Autophagy induced by exogenous bile acids is therapeutic in a model of α-1-AT deficiency liver disease.

The bile acid nor-ursodeoxycholic acid (norUDCA) has many biological actions, including antiapoptotic effects. Homozygous PIZZ α-1-antitrypsin (A1AT)-deficient humans are known to be at risk for liver disease, cirrhosis, and liver cancer as a result of the accumulation of the toxic, A1AT mutant Z protein within hepatocytes. This accumulation triggers cell death in the hepatocytes with the largest mutant Z-protein burdens, followed by compensatory proliferation. Proteolysis pathways within the hepatocyte, including autophagy, act to reduce the intracellular burden of A1AT Z protein. We hypothesized that norUDCA would reduce liver cell death and injury in A1AT deficiency. We treated groups of PiZ transgenic mice and wild-type mice with norUDCA or vehicle, orally, and examined the effects on the liver. The PiZ mouse is the best model of A1AT liver injury and recapitulates many features of the human liver disease. Mice treated with norUDCA demonstrated reduced hepatocellular death by compensatory hepatocellular proliferation as determined by bromodeoxyuridine incorporation (3.8% control, 0.88% treated, P < 0.04). Ki-67 staining as a marker for hepatocellular senescence and death was also reduced (P < 0.02). Reduced apoptotic signaling was associated with norUDCA, including reduced cleavage of caspases-3, -7, and -8 (all P < 0.05). We determined that norUDCA was associated with a >70% reduction in intrahepatic mutant Z protein (P < 0.01). A 32% increase in hepatic autophagy associated with norUDCA was the likely mechanism. norUDCA administration is associated with increased autophagy, reduced A1AT protein accumulation, and reduced liver injury in a model of A1AT deficiency.

Oxidative stress contributes to liver damage in a murine model of alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin deficiency is a genetic disorder resulting in the expression of misfolded mutant protein that can polymerize and accumulate in hepatocytes, leading to liver disease in some individuals. Transgenic PiZ mice are a well-characterized model, which express human alpha-1-antitrypsin mutant Z protein (ATZ protein) and faithfully recapitulate the human liver disease. Liver tissue expressing alpha-1-antitrypsin mutant Z protein exhibits inflammation, injury and replacement of damaged cells. Fibrosis and hepatocellular carcinoma (HCC) develop in aging PiZ mice. In this study, microarray analysis was performed comparing young PiZ (ZY) mice to wild-type (WY), and indicated that there were alterations in gene expression levels that could influence a number of pathways leading to liver disease. Redox-regulating genes were up-regulated in ZY tissue, including carbonyl reductase 3 (CBR3), glutathione S-transferase alpha 1 + 2 (GSTA(1 + 2)) and glutathione S-transferase mu 3 (GSTM3). We hypothesized that oxidative stress could develop in Z mouse liver, contributing to tissue damage and disease progression with age. The results of biochemical analysis of PiZ mouse liver revealed that higher levels of reactive oxygen species (ROS) and a more oxidized, cellular redox state occurred in liver tissue from ZY mice than WY. ZY mice showed little evidence of oxidative cellular damage as assessed by protein carbonylation levels, malondialdehyde levels and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) staining. Aging liver tissue from PiZ older mice (ZO) had elevated ROS, generally lower levels of antioxidant enzymes than younger mice and evidence of cellular damage. These data indicate that oxidative stress is a contributing factor in the development of liver disease in this model of alpha-1-antitrypsin deficiency.

Hepatic progenitor cell proliferation and liver injury in α-1-antitrypsin deficiency.

OBJECTIVES: Homozygous ZZ α-1-antitrypsin (a1AT) deficiency is a common genetic liver disease that causes liver injury and hepatocellular carcinoma (HCC). The a1AT mutant Z gene encodes a mutant protein that accumulates within hepatocytes leading to hepatocellular death and a hepatic regenerative response. However, the mechanisms linking hepatocellular injury to these responses are poorly understood. In this study, we examined liver injury and response in human liver and in transgenic mice for involvement of hepatic progenitor cells. METHODS: Liver biopsy specimens of low-grade, early-stage human ZZ liver exhibiting minimal inflammation and minimal fibrosis (grade 1 and stage 1) were examined for hepatic progenitor cell (HPC) proliferation using immunoreactivity for cytokeratin-7 (CK-7). Transgenic mouse model liver and other selected human biopsies were also examined. RESULTS: Increased CK-7-positive HPC proliferation was seen in human ZZ liver compared to normal liver, but was 5-fold less HPC proliferation than in grade- and stage-matched disease control hepatitis C-infected liver. Livers from PiZ mice, a model transgenic for the human a1AT mutant Z gene, which recapitulates the human injury, also showed HPC proliferation. Human ZZ liver and PiZ mice develop dysplasia in the liver and HCC. HCC in PiZ mice was also characterized by HPC proliferation. Progressive hepatic fibrosis with age in the PiZ mice is demonstrated for the first time in the present study. CONCLUSIONS: Chronic injury in both ZZ human and PiZ mouse liver is associated with hepatic fibrosis and a unique magnitude of HPC proliferation within the hepatic proliferative response.

Characteristics of hepatocellular carcinoma in a murine model of alpha-1-antitrypsin deficiency.

AIM: Individuals with homozygous (ZZ) alpha-1-antitrypsin (alpha1AT) deficiency are at an increased risk for liver damage, cirrhosis and hepatocellular carcinoma (HCC). The transgenic PiZ mouse, expressing the human alpha1AT mutant Z gene, is a valuable model for this disease. We studied PiZ mice in order to identify and characterize mechanisms involved in the development of HCC. METHODS: Tumor incidence and histology were studied, gene expression levels were surveyed with microarrays, RNA quantified with quantitative real time polymerase chain reaction and protein levels determined with immunoblots and immunohistochemistry. RESULTS: By 16-19 months of age, approximately 69% of the PiZ mice had developed tumors. HCC was present with no evidence of benign adenomas as pre-cancerous lesions. Tumors showed abnormal mitochondria, variable levels of steatosis, globular inclusions of alpha1AT mutant Z protein and metastases. PiZ mice that subsequently developed liver tumors had higher serum levels of alpha1AT mutant Z protein than those that did not develop tumors. Cyclin D1, a cell cycle protein, was upregulated in PiZ livers without tumors compared to Wt. cFOS, a component of AP-1 that may be involved in transforming cells and MCAM, an adhesion molecule likely involved in tumorigenesis and metastases, were elevated in tumors compared with livers without tumors. CONCLUSION: In the PiZ model, many of the histological characteristics of HCC recapitulated features seen in human HCC, whether from individuals with homozygous ZZ liver disease or from unrelated causes in individuals that were not homozygous ZZ. The accumulation of mutant Z protein altered the regulation of several genes driving proliferation and tumorigenesis.

Contribution of the HEDJ/ERdj3 cysteine-rich domain to substrate interactions.

Cytoplasmic type I DnaJ/Hsp40 chaperones contain a Cys-rich domain consisting of four CXXCXG motifs that are in a reduced state and coordinate zinc, stabilizing the intervening sequence in a loop structure. However, the Cys-rich region of the endoplasmic reticulum localized HEDJ (ERdj3/ERj3p), is considerably different in sequence and arrangement. Unlike the typical type I molecule, the HEDJ CXC, and CXXC motifs were demonstrated in this study to be predominantly oxidized in intramolecular disulfide bonds. In the native state, HEDJ bound to immobilized, denatured thyroglobulin. Unlike its binding partner GRP78, redox conditions affected the interaction of HEDJ with substrate. Substitution of the Cys-rich domain cysteine residues with serine diminished or abolished HEDJ binding in the in vitro assay. These findings suggest that the Cys-rich region of HEDJ and its oxidation state are important in maintaining the substrate interaction domain in a binding-competent conformation.

The effects of Orimulsion and Fuel Oil #6 on the hatching success of copepod resting eggs in the seabed of Tampa Bay, Florida.

A 3-month microcosm study was conducted to observe the potential effects of two fuels, Orimulsion and Fuel Oil #6, on the hatching success of copepod resting eggs in the seabed of Tampa Bay, Florida. Microcosms were dosed with one of five hydrocarbon treatments via hydrocarbon-coated sand and compared with controls. Acartia tonsa eggs were nonviable in all treatments after only a few weeks of incubation, as evidenced by a marked decline in the abundance of nauplii. However, there was no evidence that exposure to simulated spills of 700 or 7000 ppm of either fuel led to significant increases in resting egg mortality as compared with controls. The results further indicate that, regardless of environmental conditions, resting eggs of A. tonsa do not remain viable in the sediment for extended periods of time.

DIFFERENCES IN THE DURATION OF EGG DIAPAUSE OF LABIDOCERA AESTIVA (COPEPODA: CALANOIDA) FROM THE WOODS HOLE, MASSACHUSETTS, REGION.

The duration of diapause of Labidocera aestiva eggs collected from the field and reared in the laboratory was determined at 5°C. A clear seasonal trend was observed. Diapause eggs produced in the early fall required a much longer exposure to cold to yield a 50% hatch (CT50) (i.e., the duration of diapause was longer) than eggs produced later in the fall. Eggs produced by laboratory animals that were reared at 14°C, 8L-16D, required a shorter period of chilling to terminate diapause than the eggs of animals reared at 19° C, 12L-12D. Considerable variation in the CT50 value was also observed among laboratory cultures that were all reared under identical conditions, but which differed in terms of selection history. The results indicate that both the genotype of the egg and the conditions prevailing during oocyte formation influence the duration of diapause. Eggs that were stored at 5°C for periods longer than 300 days no longer hatched upon warming. It is suggested that the variation in the duration of diapause is an adaptation that promotes synchronization of hatching by ensuring that all individuals terminate diapause at approximately the same time, and survival during the winter by conferring cold-hardiness. Synchronizing the onset of post-diapause development is also discussed as an alternative mechanism for achieving synchronous hatching.