Bromine is an essential trace element for assembly of collagen IV scaffolds in tissue development and architecture.

Bromine is ubiquitously present in animals as ionic bromide (Br(-)) yet has no known essential function. Herein, we demonstrate that Br(-) is a required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks, a posttranslational modification essential for tissue development and architecture found within the collagen IV scaffold of basement membranes (BMs). Bromide, converted to hypobromous acid, forms a bromosulfonium-ion intermediate that energetically selects for sulfilimine formation. Dietary Br deficiency is lethal in Drosophila, whereas Br replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br(-), collagen IV, and peroxidasin. We establish that Br(-) is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Thus, bromine is an essential trace element for all animals, and its deficiency may be relevant to BM alterations observed in nutritional and smoking-related disease. PAPERFLICK:

Peroxidasin-mediated bromine enrichment of basement membranes.

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

Elemental imaging (LA-ICP-MS) of zebrafish embryos to study the toxicokinetics of the acetylcholinesterase inhibitor naled.

The zebrafish embryo is an important model in ecotoxicology but the spatial distribution of chemicals and the relation to observed effects is not well understood. Quantitative imaging can help to gain insights into the distribution of chemicals in the zebrafish embryo. Laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) is used to quantify the uptake and the uptake kinetics of the bromine (Br) containing organophosphate naled (Dibrom®, dimethyl-1,2-dibromo-2,2-dichloroethylphosphate) and its distribution in zebrafish embryos using Br as the marker element. During exposure, the Br amounts increase in the embryos parallel to the irreversible inhibition of the acetylcholinesterase (AChE). The final amount of Br in the embryo (545 pmol/embryo) corresponds to a 280-fold enrichment of naled from the exposure solution. However, LC-MS/MS analyses showed that the internal concentration of naled remained below the LOD (7.8 fmol/embryo); also the concentration of its known transformation product dichlorvos remained low (0.85 to 2.8 pmol/embryo). These findings indicate the high reactivity and high transformation rate of naled to other products than dichlorvos. 12C normalized intensity distributions of Br in the zebrafish embryo showed an enrichment of Br in its head region. Kernel density estimates of the LA-ICP-MS data were calculated and outline the high reproducibility between replicated and the shift in the Br distribution during exposure. The Br enrichment indicates a preferential debromination or direct covalent reaction of naled with AChE in this region. Graphical abstract ᅟ.

Halogens are key cofactors in building of collagen IV scaffolds outside the cell.

PURPOSE OF REVIEW: The purpose of this review is to highlight recent advances in understanding the molecular assembly of basement membranes, as exemplified by the glomerular basement membrane (GBM) of the kidney filtration apparatus. In particular, an essential role of halogens in the basement membrane formation has been discovered. RECENT FINDINGS: Extracellular chloride triggers a molecular switch within non collagenous domains of collagen IV that induces protomer oligomerization and scaffold assembly outside the cell. Moreover, bromide is an essential cofactor in enzymatic cross-linking that reinforces the stability of scaffolds. Halogenation and halogen-induced oxidation of the collagen IV scaffold in disease states damage scaffold function. SUMMARY: Halogens play an essential role in the formation of collagen IV scaffolds of basement membranes. Pathogenic damage of these scaffolds by halogenation and halogen-induced oxidation is a potential target for therapeutic interventions.

Emission of volatile halogenated compounds, speciation and localization of bromine and iodine in the brown algal genome model Ectocarpus siliculosus.

This study explores key features of bromine and iodine metabolism in the filamentous brown alga and genomics model Ectocarpus siliculosus. Both elements are accumulated in Ectocarpus, albeit at much lower concentration factors (2-3 orders of magnitude for iodine, and < 1 order of magnitude for bromine) than e.g. in the kelp Laminaria digitata. Iodide competitively reduces the accumulation of bromide. Both iodide and bromide are accumulated in the cell wall (apoplast) of Ectocarpus, with minor amounts of bromine also detectable in the cytosol. Ectocarpus emits a range of volatile halogenated compounds, the most prominent of which by far is methyl iodide. Interestingly, biosynthesis of this compound cannot be accounted for by vanadium haloperoxidase since the latter have not been found to catalyze direct halogenation of an unactivated methyl group or hydrocarbon so a methyl halide transferase-type production mechanism is proposed.

Microbial and Functional Biodiversity Patterns in Sponges that Accumulate Bromopyrrole Alkaloids Suggest Horizontal Gene Transfer of Halogenase Genes.

Marine sponge holobionts harbor complex microbial communities whose members may be the true producers of secondary metabolites accumulated by sponges. Bromopyrrole alkaloids constitute a typical class of secondary metabolites isolated from sponges that very often display biological activities. Bromine incorporation into secondary metabolites can be catalyzed by either halogenases or haloperoxidases. The diversity of the metagenomes of sponge holobiont species containing bromopyrrole alkaloids (Agelas spp. and Tedania brasiliensis) as well as holobionts devoid of bromopyrrole alkaloids spanning in a vast biogeographic region (approx. Seven thousand km) was studied. The origin and specificity of the detected halogenases was also investigated. The holobionts Agelas spp. and T. brasiliensis did not share microbial halogenases, suggesting a species-specific pattern. Bacteria of diverse phylogenetic origins encoding halogenase genes were found to be more abundant in bromopyrrole-containing sponges. The sponge holobionts (e.g., Agelas spp.) with the greatest number of sequences related to clustered, interspaced, short, palindromic repeats (CRISPRs) exhibited the fewest phage halogenases, suggesting a possible mechanism of protection from phage infection by the sponge host. This study highlights the potential of phages to transport halogenases horizontally across host sponges, particularly in more permissive holobiont hosts, such as Tedania spp.

Detailed spectroscopic study of the role of Br and Sr in coloured parts of the Callinectes sapidus crab claw.

The exoskeleton of crustaceans consists mainly of calcium carbonate (CaCO3) minerals and in many cases exhibits vivid colouration due to the presence of proteins rich in carotenoid chromophores. The exposure of aquatic animals in sea water results often in the incorporation of trace elements in their exoskeleton. The bonding configuration of Br and Sr trace elements in regions with different staining (white, orange and blue) of the exoskeleton of the Callinectes sapidus in crab claw are systematically investigated by a number of complementary spectroscopic techniques, including X-ray absorption fine structure spectroscopy (EXAFS), X-ray fluorescence, Raman and visible light reflectivity spectroscopies. It is found that Sr substitutes for Ca and the Sr/Ca ratio is constant along the claw. In the orange region that includes the claw fingers, CaCO3 adopts a calcite-like structure, whereas in the blue and white regions, located in the palm of the claw, an aragonite-like structure dominates. On the other hand, Br, present only in the blue and orange stained parts of the claw, is bound to phenyl and/or phenol rings of amino acid residues, most probably to phenylalanine and/or tyrosine, of the chromophore protein.

Trace Elements in Ovaries: Measurement and Physiology.

Traditionally, research in the field of trace element biology and human and animal health has largely depended on epidemiological methods to demonstrate involvement in biological processes. These studies were typically followed by trace element supplementation trials or attempts at identification of the biochemical pathways involved. With the discovery of biological molecules that contain the trace elements, such as matrix metalloproteinases containing zinc (Zn), cytochrome P450 enzymes containing iron (Fe), and selenoproteins containing selenium (Se), much of the current research focuses on these molecules, and, hence, only indirectly on trace elements themselves. This review focuses largely on two synchrotron-based x-ray techniques: X-ray absorption spectroscopy and x-ray fluorescence imaging that can be used to identify the in situ speciation and distribution of trace elements in tissues, using our recent studies of bovine ovaries, where the distribution of Fe, Se, Zn, and bromine were determined. It also discusses the value of other techniques, such as inductively coupled plasma mass spectrometry, used to garner information about the concentrations and elemental state of the trace elements. These applications to measure trace elemental distributions in bovine ovaries at high resolutions provide new insights into possible roles for trace elements in the ovary.

High-resolution mass spectrometry provides novel insights into products of human metabolism of organophosphate and brominated flame retardants.

The high resolution, accurate mass, and fast scanning features of the Orbitrap(TM) mass spectrometer, combined with the separation power of ultrahigh-performance liquid chromatography were applied for the first time to study the metabolic profiles of several organic flame retardants (FRs) present in indoor dust. To mimic real-life exposure, in vitro cultured HepG2 human hepatocyte cell lines were exposed simultaneously to various FRs in an indoor dust extract for 24 h. Target parent FRs, hexabromocyclododecanes (α-, ß-, and γ-HBCDs), tris-2-chloroethyl phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCIPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), were separated in a single run for the first time using alternating positive and negative heated ESI source. Further metabolite separation and identification was achieved using full scan (70,000 full width at half maximum (FWHM)), accurate mass (up to 1 ppm) spectrometry. Structural confirmation was performed via all ion fragmentation (AIF) spectra using the optional higher collisional dissociation (HCD) cell and MS/MS analysis. First insights into human metabolism of HBCDs revealed several hydroxylated and debrominated phase I metabolites, in addition to conjugated phase II glucuronides. Furthermore, various hydroxylated, oxidized, and conjugated metabolites of chlorinated phosphorous FRs were identified, leading to the suggestion of α-oxidation as a significant metabolic pathway for these compounds.

In vivo speciation studies and antioxidant properties of bromine in Laminaria digitata reinforce the significance of iodine accumulation for kelps.

The metabolism of bromine in marine brown algae remains poorly understood. This contrasts with the recent finding that the accumulation of iodide in the brown alga Laminaria serves the provision of an inorganic antioxidant - the first case documented from a living system. The aim of this study was to use an interdisciplinary array of techniques to study the chemical speciation, transformation, and function of bromine in Laminaria and to investigate the link between bromine and iodine metabolism, in particular in the antioxidant context. First, bromine and iodine levels in different Laminaria tissues were compared by inductively coupled plasma MS. Using in vivo X-ray absorption spectroscopy, it was found that, similarly to iodine, bromine is predominantly present in this alga in the form of bromide, albeit at lower concentrations, and that it shows similar behaviour upon oxidative stress. However, from a thermodynamic and kinetic standpoint, supported by in vitro and reconstituted in vivo assays, bromide is less suitable than iodide as an antioxidant against most reactive oxygen species except superoxide, possibly explaining why kelps prefer to accumulate iodide. This constitutes the first-ever study exploring the potential antioxidant function of bromide in a living system and other potential physiological roles. Given the tissue-specific differences observed in the content and speciation of bromine, it is concluded that the bromide uptake mechanism is different from the vanadium iodoperoxidase-mediated uptake of iodide in L. digitata and that its function is likely to be complementary to the iodide antioxidant system for detoxifying superoxide.

Kinetic bromine isotope effect: example from the microbial debromination of brominated phenols.

The increasing use of kinetic isotope effects for environmental studies has motivated the development of new compound-specific isotope analysis techniques for emerging pollutants. Recently, high-precision bromine isotope analysis in individual brominated organic compounds was proposed, by the coupling of gas chromatography to a multi-collector inductively coupled plasma mass spectrometer using strontium as an external spike for instrumental bias correction. The present study, for the first time, demonstrates an application of this technique for determining bromine kinetic isotope effects during biological reaction, focusing on the reductive debromination of brominated phenols under anaerobic conditions. Results show bromine isotope enrichment factors (ε) of -0.76 ± 0.08, -0.46 ± 0.19, and -0.20 ± 0.06 ‰ for the debromination of 4-bromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol, respectively. These values are rather low, yet still high enough to be obtained with satisfying certainty. This further implies that the analytical method may be also appropriate for future environmental applications.

Antioxidant and anti-inflammatory activities of barettin.

In this paper, we present novel bioactivity for barettin isolated from the marine sponge Geodia barretti. We found that barettin showed strong antioxidant activity in biochemical assays as well as in a lipid peroxidation cell assay. A de-brominated synthetic analogue of barettin did not show the same activity in the antioxidant cell assay, indicating that bromine is important for cellular activity. Barettin was also able to inhibit the secretion of the inflammatory cytokines IL-1ß and TNFα from LPS-stimulated THP-1 cells. This combination of anti-inflammatory and antioxidant activities could indicate that barettin has an atheroprotective effect and may therefore be an interesting product to prevent development of atherosclerosis.

Formation of THMs and HANs during bromination of Microcystis aeruginosa.

Bromine-contained disinfectants and biocides are widely used in swimming pools, recreational waters and cooling towers. The objective of this study was to evaluate the formation of thrihalomethanes (THMs) and haloacetonitriles (HANs) and their cytotoxicity in algae solutions during free bromine disinfection. Disinfection by-products formation potential experiments were conducted using model solutions containing 7 mg/L (as total organic carbon) Microcystis aeruginosa cells. Effects of free bromine dosage, pH and ammonia were investigated. The results showed that brominated disinfection by-products were the major products when free bromine was applied. The total THMs formed during bromination was much as that formed during chlorination, whereas HANs were elevated by using bromination instead of chlorination. Dibromoacetonitrice (C2H2NBr2) and bromoform (CHBr3) were the only detected species during free bromine disinfection. The production of C2H2NBr2 and CHBr3 increased with disinfectant dosage but decreased with dosing ammonia. CHBr3 increased with the pH changing from 5 to 9. However, C2H2NBr2 achieved the highest production at neutral pH, which was due to a joint effect of variation in hydrolysis rate and free bromine reactivity. The hydrolysis of C2H2NBr2 was base-catalytic and nearly unaffected by disinfectant. Finally, estimation of cytotoxicity of the disinfected algae solutions showed that HANs formation was responsible for the majority of toxicity. Considering its highest toxicity among the measured disinfection by-products, the elevated C2H2NBr2 should be considered when using bromine-related algaecide.

Growth of Dehalococcoides mccartyi strain CBDB1 by reductive dehalogenation of brominated benzenes to benzene.

Brominated aromatics are used in many different applications but occur also naturally. Here, we demonstrate organohalide respiration and growth of Dehalococcoides mccartyi strain CBDB1 with 1,2,4-tribromobenzene, all three dibrominated benzene congeners and monobromobenzene. All bromobenzenes were fully dehalogenated to benzene. Growth yields were between 1.8 × 10(14) and 2.8 × 10(14) cells per mol of bromide released. Furthermore, a newly designed high-throughput methyl viologen-based photometric microtiter plate assay was established to determine the activity of the reductive dehalogenases in resting cell assays of strain CBDB1 with brominated aromatics as electron acceptors. Activities of 2.8-13.2 nkat per mg total cell protein (0.16-0.8 units per mg total cell protein) were calculated after cultivation of strain CBDB1 on 1,2,4-tribromobenzene. Mass spectrometric analyses and activity assays with whole cell extracts of strain CBDB1 gave strong evidence that four to six reductive dehalogenases were involved in the dehalogenation of all tested brominated benzenes, including the reductive dehalogenases CbdbA80 and CbrA.

Location of chloride and its possible functions in oxygen-evolving photosystem II revealed by X-ray crystallography.

The chloride ion, Cl(-), is an essential cofactor for oxygen evolution of photosystem II (PSII) and is closely associated with the Mn(4)Ca cluster. Its detailed location and function have not been identified, however. We substituted Cl(-) with a bromide ion (Br(-)) or an iodide ion (I(-)) in PSII and analyzed the crystal structures of PSII with Br(-) and I(-) substitutions. Substitution of Cl(-) with Br(-) did not inhibit oxygen evolution, whereas substitution of Cl(-) with I(-) completely inhibited oxygen evolution, indicating the efficient replacement of Cl(-) by I(-). PSII with Br(-) and I(-) substitutions were crystallized, and their structures were analyzed. The results showed that there are 2 anion-binding sites in each PSII monomer; they are located on 2 sides of the Mn(4)Ca cluster at equal distances from the metal cluster. Anion-binding site 1 is close to the main chain of D1-Glu-333, and site 2 is close to the main chain of CP43-Glu-354; these 2 residues are coordinated directly with the Mn(4)Ca cluster. In addition, site 1 is located in the entrance of a proton exit channel. These results indicate that these 2 Cl(-) anions are required to maintain the coordination structure of the Mn(4)Ca cluster as well as the proposed proton channel, thereby keeping the oxygen-evolving complex fully active.

Micronutrients and macronutrients in the liver in experimental alimentary obesity.

Strong and very strong positive correlations differing from those in the control were found between chemical elements in the liver of rats with experimental alimentary obesity under conditions of physiological hunger and soon after fatty food intake. These findings attest to sustained metabolism disturbances in obesity and formation of a pathological system. Considering the appearance of strong and very strong correlations of Br and Rb in alimentary obesity, more attention should be paid to the investigation of the role of these poorly studied elements in lipid metabolism.

Induction of halogenated vesicle transport in cells of the red seaweed Laurencia obtusa.

In clones of the red alga Laurencia obtusa, the frequency of vesicle transport from corps en cerise (CC) to the cell wall region was evaluated in response to differences in temperature, irradiance, desiccation, bacterial fouling, and bromine (Br) availability. In addition, the morphology of the corps en cerise was analyzed. Traffic of vesicles was induced by exposing L. obtusa to low temperatures and variations in irradiance. It was also verified that bacterial fouling induced vesicle traffic. Under high temperatures and desiccation, the membranous tubular connections were lost and transport of vesicles was not seen. The morphology of the corps en cerise varied according to the availability of Br in seawater. Exocytosis of secondary metabolites by L. obtusa was shown to vary in relation to temperature, irradiance, desiccation and bacterial fouling. The data suggest that the transport of vesicles in L. obtusa may be related to the inhibition of the microfouling community on the algal surface.

Brain Bromine Levels Associated with Alzheimer's Disease Neuropathology.

BACKGROUND: Bromine is a naturally occurring element that is widely present in the human environment in various chemical forms primarily as flame retardants, pesticides, and water treatments. OBJECTIVE: In this exploratory study, we investigated the association of brain bromine concentrations on Alzheimer's disease (AD) neuropathology, cerebral infarcts, and Lewy bodies. METHODS: The study was conducted in 215 deceased participants of the Memory and Aging Project, a clinical-pathologic cohort study. Brain bromine levels were measured using instrumental neutron activation analysis. Multiple brain regions were assessed for diffuse and neuritic plaques, neurofibrillary tangles, cerebral macro-and microinfarcts, and Lewy bodies. Standardized measures of AD pathology (Braak, CERAD, NIA-Reagan, global AD pathology) were computed. RESULTS: In linear regression models, the higher brain bromine levels were associated with more AD neuropathology (Braak (p trend = 0.01); CERAD (p trend = 0.02); NIA-Reagan (p trend = 0.02). CONCLUSION: Bromine accumulation in the brain is associated with higher level of AD neuropathology. The potential deleterious effects of this element on AD need further exploration.

Fluorescence and UV resonance Raman study of peptide-vesicle interactions of human cathelicidin LL-37 and its F6W and F17W mutants.

LL-37 is a broad-spectrum human antimicrobial peptide in the cathelicidin family. Potency assays in the form of minimal inhibitory concentration and vesicle leakage indicate that the single-tryptophan mutants, F6W and F17W, are as effective at killing bacteria and disrupting membranes as the native, tryptophan-free LL-37 peptide. Steady-state fluorescence and UV resonance Raman spectroscopy of F6W and F17W reveal molecular details of these tryptophan residues. The local environment polarity, hydrogen bond strength of the indole N-H moiety, and rotational freedom decrease for both F6W and F17W in the presence of carbonate ions relative to in pure distilled water; these results are consistent with burial of the hydrophobic region of alpha-helical LL-37 in oligomeric cores induced in the presence of carbonate ions. Differences in the spectroscopic properties of the carbonate-induced alpha-helical forms of F6W and F17W reflect the presence of a local lysine residue near F6W that makes the microenvironment of F6W more polar than that of F17W. In the presence of lipid vesicles, the mutants undergo additional loss of environment polarity, hydrogen bond strength, and rotational freedom. Quenching experiments utilizing brominated lipids reveal that the tryptophan residues in both mutants are essentially equidistant from the bilayer center and that bromines closer to the bilayer center, in the 9,10 positions, quench fluorescence more efficiently than those closer to the headgroups (6,7 positions). These results support carpeting or toroidal pore mechanisms of membrane disruption by LL-37 and demonstrate that the combination of tryptophan mutants and sensitive spectroscopic tools may provide important molecular clues about antimicrobial action.

Br-rich tips of calcified crab claws are less hard but more fracture resistant: a comparison of mineralized and heavy-element biological materials.

We find that the spoon-like tips of the chelipeds (large claws) of the crab Pachygrapsus crassipes differ from the rest of the claw in that they are not calcified, but instead contain about 1% bromine--thus they represent a new example of a class of structural biological materials that contain heavy elements such as Zn, Mn, Fe, Cu, and Br bound in an organic matrix. X-ray absorption spectroscopy data suggest that the bromine is bound to phenyl rings, possibly in tyrosine. We measure a broad array of mechanical properties of a heavy-element biological material for the first time (abrasion resistance, coefficient of kinetic friction, energy of fracture, hardness, modulus of elasticity and dynamic mechanical properties), and we make a direct comparison with a mineralized tissue. Our results suggest that the greatest advantage of bromine-rich cuticle over calcified cuticle is resistance to fracture (the energy of fracture is about an order of magnitude greater than for calcified cuticle). The greatest advantage relative to unenriched cuticle, represented by ant mandible cuticle, is a factor of about 1.5 greater hardness and modulus of elasticity.The spoon-like tips gain additional fracture resistance from the orientation of the constituent laminae and from the viscoelasticity of the material. We suggest that fracture resistance is of greater importance in smaller organisms, and we speculate that one function of heavy elements in structural biological materials is to reduce molecular resonant frequencies and thereby increase absorption of energy from impacts.