A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2).

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC50 value of 1.6 µM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

Charge density distribution effect in pyrrolidine-fused chlorins on microbial uptake and antimicrobial photoinactivation of microbial pathogens.

Two novels structurally related pyrrolidine-fused chlorins were synthesized from 5,10,15,20-tetrakis(pentafluorophenyl)chlorin by nucleophilic aromatic substitution of the para-fluoro groups. The reaction with 2-dimethylaminoethanol produced TPCF16-NMe2 in 77% yield, while TPCF16-NBu was obtained using butylamine in 87% yield. The latter was extensively methylated to form TPCF16-N+Bu in 92% yield. The synthetic strategy was designed to compare the effect of charge density distribution on chlorin in the efficacy to induce photodynamic inactivation of pathogens. TPCF16-NMe2 has five tertiary amines that can acquire positive charges in aqueous medium by protonation. Furthermore, four of the cationic groups are located in amino groups linked to the chlorine macrocycle by an aliphatic structure of two carbon atoms, which gives it greater movement capacity. In contrast, TPCF16-N+Bu presents intrinsic positive charges on aromatic rings. Absorption and fluorescence emission properties were not affected by the peripheral substitution on the chlorin macrocycle. Both photosensitizers (PSs) were able to form singlet molecular oxygen and superoxide anion radical in solution. Uptake and photodynamic inactivation mediated by these chlorins were examined on Staphylococcus aureus and Escherichia coli. Both phototherapeutic agents produced efficient photoinactivation of S. aureus. However, only TPCF16-NMe2 was rapidly bound to E. coli cells and this chlorin was effective to photoinactivate both strains of bacteria using lower concentrations and shorter irradiation periods. Our outcomes reveal that the charge density distribution is a key factor to consider in the development of new PSs. Accordingly, this work stands out as a promising starting point for the design of new tetrapyrrolic macrocycles with application in PDI.

[Flaccid paralysis and abdominal pain, an approach to porphyrias: a literature review and case series]. / Dolor abdominal y parálisis flácida, una aproximación a las porfirias: serie de casos y revisión de la literatura.

Porphyrias are inherited metabolic disorders caused by enzymatic deficiencies of HEM group biosynthesis. Most common in childhood at the third and fourth decade of life. They are characterized by increased levels of porphyrins, and various cutaneous, neurological, and visceral manifestations. We describe a series of 3 cases of female patients in the third decade of life with abdominal pain and a wide range of clinical manifestations and short and long-term complications. Our review contributes to the early recognition of these diseases to establish early specific managements to impact on irreversible outcomes.

Photodynamic inactivation mediated by 5-aminolevulinic acid of bacteria in planktonic and biofilm forms.

Bacterial photodynamic inactivation (PDI) employing endogenous production of porphyrins from 5-aminolevulinic acid (ALA) - named ALA-PDI-, is a new promising tool to achieve bacteria control in non-spread infections. The technique combines the action of the porphyrins acting as photosensitisers with light, to produce reactive oxygen species to target the pathogen. To date, some clinical applications of ALA-PDI have been reported although variable responses ranging from total eradication to absence of photokilling were found. ALA-PDI conducted at suboptimal conditions may lead to misleading results and the complexity of haem synthesis in bacteria hinders the optimization of the treatment. The present work aimed to gain insight on the variables affecting ALA-PDI in Gram-positives and Gram-negatives bacteria growing on planktonic and biofilm cultures and to correlate the degree of the response with the amount and type of porphyrin synthesised. Staphylococcus epidermidis and Escherichia coli clinical isolates and Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923 strains were utilised, and the optimal conditions of concentration and time exposure of ALA, and light dose were set. In both Gram-positive species analysed, a peak of porphyrin synthesis was observed at 1-2 mM ALA in biofilm and planktonic cultures, which fairly correlated with the decrease in the number of CFU after PDI (5 to 7 logs) and porphyrin content was in the same order of magnitude. In addition, ALA-PDI was similarly effective for planktonic and biofilm S. aureus cultures, and more effective in S. epidermidis planktonic cultures at low light doses. Beyond a certain light dose, it was not possible to achieve further photosensitization. Similarly, a plateau of cell death was attained at a certain ALA incubation time. Accumulation of hydrophilic porphyrins at longer incubation periods was observed. The proportion of porphyrins changed as a function of ALA concentration and incubation time in the Gram-positive bacteria, though we did not find a clear correlation between the porphyrin type and PDI response. As a salient feature was the presence of isococroporphyrin isoforms in both Gram-positive and Gram-negative bacteria. Gram-negative bacteria were quite refractory to the treatment: P. aeruginosa was slightly inactivated (4-logs reduction) at 40 mM ALA, whereas E. coli was not inactivated at all. These species accumulated high ALA quantities and the amount of porphyrins did not correlate with the degree of photoinactivation. Our microscopy studies show that porphyrins are not located in the envelopes of Gram-negative bacteria, reinforcing the hypothesis that endogenous porphyrins fail to attack these structures.

Expression and regulation of ABCG2/BCRP1 by sex steroids in the Harderian gland of the Syrian hamster (Mesocricetus auratus).

The ATP-Binding Cassette, subfamily G, member 2 (ABCG2) transporter is associated with the regulation of protoporphyrin IX transport and of other intermediates in heme biosynthesis. Because the hamster Harderian gland (HG) exhibits high concentrations of porphyrins and sexual dimorphism, we analyzed the hamster ABCG2. Cloned cDNA [2098-base pairs (bp)] contains an open-reading frame (ORF) of 1971-bp that encodes a 656 amino-acid protein with a molecular weight of 72844.56Da. The hamster ABCG2 sequence is conserved phylogenetically and shares a high percentage of identity with mouse (89%), rat (88%), and human (84%) transporters. Within its structure, a Walker A (G-X-X-G-X-G-K-S), a C signature motif characteristic of ABC transporters, and six putative transmembrane domains (TMDs) were identified. ABCG2 mRNA was detected in all hamster tissues, with higher amounts found in HG, brain, cerebellum, kidney, gut, ovary, and testis. Harderian ABCG2 expression exhibits a sexually dimorphic pattern where females display higher mRNA levels than males. Different patterns of transcriptional profiles of ABCG2 during the estrous cycle and after gonadectomy in both sexes were also observed. The differential expression between male and female HGs suggests that ABCG2 is under the regulation of gonadal steroids. The ABCG2 transporter is likely involved in the endogenous regulation of porphyrins in hamster HGs.

Metal coordination and peripheral substitution modulate the activity of cyclic tetrapyrroles on αS aggregation: a structural and cell-based study.

The discovery of aggregation inhibitors and the elucidation of their mechanism of action are key in the quest to mitigate the toxic consequences of amyloid formation. We have previously characterized the antiamyloidogenic mechanism of action of sodium phtalocyanine tetrasulfonate ([Na4(H2PcTS)]) on α-Synuclein (αS), demonstrating that specific aromatic interactions are fundamental for the inhibition of amyloid assembly. Here we studied the influence that metal preferential affinity and peripheral substituents may have on the activity of tetrapyrrolic compounds on αS aggregation. For the first time, our laboratory has extended the studies in the field of the bioinorganic chemistry and biophysics to cellular biology, using a well-established cell-based model to study αS aggregation. The interaction scenario described in our work revealed that both N- and C-terminal regions of αS represent binding interfaces for the studied compounds, a behavior that is mainly driven by the presence of negatively or positively charged substituents located at the periphery of the macrocycle. Binding modes of the tetrapyrrole ligands to αS are determined by the planarity and hydrophobicity of the aromatic ring system in the tetrapyrrolic molecule and/or the preferential affinity of the metal ion conjugated at the center of the macrocyclic ring. The different capability of phthalocyanines and meso-tetra (N-methyl-4-pyridyl) porphine tetrachloride ([H2PrTPCl4]) to modulate αS aggregation in vitro was reproduced in cell-based models of αS aggregation, demonstrating unequivocally that the modulation exerted by these compounds on amyloid assembly is a direct consequence of their interaction with the target protein.

Effects of bovine serum albumin (BSA) on the excited-state properties of meso-tetrakis(sulfonatophenyl) porphyrin (TPPS4).

To infer changes in the photophysical properties of porphyrins due to complexation with albumin, a combination of Z-scan and conventional spectroscopic techniques was employed. We measured the characteristics of excited states of meso-tetrakis(sulfonatophenyl) porphyrin bound to bovine serum albumin and observed that the binding reduces the intersystem crossing quantum yield and increases the internal conversion one. A reverse saturable absorption process was observed in the nanosecond timescale. These results are important for prediction of the efficiency of this complex in medical and optical applications, because associating porphyrins to proteins enables better accumulation in tumors and improves its stability in optical devices, but at the same time, decreases its triplet quantum yield.

Silk fibroin hydrogels for potential applications in photodynamic therapy.

In this study, we prepared translucid hydrogels with different concentrations of silk fibroin, extracted from raw silk fibers, and used them as a matrix to incorporate the photosensitizer 5-(4-aminophenyl)-10,15,20-tris-(4-sulphonatophenyl) porphyrin trisodium for application in photodynamic therapy (PDT). The hydrogels obtained were characterized by rheology, spectrophotometry, and scattering techniques to elucidate the factors involved in the formation of the hydrogel, and to characterize the behavior of silk fibroin (SF) after incorporating of the porphyrin to the matrix. The rheology results demonstrated that the SF hydrogels had a shear thinning behavior. In addition, we were able to verify that the structure of the material was able to be recovered over time after shear deformation. The encapsulation of porphyrins in hydrogels leads to the formation of self-assembled peptide nanostructures that prevent porphyrin aggregation, thereby greatly increasing the generation of singlet oxygen. Also, our findings suggest that porphyrin can diffuse out of the hydrogel and permeate the outer skin layers. This evidence suggests that SF hydrogels could be used as porphyrin encapsulation and as a drug carrier for the sustained release of photosensitizers for PDT.

Porphyrins are increased in the faeces of patients with prostate cancer: a case-control study.

BACKGROUND: Experimental models of prostate cancer have demonstrated increased levels of protoporphyrin IX (PpIX) in the blood and faeces of mice. Hence, the quantification of these autofluorescent molecules could be hypothesized to be a potential marker for this type of tumour. In this case-control study, the autofluorescence of porphyrins in human faeces from patients with prostate cancer and control subjects was analysed using fluorescence spectroscopy. METHODS: First, 3 mL of analytical-grade acetone was added to 0.3 g of faeces, and the mixture was macerated and centrifuged at 4000 rpm for 15 min. The supernatant was analysed spectroscopically. The emission spectra from 550 to 750 nm were obtained by exciting the samples at 405 nm. RESULTS: A significant difference between the samples from control and cancer subjects was established in the spectral region of 670-675 nm (p = 0.000127), which corresponds to a significant increase in faecal porphyrins in patients with cancer. There was no statistically significant correlation between PSA levels and faecal porphyrins. CONCLUSION: In this preliminary study conducted in humans, the results show a simple and non-invasive method to assess faecal porphyrins, which have the potential to function as a tumour biomarker in patients with prostate cancer. This approach has improved sensitivity and specificity over PSA testing. Additional prospective studies with larger sample sizes are required to validate these findings.

Persistent organic pollutants and porphyrins biomarkers in penguin faeces from Kopaitic Island and Antarctic Peninsula.

Polychlorinated biphenyl (PCB) levels were determined in the faeces of three Antarctic Peninsula penguin species to assess viability as a non-invasive approach for sampling PCBs in Antarctic biota. These determinations were complemented with stable isotope and porphyrins assessments, and together this methodology determined the role of diet and metabolic disruption in penguins. Up to 60% of the collected faecal samples evidenced low molecular weight PCBs, of which, the more volatile compounds were predominant, in agreement with previous results. The highest PCB levels were reported in the gentoo penguin (Pygoscelis papua; 35.3ngg-1 wet weight average), followed by the chinstrap (Pygoscelis antarctica; 6.4ngg-1 wet weight average) and Adélie penguins (Pygoscelis adeliae; 12.9ngg-1 wet weight average). Stable isotope analyses (δ15N and δ13C) demonstrated that gentoo feeding and foraging habits differed from those of Adélie and chinstrap penguins. A strong positive correlation was found between PCB concentrations and δ15N, indicating the role of diet on the observed pollutant levels. Porphyrins metabolite levels were also directly correlated with PCB concentrations. These results suggest that PCB levels impair the health of Antarctic penguins.

Limited proteolysis of myoglobin opens channel in ferrochelatase-globin complex for iron to zinc transmetallation.

Recombinant ferrochelatase (BsFECH) from Bacillus subtilis expressed in Escherichia coli BL21(DE3) was found by UV-visible spectroscopy to bind the model substrate tetraphenylporphyrin-sulfonate, TPPS, with Ka=3.8 10(5)mol/L in aqueous phosphate buffer pH 5.7 at 30°C, and to interact with metmyoglobin with Ka=1.07±0.13 10(5)mol/L at 30°C. The iron/zinc exchange in myoglobin occurring during maturation of Parma hams seems to depend on such substrate binding to BsFECH and was facilitated by limited pepsin proteolysis of myoglobin to open a reaction channel for metal exchange still with BsFECH associated to globin. BsFECH increased rate of zinc insertion in TPPS significantly and showed saturation kinetics with an apparent binding constant of Zn(II) to the [enzyme-TPPS] complex of 1.3 10(4)mol/L and a first-order rate constant of 6.6 10(-1)s(-1) for dissociation of the tertiary complex, a similar pattern was found for zinc/iron transmetallation in myoglobin.

Aminolevulinic acid dendrimers in photodynamic treatment of cancer and atheromatous disease.

The use of endogenous protoporphyrin IX after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). We have previously reported that the conjugation of ALA dendrimers enhances porphyrin synthesis. The first aim of this work was to evaluate the ability of ALA dendrimers carrying 6 and 9 ALA residues (6m-ALA and 9m-ALA) to photosensitise cancer cells. For this aim, we employed LM3 mammary carcinoma cells. In these tumour cells, at low concentrations porphyrin synthesis from dendrimers was higher compared to ALA, whereas at high concentrations, porphyrin synthesis was similar from both compounds. Topical application of ALA dendrimers on the skin overlying a subcutaneous LM3 implanted tumour showed no diffusion of the molecules either to distant skin sites or to the adjacent tumour, suggesting a promising use of the ALA macromolecules in superficial cancer models. As a second objective, we proposed the use of ALA-dendrimers in vascular PDT for the treatment of atherosclerosis. Thus, we focused our studies on ALA-dendrimer's selectivity towards macrophages in comparison with endothelial cells. For this aim we employed Raw 264.7 macrophages and HMEC-1 microvasculature cells. Porphyrin synthesis induced in macrophages by 6m-ALA and 9m-ALA (3 h, 0.025 mM) was 6 and 4.6 times higher respectively compared to the endothelial cell line, demonstrating the high affinity of ALA dendrimers for macrophages. On the other hand, ALA employed at low concentrations was slightly selective (1.7-fold) for macrophages. Inhibition studies suggested that ALA dendrimer uptake in macrophages is mainly mediated by caveloae-mediated endocytosis. Our main conclusion is that in addition to being promising molecules in PDT of superficial cancer, ALA dendrimers may also find applications in vascular PDT, since in vitro they showed selectivity to the macrophage component of the atheromatous plaque, as compared to the vascular endothelium.

Experimental protoporphyria: effect of bile acids on liver damage induced by griseofulvin.

The effect of bile acids administration to an experimental mice model of Protoporphyria produced by griseofulvin (Gris) was investigated. The aim was to assess whether porphyrin excretion could be accelerated by bile acids treatment in an attempt to diminish liver damage induced by Gris. Liver damage markers, heme metabolism, and oxidative stress parameters were analyzed in mice treated with Gris and deoxycholic (DXA), dehydrocholic (DHA), chenodeoxycholic, or ursodeoxycholic (URSO). The administration of Gris alone increased the activities of glutathione reductase (GRed), superoxide dismutase (SOD), alkaline phosphatase (AP), gamma glutamyl transpeptidase (GGT), and glutathione-S-transferase (GST), as well as total porphyrins, glutathione (GSH), and cytochrome P450 (CYP) levels in liver. Among the bile acids studied, DXA and DHA increased PROTO IX excretion, DXA also abolished the action of Gris, reducing lipid peroxidation and hepatic GSH and CYP levels, and the activities of GGT, AP, SOD, and GST returned to control values. However, porphyrin accumulation was not prevented by URSO; instead this bile acid reduced ALA-S and the antioxidant defense enzymes system activities. In conclusion, we postulate that DXA acid would be more effective to prevent liver damage induced by Gris.

Assessment of trace metals and porphyrins in excreta of Humboldt penguins (Spheniscus humboldti) in different locations of the northern coast of Chile.

To add data on trace metal contamination of Humboldt penguins in the South Pacific, levels of trace metals (As, Hg, Pb, Cu, Zn, and Cd) and porphyrins (copro-, uro-, and proto-) in excreta of Humboldt penguins that inhabit some important nesting sites on the northern coast of Chile were determined. Fresh excreta were collected on Pan de Azúcar Island, Chañaral Island, and Cachagua Island, from December 2011 to January 2012. Concentration of metals was determined by flame atomic absorption spectrophotometry, whereas porphyrins levels were measured by fluorimetric analysis. Concentrations (dry weight) of Cu (199.67 µg g(-1)), As (7.85 µg g(-1)), and Pb (12.78 µg g(-1)) were higher (p ≤ 0.05) in Cachagua Island. Colonies from Pan de Azúcar Island showed the highest levels of Hg (0.76 µg g(-1)), Cd (47.70 µg g(-1)), and Zn (487.10 µg g(-1)). Samples from Cachagua Island showed the highest (p ≤ 0.05) levels of copro- (2.16 nmol g(-1)), uro- (2.20 nmol g(-1)), and protoporphyrins (2.23 nmol g(-1)). There was a positive correlation between the metals As, Pb, and Cu with uro-, copro-, and protoporphyrins. The results indicated that penguin colonies from Cachagua Island are more exposed to metal contamination than penguin colonies from Pan de Azúcar and Chañaral Islands, thus being more likely to develop certain diseases caused by contamination with metals. Considering biomagnification, the metals detected in the excreta of Humboldt penguins can be a source of contamination from marine environments to terrestrial ecosystems, which could also affect other living organisms.

Optimization of cellular uptake of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine for maximal photoinactivation of Candida albicans.

Cellular uptake and photodynamic action of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc4⁺) was examined in Candida albicans. In vitro investigations showed that ZnPPc4⁺ was rapidly bound to C. albicans cells. The binding of phthalocyanine to cells was dependent on ZnPPc4⁺ concentrations (1-10 µM) and cells densities (106-108 cells mL⁻¹). A high amount of ZnPPc4⁺ retained in the cells after two washing steps, indicating a strong interaction between the photosensitizer and C. albicans. The uptake was temperature dependent, although the difference between 37 °C and 4 °C was about 10 %. Also, the amount of ZnPPc bound to C. albicans was affected when the cells were incubated for a longer time with azide and 2,4-dinitrophenol (DNP) prior to treatment with ZnPP4⁺. Cell survival after irradiation was dependent on the irradiation period, ZnPPc4⁺ concentration and cells density. Photoinactivation of C. albicans cells was elevated even after two washing steps. The strong dependence of uptake on cell density reveals the strength and avidity of the binding of ZnPPc4⁺ to C. albicans cells. The accumulation behaviour of ZnPPc4⁺ suggests that mainly an affinity-mediated binding mechanism can be involved. Therefore, ZnPPc4⁺ is an interesting phthalocyanine for photodynamic inactivation (PDI) of yeasts in liquid suspensions.

Interaction of meso-tetrakis (4-N-methylpyridyl) porphyrin in its free base and as a Zn(II) derivative with large unilamellar phospholipid vesicles.

Our aim was to investigate the interaction of the cationic meso-tetrakis (4-N-methylpyridyl) porphyrin, a photosensitizer used for photodynamic therapy, in its free base form (TMPyP) and complexed with Zn(II) (ZnTMPyP), with large unilamellar vesicles (LUVs), as a model for the gram-negative bacterial cell wall. Mixtures of the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipids, at different molar percentages, were used as LUVs. A significant increase of porphyrin affinity at higher POPG molar concentrations was observed from the binding constant values, K b, estimated by optical absorption and steady-state fluorescence. Besides, as demonstrated by time-resolved fluorescence, this affinity increase is also followed by a higher fraction of vesicle-bound porphyrin in the LUVs. Moreover, based on the K b values, we have observed a higher affinity of the ZnTMPyP to the POPG containing LUVs as compared to the TMPyP. Steady-state fluorescence quenching and zeta potential studies revealed that both porphyrins are possibly located at the LUVs Stern layer region. Therefore, the electrostatic attraction between the positively charged porphyrin peripheral groups and the negatively charged outer surface of the LUVs plays an important role in porphyrin association and localization. Our results have improved the understanding of the successful application of cationic porphyrins on the photo-inactivation of gram-negative bacteria. Since a higher accumulation of the ZnTMPyP in the bacterial cell wall would be expected, this porphyrin could be a more efficient therapeutic drug for this treatment.

Photodynamic efficiency of cationic meso-porphyrins at lipid bilayers: insights from molecular dynamics simulations.

Porphyrin derivatives have applications as photoactive drugs in photodynamic therapy. However, little is known about their interactions with phospholipid membranes at the molecular level. We employed molecular dynamics simulations to model the binding between a series of cationic meso-(N-methyl-4-pyridinium)phenylporphyrins and anionic phosphatidylglycerol lipid bilayers. This was done in the presence of molecular oxygen within the membrane. The ability of various porphyrins to cause photodamage was quantified in terms of their immersion depth and degree of exposition to a higher oxygen concentration inside the membrane. Simulations showed that the photodynamic efficiency could be improved as the number of hydrophobic phenyl substituents attached to the porphyrinic ring increased. In the specific case of porphyrins containing two hydrophobic and two charged substituents, the cis isomer was significantly more efficient than the trans. These results correlate well with previous experimental observations. They highlight the importance of both the total charge and amphiphilicity of the photosensitizer for its performance in photodynamic therapy.

A survey of environmental pollutants and cellular-stress markers of Porites astreoides at six sites in St. John, U.S. Virgin Islands.

Coral communities along the coast of St. John, U.S. Virgin Islands have exhibited site-specific behavior in declines. In order to determine if these specific coral communities are stressed and whether a pollutant or environmental factor present at this site is a probable stressor, we surveyed six near-shore coral communities in St. John, USVI for environmental pollutants and to determine the cellular physiological condition of the coral, Porites astreoides. The six sites within St. John are Cruz Bay, Caneel Bay, Hawksnest Bay, Trunk Bay, Tektite Reef in Beehive Bay, and Red Point. Red Point was considered the reference site because of its abundance and diversity of species, and it was the furthest removed from down-stream and down-current anthropogenic activities. All sites showed distinct cellular-stress marker patterns, indicating that the physiological condition of each population was different. Populations at Cruz, Hawksnest, Trunk, and Tektite were stressed, as indicated by high levels of DNA lesions and expression of stress proteins. Hawksnest and Tektite were contaminated with polyaromatic hydrocarbons (PAHs), while Cruz was contaminated with semi-volatile organochlorines and nitrogen-based biocides. At least for Hawksnest and Tektite, stress-marker patterns were consistent with an exposure to PAHs. Fecal coliform levels were high in Cruz and Trunk, indicating fecal contamination, as well as consideration for management action. Results from this study serve as a justification for a more thorough and methodical investigation into the stressors responsible for declines of coral populations within St. John. Furthermore, this study supports the argument for the importance of local factors contributing to regional coral reef declines; that not all forces impacting coral are global.

Susceptibility of Candida albicans to photodynamic action of 5,10,15,20-tetra(4-N-methylpyridyl)porphyrin in different media.

The photodynamic activity of 5,10,15,20-tetra(4-N-methylpyridyl)porphyrin (TMPyP) was evaluated in vitro on Candida albicans cells under different experimental conditions. This tetracationic porphyrin binds rapidly to C. albicans cells, reaching a value of ∼1.7 nmol 10(-6) cells when the cellular suspensions (10(6) CFU mL(-1) ) were incubated with 5 µM sensitizer. The amount of cell-bound sensitizer is not appreciably changed when cultures are incubating for longer times (>15 min) but it diminishes with the number of washing steps. Photosensitized inactivation of C. albicans cellular suspensions increases with both sensitizer concentration and irradiation time, causing a ∼5 log decrease of cell survival when the cultures are treated with 5 µM TMPyP and irradiated for 30 min. However, the photocytotoxicity decreases after one washing step, with the decrease in cell-bound sensitizer. The growth of C. albicans cells was arrested when the cultures were exposed to 5 µM TMPyP and visible light. On agar surfaces, the phototoxic effect of this sensitizer, which caused an inactivation of C. albicans cells, remained high. No growth was observed in areas containing TMPyP and irradiated. Moreover, in small C. albicans colonies, C. albicans cells were completely inactivated. These studies indicate that TMPyP is an effective sensitizer for photodynamic inactivation of yeasts in both liquid suspensions or localized on a surface.