Transcriptome profile analysis reveals putative molecular mechanisms of 5-aminolevulinic acid toxicity.

5-aminolevulinic acid (5-ALA) is the first precursor of the heme biosynthesis pathway, accumulated in acute intermittent porphyria (AIP), an inherited metabolic disease characterized by porphobilinogen deaminase deficiency. An increased incidence of hepatocellular carcinoma (HCC) has been reported as a long-term manifestation in symptomatic AIP patients. 5-ALA is an α-aminoketone prone to oxidation, yielding reactive oxygen species and 4,5-dioxovaleric acid. A high concentration of 5-ALA presents deleterious pro-oxidant potential. It can induce apoptosis, DNA damage, mitochondrial dysfunction, and altered expression of carcinogenesis-related proteins. Several hypotheses of the increased risk of HCC rely on the harmful effect of elevated 5-ALA in the liver of AIP patients, which could promote a pro-carcinogenic environment. We investigated the global transcriptional changes and perturbed molecular pathways in HepG2 cells following exposure to 5-ALA 25 mM for 2 h and 24 h using DNA microarray. Distinct transcriptome profiles were observed. 5-ALA '25 mM-2h' upregulated 10 genes associated with oxidative stress response and carcinogenesis. Enrichment analysis of differentially expressed genes by KEGG, Reactome, MetaCore™, and Gene Ontology, showed that 5-ALA '25 mM-24h' enriched pathways involved in drug detoxification, oxidative stress, DNA damage, cell death/survival, cell cycle, and mitochondria dysfunction corroborating the pro-oxidant properties of 5-ALA. Furthermore, our results disclosed other possible processes such as senescence, immune responses, endoplasmic reticulum stress, and also some putative effectors, such as sequestosome, osteopontin, and lon peptidase 1. This study provided additional knowledge about molecular mechanisms of 5-ALA toxicity which is essential to a deeper understanding of AIP and HCC pathophysiology. Furthermore, our findings can contribute to improving the efficacy of current therapies and the development of novel biomarkers and targets for diagnosis, prognosis, and therapeutic strategies for AHP/AIP and associated HCC.

Analysis of Renal Cell Carcinoma Cell Response to the Enhancement of 5-aminolevulinic Acid-mediated Protoporphyrin IX Fluorescence by Iron Chelator Deferoxamine†.

As a tumor photodiagnostic agent, 5-aminolevulinic acid (ALA) is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) with fluorescence. ALA-PpIX fluorescence was evaluated in human renal cell carcinoma (RCC) cell lines and non-tumor HK-2 cell lines. We found that extracellular PpIX level was correlated with ABCG2 activity, illustrating its importance as a PpIX efflux transporter. Extracellular PpIX was also related to the Km of ferrochelatase (FECH) that chelates PpIX with ferrous iron to form heme. The Vmax of FECH was higher in all RCC cell lines tested than in the HK-2 cell line. TCGA dataset analysis indicates a positive correlation between FECH expression and RCC patient survival. These findings suggest FECH as an important biomarker in RCC. Effects of iron chelator deferoxamine (DFO) on the enhancement of PpIX fluorescence were assessed. DFO increased intracellular PpIX in both tumor and non-tumor cells, resulting in no gain in tumor/non-tumor fluorescence ratios. DFO appeared to increase ALA-PpIX more at 1-h than at 4-h treatment. There was an inverse correlation between ALA-PpIX fluorescence and the enhancement effect of DFO. These results suggest that enhancement of ALA-PpIX by DFO may be limited by the availability of ferrous iron in mitochondria following ALA administration.

Relationship between tumor cell infiltration and 5-aminolevulinic acid fluorescence signals after resection of MR-enhancing lesions and its prognostic significance in glioblastoma.

PURPOSE: This study investigated the degree of tumor cell infiltration in the tumor cavity and ventricle wall based on fluorescent signals of 5-aminolevulinic acid (5-ALA) after removal of the magnetic resonance (MR)-enhancing area and analyzed its prognostic significance in glioblastoma. METHODS: Twenty-five newly developed isocitrate dehydrogenase (IDH)-wildtype glioblastomas with complete resection both of MR-enhancing lesions and strong purple fluorescence on resection cavity were retrospectively analyzed. The fluorescent signals of 5-ALA were divided into strong purple, vague pink, and blue colors. The pathologic findings were classified into massively infiltrating tumor cells, infiltrating tumor cells, suspicious single-cell infiltration, and normal-appearing cells. The pathological findings were analyzed according to the fluorescent signals in the resection cavity and ventricle wall. RESULTS: There was no correlation between fluorescent signals and infiltrating tumor cells in the resection cavity (p = 0.199) and ventricle wall (p = 0.704) after resection of the MR-enhancing lesion. The median progression-free survival (PFS) and median overall survival (OS) were 12.5 (± 2.1) and 21.1 (± 3.5) months, respectively. In univariate analysis, the presence of definitive infiltrating tumor cells in the resection cavity and ventricle wall was significantly related to the PFS (p = 0.002) and OS (p = 0.027). In multivariate analysis, the absence of definitive infiltrating tumor cells improved PFS (hazard ratio: 0.184; 95% CI: 0.049-0.690, p = 0.012) and OS (hazard ratio: 0.124; 95% CI: 0.015-0.998, p = 0.050). CONCLUSIONS: After resection both of the MR-enhancing lesions and strong purple fluorescence on resection cavity, there was no correlation between remnant fluorescent signals and infiltrating tumor cells. The remnant definitive infiltrating tumor cells in the resection cavity and ventricle wall significantly influenced the prognosis of patients with glioblastoma. Aggressive surgical removal of infiltrating tumor cells may improve their prognosis.

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.

α-Bisabolol improves 5-aminolevulinic acid retention in buccal tissues: Potential application in the photodynamic therapy of oral cancer.

CONTEXT: 5-Aminolevulinic acid (5-ALA) is a prodrug used in photodynamic therapy (PDT) of tumors, including cancer of the oral mucosa. 5-ALA poorly penetrates oral tissues due to its high hydrophilicity, which impairs its local effects in PDT. OBJECTIVES: To examine whether α-bisabolol (α-Bis) influences the 5-ALA permeability in the porcine buccal mucosa, to an extent that improves its application in PDT (which requires low permeation and high retention in the buccal mucosa). METHODS: In vitro permeability studies with 5-ALA (1% and 10% w/w) associated with α-Bis (1% to 20% w/w) in propylene glycol were carried out at 4h and 24h using porcine buccal mucosa in a modified Franz cell system. The in vitro release profiles (0.5 to 48h) of the selected formulation and its respective control were determined using artificial membranes. Samples of buccal mucosa treated with the formulation were submitted to histopathological analysis, using a routine optical microscopy technique. RESULTS: The association of 1% 5-ALA and 5% α-Bis provided the best results; after 4h of treatment with this formulation, the 5-ALA permeation was low and its retention in the mucosa was six-fold higher than that promoted by the control formulation (5-ALA alone). Histological analysis of the porcine buccal mucosa evidenced that 5% α-Bis altered the tissue morphology, which probably promoted 5-ALA retention. We concluded that 5% α-Bis is a potential adjuvant in formulations containing 5-ALA that could improve its retention after topical oral administration for the PDT treatment of cancer.

Effect of diphenyl diselenide diet supplementation on oxidative stress biomarkers in two species of freshwater fish exposed to the insecticide fipronil.

The ability of diphenyl diselenide [(PhSe)2] to attenuate oxidative damage was evaluated in the liver, gills, brain, and muscle of carp (Cyprinus carpio) and silver catfish (Rhamdia quelen) experimentally exposed to fipronil (FPN). Initially, the fish were fed a diet without (PhSe)2 or a diet containing 3.0 mg/kg of (PhSe)2 for 60 days. After the 60-day period, the fish were exposed to 0.65 µg/L of FPN for 192 h. The results showed that carp exposed to FPN and not fed with (PhSe)2 exhibited acetylcholinesterase (AChE) inhibition in brain and muscle, and increased thiobarbituric acid-reactive substance (TBARS) in liver, gills, and brain. Furthermore, FPN decreased nonprotein thiols (NPSH) and δ-aminolevulinate dehydratase (δ-ALA-D) in carp liver and gills, and increased plasma glucose and protein levels. In silver catfish, FPN inhibited AChE and increased TBARS levels in muscle. In addition, glutathione S-transferase (GST) decreased in liver and muscle, and plasma glucose was increased. (PhSe)2 reversed some of these effects. It prevented the increase in TBARS levels in liver, gills, and brain in carp and in silver catfish muscle, and reversed the increase in plasma glucose levels in both species. Additionally, (PhSe)2 increased the NPSH levels in carp and silver catfish that had decreased in response to FPN exposure. However, (PhSe)2 was not effective in reversing the AChE inhibition in brain and muscle or the δ-ALA-D decrease in carp liver. Thus, (PhSe)2 protects tissues of both species of fish, mainly by preventing or counteracting the effects of FPN, on TBARS levels, antioxidants, and present anti-hyperglycemic property.

Bis(phenylimidazoselenazolyl) diselenide as an antioxidant compound: An in vitro and in vivo study.

The organoselenium compounds have been reported for many biological properties, especially as potent antioxidants. The compound bis(phenylimidazoselenazolyl) diselenide (BPIS) is a novel diaryl diselenide derivative, which shows antinociceptive and anti-inflammatory properties in mice, but whose antioxidant activity has not been studied. The present study aimed to investigate the antioxidant and toxicological potential of BPIS in brain of rats in vitro, and the effect of BPIS against the oxidative damage induced by sodium nitroprusside (SNP) in mouse brain. BPIS, at low molecular range, reduced lipid peroxidation (LP) and protein carbonyl (PC) content in rat brain homogenates (IC50 values of 1.35 and 0.74 µM, respectively). BPIS also presented dehydroascorbate reductase-like and glutathione-S-transferase-like, as well as DPPH and NO-scavenging activities. Related to togicological assays, BPIS inhibited δ-ALA-D and Na(+), K(+)-ATPase activities in rat brain homogenates and [(3)H]glutamate uptake in synaptosomes in vitro, but these effects were observed at higher concentrations than it had antioxidant effect (IC50 values of 16.41, 26.44 and 3.29 µM, respectively). In vivo, brains of mice treated with SNP (0.335 µmol per site; i.c.v.) showed an increase in LP and PC and a reduction in non protein thiol content, however, it was not observed significant alterations in antioxidant enzyme activities. BPIS (10 mg/kg; p.o.) protected against these alterations caused by SNP. In conclusion, the results demonstrated the antioxidant action of BPIS in in vitro assays. Furthermore, BPIS protected against oxidative damage caused by SNP in mouse brain, strengthening the potential antioxidant effect of this compound.

Effects of Silencing Heme Biosynthesis Enzymes on 5-Aminolevulinic Acid-mediated Protoporphyrin IX Fluorescence and Photodynamic Therapy.

Aminolevulinic acid (ALA)-mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA-mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA-mediated PDT. Silencing PBGS or PBGD significantly reduced ALA-stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA-stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA-stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA-PDT, while increased sensitivity to ALA-PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA-mediated PpIX fluorescence and PDT efficacy.

Assessment of ALA-induced PpIX production in porcine skin pretreated with microneedles.

Photodynamic therapy (PDT) is used for skin treatments of premalignant and cancer lesions and recognized as a non-invasive technique that combines tissue photosensitization and subsequent exposure to light to induce cell death. However, it is limited to the treatment of superficial lesions, mainly due to the low cream penetration. Therefore, the improvement of transdermal distribution of aminolevulinic acid (ALA) is needed. In this study, the kinetics and homogeneity of production of ALA-induced PpIX after the skin pre-treatment with microneedles rollers of 0.5, 1.0 and 1.5 mm length were investigated. An improvement in homogeneity and production of PpIX was shown in a porcine model. Widefield fluorescence imaging three hours after the topical application of ALA-cream in the combined treatment with microeedles rollers.

Chitosan-based mucoadhesive films containing 5-aminolevulinic acid for buccal cancer's treatment.

Photodynamic therapy (PDT) is a relatively new method to treat various kinds of tumors, including those of the oral cavity. The topical 5-ALA-PDT treatment for tumors of the oral mucosa is preferred, since when administered systemically, there is a general photosensitization drawback in the patient. However, 5-ALA is a hydrophilic molecule and its penetration and retention is limited by topical route, including oral mucosa. We propose a topical delivery system of chitosan-based mucoadhesive film, aiming to promote greater retention of 5-ALA in tissue. The chitosan (CHT) films (4% w/w) were prepared using the solvent evaporation/casting technique. They were tested without 5-ALA resulting in permeability to water vapor (W.V.P=2.15-8.54 g mm/(h cm(2)Pa) swelling ∼300.0% (±10.5) at 4 h or 24 h and in vitro residence time >24 h for all tests. CHT films containing 10.0% (w/w) 5-ALA have resulted in average weight of 0.22 g and thickness of 0.608 mm as suitable characteristics for oral application. In the presence of CHT films both in vitro permeation and retention of 5-ALA (1.0% or 10.0%) were increased. However, 10.0% 5-ALA presented highest values of permeation and retention (∼4 and 17 times respectively, compared to propylene glycol vehicle). On the other hand, in vitro mucoadhesion of CHT films was decreased (18.2-fold and 3.1-fold) by 5-ALA addition (1.0% or 10.0% respectively). However, CHT film containing 10.0% of 5-ALA can be a potential delivery system for topical use in the treatment of tumors of the oral cavity using PDT because it favored the retention of 5-ALA in this tissue and has shown convenient mucoadhesion.

Herbicide clomazone effects on δ-aminolevulinic acid activity and metabolic parameters in Cyprinus carpio.

The objective of this study was to investigate δ-aminolevulinic acid (δ-ALA-D) activity and metabolic parameters of Cyprinus carpio exposed to clomazone herbicide. Fish were exposed 2.5, 5, 10 and 20 mg L(-1) of clomazone for 192 h. Results indicated that δ-ALA-D activity was decreased in the gills at concentrations of 5 and 10 mg L(-1). Liver glycogen increased, while muscle and gill glycogen levels decreased at 5, 10 and 20 mg L(-1). Glucose was increased in the gills and plasma. Lactate decreased in the gills and liver and increased in the muscle. Protein and amino acids levels increased in the liver and gills and decreased in the muscle. At a clomazone concentration of 20 mg L(-1), ammonia increased in the gills and muscle and decreased in the liver. The results indicated that the metabolic parameters of glycogen, lactate, protein and amino acids in liver, muscle and gills, blood glucose levels, and the enzyme δ-ALA-D in gills may be useful indicators of clomazone toxicity in carp.

Effects of 5-aminolevulinic acid (ALA)-containing supernatants from selected Rhodopseudomonas palustris strains on rice growth under NaCl stress, with mediating effects on chlorophyll, photosynthetic electron transport and antioxidative enzymes

Background: Rice is globally one of the most important food crops, and NaCl stress is a key factor reducing rice yield. Amelioration of NaCl stress was assessed by determining the growth of rice seedlings treated with culture supernatants containing 5-aminolevulinic acid (ALA) secreted by strains of Rhodopseudomonas palustris (TN114 and PP803) and compared to the effects of synthetic ALA (positive control) and no ALA content (negative control). Results: The relative root growth of rice seedlings was determined under NaCl stress (50 mM NaCl), after 21 d of pretreatment. Pretreatments with 1 μM commercial ALA and 10X diluted culture supernatant of strain TN114 (2.57 μM ALA) gave significantly better growth than 10X diluted PP803 supernatant (2.11 μM ALA). Rice growth measured by dry weight under NaCl stress ordered the pretreatments as: commercial ALA N TN114 N PP803 N negative control. NaCl stress strongly decreased total chlorophyll of the plants that correlated with non-photochemical quenching of fluorescence (NPQ). The salt stress also strongly increased hydrogen peroxide (H2O2) concentration in NaCl-stressed plants. The pretreatments were ordered by reduction in H2O2 content under NaCl stress as: commercial ALA N TN114 N PP803 N negative control. The ALA pretreatments incurred remarkable increases of total chlorophyll and antioxidative activities of catalase (CAT), ascorbate peroxide (APx), glutathione reductase (GR) and superoxide dismutase (SOD); under NaCl stress commercial ALA and TN114 had generally stronger effects than PP803. Conclusions: The strain TN114 has potential as a plant growth stimulating bacterium that might enhance rice growth in saline paddy fields at a lower cost than commercial ALA.

Effect of photodynamic therapy on the skin using the ultrashort laser ablation.

Photodynamic Therapy (PDT) with 5-aminolevulinic acid (ALA) is known to be limited for applications in tumours of large volume mainly due to the limited penetration of topical photosensitization. The results show that micro-holes created using a femtosecond laser before PDT significantly increased the depth of PDT effect in the healthy tissue. The combination of ultrashort laser ablation technique with PDT showed an important scientific breakthrough related to transportation and delivery of drugs into the deeper regions of the tissue.

Effects of diphenyl diselenide and diphenyl ditellurite on chicken embryo development.

Studies of our group has demonstrated that (PhSe)2 plays some pharmacologic activities. In addition, it is possible that this compound would be an alternative source of organic selenium in animal foods. However, previous works showed that diphenyl diselenide (PhSe)2 and diphenyl ditelluride (PhTe)2 are toxic for mammals, but their undesirable effects were never tested in avian models. Then, the present study was carried to examine the possible teratogenic effects of (PhSe)2 and (PhTe)2 on chicken embryo development. The eggs were injected with (PhSe)2 at 0, 1 and 10 nmol or with (PhTe)2 at 4 nmol. The control was injected with 10 µl of soya bean oil (vehicle). In order to determine the possible toxic effect of these chemicals, we measure the embryo dimensions, the encephalon, heart and liver weight, thiobarturic acid reactive species (TBARS) level and the δ-aminonevulinate dehydratase (ALA-D) activity. (PhSe)2 and (PhTe)2 did not affect the embryo dimensions. Treatment with (PhSe)2 at 10 nmol per egg caused a increase on TBARS level and on ALA-D activity of the liver tissue, whereas (PhTe)2 decreased encephalon weight, had a tendency to increase to increase TBARS level but did not affect ALA-D activity. Taken together, these results indicate that (PhSe)2 and (PhTe)2 are slightly toxic for chicken embryos. Furthermore, (PhTe)2 caused a decrease in encephalon, which indicates its neurotoxicity. Finally, these results indicate that (PhTe)2 seems not be promissory for therapeutic applications, whereas (PhSe)2 could be of clinical and/or nutritional concern, which will be target for further researches.

Responses of hepatocytes to DDT and methyl mercury exposure.

The aim of the current work was to investigate the effects of dichlorodiphenyltrichloroethane (DDT) and monomethyl mercury (MeHg) on hepatocytes from tropical fish Hypostomus commersoni (cascudo). In order to verify DDT and MeHg impacts on the redox milieu, cells were exposed for 4 days to 50 nM of DDT, 0.25 and 2.5 microM of MeHg and to a combination of 50 nM of DDT and 0.25 microM of MeHg. These concentrations were compared with those previously published (Filipak Neto et al., 2008) for the predator fish Hoplias malabaricus (traíra). The effects were mostly noticeable on reduced glutathione concentration and delta-aminolevulinic acid dehydratase and glutathione S-transferase activity. Catalase activity increased in the group exposed to 2.5 microM of MeHg and hydrogen peroxide levels decreased in all exposed groups. Also, superoxide anion levels decreased in the groups exposed to 2.5 microM of MeHg and DDT *MeHg group. Cell viability decreased only in the DDT exposed group, demonstrating that the antioxidant defense mechanism of H. commersoni hepatocytes is more efficient than H. malabaricus. These results corroborate the resistance of H. commersoni to polluted areas and support the hypothesis that this species is more resistant to DDT and MeHg than H. malabaricus species.

Association between diphenyl diselenide and cadmium chloride attenuates the toxicity of both in tissues of mice in vitro.

The in vitro effect of association between cadmium chloride (CdCl(2)) and diphenyl diselenide (PhSe)(2) was investigated. Mouse liver, kidney and testes were used to evaluate delta-aminolevulinate dehydratase (delta-ALA-D) activity and thiobarbituric acid-reactive substances (TBARS) levels. CdCl(2) and (PhSe)(2), at different concentrations, caused an inhibition in delta-ALA-D activity of liver, kidney and testes. The inhibitory effect of (PhSe)(2) in hepatic and renal delta-ALA-D activity was attenuated by interaction of this compound with CdCl(2) at non-inhibitory concentration. Dithiothreitol (DTT), but not ZnCl(2), restored the inhibition observed in delta-ALA-D activity in all tissues. This suggests an involvement of sulfhydryl groups in delta-ALA-D inhibition. TBARS levels were increased by CdCl(2) in all tissues and (PhSe)(2) was able to abolish this increase. The results demonstrate that the association between CdCl(2) and (PhSe)(2) attenuates the toxicity of both compounds and the possible mechanisms of this protection are due to complex formation between cadmium and selenium and involves the antioxidant activity of (PhSe)(2).

Porphyrin synthesis from aminolevulinic acid esters in endothelial cells and its role in photodynamic therapy.

Photodynamic therapy (PDT) may cause tumour cell destruction by direct toxicity or by inducing microcirculatory shutdown. Protoporphyrin IX generated from 5-aminolevulinic acid (ALA) has been widely used as an endogenous photosensitiser in PDT. However, the hydrophilic nature of the ALA molecule limits its penetration through the stratum corneum of the skin and cell membranes and thus, ALA alkyl-esters have been developed to improve ALA permeation. The aim of this work was to study Protoporphyrin IX synthesis from ALA and its derivatives ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA) in the microvascular endothelial cell line HMEC-1 derived from normal skin, and to evaluate their response to PDT. We found that lower light doses are required to photosensitise HMEC-1 endothelial cells than to photosensitise PAM212 transformed keratinocytes, showing some possible selectivity of ALA-PDT for vascularisation in skin. Employed at concentrations leading to equal Protoporphyrin IX synthesis, ALA, He-ALA and Me-ALA presented the same efficacy of HMEC-1 photosensitisation. However, He-ALA was a promising compound for the use in the enhancement of Protoporphyrin IX in HMEC-1 cells employed at low concentrations at both short and long time exposures whereas Me-ALA should be employed at high concentrations and longer time periods in order to surpass the Protoporphyrin IX levels obtained with ALA. The advantage of Me-ALA over ALA was based on its lower dark toxicity. This is the first work to report vascular cell photosensitisation employing alkyl-esters of ALA, and we demonstrated that these derivatives could exert the same effect as ALA and under certain conditions enhance photosensitisation of vasculature.

Sustained and efficient porphyrin generation in vivo using dendrimer conjugates of 5-ALA for photodynamic therapy.

The use of endogenous protoporphyrin IX (PpIX) after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). However the efficacy of ALA-PDT is sub-optimal for thicker tumours and improved ALA delivery and therapeutic response are required. We have investigated the conjugation of ALA to a second-generation dxcendrimer for enhancing porphyrin synthesis in vitro and in vivo in a murine tumour model using systemic i.p. administration. In vitro, the dendrimer was more efficient than ALA for porphyrin synthesis at low concentrations in good correlation with higher cellular ALA dendrimer accumulation. In vivo, the porphyrin kinetics from ALA exhibited an early peak between 3 and 4 h in most tissues, whereas the dendrimer induced sustained porphyrin production for over 24 h and basal values were not reached until 48 h after administration. Integrated porphyrin accumulation from the dendrimer and ALA, at equivalent molar ratios, was comparable showing that the majority of ALA residues were liberated from the dendrimer. The porphyrin kinetics appear to be governed by the rate of enzymatic cleavage of ALA from the dendrimer, which is consistent with in vitro results. ALA dendrimers may be useful for metronomic PDT, and multiple low-dose ALA-PDT treatments.

Cadmium induced oxidative stress in soybean plants also by the accumulation of delta-aminolevulinic acid.

Cadmium toxicity has been extensively studied in plants, however its biochemical mechanism of action has not yet been well established. To fulfil this objective, four-weeks-old soybean nodulated plants were treated with 200 muM Cd(2+) for 48 h. delta-aminolevulinic acid dehydratase (ALA-D, E.C. 4.2.1.24) activity and protein expression, as well as delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) concentrations were determined in nodules, roots and leaves. In vitro experiments carried out in leaves were performed using leaf discs to evaluate the oxidant and antioxidant properties of ALA and S-adenosyl-L: -methinone (SAM), respectively. Oxidative stress parameters such as thiobarbituric acid reactive substances (TBARS) and GSH levels as well as superoxide dismutase (SOD, E.C. 1.15.1.1), and guaiacol peroxidase (GPOX, E.C. 1.11.1.7) were also determined. Cadmium treatment caused 100% inhibition of ALA-D activity in roots and leaves, and 72% inhibition in nodules whereas protein expression remained unaltered in the three studied tissues. Plants accumulated ALA in nodules (46%), roots (2.5-fold) and leaves (104%), respect to controls. From in vitro experiments using leaf discs, exposed to ALA or Cd(2+), it was found that TBARS levels were enhanced, while GSH content and SOD and GPOX activities and expressions were diminished. The protective role of SAM against oxidative stress generated by Cd(2+) and ALA was also demonstrated. Data presented in this paper let us to suggest that accumulation of ALA in nodules, roots and leaves of soybean plants due to treatment with Cd(2+) is highly responsible for oxidative stress generation in these tissues.