Germ-Free C57BL/6 Mice Have Increased Bone Mass and Altered Matrix Properties but Not Decreased Bone Fracture Resistance.

The gut microbiome impacts bone mass, which implies a disruption to bone homeostasis. However, it is not yet clear how the gut microbiome affects the regulation of bone mass and bone quality. We hypothesized that germ-free (GF) mice have increased bone mass and decreased bone toughness compared with conventionally housed mice. We tested this hypothesis using adult (20- to 21-week-old) C57BL/6J GF and conventionally raised female and male mice (n = 6-10/group). Trabecular microarchitecture and cortical geometry were measured from micro-CT of the femur distal metaphysis and cortical midshaft. Whole-femur strength and estimated material properties were measured using three-point bending and notched fracture toughness. Bone matrix properties were measured for the cortical femur by quantitative back-scattered electron imaging and nanoindentation, and, for the humerus, by Raman spectroscopy and fluorescent advanced glycation end product (fAGE) assay. Shifts in cortical tissue metabolism were measured from the contralateral humerus. GF mice had reduced bone resorption, increased trabecular bone microarchitecture, increased tissue strength and decreased whole-bone strength that was not explained by differences in bone size, increased tissue mineralization and fAGEs, and altered collagen structure that did not decrease fracture toughness. We observed several sex differences in GF mice, most notably for bone tissue metabolism. Male GF mice had a greater signature of amino acid metabolism, and female GF mice had a greater signature of lipid metabolism, exceeding the metabolic sex differences of the conventional mice. Together, these data demonstrate that the GF state in C57BL/6J mice alters bone mass and matrix properties but does not decrease bone fracture resistance. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Antibiotic perturbation of the murine gut microbiome introduces inter-individual susceptibility to arsenic.

Arsenic is a Group 1 human carcinogen and at least 200 million people around the world are exposed to unsafe levels of arsenic, predominantly through contaminated drinking water. Arsenic has also been used for hundreds, if not thousands, of years as an intentional poison due to its odorless/tasteless properties and the general lack of technology required to identify it. Both acute and chronic arsenic-related health outcomes are highly variable among similarly exposed individuals even after controlling for important factors, like host genetics, making the mechanisms underlying this often-made epidemiologic observation difficult to experimentally address and not fully understood. Here, we describe an experimental model of arsenic exposure in C57BL/6 mice that recapitulates key aspects of inter-individuality in disease observed in humans. We show that co-administration of the antibiotic, cefoperazone, and high-level arsenic (100 ppm, inorganic sodium arsenate) results in incomplete mortality with a ratio of 60 % lethality to 40 % survival, and that survival, at least in part, depends not only on an intact microbiome but also a regulated response involved with water transport. This work provides an experimental framework for identifying critical pathways involved in generating inter-individual variability in disease outcome following arsenic exposure.

The Human Gut Microbiome's Influence on Arsenic Toxicity.

PURPOSE OF REVIEW: Arsenic exposure is a public health concern of global proportions with a high degree of interindividual variability in pathologic outcomes. Arsenic metabolism is a key factor underlying toxicity, and the primary purpose of this review is to summarize recent discoveries concerning the influence of the human gut microbiome on the metabolism, bioavailability, and toxicity of ingested arsenic. We review and discuss the current state of knowledge along with relevant methodologies for studying these phenomena. RECENT FINDINGS: Bacteria in the human gut can biochemically transform arsenic-containing compounds (arsenicals). Recent publications utilizing culture-based approaches combined with analytical biochemistry and molecular genetics have helped identify several arsenical transformations by bacteria that are at least possible in the human gut and are likely to mediate arsenic toxicity to the host. Other studies that directly incubate stool samples in vitro also demonstrate the gut microbiome's potential to alter arsenic speciation and bioavailability. In vivo disruption or elimination of the microbiome has been shown to influence toxicity and body burden of arsenic through altered excretion and biotransformation of arsenicals. Currently, few clinical or epidemiological studies have investigated relationships between the gut microbiome and arsenic-related health outcomes in humans, although current evidence provides strong rationale for this research in the future. SUMMARY: The human gut microbiome can metabolize arsenic and influence arsenical oxidation state, methylation status, thiolation status, bioavailability, and excretion. We discuss the strength of current evidence and propose that the microbiome be considered in future epidemiologic and toxicologic studies of human arsenic exposure.

Cellular arsenic transport pathways in mammals.

Natural contamination of drinking water with arsenic results in the exposure of millions of people world-wide to unacceptable levels of this metalloid. This is a serious global health problem because arsenic is a Group 1 (proven) human carcinogen and chronic exposure is known to cause skin, lung, and bladder tumors. Furthermore, arsenic exposure can result in a myriad of other adverse health effects including diseases of the cardiovascular, respiratory, neurological, reproductive, and endocrine systems. In addition to chronic environmental exposure to arsenic, arsenic trioxide is approved for the clinical treatment of acute promyelocytic leukemia, and is in clinical trials for other hematological malignancies as well as solid tumors. Considerable inter-individual variability in susceptibility to arsenic-induced disease and toxicity exists, and the reasons for such differences are incompletely understood. Transport pathways that influence the cellular uptake and export of arsenic contribute to regulating its cellular, tissue, and ultimately body levels. In the current review, membrane proteins (including phosphate transporters, aquaglyceroporin channels, solute carrier proteins, and ATP-binding cassette transporters) shown experimentally to contribute to the passage of inorganic, methylated, and/or glutathionylated arsenic species across cellular membranes are discussed. Furthermore, what is known about arsenic transporters in organs involved in absorption, distribution, and metabolism and how transport pathways contribute to arsenic elimination are described.

Characterization of arsenic hepatobiliary transport using sandwich-cultured human hepatocytes.

Arsenic is a proven human carcinogen and is associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high-affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)(2)]. In rats, the related canalicular transporter Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)(3) and MMA(GS)(2). The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic efflux was detected only across the basolateral membrane for 9 out of 14 SCHH preparations, 5 had both basolateral and canalicular efflux. Basolateral transport of arsenic was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2-dependence. Treatment of SCHH with As(III) (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1, MRP6, or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of arsenic. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest arsenic basolateral transport prevails over biliary excretion and is mediated at least in part by MRPs, most likely including MRP4.

A novel pathway for arsenic elimination: human multidrug resistance protein 4 (MRP4/ABCC4) mediates cellular export of dimethylarsinic acid (DMAV) and the diglutathione conjugate of monomethylarsonous acid (MMAIII).

Hundreds of millions of people worldwide are exposed to unacceptable levels of arsenic in drinking water. This is a public health crisis because arsenic is a Group I (proven) human carcinogen. Human cells methylate arsenic to monomethylarsonous acid (MMA(III)), monomethylarsonic acid (MMA(V)), dimethylarsinous acid (DMA(III)), and dimethylarsinic acid (DMA(V)). Although the liver is the predominant site for arsenic methylation, elimination occurs mostly in urine. The protein(s) responsible for transport of arsenic from the liver (into blood), ultimately for urinary elimination, are unknown. Human multidrug resistance protein 1 (MRP1/ABCC1) and MRP2 (ABCC2) are established arsenic efflux pumps, but unlike the related MRP4 (ABCC4) are not present at the basolateral membrane of hepatocytes. MRP4 is also found at the apical membrane of renal proximal tubule cells, making it an ideal candidate for urinary arsenic elimination. In the current study, human MRP4 expressed in HEK293 cells reduced the cytotoxicity and cellular accumulation of arsenate, MMA(III), MMA(V), DMA(III), and DMA(V) while two other hepatic basolateral MRPs (MRP3 and MRP5) did not. Transport studies with MRP4-enriched membrane vesicles revealed that the diglutathione conjugate of MMA(III), monomethylarsenic diglutathione [MMA(GS)(2)], and DMA(V) were the transported species. MMA(GS)(2) and DMA(V) transport was osmotically sensitive, allosteric (Hill coefficients of 1.4 ± 0.2 and 2.9 ± 1.2, respectively), and high affinity (K0.5 of 0.70 ± 0.16 and 0.22 ± 0.15 µM, respectively). DMA(V) transport was pH-dependent, with highest affinity and capacity at pH 5.5. These results suggest that human MRP4 could be a major player in the elimination of arsenic.

Heavy metal detoxification in crustacean epithelial lysosomes: role of anions in the compartmentalization process.

Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. Previous studies have shown that zinc uptake by lysosomal membrane vesicles (LMV) occurred by a vanadate- and thapsigargin-sensitive ATPase that was stimulated by a transmembrane proton gradient established by a co-localized V-ATPase associated with this organelle. In the present study, hepatopancreatic LMV from the American lobster Homarus americanus were prepared by standard centrifugation methods and 65Zn2+, 36Cl-, 35SO(4)2- and 14C-oxalate2- were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO4(2-) or PO(4)3- led to a threefold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol, Cl- or oxalate2-. The stimulation of 65Zn2+ uptake by intravesicular sulfate was SO(4)2- concentration dependent with a maximal enhancement at 500 micromol l(-1). Zinc uptake in the presence of ATP was proton-gradient enhanced and electrogenic, exhibiting an apparent exchange stoichiometry of 1Zn+/3H+. 35SO4(2-) and 14C-oxalate2- uptakes were both enhanced in vesicles loaded with intravesicular Cl- compared to vesicles containing mannitol, suggesting the presence of anion countertransport. 35SO4(2-) influx was a sigmoidal function of external [SO(4)2-] with 25 mmol l(-1) internal [Cl-], or with several intravesicular pH values (e.g. 7.0, 8.0 and 9.0). In all instances Hill coefficients of approximately 2.0 were obtained, suggesting that 2 sulfate ions exchange with single Cl- or OH- ions. 36Cl- influx was a sigmoidal function of external [Cl-] with intravesicular pH of 7.0 and 9.0. A Hill coefficient of 2.0 was also obtained, suggesting the exchange of 2 Cl- for 1 OH-. 14C-oxalate influx was a hyperbolic function of external [oxalate2-] with 25 mmol l(-1) internal [Cl-], suggesting a 1:1 exchange of oxalate2- for Cl-. As a group, these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions (SO4(2-) and PO4(3-)) are known to associate with metals inside vesicles and a detoxification model is presented that suggests how these anions may contribute to concretion formation through precipitation with metals at appropriate vesicular pH.