Tumor localization of newly developed hematoporphyrin (DHP) using a bladder tumor model: a novel hematoporphyrin derivative

Application of laser irradiation with porphyrin(s) or their derivatives for the destruction of tumors in humans requires preliminary studies of their localization in normal and malignant tissues. A novel derivative of hematoporphyrin (HP) was prepared. The newly developed hematoporphyrin (DHP) was administered to Fisher rats with bladder tumors and showed greater accumulation in the tumoral tissues. Comparative data on (HP) and (DHP) are presented and discussed in light of the enhanced tumor porphyrin uptake caused by these agents. The homogeneous intense fluorescence noted with DHP-treated animals suggests that total tumor kill curative therapy will be more feasible. The study paves the way to refining increased porphyrin augment phototherapy and laser application in the field of oncology.     

The effect on photohaemolysis of variation in the structure of the porphyrin photosensitizer.

A comparison of the photosensitizing ability of a variety of porphyrins for photohaemolysis gives the following order of activity: protoporphyrin greater than deuteroporphyrin, mesoporphyrin, haematoporphyrin dimethyl ester much greater than haematoporphyrin diacetate, haematoporphyrin greater than haematoporphyrin monoacetate, coproporphyrin III, haematoporphyrin derivative, coproporphyrin III tetramethyl ester greater than uroporphyrin I, meso-tetra-(N-methyl-4-pyridinium)porphyrin tetratoluene-p-sulphonate, meso-tetra-(p-carboxyphenyl)porphyrin, protoporphyrin dimethyl ester, meso-tetra-(p-hydroxy-sulphonylphenyl)porphyrin tetrasodium salt, uroporphyrin III, deuteroporphyrin-3,8-disulphonic acid and protohaemin. The results for the metal-free porphyrins are rationalized in terms of solubility and partition properties, and a model is proposed for the incorporation of amphipathic porphyrins into the membrane lipid bilayer. Experiments with erythrocytes from patients with erythropoeitic protoporphyria and with normal erythrocytes to which porphyrin was added in a deuterium oxide medium do not lead to an increase in the rate of photohaemolysis. A possible explanation for this somewhat surprising observation is outlined.     

The one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: an electron spin resonance investigation

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, delta H = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III greater than coproporphyrin I greater than deuteroporphyrin IX greater than mesoporphyrin IX greater than Photofrin II greater than protoporphyrin IX greater than uroporphyrin I greater than uroporphyrin III greater than hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration.     

The one-electron reduction of uroporphyrin I by rat hepatic microsomes

Uroporphyrin I, which accumulates in body tissues of congenital erythropoietic porphyria patients, can undergo an enzymatic one-electron reduction to the porphyrin anion radical when a suitable reducing cofactor is present. We have demonstrated, in the absence of light, that anaerobic microsomal incubations containing NADPH and uroporphyrin I give an electron spin resonance spectrum consistent with the enzymatic formation of a porphyrin anion free radical. This radical undergoes a second-order decay (k2 approximately 10(5) M-1 s-1) due to nonenzymatic disproportionation of the radical. Aerobic microsomal incubations were also investigated for the reduction of oxygen to superoxide by monitoring oxygen consumption and the spin-trapping of superoxide. These experiments demonstrate that electron transfer from the porphyrin radical to molecular oxygen does occur, but due to the slow formation of the radical anion, no oxygen consumption above the basal level could be detected in the microsomal incubations. The photoreduction of uroporphyrin I in aerobic and anaerobic incubations was also investigated.     

Partition of porphyrins between cyclohexanone and aqueous sodium acetate as a function of pH. Determination of uroporphyrin and of hydrophilic porphyrin conjugates

1. The partition of uroporphyrins I and III, coproporphyrins I and III, haematoporphyrin IX, porphyrin c and a hydrophilic porphyrin–peptide fraction from variegate-porphyria faeces has been studied in systems of equal volumes of cyclohexanone and sodium acetate buffers of varying pH and concentration. 2. The concentration of acetate in the aqueous phase has little effect on the partition of porphyrin c, but markedly influences that of uroporphyrin. At 50% acetate saturation and pH4·5, only 5% enters the cyclohexanone phase whereas 60% of porphyrin c is extracted under similar conditions. 3. This circumstance forms the basis of a method for the determination of hydrophilic porphyrin–peptides in variegate-porphyria urine. Its reliability has been checked in model experiments. 4. At pH1·5 and an aqueous phase half-saturated with sodium acetate, an equal volume of cyclohexanone removes 95–97% of uroporphyrin and about 55% of porphyrin c. Uroporphyrin may therefore be determined as a second step in the method. 5. For the routine determination of uroporphyrin in systems free from other hydrophilic porphyrins, cyclohexanone extraction may be performed at any pH in the range 1·0–3·0.     

Modulation of porphyrin binding to serum albumin by pH

In this study, we show that the difference in acidity of functional groups in porphyrin photosensitizers provides a meaningful avenue to achieve differential localization and retention of porphyrins in tissues and cells, and in the end could be a positive factor in the photodynamic treatment of cancer (PDT). We have demonstrated that meso-tetraphenylporphyrin derivative with four phosphonate (bond P(double bond O)(bond OH)(2)) moieties exists in aqueous solutions mainly in four forms that differ by a degree of protonation of the porphyrin ring and ionization of the phosphonate group. It is shown that each porphyrin form has different affinities toward the model protein (bovine serum albumin, BSA). Thus pH of the medium significantly modulates the affinity of the phosphonate porphyrin toward BSA. At lower pH (pH 6.0), the phosphonate porphyrin and BSA form a complex with affinity constant of K(b)=6.9 x 10(5) M(-1), while at pH 7.0 the K(b)=6.1 x 10(5) M(-1). At pH 8.0 the association is significantly lower. Because cancerous cells have generally lower pH (pH approximately 6.9) compared to healthy cells (pH approximately 7.4), the pH of such cells could be a decisive factor for cellular retention of the porphyrin in the form of an associate with intracellular proteins. Moreover, we have also demonstrated that the protonation/deprotonation equilibria do not negatively affect the photophysical properties or ability of phosphonate porphyrin to generate singlet oxygen.     

Export of a heterologous cytochrome P450 (CYP105D1) in Escherichia coli is associated with periplasmic accumulation of uroporphyrin

This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid. A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E. coli. Its progressive induction in the presence of 5-aminolevulinic acid-supplemented medium was accompanied by an accumulation of a greater than 100-fold higher amount of uroporphyrin I in the periplasm relative to cells lacking CYP105D1. Expression of a cytoplasm-resident engineered CYP105D1 at a comparative level to the secreted form was far less effective in promoting porphyrin accumulation in the periplasm. Expression at a 10-fold molar excess over the exported CYP105D1 of another periplasmically exported hemoprotein, the globular core of cytochrome b5, did not substitute the role of the periplasmically localized CYP105D1 in promoting porphyrin production. This, therefore, eliminated the possibility that uroporphyrin accumulation is merely a result of increased hemoprotein synthesis. Moreover, in the strain that secreted CYP105D1, uroporphyrin production was considerably reduced by azole-based P450 inhibitors. Production of both holo-CYP105D1 and uroporphyrin was dependent upon 5-aminolevulinic acid, except that at higher concentrations this resulted in a decrease in uroporphyrin. This study suggests that the exported CYP105D1 oxidatively catalyzes periplasmic conversion of uroporphyrinogen I to uroporphyrin I in E. coli. The findings have significant implications in the ontogenesis of human uroporphyria-related diseases.     

Tumour localization of uroporphyrin isomers I and III and their correlation to albumin and serum protein binding

We were the first to report the superiority of uroporphyrin I (UROP I) as a tumour localizer when compared to haematoporphyrin derivative (HPD). In this study, we compared both isomers of UROP, i.e. I and III, in a KHJJ mammary carcinoma mouse model. Six and 18 h after UROP administration, the tumour, skin and gut porphyrin (P) content was quantitated. Tumour UROP I levels were always at least 50% higher than UROP III in tumour, whereas both isomers were barely detectable in the skin and gastrointestinal tract. We then explored the possibility that tumour P uptake might relate in part to the affinity of circulating P to mouse serum proteins (MSP), in particular, the major binding protein constituent, albumin. Copro-P III, deutero-P 2,4 disulphonic acid (DP), proto-P IX (PP) and heptacarboxylic P I (Hepta I) which in our mouse tumour model do not localize in malignant tissue, were compared to UROP I and III. The P was mixed with 0.775 microM human serum albumin (HSA) at different molar ratios (HSA:P range 2-8) and the unbound P concentration quantitated using an Amicon CF-25 membrane cone with centrifugation. The percentage free P was significantly higher for UROP I (92-98%) than III (82-95%) and significantly more than that observed with non-tumour localizing P studied. Similar data were obtained with MSP. This is consistent with the notion that enhanced uptake and retention (particularly UROP I) by malignant neoplastic tissue might reflect a higher affinity for UROP by tumour constituents than by circulating proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Manganese porphyrin reduces renal injury and mitochondrial damage during ischemia/reperfusion

Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R-related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant, Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP(5+)), using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP(5+) in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 h protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 microg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified the ATP synthase-beta subunit as a key protein induced by MnTnHex-2-PyP(5+) treatment alone and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP(5+) protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.     

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site, proved to be the preferential one, being 6.50 x l0(5), 1.94 x 10(6) and 8.94 x 10(5), respectively. In addition, it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions.     

High-performance liquid chromatography of porphyrin esters: Identification of mixed esters generated in sample preparation

Uroporphyrin and its naturally occurring decarboxylated derivatives can be conveniently separated, identified, and quantified chromatographically after formation of their methyl esters. Use of high-performance liquid chromatography has revealed the presence of previously unobserved components in such mixtures. One of these components was isolated and identified as uroporphyrin heptamethyl monoethyl ester. The family of uroporphyrin methylethyl esters was synthesized and its chromatographic behavior investigated. The quantity of ethanol present in solvents, e.g., chloroform, which are used during the standard preparation of porphyrin methyl esters is sufficient for the synthesis of significant amounts of methyl-ethyl esters. The presence of methyl-ethyl esters can lead to errors in chromatographic purification, identification, and/or quantification of components in mixtures of porphyrins.     

Aberrant porphyrin metabolism in hepatocellular carcinoma

In 15 patients with hepatocellular carcinoma (HCC) and 14 patients with liver cirrhosis (LC), urinary excretions of delta-aminolevulinic acid (ALA), porphobilinogen (PBG), uroporphyrin (UP), coproporphyrin (CP), and erythrocyte contents of CP and protoporphyrin (PP) were examined. In patients with HCC, urinary excretions of ALA and PBG and erythrocyte contents of CP and PP were not increased, but urinary excretions of UP and CP were significantly increased more than those of LC patients. Urinary excretions of UP and CP had no correlations with liver function tests and excretion of UP correlated slightly with blood hemoglobin level. After administration of ALA intravenously, urinary excretions of UP and CP were clearly increased in patients with HCC compared to normal controls. A Red fluorescent area was present at the cancerous area but not in the noncancerous cirrhotic area in a patient with HCC. These results suggest that aberrant porphyrin metabolism occurred in patients with HCC compared to other liver diseases.     

Ferritin accumulation and uroporphyrin crystal formation in hepatocytes of C57BL/10 mice: a time-course study

To establish the time-sequence relationship between ferritin accumulation and uroporphyrin crystal formation in livers of C57BL/10 mice, a biochemical, morphological and morphometrical study was performed. Uroporphyria was induced by the intraperitoneal administration of hexachlorobenzene plus iron dextran and of iron dextran alone. Uroporphyrin crystal formation started in hepatocytes of mice treated with hexachlorobenzene plus iron dextran at 2 weeks and in mice treated with iron dextran alone at 9 weeks. In the course of time, uroporphyrin crystals gradually increased in size. Uroporphyrin crystals were initially formed in hepatocytes in the periportal areas of the liver, in which also ferric iron staining was first detected. The amount and the distribution of the main storage form of iron in hepatocytes, ferritin, did not differ between the two treatment groups. Ferritin accumulation preceded the formation of uroporphyrin crystals in hepatocytes in both treatment groups. Moreover, uroporphyrin crystals were nearly always found close to ferritin iron. We conclude that uroporphyrin crystals are only formed in hepatocytes in which also iron (ferritin) accumulates. Hexachlorobenzene accelerates the effects of iron in porphyrin metabolism, but does not influence the accumulation of iron into the liver.     

δ-Aminolevulinic acid dehydratase inactivation by uroporphyrin I in light and darkness

• 1.1. The action of uroporphyrin I on erythrocytic ALA-D activity under dark and light conditions was examined.
• 2.2. Photo and non-photoinactivation of ALA-D induced by uroporphyrin I were observed.
• 3.3. Both effects were dependent on uroporphyrin concentration, temperature and time of exposure of the protein to the porphyrin.
• 4.4. Light-dependent effect of uroporphyrin I is related with the phototoxicity of porphyrins and could be produced by primary amino acid photooxidation followed by secondary cross-linking of the protein.
• 5.5. Light-dependent effect of uroporphyrin I could be ascribed to a direct enzyme inhibition due to binding of the porphyrin to the protein inducing structural changes at or near its active site.

     

A novel nuclear localization signal in human DNA topoisomerase I

DNA topoisomerase (topo) I is a nuclear enzyme that plays an important role in DNA metabolism. Based on conserved nuclear targeting sequences, four classic nuclear localization signals (NLSs) have been proposed at the N terminus of human topo I, but studies with yeast have suggested that only one of them (amino acids (aa) 150-156) is sufficient to direct the enzyme to the nucleus. In this study, we expressed human topo I fused to enhanced green fluorescent protein (EGFP) in mammalian cells and demonstrated that whereas aa 150-156 are sufficient for nuclear localization, the nucleolar localization requires aa 157-199. More importantly, we identified a novel NLS within aa 117-146. In contrast to the classic NLSs that are rich in basic amino acids, the novel NLS identified in this study is rich in acidic amino acids. Furthermore, this novel NLS alone is sufficient to direct not only EGFP into the nucleus but also topo I; and the EGFP.topo I fusion driven by the novel NLS is as active in vivo as the wild-type topo I in response to the topo I inhibitor topotecan. Together, our results suggest that human topo I carries two independent NLSs that have opposite amino acid compositions.     

Expression and localization of extracellular matrix metalloproteinase inducer in giant cell tumor of bone

Matrix metalloproteinases (MMPs) are regarded as a significant regulator in tumor invasion and metastasis. Previous studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN) in tumor cells induces the synthesis of MMPs. EMMPRIN is abundantly present on the surface of tumor cells and stimulate adjacent stromal cells to synthesize MMPs to induce tumor progression. Giant cell tumor (GCT) of bone is a benign but locally aggressive primary neoplasm of bone. The spindle-shaped mononuclear stromal cells are considered to be the tumor components of GCT, which are capable of inducing osteoclast formation by recruiting the circulating monocyte and macrophage. In this study, we proposed that EMMPRIN is associated with the biological progression and aggressiveness of GCT. We have conducted semi-quantitative RT-PCR to determine the correlation of EMMPRIN expression with the clinical stage of GCT. We have also examined the cellular localization of EMMPRIN in GCT using in-situ hybridization (ISH) and Immunohistochemistry (IH). The results showed that EMMPRIN was present in GCT and its mRNA levels were associated with the clinical stage of GCT. Higher expression level of EMMPRIN was observed in GCT with advanced stage (stage III). There was a great significance (P < 0.05) of EMMPRIN expression between stage I & II and stage III GCTs. Both ISH and IH demonstrated that EMMPRIN is present at the multinuclear osteoclast-like giant cells of GCT, with strong immunostaining on the cell membrane. The stromal-like tumor cells were also positively stained but the intensity was weaker. Interestingly, the production of EMMPRIN in osteoclast-like cells of GCT seems to be regulated by stromal-like tumor cells. Receptor activator of NF-kappaB ligand (RANKL), which has been previously shown to be produced by the stromal-like tumor cells for the recruitment of osteoclast-like giant cells in GCT, enhanced the expression of EMMPRIN mRNA during the differentiation of macrophage-like RAW(264.7) cells into osteoclasts. In short, our studies suggest that EMMPRIN may be an important regulatory factor involved in the biological behaviors of GCT.     

Development and utilization of a procedure for measuring urinary porphyrins by high-performance liquid chromatography

We describe a procedure for determining the five principal urinary porphyrins — uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, and coproporphyrin — by using high-performance liquid chromatography (HPLC). The method involves a 12- to 15-min isocratic separation on a Bondapak® Phenyl column. Studies have indicated that urine samples must be preserved to maintain porphyrin stability for extended periods. We have found that the pH of preserved samples must be adjusted into the acidic range before the samples can be accurately analyzed by this HPLC procedure. Our studies demonstrate that good reproducibility and recovery are achieved with this method. Urinary porphyrin values for normal and porphyric individuals are reported.