Taring the scales: Weight-of-evidence framework for biocompatibility evaluations.

ISO 10993-1:2018 describes evaluating the biocompatibility profile of a medical device from a risk-based approach. This standard details the battery of information that should be considered within the assessment of a device, including raw material composition data, manufacturing processes, and endpoint testing. The ISO 10993/18562 series requires worst-case assumptions and exposure scenarios to be used in the evaluation, which may result in an over-estimation of patient safety risk. Currently, biocompatibility assessments evaluate each data set independently, and the consequence of this individualized assessment of exaggerated inputs is potential false alarms regarding patient safety. To evaluate these safety concerns, the ISO standards indicate that professional judgement should be used to estimate patient risk but does not provide guidance on incorporating a holistic review of the data into the risk assessment. Recalibrating these worst-case data to evaluate them in a weight-of-evidence (WoE) approach may provide a more realistic data set to determine actual patient risk. This proposed WoE framework combines understanding data applicability with a method for gauging the strength of data that can provide additional support for the final safety conclusion. Using a WoE framework will allow risk assessors to contextualize the data and utilize it to comprehensively estimate patient safety.

An integrated benefit-risk assessment of cobalt-containing alloys used in medical devices: Implications for regulatory requirements in the European Union.

In 2017, the European Union (EU) Committee for Risk Assessment (RAC) recommended the classification of metallic cobalt (Co) as Category 1B with respect to its carcinogenic and reproductive hazard potential and Category 2 for mutagenicity but did not evaluate the relevance of these classifications for patients exposed to Co-containing alloys (CoCA) used in medical devices. CoCA are inherently different materials from Co metal from a toxicological perspective and thus require a separate assessment. CoCA are biocompatible materials with a unique combination of properties including strength, durability, and a long history of safe use that make them uniquely suited for use in a wide-range of medical devices. Assessments were performed on relevant preclinical and clinical carcinogenicity and reproductive toxicity data for Co and CoCA to meet the requirements under the EU Medical Device Regulation triggered by the ECHA re-classification (adopted in October 2019 under the 14th Adaptation to Technical Progress to CLP) and to address their relevance to patient safety. The objective of this review is to present an integrated overview of these assessments, a benefit-risk assessment and an examination of potential alternative materials. The data support the conclusion that the exposure to CoCA in medical devices via clinically relevant routes does not represent a hazard for carcinogenicity or reproductive toxicity. Additionally, the risk for the adverse effects that are known to occur with elevated Co concentrations (e.g., cardiomyopathy) are very low for CoCA implant devices (infrequent reports often reflecting a unique catastrophic failure event out of millions of patients) and negligible for CoCA non-implant devices (not measurable/no case reports). In conclusion, the favorable benefit-risk profile also in relation to possible alternatives presented herein strongly support continued use of CoCA in medical devices.

A hazard evaluation of the reproductive/developmental toxicity of cobalt in medical devices.

Cobalt (Co) is an essential element with human exposure occurring from the diet, supplement ingestion, occupational sources, and medical devices. The European Chemical Agency (ECHA) recently voted to classify Co metal as a Reproductive Hazard Category 1B; presumed human reproductive toxicant due to adverse testicular effects in male rodents. A weight of evidence evaluation of the preclinical reproductive and developmental toxicity studies and available clinical data was performed to critically evaluate the relevance of this proposed classification for Co in medical devices. Reproductive responses to Co are limited to the male testes and sperm function following high systemic exposure in rodents, only at Co concentrations/doses that result in overt toxicity (i.e., above the maximum tolerable dose (MTD)). The potential mechanisms of Co reproductive/developmental toxicity, including its indirect mode of action in the testes and relevance to humans, are discussed. The available preclinical and clincial evidence suggests that it would be more appropriate to classify Co as a Reproductive Hazard Category 2 compound: suspected human reproductive toxicant and, in the case of Co-containing medical devices, it should not be considered a reproductive hazard.

Carcinogenic hazard assessment of cobalt-containing alloys in medical devices: Review of in vivo studies.

Cobalt (Co) alloys have been used for over seven decades in a wide range of medical devices, including, but not limited to, hip and knee implants, surgical tools, and vascular stents, due to their favorable biocompatibility, durability, and mechanical properties. A recent regulatory hazard classification review by the European Chemicals Agency (ECHA) resulted in the classification of metallic Co as a Class 1B Carcinogen (presumed to have carcinogenic potential for humans), primarily based on inhalation rodent carcinogenicity studies with pure metallic Co. The ECHA review did not specifically consider the carcinogenicity hazard potential of forms or routes of Co that are relevant for medical devices. The purpose of this review is to present a comprehensive assessment of the available in vivo preclinical data on the carcinogenic hazard potential of exposure to Co-containing alloys (CoCA) in medical devices by relevant routes. In vivo data were reviewed from 33 preclinical studies that examined the impact of Co exposure on local and systemic tumor incidence in rats, mice, guinea pigs, and hamsters. Across these studies, there was no significant increase of local or systemic tumors in studies relevant for medical devices. Taken together, the relevant in vivo data led to the conclusion that CoCA in medical devices are not a carcinogenic hazard in available in vivo models. While specific patient and implant factors cannot be fully replicated using in vivo models, the available in vivo preclinical data support that CoCA in medical devices are unlikely a carcinogenic hazard to patients.

Global functions of extracellular, transmembrane and cytoplasmic domains of organic solute transporter ß-subunit.

Transport of bile acids across the basolateral membrane of the intestinal enterocyte is carried out by the organic solute transporter (Ost) composed of a seven-transmembrane domain (TMD) subunit (Ostα) and an ancillary single TMD subunit (Ostß). Although previous investigations have demonstrated the importance of the TMD of Ostß for its activity, further studies were conducted to assess the contributions of other regions of the Ostß subunit. Transport activity was retained when Ostß was truncated to contain only the TMD with 15 additional residues on each side and co-expressed with Ostα, whereas shorter fragments were inactive. To probe the broader functions of Ostß segments, chimeric proteins were constructed in which N-terminal, TMD or C-terminal regions of Ostß were fused to corresponding regions of receptor activity-modifying protein (RAMP1), a single TMD protein required by several seven-TMD G-protein-coupled receptors including the calcitonin receptor-like receptor (CLR). Ostß/RAMP1 chimeras were expressed with Ostα and CLR. As expected, replacing the Ostß TMD abolished transport activity; however, replacing either the entire N-terminal or entire C-terminal domain of Ostß with RAMP1 sequences did not prevent plasma membrane localization or the ability to support [3H]taurocholate uptake. Co-immunoprecipitation experiments revealed that the C-terminus of Ostß is a previously unrecognized site of interaction with Ostα. All chimeras containing N-terminal RAMP1 segments allowed co-expressed CLR to respond to agonists with strong increases in cyclic AMP. These results provide new insights into the structure and function of the heteromeric Ost transporter complex.

Data on the histological and immune cell response in the popliteal lymph node in mice following exposure to metal particles and ions.

Hip implants containing cobalt-chromium (CoCr) have been used for over 80 years. In patients with metal-on-metal (MoM) hip implants, it has been suggested that wear debris particles may contribute to metal sensitization in some individuals, leading to adverse reactions. This article presents data from a study in which the popliteal lymph node assay (PLNA) was used to assess immune responses in mice treated with chromium-oxide (Cr2O3) particles, metal salts (CoCl2, CrCl3, and NiCl2) or Cr2O3 particles with metal salts ("A preliminary evaluation of immune stimulation following exposure to metal particles and ions using the mouse popliteal lymph node assay" (B.E. Tvermoes, K.M. Unice, B. Winans, M. Kovochich, E.S. Fung, W.V. Christian, E. Donovan, B.L. Finley, B.L. Kimber, I. Kimber, D.J. Paustenbach, 2016) [1]). Data are presented on (1) the chemical characterization of TiO2 particles (used as a particle control), (2) clinical observations in mice treated with Cr2O3 particles, metal salts or Cr2O3 particles with metal salts, (3) PLN weight and weight index (WI) in mice treated with Cr2O3 particles, metal salts or Cr2O3 particles with metal salts, (4) histological changes in PLNs of mice treated with Cr2O3 particles, metal salts or Cr2O3 particles with metal salts, (5) percentages of immune cells in the PLNs of mice treated with Cr2O3 particles, metal salts or Cr2O3 particles with metal salts, and (6) percentages of proliferating cells in the PLNs of mice treated with Cr2O3 particles, metal salts or Cr2O3 particles with metal salts.

A preliminary evaluation of immune stimulation following exposure to metal particles and ions using the mouse popliteal lymph node assay.

The objective of this preliminary study was to evaluate the threshold for immune stimulation in mice following local exposure to metal particles and ions representative of normal-functioning cobalt-chromium (CoCr) metal-on-metal (MoM) hip implants. The popliteal lymph node assay (PLNA) was used in this study to assess immune responses in BALB/c mice following treatment with chromium-oxide (Cr2O3) particles, metal salts (CoCl2, CrCl3 and NiCl2), or Cr2O3 particles together with metal salts using single-dose exposures representing approximately 10days (0.000114mg), 19years (0.0800mg), and 40years (0.171mg) of normal implant wear. The immune response elicited following treatment with Cr2O3 particles together with metal salts was also assessed at four additional doses equivalent to approximately 1.5months (0.0005mg), 0.6years (0.0025mg), 2.3years (0.01mg), and 9.3years (0.04mg) of normal implant wear. Mice were injected subcutaneously (50µL) into the right hind foot with the test article, or with the relevant vehicle control. The proliferative response of the draining lymph node cells (LNC) was measured four days after treatment, and stimulation indices (SI) were derived relative to vehicle controls. The PLNA was negative (SI<3) for all Cr2O3 particle doses, and was also negative at the lowest dose of the metal salt mixture, and the lowest four doses of the Cr2O3 particles with metal salt mixture. The PLNA was positive (SI>3) at the highest two doses of the metal salt mixture and the highest three doses of the Cr2O3 particles with the metal salt mixture. The provisional NOAEL and LOAEL values identified in this study for immune activation corresponds to Co and Cr concentrations in the synovial fluid approximately 500 and 2000 times higher than that reported for normal-functioning MoM hip implants, respectively. Overall, these results indicate that normal wear conditions are unlikely to result in immune stimulation in individuals not previously sensitized to metals.

An exposure and health risk assessment of lead (Pb) in lipstick.

Lead (Pb) content in lipstick and other consumer products has become an increasing concern. In 2010, the United States Food and Drug Administration tested 400 lipstick samples and found a maximum Pb concentration of 7.19 ppm. To assess the safety of lipstick in adults that chronically apply lipstick as well as instances where children might incidentally ingest lipstick products, the United States Environmental Protection Agency's (US EPA) Adult Lead Model and Integrated Exposure Uptake Biokinetic Model for Lead in Children were used to determine the blood Pb concentrations of adults and children ingesting varying amounts of lipstick of different Pb concentrations. Modeled blood Pb concentrations were compared with oral ingestion guidelines and to the Centers for Disease Control and the US EPA's actionable blood Pb levels of 5 and 10 µg/dL. Background Pb exposure was the primary contributor to estimated blood Pb levels (BLLs) in children and adults, and Pb exposure from lipstick did not significantly increase estimated BLLs. These results suggest that the safety of consumer products and cosmetics should be assessed not only by the presence and amounts of hazardous contents, but also in conjunction with an assessment of estimated background exposures and comparison to health-based standards.

Toxicology-based cancer causation analysis of CoCr-containing hip implants: a quantitative assessment of genotoxicity and tumorigenicity studies.

In this paper, quantitative methods were used to evaluate the weight of evidence regarding a causative relationship between cobalt-chromium (CoCr)-containing hip implants and increased cancer risk. We reviewed approximately 80 published papers and identified no-observed-adverse-effect level (NOAEL) and/or lowest-observed-adverse-effect level (LOAEL) values for specific endpoints of interest: genotoxic effects from in vitro studies with human cell lines as well as genotoxicity and tumor formation in animal bioassays. Test articles included Co particles and ions, Cr particles and ions, and CoCr alloy particles as well as CoCr alloy implants. The NOAEL/LOAEL values were compared with body burdens of Co/Cr particles and ions we calculated to exist in systemic tissues of hip implant patients under normal and excessive wear conditions. We found that approximately 40 tumor bioassays have been conducted with CoCr alloy implants or Co/Cr particles and ions at levels hundreds to thousands of times higher than those present in hip implant patients, and none reported a statistically significant increased incidence of systemic tumors. Results from in vitro and in vivo genotoxicity assays, which are relatively less informative owing to false positives and other factors, also indicated that DNA effects would be highly unlikely to occur as a result of wear debris from a CoCr implant. Hence, the toxicological weight of evidence suggests that CoCr-containing hip implants are unlikely to be associated with an increased risk of systemic cancers, which is consistent with published and ongoing cancer epidemiology studies involving patients with CoCr hip implants.

Correlation of blood Cr(III) and adverse health effects: Application of PBPK modeling to determine non-toxic blood concentrations.

Chromium (Cr) (III) is a trace metal essential to human health and exposure typically occurs via the diet on a daily basis. Some groups of individuals, such as those consuming Cr(III) supplements or patients with Cr-containing implants, may have elevated blood Cr(III) concentrations. Although blood Cr(III) levels are thought to be an accurate metric of exposure, little is known about the relationship between these concentrations and possible adverse health risks. This study evaluated the various effects reported in animal and human epidemiological studies of Cr(III) exposure in an attempt to correlate them with blood Cr(III) concentrations. The target endpoints identified in this analysis included the hematological, hepatic, and renal systems. Animal and human physiological-based pharmacokinetic (PBPK) models were used to estimate steady state blood Cr(III) concentrations from a variety of dosing regimens. Based on the animal studies, our results suggest that blood Cr(III) concentrations as high as 480-580 µg/L are not associated with any responses. For each of the three health endpoints considered in this analysis (hematological, hepatic, and renal) no adverse effects were observed below 3,700 µg/L. Some hematological responses were observed at 3,700 µg/L, and adverse effects clearly occurred at 7,500 µg/L. These findings can be used to assess potential health risks to individuals with elevated blood Cr(III) concentrations.

The heteromeric organic solute transporter, OSTα-OSTß/SLC51: a transporter for steroid-derived molecules.

The organic solute transporter alpha-beta (OSTα-OSTß) is one of the newest members of the solute carrier family, designated as SLC51, and arguably one of the most unique. The transporter is composed of two gene products encoded by SLC51A and SLC51B that heterodimerize to form the functional transporter complex. SLC51A encodes OSTα, a predicted 340-amino acid, 7-transmembrane (TM) domain protein, whereas SLC51B encodes OSTß, a putative 128-amino acid, single-TM domain polypeptide. Heterodimerization of the two subunits increases the stability of the individual proteins, facilitates their post-translational modification, and is required for delivery of the functional transporter complex to the plasma membrane. There are no paralogues for SLC51A or SLC51B in any genome that has been examined. The transporter functions via a facilitated diffusion mechanism, and can mediate either efflux or uptake depending on the electrochemical gradient of its substrates. Overall, characterization of the transporter's substrate specificity, transport mechanism, tissue distribution, subcellular localization, and transcriptional regulation as well as the phenotype of the recently generated Slc51a-deficient mice have revealed that OSTα-OSTß plays a central role in the transport of bile acids, conjugated steroids, and structurally-related molecules across the basolateral membrane of many epithelial cells. In particular, OSTα-OSTß appears to be essential for intestinal bile acid absorption, and thus for dietary lipid absorption.

ß-Subunit of the Ostα-Ostß organic solute transporter is required not only for heterodimerization and trafficking but also for function.

The organic solute transporter, Ost/Slc51, is composed of two distinct proteins that must heterodimerize to generate transport activity, but the role of the individual subunits in mediating transport activity is unknown. The present study identified regions in Ostß required for heterodimerization with Ostα, trafficking of the Ostα-Ostß complex to the plasma membrane, and bile acid transport activity in HEK293 cells. Bimolecular fluorescence complementation analysis revealed that a 25-amino acid peptide containing the Ostß transmembrane (TM) domain heterodimerized with Ostα, although the resulting complex failed to reach the plasma membrane and generate cellular [(3)H]taurocholate transport activity. Deletion of the single TM domain of Ostß abolished interaction with Ostα, demonstrating that the TM segment is necessary and sufficient for formation of a heteromeric complex with Ostα. Mutation of the highly conserved tryptophan-asparagine sequence within the TM domain of Ostß to alanines did not prevent cell surface trafficking, but abolished transport activity. Removal of the N-terminal 27 amino acids of Ostß resulted in a transporter complex that reached the plasma membrane and exhibited transport activity at 30 °C. Complete deletion of the C terminus of Ostß abolished [(3)H]taurocholate transport activity, but reinsertion of two native arginines immediately C-terminal to the TM domain rescued this defect. These positively charged residues establish the correct N(exo)/C(cyt) topology of the peptide, in accordance with the positive inside rule. Together, the results demonstrate that Ostß is required for both proper trafficking of Ostα and formation of the functional transport unit, and identify specific residues of Ostß critical for these processes.

Neurosteroid transport by the organic solute transporter OSTα-OSTß.

A variety of steroids, including pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulfate (DHEAS) are synthesized by specific brain cells, and are then delivered to their target sites, where they exert potent effects on neuronal excitability. The present results demonstrate that [(3)H]DHEAS and [(3)H]PREGS are relatively high affinity substrates for the organic solute transporter, OSTα-OSTß, and that the two proteins that constitute this transporter are selectively localized to steroidogenic cells in the cerebellum and hippocampus, namely the Purkinje cells and cells in the cornu ammonis region in both mouse and human brain. Analysis of Ostα and Ostß mRNA levels in mouse Purkinje and hippocampal cells isolated via laser capture microdissection supported these findings. In addition, Ostα-deficient mice exhibited changes in serum DHEA and DHEAS levels, and in tissue distribution of administered [(3)H]DHEAS. OSTα and OSTß proteins were also localized to the zona reticularis of human adrenal gland, the major region for DHEAS production in the periphery. These results demonstrate that OSTα-OSTß is localized to steroidogenic cells of the brain and adrenal gland, and that it modulates DHEA/DHEAS homeostasis, suggesting that it may contribute to neurosteroid action.

Perturbations in mitochondrial dynamics induced by human mutant PINK1 can be rescued by the mitochondrial division inhibitor mdivi-1.

Mutations in the mitochondrial encoded protein PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive Parkinson disease (PD). In mammalian cells, mutant PINK1 has been reported to promote fission or inhibit fusion in mitochondria; however, the mechanism by which this process occurs remains elusive. Using an ecdysone-inducible expression system in mammalian dopaminergic neuronal cells, we report here that human mutant PINK1 (L347P and W437X) mediates an overall fission effect by increasing the ratio of mitochondrial fission over fusion proteins, leading to excessive dysfunctional fragmented mitochondria. Knocking down endogenous Pink1 produces similar effects. In contrast, overexpressing human wild type PINK1 produces a pro-fusion effect by increasing the ratio of mitochondrial fusion/fission proteins without resulting in functionally compromised mitochondria. Parkin knockdown blocks the imbalance in fission/fusion proteins. Furthermore, overexpressing parkin and ubiquitin increases degradation of the mitochondrial fission hFis1 protein, suggesting PINK1 and parkin maintain proper mitochondrial function and integrity via the fission/fusion machinery. Through genetic manipulations and treatment with the small molecule mitochondrial division inhibitor (mdivi-1), which inhibits DLP1/Drp1, both structural and functional mitochondrial defects induced by mutant PINK1 were attenuated, highlighting a potential novel therapeutic avenue for Parkinson disease.

The organic cation transporter-3 is a pivotal modulator of neurodegeneration in the nigrostriatal dopaminergic pathway.

Toxic organic cations can damage nigrostriatal dopaminergic pathways as seen in most parkinsonian syndromes and in some cases of illicit drug exposure. Here, we show that the organic cation transporter 3 (Oct3) is expressed in nondopaminergic cells adjacent to both the soma and terminals of midbrain dopaminergic neurons. We hypothesized that Oct3 contributes to the dopaminergic damage by bidirectionally regulating the local bioavailability of toxic species. Consistent with this view, Oct3 deletion and pharmacological inhibition hampers the release of the toxic organic cation 1-methyl-4-phenylpyridinium from astrocytes and protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration in mice. Furthermore, Oct3 deletion impairs the removal of the excess extracellular dopamine induced by methamphetamine and enhances striatal dopaminergic terminal damage caused by this psychostimulant. These results may have far-reaching implications for our understanding of the mechanism of cell death in a wide range of neurodegenerative diseases and may open new avenues for neuroprotective intervention.

OST alpha-OST beta: a key membrane transporter of bile acids and conjugated steroids.

The organic solute and steroid transporter, Ost alpha-Ost beta, is an unusual heteromeric carrier that appears to play a central role in the transport of bile acids, conjugated steroids, and structurally-related molecules across the basolateral membrane of many epithelial cells. The transporter's substrate specificity, transport mechanism, tissue distribution, subcellular localization, transcriptional regulation, as well as the phenotype of the recently characterized Ost alpha-deficient mice all strongly support this model. In particular, the Ost alpha-deficient mice display a marked defect in intestinal bile acid and conjugated steroid absorption; a decrease in bile acid pool size and serum bile acid levels; altered intestinal, hepatic and renal disposition of known substrates of the transporter; and altered serum triglyceride, cholesterol, and glucose levels. Collectively, the data indicate that Ost alpha-Ost beta is essential for bile acid and sterol disposition, and suggest that the carrier may be involved in human conditions related to imbalances in bile acid or lipid homeostasis.

Ostalpha-Ostbeta is required for bile acid and conjugated steroid disposition in the intestine, kidney, and liver.

Mice deficient in the organic solute transporter (Ost)-alpha subunit of the heteromeric organic solute and steroid transporter, Ostalpha-Ostbeta, were generated and were found to be viable and fertile but exhibited small intestinal hypertrophy and growth retardation. Bile acid pool size and serum levels were decreased by more than 60% in Ostalpha-/- mice, whereas fecal bile acid excretion was unchanged, suggesting a defect in intestinal bile acid absorption. In support of this hypothesis, when [3H]taurocholic acid or [3H]estrone 3-sulfate were administered into the ileal lumen, absorption was lower in Ostalpha-/- mice. Interestingly, serum cholesterol and triglyceride levels were also approximately 15% lower in Ostalpha-/- mice, an effect that may be related to the impaired intestinal bile acid absorption. After intraperitoneal administration of [3H]estrone 3-sulfate or [3H]dehydroepiandrosterone sulfate, Ostalpha-/- mice had higher levels of radioactivity in their liver and urinary bladder and less in the duodenum, indicating altered hepatic, renal, and intestinal disposition. Loss of Ostalpha was associated with compensatory changes in the expression of several genes involved in bile acid homeostasis, including an increase in the multidrug resistance-associated protein 3, (Mrp3)/Abcc3, an alternate basolateral bile acid export pump, and a decrease in cholesterol 7alpha-hydroxylase, Cyp7a1, the rate-limiting enzyme in bile acid synthesis. The latter finding may be explained by increased ileal expression of fibroblast growth factor 15 (Fgf15), a negative regulator of hepatic Cyp7a1 transcription. Overall, these findings provide direct support for the hypothesis that Ostalpha-Ostbeta is a major basolateral transporter of bile acids and conjugated steroids in the intestine, kidney, and liver.

OSTalpha-OSTbeta: a major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia.

The cellular and subcellular localization and mechanism of transport of the heteromeric organic solute transporter (OST) OSTalpha-OSTbeta was examined in human and rodent epithelia. The two subunits of the transporter were expressed together in human small intestine, kidney, and liver, tissues that also express the apical sodium-dependent bile acid uptake transporter ASBT (SLC10A2). Indirect immunofluorescence microscopy localized OSTalpha and OSTbeta to the basolateral membrane of mouse, rat, and human ileal enterocytes, renal proximal tubular cells, and cholangiocytes. Transport in OSTalpha-OSTbeta-expressing Xenopus laevis oocytes was unaffected by depletion of intracellular adenosine triphosphate, or by changes in transmembrane Na(+), K(+), H(+), or Cl(-) concentration gradients. However, the oocytes demonstrated robust substrate efflux and trans-stimulation, indicating that transport occurs by facilitated diffusion. Madin Darby canine kidney cells coexpressing mouse Ostalpha and Ostbeta exhibited enhanced apical to basolateral transport of the major glycine and taurine conjugated bile acid species. In conclusion, the selective localization of OSTalpha and OSTbeta to the basolateral plasma membrane of epithelial cells responsible for bile acid and sterol reabsorption, the substrate selectivity of the transporter, and the facilitated diffusion transport mode collectively indicate that OSTalpha-OSTbeta is a key basolateral transporter for the reabsorption of these important steroid-derived molecules.

The heteromeric organic solute transporter alpha-beta, Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter.

Bile acids are transported across the ileal enterocyte brush border membrane by the well characterized apical sodium-dependent bile acid transporter (Asbt) Slc10a2; however, the carrier(s) responsible for transporting bile acids across the ileocyte basolateral membrane into the portal circulation have not been fully identified. Transcriptional profiling of wild type and Slc10a2 null mice was employed to identify a new candidate basolateral bile acid carrier, the heteromeric organic solute transporter (Ost)alpha-Ostbeta. By Northern blot analysis, Ostalpha and Ostbeta mRNA was detected only in mouse kidney and intestine, mirroring the horizontal gradient of expression of Asbt in the gastrointestinal tract. Analysis of Ostalpha and Ostbeta protein expression by immunohistochemistry localized both subunits to the basolateral surface of the mouse ileal enterocyte. The transport properties of Ostalpha-Ostbeta were analyzed in stably transfected Madin-Darby canine kidney cells. Co-expression of mouse Ostalpha-Ostbeta, but not the individual subunits, stimulated Na(+)-independent bile acid uptake and the apical-to-basolateral transport of taurocholate. In contrast, basolateral-to-apical transport was not affected by Ostalpha-Ostbeta expression. Co-expression of Ostalpha and Ostbeta was required to convert the Ostalpha subunit to a mature glycosylated endoglycosidase H-resistant form, suggesting that co-expression facilitates the trafficking of Ostalpha through the Golgi apparatus. Immunolocalization studies showed that co-expression was necessary for plasma membrane expression of both Ostalpha and Ostbeta. These results demonstrate that the mouse Ostalpha-Ostbeta heteromeric transporter is a basolateral bile acid carrier and may be responsible for bile acid efflux in ileum and other ASBT-expressing tissues.