admission. This proportion could already be greater in some parts of the country and may increase if referrals of cases of self-poisoning increase faster than the facilities for their assessment and management. The provision of social work and psychiatric expertise in casualty departments may be one means of preventing unnecessary medical admissions without risk to the patients. Dr Blake's and Dr Bramble's figures do not demonstrate, however, that any advantage would attach to medical teams taking over assessment from psychiatrists except that, by implication, assessments would be completed sooner by staff working on the ward full time. What the figures actually suggest is that if assessment of overdoses were left to house doctors there would be an increase in admissions to psychiatric units (by 19°U), outpatients (by 5O°'), and referrals to social services (by 140o). So for house doctors to assess overdoses would provide no economy for the psychiatric or social services. The study does not tell us what the consequences would have been for the six patients who the psychiatrists would have admitted but to whom the house doctors would have offered outpatient appointments. E J SALTER

Vitamin D deficiency.

Photosensitizers of the porphyrin and phthalocyanine series for photodynamic therapy

Basic principles of photodynamic therapy

Photodynamic therapy (PDT) is an innovative and attractive modality for the treatment of small and superficial tumours. PDT, as a multimodality treatment procedure, requires both a selective photosensitizer and a powerful light source which matches the absorption spectrum of the photosensitizer. Quadra Logic's Photofrin, a purified haematoporphyrin derivative, is so far the only sensitizer approved for phase III and IV clinical trials. The major drawbacks of this product are the lack of chemical homogeneity and stability, skin phototoxicity, unfavourable physicochemical properties and low selectivity with regard to uptake and retention by tumour vs. normal cells. Second-generation photosensitizers, including the phthalocyanines, show an increased photodynamic efficiency in the treatment of animal tumours and reduced phototoxic side effects. At the time of writing of this article, there were more than half a dozen new sensitizers in or about to start clinical trials. Most available data suggest a common mechanism of action. Following excitation of photosensitizers to long-lived excited singlet and/ or triplet states, the tumour is destroyed either by reactive singlet oxygen species (type II mechanism) and/or radical products (type I mechanism) generated in an energy transfer reaction. The major biological targets of the radicals produced and of singlet oxygen are well known today. Nucleic acids, enzymes and cellular membranes are rapidly attacked and cause the release of a wide variety of pathophysiologically highly reactive products, such as prostaglandins, thromboxanes and leukotrienes. Activation of the complement system and infiltration of immunologically active blood cells into the tumorous region enhance the damaging effect of these aggressive intermediates and ultimately initiate tumour necrosis. The purpose of this review article is to summarize the up-to-date knowledge on the mechanisms responsible for the induction of tumour necrotic reactions.

Photophysical and photobiological processes in the photodynamic therapy of tumours

Reactive oxygen species (ROS) can attack a diverse range of targets to exert antimicrobial activity, which accounts for their versatility in mediating host defense against a broad range of pathogens. Most ROS are formed by the partial reduction in molecular oxygen. Four major ROS are recognized comprising superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen ((1)O2), but they display very different kinetics and levels of activity. The effects of O2•- and H2O2 are less acute than those of •OH and (1)O2, because the former are much less reactive and can be detoxified by endogenous antioxidants (both enzymatic and nonenzymatic) that are induced by oxidative stress. In contrast, no enzyme can detoxify •OH or (1)O2, making them extremely toxic and acutely lethal. The present review will highlight the various methods of ROS formation and their mechanism of action. Antioxidant defenses against ROS in microbial cells and the use of ROS by antimicrobial host defense systems are covered. Antimicrobial approaches primarily utilizing ROS comprise both bactericidal antibiotics and nonpharmacological methods such as photodynamic therapy, titanium dioxide photocatalysis, cold plasma, and medicinal honey. A brief final section covers reactive nitrogen species and related therapeutics, such as acidified nitrite and nitric oxide-releasing nanoparticles.

Antimicrobial strategies centered around reactive oxygen species - bactericidal antibiotics, photodynamic therapy and beyond

The properties of a new photosensitizer, meso-tetra-hydroxyphenyl-chlorin (mTHPC), were studied using V79 cells (Chinese-hamster lung fibroblasts). Comparisons were made with 2 other photosensitizers: photofrin II (PII) and meso-tetra-hydroxyphenyl-porphyrin (mTHPP). A main advantage of mTHPC is that it has a strong absorption at 652 nm. Maximal cellular uptake of the dye was observed after 24 hr incubation of the cells with the drug. Using a confocal laser-scanning fluorescence microscope, we observed a diffuse distribution of m THPC in the cytoplasm. Furthermore, the lipophilicity of mTHPC was compared with that of the components of PII by means of high-pressure liquid chromatography (HPLC). Absorption and fluorescence spectroscopy indicated that aggregated as well as monomeric mTHPC was bound to the cells. The action spectrum for photo-inactivation of the cells showed that aggregated mTHPC did not contribute significantly to its photosensitizing effects. In the present cellular system, the efficiency of photodynamic therapy (PDT) with mTHPC (cells were irradiated at a wavelength of 652 nm) was higher than with PII (irradiation at 630 nm) or with mTHPP (648 nm). The quantum yield for photo-inactivation of cells was smaller for mTHPC than for mTHPP and PII. The addition of 1,3-diphenylisobenzofuran (DPBF) reduced cell inactivation during PDT. Thus, PDT with mTHPC seems to act at least partly via a type-II process. © 1994 Wiley-Liss, Inc.

Evaluation of a new photosensitizer, meso‐tetra‐hydroxyphenyl‐chlorin, for use in photodynamic therapy: A comparison of its photobiological properties with those of two other photosensitizers

The response of human colon adenocarcinoma cells of the line WiDr to the combined treatment of ionizing radiation and photosensitization by 5-aminolevulinic acid-induced protoporphyrin IX was assessed by a colony-forming assay. A dose of X rays inactivating approximately 50% of the cells was used. Seventy to 85% of the cells accumulated in S and G2 + M phase 12-24 h after such a treatment as measured by flow cytometry, while the distribution of cells in the phases of the cell cycle approached that of untreated cells 48 h after X-ray treatment. Cellular photosensitization was developed by endogenous synthesis of protoporphyrin IX (PPIX) from the precursor 5-aminolevulinic acid (5-ALA). This was performed by treating the cells with 1 mM 5-ALA for 4 h in a serum-free medium. The endogenous synthesis of PPIX increased with time after the cells had been subcultured, i.e. the ability of the cells to synthesize PPIX increased 1.5-2-fold within 48 h of incubation. This was not due to effects of trypsin on the cells. Photochemotherapy with 5-ALA was given 0-48 h after X rays. The combined cytotoxic effect was analyzed by an isobologram after correction of the survival curves for microcolony formation and differences in intracellular concentration of PPIX. The results indicate that 5-ALA PCT given 0-4 h after X rays acts slightly antagonistically while 5-ALA PCT given 12-48 h after X rays acts slightly synergistically.

Combined treatment of ionizing radiation and photosensitization by 5-aminolevulinic acid-induced protoporphyrin IX.

The photodynamic effect of a photoproduct of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) was investigated in WiDr cells, a human adenocarcinoma cell line. The fluorescence excitation and emission spectra of PpIX and the photoproduct were measured. After 1, 3 or 5 min exposure of the ALA-incubated cells to 140 mW/cm 2 light at 635 nm, the photoproduct — the chlorin photoprotoporphyrin (Ppp), had an emission band around 670 nm. The Ppp excitation peak at 670 nm is well separated from the PpIX peak at 635 nm. The outcome of photodynamic therapy (PDT) was determined by measuring intracellular fluorescence intensity of propidium iodide (PI) 2 h following PDT and methylene blue (MB) staining 24 h following PDT. A significant increase in the fluorescence intensity of PI was noted when the ALA-loaded cells were exposed to 670 nm light after exposure to 635 nm, indicating enhanced cell membrane inactivation induced by the photodynamic action of the photoproduct. However, the fraction of the cells that survived following the same treatment as measured by MB staining was not significantly affected based on an analysis of variance. The fluorescence of PpIX decayed significantly during 635 nm light exposure. Exposure to light at 670 nm does not lead to any photodegradation of PpIX. The fluorescence of Ppp was bleached during 670 nm light exposure. Exposure of Ppp at 670 nm gives no PpIX back. Thus, the phototransformation of PpIX to Ppp is probably not a reversible process.

The photosensitizing effect of the photoproduct of protoporphyrin IX.

The effects of photodynamic therapy (PDT) alone and in combination with Mitomycin C (MMC) on WiDr cells, a human colon adenocarcinoma cell line, were investigated. The addition of MMC increased the cytotoxicity of PDT. The presence of MMC resulted in a reduction or a removal of the shoulder of the PDT survival curves as well as an increase in their slopes. Increasing with the concentrations of MMC from 0.01 to 0.025 micrograms/ml, the cytotoxic effects of the two treatments changed from additivity to supra-additivity as judged by comparing dose-response curves for each treatment alone with survival curves after combination therapy and by isobologram analysis. The cytotoxicity of MMC could also be enhanced by a practically nontoxic treatment of PDT (8% cell inactivation). The cytotoxicities of MMC and PDT in combination were found to be dependent on the sequence of the two treatments. When MMC (> or = 0.02 micrograms/ml) and Photofrin II were given simultaneously for 16 h and then followed by irradiation, the combination was found to be more effective than when MMC was given to the cells immediately after PDT and kept in the medium for 16 h. Possible mechanisms of the combination effects of PDT and MMC are discussed briefly.

Potentiation of Photodynamic Therapy by Mitomycin C in Cultured Human Colon Adenocarcinoma Cells

BALB/c nude mice bearing WiDr human colon adenocarcinoma were used to determine the effect of ultrasound on the production of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) both in the tumors and in skin overlying the tumors. Ultrasound (1 MHz) with pulsed irradiation at an average intensity of 3 W/cm2 was given 10 min to the tumor area 10 min after administration of ALA (20% in an oil-in-water emulsion applied topically on the surface of the tumor for 30 min to 3 hr). An approximately 45% increase in the amount of PpIX produced by ALA in the tumors was obtained within 1 to 2 hr following ultrasound treatment. In particular, 1 hr after ultrasound treatment, the amount of PpIX in the tumors was at the same level as that 3 hr after ALA application alone. However, pulsed ultrasound irradiation for 5 min or continuous irradiation for 5 or 10 min had no significant effect on the production of PpIX by the tumor 1 hr after topical ALA application. Furthermore, in most cases, the amount of PpIX in the tumors was significantly decreased when ultrasound was given immediately before ALA application. There was no significant change in the ratio of the amount of PpIX in tumor to that in skin after ultrasound treatment. Most likely, the distribution of PpIX fluorescence in the tumors treated with ultrasound was more homogeneous than that in the tumors given ALA only. Our results provide a theoretical basis for possible clinical use of ultrasound-combined ALA or ALA based photodynamic therapy. Int. J. Cancer 78:464–469, 1998. © 1998 Wiley-Liss, Inc.

Production of protoporphyrin IX induced by 5-aminolevulinic acid in transplanted human colon adenocarcinoma of nude mice can be increased by ultrasound.

Li-Wei Ma

Papers

Evaluation of a new photosensitizer, meso‐tetra‐hydroxyphenyl‐chlorin, for use in photodynamic therapy: A comparison of its photobiological properties with those of two other photosensitizers

Combined treatment of ionizing radiation and photosensitization by 5-aminolevulinic acid-induced protoporphyrin IX.

The photosensitizing effect of the photoproduct of protoporphyrin IX.

Potentiation of Photodynamic Therapy by Mitomycin C in Cultured Human Colon Adenocarcinoma Cells

Production of protoporphyrin IX induced by 5-aminolevulinic acid in transplanted human colon adenocarcinoma of nude mice can be increased by ultrasound.