THE EFFECT OF SELENITE ON THE PHYSIOLOGICAL AND MORPHOLOGICAL PROPERTIES OF THE BLUE-GREEN ALGA PHORMIDIUM LURIDUM VAR. OLIVACEA1

Phormidium luridum cultures were treated with sodium selenite in concentrations ranging from 10^?6 M to 10^?2 M. In contrast to the increasing culture turbidity of control and 10^?6 M selenite cultures, the turbidity of the other selenite cultures declined in proportion to time and selenite concentration. Chlorophyll extraction revealed similar results. Photosynthetic activity was inhibited within 6 hr in all cultures except control and 10^?6 M selenite. Phormidium at concentrations greater than 10^?6 M selenite showed a gradual loss of the bright green color and turned semitransparent. Cell-associated granules of reduced selenium were observed at higher selenite concentrations. Other structural changes observed were the presence of intracellular and intercellular spaces, spheroplast formation, and gradual cell lysis. Protein analyses of total cell samples and supernatant fractions confirmed cellular breakdown of selenite-treated algal cells.     

Reduction of Selenite to Elemental Red Selenium by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. Strain B1

A bacterium that reduces the soluble and toxic selenite anion to insoluble elemental red selenium (Se⁰) was isolated from a laboratory bioreactor. Biochemical, morphological, and 16S rRNA gene sequence alignment identified the isolate as a Rhizobium sp. that is related to but is genetically divergent from R. radiobacter (syn. Agrobacterium tumefaciens) or R. rubi (syn. A. rubi). The isolate was capable of denitrification and reduced selenite to Se⁰ under aerobic and denitrifying conditions. It did not reduce selenate and did not use selenite or selenate as terminal e⁻ donors. Native gel electrophoresis revealed two bands, corresponding to molecular weights of ∼100 and ∼45 kDa, that reduced selenite. Tungsten inhibited in vivo selenite reduction, suggesting that a molybdenum-containing protein is involved in selenite reduction. This organism, or its enzymes or DNA, might be useful in bioreactors designed to remove selenite from water.     

Bioavailability of selenium accumulated by selenite-reducing bacteria

The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (Se^o). A laboratory strain ofBacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na₂SeO₃). After harvest, the washed, lyophilizedB. subtilis andM. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of Se^o. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray Se^o had a bioavailability of 2% of selenite, but that the bioavailability of red Se^o depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray Se^o and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3–4%). These findings suggest that aerobic bacteria such asB. subtilis andM. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.     

Reduction of Selenite and Detoxification of Elemental Selenium by the Phototrophic Bacterium Rhodospirillum rubrum

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1.5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.     

Extracellular reduction of selenite by a novel marine photosynthetic bacterium

A novel purple nonsulfur bacterium strain NKPB030619, which has resistance to over 5 mM selenite, was isolated from a marine environment. An initial concentration of 1.1 mM selenite, added to the medium, was decreased to under 0.05 mM within 5 days. The color of the cell suspension turned red within 2 days. The red coloration gradually decreased and black precipitates appeared during 2 weeks of cultivation. Under these conditions, two main types of deposit were formed extracellularly. These deposits were thought to contain red amorphous selenium and black vitreous selenium. The selenite reduction to elemental selenium in this bacterium was induced by the introduction of light and l-malic acid under anaerobic conditions. These results suggest that selenite reduction is coupled with photosynthesis and l-malic acid can serve as the indirect electron donor for its reduction. Phylogenetic analysis based on the 16S rDNA sequence showed that NKPB0360619 belongs to the α subdivision of Proteobacteria and is classified into the Rhodobacter species. The highest similarity of 86.2% was observed with R. sphaeroides. Received: 13 August 1996 / Received last revision: 6 May 1997 / Accepted: 11 May 1997     

Physiological adaptations and tolerance towards higher concentration of selenite (Se+4) in Enterobacter sp. AR-4, Bacillus sp. AR-6 and Delftia tsuruhatensis AR-7

Environmental contamination with selenium is a major health concern. A few bacterial strains have been isolated that can transform toxic selenite to non-toxic elemental selenium only at low concentrations (0.001–150 mM) in recent past. We have previously reported isolation and characterization of few selenite-tolerant bacterial strains. These strains were found to be resistant to selenite at (300–600 mM) concentrations. In the present study we have characterized some physiological adaptations of strains Enterobacter sp. AR-4, Bacillus sp. AR-6 and Delftia tsuruhatensis AR-7 during exposure to higher concentration of selenite under aerobic and anaerobic environments. Adaptive responses are largely associated with alteration of cell morphology and change in total cellular fatty acid composition. Interestingly, electron microscopy studies revealed substantial decrease in cell size and intracellular deposition of Se⁰ crystals when reduction is carried out under aerobic conditions. On the other hand, cell size increased with adhesion of Se⁰ on cell surface during anaerobic reduction. Fatty acid composition analysis demonstrated selective increase in saturated and cyclic fatty acids and decrease in unsaturated ones during aerobic transformation. Changes observed during anaerobic transformation were in surprising contrast as indicated by total absence of saturated and cyclic fatty acids. Results presented here provide evidences for putative occurrence of two distinct mechanisms involved in tolerance towards higher concentrations of selenite utilization under aerobic and anaerobic conditions. Further, prior exposure to higher concentration of Se⁺⁴ enabled rapid adaptation indicating role of inducible system in adaptation.     

Reduction of Elemental Selenium to Selenide: Experiments with Anoxic Sediments and Bacteria that Respire Se-Oxyanions

A selenite-respiring bacterium, Bacillus selenitireducens, produced significant levels of Se(-II) (as aqueous HSe^?) when supplied with Se(0). B. selenitireducens was also able to reduce selenite [Se(IV)] through Se(0) to Se(-II). Reduction of Se(0) by B. selenitireducens was more rapid in cells grown on colloidal sulfur [S(0)] or Se(IV) as their electron acceptor than for cell lines grown on fumarate. In contrast, three cultures of selenate-respiring bacteria, Sulfurospirillum barnesii, B. arsenicoselenatis, and Selenihalanaerobacter shriftii either were unable to reduce Se(0) to Se(-II) or had only a very limited capacity to achieve this reduction. Biological reduction of Se(0) to Se(-II) was observed during incubation of estuarine sediment slurries, while no such activity was noted in formalin-killed controls. The majority of the Se(-II) produced was found in the sediments as a solid precipitate of FeSe, rather than in solution as HSe^?. These results demonstrate that certain anaerobic bacteria have the capacity to reduce Se(0) to Se(-II), providing a possible biological explanation for the occurrence of the selenide species in some sedimentary rocks.     

Reduction of Selenite by Azospirillum brasilense with the Formation of Selenium Nanoparticles

The ability to reduce selenite (SeO₃ ^2?) ions with the formation of selenium nanoparticles was demonstrated in Azospirillum brasilense for the first time. The influence of selenite ions on the growth of A. brasilense Sp7 and Sp245, two widely studied wild-type strains, was investigated. Growth of cultures on both liquid and solid (2 % agar) media in the presence of SeO₃ ^2? was found to be accompanied by the appearance of the typical red colouration. By means of transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS) and X-ray fluorescence analysis (XFA), intracellular accumulation of elementary selenium in the form of nanoparticles (50 to 400 nm in diameter) was demonstrated for both strains. The proposed mechanism of selenite-to-selenium (0) reduction could involve SeO₃ ^2? in the denitrification process, which has been well studied in azospirilla, rather than a selenite detoxification strategy. The results obtained point to the possibility of using Azospirillum strains as endophytic or rhizospheric bacteria to assist phytoremediation of, and cereal cultivation on, selenium-contaminated soils. The ability of A. brasilense to synthesise selenium nanoparticles may be of interest to nanobiotechnology for “green synthesis” of bioavailable amorphous red selenium nanostructures.     

A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite

The measurement of elemental selenium (Se⁰) is needed to assess the rate and magnitude of bacteria reduction of selenite or selenate. We have developed a spectrophotometric method for the measurement Se⁰ that is rapid and can be employed to measure the quantity of Se⁰ produced by bacterial cultures. This method employs the use of 1 M Na₂S to convert the insoluble elemental selenium to a red-brown solution and with this method there is a direct correlation between concentration of elemental selenium and the absorption at 500 nm. To demonstrate the utility of this assay, we have followed the reduction of selenite to Se⁰ by Moraxella bovis, and by bacterial consortia in soil and water samples.     

Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Summary Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and¹³C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 M selenate and 100 M selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.     

Selenite-stress selected mutant strains of probiotic bacteria for Se source production

Selenium deficiency is a major health problem worldwide for about 1 billion people. Bacterial cells usually possess low tolerance to selenite stress and also low ability to reduce high concentrations of toxic selenite. Here, high tolerance to selenite and selenium bioaccumulation capability were developed in mutated clones of probiotic and starter bacteria including Enterococcus faecium, Bifidobacterium animalis ssp. lactis, Lactobacillus casei and Lactococcus lactis ssp. lactis by food-level strain development process and clone selection. All mutant clones possessed increased glutathione concentration and glutathione reductase activity. The selenite treatment increased further these values in L. casei mutant strain pointing at a different selenite reduction pathway and/or stress response in this organism. Considerable conversion of selenite to cell bound selenium forms with a concomitant high biomass production was detected in E. faecium and B. animalis ssp. lactis cultures. Possible application of these strains as food and feed supplements is under investigation.     

Diminazene aceturate associated with sodium selenite and vitamin E in the treatment of Trypanosoma evansi infection in rats

The aim of this study was to evaluate the utilization of a standard treatment with diminazene aceturate against the infection caused by Trypanosoma evansi, associated to sodium selenite and vitamin E. In vitro tests showed trypanocidal effect related to the treatment with diminazene aceturate and sodium selenite, but vitamin E had no harmful effect on the trypanosomes. In vivo experiments utilized a total of 72 adult outbreed females rats, separated into 9 groups (A, B, C, D, E, F, G, H and I), 8 animals each. Group A was the uninfected group; groups B to I were infected with 0.2 mL of blood containing 10⁶ trypanosomes. Parasitemia was estimated daily by microscopic examination of blood smears. Group B served as positive control; group C was treated with diminazene aceturate; group D with sodium selenite; group E with vitamin E; group F received an association of diminazene aceturate and sodium selenite; group G received an association of diminazene aceturate and vitamin E; group H received an association of diminazene aceturate, sodium selenite and vitamin E, and group I received an association of sodium selenite and vitamin E. Diminazene aceturate was administrated in a single dose on the 3rd day post infection (PI). Sodium selenite and vitamin E were administered at the 3rd and 23rd day PI. In vivo tests showed increase of longevity in groups treated with diminazene aceturate associated with sodium selenite (groups F and H). No difference was found between groups C and E, thus the vitamin E did not increase the efficacy of treatment against T. evansi when associated to diminazene aceturate. The curative efficacy of treatments was 37.5, 87.7, 37.7 and 75% to the groups C, F, G and H, respectively. Other treatments showed no efficacy. The sodium selenite when combined with chemotherapy may represent an alternative in the treatment of trypanosomosis.     

Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3

A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO₂ with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Se⁰). Hence, SES-3 can carry out a complete reduction of selenate to Se⁰. Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of [¹⁴C]lactate to ¹⁴CO₂ coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m-chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Se⁰ by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [⁷⁵Se]selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Se⁰. This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Se⁰ may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.     

Reduction of Selenite to Red Elemental Selenium by Rhodopseudomonas palustris Strain N

The trace metal selenium is in demand for health supplements to human and animal nutrition. We studied the reduction of selenite (SeO₃ ⁻²) to red elemental selenium by Rhodopseudomonas palustris strain N. This strain was cultured in a medium containing SeO₃ ⁻² and the particles obtained from cultures were analyzed using transmission electron microscopy (TEM), energy dispersive microanalysis (EDX) and X ray diffraction analysis (XRD). Our results showed the strain N could reduce SeO₃ ⁻² to red elemental selenium. The diameters of particles were 80–200 nm. The bacteria exhibited significant tolerance to SeO₃ ⁻² up to 8.0 m mol/L concentration with an EC₅₀ value of 2.4 m mol/L. After 9 d of cultivation, the presence of SeO₃ ²⁻ up to 1.0 m mol/L resulted in 99.9% reduction of selenite, whereas 82.0% (p<0.05), 31.7% (p<0.05) and 2.4% (p<0.05) reduction of SeO₃ ⁻² was observed at 2.0, 4.0 and 8.0 m mol/L SeO₃ ²⁻ concentrations, respectively. This study indicated that red elemental selenium was synthesized by green technology using Rhodopseudomonas palustris strain N. This strain also indicated a high tolerance to SeO₃ ⁻². The finding of this work will contribute to the application of selenium to human health.     

High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae

Background

Microorganisms that are exposed to pollutants in the environment, such as metals/metalloids, have a remarkable ability to fight the metal stress by various mechanisms. These metal-microbe interactions have already found an important role in biotechnological applications. It is only recently that microorganisms have been explored as potential biofactories for synthesis of metal/metalloid nanoparticles. Biosynthesis of selenium (Se⁰) nanospheres in aerobic conditions by a bacterial strain isolated from the coalmine soil is reported in the present study.

Results

The strain CM100B, identified as Bacillus cereus by morphological, biochemical and 16S rRNA gene sequencing [GenBank:GU551935.1] was studied for its ability to generate selenium nanoparticles (SNs) by transformation of toxic selenite (SeO₃ ^2-) anions into red elemental selenium (Se⁰) under aerobic conditions. Also, the ability of the strain to tolerate high levels of toxic selenite ions was studied by challenging the microbe with different concentrations of sodium selenite (0.5 mM-10 mM). ESEM, AFM and SEM studies revealed the spherical Se⁰ nanospheres adhering to bacterial biomass as well as present as free particles. The TEM microscopy showed the accumulation of spherical nanostructures as intracellular and extracellular deposits. The deposits were identified as element selenium by EDX analysis. This is also indicated by the red coloration of the culture broth that starts within 2-3 h of exposure to selenite oxyions. Selenium nanoparticles (SNs) were further characterized by UV-Visible spectroscopy, TEM and zeta potential measurement. The size of nanospheres was in the range of 150-200 nm with high negative charge of -46.86 mV.

Conclusions

This bacterial isolate has the potential to be used as a bionanofactory for the synthesis of stable, nearly monodisperse Se⁰ nanoparticles as well as for detoxification of the toxic selenite anions in the environment. A hypothetical mechanism for the biogenesis of selenium nanoparticles (SNs) involving membrane associated reductase enzyme(s) that reduces selenite (SeO₃ ^2-) to Se⁰ through electron shuttle enzymatic metal reduction process has been proposed.     

Reduction of selenium oxyanions by unicellular,polymorphic and filamentous fungi: Cellular location of reduced selenium and implications for tolerance

Summary The ability of several filamentous, polymorphic and unicellular fungi to reduce selenite to elemental selenium on solid medium was examined.Fusarium sp. andTrichoderma reeii were the only filamentous fungi, of those tested, which reduced selenite to elemental selenium on Czapek-Dox agar resulting in a red colouration of colonies. Other organisms (Aspergillus niger, Coriolus versicolor, Mucor SK, andRhizopus arrhizus) were able to reduce selenite only on malt extract agar. Several fungi were able to grow in the presence of sodium selenite but were apparently unable to reduce selenite to elemental selenium, indicating that other mechanisms of selenite tolerance were employed, such as reduced uptake and/or biomethylation to less toxic, volatile derivatives. Sodium selenate was more toxic toFusarium sp. than selenite, and the toxicity of both oxyanions was increased in sulphur-free medium, with this effect being more marked for selenate. Scanning electron microscopy ofAspergillus funiculosus andFusarium sp. incubated with sodium selenite showed the presence of needle-like crystals of elemental selenium on the surfaces of hyphae and conidia, while transmission electron microscopy ofA. funiculosus revealed the deposition of electron-dense granules in vacuoles of selenite-treated fungi. Several yeasts were able to grow on MYGP agar containing sodium selenate or sodium selenite at millimolar concentrations. Sone, notablyRhodotorula rubra andCandida lipolytica, and the polymorphic fungusAureobasidium pullulans were also effective at reducing selenite to elemental selenium, resulting in red-coloured colonies.Schizosaccharomyces pombe was able to grow at selenite concentrations up to 5 mmol L^–1 without any evidence of reduction, again indicating the operation of other tolerance mechanisms.     

Reduction of grasshopper populations following field application of the fungus Beauveria bassiana

Conidia of the Hyphomycete fungus Beauveria bassiana (Bals.) were applied in an attempt to reduce field populations of grasshoppers, primarily the migratory grasshopper Melanoplus sanguinipes (Fabricius). Dry spores were applied with wheat bran carrier to three fallow fields at a rate of 2.0 × 10¹³ spores ha^?1 in 10 kg bait ha^?1. Examination of culture plates that had been placed in the field to capture spores and of bran carrier with scanning electron microscopy indicated that a substantial portion of the B. bassiana colony—forming units (spores and clumps of spores) did not adhere to the bran and were applied in the field as free particles. Grasshoppers collected from the treated plots at intervals after application were assayed for infection by B. bassiana. The observed rate of mycosis in the treated populations was 70% of those collected after 2 days, declining to 41% by 13 days and 5% by 19 days after application. Analysis of reductions in population density gave results in agreement with the infection data. Treated populations declined 60% and 33% by 9 and 15 days after application respectively. The reductions were significant on both post—treatment sampling dates (p < 0.05) and the three replicated fields gave comparable results. This is the first field demonstration of effectiveness of this fungus as a microbial control agent of grasshoppers.     

High-dose sodium selenite can induce apoptosis of lymphoma cells in adult patients with non-Hodgkin's lymphoma

The present study was undertaken to explore the effect of administration of high doses of sodium selenite on the apoptosis of lymphoma cells in patients with non-Hodgkin’s lymphoma (NHL). Forty patients with newly diagnosed NHL were randomly divided into two groups. Group I received standard chemotherapy, whereas group II received adjuvant sodium selenite 0.2 mg kg⁻¹ day⁻¹ for 7 days in addition to chemotherapy. Flow cytometry was used for monitoring of lymphoma cells apoptosis at the time of diagnosis and after therapy in the two groups. Sodium selenite administration resulted in significant increase in percentage of apoptotic lymphoma cells after therapy in group II (78.9 ± 13.3% versus 58.9 ± 18.9%, p < 0.05). In addition, patients who received sodium selenite treatment demonstrated statistically significant increase in percentage of reduction of cervical and axillary lymphadenopathy, decrease in splenic size, and decreased percentage of bone marrow infiltration. Also, we found a statistically significant decrease in cardiac ejection fraction (CEF) in group I and no reduction in CEF in patients who received sodium selenite ‘group II’, denoting the cardioprotective effect of selenium. It is concluded that sodium selenite administration at the dosage and duration chosen has synergistic effect to chemotherapy in inducing apoptosis and, consequently, could improve clinical outcome.     

Tissue and concentration-dependent effects of sodium selenite on muscle contraction

In this study, we demonstrated that sodium selenite with high doses (≥ 10^-3 M) were potent in inducing a contracture type effect on heart and smooth muscles. Selenite (Se), at a concentration of 10^-3 M, caused a contracture effect in heart preparations. Also, low Se concentrations did not have any significant effect. Although low concentrations of Se (≥ 10^-5 M) had a biphasic effects on acetylcholine (ACh) induced and spontaneous ileum contractions, 10^-3 M selenite enhanced once more a contracture effect similar to that of the heart preparations. Replacing Ca²⁺ concentration of the bathing solution by twofold Ca²⁺ or Ca²⁺-free did not change the effects of selenite (10^-5 M) on contractility of ileum preparations. In vascular smooth muscle, low concentration of selenite (<10^-4) had no significant effects on KC1, and phenylephrine-induced contractions and acethylcholineinduced endothelium-dependent relaxations of isolated rabbit aorta. However, the contractions induced by phenylephrine and the relaxations induced by acetylcholine in rabbit aorta were depressed significantly by high concentration of selenite (10^-3 M). The results obtained by selenite exposure from these three different types of tissue preparations first suggest that the high concentration of selenite exposure induces some alterations in the functions of muscles and endothelium in a tissue and dose-dependent manner. Second, this observed irreversible type of dysfunction of tissues induced by l0^-3 M selenite is not directly dependent on the Ca²⁺ entrance into the cytosol, but might be induced by the increase of intracellular Ca²⁺ with the disturbance of Ca²⁺ regulation.