Comparative in situ study of the intestinal absorption of aluminum, manganese, nickel, and lead in rats

This comparative study of the intestinal absorption of four toxic metals (aluminum, manganese, nickel, and lead) carried out in rats using the in situ intestinal perfusion technique was able to measure the partition of each metal between the intestine (intestinal retention), the blood circulation, and target tissues after 1 h. The perfused metal solutions were at concentrations likely to occur during oral intoxication. It was found that aluminum (48 and 64 mM), even as a citrate complex, crossed the brush border with difficulty (0.4% of the perfused amount); about 60% of this was retained in the intestine and the remainder was found in target tissues (about 36%). Conversely, lead (4.8–48 μM) penetrated the intestine more easily (about 35% of the perfused amount), was slightly retained (about 12% of the input), and was soon found in the tissues (about 58% of the input) and to a lesser degree in circulation (about 29%). Within the same concentration range, nickel and manganese showed certain similarities, such as a reduced crossing of the brush border proportional to the increase in the concentration perfused (0.17–9.5 mM). There was similar intestinal retention and absorption (about 80% and 20% of the input, respectively). Manganese crossed the brush border more easily and was diffused more rapidly into tissues. Finally, the addition of equimolar amounts of iron (4.7 mM) produced opposite effects on the absorption of the two elements, inhibiting manganese and showing a trend to increase in nickel absorption. This could be the result of competition between Fe²⁺ and Mn²⁺ for the same transcellular transporters and the slight predominance of paracellular mechanism in the event of “Fe²⁺-Ni²⁺” association.     

Airborne manganese exposure differentially affects end points of oxidative stress in an age-and sex-dependent manner

Juvenile female and male (young) and 16-mo-old male (old) rats inhaled manganese in the form of manganese sulfate (MnSO₄) at 0, 0.01, 0.1, and 0.5 mg Mn/m³ or manganese phosphate at 0.1 mg Mn/m³ in exposures of 6h/d, 5d/wk for 13 wk. We assessed biochemical end points indicative of oxidative stress in five brain regions: cerebellum, hippocampus, hypothalamus, olfactory bulb, and striatum. Glutamine synthetase (GS) protein levels, metallothionein (MT) and GS mRNA levels, and total glutathione (GSH) levels were determined for all five regions. Although most brain regions in the three groups of animals were unaffected by manganese exposure in terms of GS protein levels, there was significantly increased protein (p<0.05) in the hippocampus and decreased protein in the hypothalamus of young male rats exposed to manganese phosphate as well as in the aged rats exposed to 0.1 mg/m³ MnSO₄. Conversely, GS protein was elevated in the olfactory bulb of females exposed to the high dose of MnSO₄. Statistically significant decreases (p<0.05) in MT and GS mRNA as a result, of manganese exposure were observed in the cerebellum, olfactory bulb, and hippocampus in the young male rats, in the hypothalamus in the young female rats, and in the hippocampus in the senescent males. Total GSH levels significantly (p<0.05) decreased in the olfactory bulb of manganese exposed young male rats and increased in the olfactory bulb of female rats exposed to manganese. Both the aged and young female rats had significantly decreased (p<0.05) GSH in the striatum resulting from manganese inhalation. The old male rats also had depleted GSH levels in the cerebellum and hypothalamus as a result, of the 0.1-mg/m³ manganese phosphate exposure. These results demonstrate that age and sex are variables that must be considered whenassessing the neurotoxicity of manganese.     

Differential Lowering by Manganese Treatment of Activities of Glycolytic and Tricarboxylic Acid (TCA) Cycle Enzymes Investigated in Neuroblastoma and Astrocytoma Cells Is Associated with Manganese-Induced Cell Death

Manganese (Mn) is a trace metal required for normal growth and development. Manganese neurotoxicity is rare and usually associated with occupational exposures. However, the cellular and molecular mechanisms underlying Mn toxicity are still elusive. In rats chronically exposed to Mn, their brain regional Mn levels increase in a dose-related manner. Brain Mn preferentially accumulates in mitochondria; this accumulation is further enhanced with Mn treatment in vivo. Exposure of mitochondria to Mn in vitro leads to uncoupling of oxidative phosphorylation. These observations prompted us to investigate the hypothesis that Mn induces alterations in energy metabolism in neural cells by interfering with the activities of various glycolytic and TCA cycle enzymes using human neuroblastoma (SK-N-SH) and astrocytoma (U87) cells. Treatments of SK-N-SH and U87 cells with MnCl2 induced cell death in these cells, in a concentration- and time-dependent manner, as determined by MTT assays. In parallel with the Mn-induced, dose-dependent decrease in cell survival, treatment of these cells with 0.01 to 4.0 mM MnCl2 for 48 h also induced dose-related decreases in their activities of hexokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, and malate dehydrogenase. Hexokinase in SK-N-SH cells was the most affected by Mn treatments, even at the lower range of concentrations. Mn treatment of SK-N-SH cells affected pyruvate kinase and citrate synthase to a lesser extent as compared to its effect on other enzymes investigated. However, citrate synthase and pyruvate kinase in U87 cells were more vulnerable than other enzymes investigated to the effects of Mn. The results suggest the two cell types exhibited differential susceptibility toward the Mn-induced effects. Additionally, the results may have significant implications in flux control because HK is the first and highly regulated enzyme in brain glycolysis. Thus these results are consistent with our hypothesis and may have pathophysiological implications in the mechanisms underlying Mn neurotoxicity.     

Specific intra-tissue responses to manganese in the floating lamina of Trapa natans L

Plant tolerance to heavy metals requires morpho-physiological mechanisms that are still poorly understood, especially in hydrophytes. This study focuses on the young floating lamina of the rhyzophyte Trapa natans exposed for 10 d to 130 microM Mn. The lamina has the ability to bioaccumulate Mn (> 3000 microg g(-1)). X-ray microanalysis of Mn cellular distribution revealed accumulation in the upper epidermis, in the first palisade layer, and in the idioblasts of the spongy tissue, which were shown with electron microscopy to contain osmiophilic vacuolar deposits, also observed to a minor extent in the control leaves. On the basis of biochemical and histochemical tests, these deposits were attributed to phenolic compounds that were probably able to chelate Mn. Net photosynthesis, photosynthetic pigments, room temperature microspectrofluorimetric analyses, and ultrastructural studies of plastids were performed to evaluate the status of the photosynthetic apparatus. A greater development of thylakoid membranes was observed in plastids of the second palisade and spongy tissue, which, however, did not accumulate Mn. Only the spongy tissue experienced inadequate assembly of PS II, but this did not significantly influence the photosynthetic yield of the whole lamina. It was concluded that T. natans can optimise productivity in the presence of Mn by means of specific intra-tissue responses within the framework of the floating lamina.     

Improvements in the determination of manganese peroxidase activity

The existing method of determining the activity of manganese peroxidase (MnP), produced by Phanerochaete chrysosporium, was improved. 2,6-Dimethoxyphenol at 80 mM was used as a substrate and, after the decolorization of the reaction mixture, H₂O₂ was added and the initial reaction rate was used to determine MnP activity.     

Bauxite residue sand has the capacity to rapidly decrease availability of added manganese

Bauxite residue sand, even though a poor substrate for plant growth because of very high pH, salinity and sodicity, is required to be revegetated. Manganese deficiency is observed in residue-grown plants because broadcast applications of manganese fertiliser to the surface of residue deposits have a low residual value. In a laboratory experiment, manganese (as MnSO₄) was added to fresh and 4-year-old residue sand and a sequential fractionation procedure performed at 0, 1, 4, 8 and 24 h and 6, 14, 21, 43, 73, 103 and 130 d. Extraction with DTPA estimated plant-available Mn, while sequential fractionation with various extractants yielded the following fractions: readily soluble [Ca(NO₃)₂]; weakly adsorbed [CaDTPA-B₄O₇]; carbonate-bound [HNO₃]; and oxide-bound [NH₂OHHCl]. Residual Mn was calculated as a difference between the sum of all these forms and total Mn in residue sand. Transformation of manganese from the initially dominant readily soluble form to the less-available forms was very rapid (< 24 h). A change to fertilisation strategies is required if better efficiency of manganese application and uptake is to be achieved for plants growing on bauxite residue.     

Chlorophyll fluorescence transients of Photosystem II membrane particles as a tool for studying photosynthetic oxygen evolution

The rise of the chlorophyll fluorescence yield of Photosystem II (PS II) membranes as induced by high-intensity actinic light comprises only two distinct phases: (1) the initial O-J increase and (2) the subsequent J-P increase. Partial inhibition of the PS II donor side by heating or washing procedures which remove peripheral PS II proteins or cofactors of the oxygen-evolving complex results in decrease of magnitude and rate of the J-P phase. The rate constant of the J-P increase is directly proportional to the steady-state rate of oxygen evolution; complete suppression of the J-P phase corresponds to full inhibition. A characteristic dip after J-level is observed only in Tris-washed or severely heated PS II membranes; manganese release correlates with appearance of the dip after J-level as verified by EPR spectroscopy. Presence of stabilizing cosolutes (glycine betaine, sucrose) or addition of donor-side cofactors (bicarbonate, chloride, calcium) to PS II membranes before heating (47 °C, 5 min) diminishes J-P phase suppression and prevents dip appearance, whereas the addition after heating is without effect. In conclusion, analysis of chlorophyll fluorescence transients of PS II membranes is a potentially useful tool for investigations on photosynthetic oxygen evolution. A decreased rate of the J-P phase can be employed as a convenient indicator for partial inhibition of oxygen-evolution activity; the appearance of a dip after J-level is suggestive of manganese release. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative study of effects of artificial electron donors on the AT-band of photosystem II thermoluminescence

Extraction of the Mn-cluster from photosystem II (PS II) inhibits the main bands of thermoluminescence and induces a new A_T-band at –20°C. This band is attributed to the charge recombination between acceptor Q_A ^– and a redoxactive histidine residue on the donor side of PS II. The effect of Mn(II) and Fe(II) cations as well as the artificial donors diphenylcarbazide and hydroxylamine on the A_T-band of thermoluminescence was studied to elucidate the role of the redoxactive His residue in binding to the Mn(II) and Fe(II). At the Mn/PS II reaction center (RC) ratio of 90 : 1 and Fe/PS II RC ratio of 120 : 1, treatment with Mn(II) and Fe(II) causes only 60% inhibition of the A_T-band. Preliminary exposure of Mn-depleted PS II preparations to light in the presence of Mn(II) and Fe(II) causes binding of the cations to the high-affinity Mn-binding site, thereby inhibiting oxidation of the His residue involved in the AT -band formation. The efficiency of the A_T-band quenching induced by diphenylcarbazide and hydroxylamine is almost an order of magnitude higher than the quenching efficiency of Mn(II) and Fe(II). Our results suggest that the redox-active His is not a ligand of the high-affinity site and does not participate in the electron transport from Mn(II) and Fe(II) to YZ . The concentration dependences of the A_T-band inhibition by Mn(II) and Fe(II) coincide with each other, thereby implying specific interaction of Fe(II) with the donor side of PS II.     

In vitro degradation of a polymeric dye (Poly R-478) by manganese peroxidase

The aim of this study is the evaluation of the enzymatic action of the ligninolytic enzyme manganese peroxidase (MnP), through a suitable addition of H(2)O(2), as a feasible system for the in vitro degradation of complex structures. For this purpose, a highly recalcitrant polymeric dye (Poly R-478) was selected as a model compound. An amperometric technique was used to determine the H(2)O(2) requirement in the decolorization by nonpurified MnP. Two H(2)O(2) supply strategies-fed-batch (every hour) or semicontinuous (every 5 min)-were applied. The addition of H(2)O(2) in pulses led to a limited decolorization after the pulses and the instantaneous consumption or decomposition of H(2)O(2). Therefore, this way of addition may limit the actual H(2)O(2) concentration in the reaction mixture. In contrast, the semicontinuous strategy maintained lower and prolonged concentrations of H(2)O(2), which allowed a clearly greater decolorization (48% after 2 h). In addition, the effect of Mn(+2) concentration on the decolorization efficiency was investigated to establish the optimal application of the MnP-oxidative system. The enzymatic treatment provoked not only the destruction of the chromophoric groups but also a noticeable breakdown of the chemical structure of the dye. In experiments with pure enzyme, MnP proved to be the main factor responsible for the dye decolorization.     

Synthesis and antimicrobial activity of copper(II) and manganese(II) α,ω-dicarboxylate complexes

Copper(II) ,-dicarboxylate complexes of general formulae, [Cu(O₂C(CH₂)_nCO₂)]·xH₂O, [Cu(O₂C(CH₂)_nCO₂) (phen)₂]·xH₂O and [Cu(O₂C(CH₂)_nCO₂)(bipy)_y]·xH₂O (n=1–8; y=1, 2; phen = 1,10-phenanthroline; bipy = 2,2-bipyridine) were synthesised. These copper complexes, some related manganese(II) complexes and the metal-free ligands were screened in vitro for their ability to inhibit the growth of Candida albicans. Metal-free 1,10-phenanthroline and all of the copper(II) and manganese(II) phenanthroline complexes were potent growth inhibitors, with only one bipyridine complex, [Cu(O₂C(CH₂)CO₂)(bipy)₂]·2H₂O, having moderate activity. The remaining substances were effectively inactive. Complexes which were active against C. albicans also proved effective against C. glabrata, C. tropicalis and C. kreusi with the manganese complexes retaining superior activity. For the phenanthroline complexes the active drug species is thought to be the dication [M(phen)₂(H₂O)_n]²⁺ (M = Cu, Mn). Escherichia coli and Staphylococcus aureus were resistant to all of the metal complexes and also to metal-free 1,10-phenanthroline. Only the copper phenanthroline complexes showed intermediate activity against Pseudomonas aeruginosa.