Preface

BATLLE, ALCIRA; ROSSETTI, MARIA VICTORIA

PORPHYRIAS AND ASSOCIATED PATHOLOGIES. BIOCHEMISTRY AND MOLECULAR BIOLOGY” CELLULAR AND MOLECULAR BIOLOGY, VOL. 55, PART II

CMB edition

Lugar: Noisy Le Grand; Año: 2009; p. 1 - 7

To begin with, we wish to thank again Professor Dr. Raymond Wegmann “The Founding President” and Professor Dr. Jean-Michel Maixent, “The President”, Editors-in-Chief of Cellular and Molecular BiologyTM for their generous invitation to become Guest- Editor and Co-Guest Editor of this special theme issue on “Porphyrias and associated pathologies. Biochemistry and Molecular Biology”, which has been published in two Volumes, Part I, appeared in February 16 th, 2009 and now, Part II, scheduled for July 1 st 2009. Although Cellular and Molecular BiologyTM has already published three others special issues on this subject, one in 1997, and two in January and December 2002, …”to have another issue on “Porphyrins and Porphyrias” is not too much, since it belongs to one of the modern problems for which orphan drugs are requested”… (quoted from Professor Dr. Raymond Wegmann, Cellular and Molecular BiologyTM, , 2002, 48, 823). We also wish to thank again Mr Mourad Fares, Executive Editor of CMB, for his support, composition and editing of the papers. The articles put together in Part II of this theme issue, come from the highest experts in the world in this field, biologists, physicians, chemists, physics, all of them leading porphyrinologists, which will assure, to make Part II as well as it was with Part I, a highly important reference issue for the future. Therefore we are also deeply thankful to all the authors for their contributions and most important for their collaboration in making possible the production of this theme CMB issue on “Porphyrias and associated pathologies. Biochemistry and Molecular Biology” “Porphyrins and Porphyrias”, since the middle of the 50’s, porphyrinologists from all over the world have been meeting at least once or later more than once a year, mostly in Europe and USA, to discuss about the latest innovative results on the biochemistry, molecular biology, regulation and functional mechanisms of the enzymes involved at each step of the porphyrins biosynthesis, which have been isolated, cloned and even chrystalized, and its related diseases: the Porphyrias. In 1997, Professor Robert Aquaron, attempted to list a number of the International Porphyrins and Porphyrias Meetings, mostly hold in Europe, since 1955, until then. However we should also recall and include the Gordon Research Conferences, started in 1968, occurring in USA, every two years ( about 22) which were just mentioned as well as the Tetrapyrrole Discussion Group Meetings, gathering somewhere in Europe, much more often in UK, every nine months since 1967 (by now there have been about 33 meetings). Porphyrins and Porphyrias, certainly, one of the most important and endless theme for discussion. Some of us have had the honour and pleasure of having been closely associated, known and being personally involved in Porphyrins and Porphyrias research for nearly 50 years by now, also having had the pionners in this field as our mentors, something to celebrate, so we will, after reading this special issue of CMB. Porphyrins synthesis is one of the most fundamental attributes of all living cells. Central to the fundamental processes of photosynthesis and respiration, are chlorophylls and haem, respectively, which are porphyrins, that is the reason why, Lemberg and Ledge in 1949, coined the expression “porphyrins are the pigments of life”, could it be anything more important than that?. We would wish to partially quote one of us (Batlle, CMB TM, 2002, 48, 823), when saying that porphyrins are unique and intriguing molecules, historically having a geologic and a biologic medical chapter. The former going back many millions of years, when the formation of porphyrin-like compounds and porphyrins was contemporary with the development of life on earth. The latter started at the beginning of nineteen century when iron-free hematin was obtained by Scherer in 1941, after treating dried blood with concentrated sulphuric acid. This pigment was later purified by Thudichum in 1867, who named it “cruentine” and described for the first time its “splendid blood-red” fluorescence. The term “porphyrin” was soon after coined by Hoppe Seyler in 1871. As already stated, porphyrin biosynthesis is one of the most fundamental attributes of all living cells. Classical isotope tracer studies from the laboratories of Shemin, Rimington, Granick and Neuberger, identified the precursors and intermediates in the haem biosynthetic pathway marking the beginning of a whole new field of research in the biogenesis of these pigments. Then, most of the eight enzymatic steps involved were identified. Today all the enzymes have been cloned and sequenced and most of them have also been crystallized. The porphyrin pathway is very finelly controlled. In most tissues and species, Aminolevulic Acid Synthetase (ALA-S) is the regulatory enzyme. Regulation occurs by feed back inhibition of ALA-S, so haem deficiency, owing to blocking the pathway at some step, as it happens in the Porphyrias, releases this inhibition. The term “porphyria”, gradually emerged after Stokvis(1889) reporting the death of an elderly women, excreting dark red urine after having received sulfonal. Human porphyrias are specific inherited or acquired defects, each representing a partial failure of one of the seven enzymes beyond ALA-S and they are characterised by a typical excretion pattern of porphyrin intermediates. We would like to recall that porphyrins are the only photosensitizers synthesized in the cells and the best examples of these endogenous sensitizers are the porphyrin intermediates formed and accumulated in the cutaneous porphyrias, producing the characteristic skin photosensitization. Photodynamic Therapy (PDT) is a promising new modality of cancer treatment, which involves the combination of a photosensitizing agent, which is taken up selectively and retained by tumoural cells, and light of an appropriate wavelength. Separately, each of these factors is harmless by itself, though, when combined, in the presence of oxygen, cytotoxic reactive oxygen species are produced, leading to irreversible cellular damage, causing cell death and tumor destruction. After either exogenous administration or endogenous synthesis, porphyrins finally accumulate in higher proliferative cells. Light energy absorbed by the photosensitizer (PS) can produce fluorescence. The tumour localizing properties of the PS have been extensively employed for the Photodetection (PD) and diagnosis, as well as for the PDT of tumours. Photodynamic properties of porphyrins are a well known characteristic of these compounds. As already stated, a clear demonstration of their powerful photosensitizing properties, can be seen in the cutaneous porphyrias, where abnormal quantities of circulating porphyrins result in the typical skin photosensitivity. One of the main developments in PDT, has been the novel approach using the precursor 5- Aminolevulinic Acid (ALA), leading to the endogenous synthesis of the active photosensitizer (PS) Protoporphyrin IX (PPIX), the so called ALA based PDT or ALA-PDT. Besides PPIX, other PSs have been synthesized or extracted from natural products, to be used in PD or PDT. In the paper “Endogenous and exogenous Porphyrins as Photosensitizers in the HEP-2 Human Carcinoma Cell Line” by M.G.Alvarez, N.E. Milanesio, V. Rivarola, E. Durantini, A. Battle and H. Fukuda, the photodynamic activity of three PSs: ALA-induced PPIX, the porphyrin derivative 5-(4-trimethylammoniumphenyl)-10, 5, 20-tris (2,4,6-trimethoxyphenyl) porphyrin (CP) and the molecular dyad porphyrin-C60 (P-C60), the last two incorporated into liposomal vesicles, was evaluated on Hep-2 human larynx carcinoma cell line. When photosensitized with ALA and P-C60, chromatine condensation characteristic of apoptotic cell death was found; instead, 58 % of necrotic cells were observed with CP. The results show that in the Hep-2 cells, of the three PSs analyzed, the molecular dyad P-C60 was more efficient than CP and PPIX, and confirm that PDT can induce different mechanisms of cell death depending on the PS and the irradiation dose. In the next paper “ROS production by endogenously generated Protoporphyrin IX in murine leukemia cells”, by B. Diez, R. Cordo Russo, M.J. Teijo, S. Hajos, A. Batlle and H. Fukuda, they studied the efficiency of PpIX synthetized from ALA on ROS generation, in the Vincristine resistant (LBR-V160), Doxorubicin resistant (LBR-D160) and sensitive (LBR-) murine leukemia cell lines. Cells were incubated with 1 mM ALA and then irradiated during different times with fluorescent light. Then, production of ROS was analyzed by flow cytometry using different fluorescent probes. It was found that superoxide anion production in the three cell lines increased with irradiation time whereas no peroxide hydrogen was detected. Mitochondrial damage also increased in an irradiation time dependent manner, being higher in the Vincristine resistant line. Because the apoptotic cell death increased with irradiation time, the authors have shown that ROS are critical in ALA-PDT efficiency to kill malignant cells. Clinical manifestation of porphyrias are often associated with exposure to precipitating agents, including polyhalogenated aromatic hydrocarbons, alcohol abuse, estrogens and steroids ingestion, stress and infection with Hepatitis C virus (HCV), less frequently, Hepatitis B virus (HBV). and association with infection with the Human Immunodeficiency virus (HIV). The role that steroids play in the expression of housekeeping 5-Aminolevulic Acid Synthetase (ALA-S1) has been the subject of scrutiny for a number of years. The very earlier studies from Granick and others, showed that in addition to a female preponderance of acute attacks, numerous steroids of both natural and pharmaceutical origins could be linked to induction of attacks and to changes in the activity of ALA-S. In the paper “Functional analysis of the 5’ regulatory region of the 5-aminolevulinate synthase (ALAS1) gene in response to estrogen” BY N. du Plessis, M Kimberg, M G Zaahl, A Sadie, M Venter, L van der Merwe, A Louw & L Warnich, the authors investigated some factors influencing the clinical expression of porphyrias, primarily by altering the rate of heme synthesis. To date, no genotypephenotype correlation has been made to explain the variable penetrance observed in variegate porphyria (VP) and other acute hepatic porphyrias. As first and rate determining gene in the heme pathway, ALAS1, appears to be an ideal candidate modifier. Previous studies established critical mechanisms for ALAS1 regulation and a direct transcriptional response to drugs by defined drugresponsive enhancer sequences. They evaluated the effects of a possible oestrogen binding receptor at the five-prime end of ALAS1 gene. Its role appears to be in increasing the transcriptional response when oestradiol is present. It is also known that in some porphyrias, mild to moderate hepatic iron overload plays a key role in its pathogenesis. Hemochromatosis is the commonest cause of primary iron overload and some mutations in the hemochromatosis gene (HFE), associated with hereditary Hemochromatosis, have been found to be more frequent in PCT. M.V. Rossetti, M. Méndez, S. Afonso, E. Gerez, A. Batlle, A. Muñoz and V. Parera in their paper “HFE gene mutations in patients with altered iron metabolism in Argentina“ have investigated the prevalence of C282Y, H63D and S65C mutations in 95 individuals bearing iron metabolism alterations to establish an early diagnosis of Hereditary Hemochromatosis (HH). Among this population, 58% carried mutations in the HFE gene. H63D mutation was found in 32.6% of the subjects (29.5% in heterozygocity, 3.15% in homozygocity). S65C mutation was only detected in the heterozygous form (5.3% of the patients), 2 of them carried also H63D mutation. C282Y in heterozygocity was found in 15.8% of the individuals. Their findings were consistent with the Mediterranean origins of the population of Argentina, where the commonest mutation was H63D. Association of porphyrias with other pathologies, such as diabetes, lupus, leukaemia, Hansen’s disease, cancer and autism has been reported. There is also association of porphyrias with the treatments used for other pathologies, such as estrogen-therapy in prostate cancer and hemodialysis in patients with renal failure. Porphyrias are often multifactorial, therefore, knowledge of all risk or etiological factors in each patient is most important for the management of the disease. The paper “Association between Porphyria Cutanea Tarda and Beta Thalassemia Major” By L. Barbieri, A. Macri, G.L. Palmieri, C. Aurizi and G. Biolcati, describes the first two cases of porphyria cutanea tarda (PCT) associated with beta-thalassemia major. The clinical course of two female patients affected by beta-thalassemia major was complicated by the onset of PCT. Both patients were also suffering from hepatitis C virus infection, iron overload and anaemia. The authors discuss about the role performed by some of these conditions in triggering overt PCT. An improvement of the clinical and biochemical picture of PCT was obtained with chloroquine therapy. Porphyria cutanea tarda (PCT) is caused by inhibition of uroporphyrinogen decarboxylase (URO-D) activity in hepatocytes. Subnormal URO-D activity results in accumulation and urinary excretion of highly carboxylated porphyrins, uroporphyrin and heptacarboxyl porphyrin. Heterozygosity for mutations in the URO-D gene can be found in the familial form of PCT (F-PCT). Over 70 mutations of URO-D have been described so far, but very few have been characterized structurally. In the paper “Structural and kinetic characterization of mutant human uroporphyrinogen decarboxylases”, C. A. Warby, J. D. Phillips, H. A. Bergonia, F. G. Whitby, C. P. Hill, and J. P. Kushner, characterized 3 mutations in the URO-D gene found in patients with F-PCT. G318R, K297N and D306Y mutations. Expression of the D306Y mutation resulted in an insoluble recombinant protein. G318R and K297N have little effect on the structure or activity of recombinant URO-D, but the proteins displayed reduced stability in vitro. A number of murine models of PCT centered around the administration of iron and compounds that induce transcription of cytochrome P450s have been developed. So, the influence of porphyrins and porphyrinogens themselves on metabolic alterations in hepatocytes has been difficult to distinguish from the effects of the compounds precipitating the porphyric state. The Utah team had previously developed a genetic murine model of PCT by crossing porphyria-susceptible Urod+/- mice with mice homozygous for deletion of the hemochromatosis gene (Hfe). The Urod+/-, Hfe-/- mouse spontaneously develops uroporphyria and allows studying the effects of porphyrinogen and porphyrin accumulation on enzymes and transporters involved in tetrapyrrole disposition without the confounding effects of exogenous factors. In their paper "Longitudinal Study of a Mouse Model of Familial Porphyria Cutanea Tarda", D-D.Arch, H.A. Bergonia, L. Hathaway, J. P. Kushner, J.D. Phillips and M.R. Franklin, describe for the first time, a longitudinal study of Urod+/-, Hfe-/- from 8 weeks to 1 year. The authors have shown that the first sigh of abnormal porphyrin biosynthesis in the Urod+/-,