Document KzKGkk8R2qOJOXaBZpb4e23Lr

ABSTRACT MUST BE RECEIVED AT SOCIETY OFFICE BY THURSDAY, DECEMBER 1. 1977 DO NOT FOLD THIS FORM 1 Indicate below the numbers and titles of sessions in which your abstract might be programed (see Topic Category List); CAREFUL SELECTION IS CRITICALLY IMPORTANT. Unified 1978 FASEB Moil to: Dr. Kenneth M. Endicott, Executive Officer American Association of Pathologists 1 st #...12.8................... 2nd #..Q.2.6................... 3rd #..127................. Tide . ImffiURSES.Chology................... Title..X.Vffio.r.Iniinunglogy................ Title .JSS&uaobiologX......................... Abstract Form 9650 Rockville Pike Bethesda, Maryland 20014 (See paragraph below for the number of photocopies to be submitted with abstracts.) -'L`C ` '"-V . in ` 4637 T _"" c='nrt --::sia.na PRESENTATION PREFERENCE Preferred choice (CHECK ONE ONLY) 30C Poster presentation Slide presentation Indifferent 16 mm. films (silent or optical sound) are permitted if essential to 10-minute slide session presentation. MOVIE YES.......... NO.......... Submit justification by letter to Soci ety Office, with abstract. IMPORTANT: Sec sample abstracts, typing and mail ing instructions on reverse side; use enclosed Check List for preparation of abstract. PATHOLOGY . LYMPHOCYTE TRANSFORMATION TESTS IN VINYL CHLORIDE (VC) WORKERS. H. Philip Fortwengler*, Michael E. Dever*, Carlo H. Tamburro*', and Enrique Espinosa, Univ. of Louisville School of Medicine, Louisville, KY 40201 Previous reports have shown circulating immune complexes in VC workers and a tumor-associated antigen in VC-related liver angiosarcoma. This suggests immune stimulation by a tissue or plasma antigen induced by or conjugated with VC or a metabo lite. In this work, the in vitro lymphocyte reactivity for such antigens was tested in 79 VC workers including 25 with liver abnormalities, and 20 normal individuals having no ex posure to VC. The liver angiosarcoma antigen preparation in cluded the tumor-associated antigen and other tissue antigens as shown by immunodiffusion. Stimulation indices (SI) were calculated from cellular incorporation of tritiated thymidine. Mean SI in the normal individuals for antigens of angiosarcoma and normal liver tissues were 4.7 (SE1.7) and 3.8 (-0.9) and in VC workers, 1.8 (0.2) and 2.5 (0.3) respectively. Mean SI for PHA and Con A in the normal individuals were 235 (35) and 209 (30) and in VC workers 201 (23) and 180 (19) re spectively. Thus, these results suggest that VC 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. (Supported in part by the Manufacturing Chemists Association) The original typed copy of this abstract )rm (for reproduction by photo-offset in EDERATION PROCEEDINGS) must be ibmitted together with 9 photocopies, one :t of author index cards, three abstract lentification cards, and return postal. All laterial must reach the Society office NO ATER THAN THURSDAY, DECEMBER , 1977. All compounds that are designated by code or initial letters must be identified adequately in the abstract. e.g., MJ-1999: 4-d-isopropyUmino-l-hydroxyethyl) methanesulfonanilide hydrochloride. Each Abstract Form submitted MUST BE SIGNED by a member of the AMERICAN ASSOCIATION OF PATHOLOGISTS.* [AILING ADDRESS OF FIRST AUTHOR (Please Print or Type) H. Philip Fortwengler........................... Univ. of Louisville Sch. Med Enrique Espinosa. M.D. (Member's Neme: P(M*e flrint or Type) (Members Signature) ...^.5^.sylle,...Ke.nJt.VSJS3;........ Zip ..AQ2Q1........ Member's telephone no.: Area Code .....$.Q?.... # ..56.8..5525 Telephone no.: Area Code ...502 # 588-5.25.1 *See the enclosed unified rules for eligibility of papers. CMA 003885 ABSTRACT MUST BE RECEIVED AT SOCIETY OFFICE BY TUESDAY, DECEMBER 19 Please consider this jbstract for inclusion in ihe tentatively listed minisymposium. M_____: . ... ____________________ __________ Indicate below the numbers and titles of sessions in which your abstract m1/ijh'bMe programed (set ToOppitc Ccaatteeggory List); Liver Pathophysiology 2# . 3/ 1979 FASEB Abstract Form DO NOT FOLD THIS FORM Mail to: Dr. Kenneth M. Endicott, Executive ' American Association of Pathologists 9650 Rockville Pike Bethesda, Maryland 20014 CONFIDENTIAL Subject to rroteco' r r' rlT. Ross v. Conor:',_Tv- Lo. SO-1837 14th Jut :i:i 7:st riot Court Celcj^sieu Pv rh, I oui .si PATHQLOG" FACTOR VIII CONTENT AS EVIDENCE FOR ENDOTHELIAL ORIGIN OF VINYL CHLORIDE ASSOCIATED LIVER ANGIOSARCOMA (VCA). H. Philip Fortwengler*, Douglas Jones*, Carlo H. Tamburro* and Enrique Espinosa (SPON: G. Randolph Schrodt). Univ. of Louisville School of Medicine, Louisville, KY. 40232 To ascertain the endothelial cell origin of VCA we have looked for Factor VIII in the tumor since this factor ap pears to be specific for such cells (Hoyer et al., 1973). Frozen sections of three VCA and one idiopathic case were examined for presence of Factor VIII by indirect immuno fluorescence. Sections of angiosarcoma demonstrated a strikingly increased specific fluorescence which lined enlarged sinusoids. This intense fluorescence was easily seen on low power(lOOx) as an irregular or splotchy pat tern. Occasional striations of linear fluorescence which did not follow hepatic cords were also present. A similat pattern of staining was also given by the idiopathic ana 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 tal angiosarcoma tissue resulted in complete inhibition of immunofluorescence. These findings demonstrate that VCA and idiopathic angiosarcoma include proliferating cells containing Factor VIII and therefore strongly suppoi an endothelial cell origin of this tumor. (Supported in part by the Manufacturing Chemists Association.) All compounds that are designated by code or initial letters must be identified adequatet in the abstract, e.g., MJ-I999: 4-(2-isopropyUmmo-l-hydroxyethyl) methanesulfonar lide hydrochloride. MAILING ADDRESS OF FIRST AUTHOR (Please Prim or Type. Provide full name rather than initals.) .. .Philip, .For.tv.engler* ,M.S......................... 538 MDR Building 511 South Floyd Street............................... University of Lou'isTilie Med. School Louisville, KY Zip !*.9.2.??..... Telephone No.: Area Code. 5.0?#.. .58.8r5.251.......... Each Abstract Form submitted MUST BE SIGNED by a membe of the AMERICAN ASSOCIATION OF PATHOLOGISTS. G, Randolph Schrodt ~0~A (Memo's Narrje Please Prim o* Typo/Proitae Ml J- MtouU/'/'J; tJc/Z-2/^ yfMtmbei's S*oniui*\ Member's telephone no.: Area Code........502............ #----- 588-^5341 - Signing member, are you willing to chair a session? ( ) yes, category #---------- ( ) no If yes, are you also willing to give an overview or introductory lecture?1 ( ) yes ( ) no CMA 003886 ABSTRACT must BE RECEIVED AT SOCIETY OFFICE BY TUESDAY, DECEMBER 19 p'.cue consider this jbsuact fcr inclusion in ihe -eniatively listed mmiiymposium. M___ : -------------------------------- ------------------------------------------ Indicate below the numbers and titles of sessions m which your abslrao mith be protramed (see Topic Caiegory List);, , , if Till- Liver Rathcohvsi ________ Jf -riile Carcinogenesis ; ucemica-t., " > if) 1979 FASEB Abs+tr"a3cf'e+ adii FO \'0T FOLD T'-!'S FORM Mail to: Dr. Kenneth M. EndiCott, Executive Off American Association of Pathologists 9650 Rockville Pike Ir.h-yds, L Urtti 20014 ------------------------- s?.~':rsv ^ S\:bif-ct co ? . s', s.-'v. V 'i'*v .'n C'tjtc v. ". ;r o. r-' - , ; ?"-7 C 4. I C S S1 v: a s ii i11E( .L 0` 'J D J .. C. 'i E. PATHOEOC FACTOR VIII CONTENT AS EVIDENCE FOR ENDOTHELIAL ORIGIN OF VINYL CHLORIDE ASSOCIATED LIVER ANGIOSARCOMA (VCA). H. Philip Fortwengler*, Douglas Jones*, Carlo H. Tamburro* and Enrique Espinosa (SPON: G. Randolph Schrodt), Univ. of Louisville School of Medicine, Louisville, KY. 40232 To ascertain the endothelial cell origin of VCA we hav looked for Factor VIX1 in the tumor since this factor ap pears to be specific for such cells (Hoyer et al., 1973). Frozen sections of three VCA and one idiopathic case were examined for presence of Factor VIII by indirect immuno fluorescence. Sections of angiosarcoma demonstrated a strikingly increased specific fluorescence which lined enlarged sinusoids. This intense fluorescence was easily seen on low power(lOOx) as an irregular or splotchy pat tern. Occasional striations of linear fluorescence which did not follow hepatic cords were also present. A simila pattern of staining was also given by the idiopathic angi 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 experimen tal angiosarcoma tissue resulted in complete inhibition of immunofluorescence. These findings demonstrate that VCA and idiopathic angiosarcoma include proliferating cells containing Factor VIII and therefore strongly suppe an endothelial cell origin of this tumor. (Supported in part by the Manufacturing Chemists Association.) All compounds that art designated by code or initial letters must be identified adequatt in the abstract, e.g., MJ-1999: 4-(2-isopropylam ino-J -hydroxyethyl) methanesulfona lide hydrochloride, ,- MAILING ADDRESS OF FIRST AUTHOR (Please Prim or Type. Provide full name rather than initals.) .. .Philip. .For.tv.engier.x. M.S-. 538 MDR Building 511 South Floyd Street University of Louisville Med. School Louisville, KY 40202 Telephone No.; Area Code. 5.02#.. .58.8r5.251.......... Each Abstract Form submitted MUST BE SIGNED by a memb of the AMERICAN ASSOCIATION OF PATHOLOGISTS. G. Randolph Schrodt ________________________________ Wa (MefKBwt PImm Print o> TytsrfProvloe lull Member's telephone no.: Area Code........502............#----- 588-5341 Signing member, arc you willing to chair a session? ( ) yes, category #--------- ( ) no If yes. are you also willing to give an overview or introductory lecture? ( ) yes ( ) no CMA 003887 r .* i CLINICAL RESEARCH Abstract Reproduction Form _coTn?r 3 TYPE name, address, and telephone number of author who should receive correspondence. Telephone 502/532-22X1 ex. 366___ Subj 9 Poss v. ""ot-ve C'r:?or in 14th cm 1' o u * j : 502/895-2955jgh- Leu home S0'^37 V- ln-IL-, ZT` 1 THIS FORM MUST BE SIGNED BY A MEMBER Name ------------------------------------------------------------------------------------------------------------------------------- Carlo H. Tamburro, M.D. ... . University of Louisville..School of Medicine Department of Medicine Louisville, Kentucky 40201 FOR OFFICE USE ONLY Date Payment 1510.00) .................... TYPE ABSTRACT MERE/ BE SURE TO STAY UV77/AV BORDER ----- CHECK Preferred Sub-Specialty Classification: ____ Cardiovascular Clinical ___ Epidemiology Clinical ____ Pharmacology ____ Dermatology ____ Endocrinology* ____ Gastroenterology ____Genetics X._ Health Care Research ____Hematology ___ Immunology & ___ Conn. Tissue ____ Infectious Disease ____ Metabolism* ____ Oncology .___ Pulmonary ____. Renal & Electrolyte Traditionally, Endocrinology has included papers dealing wIh th* thyroid, jdmul ond pituitary glands, and gonads, while ab stracts dealing with the parathy roids, calcium and phosphorus metabolism, hones. th> roCalcitonin, diabetes, insulin, glucagon, and growth hormone have Inren considered under Mvtoboltsin. USE OF DYE CLEARANCE IN DETECTION OF HEPATOCELLULAR INJURY AMONG VINYL CHLORIDE WORKERS. P. Fortwengler* and C. H. Tamburro. Department of Medicine, Digestive Diseases & Nutrition Section, University of Louisville, School of Medicine, Louisville, Kentucky. Serious hepatic injury associated with exposure to potentially harmful indus trial chemicals and the need for a sensitive method of early detection has been previously noted among vinyl chloride workers. (Creech, J. L., et al, Gastroent., 67:786, 1974). Forty-three of the 1,183 employees of this poly vinyl chloride production plant with biochemical, radioisotopic, and histologi cal evidence of liver dysfunction were evaluated using indocyanine green (ICG) dye clearance, at both the 0. 5 mg/Kg and 5.0 mg/Kg dose. Percentage dis appearance rates (PDR) were determined by both blood and ear densitometry with automatic computer calculation of PDR. Liver histology was normal in 9, minor changes in 10, fat and minimal fibrosis in 14, portal fibrosis in 4, fibrosis and lobular distortion in 2, and angiosarcoma in 4. Individual bio chemical tests correctly indicated hepatic status in 80-83% of the cases with 8% false positives and 10-15% false negatives. Radioisotopic studies alone correctly indicated liver status in only 55%* with,45% either false positive or false negative. The 0.5 mg/Kg dose of ICG correctly indicated liver status in 80% with no false positives, but failed to indicate liver dysfunction in 20%. However, at the 5.0 mg/Kg dose, all cases of liver dysfunction were detected. Ear densitometry values were essentially the same as blood values. Ear densitometry with electronic calculator allows a dye clearance determination over 10 minutes without drawing blood. This data demonstrates an effective means for early detection of industrial chemical hepatotoxicity. PLEASE CHECK ABSTRACT CAREEULLY EOR APPEARANCE BEFORE MAILING IMPORTANT The instructions accompanying this form must be followed COMPLETELY for all abstracts which are to appear in CLINICAL RESEARCH. Ab stracts which do not conform either will be re typed by the publisher at a cost of S 15.00 to the author, or rejected. Revised June 1974 "The sponsoring member affirms that the material herein will not have been previously published or presented at any national meeting, that any animal stud ies conform with the "Guiding Principles in the Care and Use ot Animals' ot the American Physiological Society and that any human experimentation has been conducted according to a protocol approved by the institutional commitiee on ethics of human investigation or-if no such committee exists--that it conforms with the principles of the Declaration of Helsinki of the World Medical Associa tion (CLINICAL RESEARCH 14:193, 1966). 6 /MEMBER'S SIGNATURE.. 11.(,///. ]) v t j.-/. lJAl U .7.77. CMA 003888 ^BE RECEIVED AT iGCJEJ Y UPMUt Of i mum^uat, ctuMUAMT iz, ta/o : ________ A\li4 .ambcn mnd title* of sessions in which your .btct ^.mmrd ( Topic Category Lin); ^ wfochemlcal Pharmacology Tif|r F.nzvmes - General __ rule ---.------------------------------------------- lioJul Technique! DO NOT FOLD THIS FORM 197S AS3C Abstract Form rfcliul typed copy of this abstract form must be submitted together photocopies- N^ v N\A<> \<r \ \> N ' \ \\ RTANT: intple abstracts, typing and mailistructionj on reverse side; use ;ed Check List for preparation of ct. e original typed copy of this ct form (for reproduction by -offset in FEDERATION PRO>INGS) must be submitted to with 7 photocopies. stracts submitted for the "Edulal Techniques" poster session do revent a member from submitting tonsoring an abstract for the r sessions. ard projectors for 2" x 2" and t 4" slides will be available'in all sessions. Other audio-visual aids e provided at cost if the request ustification accompany this ab. Authors will be billed following eeting. DECREASED GLUC0SE-6-PH0SPHATASE ACTIVITY IN LIVER IN VINYL CHLORIDE EXPOSED RATS. J.T. Du* and C.H. Tamburro* (SPON: M. Fonda) Dig. Dis. & Nutt. Sect., Dept. Med., Cancer Center, Unlv. 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. Khim 19:568, 1973) have been shown. We exposed adult SpragueDawley rats to 10,000-20,000 ppm of vinyl chloride (VC), 4-8 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 micro somal 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.(B.F.Goodrich Grant) Supported by NCI Contract //NOl-CN-55212 All compounds that are designated by code or initiai letters must be identified adequately in the abstract, e.g., N1J-1999:4-(2-isoptopylamino-l-hydroxyethyl) methanejulfonamlide hydrochloride. Each Abstract Form submitted MUST BE SIGNED by a member of the American Society of Biological Chemists. Member's Signature MG ADDRESS OF FIRST AUTHOR f... Du.,. Fh.D........................................................... rersity of Louisville, MDR Bldg., Rm.535 Telephone No.: Area Code Mail to: - 588-5228 S... .Floyd. .Street;,.Ky.-. Zip AQ2P.1.. ane no.: Area Code 502 # 588-5251 CONFIDENTIAL CMA 003889 Subject to Protective Order in Boss v. Conoco, Inc., No. 90-4837 Robert A. Harte, Executive Officer American Society of Biblogical Chemists 9650 Rockville Pike Bethesda, Maryland 20014 14th Judicial District Court Calcasieu Parish, Louisiana ' - ( qs BE-'CIVED at f" " V Ui-MUfc or imukiuat, r;wi,-AMT i^, ia/o /fth* .umixr> *nd miss of -ssioal ,n "hich yQut lbutlI:l ^mm^(wToP.cO:et<=ry Ua>; DO NOT FOLD THIS FORM d - n-'orhemical Pharmacology Ai------------- * as _ (Janar, ' JJ-l Title .Tik Liui Tecfinjq^el. 1Q7C ACnP iu/u n\jJu ^hcrrppr form ..Uw '.) i-iUi 1(ul t jpel copy of :.*iii photocopies. form must be rubmitted *.o;e:b;r N N'\f . <>, \S \o. 4 \% svr s RIANT: anple abstracts, typing and maiF istractions on reverse side; use ,ed Check List for preparation of ct* e original typed copy of this ct form (for reproduction by -offset in FEDERATION PROINGS) must be submitted to with 7 photocopies. streets submitted for the "Edual Techniques" poster session do .-event a member from submitting tonsoring an abstract for the r sessions. ard projectors for 2" x 2" and t 4" slides will be available in all sessions. Other audio-visual aids e provided at cost if the request ustification accompany this ab. Authors will be billed following eeting. DECREASED GLUC0SE-6-PH0SPHATASE ACTIVITY IN LIVER IN VINYL CHLORIDE EXPOSED RATS. J.T. Du* and C.H. Tamburro* (S?ON: M. Fonda) Dig. DIs. & Nutr. Sect., Dept. Med., Cancer Center, Dniv. 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. Xhim J_9:568, 1973) have been shown. We exposed adult SpragueDawley rats to 10,000-20,000 ppm of vinyl chloride (VC), 4-8 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 micro somal fraction, decreased 253 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. (B.F,Goodrich Grant) Supported by NCI Contract /r`NOl-CN-55212 All compounds that arc designated by code or initial letters must be identified adequately bi the abstract, e.g., MJ-1999; 4-(2-isopropyUmino-l-hydroxyeihyl) metluneiulfonantiide hydrochloride. Each Abstract Form submitted MUST BE SIGNED by a member of the American Society of Biological Chemists. NG ADDRESS OF FIRST AUTHOR r... Du., Ph.D. /ersity of Louisviiie, MDR Bidg., Sm.535 S... .Floyd. .Street;iLou...! .Ky... Zip AQ2P.1. one no.: Area Code 502 # 588-5251 'CONPiDENTIAL ` Subj-ect to Protective Order in Fogs v. -Conoco, Inc.. No. 90-4937 14th Judicial District Court Csl''1'si *=i] Parish, Louisiana Member's Signature Telephone No.: Area Code ,d9?... -- ............ to: Robert A. Hart*. Executive Officer American Society of Biological Chemists 9650 Rockville Pike Bethesda, Maryland 20014 CMA 003890 jMinrtbi I.tuiji oc ncuavEU A I ttUClfcl r (JM-ICfc BY WfcUNESDAY, n JANUARY 1978 Please consider this abstract for inclusion in the tentatively listed minisymposium, M 27 ; Chemfral Carr innffanpaf a Indicate below the numbcri and titles of sessions in which your abstract might be programed (see Topic Category List); 1 jt# M2 7TjtleChemical Carcinogenesis 2nd #__LfiiTitle Riochemiral Pharmacol ngv 3rd --------.----- _ Title Educational Techniques poster session The original typed copy of this abstract form must be submitted together with 8 photocopies. DO NOT FOLD THIS FORM 1978 ASBC/AA! Abstract Form If abstract is scheduled for regular ses sion I prefer: Poster presentation (XSlide presentation Final decision will be made by Program Committee IMPORTANT: See sample abstracts, typing and mail ing instructions on reverse side; use enclosed Check List for preparation of abstract. The original typed copy of this abstract form (for reproduction by photo-offset in FEDERATION PRO CEEDINGS) must be submitted to gether with 8 photocopies. Abstracts submitted for the "Edu cational Techniques" poster session do not prevent a member from submitting or sponsoring an abstract for the regular sessions. Standard projectors for 2" x 2" and 3`A" x 4" slides will be available in all slide sessions. Other audio-visual aids can be provided at cost if the request and justification accompany this ab stract. Authors will be billed following the meeting. ELEVATED GLUTATHIONE CONTENT, GLUTATHIONE-S-TRANSFERASE AND GLUTATHIONE REDUCTASE IN LIVER OF RATS EXPOSED TO VINYL CHLORIDE Du, J.T.* and Tamburro. C.H.* (SPON: McGeachin, R. L.) Dig. Dis. & Nutr. Sect-, Dept. Med., Cancer Center, Univ. Lou. Med. Sch., Loy., Ky., 40232 Vinyl chloride (VC) is believed to be metabolized to chloroethylene oxide (CEO) and chloroacetaldehyde, and detoxified by way of glutathione. Rats were exposed to 28,000 ppm VC, 7hrs/ day, 5days/wk for 4 and 6 weeks, the activity of glutathione epoxide-^-transferase (GEST) was elevated 30 to 54% over normal control and air control (9.71+0.68 vs 7.52+0.97 and 6.30+ 0.68) respectively. However, the activity of glutathione aralky1-S-transferase (GAST) was not significantly elevated until 6 weeks of exposure to VC. The content of reduced glutathione was also elevated 45% in the VC treated group and the activity of the glutathione reductase, the enzyme to re generate glutathione from the oxidized form was elevated 50%. These results demonstrate that VC exposed rats have the capa city to maintain glutathione reductase activity and glutathione concentration for detoxification. Further, it suggests that the primary route of VC metabolism is initial oxidation to CEO and then detoxification by GEST directly. With longer exposure and probable saturation of the direct route, there is greater rearrangement of CEO to chloroacetaldehyde, and de toxification with glutathione as supported by the delayed in duction of GAST (Supported by a grant from Manufacturing Chemists Association). All compounds that arc designated by code or initial letters must be identified adequately in the abstract, e.g., MJ-1999: 4-{2*isopropylamino-I-hydroxyethyl) methanesulfonanilide hydrochloride. Each Abstract Form submitted MUST BE SIGNED by a member of the American Society of Biological Chemists. McGeachin! .R. ,L........................................... ...................... (Member's Name: Please Print or 'Type) MAILING ADDRESS OF FIRST AUTHOR ( Please print or type ) Du, Julie T. ' "535 MDR' Building,' 'p" 67 Box' '35260........... U. of. .L.Med. Sch. , Lou., .Ky.... Zip. .40232 Telephone no.: Area Code . . .59.2... .#. 588^525,1......... CONFIDENT! AT. Subject~to Protective 0rdr i Koss_ v. J^onor^inc^ No. 9o-4( 14th J'ldicinTDi^triot Court CalnastPu Parish, (Member's Signature) Telephone No.: Area Code. .502 _ .5.88-5.290 ... Mail to: Russell J. Hilinoe, Executive Officer American Society of Biological Chemists 9650 Rockville Pike Bethesda, Maryland 20014 CMA 003891 ASPET/SOT 1982 Louisville, Kentucky Deadline for Receipt! May 7, IMS Abstract Processing Fee: $20.00 Select Category Numbers and Titles (See list over) i- Zh. Presentation Preference _ Poster X_ Oral (slides) _ Indifferent If your choice is not available Willingly accept the alternative __Grudgingly accept the alternative Withdraw the abstract Poster boards are 6' wide x 4* high. Serum Bile Acids (SBA) Screening For Chemical Hepatotoxicity. Gary Liss* and Carlo H. Tamburro* (SPON: William Waddell). NIOSH Robert Taft Laboratory, Cincin., OH. & Liver Research Center, Div. Occupational Health, Depts. of Medicine & Community Health, Univ. of Louisville, Lou., Ky. 40292. Standard liver tests have limited ability to detect latent liver disease (Environ. Health Perspect. 1981; 41:117-112). Serum bile acids are more sensitive indicators of liver in jury. During the medical surveillance of 1000 chemical work ers 67 liver biopsies were investigated for histological evi dence of chemical injury (Gastroenterology 1979; 77:A33); 15 had chemical liver disease (CLD), 27 nonchemical liver disease (NCLD) & 25 had normal biopsies (NBX). Fasting SBA-cholylglycine (CG) & conjugates of cholic acid (CCA) were studied by 125i_radioimmunoassay in these groups and 416 "normal" work ers. Mean + S.E.M. for CG in the CLD, NCLD, NBX & normal groups were 95.17 + 28.75, 27.25 + 4.43, 34.60 + 7.13 & 14.9 + 0.88 ug/ml, respectively. Analysis of variance showed sig nificant differences (p<0.035) in the 3 biopsy groups; p<0.001 for all 4 groups. The CLD values were significantly differ ent in CG levels of the NBX (p<0.02). No significant differ ence in CG levels in NCLD and NBS groups; CCA showed similar results. Fasting SBA showed promise in detecting latent chemical liver injury. Prepare your abstract carefully. It will be printed by photo-offset exactly as received. See the example (over) for lay-out and style. For elite typewriters (12 pitch), set the margins at 36 and 98 (62 spaces). Use 25 lines. For pica typewriters (10 pitch) set margins at 30 and 61 (51 spaces). Use 25 lines. The first typed letter should nearly or just touch the top and left type box (blue margin lines). Title. Leave no margins. Use a short, descriptive title. Use upper and lower case letters. Authorship. Underline names. Place an asterisk* after the name of each author not a member of ASPET or SOT. If no author is a member, type the following after the name of the last author: (SPON: sponsor's name). Continue on the same line with the authors' institutional affUiation(s), city, state and zip code. Text. Start the text on the next line, indenting 3 spaces. Subsequent lines should extend the entire width of the type box. The text mould have all of the elements of a report: introduction, method, result(s) and conclusion. It is improper to substitute "The results will be discussed" for the results and conclusion. Adequately identify all chemical compounds used. Do not fold abstract. Be certsin abstract is prepared securely for mailing. All subsequent correspondence must reference the first author to enable us to identify the abstract. The Program Committee selects chairmen and overview ers from volunteers. See below. First author phone #: (502) 588-5251 Membership of author or sponsors. ASPET^Xj SOT _X_ 1. Member signature /VX^\ _________ 2. Name (Type or print) Wl/l^am Waddell, M.D. 3. Will you chair a session? __ yjs; Xno In what category Will you give a 25 minute overview? __ yes; no; In what category the original, 3 copies, a self-addressed post card for program confirmation and 1 check to Kay A. Croker, Acting Executive Officer, ASPET, 9650 Rockville Pike, Bettiesda, MD 20814, USA. Reprints cost $15 per 100, lots of 100 only. Order below and fill in the mailing label, below left. Invoice: Abstract Processing Fee Reprints: Quantity ordered__coat: TOTAL $ 20.00 _______ i Mailing label for reprints (Name and Address): Mailing label to the first author (Name and Adcfress): CMA 003892 Bom v l4tJi "s2oo, i;v" 0rfer ln -------- iRc` No. 90-4837 p,, Court T om' ASPET/SOT 1932 Louisviilc, Kcituclty Deadline for Receipts May 7, 1982 *bs;rset Processing Fee: $20.00 Select Category Numbers end Titles iiee ust over) 1- 2A. Presentation Preference _ Poster X_ Oral (slides) _ Indifferent If your choice is not available Willingly accept the alternative _Grudgingly accept the alternative Withdraw the abstract Poster boards are S' wide x 4' high. Serua Bile Acids (S3A) Screening For Chemical Hap, - v* J _ _ Gary L13S* and Carlo H. TonburroJ1 (3?CN: William adviel lv' NIOSH Sober: Taf; Laboratory, Cir.cin. , OH. 0 1 r r. = icw;; Center, Div. Occupational Health, Depcs. of Medicine .) Car,- munity Health, Univ. of Louisville, Lou., Ky. 4G292. i i ver C'SSC3 h 2 vs liT.iC'^d 3biii2'r zo br ^ . *'' * 1 J -. -- J p*^ ^ ,J .3 ' * U 1 O ^ 1 .1 1 ` ` _ 1 ' ' ' Serum bile acids are more sensitive indicators of liver in jury. Curing the medical surveillance of 1CC0 chemical ..cri ers 67 liver biopsies were investigated for hiscological evi dence of chemical injury (Gastroenterology 1979; 77:A33); 15 had chemical liver disease (CLD), 27 nonchemical liver disease (NCLD) & 25 had normal biopsies (NBX). Fasting SBA-cholylglycine (CG) & conjugates of cholic acid (CCA) were studied by l^I-radioimmunoassay in these groups and 416 "normal" work ers. Mean + S.E.M. for CG in the CLD, NCLD, NBX 6 normal groups were 95.17 + 28.75, 27.25 + 4.43, 34.60 + 7.13 6 14.9 + 0.88 ug/ml, respectively. Analysis of variance showed sig nificant differences (p<0.035) in the 3 biopsy groups; p<0.001 for all 4 groups. The CLD values were significantly differ ent in CG levels of the NBX (p<0.02). No significant differ ence in CG levels in NCLD and NBS groups; CCA- showed similar results. Fasting SBA showed promise in detecting latent chemical liver injury. Prepare your abstract carefully. It will be printed by photo-offset exactly as received. See the example (over) for lay-out and style. For elite typewriters (12 pitch), set the margins at 38 and 98 (62 spaces). Use 28 lines. Far pice typewriters (10 pitch) set margins at 30 and 81 (51 spaces). Use 28 lines. The first typed letter should nearly or just touch the top and left type box (blue margin lines). Title. Leave no margins. Use a short, descriptive title. Use upper and lower case letters. Authorship. Underline names. Place an asterisk* after the name of each autW not a member of ASPET or SOT. If no author is a member, type the following after the name of the last author: (SPON: sponsor's nama). Continue on the same line with the authors' institutional affiliation(s), city, stata and zip coda. Text. Start the text on the next line, indenting 3 spaces. Subsequent lines should extend the entire width of the type box. The text Mwuld have ail of the elements of a report: introduction, method, raaultU) and conclusion. It is improper to substitute "The results will be discussed" for the results and conclusion. Adequately identify all chemical compounds used. Do not fold abstract. Be certain abstract is prepared securely for mailing. All subsequent correspondence mimt reference the first author to enable us to identify the abstract. The Program Committee selects chairmen and overview ers from volunteers. See below. First author phone #: (502) 588-5251 Membership of author or sponsors. ASPET^Xj SOT ^ 1. Member signature jyvJKOv\Vr-^~Kjy 2. Name (Type or print) Wl/liam Waddell, M.D. 3. Will you chair a session? __ yqst Xno In what category Will you give a 28 minuta overview? _y.X no; In what category Mail the original, 3 copies, a self-addressed post card for program confirmation and 1 check to Kay A. Croker, Acting Executive Officer, ASPET, 9880 Rockville Pike, Bethesda, MD 20814, USA. Reprints coat $18 per 100, lots of 100 only. Order below and fill in the mailing label, haloes left. Invoices Abetract Proceesing Fee Reprints: Quantity ordered TOTAL cost: $ 20.00 _____ * Mailing label for reprtsts (Name and Address): Mailing label to the first author (Name and Address): CMA 003893 FORM FOR ABSTRACTS TO BE PUBLISHED IN GASTROENTEROLOGY Type Abstract in Space Below 7 Check the most appropriate cate gory below: Absorption Secretion Motility Liver/Biliary/Bile Salt G.L Hormones Morphology Clinical Immunology/Microbiology I prefer presentation at: Poster Session (2 Regular Forum 3 Please publish this abstract in GASTROENTEROLOGY at a cost of S25.00 (S3Q.00 if bill ing required) 13 My check payable to the Amer ican Gastroenterological Asso ciation is enclosed. Please bill me--instructions en closed. Please do not publish this ab stract in GASTROENTER OLOGY. 7T5SWE AND URINARY GLYCOSAMINOGLYCANS (GAG) CHANGES IN EEEAXIC FIBROSIS C. E. Kupchella, J. 0. Jarvis, K. L. Curran, R. A. Greenberig, and C. H. Tamburro Cancer Center and Digestive Diseases and Nutrition Section, University of Louisville School of Medicine, Louisville, Kentucky. GAG's are felt to play an important role in the collagen fibril formation and collagen bundle stabilization. It has been assumed that hepatic fibrogenesis contributes little to the total body connective tissue and would not be reflected in urinary excretion of GAG degradation or syn thetic products. We have previously found, however, that increased urinary GAG's occur in humans with cirrhosis, hepatitis, and hepatic angiosarcoma (Kupchella, C.E., Tamburro, C. H., Clin. Res. 25:329, 1977; Curran, K. L., Kupchella, C. E., Tamburro, C. H., Cancer (In press), 1977). In the present study, changes in urinary and hepatic GAG's were measured in rats with CCL4~induced hepatic damage. Highly significant increases (P <.001) in total GAG's and in the hyaluronic acid (HA), chrondrotin sulfate (CS), and the heparin (H) fractions were found in hepatic tissue after three weeks and persisted through nine weeks of ex posure. Histological examination after three weeks showed hepatic necrosis with extensive fatty metamorphosis without histochemical (Trichrome-Alcian Blue-PAS) evidence of fibrosis. Control hepatic tissue GAG levels were 31+5 Ug (of uronic acid) per gram of dry defatted liver. CCL4 treated livers showed a 2 to 4 fold increase in GAG levels; 81.4 pg at 3; 98 pg at 6; and 113 pg/g at nine weeks, re spectively. Increases occurred mainly in the CS and H frac tions. The urinary CS fraction was significantly (P < .05) elevated over the nine weeks of exposure; the HA fraction was elevated during the first two weeks only, and the H fraction excretion appeared to be directly related to CCL4 injections. These data support our previous observations in humans and demonstrate that urinary GAG excretion pattern changes occur before histochemical evidence of collagen formation and suggest that urinary GAG patterns may be a useful Indicator of early hepatic fibrogenesis. MAILING ADDRESS OF PRINCIPAL AUTHOR Carlo H. Tamburro, M.D. 511 South Floyd Street Room 535 MDR Building Louisville, Kentucky 40201 zip CONFIDENTIAL Subject to Protective Order in Ross v. Conoco, Inc,, No. 90-4837 14th Judicial District Court Co 1 ^ s'- on P-5H sh , T.o'ii r * pTiq. If, in the conduct of these studies, human subjects were exposed to risks not required by their medical needs, the author affirms that the study was approved by an appro priate committee, or, if no such committee was available and informed consent was needed, it was obtained in accordance with the principles set forth in "The Institu tional Guide to DHEW Policy on Protection of Human Subjects". CMA 003894 CLINICAL RESEARCH Abstract Reproduction Form co;r?n 11AL TYPE name, address, and telephone number of author who should receive correspondence in Box A and complete Box B. Telephone 502-588-5245 (Area code) office 502-239-1818 (Area code) home Subject to Protective Order in Sos5_ v. Conoco, Inc.^ K0. 9Q-4537 14th Judicial District Ccu-t Calcasieu Parish, Louisi="'a % Name Address . Charles E. Kupchella, Ph. D. Associate Director, Cancer Center 213 MDR Building Health Sciences Center Louisville, Kentucky 40201 Date Payment (SI0.00) Check number Puichaie order October 1. ] CHECK Preferred Sub-Specialty Oaaiftcation: ____ Cardiovascular Clinical ____ Epidemiology Clinical ____ Pharmacology ____ Dermatology ____ Endocrinology* ____ Gastroenterology ____ Genetics ____ Health Care Research ____ Hematology Immunology & ___ Conn. Tissue ____ Infectious Disease ____ Metabolism* ____ Oncology ____ Pulmonary ____ Renal & Electrolyte *TrmditionaUyT Endocrinology hi* iaduded piper* dealing with the thyroid, adrenal and pituitary gland*, and gonad*, while Ab stract* dealing with the parathy* rokb, calcium and phosphorus aetaboiiam, bone*, thyrocalcitooin, diabetes, insulin, glucagon, and growth hormone have been considered under Mtmbolljn. URINARY CH0NDR0ITIN SULFATE FRACTION PATTERNS IN HEPATIC ANGI0SARCC C. E. Kupchella, K. L. Curran, and C. H. Tamburro. Cancer Center, University of Louisville, Louisville, Kentucky. This study was undertaken to evaluate the usefulness of urinary glycosaminoglycan patterns in the detection of hepatic angiosarcomc and in monitoring the course of this disease. Glycosaminoglycans t tracted from 24 hr urines from 2 patients with angiosarcoma of the liver and from normal controls were separated as cetylpyridintum cc plexes into "hyaluronic acid," "chondroitin sulfate," and "heparin1 fractions. These fractions were further purified by anion-exchange chromatography. The "chondroitin sulfate" fraction isolated fro4fc angiosarcoma patients consistently demonstrated an increase in t^^ amount of glycosaminoglycan eluted in 1.25 M NaCl and a concomitant decrease in the 1.5 M NaCl eluticn peak. This "shift" was apparent related to the course of the disease and was unaffected by chemo therapy. As indicated by the following results, the ratio of 1.25 to 1.5 M elution peaks may be of value in monitoring the course of angiosarcoma: 2 normal controls, .364 and .316; angiosarcoma (pre chemotherapy), .843; angiosarcoma (post-chemotherapy), .971; angio sarcoma (advanced), 5.00. Characterization of the 1.5 M and 1.25 b NaCl elution peaks by hyaluronidase susceptibility and comparison w elution patterns reported in the literature suggest that the observe shift was from chondroitin 4 and/or chondroitin 6 sulfate to heparar sulfate. Preliminary data indicate that the excretion pattern in vinyl-chloride-associated liver injury other than angiosarcoma is characteristically different from patterns associated with hepatiti cirrhosis, or metastases to the liver. These observations may be related to progressive connective tissue proliferation in angiosarc IMPORTANT The instructions accompanying this form must be followed COMPLETELY for all abstracts which are to appear in CLINICAL RESEARCH. Ab stracts which do not conform either will be re typed by the publisher at a 'cost of $ 15,00 to the author, or rejected. Revised June 1976 CMA 003895 THIS FORM AS WELL AS THE FORM LETTER OF TRANSMrTTAL MUST BE SIGNED BY A MEMBER MEMBER'S SIGNATURE > CLINICAL RESEARCH Abstract Reproduction Form r coKFirz'i'riA; TYPE name, address, and telephone number of author who should receive correspondence in 2c.x A and romoleie 3o.x B. Subject to Protective eras'- in Ross_ v. Conoco, Inc.,_ Ho. SO-4837 14th Judicial Dijtrict Court C^lonsipu Parish, j 0 U1; tana (Ana idle) office (Arts code) home Name Address Charles E. Kupchella, Ph. D. Associate Director, Cancer Center 213 MOR Building Health Sciences Center Louisville. Kentucky 4Q2Q1 Data -October 1. PiynNnt (J 10.00) ______________ Cluck number _________________ rurduaa order _________________ CHECK Prtferrtd Sub-Sp*daity Ckmflcanon: ____ Cardiovascular Clinical ____ Epidemiology Cliniroi ____ Rtarmacology ____ Dermatology ____ Endocrinology* ____ Gastroenterology ____ Genetics ____ Health Care Research ___ _ Hematology Immunology A ____ Conn. Tissue ____ Infectious Disease ____ Metabolism* ____ Oncology ____ Pulmonary ____ Renal A Electrolyte *Tr*dltioiuUy, Endocrtnoiofy ha iAdodad popon daalinf with tha thyretd, *dnnai and pituitary ftlantfc, red gonad* white lb' creeta darilng with tha parAthyrote*, eddim red phrephotre --nhniiam, boo. tfcyrectetetoteo* dtebataa, tnvuiio, teorepoo, red powth itoreoM fehre boon i ureiilwail uniat iifnahoflu URINARY CHONDROITIN SULFATE FRACTION PATTERNS IN HEPATIC ANGIOSARC C. E. Kupchella, K. L. Curran, and C. H. Tamburro, Cancer Center, University of Louisville, Louisville, Kentucky. This study was undertaken to evaluate the usefulness of urinary glycosaminog1yean patterns in the detection of hepatic angiosarcom and in monitoring the course of this disease. Glycosamlnoglycans tracted from 24 hr urines from 2 patients with angiosarcoma of the liver and from normal controls were separated as cetylpyridinium c plexes into "hyaluronic acid," "chondroltin sulfate," and "heparin fractions. These fractions were further purified by anion-exchang chromatography. The "chondroitin sulfate" fraction isolated from angiosarcoma patients consistently demonstrated an increase in the amount of glycosaminoglycan eluted in 1.25 M NaCl and a concomitan decrease in the 1.5 M NaCl elution peak. This "shift" was apparen related to the course of the disease and was unaffected by chemo therapy. As indicated by the following results, the ratio of 1.25 to 1.5 M elution peaks may be of value in monitoring the course of angiosarcoma: 2 normal controls, .364 and .316; angiosarcoma (pre chemotherapy), .843; angiosarcoma (post-chemotherapy), .971; angio sarcoma (advanced), 5.00. Characterization of the 1.5 M and 1.25 i NaCl elution peaks by hyaluronidase susceptibility and comparison v elution patterns reported in the literature suggest that the obser shift was from chondroitin 4 and/or chondroitin 6 sulfate to hepar; sulfate. Preliminary data indicate that the excretion pattern in vinyl-chloride-associated liver injury other than angiosarcoma is characteristically different from patterns associated with hepatit* cirrhosis, or metastases to the liver. These observations may be related to progressive connective tissue proliferation in angiosarc IMPORTANT Hu instruction* accompanying tiro form mrot be followed COMPLETELY for all abstracts which are to appear in CLINICAL RESEARCH. Ab stracts which do not conform either will be re typed by the publisher at a`cost of S15.00 to the author, or rejected. Rntiaed June 1976 THIS FORM AS WELL AS THE FORM LETTER OF TRANSMITTAL MUST BE SIGNED BY A MEMBER MEMBER'S SIGNATURE CMA 003896 CLINICAL RESEARCH Abstract Reproduction Form C 0Nx i jjEii i i An 9 TYPE name, address, and telephone number of author who should receive correspondence in Box A and complete Box B. Telephone (502) 588-5253(502) 895-2955 (Are* code) office (Are* code) home Subject to Protective Order in Ross v. Conoco , Inc. , No. 90--1337 14th Judicial District Court Calcasieu Parish, Louisiana A Name ad/irM, Carlo H. Tamburro. M.D. University of Louisville Cancer Center 511 South Floyd Street Room 535 MDB Rtrlldlnv Louisville, Kentucky 40201 B imv --jL/ kMhrl Payment fllO.OO) Check number PuidiaMorder __17--1185 CHECH Preferred Sub-Specialty Claaiftcation: ____ Cardiovascular Clinical ____ Epidemiology Clinical ____ Pharmacology ____ Dermatology ____ Endocrinology* ____ Gastroenterology ____ Genetics y Health Care Research ____ Hematology Immunology & ____ Conn. Tissue ____ Infectious Disease ____ Metabolism* ____ Oncology ____ Pulmonary ____ Renal & Electrolyte "Traditionally, Endocrinology hu included piper* dealing with the thyroid, adrenal and pituitary glands, and gonads, while ab stracts dealing with the parathy roids, calcium and phoaphoma metabolism, bones, thyrocalcitonin, diabetes, insulin, glucagon, and growth hormone have been considered under Metabolism, URINARY GLYCOSAMINOGLYCAN EXCRETION PATTERNS IN CHEMICALLY INDUCED LIVER INJURY AND CANCER C. E. Kupchella*and C. H. Tamburro**, Cancer Center, University of Louisville, Louisville, Kentucky, Glycosaminoglycans (GAG) are essential compounds of connective tissue (CT) matrix and are increased with CT proliferation. Urinary GAG pat terns were studied in 9 individuals with vinyl chloride (VC) induced chemical injury, 2 VC induced angiosarcomas, 8 viral hepatitis, 6 alco holic cirrhosis, 7 non-hepatic cancers, and 9 controls. Hepatic histo logical and electron microscopic (EM) studies were obtained in all but normal controls which were studied biochemically, radioisotopically, and physically. Urines were analyzed for creatinine, total GAG, and uronic acid content. GAG levels (ug uronle acid/mg creatinine) in con trols were 3,2 + .4, in VC exposed 4.1 +4, and in alcoholic liver dis ease 5.1 + 0.8. In contrast, in angiosarcoma they were 7.6 + 1.6 and hepatic metastasis, 13.8 + .9. Total GAG's were further separated into hyaluronic acid (HA), chondroitin sulfate (CS) and heparin (H) frac tions. Although no significant differences were found in total GAG's 78Z (7/9) of VC exposed had positive CS and negative HA and H fractions in contrast to 9X (3/32) of the other cases. Further characterization by anion-exchange chromatography showed a shift in the CS fraction composition. Total GAG's eluted at 1.25 M NaCl, were increased and at 1.50 M NaCl decreased. These changes became more pronounced as the disease developed. Enzyme digestion indicates that 1.25 M fraction is heparan sulfate (HS). EM studies showed increased sinusoidal collagen. Since HS is associated with blood vessels, its increased urinary excre tion in early VC injury and angiosarcoma (vascular injury and sinusoida cell cancer) may be used as an early indicator of chemical injury and liver cancer formation. IMPORTANT The instructions accompanying this form must be followed COMPLETELY for ail abstracts which are to appear in CLINICAL RESEARCH. Ab stracts which do not conform either will be re typed by the publisher at a cost of 515.00 to the author, or rejected. Revised June 1976 THIS FORM AS WELL AS THE FORM LETTER OF TRANSMITTAL MUST BE SIGNED BY A MEMBER MEMBER'S SIGNATURE JOML I'to CMA 003897 Check the most appropriate category below: Absorption Secretion O Motility G Liver/Biliary/Bile Salt Q G.l. Hormones Morphology Clinical Immunology/Microbiology 0 Publish this abstract in GASTRO* ENTEROLOGY at a cost of S25.00 (S30.00 if billing required) 0 Check payable to the American Gastroenterological Association enclosed. Bill me--instructions enclosed. Do not publish this abstract in GASTROENTEROLOGY. CONFIDENTIAL Subject to Protective Order in Conoco, Inc. , No. 90-4837 l4th Judicial District Court p-.^^ Louisiana 1C Type Abstract in Space Below TISSUE AND URINARY GLYCOSAMINGLYCANS IN TRANSPLANTABLE HEPATOMAS. C. E. Kupchella, K. L. Curran, E. Drake. J. Kennedy, and C. H. Tamburro. Cancer Center, and Division of Digestive Diseases and Nutrition, University of Louisville, School of Medicine, Louisville, Kentucky. , 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 t`. .'livers of tumor-bearing animals, and d) urinary GAG ex cretion as a function of tumor growth. Three types of Morris hepatomas, 7777, 5123tc, and 9618A, which differ ii jrates of growth, metastatic potential, fibrosis, and ^ecrosis, were studied. Urinary and tissue GAGs were ex tracted as cetylpyridinium complexes and measured as uron: jacid. Tissue GAGs were also evaluated histochemically jusing alcian blue staining with and without enzyme preitreatment. Tumor tissue exhibited four- to six-fold greater GAG levels in the hyaluronic acid (90 +10, 91 + 112, and 111 + 9 vs. 25 + 3 fig uronic acid/g dry liver, rejspectively) and chondroitin sulfate (261 + 29, 217 + 51, jand 208 + 22 v. 47 + 7 ng uronic acid/g, respectively) fractions than normal liver; the heparin fractions did not differ significantly (31 + 7, 17 + 4 and 67 + 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 afte jtwo weeks in animals bearing fast-growing tumors. The GAC (tissue levels in fast vs. slow-growing tumors were not si jnificantly different. This further supports our previous] ,reported studies of urinary GAG excretion in human hepatic angiosarcoma (Curran, K. L., et al., Cancer 40 (6): 30503053) in suggesting that urinary GAG analyses may be usefu in the detection, screening and diagnosis of hepatic cance. TYPE name, address, and telephone number of author who should receive correspondence: Name Charles E. Kupchella, Ph.P., Associate Director, Cancer Center 213 MDR Building, University of Louisville, P. 0. Box 35260, Louisville, Ky. Address ------------------------------- --------------------------------------------------------------------------------------------------- ---------- 40232" Telephone (502) 588-5245________________ ______________________________________________ IMPORTANT The principal author affirms that the material herein will not have been previously published or presented at any meeting of a national society and that if in the conduct of these studies, human subjects were exposed to risks not re quired by their medical needs, that the study was approved by an appropriate committee or, if no such committee was available and informed consent was needed, it was obtained in accordance with the principles set forth in The Inslitu- Guide to DilliW Policy on Protection of Human Subjects." CMA 003898 Check the most appropriate category CiiO'.v; Absorption G Secretion Motility C Liver/ Biliary/' Bile Salt O G.l. Hormones Q Morphology Clinical Immunology/Microbiology 13 Publish this abstract in GASTRO ENTEROLOGY at a cost of 525.00 {530.00 if billing required) B Check payable to the American Gastroenterological Association enclosed. O Bill me--instructions enclosed. Do not publish this abstract in GASTROENTEROLOGY. CONFIDENTIAL to Protective Order in v- Conoco , Inc , , No. 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana Type Abstract in Space Below TISSUE AND URINARY GLYCOSAMINGLYCANS IN TRANSPLANTABLE HEPATOMAS. C. E. Ku^chella, K. L. Curran, E. Brake, J. Ksr.r.adv, ar.d C, H. iiaburro. Cancer Center, anc Division of Digestive Diseases and Nutrition, University of Louisville, Scnooi of Medicine, Louisville, Kentucky. i The purpose of this investigation was to evaluate: a) :the glycosarainoglycans (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 t' 'livers of tumor-bearing animals, and d) urinary GAG ex cretion as a function of tumor growth. Three types of Morris hepatomas, 7777, 5123tc, and 9618A, which differ i: ]rates of growth, metastatic potential, fibrosis, and necrosis, were studied. Urinary and tissue GAGs were ex tracted as cetylpyridinium complexes and measured as uron: jacid. Tissue GAGs were also evaluated histochemically using alcian blue staining with and without enzyme preItreatment. Tumor tissue exhibited four- to six-fold greater GAG levels in the hyaluronic acid (90 +10, 91 + 12, and 111 + 9 vs. 25 + 3 uronic acid/g dry liver, re spectively) and chondroitin sulfate (261 + 29, 217 + 51, and 208 + 22 vs. 47 + 7 yg uronic acid/g, respectively) fractions than normal liver; the heparin fractions did not differ significantly (31 +7, 17+4 and 67+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 aftt two weeks in animals bearing fast-growing tumors. The GAC tissue levels in fast vs. slow-growing tumors were not sig Inificantly different. This further supports our previous! reported studies of urinary GAG excretion in human hepatic angiosarcoma (Curran, K. L., et al., Cancer 40 (6): 30503053) in suggesting that urinary GAG analyses may be usefc in the detection, screening and diagnosis of hepatic cance TYPE name, address, and telephone number of author who should receive corrcspo^encc: _________ Namc Charles E. Kupchella, Ph.D., Associate Director, Cancer Center 213 MDR Building. University of Louisville, P. 0. Box 35260, Louisville, Ky, Address------------------------------- ---------------------- --------------------- -------------- ----------------------------------------------------- 40252~ Telephone (502) 588-5245_--------------------------------------------------i------ ------------------------------------------------------------ IMPORTANT The principal author affirms that the material herein will not have been previously published or presented at any meeting of a national society and that if in the conduct of these studies, human subjects were exposed to risks not re quired by their medical needs, that the study was approved by an appropriate committee or, if no such committee was available and informed consent was needed, it was obtained in accordance with the principles set f rth in "The Institu- Guide to DHEW Policy on Protection of Human Subjects." CMA 003899 Check the most appropriate category below: Absorption Secretion Motility C Liver/Biliary/Bile Salt G.I. Hormones Morphology Clinical Immunology/Microbiology XI Publish this abstract in GASTRO ENTEROLOGY at a cost of S25.00 (S30.00 if billing required) B Check payable to the American Gastroenterological Association enclosed. Bill me--instructions enclosed. Do not publish this abstract in GASTROENTEROLOGY. CONFIDENTIAL iuo.ject to Protective Od<=r in c`r,, tMaic3al District Court Cal ,,3sieu Parish, Louistq^q, Type Abstract in Space Below 11 URINARY GLYCOSAMINOGLYCAN PATTERNS IN HUMAN HEPATIC ANGIO SARCOMA, HEPATOMA, AND IN WORKERS AT RISK FOR ANGIOSARCOMA. K. L. Curran, C. E. Kupchella, J. Sandoz, and C. H. 1 .Tamburro. Cancer Center and Division of Digestive Diseases and Nutrition, University of Louisville, School of Medicine, [Louisville, Kentucky. i j A previous study reported an abberatlon in glyccsamino- ( !glycans (GAGs) eluted from anion-exchange columns with 1.251 jand 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 indijviduals with a history of high vinyl-chloride exposure and 'documented liver disease were paired with individuals with |a high exposure index to vinyl-chloride but no clinical jliver disease. Similarly six persons with low exposure jbut abnomal liver function were paired with low exposure/ | | ' ! j I | jnormal liver function individuals. A 24-hour urine was collected from each individual and the GAGs analyzed by ' |anion-exchange chromatography. Individuals with clinically jactlve liver disease at the time of this Btudy were found |to have urinary GAG excretion patterns which were similar jto those described in angiosarcoma. No significant dif- j | jferences 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 f liver disease in humans. 1 ! I I TYPE name, address, and telephone number of author who should receive correspondence: tCaiw^ Charles E. Kunche'Ma. Ph.D.. Associate Director Cancer Center-------------------------------A 213 MDR Building, University of Louisville, P. 0.-Box 35260/ Louisville, Ky. 40235 .-- f502) 588-5245 IMPORTANT The principal author affirms that the material herein will not have-been previously published or presented at any mrciinp of a nation;.' society and that if ;n *w-.- ' - ,/studies, human sii*"'* \poscd to risks not re- CMA 003900 CLINICAL RESEARCH Abstract Reproduction Form ''YTE name, address, and telephone number of luthur who should receive correspondence in Box A and complete Boxes B, C and D. Telephone ^02588--5245 (Area code) office 502 (Area code) A Name _ Address Charles E. Kupchella Cancer Center University of Louisville Louisville, Ky. 40232 N0 This abstract is submitted to: American Federation for Clinical Research (name of organization, selected from list on form letter of t.-ansm| 239-1818 home B (See Rule 15) Dare______ Payment (SI5.00) Check number __ Purchase order number. Issued by. (name of institution) A copy of this abstract must be attact to original purchase order ;o aid in id tinea tion. TYPE ABSTRACT HERE/BE SURE TO STAY WITHIN BORDER IC CHECK OSE i for national meeting] only) ` Prefer poster session presentation sI Consider for poster session if not | selected for oral presentation Do not consider for poster session under any circumstances ID J CHECK SISCLE SVBSPECIALTY CLASSmCA T!ON: code Cardiovascular*no.: () Clinical Epidemiology......... ......... Clinical Nutrition.................. ......... Clinical Pharmacology___ _____ Dermatology........................... ......... Endocrinology (see Rule }) ____ Gastroenterology.................. ......... I Genetics.................................... ......... Health Care Research......... ......... Hematology ........................... ......... Hypertension ......................... ......... Immunology 8c Rheumatology___ Infectious Disease................ ......... I Metabolism (see Rule 5)... ____ Oncology.................................. X Pulmonary............................. ......... Renal it Electrolyte............. ......... GLYC0SAMIN0GLYCAN 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 associatet^B 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 peritonea; 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. Clnv.Re/. For abstracts submitted to C.lrdIO\ ascular only, select single subcategory and enter code no. (1-6) inspace above: (1) Clinical; ll) Basic Suence: 13) Electrophysiology- Dysrhythmias; (41 Echo cardiography: (51 Radiologv-Radionudides. (61 Other. Subclasstfication is designed to aid in reviewing process cnlv and is independent of program selection. PLEASE CHECK ABSTRACT CAREFULLY FOR APPEARANCE BEFORE MAILING ^? IA L BOTH THIS FORM AND THE FORM LETTER Subject to Protect^TVA OF TRANSMITTAL MUST BE SIGNED Rose 4ut.ecri.ve Order in y. _Cpnoco. Inn w,, 9n BY A MEMBER (RULE 2) OourT7 Calcasieu Parish, Louisiana---- Charles E. Kupchella, Ph.D. (please type name) Revised May 1978 MEMBER'S SIGNATURE CMA 003901 CLINICAL RESEARCH Absirac: Reoroduction Form *V?E rums, addreas, and telephone number of uthor Arto should receive correspondence in Box .and ram.ciese 3oxe: 2. C acid D. 502 538-3245 i A.-eu cciiej oiuce 302 race) name Adccess Charles E. Kupchella Cancer Center University of Louisville Louisville, Xy. 40232 r\ 'j This abstract is iubm.ttid :c: isritan Pecerztior. for Clir.dzo.il .'osaorzh * sur,* st aryani;i:;on, a:s.d ;';am Arm 239-1313 j B (See Rule 15) Payment (SI 3,00) Check number _ Purchaie order number. Issuei by . (name of institution) A copy of thu abstract must be attic to ongiiui purchue order to ltd in i< tificatton. TYPE ABSTRACT HERE/BE SURE TO STAY WITHIN BORDER ,u ' CHECK OSE j i.'er nano not meetings only) j Prefer poster session presentation __1 I Consoler for poster session if not ' | selected tor oral presentation 2 ! Z\> oot consider for poster ___ i session under any circumstances i__; iD j CHECK SINGLE SL'SSFECIALTY CLASSIFICATION. ! cede j Cardiovascular*______no.: (_____ ) ; Clinical Epidemiology......... ......... | Clinical Nutrition.................. ......... j Clinical Pharmacology .... ____ j Dermatology........................... ......... 1 Endocrinology (see Rule 5) ____ | Gastroenterology.................. ......... : Genetics.................................... ......... I Health Care Research......... ......... | Hematology ........................... ..... Hypertension ......................... . . Immunology St Rheumatology___ Infectious Disease................ __ i Metabolism (see Rule J)... -----; Oncology.................................. X | Pulmonary ............................. __ Renal St Electrolyte.............. __ CLYCOSAM.INOGLYCAN CHANCES ASSOCIATED VITH HEPATIC TUMORS: THE CONTRIBUTIONS OF REGENERATION AND NECROSIS. C. E. Kupchella, . M. Secskas,* J. S. KannedV,* and E. Espinosa*. Cancer Cancel and Department of Pathology, University of Louisville, School of Medicine, Louisville, Kentucky. Although 3lycosanlnoglycans (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 mala Sprague Dawley rats by partial hepatectocry and hepatic GAGs were evaluated at 4, 8 and 12 days post operatively. Necrosis was induced by: a) ligating the nedrum lobe, b) by resecting and placing median lobes in the peritonea 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. CtiN.Ru. 27 (*-)'38* `For abstracts submitted to Cardio vascular only, select single subcategory and enter code no. (l-bi in space above: (1) Clinical; (Z1 3asic Science (3) Eiectrophysiology-Oysrhytbmiaa: (!) Echotirdiograpnv-. (5) Radtology-Radiomidida: (5) Other. Subciassification | a designed to Sul in reviewing process I enlv and it independent of program I selection. PLEASE CHECK ABSTRACT CAREFULLY FOR APPEARANCE BEFORE MAILING CONFIDENTIAL Subject to Protective Order in ^ss_v. Conoco. Inc.. No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisian*------ BOTH THIS FORM AND THE FORM LETTER OF TRANSMITTAL MUST BE SIGNED BY A MEMBER I RULE 2) Charles E. Kupchella, Ph.D. Ipkuc type name) Revised May 1978 MEMBER'S SIGNATURE: ... . ______ .-r CMA 003902 t\ff\R\QM PU/3UC HfiACm ASteCWnoiJ oecemc^ 1^18 S/|n 0cgo 3097 Early Detection of Disease in Individuals Ex posed lo Vinyl Chloride. RichnrU Greenberg, MD. and Carlo Tamburro. MD, University of Loinsuile. Louis ville. Kentucky This is a collaborative comparison study of the effectiveness of ultrasound, naii bed capillary study and urinary glycosnmmcglycan excretions in vinyl chloride workers with biochemical dysfunction or histopathologicaliy documented injury. it represents an effort to detect early mor bidity in vinyl chloride workers. Included is Dr. Maricq's method cf investigating capil laries of the middle and distal phalanges of the fingers, including the nailfold, as a screening test for early changes from v iny I 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 unne. The results of the massive clinical study in Lcuisvtlle 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. 1 193 CONFIDENTIAL Subject to Protective Order in -ftQaa v Conoco, Inc,. No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003903 Presentation choice: Platform (Ji or Poster Session \d} V 5CIENTIHC PAPER Title: Computer Assisted Morphologic Quantitation of Collagen in Human Liver Biopsies. Kame(s) of authors, institutional affiliations and addresses: G. H. Barrows, M.D., M.J. Joyce, G.R. Schrodt, M.D., R. Greenberg, Ph.D., C.H. Tamburro, M.D. Departments of Pathology, Epidemiology and Biostatistics, and Medicine, University of Louisville, Louisville, Kentucky 40202. Underline name of author to notify and give Telephone (Area) 502-588-5341 xt _ Department of Pathology, University of Louisville School of Medicine, P.0. Box 40232, Louisville, Kentucky 40232 ------------------------------------------------------------------------------------------------------------ -------------------------------- Zip------------------- Abstract -- Double space. Because unfixed liver is rarely available for analytical studies, appraisal oi the extent of collagen must be made from appropriately stained sections of live biopsy material leading to judgemental as well as sampling error. We have de veloped a computer assisted morphologic technique to evaluate the distribution of collagen in liver biopsy material. We examined multiple biopsy samples from patients dying suddenly with no history of liver disease to establish normal values. A Hewlett-Packard 9864-A digitizer and 9815-A micro computer were used to quantitate areas of trichrome stainable collagen within the biopsy samples. Random area selection and statistical analysis proved to be important consider ations in determining the experimental model and these factors were readily in corporated into the calculator program. Stainable collagen estimates in normal liver varied from 0 to 6.1% (mean = 1.25%) in patients with no evidence of li\er disease and showed considerable variation even in different biopsies from the same patient. As anticipated, subcapsular biopsies had more collagen than deep biopsy; however, the specimens from several deep biopsies often had significant variation in collagen content. Differences in central, mid-zonal and portal collagen could be obtained using this method. The computer assisted morpholog ical technique for evaluating hepatic collagen appears to be readily adaptable to clinical studies. This study of normal liver implies considerable care must hp taltpn hefnre the clinical diagnosis of significant fibrosis can be.made.----- (Limit abstract to 250 words or less) CMA 003904 (OVER) Prc^i'iil.Ttiun choice: J laliunn M r ur I'liMur .sci'hjm c_, SCIENTIFIC PAPER :tnputer Assisted Morphologic vjan 11 tati cn of Collagen in Homan Liver 3icpsi2s. .Vame(s) of authors, institutional affiliations and nddrcsscs: G. H. Barrows, M.D., M.J. Joyce, G.R. Schrodt, M.D., R. Greenberg, Ph.D., C.H. Tamburro, M.D. Departments of Pathology, Epidemiology and Biostatistics, and Medicine, University of Louisville, Louisville, Kentucky 40202. Underline name of author to notify and give Telephone (Area) 502-588-5341 Exf^ Department of Pathology, University of Louisville School of Medicine, P.0. Box 40232, Louisville, Kentucky 40232" ------------------------------------------------------------------------------------------------------------------------------------------- Zip----------------- Abstract -- Double space. Because unfixed liver is rarely available for analytical studies, appraisal ot the extent of collagen must be made from appropriately stained sections of live biopsy material leading to judgemental as well as sampling error. We have de veloped a computer assisted morphologic technique to evaluate the distribution of collagen in liver biopsy material. We examined multiple biopsy samples from patients dying suddenly with no history of liver disease to establish normal values. A Hewlett-Packard 9864-A digitizer and 9815-A micro computer were used to quantitate areas of trichrome stainable collagen within the biopsy samples. Random area selection and statistical analysis proved to be important consider ations in determining the experimental model and these factors were readily in corporated into the calculator program. Stainable collagen estimates in normal liver varied from 0 to 6.11 (mean = 1.25%) in patients with no evidence of liver disease and showed considerable variation even in different biopsies from the same patient. As anticipated, subcapsular biopsies had more collagen than deep biopsy; however, the specimens from several deep biopsies often had significant variation in collagen content. Differences in central, mid-zonal and portal collagen could be obtained using this method. The computer assisted morpholog ical technique for evaluating hepatic collagen appears to be readily adaptable to clinical studies. This study of normal liver implies considerable care must be taken before the clinical diagnosis of sionificant fibrosis can be made. (Limit abstract to 250 words or less) (OVER) 003905 CMA \:'iL-r ;cnn Association f or The Study of l.ncr IVeases ABSTRACT FORM IS Type name. .iJdrcss. ;md telephone number of author u ho should receive correspondence tn Box C. and complete Boxes A and B. TYPE ABSTRACT BELOW. BE SURE TO STAY WITHIN BORDER CONFIDENTIAL Subject to Protective Order in Ross v. Conoco, Inc. , No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana A Cost of Publication Payment enclosed (S25.00) Check no. ____ JtZ (Payable to Elsevier Publishing Co.) nr Payment delayed ($10.00) Purchase order no Issued by.___________ ,, (Institution Name) A copy of this abstract must be at tached to original purchase order to aid in identification. B IMPORTANT The principal author affirms that the material herein: I) will not have been previously published or presented at any national meeting of a national so ciety: 2) that if human subjects were exposed to risks not required by their medical needs, the study was ap proved by an appropriate committee or. if no such committee was available and informed consent was needed, it vs as obtained in accordance with the principles enunciated in "The Institu tional Guide to DHFAV Policy on Pro tection of Human Subjects": or 3) any animal studies Conform with the ' Guiding Principles in the Care and Use of Animals" of the American Physiological Soytgty / sv x. (^/CiIPJT&'T) < Siwn it,:, u t*t I'nnvif.il -Nuihor) ) I'VJ EARLY HEPATIC HISTOLOGICAL ALTERATIONS AMOSC CHEMICAL (VINYL MONOMER) WORKERS, C.H. Tamburro, L. Hakk, and H. Popper, Division of Digestive Diseases and Nutrition and St. Anthony Hospital, University of Louisville, Louisville,- Ky., and Stratton Laboratory for Study of Liver Disease, ' Mt. Sinai School of Medicine, New York, N.T. Industrial vinyl chloride exposure is associated with hepatic subcapsular, portal, and perisinusoidal fibrosis land hyperplasia of both sinusoidal cells ani hepatocytes. Earlier histological studies, mainly on autopsy material, I lindicated that focal mixed hyperplasia (of hepatocytes and :sinusoidal cells) is the early histologic alteration indi cative of exposure. To substantiate this observation and ]its potential use in screening workers, liver biopsies from ,55 persons were investigated in double blind duplicative fashion; 34 were workers exposed to chemicals with hepatic biochemical abnormalities, and 21 were a coeparison group composed of 8 non-exposed persons and 13 exposed workers without hepatic abnormalities who had biopsies for non- Oliver related reasons. Of the exposed workers with abnor malities, 12 (35%) had a hepatic lesion consistent with 1 jexposure; 6 (18%) had focal hepatocytic hypaplasia; 6 (18ZJ had focal mixed hyperplasia. In contrast, <nly 5 of the .comparison group (2 exposed and 3 non-exposed persons) had .similar findings; 1 exposed and 2 non-exposed had only ; (focal hepatocytic hyperplasia. The other exposed worker j had focal mixed hyperplasia as did the other non-exposed j (individual who, subsequent to the biopsy, was found to have j angiosarcoma. Thus, only 19% of the comparison group i demonstrated hepatic lesions with chemical sposure, half of whom had worked with chemicals. Of the 55 biopsies, 22 were re-read double blindly 2 to 4 times over a 2-year jperlod. In addition., 9 of these individuals had second or third biopsies also read double blindly. Duplicate readings were identical in 86%, 9% had 1 positive and 1 questionable [ reading, and only 5% were read differently. Focal hepato- 1 cytic hyperplasia, in addition to the previously described ! pixed hyperplasia, appears to be the earliest identifiable changes consistent with chemical exposure and seem to be precursors of angiosarcoma. They are useful in the screening of chemical workers. I r Name Carlo H. Tamburro. M.D.. Professor of Medicine Address,,5_1__1__S_.__ FloyJd, Room 535 Louisville, KY. 40202 ______ Telephone, Office: 588-5252 (502) H,,mr. 895-2955_(502) _________ ___________ IIIHM (area cvJcl____________________________________________ __________ CMA 003906 16 Chloroacetaldehyde-induced Damage to Bacillus subtilis. A.D. Laumbach, U.N. Streips* and J.L. Wong. Bureau Foods, FDA and U. Louisville, Louis ville, Ky. Chloroacetaldehyde (CAA), a proposed metabolite of vinyl chloride meta bolism, has been shown to be mutagenic in the Salmonella assay system, and to specifically inhibit the growth of a recombination-deficient mutant of Bacillus subtilis (Ellmore, et^ al BBA, 442: 405, 1976). We have examined further the biological activity of this compound. First, CAA caused a significant increase in induced mutations to streptomy cin resistance. Using B^. subtilis 168, the relative mutation frequency (treated 5mM CAA/control) was 13.6, while using an her mutant of B. subtilis the frequency was 14.2. Secondly, a survey of the available repair-deficient mutants of B^. subtilis revealed, that the only strains sensitive to CAA were of the _rec-genotype. Furthermore, the several rec-mutants, show three types of response to CAA. Strong killing by CAA (inhibition of growth of 10 mm or more) was exhibited on the recA, recB, rec-4 (GSY1616), and recD27 (GSY1627) strains. Intermediate killing (3mm-8mm) was shown with strains bearing the recE, recF, and rec-13 (BD246) loci. All other strains examined (recC, recH, mtc-41, uvr, her, polA, as well as several repair-proficient cultures) grew in the presence of 1.1M CAA. These studies are part of an ongoing program sponsored by the MCA to determine the molecular basis for vinyl-chloride induced carcinogenesis. CMA 039q7 CONFIDENTIAL Subject to Protective Order in --s--v- Conoco, Inc.. No. 90-4837 I4th Judicial District Court Calcasieu Parish, Louisiana 16 Chloroacetaidehyde-mduced Damage do Bacillus subtilis. A.3. Laumbach, c Chioraa^ctalaeayae (CAA), a proposed metabolite of vinyl chloride meta bolism, has been shown to be mutagenic in the Salmonella assay svstem, and to specifically inhibit the growth of a recombination-deficient mutant of Bacillus subtilis (Ellmore, et_ al 3BA, 442: 405, 1976). Ue have examined further the biological activity of this compound. First, CAA caused a significant increase in induced mutations to streptomy cin resistance. Using B_. subtilis 168. the relative mutation frequency (treated 5mM CAA/control) was 13.6, while using an her mutant of B. subtilis the frequency was 14.2. Secondly, a survey of the available repair-deficient mutants of B^. sub til is revealed, that the only strains sensitive to CAA were of the rec-genotype. Furthermore, the several rec-mutants, show three types of response to CAA. Strong killing by CAA (inhibition of growth of 10 mm or more) was exhibited on the recA, recB, rec-4 (GSY1616), and recD27 (GSY1627) strains. Intermediate killing (3mm-8mm) was shown with strains bearing the recE, recF, and rec-13 (BD246) loci. All other strains examined (recC, recH, mtc-41, uvr, her, polA, as well as several repair-proficient cultures) grew in the presence of 1.1M CAA. These studies are part of an ongoing program sponsored by the MCA to determine the molecular basis for vinyl-chloride induced carcinogenesis. CMA 003908 17 Chloroacetaldehyde-induced Damage Co Bacillus subtilis. A.D. Laumbach, U.N. Streips*, and J.L. Wong. Bureau Foods, FDA, and University of Louisville, School of Medicine, Health Sciences Center, Louisville, Ky. 40232 (USA). Chloroacetaldehyde (CAA), a proposed metabolite of vinyl chloride metabo lism, has been shown to be mutagenic in the Salmonella assay system, and to specifically inhibit the growth of a recombination-deficient mutant of Bacillus subtilis (Ellmore, et al., BBA, 442:405, 1976). We have examined further the biological activity of this compound. First, CAA caused a significant increase in induced mutations to streptolycin resistance. Using 1J. subtilis 168, the relative mutation frequency (treated 5mM CAA/control) was 13.6, while using an her mutant of 1$. subtilis the frequency was 14.2. Secondly, a survey of the available repair-deficient mutants of B^. subtilis revealed, that the only strains sensitive to CAA were of the rec-genotype. Furthermore, the several rec-mutants, show three types of response to CAA. Strong killing by CAA (inhibition of growth of 10 ram or more) was exhibited on the recA, recB, rec-4 (GSY1616), and recD27 (GSY1627) strains. Intermediate killing (3tnm-8mm) was shown with strains bearing the recE, recF, and rec-13 (BD246) loci. All other strains examined (recC, recH, mct-41, uvr, her, polA, as well as several repair-proficient cultures) grew in the presence of 1.1M CAA. The effects of CAA on the biological activity of DNA molecules in vitro are being studied. These studies are part of an ongoing program sponsored by the Manufacturing Chemists Association to determine the molecular basis for vinyl-chloride induced carcinogenesis. CMA 003909 AD'oTtf j'.'i fn 2 or 5 It INHIBITION OF INTERFERON INDUCTION AS A SCREEN FOR THE CARCINOGENIC ' POTENTIAL OF CHEMICALS. Gerald Sonnenfeld," Mary Carol BarneS, and Uldis N. Streips, Department of Microbiology and Immunology, Univ. of Louisville School of Medicine, Louisville, Kentucky 40232. Type I interferon was produced after challenge of mouse embryo fibroblasts with Newcastle disease virus. This production of interferon could be sharply reduced by pretreatment of the cells with the known car- : cinogens benz-o-pyrene, aflatoxin, 2-aminofluorine, and the #4 fraction of tobacco smoke condensate. This finding suggested that inhibition of interferon induction (I-I-I) may serve as an indicator for the carcino genic potential of chemicals. We tested the screening potential of this assay by testing two chemical analogs, methylmethanesulfonate (MMS) a car cinogen and mutagen and ethylmethanesulfonate (EMS) a non-carcinogenic mutagen. Both of these chemicals are active in the Ames Salmonella assay but can be separated in "SOS" induction assays such as the Comptest. In ' the I-I-I assay, MMS was highly positive while EMS showed no inhbition of interferon induction. Thus, the I-I-I assay appears to have the potential to screen for carcinogens. Preliminary results using the carcinogen chlor-^| acetaldehyde and its non-carcinogenic analogs chloroethanol and chloroacetic acid support this idea. Following further verification of the dis crimination potential of the 1-1*1 assay, this test could become an integ ral part of a comprehensive battery of tests for chemical carcinogenicity_ A'lSl . be ' t 1 r l.'iU I 4,.v. J, .'..ilnp.isiio;: iV early date "ill 1c- appreciate'. I :!S I: tlie Ah,- '.rig! in i i Ke-.i :ip,' lOfite.ruii = L11.i- tile Line lines. Your abj-tiect will appear i: ,s.e .if 1:-. .ii.i.ii'iu'.! ef ( l.i.i .it Hfinr.icl-',ir.d Oncolc-v exactly as s.'.iL-.riiUod. I'Ur.se sen; ele;-, ;. . .. Ahw :i i ilions are printed on the l'c-eeroe side of this page. IndichU- 1 ' - >is..\me m.i.v. ...i into ce.trg'Oi'y for your abstract. : ini;'-. .i i i. i* u *',*'O ji vioe ' ti ///.) i \ ii^' .- .(ii- ! - i * I , ic.e 1 . , !i. i i.r;,..; Gerald Sonnenfeld, Ph.P. Department of Microbiology _ and Immunology, University of Louisville School of Medicine, Mealth Sciences Center Louisville, KY 40232. CONFIDENTIAL \: Subject to Protective Order in ` rs t Ross_v. Conoco, Inc., No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003910 It CO^FIBEN'VT iT i,, SH. v. _ConoooI_jnc^lVo> S0.4 n ctlc JlidlClal District Court 7 Calcasieu Parish, Louisiana fa!-. 2 r INHIBITION 0? INTERFERON INDUCTION AS A SCREEN FOR TH^ CARCINOGENIC POTENTIAL OF CHEMICALS. Geral4 Sonnenfeld,* Mary Caxo BarneS, and Uldis N. Streips, Department of Microbiology and Immunology Univ. of Louisville School of Medicine, Louisville, Kentucky 40232. Type I interferon was produced after challenge of moutie embryo fibroblasts with Newcastle disease vijrus. This production of interferon could be sharply reduced by pretreatment of the cells with 1 he known car cinogens ben2-o-pyrene, aflatoxin, 2-aminofluorine, and the #4 fraction of tobacco smoke condensate. This finding suggested that inhibition of interferon induction (I-I-I) may serve as an indicator for the carcino genic potential of chemicals. We tested the screening potential of this assay by testing two chemical analogs, methylmethanesulfonace (MMS) a car cinogen and mutagen and ethylmethanesulfonate (EMS) a non-carcinogenic mutagen. Both of these chemicals are active in the Ames Salmonella assay but can be separated in "SOS" induction assays such as the Qomptest. In the I-I-I assay, MMS was highly positive while EMS showed no inhbition of Interferon induction. Thus, the I-I-I assay appears to have the potential to screen for carcinogens. Preliminary results using the carcinogen chloracetaldehyde and its non-carcInogenic analogs chloroethanol and chloro- j acetic acid support this idea. Following further verification of the dis crimination potential of the I-I-I assay, this test could become an integ ral part of a comprehensive battery of tests for chemical carcinogenicity - .A fiS*.' lit- ; >> in- !.iu-r H iu,v. i. )$ift. is.ilmiissio;; a1 tm early (Utc nill l,o appreciate* This i: ? , the Abr.tiorijrinai iv-n-ly i |V ! l-.vi-eiiai ,c!t:.;is the 'olia.i lines. Your nbatract will appear i. ssna >:' !.`i.. . i;i.] or (`l.-.i- ..! tternaiol' ;:y ar.d Once'.oay exactly : aiiluniuod. Vlwise son: . .. Ah..':i\"'r !, :i; iicMons a: j i n.tist on the ricciae sklfe of this page. tmlicislv. ` ftpo.fi; u-o ;`.i<;..,...vrialo category for your abstract. ^ i i i...iva.-t a.i. ;;.i`l t.ii.ua*ian (!_-i :,i riii.t r.: ! .-i (; ' :: oca! Tr:v; > ' .c.. N'. r.n- i. . ... . r ;ii:i : Gerald Sonnenfeld, Ph.D. Department af Microbiology _ and Immunology, University of Louisville School of Medicine, Neale ti Sciences Center Louisville, KY 40232. i . , ;* -. . . 0 , ;. >t-.*! ; ..i-.'i i-1;-1 1 ... .. I'.S.A. CMA 0039H 19 Reproduction Copy tor PROGRAM AND ABSTRACTS of the 30TH ANNUAL MEETING Of-THE TISSUE CULTURE ASSOCIATION, INC. Properties of 14 Peek 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 RPM allowed maintenance for at least 3,4 weeks with cells exhibiting excellent morphology without sloughing. After treating monolayers with Hoechst 33258 DN'A stain, intense cytoplasmic 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 RIA assay revealed.31 to 47 ratio-units/0.2 ml after 2 to 14 weeks in medium with 10% fetal calf serum. ' The 14 week old cultures were extremely well adapted to maintenance in serum free medium for 14 additional days when supplementing with 300 ng/ral of insulin, in contrast to 1 week cultures which survived well for only 5 days wi Lhout serum.. The HDsAg-titers of supernaf.es from these 1 week or 14 week cultures, were very similar to the previous harvests in 30% scrum. In addition, both young and 1.4 week cultures were similar in their rate of acid production; and in that both elaborated serum albumin, fibrinogen, transferrir '-nd alpha-2 macroglohulj n, the proteins tested to date. CMA 003912 ABSTRACT MUST BE RECEIVED AT SOCIETY OFFICE 11Y rUESQAY, DECEMBER 19 Plraie consider this abstract for inclusion in (he tentatively listed minisymposium, m&L_ ; Chernical_axciii Qgeii esi Indicate below the numberj and titles of sessions in which yout abstract migh be programed (see Topic Category List); if A30------- Title &a-tTso{shysi-Qloy---------------------- 3/ M Title Tuao laacTfjU 1979 FASEB Abstract Form .2 DO NOT HOLD THIS FORM Mail to: American Association of Immunologist 9650 Rockville Pike Bethesda, Maryland 20014 IMMUNOLOt LIVER-SPECIFIC.F ANTIGEN IN TRANSPLANTABLE HEPATOMAS HAVINl DIFFERENT GROWTH RATES. Enrique Espinosa, Sue Chia*, Sam Caple* and Charles Kupchella*. Dept. Pathology and Cancer Center, Univ. of Louisville School of Medicine, Louisville KY 40232 In the course of studies of antigenic changes associatec with liver neoplasia we measured the relative concentratioi of liver-specific F antigen in fast (7777), slow (9618A) at medium (5l23tc) growing Morris hepatomas in comparison wit! its concentration in normal adult rat liver. F antigen va: solubilized from each tissue by aqueous homogenization. T! supernatant containing the antigen was then tested by a double diffusion dilution assay capable of detecting a min mum of 1-2% the concentration of antigen present in normal rat liver. The F antigen antiserum used was prepared ac cording to Fravi and Lindenmann by immunizing CBA mice wi BALB/c mouse liver extract. F antigen was undetectable the fast growing hepatoma and ranged from less than 2% t 10% 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 wi their rates of growth. (Supported-^.* -fey-j-the Manu facturing Chemists Association^ -------- Subject to Protective Order in Ross v. Conoco, Inc. , No. 90-4S37 14th Judicial District Court Calcasieu Parish, Louisiana All compounds lhal are designated by code or initial letters must be identified adequat. in the abstract, e.g., MJ-J999: 4-(2-isopropyl3mino-l-hydroxyelhyl) methanesulfona lide hydrochloride. MAILING ADDRESS OF FIRST AUTHOR (Please Prim or Type. Provide full name rather than initals.) Enrique Espinosa, M.D. .... 511. .South .Floyd .Street .... Louisville..... JOf... zip .40.232 Telephone No.: AreaCod5P.2.. #5.88-5525........ CMA 003913 Each Abstract Form submitted MUST BE SIGNED by a member of the AMERICAN ASSOCIATION OF IMMUNOLOGISTS. Enriqye Espinosa iMerrfet'i Name Please Print or rvtde full name ) / / (Member s Signature) Member's telephone no.: Area Code.. 502 .. .*.588.-5525... ABSTRACT MUST BE RECEIVED AT SOCIETY OFFICE UY lUtSDAY. DECEMBER 19 2 Plcaif conuder ihu abstract for inclusion in ihe tentatively luted minis) mposiura, w3 ; __hemic_aL_C3HLinagnesis------------------------- Indicate below 'he numbns and lilies of sessions in winch >out abstract mijh be programed (set Topie Category List); 1/ 4.3Q-------- Title i.i-vqr Pat-bophys-i-a-l-ogy----------- -- li 402------- Tlllt T-unsoC.-5i-oaU>gv-------------------------~--- 3/ hOy Title T'"""r `jpo'-Ole-tnripji;---------------- 1979 DO NOT FOLD THIS FORM FASEB Abstract rr--orm Mail to: American Association of S.Ttmunologist 9SS0 Rockville Pike SeifiL'tda, Maryland 20014 3!TT 14T. Subject to , - --S Ross v. c.'-; . Ir.c, , Lo , ..; ;J 7 14 N" ' '1 ' 1 District Court Calccsleu Parish,, Louisiana immunolo presentation preference Preferred choice (CHECK ONE ONLY) (J Ponce presentation Slide presentation Indifferent If your first choice is unavailable, you will SI Accept the alternative Withdraw the abstract 16 rum. films (silent or optical sound) are permitted if essential to 10-minute slide session presentation. MOVIE YES....,,... NO...)?.... Submit justification by letter to Society Office, with abstract. IMPORTANT Before you complete this form: See the enclosed rules for eligibility of Papers- See sample abstracts and typing instruc tions on reverse side. Use enclosed Check List for preparation of abstract. LIVER-SPECIFIC F ANTIGEN IN TRANSPLANTABLE HEPATOMAS HAVIN DIFFERENT GROWTH RATES. Enrique Espinosa, Sue Chia*, Sam Caple* and Charles Kupchella*. Dept. Pathology and Cancer Center, Univ. of Louisville School of Medicine, Louisville KY 40232 In the course of studies of antigenic changes associate with liver neoplasia we measured the relative concentratio of liver-specific F antigen in fast (7777), slow (9618A) a medium (5123tc) growing Morris hepatomas in comparison wit its concentration in normal adult rat liver. F antigen wa solubilized from each tissue by aqueous homogenization. T supernatant containing the antigen was then tested by a double diffusion dilution assay capable of detecting a min mum of 1-22 the concentration of antigen present in normal rat liver. The F antigen antiserum used was prepared ac cording to Fravi and Lindenmann by immunizing CBA mice wit BALB/c mouse liver extract. F antigen was undetectable in the fast growing hepatoma and ranged from less than 22 to 102 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 wi their rates of growth. (Supported in part by the Manu facturing Chemists Association) The original typed copy of this abstract form (for reproduction by photo-offset) with - 8 photocopies. - one set of author index cards, - three abstract identification ante. and rBurn Program Confirmation Postal All compounds that are designated by code or initial letters must be identified adcquai in the abstract, e.g., MJ-1999: 4-(2-isopropyIamino-l-hydroxyethyt) melhartesulfon, tide hydrochloride. must reach the Society office NO LATEX THAN TUESDAY. DECEMBER 19. Each Abstract Form submitted MUST BE SIGNED by a member of the AMERICAN ASSOCIATION OF IMMUNOLOGISTS. MAILING ADDRESS OF FIRST AUTHOR (Please Print or Type. Provide full name rather than inital*-) __________ Enriqye Espinosa IMlei mber's Name, please Print o' Vidreit tun wr* \ Enrique Espinosa, M.D. .... 51L .South. .Floyd .S.treet. / 9/ (Member's S*QAr*tu<J Member's telephone no.: Are* Code.. 5Q2................ #. 388.-55.25. ....Louisville...... KY___ Zip .40.23.2. Telephone No.; Area Cod^.02,, #588-5525.......... CMA 003914 ABSTRACT MUST BE RECEIVED AT SOCIETY OFFICE BY TUESDAY, DECEMBER 19 21 Please consider this abstract for inclusion in the tentatively listed minisymposium* m Ai : Chemical Carcinogenesis Indicate below the numbers and titles of sessions in which yout abstract migh be programed (sec Topic Category List); if 4 30 Title Liver Pathophysiology 21 MQl--------Title 'Fumor--Biology------ -------------- 3/ -------Ti,le Tpmor Specif ic~AnLlgeinj-------------- 1979 FASEB Abstract Form DO NOT FOLD THIS FORM Mail to; Dr. Kenneth M. Endicott, Executive Office American Association of Pathologist* 9650 Rockville Pike Bethesda, Maryland 20014 C ONFIDiri'J T1XL------------------- v'LO^GC't uO Protective Ordar in Foil v. Conoco, Ino , , Ho. 90 -- 4337 14th Judicial District Court Calcasieu Parish, LouisiarratTHOLOGY PRESENTATION PREFERENCE Preferred choice (CHECK ONE ONLY) & Poster presentation Slide presentation G Indifferent If your first choice is unavailable, you will dF Accept the alternative Withdraw the abstract 16 mm. films (silent or optical sound) are permitted if essential to 10-minute slide session presentation,, MOVIE YES.......... NO.x....... Submit justification by letter to Society Office, with abstract. IMPORTANT Before you complete this form: See the enclosed rules for eligibility of paper*. See sample abstiacts and typing instruc tions on reverse side. Use enclosed Check List for preparation of abstract. TWO LIVER ANTIGENS UNDETECTABLE IN A FAST GROWING LINE OF TRANSPLANTED HEPATOMA (MORRIS HEPATOMA 7777). Enrique Espinosa, Sam Caple*, Charles Kupchella* and Sue Chia*. Dept. Pathology and Cancer Center, Univ. of Louisville School of Medicine, Louisville, KY 40232 During investigations of antigenic changes in liver tumor: we examined fast (7777), medium (5123tc) and slow (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 5123tc and 9618A gave immunoelectrophoretii patterns similar to normal liver. In contrast, hepatoma 777 showed 2 arcs of precipitation missing. This was confirmed by the finding that after absorption of antiserum with hepa toma 7777 two lines of precipitation given by normal liver extract persisted whereas absorption with extract of norm^^h liver, hepatomas 5l23tc or 9618A abolished all lines of 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 Sephadex G-200 and Bio-Gel A-5m columns these antigens eluted as proteins of approximately 51,000 and 240,000 daltons respectively. The fact that 7777 is the fastest growing and least differentiated of the tumors studi. suggests a possible functional relationship between the absent antigen and these properties. (Supported in part by the Manufacturing Chemists Association) The original typed copy of this abstract form (for reproduction by photo-offset) with 8 photocopies, one set of author index cards, - three abstract identification cards, - and return Program Confirmation Postal must reach the Society office NO LATER THAN TUESDAY,DECEMBER 19. All compounds that are designated by code or initial letters must be identified adequately in the abstract, e.g., MJ-1999: 4-(2-isopropyIamino-l-hydroxyeth>i) methanesulfonanilide hydrochloride. Each Abstract Form submitted MUST BE SIGNED by a member of the AMERICAN ASSOCIATION OF PATHOLOGISTS. MAILING ADDRESS OF FIRST AUTHOR (Please Print or Type. Provide full name rather than initali.) Enrique Espinosa, M.D. 511 South Floyd Street Enrique Espinosa (Member's Name Please Print aigvoe ft'ovrtte full name.) (Member's 5 tone'tore) ^502 588-5525 Member's telephone no.: Area Code. ..........#................. Louisville KY 40232 ......................................................... Zip................. 502 Telephone No.: Area Code 588-5525 Signing member, arc you willing to chair a session? ( ) yes. category #______, C ) no Jf yes, are you also willing to give an overview or introductory lecture? ( ) yes ( ) no CMA 003915 Alilei i 1 International Association For Tile Study Of The Liver Abstract Submission Dates: IASL - June 15, 1980 AASLD-July 15, 1980 ABSTRACT FORM 2 Abstract submitted to: AASLD M IASL Type name, address, and telephone number of author who should receive correspondence in Box D, and complete Boxes A and B and C. < For office use) CATEGORIES - Indicate category of choice 1. Immunology 2. Collagen Fibrosis 3. Morphology 4. Viral Hepatitis 5. Chronic Hepatitis @ Metabolic Liver Disease 7. Complications of Liver Disease 8. Alcohol and the Liver 9. Bile Acids 10. Bilirubin Cholestasis 11. Pediatric Liver Disease 12. Miscellaneous A Cost of Publication Payment enclosed ($25.00) Check no. ...... 7^ (Payable to Elsevier Publishing Co.) or Payment delayed ($30.00) Purchase order no Issued by (Institution Name) A copy of this abstract must be at tached to original purchase order to aid in identification. Payment must be made in U.S. Currency. B IMPORTANT The principal author affirms that the material herein: I) will not have been previously published or presented at any national meeting of a national so ciety: 2) that if human subjects were exposed to risks not required by their medical needs, the study was ap proved by an appropriate committee or. if no such committee was available and informed consent was needed, it was obtained in accordance with the principles enunciated in "The Institu tional Guide to DHEW Policy on Pro tection of Human Subjects": or 3) any animal studies conform with the "Guiding Principles in the Care and Use of Animals" of the American Physiological Society. (sUr/b (Signuiure of Principal Author) TYPE ABSTRACT BELOW. BE SURE TO STAY WITHIN BORDE OXIDATIVE AND DETOXIFYING ABILITY OF LIVER MESENCHYMf PARENCHYMAL CELLS IN THE METABOLISM OF XENOBIOTICS Du, J. and Tamburro, C.H., Liver Research Laboratorit Dept, of Medicine, University of Louisville, Louisvil The metabolism of xenobiotics, by the liver most ofte volves oxidation via mixed function oxidase (MFO) anc hydryl detoxification with glutathione. Hepatic tox: and carcinogenicity are modified by these two pathway Vinyl monomer (e.g. vinyl chloride) exposure is assc with liver parenchymal cell (hepatocytic cell, HC) tc and mesenchymal cell (sinusoidal cell, SC) carcinoger even though the hepatocyte is the main cell for xenot oxidation. The metabolic ability of these two cell g was assessed by isolation of SC using pronase digest! and HC using collagenase profusion. Viability assess 90% by trypan blue exclusion and cross contamination mated (< 1%) by pryuvate kinase activity. MFO activi (demethylation benzphetamine), glutathione transferas (E) using (1,2, epoxy-(P-nitro nitrophenoxy) propane substrate, glutathione transferase A,C and E (A) nitrobenzyl chloride) as a substrate and glutathio^M tase (GR), were determined in subcellular isolated HC SC fractions. Results based on protein content (N mo mln/mg protein) were: EA SC 42.9 + 17 18.5 + 6 HC 82.5+11 215.0 + 61 GR 47.4 + 2 65.0 + 7 MFO 7.49 + 16.0 + and activity in mole/10 cell were: E A GR SC 1.16 0.5 1.28 HC 65.6 220. 52. MFO 0.026 4.7 These results demonstrate both the greater average, as as total, cellular ability of HC to oxidize xenobiotic More importantly, it demonstrates an increased abilit' detoxify the xenobiotic's toxic metabolites. This gr ability may account for less severe HC injury and pla important role in preventing HC malignant, transformat In contrast, SC ability to oxidize but its lesser abi to detoxity xenobiotic. may account for SC's potential maligBetvt---fe-gansfennafcien.--------------------------------------------------------- D Nam* Carlo H. Tamburro. M.D. Division of Digestive Diseases & Nutriti Address Oil South Floyd - MDK Bldg. Km. 535 Louioville, Kentucky--40292 C Indicate if the Presentor is: Fellow Student March 19*0 CMA 003916 502-588-5252 Hnm<., 502-895^ Telephone, Office: CONFAB ITT IAL Urea code) Subje''+ ''roteot^v') 0 -'-i- ^ Pops v .ono.,0. In:. , "s. ' ^ "iZ'l 14th Judicial District Court Calcasieu Parish,, Louisiana c *2 O <O75 Oo d_ olS o , n) > jn -: ^ urt 2 WM rl'il*u^ Uiici / . v .t International Association l-or The Study O' i he L,'-.;- AcJ:::c: Subniiision Daces: lASL - June 15, 190 AASLD July 15, 1980 ABSTRACT FORM 2 Abstract submitted to: AASLD "E IASL O Type name, address, and telephone number of author ?'"''itder.ee in Box D, ar.d rcmplete Boxes \ ,c si'.ou.d receive CATEGORIES - Indicate category of choice TYPE ABSTRACT BELOW. BE SURE TO STAY WITHIN 3CRD 2. Collagen Fibrosis 3. Morphology 4. Viral Hepatitis 5. Chronic Hepatitis (0 Metabolic Liver Disease 7. Complications of Liver Disease 8. Alcohol and the Liver 9. Bile Adds 10. Bilirubin Cholestasis 11. Pediatric Liver Disease 12. Miscellaneous Cost of Publication Payment enclosed (S25.00) Check no._____^2 (Payable to Elsevier Publishing Co.) nr Payment delayed (S30.00) Purchase order no Issued by I institution Name) A copy of this abstract must be at tached to original purchase order to aid in identification. Payment must be made in U.S. Currency. B IMPORTANT The principal author affirms that the material herein: I) will not have been previously published or presented at any national meeting of a national so ciety; 2) that if human subjects were exposed to risks not required by their medical needs, the study was ap proved by an appropriate committee or. if no such committee was available and informed consent was needed, it was obtained in accordance with the principles enunciated in "The Institu tional Guide to DHEW Policy on Pro tection of Human Subjects ": or 3) any animal studies conform with the "Guiding Principles in the Care and Use of Animals" of the American OXIDATIVE AND DETOXIFYING ABILITY OF LIVER MESENCHY1 PARENCHYMAL CELLS IN THE METABOLISM OF XENOBIOTICS Du, J, and Tamburro, C.H., Liver Research Laboratori Dept, of Medicine, University of Louisville, Louisvi The metabolism of xenobiotics, by the liver most oft volves oxidation via mixed function oxidase (MFO) an hydryl detoxification with glutathione. Hepatic tox and carcinogenicity are modified by these two pathwa Vinyl monomer (e.g. vinyl chloride) exposure is ass with liver parenchymal cell (hepatocycic cell, HC) c and mesenchymal cell (sinusoidal cell, SC) carcinoge even though the hepatocyte is the main cell for xeno oxidation. The metabolic ability of these two cell was assessed by isolation of SC using pronase digest and HC using collagenase profusion. Viability asses 90% by trypan blue exclusion and cross contamination mated (< 1%) by pryuvate kinase activity. MFO activ (demethylation benzphetamine), glutathione transfers: (E) using (1,2, epoxy-(P-nitro nitrophenoxy) propane substrate, glutathione transferase A,C and E (A) usi: nitrobenzyl chloride) as a substrate and glutathione tase (GR), were determined in subcellular isolated H< SC fractions. Results based on protein content (N me min/mg protein) were: E A GR MFO SC 42.9 + 17 18.5 + 6 47.4 + 2 7.49 + HC 82.5+11 215.0+61 65.0+7 16.0 + and activity in mole/10^ cell were: E SC 1.16 HC 65.6 A 0.5 220. GR 1.28 52. MP0 0.026 4.7 These results demonstrate both the greater average, a as total, cellular ability of HC to oxidize xenobioti More importantly, it demonstrates an increased abilit detoxify the xenobiotic's toxic metabolites. This gi ability may account for less severe HC injury and pla important role in preventing HC malignant transformat In contrast, SC ability to oxidize but its lesser abl to detoxity xenobiotic may account for SC's potentiaJ (Signature of Principal Author) D Name__ Carlo H. Tamburro. M.D. Division of Digestive Diseases & Nutriti Address 511 'South Floyd -"THDK' BT3g'l" Km. 5JS bouiovilicy Kentucky--40293 C Indicate if the Presentor is: Fellow Student Marcn lSO Telephone, Office: 502-588-5252 Home: 502-895-25 urea co<te> Urea code) CMA 003917 ASPET/SOT 1982 Louisville, Kentucky Deadline far Receipts May 7,1982 Abstract Processing Fee: $20.00 Select Category Numbers and Titles (See list over) 1. 79_ .2 15 Presentation Preference _ Poster X_ Oral (slides) _ Indifferent If your choice is not available _ Willingly accept the alternative X_ Grudgingly accept the alternative Withdraw the abstract Poster boards are 6' wide x 4' high. 23 Toxic, Carcinogenic & Safe Exposure Levels in Vinyl Chloride Induced Hepatic Angiosarcoma. Carlo H. Tamburro* and Richard A. Greenberg* (SPON: William Waddell). Depts. of Medicine & Community Health, Univ. of Louisville, Lou., Ky, 40292. Vinyl chloride (VC) induced hepatic angiosarcoma (HA) has rapidly decreased since 1974. The relationship of latency & duration of exposure was studied in 21 North American (NA) cases and 62 worldwide (WW) reported cases; 92Z of NA 6 WW cases had long exposures (LE) (10-33 yrs); 7 cases (8%) had short exposures (SE) (3.5-7 yrs). The mean duration in both LE & SE groups was 18.5 yrs. with 2 peaks at 15 & 25 yrs. Deaths from HA began in 1955, peaked in 1976 & rapidly fell thereafter. HA peaked in Canada (CA) in 1973 and USA 1974, France (FR) 1976, & Germany (GE) 1978. Operations began in CA in 1941 & USA 1939-44; FR 1941-57, & GE 1952-60. Exposure levels reported in 1940's of ^2000 ppm; in 1950's ^200-500 ppm, in early 1960's ^50-500 ppm, late '60's & early '70's ^50-250 ppm, & 1-10 ppm after '74. No relationship was found in total average exposures (TAE) range of 50 ppm; 200-1000 ppm range showed a direct relation of lower TAE to shorter latency, while higher TAE (1000-2000 ppm) had longer laten cies. The data suggests VC has a biological threshold level, an optimum carcinogenic level and subcarcinogenic toxic level for occurrences of HA. Prepare your abstract carefully. It will be printed by photo-offset exactly as received. See the example (over) for lay-out end style. For elite typewriters (12 pitch), set the margins at 36 and 98 (62 spaces). Use 25 lines. For pica typewriters (10 pitch) set margins at 30 and 81 (51 spaces). Use 25 lines. The first typed letter should nearly or just touch the top and left type box (blue margin lines). Title. Leave no margins. Use a short, descriptive title. Use upper and lower case letters. Authorship. Underline names. Place an asterisk* after the name of each author not a member of ASPET or SOT. If no author is a member, type the following after the name of the last author: (SPON: sponsor's name). Continue on the same line with the authors' institutional affiliation(s), city, state and zip code. Text. Start the text on the next line, indenting 3 spaces. Subsequent lines should extend the entire width of the type box. The text should have all of the elements of a report: introduction, method, result(s) and conclusion. It is improper to substitute "The results will be discussed" for the results and conclusion. Adequately identify all chemical compounds used. Do not fold abstract. Be certain abstract is prepared securely for mailing. AU subsequent correspondence must ref rence the first author to enable us to identify the abstrset. The Program Committee selects chairmen and overview ers from volunteers. See below. First author phone #: (502) 588-5251 Membership of author or sponsorC^SPET^C; SOT 1. Member signature 2. Name (Type or print) I ______ William Waddell. M.D. 3. Will you chair a session? __ yes; \ no In what category Will you give a 25 minute overview? __ yes; _^no; In what category Mail the original, 3 copies, a self-edtkessed post card for program confirmation and 1 check to Kay A. Croker, Acting Executive Officer, ASPET, 9650 Rockville Pike, Bethesda, MD 20814, USA. Reprints cost $15 per 100, lots of 100 only. Order below and fill in the mailing label, below left. Invoice: Abstract Processing Fee Reprints* Quantity ordered__cost: TOTAL $ 20.00 _______ $ Mailing label for reprints (Nsme and Address): CONFIDENT T 4 T. BoSSUsbJvCtrt0 Frotectlve Order In ~J- gjZSZzJ.n^ No. 90-4637 14.h Judicial District Court Calcasieu Parish. Louisiana Mailing label to the first author (Name and Adtk-ess): CMA 003918 PREPRINTS CONFIDENTIAL Subject to Protective Order in Poss v. Conoco, Inc., No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana 003919 The reproducibility in identifying these histological lesions is quite high. Under double blind conditions there was consistent histological identification of chemical injury in both duplicate readings of single biopsies as well as single readings of multiple biopsies in the same individual. Inconsistency appears to occur more frequently among the duplicate readings of wedge biopsies and between wedge and needle biopsies than between duplicate readings of needle biopsies or readings of multiple needle or wedge biopsies in the same individual. This may be due to the increased time and attention needed for wedge section review in order to determine the absence or presence of these lesions anywhere in the specimen. There are, however, some pitfalls that require caution. The quality of the histological preparation is of vital importance. Technical artifact, excessive thickness, torn or fragmented spec Tv 'C ^ ` *''* 03 o^-f-H z^y c " <V O ctf ^ ^ -TMj HA , ft I'v s t? 5 ;'/-V?V -/ C *-? f ^ 1_^ f -- -c -tO''^/I 'rw~j a7^ ^ 1/ j C/'~, a > rC?j// ^zj *-* ,ha eg >` -eg oeg ?/, \+p-1 <-v imens will greatly reduce the ability to correctly identify these lesions. The use of silver impregnation for assessment of the reticular structure is very useful in verifying focal areas of hepatocellular hyperplasia. The presence of fatty infiltration and/or chronic disease (hepatitis, granuloma, etc.) may prevent the identification of these lesions. We were, however, able to correctly identify the presence or absence of these early lesions in the six individuals who also had a concomitant granulomatous process (four individuals with granuloma were repeatedly read as negative and two individuals with gran uloma were repeatedly read as positive for chemical liver injury). The more frequent elevation of alkaline phosphatase in those individuals with histological evidence of chemical injury may provide some guidance in sequential uses of laboratory screening tests. During the first phase, tests with the greater sensitivity for identifying liver injury, such as the ICG and ALT can be used. Positive results can be followed up by using tests of greater specificity, such as the alkaline phosphatase, to further identify those at high risk of chemical injury. CMA 003920 10 of acute or chronic injury, appear to be the earliest change associated with chemical exposure. Subsequently, there occurs perisinusoidal fibrosis associated with a proliferation of fibroblasts and/or its precursor cell, the Ito cell. In later stages sinusoidal cell activation occurs involving both the macrophagic and endothelial lining cells. These changes are associated with focal sinusoidal enlargement. In the late stage mixed hyperplasia (hepatocyte and sinusoidal cells) becomes prominent, followed by sinusoidal cell dysplasia with palasading of cells, and finally malignant transformation. Our data demonstrates that focal hepatocytic hyperplasia is the earliest consistently identifiable histological finding in industrial workers exposed to vinyl monomer chemicals. The very limited occurrence of these findings in non-chemical workers and the fact that focal mixed hyperplasia, a more advanced lesion, was only found in chemically exposed workers and in a nonchemical worker with vinyl chloride exposure (via hair spray), adds further confirmation that these lesions are related to prolonged or repeated periods of chemical exposure. Whether these lesions should be viewed as precursors to the development of cancer or only as an early reflection of the degree and duration of chemical exposure cannot be determined at this point in time. The unchanging and stable status of screening studies over a seven year follow-up period and the histo logical similarities among the serial biopsies of these workers suggest that these early lesions are non-progressive when the affected individuals are removed from exposure (7). The presence of sinusoidal cell dysplasia, however, has been associated with the later development of angiosarcoma as illustrated in one of our cases and may represent a true precancerous or a non-reversible lesion. CMA 003921 this worker cohort had shown that all the cases of angiosarcomas had an average vinyi chloride exposure ranking (AER) of 3.5 or greater. AES's of 3.5 or greater occurred in 455 of the CLI group. In contrast, those with liver diseas (LD) alone, only 2215 had a vinyl chloride exposure rating of 3.5 or greater and in the chemically exposed workers with no evidence of liver disease, 325 had a rating of 3.5 or greater. Biochemical Studies A study of the biochemical abnormalities among the two histological groups of chemical workers with evidence of liver disease is illustrated in Figure 7. The five screening tests with the best degree of sensitivity and specificity in identifying latent liver injury were reviewed. Indocyanine green clearances, at the 0.5 mg/kg dose, provides the most sensitive and the most specific of the laboratory screening study, followed by the alanine amiontransferase and aspartase aminotransferase, gamma glutamyl transpeptidase and alkaline phosphatase (6). However, specificity for chemical injury appeared to be associated more with abnormalities in the alkaline phosphatase, which was far more frequently abnormal in workers with chemical liver disease than in those with non-chemical liver injury. In contrast, the ALT, AST, GGT, and ICG were more frequently abnormal in those workers with liver disease of a non-chemical origin. DISCUSSION Figure 8 provides the most likely progression in the development of vinyl chloride and other vinyl monomers hepatic injury and cancer development. These findings confirm those previously reported by Thomas and Popper (2) in their original pathological studies of vinyl chloride associated hepatic angiosarcoma and hepatic injury. Focal hepatocytic megalocytosis associated with focal increases in reticulum structure, in the absence of other evidence CMA 003922 this worker cohort had shown that all the cases of angiosarcomas had an average vinyl chloride exposure ranking (AER) of 3.5 or greater. AER's of 3.5 or greater occurred in 45% of the CL I group. In contrast, those with liver disease (LD) alone, only 22% had a vinyl chloride exposure rating of 3.5 or greater and in the chemically exposed workers with no evidence of liver disease, 32% had a rating of 3.5 or greater. Biochemical Studies A study of the biochemical abnormalities among the two histological groups of chemical workers with evidence of liver disease is illustrated in Figure 7. The five screening tests with the best degree of sensitivity and specificity in identifying latent liver injury were reviewed. Indocyanine green clearances, at the 0.5 mg/kg dose, provides the most sensitive and the most specific of the laboratory screening study, followed by the alanine amiontransferase and aspartase aminotransferase, gamna glutamyl transpeptidase and alkaline phosphatase (6). However, specificity for chemical injury appeared to be associated more with abnormalities in the alkaline phosphatase, which was far more frequently abnormal in workers with chemical liver disease than in those with non-chemical liver injury. In contrast, the ALT, AST, GGT, and ICG were more frequently abnormal in those workers with liver disease of a non-chemical origin. DISCUSSION Figure 8 provides the most likely progression in the development of vinyl chloride and other vinyl monomers hepatic injury and cancer development. These findings confirm those previously reported by Thomas and Popper (2) in their original pathological studies of vinyl chloride associated hepatic angiosarcoma and hepatic injury. Focal hepatocytic megalocytosis associated with focal increases in reticulum structure, in the absence of other evidence CMA 003923 A comparison of the reproducibility of the biopsy readings is shown in Tables 3, 4 and 5. Table 3 illustrates the high degree of consistent readings between multiple biopsies in the same individual. In only one individual was one of the four biopsies read differently--the wedge was read differently from the three needle biopsies. Table 4 makes a comparison between readings of multiple needle biopsies or multiple wedge biopsies from a single individual. In these cases there was consistent agreement in all readings. Table 5 illustrates the consistent readings of the same biopsies over the three-year period. All the needle biopsy duplicate readings and 12 of the 14 wedge duplicate readings were the same. At least 96% (43/45) of all readings, whether duplicate or from multiple biopsies, provided reproducible and consistent findings. Correlation of Histological Lesions With Exposure Biopsies from all the chemical workers were divided into three categories on the basis of final diagnosis: 1) individuals with no evidence of histological liver disease (N) 2) individuals who had liver disease but no evidence of chemical injury (LD) 3) individuals who had chemical liver injury identified by the histolog ical criteria specified (CLI). A comparison was made between the average vinyl chloride exposure ranking of each worker and his/her final histological diagnosis. The exposure rankings were not known to those making the final histological determination at any time during the three year period. As illustrated in Figure 6 the group of workers with evidences of chemical liver injury had the highest percentage of individuals with the highest ranking of exposure to vinyl chloride. Previous studies of district Court sn- Louisiana for each job for each year based on the best available information and i "tjjtr-'ai a-:e'tis = . Z:;r .cr.-^r's cv tuI a :i ve e,::c;jra :c =acr. cf toe 22 chemicals was based on one various job classifications he/she held during their employment. A detailed explanation of this system and an actual in-the-field demonstration of the ability of this system to identify work-related liver injury (angiosarcoma) has been published elsewhere (4,5). RESULTS Thirty-seven percent (13/35) of the exposed workers with screening test abnormalities had hepatic lesions consistent with chemical exposure. Among the exposed workers without biochemical abnormalities 23% (3/13) had hepatic lesions consistent with chemical exposure. In the non-worker comparison group none of those with normal biochemical screening tests and 17/19 (89%) with abnormal biochemical screening tests had hepatic lesions consistent with chemical expo sure. Of the remaining two, one had focal hepatocellular hyperplasia; the other had focal mixed hyperplasia and early peliosis hepatis. This latter individual was later found to have angiosarcoma possibly from vinyl chloride hair spray exposure. The former individual had no history of chemical exposure .and/or alcohol consumption, but did have hepatobiliary tract disease in the form of biliary stones. Table 1 lists the hepatic lesions found in those with and without biochemical abnormalities for the entire group. Reproducibility An evaluation of the reproducibility of these biopsy readings was carried out in 17 individuals who had 32 biopsies. Seven individuals had only one biopsy (2 needle, 5 wedged) and 10 individuals had their biopsies read in dupli cate. Four had needle biopsies with 9 readings and 6 had wedge biopsies with 14 readings (Table 2). CMA 003925 for each job for each year based on the best available information and industrial expertise. Each worker's cumnulative exposure to each of the 22 chemicals was based on the various job classifications he/she held during their employment. A detailed explanation of this system and an actual in-the-field demonstration of the ability of this system to identify work-related liver injury (angiosarcoma) has been published elsewhere (4,5). RESULTS Thirty-seven percent (13/35) of the exposed workers with screening test abnormalities had hepatic lesions consistent with chemical exposure. Among the exposed workers without biochemical abnormalities 23% (3/13) had hepatic lesions consistent with chemical exposure. In the non-worker comparison group none of those with normal biochemical screening tests and 17/19 (89%) with abnormal biochemical screening tests had hepatic lesions consistent with chemical expo sure. Of the remaining two, one had focal hepatocellular hyperplasia; the other had focal mixed hyperplasia and early peliosis hepatis. This latter individual was later found to have angiosarcoma possibly from vinyl chloride hair spray exposure. The former individual had no history of chemical exposure and/or alcohol consumption, but did have hepatobiliary tract disease in the form of biliary stones. Table 1 lists the hepatic lesions found in those with and without biochemical abnormalities for the entire group. Reproducibility An evaluation of the reproducibility of these biopsy readings was carried out in 17 individuals who had 32 biopsies. Seven individuals had only one biopsy (2 needle, 5 wedged) and 10 individuals had their biopsies read in dupli cate. Four had needle biopsies with 9 readings and 6 had wedge biopsies with 14 readings (Table 2). CMA 003926 6 hepatocellular hyperplasia; b) focal increased reticulum associated with the hepatocytic hyperplasia; c) perisinusoidal fibrosis; d) focal hyperplasia; e) focal mixed hyperplasia associated with increased reticulum deposition; f) sinusoidal cell activation; g) sinusoidal dilitation; h) portal and capsular fibrosis; i) sinusoidal dysplasia; and, j) hepatic angiosarcoma. The presence of focal hepatic cellular hyperplasia with focal increases in reticulum, in the absence of other evidences of hepatocellular disease or steatosis, were considered the minimum presumptive evidence of chemical exposure. The presence of focal mixed hyperplasia with increased reticulum and/or peri sinusoidal fibrosis was considered evidence of chemical injury of a more advanced stage. Sinusoidal cell activation and dilitation of the sinusoids were con sidered evidences of chemical injury of an even further stage of advancement. The presence of subcapsular fibrosis, (on wedge biopsies only) and/or increases in both portal and sinusoidal fibrosis, with sinusoidal cell activation and sinusoidal dilitation was considered the most advanced stage of chemical injury characteristic of vinyl chloride or vinyl monomer exposure. Finally, sinu soidal cell dysplasia and/or evidence of malignant transformation, constituted definitive evidence of chronic vinyl chloride exposure and the terminal stages of injury. Work and exposure histories were available for all the chemical workers. The work histories provide a total and average exposure data for 22 chemicals based on rank order exposure estimates. These exposure work histories consisted of an estimate of exposure to each of 22 different chemicals for a.ll of the 350 job classifications used since the start of the chemical plant. Each of the chemicals exposure was ranked on a scale of zero to six for each of the 22 chemicals. These exposure ranking estimates were done separately CMA 003927 confidential Subject to .Proten-M^ " In Wo. 90-4837 nucleoli with a moderate amount of cytoplasm which reacted on PAS staining and persisted after diastase reaction. These cells are interpreted as activated fibroblasts, are associated with an increase in fat storing sinusoidal cells and augmented perisinusoidal fibrosis tissue (Figure ). 3. Fibrotic changes consisting of an excel! of irregular connective tissue in the periportal zone, frequently arranged around proliferating bile ductules, and focal thickened subcapsular accumnulation of corrective tissue into the parencyma (Figure 7). These histological lesions are believed to be the three earliest hepatic findings associated with vinyl monomer chemical injury. The hepatocytic changes are referred to as focal hepatocytic hyperplasia or FHH. The sinu soidal cell changes (sinusoidal cell hyperplasia) associated with the hepatocytic changes are referred to as focal mixed hyperplasia or FMH. For purposes of this study, early fibrotic changes were not classified since the majority of liver biopsies were of needle type, which could not be evaluated for subcaps ular changes. The liver biopsies were coded and read blindly to identify (1) histologi cal evidence of hepatic disease; (2) any characteristics of non-chemical injury excluding steatosis; (3) steatosis with or without fibrosis and, (4) evidence of chemical injury identified by the following histological findings; a) focal CONFIDENTIAL Subject to Protective Order iff P.oss v. Conoco, Inc. No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003928 A group of 30 individuals who were not chemical workers but had undergone abdominal surgery with liver biopsies, were used as a comparison population. All of the comparison population's liver biopsies were performed during the same three-year period, at the same hospital, by members of the same medical- surgical team, with the informed consent of the individuals. This group had similar hepatic biochemical studies with the exception of the GGT and ICG clearance. None had a history of extensive exposure of working with halo- genated hydrocarbons, although one patient had a history of household contact with vinyl chloride via conmercial home products. Pathology Earlier experiences with autopsy and animal material from vinyl chloride associated angiosarcoma have recognized certain hepatic lesions in the non- tumorous area of the liver parenchyma ( ). They include: 1. Hepatocytic changes consisting of foci of enlarged hepatocytes with increased amount of cytoplasm, and large hyperchromatic nuclei (Figure 1). These cells are intermixed with hepatocytes of normal and smaller sizes and create focal, indistinct nodular areas (Figure 2a). These areas are often associated with very slight increased sinusoidal dilitation and focal increase in the reticulin framework illustrated best by silver impregnation (Figure 2b). 2. Sinusoidal lining cell changes consisting of an increase in cell number associated with nuclear changes, particularly conspicuous in areas of sinusoidal widening. This proliferation of sinusoidal cells involves (a) Kupffer cells (macrophages) with PAS-positive granules; (b) fibroblasts with elongated, almost rectangular, nuclei with loose vesicular chromatin patterns and inconspicuous CONFIDENTIAL Subject to Protective Order in Ross v. Conoco, Inc.._ No. 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana 4 t:4T9CO'JCT [ 3fi with various heoatic histologies I abnormalities. These include subcaosular, portal, and perisinusoidal fibrosis as well as hyperplasia of both hepatocytes and sinusoidal cells. Early histological studies, mainl/ on autopsy material, had shown focal mixed hyperplasia (hyperplasia of hepatocytes and sinusoidal cells) to be an early histological alteration associated with vinyl chloride exposure (1,2,3). To sinstantiate this observation and determine its potential use in medical surveillance screening of the exposed workers, liver bioosies from 73 individuals were studies in duplicate blind fashion to determine 1) if these early histological findings were associ ated with extensive vinyl chloride exposure, and 2) if these histological findings occur in non-exposed popula tions or were associated with other diseases. MATERIALS AND METHODS Liver biopsies from 48 vinyl monomer chemical workers with and without hepatic biochemical abnormalities were investigated. All 48 chemical workers had had hepatic biochemical studies performed on an annual or semi-annual basis. These included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), total bilirubin (TB), gamma glutamyl transpeptidase (GGT), prothrontin time (PT), and indocyanine green clearance (ICG). In addi tion, history and physical examinations, liver-spleen scans, and abdominal plain films had been performed annually. Thirteen of the chemical workers had hepatic biopsies for non-liver related reasons while 35 had biopsies performed because of screening biochemical and/or liver-spleen radioisotopic scan abnor malities. CMA 0393o INTRODUCTION Industrial vinyl chloride and other vinyl monomer exposure is associated with various hepatic histological abnormalities. These include subcapsuiar, portal, and perisinusoidal fibrosis as well as hyperplasia of both hepatocytes and sinusoidal cells. Early histological studies, mainly on autopsy material, had shown focal mixed hyperplasia (hyperplasia of hepatocytes and sinusoidal cells) to be an early histological alteration associated with vinyl chloride exposure (1,2,3). To substantiate this observation and determine its potential use in medical surveillance screening of the exposed workers, liver biopsies from 78 individuals were studies in duplicate blind fashion to determine 1) if these early histological findings were associated with extensive vinyl chloride exposure, and 2) if these histological findings occur in non-exposed popula tions or were associated with other diseases. MATERIALS AND METHODS Liver biopsies from 48 vinyl monomer chemical workers with and without hepatic biochemical abnormalities were investigated. All 48 chemical workers had had hepatic biochemical studies performed on an annual or semi-annual basis. These included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), total bilirubin (TB), gamma glutamyl transpeptidase (GGT), prothrontin time (PT), and indocyanine green clearance (ICG). In addi tion, history and physical examinations, liver-spleen scans, and abdominal plain films had been performed annually. Thirteen of the chemical workers had hepatic biopsies for non-liver related reasons while 35 had biopsies performed because of screening biochemical and/or liver-spleen radioisotopic scan abnor malities. Subject to Protective Order in Boss v. Cor.o Wo. 90-4837 1 th Judicial District Court Calcasieu Parish, Louisiana CMA 003931 ABSTRACT Earlier histological studies of industrial vinyl chloride exposure, obtained mainly on autopsy material, indicated that focal mixed (hepatocytes and sinu soidal cells) hyperplasia is the earliest histological alteration indicative of exposure. To substantiate this observation and its potential use in screening workers, 93 liver biopsies from 78 persons were investigated in double blind duplicative fashion; 35 were exposed chemical workers with hepatic screening tests abnormalities, and 13 were exposed workers without hepatic abnormalities who had liver biopsies for non-liver related reasons. A comparison group consisted of 30 individuals who were not chemical workers, but had liver biop sies for non-hepatic related reasons during the same time period. Of the exposed workers, 23 (48%) had a hepatic lesion consistent with exposure; 17 (35%) had only focal hepatocytic hyperplasia; six (13%) had focal mixed hyper plasia or more advanced lesions. In contrast, only five of the comparison group had similar findings; four (13%) had only focal hepatocytic hyperplasia and one (3%) had focal mixed hyperplasia and sinusoidal dilitation. On a subsequent biopsy, this individual was found to have angiosarcoma and a history of using hair spray containing vinyl chloride as propellant. Ten individuals had 28 multiple biopsies also read double blindly, and 10 individuals had 23 readings of the same biopsy; 21/23 (91%) duplicate readings and 27/28 (96%) multiple biopsy readings in the same individuals were identical. Only 18% of either duplicate and/or multiple biopsy readings had disagreements in their biopsy assessment. Focal hepatocytic hyperplasia, in addition to the previously described mixed hyperplasia, appears to be the earliest identifiable change consistent with chemical exposure and both lesions are present prior to the ' development of angiosarcoma. These lesions can be consistently identified and are useful in the screening of chemical workers for evidence of exposure. CMA 003932 T IAL Subject to Protective Order in JTSS v. Conoco, Inc,, No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana EARLY HEPATIC HISTOLOGICAL ALTERATIONS AMONG CHEMICAL (VINYL MONOMER) WORKERS 1 23 Carlo H. Tamburro, Laszlo Makk, and Hans Popper l Liver Research Center, Department of Medicine 21 and St. Anthony Hospital, University of Louisville 3 Louisville, Ky., Stratton Laboratory for Study of Liver Diseases, Mt. Sinai Hospital fo Medicine, New York, New York 1 Portions of this work supported by NCI Contract No-l-CN-55212 and the Manufacturing Chemists Association Grant VC7.0 JSpunodco^^Inr^^oVe -Or9d0e-r48ln37 rlU * c,,,,4837 ' Lo"isiana CMA 003933 fh. :m i ro jj ^i -j, cnJ oo .) +J 3 ~ "5 -| ^ ^ CJ,1 M _* si i52 g!^~ M -p 0I ^ 1 HO The reproducibility in identifying these histological lesions is ounce high. Under double bl'nj conditions t'-ere ..as consistent histological cenci fication of chemical injury in both duplicate readings of single biopsies as well as single readings of multiple biopsies in the same individual. Inconsistency appears to occur more frequently among the duplicate readings of wedge biopsies and between wedge and needle biopsies than between duplicate readings of needle biopsies or readings of multiple needle or wedge biopsies in the same individual. This may be due to the increased time and attention needed for wedge section review in order to determine the absence or presence of these lesions anywhere in the specimen. There are, however, some pitfalls that require caution. The quality of the histological preparation is of vital importance. Technical artifact, excessive thickness, torn or fragmented spec imens will greatly reduce the ability to correctly identify these lesions. The use of silver impregnation for assessment of the reticular structure is very useful in verifying focal areas of hepatocellular hyperplasia. The presence of fatty infiltration and/or chronic disease (hepatitis, granuloma, etc.) may prevent the identification of these lesions. We were, however, able to correctly identify the presence or absence of these early lesions in the six individuals who also had a concomitant granulomatous process (four individuals with granuloma were repeatedly read as negative and two individuals with gran uloma were repeatedly read as positive for chemical liver injury). The more frequent elevation of alkaline phosphatase in those individuals with histological evidence of chemical injury may provide some guidance in sequential uses of laboratory screening tests. During the first phase, tests with the greater sensitivity for identifying liver injury, such as the ICG and ALT can be used. Positive results can be followed up by using tests of greater specificity, such as the alkaline phosphatase, to further identify those at high risk of chemical injury. CMA 003934 CONCLUSIONS Focal hepatocellular hyperplasia, in addition to the previously described focal mixed hyperplasia, appear to be the earliest identifiable changes consistent with chemical exposure. These histological lesions are useful in identifying high risk exposed chemical workers as part of a medical screening surveillance program. Biochemical studies may help identify which individuals warrant a liver biopsy for further identification of the cause of the liver abnormalities. 'O. 90-4337 lot CMA 003935 REFERENCES 1. Popper H, and Thomas LB. Alterations of liver and spleen among workers exposed to vinyl chloride. Annals of the New York Academy of Sciences 1975; 245:172-194. 2. Thomas LB and Popper H. Pathology of angiosarcoma of the liver among vinyl chloride-polyvinyl chloride workers. Annals of the New York Academy of Sciences 1975; 246:268-277. 3. Gedigk P, Muller R and Bechtelsheimer H. Morphology of liver damage among polyvinyl chloride production workers. A report on 51 cases. Annals of the New York Academy of Sciences 1975; 246:278-285. 4. Greenberg RA and Tamburro CH. Exposure indices for epidemiological sur veillance of carcinogenic agents in an industrial chemical environment. Journal of Occupational Medicine 1981; 23:No. 5, 353-358. 5. Greenberg RA and Tamburro CH. Monitoring exposure to hazardous chemicals in an industrial setting: a method of demonstrated utility. Journal of Occupational Medicine ,198 6. Tamburro CH and Greenberg RA. Effectiveness of federally-required medical laboratory screening in the detection of chemical liver injury. Environmental Rt?4pectives, 1981 , 41:117-122 7. Tamburro CH, Davidson CS, Fisher W, et al. Medical surveillance for chemical hepatotoxicity. Guidelines for D etection of Hepatotoxicity Hie to Drugs and Chemicals. Davidson CS, Leevy CM and Chamberlain EC (ed.) U.S. Department of HEW, NIH Pub. No. 79-313, 1979, Chapter 5; 60-80. CMA 003936 CONFIDENTIAL Subject to Protective Order in Boss v. Conoco, Inc., No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana REFERENCES 1. Pepper H, end Thomas L3. -Iterations or liver and scleen among workers exposed to vinyl chloride. Annals of :he Jew York Academy of Sciences 1975: 245:172-194. 2. Thomas LB and Popper H. Pathology of angiosarcoma of the liver among vinyl chloride-polyvinyl chloride workers. Annals of the New York Academy of Sciences 1975; 246:268-277. 3. Gedigk P, Muller R and Bechtelsheimer H. Morphology of liver damage among polyvinyl chloride production workers. A report on 51 cases. Annals of the New York Academy of Sciences 1975; 246:278-285. 4. Greenberg RA and Tamburro CH. Exposure indices for efAamiological sur veillance of carcinogenic agents in an industrial chemical environment. Journal of Occupational Medicine 1981; 23:No. 5, 353-358. 5. Greenberg RA and Tamburro CH. Monitoring exposure to hazardous chemicals in an industrial setting: a method of demonstrated utility. Journal of Occupational Medicine,198 6. Tamburro CH and Greenberg RA. Effectiveness of federally-required medical laboratory screening in the detection of chemical liver injury. Environmental pr4pectives, 198S , 41:117-122 7. Tamburro CH, Davidson CS, Fisher W, et al. Medical surveillance for chemical hepatotoxicity. Guidelines for Detection of Hepatotoxicity rue to Drugs and Chemicals. Davidson CS, Leevy CM and Chamberlain EC (ed.) U.S. Department of HEW, NIH Pub. No. 79-313, 1979, Chapter 5; 60-80. CONFIDENTIAL Subject to Protective Order in Bess v. Conoco, Inc., No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003937