Search

Anemia is a major cause of morbidity and mortality worldwide resulting from a wide variety of pathological conditions. In severe cases it is treated by blood transfusions or injection of erythroid stimulating agents, e.g., erythropoietin (Epo), which can be associated with serious adverse effects. Therefore, there is a need to develop new treatment modalities. We recently reported that treatment of erythroleukemic cells with the novel the bi-functional prodrugs of 5-aminolevulinic acid (ALA) and butyric acid (BA), AN233 and AN908, enhanced hemoglobin (Hb) synthesis to a substantially higher level than did ALA and BA individually or their mixture. Herein, we describe that these prodrugs when given orally to mice induced histone deacetylase inhibition in the kidneys, bone marrow and spleen, thus, indicating good penetrability to the tissues. In mice where anemia was chemically induced, treatment with the prodrugs increased the Hb, the number of red blood cells (RBCs) and the percentage of reticulocytes to normal levels. The prodrugs had no adverse effects even after repeated treatment at 100-200mg/kg for 50days. The lack of increased levels of Epo in the blood of mice that were treated with the prodrugs suggests that AN233 and AN908 affected the Hb and RBC levels in an Epo-independent manner. Taken together with our previous studies, we propose that the prodrugs increase globin expression by BA inhibition of histone deacetylase and elevation heme synthesis by ALA. These results support an Epo-independent approach for treating anemia with these prodrugs.

A new type of nanoparticles, Pdots, and a new methodology of photosensitization are developed to achieve a more efficient photodynamic effect in aqueous solutions and in cells. Pdots are nano-sized particles, composed of conjugated chromophoric polymers coated with PEGylated phospholipids. They exhibit good aqueous colloidal properties, a broad absorption band and a strong and narrow emission band. We show that these characteristics improve biological photosensitization, which is employed in the photodynamic therapy of cancer. Amphiphilic photosensitizers such as Rose Bengal partition, non-covalently but with a high affinity, into the amphiphilic coating of the Pdots, without necessitating covalent attachment. At this close contact, very efficient fluorescence resonance energy transfer (FRET) occurs between the Pdot donor and the sensitizer acceptor. The Pdots serve as broad-band collectors of light, which is funneled, via energy transfer, to the photosensitizer. Therefore, energy transfer from them can additively assist in the activity of the acceptors of energy. The energy transfer mechanism, strong uptake of the Pdot-sensitizer dyads by MCF-7 adenocarcinoma cells and their enhanced photosensitized killing are demonstrated.

Background Staphylococcus aureus is a major pathogen in clinical microbiology. It is known to cause infections at various body sites and can be life-threatening. The development of resistance to many well-established antibiotic treatments and the prevalence of methicillin-resistant S. aureus (MRAS) among hospital patients and the general community pose challenges in treating the pathogen. The antimicrobial effect of photodynamic therapy (PDT) has been a subject of study for a long time and can offer new strategies for dealing with resistant strains. Objective In our study, we searched for a positive synergistic relationship between PDT and the standard antibiotics used to treat S. aureus and MRSA infections. Materials and methods The phototoxic profile of deuteroporphyrin (DP) in both resistant and susceptible clinical strains of S. aureus was determined by plating of treated and untreated broth cultures. Electron microscopy imaging was done to explore possible sites of damage and free-radical accumulation in the cells during DP-PDT. Minimal inhibitory concentration (MIC) of oxacillin, gentamicin, vancomycin, rifampin, and fusidic acid was determined using the broth dilution method, and the checkerboard method was used to detect and evaluate the synergistic potential of DP-PDT and antibiotic combinations. A synergistic combination was further characterized using broth cultures and plating. Results DP-PDT using a light dose of 15 J/cm2 showed a bactericidal effect even with a small concentration of 17 μM DP. Transmission electron microscopy indicated profound damage in the cell wall and cell membrane, and the appearance of mesosome-like structures. Free radicals tend to localize in the cell membrane and inside the mesosome. No synergistic effect was detected by combining PDT with gentamicin, vancomycin, rifampin, and fusidic acid treatments. A positive synergistic effect was observed only in DP-PDT-oxacillin combined treatment using the checkerboard method. The effect was observed in clinical antibiotic-resistant isolates after DP-PDT using a light dose of 46 J/cm2 and small concentrations of DP. Oxacillin MIC decreased below 2 μg/ml in resistant strains under such conditions. Cultures which did not undergo new cycles of DP-PDT recovered their original oxacillin resistance after a few generations. Conclusions PDT with porphyrins shows possible new therapeutic options in treating drug-resistant S. aureus at body sites suitable for irradiation. The synergistic effect of DP-PDT with oxacillin on clinical strains illustrates the potential of PDT to augment traditional antibiotic treatment based on cell wall inhibitors. Lasers Surg. Med. 50:535-551, 2018. © 2018 Wiley Periodicals, Inc.

Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

Anemia is a major clinical symptom of a wide variety of pathological conditions a common related to reduced erythropoiesis. Whereas erythropoietin treatment showed an improvement in the patients' condition, it revealed increased risks of thromboembolic and cardiovascular events. Herein we describe stimulation of erythropoiesis by the multifunctional 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu), a 5-aminolevulinic-acid (ALA) derivative, which undergoes metabolic hydrolysis yielding two erythroid differentiation inducers, ALA and butyric acid (BA), each acting through a different mechanism. ALA, the first precursor in the heme biosynthesis, accelerates heme synthesis and BA, a histone deacetylase inhibitor (HDACI) that activates the transcription of globin mRNA. Our results show that the AlaAcBu mutual prodrug is a potent chemical differentiation inducer of K562 human erythroleukemia cells manifested by augmentation of heme and globin synthesis and assembly of hemoglobin. Exposure of K-562 cells to AlaAcBu resulted in an increase in heme synthesis and globin expression. Stimulation of the heme pathway was evident by the over-expression of porphobilinogen deaminase (PBGD) and ferrochelatase. AlaAcBu promoted cellular erythroid differentiation depicted by the expression of the marker glycophorin A and cellular maturation characterized by cytoplasm hemoglobinization, polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis presented in this report.

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.

Successful 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is dependent on efficient porphyrin synthesis in the inflicted cancer tissue, which is regulated by several enzymes. Irradiation of the tumor excites the light-sensitive porphyrins and results in ROS production and cell death. In this study we investigated the effect of the expression levels of two main enzymes in heme biosynthesis, ALA dehydratase (ALAD) and porphobilinogen deaminase (PBGD), on the capacity of K562 cells to undergo cell death following ALA-PDT. We manipulated PBGD and ALAD expression levels by shRNAs and PBGD overexpressing plasmid. PBGD down-regulation induced an elevation in ALAD activity, while overexpression of PBGD reduced ALAD activity, indicating a novel regulation feedback of PBGD on ALAD activity. This feedback mechanism enabled partial PpIX synthesis under PBGD silencing, whereas ALAD silencing reduced PpIX production to a minimum. ALA-PDT efficacy was directly correlated to PpIX levels. Thus, only ALAD-silenced cells were not affected by ALA+ irradiation, while following PBGD silencing, the accumulated PpIX, though decreased, was sufficient for successful ALA-PDT. The alterations in ALAD activity level initiated by changes in PBGD expression indicates PBGD's central role in heme synthesis. This enables efficient ALA-PDT, even when PBGD is not fully active. Conversely, ALAD loss resulted in reduced PpIX synthesis and consequently failure in ALA-PDT, due to the absence of compensation mechanism for ALAD.

Herein we describe a series of multifunctional 5-aminolevulinic-acid (ALA) prodrugs for photodynamic dependent and independent cancer therapy (PDT). We studied the cell-death mechanisms in glioblastoma U251 cells treated with four ALA-prodrugs: (1) AlaAcBu, that releases ALA, acetaldehyde, and butyric acid; (2) AlaFaBu, that releases ALA, formaldehyde, and butyric acid; (3) AlaFaPi, that releases ALA, formaldehyde and pivalic acid (4) AlaAcPi that releases ALA, acetaldehyde and pivalic acid. We examined the light-activated and dark cell-death mechanisms of the active metabolites released from the prodrugs by unspecific cellular hydrolases. The active moieties accelerated biosynthesis of protoporphyrin IX (PpIX) due to upregulated porphobilinogen deaminase (PBGD) activity. AlaAcBu was found to be the superior prodrug for PDT due to its ability to induce the highest PpIX synthesis. Photo-irradiation of AlaAcBu-treated cells led to dissipation of the mitochondrial membrane potential and reduction in the mitochondria metabolic activities; apoptosis and necrosis. Electron microscopy analyses of these cells revealed mitochondrial and endoplasmic reticulum swelling, membrane blebbing, apoptotic bodies and necrotic cell rupture. The formaldehyde-releasing prodrugs AlaFaBu and AlaFaPi induced low PDT efficacy, moreover sequestering the formaldehyde with semicarbazide resulted in high PpIX synthesis, suggesting that formaldehyde inhibited its synthesis. ALA and AlaAcBu phototherapy resulted in a dramatic accumulation of ubiquitinated proteins due to reduced proteasome activity and expression. In conclusion, the PDT potency of the prodrugs was in the order: AlaAcBu, AlaAcPi > AlaFaBu ≥ ALA > AlaFaPi, and the superiority of AlaAcBu stems from lower molar concentrations of AlaAcBu and lower light intensity needed to activate cell death following PDT.

The involvement of environmental heavy metals in Parkinson's disease (PD) has been suggested by epidemiologic studies; however, the mechanism of this effect is unknown. PD is characterized by the aggregation of alpha-synuclein in Lewy bodies. We previously showed that Pb2+ accelerates proteasomal activity. Therefore, we examined the effect of Pb2+, Ga3+, and Cu2+ on alpha-synuclein in human SH-SY5Y cells. The heavy metals induced an increase in heme-oxygenase-1 levels without significant cell death or ROS generation. The metals inhibited ALA-dehydratase, which is the inhibitory subunit of the proteasome, thereby accelerating proteasomal activity and decreasing protein levels of CDK-1 and PBGD. However, alpha-synuclein protein levels increased after exposure to metals, similar to the effect obtained with the proteasome inhibitor, hemin, suggesting that alpha-synuclein is inaccessible to proteasomal degradation. Indeed, electron microscopy revealed the formation of aggresomes in Pb2+- or hemin-treated cells. Thus, although heavy metals enhance proteasomal activity, alpha-synuclein is protected from degradation, and its protein levels and aggregation are increased.

Absorption and fluorescence spectra, photosynthetic rates, pigment concentrations, and cell size were investigated during photoacclimation of Porphyra leucosticta, a red marine macroalga that thrives during winter in the eastern Mediterranean. Optical properties were measured on intact thalli using image analysis or from crude extracts after disrupting algal tissue. Absorption spectra from crude extracts showed major peaks at 485, 620, and 664 nm for chlorophyll a (chl a) and 500, 564 and 615 nm for phycoerythrin (PE) and phycocyanin (PC). Absorption peaks from intact thalli were less conspicuous than those from crude extracts, possibly due to PE contributing to the bulk absorption of light by chl a in intact cells. Fluorescence emission of intact thalli was about threefold higher than that of crude extracts at 420 and 560 nm. Image analysis revealed differences in maximal fluorescence emissions as related to cell size, cell age, and thallus functional areas, with female cells having nearly a twofold high...

Multifunctional acyloxyalkyl ester prodrugs of 5-aminolevulinic acid in cancer cell lines inhibited the proteasome and induced apoptosis and heme synthesis. The most potent prodrug was butyryloxymethyl 5-amino-4-oxopentanoate (1a). The metabolically released formaldehyde from the prodrugs was the dominant factor affecting cell viability by a ROS-dependent mechanism and was responsible for rapid phosphorylation of H2AX, suppression of the cell survival protein c-myc, and transient elevation in the expression of p21. 1a, which differs from 2a by releasing butyric instead of pivalic acid, was a more potent inducer of heme and acetylated H4 expression and induced apoptosis through activation of caspase 9. 1a and 1b specifically increased the level of the photosensitizer protoporphyrin 9, leading to enhancement of cell death by photodynamic therapy (PDT). The advantage of these multifunctional prodrugs over 5-ALA is their greater potency in the non-PDT mechanism of cancer cell killing and their ability to also augment PDT.

5-Aminolevulinic acid (ALA)-induced photodynamic therapy (ALA-PDT) has achieved remarkable research accomplishments over the past 30 years, yet its application in medical oncology still awaits clear recognition as a valid alternative therapeutic modality. It is well documented that topical ALA-PDT enables the treatment of multiple skin lesions simultaneously, and provides excellent cosmetic results with no acquired multi-drug resistance (MDR). Furthermore, upon disease recurrence the treatment can be repeated resulting in the same therapeutic efficacy. Additionally, in oncological surgery, ALA fluorescence-guided resection is a practical and simple method for visualizing intra-operative brain and urological tumors with millimeter accuracy. The urgent challenge is to direct future research of ALA-phototherapy and fluorescence diagnosis to the maturation of their medical status in oncology. Therefore, the future objectives are to amplify critical evidence-based results of ALA-PDT safety and efficacy and to validate its unique advantages over other technologies. Strong statistical PDT documentation and the positive predictive values of protoporphyrin IX (PpIX)-guided surgery will persuade the medical community to implement ALA-based therapeutics into standard clinical and surgical oncology practice. Research must address the phenomenon that no MDR develops as a consequence of PDT, since MDR is the major stumbling block in oncological therapeutics. A feasible goal should be to improve ALA administration protocols based on recent knowledge that preactivation of the enzyme porphobilinogen deaminase enhances PpIX accumulation in cancer cells and photodestruction. Moreover the recent introduction of multifunctional ALA prodrugs that maximize photosensitizer biosynthesis, targeting multiple sub-cellular targets, may increase PDT anti-cancer efficacy in additional disease settings. In conclusion, well-documented clinical results, new ALA delivery protocols, and novel multifunctional ALA prodrugs may advance ALA-PDT to becoming a front-line cancer therapy.

Acute intermittent porphyria (AIP) is a neuropathic disease caused by a dominant inherited deficiency in porphobilinogen deaminase (PBGD). We investigated the expression and the degradation of the human PBGD-mutations G748A, G748C and 887insA following transfection into human SH-SY5Y neuroblastoma cells. Mutant proteins exhibited reduced protein expression compared to transfected wild-type (wt) PBGD as revealed by Western blotting. The transcription levels assessed by real-time PCR of these mutant species were identical to those of the wild type. Immuno-fluorescence microscopy revealed reduced cellular distribution of the mutated PBGDs in the cytosol and the nucleus in comparison to the wild-type PBGD. Enhanced cellular accumulation of the mutated and wild-type PBGDs was detected following inhibition of the proteasome by the inhibitors CLBL and hemin. Elevated expression of wt and mutated PBGD protein levels was either achieved by hemin or heme-arginate treatment. On the other hand, enhanced PBGD degradation was achieved by lead poisoning of ALAD in the SH-SY5Y cells concomitant with acceleration of proteasomal activity, most probably by ALAD participation in proteasomal regulation [G.G. Guo, M. Gu, J.D. Etlinger, 240-kDa proteasome inhibitor (CF-2) is identical to delta-aminolevulinic acid dehydratase. J Biol Chem 1994; 269:12399–402.] Our results suggest that the difference in expression between the wild-type and mutant proteins appears to be regulated on the level of protein degradation. In conclusion, we demonstrate that the PBGD cellular pool is controlled by the proteasome activity, which in turn is down regulated by hemin or up-regulated by Pb-ALAD.

Photodynamic therapy (PDT) involves a two-stage process. A light-absorbing photosensitiser (Ps) is endocytosed and then stimulated by light, inducing transfer of energy to a cytoplasmic acceptor molecule and the generation of reactive oxygen species that initiate damage to cellular membrane components and cytolysis. The expanded use of PDT in the clinic is hindered by the lack of Ps target-cell specificity and the limited tissue penetration by external light radiation. This study demonstrates that bioconjugates composed of transferrin and haematoporphyrin (Tf–Hp), significantly improve the specificity and efficiency of PDT for erythroleukemic cells by a factor of almost seven-fold. Fluorescence microscopy showed that the conjugates accumulate in intracellular vesicles whereas free Hp was mostly membrane bound. Experiments with cells deliberately exposed to Tf–Hp at

Formation of protoporphyrin IX (PpIX) after topical application of 5-aminolevulinic acid (ALA) and its hexylester derivative (ALA-Hex) was studied on healthy human skin. Temperature, density of hai ...

Pieces cut from colonies of the soft coral Dendronephthya hemprichi exhibited rapid and effective attachment to hard surfaces. Attachment involved development of root-like processes (RLPs), which appeared at the basal part of the fragment 4 days after its removal from the colony. The fine structural changes and cascade of cellular events occurring in the RLP before and after attachment were studied using SEM, TEM, and LM. The epidermis of the RLPs is actively involved in the attachment process and several distinct phases are documented: appearance of numerous oval vesicles, extrusion of these vesicles resulting in the formation of an outer layer composed of extracellular organic matrix and organellar debris, which functions as an adhesive device leading to initial attachment. The latter phase was followed by the formation of desmocytes, which develop in the RLP epidermis and function as anchoring devices, mediating the firm attachment of the fragment to the substrate. This is the first evidence among anthozoans that desmocytes play an active role in anchoring tissue to substrate and thus extends the range of functions exhibited by desmocytes among anthozoans.

The mechanistic aspects of Escherichia coli photodynamic inactivation (PDI) have been studied in bacteria expressing the reporter protein GFP, following transfection with wild type pGFP plasmid and treatment with the hydrophilic cationic sensitizer tetra-meso(N-methyl-4-pyridyl)porphine tetratosylate (TMPyP). Cell survival and morphology during PDI were correlated with plasmid-GFP degradation in comparison to DNA and RNA strand-breaks, while photobleaching of the GFP chromophore was used to monitor protein photodamage. Singlet oxygen generated upon TMPyP photoactivation interacted with target nucleic acid polymers in a drug-and light-dose dependent manner. The hierarchy and cascade of the photodamage was in the order: genomic-DNA > total RNA > plasmid-DNA, as revealed by specific extraction and agarose electrophoresis. The notable resistance of the plasmid DNA in comparison to genomic DNA has implications for PDI of antibiotic-resistant bacteria. Re-growth of the treated cells in fresh medium showed structural features of an SOS response. Under these conditions, DNA repair machinery was initiated by typical alignment of DNA–protein co-aggregates accompanied by lateral assembly of ribosomes, apart from damaged DNA-arrays, as depicted by electron microscopy. GFP–TMPyP interactions were demonstrated by double green and red fluorescence on electrophoresis plates analyzed by spectral imaging. Photobleaching measurements revealed specific GFP photodamage directly related to PDI of the E. coli. The kinetics of both the GFP photobleaching and the K+ efflux, representing photodamage to cytosolic proteins and membrane damage, respectively, were found to be similar. The survival curves were correlated to chromosomal degradation and ultrastructural damage. We conclude that TMPyP-dependent PDI of E. coli is primarily dependent on genomic DNA photodamage rather than on protein or membrane malfunctions.

Propionibacterium acnes is a Gram-positive, microaerophilic bacterium that causes skin wounds. It is known to naturally produce high amounts of intracellular porphyrins. The results of the present study confirm that the investigated strain of P. acnes is capable of producing endogenic porphyrins with no need for any trigger molecules. Extracts from growing cultures have demonstrated emission peaks around 612 nm when excited at 405 nm, which are characteristic for porphyrins. Endogenic porphyrins were determined and quantified after their extraction from the bacterial cells by fluorescence intensity and by elution retention time on high-performance liquid chromatography (HPLC). The porphyrins produced by P. acnes are mostly coproporphyrin, as shown by the HPLC elution patterns. Addition of delta-aminolevulinic acid (ALA) enhanced intracellular porphyrin synthesis and higher amounts of coproporphyrin have been found. Eradication of P. acnes by its endogenic porphyrins was examined after illumination with intense blue light at 407-420 nm. The viability of 24 h cultures grown anaerobically in liquid medium was reduced by less than two orders of magnitude when illuminated once with a light dose of 75 J cm(-2). Better photodynamic effects were obtained when cultures were illuminated twice or three times consecutively with a light dose of 75 J cm(-2) and an interval of 24 h between illuminations. The viability of the culture under these conditions decreased by four orders of magnitude after two illuminations and by five orders of magnitude after three illuminations. When ALA-triggered cultures were illuminated with intense blue light at a light dose of 75 J cm(-2) the viability of the treated cultures decreased by seven orders of magnitude. This decrease in viability can occur even after a single exposure of illumination for the indicated light intensity. X-ray microanalysis and transmission electron microscopy revealed structural damages to membranes in the illuminated P. acnes. Illumination of the endogenous coproporphyrin with blue light (407-420 nm) apparently plays a major role in P. acnes photoinactivation. A treatment protocol with a series of several illuminations or illumination after application of ALA may be suitable for curing acne. Treatment by both pathways may overcome the resistance of P. acnes to antibiotic treatment.

Recently, considerable interest has been directed to red-fluorescence photodiagnosis of brain and other tumours during surgery using the protoporphyrin IX natural precursor, 5-aminolaevulinic acid. In the present study we focused on the role of the rate-limiting enzyme porphobilinogen deaminase in glioma C6 cell activity, differentiation and sub-cellular distribution. Over-expression of the human housekeeping porphobilinogen deaminase in the glioma cells, using the housekeeping-porphobilinogen deaminase plasmid, induced a G1 cell cycle attenuation accompanied by increases in enzyme activity and c6 differentiation toward astrocytes. Visualisation of subcellular localisation of the porphobilinogen deaminase using the independent techniques of fluorescence immuno-staining with specific anti-human porphobilinogen deaminase antibodies and cellular expression of porphobilinogen deaminase fused to green fluorescent protein, revealed (unexpectedly) a major fraction of porphobilinogen deaminase in the nucleus and only a minor fraction in the cytoplasm. Both C and N terminals of porphobilinogen deaminase fused to green fluorescent protein revealed a major fraction of the newly synthesized fused porphobilinogen deaminase in the nucleus. Furthermore, newborn rat brain cells grown in a primary culture showed the same localisation pattern of porphobilinogen deaminase in the nuclei. Stimulation of C6 glioma cell differentiation by butyrate induced a marked decrease in porphobilinogen deaminase both in the nucleus and in the cytoplasm as determined by Western blotting and fluorescence immuno-localisation. These findings suggest a possible dual role for housekeeping porphobilinogen deaminase in fast dividing glioma cells, one related to the porphyrin synthesis pathway and another coupled to nuclear function, which might be linked to tumorigenesis. British Journal of Cancer (2002) 86, 1006–1011. DOI: 10.1038/sj/bjc/6600173 www.bjcancer.com © 2002 Cancer Research UK

Cancer cells are considered to express primary drug resistance, yet chemotherapeutics are the front line of cancer treatments and have achieved great successes. In time, most neoplastic cells develop secondary resistance to chemotherapy, which remains the major obstacle to cancer treatment. Progressive multi-drug resistance develops by upregulation of a large family of drug ABC transporters, such as the P-glycoprotein.

SUMMARY We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G1-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147–153, 2001)

The subcellular localization sites of TPPS4 and TPPS1 and the subsequent cellular site damage during photodynamic therapy were investigated in CT-26 colon carcinoma cells using spectroscopic and electron microscopy techniques. The association of both porphyrins with the mitochondria was investigated and the implications of this association on cellular functions were determined. Spectrofluorescence measurements showed that TPPS4 favors an aqueous environment, while TPPS1 interacts with lipophilic complexes. The subcellular localization sites of each sensitizer were determined using spectral imaging. Mitochondrial-CFP transfected cells treated with porphyrins revealed localization of TPPS1 in the peri-nuclear region, while TPPS4 localized in the mitochondria, inducing structural damage and swelling upon irradiation, as shown by transmission electron microscopy. TPPS4 fluorescence was detected in isolated mitochondria following irradiation. The photodamage induced a 38% reduction in mitochondrial activity, a 30% decrease in cellular ATP and a reduction in Na(+)/K(+)-ATPase activity. As a result, cytosolic concentrations of Na(+) and Ca(2+) increased, and the level of K(+) decreased. In contrast, the lipophilic TPPS1 did not affect mitochondrial structure or function and ATP content remained unchanged. We conclude that TPPS4 induces mitochondrial structural and functional photodamage resulting in an altered cytoplasmic ion concentration, while TPPS1 has no effect on the mitochondria.

Pulsed photothermic treatment (PTT) of pigmented B16 mice melanoma tumors was carried out using a Photodyne incoherent light delivery system. Tumor heating with average temperature of 41-44 degrees C was observed during broadband photoirradiation (600-800 nm) at light doses of 60-120 J/cm(2) delivered using 0.6 J/cm(2) pulses (2 ms) at 1 Hz repetition rate. Electron microscopy of tumor samples revealed pronounced structural changes in microvasculature and melanosomes. Pulsed PTT caused damage to endothelial cells and vascular walls, swelling of mitochondria and melanosomal disruption without nuclear alteration. Significant tumor response with necrosis formation followed by tumor regression was observed by a tumor growth study after PTT at 120 J/cm(2).

The interaction of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) and hyperthermia is not well understood. In the present study, significant enhancement of tumor damage was observed after simultaneous application of ALA-PDT and IR-induced hyperthermia using a broad-band incoherent light source. One hour after systemic administration of ALA at a dose of 200 mg/kg, subcutaneously transplanted C26 colon carcinoma tumors were irradiated with two bands of the VersaLight system, red (R, 580-720 nm) and red plus IR (R + IR, 580-720 nm and 1250-1600 nm). Photoirradiation using the R + IR band at different fluence rates and exposures caused mild heating of the tumor to 39-43 degrees C at a 3 mm depth. Electron microscopy after ALA + R, ALA + R + IR and R + IR treatments showed early mitochondrial swelling that was more pronounced in the ALA + R + IR group. Tumor necrosis assessment, using histology and vital staining, revealed an enhancement of tumor necrosis depth in the ALA + R + IR group compared to ALA + R and R + IR. The results showed that subhyperthermic heating to 39-39.5 degrees C in the ALA + R + IR group decreased the threshold light dose required for 100% tumor necrosis from 210 J/cm2 (observed in the ALA + R group) to 140 J/cm2. A tumor growth delay test, based on tumor volume measurement, also revealed significant enhancement of antitumor effect after application of ALA + R + IR compared to ALA + R.

Neuromelanin in the substantia nigra may be associated with the pathogenesis of nigral cell death in Parkinson's disease. We used synthetic dopamine-melanin (DA-M) as a model compound for neuromelanin and examined its toxic effects on mice cerebellar granule cells and a rat pheochromocytoma cell line (PC12). The DA-M and dopamine-treated cells showed an accumulation of black deposits when examined by light microscopy. Electron microscopy revealed different stages of DA-M phagocytosis, starting with DA-M binding, engulfment of the particles and the formation of phagosomes located in the cytoplasm. Using absorption assays, we found that NaN3 and low temperature inhibit the internalization of DA-M, pointing to an energy-dependent phagocytosis mechanism. These results suggest that neuromelanin can be phagocytised by neuronal cells which may thus be subjected to its toxic effects. These findings may contribute to our understanding of the formation and disposition of neuromelanin and its possible role in the etiology of Parkinson's disease.

The spectroscopic and biological properties of the new photosensitizer lutetium texaphyrin (Lu-Tex) were assessed in vitro and in vivo on a C26 colon carcinoma model, in comparison with hematoporphyrin (Hp), photofrin II (PII) and chlorin e 6( Chl ). Strong binding of Lu-Tex to lipid bilayer membranes was observed. The results of confocal fluorescence microscopy on C26 cells showed that Lu-Tex was localized in small vesicles in the cytoplasm, possibly in the lysosomes, while Chl and Hp were distributed in larger cytoplasmic vesicles attributed to mitochondria. Scanning electron microscopy and X-ray microanalysis revealed that photodynamic therapy with Lu-Tex induced only slight damage to the cell membrane, leading to a delayed cell response. Chl and Hp caused significant structural damage to the outer cell membrane, resulting in ionic imbalance and fast cell death. The in vitro quantitative assessment of the relative efficiency per absorbed photon of the sensitizers revealed that Lu-Tex was less effective than Chl and Hp . However, the results of our in vivo study showed that at the same light and drug doses the anti-tumor efficiency of the agents was in the following order: Lu-Tex > Chl > PII . The strong in vivo anti-tumor effect of Lu-Tex can be explained by its higher integrated absorption in the long-wavelength range.

>> SUMMARY Spectral morphometric characterization of typical chronic lymphocytic leukemia (B-CLL) cells vs normal small lymphocytes stained by May–Grunwald–Giemsa was carried out by multipixel spectral imaging. The light intensity (450–850 nm of 10 4 pixels) from nuclear domains of each stained cell was recorded and represented as light transmittance spectra and optical density. Transmitted light spectra of two nuclear domains were determined, one with low-intensity light transmittance (LIT) and the other with high-intensity light transmittance (HIT). A spectral library was constructed using the four transmitted light spectra representing the HIT and LIT domains of the normal human lymphocytes and the LIT and HIT domains of the CLL cells. The spectral library served to scan CLL lymphocytes from 10 cases of CLL and the lymphocytes of 10 healthy individuals. Each spectrally similar domain in the nuclei of the lymphocytes was assigned an arbitrary color. The morphometric analysis of the spectrally classified nuclei showed specific spectral patterns for B-CLL in 92% of the cells. The specific spectral characteristics of each of the two cell populations were also observed by their optical density light absorbance spectra. We propose that spectral morphometric analysis may serve as an additional diagnostic tool for detection of CLL lymphocytes in a hematological specimen. (J Histochem Cytochem 46:1113–1118, 1998)

The spectral characteristics of erythropoietic cellular inclusions stained by May-Grunwald Giemsa (MGG) were determined by spectrally resolved imaging. Multipixel spectra were obtained from Cabot rings and Howell-Jolly (HJ) bodies, displaying a range of wavelengths of transmitted light. The spectral characteristics of these inclusions were compared with those of isolated DNA, histones (type II) and arginine-rich histones (type VI), all stained by MGG. Results of single-cell spectroscopy show that the spectra of Cabot rings and HJ bodies share spectral characteristics with the type II and type VI histones. However, no resemblance was found between Cabot rings and DNA spectra. The spectral analysis of heterochromatin displayed a spectral pattern with characteristics of both DNA and histones, while the euchromatin showed a major contribution of the DNA component.

The photodynamic inactivation of herpes simplex virus type 1 (HSV-1) by two phthalocyanines (PCS), the cationic dye HOSiPcOSi( CH,),( CH,),N+ (CH,) 31- (Pc5) and the amphiphilic dye aluminum dibenzodisulfophthalocyanine hydroxide ( AlN,SB,POH), has been compared with that by the anionic dye, Merocyanine 540 (Mc540). Both PC derivatives demonstrate a remarkable virucidal activity upon light activation even 3 h after the onset of HSV-1 adsorption, while Mc540 is effective for only 30 min after adsorption. Since fusion and virus penetration are promoted by membrane glycoproteins, we have studied the damage to viral proteins following photodynamic treatment (PDT) of HSV-1 and its relation to inactivation. The effect of AlN,SB,POH PDT is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Major changes are found in the protein profile of PDT-treated HSV- 1. A reduced ability of specific antibodies to react with HSV-1 major envelope proteins is detected by employing the Western blot assay. In particular, we demonstrate the related changes of glycoprotein D (gD) , a structural protein of the HSV envelope. Since the envelope proteins participate in viral entry into the host cell, these alterations to viral envelope proteins may impair their ability to participate in early events of viral entry, leading to reduced infectivity of HSV-I. In contrast, no significant changes in the proteins’ electrophoretic mobility could be seen after PDT with Mc540 or with Pc5. When HSV-1 purified proteins are subjected to combined electrophoretic and electro-osmotic forces on cellulose acetate, there is a shift in their cathode mobility, which may indicate changes in the protein mass and protein net charges following AlN,SB,POH photosensitization. There are only minor changes in the virus proteins, assayed as above, when HSV-1 is treated with Pc5. 0 1998

A novel spectral image-analysis system based on Fourier transformed spectroscopy combined with image processing has been used for the in vivo study of porphyrin localisation in human skin lesions. Fluorescence measurements were performed on patients with basal cell carcinomas (BCC) and acne. BCC lesions were examined after a 16-hour topical application of aminolaevulinic acid (ALA); for acne lesions, autofluorescence was studied. The results of fluorescence microscopy of BCC lesions revealed red fluorescence in the stratum corneum, epidermis, pilosebaceous units and in the tumour sites. The in vivo macroscopic examination showed higher protoporphyrin IX (PP) fluorescence in BCC lesions and in acne as compared to the surrounding normal tissue. An enhanced demarcation of the lesions' borders was obtained using the spectral similarity-mapping function.

A novel concept of spectrally resolved morphometry for histological specimens was developed using light microscopy combined with spectrally resolved imaging. The spectroscopic characteristics of rat hepatocytes stained by Haematoxylin and Eosin, Romanowsky–Giemsa, periodic acid–Schiff and Masson's trichrome were assessed. Light intensity in the range 450–850 nm was recorded from 10000 pixels of nuclear domains of each stained cell and represented as light transmittance spectra and optical density. In order to identify spectral shifts caused by stain– macromolecule interactions, we compared the spectra of individual stain components with those of DNA and bovine serum albumin. Chromatin and interchromatin areas were classified spectrally using a chosen spectral library followed by morphometric calculations of nuclear domains for each staining method. The spectral fingerprints of Masson's trichrome stain distinguished the nucleolus from the rest of the nuclear c hromatin, enabling the demarcation and calculation of the nucleolar area. Spectrally resolved imaging of human hepatocytes stained by Masson's trichrome stain revealed marked differences between the nucleolar area in normal human hepatocytes compared with hepatocellular carcinoma. Masson's trichrome stain also distinguished the nucleolar area in human breast carcinoma cells and keratinocytes.

The assessment of deuteroporphyrin-hemin complex as an agent for the treatment of burn wounds infected with a multiple-drug resistant strain of Staphylococcus aureus was performed. The effect of the porphyrin on the survival of the infectious bacteria was first assayed in culture, and later tested as well in a burned infected animal model. The addition of deuteroporphyrin and hemin, separately or together (as a complex) to a growing culture of S. aureus was monitored during 8 hours. It was found that deuteroporphyrin alone was strongly bactericidal only after photosensitization. On the other hand, hemin alone was moderately bactericidal but light independent. A combination of both deuteroporphyrin and hemin was extremely potent even in the dark and did not require illumination to eradicate the bacteria. The in vivo experiments by application of the above porphyrins in combination to infected burn wounds in guinea pigs was an effective way to reduce dramatically the contaminating S. aureus. Reduction of more than 99% of the viable bacteria was noted after the porphyrin mixture was dropped on the eschar or injected into the eschar, an effect that lasted for up to 24 hours. The deuteroporphyrin-hemin complex may be suggested as a new bactericidal treatment of S. aureus infected burns since it was found to be a potent and promising anti-Staphylococcal agent.

The novel method of Fourier transform multi-pixel spectroscopy was used for the nondestructive analysis of and comparison of pigmentation in different regions of live thalli of the red alga Porphyra linearis. Because the thallus in this alga consists of a monolayer of nonoverlapping cells, we were able to analyze the pigmentation of single cells by combining light absorbance with natural fluorescence data. From the image of each cell in the vegetative male and female reproductive and holdfast regions, more than 4 ± 104 fluorescence and absorbance spectra were obtained. Specific pigments in the different regions were localized by the use of a software program of similarity mapping followed by image construction. The reconstructed images revealed subcellular localization of each pigment according to specific spectroscopic fingerprints. The results showed that the vegetative and female reproductive cell types had a significantly higher content of phycoerythrin than of phycocyanin, and quite similar chlorophyll a levels. Most of the holdfast cells were poorly pigmented, but had more chlorophyll a than phycoerythrin or phycocyanin. The male reproductive cells contained only traces of pigments. Thus, by using Fourier transform multipixel spectroscopy, we were able to characterize the pigmentation of different regions of the thallus and follow the distribution patterns of the different pigments on the subcellular level along the differentiation gradient of the alga.

An in vivo fluorescence monitoring and photodynamic therapy (PDT) study was performed using the new photosensitizer lutetium texaphyrin (Lu-Tex). This photosensitizer is water soluble and has the additional advantage of strong absorption near 730 nm. C26 colon carcinoma was transplanted in the foot of BALB/c mice. In vivo fluorescence spectroscopy was applied to study Lu-Tex tissue distribution kinetics. For this purpose, fluorescence intensity both in the foot with the tumor and in the normal foot was measured in vivo by the laser-induced fluorescence (LIF) system. For PDT, both feet of the mice were irradiated simultaneously with the use of a new high intensity pulsed light delivery system, the Photodyne. The results of the LIF measurements showed that the maximal fluorescence intensity ratio between the normal and tumor bearing foot (FIR) was observed 24–48 h after the agent injection. Photoirradiation with doses from 90 to 240 J cm −2 (0.6 J cm −2 per 2 ms pulse, 1 Hz) 24 h after injection of Lu-Tex at a dose of 10 mg kg −1 caused significant tumor necrosis and delay in the tumor growth rate. The antitumor effect was enhanced with increasing light doses. Normal tissue response to PDT with Lu-Tex was determined as the damage index of the normal foot, which was irradiated simultaneously with the tumor bearing foot. The normal tissue response after PDT with Lu-Tex was compared with 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PP), chlorin e 6 (Chl) and Photofrin (PII) at the same values of antitumor effect. The results showed that at 50, 80 and 100% inhibition of tumor growth the orders of the values of normal foot damage indexes were as follows: ALA > Lu-Tex ≥ PII > Chl, PII > ALA > Lu-Tex > Chl and PII > Lu-Tex > ALA > Chl respectively.

A Fourier transform multipixel spectroscopy system was set up and applied to fluorescence microscopy of single living cells. Continuous fluorescence spectra for all pixels of the cell image were recorded simultaneously by the system. Multiple frames of data were first acquired and stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achieved, typically, for 104 pixels in a single cell. The net result was I(xy,λ), the fluorescence intensity (I) for each pixel of the image (xy), as function of wavelength (λ). The present study demonstrates that multipixel spectroscopy can reveal dynamic processes of the food-digestive cycle in the unicellular Paramecium vulgaris fed with algae. Spectral variability of fluorescence intensity at different cytoplasmic sites pinpointed the location of cellular deposits of chlorophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll. Localization of compartmental spectral changes was achieved using a ‘similarity mapping’ algorithm, followed by enhanced image construction. Similarity mapping based on the fluorescence spectrum of native chlorophyll revealed a highlighted image of the cell cytopharynx structure where algae were ingested. Phagolysosomes, migrating vacuoles and the cytoproct, each containing different ratios of pheophytin, were similarly imaged.

An in vivo study of tissue distribution kinetics and photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA), chlorin e6 (Chl) and Photofrin (PII) was performed to evaluate the selectivity of porphyrin accumulation and tissue damage effects in a tumour model compared with normal tissue. C26 colon carcinoma of mice transplanted to the foot was used as a model for selectivity assessment. Fluorescence measurements of porphyrin accumulation in the foot bearing the tumour and in the normal foot were performed by the laser-induced fluorescence (LIF) system. A new high-intensity pulsed light delivery system (HIPLS) was used for simultaneous irradiation of both feet by light in the range of 600-800 nm, with light doses from 120 to 300 J cm-2 (0.6 J cm-2 per pulse, 1 Hz). Photoirradiation was carried out 1 h after injection of ALA, 3 h after injection of Chl and 24 h after injection of PII. A ratio of porphyrin accumulation in tumour vs normal tissue was used as an index of accumulation selectivity for each agent. PDT selectivity was determined from the regression analysis of normal and tumour tissue responses to PDT as a function of the applied light dose. A normal tissue damage index was defined at various values (50, 80 and 100%) of antitumour effect. The results of the LIF measurements revealed different patterns of fluorescence intensity in tumour and normal tissues for ALA-induced protoporphyrin IX (ALA-PpIX), Chl and PII. The results of PDT demonstrated the differences in both anti-tumour efficiency and normal tissue damage for the agents used. The selectivity of porphyrin accumulation in the tumour at the time of photoirradiation, as obtained by the LIF measurements, was in the order ALA-PpIX > Chl > PII. PDT selectivity at an equal value of anti-tumour effect was in the order Chl > ALA-PpIX > PII. Histological examination revealed certain differences in structural changes of normal skin after PDT with the agents tested. The results of PDT selectivity assessment with respect to differences in mechanisms of action for ALA, Chl and PII are discussed. Images Figure 3 Figure 4

The aim of our study was to find out if Photofrin II, a cytotoxic drug used routinely in photodynamic therapy (PDT), can induce immune responses in vitro, and to compare its effects with those of the protoporphyrin 9, hemin, which also has antitumor properties. We tested the effect of these porphyrins on lymphocyte proliferation and secretion of interleukin-2, interleukin-3, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma), by human or murine mononuclear cells (MNC) without an activating light. Both the Photofrin II- and hemin-treated cells showed a significant increase in cytokine secretion in the presence of suboptimal concentrations of mitogen. Moreover, Photofrin II and hemin significantly increased production of TNFalpha and IFNgamma even in the absence of mitogen. The cellular binding sites of Photofrin II and hemin to MNC were localized by electromicroscopy or fluorescence. Combined stimulation of cells by mitogens and porphyrins maintained optimal vital ionic balance of potassium, sodium and chlorine in the lymphocytes. In the cells thus treated there was a significant increase in intracellular calcium, a vital second messenger for lymphokine secretion. We demonstrate that the effect of Photofrin II on the immune system involves enhanced cytokine secretion which may account for the subsequent tumor eradication by PDT.

Exposure of mouse thymocytes to dopamine caused apoptosis (programmed cell death). This was manifested by cellular condensation and membrane damage shown by flow cytometry measurements and scanning electron microscopic study. Dopamine also affected thymocytic nuclei and their genomic DNA integrity. Most of the DNA molecules accumulated in a subdiploid peak in flow cytometry analysis, indicating DNA fragmentation to small particles. DNA analysis showed the typical pattern of ‘DNA ladder’ caused by internucleosomal DNA cleavage. X-ray microanalysis of the cellular elements of dopamine-treated cells showed elevation of sodium (Na), chloride (Cl) and calcium (Ca) peaks, accompanied by reduction in phosphate (P) concentrations. Comparison of the potassium (K) and P concentrations showed significant differences between the two major death processes: necrosis (induced by exposure to sodium azide (NaN3)) and apoptosis (induced by dopamine). High concentrations of K indicated cell viability while reductions in P and elevations in Ca levels were found to be typical of apoptotic cell death. The antioxidant dithiothreitol (DTT) suppressed dopamine-induced apoptosis in thymocytes, suggesting that its toxicity may be mediated via generation of reactive oxygen radicals. Our study suggests that under certain circumstances, dopamine and/or its metabolites, may induce a process of apoptotic cell death of the dopamine-producing cells in the substantia nigra. Increased accessibility of dopamine to the nigral cell nucleus or inability to scavenge excess free radicals generated from dopamine oxidation triggering programmed cell death, may cause the progressive nigral degeneration in Parkinson's disease.

The antibacterial photodynamic effects of uncharged (o-tetrahydroxyphenyl porphine [THPP], m-THPP and p-THPP), cationic (5,10,15,20-tetra[4-N-methylpyridyl]porphine [TMPyP]) and anionic (5,10,15,20-tetra[4-sulfonatophenyl porphine] [TPPS4]) porphines on Staphylococcus aureus and Escherichia coli bacteria inactivation were examined. The results show that uncharged porphines provoked antibacterial photodynamic activity on S. aureus, and also on E. coli in the presence of the membrane-disorganizing peptide polymixin B nonapeptide (PMNP). The TMPyP compound was highly photoactive toward gram-positive bacteria but only marginally effective on gram-negative cells, whereas TPPS4 showed no activity on either gram-positive or gram-negative bacteria. The photoactivity of TMPyP is due to the electrostatic attraction between the positively charged sensitizer molecule and the negatively charged membrane of the gram-positive target cells. For TPPS4, the inactivity toward gram-positive bacteria is due to electrostatic repulsion between the charged sensitizer molecule and the cell membrane. For gram-negative bacteria, the inactivity is conceivably due to preferential (electrostatic) binding to the positively charged PMNP, which is an adjuvant for membrane disorganization, but has no effect on cell viability. For hydrophobic sensitizers, the photoactivity depends on the state of aggregation. The extent of deaggregation of the different THPP isomers was determined by fluorescence measurements of bound sensitizers and could be positively correlated with their photoinactivation capacity. We conclude that the structure-activity relationships of these porphines are affected by their net charge and by aggregation.

Topical 5-aminolevulinic acid (ALA) application in three different creams was carried out on mice bearing subcutaneously transplanted C26 colon carcinoma. The creams contained (a) 20% ALA alone, (b) ALA with 2% dimethylsulphoxide (DMSO) and (c) ALA, DMSO and 2% edetic acid disodium salt (EDTA). Protoporphyrin IX (PP) production in the tumour and in the skin overlying the tumour was studied by two methods: laser-induced fluorescence (LIF) and chemical extraction. The kinetics of PP production in the skin and in the tumour, as studied by the LIF method, was similar for all three cream preparations. The PP fluorescence intensity in the tissues reached its maximum 4-6 h after application of the creams. Quantitative analysis showed that the PP concentration after treatment was more pronounced in the skin than in the tumour. The efficiency of porphyrin production in the skin by the creams used was in the following order: ALA-DMSO-EDTA > ALA-DMSO > ALA. In the tumour the enhancing effect of DMSO and EDTA on PP accumulation induced by ALA was observed mainly in the upper 2 mm section. However, the concentration of PP in the tumour was found to be approximately the same for ALA-DMSO and ALA-DMSO-EDTA cream combinations. The possible mechanisms of the effect of DMSO and EDTA are discussed.

The ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), insulin, 12-O-tetradecanoylphorbol-13-acetate (TPA), and aurintricarboxylic acid (ATA) to protect the human breast cancer cell line MDA-231 from death induced by the anticancer drug adriamycin was investigated. Cell death was induced in the MDA-231 cells either by a short-time exposure to a high dose of adriamycin (2 micrograms.ml-1.1h-1) and further culturing in the absence of the drug, or by continuous exposure to a low dose of adriamycin (0.3 micrograms/ml). Cell death was evaluated after 48 h of incubation by several techniques (trypan blue dye exclusion, lactic dehydrogenase activity, cellular ATP content, transmission electron microscopy, and DNA fragmentation). EGF, TPA, and ATA, each at an optimal concentration of 20 ng/ml, 5 ng/ml, and 100 micrograms/ml respectively, substantially enhanced survival of cells exposed either to a high or low dose of adriamycin. Neither IGF-1 nor insulin, each at concentrations of 20 ng/ml, had an effect on cell survival. The three survival factors enhanced protein synthesis in the untreated cells and attenuated the continuous decrease in protein synthesis in the adriamycin-treated cells. Moreover, the three survival factors protected the MDA-231 cells from death in the absence of protein synthesis (cycloheximide 30 micrograms/ml). These results suggest that EGF, TPA, and ATA promote survival of adriamycin pretreated cells by at least two mechanisms: enhancement of protein synthesis and by a protein synthesis independent process, probably a posttranslational modification effect.

In the present study, we investigated the ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin to protect the human breast cancer cell line MDA-231 from death induced by the antitumor drug actinomycin D (ACT-D). ACT-D is an inhibitor of RNA and protein synthesis, and its cytotoxicity may result due to continuous depletion in some vital protein molecules. Cell death was induced in the MDA-231 cells by either continuous exposure to a low dose of ACT-D (0.2 microgram/ml), or by a short-time exposure to a high dose of ACT-D (2 micrograms/ml) and further culturing in the absence of the drug. Cell death was evaluated by the trypan blue dye exclusion test, the release of lactic dehydrogenase into the culture medium, and the depletion in the cellular ATP content. EGF and IGF-1, each at an optimal concentration of 20 ng/ml, enhanced substantially survival of cells exposed either to a low or a high dose of ACT-D. The combination of EGF (10 ng/ml) and IGF-1 (10 ng/ml) had an additive survival effect, which proposes that each of the growth factors enhanced survival by a distinct pathway. Insulin up to 40 ng/ml had no effect on cell survival. Pretreatment of the cells for 1 to 5 h with EGF and IGF-1 protected cells from the cytotoxic effect of ACT-D. Exposure of the cells to 2 micrograms/ml of ACT-D for 1 h resulted in a drastic inhibition in uridine incorporation and only in a slight inhibition in leucine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

1. 1. The immediate and fast ionic fluxes in Friend erythroleukemic cells (FELC), erythrocytes and Staphylococcus aureus during short intervals of porphyrin mediated photosensitization were determined uniquely by X-ray microanalysis (XRMA) combined with electron microscopy. 2. 2. Photodynamic inactivation of FELC was mediated by either endogenous protoporphyrin induced by 5-amino levulinic acid (5-ALA), or Photofrin-II. We describe the predominant phenomena of >85% K-loss within 2–10 min of photoactivation. However the accompanied Na inflow and the collapse of the cellular balance of elemental-composition were inconsistent and acted as a function of cell damage. 3. 3. Erythrocytes treated with hematoporphyrin (HP) lost most of their intracellular K yet instantly gained Na. Nevertheless the K/Na molar ratio of the control erythrocytes was nearly 12/1 while after photosensitization and K loss it changed to 1/1. 4. 4. The S. aureus bacteria photosensitized with HP showed entire K-loss as well as marked Na efflux which increased with irradiation time; this was accompanied by the decline of other cell elements. 5. 5. The prevailing K loss in FELC, erythrocytes and bacteria during the first minutes of photosensitization is deduced to be an immediate primary consequence of the photodynamic effect, while other ionic changes are joined in order with the development of cellular damage.

We have monitored several photosensitized reactions in proteins, liposomes and cells under similar conditions. We found that the depolarization of K(+)-diffusion potential of liposomes or the leakage of an entrapped molecule, calcein, progress at a much slower rate than the photosensitized damage to proteins and the photosensitized killing of bacterial and leukemic cells. X-ray microanalysis revealed that upon light exposure of HP-treated leukemic cells and bacteria, they totally lost their cellular potassium. We deduce that the direct photosensitized oxidation of lipid components cannot cause the depolarization of cells, which in turn could be responsible for their death. A photosensitized damage to protein sites in the cell, probably in the membrane, is a more likely reason for the depolarization, the loss of potassium ions and cell death that is caused in light-activated photodynamic processes.

Environmental pollution of heavy metals is very abundant nowadays from industry, chemicals, old paints, and pipes or resulting from previous contaminants accumulating in the food chain. Most of the iron demands of the body are needed for heme synthesis and assembly, but iron is also required for Fe-S cluster proteins and other redox enzymes. Heme is an essential, iron-binding molecule used as a prosthetic group of hemoproteins or as a regulator in multiple cellular pathways. In this review, we focused on the effect of exposure to heavy metals, such as Pb, Ga, Cu, Kd, Hg and Al, on heme synthesis as the main iron-sequestering process of the human body. These metals compete with iron on transporters, reduce the cellular iron pool and moreover, bind to proteins, and cause physical and mental disturbances. Heavy metals mainly impair various aspects of the heme synthesis pathway: gene expression, enzyme activity, and iron integration into protoporphyrin IX. Main risk factors are described as well as effects on iron dependent processes in order to increase public awareness to the distribution of heavy metals in our close environment and the harsh consequences of exposure, even in low doses.