Role of Multidrug-Resistance Protein 2 in coproporphyrin transport: results from experimental studies in bile fistula rat models.

Coproporphyrin (CP) is one of the main by-products of heme biosynthesis and its abnormal accumulation is associated with different forms of porphyria. Indirect data obtained from animal and human models have suggested a possible role for Multidrug Resistance-associated Protein 2 (MRP2) and other MRPs in hepatocyte excretion of CP. Using normal, MRP2-deficient and a cholestatic rat model, we have assessed the role of MRPs in CP disposition. MRP levels were assayed using immunofluorescence. Biliary and urinary excretion patterns of CP and conjugate bilirubin were measured during equimolar infusions of CP isomers with and without phenoldibromopthalein sulfonate (BSP), a well-known MRP2 substrate. Our results suggest a role for the MRP system as a possible regulator of CP traffic and accumulation in normal and pathological conditions. Alteration in this systems (as observed in cholestatic disease) may play an important role in triggering clinical expression of porphyria in individuals with underlying mutations leading to porphyrin accumulation and may help explain the phenotypic heterogeneity in patients affected by different forms of porphyrias.

Clinical, biochemical and genetic characteristics of Variegate Porphyria in Italy.

Variegate Porphyria (VP) is an autosomal dominant disorder found worldwide but is rare in Italy. In this study we provide an overview of clinical, biochemical and genetic background of 33 Italian VP patients diagnosed in the last fifteen years. About 70% of patients had experienced clinical symptoms: 43.4% had photosensivity, 8.7% acute attacks and 47.8% both. Among the 33 patients, 14 different mutations were identified. Of these only 6 defects have been previously described in other countries and 8 are unique having been identified for the first time in Italy. Two of these, the c.851G>T and the c.1013C>G, were found in two and four unrelated families respectively. No mutation has been found in homozygosis and no significant correlation has been observed between specific clinical and biochemical manifestations and the type of mutation. In contrast, normal faecal protoporphyrin excretion was high predictive of silent phenotype. Normal urinary excretion of PBG and ALA, predicted absence of neurovisceral symptoms. This paper represents the first compilation of data on genotype-phenotype relation in Italian patients with VP.

Anti- and pro-oxidant factors and endothelial dysfunction in chronic cigarette smokers with coronary heart disease.

BACKGROUND: Endothelial dysfunction in cigarette smokers has been ascribed to increased oxidative damage. The aims of the present study were to compare the endothelial function of normotensive smokers with that of non-smokers and to examine its relation to some parameters representative of oxidative damage and of antioxidant capacity. METHODS: We investigated 32 chronic smokers (15-30 cigarettes daily) affected by coronary heart disease, ranging from acute myocardial infarction to instable angina pectoris, and 28 matched non-smokers without any definite risk factors. All subjects underwent assessment of nitric oxide (NO)-dependent endothelial function, measured as brachial artery vasodilatation in response to reactive ischemia, using a standardized echographic method. Plasma and urinary levels of NO were also measured in all subjects, as were urinary 15-isoprostane F(2t), plasma serum lipids, homocysteine (Hcy), ascorbic acid, retinol, tocopherol, and alpha- and beta-carotene (by high-performance liquid chromatography). RESULTS: Smokers showed a significantly lower NO-mediated vasodilatation response (3.50% vs. 6.18%, p<0.001) and higher levels of urinary NO metabolites and 15-isoprostane F(2t). They also had higher levels of Hcy (p<0.001); these values were significantly and inversely related to NO serum levels (r=-0.512, p<0.001). Moreover, smokers had a significant and corresponding reduction in circulating levels of ascorbic acid, tocopherol, and alpha- and beta-carotene. CONCLUSIONS: The present study shows a clear relation between endothelial dysfunction (NO production impairment) and cigarette smoking, especially in the presence of high levels of LDL-cholesterol. It also defines some markers of both oxidative damage and antioxidant protective capacity in this condition. The monitoring of these factors may be advisable in order to assess the amount of endothelial damage.

Pro-oxidant and antioxidant factors in acute intermittent porphyria: family studies.

Given the crucial role of iron and porphyrins in oxidative cellular damage in the chronic porphyrias, we undertook an extensive study in families with acute porphyrias to evaluate the possible role of similar oxidative damage in these diseases, whose natural history is often also complicated by neoplastic evolution. Four unrelated patients with acute intermittent porphyria (AIP) were studied together with 37 members of four different families. Aminolevulinic acid and porphobilinogen were measured in urine, and porphyrins in urine, plasma and stools. The activity of the congenitally deficient enzyme, porphobilinogen deaminase, and the concentrations of plasma iron, transferrin, ferritin, and various antioxidants (ascorbic acid, retinol, tocopherol, alpha- and beta-carotene, by a personal HPLC method) and the urinary and plasma metabolites of nitrous oxide were also assayed. The results showed no relationship between the observed increase of porphyrin metabolites and the presence of markers of oxidative damage or the decrease of circulating antioxidants: however, when such a decrease was registered, it depended on spontaneous or iatrogenic iron accumulation. We conclude that family screening, recommended for the identification of AIP carriers, must also include evaluation of iron stores with a view to preventing the oxidative damage and in order to forestall the neoplastic evolution of the disease.

Molecular characterization of porphyrias in Italy: a diagnostic flow-chart.

The porphyrias are disorders associated with inherited or acquired enzyme deficiencies in the heme biosynthetic pathway. The differential diagnosis is often difficult since the phenotype is very similar in some forms and the biochemical tests are not commonly available. Here we provide an update on the molecular diagnosis of porphyrias in Italy and a flow-chart to facilitate the identification of mutations in heme biosynthetic genes. The molecular analysis has allowed us to identify the molecular defect underlying the disease in 66 probands with different porphyrias [acute intermittent porphyria (AIP), variegate porphyria (VP), porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP)]. No Italian patients with defects in coproporphyrinogen oxidise (CPOX) gene, responsible for hereditary coproporphyria (HCP), have been detected. The molecular characterization has been extended to 115 relatives with the identification of 55 asymptomatic mutation carriers and 60 normal subjects. We have so far identified 50 different mutations among 4 genes associated with the most common porphyrias showing a high molecular heterogeneity: 22 in the hydroxymethylbilane synthase (HMBS) gene (AIP), 7 in the protoporphyrinogen oxidase (PPOX) gene (VP), 16 in the uroporphyrinogen decarboxylase (UROD) gene (PCT) and 5 in the ferrochelatase (FECH) gene (EPP). Among the 50 molecular defects, 29 seem to be restricted to the Italian population.

Antioxidant liposoluble vitamins and carotenoids in chronic hepatitis.

Background: It is known that antioxidant liposoluble vitamins and carotenoids are reduced in liver cirrhosis, but little is known about chronic viral hepatitis, where oxidative damage has to be taken into account. Methods: Fifty-five patients with chronic hepatitis, mainly C virus-related, were matched with 16 patients with biliary stones and 20 healthy controls. Plasma and liver analyses were carried out using a well-tried HPLC technique that affords an accurate quantification of retinol, tocopherol, alpha- and beta-carotene, cryptoxanthin, and lycopene. Results: Plasma concentration of retinol, tocopherol, beta-carotene, and lycopene was significantly decreased in both patient groups, particularly in those with chronic hepatitis. In contrast, liver concentration of both esterified and free retinol, tocopherol, and some carotenoids was better preserved in the hepatitis group than in the cholelithiasis group. A strict correspondence between aminotransferases and the amount of liver-stored retinol was documented. Conclusions: Plasma vitamin and carotenoid depletion co-existing with preserved liver storage may indicate a functional defect in liver pool mobilization or even a real depletion of the antioxidant defenses, which play a key role in averting cellular damage. The implications for nutrition and therapy need to be taken into account.

Prognostic features and survival of hepatocellular carcinoma in Italy: impact of stage of disease.

The aim of this study was to evaluate the prognostic factors at presentation and survival in Italian patients with hepatocellular carcinoma (HCC). Clinical and demographic data of 176 patients consecutively observed from 1993 to 1997 were evaluated by univariate and multivariate analyses. Overall median survival was 18 months. At univariate analysis, low albumin, high bilirubin, high alkaline phosphatase, high alpha-fetoprotein (AFP); high platelet count, hepatitis B surface antigen (HBsAg)-positivity, the presence of ascites, of encephalopathy, of portal vein thrombosis (PVT), male sex, no treatment, poor differentiation, untreatable tumours and incidental diagnosis were each associated with shorter survival. HBsAg-positive subjects more often presented with untreatable lesions or diffuse tumours (P=0.001 and P=0.007, respectively) and had significantly worse survival (P=0.0057). By multiple regression analysis, low albumin, high bilirubin, abnormal AFP, presence of PVT and of untreatable lesions were independent risk factors for worse survival. Thus, the most important factors influencing survival are the degree of functional impairment of the liver, the presence of hepatitis B viral (HBV) infection, the type of diagnosis and the aggressiveness of the tumour.

Acute intermittent porphyria: heterogeneity of mutations in the hydroxymethylbilane synthase gene in Italy.

Acute intermittent porphyria (AIP) is an autosomal disorder caused by molecular abnormalities in the gene coding for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. So far, more than 170 different mutations responsible for AIP have been identified worldwide in the HMBS gene. In this study we have performed molecular characterization in 14 patients with suspected diagnosis of AIP and in 29 family members of Italian ancestry. Molecular analysis of the HMBS gene allowed us to identify 13 different mutations among 14 patients with reduced HMBS activity: 5 splicing defects (IVS9+22 G>A, 612 G>T, IVS11-2 delA, IVS12+2 T>C, and IVS13-1 G>A), 1 small insertion (182 insGA), 1 small deletion (730-731 delCT), and 6 missense/nonsense mutations (76 C>T, 295 G>A, 331 G>A, 580 C>T, 673 C>T, and 874 C>T), resulting in single-amino-acid substitutions or protein truncations. Six of these molecular abnormalities had already been described and 7 are new findings. In a previous work on an Italian population we detected 7 different mutations among 8 AIP patients. This study has raised to 18 the number of different mutations so far found among the Italian AIP population, 11 of which are new findings. We can conclude that the mutation screening in the Italian population contributes to improvement of the diagnostic approach of AIP and to establishing possible clustering of mutations in the Mediterranean area.

Mortality in a population with long-term exposure to inorganic selenium via drinking water.

We analyzed the 1986-1997 mortality in a cohort of 2065 residents of an Italian municipality which had been exposed to drinking water with a high content of inorganic selenium over a long period of time, and compared it with mortality in the remainder of the municipal population. Mortality from malignant neoplasms increased [standardized mortality ratio (SMR) 1.17, 95% confidence interval (CI) 0.96-1.42], mainly due to an excess mortality from melanoma and colorectal cancer in both sexes, kidney cancer in men, and lymphoid malignancies in women. Overall cardiovascular mortality changed little (SMR 1.05, 95% CI 0.89-1.23), despite the higher cerebrovascular mortality (SMR 1.43, 95% CI 1.03-1.93). Coronary disease mortality slightly decreased (SMR 0.87, 95% CI 0.63-1.16), due to a low mortality among women. We also noted an excess mortality from Parkinson's disease in men and from motor neuron disease in women. Evaluation of these findings is, however, hampered by the lack of information about potential lifestyle confounders, the fact that the exposure could only be characterized by a simple dichotomization, and the inconsistencies of most estimates between the two sexes.

BCRP/MXR/ABCP expression in topotecan-resistant human breast carcinoma cells.

We have previously described a mitoxantrone-resistant MCF7 cell line that is cross-resistant to topotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (CPT-11), and 9-aminocamptothecin, but not to camptothecin. A novel mechanism that resulted in decreased topotecan accumulation in MCF7/MX cells was proposed (Yang et al. Cancer Res 55: 4004-4009, 1995). We now have developed a topotecan-resistant cancer cell line from wild-type MCF7 cells. MCF7/TPT300 cells were 68.9-fold resistant to topotecan, 68.3-fold to 10-hydroxy-7-ethylcamptothecin (SN-38), and 116-fold to mitoxantrone, but only 4.1-fold to camptothecin. Topotecan efflux was increased in MCF7/TPT300 cells compared with MCF7/WT cells, and this increase was reversed upon ATP depletion by sodium azide, suggesting an energy-dependent drug efflux mechanism. However, MCF7/TPT300 cells did not overexpress P-glycoprotein or the multidrug resistance-associated protein (MRP1). In contrast, overexpression of the breast cancer resistance protein (BCRP/MXR/ABCP) was observed in MCF7/TPT300 cells as well as DNA topoisomerase I down-regulation. Our data suggest that enhanced topotecan efflux contributes partly to topotecan resistance in MCF7/TPT300 cells, possibly mediated by BCRP/MXR/ABCP.

Multiwavelength videomicrofluorometric study of cytotoxic effects of a marine peptide, didemnin B, on normal and MDR resistant CCRF-CEM cell lines.

The development of multidrug resistance (MDR) in heterogeneous cell sensitive and resistant populations to a variety of clinically important cytotoxic drugs poses a major obstacle to cancer chemotherapy. Didemnin B, a marine cyclic depsipeptide, displays interesting biological properties: antiviral activity, inhibition of DNA, RNA and protein synthesis, initiation of apoptosis and ability to block the cell cycle. As very little is known about its mode of action, we studied the effect of increasing doses of Didemnin B on sensitive and resistant human leukemic lymphoblast cell lines. The fluorescence of living cells simultaneously stained with Hoechst 33,342, Rhodamine 123 and Nile Red, were analyzed in a multiparametric approach involving multiwavelength microfluorometry. High concentrations of Didemnin B induced, in the sensitive cell line, a very early decrease in the energetic state of the mitochondria that occurs before a significant decrease of nuclear DNA content, observed simultaneously on sensitive and resistant cells, that could be related to an apoptosis process. Furthermore low Didemnin doses (50 nM) affected CEM-WT and CEM VLB differently, while higher doses (200 nM-250 nM and over) affected the two cell lines in the same way. This indicated that, at these doses, the membranar Pgp has no effect on the mode of action of Didemnin, suggesting that Didemnin does not need to be internalized to be active.

Peculiar facies, obesity, cleft lip and palate, growth hormone deficiency and mental retardation: a new syndrome?

A female child with peculiar facies, obesity, cleft lip and palate, growth hormone deficiency and mental retardation is described. The present case does not appear to fit any of the known syndromes.

The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane.

The products of the ABC gene family can be generally classified as either full-transporters of half-transporters. Full-transporters are expressed in the plasma membrane, whereas half-transporters are usually found in intracellular membranes. Recently, an ABC half-transporter, the ABCG2 gene product Breast Cancer/Mitoxantrone Resistance Protein (BCRP/MXR), has been shown to cause mitoxantrone and topotecan resistance. The purpose of this study was to determine the expression and the intracellular localization of this protein in various drug-resistant cell lines. BCRP/MXR expression was determined by Western blot and immunohistochemistry. This protein is highly overexpressed in several drug-resistant cell lines and localizes predominantly to the plasma membrane, instead of to intracellular membranes as seen with all other known half-transporters. Therefore, BCRP/MXR is unique among the ABC half-transporters by being localized to the plasma membrane.

Multiwavelength videomicrofluorometric study of cytotoxic properties of a marine peptide, didemnin B, using adriamycin as reference compound.

Didemnin B (DB), a marine natural product, has very encouraging biological activity in vitro (Antineoplastic, immunosuppressive, antiviral). To learn more about its intracellular effects and targets, videomicrofluorometry on single living cells and a protocol of multiple labeling: Hoechst 342 for nuclear DNA, Rhodamine 123 for mitochondria and Nile Red for plasma membrane, have been used. DB behaves differently from Adriamycin, inducing at its IC50 dose of (20 nM) an accumulation of the CEM-WT lymphoblasts in the S phase of the cell cycle while we observed a 50% decrease of the mitochondrial labeling by R123, showing a decrease of the mitochondrial energetic state. Cytostatic dose of DB (250 nM) confirms these observations. However the treatment with a dose reported as apoptotic (1000 nM) induces a much faster effect (corresponding to that of 72 hours at the IC50 dose), 24 hours incubation induced a drastic decrease of nuclear DNA content as well as of the mitochondria energetic state. The evolution of NAD(P)H cellular content exhibited an increase that seems to indicate that the decrease of mitochondrial energetic state was dependent on inhibition of the mitochondrial activity due to an effect of DB at the mitochondrial level, either direct or mediated. Furthermore, the decrease of mitochondrial labeling appears as a very early event in the mechanisms leading to apoptosis.

Circulating pro- and antioxidant factors in iron and porphyrin metabolism disorders.

BACKGROUND: Porphyria cutanea tarda and haemochromatosis are taken to be spontaneous human models of oxidative cellular damage, with an increased risk of fibrosis and cancer evolution. AIM: To define the relative pro-oxidant roles of porphyrin and iron, in their different molecular forms, and their effects on antioxidant biological systems. PATIENTS: A group of 17 patients with porphyria cutanea tarda and a group of 14 patients with primary and secondary haemochromatosis, were compared with 21 healthy controls. METHODS: Plasma retinol, tocopherol, alpha- and beta-carotene, ascorbic acid, glutathione, malonyldialdehyde and red blood cell free iron were determined using high performance liquid chromatography. RESULTS: Only a modest increase in iron stores was demonstrated in the porphyria cutanea tarda group; in the haemochromatosis patients ferritin levels were almost seven times higher. By contrast, there was a sharp and virtually identical increase in red blood cell free iron and malonyldialdehyde in both the patient groups. A significant reduction was observed in retinol, alpha-, beta-carotene and red blood cell glutathione levels being more marked in porphyria cutanea tarda than in haemochromatosis patients. CONCLUSIONS: The study confirms the strong pro-oxidant effects of porphyrins in vivo, through an induction of the free toxic iron form, even though the total iron pool is not greatly expanded. The additional free-iron and porphyrin oxidant effects are documented both in red blood cell and plasma in the porphyria cutanea tarda group. It confirmed that aging exerts a negative influence in terms of pro- and antioxidant balance in all cases, but particularly in the haemochromatosis group.

Multiwavelength videomicrofluorometric study of some human leukemic lymphoblasts: effect of adriamycin on some biological parameter.

The development of multidrug resistance (MDR) in heterogeneous cell sensitive and resistant populations to a variety of clinically important cytotoxic drugs poses a major obstacle to cancer chemotherapy. The MDR phenotype is characterized by a decrease the intracellular drug accumulation and by an overexpression of the MDR1 gene which encodes the membrane protein, P-glycoprotein (Pgp). To evaluate the MDR phenotype, rationale investigations of the cytotoxic processes and effect,s of Adriamycin (ADR) were done to obtain information on individual cells. Such information could be obtained through a multiparametric approach involving multiwavelength microfluorometry and numerical image analysis on single living cells. To achieve this, cells should be simultaneously stained with Hoechst 33342 (nuclear staining), Rhodamine 123 (mitochondria staining) and Nile Red (cell contour delineation). Changes in the biological parameters accessible from R123, Ho33342 and C-SNARF-1/AM (probe used for the pHi measurements) labelling were found more informative than changes in morphological parameters for the discrimination of sensitive and resistant cells. Furthermore, this approach allows the discrimination between two resistant cell lines expressing different mechanisms of resistance.

Hepatic tocopherol content in primary hepatocellular carcinoma and liver metastases.

The high incidence of hepatocellular carcinoma (HCC) in cirrhosis, where previous studies have indicated a severe reduction in several antioxidant vitamin factors, prompted us to compare plasma liposoluble vitamins with tocopherol content in healthy and neoplastic liver tissue in humans. This, with a view to a more positive preventive dietary approach, given the conflicting results obtained by liposoluble vitamin dietary supplementation in different malignancies. Eleven patients with cirrhosis, 18 patients affected by cirrhosis with HCC, and 10 patients with liver metastases (LM) from digestive tract adenocarcinomas were compared with controls who had undergone perlaparoscopic cholecistectomy. Plasma alpha- and beta-carotene, retinol and tocopherol, together with liver tocopherol, from both nonmalignant portions and malignant nodules of the same organ, were determined by high-performance liquid chromatography following a well-assessed technique. The results confirm a trend towards a reduction in circulating carotenoids and tocopherol in cirrhosis and in patients affected by cirrhosis with HCC. Tocopherol content in liver tissue is significantly decreased in cirrhosis (0.26 + 0.03 micromol/g prot., mean + SEM, P < .001) and in cirrhotic areas of the HCC group (0.31 + 0.02, P < .002), with respect to its content in liver specimens of healthy controls (0.46 + 0.03) and in healthy areas of the same organ in patients with LM (0.41 + 0.03). Tocopherol concentration is further reduced by 50% in malignant liver nodules of HCC, with respect to surrounding cirrhotic tissue, whereas in metastatic liver nodules from digestive neoplasms the tocopherol content is almost twice that of healthy surrounding areas. This unpredictable tocopherol behavior in liver specimens, of secondary as opposed to primary malignancies of the liver, affords further insight into the conflicting effects of liposoluble vitamins employed in the chemopreventive treatment of different malignant diseases, where hepatic tocopherol concentration show opposite trends: halved in primary HCC and doubled in LM of digestive adenocarcinomas, with respect to healthy controls.

Multi-frequency bioelectric impedance measurements for predicting body water compartments in patients with non-ascitic liver cirrhosis.

We assessed total body water (TBW) and extracellular water (ECW) in thirty-four non-ascitic cirrhotics and twenty healthy controls by 2H2O and Br dilution. In the same subjects, bioelectric impedance (BI) was recorded at multiple frequencies. Body hydration was similar for controls (mean 55.6 (SD 6.7)), less-severe cirrhotics (Child-Pugh classification A; CPA; n 21, mean 56.2 (SD 6.2)) and moderately-severe cirrhotics (Child-Pugh classification B; CPB; n 13, mean 57.2 (SD 5.4)). However, intracellular water standardized per litre TBW was significantly higher in CPB subjects (mean 27.0 (SD 7.5); P < 0.01) compared with CPA (mean 21.3 (SD 10.6)) and control subjects (mean 18.0 (SD 9.8)). Published formulas for predicting TBW and ECW from BI at multiple frequencies were applied to the cirrhotics. These formulas gave accurate predictions of TBW and ECW, although standard errors of estimates were higher for CPB subjects (TBW < or = 2.5 and ECW < or = 2.1 l) than those for CPA (TBW < or = 2.0 and ECW < or = 1.8 l) and control (TBW 1.4 and ECW 0.9 l) subjects.

Thyroid hormone promotes BCL-2 expression and prevents apoptosis of early differentiating cerebellar granule neurons.

Programmed cell death is a basic cellular process that has aroused much interest in recent years. Like immune cells, cultures of cerebellar granule neurons are very homogeneous and provide a unique opportunity for quantifying by flow cytometry one form of programmed cell death in the CNS, the apoptosis, and for studying its regulation by neurotrophic factors. We found that thyroid hormone promoted postmitotic survival by preventing the apoptosis of newly formed and early differentiated granule neurons in a dose-dependent manner. This regulation could be through the protein bcl-2, which is known to prevent cell death. This protein was present at all stages of granule neuron differentiation and appeared to be developmentally regulated. It was underexpressed in apoptotic granule neurons. The protein content of the cerebellum in hypothyroid rats was drastically reduced. In contrast, thyroid hormone caused a marked dose-dependent increase in the amounts of this protein in granule neuron cultures. The possibility that thyroid hormone may be directly or indirectly required to promote cell survival is discussed, in terms of the hormone control of the local delivery of neurotrophins, such as NGF and NT-3, as well as the expression of their low affinity receptors, gp75. We suggest that thyroid hormone has a permissive action on the developing CNS.