Released fibroblast growth factor18 from a collagen membrane induces osteoblastic activity involved with downregulation of miR-133a and miR-135a.

We have developed a unique delivery system of growth factors using collagen membranes (CMs) to induce bone regeneration. We hypothesized that fibroblast growth factor18 (FGF-18), a pleiotropic protein that stimulates proliferation in several tissues, can be a good candidate to use our delivery system for bone regeneration. Cell viability, cell proliferation, alkaline phosphatase activity, mineralization, and marker gene expression of osteoblastic differentiation were evaluated after mouse preosteoblasts were cultured with a CM containing FGF-18, a CM containing platelet-derived growth factor, or a CM alone. Furthermore, expression of microRNA, especially miR-133a and miR-135a involving inhibition of osteogenic factors, was measured in preosteoblasts with CM/FGF-18 or CM alone. A sustained release of FGF-18 from the CM was observed over 21 days. CM/FGF-18 significantly promoted cell proliferation, alkaline phosphatase activity, and mineralization compared to CM alone. Gene expression of type I collagen, runt-related transcription factor 2, osteocalcin, Smad5, and osteopontin was significantly upregulated in CM/FGF-18 compared to CM alone, and similar to CM/platelet-derived growth factor. Additionally, CM/FGF-18 downregulated expression of miR-133a and miR-135a. These results suggested that released FGF-18 from a CM promotes osteoblastic activity involved with downregulation of miR-133a and miR-135a.

Ex vivo nonviral gene delivery of µ-opioid receptor to attenuate cancer-induced pain.

Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 (∼45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the µ-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified ß-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more ß-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.

The potential of stromal cell-derived factor-1 delivery using a collagen membrane for bone regeneration.

Stromal cell-derived factor-1 (SDF-1) is a cytokine that is important in stem and progenitor cell recruitment in tissue repair after injury. Regenerative procedures using collagen membranes (CMs) are presently well established in periodontal and implant dentistry. The objective of this study is to test the subsequent effects of the released SDF-1 from a CM on bone regeneration compared to platelet-derived growth factor (PDGF) in vitro and in vivo. For in vitro studies, cell proliferation, alkaline phosphatase activity, and osteoblastic differentiation marker genes were assessed after MC3T3-E1 mouse preosteoblasts were cultured with CMs containing factors. In vivo effects were investigated by placement of CMs containing SDF-1 or PDGF using a rat mandibular bone defect model. At 4 weeks after the surgery, the new bone formation was measured using micro-computed tomography (µCT) and histological analysis. The results of in vitro studies revealed that CM delivery of SDF-1 significantly induced cell proliferation, ALP activity, and gene expression of all osteogenic markers compared to the CM alone or control, similar to PDGF. Quantitative and qualitative µCT analysis for volume of new bone formation and the percentage of new bone area showed that SDF-1-treated groups significantly increased and accelerated bone regeneration compared to control and CM alone. The enhancement of bone formation in SDF-1-treated animals was dose-dependent and with levels similar to those measured with PDGF. These results suggest that a CM with SDF-1 may be a great candidate for growth factor delivery that could be a substitute for PDGF in clinical procedures where bone regeneration is necessary.

Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex.

A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP·DNA(8:1)·FG lipoproteoplex complex demonstrated enhanced transfection of ß-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat·FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP·DNA(8:1)·FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery.

Efficient in vivo gene delivery using modified Tat peptide with cationic lipids.

A combination of modified HIV-1 Tat (mTat) peptide and cationic lipids, FuGENE HD (FH), dramatically enhanced transfection efficiency across a range of cell lines when compared to mTat or FH alone (Biomaterials 35:1705-1715 2014). The efficiency of this Tat peptide combination was significantly higher than many commercial non-viral vectors. In this present study, we tested the feasibility of this non-viral vector, mTat/FH, in vivo using plasmid DNA encoding a luciferase gene. The results of the in vivo studies showed that animals administered mTat/FH/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, FH/DNA, or DNA alone. Histological evaluation showed little immune response in the muscles, livers, and kidneys of mice administered with the mTat/FH. The combination of mTat with FH could significantly improve transfection efficiency, expanding the potential use of non-viral gene vectors in vivo.

The effect of a bioactive collagen membrane releasing PDGF or GDF-5 on bone regeneration.

Regenerative procedures using barrier membrane technology are presently well established in periodontal/endodontic surgery. The objective of this study was to compare the subsequent effects of the released platelet-derived growth factor (PDGF) and growth/differentiation factor 5 (GDF-5) from collagen membranes (CMs) on bone regeneration in vitro and in vivo. In vitro studies were conducted using MC3T3-E1 mouse preosteoblasts cultured with or without factors. Cell viability, cell proliferation, alkaline phosphatase (ALP) activity and bone marker gene expression were then measured. In vivo studies were conducted by placing CMs with low or high dose PDGF or GDF-5 in rat mandibular defects. At 4 weeks after surgery new bone formation was measured using µCT and histological analysis. The results of in vitro studies showed that CM/GDF-5 significantly increased ALP and cell proliferation activities without cytotoxicity in MC3T3-E1 cells when compared to CM/PDGF or CM alone. Gene expression analysis revealed that Runx2 and Osteocalcin were significantly increased in CM/GDF-5 compared to CM/PDGF or control. Quantitative and qualitative µCT and histological analysis for new bone formation revealed that although CM/PDGF significantly enhanced bone regeneration compared to CM alone or control, CM/GDF-5 significantly accelerated bone regeneration to an even greater extent than CM/PDGF. The results also showed that GDF-5 induced new bone formation in a dose-dependent manner. These results suggest that this strategy, using a CM carrying GDF-5, might lead to an improvement in the current clinical treatment of bone defects for periodontal and implant therapy.

Beta-type calcium phosphates with and without magnesium: From hydrolysis of brushite powder to robocasting of periodic scaffolds.

Several approaches have attempted to replace extensive bone loss, but each of them has their limitation. Nowadays, additive manufacture techniques have shown great potential for bone engineering. The objective of this study was to synthesize beta tricalcium phosphate (ß-TCP), beta tricalcium phosphate substituted by magnesium (ß-TCMP), and biphasic calcium phosphate substituted by magnesium (BCMP) via hydrolysis and produce scaffolds for bone regeneration using robocasting technology. Calcium deficient apatites, with and without magnesium were obtained by hydrolysis, calcined and physico-chemically characterized. Colorimetric cell viability assay, calcium nodule formation, and the expression of alkaline phosphatase, osteocalcin, transforming growth factor beta-1 and collagen were assessed using a mouse osteoblastic cell line (MC3T3-E1). Direct-write assembly of cylindrical periodic scaffolds was done via robotic deposition using ß-TCP, ß-TCMP, and BCMP colloidal inks. The sintered scaffolds were characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, Archimede's method, and uniaxial compression test. According to the cell viability assay, the powders induced cell proliferation. Calcium nodule formation and bone markers activity suggested that the materials present potential value in bone tissue engineering. The scaffolds built by robocasting presented interconnected porous and exhibited mean compressive strength between 7.63 and 18.67 MPa, compatible with trabecular bone.

Long-term efficient gene delivery using polyethylenimine with modified Tat peptide.

Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (≈5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.

Menopause increases the iron storage protein ferritin in skin.

Menstruation and desquamation are important routes for humans to excrete iron. Because menstruation is no longer available in postmenopausal women, in the present study, we examined whether iron accumulates more in postmenopausal skin than in premenopausal skin. Skin biopsy samples were obtained from six pre- and six postmenopausal Caucasian women. Iron levels in the form of ferritin were 42% higher, but vascular endothelial growth factor and total antioxidant capacity were 45% and 34% lower in postmenopausal skin (58.8 ± 1.3 years old) than in premenopausal skin (41.6 ± 1.7 years old), respectively. Moreover, in vitro cultured normal human epidermal keratinocytes had surprisingly high levels of ferritin when compared to immortalized human breast epithelial MCF-10A cells or human liver HepG2 cancer cells. Our results indicate that skin is a cellular repository of iron and that menopause increases iron in skin and, thus, may contribute to the manifestation of accelerated skin aging and photo aging after menopause.

Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis.

The HIV-1 Tat peptide has been successfully used for intracellular gene delivery. Likewise, various lipid-based methods have shown increased endocytosis and can influence endosomal escape. This study combines the favorable properties of Tat peptide with that of lipid systems for DNA delivery. We combined the lipid FuGENE HD (FH) with the Tat peptide sequence modified with histidine and cysteine residues (mTat). mTat/FH transfection was evaluated by luciferase expression plasmid in five cell types. mTat/FH produced significant improvement in transfection efficiency of all cell lines when compared to FH or mTat. Treatment with chloroquine, associated with energy-dependent endocytosis, significantly increased transfection efficiency with mTat/FH while incubation at low temperature decreased it. The zeta potential of mTat/FH/DNA was significantly higher compared to FH, mTat, or their DNA combination in the presence of serum, and it was correlated with transfection efficiency. The particle size of the FH/DNA complex was significantly reduced by addition of mTat. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/FH transfection, but transfection was increased by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. These findings demonstrated the feasibility of using a combination of mTat with lipids, utilizing temperature-dependent and caveolae-mediated endocytosis, as a potentially attractive non-viral gene vector.

Effects of iron deficiency and iron overload on angiogenesis and oxidative stress-a potential dual role for iron in breast cancer.

Estrogen alone cannot explain the differences in breast cancer (BC) recurrence and incidence rates in pre- and postmenopausal women. In this study, we have tested a hypothesis that, in addition to estrogen, both iron deficiency due to menstruation and iron accumulation as a result of menstrual stop play important roles in menopause-related BC outcomes. We first tested this hypothesis in cell culture models mimicking the high-estrogen and low-iron premenopausal condition or the low-estrogen and high-iron postmenopausal condition. Subsequently, we examined this hypothesis in mice that were fed iron-deficient and iron-overloaded diets. We show that estrogen only slightly up-regulates vascular endothelial growth factor (VEGF), an angiogenic factor known to be important in BC recurrence. It is, rather, iron deficiency that significantly promotes VEGF by stabilizing hypoxia-inducible factor-1α. Conversely, high iron levels increase oxidative stress and sustain mitogen-activated protein kinase activation, which are mechanisms of known significance in BC development. Taken together, our results suggest, for the first time, that an iron-deficiency-mediated proangiogenic environment could contribute to the high recurrence of BC in young patients, and iron-accumulation-associated pro-oxidant conditions could lead to the high incidence of BC in older women.

Effects of cellular iron deficiency on the formation of vascular endothelial growth factor and angiogenesis. Iron deficiency and angiogenesis.

BACKGROUND: Young women diagnosed with breast cancer are known to have a higher mortality rate from the disease than older patients. Specific risk factors leading to this poorer outcome have not been identified. In the present study, we hypothesized that iron deficiency, a common ailment in young women, contributes to the poor outcome by promoting the hypoxia inducible factor-1α (HIF-1α and vascular endothelial growth factor (VEGF) formation. This hypothesis was tested in an in vitro cell culture model system. RESULTS: Human breast cancer MDA-MB-231 cells were transfected with transferrin receptor-1 (TfR1) shRNA to constitutively impair iron uptake. Cellular iron status was determined by a set of iron proteins and angiogenesis was evaluated by levels of VEGF in cells as well as by a mouse xenograft model. Significant decreases in ferritin with concomitant increases in VEGF were observed in TfR1 knockdown MDA-MB-231 cells when compared to the parental cells. TfR1 shRNA transfectants also evoked a stronger angiogenic response after the cells were injected subcutaneously into nude mice. The molecular mechanism appears that cellular iron deficiency elevates VEGF formation by stabilizing HIF-1α. This mechanism is also true in human breast cancer MCF-7 and liver cancer HepG2 cells. CONCLUSIONS: Cellular iron deficiency increased HIF-1α, VEGF, and angiogenesis, suggesting that systemic iron deficiency might play an important part in the tumor angiogenesis and recurrence in this young age group of breast cancer patients.

Comparison of transfection efficiency of nonviral gene transfer reagents.

This study compared six commercially available reagents (Arrest-In, ExpressFect, FuGENE HD, jetPEI, Lipofectamine 2000, and SuperFect) for gene transfection. We examined the efficiency and cytotoxicity using nine different cell lines (MC3T3-E1 mouse preosteoblasts, PT-30 human epithelial precancer cells, C3H10T1/2 mouse stem cells, MCF-7 human breast cancer cells, HeLa human cervical cancer, C2C12 mouse myoblasts, Hep G2 human hepatocellular carcinoma, 4T1 mouse mammary carcinoma, and HCT116 human colorectal carcinoma), and primary cells (HEKn human epidermal keratinocytes) with two different plasmid DNAs encoding luciferase or ß-galactosidase in the presence or absence of serum. Maximal transfection efficiency in MC3T3-E1, C3H10T1/2, HeLa, C2C12, Hep G2, and HCT116 was seen using FuGENE HD, in PT-30, 4T1, and HEKn was seen using Arrest-In, and in MCF-7 was seen using jetPEI. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetPEI and ExpressFect. These results suggest that FuGENE HD is the most preferred transfection reagent for many cell lines, followed by Arrest-In and jetPEI. These results may be useful for improving nonviral gene and cell therapy applications.

Roles of hormone replacement therapy and iron in proliferation of breast epithelial cells with different estrogen and progesterone receptor status.

Estrogen and iron play critical roles in a female body development and were investigated in the present study in relation to in vitro cell proliferation. Prempro, a hormone replacement therapy drug, and 17beta-estradiol (E2) were shown to increase cell proliferations in estrogen receptor positive (ER+) cells independent of progesterone receptor (PR) status. For example, increased cell proliferation was observed in ER+/PR+ human breast cancer MCF-7, its matching non-cancerous human breast epithelial MCF-12A, and ER+/PR+ murine mammary cancer MXT+ cells, but not in ER-/PR- MDA-MB-231, its matching non-cancerous MCF-10A, and MXT- (ER-/PR+) cells. By mimicking post-menopausal conditions of high estrogen in local breast tissue and increased iron levels due to cessation of menstrual periods, E2 and iron were shown to exert synergistic effects on proliferation of MCF-7 cells and significantly increased Ki67 and proliferating cell nuclear antigen. Western blotting of E2-treated ER+ but not ER- cells showed that E2 also increased transferrin receptor (TfR). Further studies are needed to assess the mitogenic effects of iron and estrogen in normal post-menopausal breast.

Deferoxamine synergistically enhances iron-mediated AP-1 activation: a showcase of the interplay between extracellular-signal-regulated kinase and tyrosine phosphatase.

Deferoxamine (DFO) is a drug widely used for iron overload treatment to reduce body iron burden. In the present study, it was shown in mouse epidermal JB6 cells that all iron compounds transiently induced extracellular signal-regulated kinases (ERK) phosphorylation, whereas DFO further enhanced ERK phosphorylation over long periods. The ERK phosphorylation by DFO treatment appears to be due to the inhibition of MAPK phosphatases (MKP) by DFO. The combined effects of iron-initiated MAPK activation and DFO-mediated MKP inhibition resulted in a synergistic enhancement on AP-1 activities. The results indicate that the interplay between MAPK and MKP is important in regulating the extent of AP-1 activation. It is known that administration of DFO in iron overload patients often results in allergic responses at the injection sites. The results suggest that this synergistic AP-1 activation might play a role in DFO-induced skin immune responses of iron overload patients.

Reliability of serum assays of iron status in postmenopausal women.

PURPOSE: The aim of the study is to determine the reliability during a 2-year period of several newly developed iron-related assays to assess their potential for use in prospective epidemiologic studies. METHODS: We assessed the temporal reliability of several iron-related assays by using three serum samples collected at yearly intervals from 50 postmenopausal participants in a large prospective study. RESULTS: We observed high reliability coefficients for ferritin (0.78; 95% confidence interval [CI], 0.67-0.86), soluble transferrin receptor (sTfR; 0.79; 95% CI, 0.69-0.87), sTfR/ferritin ratio (0.74; 95% CI, 0.62-0.83), and hepcidin (0.89; 95% CI, 0.84-0.94). In a subset of 30 women, lower reliability was observed for serum iron (0.50; 95% CI, 0.29-0.70), unsaturated iron-binding capacity (0.55; 95% CI, 0.34-0.73), total iron-binding capacity (0.60; 95% CI, 0.40-0.76), and serum transferrin saturation rate (0.44; 95% CI, 0.22-0.65). The reliability of anti-5-hydroxymethyl-2'-deoxyuridine autoantibody titers, a biomarker of oxidized DNA damage, one of the mechanisms by which iron is thought to impact disease risk, was very high (0.97, 95% CI, 0.5-0.99). CONCLUSIONS: Our results show that some newly developed iron-related assays could be useful tools to assess iron-disease associations in prospective cohorts that collect a single blood sample.

Iron-induced interleukin-6 gene expression: possible mediation through the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways.

Increased iron store in the body may increase the risk of many diseases such as cancer and inflammation. However, the precise pathogenic mechanism of iron has not yet been elucidated. In the present study, the early biological responses of cells to iron treatment were investigated in AP-1 luciferase reporter stably transfected mouse epidermal JB6 cells and primary rat hepatocytes. It was shown that water-soluble iron compounds, such as FeSO4 and Fe2(SO4)3, were more active in inducing AP-1 in JB6 cells than water-insoluble iron compounds, such as Fe2O3 and FeS. Iron stimulated mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-jun NH2 terminal kinases (JNKs), both in JB6 cells and in primary rat hepatocytes, as determined by the phosphorylation assay. Interestingly, the increase in AP-1 luciferase activity by iron was inhibited by the pretreatment of the cells with PD98059, a specific MEK1 inhibitor, and SB202190, a p38 kinase inhibitor. Levels of interleukin-6 (IL-6), a pro-inflammatory cytokine, were increased in JB6 cells by iron in a dose-dependent manner. The increase in IL-6 and its mRNA by iron was also eliminated by the pretreatment of the cells with PD98059 and SB202190. Since the IL-6 promoter contains an AP-1 binding site, our studies indicate that the iron-induced IL-6 gene expression may be mediated through ERKs and p38 MAPK pathways, possibly one of the important mechanisms for the pathogenesis of iron overload.

Calcein as a fluorescent iron chemosensor for the determination of low molecular weight iron in biological fluids.

The fluorescence quenching of calcein (CA) is not iron specific and results in a negative calibration curve. In the present study, deferoxamine (DFO), a strong iron chelator, was used to regenerate the fluorescence quenched by iron. Therefore, the differences in fluorescence reading of the same sample with or without addition of DFO are positively and specifically proportional to the amounts of iron. We found that the same iron species but different anions (e.g. ferric sulfate or ferric citrate) differed in CA fluorescence quenching, so did the same anions but different iron (e.g. ferrous or ferric sulfates). Excessive amounts of citrate competed with CA for iron and citrate could be removed by barium precipitation. After optimizing the experimental conditions, the sensitivity of the fluorescent CA assay is 0.02 microM of iron, at least 10 times more sensitive than the colorimetric assays. Sera from 6 healthy subjects were tested for low molecular weight (LMW) chelator bound iron in the filtrates of 10 kDa nominal molecular weight limit (NMWL). The LMW iron was marginally detectable in the normal sera. However, increased levels of LMW iron were obtained at higher transferrin (Tf) saturation (1.64-2.54 microM range at 80% Tf saturation, 2.77-3.15 microM range at 100% Tf saturation and 3.09-3.39 microM range at 120% Tf saturation). The application of the assay was further demonstrated in the filtrates of human liver HepG2 and human lung epithelial A549 cells treated with iron or iron-containing dusts.

Roles of bioavailable iron and calcium in coal dust-induced oxidative stress: possible implications in coal workers' lung disease.

Marked regional differences in prevalence of pneumoconiosis are apparent in the US despite comparable dust exposure. In the present study, we examined the ability of 28 coal samples to release bioavailable iron (BAI) and calcium, as well as other metals such as Cr, Ni, Cu, and Co, from three coal mine regions in Utah (UT), West Virginia (WV), and Pennsylvania (PA), respectively. BAI is defined as iron (both Fe2+ and Fe3+) released by the coals in 10 mM phosphate solution, pH 4.5, which mimics conditions of the phagolysosomes in cells. We found that coals from the UT, WV, and PA regions released average levels of BAI of 9.6, 4658.8, and 12149 parts per million (ppm, w/w), respectively, which correlated well with the prevalence of pneumoconiosis from that region (correlation coefficient r = 0.92). The low levels of BAI in the UT coals were due to the presence of calcite (CaCO3), which was shown to be preferentially acid solubilized before iron compounds. Release of iron by two coal samples from the PA and UT regions was further examined in vitro in human lung epithelial A549 cells. We found that the coal from PA, with a high prevalence of pneumoconiosis, released BAI in a dose-dependent manner, both in tissue culture media and in A549 cells. At 2 microg/cm2, levels of lipid peroxidation induced by the PA coal were increased 112% over control cells at 24 h treatment, and were sustained at this level for 3 days. The coal from UT, with a low prevalence of pneumoconiosis, induced a marginal increase in cellular iron at 5 and 10 microg/cm2 treatments and had no effect on lipid peroxidation. Calcium levels in the cells treated with the PA and UT coals were 8.6 and 11.5 micromoles/10(6) cells, respectively, and were significantly higher than that in the controls (5.3 micromoles/10(6) cells) [corrected]. Our results suggest that the differences in the BAI content in the coals may be responsible for the observed regional differences in the prevalence of pneumoconiosis. Therefore, BAI may be a useful characteristic of coal for predicting coal's toxicity.

Ferrous ion autoxidation and its chelation in iron-loaded human liver HepG2 cells.

Ferrous ion (Fe(2+)) is long thought to be the most likely active species, producing oxidants through interaction of Fe(2+) with oxygen (O(2)). Because current iron overload therapy uses only Fe(3+) chelators, such as desferrioxamine (DFO), we have tested a hypothesis that addition of a Fe(2+) chelator, 2,2'-dipyridyl (DP), may be more efficient and effective in preventing iron-induced oxidative damage in human liver HepG2 cells than DFO alone. Using ferrozine as an assay for iron measurement, levels of cellular iron in HepG2 cells treated with iron compounds correlated well with the extent of lipid peroxidation (r = 0.99 after log transformation). DP or DFO alone decreased levels of iron and lipid peroxidation in cells treated with iron. DFO + DP together had the most significant effect in preventing cells from lipid peroxidation but not as effective in decreasing overall iron levels in the cells. Using ESR spin trapping technique, we further tested factors that can affect oxidant-producing activity of Fe(2+) with dissolved O(2) in a cell-free system. Oxidant formation enhanced with increasing Fe(2+) concentrations and reached a maximum at 5 mM of Fe(2+). When the concentration of Fe(2+) was increased to 50 mM, the oxidant-producing activity of Fe(2+) sharply decreased to zero. The initial ratio of Fe(3+):Fe(2+) did not affect the oxidant producing activity of Fe(2+). However, an acidic pH (< 3.5) significantly slowed down the rate of the reaction. Our results suggest that reaction of Fe(2+) with O(2) is an important one for oxidant formation in biological system, and therefore, drugs capable of inhibiting redox activity of Fe(2+) should be considered in combination with a Fe(3+) chelator for iron overload chelation therapy.