against disseminated tuberculosis by Mycobacterium bovis bacillus Calmette-Guerin secreting murine GM-CSF is associated with expansion and activation of APCs

Modulating the host-immune response by the use of recombinant vaccines is a potential strategy to improve protection against microbial pathogens. In this study, we sought to determine whether secretion of murine GM-CSF by the bacillus Calmette-Guérin (BCG) vaccine influenced protective immunity against Mycobacterium tuberculosis. BCG-derived GM-CSF stimulated the in vitro generation of functional APCs from murine bone marrow precursors, as demonstrated by the infection-induced secretion of IL-12 by differentiated APCs, and the ability of these cells to present Ag to mycobacterium-specific T cells. Mice vaccinated with BCG secreting [corrected] murine GM-CSF (BCG:GM-CSF) showed increased numbers of CD11c+MHCII+ and CD11c-CD11b+F480+ cells compared with those vaccinated with control BCG, and this effect was most apparent in the draining lymph nodes at 7 and 14 days postvaccination. Vaccination with BCG:GM-CSF also resulted in enhanced expression of costimulatory molecules on migratory dendritic cells in the draining lymph nodes. The increased APC number was associated with an increase in the frequency of anti-mycobacterial IFN-gamma-secreting T cells generated after BCG:GM-CSF vaccination compared with vaccination with control BCG, and this effect was sustained up to 17 wk in the spleens of immunized mice. Vaccination with BCG:GM-CSF resulted in an approximately 10-fold increase in protection against disseminated M. tuberculosis infection compared with control BCG. This study demonstrates the potential of BCG secreting [corrected] immunostimulatory molecules as vaccines to protect against tuberculosis and suggests BCG:GM-CSF merits further appraisal as a candidate to control M. tuberculosis infection in humans.     

带有功能性多肽的光敏剂及其应用

光动力治疗( photodynamic therapy,PDT )是光敏剂在特定波长光源的激发下、在氧分子存在下产生细胞毒性物质的一种治疗方法,主要用于抗肿瘤治疗．目前临床应用的光敏剂对肿瘤细胞的靶向性比较有限,近来的一个热门研究方向是靶向性光敏剂．结合作者多年来在该方向的工作,综合近年来光敏剂研究的发展,比较全面地阐述了带有功能性多肽的靶向性光敏剂及其在光动力治疗中的应用．阐述多肽作为靶向基团的优势,总结了包括透膜多肽、血管靶向多肽、细胞受体靶向多肽等功能多肽与光敏剂偶合物的生物效应,说明了多肽能够实现光敏剂的靶向作用．     

Photodynamic activities of sulfonamide derivatives of porphycene on nasopharyngeal carcinoma cells

Two sulfonamide derivatives of porphycene, namely PS6 and PS6A, were synthesized, and their photodynamic efficacies on the nasopharyngeal carcinoma (NPC) cell line NPC/CNE-2 were evaluated. By comparing the 50% lethal concentrations (LC(50)) of these photosensitizers, we found that PS6A with a cationic ammonium group on the side chain exhibited potent photocytotoxicity on the NPC cell line. At a light dose of 1 J/cm(2), the LC(50) values of PS6 and PS6A for NPC cells were 11.6 and 1.92 microM, respectively. CNE-2 was found to rapidly take up PS6A in the first hour of incubation, and the uptake kinetics steadily increased to a plateau level after 18 h of incubation. The uptake of PS6A was temperature dependent. Over 99% of CNE-2 cells were sensitized by PS6A 24 h after drug treatment. Collapse of the mitochondrial membrane potential was also observed in PS6A photodynamic therapy (PDT)-treated CNE-2 cells 1.5 h after PDT. Confocal microscopy revealed that PS6A was predominantly localized in the mitochondria, lysosomes and Golgi bodies of NPC cells. Significant genotoxicity was not observed in CNE-2 cells. In functional studies, the in vitro formation of a capillary-like network of human umbilical vein endothelial cells in Matrigel was greatly inhibited by PS6A PDT in a dose-dependent manner. In conclusion, PS6A mediates both in vitro antitumor and antiangiogenic activities. PS6A might be a candidate for photodynamic treatment of NPCs.     

DTD, an anti-inflammatory ditriazine, inhibits angiogenesis in vitro and in vivo

The ditriazine derivative DTD (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d':3,2-d]-1,2,3-ditriazine) has been previously reported to reduce the degree of granulomatous inflammation and vascular density in a murine air pouch granuloma model. The aim of this study was to test whether DTD affects angiogenesis. Our results show that DTD inhibits in vivo angiogenesis in the chorioallantoic membrane (CAM) assay at doses equal or lower than 0.3 nmol/egg. Different in vitro assays were used to study the potential effects of this compound on key steps of angiogenesis, namely, a colorimetric assay of cell proliferation/viability, a morphogenesis on Matrigel assay, zymographic assays for gelatinases and nuclear morphology and cell cycle analysis for apoptosis induction. Our data indicate that DTD inhibits proliferation but does not induce apoptosis in endothelial cells in vitro. DTD suppresses the endothelial capillary-like chord formation at concentrations lower than those required to inhibit proliferation. DTD treatment inhibits the matrix metalloproteinase-2 production in endothelial and fibrosarcoma cells, but does not affect the cyclooxygenase-2 expression in endothelial cells, as assessed by western blot analysis. Taken together, results here presented indicate that DTD exhibits an anti-angiogenic activity that is independent of inflammatory processes and make it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.     

Metabolism of chrysene, 5-methylchrysene, 6-methylchrysene and 5,6-dimethylchrysene in rat liver cytosol, in vitro, and in rat subcutaneous tissue, in vivo

The polynuclear aromatic hydrocarbon chrysene undergoes a bioalkylation substitution reaction in vitro, in rat liver cytosol preparations, and in vivo, in rat dorsal subcutaneous tissue to yield 6-methylchrysene as a metabolite. In addition, both 5-methyl- and 6-methylchrysene were found to undergo a dealkylation reaction in these tissues to yield chrysene as well as both a biooxidation reaction to yield the corresponding hydroxyalkyl substituted chrysene and a bioalkylation reaction to give a dimethyl substituted chrysene. 5-Methylchrysene enzymatically cyclized to the 4,5-methylenechrysene derivative, an analog of benzo[a]pyrene in these tissues. 5,6-Dimethylchrysene was metabolized to monomethyl chrysenes, chrysene, and the hydroxyalkyl substituted chrysenes. The results suggest that chemical or biochemical substitution of a methyl group at the center of highest biochemical reactivity may be a necessary step in the metabolic activation and carcinogenicity of these compounds and their methylene bridged metabolites.