4-Nitrochalcone as a potential drug in non-clinical breast cancer studies.

Breast cancer is a high-magnitude public health problem, continually challenging physicians and scientists worldwide in the field of drug therapy. 4-nitrochalcone (4NC) is a phenolic compound that has promising antitumor activity in vitro, but its application in breast cancer treatment is still poorly explored. This study aimed to evaluate the action of 4NC in vitro and in vivo breast cancer models. The cytotoxic potential of 4NC was tested towards MCF-7 and MDA-MD-231 breast cancer cells, with a lower impact in the non-tumor lineage HB4a. For in vivo studies, solid Ehrlich carcinoma (SEC) was used, a syngeneic mouse model with non-nuclear estrogen and progesterone positivity, characterized by immunohistochemistry. Daily oral administration of 4NC (25 mg kg-1) for 21 days led to a consistent reduction in tumor growth compared to the vehicle group. No signs of toxicity evaluated by hematological, biochemical, histological, and oxidative stress parameters were observed in mice, and the DL50 was >2000 mg kg-1. The effectors Raptor and S6K1 showed decreased activation, with a consequent reduction in protein synthesis; concomitantly, there was an increase in LC3-II levels, but the protective autophagic response was not completed, with the maintenance of p62 levels and cell death. These results open new possibilities for the use of 4NC as a tumor cell metabolism modulating agent.

Cholangiocytes express an isoform of soluble adenylyl cyclase that is N-linked glycosylated and secreted in extracellular vesicles.

Soluble adenylyl cyclase (sAC)-derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85 ) or 75 kD (sAC75 ) sAC protein band under glucose-sufficient or glucose-deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase-F demonstrated that both sAC75 and sAC85 are N-linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo-H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N-linked glycosylated sAC (sACEV ) in extracellular vesicles under conditions that support intracellular sAC85 (glucose-sufficient) as opposed to sAC75 (glucose-deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N-linked glycosylated sAC isoform that is rapidly released into extracellular vesicles.

The phospholipid flippase ATP8B1 is required for lysosomal fusion in macrophages.

ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases (P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.

Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells.

Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression.

Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na(+) -taurocholate cotransporting polypeptide knockout mice.

UNLABELLED: The Na(+) -taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte-specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1(-/-) hepatocytes showed absence of sodium-dependent taurocholic acid uptake, whereas sodium-independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1(-/-) mice, explained by increased Asbt and Ostα/ß expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down-regulation of OATP1A1 and up-regulation of OATP1A4. Furthermore, sodium-dependent taurocholic acid uptake was inhibited by Myrcludex B in wild-type hepatocytes, while Slc10a1(-/-) hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1(-/-) mice. CONCLUSION: The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation.

P4 ATPases: flippases in health and disease.

P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed "lipid flipping". Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse) of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role.

Increased efflux of oxidized glutathione (GSSG) causes glutathione depletion and potentially diminishes antioxidant defense in sickle erythrocytes.

Erythrocytes are both an important source and target of reactive oxygen species in sickle cell disease. Levels of glutathione, a major antioxidant, have been shown to be decreased in sickle erythrocytes and the mechanism leading to this deficiency is not known yet. Detoxification of reactive oxygen species involves the oxidation of reduced glutathione (GSH) into glutathione-disulfide (GSSG) which is actively transported out of erythrocyte. We questioned whether under oxidative conditions, GSSG efflux is increased in sickle erythrocytes. Erythrocytes of 18 homozygous sickle cell patients and 9 race-matched healthy controls were treated with 2,3-dimethoxy-l,4-naphthoquinone, which induces intracellular reactive oxygen species generation, to stimulate GSSG production. Intra- and extracellular concentrations of GSH and GSSG were measured at baseline and during 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. While comparable at baseline, intracellular and extracellular GSSG concentrations were significantly higher in sickle erythrocytes than in healthy erythrocyte after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation (69.9 ± 3.7 µmol/l vs. 40.6 ± 6.9 µmol/l and 25.8 ± 2.7 µmol/l vs. 13.6 ± 1.7 µmol/l respectively, P<0.002). In contrast to control erythrocytes, where GSH concentrations remained unchanged (176 ± 8.4 µmol/l vs. 163 ± 13.6 µmol/l, NS), GSH in sickle erythrocytes decreased significantly (from 167 ± 8.8 µmol/l to 111 ± 11.8 µmol/l, P<0.01) after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. Adding multidrug resistance-associated protein-1 inhibitor (MK571) to erythrocytes blocked GSSG efflux in both sickle and normal erythrocytes. GSSG efflux, mediated by multidrug resistance-associated protein-1, is increased in sickle erythrocytes, resulting in net loss of intracellular glutathione and possibly higher susceptibility to oxidative stress.

The biliary HCO(3)(-) umbrella: a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies.

This review focuses on the hypothesis that biliary HCO(3)(-) secretion in humans serves to maintain an alkaline pH near the apical surface of hepatocytes and cholangiocytes to prevent the uncontrolled membrane permeation of protonated glycine-conjugated bile acids. Functional impairment of this biliary HCO(3)(-) umbrella or its regulation may lead to enhanced vulnerability of cholangiocytes and periportal hepatocytes toward the attack of apolar hydrophobic bile acids. An intact interplay of hepatocellular and cholangiocellular adenosine triphosphate (ATP) secretion, ATP/P2Y- and bile salt/TGR5-mediated Cl(-)/ HCO(3)(-) exchange and HCO(3)(-) secretion, and alkaline phosphatase-mediated ATP breakdown may guarantee a stable biliary HCO(3)(-) umbrella under physiological conditions. Genetic and acquired functional defects leading to destabilization of the biliary HCO(3)(-) umbrella may contribute to development and progression of various forms of fibrosing/sclerosing cholangitis.

Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs.

Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b-/- mice, as SLCO genes encode OATPs). Slco1a/1b-/- mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b-/- mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b-/- mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b-/- mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.

P4 ATPases--the physiological relevance of lipid flipping transporters.

P4 ATPases are integral transmembrane proteins implicated in phospholipid translocation from the exoplasmic to the cytosolic leaflet of biological membranes. Our present knowledge on the cellular physiology of P4 ATPases is mostly derived from studies in the yeast Saccharomyces cerevisiae, where P4 ATPases play a pivotal role in the biogenesis of intracellular transport vesicles, polarized protein transport and protein maturation. In contrast, the physiological and cellular functions of mammalian P4 ATPases are largely unexplored. P4 ATPases act in concert with members of the CDC50 protein family, which are putative beta-subunits for P4 ATPases. This review highlights the current status of a slowly emerging research field and emphasizes the contribution of P4 ATPases to the vesicle-generating machinery.

Progressive familial intrahepatic cholestasis type 1.

Progressive familial intrahepatic cholestasis type 1 is a rare genetic liver disease that presents in the first year of life. Bile salts are elevated and these patients are often jaundiced. Despite the cholestasis, serum gamma-glutamyltransferase activity is normal or reduced. Pruritus is a major symptom in these patients. Partial external biliary diversion is helpful in several patients as it reduces the pruritus and postpones or even avoids liver transplantation. The disease is caused by mutations in the gene ATP8B1 that preclude the normal expression of ATP8B1. ATP8B1 is a protein that acts as a lipid flippase, transporting phosphatidylserine from the exoplasmic to the cytoplasmic leaflet of the canalicular membrane of hepatocytes. The authors have shown that the canalicular membrane of ATP8B1-deficient hepatocytes is less stable as evidenced by enhanced extraction of membrane constituents by bile salts. Recent evidence suggests membrane instability in ATP8B1-deficient hair cells of the ear, providing an explanation for hearing loss in ATP8B1 deficiency. Although the exact etiology of cholestasis is incompletely understood, it is hypothesized that ATP8B1 deficiency results in enhanced cholesterol extraction from the canalicular membrane, which impairs the function of the bile salt export pump (BSEP), resulting in cholestasis. Mutations in ATP8B1 also cause benign recurrent intrahepatic cholestasis, a milder variant of the disease characterized by episodes of cholestasis. The onset and resolution of the cholestatic episodes in these patients is still not well understood.

Strain background modifies phenotypes in the ATP8B1-deficient mouse.

BACKGROUND: Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease.

Reduction of liver macrophage transduction by pseudotyping lentiviral vectors with a fusion envelope from Autographa californica GP64 and Sendai virus F2 domain.

BACKGROUND: Lentiviral vectors are well suited for gene therapy because they can mediate long-term expression in both dividing and nondividing cells. However, lentiviral vectors seem less suitable for liver gene therapy because systemically administered lentiviral vectors are preferentially sequestered by liver macrophages. This results in a reduction of available virus and might also increase the immune response to the vector and vector products.Reduction of macrophage sequestration is therefore essential for efficient lentiviral liver gene therapy. RESULTS: Fusions were made of Autographa californica GP64 and the hepatocyte specific Sendai Virus envelope proteins. Lentiviral vectors were produced with either wild type GP64, Sendai-GP64, or both wild type GP64 and Sendai-GP64 and tested in vitro and in vivo for hepatocyte and macrophage gene transfer.Sendai-GP64 pseudotyped vectors showed specific gene transfer to HepG2 hepatoma cells, with no detectable transduction of HeLa cervical carcinoma cells, and a decreased affinity for RAW mouse macrophages. Co-expression of wild type GP64 and Sendai-GP64 resulted in improved viral titers while retaining increased affinity for HepG2 cells.In vivo, the Sendai-GP64 vectors also showed decreased transduction of murine liver macrophages. CONCLUSION: We demonstrate reduced macrophage transduction in vitro and in vivo with GP64/Sendai chimeric envelope proteins.

P4 ATPases - lipid flippases and their role in disease.

P4 ATPases (type 4 P-type ATPases) are multispan transmembrane proteins that have been implicated in phospholipid translocation from the exoplasmic to the cytoplasmic leaflet of biological membranes. Studies in Saccharomyces cerevisiae have indicated that P4 ATPases are important in vesicle biogenesis and are required for vesicular trafficking along several intracellular vesicular transport routes. Although little is known about mammalian P4 ATPases, some members of this subfamily appear to be associated with human disease or mouse pathophysiology. ATP8B1, a phosphatidylserine translocase, is the most extensively studied mammalian P4 ATPase. This protein is important for maintaining the detergent resistant properties of the apical membrane of the hepatocyte. Mutations in ATP8B1 give rise to severe liver disease. Furthermore, a role for Atp8b3 in mouse sperm cell capacitation has been suggested, whereas deficiency of Atp10a and Atp10d leads to insulin resistance and obesity in mice. Here we review the present status on the pathophysiological consequences of P4 ATPase deficiency.

Activity of the bile salt export pump (ABCB11) is critically dependent on canalicular membrane cholesterol content.

Mutations in ATP8B1 cause severe inherited liver disease. The disease is characterized by impaired biliary bile salt excretion (cholestasis), but the mechanism whereby impaired ATP8B1 function results in cholestasis is poorly understood. ATP8B1 is a type 4 P-type ATPase and is a flippase for phosphatidylserine. Atp8b1-deficient mice display a dramatic increase in the biliary extraction of cholesterol from the canalicular (apical) membrane of the hepatocyte. Here we studied the hypothesis that disproportionate cholesterol extraction from the canalicular membrane impairs the activity of the bile salt transporter, ABCB11, and as a consequence causes cholestasis. Using single pass liver perfusions, we show that not only ABCB11-mediated transport but also Abcc2-mediated transport were reduced at least 4-fold in Atp8b1 deficiency. We show that canalicular membranes of cholestatic Atp8b1-deficient mice have a dramatically reduced cholesterol to phospholipid ratio, i.e. 0.75 +/- 0.24 versus 2.03 +/- 0.71 for wild type. In vitro depletion of cholesterol from mouse liver plasma membranes using methyl-beta-cyclodextrin demonstrated a near linear relation between cholesterol content of the membranes and ATP-dependent taurocholate transport. Abcc2-mediated transport activity was not affected up to 30% of membrane cholesterol depletion but declined to negligible levels at 70% of membrane cholesterol depletion. These effects were reversible as cholesterol repletion of the liver membranes completely restored Abcb11- and Abcc2-mediated transport. Our data demonstrate that membrane cholesterol content is a critical determinant of ABCB11/ABCC2 transport activity, provide an explanation for the etiology of ATP8B1 disease, and suggest a novel mechanism protecting the canalicular membrane against luminal bile salt overload.

Activation of cyclic AMP Signaling in Ae2-deficient mouse fibroblasts.

Anion exchanger 2 (AE2, SLC4A2) is a ubiquitously expressed membrane solute carrier that regulates intracellular pH (pH(i)) by exchanging cytosolic bicarbonate for extracellular chloride. We used fibroblasts from Ae2-deficient (Ae2(a,b)(-/-)) mice to study the effects of an alkaline shift in resting intracellular pH (pH(i)) on the activation of cAMP signaling and gene expression. Ae2(a,b)(-/-) fibroblasts show increased pH(i) (by 0.22 +/- 0.03 unit) compared with wild type cells at extracellular pH (pH(o)) 7.4 and 37 degrees C. This shift in resting pH(i) is associated with an up-regulation of bicarbonate-activated soluble adenylyl cyclase expression, increased cAMP production, Creb phosphorylation, inducible cAMP early repressor 1 mRNA expression, and impaired activation of c-Fos transcription by forskolin. These results highlight the importance of bicarbonate transport via Ae2 in maintaining pH(i) homeostasis in cultured mouse fibroblasts and unveil the role of cAMP in the cellular response to chronic alkalization, which putatively includes an inducible cAMP early repressor 1-mediated attenuation of phosphorylated Creb activity.

Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity.

BACKGROUND: The presence of cholesterol in the Human Immunodeficiency Virus (HIV) lipid envelop is important for viral function as cholesterol depleted viral particles show reduced infectivity. However, it is less well established whether other viral membrane lipids are also important for HIV infection. The ABCB4 protein is a phosphatidyl choline (PC) floppase that mediates transport of PC from the inner to the outer membrane leaflet. This property enabled us to modulate the lipid composition of HIV vectors and study the effects on membrane composition and infection efficiency. RESULTS: Virus generated in the presence of ABCB4 was enriched in PC and cholesterol but contained less sphingomyelin (SM). Viral titers were reduced 5.9 fold. These effects were not observed with an inactive ABCB4 mutant. The presence of the ABC transport inhibitor verapamil abolished the effect of ABCB4 expression on viral titers. The ABCB4 mediated reduction in infectivity was caused by changes in the viral particles and not by components co purified with the virus because virus made in the presence of ABCB4 did not inhibit virus made without ABCB4 in a competition assay. Incorporation of the envelope protein was not affected by the expression of ABCB4. The inhibitory effect of ABCB4 was independent of the viral envelope as the effect was observed with two different envelope proteins. CONCLUSION: Our data indicate that increasing the PC content of HIV particles reduces infectivity.

A mouse genetic model for familial cholestasis caused by ATP8B1 mutations reveals perturbed bile salt homeostasis but no impairment in bile secretion.

Mutations in ATP8B1, a broadly expressed P-type ATPase, result, through unknown mechanisms, in disorders of bile secretion. These disorders vary in severity from mild and episodic to progressive with liver failure. We generated Atp8b1G308V/G308V mutant mice, which carry a mutation orthologous to that present in homozygous form in patients from the Amish index kindred for severe ATP8B1 disease. In contrast to human patients, Atp8b1(G308V/G308V) mice had unimpaired bile secretion and no liver damage, but showed mild abnormalities including depressed weight at weaning and elevated serum bile salt levels. We challenged the hepatobiliary metabolism of Atp8b1G308V/G308V mice by administering exogenous bile salts. Upon bile salt feeding, Atp8b1G308V/G308V mice, but not wild-types, demonstrated serum bile salt accumulation, hepatic injury and expansion of the systemic bile salt pool. Unexpectedly, this failure of bile salt homeostasis occurred in the absence of any defect in hepatic bile secretion. Upon infusion of a hydrophobic bile salt, wild-type mice developed cholestasis while Atp8b1G308V/G308V mice maintained high biliary output and more extensively rehydroxylated the infused bile salt. Increased bile salt hydroxylation, which reduces bile salt toxicity, may explain the milder phenotype in Atp8b1G308V/G308V mice compared with humans with the equivalent mutation. These results demonstrate the key role of Atp8b1 in bile salt homeostasis and highlight the importance of bile salt hydroxylation in the prevention of cholestasis. The mouse phenotype reveals that loss of Atp8b1 disrupts bile salt homeostasis without impairment of canalicular bile secretion; in humans this process is likely to be obscured by early onset of severe liver disease.

Anion exchanger 2 is essential for spermiogenesis in mice.

Na+-independent anion exchangers (AE) mediate electroneutral exchange of Cl- for HCO3- ions across cell membranes, being involved in intracellular pH and cell volume regulation and in transepithelial hydroionic fluxes. Bicarbonate activation of adenylyl cyclase is known to be necessary for sperm motility and sperm capacitation, and a few studies have suggested a possible role of AE carriers in reproduction. Among the four AE genes identified in mammals thus far, only Ae2 (Slc4a2) has been determined to be expressed in the male reproductive system, especially in developing spermatozoa and in epididymal epithelium. Most AE genes drive alternative transcription, which in mouse Ae2 results in several Ae2 isoforms. Here, we generated mice carrying a targeted disruption of Ae2 that prevents the expression of the three AE2 isoforms (Ae2a, Ae2b1, and Ae2b2) normally found in mouse testes. Male Ae2-/- mice (but not female Ae2-/- mice) are infertile. Histopathological analysis of Ae2-/- testes shows an interruption of spermiogenesis, with only a few late spermatids and a complete absence of spermatozoa in the seminiferous tubules. The number of apoptotic bodies is increased in the seminiferous tubules and in the epididymis, which also shows squamous metaplasia of the epididymal epithelium. Our findings reveal an essential role of Ae2 in mouse spermiogenesis and stress the recently postulated involvement of bicarbonate in germ-cell differentiation through the bicarbonate-sensitive soluble-adenylyl-cyclase pathway.

The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria.

The breast cancer resistance protein (BCRPABCG2) is a member of the ATP-binding cassette family of drug transporters and confers resistance to various anticancer drugs. We show here that mice lacking Bcrp1Abcg2 become extremely sensitive to the dietary chlorophyll-breakdown product pheophorbide a, resulting in severe, sometimes lethal phototoxic lesions on light-exposed skin. Pheophorbide a occurs in various plant-derived foods and food supplements. Bcrp1 transports pheophorbide a and is highly efficient in limiting its uptake from ingested food. Bcrp1(-/-) mice also displayed a previously unknown type of protoporphyria. Erythrocyte levels of the heme precursor and phototoxin protoporphyrin IX, which is structurally related to pheophorbide a, were increased 10-fold. Transplantation with wild-type bone marrow cured the protoporphyria and reduced the phototoxin sensitivity of Bcrp1(-/-) mice. These results indicate that humans or animals with low or absent BCRP activity may be at increased risk for developing protoporphyria and diet-dependent phototoxicity and provide a striking illustration of the importance of drug transporters in protection from toxicity of normal food constituents.