Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+). In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 microM Hg(2+). When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.     

Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.     

Ferrous Iron-Dependent Volatilization of Mercury by the Plasma Membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe²⁺ medium (pH 2.5) supplemented with 6 μM Hg²⁺. In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 μM Hg²⁺. When incubated for 3 h in a salt solution (pH 2.5) with 0.7 μM Hg²⁺, resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe²⁺ was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30°C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe²⁺-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 μM Hg²⁺ and 1 mM Fe²⁺, plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe²⁺-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe²⁺-dependent mercury volatilization activity of the plasma membrane.     

Development of a biological mercury removal-recovery system

A mercury removal-recovery system was developed for collection of elemental mercury volatilized by biological mercuric ion reduction. Using the mercury removal-recovery system, removal of mercuric chloride from mercury-containing buffer without nutrients by resting cells of mercury-resistant bacterium, Pseudomonas putida PpY101/pSR134 was tested. Optimum temperature, pH, thiol compounds and cell concentration on removal of mercuric chloride were determined, and 92 to 98% of 40 mg Hg l^–1 was recovered in 24 h. The efficiency of mercuric chloride removal from river water and seawater was as high as that observed when using a buffered solution.     

Ferrous-iron-dependent uptake of L-glutamate by a mesophilic,mixotrophic iron-oxidizing bacterium strain OKM-9

Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol x min(-1) x mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 microM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.     

Development of microbial mercury detoxification processes using mercury-hyperresistant strain of Pseudomonas aeruginosa PU21

A mercury-hyperresistant strain of Pseudomonas aeruginosa PU21 harboring plasmid Rip64 was utilized to develop bioprocesses able to detoxify and recover soluble mercuric ions in aquatic systems. The kinetics of mercury detoxification was investigated to determine the parameters needed for the design of the bioprocesses. Batch, fed-batch, and continuous bioreactors were utilized to evaluate the efficiency and feasibility of each mode of operation. The results showed that the specific mercury detoxification rate was dependent on cell growth phases, as well as the initial mercury concentrations. Cells at the lag growth phase exhibited the best specific detoxification rate of approximately 1.1 x 10(-6) microg Hg/cell/h, and the rate was optimal at an initial mercury concentration of 8 mg/L. In batch operations with initial mercuric ions ranging from 2 to 10 mg/L, the mercuric ions added were rapidly volatilized from the media in less than 2-3 h. With periodic feeding of 3 or 5 mg Hg/L at fixed time intervals, the fed-batch processes had mercury removal efficiencies of 2.9 and 3.3 mg Hg/h/L, respectively. For continuous operations, the effluent cell concentration (Xe) was essentially invariant at 527 and 523 mg/L with the dilution rates (D) of 0.18 and 0.325 h-1, respectively. The increase in mercury feeding concentrations (Hgf) from 1.0 to 6.15 mg Hg2+/L did not affect the steady-state cell concentration (Xe) but forced the effluent mercury concentration (Hge) to increase. The decrease in the dilution rate, however, resulted in lower Hge values. It was also found that sequential mercury vapor absorption columns recovered over 80% of the Hg degrees released from the bioreactor while the residual mercury vapor was subsequently immobilized by an activated carbon trap in the down stream of the absorption column.     

Effects of time and dietary iron on tissue iron concentration as an estimate of relative bioavailability of supplemental iron sources for ruminants

Two experiments were conducted to investigate the effects of time and dietary Fe on tissue Fe concentrations following short-term, high level supplementation for use as a bioassay procedure for supplemental Fe sources for ruminants. In Experiment 1, 28 wethers were allotted randomly to four experimental diets which were fed for 15 or 30 days. The basal maize–soyabean meal–cottonseed hulls diet (193 mg kg⁻¹ Fe) was supplemented with 0, 400, 800 or1200 mg kg⁻¹ added Fe from reagent grade ferrous sulfate (FeSO₄·7H₂O). Iron concentrations in liver, kidney, and spleen increased (P<0.05) as dietary Fe increased; however, muscle, heart, and bone Fe concentrations were unaffected. A logarithmic transformation of liver or kidney Fe concentrations at 30 days regressed on added dietary Fe produced the best fits to a linear model. In Experiment 2, bioavailability of Fe from three feed grade ferrous carbonates known to differ (carbonates A, B, and C) was compared to that from reagent grade ferrous sulfate. The dietary treatments fed for 30 days included the above basal diet (90 mg kg⁻¹ Fe) supplemented with 0, 300, 600 or 900 mg kg⁻¹ added Fe from ferrous sulfate or 600 mg kg⁻¹ Fe from ferrous carbonates A, B, or C. Liver Fe concentrations from sheep fed ferrous sulfate were numerically greater than those of animals fed the carbonate sources or control diet. Kidney Fe concentrations from lambs fed ferrous sulfate at 600 mg kg⁻¹ Fe or carbonate-A were greater (P<0.05) than those fed carbonates B or C. Iron concentrations in spleen were lower (P<0.05) in lambs fed carbonate-B than for those fed 600 mg kg⁻¹ Fe as ferrous sulfate, but were similar to other carbonates. Overall average bioavailability estimates based on multiple regression slope ratios for the three tissues were ferrous sulfate 1.00, carbonate-A 0.55, carbonate-B 0.00, and carbonate-C 0.20. Estimates for carbonates A and C were similar to those based on hemoglobin concentrations reported previously for young swine supplemented at dietary concentrations near the requirement.     

Effect of Zinc Sulfate Fortificant on Iron Absorption from Low Extraction Wheat Flour Co-Fortified with Ferrous Sulfate

The co-fortification of wheat flour with iron (Fe) and zinc (Zn) is a strategy used to prevent these deficiencies in the population. Given that Zn could interact negatively with Fe, the objective was to assess the effect of Zn on Fe absorption from bread prepared with wheat flour fortified with Fe and graded levels of Zn fortificant. Twelve women aged 30–43 years, with contraception and a negative pregnancy test, participated in the study. They received on four different days, after an overnight fast, 100 g of bread made with wheat flour (70 % extraction) fortified with 30 mg Fe/kg as ferrous sulfate (A) or prepared with the same Fe-fortified flour but with graded levels of Zn, as zinc sulfate: 30 mg/kg (B), 60 mg/kg (C), or 90 mg/kg (D). Fe radioisotopes (⁵⁹Fe and ⁵⁵Fe) of high specific activity were used as tracers and Fe absorption iron was measured by the incorporation of radioactive Fe into erythrocytes. Results: The geometric mean and range of ±1 SD of Fe absorption were: A?=?19.8 % (10.5–37.2 %), B?=?18.5 % (10.2–33.4 %), C?=?17.7 % (7.7–38.7 %), and D?=?11.2 % (6.2–20.3 %), respectively; ANOVA for repeated measures F?=?5.14, p?<?0.01 (Scheffè’s post hoc test: A vs D and B vs D, p?<?0.05). We can conclude that Fe is well absorbed from low extraction flour fortified with 30 mg/kg of Fe, as ferrous sulfate, and up to 60 mg/kg of Zn, as Zn sulfate. A statistically significant reduction of Fe absorption was observed at a Zn fortification level of 90 mg Zn/kg.     

Enhanced biosorption of mercury(II) and cadmium(II) by cold-induced hydrophobic exobiopolymer secreted from the psychrotroph <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> BM07

The cells of psychrotrophic Pseudomonas fluorescens BM07 were found to secrete large amounts of exobiopolymer (EBP) composed of mainly hydrophobic (water insoluble) polypeptide(s) (as contain approximately 50 mol% hydrophobic amino acids, lacking cysteine residue) when grown on fructose containing limited M1 medium at the temperatures as low as 0-10 degrees C but trace amount at high (30 degrees C, optimum growth) temperature. Two types of nonliving BM07 cells (i.e., cells grown at 30 degrees C and 10 degrees C) as well as the freeze-dried EBP were compared for biosorption of mercury (Hg(II)) and cadmium (Cd(II)). The optimum adsorption pH was found 7 for Hg(II) but 6 for Cd(II), irrespective of the type of biomass. Equilibrium adsorption data well fitted the Langmuir adsorption model. The maximum adsorption (Q (max)) was 72.3, 97.4, and 286.2 mg Hg(II)/g dry biomass and 18.9, 27.0, and 61.5 mg Cd(II)/g dry biomass for cells grown at 30 degrees C and 10 degrees C and EBP, respectively, indicating major contribution of heavy metal adsorption by cold-induced EBP. Mercury(II) binding induced a significant shift of infrared (IR) amide I and II absorption of EBP whereas cadmium(II) binding showed only a very little shift. These IR shifts demonstrate that mercury(II) and cadmium(II) might have different binding sites in EBP, which was supported by X-ray diffraction and differential scanning calorimetric analysis and sorption results of chemically modified biomasses. This study implies that the psychrotrophs like BM07 strain may play an important role in the bioremediation of heavy metals in the temperate regions especially in the inactive cold season.     

Effect of Increasing Levels of Zinc Fortificant on the Iron Absorption of Bread Co-Fortified with Iron and Zinc Consumed with a Black Tea

Iron (Fe) and zinc’s (Zn) interaction at the absorptive level can have an effect on the success of co-fortification of wheat flour with both minerals on iron deficiency prevention. The aim of the study was to determine the effect of increasing levels of zinc fortificant on the iron absorption of bread co-fortified with iron and zinc consumed with a black tea. Twelve women aged 33–42 years participated in the study. They received on four different days 200 mL of black tea and 100 g of bread made with wheat flour (70 % extraction) fortified with either 30 mg Fe/kg alone, as ferrous sulfate (A), or with the same Fe-fortified flour, but with graded levels of Zn, as zinc sulfate: 30 mg/kg (B), 60 mg/kg (C), or 90 mg/kg (D). Fe radioisotopes (⁵⁹Fe and ⁵⁵Fe) of high specific activity were used as tracers, and Fe absorption iron was measured by the incorporation of radioactive Fe into erythrocytes. The geometric mean and range of ±1 SD of Fe absorption were as follows: A?=?6.5 % (2.2–19.3 %), B?=?4.6 % (1.0–21.0 %), C?=?2.1 % (0.9–4.9 %), and D?=?2.2 % (0.7–6.6 %), respectively; ANOVA for repeated measures F?=?10.9, p?<?0.001 (Scheffè’s post hoc test: A vs. C, A vs. D, B vs. C, and B vs. D; p?<?0.05). We can conclude that Fe absorption of bread made from low-extraction flour fortified with 30 mg/kg of Fe, as ferrous sulfate, and co-fortified with zinc, as zinc sulfate consumed with black tea is significantly decreased at a zinc fortification level of ≥60 mg/kg flour.     

Mercury uptake and removal by Euglena gracilis

The uptake and removal of mercury (added as HgCl2) from the culture medium by Euglena gracilis was studied. In cultures initiated in the light, cells accumulated a small fraction of the added heavy metal (5-13%). Mercury was both biologically and nonbiologically volatilized, and cell growth was partially inhibited; under these conditions the glutathione content was 3.2 nmol/10(6) cells. In contrast, in cultures initiated in the dark, mercury uptake by cells was two to three times higher, biological volatilization remained unchanged and nonbiological volatilization and growth were negligible; the glutathione content diminished to 1.4 nmol/10(6) cells. Biological mercury volatilization depended on cell density and metal concentration, but was light-independent. Thus, volatilization of mercury by Euglena appeared not to be an effective mechanism of resistance, whereas a high intracellular level of glutathione and a low mercury uptake seemed necessary for successful tolerance.     

Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans.

Cell-free mercury volatilization activity (mercuric reductase) was obtained from a mercury-volatilizing Thiobacillus ferrooxidans strain, and the properties of intact-cell and cell-free activities were compared with those determined by plasmid R100 in Escherichia coli. Intact cells of T. ferrooxidans volatilized mercury at pH 2.5, whereas cells of E. coli did not. Cell-free enzyme preparations from both bacteria functioned best at or above neutral pH and not at all at pH 2.5. The T. ferrooxidans mercuric reductase was a soluble enzyme that was dependent upon added NAD(P)H. The enzyme activity was stable at 80 degrees C, required an added thiol compound, and was stimulated by EDTA. Antisera against purified mercuric reductases from transposon Tn501 and plasmid R831 (which inactivated mercuric reductases from a wide range of enteric and pseudomonad strains) did not inactivate the enzyme from T. ferrooxidans.     

Acclimation of aquatic microbial communities to Hg(II) and CH3Hg+ in polluted freshwater ponds

The relationship of mercury resistance to the concentration and chemical speciation of mercurial compounds was evaluated for microbial communities of mercury-polluted and control waters. Methodologies based on the direct viable counting (DVC) method were adapted to enumerate mercury-resistant communities. Elevated tolerance to Hg(II) was observed for the microbial community of one mercury-polluted pond as compared to the community of control waters. These results suggest an in situ acclimation to Hg(II). The results of the methylmercury resistance-DVC assay suggested that minimal acclimation to CH₃Hg⁺ occurred since similar concentrations of CH₃HgCl inhibited growth of 50% of organisms in both the control and polluted communities. Analyses of different mercury species in pond waters suggested that total mercury, but not CH₃Hg⁺ concentrations, approached toxic levels in the polluted ponds. Thus, microbial acclimation was specific to the chemical species of mercury present in the water at concentrations high enough to cause toxic effects to nonacclimated bacterial communities.     

Characterization by Hg2+ of two different pathways for mitochondrial Ca2+ release

The addition of Hg2+ to loaded kidney mitochondria induces the fast release of the accumulated cation. The Ca2+-efflux reaction exhibits kinetics characteristics that depend on the extent of the binding of Hg2+ to the membrane. At high levels of Hg2+ bound (approx. 11 nmol/mg), Ca2+ efflux rate is highly insensitive to the temperature of incubation, and the efflux seems to be directly related to the internal free Ca2+ concentration. At these levels of bound Hg2+, accumulated Sr2+ is released with characteristics similar to those observed with Ca2+. At lower levels of Hg2+ binding (2.5 nmol/mg), the efflux reaction is highly dependent on the incubation temperature and on the internal free Ca2+ concentration; under these conditions Sr2+ is not released. NAD(P)H oxidation as induced by the low Hg2+ concentration is inhibited at the lower temperatures. Radiolabeled Hg2+ incorporates into two clearly defined regions of membrane proteins separated through sodium dodecyl sulfate gel electrophoresis. One of the regions corresponds to proteins of apparent high molecular mass (i.e., 150 kDa), and the other to proteins with apparent molecular masses of 37-25 kDa. Mitochondria incubated with 2 microM 203Hg2+ incorporate the radionuclide in proteins that have molecular masses of around 41 and 26 kDa. The results indicate that, depending on the amount of Hg2+ bound to the inner membrane, two clearly distinct Ca2+ release mechanisms can be distinguished.     

Glucose transport system in a facultative iron-oxidizing bacterium, Thiobacillus ferrooxidans

Properties of a heat-labile glucose transport system in Thiobacillus ferrooxidans strain AP-44 were investigated with iron-grown cells. [14C]glucose was incorporated into cell fractions, and the cells metabolized [14C]glucose to 14CO2. Amytal, rotenone, cyanide, azide, 2,4-dinitrophenol, and dicyclohexylcarbodiimide strongly inhibited [14C]glucose uptake activity, suggesting the presence of an energy-dependent glucose transport system in T. ferrooxidans. Heavy metals, such as mercury, silver, uranium, and molybdate, markedly inhibited the transport activity at 1 mM. When grown on mixotrophic medium, the bacteria preferentially utilized ferrous iron as an energy source. When iron was exhausted, the cells used glucose if the concentration of ferrous sulfate in the medium was higher than 3% (wt/vol). However, when ferrous sulfate was lower than 1%, both of the energy sources were consumed simultaneously.     

Effect of Increasing Concentrations of Zinc on the Absorption of Iron from Iron-Fortified Milk

The cofortification of milk with iron (Fe) and zinc (Zn) is a strategy used to prevent these deficiencies during childhood. Given that Zn can negatively interact with iron in aqueous solutions, the objective of the present study was to determine the effect of Zn on Fe absorption of milk fortified with Fe and Zn. Twenty-eight women between 33 and 47?years of age, with contraception and a negative pregnancy test, participated in one of two absorption studies. They received on four different days, after an overnight fast, 200?mL of milk (26?% fat) fortified with 10?mg Fe/L, as (A) ferrous sulfate, or the same milk but with graded doses of added Zn, as Zn sulfate of (B) 5, (C) 10, and (D) 20?mg/L (study 1, n?=?15). In study 2 (n?=?13), subjects received the same milk formulations, but these were also fortified with ascorbic acid (70?mg/L). Milk was labeled with radioisotopes ⁵⁹Fe or ⁵⁵Fe, and the absorption of iron was measured by erythrocyte incorporation of radioactive Fe. The geometric mean and range of ±1 SD of Fe absorption in study 1 were as follows: formula A?=?6.0?% (2.8?C13.0?%); B?=?6.7?% (3.3?C13.6?%); C?=?5.4?% (2.2?C13.2?%); and D?=?5.2?% (2.8?C10.0?%) (ANOVA for repeated measures, not significant). For study 2, data are as follows: 8.2?% (3.6?C18.7?%); B?=?6.4?% (2.5?C16.4?%); C?=?7.7?% (3.2?C18.9?%); and D?=?5.2 (1.8?C14.8?%) (ANOVA for repeated measures, not significant). In conclusion, according to the results from this study, it appears that the addition of zinc up to 20?mg/L does not significantly inhibit iron absorption from milk fortified with 10?mg/L of iron.     

Uptake of ferrous iron histidinate, a promoter of lipid peroxidation, by Ehrlich ascites tumor cells

The kinetics of the uptake of Fe(II)-histidinate, a known promoter of lipid peroxidation, into Ehrlich ascites tumor (EAT) cells and the intracellular binding of iron were studied in vitro. EAT cells (27.10(6)/ml) were incubated in Hanks' balanced salts solution at 37 degrees C for various time intervals in the presence of FeSO4 (1 mM) and L-histidine (10 mM). Total iron was determined by the 1,10-phenanthroline/ascorbate method and ferric iron by reaction with 5-sulfosalicylic acid; the difference was ascribed to ferrous iron. Total iron decreased rapidly in the medium (242 nmol within the first 10 min), and a corresponding increase of total iron (saturation value 376 nmol after 60 min) was determined within the cells, after the cellular proteins had been solubilized with 6 M urea. In the absence of EAT cells, Fe(II)-histidinate was readily oxidized to Fe(III)-histidinate by oxygen, but this reaction was strongly retarded by the tumor cells. The uptake of iron histidinate occurred in the oxidized state, while an uptake of ferrous iron could not be proven unambiguously. When EAT cells were saturated with iron, it was found that 93% of intracellular iron was bound to water-insoluble proteins and 7% was associated with soluble proteins, while no unbound iron was detectable by the method used. It was concluded that, despite the high uptake of total iron, only a very small portion of the intracellular iron was available as a redox catalyst for lipid peroxidation.     

Pathophysiological consequences of enhanced intracellular superoxide formation in isolated perfused rat liver.

The potential toxicity of enhanced intracellular reactive oxygen formation was investigated in isolated perfused livers of male Fischer rats. The presence of the redox-cycling agent diquat in the perfusate (200 microM) increased the basal efflux of glutathione disulfide (GSSG) into bile (2.65 +/- 0.26 nmol GSH-equivalents/min per g liver wt.) and perfusate (0.55 +/- 0.15 nmol/min per g) approximately 10-fold. Since no evidence was found for degradation of GSSG in the biliary tract of these animals, it could be estimated that diquat induced a constant O2- generation of approximately 1000 nmol/min per g liver wt for 1 h. Thus, reactive oxygen formation under these conditions was 1-2 orders of magnitude higher than under various pathophysiological conditions. Only minor liver injury (release of lactate dehydrogenase activity) was observed. To increase the susceptibility of the liver to the oxidant stress, animals were pretreated in vivo with 200 mg/kg body wt. phorone, which caused a 90% depletion of the hepatic glutathione content, 100 mg/kg ferrous sulfate, a combination of phorone and ferrous sulfate, or 40 mg/kg BCNU, which caused a 60% inhibition of hepatic GSSG reductase. Only the combined treatment of phorone + ferrous sulfate or BCNU caused a significant increase of the diquat-induced liver injury. Our results demonstrated an extremely high resistance of the liver against intracellular reactive oxygen formation (even with impaired detoxification systems) and can serve as reference for the evaluation of potential contributions of reactive oxygen to liver injury in various disease states.     

Iron and gallium increase iron uptake from transferrin by human melanoma cells: further examination of the ferric ammonium citrate-activated iron uptake process

Previously we showed that preincubation of cells with ferric ammonium citrate (FAC) resulted in a marked increase in Fe uptake from both (59)Fe-transferrin (Tf) and (59)Fe-citrate (D.R. Richardson, E. Baker, J. Biol. Chem. 267 (1992) 13972-13979; D.R. Richardson, P. Ponka, Biochim. Biophys. Acta 1269 (1995) 105-114). This Fe uptake process was independent of the transferrin receptor and appeared to be activated by free radicals generated via the iron-catalysed Haber-Weiss reaction. To further understand this process, the present investigation was performed. In these experiments, cells were preincubated for 3 h at 37 degrees C with FAC or metal ion solutions and then labelled for 3 h at 37 degrees C with (59)Fe-Tf. Exposure of cells to FAC resulted in Fe uptake from (59)Fe-citrate that became saturated at an Fe concentration of 2.5 microM, while FAC-activated Fe uptake from Tf was not saturable up to 25 microM. In addition, the extent of FAC-activated Fe uptake from citrate was far greater than that from Tf. These results suggest a mechanism where FAC-activated Fe uptake from citrate may result from direct interaction with the transporter, while Fe uptake from Tf appears indirect and less efficient. Preincubation of cells with FAC at 4 degrees C instead of 37 degrees C prevented its effect at stimulating (59)Fe uptake from (59)Fe-Tf, suggesting that an active process was involved. Previous studies by others have shown that FAC can increase ferrireductase activity that may enhance (59)Fe uptake from (59)Fe-Tf. However, there was no difference in the ability of FAC-treated cells compared to controls to reduce ferricyanide to ferrocyanide, suggesting no change in oxidoreductase activity. To examine if activation of this Fe uptake mechanism could occur by incubation with a range of metal ions, cells were preincubated with either FAC, ferric chloride, ferrous sulphate, ferrous ammonium sulphate, gallium nitrate, copper chloride, zinc chloride, or cobalt chloride. Stimulation of (59)Fe uptake from Tf was shown (in order of potency) with ferric chloride, ferrous sulphate, ferrous ammonium sulphate, and gallium nitrate. The other metal ions examined decreased (59)Fe uptake from Tf. The fact that redox-active Cu(II) ion did not stimulate Fe uptake while redox-inactive Ga(III) did, suggests a mechanism of transporter activation not solely dependent on free radical generation. Indeed, the activation of Fe uptake appears dependent on the presence of the Fe atom itself or a metal ion with atomic similarities to Fe (e.g. Ga).     

Acid-bacterial and ferric sulfate leaching of pyrite single crystals

Pyrite single-crystal cubes were cut, polished. and x-rayed to produce orientations of (100), (110), (111), and (112). These crystallographically developed surfaces then were prepared to expose an area of 1 cm(2), and the remainder of the crystal was coated with an acid-resistant silicone cement. Crystals with representative orientations then were leached in ferric sulfate solutions adjusted to a pH of 2.3 with H(2)SO(4) containing up to 6 x 10(3) ppm of Fe(3+) at 30 and 55 degrees C. Leaching was also conducted in acid-bacterial lixiviants containing Thiobacillus ferrooxidans at 30 degrees C and a thermophilic microorganism at 55 degrees C. Surface corrosion and pitting associated with pyrite leaching were examined by scanning electron microscopy. Pyrite leaching in ferric sulfate solutions was observed to be different when compared to acid-bacterial leaching. Ferric sulfate leaching required nearly 2 x 10(3) ppm of Fe(3+) at 30 degrees C while acid-bacterial leaching at 30 degrees C occurred without additions of Fe(3+), and values of Fe(3+) never exceeded 10(2) ppm. Because of precipitate formation, an accurate assessment of the role of crystallographic orientation on the leaching of pyrite is difficult.