Altered liver parameters in a genetic mouse model of porphyria cutanea tarda and possible involvement of ABC transporters in the disease

The project sought to examine the changes in the liver associated with porphyria and whether ATP binding cassette (ABC) transporters in the hepatocyte might in some manner be connected with the disruption of porphyrin homeostasis. The animal model of porphyria selected for the study was a genetic mouse model of porphyria cutanea tarda (PCT) that spontaneously develops the disorder with maturity. This model affords the opportunity to comprehensively evaluate liver changes without the administration of any exogenous compounds, which in other animal models are used to precipitate PCT. Many changes in hepatic parameters were present in the porphyric mouse model. Total liver heme concentration was increased, select cytochrome P450 activities were decreased while others were unchanged, UDP-glucuronosyltransferase activity was unchanged while glutathione S-transferase activity was elevated. Because of their broad and overlapping substrate selectivities, changes in specific transporters are most easily investigated through changes in mRNA expression.

ALTERED LIVER PARAMETERS IN A GENETIC MOUSE MODEL OF PORPHYRIA CUTANEA TARDA AND POSSIBLE INVOLVEMENT OF ABC TRANSPORTERS IN THE DISEASE by Dorinda Deana Arch A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pharmacology and Toxicology The University of Utah August 2010 Copyright © Dorinda Deana Arch 2010 All Rights Reserved STATEMENT OF DISSERTATION APPROVAL The dissertation of Dorinda Deana Arch has been approved by the following supervisory committee members: Michael R. Franklin , Chair 11/19/09 Date Approved John D. Phillips , Member 11/19/09 Date Approved Philip J. Moos , Member 11/19/09 Date Approved Christopher A. Reilly , Member 11/19/09 Date Approved Douglas E. Rollins , Member 11/19/09 Date Approved and by William Crowley , Chair of the Department of Pharmacology and Toxicology and by Charles A. Wight, Dean of The Graduate School. ABSTRACT The project sought to examine the changes in the liver associated with porphyria and whether ATP binding cassette (ABC) transporters in the hepatocyte might in some manner be connected with the disruption of porphyrin homeostasis. The animal model of porphyria selected for the study was a genetic mouse model of porphyria cutanea tarda (PCT) that spontaneously develops the disorder with maturity. This model affords the opportunity to comprehensively evaluate liver changes without the administration of any exogenous compounds, which in other animal models are used to precipitate PCT. Many changes in hepatic parameters were present in the porphyric mouse model. Total liver heme concentration was increased, select cytochrome P450 activities were decreased while others were unchanged, UDP-glucuronosyltransferase activity was unchanged while glutathione S-transferase activity was elevated. Because of their broad and overlapping substrate selectivities, changes in specific transporters are most easily investigated through changes in mRNA expression. Of twelve ABC transporters examined, mRNA expression of seven were unchanged (Abcb1a, Abcb1b, Abcb6, Abcb11, Abcc3, Abcc5 and Abcg2), three were elevated (Abca3, Abcc1 and Abcc4) and two were depressed (Abcc2 and Abcc6). The possible association of ABC transporters with porphyria arises from a consideration of how highly anionic porphyrins might or might not move across the lipophilic cellular membrane of the hepatocyte. Depressed levels of relevant transporters could contribute to the initial accumulation of porphyrins characteristic of the disease. However, the eventual excretion of porphyrins in bile and urine indicates an ability to ultimately leave the hepatocyte, and the mechanism of how this might occur required an ability to measure this efflux under controlled conditions. To this end, a procedure to isolate and maintain viable hepatocytes from a porphyric mouse was developed and used to evaluate possible perturbing influences. Isolated hepatocytes revealed that while cells contained 70% porphyrinogen, 30% porphyrin, only the porphyrin was effluxed. A variety of approaches failed, however, to provide conclusive evidence that this efflux was mediated by ABC transporters. Interesting preliminary evidence suggested that lysosomal secretion might be a mechanism responsible for the porphyrin efflux in isolated cells. iv TABLE OF CONTENTS ABSTRACT.......................................................................................................................iii LIST OF FIGURES...........................................................................................................vii LIST OF TABLES..............................................................................................................ix Chapters 1 INTRODUCTION………………………………………………………………………..1 References………………………………………………………………………………8 2 HEPATIC CHANGES IN A SPONTANEOUSLY PORPHYRIC GENETIC MOUSE MODEL OF PORPHYRIA CUTANEA TARDA………….……………….12 Introduction..........................................................................................................12 Materials and Methods ........................................................................................ 13 Results ................................................................................................................19 Discussion...........................................................................................................32 Summary.............................................................................................................36 References..........................................................................................................37 3 ISOLATION OF VIABLE HEPATOCYTES: METHOD DEVELOPMENT………..42 Introduction..........................................................................................................42 Methods...............................................................................................................42 Results and Discussion .......................................................................................43 Summary.............................................................................................................59 References..........................................................................................................59 4 PORPHYRIN TRANSLOCATION ACROSS CELLULAR MEMBRANES…….....61 Introduction..........................................................................................................61 Methods...............................................................................................................62 Results and Discussion .......................................................................................63 Summary.............................................................................................................94 References..........................................................................................................95 5 CONCLUSIONS…………………………………………………………………….....99 Reiteration...........................................................................................................99 Alternate Mechanisms of Porphyrin Efflux ........................................................ 101 References........................................................................................................106 vi LIST OF FIGURES Figure Page 1.1 The Heme Biosynthetic Pathway……………………………………………………….3 2.1 Relative mRNA Expression of ABC Transporters Showing Statistically Significant Differences Between Urod+/-, Hfe-/- and Wild-type Animals................................................................................................................ 31 3.1 Flow Cytometric Data Comparing Isolated Primary Mouse Hepatocytes............ 47 3.2 Photomicrographs (40X magnification) of Hepatocyte Preparations from a Nonporphyric and Porphyric Mouse .................................................................... 51 3.3 Overall Cell Loss and Viability Gains of Percoll Purified Murine Hepatocyte Preparations..................................................................................... 52 3.4 Isolated Hepatocyte Viability Over Time in the Presence and Absence of Fetal Bovine Serum............................................................................................. 54 3.5 Reduced and Oxidized Glutathione Concentrations in Isolated Hepatocytes In the Presence and Absence of Fetal Bovine Serum......................................... 55 3.6 Effect of δ-Aminolevulinic Acid for 7, 16 and 23 Hours on Hepatocyte Porphyrins and Porphyrinogens .......................................................................... 56 4.1 Effect of Temperature on Hepatocyte Porphyrin Efflux ....................................... 64 4.2 Determination of the Substrate Redox State of Effluxed Porphyrin .................... 65 4.3 The Stability of Effluxed Porphyrins from Porphyric Hepatocytes ....................... 69 4.4 Determination of Synthetic Uroporphyrinogen Half-life (t1/2) in Cell Culture Medium................................................................................................... 70 4.5 Determination of Isolated Hepatocyte Viability Over Time .................................. 72 4.6 Determination of Residual and Effluxed Porphyrins of Hepatocytes in the Presence (Panel A) and Absence (Panel B) of Fetal Bovine Serum................... 73 4.7 A Side-by-Side Comparison of Cellular and Effluxed Porphyrins of Isolated Hepatocytes in the Presence and Absence of Fetal Bovine Serum in the Medium .............................................................................. 74 4.8 The Effects of Varying Medium pH on Porphyrin Efflux from Isolated Porphyric Hepatocytes ........................................................................................ 78 4.9 The Effect of GSH Depleting Agents on Isolated Hepatocyte Viability and Porphyrin Efflux ............................................................................................ 80 4.10 Cell Viability of Electroporated Primary Hepatocytes .......................................... 90 4.11 Mouse Primary Hepatocytes in the Presence of Propidium Iodide ..................... 91 5.1 Localization of Hepatocyte Porphyrins to Lysosomes....................................... 102 5.2 Time-lapsed Photomicrographs of Porphyrin Efflux ("Extrusion") from Hepatocytes .............................................................................................. 104 5.3 Proposed Scheme for Porphyrin Elimination from a Porphyric Mouse Hepatocyte ............................................................................................ 107 viii LIST OF TABLES Table Page 2.1 Experimental Conditions and Primer Sequences Utilized for Gene Expression Determinations ................................................................................. 20 2.2 Age-related Changes in Hepatic Porphyrins in Male and Female Wild-type and Urod+/-, Hfe-/- Mice...................................................................................... 21 2.3 Age-related Changes in Urinary Porphyrins of Male and Female Wild-type and Urod+/-, Hfe-/- Mice...................................................................................... 22 2.4 Isolated Hepatocyte Porphyrin and Porphyrinogen Levels in Male and Female Wild-type and Urod+/-, Hfe-/- Mice......................................................... 24 2.5 Hepatic Heme and Cytochrome P450 Levels in Female Urod+/-, Hfe-/- and Wild-type Mice..................................................................................................... 25 2.6 Hepatic Cytochrome P450 Activities in Urod+/-, Hfe-/- and Wild-type Mice........ 26 2.7 Hepatic Phase II Enzyme Activities in Urod+/-, Hfe-/- and Wild-type Mice ......... 28 2.8 Relative mRNA Expression of ABC Transporters Not Showing a Statistically Significant Change Between Urod+/-, Hfe-/- and Wild-type Mice..................................................................................................... 30 4.1 Determination of the Oxidation State of Effluxed and Residual Hepatocyte Porphyrins ........................................................................................ 67 4.2 Effects of Chloroquine on Hepatocyte Porphyrin Efflux ...................................... 81 4.3 Effects of Sodium Azide on Hepatocyte Porphyrin Efflux.................................... 82 4.4 Effects of Rifampicin on Hepatocyte Porphyrin Efflux ......................................... 84 4.5 Effects of Verapamil on Hepatocyte Porphyrin Efflux.......................................... 84 4.6 Effects of Indomethacin on Hepatocyte Porphyrin Efflux .................................... 85 4.7 Effects of Probenecid on Hepatocyte Porphyrin Efflux........................................ 85 4.8 Effects of Sodium Orthovanadate on Hepatocyte Porphyrin Efflux..................... 87 4.9 Effects of Solute Carrier Protein Superfamily Cosubstrates on Hepatocyte Porphyrin Efflux................................................................................ 88 4.10 Effect of Electroporation in the Presence of Sodium Orthovanadate on Hepatocyte Porphyrin Efflux.......................................................................... 93 4.11 Effect of Electroporation in the Presence of Probenecid on Hepatocyte Porphyrin Efflux ................................................................................................... 93 x CHAPTER 1 INTRODUCTION The liver is an organ with numerous important functions related to whole body physiology. These functions include carbohydrate storage and metabolism (i.e., gluconeogenesis and glycogenolysis), urea formation, metabolism of fats, bile formation to aid in digestion, the synthesis of various blood proteins most notably albumin, the metabolism of hormones and the metabolism of drugs and other xenobiotics. The latter function is key in the protection of the body from the accumulation of potentially toxic, foreign (xenobiotic) chemicals. To accomplish this protection, an elaborate system of enzymes has evolved for biotransformation (Phase I and Phase II metabolism) and disposition (Phase III or transmembrane transport). The elaborate system is collectively designed to increase water solubility and facilitate excretion of foreign compounds. For ease of understanding, these proteins and enzymes have been grouped into the three phases mentioned above. Phase I, or nonsynthetic reactions, typically precede Phase II reactions and introduce or uncover polar groups on the xenobiotic resulting in a more polar compound than the original. Phase I reactions include oxidations, reductions and hydrolyses and metabolites produced can be biologically active or inactive. The most common reactions are oxidations. Phase II metabolism, or conjugation reactions, include methylation, acetylation, sulfation, and most commonly glucuronidation. These reactions involve conjugations with polar groups or reactive centers present on xenobiotics, or as a result of Phase I metabolism. The products of Phase II metabolism 2 often possess greater polarity and are in most cases, biologically inactive. Phase III does not involve metabolism of the xenobiotics; it relates to the translocation of polar chemicals across a membrane containing a lipid bilayer. In the hepatocyte, efflux transporters of the hepatocyte are critical for preventing the accumulation of the highly polar metabolites produced by Phase I and/or Phase II reactions inside the cell. The dominance of oxidation as a Phase I reaction, and the hemoprotein nature of the cytochrome P450s that perform this reaction places a unique demand on the liver for the synthesis of heme. In humans, approximately 15% of the total heme synthesized daily is produced in the liver for the purpose of incorporation into heme based enzymes with more than half this production used for the formation of microsomal P450 cytochromes (Thunell, 2000). Heme is a complex of an atom of iron and protoporphyrin IX (1,3,5,8 methyl, 2,4-vinyl, 6,7 proprionate porphyrin). Formation of protoporphyrin IX is a highly complex biosynthetic scheme (Figure 1.1). The first step occurs in the mitochondrial matrix, where a condensation of glycine and succinyl coenzyme A produces δ-aminolevulinic acid (ALA). In the liver, this reaction is catalyzed by ALA-synthase 1 (ALAS-1). The ALA is exported into the cytosol where in a reaction catalyzed by ALA dehydratase two molecules condense to yield the monopyrrole, porphobilinogen. Porphobilinogen is the basic building block for all natural tetrapyrrole containing moieties, including heme, the cobalamines, and in plants the chlorophylls. In the formation of protoporphyrin IX, four molecules of porphobilinogen polymerize to form a linear tetrapyrrole, hydroxymethylbilane, catalyzed by porphobilinogen deaminase, followed by ring closure catalyzed by uroporphyrinogen III synthase (UROS). A nonenzymatic ring closure can occur but this forms uroporphyrinogen I, an isomer which is not a substrate for the next enzyme in the heme biosynthetic pathway. The enzymatically generated uroporphyrinogen III then undergoes four sequential 3 Glycine + Succinyl CoA ↓ Delta-aminolevulinic Acid (ALA) ↓ Porphobilinogen (PBG) ↓ Hydroxymethylbilane ↓ Uroporphyrinogen III ↓ Coproporphyrinogen III ↓ Protoporphyrinogen IX ↓ Protoporphyrin IX ↓ Heme MITOCHONDRION CYTOSOL ALA synthase ALA dehydratase PBG deaminase Uroporphyrinogen III cosynthase Uroporphyrinogen decarboxylase Coproporphyrinogen oxidase Protoporphyrinogen oxidase Ferrochelatase ALA-dedydrat deficiency por ase phyria Acute interm porphyria ittent Porphyria c tarda (PCT) Figure 1.1. The Heme Biosynthetic Pathway. The heme biosynthetic pathway, shown on the left portion of the diagram, depicting the resulting disorders, shown on the right side of the diagram, with disruption to the corresponding enzymes of the pathway, shown in the center of the diagram. utanea Hereditary coproporphyria Variegate porphyria 4 decarboxylation steps, all catalyzed by uroporphyrinogen decarboxylase (UROD), to yield coproporphyrinogen III. Coproporphyrinogen III is transported across the outer mitochondrial membrane where it is oxidatively decarboxylated by coproporphyrinogen oxidase to yield protoporphyrinogen IX. Protoporphyrinogen IX is oxidized by protoporphyrinogen oxidase to protoporphyrin IX (PPIX), which is transported into the mitochondrial matrix where an atom of iron is inserted into the tetrapyrrole macrocycle by ferrochelatase to yield the final product, heme. Following synthesis, heme is translocated out of the mitochondria to other subcellular organelles where among other functions, it is incorporated into hemoproteins. In the microsomes (endoplasmic reticulum) the dominant hemoproteins are cytochrome b5 and cytochrome P450. Disruption of the heme biosynthetic pathway can result in many disease states, including a range of porphyrias (Figure 1.1). The classification of the porphyrias is related to the location of the defect site (hepatic or erythropoietic), by the enzyme involved, and whether they cause acute, neuropsychiatric symptoms or not (James and Hift, 2000). Focusing on the hepatic forms only, a defect in ALA dehydratase produces the disorder δ-aminolevulinate dehydratase deficiency porphyria or plumboporphyria (this enzyme is highly sensitive to lead and clinical symptoms of lead poisoning are similar to those of hereditary ALA-D deficiency suggesting that ALA-D inhibition may be responsible for most of the clinical effects of lead poisoning; Wyckoff and Kushner, 1994); a defect in porphobilinogen deaminase produces acute intermittent porphyria; a defect in UROD produces porphyria cutanea tarda (PCT); a defect in coproporphyrinogen oxidase results in hereditary coproporphyria; and a defect in protoporphyrinogen oxidase results in variegate porphyria. All the hepatic porphyrias except for PCT are acute porphyrias (James and Hift, 2000). PCT, the most common porphyria in humans (Wyckoff and Kushner, 1994; Bulaj et al., 2000), is characterized 5 clinically by a photosensitive dermatosis and biochemically by supranormal excretion of highly carboxylated porphyrins (uroporphyrin and heptacarboxylic porphyrins) in the urine and tetracarboxylated porphyrins (coproporphyrin and isocoproporphyrin) in the stool. The highly carboxylated porphyrins arise because of a marked reduction in the activity of UROD (Wyckoff and Kushner, 1994; Phillips et al., 2001). The photosensitive skin lesions relate to the respective solubilities with the more lipophilic protoporphyrin accumulating in the cell membrane while the other more soluble porphyrins are confined to the intercellular and intracellular aqueous phase and cause the delayed bullous lesions observed. The accumulated porphyrins in the skin cells interact with molecular oxygen in the presence of UV light to produce activated oxygen and peroxide radicals. The free radicals damage cellular structures such as lysosomes, which then release cytotoxic enzymes and result in the cutaneous photosensitivity (Cockayne, 2003). There are four variants of PCT, and a genetic basis has only been established for two of them. The first variant is called familial PCT and its UROD defect is due to mutations of the UROD gene which is transmitted as an autosomal dominant trait (Kushner et al., 1976). This variant accounts for 25 to 50% of all PCT cases (Held et al., 1989; Doss et al., 1991; Koszo et al., 1992). Patients do not exhibit clinical symptoms until later in life and most carriers of these mutant UROD alleles do not express clinical manifestations even though enzyme activity is half normal in all tissues. The second genetic variant called hepatoerythropoietic porphyria (HEP) is rare. HEP occurs in individuals with two mutant UROD alleles either homozygotes for a single mutation or as compound heterozygotes with different mutant alleles (Elder et al., 1981). The clinical phenotype of this variant appears during childhood and can be quite severe. The third variant accounts for about half of the PCT cases and has been called sporadic PCT. The UROD defect in sporadic PCT is restricted to the liver (de Verneuil et al., 1978; 6 Elder et al., 1978; Elder et al., 1980; Felsher et al., 1982). Within the hepatocyte of patients with sporadic PCT, the amount of UROD protein is normal even though the catalytic activity is reduced (Elder et al., 1985) which suggests the presence of an inhibitor (Wyckoff and Kushner, 1994; Phillips et al., 2007). In most cases of sporadic PCT, hepatic siderosis is present and is due to heterozygous or homozygous mutant of the hemochromatosis gene (Wyckoff and Kushner, 1994; Lamoril et al., 2002). The fourth variant is toxic PCT and is associated with the exposure to halogenated aromatic hydrocarbons (e.g., hexachlorobenzene, pentachlorophenol, 2,3,7,8-tetrachlorodibenzo-p- dioxin, and the polychlorinated biphenyls). The exact mechanism by which this class of chemicals inhibit UROD activity is not known, but animal models of this disorder suggest that both iron and the induction of specific isozymes of cytochrome P450 are involved (Smith and Francis, 1983; Lambrecht et al., 1992). The manner by which the tetrapyrroles of porphyria leave the hepatocyte to reach the skin and urine (via the blood) and stool (via the bile) has not been determined. Because of their tendancy towards being mostly anionic at physiological (near neutral) pH, efflux transporters have been hypothesized to play a key role. Transporters possibly involved are members of the two transporter superfamilies; the ATP binding cassette (ABC) transporters, which rely on the hydrolysis of ATP to produce the energy for the translocation of their substrates, and the solute carrier (SLC) proteins which may be facilitated transporters or secondary active transporters or antiporters (Hediger et al., 2004; Giacomini and Sugiyama, 2006), and do not require ATP for translocation but rely on passive transport and chemiosmotic ion gradients for sym- and antiport (He et al., 2009). The ABC transporters include multidrug resistance proteins (ABCB1s/MDRs), multidrug resistance associated proteins (ABCCs/MRPs), breast cancer resistance protein (ABCG2/BCRP) and the bile salt export protein (ABCB11/BSEP). Members of 7 the SLC superfamily of transporters include the organic cation transporters (OCTs) and the organic anion transporters (OATs). However, in the hepatocyte, SLCs are generally considered to be uptake transporters (Koepsell, 1998; Sekine et al., 1998; Abe et al., 1999; Konig et al., 2000; Giacomini and Sugiyama, 2006), and members of the ABC superfamily are considered to be efflux transporters (Giacomini and Sugiyama, 2006) and therefore are more likely involved in the transport and efflux of porphyrins. Compounds transported into the bile by ABCB1/MDR1 tend to be neutral or cationic with a bulky structure (Giacomini and Sugiyama, 2006). In contrast, compounds transported by ABCC2/MRP2 (into the bile), ABCC1/MRP1 and ABCC3/MRP3 (into the blood) generally bear a negative charge (Giacomini and Sugiyama, 2006), and are therefore those more likely involved in porphyrin efflux. A defect in ABCC2/MRP2 results in mild jaundice (Dubin-Johnson syndrome; Toh et al., 1999) subsequent to the impairment of the biliary excretion of the glucuronide conjugate of the linear tetrapyrrole, heme breakdown product bilirubin. Other ABC transporters involved in the translocation of porphyrins and tetrapyrroles across biological membranes have been documented. ABCB6 in the outer mitochondrial membrane has a demonstrated role in the intracellular movement of porphyrins, from cytosol into the mitochondria (Krishnamurthy et al., 2006), and ABCG2/BCRP in the export of protoporphyrin IX from the cell (Jonker et al., 2002; Zhou et al., 2005; Tamura et al., 2006). ABC-mitochondrial erythroid (ABC-me), now designated ABCB10, may transport porphyrin into the mitochondria for heme biosynthesis (Shirihai et al., 2000). To date, no studies in man have linked any of these known tetrapyrrole ABC transporters nor any genetic variations to PCT. If transporters which are known to transport anionic drugs and drug metabolites, and certain tetrapyrroles also transport the highly carboxylated porphyrins that accumulate during PCT, PCT may arise or be exacerbated by limitations in their activity. 8 Compromised activity may result from competition and saturation, by altered formation or the mRNA expression of the transporters being downregulated. To consider these possibilities, a hypothesis was developed which was "among changes in the porphyric liver, alterations in hepatic membrane efflux transporters, generally considered to be important in the distribution and excretion of drugs and drug metabolites, play a role in the heme biosynthetic disorder porphyria cutanea tarda with their involvement in translocation of the disorder's resultant highly carboxylated porphyrins in the hepatocyte." Specific aims proposed to assist in proving or disproving the hypothesis included identifying the entity transported across the hepatocyte membrane; and identifying transporter(s) possibly involved by use of inhibitors, by glutathione sensitivity and by transcriptional changes. References Abe T, Kakyo M, Tokui T, Nakagomi R, Nishio T, Nakai D, Nomura H, Unno M, Suzuki M, Naitoh T, Matsuno S and Yawo H (1999) Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1. J Biol Chem 274:17159-17163. Bulaj ZJ, Phillips JD, Ajioka RS, Franklin MR, Griffen LM, Guinee DJ, Edwards CQ and Kushner JP (2000) Hemochromatosis genes and other factors contributing to the pathogenesis of porphyria cutanea tarda. Blood 95:1565-1571. 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Konig J, Cui Y, Nies AT and Keppler D (2000) Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J Biol Chem 275:23161-23168. Koszo F, Morvay M, Dobozy A and Simon N (1992) Erythrocyte uroporphyrinogen decarboxylase activity in 80 unrelated patients with porphyria cutanea tarda. Br J Dermatol 126:446-449. 10 Krishnamurthy PC, Du G, Fukuda Y, Sun D, Sampath J, Mercer KE, Wang J, Sosa- Pineda B, Murti KG and Schuetz JD (2006) Identification of a mammalian mitochondrial porphyrin transporter. Nature 443:586-589. Kushner JP, Barbuto AJ and Lee GR (1976) An inherited enzymatic defect in porphyria cutanea tarda: decreased uroporphyrinogen decarboxylase activity. J Clin Invest 58:1089-1097. Lambrecht RW, Sinclair PR, Gorman N and Sinclair JF (1992) Uroporphyrinogen oxidation catalyzed by reconstituted cytochrome P450IA2. Arch Biochem Biophys 294:504-510. Lamoril J, Andant C, Gouya L, Malonova E, Grandchamp B, Martasek P, Deybac JC and Puy H (2002) Hemochromatosis (HFE) and transferrin receptor-1 (TFRC1) genes in sporadic porphyria cutanea tarda (sPCT). Cell Mol Biol (Noisy-le-grand) 48:33- 41. Phillips JD, Bergonia HA, Reilly CA, Franklin MR and Kushner JP (2007) A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda. Proc Natl Acad Sci U S A 104:5079-5084. Phillips JD, Jackson LK, Bunting M, Franklin MR, Thomas KR, Levy JE, Andrews NC and Kushner JP (2001) A mouse model of familial porphyria cutanea tarda. Proc Natl Acad Sci U S A 98:259-264. Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y and Endou H (1998) Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 429:179-182. Shirihai OS, Gregory T, Yu C, Orkin SH and Weiss MJ (2000) ABC-me: a novel mitochondrial transporter induced by GATA-1 during erythroid differentiation. Embo J 19:2492-2502. Smith AG and Francis JE (1983) Synergism of iron and hexachlorobenzene inhibits hepatic uroporphyrinogen decarboxylase in inbred mice. Biochem J 214:909-913. Tamura A, Watanabe M, Saito H, Nakagawa H, Kamachi T, Okura I and Ishikawa T (2006) Functional validation of the genetic polymorphisms of human ATP-binding cassette (ABC) transporter ABCG2: identification of alleles that are defective in porphyrin transport. Mol Pharmacol 70:287-296. Thunell S (2000) Porphyrins, porphyrin metabolism and porphyrias. I. Update. Scand J Clin Lab Invest 60:509-540. Toh S, Wada M, Uchiumi T, Inokuchi A, Makino Y, Horie Y, Adachi Y, Sakisaka S and Kuwano M (1999) Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome. Am J Hum Genet 64:739-746. 11 Wyckoff EE and Kushner JP (1994) Heme Biosynthesis, the Porphyrias, and the Liver, in The Liver: Biology and Pathobiology (Arias IM, Boyer JL, Fausto N, B. JW, Schacher DA and Shafritz DA eds) pp 505-527, Raven Press, New York. Zhou S, Zong Y, Ney PA, Nair G, Stewart CF and Sorrentino BP (2005) Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels. Blood 105:2571-2576. CHAPTER 2 HEPATIC CHANGES IN A SPONTANEOUSLY PORPHYRIC GENETIC MOUSE MODEL OF PORPHYRIA CUTANEA TARDA Introduction An overview of the myriad functions of the liver were discussed in Chapter 1. Porphyria cutanea tarda (PCT) is a liver based disorder, and as such is likely accompanied by changes in other functions and parameters in this highly metabolic organ. Given the importance of the liver in metabolism and disposition of drugs, changes in Phase I and Phase II drug metabolizing enzymes, and Phase III transporters were of likely considerable clinical relevance. Therefore, changes in Phase I, II and III parameters within the genetic mouse model of PCT to be used in investigating hepatocyte porphyrin efflux were investigated. PCT and the porphyrin accumulation is caused by a limitation in the activity of the fifth enzyme in the heme biosynthetic pathway, uroporphyrinogen decarboxylase (Urod). However, mice that have half-normal activity of Urod as a result of a heterozygous deletion (i.e., Urod+/-) do not develop a porphyric phenotype (Franklin et al., 2001). When Urod+/- mice are crossed with mice homozygous for deletion of the hemochromatosis gene (Hfe) both iron and porphyrins increasingly accumulate in the liver with age (Franklin et al., 2001; Phillips et al., 2001). The liver iron accumulation with the Hfe deletion arises from the hyperabsorption of iron by the duodenal enterocyte. This spontaneously porphyric genetic model (Urod+/-, Hfe-/-), which most closely 13 represents patients possessing a combination of familial and sporadic PCT, has some clear advantages over other animal models for the study of hepatic enzyme alterations associated with PCT. Most, if not all other models use chemicals such as polychlorinated biphenyls (PCBs) to precipitate the uroporphyria. The confounding effects such precipitating agents have on the expression and activity of hepatic drug metabolizing enzymes and ABC transporters (Klaassen and Slitt, 2005), are avoided in a genetic model. In the future, if specific transporter involvement has been implicated in the genetic model, it would be of interest to track similar involvement in the more complicated animal model. Cytochrome P450s are key enzymes in the first step of conversion of lipophilic parent drugs and xenobiotics to more polar and more easily excreted metabolites. They are hemoproteins and likely to be affected by changes in the heme biosynthetic pathway. Equally important in the ability to eliminate drugs is the conjugation of these P450 generated metabolites by nonheme containing Phase II enzymes, and the subsequent transfer out by Phase III transporters. Therefore enzyme changes in all of these phases were targeted for investigation. The study of transporters gained particular importance because in addition to the transport of anionic drug conjugates they may also be key in the elimination of anionic porphyrin(ogen)s. Materials and Methods Animals Combined genotype Urod +/-, Hfe-/- (C57BL/6J) mice were generated as previously described (Phillips et al., 2001). All procedures involving animals were approved by the University of Utah Animal Care and Use Committee and were in concordance with NIH guidelines for the humane care of laboratory animals. 14 Liver and Urine Porphyrin Analysis A sample of liver tissue, ~50 mg, was taken at the time of sacrifice and immediately frozen. Total hepatic porphyrin analysis was performed as described previously (Franklin et al., 1997). The frozen liver pieces were weighed, homogenized in 0.1 M Tris-chloride buffer, pH 6.8, at 4oC and centrifuged at 100,000g for 30 min. The supernatant was diluted with an equal volume of 3 M HCl (1.5 M final concentration), and centrifuged at 1,200g for 10 min. The porphyrin composition of the sample was determined using reverse phase HPLC with a fluorescence detector (Ex 404 nm, Em 618 nm) using a linear gradient system of a mobile phase consisting of sodium phosphate buffer, pH 4.5, and methanol (Ford et al., 1981). Peak quantitation used the Empower (Waters Corp., Milford, MA) software package. Total porphyrin content was determined from the sum of the 8-, 7-, 6-, 5- and 4- COOH peak components. Urine samples (50 μl) were analyzed in a similar manner. Hepatocyte Isolation Where the preparation of hepatocytes was required, a modification of methods described by Berry and Friend (1969) and Seglen (1972; 1973b; 1973a) using an in situ two step EGTA/collagenase perfusion was utilized. Animals were anesthetized with Avertin (0.6 mg/g, i.p.; 2,2,2-tribromoethanol in tert-amyl alcohol [Sigma-Aldrich®, St. Louis, MO]) and the liver perfused via the vena cava with a Ca++-, Mg++- and indicator-free Hank's Balanced Salt Solution (HBSS; Hyclone® Laboratories, Inc., Logan UT) containing 0.2 mM EGTA (Sigma-Aldrich®, St. Louis, MO) at a rate of ~6 ml/min until adequate tissue blanching occurred. The perfusion medium was then switched to HBSS perfusion buffer containing 100 U/ml collagenase (Type II, Worthington Biochemical Corp., Lakewood, NJ) and 2 mM CaCl2 (Mallinckrodt, St. Louis, MO) and the perfusion 15 continued until adequate tissue dissociation had occurred, usually for 3-6 min. Following gentle excision of the liver to avoid disintegration, the gallbladder was removed and the liver was placed into a dish with chilled Dulbecco's Modified Eagle's Medium (DMEM; Hyclone® Laboratories, Inc., Logan UT) with high glucose and without phenol red or L-glutamine (sodium pyruvate [Invitrogen, Carlsbad, CA] was supplemented into the medium), and gently macerated with forceps. The resulting suspension was strained through chiffon cloth, and the eluate clarified by centrifugation at 4oC for 3 min at 50g. The sedimented material was subjected to two more washes with DMEM, with the suspension being screened through a 70 μm nylon mesh cell strainer (BD Biosciences, San Jose, CA) prior to the final wash. Cell yield and viability were determined by trypan blue exclusion using a hemocytometer. To further eliminate nonviable cells, the cells were subjected to low speed, iso-density Percoll (GE Healthcare Life Sciences, Piscataway, NJ) centrifugation (Kreamer et al., 1986). Only hepatocyte preparations with >85% viability were utilized for the reduced and oxidized porphyrin determinations. Hepatocyte Porphyrin Determination For determination of reduced and oxidized porphyrins, hepatocytes were diluted in DMEM to a concentration of 105 viable cells/ml and 104 viable cells were aliquoted into wells of a 96 well plate. The fluorescence (Ex 390 nm, Em 620 nm) of oxidized porphyrins was then determined in a fluorometer (Molecular Devices, Sunnyvale, CA). The fluorescence was redetermined following the oxidation of any porphyrinogen present to the porphyrin by the addition of 30% hydrogen peroxide (Sigma-Aldrich®, St. Louis, MO) to a final concentration of 0.6%. The difference between the pre- and postperoxide fluorescence was the amount of reduced porphyrinogen present. 16 Hepatic Microsome and Cytosol Preparation Livers were excised, and after gall bladder removal, homogenized as a 20% suspension in 0.25 M sucrose. The homogenate was successively centrifuged at 10,000g and 18,000g, discarding the precipitate each time. The resultant supernatant was centrifuged at 105,000g, the cytosol removed, and the microsomal pellet resuspended in 50 mM Tris-buffered 0.25 M sucrose, pH 7.4 (El-Sayed and Franklin, 2006). The protein content of both microsomes and cytosol was determined by the method of Lowry et al. (1951). Liver and Microsome Heme Analysis Livers were perfused in situ with ~25 ml of ice cold saline via the hepatic portal vein prior to homogenization. The heme content of whole homogenate, and subsequently isolated microsomes was determined from the alkaline pyridine hemochromagen absorption spectrum utilizing the extinction coefficient of 32.4 mM-1cm-1 for the 557 vs. 575 nm wavelengths (Strittmatter and Velick, 1956). Microsome and Cytosol Enzyme Assays The microsomal cytochrome P450 concentration was quantified from the dithionite reduced (ferrous) heme carbon monoxide complex using the extinction coefficient of 91 mM-1cm-1 for the 450 to 490 nm wavelength pair (Omura and Sato, 1964). P450 monooxygenase activities towards methoxy-, ethoxy- (Sigma-Aldrich®, St. Louis, MO), pentoxy- and benzoxyresorufin (Invitrogen, Carlsbad, CA) were determined from the rate of fluorescence increase due to the formation of the resorufin product (Ex 544 nm, Em 612 nm; Prough et al., 1978). Assays were performed in 96 well plates at 37oC with 50 ng of protein (500 ng for pentoxyresorufin) and excess 17 NADPH (>1.5 mM; Sigma-Aldrich®, St. Louis, MO). 7-Ethoxytrifluoromethylcoumarin (Sigma-Aldrich®, St. Louis, MO) O-dealkylation, utilizing 500 ng of protein per well in 96 well plates at 37oC, was determined from the rate of fluorescence increase due to the formation of the 7-hydroxytrifluoromethylcoumarin metabolite (Ex 409 nm, Em 550 nm) in the presence of excess NADPH (Franklin and Hathaway, 2008). Bufuralol (BD Biosciences, San Jose, CA) -1΄-hydroxylation, utilizing 2 μg of protein, and testosterone (Sigma-Aldrich®, St. Louis, MO) 6β-hydroxylation, utilizing 500 ng of protein, were determined by HPLC analysis (Franklin and Constance, 2007). Briefly, the 6β- hydroxylation of testosterone was measured by extracting the incubation reaction with dichloromethane, utilizing 11β-hydroxytestosterone as an internal standard, and subjecting the extract to HPLC separation (absorbance detection at 236 nm). The 1΄- hydoxylation of bufuralol was determined (Ex 285 nm, Em 310 nm) by HPLC separation of the acid treated incubation reaction, utilizing propranolol as an internal standard. Quantification of both HPLC assays was achieved by integration of peak area and comparison with authentic metabolite standards. HPLC separations were performed on a Supelco Discovery C18 column (250 X 4.6 mm, 5 μm) with flow rates of 1 ml/min. Gradients for bufuralol-1΄-hydroxylation and testosterone 6β-hydoxylation were acetonitrile and 2 mM perchloric acid, and acetonitrile and 0.1% trifluoroacetic acid, respectively. Microsomal NADPH-cytochrome c (P450) reductase activity was determined by the rate of reduction of cytochrome c monitored by absorbance at 550 nm (Masters et al., 1971). Microsomal UDP-glucuronosyltransferase activity was determined with 4-nitrophenol as the aglycone. The rate of glucuronidation by detergent activated microsomes was determined from the consumption of aglycone monitored spectrophotometrically at 417 nm (Franklin and Finkle, 1986). Cytosolic glutathione S- 18 transferase activity was determined from the rate of formation of the 1-chloro-2,4- dinitrobenzene glutathione conjugate monitored at 340 nm (Le and Franklin, 1997). Quantitative Real-Time PCR Determinations For quantitative real-time PCR (qPCR), a 50-100 mg liver sample was taken at the time of sacrifice and immediately homogenized in 1 ml of TRIzol Reagent (Invitrogen, Carlsbad, CA). For RNA isolation, an aliquot of TRIzol homogenate stored at -80oC was thawed to room temperature and extracted using the QIAGEN RNeasy Mini Kit (QIAGEN, Valencia, CA) following the manufacturer's instructions. Concentration and purity of the RNA extract was determined spectrophotometrically at 260 and 280 nm, respectively (NanoDrop, Thermo Fisher Scientific, Pittsburgh, PA), and the RNA integrity verified using the Experion Bio-Analyzer (Bio-Rad Laboratories, Hercules, CA). Total RNA samples with an A260:A280 value equal to or greater than 1.8, and with a 28S:18S value equal to or greater than 1.0 were considered acceptable. One μg of total RNA was reverse transcribed using SuperScript II Reverse Transcriptase reagent, according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Appropriate dilutions were made prior to qPCR. qPCR was performed on a Roche Lightcycler 480 (Roche Diagnostics, Indianapolis, IN) or ABI Prism 7900H sequence detector system utilizing either TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) or SYBR Green I Master reagent (Roche Diagnostics, Indianapolis, IN) with primers constructed with Vector NTI software (Invitrogen, Carlsbad, CA). Mus musculus proteasome subunit, beta-type 6 (NM_008946) was used as the invariant gene for normalization (Rubie et al., 2005). The cDNA dilutions utilized, 19 the known primer sequences for the transporters examined, and the reagent system employed are shown in Table 2.1. Results Liver and Urine Porphyrin Analysis The hepatic and urinary porphyrins of the spontaneously porphyric Urod+/-, Hfe- /- animals varied with age and gender. Porphyrin concentrations of both liver (Table 2.2) and urine (Table 2.3) in mature females are greater than those observed in mature males. In early immature animals, porphyrin concentrations are similar to those in wild-type animals, but porphyrin levels increase as animals gain maturity. Hepatic porphyrin levels in both male and female Urod+/-, Hfe-/- mice show a plateau of accumulation by 16-20 weeks of age (Table 2.2). No such accumulation occurs in wild-type animals. Urinary porphyrins of mature wild-type mice are 1-2 μM, with the 6-carboxyl component being the major porphyrin present (~70%; Table 2.3). Immature (10.5 week) Urod+/-, Hfe-/- mice have a slightly elevated urine porphyrin concentration of ~3 μM. As Urod+/-, Hfe-/- animals mature, the major porphyrin component becomes the 8-carboxyl form. This form then remains dominant throughout their lifetime. Hepatocyte Porphyrin Determinations In freshly isolated hepatocytes from mature porphyric animals, ~70% of the accumulated porphyrin was present in the reduced form. Such information was previously unknown; from the generalized pink fluorescence of porphyric liver it had largely been assumed to exist as oxidized. The percentage was determined by utilizing the fluorescent property of oxidized porphyrin; the difference between pre- and postoxidized preparations revealed the amount of porphyrinogen initially present within 20 Transporter Reagent cDNA Dilution Catalog No. or Primer Sequences Abca3 TaqMan 1:20 4331182 Mm_00550501_m1 Abcb1a SYBR 1:2.5 sense: 5'-cttacagccagcattctccgtaa-3' antisense: 5'-tcccaaggatcagaaacaacaa-3' Abcb1b SYBR 1:2.5 sense: 5'-aggagatcaatttacagaagtgtcca-3' antisense: 5'-ctatggcaaacactggttgtatgca-3' Abcb6 TaqMan 1:20 4331182 Mm_00470049_m1 Abcb11 SYBR 1:50 sense: 5'-gtctacttcatgcttgtgaccct-3' antisense: 5'-cctgatagctgcctctggaa-3' Abcc1 TaqMan 1:20 4331182 Mm_00456156_m1 Abcc2 SYBR 1:50 sense: 5'-gagaggctacagtcgataacga-3' antisense: 5'-agctgcatcgtcaggaattt-3' Abcc3 SYBR 1:50 sense: 5'-agcacgccgctcttcat-3' antisense: 5'-ttccagccgcttgagct-3' Abcc4 TaqMan 1:20 4351375 Mm_01226379_m1 Abcc5 TaqMan 1:20 4331182 Mm_00443360_m1 Abcc6 TaqMan 1:20 4331182 Mm_00497685_m1 Abcg2 SYBR 1:50 sense: 5'-tttcctgactaccaaccagtgt-3' antisense: 5'-cagacatcacctttccaaagaa-3' Psmb6 TaqMan 1:20 4331182 Mm_00833555_g1 SYBR 1:50 & 1:2.5 sense: 5'-tggcaggaatcatcattgca-3' antisense: 5'-ccgatggcaaaggactgtct-3' Experimental Conditions and Primer Sequences Utilized for Gene Expression Determinations. Table 2.1 21 Gender & Age Liver Porphyrins Genotype (weeks) (nmol/g liver) % 8COOH % 7COOH MALE Wild-type 11 0.14 + 0.05 (4) 100 + 0 0 + 0 28 0.10 + 0.02 (4) 100 + 0 0 + 0 Urod+/-, Hfe -/- 16 298.9 + 75.4 (2) 91 + 1 9+ 1 38 289.3 + 16.5 (3) 85 + 1 14 + 1 FEMALE Wild-type 8 0.10 + 0.00 (2) 100 + 1 0 + 0 10 0.37 + 0.16 (6) nd nd 16.5 0.58 + 0.18 (6) 98 + 2 2+ 2 Urod+/-, Hfe-/- 5.5 0.63 + 0.13 (4) 54 + 5 46 + 5 20.5 454.8 + 56.0 (5) 89 + 3 11 + 1 59 406.0 + 58.6 (6) 85 + 2 15 + 2 Age-related Changes in Hepatic Porphyrins in Male and Female Wild-type and Urod+/-, Hfe -/- Mice. All data are expressed as mean + SEM. Table 2.2 nd = not determined. 22 Age Urine (weeks) Porphyrins and (n)* (μM) MALE Wild-type 28 (4) 1.2 + 0.06 7.9 + 0.7 6.3 + 0.5 77.3 + 0.9 1.6 + 0.1 7.1 + 04 Urod+/-, Hfe-/- 10.5 (4) 3.05 + 0.78 49.7 + 8.2 9.6 + 1.3 35.5 + 5.8 1.5 +0.2 3.8 + 0.9 21.5 (9) 21.5 + 0.64 63.4 + 2.9 10.9 + 1.7 16.0 + 1.6 2.7 + 0.2 6.9 + 1.0 23.5 (9) 8.22 + 0.96 67.9 + 1.4 9.3 + 0.5 13.2 + 1.0 2.8 + 0.2 6.8 + 0.6 30 (10) 8.62 + 1.01 73.7 + 3.2 7.5 + 1.0 10.9 + 1.2 2.1 + 0.2 5.9 + 1.1 35 (9) 11.22 + 1.71 72.6 + 2.1 7.6 + 0.6 12.6 + 1.3 2.2 + 0.3 5.0 + 0.6 41 (7) 10.80 + 0.73 74.5 + 1.7 7.5 + 0.7 11.5 + 1.0 1.9 + 0.2 4.6 + 0.5 FEMALE Wild-type 19 (2) 1.85 + 0.07 4.1 + 0.5 2.6 + 0.6 67.9 + 5.8 1.7 + 0.1 24.0 + 5.8 Urod+/-, Hfe-/- 10.5 (2) 2.84 + 0.41 25.3 + 8.2 9.6 + 0.9 48.3 + 13.1 1.7 + 0.2 15.3 + 4.0 21.5 (2) 19.96 + 2.81 71.3 + 0.3 8.2 + 0.3 9.0 + 0.5 2.3 + 0.2 9.4 + 0.2 23.5 (4) 19.25 + 5.00 77.7 + 1.7 6.1 + 0.1 7.4 + 0.9 1.7 + 0.1 7.1 + 0.7 All data are expressed as mean + SEM. The dominant porphyrin (%) is shown in bold. Mice considered, without statistical analysis, to be porphyric by virtue of their total urine porphyrins are also shown in bold. * (n) = number of animals/group. Table 2.3 Age-related Changes in Urinary Porphyrins of Male and Female Wild-type and Urod+/-, Hfe -/- Mice. %8COOH %7COOH %6COOH %5COOH %4COOH 23 the cell (Table 2.4). The presence of such a high proportion of the porphyrin in the reduced state could have ramifications should any transporters have a preference for either a reduced or oxidized porphyrin. Liver and Microsome Heme Analysis Despite the altered heme precursor status, total hepatic heme levels of the Urod+/-, Hfe-/- animals were not different from wild-type controls; the 37% increase was not statistically significant (Table 2.5). However, the 31% drop in microsomal P450 levels was statistically significant and the decrease (0.20 nmol/mg protein) was reflected in the decrease in the microsomal heme level (0.22 nmol/mg). The other major microsomal hemoprotein, cytochrome b5, was therefore not altered. The location of the above normal levels of extra microsomal heme was not ascertained. Hepatic Phase I (P450 Oxidation) Drug Metabolizing Enzyme Activities Whether the decrease observed in the microsomal P450 concentration represented a global decline of all P450s present within the mouse microsomes, or was selective for only some monooxygenase activities representing subfamilies of P450, was determined (Table 2.6). In the second group of animals, those in which activities were determined (Table 2.6), total cytochrome P450 concentration was depressed to a greater extent (56% vs. 31%) than previous (Table 2.5). There was no significant shift in the absorbance maximum of the carbon monoxide complex hinting at no selectivity for Cyp1a family members; a selective loss of these would elevate the average maximum absorbance above 450.1 nm. The activities generally ascribed (from human studies, and to date no species exhibits similar P450 activities to that of man; Bogaards et al., 24 Gender Age in Cell Pre-Peroxide Post-Peroxide and weeks Viability Porphyrins Porphyrins % Genotype and (n)* % (nM)** (nM)** Porphyrinogen MALE Wild-type 28.0 + 2.8 (2) 87.0 + 7.1 nd nd Urod+/-, Hfe -/- 29.7 + 3.3 (6) 90.5 + 1.8 5.1 + 0.3 22.5 + 7.6 68.7 + 6.1 FEMALE Wild-type 57.7 + 3.3 (2) 90.0 + 7.1 nd Urod+/-, Hfe-/- 30.7 + 7.5 (8) 90.3 + 2.5 10.2 + 3.7 36.9 + 14.1 68.3 + 4.8 All data are expressed as mean + SEM. nd = not detected. Isolated Hepatocyte Porphyrin and Porphyrinogen Levels in Male and Female Wild-type and Urod+/-, Hfe -/- Mice. Table 2.4 *(n) = number of animals/group. **Cell suspension at 105 viable cells/mL. 25 Parameter Wild-type* Urod+/-, Hfe -/-* Change Liver heme (nmol/g wet tissue) 76 + 13 104 + 7 28 Microsomal heme (nmol/mg protein) 1.14 + 0.17 0.92 + 0.055 -0.22** Microsomal P450 (nmol/mg protein) 0.64 + 0.03 0.44 + 0.018 -0.20** Microsomal non-P450 heme (calculated) (nmol/mg protein) 0.50 + 0.04 0.48 + 0.04 -0.02 Hepatic Heme and Cytochrome P450 Levels in Female Urod+/-, Hfe-/- and Wild-type Mice. *(n)=3 animals/group and data are expressed at mean + SEM. **Statistically significant, p<0.05. Table 2.5 26 Change Phase I: Cytochrome P450 Parameter* Wild-type** Urod+/-, Hfe-/-** P450 concentration (nmol/mg) 0.549 + 0.056 (4) 0.244 + 0.022 (5) P450-CO absorbance max. (nm) 450.1 + -56%*** 0.2 (4) 449.9 + 0.4 (6) Testosterone 6β-hydroxylation 2837 + -0.2 nm 125 (5) 2564 + 117 (6) Pentoxyresorufin O-dealkylation 2.29 + -10% 0.11 (4) 2.18 + 0.44 (6) Benzoxyresorufin O-dealkylation 33.8 + -5% 3.3 (4) 15.3 + 3.3 (6) Bufuralol 1'-hydroxylation 73.8 + -55%*** 7.1 (4) 37.6 + 4.1 (5) Ethoxyresorufin O-deethylation 32.7 + -49%*** 3.3 (5) 21.8 + 3.3 (6) -43%*** 7-EFC-O-dealkylation**** 296.7 + 32.5 (5) 143.6 + 19.7 (6) -52%*** Mice. *Monooxygenase activities in pmol/mg microsomal protein/min. **(n) = number of mature female mice/group and data are expressed as mean + Table 2.6 Hepatic Cytochrome P450 Activities in Urod+/-, Hfe-/- and Wild-type SEM. ***Statistically significant, p<0.05. ****7-ethoxy-4-trifluoromethylcoumarin O-deethylation 27 2000) to Cyp3a and Cyp2b subfamilies, testosterone 6β-hydroxylation and pentoxyresorufin O-dealkylation respectively, showed no significant decrease. However, a significant decrease was observed in benzoxyresorufin O-dealkylation activity, an activity also generally ascribed to Cyp2bs. The discrepancy between these two assays would implicate differences in the Cyp2bs contributing to the two reactions. Bufuralol 1΄- hydroxylation, an activity ascribed to human CYP2D subfamily, was depressed by nearly 50%, and activities attributed to human CYP1A subfamilies, methoxy- and ethoxyresorufin dealkylations, were also decreased by 33 and 43%, respectively. 7- ethoxy-4-trifluoromethylcoumarin O-deethylation, a widely used marker for assessing human CYP1A2 activity, was also profoundly depressed (52%). It should be noted that the selectivity of certain human cytochrome P450s for a given reaction does not necessarily indicate that the reaction can be applied to a mouse ortholog. A recent study has indicated that mouse cytochrome P450s are in general less substrate selective than their human counterparts (McLaughlin et al., 2008). Hepatic Phase II (Conjugation) Drug Metabolizing Enzyme Activities To determine if the genetic modifications resulting in spontaneous uroporphyria development also affected nonhemea containing Phase II drug metabolizing enzymes, UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) activities were determined (Table 2.7). There was no effect observed on the microsomal UGT activity, but there was an 81% increase in the cytoplasmic GST activity. 28 Phase II enzyme activities: Wild-type Urod+/-, Hfe-/- Change UDP-glucuronosyltransferase [4-NP] (nmol/mg microsomal protein/min) 1.30 + 0.10 (4) 1.49 + 0.09 (5) 14% Glutathione S-transferase [CDNB] (nmol/mg cytosolic protein/min) 2575 + 75 (6) 4658 + 258 (6) +81%* 4-NP = 4-nitrophenol CDNB = 1-chloro-2,4-dinitrobenzene *Statistically significant, p<0.05. Hepatic Phase II Enzyme Activities in Urod+/-, Hfe-/- and Wild-type Mice. (n) = number of mature female mice/group. All data are expressed as mean + SEM. Table 2.7 29 Hepatic Phase III (ABC) Drug Metabolite (and Possibly Porphyrin) Transporter Expression The mRNA expression of 12 hepatic ABC transporters in mature wild-type and spontaneously porphyric (Urod+/-, Hfe-/-) mice was evaluated by qPCR. Expression of seven of the examined transporters, Abcb6, Abcb11, Abcg2, the Mrp transporters Abcc3/Mrp3 and Abcc5/Mrp5, and the multidrug resistance (Mdr) transporters Abcb1a/Mdr1a and Abcb1b/Mdr1b, were no different between groups (Table 2.8). The most abundantly expressed transporter (highest Abc/Psmb6 ratio) of those examined,Abcb11, showed the largest change, a 29% decrease, but this decrease failed to achieve statistical significance (p=0.11). Other ABC transporters showed increased and decreased expression (Figure 2.1). There was a significant increase over wild-type in Abca3, Abcc1/Mrp1 and Abcc4/Mrp4, and a significant decrease versus wild-type in Abcc6/Mrp6 and the highly expressed Abcc2/Mrp2. The difference in Abcb1a/Mdr1a expression, 80% higher in Urod+/-, Hfe-/- animals, was not considered statistically significant (p=0.07). Careful consideration must be given when evaluating the mRNA expression of transporters in the mouse; ontogeny is a known factor affecting expression as well as tissue distribution (Maher et al., 2005b). The expression levels of Abcc1/Mrp1 mRNA shown in Figure 2.1 were considerably higher than had been previously reported (Maher et al., 2005b). These data were obtained using probe 4331182 Mm_00456156_m1 (See Table 2.1). Similar (high) levels of expression were seen with two additional probes covering different regions of the gene (data not shown). 30 Phase III ABC Wild-t ype Urod+/-, Hfe-/- Change** Abcb1a/Mdr1a 0.409 + 0.078 0.738 + 0.161 80% Abcb1b/Mdr1b 0.001 + 0.0002 0.002 + 0.0003 100% Abcb6 0.804 + 0.127 0.720 + 0.078 -10% Abcb11 3.114 + 0.319 2.206 + 0.223 -29% Abcc3/Mrp3 1.272 + 0.125 1.252 + 0.051 -2% Abcc5/Mrp5 0.772 + 0.102 0.792 + 0.051 3% Table 2.8 Relative mRNA Expression of ABC Transporters Not Showing a Statisically Significant Change Between Urod+/-, Hfe-/- and Wild-type Mice. **No changes were statistically significant, p<0.05. *qPCR mRNA expression normalized to the mRNA expression of Psmb6. Each analyzed group contained 4-5 mature female animals and the data are expressed as mean + SEM. 31 0 0.5 1 1.5 2 2.5 3 3.5 Abca3 Abcc1 Abcc2 Abcc4 Abcc6 mRNA Expression Relative to Psmb6 Urod+/+, Hfe+/+ Urod+/-, Hfe-/- Figure 2.1. Relative mRNA Expression of ABC Transporters Showing Statistically Significant Differences Between Urod+/-, Hfe-/- and Wild-type Animals. A graphical representation of the relative expression of five transporters compared to that of Psmb6, as determined by qPCR. Each analyzed group contained 4-5 mature female animals and the data are expressed as mean + SEM. Significant change (p<0.05) compared to wild-type. 32 Discussion The initial sign of abnormal porphyrin status in the Urod+/-, Hfe-/- mouse occurs between 8 and 10 weeks of age as demonstrated by an altered urinary porphyrin profile (increased levels of the 8-carboxyl and decreases in the 6-carboxyl form). By 16 weeks, hepatic porphyrin content is noticeably elevated. Mature animals (>16 weeks) have a slightly increased total liver heme content but a reduced microsomal heme concentration (Table 2.5). The absence of a decrease in overall heme indicates that although much of the flux through the heme biosynthetic pathway has only progressed as far as the uroporphyrinogen synthesis, where it has accumulated, sufficient amounts pass through to support normal or even slightly elevated heme levels in the hepatocyte. The observed loss of microsomal heme is therefore a selective and localized loss. This depression in microsomal heme corresponded to a reduction in several cytochrome P450 activities and overall P450 content (Table 2.6). Since two hemoproteins contribute to the heme in microsomes, cytochrome b5 and cytochrome P450 (and the heme loss corresponds to the P450 loss), the microsomal heme loss accompanying uroporphyria appears selective for only one of these hemoproteins. Additionally, since some P450 activities remain at normal levels, the loss of P450 appears selective for only certain isoforms. Thus the suppression of cytochrome P450s has a major degree of selectivity to it. Surprising among the P450 activities depressed are those attributed to the Cyp1a family since in chemically induced models of PCT, elevation of these activities, especially the contribution of Cyp1a2, appears to be a prerequisite (Smith et al., 2001). Selective loss of P450s accompanying uroporphyria could arise through a variety of mechanisms; selective destruction or catabolism and selective downregulation of transcription or translation, being most likely. The literature provides no information of effects of porphyrin(ogen)s on any of these processes. Indeed, while the literature is 33 replete with studies and mechanisms of upregulation of cytochrome P450s, scant attention has been paid to mechanisms of cytochrome downregulation (repression). However, it has been noted that many organic compounds can degrade the heme moiety of hepatic microsomal cytochome P450 without affecting the levels of the other microsomal enzymes such as cytochrome b5 and NADPH-cytochrome c reductase (Ivanetich et al., 1978). Selectivity for only specific cytochromes could suggest that whatever the mechanism, it is related to a property or metabolic activity that only these isoforms possess. The increase in activity of the nonheme containing cytosolic Phase II conjugation family of enzymes, the GSTs, that accompanies uroporphyria was not altogether surprising. The mRNA transcription of most GSTs is known to be regulated by an extensive array of enhancer mechanisms. An exhaustive study of mouse GSTs by Knight et al. (2008), showed them upregulated by AhR (Gstm1), constitutive androstane receptor (CAR) and pregnane X receptor (PXR; Gsta1/2, Gstm1, Gstm2, Gstm3), peroxisome proliferator activated receptor  (PPAR; Gstk1, Gstp1/2, Gstt2) and nuclear factor E2-related factor 2 (Nrf2; Gsta1/2, Gsta4, Gstm1, Gstm2, Gstm3, Gstm4). The Nrf2-Kelch-like ECH-associated protein 1 (Keap1) system is a response pathway activated by redox changes within the cell (Dinkova-Kostova et al., 2002; McMahon et al., 2004; Imhoff and Hansen, 2009). This may be of relevance in the uroporphyria under consideration in Urod+/-, Hfe-/- mice where there appears to be a redox balance between the porphyrinogen (the major component at 68%) and its porphyrin oxidation product (Table 2.4). Transcriptional regulation of certain family members of the Abcc/Mrp transporters is modulated by the Nrf2-Keap1 pathway (Maher et al., 2005a). While mRNA transcription of Abcc2-6/Mrp2-6 are each elevated, Abcc1/Mrp1 transcription is depressed by treatment of mice with prototypical activators butylated 34 hydroxyanisole, oltipraz and ethoxyquin. The direction of these changes differs markedly from the observations in porphyric mice, where only the Abcc4/Mrp4 change (elevation) coincided. The transcription of Abcc2/Mrp2 and Abcc6/Mrp6 was depressed, Abcc3/Mrp3 and Abcc5/Mrp5 was unaltered and Abcc1/Mrp1 was increased alongside Abcc4/Mrp4 (Table 2.8 and Figure 2.1). From the statistically significant hepatic transporter changes observed, a putative transcription binding factor which may be responsible for the repression of Abcc2/Mrp2 and Abcc6/Mrp6 transcription is LBP-1. This protein has also been implicated in the repression of HIV transcription (Toohey and Jones, 1989; Kato et al., 1991; Yoon et al., 1994). Multiple computational systems have been developed, and were utilized, to aid in identifying putative transcription factor binding sites (TFBS) within the known DNA sequences (Elnitski et al., 2006). The Transcriptional Regulatory Element Database was initially employed to uncover any regulatory elements within the sequences that had exhibited statistically significant changes. Results from this search were proceeded by a search within the Multiple EM for Motif Elicitation (MEME) site. MEME allows for the discovery of signals, or "motifs", within DNA sequences of interest (Bailey and Elkan, 1994). Lastly, the Transcriptional Element Search Software web based tool was employed which utilizes TRANSFAC to create tables which attempt to align model binding sites with the user defined DNA sequence, allowing for identification of possible TFBS in the supplied sequences (Schug and Overton, 1997). Although additional laboratory analyses will be needed to verify this protein is involved with the repression of these genes, it is a start. Intense interest in Abcc/Mrp transporters derives from their ability, as a generalization (since each family member appears to have some, but often overlapping substrate selectivity), to transport organic anions (Giacomini and Sugiyama, 2006), a 35 chemical feature of porphyrin(ogen)s. Abcc1/Mrp1 transports many organic anions, ranging from glutathione conjugates, glutathione disulfide, glucuronides, unconjugated anionic drugs, dyes and amphipathic neutral/basic drugs (Choudhuri and Klaassen, 2006). In vesicular preparations, unconjugated bilirubin, a noncyclized dicarboxylic tetrapyrrole, also has a high affinity for this transporter (Rigato et al., 2004). Abcc2/Mrp2 is involved in the export of a variety of both conjugated and unconjugated anionic compounds into bile, and has a substrate selectivity similar to that of Abcc1/Mrp1 (Jedlitschky et al., 1997; Madon et al., 1997; Choudhuri and Klaassen, 2006). Regarding species differences, human MRP2 and mouse Mrp2 have largely overlapping substrate specificities, but there are important differences in transport efficiency of MRP2/Mrp2 substrates and in the modulation of transport by other compounds (Zimmermann et al., 2008). Abcc4/Mrp4 is also an organic anion transporter, whose substrates include conjugated steroids, cyclic nucleotides, methotrexate, bile salts and miscellaneous compounds such as folate, urate, and p-aminohippurate (Borst et al., 2007). Abcc6/Mrp6 transports a different spectrum of substrates compared to other Mrps (Madon et al., 2000), although human MRP6 shares with ABCC1/MRP1 the ability to transport several glutathione conjugated substrates in vesicular preparations (Belinsky et al., 2002). Abcc2/Mrp2 is unique among this family of transporters in being localized to the apical (biliary canalicular) membrane of hepatocytes (Buchler et al., 1996; Paulusma et al., 1996). Abcc1/Mrp1 (Cherrington et al., 2002), Abcc4/Mrp4 (Rius et al., 2003) and Abcc6/Mrp6 (Madon et al., 2000; Borst and Elferink, 2002) are localized on the basolateral (sinusoidal) side of hepatocytes. Dependent on the extent, if any, to which these transporters are involved in the translocation of the accumulated porphyrins, porphyrinogens or even the partially oxidized uroporphomethene inhibitor of UROD 36 (Phillips et al., 2007) out of the hepatocyte, their expression in the Urod+/-, Hfe-/- animal (Abcc2/Mrp2 and Abcc6/Mrp6 downregulated and Abcc1/Mrp1 upregulated) could alter from normal, the extent to which the various tetrapyrroles appear in the urine and bile. The extent to which the downregulation of transporters might contribute to the intracellular accumulation of porphyrin(ogen)s characteristic of the disease state is a consideration that must await elucidation of the substrate characteristics of the various transporters. Summary As a consequence of a combination of the complete deletion of the Hfe gene and the heterozygosity with respect to the Urod gene, mice develop uroporphyria as they gain maturity. Changes in hepatic and urinary porphyrin profiles (composition) are observed earlier (10.5 and 5.5 weeks, respectively) than the dominant feature of the disease, high concentrations of highly carboxylated porphyrins in the liver and urine. The porphyrins are mainly present (70%) as the reduced (porphyrinogen) form when determined in an isolated porphyric hepatocyte. This genetic model of PCT also shows changes in many other hepatic parameters likely to affect drug metabolism and disposition. Overall, hepatic heme is unaffected, but microsomal heme is depressed; several but not all cytochrome P450s are depressed, and glutathione conjugation, but not glucuronidation activity is elevated. Of the 12 hepatic ABC transporters evaluated, mRNA expression of some are elevated, some are unchanged and others are depressed. Given the development of porphyria in the genetic mouse model with age, it would be of interest in future experiments to evaluate transporter expression changes during this period to seek possible correlations. Given the differences between males and females, it would also be of interest to examine hormonal involvement. Given the 37 present paucity of information of the function (substrates) of the transporters showing changes in mRNA expression, it is difficult to speculate on support or not of the dissertation hypothesis, but the presence of changes makes further experimentation a viable option. Much of the data in Chapter 2 was included in the publication "Longitudinal study of a mouse model of familial porphyria cutanea tarda", DD Arch, H Bergonia, L Hathway, JP Kushner, JD Phillips and MR Franklin, Cellular and Molecular Biology (Noisy-le-grand), 2009 Jul 1;55(2):46-54. 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Paulusma CC, Bosma PJ, Zaman GJ, Bakker CT, Otter M, Scheffer GL, Scheper RJ, Borst P and Oude Elferink RP (1996) Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science 271:1126-1128. Phillips JD, Bergonia HA, Reilly CA, Franklin MR and Kushner JP (2007) A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda. Proc Natl Acad Sci U S A 104:5079-5084. Phillips JD, Jackson LK, Bunting M, Franklin MR, Thomas KR, Levy JE, Andrews NC and Kushner JP (2001) A mouse model of familial porphyria cutanea tarda. Proc Natl Acad Sci U S A 98:259-264. Prough RA, Burke MD and Mayer RT (1978) Direct fluorometric methods for measuring mixed function oxidase activity. Methods Enzymol 52:372-377. Rigato I, Pascolo L, Fernetti C, Ostrow JD and Tiribelli C (2004) The human multidrug-resistance- associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin. Biochem J 383:335-341. Rius M, Nies AT, Hummel-Eisenbeiss J, Jedlitschky G and Keppler D (2003) Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38:374-384. Rubie C, Kempf K, Hans J, Su T, Tilton B, Georg T, Brittner B, Ludwig B and Schilling M (2005) Housekeeping gene variability in normal and cancerous colorectal, pancreatic, esophageal, gastric and hepatic tissues. Mol Cell Probes 19:101-109. Schug J and Overton GC (1997) Modeling transcription factor binding sites with Gibbs sampling and minimum description length encoding. Proc Int Conf Intell Syst Mol Biol 5:268-271. Seglen PO (1972) Preparation of rat liver cells. I. Effect of Ca 2+ on enzymatic dispersion of isolated, perfused liver. Exp Cell Res 74:450-454. 41 Seglen PO (1973a) Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion. Exp Cell Res 82:391-398. Seglen PO (1973b) Preparation of rat liver cells. II. Effects of ions and chelators on tissue dispersion. Exp Cell Res 76:25-30. Smith AG, Clothier B, Carthew P, Childs NL, Sinclair PR, Nebert DW and Dalton TP (2001) Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl Pharmacol 173:89-98. Strittmatter P and Velick SF (1956) The isolation and properties of microsomal cytochrome. J Biol Chem 221:253-264. Toohey MG and Jones KA (1989) In vitro formation of short RNA polymerase II transcripts that terminate within the HIV-1 and HIV-2 promoter-proximal downstream regions. Genes Dev 3:265-282. Yoon JB, Li G and Roeder RG (1994) Characterization of a family of related cellular transcription factors which can modulate human immunodeficiency virus type 1 transcription in vitro. Mol Cell Biol 14:1776-1785. Zimmermann C, van de Wetering K, van de Steeg E, Wagenaar E, Vens C and Schinkel AH (2008) Species-dependent transport and modulation properties of human and mouse multidrug resistance protein 2 (MRP2/Mrp2, ABCC2/Abcc2). Drug Metab Dispos 36:631-640. CHAPTER 3 ISOLATION OF VIABLE HEPATOCYTES: METHOD DEVELOPMENT Introduction To study the efflux of porphyrin from the hepatocyte without the complications of downstream elimination processes, it was necessary to develop a technique for the isolation of hepatocytes from porphyric animals and study the process in vitro. Isolation from porphyric animals was required because there was no known method or current technology for generating a porphyric hepatocyte from a normal primary hepatocyte or hepatoma cell line in vitro. While a method for isolating hepatocytes from rats has been available for several decades (Berry and Friend, 1969), there is no rat model of PCT currently available that does not involve the use of precipitating chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and the polychlorinated biphenyls. Therefore it was necessary to work with a mouse model. The surgical requirements with an animal 1/10th the size of a rat have dissuaded all but the most persistent attempts at isolating hepatocytes in this species. This paucity of information made it necessary to investigate many deviations from rat protocols, and the outcomes of these explorations and modifications form the basis of this chapter. Methods The hepatocyte isolation procedure finally developed was described in Chapter 2. To arrive at the standard protocol, anesthetic choices, tissue dissociation techniques, 43 media to be used for perfusion and culturing, along with the conditions of use and methods for cell viability enhancement were evaluated. Results and Discussion Anesthetic Choice For the isolation of viable mouse hepatocytes, it was imperative the animal retain adequate cardiac function, during the surgical placement of catheters and until perfusion was initiated. Any cessation of adequate hepatic circulation prior to perfusion results in poor quality/low viability hepatocytes (Purdue University, 2002). Although the surgical procedure results in eventual exsanguination and death, it was imperative that the animal suffer no unnecessary pain or discomfort during the initial stages of the procedure. The most useful test for full and adequate anesthesia under which surgery can proceed was found to be the absence of a reaction to a paw pinch. If anesthesia is inadequate, a firm pinch to the hind paw results in a quick withdrawing reflex (referred to hereafter as the "pinch reflex"). A combination of ketamine (0.1 mg/g; Hospira, Inc., Lake Forest, IL) and xylazine (0.005 mg/g) was originally employed based on an online Amaxa (now part of Lonza) protocol but adequate anesthesia was not obtained. An increase of xylazine to 0.01 mg/g within the combination also was without success. Other investigative groups (laboratories of A. A. Elfarra, University of Wisconsin-Madison, J. E. Manautou, University of Connecticut; both personal communications) have utilized this combination in their mouse hepatocyte isolation procedures. In our experience this intraperitoneally (i.p.) injected combination eventually does provide adequate anesthesia, however, a higher dose was required and the time of onset was much greater than the information stated in the literature (2-3 min induction with an 80 min surgical anesthesia; Otto, 2004). 44 An extended time of onset and the need for a higher dose was also evident during onsite training received at the University of Connecticut. Short-acting barbiturates such as pentobarbital and hexobarbital have hypnotic and sedative effects and were used in the 1940s-1950s as agents for inducing anesthesia in short surgical, diagnostic, or therapeutic procedures associated with minimal painful stimuli. Pentobarbital (0.1 mg/g) has been used by Purdue University, the Cell Culture Core, University of Southern California (personal communication), and in the original hepatocyte isolation protocols of Berry and Friend (1969). Hexobarbital, like pentobarbital has a relatively fast onset of effect and short duration of action, but it can be difficult to control the depth of anesthesia. Our anesthesia attempts with hexobarbital (0.1 mg/g, i.p.; Winthrop Laboratories, NY, NY) suffered similar drawbacks to those encountered with the ketamine/xylazine combination; slow onset/high dosing requirements, and recurrent incomplete anesthesia. Isoflurane, utilized in the laboratory of D. J. Drucker, University of Toronto for mouse hepatocyte isolations (personal communication), and methoxyflurane (Pitman- Moore, Mundelein, IL; used previously as a rodent anesthetic in the Franklin research group, University of Utah) are fluorinated inhalation anesthetics which produce their effects rapidly and effectively. With methoxyflurane administration, the animal was placed into an enclosed chamber with ~500 μl of compound applied to a square of gauze. After a few minutes the animal appeared unresponsive, and tested negative with the pinch reflex. A nose cup, containing another piece of saturated gauze, was placed over the animal's snout to maintain anesthesia during the surgical procedure. Drawbacks to the use of this anesthetic were adverse effects of the agent on the operator once the animal was removed from the enclosed chamber, and reduced 45 hepatic blood flow, the latter a characteristic of all currently used inhalation anesthetics (Suttner et al., 2000). Avertin (2,2,2-tribromoethanol in tert-amyl alcohol [Sigma, St. Louis, MO]), an agent utilized for rat anesthesia in the laboratory of S. L. Bealer, University of Utah, was the anesthetic agent ultimately utilized. The preparation of this agent is somewhat arduous. A 60 mg/ml working solution in saline is prepared monthly and stored in the dark at 4oC. This is made from a 1.6 g/ml stock solution prepared yearly which is also maintained in the dark but at room temperature. The working solution required stirring overnight in the dark, to dissolve crystals which formed upon cooling. For surgery, approximately 3 ml of the stirred solution was filter sterilized and kept at room temperature in an amber vial and 0.6 mg/g (i.p.) was used to provide anesthesia. The original Amgen protocol, which has since been adopted by the Veterinary Office of Washington State University, indicated an effective dose for mice of between 0.4 and 0.6 mg/g. We conducted a dose response investigation on two strains of mice, CF-1 and C57BL6. At doses of 0.4 and 0.5 mg/g, a pinch reflex was still observable 10 min postadministration, and recovery began 20 min postadminstration. At 0.6 mg/g, the final adopted dose, animals had no pinch reflex 5 min postadministration, and recovery began at 35 min (C57BL6) and 50 min (CF-1). The procedures needed for mouse hepatocyte isolation (surgery and perfusion) can, with practice, be completed within 15 min. In the procedure, cardiac cessation occurs within 5 to 10 min of initiation. Tissue Dissociation Techniques A commercially available TissuePress (Biospec Products, Inc., Bartlesville, OK) advertised to be "used to prepare viable single cell suspensions" of fresh soft tissues such as liver was first utilized in attempts to rapidly isolate hepatocytes in a single cell 46 suspension. The majority of isolated cells were not able to exclude trypan blue and had viability yields of 13% as determined by flow cytometry with propidium iodide (Figure 3.1). As a positive control of "good" cells, commercially available primary mouse hepatocytes were also evaluated by flow cytometry (Figure 3.1). Flow cytometry was not revisited when isolation procedures were ultimately perfected. With less than sympathetic assistance from Biospec Products technical support and a note that cells obtained by mechanical dispersion techniques resulted in cells which are uniformly damaged and dead (Seglen, 1976), mechanical dispersion was deemed an unacceptable method for our needs. Enzymatic digestion of thinly sliced liver tissue in vitro also met with little success in yielding viable hepatocytes. Schwab et al. (2007) had described such a protocol which they utilized to evaluate the function of a transporter from surgical samples of solid tumors. A longer more complicated procedure using in situ, dual stage enzymatic digestion of the liver therefore appeared to be the most suitable way to obtain an adequate suspension of viable hepatocytes to conduct studies of porphyrin efflux under controlled in vitro conditions. Surgical Placement and Choice of Catheter Vessel catheterization was initially attempted for in situ dual stage enzymatic perfusion via the portal vein, with a catheter constructed from a short piece of PE-10 (i.d. 0.28 mm, o.d. 0.61 mm) inserted into and heat fused to a longer piece of PE-60 (i.d. 0.76 mm, o.d. 1.22 mm) polyethylene tubing and secured with a 2-0 silk suture. Suture material finer than 2-0 was found to sever the vessel upon tightening. The portal vein was the vessel employed in the online Amaxa mouse hepatocyte isolation protocol and in rats by Berry and Friend (1969). To catheterize this vessel it must be separated from 47 Region Diameter % Gated Count Region Diameter % Gated Count Viables 5.33 13.39% 293 Viables 27.44 84.27% 8,427 Dead 5.32 86.61% 1,895 Dead 30.24 15.73% 1,573 Figure 3.1. Flow Cytometric Data Comparing Isolated Primary Mouse Hepatocytes. The middle panels are histograms depicting the amount of fluorescence from propidium iodide staining, as shown on the x-axis. The x-axis is defined by its decades (i.e., 100, 101, 102, etc.) and fluorescence detection of the propidium iodide is set for all decades other than the first. The y-axis of these panels reflect the number of events/cell number (counts). The lower panels are scattergrams depicting the number of recognized events/cells as registered by detection of the emitted side scatter (ss, as shown on the y-axis) and their electronic volume (EV) or size, depicted on the x-axis. The upper most panels are numerical summaries of the graphical data showing the total number of counted events in the defined regions of viability, also given as the percent (%) gated, and the average size (as diameter) of the populations. The left most series of data were acquired from cells obtained from a liver preparation utilizing the Biospec TissuePress. The right most series of data were acquired from a preparation of primary mouse hepatocytes kindly donated by CellzDirect. 48 its surrounding tissue so the suture can be inserted under it in preparation for securing the catheter once it is placed. Catheter insertion requires that a small puncture be made in the vessel wall with a dulled 26 G hypodermic needle and the PE-10 portion of the catheter inserted into the puncture site and secured with the suture, a procedure that required the use of a dissection microscope to aid in visualization. While perfusion via the portal vein was achieved, successful catheter placement was inconsistent. Perfusion via the inferior (caudal) vena cava is described in the Purdue University online mouse hepatocyte isolation protocol. Use of the catheter described above at this site was unsuccessful, largely due to the puncture site becoming completely obscured by the exiting blood. As alternatives, a spring loaded catheter (22 G x 1" BD Insyte Autoguard shielded IV catheter) secured with a 2-0 silk suture placed by a curved surgical needle or a 22 G needle held in place by a 1 1/8" smooth electrical copper test clip (RadioShack©; J. Dever, University of Wisconsin-Madison, personal communication) were utilized. Catheterization at this site had some success, and resulted in hepatocytes with initial viabilities of >85% by trypan blue exclusion, but all too often, resulted in the kidney capsule filling with perfusion medium and interfering with adequate liver perfusion. The most consistent, successful perfusion of the liver was obtained with a catheter (Surflash® polyurethane IV, 20 G x 1") inserted through the right atrium and into the inferior vena cava, a procedure employed at the University of Connecticut. Although consistently successful, this perfusion route generated hepatocytes with lower initial viabilities than the above, and required the use of a Percoll iso-density gradient step to increase the viability to >85%. This level of viability was observed over a range of flow rates and collagenase concentrations. 49 Perfusion Medium The use of Krebs-Henseleit Buffer 1 & 2 (KHB 1 & 2) as the perfusion medium, as utilized in the Amaxa mouse isolation protocol resulted in excessively inconsistent viabilities and dramatic viability reductions when the temperature was elevated above 4oC. Hank's Buffered Salt Solution lacking calcium, magnesium and phenol red (HBSS; Hyclone, Logan, UT), is the medium described by Berry and Friend (1969), and used at the University of Southern California, the University of Connecticut, and the University of Wisconsin-Madison. HBSS was used as the base of the dual perfusion buffers (the first contains a calcium chelator, the second contains collagenase and calcium sufficient to support collagenase activity) and has provided satisfactory results. Although the Amaxa protocol indicated the need for 2% (w/v) bovine albumin (Sigma-Aldridge, St. Louis, MO) in the second perfusion medium, its addition carried with it two drawbacks. Addition of albumin necessitated the back adjustment of the medium pH, and was liable to result in excess frothing when the medium was gassed with Carbogen (see below) prior to and during perfusion, and was therefore discontinued. Gassing of perfusion buffers with Carbogen (95% O2, 5% CO2) prior to and during perfusion was indicated in the Amaxa isolation protocol. In our hands, gassing and not gassing resulted in similar outcomes, and gassing was eventually discontinued for streamlining of the hepatocyte isolation procedure. It had been noted previously that while continuous oxygenation is of vital importance during long term perfusions of rat liver, short periods of anoxia were tolerable (Seglen, 1976). 50 Percoll Iso-density Gradient Enrichment of Viable Hepatocytes As indicated above, Percoll iso-density gradient enrichment of viable cells is required subsequent to hepatocyte isolation when performing the perfusion with a catheter through the atrium and into the inferior vena cava. A procedure (University of Connecticut) utilizing a 10 ml 40% Percoll cushion in a 50 ml conical tube was unsuccessful in our hands. Modification of a protocol described by Kreamer et al. (1986) resulted in cells with >85% viability and Figure 3.2 shows photomicrographs of hepatocytes, from both porphyric and nonporphyric mice, isolated with the above mentioned technique. Briefly, isolated cells (2.5 x 106/ml of 40% Percoll solution prepared with Dulbecco's Modified Eagle's Medium [DMEM; see below]) were placed into a 15 ml conical tube and centrifuged at 50g for 10 min with minimal braking. The Percoll solution was removed immediately following centrifugation, the cell pellet resuspended into DMEM, and washed twice (~25 ml DMEM, 4oC, 50g). The Percoll enrichment produced a gain in viability and as anticipated, the gain in viability was greatest for cell preparations with low initial viabilities. The loss of cell number was also greatest for cell preparations with low initial viabilities (Figures 3.3A & B). The Percoll iso-density gradient exposure had no effect on intracellular glutathione content, reduced and oxidized (GSH and GSSG), of the isolated hepatocytes (data not shown). The concentrations were determined utilizing a commercially available kit from Promega, the GSH-Glo™ Glutathione Assay. This assay determines native GSH and following a GSSG reduction step using (tris(2-carboxylethyl)phosphine), total GSH and GSSG. The assay is based on the conversion of a luciferin derivative into luciferin in the presence of GSH, catalyzed by the addition of glutathione S-transferase (GST). The luminescence signal generated from luciferin following the addition of luciferase is proportional to the amount of GSH present within the sample. 51 Figure 3.2. Photomicrographs (40X magnification) of Hepatocyte Preparations from a Nonporphyric and Porphyric Mouse. Panel A: isolated hepatocytes from a nonporphyric animal imaged with transmitted light; Panel B: hepatocytes from a porphyric animal imaged with transmitted light. 52 A. 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 10 Cell Loss with Percoll (%) Pre-Percoll Viability (%) 0 B. -10 0 10 20 30 40 50 60 0 20 40 60 80 10 Percoll Viability Gain (%) Pre-Percoll Viability (%) 0 Figure 3.3. Overall Cell Loss and Viability Gains of Percoll Purified Murine Hepatocyte Preparations. A graphical representation of the percentage of cell loss (Figure A) and the percent increase of viability (Figure B) with hepatocyte preparations with low initial viabilities upon purification with Percoll iso-density gradient centrifugation. 53 Hepatocyte Maintenance In vitro maintenance of isolated hepatocytes was first evaluated using Williams E medium based upon information in the Amaxa isolation protocol and information that the formulation was suitable for long-term cultures of adult rat liver epithelial cells (Invitrogen). However, this medium resulted in excessive loss of cell viability over time (determined from intact cell protease activity using the Promega MultiTox-Fluor Multiplex Cytotoxicity Assay). Addition of 10% fetal bovine serum (FBS) to Williams E medium resulted in an improvement in viability maintenance over time but was inferior to the maintenance of viability of cells placed in DMEM, with or without FBS supplementation (Figure 3.4). DMEM is the recommended medium in the University of Wisconsin-Madison protocol. In DMEM, FBS also produced a slight improvement in the maintenance of viability in this medium over time. A dramatic effect of FBS addition, however, was on the intracellular concentration of GSH and GSSG (Figure 3.5). Within a 4 h time course, there was ~2.5 fold higher concentration of both GSH and GSSG in hepatocytes supplemented with FBS. δ-Aminolevulinic Acid Addition to Isolated Hepatocyte Medium The ability of hepatocytes isolated from spontaneously porphyric mice (Urod+/-, Hfe-/-) to continually efflux porphyrins into the medium is potentially dependent on their ability to maintain synthesis of the porphyrins. The influence of the porphyrin precursor, δ-aminolevulinic acid (ALA), added to the medium on the porphyrin concentration in cells was therefore investigated (Figure 3.6). For each hepatocyte preparation, porphyrin concentration in cells is reported after 7, 16 and 23 h. 54 0 200 400 600 800 1000 1200 1400 1600 1800 DMEM + DMEM - WE + WE - DMEM + DMEM - WE + WE - DMEM + DMEM - WE + WE - DMEM + DMEM - WE + WE - 20,000 cells / well 15,000 cells / well 10,000 cells / well 7,500 cells / well Viability RFU 3 h 6 h 9 h 24 h . illiams E Medium (WE), with (+) or without (-) the presence of 10% fetal bovine serum was monitored for 24 h. Varying cell densities were aliquoted into 96 well plates and the viabilities evaluated every 3 h from intact protease activity determined using the Promega MultiTox-Fluor Multiplex Cytotoxicity Assay. Figure 3.4. Isolated Hepatocyte Viability Over Time in the Presence and Absence of Fetal Bovine Serum Viability of isolated hepatocytes maintained either in Dulbecco's Modified Eagle's Medium (DMEM) or W 12 h 15 h 18 h 21 h 55 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1 h 2 h 3 h 1 h 2 h 3 h Without 10% Fetal Bovine Serum With 10% Fetal Bovine Serum [Glutathione] (nmol/10,000 cells) [GSH] Total [GSH] & [GSSG] Figure 3.5. Reduced and Oxidized Glutathione Concentrations in Isolated Hepatocytes in the Presence and Absence of Fetal Bovine Serum. Glutathione concentrations of isolated mouse heptatocytes maintained in Dulbecco's Modified Eagle's Medium, with or without the presence of 10% fetal bovine serum was monitored over time. 104 viable cells were aliquoted into 96 well plates and the reduced (GSH) and oxidized (GSSG) glutathione concentrations were determined hourly via the Promega GSH-Glo™ Glutathione Assay. 56 0 10 20 30 40 50 60 70 80 90 100 7 h 16 h 23 h Porphyrin RFU Additional Cellular Porphyrins Revealed w/ Oxidation Constitutive Oxidized Cellular Porphyrins Figure 3.6. Effect of δ-Aminolevulinic Acid for 7, 16 and 23 Hours on Hepatocyte Porphyrins and Porphyrinogens. Isolated porphyric hepatocytes were aliquoted (104 viable cells/well) in medium with or without the addition of 250 μM δ-aminolevulinic acid (ALA). After 7, 16 and 23 h porphyrin(ogen) concentrations in the adhered cells was determined by fluorescence (Ex 390 nm, Em 620 nm). In each histogram stack, constitutive oxidized cellular porphyrins are depicted in the lower component (light gray) and additional cellular porphyrins revealed with oxidation are shown in the upper component (dark gray). Each time period contains values obtained without (left) and with (right) 250 μM ALA. 57 The left bars at each time period show values from cells in medium containing no additional ALA, and the right bars from cells maintained in the presence of 250 μM ALA. At time periods after 7 h, hepatocytes maintained in medium containing 250 μM ALA had increased intracellular concentrations of porphyrins present as such (light gray blocks), as well as porphyrinogens (dark gray blocks), the latter revealed by the addition of hydrogen peroxide which oxidizes all the porphyrinogens to porphyrins (Figure 3.6). These determinations were performed on adhered cells with the original incubation medium removed. ALA addition appears to prevent a post-7 h decline in porphyrins. In the short term (0-7 h), the isolated hepatocytes appear able to maintain similar high levels of porphyrin(ogen)s without regard to the presence or absence of ALA. Whether this level is the same as when the hepatocytes were freshly isolated is not known since these starting levels were not determined, but after seven hours, the presence of ALA in the medium is essential to maintain the 7-h levels. This observation would be crucial if future experiments required long(er) culturing of Urod+/-, Hfe-/- hepatocytes. Hepatocyte Viability Assessment The viability of cells harvested following in situ perfusion with collagenase was determined by the cell's ability to exclude the dye, trypan blue. Live cells that possess intact membranes are able to exclude trypan blue or, other dyes, while the dye penetrates into the interior of dead cells and they appear blue under the microscope. Other such dyes utilized for this purpose include eosin and propidium iodide and with these, the cells appear fluorescent red and yellow, respectively. Typically, ~72% of freshly isolated cells were able to exclude dye and this was increased to ~89% following Percoll enrichment. Trypan blue exclusion was not a satisfactory method for quantifying viabilities for cells that had been plated and had adhered to their substrate. Isolated 58 hepatocytes were largely adherent within one hour of plating. Physical dislodgement either utilizing a cell scraper or by sharp impact of the culture flask resulted in viabilities of 9-22% and 26%, respectively, in the released cells (scraped cells had been plated for 24 h, sharp impact cells for 4). Trypsinization and scraping of adherent cells (24-h culture) yielded cells with no better viability than those released by scraping alone, 9- 22%. Ethylenediaminetetraacetic acid (EDTA) release of plated cells yielded viabilities of 41%, and 0%, respectively, for adherent cells cultured for 2 and 24 h. Although not as robust as when freshly isolated, it was established that cells were not dead prior to their attempted release. Prior to release, the 24-h adherent cells showed ~50% staining with YO-PRO®-1 (Invitrogen™, Carlsbad, CA), a proprietary carbocyanine nucleic acid stain used to identify apoptotic cells. From the foregoing, it was evident that attempted removal of adherent primary hepatocytes resulted in great losses of viability, much more than experience has shown with cultured hepatoma cell lines, and a method for determining viability that did not require release was necessary. The MultiTox-Fluor Cytotoxicity Assay from Promega (Madison, WI) provided such a method of determining cell viability in a 96 well plate. This simple, rapid and sensitive (1 x 104 cells required) assay measures live cell protease activity using a fluorogenic, cell permeant, peptide substrate (glycyl-phenylalanyl-amino-fluorocoumarin; GF-AFC) that is cleaved by a live cell protease to generate a fluorescence signal (Ex 400 nm, Em 505 nm) proportional to the number of living cells. The protease becomes inactive with loss of cell membrane integrity and leakage into the culture medium. Briefly, 100 ul of a 1 x 105/ml dilution of viable cells were aliquoted into wells of a 96 well tray. Thirty min prior to the end of an experiment, 100 ul of the MultiTox-Fluor Multiplex Cytotoxicity Assay Reagent were added to the wells, and incubated at 37oC for the 59 remaining 30 min. Viability was then determined by measuring the resulting fluorescence (Ex 400 nm, Em 505 nm). Summary A method of isolating murine hepatocytes suitable for investigating porphyrin efflux under controlled in vitro conditions has been developed. Avertin proved to be the best anesthetic for the surgery and cannulation of the inferior vena cava via the right atrium provided the most consistent hepatic perfusion for in situ digestion. A Percoll iso-density purification step was necessary to achieve the desired 85% viability cutoff. After plating, cells maintained viability better in DMEM compared to WE, and better if the medium was fortified with FBS. Cells lost viability if detachment was attempted, had higher levels of glutathione if FBS was present in the medium and maintained cell porphyrin levels better over the longer term culture if ALA was present. References Berry MN and Friend DS (1969) High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol 43:506-520. http://campusvet.wsu.edu/infofac/avertinuse.htm http://www.cyto.purdue.edu/hmarchiv/2002/0332.htm http://www.lonzabio.com/fileadmin/groups/marketing/Downloads/Protocols/Cell_lines/am axa_OP_Hepatocyte_mouse_DPL-1002.pdf https://commerce.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewCategories&pc =43&npc=2&nc=3& Kreamer BL, Staecker JL, Sawada N, Sattler GL, Hsia MT and Pitot HC (1986) Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell Dev Biol 22:201-211. Otto K (2004) Anesthesia, Analgesia and Euthanasia, in The Laboratory Mouse (Hedrich H ed) pp 555-556, Elsevier Academic Press, London, San Diego. 60 Schwab R, Micsik T, Szokoloczi O, Schafer E, Tihanyi B, Tihanyi T, Kupcsulik P, Diofalvi K, Mersich T, Besznyak I, Jr., Zarand A, Mihalik R, Sarkadi B, Keri G, Pap A, Jakab F, Kopper L and Petak I (2007) Functional evaluation of multidrug resistance transporter activity in surgical samples of solid tumors. Assay Drug Dev Technol 5:541-550. Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29-83. Suttner SW, Schmidt CC, Boldt J, Huttner I, Kumle B and Piper SN (2000) Low-flow desflurane and sevoflurane anesthesia minimally affect hepatic integrity and function in elderly patients. Anesth Analg 91:206-21 CHAPTER 4 PORPHYRIN TRANSLOCATION ACROSS CELLULAR MEMBRANES Introduction In porphyria cutanea tarda (PCT), an oxidation reaction results in the generation of an inhibitor of hepatic UROD activity (Phillips et al., 2007), causing the accumulation to superphysiologic levels of highly carboxylated porphyrinogens and/or porphyrins in the liver. Subsequently some of the porphyrin(ogen)s enter plasma and bile and those in the plasma are then excreted into the urine, again at superphysiologic levels. The highly carboxylated porphyrins have molecular weights of 655-837 Da, have calculated dimensions of 15x13x6 Å and exist mainly in the ionized state at physiological pHs. Carrier mediated transport is likely required for the movement of these large, anionic molecules across lipophilic hepatocyte membranes. Subsequent urinary excretion could also occur by active secretion in the proximal convoluted tubule, but elimination in the kidney could occur via glomerular filtration without the need for carrier mediated transport. Translocation of porphyrin(ogen)s out of the hepatocyte, the first critical step in the elimination process, was in need of examination. The successful isolation of hepatocytes from spontaneously porphyric mice described in the foregoing chapter has provided a unique opportunity for the investigation of the characteristics of any porphyrin(ogen)s effluxed to be evaluated in vitro, where conditions can be closely controlled. 62 The initial direction of the research was based on the physio-chemical properties of porphyrin(ogen), and that others have demonstrated the involvement of ATP binding cassette (ABC) transporters with the translocation of other tetrapyrroles. As noted in Chapter 2, there were statistically significant changes in several ABC transporters. To evaluate the possible involvement of these proteins, several known inhibitors, both general and semispecific, were added to cell suspensions of porphyric hepatocytes and subsequent efflux was monitored. Additionally, compounds which are known to perturb metabolic processes, i.e., ATP generation or glutathione concentration, were added to the porphyric cell suspensions and subsequent efflux was monitored. Furthermore, compounds and ions known to be translocated by members of the solute carrier (SLC) protein superfamily were also added to the porphyric cell suspensions and efflux was also monitored. As part of these investigations, consideration needed to be given as to the nature of the translocated substrate, whether it was the reduced (porphyrinogen) or oxidized (porphyrin) form. Methods The hepatocyte isolation procedure finally developed was described in Chapter 2. However, the additional step with Percoll viability enrichment (Percoll iso-density gradient centrifugation) was required for any examination subsequent to the dual stage enzymatic digestion with catheterization through the atrium. This would include the majority of the studies described within this chapter. A trypan blue exclusion count and viability determination was conducted following the media wash of the Percoll purified cell pellet. At this point, cellular suspensions were made (1 x 105 viable cells/ml) containing the compound of interest for altering porphyrin(ogen) efflux and aliquoted in triplicate into wells of a 96 well plate utilizing a multichannel pipette. Suspensions 63 required frequent mixing due to the relatively rapid sedimentation rate demonstrated by this cell type. Following the allotted incubation time, all porphyrin concentrations were determined by fluorescence measurements. Results and Discussion Porphyrin Efflux: Temperature Dependence It is important to stress here that the isolated cells were held at 4oC in all steps following digestion, beginning with masceration. Isolated heptatocytes from spontaneously porphyric (Urod+/-, Hfe-/-) mice efflux porphyrins while held in DMEM medium. Effluxed porphyrins were determined from the fluorescence (Ex 390 nm, Em 620 nm) of 100 uL of a 1 x 105/ml viable cell suspension from which the cells had been removed by centrifugation. Over the 1-h period for which efflux was observed, the rate of efflux was temperature dependent (Figure 4.1). At 37oC, the rate of efflux was twice that observed at 4oC (145 vs. 70 RFU/h). Porphyrin Efflux: Porphyrin or Porphyrinogen? Hepatocytes from porphyric mice contain both porphyrinogens and porphyrins, but only porphyrins are fluorescent. The porphyrinogens can be quantified following their conversion (oxidation) to porphyrins by treatment with hydrogen peroxide (H2O2). It is unknown whether porphyrins, porphyrinogens, or both are substrates for the proposed ABC transporters. To ascertain the redox state of the effluxed porphyrin(s), the initial temperature dependence experiment was repeated but with the addition of an oxidation step to the medium from which cells had been removed. As before, efflux was observed at both 4 and 37oC temperatures and the rate was faster at 37oC (Figure 4 The fluorescence of the cell suspension media from which the cells had been remov .2). ed 64 0 50 100 150 200 250 300 0 20 40 60 8 Porphyrin RFU Time (min) 0 Figure 4.1. Effect of Temperature on Hepatocyte Porphyrin Efflux. Effluxed porphyrins were monitored by fluorescence (Ex 390 nm, Em 620 nm) in the culture medium at 5 min intervals for 1 h. Aliquots of a hepatocyte suspension from a porphyric animal (1 x 105 viable cells/ml) were maintained at 4oC or 37oC. At intervals the suspension was centrifuged to remove cells, and 100 ul of the resulting cell free medium were assayed for porphyrins. (4oC = ●, 37oC = ■). 65 0 50 100 150 200 250 300 350 400 450 0 20 40 60 8 Porphyrin RFU Time (min) 0 ). Figure 4.2. Determination of the Substrate Redox State of Effluxed Porphyrin. Fluorescence (Ex 390 nm, Em 620 nm) of medium porphyrins which were effluxed over an hour was determined at 5 min intervals. Aliquots of a hepatocyte suspension from a porphyric animal (1 x 105 viable cells/ml) were maintained at 4oC or 37oC. At intervals the suspension was centrifuged to remove cells, and 100 ul of the resulting cell free medium were assayed for porphyrins (4oC = ●, 37oC = ■). H2O2 was then added in small aliquots to the 100 ul of medium to totally oxidize the porphyrinogens to porphyrins and the fluorescence was redetermined (4oC = O, 37oC = □ 66 showed no appreciable increase following the addition of H2O2 (Figure 4.2) indicating that only porphyrins are present, and therefore likely the entity selectively effluxed from the isolated hepatocytes. Although the intracellular levels of porphyrin/porphyrinogen initially present in the mouse were not recorded, the higher rate of efflux at 37oC in Figure 4.2 vs. Figure 4.1 can be attributed to the different levels present in the individual animals. Hepatocyte porphyrin efflux was maintained over at least 3 h (Table 4.1). In this determination, a hepatocyte suspension from a porphyric animal was aliquoted into a 96 well plate (104 viable cells/well), and at hourly intervals the porphyrin fluorescence of the well contents, the removed medium and the adhered cells were individually determined. The porphyrin fluorescence of these fractions was redetermined after 24 h when any porphyrinogen initially present would have undergone oxidation to porphyrin as a result of prolonged exposure to oxygen and UV light. There was no appreciable gain in fluorescence of the medium after 24 h (changes ranged from 15% gain for the 2- h sample to 11% loss for the 4-h sample). [By HPLC analysis, the effluxed porphyrins from isolated hepatocytes of mature porphyric animals were largely 7 and 8 carboxyl porphyrins (data not shown), a composition similar to that of liver tissue and urine from porphyric mice (Arch et al., 2009) Chapter 2]. At all time points of sampling the adhered cells showed a 79 + 3% gain in fluorescence after 24 h indicating that initially only ~20% of the total porphyrins present in the cells exist as porphyrin; most (~80%) exist as porphyrinogens. This percentage was slightly higher than the 72% in the original cell suspension, a likely consequence of the selective loss/efflux of porphyrins. An increase in porphyrin RFU can be observed in medium up to 3 h with the total amount of porphyrin (post-oxidation) in adhered cells decreasing over that time frame. 67 Time Well Contents (Cells and Medium)* Cells* Medium Time 0 476/1710 (72%) 91/87 1 h 412/1557 241/1422 (83%) 181/193 2 h 577/1551 232/1189 (80%) 320/369 3 h 741/1506 209/982 (79%) 538/507 4 h 792/1565 337/1229 (73%) 447/397 Porphyrin RFU (pre-oxidation/post-oxidation) * Percentages shown in parentheses indicate the amount of reduced porphyrinogen present. Table 4.1 Determination of the Oxidation State of Effluxed and Residual Hepatocyte Porphyrins 68 Uroporphyrin Stability and Uroporphyrinogen Half-life To evaluate whether the 7-carboxy porphyrin and uroporphyrin (8-carboxy porphyrin) effluxed from isolated porphyric heptocytes are chemically stable in cell culture medium, the medium removed from adhered cells after 1 h was monitored for fluorescence at 5 min intervals for 80 min (Figure 4.3). The negligible loss of fluorescence over the time period observed indicated that porphyrin effluxed from isolated porphyric hepatocytes is stable and available for detection for extended periods in the cell culture medium. If uroporphyrinogen has a very short half-life (t1/2) in cell culture medium it could be that the reduced form is also effluxed but is sufficiently unstable to render it nondetectable. To determine the t1/2 of uroporphyrinogen in cell culture medium, uroporphyrinogen was synthesized from uroporphyrin using palladium on carbon reduction in a hydrogen atmosphere at ambient temperature and pressure in darkness (Bergonia et al., 2009) and the uroporphyrinogen so formed was diluted in cell culture medium to concentrations equivalent to those of porphyrins normally achieved from hepatocyte efflux. Aliquots in a 96 well plate, were monitored for fluorescence at 5- min intervals for 3 h (Figure 4.4). At ~20 min, the fluorescence from oxidation to porphyrin reached its half maximal value, indicating this to be the t1/2 for oxidation of uroporphyrinogen in DMEM. Hence if uroporphyrinogen was effluxed, in hepatocyte experiments an increase in fluorescence in the effluxed medium over time would always be detectable, as uroporphyrinogen spontaneously oxidized to porphyrin. No increases were ever seen, indicating that efflux appears to be selective for the oxidized or porphyrin form. 69 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 8 Porphyrin RFU Time (min) 0 Figure 4.3. The Stability of Effluxed Porphyrins from Porphyric Hepatocytes. Medium was removed from aliquoted 104 porphyric cells/well following 1 h of efflux. The medium fluorescence (Ex 390 nm, Em 620 nm) was then monitored for 80 min, at 5 min intervals. Fluorescence of the medium containing the porphyrin is shown as a solid circle ● and a medium (DMEM) blank is shown as an X. 70 -20 0 20 40 60 80 100 120 140 160 0 50 100 150 200 Porphyrin RFU ~20 min Time (min) Figure 4.4. Determination of Synthetic Uroporphyrinogen Half-life (t1/2) in Cell Culture Medium. Dilutions of synthetic uroporphyrinogen in cell medium (DMEM) were aliquoted into wells of a 96 well plate and fluorescence (Ex 390 nm, Em 620 nm) was monitored for 3 h, at 5 min intervals. Uroporphyrinogen concentrations were 875 nM (▲), 438 nM ( ) and the medium (DMEM) blank is shown as an X. The arrow indicates the time at which fluorescence reached the half maximum value. 71 Porphyrin Efflux: Effects of Fetal Bovine Serum and Time As noted above (Table 4.1) there was little further increase in the fluorescence of the medium (DMEM), and therefore of porphyrins effluxed from porphyric hepatocytes between 3 and 4 h of plating, so 0-3 h was eventually adopted as the standard time course over which efflux was evaluated. DMEM lacks the high protein content and nutrients necessary for optimal long-term cell culture. Experiments conducted with and without the addition of 10% fetal bovine serum (FBS) to DMEM showed it had little effect on viability preservation (Figure 4.5) but served to maintain, or enhance, the glutathione concentrations of isolated hepatocytes (Figure 3.5). The efflux of porphyrins from isolated hepatocytes from porphyric mice over 3 h in the presence and absence of FBS is shown in Figures 4.6A and 4.6B. Individual hepatocyte isolations are arranged from left to right according to the increasing amount of total porphyrins, i.e., the summation of cellular and effluxed porphyrins and porphyrinogens. The reasons for such considerable variations in genetically identical animals have yet to be resolved. Variations similar to those shown here are known to occur in littermates cohabitating in the same cage. The four identical hepatocyte preparations that were evaluated in the presence and absence of FBS are shown with an asterisk. Some of these data are duplicated in side-by-side comparisons in Figure 4.7. For each hepatocyte preparation, porphyrin concentration in cells and medium are reported after 1, 2 and 3 h, or in two instances, mice D4.48 and D5.4, 1 and 3 h. The clustered column charts which depict the results from the efflux experiments are comprised as follows: Each cluster consists of three separate stacked columns. The left most column is data collected from the earliest time point within the time course, usually at 1 h. The middle stacked column is from the second time point, usually 2 h, 72 60 70 80 90 100 110 120 130 1h 2 h 3 h 4h % Viability Figure 4.5. Determination of Isolated Hepatocyte Viability Over Time. Isolated hepatocytes (104 cells) were aliquoted into wells of 96 well plates, either with (▲; n=7) or without (■; n=3) 10% fetal bovine serum supplementation. At hourly intervals up to 4 h the viabilities of the cells was determined from the cellular protease activity using the MultiTox-Fluor Cytotoxicity Assay reagents. Viability is normalized to the corresponding first hour value. Values are mean + SEM of duplicate determinations. 73 0 50 100 150 200 250 300 350 400 Porphyrin RFU 0 50 100 150 200 250 300 350 A. B. Figure 4.6. Determination of Residual and Effluxed Porphyrins of Hepatocytes in the Presence (Panel A) and Absence (Panel B) of Fetal Bovine Serum. Isolated porphyric hepatocytes were aliquoted (104 viable cells/well) in medium supplemented with (Panel A) or without (Panel B) 10% fetal bovine serum and assayed after 1, 2, and 3 h. Data from each time is shown as a column and the three time points are grouped as a cluster. At each time point, porphyrin concentrations in the adhered cells and removed medium were determined. The fluorescence (Ex 390 nm, Em 620 nm) of the removed medium, which contains the effluxed porphyrins, and the cells to which had been added fresh medium, was determined prior to, and again after treatment with H2O2 to oxidize any porphyrinogens to porphyrins. In each column, constitutive oxidized cellular porphyrins are depicted in the lower component (light gray); additional cellular porphyrins revealed with oxidation are shown in dark gray; the initial reading of medium porphyrin is shown hatched; and medium RFU gains or losses following oxidation are shown with the uppermost component in black and white, respectively. Each hepatocyte preparation is labeled with the animal identification number and gender (M or F) and those which were evaluated both with and without fetal bovine serum in the medium bear an asterisk (*). Hepatocyte preparations are arranged left to right in order of increasing levels of total porphyrin. Each stack in a cluster are values obtained after 1, 2 and 3 h. 74 0 50 100 150 200 250 300 350 400 D5.9 (F) -FBS D5.9 (F) +FBS 0 50 100 150 200 250 300 D5.5 (F) -FBS D5.5 (F) +FBS Porphyrin RFU 0 20 40 60 80 100 120 140 160 D5.7 (F) -FBS D5.7 (F) +FBS Porphyrin RFU 0 20 40 60 80 100 120 140 160 180 D4.90 (M) -FBS D4.90 (M) +FBS Figure 4.7. A Side-by-Side Comparison of Cellular and Effluxed Porphyrins of Isolated Hepatocytes in the Presence and Absence of Fetal Bovine Serum in the Medium. Isolated porphyric hepatocytes were aliquoted (104 viable cells/well) in medium supplemented with (right cluster of each panel) or without (left cluster of each panel) 10% fetal bovine serum (FBS) and assayed after 1, 2, and 3 h. Data from each time point are shown as a column and the three time points are grouped as a cluster. At each time point, porphyrin concentrations in the adhered cells and removed medium were determined. The fluorescence (Ex 390 nm, Em 620 nm) of the removed medium, which contains the effluxed porphyrins, and the cells to which had been added fresh medium, was determined prior to, and again after treatment with H2O2 to oxidize any porphyrinogens to porphyrins. In each column, constitutive oxidized cellular porphyrins are depicted in the lower component (light gray); additional cellular porphyrins revealed with oxidation are shown in dark gray; the initial reading of medium porphyrin is shown hatched; and medium RFU gains or losses following oxidation are shown with the uppermost component in black and white, respectively. Each hepatocyte preparation is labeled with the animal identification number and gender (M or F) and hepatocyte preparations are arranged in four panels. 75 and the right most column of the cluster represents the data collected from the final time point, generally at 3 h. Components in each column represent the relative fluorescence units of the cellular fraction and the medium fraction. The bottom most component (light gray) in the stack indicates the constitutive oxidized cellular porphyrins. This is the porphyrin fluorescence of the adhered cell fraction with the original medium from incubation removed. The second component (dark gray) is the additional fluorescence detected in the cellular fraction following complete oxidation of the porphyrinogen to porphyrin with H2O2; essentially the amount of reduced porphyrinogen of the cells. The third component (hatched) is the fluorescence of the incubation medium which was removed from the adhered cells containing the effluxed porphyrins. The small uppermost component, if present, represents the change in fluorescence observed following H2O2 treatment of the medium. The uppermost component is black if there was a gain in fluorescence, and white if there was a decrease in fluorescence. Hepatocytes from the female (F) mouse, D4.35 (third cluster, Figure 4.6A) is a "typical" experimental outcome. Cellular porphyrinogens (revealed with H2O2 oxidation) constituted a larger percentage than porphyrins, and the total (oxidized and reduced) dec