Regulation of endothelial heme oxygenase activity during hypoxia is dependent on chelatable iron. (41/636)

The regulation of heme oxygenase (HO) activity and its dependence on iron was studied in bovine aortic endothelial cells (BAEC) subjected to hypoxia-reoxygenation (H/R). HO activity was induced by hypoxia (10 h) and continued to increase during the reoxygenation phase. HO-1 protein levels were strongly induced by hypoxia from undetectable levels and remained elevated at least 8 h postreoxygenation. Addition of the Fe(3+) chelator desferrioxamine mesylate (DFO) or the Fe(2+) chelator o-phenanthroline during hypoxia alone or during the entire H/R period inhibited the induction of HO activity and HO-1 protein levels. However, DFO had no effect and o-phenanthroline had a partial inhibitory effect on HO activity and protein levels when added only during reoxygenation. Loading of BAEC with Fe(3+) enhanced the activation of the HO-1 gene by H/R, whereas loading with L-aminolevulinic acid, which stimulates heme synthesis, had little effect. These results suggest that chelatable iron participates in regulating HO expression during hypoxia.  (+info)

A mouse model of familial porphyria cutanea tarda. (42/636)

Approximately one-third of patients with porphyria cutanea tarda (PCT), the most common porphyria in humans, inherit a single mutant allele of the uroporphyrinogen decarboxylase (URO-D) gene. PCT associated with URO-D mutations is designated familial PCT. The phenotype is characterized by a photosensitive dermatosis with hepatic accumulation and urinary excretion of uroporphyrin and hepta-carboxylic porphyrins. Most heterozygotes for URO-D mutations do not express a porphyric phenotype unless hepatic siderosis is present. Hemochromatosis gene (HFE) mutations are frequently found when the phenotype is expressed. We used homologous recombination to disrupt one allele of murine URO-D. URO-D(+/-) mice had half-wild type (wt) URO-D protein and enzymatic activity in all tissues but did not accumulate hepatic porphyrins, indicating that half-normal URO-D activity is not rate limiting. When URO-D(+/-) mice were injected with iron-dextran and given drinking water containing delta-aminolevulinic acid for 21 days, hepatic porphyrins accumulated, and hepatic URO-D activity was reduced to 20% of wt. We bred mice homozygous for an HFE gene disruption (HFE(-/-)) to URO-D(+/-) mice, generating mice with the URO-D(+/-)/HFE(-/-) genotype. These animals developed a porphyric phenotype by 14 weeks of age without ALA supplementation, and URO-D activity was reduced to 14% of wt. These data indicate that iron overload alone is sufficient to reduce URO-D activity to rate-limiting levels in URO-D(+/-) mice. The URO-D(+/-) mouse serves as an excellent model of familial PCT and affords the opportunity to define the mechanism by which iron influences URO-D activity.  (+info)

Photodynamic diagnosis of breast tumours after oral application of aminolevulinic acid. (43/636)

Photodynamic diagnosis is of increasing interest for diagnosis in oncology. It is based on a more intense incorporation of a fluorescent dye in tumours compared to normal tissue. As a feasibility study we investigated the effectiveness of oral application of 5-aminolevulinic acid for photodynamic diagnosis of human primary mammary tumours. The study included 16 patients with palpable breast tumours. Aminolevulinic acid was administered at a concentration of 40 mg kg(-1)bodyweight 150-420 min prior to tumourectomy. Intraoperatively blue light (405 nm) was applied to the operation site. Sections of the excised tumour and some lymph nodes were prepared and analysed with a fluorescent microscope. All primary mammary tumour tissues showed significantly higher fluorescence intensity than surrounding normal mammary tissue. Fluorescence of the mammary tumours could also be discriminated macroscopically and intraoperatively. Fluorescence intensity in nonmetastatic lymph node tissue was higher in 2 out of 3 patients than in primary tumour tissue. By photodynamic diagnosis using aminolevulinic acid we were able to reliably distinguish primary mammary tumours from normal mammary tissue microscopically and macroscopically in all our patients. We suggest that photodynamic diagnosis with aminolevulinic acid for breast tumours should be further investigated and developed for intraoperative use and may well be a simple tool for better intraoperative diagnosis and recognition of tumour margins. We hypothesize that lymph node metastasis of breast tumours will not be detectable by this method.  (+info)

The degradative effects of porphyrins and heme compounds on components of the microsomal mixed function oxidase system. (44/636)

The effects of in vitro treatment of the hepatic microsomal fraction with various porphyrin compounds on the activity and the content of the heme-containing components of the mixed function oxidase system were studied. The compounds examined were hematin, methemalbumin (with heme to protein molar ratio of 13:1 or 1:1), mesohemalbumin, bilirubin, biliverdin, mesoporphyrin IX, and protoprophyrin IX. The activity of the system was monitored by measuring its oxidative activity for the type I and type II substrates, ethylmorphine and aniline, respectively; as well as the microsomal contents of cytochrome P-450 and b5 and 14C-labeled heme, Mesoporphyrin IX was found to be most effective in inhibiting the oxidative activity of the mixed function oxidase system as well as in decreasing the microsomal contents of cytochromes P-450, b5, and heme. Biliverdin exerted no effect on these parameters. Hematin and the other compounds studied exerted variable inhibitory effects on the system. The degradative and inhibitory effects of protoporphvrin IX and mesoporphyrin IS could be blocked significantly by conducting the studies in the dark. The presence of biliverdin decreased the inhibitory effects of the porphyrins on the system; conversely the effects could be magnified in the presence of deuterium oxide. It is suggested that the mechanism by which porphyrins inhibit the mixed function oxidase system is through porphyrin-sensitized photo-oxidation of various constituents of the hepatic microsomal fraction and that the formation of singlet oxygen molecules is most likely involved in this process. Moreover the destructive effects of heme compounds on the microsomal components and activities of the drug-metabolizing mixed function oxidase system raise questions concerning the hypothesis that the components of this system, and specifically cytochrome P-450, are involved in the activity of the heme oxygenase system.  (+info)

Endoscopic fluorescence detection of low and high grade dysplasia in Barrett's oesophagus using systemic or local 5-aminolaevulinic acid sensitisation. (45/636)

BACKGROUND AND AIMS: Barrett's oesophagus is associated with an increased risk of cancer. As dysplasia is not visible during routine endoscopy, random biopsies in the four quadrants every 1-2 cm are recommended. Endoscopic fluorescence detection (EFD) after sensitisation with 5-aminolaevulinic acid (5-ALA) with different modes and concentrations was assessed to optimise the technique for detection of dysplasia or early cancers. 5-ALA is converted intracellularly to protoporphyrin IX which accumulates in malignant tissue and can be detected by typical red fluorescence after illumination with blue light. METHODS: In 47 patients with Barrett's oesophagus, 10 with known dysplasia, 58 fluorescence endoscopies were performed after sensitisation with different concentrations of 5-ALA given orally (5, 10, 20, 30 mg/kg) or locally (500-1000 mg) by spraying the mucosa via a catheter. EFD was performed 4-6 hours after systemic and 1-2 hours after local sensitisation using a special light source delivering white or blue light. A total of 243 biopsies of red fluorescent (n=113) and non-fluorescent areas (n=130) were taken. RESULTS: In three patients, two early cancers and dysplasia, not visible during routine endoscopy, were detected by EFD. Thirty three biopsies revealed either low or high grade dysplasia. Sensitivity for detection of dysplastic lesions ranged from 60% after local sensitisation with 500 mg to 80%, 100%, and 100% after systemic application of 5-ALA 10, 20, and 30 mg/kg, respectively. However, specificity was best for local sensitisation (70%) while systemic administration revealed values between 27% and 56%. Using 5 mg/kg, no red fluorescence in dysplastic lesions was found. No severe side effects were noted. CONCLUSION: EFD is a promising tool to detect non-visible dysplastic lesions in Barrett's oesophagus using 5-ALA sensitisation. A randomised controlled study is now indicated to compare the efficacy of EFD with the standard technique of four quadrant random biopsies.  (+info)

Immunological and viral factors associated with the response of vulval intraepithelial neoplasia to photodynamic therapy. (46/636)

Topical 5-aminolevulinic acid-based photodynamic therapy (PDT) has produced complete response rates of >90% for nonmelanoma skin carcinomas, which are mostly human papillomavirus (HPV) negative. Using a similar treatment protocol, we observed a short-term response in only one third (10 of 32) of high-grade vulval intraepithelial neoplasia (VIN 2-3) lesions. Unifocal lesions were found more responsive than multifocal and pigmented lesions. Animal model studies have suggested that long-term PDT response involves an immune reaction in which CTLs play a crucial role. In this study, we have assessed: (a) HPV infection; (b) HLA expression; and (c) immune infiltrating cells in VIN biopsies from responders and nonresponders to determine whether these factors may limit response to topical 5-aminolevulinic acid-based PDT. Tissues from normal vulva (n = 9), vulval carcinoma (n = 11), and VIN (32 patients from which 19 pre- and 43 post-PDT biopsies were taken) were investigated for immune cell infiltration and HLA class I expression by immunohistochemistry and HPV infection by PCR. There was a greater likelihood of HPV positivity associated with a lack of response of VIN to PDT (P = 0.002), and VIN nonresponders were more likely to show HLA class I loss compared with responders (P = 0.030). HLA class I down-regulation was significantly greater in the carcinomas (82%, total loss) than the VIN (28%, 19%, total loss; and 9%, allele loss; P = 0.004). None of the cases with class I down-regulation responded to PDT, whereas 3 of 6 (50%) of cases that showed total class I loss subsequently developed superficial invasion. Compared with normal vulval skin, VIN lesions showed increased infiltration by CD4 (T-helper) and CD68 (macrophages) but not CD1a (Langerhans cells) or CD8 (CTLs). There was, however, a significant increase of CD8 infiltration in posttreatment VIN responders compared with nonresponders (P = 0.0001). These data clearly support the contention that high-risk HPV infection and lack of cell-mediated immunity may play a role in the observed poor response of lower genital lesions to topical PDT.  (+info)

Expression of coproporphyrinogen oxidase and synthesis of hemoglobin in human erythroleukemia K562 cells. (47/636)

Coproporphyrinogen oxidase (CPOX), the sixth enzyme in the heme-biosynthetic pathway, catalyzes oxidative decarboxylation of coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA-transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with delta-aminolevulinic acid (ALA), most became benzidine-positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA-treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme-biosynthetic enzymes, including CPOX, ferrochelatase and erythroid-specific delta-aminolevulinic acid synthase, as well as of alpha-globin synthesis, were observed when cells were treated with transforming growth factor (TGF)beta 1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGF beta 1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGF beta 1-treated K562 cells led to a decrease in heme production compared with sense oligonucleotide-transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.  (+info)

Determination of 5-aminolevulinic acid (ALA) by HPLC method. (48/636)

5-Aminolevulinic acid is at present most popular precursor of photosensitizer for photodynamic therapy and diagnostics. The effect of accumulation of PpIX induced by exogenous ALA is based on the avoidance of feedback control in the pathway of heme biosynthesis. For use ALA in medicine, development of analytical methods that will enable precise determination of content and purity is necessary. The ALA content is determined i. a. by titration with perchloric acid. Much greater problems are connected with purity determination by HPLC method. There are rather scarce literature data concerning purity determination of ALA by HPLC.  (+info)