Cytotoxicity of ammonium hexafluorosilicate on human gingival fibroblasts.

Ammonium hexafluorosilicate (SiF), which is claimed to significantly improve occlusion of dentinal tubules, was proposed as a novel desensitizer for dentine hypersensitivity (DH). However, the cytotoxicity of SiF on oral cells is lacking. The purpose of this study was to investigate the cytotoxicity of SiF on human gingival fibroblasts (hGFs) under different dosages (0.001%, 0.01%, 0.1%, and 1%) and treatment durations (1, 5, 10, and 30min). Cell proliferation, mitochondrial membrane potential (MMP) and cell cycle were tested by MTT assay, JC-1 staining and flow cytometry, respectively. Glutathione (GSH) depletion was analyzed to further investigate the underlying mechanism of SiF-induced cytotoxicity. MTT assay showed that there was significantly lower number of viable cells when the hGFs were treated with 0.01% (10min), 0.1% (10 and 30min) and 1% (5, 10, and 30min) SiF than the control group (p<0.05). MMP decreased and GSH depletion increased dramatically along with higher concentrations (0.1% and 1% SiF) and prolonged times (10 and 30min). DNA synthesis [S (%)] of cells treated with 0.1% and 1% SiF (5, 10, and 30min) was significantly lower than the control group (p<0.05). Our results indicate exposure to up to 0.01% SiF for less than 5min causes low or no cytotoxicity in vitro.

A novel semiquantitative polymerase chain reaction/enzyme digestion-based method for detection of large scale deletions/conversions of the CYP21 gene and mutation screening in Turkish families with 21-hydroxylase deficiency.

21-Hydroxylase deficiency is a recessively inherited disorder resulting from mutations in the CYP21 gene. The CYP21 gene is located along with the CYP21P pseudogene in the human leukocyte antigen major histocompatibility complex region on chromosome 6. Molecular diagnosis is difficult due to the 98% similarity of CYP21 and CYP21P genes and the fact that almost all frequently reported mutations reside on the pseudogene. Allele-specific PCR for the 8 most frequently reported point mutations was performed in 31 Turkish families with at least a single 21-hydroxylase-deficient individual. The allele frequencies of the point mutations were as follows: P30L, 0%; IVS2 (AS,A/C-G,-13), 22.5%; G110delta8nt, 3.2%; I172N, 11.4%; exon 6 cluster (I236N, V237E, M239K), 3.2%; V281L, 0%; Q318X, 8%; and R356W, 9.6%. Large deletions and gene conversions were detected by Southern blot analysis, and the allele frequencies were 9.6% and 22.5%, respectively. Sequence analysis of the gene, performed on patients with only 1 mutated allele, revealed 2 missense mutations (R339H and P435S). A novel semiquantitative PCR/enzyme digestion-based method for the detection of large scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.

Hemopexin decreases spontaneous chemiluminescence of cold preserved liver after reperfusion.

Hemopexin is a plasma protein with exceptionally high affinity for heme. During liver transplantation heme is released via lysis of transfused blood. This heme may catalyze peroxidative reactions that contribute to "reperfusion" injury of the organ. Using a rat liver model of cold storage and reperfusion we tested the potential anti-oxidant effects of hemopexin. After 3 h of cold storage rat liver was reperfused with warm oxygenated buffer. Spontaneous liver chemiluminescence, which is a parameter of oxyradical production, was measured during reperfusion and expressed as an index of free radical production (IFRP). Chemiluminescence reached a maximum within 5 min of reperfusion and decreased to baseline within 30 min. Addition of hemopexin to the perfusate (5 microM) significantly decreased the IFRP. By contrast, the control proteins albumin and gamma-globulin (10 microM) had a smaller non-significant effect. The data suggest that heme could be complexed by hemopexin during reperfusion, thus inhibiting heme mediated cellular injury.

EPR studies on the effects of complexation of heme by hemopexin upon its reactions with organic peroxides.

Hemopexin, a heme-binding serum glycoprotein, is thought to play an important role in the prevention of oxidative damage that may be catalysed by free heme. Through the use of EPR techniques, the generation of free radicals from organic hydroperoxides by heme and heme-hemopexin complexes, and the concomitant formation of high oxidation-state iron species has been studied; these species are implicated as causative agents in processes such as cardiovascular disease and carcinogenesis. From the rates of production of these species from both n-alkyl and branched hydroperoxides, it has been inferred that the dramatic reduction in the yield of oxidising species generated by heme upon its complexation with hemopexin arises from steric hindrance of the access of hydroperoxide to the bound heme.

Purification, characterization, and cloning of a heme-binding protein (23 kDa) in rat liver cytosol.

A heme-binding protein (designated HBP23) has been purified from rat liver cytosol using heme-affinity chromatography and either reverse-phase high-performance liquid chromatography or sequential ion-exchange chromatography. The protein (23 kDa) binds heme with an affinity (Kd = 55 nM) higher than that of the abundant cytosolic heme-binding proteins, heme-binding protein (HBP)/liver fatty acid-binding protein (L-FABP) and the glutathione S-transferases (GSTs) (Kd = 100-200 nM). HBP23 is present in the cytosol of liver, kidney, spleen, small intestine, and heart, with the liver showing the highest content. A cDNA coding the 23-kDa protein was cloned using reverse transcription polymerase chain reaction with degenerative oligonucleotides derived from partial amino acid sequences. The cloned cDNA encoded 199 amino acids, and its amino acid sequence showed no homology to HBP/L-FABP, GSTs, or any other heme-binding proteins or hemeproteins. Homology search showed that HBP23 is highly homologous to mouse macrophage 23-kDa stress protein, which is inducible by oxidant stress in peritoneal macrophages [Ishii, T., Yamada, M., Sato, H., Matsue, M., Taketani, S., Nakayama, K., Sugita, Y., and Bannai, S. (1993) J. Biol. Chem. 268, 18633-18636]. Thioredoxin peroxidase as well as HBP23 and the mouse macrophage 23-kDa stress protein are members of the peroxiredoxin family, a recently recognized class of antioxidant proteins [Chae, H. Z., Chung, S. J., & Rhee, S. G. (1994) J. Biol. Chem. 269, 27670-27678]. An increase in HBP23 mRNA was observed in Hepa 1-6 cells after treatment with heme and cadmium and during liver regeneration after partial hepatectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of haemopexin receptors by cultured human cytotrophoblast.

The expression of cell-surface haemopexin (Hx) receptors on human cytotrophoblasts was assessed by using four different Hx species purified from plasma: human Hx isolated by wheatgerm-affinity chromatography, human Hx isolated by haem-agarose-affinity chromatography and rabbit and rat Hx, also isolated by haem-agarose-affinity chromatography. About 3500-7000 high-affinity (Kd 0.34-0.85 nM) receptors per cell were measured by Scatchard-type analysis at 4 degrees C using human (species obtained by both methods) or rabbit 125I-labelled haem-Hx. Measured simultaneously, transferrin receptor number and affinity were 40,000/cell and 0.83 nM respectively. In contrast with transferrin receptors, the number of Hx receptors did not increase during 24 h in cytotrophoblast culture. Rat Hx showed no specific binding to human Hx receptors in cytotrophoblast cultures.

The type II hemopexin interleukin-6 response element predominates the transcriptional regulation of the hemopexin acute phase responsiveness.

Hemopexin (Hx) is induced during the acute phase response (APR) by the cytokine interleukin (IL)-6. A type II IL-6 response element (RE) of the Hx gene has been characterized recently (J. Biol. Chem. (1994); 269, 12654-12661). To assess Hx gene regulation by other agents, various cytokines and growth factors were tested for their ability to induce Hx in rat hepatoma H-35 cells. IL-6-type cytokines, IL-1 beta and TNF-alpha, in contrast to transforming growth factor-beta (TGF-beta), hepatocyte growth factor and insulin significantly increased Hx gene expression. Chloramphenicol acetyltransferase (CAT) activity in H-35 cells transfected with constructs that contained the 5'-flanking Hx promoter region or multiple copies of the Hx IL-6-RE fused to the CAT gene was upregulated only by IL-6-type cytokines, although to varying degrees. These data indicate that signal transduction pathways mediated by IL-6-type cytokines but not those by IL-1 beta and TNF-alpha converge on the common Hx IL-6-RE.