Immunocytochemical investigation of catalase and peroxisomal lipid β-oxidation enzymes in human hepatocellular tumors and liver cirrhosis

A significant reduction of catalase activity, a peroxisomal marker enzyme, occurs in human hepatic neoplasias, but no information is available on other peroxisomal proteins. We have studied by means of immunohistochemistry four specific proteins of peroxisomes (catalase and three enzymes of lipid β-oxidation) in human hepatocellular tumors of various differentiation grades from adenoma to anaplastic carcinoma. In all tumors, except the adenomas, the tumor cells contained fewer peroxisomes than extrafocal hepatocytes and the reduction of antigenic sites in the tumor types generally correlated with the degree of tumor dedifferentiation as assessed by classical histopathological criteria. Two poorly differentiated tumors had no detectable peroxisomes at all. There were no major differences in the intensities of the immunocytochemical staining for all four studied peroxisomal antigens in different tumors, suggesting that the neoplastic transformation affects the biogenesis of the entire organelle and not merely the individual peroxisomal enzyme proteins. Some tumors exhibited a distinct peripheral distribution of peroxisomes. In cases with associated liver cirrhosis, the hepatocytes in the adjacent liver showed marked peroxisome proliferation, forming large perinuclear aggregates, occupying occasionally the entire cytoplasm. Taken together, our observations indicate that peroxisomes are significantly altered in both hepatocellular tumors and liver cirrhosis and, thus, could be responsible for some of the metabolic derangements observed in those disease processes. Received: 19 April 1999 / Accepted: 28 June 1999     

Genetic polymorphisms in three Iranian populations with different risks of esophageal cancer, an ecologic comparison

The age-standardized incidence of esophageal cancer (EC) varies from 3 to >100/100,000 per year in different provinces of Iran. This striking variation of incidence is associated with differences in ethnic backgrounds, raising the possibility that genetic factors are involved in the pathogenesis of EC. We compared the frequencies of polymorphisms in ten genes that have been hypothesized to have a role in risk of EC (CYP1A1, CYP2A6, CYP2E1, GSTM1, GSTP1, GSTT1, ADH2, ADH3, ALDH2, and O6-MGMT) among three Iranian ethnic groups with highly varying rates of EC. These three groups included high-risk Turkomans, medium-risk Turks, and low-risk Zoroastrian Persians. Compared to Zoroastrians, Turkomans had higher frequency of four alleles that are speculated to favor carcinogenesis (CYP1A1 m1, CYP1A1 m2, CYP2A6*9, and ADH2*1); these results are consistent with an influence of these allele variants on the population risk of EC. However, none of these four alleles had a high enough prevalence in Turkomans to explain the high rates of EC in this group. Three of these four alleles (CYP1A1 m1, CYP1A1 m2, CYP2A6*9) were less frequent among Turkomans than in some Asian populations with lower risks of EC. We conclude that it is unlikely that variations in these polymorphic genes are major contributors to the high incidence of EC among Turkomans in Iran.     

Candidate DNA replication initiation regions at human trinucleotide repeat disease loci

The positions of DNA replication initiation regions (IRs) at three human trinucleotide repeat (TNR) disease loci were examined in order to characterize the role played by IRs in explaining the known locus-specific variation in TNR instability levels. Using three different normal cell lines, candidate IRs were identified at the HD, SCA-7 and SBMA loci. At each locus the IR is less than 3.6 kb from the CAG/CTG repeat tract. Preliminary studies with a cell line homozygous for an HD disease mutation indicated no change in the position of the candidate IR in spite of the mutation. Comparison with experimental results from model systems suggests that a complex relationship may exist between instability and the proximity and/or orientation of the repeats with respect to an IR.     

Correlation of testicular sperm extraction with morphological, biophysical and endocrine profiles in men with azoospermia due to primary gonadal failure

To identify the predictive factors for testicular sperm extraction (TESE) and to understand the pathology associated with TESE, we carried out a prospective study in 40 consecutive men with azoospermia due to primary gonadal failure. The main outcome measure was the retrieval of at least one testicular spermatozoon. Endocrine and biophysical profiles, testicular histology, Johnsen score and testicular spermatids were used as predictors of sperm extraction. Spermatogenesis was quantified with the Johnsen score. A variable pattern of spermatogenesis was common, being present in 20 (50%) patients. Visualisation of testicular spermatids on testicular histology showed a strong association with TESE (P < 0.0001). Statistically significant differences were detected in plasma follicle stimulating hormone (FSH) and testicular volume between patients who had hypospermatogenesis and Sertoli cell-only or maturation arrest. There were no significant differences in Johnsen score, biophysical and endocrine profiles between the groups with successful and failed TESE. However, a statistically significant trend occurred with changes in histological pattern [chi2 for trend, P = 0.001; Pearson's coefficient (r) = 0.6], Johnsen score (P = 0.022; r = 0.5), testicular volume (P = 0.01; r = 0.5) and plasma FSH concentrations (P = 0.044; r = 0.4), albeit to a limited degree. Difference in the interpretation of histological patterns with different assessors was observed. The type of occupation or risk factors for azoospermia showed no association with testicular pathology or TESE. Variable histological patterns in different tubules in the same individual may explain the poor correlation of TESE with endocrine and biophysical profiles, Johnsen score and histological pattern. Differences in the amount of tissue used for TESE and histopathology, and misinterpretation of testicular histology rather than failure to quantify spermatogenesis may explain the poor correlation between histological patterns and TESE. Testicular spermatids predicted TESE. However, considerable overlap in values means that no single variable can provide a perfect discrimination between the groups with successful and failed TESE.      

Mitochondrial alterations caused by defective peroxisomal biogenesis in a mouse model for Zellweger syndrome (PEX5 knockout mouse)

Zellweger syndrome (cerebro-hepato-renal syndrome) is the most severe form of the peroxisomal biogenesis disorders leading to early death of the affected children. To study the pathogenetic mechanisms causing organ dysfunctions in Zellweger syndrome, we have recently developed a knockout-mouse model by disrupting the PEX5 gene, encoding the targeting receptor for most peroxisomal matrix proteins (M Baes, P Gressens, E Baumgart, P Carmeliet, M Casteels, M Fransen, P Evrard, D Fahimi, PE Declercq, D Collen, PP van Veldhoven, GP Mannaerts: A mouse model for Zellweger syndrome. Nat Genet 1997, 17:49-57). In this study, we present evidence that the absence of functional peroxisomes, causing a general defect in peroxisomal metabolism, leads to proliferation of pleomorphic mitochondria with severe alterations of the mitochondrial ultrastructure, changes in the expression and activities of mitochondrial respiratory chain complexes, and an increase in the heterogeneity of the mitochondrial compartment in various organs and specific cell types (eg, liver, proximal tubules of the kidney, adrenal cortex, heart, skeletal and smooth muscle cells, neutrophils). The changes of mitochondrial respiratory chain enzymes are accompanied by a marked increase of mitochondrial manganese-superoxide dismutase, as revealed by in situ hybridization and immunocytochemistry, suggesting increased production of reactive oxygen species in altered mitochondria. This increased oxidative stress induced probably by defective peroxisomal antioxidant mechanisms combined with accumulation of lipid intermediates of peroxisomal beta-oxidation system could contribute significantly to the pathogenesis of multiple organ dysfunctions in Zellweger syndrome.     

Influence of ischaemia and reperfusion on cardiac signal transduction. G protein content, adenylyl cyclase activity, cyclic AMP content, and forskolin and dibutyryl cyclic AMP-induced inotropy in the rat Langendorff heart

Summary— We investigated whether post-receptor alterations contribute to the diminished β-adrenergic inotropic effects in the rat Langendorff heart following ischaemia (I) and reperfusion (R). We quantitated immunodetectable G_s and G_i protein α-subunit content, basal and stimulated adenylyl cyclase activity and cyclic AMP (cAMP) content in normoxic, ischaemic (30 min) and ischaemic reperfused (30 min) hearts. In addition, we measured the inotropic response of normoxic and reperfused Langendorff hearts to forskolin and dibutyryl cAMP (db-cAMP). Immunodetectable G_s and G_i α-subunits were unaltered by I or R. Basal adenylyl cyclase activity was decreased during I, but recovered during R. In membranes from normoxic hearts, isoprenaline, GTP, Gpp(NH)p, NaF, forskolin or Mn²⁺ enhanced adenylyl cyclase activity. This increase in activity was diminished in ischaemic hearts, but could be restored by R. cAMP content decreased time-dependently during I and did not recover by R, indicating ATP depletion. Forskolin and db-cAMP induced an inotropic response in normoxic hearts, which was virtually abolished after I and R. We conclude that adenylyl cyclase responsiveness is impaired during I. Since adenylyl cyclase responsiveness recovers during R, whereas inotropic responses to forskolin and db-cAMP are virtually absent in reperfused hearts, an additional mechanism downstream of cAMP formation appears to be defective during R, which prevents recovery of inotropic responses to hormonal stimulation.     

Expression of peroxisome proliferator-activated receptors in the liver of gray mullet (Mugil cephalus)

During the last decade, peroxisome proliferation has emerged as a novel biomarker of exposure to certain organic chemical pollutants in aquatic organisms. Peroxisome proliferation is mediated by nuclear receptors, peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described in mammals: PPAR alpha, PPAR beta and PPAR gamma. PPARs have also been discovered in several fish species. The aim of the present study was to investigate the expression of PPAR subtypes and their cellular distribution patterns in the liver of gray mullet Mugil cephalus, a fish species widely distributed in estuaries and coastal areas in Europe and used as sentinel of environmental pollution. For this purpose, antibodies were generated against the three subtypes of mouse PPARs and different protocols of antigen retrieval were used. In western blots, main bands were detected of approximately 44 kDa for PPAR alpha, two bands of 44 and 58 kDa for PPAR beta and a single band of 56 kDa for PPAR gamma. Similar results were obtained in mouse liver and may indicate antibody recognition of two forms of the protein in certain cases. PPAR alpha was the subtype most markedly expressed in gray mullet liver, being expressed mainly in melanomacrophages, nuclei of hepatocytes and sinusoidal cells and connective tissue surrounding bile ducts. PPAR beta was expressed in the same cell types but immunolabeling was generally weaker than for PPAR alpha. PPAR gamma showed very weak expression; positivity was mainly found in melanomacrophages and connective tissue surrounding bile ducts. Our results demonstrate that all the three PPAR subtypes are expressed in gray mullet liver but in different intensities. The cellular distribution patterns of PPAR subtypes in gray mullet liver resembled partly those found in mouse liver with PPAR alpha as the main subtype expressed in hepatocytes. The fact that melanomacrophages, cells of the immune system in fish, show strong expression of both PPAR alpha and PPAR beta whereas PPAR gamma expression is almost restricted to this cell type suggest a significant role of PPAR-mediated regulation of cell function in melanomacrophages.