J. Malemud Charles

Matrix metalloproteinases (MMPs) are members of an enzyme family that require a zinc ion in their active site for catalytic activity. MMPs are critical for maintaining tissue allostasis. MMPs are active at neutral pH and can therefore catalyze the normal turnover of extracellular matrix (ECM) macromolecules such as the interstitial and basement membrane collagens, proteoglycans such as aggrecan, decorin, biglycan, fibromodulin and versican as well as accessory ECM proteins such as fibronectin. Members of the MMP family include the "classical" MMPs, the membrane-bound MMPs (MT-MMPs) the ADAMs (a disintegrin and metalloproteinase; adamlysins) and the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif). There are more than 20 members in the MMP and ADAMTS family including the collagenases, gelatinases, stromelysins, some elastases and aggrecanases. Adamlysins are membrane-bound MMPs that also degrade aggrecan, but more importantly, one ADAM family member (i.e.ADAM-17) is a tumor necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE) that activates pro-TNF-alpha. Most of the MMPs are synthesized as inactive latent enzymes. Conversion to the active enzyme is generally mediated by activator systems that include plasminogen activator or the pro-hormone convertase, furin. MMP activity is regulated by a group of endogenous proteins, called, tissue inhibitor of metalloproteinases (TIMPs) that bind to active and alternative sites of the activated MMP. Significant advances have occurred in the understanding of the regulation of MMPs, ADAMs and ADAMTSs gene expression. In addition, development of MMP inhibitors to study MMP structure/function relationships spawned many studies to determine the effectiveness of MMP inhibitors in regulating abnormal connective tissue turnover. In addition, development of MMP null mice carrying specific MMP deletions has provided an opportunity to explore the role of MMPs in normal development as well as in such diverse conditions and diseases as skeletal dysplasias, coronary artery and heart disease, arthritis, cancer, and brain disorders.

Differentiation is the cellular process that regulates development of long bones and joint surface cartilage of synovial cavities. Growth plate cartilage development is commonly referred to as endochondral ossification which is the end stage of long bone development. Endochondral ossification proceeds as a continuum of chondrocyte proliferation cycles followed by non-proliferative phases coupled to extracellular matrix protein transformations that are regulated by proteins of the hedgehog family and by parathyroid-hormone-related peptide and its receptor, the parathyroid-hormone-related peptide receptor. A compelling body of evidence has now emerged implicating matrix metalloproteinases (MMPs) in the process of long bone lengthening and endochondral ossification. Among the MMPs, MMP-13 (collagenase-3), MMP-9 (92-kDa gelatinase; gelatinase B) and MMP-14 (MT1-MMP) are the most abundant proteinases that regulate cellular migration, alterations in the extracellular matrix and apoptosis in growth plate cartilage. Murine mutation or ablation models of growth plate development that target MMPs often result in skeletal abnormalities, indicating the critical role that MMPs play in these animal models and in skeletal maturation. Many of the MMPs that have been identified as regulating the spatial and temporal changes in rodent and rabbit endochondral ossification have also been identified by in situ hybridization and immunohistochemical analysis of human long bone development. Genetic manipulation to correct defective or dysfunctional MMP genes or MMP activity found in certain human chondrodysplasias may provide a novel strategy for treating medical disorders characterized by skeletal anomalies.

Zebrafish have been used for many years in developmental and reproductive biology, yet little is still known about the adult zebrafish liver. Additionally, many of the mammalian liver cell types have yet to be characterized in zebrafish, including Kupffer cells, oval cells, and stellate cells. Also, there is minimal information available regarding zebrafish liver transporters and nuclear receptors. Therefore, it is important to understand the mechanism of xenobiotic metabolism in zebrafish to support their use as a model organism for studying the fate and biological effects of xenobiotics. The value of zebrafish as a model organism is based on a broad set of gene similarities (among fish species and humans) involved in metabolism and transportation of endogenous and exogenous chemicals. While there are molecular similarities between zebrafish and humans, there are also differences that may impact how compounds are metabolized. Therefore, it is absolutely necessary to investigate and validate the assumptions that zebrafish possess conserved genes, enzymes, and processes involved in all stages of metabolism. This study investigates the impact of ethanol exposure on liver toxicity markers in adult Danio rerio . Liver tissue from sham and ethanol injected fish were collected, pooled and RNA was isolated for qPCR. Relative expression levels for each gene were determined and normalized to βactin. Of the seventeen genes that were assessed, our results demonstrate a significant upregulation of the following genes following ethanol treatment: abcb4 (mdr3), abcc1(mrp1), abcc4(mrp4), abcc9(sur2), abcg5, mate1, oatp1 and slc4a2(ae2). Alcohol metabolism increases acetate production, a precursor for cholesterol and fatty acid synthesis. Historically, this increase in acetate levels driven by alcohol, is a contributor of hepatic steatosis (fatty liver). In a previous study conducted in the lab, zebrafish chronically exposured to ethanol displayed hallmark signs of hepatic steatosis. An upregulation in slc4a2, a sterol and cholesterol transporter aimed to eliminate dietary cholesterol, was found, consistent with the digestion of high levels of acetate. Furthermore, upregulation of abcc4, a prostaglandin transporter, is indicative of oxidative stress within the liver, caused by ethanol metabolism. Additionally, the upregulation of the other liver transporters would be consistent with normal hepatic ion flow during xenobiotic methabolism. Support or Funding Information Quinnipiac University: College of Arts and Sciences

Zebrafish have been used for many years in developmental and reproductive biology, yet little is known about the adult zebrafish liver. Additionally, many of the mammalian liver cell types have yet to be characterized in zebrafish, including Kupffer cells, oval cells, and stellate cells. There is minimal information available regarding zebrafish liver transporters and nuclear receptors. Therefore, it is important to understand the mechanism of xenobiotic metabolism in zebrafish to support their use as a model organism for studying the fate and biological effects of xenobiotics. The value of zebrafish as a model organism is based on a broad set of gene similarities (among fish species and humans) involved in metabolism and transportation of endogenous and exogenous chemicals. While there are molecular similarities between zebrafish and humans, there are also differences that may impact how compounds are metabolized. Therefore, it is absolutely necessary to investigate and validate the assumptions that zebrafish possess conserved genes, enzymes, and processes involved in all stages of metabolism. This study investigates the impact of ethanol exposure on liver toxicity markers in adult Danio rerio and the impact of ethanol treatment on developing zebrafish. For the adult study, liver tissue from sham and ethanol injected fish were collected, pooled and RNA was isolated for qPCR. Relative expression levels for each gene were determined and normalized to β‐actin. Of the seventeen genes that were assessed, our results demonstrate a significant up‐regulation of the following genes following ethanol treatment: abcb4 (mdr3), abcc1 (mrp1), abcc4 (mrp4), abcc9 (sur2), abcg5, mate1, oatp1 and slc4a2 (ae2). Alcohol metabolism increases acetate production, a precursor for cholesterol and fatty acid synthesis. Historically, this increase in acetate levels driven by alcohol, is a contributor of hepatic steatosis (fatty liver). This was demonstrated in a chronic ethanol exposure study of developing zebrafish from 10dpf to 90dpf, which displayed hallmark signs of hepatic steatosis. An upregulation in slc4a2, a sterol and cholesterol transporter aimed to eliminate dietary cholesterol, was found, consistent with the digestion of high levels of acetate. Furthermore, upregulation of abcc4, a prostaglandin transporter was found, indicating oxidative stress within the liver. Additionally, the upregulation of the other liver transporters is consistent with normal hepatic ion flow during xenobiotic metabolism. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .