Document bygeo1YnX6Z9JQVOJ533vL9vk

MANUFACTURING CHEMISTS ASSOCIATION 1825 CONNECTICUT AVENUE, N W., WASHINGTON, 0 C. 20009 TELEPHONE. (202) 328-4200 TELEX: 89617 (MCA WSH) Caluasieu Parish July 31, 1978 TO: Vinyl Chloride Technical Panel Vinyl Chloride Research Coordinators Vinyl Chloride Medical Subcommittee -- ------ -- Gentlemen: As reported in Section 6.0 of the Record of the University of Louisville Meeting on June 14, 1978, Drs. Tamburro. and O'Connell promised to send the Project Manager complete abstracts of all research reviews presented on all component studies. We are pleased to report that they have exceeded their promise by sending us complete transcripts of the actual research reviews covering Dr. Tamburro's "Overview of the University of Louisville Multi-Disciplinary Approach to the Study of Chemical Carcinogenesis" and the reports on the component research projects as well. Included in the enclosed collection of papers are copies of all slides which were presented during the June 14th meeting. We trust you will find these research summaries helpful and, for the benefit of those of you who were unable to attend, they afford an 'excellent insight into the overall value of this research program. Sincerely JTS:ep Enclosure J. T. Seawell Project Manager Vinyl Chloride Research CMA 002212 THE WRITER'S DIRECT DIAL NUMBER IS (202) 328-4258 ......... " COHFIDBHTIAL Subject to Protective Crior it Boss v. Conoco. Ire., i'o. ?0-ir 14th Juiicinl district Court . ^ . Calcasieu Pariah *. Louisiana APPENDIX 0022^3 a& CONFIDENTIAL Subject to Protective Crror in Ross v. Cor.ocolr.o_^ "o . SO - 3,337 14th Judicial 5istrict Court Calcasieu Parish,, Louisiana ABSTRACT LYMPHOCYTE RESPONSE TO ANTIGENS OF LIVER ANGIOSARCOMA IN VINYL CHLORIDE WORKERS. H. Philip Fortwengler, Michael E. Dever, Carlo H. Tamburro, and Enrique Espinosa, University of Louisville School of Medicine, Louisville, Kentucky 40232. Angiosarcomatous liver tissue of vinyl chloride workers was shown by immunodiffusion and iromunofluorescent procedures to include a tumor-associated antigen, antigens shared with normal liver and other tissues and tumor-bound immunoglobulin G. In addition, evidence has been presented for the existence of a tissue or plasma antigen induced by, or conjugated with vinyl chloride or a vinyl chloride metabolite. Possible lympho cyte sensitization to such antigens in 79 vinyl chloride workers including 26 with liver abnormalities was assayed by the lympho cyte transformation test. Antigen extracts used were prepared from the angiosarcomatous tumor and normal liver tissues. Twenty normal Individuals having no exposure to vinyl chloride served as controls. Stimulation indices were calculated from cellular incorporation of tritiated thymidine. Mean stimulation indices in the normal individuals for' antigens of angiosarcoma and normal liver tissues were 4.7 (SE*1.7) and 3.8 (*0.9) and in vinyl chloride workers, 1.8 (*0.2) and 2.5 (*0.3) respec tively. Mean stimulation indices for PHA and concanavalin A in the.normal individuals were 235 (*35) and 209 (*30) and in vinyl chloride workers 201 (*23) and 180 (*19) respectively. Thus, these results suggest that vinyl chloride workers.have a decreased lymphocyte response to antigens of liver angiosar coma and normal liver tissues rather than the hypothesized in creased reactivity. This appears to be due to a lower over all lymphocyte responsiveness in these chemical workers in view of their decreased reactivity to PHA and concanavalin Ai (Supported in part by the Manufacturing Chemists Association) Pc's'A -t v cMa 23l4 ^ V4.I x- J.Ujrt ^ Sogs^y. .Conoco, Ino gQ. 90-4337 4th Judicial District Court ^^sieu Parish,, Louisiana .1 jc-idAi <>r Tlirvr vra.vrO^.C0^r *822.1' Csl0-' ' t * vsas T^ siisi* Afeftrasfts 63rd Annual Meeting Dallas, Texas April 1-10, 1979 Society Grouping II--APS, AAP, and AAI (April 6-10) FED5RA7SON Or AMERICAN SGC2E73SS FOR EXPERIMENTAL B30L0GY ENDOTHELIUM AND SMOOTH MUSCLE <4061--10661 W CMA 002215 4062 - pathology FACTOR VIII CONTENT AS EVIDENCE FOR ENDOTHELIAL ORIC1N OF VINYL CHLORIDE ASSOCIATED LIVER ANGIOSARCOMA (VCA). H. Philip Fortwengler*, Douglas Jones*, Carlo II. Tamburro* and Enrlqtii.* Cjipluoia (SPON: C. Randolph Sehrodt). Univ. of Louisville School of Medicine, Louisville, Y, 40232 To ascertain the endothelial cell origin of VCA wo have looked for Factor VIII in the tumor since thin factor ap pears to be specific for such eells (Moyer et al.t 1973). Frozen sections of three VCA and one idiopathic case were examined for presence of factor Vtlt by Indirect lestunofluoreseence. Sections of angiosarcoma demonstrated a strikingly increased specific fluorescence which lined enlarged sinusoids. This intense fluorescence was easily seen on low power(lOOx) ns an Irregular or splotchy pat tern. Occasional striations of linear fluorescence which did not follow hepatic cords Were also present. A similar pattern of staining was also given by the idiopathic angio sarcoma but was never seen in sinusoids of normal liver. These observations were further supported by the finding that absorption of Factor VIII antiserum with experiment * talanglosarcoma tissue resulted In complete inhibition of immunofluorescence. These finding demonstrate that VCA and idiopathic anglosarcoflii Include proliferating cells containing Factor VIII end therefore strongly support an cndothel ial cell origin of this tu.mor. (Supported in part by the Manufacturing Chemists Association.) uuixe i. Du, iii.i). V-- CONFIDENTIAL Subject to Protective Or--r in Boss v. Conoco, Trc. , 7c. io-43 14th. Judicial District Court Calcasieu Bariah,, la& s DECREASED GLUCOSE-6-PHOSPHATASE ACTIVITY IN LIVER IN VINYL CHLORIDE EXPOSED RATS. J.T. Du* and C.H. Tamburro* (SFON: M. Fonda) Dig. Dis. & Nutr. Sect., Dept. Med., Cancer Center, Univ. of Louisville Med. Sch., Lou., Ky. 40201. Increases in key glycolytic enzymes paralleling hepatoma tumor growth (Heinrich, et al., FEBS Letters, 42:145, 1974) and decreases in key gluconeogenic enzymes prior to and with the development of hepatomas (Isok, et al., Voprosy. Med. Khia 19:568, 1973) have been shown. We exposed adult SpragueDawley rats to 10,000-20,000 ppm of vinyl chloride (VC), 4-S hrs./day, 5 days/wk. for 3-4 wks. (40-140 hrs. exposure) to induce liver injury and angiosarcoma formation. Glucose-6phosphatase, a key gluconeogenic enzyme in the liver microsomal fraction, decreased 25% over control (P<0.05 from com bined data). Other microsomal proteins and enzymes related to VC metabolism, i.e., P450, NADPH-cytochrome c reductase and mixed function oxidase were unchanged in the same microsomal fraction with no differences in either mitochondrial cyto chrome oxidase or blood transaminases. The decrease in glucose-6-phosphatase is similar to the lower gluconeogenic enzyme findings in hepatomas. This may reflect an increase in glycolysis and ribose-5-phosphate production associated with de novo purine biosynthesis prior to tumor development, inhi bition in enzyme synthesis, or increased breakdown due to VC exposure. Work is under way to determine the mechanism. The decrease in glucose-6-phosphatase may be an early biochemical lesion usable as an indicator of liver injury associated with the subsequent development of angiosarcoma. ' Fed. Proc., 35, 1422 (1976). 1 !' | } [ f ' ] ] ; I j CMA 002216 nOHFIDMTIAL Uth Judicial District Court Calcasieu Parish,. Louisian^ ABSTRACT INCREASED CAPABILITY OF DETOXIFICATION BY ELEVATION OF GLUTATHIONE-S-TRANSFERASE AND GLUTATHIONE IN RAT LIVER EXPOSED TO VINYL CHLORIDE. Du, J.T.; and Tamburro, C.H. Digestive Diseases and Nutrition Section, Department of Medicine, Cancer Center, University of Louisville Medical School, Louisville, Kentucky 40201. Vinyl Chloride (VC) is believed to be metabolized in the liver to mutagenic compounds chloroethylene oxide and chloroacetaldehyde and detoxified by gluta thione transferase leading to the formation of mercapturic acid. Adult male rats were exposed to 28,000 ppm VC, 7 hrs/day, 5 days/week for 2, 4, and 6 weeks. During the 6 weeks, there was no eivdence of abnormality by clinical liver func tion tests in blood. However, the activity of glutathione epoxide-B-transferase (GEST, l,2-epoxy-3-(-nitrophenoxy) propane as substrate) and glutathione aralkylS^transferase (GAST, 2.-I1itrobenzyl chloride as substrate) in liver were elevated significantly over controls after 4 and 6 weeks respectively. GEST increased 17%, 28% and 42% and GAST increased 24%, 28% and 35% over controls at 2, 4 and 6 weeks suggesting enzyme induction by VC exposure. The content of reduced glutathione (GSH) was also elevated in the VC treated group (21%, 45% and 49% over controM^ at 2, 4 and 6 weeks of exposure), and the activity of glutathione reductase, enzyme to regenerate GSH from its oxidized form was also increased (50% to 86% after 2-6 weeks of exposure). The content of P-450, in the meantime was reduced in the exposed group. These results suggest that VC exposed rats had increased capacity of detoxification by induction of glutathione-S-transferase and elevation of glutathione. CMA 002217 C0WIBS52i^L -i--^-r'iuTv^^9T ln Subject to -- V_'c,'i v,. CO-4337 Boss. 7. SS^_-^rVt-i.ct Court Type Abstract in Space Beidw TISSUE AXD UKINAKY GLYCOSAMIXGLYCANS IK TKAUSPLAUTASLZ HEPATOMAS. C. E. Xupchella, K. L. Curran. S. Drake, J. Kennedy, arid C. K. Tanburro. Cancer Center, and Division. :of Digestive Diseases and liutririon. University or ^Louisville, School of Medicine, Louisville, Kentucky. i The purpose of this investigation was to evaluate: a) the glycosaminoglycans (GAGs) in different behavioral/histological types of intermuscularly transplanted hepa tomas, b) GAG patterns in tumor tissue in relationship to degrees of fibrosis and necrosis, c)' the GAG changes in th livers of tumor-bearing animals, and d) urinary GAG ex cretion as e function of tumor growth. Three types of Morris hepatomas, 7777, 5123tc, and 96ISA, which differ in rates of growth, metastatic potential, fibrosis, "and necrosis, were studied. Urinary and tissue G.AGs were ex tracted as cetylpyridir.ium complexes and measured as urcr.i: acid. Tissue GAGs were also evaluated histochsmically using aleian blue staining with and without enzyme pretreatment. Tumor tissue exhibited four- to site-fold greater GAG levels in the hyaluronic acid (90 gy 10, 91 -r 12, and ill '+ 9 vs. 25 + 3 ;ig uronic acid/g dry liver, re spectively) and chendroirin sulfate (25i A 29, 217 ^ 51, and 208 m 22 vs. 47 A 7 tg uronic acid/g, respectively) fractions than normal liver; the heparin fractions did. net differ significantly (31 A 7, 17 r 4 and 67 3 11 vs. 39 -- ;10). The livers of tumor-bearing animals exhibited slightly greater hyaluronic acid levels than normal livers. Moreover, increased urinary GAG excretion was evident after jtwo weeks in animals bearing fast-growing turners. Ike GAG tissue levels in fast vs. slow-growing tumors were not sig nificantly different. This further supports cur previously reported studies of urinary GAG excretion in human hepatic Jangiosarcoma (Curran, K. L., st al., Cancer 40 (6): 2050.3053) an suggesting mat urinary u.-iu analyses may oe useru-- in the detection, screening and diagnosis of hepatic cancer yo l-l. 7 r . CMA 002218 nOrlFIDSrlTIAL SubjecftTF^tectiva C^r in Boss v. Conoco. Ir.o^iiQ- 90 48 . 14tli Judicial District Court Parish., Louisiana GLYCOSAMINOGLYCAN CHANGES ASSOCIATED WITH HEPATIC TUMORS: THE CONTRIBUTIONS OF REGENERATION AND NECROSIS. C. E. Kupchella, E. M. Secskas,* J. S. Kennedy,* and E. Espinosa*. Cancer Center and Department of Pathology, University of Louisville, School of Medicine, Louisville, Kentucky. Although glycosaminoglycans (GAGs) have been shown to be elevated in many types of animal and human tumors including hepatic tumors, the cause and significance of these changes in neoplasia are still open questions. Regeneration and necrosis are operative in hepatic cancer and the purpose of this investigation was to evaluate the GAG changes associated with hepatic regeneration and hepatic necrosis. Regeneration was induced in male Sprague Dawley rats by partial hepatectomy and hepatic GAGs were evaluated at 4, 8 and 12 days post operatively. Necrosis was induced by: a) ligating the medium lobe, b) by resecting and placing median lobes in the peritoneal cavity and c) by resecting median lobes and incubating them in vitro in sterile saline. Analyses were carried out after 5 days of treatment. While regenerating livers exhibited GAG levels that were not statistically different from sham operated controls or non-operated controls, in vivo necrosis was accompanied by 3-4 fold Increases in tissue GAGs. These data suggest that necrosis may make a substantial contribution to the elevated GAG levels found in some tumors. 0o**19 AmftlCArJ PWSUC HAkTW A-ircClADorJ OtCtfTQ&l 1976 S/)N OteGQ. 3097 Subject to Prots'.i-.-? Cr-^or in Boss v. Conoco, ,*;. ?0- `3J - }4t6 Judicial 3ist;riot Court ' ^t.lcasie'A Parish,, Louisiana. \\.v " Early Detection of Disease in Individuals Ex posed to Vinyl Chloride. Richard Greenberg. MD, and Carla Tamburro, MD. University of Louisville, Louis ville, Kentucky This is a collaborative comparison study of the effectiveness of ultrasound, naii bed capillary study and urinary glycossmtneglycan excretions in vinyl chloride workers with biochemical dysfunction or histopathologically documented injury. It represents an effort to detect early mor bidity in vinyl chloride workers- Included is Dr. Maricq's method of investigating capil laries of the middle and distal phalanges of the fingers, including the nailfold, as a screening test for early changes from vinyl chloride exposure. Another new method is Dr. Taylor's "grey scale" ultrasonographic method which has not yet been widely adopted for diagnosis of liver disease. The University of Louisville group reported that the first indicator of liver changes occurred in the vascular sinusoids and was reflected by the appearance of mucopolysaccharides in the urine. The results of the massive clinical study in Louisville should soon be able to indicate whether more sensitive criteria of early dis ease may be forthcoming, especially from the liver function testing'. The special value of the capillary and ultrasound method re main doubtful at this time for early detection of the effects of exposure to vinyl chloride. 193 CMA 002220 *#*-r0'5c............" Vo Soss v Con: L1 . c --* A ^ sial - ^* C oM'+ Type Abstract in Space Beiow .URINARY GLYCOSAMINGGLYCAN PATTERS IN HUMAN HEPATIC ANCIO-" SARCOMA, HEPATOMA, AND IN WORKERS AT RISK FOR ANGIOSARCOMA. K. L. Curran, C. E. Kupchella, J. Sandoz, and C. H. .Taiaourro. Cancer Cancer and Division of Digestive Diseases and Nutrition, University of Louisville, School of Medicine '.ouisville, Kentucky. A previous study reported an abberation in glycosaaino- jglycans (GAGs) eluted from anion-exchange columns with 1.25 ;and 1.5 M NaCl in the urine of patients with hepatic angio sarcoma. A controlled pilot study examined urinary GAG ^patterns in workers at risk for angiosarcoma. Six indi viduals with a history of high vinyl-chloride exposure and 'documented liver disease were paired with individuals with [a high exposure index to vinyl-chlorice but no clinical fiver disease. Similarly six persons with low exposure :but abnormal liver function were paired with low exposure/ |normal liver function individuals. A 24-hour urine w'as .'collected from each individual and the GAGs analyzed by ' janion-exchange chromatography. Individuals with clinically 'active liver disease at the time of this study were found ito have urinary GAG excretion patterns which were similar .'to those described in angiosarcoma. No significant dif ferences were found between high vs. low vinyl-chloride exposure or between those with inactive liver disease and those with normal liver function. GAG excretion was also studied in an additional hepatic angiosarcoma and human hepatoma and confirm the reported urinary changes. These findings support the concept that urinary GAGs are in creased only in active hepatic disease and may be useful in evaluating the'degree of activity at the various stages of liver disease in humans. ' .r;Troc N'/fv : ; I CMA 002221 hftt-AcD)i\!<uO SIXTH ANNUAL MEETING SOUTHEASTERN KIAVAH ISLAND SOUTH CAROLINA NOVEMBER 15-17,1978 CANCER RESEARCH TISSUE AND URINARY GLYCO SAMINCC-LYCAN'S IN ^ TRANSPLANTA3IZ HEPATOMAS CF DIFFERING GROWTH RATES, Kupchsila, C.E., Curran, K.L., Drake, E., Kennedy, J., and Tamburro, C.H. Cancer Center and Department of Medicine, University of Louisville, Louisville, KY. 40232. The purpose of this investigation was to evaluate the glycosaminoglycans (GAGs) in dif ferent behavioral/histological types of intermusculzrly transplanted hepatomas and in the livers and urine of animals bearing these tumors. Fast (7777), medium (5123tc), and slow (961SA) growing Morris Hepatomas were studied. Urinary and tissue GA.Gs were extracted as cetylpryiainiun complexes and measured as uronic acid. Tissue GAGs were also evaluated histochesically. Tissue from fast,`medium, and slow growing tumors exhibited 4 to 6 fold greater GAG levels than nopaal liver in the hyaluronic acid (90 A 10, 91 + 12, and 111 + 9 vs. 25 + 3 /ig uronic acid/g dry liver, respectively) and chcndrcitin sulfate/ heparan sulfate (261 + -9, 217 + 51, and 2CS + 22 vs. 47 + 7 >ug uronic acid/g, respectively) frac tions; the heparin fractions did not differ significantly. The livers cf tumor-bearing animals exhibited slightly greater hyaluronic acid levels than normal livers. Increased urinary GAG excretion was evident after two weeks in animals bearing fast-growing tumors. These data together with those of our previously reported studies of GAG excretion in human hepatic angiosarcoma (Curran, X.L., at ai., Cancer 40(6): 3C50-3053) suggest chat urinary GAG analyses may be useful in the detection and diagnosis of hepatic cancer. Uth >.a': 03 SOUTHEASTERN CANCER RESEARCH ASSOCIATION SIX TH ANNUAL MEETING NOVEMBER 15-17,1978 or GLYCOSAMIN'CGLTCAN CHANGES ASSOCIATED WITH HEPATIC TUMORS: THE CONTRIBUTIONS OP REGENERATION' and NECROSIS. Kupchella, Charles E., Secskas, Elaine, Kennedy, Jeffery, and Espinosa, Enrique, Cancer Cancer and Department of Pathology, University of Louisville, School of Medicine, Louisville, KY. 40232. Although glycosanineglycans (GAGs) have been shewn to be elevated in nany types of animals and human tumors including hepatic tuners, the causa and significance of these changes in neoplasia are still open questions. Regen eration and necrosis are operative in hepatic cancer and the purpose of this investigation was to evaluate the GAG changes associated with hepatic regeneration and hepatic necrosis. Regeneration was induced in male Sprague Davley rats by partial hepatectony and hepatic GAGs were evaluated at i, 3 and 12 days post operatively. Necrosis was induced by: a) ligating the median lobe, b) by resecting and placing nedian lobes in the peritoneal cavity and c) by resecting nedian lobes and incubating them _in vitro in sterile saline. Analyses ware carried out after 5 days of treatment. While regenerating livers exhibited GAG levels that were not statistically different from sham operacad controls or nan-operated controls, in vivo necrosis was accompanied by 3-4 fold increases in tissue GAGs. These data suggest that necrosis is a substantial contri butor to the elevated GAG levels found in sene tumors. ! j KIATTAH ISLAND SOUTH CAROLINA CMA 002223 T.nlw>mtory Investigation Vul. AO, Nu,1!I7* " ' 122l12ct'tiai ANNUAL MEETING ABSTRACTS Subject to Boss y. Ccr-.c-c -V/" , in llu-se liinmrH (-(insisted primarily of fusi- assisted morphbldffrifa Ccchn+queUbr evaluating'hepirtfc I'll in ni-vu-i Ctrl I-i wit.h iiidiniiierit. ciisiMii|iliiIic cytoplasm emlieddcd in n dense connective tissue struma. Junc tional activity, theque formation, and pigment were un common features. As a result desmoplastic nevi may be collagen iiiipcarsflailla.'iewuljlyawiniU.qble-tp c.linrcnl stud ies, This .study of normal liver implies that considerable care must be taken before the clinical diagnosis of sig nificant fibrosis can be made. confused with reticulohistiocytoma, atypical fibroxan thoma, dermatofibroma, and even necrobiotic granu Uptake and Nuclear Transfer of 17-Fluorcscein-La- lomas. Well defined intranuclear invaginations of cyto beled Estradiol in-Normal and Abnormal Human plasm occurred in 12 cases, and this was an important Endometrium feature differentiating desmoplastic nevi from these fi- G. H. Barrows, J. D. Riehm, and M. Riley. Depart brohistiocytic lesions. Desmoplastic malignant mela ment of Pathology, University of Louisville School of noma must also be considered in the microscopic differ-' Medicine, P.O. Box 35260, Louisville, Kentucky 40232, ential diagnosis, but distinguishing features of desmo We have developed a 17-fluorescein-labeled estradiol plastic melanoma include indentification of preexisting which is specifically transferred to nucleii of target tissue ' malignant lentigo or acral lentiginous melanoma, atypical (breast, endometrium) and inhibited by synthetic and mitotic figures, and foci of necrosis. Clinically most des natural estrogens. By in vitro incubation, patterns of moplastic nevi were dome-shaped nodules. The age range temperature-dependent nuclear uptake may be observed. was from 7 to 53 years (mean 28); duration from 2 months Marked cyclic variation is observed in normal human to 15 years; male to female ratio, 6/8; and four patients endometrium. With proliferative and early secretory en gave a positive history of preceding trauma. Five lesions dometrium, temperature-dependent nuclear labeling is were located on the thigh or leg; and the rest, with the present and appears to peak at late proliferative stages. exception of sparing the palms and soles, were widely Mid and late secretory endometrium, while having spe distributed. Desmoplastic nevi were compared to the cific cytoplasmic labeling, do not exhibit temperature ordinary variants of mixed spindle cell and epithelioid dependent nuclear labeling. This suggests that estradiol cell nevi in an attempt to define etiologic factors explain transport during mid and late secretory phases is in ing a desmoplastic reaction. Initially, it was thought that hibited or minimal. Abnormal endometrial samples ex patient age or preceding trauma might be important. No hibit a variety of patterns with the fluorescein-labeled satisfactory explanation could be found since, with the estradiol. Endometrium with cystic hyperplasia reveals exception of a predilection for occurrence on the lower very little nuclear glandular staining, whereas in adeno extremities, the other clinical variables were not statis matous hyperplasia strong nuclear uptake of the fluores- tically different. cein-labeled compound is observed. Some cases of well differentiated endometrial carcinoma have strong nu Computer-Assisted Morphologic Quantitation of clear uptake of the fluorescein-labeled estradiol whereas Collagen in Human Liver Biopsies poorly differentiated endometrial carcinoma has little or G. H. Barrows, M. J. Joyce, G. R. Schrodt, R. no nuclear uptake. The method provides a good means Greenberc, and C. H. Tamburro. Departments of of determining the endocrinologic behavior of glandular Pathology, Epidemiology and Biostatistics, and Medi versus stomal elements of endometrium and suggests cine, University of Louisville, Louisville, Kentucky that in abnormal endometrium either variation in steroid 40202. uptake mechanisms or hormonal milieu may be impor Because unfixed liver is rarely available for analytical tant in the development of the lesion. studies, appraisal of the extent of tollagen must be made from appropriately stained sections of liver biopsy ma Aortic Infarction following Dissecting Aortic terial leading to judgemental as well as sampling error. Aneurysm We have developed a computer-assisted morphologic Sanford H. Barsky. Departments of Pathology, Beth technique to evaluate the distribution of collagen in liver Israel Hospital and Harvard Medical School, Boston, biopsy material. We examined multiple biopsy samples Massachusetts 02215. j from patients dying suddenly with no history of liver Aortic infarction was observed in 21 of 34 cases of disease to establish normal values. A Hewlett-Packard dissecting aortic aneurysm. This lesion occurred as a 9864-A digitizer and 9815-A microcomputer were used to central zone of necrosis, sparing media adjacent to the quantitate areas of trichrome stainable collagen within true and false lumina. This infarct followed rather than the biopsy samples. Random area selection and statistical preceded dissection, took approximately 48 hours after analysis proved to be important considerations in deter dissection to develop, and did not organize with time. mining the experimental model and these factors were Lesions months to years in duration were similar histo readily incorporated into the calculator program. Stain- logically to those only several days old, and although able collagen estimates in normal liver varied from 0 to there was some tendency to calcify in the older lesions, 6.1 per cent (mean = 1.25 per cent) in patients with no inflammation was consistently absent. The lesion oc evidence of liver disease and showed considerable varia curred exclusively in the thoracic aorta. Histologically, tion even in different biopsies from the same patient. As aortic infarction was' completely distinct from medial anticipated, subcapsular biopsies had more collagen than cystic necrosis (MCN). Aortic infarction was character deep biopsy; however, the specimens from several deep ized by preserved elastic laminae with no increase in biopsies often had significant variation in collagen con mucopolysaccharides, in contrast to MCN which was tent. Differences in central, micf-zonal, and portal colla characterized by elastica disruption and increased mu gen could be obtained using this method. The computer- copolysaccharides. When both MCN and aortic infarc- CMA 002224 ABSTRACTS OF THE ANNUAL MEETING-1978 125 Subject to Proiective Order in Ross ?. Ccnooo, Inc., Eo, 90-4331' 14tii Judicial District Court Calcasieu Rarlsii. Louisiana, Abstracts of the Annual Meeting of the SeSs/ ton* ri3Scs,oBSo3(Dj^ 1978 H 128 Chloroaccealdthyde-induced Damage to Bacillus ubcllis- A.D. LAUMBACH, U.N* STKEIPS* and J.L. VON'C. Bureau Foods, TDA and U* Louisville, Louisville, KY, Chloroaceealdehyde (CAA), a proposed oecabollte of vinyl chloride netaboliam, has been shown to be mutagenic In ehe Saleanella assay system, and to specifically Inhibit the growth of a recooblaatlon-defIcient ctutanc of Bacillus subtills (tllnore, et al BBA, 442: 405, 1976). Ue have examined further the biological activity of this compound. First, CAA caused a significant Increase in in duced outations to streptomycin resistance. Using B, sub tills 163, the relative mutation frequency (treated 5kM CAA/eontrol) was 13.6, while using an her mutant of _B. sub- tllis the frequency was*14.2. Secondly, a survey of the available repair-deficient mutants of subtilis revealed, that the only strains sensitive to CAA were of the roc*" genotype. Furthernore, the several rec" mutants, show three types of response to CAA. Strong killing by CAA (in hibition of growth of 10 ran or more) was exhibited on the rerA, rc-c5, roc-4(C5Y1616) t and rcrD27(C5Yl627) strains- Intcrt-ediatc killing (3csn-8"*.'r) was shown with strains bearing tha rccE recF. and roc-"l3(BD246) loci. All other strains examined (rccC. rccll, nt_c-4l, uvr. her, olA, as well as several repair-proficient cultures) grew in the presence of 0.1! CAA. Tht$e studies arc part of an ..ngoing progra m sponsored by the KCA to dctcrnlnc the molecular basts for vinyl- chloridc induced carcinogenesis. H 131 Influence of Mutagens on the Nucleoid Structure of the Chromosome in Escherichia coll, K.KLEPPE*and I. LOSSlUS,Oepartnent of Bloche/n 151ry, University of Bergen, Bergen, Norway. The chromosome in a bacterium exists in a tightly supereoiled structure termed the nucle oid. We have investigated the effect of two mutagenic compounds, methyl methane sulfonate and mitomycin C, on the nucleoid structure of the chromosome in Escherichia coll K -12. Treat ment of cell envelope-free nucleoids with the mutagens caused no changes in the structure as measured by their sedimentation coefficients. The nucleoids from mutagen treated cells,hoveveg were found to be partially unfolded, having sedimentation coefficients of approximately 800% In some mutants, defective in repair or recombi nation, even greater changes in the nucleoid structure were seen* The altered sedimentation behaviour was shown to be'due to relaxation of supercoils in the chromosome caused by nicks in the DN'A strands. During the repair period the intact nucleoid structure was partially restore!* The mutagens also caused considerable changes In the structure of the cell envelope-associated nucleoid. CMA 002225 K.6 Jntn-.sl; to i'cnlcl 1 i in tn *; iv/locot-ras a-a'L'it: rrwii!c* of Cvtcv liv'c *i*.-i-f.no 5*r jst'ir*.-, 'J.VirvFlEX. .G rsi V':, >*. ST"*'J'ir\CM . Pharr lkologis^h^s Tn.tt i tun, Univ^rsitnt Vjin, n -5-3 K*ilr, 4t. Kc.Jvrst PupubMi: of ^ifasy la grir-n'j^tivi* bacteria the cnrvme-i-, ic-pvndunt tvp*s of p-rnicll- Ilr rvaistanc-* l*1 aAnj.-*d to fev l-aur^i or. a &ermr*M li ty harrier ii the outtr r*onhrano. in i 11 v#s, hc-ever, a similar fraction of thf; cell wall could not he vjiabllshe:! until row. From earlier otcUcs vc receive o-p evidence foe a possible ro.r of the cvtovlasnlc r^r-LrariO In tljla resistance p-eha.nism. tJ erefere studied the binding of ^C-bcntyl pan IcLI 1 if. (l^c-TP) ta the *'r,-brfine of resistant nr* 1 sensitive stonhyloeocci. First whole edli vote incubate-l In buffer with increasing con centrations of Hc-CF at 3?C for 30 r-ln and then cytoplasmic ne-bra.naa were prepared following the ct>od of r.ft.rxfeACX (Y-ath.iniyrol.xxn, 95-i2o (I9?l)). In membranes of sensitive taphyloroecl the binding increases sharply even at low tentell1 in-concentration* end levels out above o.3 nmoles l*C-SP/nl, In contrast, the binding to the resistant cells is reduced to a high degree, tt reaches only 1-23 at o.J nmoles/nl. If the bln* ding constants are calculated it can be seen that tho number of binding sites is the sane both in the resistant and in the sensi tive ner.branes (2o rviolcs 6P/g membrane protein). however with the resistant membranes the affinity to penicillin Is decreased ty a factor of 25o to 7oo. To exclude a permeability barrier In the cell wall the binding of penicillin to Isolated membrane* was examined. The incubation of the nenbraneS with Hc-dp vs carried out under the same condi tions as already mentioned. In this case. too. the binding to the resistant membranes vas greatly reduced. This Is also due to s high loss of affinity by approx, the sane factor as above, rron these results a permeability barrier in the cell wall can be excluded, Th Cause for this type of resistance must thus be located in the membrane itself. Therefore the membrane lipids were analysed. Differences were observed in the phospholipid fraction. The resistant membranes contain 4o-So3 more cacdlollaln and more phosphatidyl- glycerol than tUa sensitive ones. Additionally a cardlolipln derivative is found with the hydroxyl group of the central gly cerol molecule acylatcd. The other lipids arc unchanged in Quan tity and in fatty acid composition. From this it can be concluded that the intrinsic resistance of staphylococci is based on an altered membrane structure caused by Increased llpophlllty. - * e^c K.4 ChloreACCtaldehyde-induccd Dnruge to Bacillus mbtllli. A. D. JJIUXBAC!. r, s, iuitfs*, end J. l. (unu. iurua reads, FDA, and Cniwraicy of Uiv,'llle, Schdul of Medicine, Kuilth fcitlXfl Ceatef, Louisville, Ce.i:u.'ky 402)2 (USA), ChLoruaartaldehyde (CAA), a proposed wtabolit* of vinyl chloride eetsbelfis, has been shown to be outagealc in Che Salnanella assay svsctQ, sad co specifically inhibit the |Wth of a ncuiAiiuitIwi-dclItlnt rutaot Sf Bacillus SuSttlls (Elloere, el., BBA, 4425 40S, 1976). Ve have examined further Che biological activity of this Compound, First, CAA caused a significant increase In inJoced Rotations to Streptolycla mlitiMi. Using B. subtil 1* 163, tin relative eutacioo frequency (Created $ oX CAA/eentrol) was 13.6, while usinf an her mutant of B. aubtHls tho frequency vas 1*.2. Secondly, a Survey of the available repair-deficient Miiaoc* of 6. subtilis revealed, thae tho only attains sensitive to CAA ver* of tho rsc~ genotype. Fufthsrnors, the several ft* nutSAC*. shew throe typea of respond* to CAA. Strong kilting by CAA (inhibition of growth of 10 Cm Or care) was exhibited On the recA. rCB, rec-4 (CSU616). and fee027 (C5Y1677) Strains. Interaodlat* killing (3=^6sm) Vas shown with strains bearing the re^c* r*cF. aoJ rye-13 (60246) loci, Atl Other strains examined (recC. tseH, art-41, uvr, her, ooiA. as wall as several rspalr-proficiaac cultures) grew in the presence of O.lh CAA, The effects of CAA on'the biological activity of DXA oolocules in vitro are be log studied. These atuitvS are pirt of at ongoing proAfa? sponsored by the Manufacturing Ch?sis* Association to docereine tire olitutir basis for vtnyl-chlorijo ieJuced carciniiraesis. ^W.:-:-.:--.-co??7II)3tTTXAI| Subject to Protective CrJ.cr tn 1?0S8 v. Conoco, Ir.c., io- SO-4337 14th Judicial Uls'Crict Court Calcasieu Parish*.Louisiana.----- CMA 00222* - J-J5 J .* t i - i t j j i\ Iti I \ I ,1 r m -?m y&&. "li ..a *--? /vA-j., an) .. ,'.Vf^ -'_V'^tfS`J ^'.m4t >i "*'i MARCH 1, 1979 VOLUME 38, NUMBER 3 'PARI of 1 lirt-r Piirt.s a fl tr o CONFIDENTIAL Subject to Protective Order in Ross v. Conoco, Inc., Do. 90-4337 A4tb Judicial District Court iVv- Calcasieu Parish^ Loul .glana -------- 63rd Annual Meeting Dallas, Texas April 1-10, 1979 Society Grouping II--APS, AAP, and AAI (April 6-10) \ ~??i SATURDAY. PM TUMOR BIOLOGY IV; ROLE OF ANTIGENS (4449-4453) 1069 4449 Pathology T**0 LIVER ANTIGEN'S UNDETECTABLE IN A FAST CROWING LINE OF TRANSPLANTED HEPATOMA (MORRIS HEPATOMA 7777), Enrique Fsoinosa, Saa Canle*. Charles Kupchella* end Sue Chla*. Dept. Pathology and Cancer Center, Untv. of Louisville School of Medicine, Louisville, 1Y 40232 During investigation* of antigenic changes in liver tumors v exaMned fast (7777), medium (5123tc) and alov (9618a) growing Morris hepatomas for presence of liver antigens. Analyses of normal liver and hepatoma tissue saline extracts were performed by immunodiffusion methods using rabbit anti serum to rat liver extract absorbed with pooled normal rat plasma- Hepatoma 5l23tc and 9613A gave lmmunoelectrophoretlc patterns similar to normal liver. In contrast, hepatoma 7777 shoved 2 arcs of precipitation missing. This was confirmed by Che finding tint after absorption of antiserum with hepa toma 7777 two lines of precipitation given by normal liver extract persisted whereas absorption with extract of normal liver, hepatomas 5l23te or 9618A abolished all lines of pre cipitation. The liver antigens found to be absent in hepa toma 7777 were characterized as proteins relatively unstable to heating and acid pH and unrelated to liver-specific F antigen. In Sfphadex G--200 and Bio-Gel A-5ra columns these antigens eluted as proteins of approximately 51,000 and 240,030 dalcons respectively. The fact that 7777 is the fastest growing and least differentiated of the tunors studied suggests a possible functional relationship between the absent antigen and these properties. (Supported in part by the Manufacturing Chemists Association) 44$0 UVWUNOLOGY LIVER-SPECIFIC F ANTIGEN IN TRANSPLANTABLE HEPATOMAS HAVING DIFFERENT GROWTH RATES. Enrique Espinosa, Sue Chla*. Safl Caple* and Charles Kunchella*. Dept. Pathology and Cancer Center, Untv. of Louisville School of Medicine, Louisville, KY 40232 In the course of studies of antigenic changes associated with liver neoplasia we measured the relative concentration of liver-specific F antigen in fast (7777), slow (9516A) and medium (S123te) growing Morris hepatomas In comparison with its concentration 1q normal adult rat liver. F antigen was solubilized from each tissue by aqueous homogenization. The supernatant containing the antigen Vas then tested by a double diffusion dilution assay capable of detecting a mini mum of 1-22 the concentration of antigen present In normal rat liver. The F antigen antiserum used was prepared ac cording to Fravt and Llftdenmann by liraunlzlftg CSA mice with BALB/c mouse liver extract. F antigen was undetectable in the fast growing hepatoma and ranged frost less than 2Z to 10Z of the normal liver concentration In the slow growing one. The medium growing hepatoma showed similar or higher concentration than normal rat liver. Thus, the level of f antigen in these tumors does not appear to correlate with their rates of growth. (Supported in part by the Manu facturing Chemists Association) CMA 002227 CONFIDENTIAL 1 CONFIDENTIAL Subject to Protective Order in Boss v. Conoco, Icc , lie. 00-4337 14th Judicial Oiotj-ict Cnurt Subject to "rotoctivg Order In Pos3 v. _N:!fo, 90-4037 14th Cwrfe ^ Louisians ,0 .... Calcasieu Parish,, Louisiana, E KcibiocJuction Copy for Pt-'CGPlAf/i A.tJD ABC! P.AC iS of fhs 30TH ANNUAL MEETING OPTHETISSUB CULTURE ASSOCIATION', INC. Properties of JA Week Maintenance Cultures of'PLC/PRF/5 Cells. P.B. JOHNSTON'", E.. Espinosa, S. Chia and S. Caple. Univ. of: .Louisville, Louisville, Ky. 40232;.__^ This established cell line was originally described as being short lived with in a given passage, with considerable cell loss by day 6 to 8. However,after 80 passages, we found that culturing in glass roller bottles at 0.25 PPM. allowed maintenance for at least 14. weeks with cells exhibiting excellent morphology'without sloughing. After treating monolayers with Hoechst 33258 DNA stain, intense cytoplasmc fluorescence was seen in a proportion of the cells, possibly a function- of the subviral hepatitis B genome. HBsAg of culture supernates was monitored and solid phase KIA assay revealed.31 to 47 ratio-units/0.2 ml after 2 to 14 weeks in medium with 10% fetal calf sei'um. ' The 14 week old cultures were extremely well adapted to maintenance in serum free medium for 14 additional days when supplementing with 100 ng/ml of insulin, in contrast to 1 week cultures which survived well for only 5 days without serum.- The HBsAg-titers of supernates from these 1 week or 14 week cultures, were very similar to the previous harvests in 10% scrum. In addition, both young and 14 week cultures were similar in their rate of acid production; and in that both elaborated serum albumin, fibrinogen, transferrir and alpha-2 macroglobulin, the proteins tested to date. CMA 002228