Oral cadmium in mice carrying 5 versus 2 copies of the Slc39a8 gene: comparison of uptake, distribution, metal content, and toxicity.

The highly conserved human and mouse SLC39A8 gene encodes the divalent cation/bicarbonate symporter ZIP8 expressed ubiquitously in most cell types. Our bacterial artificial chromosome-transgenic BTZIP8-3 line has 3 additional copies of the Slc39a8 gene in addition to its constitutive diploid pair found in wild-type (WT) mice. In liver, kidney, lung, testis, gastrointestinal tract, and brain, BTZIP8-3 mice are known to express ∼2.5 times greater amounts of ZIP8, compared with WT mice. Herein we administered cadmium chloride (CdCl2) in drinking water (100 mg/L through week 2, 200 mg/L through week 4, 400 mg/L through week 8, 800 mg/L through week 12, and 1600 mg/L through week 20, when the experiment was concluded). We postulated that Cd uptake and distribution--and, therefore, toxicity in certain tissues--would be enhanced in BTZIP8-3, compared with WT mice. BTZIP8-3 and WT groups ingested comparable amounts of Cd. Compared with WT, BTZIP8-3 mice showed tissue specific: increases in Cd, zinc, and manganese content and decreases in calcium content. Both Cd-exposed BTZIP8-3 and WT were similar in lower urinary pH; increased plasma alanine and aspartate aminotransferase activities; elevated iron and copper content in liver, kidney, lung, and testis; and higher blood urea nitrogen and kidney weight. Histological changes in liver, kidney, lung, and testis were minimal. In summary, at the daily oral Cd exposures chosen for this study, 5 versus 2 Slc39a8 gene copies result in no differences in Cd toxicity but do cause differences in tissue-specific content of Cd, zinc, manganese, calcium, iron, and copper.

Loss of NHE1 activity leads to reduced oxidative stress in heart and mitigates high-fat diet-induced myocardial stress.

Acute inhibition of the NHE1 Na(+)/H(+) exchanger protects against ischemia-reperfusion injury and chronic inhibition attenuates development of cardiac hypertrophy and failure. To determine the cardiac effects of chronic inhibition of NHE1 under non-pathological conditions we used NHE1-null mice as a model of long-term NHE1 inhibition. Cardiovascular performance was relatively normal in Nhe1(-/-) mice although cardiac contractility and relaxation were slightly improved in mutant mice of the FVB/N background. GSH levels and GSH:GSSG ratios were elevated in Nhe1(-/-) hearts indicating an enhanced redox potential. Consistent with a reduced need for antioxidant protection, expression of heat shock proteins Hsp60 and Hsp25 was lower in Nhe1(-/-) hearts. Similarly, expression of mitochondrial superoxide dismutase 2 was reduced, with no increase in expression of other ROS scavenging enzymes. GLUT1 levels were increased in Nhe1(-/-) hearts, the number of lipid droplets in myocytes was reduced, and PDK4 expression was refractory to high-fat diet-induced upregulation observed in wild-type hearts. High-fat diet-induced stress was attenuated in Nhe1(-/-) hearts, as indicated by smaller increases in phosphorylation of Hsp25 and α-B crystallin, and there was better preservation of insulin sensitivity, as evidenced by PKB/Akt phosphorylation. Plasma glucose and insulin levels were lower and high-fat diet-induced hepatic lipid accumulation was reduced in Nhe1(-/-) mice, demonstrating extracardiac effects of NHE1 ablation. These data indicate that long-term ablation of NHE1 activity increases the redox potential, mitigates high-fat diet-induced myocardial stress and fatty liver disease, leads to better preservation of insulin sensitivity, and may alter both cardiac and systemic metabolic substrate handling in mice.

Oral benzo[a]pyrene in Cyp1a1/1b1(-/-) double-knockout mice: Microarray analysis during squamous cell carcinoma formation in preputial gland duct.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1, CYP1B1) and other enzymes can activate PAHs to reactive oxygenated intermediates involved in mutagenesis and tumor initiation; also, CYP1 enzymes can detoxify PAHs. Cyp1(+/+) wild-type (WT) and Cyp1b1(-/-) knockout mice receiving oral BaP (12.5 mg/kg/day) remain healthy for >12 months. In contrast, we found that global knockout of the Cyp1a1 gene (1a1KO) results in proximal small intestine (PSI) adenocarcinoma within 8-12 weeks on this BaP regimen; striking compensatory increases in PSI CYP1B1 likely participate in initiation of adenocarcinoma in 1a1KO mice. Cyp1a1/1b1(-/-) double-knockout (DKO) mice on this BaP regimen show no PSI adenocarcinoma, but instead preputial gland duct (PGD) squamous cell carcinoma (SCC) occurs by 12 weeks. Herein, we compare microarray expression of PGD genes in WT, 1a1KO and DKO mice at 0, 4, 8, 12 and 16 weeks of oral BaP; about four dozen genes up- or down-regulated during most critical time-points were further verified by qRT-PCR. In DKO mice, CYP3A59 was unequivocally identified as the BaP-inducible and BaP-metabolizing best candidate responsible for initiation of BaP-induced SCC. Striking increases or decreases were found in 26 cancer-related genes plus eight Serpin genes in DKO, but not in 1a1KO or WT, mice on this BaP regimen; of the 26, 8 were RAS-related oncogenes. The mechanism by which cancer-related genes are responsible for SCC tumor progression in the PGD remains to be elucidated.

Loss of the AE3 anion exchanger in a hypertrophic cardiomyopathy model causes rapid decompensation and heart failure.

The AE3 Cl(-)/HCO(3)(-) exchanger is abundantly expressed in the sarcolemma of cardiomyocytes, where it mediates Cl(-)-uptake and HCO(3)(-)-extrusion. Inhibition of AE3-mediated Cl(-)/HCO(3)(-) exchange has been suggested to protect against cardiac hypertrophy; however, other studies indicate that AE3 might be necessary for optimal cardiac function. To test these hypotheses we crossed AE3-null mice, which appear phenotypically normal, with a hypertrophic cardiomyopathy mouse model carrying a Glu180Gly mutation in α-tropomyosin (TM180). Loss of AE3 had no effect on hypertrophy; however, survival of TM180/AE3 double mutants was sharply reduced compared with TM180 single mutants. Analysis of cardiac performance revealed impaired cardiac function in TM180 and TM180/AE3 mutants. TM180/AE3 double mutants were more severely affected and exhibited little response to ß-adrenergic stimulation, a likely consequence of their more rapid progression to heart failure. Increased expression of calmodulin-dependent kinase II and protein phosphatase 1 and differences in methylation and localization of protein phosphatase 2A were observed, but were similar in single and double mutants. Phosphorylation of phospholamban on Ser16 was sharply increased in both single and double mutants relative to wild-type hearts under basal conditions, leading to reduced reserve capacity for ß-adrenergic stimulation of phospholamban phosphorylation. Imaging analysis of isolated myocytes revealed reductions in amplitude and decay of Ca(2+) transients in both mutants, with greater reductions in TM180/AE3 mutants, consistent with the greater severity of their heart failure phenotype. Thus, in the TM180 cardiomyopathy model, loss of AE3 had no apparent anti-hypertrophic effect and led to more rapid decompensation and heart failure.

Loss of parietal cell expression of Sonic hedgehog induces hypergastrinemia and hyperproliferation of surface mucous cells.

BACKGROUND & AIMS: Sonic Hedgehog (Shh) is expressed in the adult stomach, but its role as a gastric morphogen is unclear. We sought to identify mechanisms by which Shh might regulate gastric epithelial cell function and differentiation. METHODS: Mice with a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) were created. Gastric morphology and function were studied in control and HKCre/Shh(KO) mice between 1 and 8 months of age. RESULTS: In contrast to control mice, HKCre/Shh(KO) mice developed gastric hypochlorhydria, hypergastrinemia, and a phenotype that resembled foveolar hyperplasia. The fundic mucosa of HKCre/Shh(KO) mice had an expanded surface pit cell lineage that was documented by increased incorporation of bromodeoxyuridine and was attributed to the hypergastrinemia. Compared with controls, numbers of total mucous neck and zymogen cells were significantly decreased in stomachs of HKCre/Shh(KO) mice. In addition, zymogen and neck cell markers were coexpressed in the same cell populations, indicating disrupted differentiation of the zymogen cell lineage from the mucous neck cells in the stomachs of HKCre/Shh(KO) mice. Laser capture microdissection of the surface epithelium, followed by quantitative reverse-transcription polymerase chain reaction, revealed a significant increase in expression of Indian Hedgehog, glioma-associated oncogene homolog 1, Wnt, and cyclin D1. Laser capture microdissection analysis also showed a significant increase in Snail with a concomitant decrease in E-cadherin. CONCLUSIONS: In the stomachs of adult mice, loss of Shh from parietal cells results in hypochlorhydria and hypergastrinemia. Hypergastrinemia might subsequently induce increased Hedgehog and Wnt signaling in the surface pit epithelium, resulting in hyperproliferation.

Loss of the NHE2 Na+/H+ exchanger in mice results in dilation of folliculo-stellate cell canaliculi.

Genetic ablation of the NHE2 Na+/H+ exchanger causes gastric achlorhydria, absorptive defects in kidney and colon, and low fertility. Here we show that NHE2 is expressed in the pituitary, with the highest mRNA expression in pars distalis and lower expression in pars intermedia. In pars distalis of NHE2-null mice, prominent cyst-like dilatations of folliculo-stellate (FS) cell canaliculi developed with age, and there were increased FS cell area, accumulation of lipid in FS cell cytoplasm, redundancies in FS cell basement membrane, and other changes. The expansion of the canaliculi indicates that NHE2 is a major absorptive Na+/H+ exchanger in the luminal membranes lining the extensive network of channels formed by FS cells, which may provide a means of intrapituitary communication. The results suggest that NHE2 contributes to homeostatic regulation of the volume and composition of the canalicular fluid and may counter the secretory activity of the CFTR Cl⁻ channel, which is known to be expressed in pituitary.

Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells and impairs acid secretion in the stomach.

Slc26a9 is a recently identified anion transporter that is abundantly expressed in gastric epithelial cells. To study its role in stomach physiology, gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant (Slc26a9(-/-)) mice appeared healthy and displayed normal growth. Slc26a9 deletion resulted in the loss of gastric acid secretion and a moderate reduction in the number of parietal cells in mutant mice at 5 weeks of age. Immunofluorescence labeling detected the H-K-ATPase exclusively on the apical pole of gastric parietal cells in Slc26a9(-/-) mice, in contrast to the predominant cytoplasmic localization in Slc26a9(+/+) mice. Light microscopy indicated that gastric glands were dilated, and electron micrographs displayed a distinct and striking absence of tubulovesicles in parietal cells and reductions in the numbers of parietal and zymogen cells in Slc26a9(-/-) stomach. Expression studies indicated that Slc26a9 can function as a chloride conductive pathway in oocytes as well as a Cl(-)/HCO(3)(-) exchanger in cultured cells, and localization studies in parietal cells detected its presence in tubulovesicles. We propose that Slc26a9 plays an essential role in gastric acid secretion via effects on the viability of tubulovesicles/secretory canaliculi and by regulating chloride secretion in parietal cells.

Organ-specific roles of CYP1A1 during detoxication of dietary benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental toxicants derived from sources that include cigarette smoke, petroleum distillation, gas- and diesel-engine exhaust, and charcoal-grilled food. The gastrointestinal tract is the principal route of PAH exposures, even when inhaled. The most thoroughly studied prototype of PAHs is benzo[a]pyrene (BaP), well known to be toxic, mutagenic, and carcinogenic in various tissues and cell types. This lab has previously shown that Cyp1a1(-/-) global knockout mice treated by oral administration of BaP die at 28 to 32 days with immunosuppression, whereas wild-type mice remain healthy for 1 year on high BaP doses (125 mg/kg/day). Thus, for oral BaP, CYP1A1 is more important in detoxication than in metabolic activation. After several days of oral BaP, we found surprisingly low CYP1A1 levels in liver, compared with that in small intestine; we postulated that this finding might reflect efficient detoxication of oral BaP in proximal small intestine such that significant amounts of the inducer BaP no longer reach the liver. In the present study, many parameters were therefore compared in wild-type, Cyp1a1(-/-) global knockout, intestinal epithelial cell-specific Cyp1a1 knockout, and hepatocyte-specific Cyp1a1 knockout mice as a function of long-term oral exposure to BaP. The peak of CYP1A1 (mRNA, protein) expression in liver occurred at 12 h, whereas highly induced CYP1A1 in small intestine persisted throughout the 30-day experiment. Hepatocyte-specific Cyp1a1 knockout mice remained as healthy as wild-type mice; intestinal epithelial cell-specific Cyp1a1 knockout mice behaved like Cyp1a1(-/-) mice, dying with immunosuppression approximately 30 days on oral BaP. We conclude that small intestine CYP1A1, and not liver CYP1A1, is critically important in oral BaP detoxication.

Oral benzo[a]pyrene-induced cancer: two distinct types in different target organs depend on the mouse Cyp1 genotype.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1 and CYP1B1) can both detoxify PAHs and activate them to cancer-causing reactive intermediates. Following high dosage of oral BaP (125 mg/kg/day), ablation of the mouse Cyp1a1 gene causes immunosuppression and death within ∼28 days, whereas Cyp1(+/+) wild-type mice remain healthy for >12 months on this regimen. In this study, male Cyp1(+/+) wild-type, Cyp1a1(-/-) and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) double-knockout mice received a lower dose (12.5 mg/kg/day) of oral BaP. Tissues from 16 different organs-including proximal small intestine (PSI), liver and preputial gland duct (PGD)-were evaluated; microarray cDNA expression and >30 mRNA levels were measured. Cyp1a1(-/-) mice revealed markedly increased CYP1B1 mRNA levels in the PSI, and between 8 and 12 weeks developed unique PSI adenomas and adenocarcinomas. Cyp1a1/1b1(-/-) mice showed no PSI tumors but instead developed squamous cell carcinoma of the PGD. Cyp1(+/+) and Cyp1b1(-/-) mice remained healthy with no remarkable abnormalities in any tissue examined. PSI adenocarcinomas exhibited striking upregulation of the Xist gene, suggesting epigenetic silencing of specific genes on the Y-chromosome; the Rab30 oncogene was upregulated; the Nr0b2 tumor suppressor gene was downregulated; paradoxical overexpression of numerous immunoglobulin kappa- and heavy-chain variable genes was found-although the adenocarcinoma showed no immunohistochemical evidence of being lymphatic in origin. This oral BaP mouse paradigm represents an example of "gene-environment interactions" in which the same exposure of carcinogen results in altered target organ and tumor type, as a function of just 1 or 2 globally absent genes.

Oral N-acetylcysteine rescues lethality of hepatocyte-specific Gclc-knockout mice, providing a model for hepatic cirrhosis.

BACKGROUND & AIMS: Certain liver diseases have been associated with depletion of glutathione (GSH), the major antioxidant in the liver. A recent report about Gclc(h/h) mice with a hepatocyte-specific ablation of Gclc (the gene encoding the catalytic subunit of the rate-limiting enzyme in GSH synthesis) has shown an essential role of GSH in hepatic function. Gclc(h/h) mice develop severe steatosis and die of liver failure within one month, due to ~95% depletion of hepatic GSH; mitochondria are the major affected organelles, displaying abnormal ultrastructure and impaired functioning. METHODS: Gclc(h/h) mice were fed with L-N-acetylcysteine (NAC; 10 g/L) in drinking water, starting at postnatal day 18. RESULTS: Gclc(h/h) mice were rescued by use of NAC supplementation, and survived until adulthood. NAC replenished the mitochondrial GSH pool and attenuated mitochondrial damage, with accompanying diminished hepatic steatosis; however, abnormal liver biochemical tests, hepatocyte death, and hepatic oxidative stress persisted in the rescued mice. At 50 days of age, the liver from rescued Gclc(h/h) mice started to display characteristics of fibrosis and at age 120 days, macronodular cirrhosis was observed. Immunohistostaining for liver-specific markers as well as the expression profile of hepatic cytokines indicated that the repopulation of hepatocytes in the cirrhotic nodules involved the expansion of oval cells. CONCLUSIONS: Replenishment of mitochondrial GSH and restoration of mitochondrial function by NAC prevents mortality caused by the loss of hepatocyte GSH de novo synthesis, allowing steatosis to progress to a chronic stage. Thus, with NAC supplementation, Gclc(h/h) mice provide a model for the development of liver fibrosis and cirrhosis.

Hedgehog signaling regulates E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions.

In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5(Ski)) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5(Ski) cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/ß-catenin and ZO-1/occludin protein complexes was observed in HKCre/Shh(KO) mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1.

CLIC5 mutant mice are resistant to diet-induced obesity and exhibit gastric hemorrhaging and increased susceptibility to torpor.

Chloride intracellular channel 5 (CLIC5) and other CLIC isoforms have been implicated in a number of biological processes, but their specific functions are poorly understood. The association of CLIC5 with ezrin and the actin cytoskeleton led us to test its possible involvement in gastric acid secretion. Clic5 mutant mice exhibited only a minor reduction in acid secretion, Clic5 mRNA was expressed at only low levels in stomach, and Clic5 mutant parietal cells were ultrastructurally normal, negating the hypothesis that CLIC5 plays a major role in acid secretion. However, the mutants exhibited gastric hemorrhaging in response to fasting, reduced monocytes and granulocytes suggestive of immune dysfunction, behavioral and social disorders suggestive of neurological dysfunction, and evidence of a previously unidentified metabolic defect. Wild-type and mutant mice were maintained on normal and high-fat diets; plasma levels of various hormones, glucose, and lipids were determined; and body composition was studied by quantitative magnetic resonance imaging. Clic5 mutants were lean, hyperphagic, and highly resistant to diet-induced obesity. Plasma insulin and glucose levels were reduced, and leptin levels were very low; however, plasma triglycerides, cholesterol, phospholipids, and fatty acids were normal. Indirect calorimetry revealed increased peripheral metabolism and greater reliance on carbohydrate metabolism. Because Clic5 mutants were unable to maintain energy reserves, they also exhibited increased susceptibility to fasting-induced torpor, as indicated by telemetric measurements showing episodes of reduced body temperature and heart rate. These data reveal a requirement for CLIC5 in the maintenance of normal systemic energy metabolism.

Volume density, distribution, and ultrastructure of secretory and basolateral membranes and mitochondria predict parietal cell secretory (dys)function.

Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd) of canalicular and apical membranes (CAMs) to tubulovesicular (TV) membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT) mice and in NHE2-/- mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2-/-, GHKAalpha-/-, and NHE4-/- mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes.

Knock-in mouse lines expressing either mitochondrial or microsomal CYP1A1: differing responses to dietary benzo[a]pyrene as proof of principle.

In the past, CYP1A1 protein was known to be located in the endoplasmic reticulum (ER; microsomes). More recently, CYP1A1 was shown also to be targeted to the inner mitochondrial membrane; mitochondrial import is dependent on NH(2)-terminal processing that exposes a cryptic targeting signal. It is interesting that microsomal and mitochondrial CYP1A1 enzymes exhibit different substrate specificities, electron donors, and inducer properties. To understand the physiological functions of microsomal versus mitochondrial CYP1A1, we have generated three knock-in lines by altering the CYP1A1 NH(2) terminus. Cyp1a1(mtt/mtt) mice encode an NH(2)-terminal 31-amino acid-truncated protein, deleting the ER-targeting signal and exposing the cryptic mitochondrial-targeting signal. Cyp1a1(mtp/mtp) mice encode a protein carrying L7N and L17N mutations; this mutant lacks the signal recognition particle (SRP)-binding site and subsequent ER-targeting, but requires proteolysis by a cytosolic peptidase for mitochondrial import. Cyp1a1(mc/mc) mice encode a microsomal protein having R34D and K39I mutations, which abolish the mitochondrial targeting signal. After dioxin or beta-naphthoflavone treatment of these mouse lines, the CYP1A1 protein was shown to be located in the mitochondria of the Cyp1a1(mtp/mtp) and Cyp1a1(mtt/mtt) lines and in microsomes of the Cyp1a1(mc/mc) line. To test for differences in function, we compared the response to dietary benzo[a]pyrene (BaP). After 18 days of daily oral BaP, wild-type and Cyp1a1(mc/mc) mice were completely protected, whereas Cyp1a1(-/-) and Cyp1a1(mtp/mtp) mice showed striking toxicity and compensatory up-regulation of CYP1A2 and CYP1B1 mRNA in several tissues. Our data support the likelihood that it is the microsomal rather than mitochondrial CYP1A1 enzyme that protects against oral BaP toxicity.

Gene expression profiling of blood to predict the onset of leukemia.

No studies have tested the hypothesis that the onset of a disease can be predicted by gene expression profiling. The AKR/J mouse strain, which spontaneously develops acute T cell lymphatic leukemia, was used to implement a novel strategy to generate global gene expression profiles of WBCs at different time points. The experimental approach was bias free because it was unknown as to which individuals in the mouse population would eventually develop the disease. Our results suggest that profiling WBC gene expression may be an effective means for the very early diagnosis of disease in humans.

Fundic gland cysts in Atp4a-/- mice mimic fundic gland polyps in humans.

BACKGROUND: Innumerable fundic gland polyps (FGPs) (that is massive FGP polyposis) may be found in the human stomach. Innumerable fundic gland cysts (FGCs) develop in mice lacking the gene Atp4a (Atp4a((-/-))), that encodes the enzyme ATPase. The aim was to monitor the evolution of FGCs in Atp4a((-/-)) mice. MATERIALS AND METHODS: Six pairs (male-female) of Atp4a((-/-)) mice, 8, 12 and 20 months old, were investigated. Wild-type Atp4a((+/+)) mice were used as controls. RESULTS: In Atp4a((-/-)) female mice, the mucosal thickness increased both at 8 months (943+/-72 mm) (p<0.02) and at 12 months (p<0.0008) and even further at 20 months (p<0.0001). The FGC area was 13.183+/-7.218 mm(2) at 12 months and 73.361+/-50.591 mm(2) at 20 months (p<0.0008). FGCs in Atp4a((-/-)) mice were lined by parietal cells, mucous pit-foveolar cells and chief cells, thus mimicking FGPs in humans. CONCLUSION: Older female Atp4a((-/-)) mice developed innumerable FGCs, a phenomenon very similar to massive FGPs seen in humans. In the light of these similarities, the Atp4a((-/-)) mouse emerges as a suitable animal model to investigate the series of events taking place during the evolution of FGPs in humans.

Basal and inducible CYP1 mRNA quantitation and protein localization throughout the mouse gastrointestinal tract.

The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice. Oral BaP was compared with intraperitoneal TCDD. In general, maximal CYP1A1 mRNA levels were 3-10 times greater than CYP1B1, which were 3-10 times greater than CYP1A2 mRNA levels. Highest inducible concentrations of CYP1A1 and CYP1A2 occurred in proximal small intestine, whereas the highest basal and inducible levels of CYP1B1 mRNA occurred in esophagus, forestomach, and glandular stomach. Ablation of either Cyp1a2 or Cyp1b1 gene resulted in a compensatory increase in CYP1A1 mRNA - but only in small intestine. Also in small intestine, although BaP- and TCDD-mediated CYP1A1 inductions were roughly equivalent, oral BaP-mediated CYP1A2 mRNA induction was approximately 40-fold greater than TCDD-mediated CYP1A2 induction. CYP1B1 induction by TCDD in Cyp1(+/+) and Cyp1a2(-/-) mice was 4-5 times higher than that by BaP; however, in Cyp1a1(-/-) animals CYP1B1 induction by TCDD or BaP was approximately equivalent. CYP1A1 and CYP1A2 proteins were generally localized nearer to the lumen than CYP1B1 proteins, in both squamous and glandular epithelial cells. These GI tract data suggest that the inducible CYP1A1 enzyme, both in concentration and in location, might act as a "shield" in detoxifying oral BaP and, hence, protecting the animal.

The frequency of gastric pyloric cells with glassy cytoplasm in baboons. A comparison with human subjects.

BACKGROUND: Pyloric cells with "glassy" cytoplasm were detected 20 years ago in the human gastric mucosa, and subsequently in the gastric mucosa of two baboons and of transgenic mice. These pyloric cells, called glassy cells (GCs) show a homogeneous, eosinophilic material that pushes the nucleous towards the free border of the cell. MATERIALS AND METHODS: The frequency of baboons having pyloric glands with GCs was assessed in the stomachs of 92 consecutive animals, dying of non-gastrointestinal disorders. RESULTS: High power examination of the apparently normal gastric mucosa revealed that 10.9% (n=10) of the 92 baboons had GCs. Periodic acid Schiff (PAS) and PAS-diastase stains suggested that the glassy material might be a glycoprotein. CONCLUSION: The relatively high frequency of GCs in the gastric mucosa of baboons suggests that this animal might be a useful model for investigating the effect of the environment in the development of GCs, as well as ascertaining the true nature of the retained "glassy" material.

Ultrastructure of Highly Ordered Granules in Alveolar Type II Cells in Several Species.

Alveolar Type II cells from seven mammalian species were examined for a protein in the rough endoplasmic reticulum (RER), which showed a multilayered, repeating motif. Each motif, 100 nm in width, comprised two parallel outer dense layers, a less dense central layer, and often 1-3 faint layers on either side of the latter. Outer layers showed periodicities at 3-4 densities/100 nm of width, while layers on either side of the central layer showed 5-7 densities/100 nm of width. RER membranes were ribosome-free when parallel to these layers, but showed four ribosomes per motif at the growing ends: one ribosome at each outer dense layer, and one on either side of the less dense central layer. Granules appeared as single or as multiple motifs, stacked, curved, folded, or branching together within the same RER profile. Hexagons of around 30 nm in diameter with central densities were seen in tangential cuts of outer dense layers. Granule incidence varied: guinea pig > ferret > dog. Possible homologous structures occurred in rabbit and cat, but not in rat or mouse. Surfactant protein A (SP-A), a C-type lectin produced in Type II cells, forms trimers and bouquet-like 18-mer and can oligomerize further. Two pairs of SP-A 18-mers with carbohydrate recognition domains pointing inwardly and outwardly, stacked vertically as a column of four molecules, then repeated side by side in rows, approximated the size and layering patterns observed in these granules. Sequence analyses of SP-A from these species showed phylogenetic distances consistent with the observed occurrence and frequency of patterned granules. Anat Rec, 301:1290-1302, 2018. © 2018 Wiley Periodicals, Inc.

Haploinsufficiency of Atp2a2, encoding the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump, predisposes mice to squamous cell tumors via a novel mode of cancer susceptibility.

A null mutation in one copy of the Atp2a2 or ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), leads to squamous cell tumors in mice and to Darier disease in humans, a skin disorder that also involves keratinocytes. Here, we examined the time course and genetic mechanisms of tumor development in the mutant animals. Atp2a2+/- mice overexpressed keratins associated with keratinocyte hyperactivation in normal forestomachs as early as 2 months of age. By the age of 5 to 7 months, 22% of mutants had developed papillomas of the forestomach, and 89% of mutants older than 14 months had developed squamous cell papillomas and/or carcinomas, with a preponderance of the latter. Tumors occurred in regions that had keratinized epithelium and were subjected to repeated mechanical irritation. The genetic mechanism of tumorigenesis did not involve loss of heterozygosity, as tumor cells analyzed by laser capture microdissection contained the wild-type Atp2a2 allele. Furthermore, immunoblot and immunohistochemical analysis showed that tumor keratinocytes expressed the SERCA2 protein. Mutations were not observed in the ras proto-oncogenes; however, expression of wild-type ras was up-regulated, with particularly high levels of K-ras. Loss of the p53 tumor suppressor gene occurred in a single massive tumor, whereas other tumors had increased levels of p53 protein but no mutations in the p53 gene. These findings show that SERCA2 haploinsufficiency predisposes mice to tumor development via a novel mode of cancer susceptibility involving a global change in the tumorigenic potential of keratinized epithelium in Atp2a2+/- mice.