dpp genes of Rhizobium leguminosarum specify uptake of delta-aminolevulinic acid. (73/636)

An operon with homology to the dppABCDF genes required to transport dipeptides in bacteria was identified in the N2-fixing symbiont, Rhizobium leguminosarum. As in other bacteria, dpp mutants were severely affected in the import of delta-aminolevulinic acid (ALA), a heme precursor. ALA uptake was antagonized by adding dipeptides, indicating that these two classes of molecule share the same transporter. Mutations in dppABCDF did not affect symbiotic N2 fixation on peas, suggesting that the ALA needed for heme synthesis is not supplied by the plant or that another uptake system functions in the bacteroids. The dppABCDF operon of R. leguminosarum resembles that in other bacteria, with a gap between dppA and dppB containing inverted repeats that may stabilize mRNA and may explain why transcription of dppA alone was higher than that of dppBCDF. The dppABCDF promoter was mapped and is most likely recognized by sigma70.  (+info)

Oxygen-mediated regulation of porphobilinogen formation in Rhodobacter capsulatus. (74/636)

A Rhodobacter capsulatus hemC mutant has been isolated and used to show that oxygen regulates the intracellular levels of porphobilinogen. Experiments using a hemB-cat gene fusion demonstrated that oxygen does not transcriptionally regulate hemB transcription. Porphobilinogen synthase activity is not regulated by oxygen nor is the enzyme feedback inhibited by hemin or protoporphyrin IX. It was demonstrated that less than 20% of [(14)C]aminolevulinate was incorporated into bacteriochlorophyll, suggesting that the majority of the aminolevulinate is diverted from the common tetrapyrrole pathway. Porphobilinogen oxygenase activity was not observed in this organism; however, an NADPH-linked aminolevulinate dehydrogenase activity was demonstrated. The specific activity of this enzyme increased with increasing oxygen tension. The results presented here suggest that carbon flow over the common tetrapyrrole pathway is regulated by a combination of feedback inhibition of aminolevulinate synthase and diversion of aminolevulinate from the pathway by aminolevulinate dehydrogenase.  (+info)

ALA and ALA hexyl ester in free and liposomal formulations for the photosensitisation of tumour organ cultures. (75/636)

In spite of the wide range of tumours successfully treated with 5-aminolevulinic acid mediated photodynamic therapy, the fact that 5-aminolevulinic acid has low lipid solubility, limits its clinical application. More lipophilic 5-aminolevulinic acid prodrugs and the use of liposomal carriers are two approaches aimed at improving 5-aminolevulinic acid transmembrane access. In this study we used both 5-aminolevulinic acid and its hexyl ester in their free and encapsulated formulations to compare their corresponding endogenous synthesis of porphyrins. Employing murine tumour cultures, we found that neither the use of hexyl ester nor the entrapment of either 5-aminolevulinic acid or hexyl ester into liposomes increase the rate of tumour porphyrin synthesis. By light and electronic microscopy it was demonstrated that exposure of tumour explants to either free or liposomal 5-aminolevulinic acid and subsequent illumination induces the same type of subcellular damage. Mitochondria, endoplasmic reticulum and plasma membrane are the structures mostly injured in the early steps of photodynamic treatment. In a later stage, cytoplasmic and nuclear disintegration are observed. By electronic microscopy the involvement of the endocytic pathway in the incorporation of liposomal 5-aminolevulinic acid into the cells was shown.  (+info)

Delta-aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines. (76/636)

BACKGROUND: Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, delta-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. RESULTS: We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.  (+info)

The role of reperfusion injury in photodynamic therapy with 5-aminolaevulinic acid--a study on normal rat colon. (77/636)

Reperfusion injury can occur when blood flow is restored after a transient period of ischaemia. The resulting cascade of reactive oxygen species damages tissue. This mechanism may contribute to the tissue damage produced by 5-aminolaevulinic acid-induced photodynamic therapy, if this treatment temporarily depletes oxygen in an area that is subsequently reoxygenated. This was investigated in the normal colon of female Wistar rats. All animals received 200 mg kg(-1) 5-aminolaevulinic acid intravenously 2 h prior to 25 J (100 mW) of 628 nm light, which was delivered continuously or fractionated (5 J/150 second dark interval/20 J). Animals were recovered following surgery, killed 3 days later and the photodynamic therapy lesion measured macroscopically. The effects of reperfusion injury were removed from the experiments either through the administration of free radical scavengers (superoxide dismutase (10 mg kg(-1)) and catalase (7.5 mg kg(-1)) in combination) or allopurinol (an inhibitor of xanthine oxidase (50 mg kg(-1))). Prior administration of the free radical scavengers and allopurinol abolished the macroscopic damage produced by 5-aminolaevulinic acid photodynamic therapy in this model, regardless of the light regime employed. As the specific inhibitor of xanthine oxidase (allopurinol) protected against photodynamic therapy damage, it is concluded that reperfusion injury is involved in the mechanism of photodynamic therapy in the rat colon.  (+info)

Aminolaevulinic acid-induced photodynamic therapy: cellular responses to glucose starvation. (78/636)

Photodynamic therapy is a cancer treatment based on the interaction of light, oxygen and a photosensitiser. Protoporphyrin. IX is an endogenous photosensitiser derived from the pro-drug aminolaevulinic acid. Tumours contain areas of hypoxia and hypoglycaemia. Tumour cells adapt to these conditions by stress protein induction which may induce resistance to cancer therapies. The effect of chronic hypoglycaemia on sensitivity to aminolaevulinic acid-induced photodynamic therapy in vitro was studied in MCF-7, human breast cancer cells. Following chronic exposure to 0, 1 or 25 mM, glucose, cells were treated with aminolaevulinic acid and the generation of intracellular protoporphyrin. IX measured by spectrofluorimetry. Aminolaevulinic acid-induced photodynamic therapy sensitivity was compared between cells following chronic exposure to 0, 1 or 25 mM glucose. Percentage cell survival was determined by clonogenic assay. Cells cultured in low glucose generated higher levels of protoporphyrin IX compared to standard glucose medium (0 mM glucose: 0.88 x 10(-5) ng cell(-1), 1 mM: 0.86 x 10(-5) ng cell(-1), 25 mM: 0.60 5x 10(-5) ng cell(-1), P<0.05). However, photodynamic therapy sensitivity was reduced in glucose deprived cells (0 mM glucose: 61% survival, 1 mM: 80.5% and 25 mM: 39.6%, P<0.05). Chronic exposure to low glucose induces photodynamic therapy resistance despite increased intracellular concentrations of protoporphyrin IX and may reflect cellular adaptation to chronic glucose deprivation.  (+info)

Clinical pharmacokinetics of 5-aminolevulinic acid in healthy volunteers and patients at high risk for recurrent bladder cancer. (79/636)

5-Aminolevulinic acid (ALA) is a precursor of protoporphyrin IX (PpIX) that is being evaluated for use in photodiagnosis and phototherapy of malignant and nonmalignant disorders. Previous clinical studies using topical, oral, and intravesical administration have been conducted in attempts to determine the optimal route of administration for ALA. The purpose of these studies was to examine the systemic pharmacokinetics and elimination of ALA, the bioavailability of ALA after oral and intravesical doses, and the factors that affect ALA concentrations in the bladder during intravesical treatment. The disposition of ALA was evaluated in six healthy volunteers receiving single intravenous and oral doses (100 mg) and eight patients at high risk for recurrent bladder cancer receiving an intravesical dose (1.328 g) of ALA. The mean (+/-S.D.) plasma area under the plasma concentration-time curve from time 0 to infinity of PpIX (0.20 +/- 0.11 microg small middle dot h/ml) after intravenous administration of ALA was not significantly different from that observed after oral administration of ALA (0.15 +/- 0.11 microg*h/ml; P = 0.49). ALA terminal half-life was approximately 45 min after intravenous or oral administration. The oral bioavailability of ALA was approximately 60%. After intravesical administration, urine production was largely responsible for decreases in ALA concentration in the bladder, with less than 1% being absorbed into the systemic circulation. In summary, oral and intravenous administration of ALA at these doses results in modest plasma levels of PpIX. Regional administration (i.e., intravesical) of ALA resulted in a significant pharmacokinetic advantage, with urinary bladder being exposed to concentrations approximately 20,000-fold higher than systemic circulation.  (+info)

Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms. (80/636)

Degradation of intrinsic hepatic [(14)C]haem was analysed as (14)CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16h after pulse-labelling with 5-amino[5-(14)C]laevulinic acid, a precursor that labels bridge carbons of haem in non-erythroid tissues. NADPH-catalysed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [(14)C]haem (largely cytochrome P-450 haem), but little (14)CO formation. No additional (14)CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [(14)C]haem. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl(2) or phenyl-t-butylnitrone(a spin-trapping agent for free radicals), it was concluded that carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide, may promote loss of cytochrome P-450 haem through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic haem, catabolism of [(14)C]methaemalbumin by microsomal haem oxygenase in vitro produced equimolar quantities of (14)CO and bilirubin, although these catabolites reflected only 18% of the degraded [(14)C]haem. This value was increased to 100% by addition of MnCl(2), which suggests that lipid peroxidation may be involved in degradation of exogenous haem to products other than CO. Phenyl-t-butylnitrone completely blocked haem oxygenase activity, which suggests that hydroxy free radicals may represent a species of active oxygen used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [(14)C]haem was decreased and haem oxygenase activity was unchanged; however, (14)CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [(14)C]haem and (14)CO excretion, one may infer that an important fraction of hepatic [(14)C]haem in normal rats is degraded by endogenous pathways not involving CO. We conclude that carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 haem through mechanisms that do not yield CO as an end product, and that are insensitive to cycloheximide and independent of haem oxygenase activity.  (+info)