University of Rhode Island DigitalCommons@URI Open Access Dissertations 1980 A MECHANISM OF ACTION OF GOLD IN RHEUMATOID ARTHRITIS. A BIOCHEMICAL AND MORPHOLOGICAL INVESTIGATION OF THE DIRECT EFFECT OF GOLD SODIUM THIOMALATE (GST) ON HUMAN SYNOVIAL CELLS IN CULTURE Ronald Lee Goldberg University of Rhode Island Follow this and additional works at: https://digitalcommons.uri.edu/oa_diss Recommended Citation Goldberg, Ronald Lee, "A MECHANISM OF ACTION OF GOLD IN RHEUMATOID ARTHRITIS. A BIOCHEMICAL AND MORPHOLOGICAL INVESTIGATION OF THE DIRECT EFFECT OF GOLD SODIUM THIOMALATE (GST) ON HUMAN SYNOVIAL CELLS IN CULTURE" (1980). Open Access Dissertations. Paper 150. https://digitalcommons.uri.edu/oa_diss/150 This Dissertation is brought to you for free and open access by DigitalCommons@URI. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. A MECHANISM OF ACTION OF GOLD IN RHEUMATOID ARTHRITIS. A BIOCHEMICAL AND MORPHOLOGICAL INVESTIGATION OF THE DIRECT EFFECT OF GOLD SODIUM THIOMALATE (GST) ON HUMAN SYNOVIAL CELLS IN CULTURE BY RONALD LEE GOLDBER~ A DISSERTATION SUBMITTED IN PARTIAL - FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHTLOSOPHY IN PHARMACEUTICAL SCIENCES (PHARMACOLOGY AND TOXICOLOGY) UNIVERSITY OF RHODE ISLAND 19 80 DOCTOR OF PHILOSOPh'Y DISSERTATION OF RONALD LEE GOLDBERG Approved: Dissertation Major Dean of the Graduate School UNIVERSITY OF RHODE ISLAND 1980 ACKNOWLEDGEMENT The author wishes to express his sincere appreciation to Dr. George Fuller for his continuous guidance, encourage­ ment and understanding throughout the author's graduate studies. Sincere appreciation is expressed to Dr. Stephen Kaplan for his enthusiasm and direction on this work. Spe­ cial thanks are conveyed to Dr. Elizabeth La Due for her assistance on the morphological work. Acknowledged are Grant Jolly, John Ewell and Philip McGrath for the electron microscope and X-ray probe analysis work. Thanks and grati­ tude are extended to Dr. Dave Parrott for his instructions and assistance with the cell culture work. Gratitudes are extended to Lynn Matoney and Hank Turner for their assist­ ance with the cell culture work. To the author's parents, many thanks for their eternal support, confidence and en­ couragement. Financial support for this investigation was provided by a grant from the Rhode Island Chapter of The Arthritis Fou.~dation awarded to Dr. Stephen Kaplan and by a NIH Grant HL 16904 awarded to Dr. George Fuller. iv ABS.TRACT Goldberg, Ronald Lee. Ph.D., University of Rhode Island, 1980. A mechanism of action of gold in rheumatoid arthritis: a bio­ chemical and morphological investigation of the direct effect of gold sodium thiomalate (GST) on human synovial cells in cul­ ture. Major Professor: Dr. George C. Fuller. Gold sodium thiomalate (GST) effects on cultured rheuma- toid synovial cells were used as a model to investigate a mech- anism which can explain clinical benefits gained by patients from gold therapy. Synovial tissue obtained from patients with rheumatoid arthritis who were undergoing reconstructive joint surgery was used to obtain explant culture of synovial cells. The experiments described were performed on growing monolayer cultures during the second to fifth passages. Synovial cells were exposed to GST at pharmacologically relevant concentra- tions (10-100 uM). Vacuoles were found by phase contrast micro- scopy in synovial cells in culture after 2 days of exposure to 100 uM GST. The vacuoles,aurosomes, were found to contain electron dense material by electron microscopy that contained gold as identified by X-ray probe analysis. The structure of the aurosomes, electron dense lamellae structures in membrane bound vesicles, was found to be similar to those found in synovial tissue of patients with rheumatoid arthritis who had received gold therapy. The time course for gold accumulation in the cell layer paralleled with aurosome formation and the degree of inhibition of [ 3H]thymidine incorporation into DNA. ii DNA synthesis, DNA content and cell number were decreased in a dose dependent manner after synovial cells were exposed to GST containing medium. The synthesis of medium proteins per flask by synovial cells was decreased in a dose dependent manner with collagen decreased to a greater extent. The per­ centage of collagen to total protein synthesized by synovial cells was decreased in a dose dependent manner. A biphasic response of an increase followed by a decrease of proteins synthesized per cell or per DNA unit was found due to GST ex­ posure. The increase in collagen protein per cell was found to be predominantly type I collagen. The percentage of type III collagen decreased due to synovial cells exposure to GST containing medium. Removal of GST from the medium resulted in recovery of biochemical parameters at low concentrations and a partial recovery at high concentration. Neither auro­ somes nor gold could be completely removed from synovial cells after medium changes and subculturing. An important thera­ peutic mechanism of action of gold in the treatment of pa­ tients with rheumatoid arthritis may be by directly inhibit­ ing synovial cells proliferation and altering protein syn­ thesis by synovial cells to a nonconducive matrix for pannus expansion. iii TABLE OF CONTENTS PAGE ABSTRACT ••... ii ACKNOWLEDGEMENTS .• ~ . iv TABLE OF CONTENTS . v LIST OF TABLES. vii LIST OF FIGURES . viii CHAPTER I. INTRODUCTION ... ~ 1 II. LITERATURE SURVEY:' . 3 A. Rh.eumatoid Arthritis • 3 1. Etiology· of rheumatoid arthritis 3 2. pathogenesis of rh.eumatoid arthritis 5 3. Pharmacological ·management of rh.eurna to id arthri ti.s . 8 a~ First line drugs . • . 8 5. Steroids .. ~ . • . 9 c. Second line drugs. • . 10 B. Animal Models for Rheumatoid Arthritis • 11 1. Adjuvant rat -.model . • • . 11 2. Antigen rabbit model • . 11 3. Collagen induced arthritis . 12 C. Human synovial Tissue and Cells in Culture. 12 1. Tissue . • . • . • • • 12 2. Cells and culture models . 13 a. Enzymatic release.... .adhesion . 13 b. Explant. • . 14 c. :Rheumatoid disease in culture. 16 3. Peptide modulators effects in culture 17 4. Antirheumatic drugs effects in cul- ture . • . ~ . • . 18 D. Role of Collagen . 19 1. Structure and synthesis .. 19 2. Types of collagens ••. 20 iv PAGE 3. Alteration of collagen types with di- sease. 4! • 23 4. Synovial collagen ...... 24 E. Gold in Rheumatoid Arthritis . 25 1. Efficacy and Toxicity-. 25 2. ~harmacokinetics . 26 a. Whole body-blood and tissue ..... 26 b. Cellular-aurosomes . 27 3. Pharmacodynami c..,.-mechani sm of action. 28 a. Protein interaction ........ 28 i. Albumin .... 28 ii. Collagen .... .•. 29 iii. Ly·sosomal enzymes 29 iv. Compliment ..... 29 b. Cell interaction . .. 30 i. Neutrophils . 30 ii. Macrophages ........ 30 iii. Lymphocytes . .... 31 4. Other gold compounds . .. 31 a. Gold thioglucose ......... 32 b. Auranofin. • .... 32 III. METHODS. 34 A. Materials . 34 B. Culture Method. 35 C. Cell Number Determinations .. 37 1. Hemocytometer ...... 37 2. Coulter counter 37 D. DNA Assays ..... 37 1. Ethidium- bromide. 37 2. [3H] thymidine . 38 E. Gold Determination - Atomic Absorption. 38 F. Collagen and Noncollagen Protein Synthesis. 39 G. Medium Collagen Assay for Type I and III Collagen . 40 1. Extraction. • . 40 2. Sample preparation for electrophoresis. 41 3. Electrophoresis procedure . 42 4. Quantitation. 43 a. Scanning procedure. 43 b. Fluorescence of band. 44 c. Radioactivity of bands. 45 v PAGE H. Carboxy Methyl Cellulose Chromatography of Collagen. 45 I. Purification of Type I and III Collagen Standards. 46 J. Phase Contrast PHotomicrographs. 48 K. Electron Microscope .... 49 1. Non-osmium procedure . 49 2. Osmium procedure .. 49 3. X-ray probe analysis . 50 L. Statistics . 50 IV. EXPERIMENTAL RESULTS . 53 A. MORPHOLOGY . 53 1. Light microscope . .... 53 2. Electron microscope ..... 56 a. In vivo and in vitro aurosomes 56 b. Genesis of the aurosome in culture. 59 c. Nuclear change . .. 59 B. BIOCHEMISTRY . 62 1. Proliferation .... 62 a. Cell number and size ..... 62 b. DNA content and rate of synthesis .•...... 65 2. Protein synthesis ...... 70 a. Noncollagen protein and colla- gen synthesis. .. 70 b. Collagen types . .. 74 i. Electrophoresis ... 74 11. Chromatography. .. 76 3. Reversibility .......... 80 4. Effect of GST on synovial cells and skin fibroblasts during log phase and confluency . 86 5. Anti-rheumatic drugs effect on synovial cells . 87 v. DISCUSSION . 90 VI. CONCLUSIONS. 105 VII. APPENDIX .. 107 VIII. REFERENCES 108 vi LIST OF TABLES TABLE PAGE 1 Effect of gold sodium thiomalate (GST) on cell viability. 55 2 Effect of latex beads and gold sodium thiomalate (GST) on proliferation and cell size . • . 3 Effect of gold sodium thiomalate (GST) on the percent of type III collagen synthesized by synovial cells . 81 4 Quantitation of gold associated with synovial and skin cell layers after exposures to gold sodium thiomalate (GST) containing medium •... 84 5 Recovery of biochemical parameters after gold sodium thiomalate (GST) exposed synovial cells are grown in medium without GST ..••••... 85 6 Effect of gold sodium thiomalate (GST) on bio­ chemical parameters of synovial and skin cells during log phase and confluency . • . • . 88 7 Effect of anti-rheumatic drugs on percent type III collagen synthesized by cultured synovial eel 1 s . 89 vii LIST OF FIGURES FIGURE PAGE 1 Phase contrast photographs of synovial cells ex- posed to gold sodium thiomalate (GST). 54 2 Electron microscope photomicrograph @f aurosomes and X-ray probe analysis . 5 7 3 In vivo and in vitro aurosomes • • • 5 8 4 Genesis of aurosomes .... 60 5 GST induced nuclear change. 61 6 Effect of gold sodium thiomalate (GST)on cell growth curve. 6 3 7 Effect of gold sodium thiomalate (GST) on cell number and size .......... 64 8 Dose dependent effect of gold sodium thiomalate on DNA accumulation . 68 9 Dose dependent effect of gold sodium thiomalate on DNA synthesis . 69 10 Dose dependent effect of gold sodium thiomalate on collagen and noncollagen protein synthesis . 71 11 Effect of gold sodium thiomalate on [3H]proline incorporation per cell and DNA.