Transferrin C subtypes and occupational photodermatosis of the face

In a factory in northern Sweden where 120 workers were uniformly exposed to photoactive substances 73 developed occupational facial eczema while 47 showed no reaction. The workers were examined with respect to 16 genetic marker systems: HLA, blood groups (ABO, Rh, MNSs, P, K, Le and Fy) and serum groups (Hp, Tf, Gc, Pi, Bf, C3, C4 and C6). Between reactors and nonreactors the following differences were found: (1) a significant decrease (p less than 0.05) of HLA A11 among the reactors; (2) a significant increase (p less than 0.05) of the C3 FS type among the reactors; (3) a highly significant increase (p less than 0.001) of the transferrin C2 gene and of the C2 variant among the reactors. The association with Tf C2 remained significant also after correction for number of significance tests. Since transferrin (iron) is known to catalyze the formation of hydroxyl radicals we hypothesize that the Tf C2 variant is more efficient in promoting radical formation and thereby cell damage. Other results supporting the notion that transferrin C2 may be associated with an increased susceptibility to toxic damage are discussed.     

Kinetics of chromosomal aberrations and first mitosis division in human lymphocytes exposed to mitomycin C

In order to understand the relationship between the chromosomal damage detectable at the first mitosis after mutagen treatment and the induced mitotic delay we studied the time pattern of both mitotic indices and chromosomal aberration frequencies in human lymphocytes treated in G1 with mitomycin C (2.5 microM) and cultured in vitro in the presence of 5-bromo-2'-deoxyuridine. Mitotic delay was observed in treated cells cultured for 81 h. At this point an increase in the frequency of chromosomal aberrations is evident and a higher proportion of abnormal cells enters mitosis, the long delay being due to the extensiveness of DNA damage. The importance of cell cycle progression for the detection of the maximal amount of induced chromosomal damage is discussed.     

Decrease of transferrin C2 frequency with age

In previous studies, transferrin C2 has been found to be associated with spontaneous abortion, prematurity, phototoxic eczema and rheumatoid arthritis. We have suggested that the mechanism behind these negative effects may be that transferrin C2 increases the risk for damage through hydroxyl radicals. This hypothesis predicts that the C2 frequency should decrease with age. Such an effect is demonstrated in this report. In a population from northern Sweden the C2 gene frequency was found to decrease from 0.173 in newborns to 0.099 in 70-year-old healthy individuals.     

Cell Cycle Kinetics and Radiation-Induced Chromosomal Aberrations Studied with C14 and H3 Labels

Chinese hamster cells in vitro were double labeled with C(14)TdR and H(3)TdR. At the time of irradiation with Co(60) gamma rays (600 rad), the cells in the G(2) phase were labeled only with C(14), whereas cells in the late and middle S phases were labeled with both C(14) and H(3). The cells in early S phase were labeled only with H(3) and the G(1) cells were unlabeled. Samples were fixed at various time intervals following irradiation and the metaphases were analyzed for chromosomal damage. The phase in which the cell was located at the time of irradiation was determined by counting grains in the first and second layers of autoradiographic film. In both control and irradiated cells some G(1) cells divided prior to some of the cells which were in the S phase denoting mixing of the populations. The G(2) phase sustained three times more chromosomal damage than the S phase. Little difference in chromosomal damage was found between the G(1) and S phases or among the different parts of the S phase. Cells in G(2) sustained a mitotic delay of 4 hr, while the other phases sustained a delay of 2 to 3 hr. Chromatid and chromosome (dicentrics) exchanges were induced in G(1) cells but only chromatid exchanges were induced in S and G(2) cells; this is consistent with the hypothesis that the chromosome consists of two subunits which separate either slightly before or immediately as the cell enters the S phase.     

Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization

Incubation of 3T3-L1 adipocytes with insulin at 37 degrees C resulted in a 2-fold increase in specific binding of transferrin to cell-surface receptors, as measured by a subsequent incubation of cells at 4 degrees C with 125I-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 s. By comparison, insulin stimulated hexose transport in 3T3-L1 adipocytes with a half-maximal effect at 8 nM and a half-time of 105 s. Scatchard analysis of 125I-transferrin binding to cells at 4 degrees C showed that the insulin-induced increase in transferrin receptor binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 degrees C with 125I-transferrin to achieve steady-state binding and then exposed to insulin, there was a 1.7-fold increase in surface-bound transferrin (acid-sensitive) and a corresponding decrease in intracellularly bound transferrin (acid-insensitive). Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin, there was a 2-fold increase in the rate of 59Fe3+ uptake from 59Fe3+-loaded transferrin. The rate of externalization of the intracellular 125I-transferrin-receptor complex at 37 degrees C was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 1.7-fold. The effect of insulin on the rate of externalization is sufficient to account for the increase in surface transferrin receptors.     

Anti-genotoxic effects of tea catechins against reactive oxygen species in human lymphoblastoid cells

Using the cytokinesis-block micronucleus assay in WIL2-NS cells, we investigated the effects of six tea constituents, (-)-epigallocatechin-3-O-gallate (EGCg), (-)-epicatechin-3-O-gallate (ECg), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (+C) and gallic acid (GA), on chromosomal damage in two ways; induction by each component on its own and prevention against treatment of reactive oxygen species (ROS). None of the tea constituents induced chromosomal damage at <10 microM. On the other hand, EGCg, EGC, ECg, +C and GA prevented H(2)O(2)-induced chromosomal damage in a dose-dependent manner with a significant effect detected at 1 microM. Chromosomal damage induced by tert-butylhydroperoxide was apparently prevented by EGCg and ECg at 0.3 microM, but not by EGC and GA even at 10 microM, suggesting that the galloyl group linked to flavan-3-ol is needed for the observed protective effect. These results suggest that physiological concentration of tea constituents are not genotoxic but rather anti-genotoxic against ROS, although their preventive effects are slightly different depending on their chemical structure.     

Protective effects of quercetin and its metabolites on H2O2-induced chromosomal damage to WIL2-NS cells

We investigated chromosomal damage caused by a typical flavonoid, quercetin, and its two conjugates, quercetin-3-O-sulfate and isorhamnetin, and their protective effects against chromosomal damage induced by H2O2. The chromosomal damage was detected by the cytokinesis-block micronucleus (CBMN) assay using a lymphoblastoid cell line, WIL2-NS. We found that quercetin itself induced chromosomal damage at 10 microM, but quercetin-3-O-sulfate and isorhamnetin did not induce damage up to 30 microM. In the medium used for the CBMN assay, quercetin (at 100 microM) generated a high concentration of H2O2, but the two conjugates did not at the same concentration. On the other hand, pretreatment with quercetin (at 1 microM), quercetin-3-O-sulfate (at 10 microM), and isorhamnetin (at 5 microM) prevented H2O2-induced chromosomal damage to WIL2-NS cells. These findings suggest that the induction and prevention of H2O2-induced chromosomal damage are different between quercetin and its metabolites.     

Iron chelators reduce chromosomal breaks in ataxia-telangiectasia cells

Ataxia-telangiectasia (A-T) is characterized by ataxia, genomic instability, and increased cancer incidence. Previously, iron chelator concentrations which suppressed normal cell colony formation increased A-T cell colony formation. Similarly, iron chelators preferentially increased A-T cell colony formation following peroxide exposure compared to normal cells. Last, A-T cells exhibited increased short-term sensitivity to labile iron exposure compared to normal cells, an event corrected by recombinant ATM (rATM) expression. Since chromosomal damage is important in A-T pathology and iron chelators exert beneficial effects on A-T cells, we hypothesized that iron chelators would reduce A-T cell chromosomal breaks. We treated A-T, normal, and A-T cells expressing rATM with labile iron, iron chelators, antioxidants, and t-butyl hydroperoxide, and examined chromosomal breaks and ATM activation. Additionally, the effect of ATM-deficiency on transferrin receptor (TfR) expression and TfR activity blockage in A-T and syngeneic A-T cells expressing rATM was examined. We report that (1) iron chelators and iron-free media reduce spontaneous and t-butyl hydroperoxide-induced chromosomal breaks in A-T, but not normal, or A-T cells expressing rATM; (2) labile iron exposure induces A-T cell chromosomal breaks, an event lessened with rATM expression; (3) desferal, labile iron, and copper activate ATM; (4) A-T cell TfR expression is lowered with rATM expression and (5) blocking TfR activity with anti-TfR antibodies increases A-T cell colony formation, while lowering chromosomal breaks. ATM therefore functions in iron responses and the maintenance of genomic stability following labile iron exposure.     

Characterization of IIB-MEL-J: a new and highly heterogenous human melanoma cell line

A highly heterogeneous cell line, IIB-MEL-J, was established from a human metastatic melanoma. This cell line contains small cells, dendritic cells, and megacells with multiple nuclei. IIB-MEL-J expresses S 100, cytokeratin intermediate filaments and the gangliosides GD2 and GD3. It requires growth factors (insulin, EGF, and transferrin) and antioxidants for optimal growth. When plated under optimal conditions, IIB-MEL-J grows with a doubling time of 70-80 hours. The cells may be fractionated by Percoll gradient centrifugation into several subpopulations (A, B, and C) with different characteristics. Subpopulation A is the slowest growing, and most of the DNA-synthesizing cells are concentrated in fractions B and C. Every subpopulation expresses S 100 and cytokeratin intermediate filaments, whereas only subpopulation B and C express GD2 and GD3. Pigmented cells are concentrated mainly in subpopulation C. Cytogenetic analysis of IIB-MEL-J revealed extensive chromosomal alterations, including a highly heterogeneous chromosome number and chromosomal rearrangements, gains, losses, isochromosomes, and double minutes. This highly heterogeneous cell line may be helpful to study cellular differentiation and interaction between different subpopulations in human melanoma.     

Subtypes of transferrin C.

Subtypes of transferrin C were studied by means of isoelectric focusing after complete desialylation of transferrin. Family data were consistent with an autosomal co-dominant mode of inheritance. Studies of serum samples from 75 individuals heterozygous for C and another (B or D) variant showed that the genes (C1 and C2) controlling the C subtypes are allelic to the B and D genes. The C2 gene frequency in Swedes and Swedish Lapps was similar to that found previously in Danes and Germans.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Evaluation of chromosomal damage by flow cytometry in turbot (Scophthalmus maximus L.) exposed to fuel oil

Flatfishes, turbots (Scophthalmus maximus), were injected intraperitoneally with two doses of fuel oil number 2. Biliary metabolites were evaluated by fixed fluorescence to verify the efficiency of intoxication. Ethoxyresorufin-O-deethylase (EROD) activity was compared with chromosomal damage measured by flow cytometry. The analysis of biliary metabolites showed a good dose-response relation and constitutes a clear reference for the subsequent measurements. Comparing flow cytometry and EROD results, a shorter delay of response for EROD activity was obtained, but chromosomal damage was significant only after 1 week. The persistence of the EROD response was shorter, while the genotoxic signal still persisted after 1 month. The measurement of chromosomal damage allowed a good differentiation between the two tested doses. In the case of EROD activity, the results were less clear. The results suggest that within a few weeks after exposure to fuel oil number 2, the measurements of chromosomal damage by flow cytometry can be used to detect a dose-dependant genotoxic response in fish.     

Evaluation of chromosomal damage by flow cytometry in turbot (Scophthalmus maximus L.) exposed to fuel oil

Flatfishes, turbots (Scophthalmus maximus), were injected intraperitoneally with two doses of fuel oil number 2. Biliary metabolites were evaluated by fixed fluorescence to verify the efficiency of intoxication. Ethoxyresorufin-O-deethylase (EROD) activity was compared with chromosomal damage measured by flow cytometry. The analysis of biliary metabolites showed a good dose–response relation and constitutes a clear reference for the subsequent measurements. Comparing flow cytometry and EROD results, a shorter delay of response for EROD activity was obtained, but chromosomal damage was significant only after 1 week. The persistence of the EROD response was shorter, while the genotoxic signal still persisted after 1 month. The measurement of chromosomal damage allowed a good differentiation between the two tested doses. In the case of EROD activity, the results were less clear. The results suggest that within a few weeks after exposure to fuel oil number 2, the measurements of chromosomal damage by flow cytometry can be used to detect a dose-dependant genotoxic response in fish.     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

Iron-transferrin-induced increase in protein kinase C activity in CCRF-CEM cells

Iron transferrin has been found to induce a mean 10-fold increase in the activity of protein kinase C in CCRF-CEM cells. This increase was not detectable up to 45 min after treatment of cells with iron transferrin, although after 60 min, a maximal increase in enzyme activity was observed. Similarly, iron transferrin at concentrations of 0.1-0.5 microgram/ml did not alter protein kinase C activity, while concentrations of iron transferrin of 1-100 micrograms/ml induced a maximal increase in enzyme activity. Apotransferrin and iron in the form of ferric citrate, as well as complexes of transferrin with copper, nickel, zinc, manganese, and cobalt did not increase protein kinase C activity. Additionally, CCRF-CEM cells pretreated with either actinomycin D or cycloheximide and then incubated with iron transferrin did not exhibit increased enzyme activity. Treatment with iron transferrin was found to have no effect on protein kinase C activity in normal human peripheral blood lymphocytes and in HL60, Daudi, and U937 cells. However, normal lymphocytes stimulated with phytohemagglutinin for 48 hr exhibited a 2-fold increase in protein kinase C activity following treatment with iron transferrin. These results indicate a specific effect of iron transferrin on protein kinase C activity in CCRF-CEM cells and in mitogen-stimulated human lymphocytes that may occur through increased synthesis of the enzyme.     

Reconstitution of the transferrin receptor in lipid vesicles. Effect of cholesterol on the binding of transferrin

Purified rabbit reticulocyte transferrin receptors were incorporated into phosphatidylcholine vesicles containing varying amounts of cholesterol. The binding of transferrin to the receptor in the reconstituted vesicles had three distinct characteristics: (1) The binding of transferrin exhibited the two components characteristic of transferrin binding to erythroid cells, a saturable, specific component and a nonsaturable, nonspecific component. (2) Transferrin binding exhibited positive cooperativity at low cholesterol/phospholipid (C/P) molar ratios. However, the cooperativity diminished and then disappeared as the C/P molar ratios were increased to the levels found in circulating red blood cells. (3) The amount of specific transferrin binding to the reconstituted vesicles also decreased as the C/P molar ratio was increased. These results indicate that in the reconstituted system the lipid environment plays a significant role in the expression of transferrin receptors.     

The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.     

Mapping of aminoacylase-1 and beta-galactosidase-A to homologous regions of human chromosome 3 and mouse chromosome 9 suggests location of additional genes.

Conserved linkage groups have been found on the X and autosomal chromosomes in several mammalian species. The identification of conserved chromosomal regions has potential for predicting gene location in mammals, particularly in humans. The genes for human aminoacylase-1 (ACY1, N-acylamino acid aminohydrolase, E.C.3.5.1.14), an enzyme in amino acid metabolism, and beta-galactosidase-A (GLB1, E.C.3.2.1.23), deficient in GM1-gangliosidosis, have been assigned to human chromosome 3. Using human-mouse somatic cell hybrids segregating translocations of human chromosome 3, expression of both ACY1 and GLB1 correlated with the presence of the p21 leads to q21 region of chromosome 3. In a previous study, assignment of these genes to mouse chromosome 9 used mouse-Chinese hamster somatic cell hybrids, eliminating mouse chromosomes. To approximate the size of the conserved region in the mouse, experiments were performed with recombinant inbred mouse strains. An electrophoretic variant of ACY-1 in mouse strains was used to map the Acy-1 gene 10.7 map U from the beta-galactosidase locus. These data suggest that there is a region of homology within the p21 leads to q21 region of human chromosome 3 and a segment of mouse chromosome 9. Since the mouse transferrin gene (Trf) is closely linked to the aminoacylase and beta-galactosidase loci, we predict that the human transferrin (TF) gene is on chromosome 3.     

The sequence of chromosome 3 loci AHSG:TF:CHE1

A large Hutterite kindred was examined for possible linkage between the chromosome 3 markers; cholinesterase (CHE1), transferrin (TF), and alpha-2HS glycoprotein (AHSG). Linkage between TF and AHSG was suggested in males (z = 1.515, theta = 0.08) and between CHE1 and TF(z = 0.661, theta = 0.21). However, linkage between CHE1 and AHSG in males was not established. Based on lods and a nuclear family informative for all three loci a possible chromosomal alignment for the loci is presented.