Richard E. Edwards

Potentiation of iron accumulation in cardiac myocytes during the treatment of iron overload in gerbils with the hydroxypyridinone iron chelator CP94

Gerbils administered iron dextran are the only animal species which have been shown to develop hemochromatosis of the liver and heart in the same manner as transfusion dependent homozygous thalassemics. The iron chelating hydroxypyridinone, CP94, has been administered prophylactically to iron overloaded gerbils in a dosing regime which favors the formation of bidentate chelated iron, to examine the possibility of additional toxicity being caused to the liver and heart by the bidentate chelated iron complex. Hepatic iron accumulation was inhibited by CP94 administration for up to 6 weeks, but not after 20 weeks. Iron accumulation in the heart was increased significantly after 6 and 20 weeks of chelator treatment. Pathological changes in both organs were markedly more severe after 20 weeks in chelator treated animals. There was a higher incidence of cardiofibrosis and more extensive liver fibrosis in iron overloaded, chelator treated animals after 20 weeks.

The role of cell death and cell proliferation in the promotion of rat liver tumours by tamoxifen

Administration of tamoxifen to rats results in liver tumours with a latency time that is dependent on the strain of rat used. Wistar and Lewis rats develop liver tumours more rapidly than Fischer rats. Significant increases in the number of apoptotic hepatocytes were found in the Wistar and Lewis strains of rats after they were fed tamoxifen for up to 6 months, but not in Fischer rats. By 6 months of exposure to tamoxifen there were liver tumours in the Wistar and Lewis rats, but not the Fischers. Sustained elevations of the PCNA labelling index were found in the livers of tamoxifen-treated Wistar and Lewis rats, over the first 6 months of tamoxifen treatment, but not Fischers. It is proposed that sustained cell death by apoptosis may play a role in the mechanism of promotion of tamoxifen-induced liver tumours, by causing liver hyperplasia. To support this concept it has been shown that cyloheximide, which causes apoptosis but not necrosis in the rat liver, causes DNA synthesis and cell division in hepatocytes.

Intrapleural administration of fibres induces mesothelioma in rats in the same relative order of hazard as occurs in man after exposure

1 The dose—response data for the induction of mesothelioma, in rats, by the intrapleural administration of the fibrous zeolite, erionite, has been compared to the published data for the crocidolite and chrysotile forms of asbestos. Erionite is more than two orders of magnitude more carcinogenic than either of the two forms of asbestos examined. 2 The relative sensitivity of the intrapleural and intraperitoneal routes of injection were also examined. The sensitivity of the intraperitoneal over the intrapleural route of administration was considerably greater for all the forms of asbestos examined but not for erionite. 3 The relationship for different fibres, between the number of fibres required to give animals mesothelioma, at the 50% or 10% observable tumour effect level (OTEL) was examined, and a ranking of relative carcinogenicity was made. 4 This showed that the data derived from the dose responses obtained by the intrapleural administration of fibres to rats ranked the relative carcinogenicity of erionite, crocidolite and chrysotile in accord with the known clinical mesothelioma induction in man after exposure to these fibres. Examination of the carcinogenicity ranking from data derived from intraperitoneal injections of fibres was not in accord with the known clinical mesothelioma induction in man for the various asbestos types examined.

A GC–MS metabolic profiling study of plasma samples from mice on low- and high-fat diets☆

Metabolic profiling of biofluids, based on the quantitative analysis of the concentration profile of their free low molecular mass metabolites, has been playing increasing role employed as a means to gain understanding of the progression of metabolic disorders, including obesity. Chromatographic methods coupled with mass spectrometry have been established as a strategy for metabolic profiling. Among these, GC-MS, targeting mainly the primary metabolism intermediates, offers high sensitivity, good peak resolution and extensive databases. However, the derivatization step required for many involatile metabolites necessitates specific data validation, normalization and analysis protocols to ensure accurate and reproducible performance. In this study, the GC-MS metabolic profiles of plasma samples from mice maintained on 12- or 15-month long low (10 kcal%) or high (60 kcal%) fat diets were obtained. The profiles of the trimethylsilyl(TMS)-methoxime(MeOx) derivatives of the free polar metabolites were acquired through GC-(ion trap)MS, using [U-(13)C]-glucose as the internal standard. After the application of a recently developed data correction and normalization/filtering protocol for GC-MS metabolomic datasets, the profiles of 48 out of the 77 detected metabolites were used in multivariate statistical analysis. Data mining suggested a decrease in the activity of the energy metabolism with age. In addition, the metabolic profiles indicated the presence of subpopulations with different physiology within the high- and low-fat diet mice, which correlated well with the difference in body weight among the animals and current knowledge about hyperglycemic conditions.

Essential Role of the AH Receptor in the Dysfunction of Heme Metabolism Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The dysfunction of hepatic heme synthesis by 2,3,7,8-tetrachlordibenzo- p-dioxin (TCDD) in mice, enhanced by iron, leads to accumulation of uroporphyrins I and III (uroporphyria) and resembles the human disorder porphyria cutanea tarda (PCT) precipitated by alcohol and estrogenic drugs. Although consequences of TCDD are considered entirely dependent on the aryl hydrocarbon receptor (AHR), this is not proven for uroporphyria. Administration of TCDD (75 microg/kg) caused uroporphyria in susceptible C57BL/6J mice with high-affinity AHR after 5 weeks (>600-fold increase in hepatic uroporphyrins). Transcriptomics showed significant modified gene expressions for intermediary, heme, and iron metabolism as well as for oxidative stress and cell injury. Resistant low-affinity AHR DBA/2 mice (no increase in porphyrins) showed far fewer changes. At this dose of TCDD, persistent up-regulation of some traditional AH battery genes occurred in both strains. Essentiality of AHR was demonstrated with C57BL/6 Ahr knockout mice. Elevation of hepatic uroporphyrins was 964-fold in Ahr (+/+) mice, lower in Ahr (+/-) (60-fold), but undetectable with Ahr (-/-) . Consistent with an oxidative mechanism, iron overload enhanced porphyria as well as general liver injury in Ahr (+/+) and Ahr (+/-) mice but had no interactive effect in Ahr (-/-) . In contrast, when iron-treated mice received, instead of TCDD, the heme precursor 5-aminolevulinic acid (ALA), causing uroporphyia in Ahr (+/+) mice (242-fold rise in uroporphyrins), elevation of uroporphyrins I and III (42-fold) also occurred in Ahr (-/-) mice and was seemingly associated with AHR-independent expression of Cyp1a2. The findings prove that AHR is a key factor in porphyria induced in mice by TCDD. However, in other models of human PCT, participation of AHR may not be an essential requirement.

Multiple polymorphic loci determine basal hepatic and splenic iron status in mice

Polymorphisms of genes linked to iron metabolism may account for individual variability in hemochromatosis and iron status connected with liver and cardiovascular diseases, cancers, toxicity, and infection. Mouse strains exhibit marked differences in levels of non‐heme iron, with C57BL/6J and SWR showing low and high levels, respectively. The genetic basis for this variability was examined using quantitative trait loci (QTL) analysis together with expression profiling and chromosomal positions of known iron‐related genes. Non‐heme iron levels in liver and spleen of C57BL/6J × SWR F2 mice were poorly correlated, indicating independent regulation. Highly significant (P < .01) polymorphic loci were found on chromosomes 2 and 16 for liver and on chromosomes 8 and 9 for spleen. With sex as a covariate, additional significant or suggestive (P < 0.1) QTL were detected on chromosomes 7, 8, 11, and 19 for liver and on chromosome 2 for spleen. A gene array showed no clear association between most loci and differential iron‐related gene expression. The gene for transferrin and a transferrin‐like gene map close to the QTL on chromosome 9. Transferrin saturation was significantly lower in C57BL/6J mice than in SWR mice, but there was no significant difference in the serum level of transferrin, hepatic expression, or functional change in cDNA sequence. β2‐Microglobulin, which, unlike other loci, was associated with C57BL/6J alleles, is a candidate for the chromosome 2 QTL for higher iron. In conclusion, the findings show the location of polymorphic genes that determine basal iron status in wild‐type mice. Human equivalents may be pertinent in predisposition to hepatic and other disorders. (HEPATOLOGY 2006;44:174–185.)

Isolation of Aspergillus fumigatus from sputum is associated with elevated airborne levels in homes of patients with asthma

Indoor bioaerosols, such as mold spores, have been associated with respiratory symptoms in patients with asthma; however, dose-response relationships and guidelines on acceptable levels are lacking. Furthermore, a causal link between mold exposure and respiratory infections or asthma remains to be established. The aim of this study was to determine indoor concentrations of Aspergillus fumigatus and a subset of clinically relevant fungi in homes of people with asthma, in relation to markers of airways colonization and sensitization. Air and dust samples were collected from the living room of 58 properties. Fungal concentrations were quantified using mold-specific quantitative PCR and compared with traditional microscopic analysis of air samples. Isolation of A. fumigatus from sputum was associated with higher airborne concentrations of the fungus in patient homes (P = 0.04), and a similar trend was shown with Aspergillus/Penicillium-type concentrations analyzed by microscopy (P = 0.058). No association was found between airborne levels of A. fumigatus and sensitization to this fungus, or dustborne levels of A. fumigatus and either isolation from sputum or sensitization. The results of this study suggest that the home environment should be considered as a potential source of fungal exposure, and elevated home levels may predispose people with asthma to airways colonization.

Method for Determining Whether the Number of Hepatocytes in Rat Liver is Increased after Treatment with the Peroxisome Proliferator Gemfibrozil

A histological method has been developed for determining the absolute numbers of rodent hepatocytes after treatment with the hypolipodemic drug gemfibrozil. It can be applied to distinguish between the enlargement of the liver that commonly occurs in rodents after treatment with chemicals, due to changes in the size of cells (hypertrophy), rather than an increase in the number of cells caused by cell division (hyperplasia). In the case of gemfibrozil the liver enlargement was found to be partly due to hypertrophy and partly to hyperplasia. The induction of hyperplasia can be associated with an increased risk of eventual liver tumour formation, and the distinction of hypertrophy from hyperplasia using a purely histological method, for the determination of increases in hepatocyte cell numbers, will be useful in the assessment of compounds which cause liver enlargement that could precede neoplasia. © 1997 by John Wiley & Sons, Ltd.

“Have You Seen This?” Peliosis Hepatis

Peliosis hepatis, characterized by the presence of blood-filled spaces within hepatic parenchyma, developed in C57B1 mice implanted subcutaneously with melanoma cells 23 days previously. The peliosis was associated with dilated hepatic sinusoids that were lined by prominent, proliferating endothelial cells. The development of peliosis hepatis was completely abrogated when melanoma growth was inhibited by administration of dexamethasone. These features support the concept that peliosis hepatis can be induced by a circulating tumor-derived endothelial growth factor such as vascular endothelial growth factor.

The preparation of highly enriched fractions of binucleated rat hepatocytes by centrifugal elutriation and flow cytometry.

A procedure is described for the isolation of highly enriched fractions of binucleated hepatocytes from rat liver. Liver cells isolated by EGTA and collagenase perfusion were initially subjected to centrifugal elutriation and second to flow cytometry coupled with Hoechst 33342 staining. The elutriation step yielded hepatocyte fractions which contained almost entirely mononuclear diploid cells and fractions enriched in binucleate hepatocytes. The fractions with the highest proportion of binucleated hepatocytes contained between 50 and 56% of these cells. Subsequent flow cytometric cell sorting yielded fractions which contained greater than 80% binucleated cells. These cells were viable in culture as demonstrated by the immunohistochemical detection of bromodeoxyuridine incorporation.