Cyclopropenoid fatty acids (CPFA) are unique compounds that contain a highly strained and reactive cyclopropene ring structure. These compounds have been shown to cause a number of toxic effects in a variety of animals. Rainbow trout (Salmo gairdneri) have proven to be particularly sensitive to CPFA. Studies have revealed that CPFA are both carcinogenic and cocarcinogenic in rainbow trout. However, the mechanism(s) of these adverse biological effects are not understood. In the present report a series of studies were performed in order to determine the effect of CPFA on the levels and synthesis rates of trout hepatocyte proteins. In the first study, the influence of dietary CPFA on protein synthesis was measured via the use of amino acid double labeling experiments in isolated hepatocytes. Both the microsomal and cytosolic subcellular fractions were examined in these studies after separation by lithium dodecyl sulfate polyacrylamide gel electrophoresis. In the cytosolic fraction, the synthesis of proteins with apparent molecular weights in the range of 68,000 to 74,000 were significantly decreased. A marked depression in both the level and synthesis rate of microsomal proteins was observed for proteins that migrate in the 200,000 to 240,000 relative molecular mass region in polyacrylamide gels. These high molecular weight proteins do not appear to be membrane proteins and one of them has biotin associated with it. Using avidin-peroxidase staining, it was shown that the mass of this protein was reduced in CPFA-fed trout by 80%. The possible identity of these proteins is discussed. In the second study, initial attempts were made to use two dimensional gel electrophoresis to study alterations in individual liver microsomal polypeptides from trout fed CPFA. In order to effectively resolve membrane proteins in the first dimension (isoelectric focusing) changes in the standard techniques were needed. Replacement of the detergent nonidet-40 with 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS) in isoelectric focusing of trout liver microsomes have greatly increased resolution. These results have allowed effective resolution of complex polypeptide patterns for comparative purposes. In the third study, antibodies against β-napthoflavone-fed rainbow trout cytochrome P-450 (LM₂) were employed to localize the corresponding polypeptide(s) via protein blotting and immunochemical staining. Microsomes isolated from β-napthoflavone-fed trout contained only a single polypeptide. In contrast, control microsomes contained two distinct polypeptides differing only in their isoelectric points. Thus, an additional P-450 isozyme in rainbow trout was tentatively identified. CPFA treatment caused a preferential decrease in only one of the isozymes found in the control samples. The presence of concanavalin A binding glycopolypeptides was determined. The two P-450 isozymes localized on control microsomal gels were found to bind concanavalin A, suggesting that these isozymes are giycoproteins. Another result of CPFA treatment was a shift in a closely related group of membrane glycopolypeptides, labeled gp80, gp82, gp80₁, and gp82₁. A decrease in the mass of gp80 and gp82, and a corresponding increase in mass of gp80₁ and gp82₁ was observed. / Graduation date: 1985

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis - Wisconsin. / Vita. Bibliography : leaves 150-154.

Thesis--University of Wisconsin--Madison. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1985. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies.

A single injection of thioacetamide (TAA) induces a significant increase in hepatocytes' DNA synthesis and mitosis within 48 hours without accompanying pathology. The present study explores this mitogen system by characterizing several changes in the livers of young adult SD rats following an injection of TAA under controlled conditions. Radioautography of ('3)H-thymidine labeled livers showed an initial depression in the number of hepatocytes in S phase after TAA. This was followed by a significant increase in DNA synthesis beginning by 24 hours. The hepatocytes entering DNA synthesis and subsequently undergoing mitosis were localized initially to zone 1, followed by zone 2. The labeled hepatocytes remained localized for at least 8 weeks. The hepatocyte mitotic index was slightly depressed for 30 hours after TAA and then increased for 36 hours. The increase in the mitotic index followed that in the DNA synthetic index by 8 to 12 hours. The natural synchrony of hepatocytes' cell cycles was verified. The increased proliferative response after TAA was also synchronous, although a slight lengthening of the S + G(,2) interval may have occurred. The percentage of binucleated hepatocytes decreased between 24 and 30 hours after TAA; possibly indicating that binucleated cells enter a round of cell division more readily than other hepatocytes. Plasma alpha-fetoprotein (AFP) concentrations increased significantly during the first and third days after TAA. The first wave was a G(,1) event indicating that hepatocytes in young adult rats remain capable of synthesizing AFP and that they need not have entered a round of DNA synthesis and mitosis prior to secreting AFP. Immunofluorescent labeling specific for AFP verified that virtually all of the differentiated hepatocytes were responsible for the AFP synthesis. This mitogen system to explore liver proliferation and growth control is proposed as an alternative to partial hepatectomy. The regimen of a single low dose of TAA creates a model system of cellular proliferation with which to study the earliest changes leading to DNA synthesis and mitosis and the enhanced expression of AFP genes without the requirement of extended time periods as with chronic administration of hepatocarcinogens.

Liver cells from lymphosarcoma-bearing DBA/1J mice were shown, by parabiotic culture with normal liver cells from isologous mice, to elaborate an agent which could pass a 25 mu filter and transform the normal cells to a malignant state.

Thesis (M. Phil.)--University of Hong Kong, 1982.

published_or_final_version / Biochemistry / Master / Master of Philosophy

The organic anion transporting polypeptides (OATP/Oatp) are products of the solute carrier organic anion (<i>SLCO/Slco</i>) transporter gene superfamily and constitute a major class of polyspecific membrane solute carriers at the basolateral membrane of hepatocytes.  Alterations in Oatp hepatic drug uptake have the potential to alter the clinical pharmacokinetics, efficacy and safety of a given drug.  Similarly to their regulation of cytochrome P450 enzymes (CYP450) nuclear receptors (NR), such as the pregnane x receptor (PXR) and constitutive androstane receptor (CAR) have been implicated in the transcriptional regulation of Oatps. Dose response experiments were performed using known PXR and CAR activators in male Sprague Dawley rats to ascertain the effects of NR activation on hepatic Oatp expression.  Dexamethasone, pregnenolone-16α-carbonitrile (PCN), 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or Phenobarbital (PB) were administered for 72h and Taqman real time PCR used to quantify transcript levels and Western blots used to quantify transporter proteins.  Only Oatp1a4 displayed responsiveness to NR activation, Oatp1a1 and Oatp1b2 levels are not altered by NR regulation.  Oatp1a4 mRNA and protein levels were only increased by the PXR ligands, dexamethasone and PCN, dexamethasone being the more potent PXR activator compared to PCN.  Dexamethasone resulted in a statistically significant 8-fold induction of Oatp1a4 mRNA with a maximal increase in response occurring at 50mg/kg (12703 ± 1985 copies/ng RNA compared with control 1540 ± 193 copies/ng RNA, p &lt; 0.001). Increasing doss of 100 mg/kg and 150 mg/kg did not result in increased Oatp1a4 levels.  Western blotting showed that dexamethasone and PCN resulted in an increase of Oatp1a4 protein.  The CAR activator, PB, increased Oatp1a4 protein levels; however this is likely to be via a post transcriptional mechanism as there was no concurrent increase in Oatp1a4 mRNA.