Interaction between haemochromatosis and transferrin receptor genes in hepatocellular carcinoma.

In hepatocellular carcinoma (HCC) iron has been implicated as a risk factor primarily in patients with hereditary haemochromatosis (HH) and cirrhosis. The wild-type HH (HFE) protein complexes with the transferrin receptor (TFR), and two HFE mutations (Cys282Tyr and His63Asp) have been found to increase the affinity of the TFR for transferrin resulting in an increased cellular uptake of iron. In previous studies we found an interaction between HFE and TFR genotypes in multiple myeloma and breast and colorectal carcinomas. In the present investigation we have studied HFE and TFR genotypes in 54 Swedish patients with HCC, using DNA from archival samples of paraffin wax blocks. The same HFE-TFR interaction as in the previously studied neoplastic disorders was found. Individuals carrying the HFE282Tyr allele (homo- and heterozygotes) in combination with homozygosity for the TFR Ser allele showed an increased risk for HCC (OR = 3.5; 95% confidence interval, CI = 1.3-9.3), which was further increased in HFE Tyr homozygotes and compound (Tyr/Asp) heterozygotes in combination with TFR 142Ser homozygosity (OR = 17.2; 95% CI = 1.8-168.9). The presence of liver cirrhosis could only be assessed in part of the patient material. In patients with verified liver cirrhosis the risk figures were substantially increased: for HFE 282 Tyr carriers in combination with TFR 142 Ser/Ser OR = 7.2; 95% CI = 2.0-25.5 and for HFE 282Tyr homozygotes and compound heterozygotes in combination with TFR 142Ser homozygosity, OR = 62.8; 95% CI = 6.1-642.5.

Transferrin-mediated uptake of aluminium by human parathyroid cells results in reduced parathyroid hormone secretion.

BACKGROUND: The present study investigates whether aluminium-transferrin (Al-Tf) uptake by Tf receptor-mediated endocytosis induces hypoparathyroidism and thus might contribute to the increasing prevalence of adynamic bone disease (ABD) in the current dialysis population. METHODS AND RESULTS: Human parathyroid glands as well as in vitro cultured human parathyroid cells were shown to express Tf receptors. Five-day-old cultures of parathyroid cells were incubated for 48 h in serum-free DMEM/F12 supplemented with 12 microM apo-Tf: 12 microM Tf to which 150 microg/l Al or 150 microg/l Al-citrate (Al-ci) was bound. The amount of Al taken up by the parathyroid cells either as Al-Tf or Al-ci did not differ. However, incubation of cell cultures with Al-Tf showed a significant proportional decrease (mean+/-SEM, -23.1+/-4.5%) in iPTH secretion as compared to the reference apo-Tf cultures. Al-ci did not suppress PTH secretion (+3.4+/-6.5%). The Al uptake after incubation with Al-Tf was found to be dose-dependent. With regard to iPTH secretion, a tendency toward a dose response relationship was observed. Northern blot analysis of parathyroid cells incubated in 12 microM apo-Tf or 12 microM Al-Tf demonstrated that the PTH mRNA synthesis was unaffected by the Tf-mediated uptake of Al. These observations suggest an effect of Al on PTH release rather than on PTH synthesis. Since the cytoskeleton can play an important role in the release of secretory vesicles, the influence of Al on the structure of actin, beta-tubulin and vimentin was investigated by confocal microscopy. Comparison of cultures incubated with apo-Tf and Al-Tf revealed no difference in the organization of these cytoskeletal proteins in relation to the inhibitory effect of Al-Tf on PTH secretion. CONCLUSION: In summary, data in the present paper demonstrate that the (i) human parathyroid gland/parathyroid cells exhibit Tf receptors; (ii) Al-Tf complex is taken up by the parathyroid gland in a dose-dependent manner; and (iii) uptake of Al by Tf receptor-mediated endocytosis reduces the secretion of PTH but not its synthesis. These in vitro findings allow us to suggest that Tf receptor-mediated uptake of Al might, besides other factors such as vitamin D, high calcium dialysate or CaCO(3) intake, play a role in the development of hypoparathyroidism associated with ABD. The exact mechanism by which Al-Tf suppresses iPTH secretion remains to be elucidated.

Manganese-containing superoxide dismutase signal sequence polymorphism associated with sporadic motor neuron disease.

An alanin-9valin (Ala-9Val) polymorphism in the mitochondrial targeting sequence of manganese-containing superoxide dismutase (Mn-SOD) has recently been described. We studied this polymorphism in 72 Swedish patients with sporadic motor neuron diseases (MND) and controls using an oligonucleotide ligation assay. There were significant differences in genotype between MND patients and controls (P = 0.025), and between male and female MND patients (P = 0.009). Individuals homozygous for the Ala allele had a higher risk for MND [odds ratio, 2.9; 95% confidence interval (CI), 1.3-6.6], which was increased when including only females in the analysis (odds ratio, 5.0; 95% CI, 1.8-14.0). In classical amyotrophic lateral sclerosis, the odds ratio was 3.8 (95% CI, 1.3-10.0), and 5. 5 (95% CI, 1.5-19.9) when including only females. The results suggest that mutations influencing the cellular allocation of Mn-SOD may be a risk factor in MND, especially in females, and that MND may be a disease of misdistribution of the superoxide dismutase enzymes.

Ethnic variation in the mitochondrial targeting sequence polymorphism of MnSOD.

In contrast to CuZn superoxide dismutase (SOD), only a very limited number of mutations have been described in MnSOD. One interesting example is a polymorphism (Ala-9Val) in the mitochondrial targeting sequence of this radical-scavenging enzyme. We have studied the Ala-9Val polymorphism in various ethnic groups by means of the oligonucleotide ligation assay. There were significant variations in this unique polymorphism between three different language groups: Baltic (Lithuanians), Finnic (Finns and Saamis) and Germanic (Swedes). The Ala frequency in an Asiatic population (Chinese) was significantly lower than in most European populations. This polymorphism may affect the mitochondrial targeting rate of MnSOD which may result in mitochondrial damage with implication in various late-onset neurological diseases.

Interaction between haemochromatosis and transferrin receptor genes in different neoplastic disorders.

A number of genes are involved in iron metabolism, including the transferrin receptor (TFR) and haemochromatosis (HFE) genes. In previous investigations an increased risk for neoplastic disease has been observed in individuals homo- and heterozygous for hereditary haemochromatosis. The HFE wild-type gene product complexes with the transferrin receptor (TF) and two different HFE mutations (Cys282Tyr and His63Asp) have been found to increase the affinity of TFR for TF and increase cellular iron uptake. In a recent study we found no associations for HFE and TFR separately, but an interaction between HFE and TFR genotypes in multiple myeloma. Individuals carrying the HFE Tyr282 allele (homo- and heterozygotes) in combination with homozygosity for the TFR Ser142 allele had an increased risk. In the present study the same association was found in breast and colorectal cancer. The odds ratio for all three neoplasms combined was 2.0 (95% CI 1.0-3.8). The risk for neoplastic disease was further increased (OR 7.7, 95% CI = 1.0-59.9) when the analysis was restricted to HFE Tyr homozygotes and compound heterozygotes in combination with TFR Ser homozygosity. Thus, an interaction between HFE and TFR alleles may increase the risk for different neoplastic disorders.

Protective effect of transferrin C3 in lung cancer?

In a previous study of lung cancer patients and controls from the Stockholm area in central Sweden, we found a significantly decreased frequency of the transferrin (TF) variant C3 in small cell and squamous epithelial lung cancer but not in adenocarcinoma, suggesting a protective effect of TF C3 in small cell and squamous lung cancer. In an attempt to replicate this association we studied TF types in lung cancer patients and controls from two additional Swedish subpopulations, viz. northern Sweden and southwestern Sweden. We were able to confirm the significantly decreased frequency of TF C3, especially in small cell lung cancer, in northern Sweden but not in southwestern Sweden. Thus the eventual protective effect of TF C3 in small cell lung cancer is an open question. We hypothesize that the association between TF C alleles and lung cancer may be secondary and dependent on linkage disequilibrium with allelic variants of newly discovered tumor-associated genes known to map to the same position (3q21) as TF, e.g. NCK and H-RYK.

New DNA polymorphisms define ethnically distinct haplotypes in the human transferrin receptor gene.

In a study of transferrin receptor (TFR) polymorphism in different ethnic groups using PCR and restriction cleavage we found a new Hin6I polymorphism in intron 7 and confirmed a tentative BanI polymorphism in exon 4 reported by Evans and Kemp [Gene 1997;199:123-131]. In all ethnic groups there was a complete and highly significant (p < 10(-10)) linkage disequilibrium where all BanI 1 alleles were linked to Hin6I 1 alleles. Furthermore in the European populations, but not in the Chinese, there was a close correlation between the three BanI-Hin6I haplotypes and the alleles of a previously described three-allelic RsaI polymorphism in the TFR gene studied by Southern blotting. There were distinct ethnic differences in TFR allele and haplotype frequencies. Thus the Saamis were significantly different from the other European ethnic groups, and the Lithuanians had a significantly increased frequency of the BanI 2-Hin6I 1 haplotype, suggesting that this marker may be informative in tracing prehistoric migrations and admixture by Baltic peoples. The new TFR polymorphisms and haplotypes may also be useful markers in studies of interactions with the transferrin and hemochromatosis genes, the genetic influence on body iron stores and disease associations.

Low serum aluminum values in dialysis patients with increased bone aluminum levels.

BACKGROUND: Using an HPLC/ETAAS hybrid speciation technique we previously demonstrated iron to have a multifold effect on the binding of aluminum to transferrin by limiting the number of available binding sites and decreasing the affinity of transferrin for aluminum. Theoretically, at a 60% iron-transferrin saturation the aluminum-transferrin fraction in serum should not exceed 30 microg/l. In the present study previous experimental data were confronted with recent clinical observations in patients with either normal iron status or iron overload. PATIENTS AND RESULTS: Serum aluminum levels and iron overload: In 38 dialysis patients with a normal iron status and of whom 63% received Al(OH)3 for phosphate binding 26 (68%) had a serum aluminum level >30 microg/l. On the other hand out of 28 transfusional iron overloaded patients; 68% of them taking Al(OH)3, only 1 subject (4%) had a serum aluminum value in excess of 30 microg/l. Taking patients of both groups receiving Al(OH)3 together a significant (p = 0.001) negative correlation (r = -0.5017) was found between the iron-transferrin saturation and the serum aluminum levels. Iron status and parenteral aluminum loading: Also could a significant (p = 0.001) negative correlation (r = -0.6383) between these parameters be found in an independent group of 44 patients which were acutely intoxicated by the use of aluminum-contaminated dialysis fluids. Since in this population aluminum loading occurred parenterally and not via the gastrointestinal tract, a direct effect of iron on the transferrin binding of aluminum rather than on the element's gastrointestinal absorption must have been responsible for the inverse relationship. Bone aluminum and iron overload: Out of 22 patients with a normal iron status (mean + SD serum ferritin: 216 +/- 245 microg/l; iron-transferrin saturation 20.4 +/- 9.6%), all of them having aluminum overload (bone aluminum level >15 microg/g and/or positive Aluminon staining) none of them presented with a serum aluminum <30 microg/l (mean +/- SD: 82.2 +/- 51.6 microg/l). On the other hand out of 13 iron overloaded patients (serum ferritin >800 microg/l; iron-transferrin saturation 61.4 +/- 17.6%) 10 (77%) presented the proposed criteria of aluminum overload in the presence of a serum aluminum level <30 microg/l. CONCLUSIONS: Our data indicate that in dialysis patients with iron overload (iron-transferrin saturation >60%; serum ferritin >800 microg/l) serum aluminum levels are low (<30 microg/l) despite exposure to aluminum by the intake of Al(OH)3 or the use of aluminum-contaminated dialysis fluids. Low serum aluminum nevertheless may be associated with aluminum overload and even aluminum-related bone disease. An effect of iron on the serum aluminum speciation may at least in part explain our observations. Our findings allow a more accurate interpretation of baseline serum aluminum values.

Al and Si: their speciation, distribution, and toxicity.

OBJECTIVES: In dialysis patients both aluminum (AI) and silicon (Si) may accumulate. Whereas the toxic effects of AI within this population are clearly established, little is known on the role of Si in the development/protection of particular dialysis-related diseases. A clear insight in the protein binding and speciation of trace elements is important to better understand the mechanisms underlying their toxicity/essentiality. Research in this field however is complex and often prone to analytical difficulties and inaccuracies. DESIGN AND METHODS: In the first part of this review techniques used for speciation studies of AI and Si in biological fluids are discussed. Notwithstanding recent technical advances (a) extraneous metal contamination, (b) unrecognized aspecific binding of metals to proteins, and (c) unwanted interactions with separation equipment such as chromatography columns and ultrafiltration membranes remain important pitfalls and often lead to erroneous conclusions. The factors that determine the speciation of AI and Si and their ultimate tissue distribution and toxicity are dealt with in the second part. Here, experimental data obtained with various speciation techniques are linked to in vivo data on the tissue distribution, localization/toxicity of both elements. CONCLUSIONS: A model in which the AI tissue distribution/toxicity is mediated by either its citrate or transferrin bound form is proposed.

DNA polymorphisms and haplotypes in the human transferrin gene.

Although a large number of human serum transferrin (TF) variants have been described, only one RFLP (AvaI) has so far been found. Here we report three new RFLPs (MvaI in intron 5 and exon 7, BbvI in exon 7) and correlations between RFLPs and between RFLPs and serum TF types. There were strong, but not always complete, disequilibria between RFLP and serum protein alleles. Thus, the most common serum TF variant, C1, was heterogeneous and could be subdivided into two common haplotypes, whereas the C2, C3, and DCHI variants were completely or almost completely (C2) homogeneous. There was a total genotypic agreement between the BbvI polymorphism and the presence/absence of the TF C3 variant, and the mutation that creates the BhvI site was found to lead to a G258S amino acid substitution.

Transferrin C2, metal binding and Alzheimer's disease.

Significant associations between the transferrin (TF) variant C2 and a number of disorders suspected to be caused by oxygen free radicals have been reported. Thus an increased frequency of the TFC2 variant has been found in patients with Alzheimer's disease (AD), and it has been hypothesized that AD is caused by free radical damage due to defective binding of iron and aluminium by TFC2. In a study of 64 patients with AD from northern Sweden we were able to confirm the association between TFC2 and AD, but there were no significant differences between TFC2 and other TF variants with respect to the binding of iron and aluminium.

Ethnic differences in the HFE codon 282 (Cys/Tyr) polymorphism.

Recent studies have shown that hereditary hemochromatosis (HH) is likely to be caused by homozygosity for a Cys282Tyr mutation in the HFE gene located 4.5 Mb telomeric to HLA-A. Population studies of this polymorphism are facilitated by the fact that the Cys282Tyr mutation creates a Rsal restriction site. We have studied the codon 282 (Cys/Tyr) polymorphism in different ethnic groups. In agreement with previous observations the Tyr allele appeared to be rare or absent in Asiatic (Indian, Chinese) populations. The highest allele frequency (7.5%) was found in Swedes. Saamis (2%) and Mordvinians (1.8%) had significantly lower frequencies of the Tyr allele. Comparisons with allele frequencies based on prevalence estimates of HH showed some disagreements with the RFLP data, particularly in Finns. The newly described HFE marker provides a new approach to the screening of HH as well as studies of the relationship between the HFE Tyr allele and different disorders including cancer.

Aluminium speciation in cerebrospinal fluid of acutely aluminium-intoxicated dialysis patients before and after desferrioxamine treatment; a step in the understanding of the element's neurotoxicity.

BACKGROUND: The association between aluminium and dialysis encephalopathy and deterioration of the neurological state during desferrioxamine treatment of dialysis patients is well established. At present little is known about the speciation and the mechanisms underlying the element's neurotoxicity. METHODS: Aluminium speciation was performed in cerebrospinal fluid samples of acutely aluminium-intoxicated dialysis patients using a recently developed high-performance liquid chromatographic/electrothermal atomic absorption spectrometric hybrid method. RESULTS: Baseline cerebrospinal fluid aluminium levels of samples taken shortly after the intoxication were low but elevated (5.0 +/- 2.0 micrograms/l, n = 3) as compared to subjects with normal renal function (< 1 microgram/l). In contrast to the situation noted in serum and to the iron speciation in cerebrospinal fluid, aluminium was not bound to transferrin but appeared as two distinct compounds, the main fraction eluting at the elution volume of aluminium-citrate/silicate. The second compound was not identified. Forty-four hours after desferrioxamine administration the cerebrospinal fluid aluminium levels had increased up to a concentration of 10.3 +/- 2.5 micrograms/l (n = 3). This was accompanied by a change in the speciation profile with aluminium appearing at the elution volume of aluminoxamine. CONCLUSIONS: Our findings may contribute to a better understanding of the neurotoxic effects of aluminium and its desferrioxamine chelate in dialysis patients.

Competition of iron and aluminum for transferrin: the molecular basis for aluminum deposition in iron-overloaded dialysis patients?

In the recent literature an inverse relationship between iron status and serum aluminum levels has repeatedly been reported in dialysis patients. To check whether this observation is, at least in part, due to an interference of iron with the protein binding of aluminum, we studied the effect of the latter element on both the number of free binding sites on transferrin (Tf) and on the affinity of the protein for aluminum. For the purpose of this, a recently developed HPLC-ETAAS hybrid method was used, allowing protein-binding studies at clinical relevant metal concentrations and under contamination-free conditions. After we incubated apo-Tf with iron and aluminum which were added in amounts equivalent to the calculated number of metal-binding sites on the protein (i.e., 2 mol metal/mol Tf), we found that Tf can be saturated for 100% with iron. However, for aluminum only a 23% aluminum-Tf saturation was observed. In Tf solutions with iron saturations ranging between 0 and 60% as well as in the serum of 15 subjects with iron-Tf saturations varying between 12 and 48%, a significant (p < 0.001) negative correlation between the degree of iron-Tf saturation and the percentage of aluminum (added in amounts equivalent to the number of the remaining binding sites on Tf) bound to Tf was noted (y = -0.26x + 24.5, r = -0.87 in serum). It is concluded that the iron-Tf saturation influences the Tf binding of aluminum not only by occupying binding sites otherwise available for aluminum, but also by lowering the affinity of Tf for aluminum. The effects of iron on serum aluminum levels and bone aluminum deposition are discussed.

Measurement of strontium in serum, urine, bone, and soft tissues by Zeeman atomic absorption spectrometry.

To study the possible accumulation of Sr in chronic renal failure patients, methods were developed for the determination of the element in serum, urine, bone, and soft tissues by using Zeeman atomic absorption spectrometry. Serum samples were diluted 1:4 with a Triton X-100-HNO3 mixture, whereas urine samples were diluted 1:20 with HNO3. Bone samples were digested with concentrated HNO3 in stoppered polytetrafluoroethylene (Teflon) tubes, whereas soft tissues were dissolved in a tetramethylammonium hydroxide solution in water. For serum and urine we used matrix-matched calibration curves, whereas bone and tissue samples were measured against aqueous calibrators. Atomization was performed from the wall of pyrolytically coated graphite tubes for all of the matrices under study. Both inter- and intraassay CVs were <6% (n = 12, n = 10, respectively), and the recovery of added analyte was close to 100% for all of the biological matrices under study. Detection limits were 1.2 microg/L (serum), 0.3 microg/L (urine), 0.4 microg/g (bone), and 2.2 ng/g (soft tissues), whereas the sensitivity determined by the slope of the calibration curve, i.e., the amount of Sr producing a 0.0044 integrated absorbance change in signal, was 2.4 pg, 2.4 pg, 3.9 pg, and 2.6 pg for these matrices respectively. We conclude that the present methods are precise and accurate and easily applicable for both routine use and research investigations. They will allow us to study the metabolism of the element in chronic renal failure patients and shed some light on the association that was recently noted between increased bone Sr concentrations and the development of osteomalacia in these individuals.

Quantitative determination of transferrin in cerebrospinal fluid using an enzyme linked immunosorbent assay.

An enzyme linked immunosorbent assay (ELISA) based method for the determination of transferrin in cerebrospinal fluid (CSF) is described. This method allows transferrin determinations in ultra-small (

An in vitro study on the binding of Al(III) to human serum transferrin with the isoelectric focusing technique.

Transferrin saturated with Al3+ subjected to isoelectric focusing (IEF) in a pH gradient can be separated into four fractions, representing the apotransferrin, transferrin with aluminum at the metal binding site in the C- or N-terminal lobe, or both. The electrophoretic mobilities of these four fractions are identical to those of the iron-transferrin counterparts. Simultaneous binding of aluminum and iron to transferrin can also be demonstrated. The decreased saturation after IEF indicates that the affinity of transferrin for aluminum is low compared with its affinity for iron. This effect is particularly evident when bicarbonate is used as the synergistic anion in the loading procedure. In contrast, loading of transferrin with aluminum in the presence of oxalate produces a di-aluminum-transferrin complex that is stable during IEF.

Cytotoxicity of mercury compounds in LLC-PK1, MDCK and human proximal tubular cells.

Six mercury compounds [HgCl2 (MC), Hg(CH3COO)2 (MA), Hg(NO3)2 (MN), C2H5HgSC6H4COONa (EMT), C6H5HgOCOCH3 (PMA) and CH3CIHg (MMC)] were studied using two kidney cell lines (MDCK and LLC-PK1), primary cultures of human proximal tubular cells (hPTC) and nonrenal cell lines (SAOS and Hep G2). Cell damage was measured with four different tests: neutral red uptake, mitochondrial dehydrogenase activity (MTT conversion), thymidine incorporation and protein content. Relative toxicity was established by the determination of the concentration of test compound inducing a 50% reduction of the parameter considered (EC50 value). Two groups could be distinguished: PMA, EMT and MMC are one order of magnitude more toxic than MC, MN and MA. Cellular uptake was measured by the HPLC-hybrid generation AAS after 24 hours treatment with 1.5 microM MC, MMC, PMA or EMT in MDCK cells, revealing Hg concentrations of 42.8 +/- 2.5 ng/mg protein for MC, 596.9 +/- 87.8 ng/mg protein for MMC, 269.8 +/- 75.7 ng/mg protein for PMA and of 115.9 +/- 25.2 ng/mg protein for EMT. Cytotoxicity was positively correlated with cellular uptake. The effect of the cellular GSH content on the toxicity of mercury was studied using the GSH synthesis inhibitor L-buthionine sulfoximine (BSO). In all cases an enhanced cytotoxicity was observed after BSO treatment. 2-Oxo-4-thiazolidine carboxylic acid (OTC) was used as a substrate for the GSH synthesis. Although OTC did not enhance the GSH content, the cytotoxicity of MC, MN and MA decreased significantly, no changes were observed for the other mercurials.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative HPLC/ETAAS hybrid method with an on-line metal scavenger for studying the protein binding and speciation of aluminum and iron.

A quantitative metal-free high-performance liquid chromatographic/electrothermal atomic absorption spectrometric (HPLC/ETAAS) technique allowing an adequate separation of the proteins and of the inorganic/organic metal species of interest was developed. A silica-based scavenger was placed proximal to the injection valve retaining any remaining Al and Fe originating from buffer solutions and recipients. ETAAS instrumental conditions and matrix modifiers were carefully selected to eliminate interferences occurring due to the salt gradient elution. The postelution Al recovery (143-1428 micrograms L-1; 100-microL sample volume) was found to be 100.1 +/- 15.3% (N = 50) whereas that of Fe was 97.3 +/- 2.0% (N = 20; 442-2653 micrograms L-1). The protein recovery varied between 95 and 105%. The HPLC/ETAAS intraassay CV (N = 3) was 8.0% for Al (eluted amount of Al bound to transferrin (Tf), 7.3 ng) whereas that of Fe was 1.4% (eluted amount of Fe bound to Tf, 124.8 ng). The intraassay CV of the HPLC Tf determination was 1.6%. The proposed method is sensitive and accurate, allowing for the first time quantitative evaluation of the competition between Al and Fe for protein binding at clinical relevant concentrations. The technique will also be used for studying the protein binding of elements other than Al and Fe.