Document G54Ezw7Oak31w8y7Y9OE87YYq

STATEi:r;ivT- cii. _viia'l.,- chloride.. research It is well known that toxic effects are caused by absorption into the body of excessive amounts of any substance. When this occurs the capacity of the body's normal defense mechanisms are exceeded and this may result in more or less serious consequences - as with an excessive intake of alcohol. However, when the amount absorbed does not exceed this capacity the body is capable of dealing with it without toxic effects. Research on vinyl chloride (VC) in rats has demonstrated that high doses of VC are handled in the body differently than low doses. The results indicate that low doses of VC are readily detoxified, but high doses of VC exceed the body's normal defense mechanisms and detoxification is slowed and perhaps changed. Therefore, exposure to high levels of VC in rats results in an increased susceptibility to toxic effects including cancer. From data available, it appears that induction of cancer is correlated with high doses of VC where detoxification is slowed, altered or incomplete, and that cancer is not induced at low doses where the body's normal defense mechanisms remain fully operative. by P. G. Watanabe et al. April 28, 1976 BOR 010670 ' if*y **l* hi i* f .-*>tTTf' Summary of the Studies on the Pharmacokinetics of Vinyl Chloride in Rats and its possible Relationship to Oncogenicity V' P. G. v.-atanabo, G. "cGov/an, J. A. Zempel and P. J. Gehring ABSTRACT Studies on the fate of 14 C-labeled vinyl chloride (VC) following oral administration and inhalation exposure in rats demonstrated that the disposition of VC in the body is a function of the dose. As the dose or exposure level of VC is increased the metabolism or detoxification pathways become saturated. VC is detoxified primarily by conjugation with nonprotein sulfhydryl groups in the liver (primarily glutathione, GSH). Studies to charac terize the depression of liver GSH as a function of magnitude and duration of exposure to VC have shown that a single 7 hour exposure of rats to 100 ppm or greater results in an unequivocal depression of liver GSH. Exposure to 10 ppm VC for 7 hours did not result in any depression of liver GSH. These results suggest that increasing doses of VC deplete liver GSH and consequently lower the bodys defense mechanism for detoxification of the reactive metabolites of VC. This increased susceptibility may BOR 010671 result in induction of carcinogenesis, and from data available, it appears that a'correlation exists between doses of VC which cause tu:. ers and those that saturate metabolic or detoxifying pathways. Conceptually, the results of these investigations indicate that statistical projections utilizing data collected from rats exposed to high doses of VC are invalid for pre dicting the hazard of low level exposure because such projections assume that the dynamics governing the fate of VC in the body are unaltered; and in fact, the data presented demonstrate that the fate of VC in rats is highly dependent on the dose administered. April 28, 1976 BOR 010672 l.uLid-uJ. B I O * T E S T Aww/jwi, 3rte. Inhalation Studies in Rats, Mice and Hamsters With Vinyl Chloride At Industrial BIO-TEST Laboratories, Inc. , a study was conducted to determine the effects from inhalation of vinyl chloride monomer to rats, mice and hamsters. The study was supported by the voluntary contributions of thirty-one VCM/PVC producing com panies and is administered by the Manufacturing Chemists Association with the advice and guidance of a technical panel of scientists. In this study, 200 rats, hamsters and mice (100 of each sex or 600 animals total per exposure group) were exposed to 50, 200 or 2,500 ppm vinyl chloride vapor 7 hours per day, 5 days per week. Mice were exposed for 9 months while rats and hamsters were exposed for 12 months. All animals were then kept for the remainder of their natural lives (up to 26 months of mice and 30 months for rats and hamsters). There was an increased incidence in neoplasms in the liver of all 3 species at all 3 doses and the incidences are dose-related. In general, the only significant increases in rats or hamsters were in the liver. For mice there appears to be an increased incidence of all neoplasms, but particularly in the lung as well as the liver. BOR 010673 n 1 i t C I O - T E S T xa.ittyuU&ue'S., Jrjz. Vinyl Chloride Inhalation Study Incidence (percent) of Neoplasms - Males CV`VC!vL ar d Oil an Control Experiment;al Groups T-I T-H f50 Dorrs) (200 pom) T-IZ (2500 op RATS Liver 6.4 30. 9 73. 7 63. 3 Kidney 0 7.4 0 0 Skin 19.0 11.2 5.0 2. 0 Misc. Pituitary Brain Mesentery Adrenals Thymus Lymph Node Genital 6.4 18. 5 10. 5 4. 4 MICE Liver Lung 1.8 27.0 90.2 90. 8 1. 8 30.2 67. 1 87. 7 Skin 0 0 1.0 4.0 Misc. Kidney Mesentery Lymph Node Genital 1.8 1. 1 9.3 10. 8 HAMSTERS Liver 0 23.9 17.5 62.5 Skin 0 5.0 4.0 1. 1 Misc. Kidney Mesentery Lymph Node Thymus Genital 0 15.2 7. 5 8.9 ' bor 010674 Vinyl Chloride Inhalation Study Species and O'--r RATS Liver Kidney Skin Misc. Pituitary Brain Mesentery Adrenals Thymus Lymph Node Incidence (percent) of Neoplasms - Females Control Exp e r im e nt al G r oups T-I ,'5n -r T-II f ? ? G -\rn) T-III (250C pom) T -IV (25CP xr:! 4. 8 0 64. 0 17. 9 39- 5 1.3 56. 0 13.2 76. 3 1. 3 23. 0 9.2 78.5 2.6 13.1 8.9 76.7 0 25.0 6.8 MICE Liver Lung Skin Misc. Kidney Mesentery Lymph Node Genital 0 4. 3 2. 0 2. 1 36.1 43. 1 37.0 38.9 82.6 60. 5 17. 0 5. 8 85.9 81.2 20.0 8.2 HAMSTERS Liver Skin Misc. Kidney Mesentery Lymph Node Thymus Genital 11.4 0 11.4 18.2 2.0 9. 1 26.6 15. 3 8.9 60. 6 1.0 6.1 BOR 010675 * Food Available During Exposure. ITEM 3 Interim Report on Mortality and Gross Observations in Rats, Mice and Hamsters V7ith Vinvl chloride M. L. Keplinger, J. W. Goode, D. E. Gordon and J. C. Calandra Industrial BIO-TEST Laboratories, Inc. 1810 Frontage Road Northbrook, Illinois 60062 BOR 010676 3natu&tual B I O - T E S T jfaua'iafrvujii, 'Jhc. Interim Report on Mortality and Gross Gnservations in Lac:, Rice and Hamsters V, Lh Yin's! Chloride M. L. lieplin-e r, j. \V. Goode, D. E. Ceido.i t.nd J. C. Calandra Industrial DIG-TEST Laboratories, Inc. 1SI0 Frcrcagc R.oad Northbroo!:, Illinois 60062 At Industrial BIO-TEST Laboratories, Inc. , a study was conducted to determine the effects from inhalation of vinyl chloride mouomr to rats, mice anu hainaters. The study was supported by the voluntary contributions of-thirty VCM/PVC producing companies It is administered by the Manufacturing Chemists Association (MCA), with the advice and guidance of a technical panel of scientists from the sponsoring firms. The protocol for the study was designed by the technical panel. The study was designed as a life-span study in these species which usually means 1-1/2 to 2 years for the "in-life" portion, with tissue examination to follow this period of time. All animals were allowed to live the remainder of their natural lives. Therefore, the total time of the study was longer than 2 years. BOR 010677 In this study, 200 rats, hamsters and mice (100 of each sex or 600 animals total per exposure group) were exposed to 50, 200 or 2,500 ppm vinyl chloride vapor 7 hours per day, 5 days per week. Mice were exposed for 9 months while rats and hamsters were exposed for 12 months. All animals were then kept for the remainder of their natural lives. The rats were COBS Charles River; mice were CDI Swiss Charles River; and the hamsters were Golden Syrian from the same source. Food was removed during exposure, water was present and no bedding was used at any time. The usual procedure in conducting inhalation studies in not to have food present in the chamber during exposure. In at least some of Dr. Maltoni's studies, food was present in the chamber. The question contaminated food was raised as to whether/^ would influence the onset or incidence of tumors. Therefore, a separate group of 100 female rats was exposed to 2,500 ppm VCM while food was in the chamber. All 600 animals per level were exposed in a single chamber. The chambers were operated under dynamic conditions. The chambers were sealed and were operated under slight negative pressure. This was done so that, in case of an accidental leak, room air would flow into the chamber instead of contaminated air flowing out of the chamber. BOR 010678 9nOM.iinial B I O * T E S T jlakviaiyUei, One., 3 The concentrations in the chamber were analyzed using gasliquid chromatography. An automatic sampling device allowed samples to be d;r.v,-;i fro:.'. location.- it; each chare' er. The concentrations at all location: in each chamber were monitored every day during the total period of operation of the chamber. Moribund and dead animals received gross autopsies and many gross lesions were photographed. The major organs of all animals were fixed for histological examination. There were no particularly adverse effects on body weights or, elements or enzymes measured in the blood. The data for mortality or survival are presented in Tables I, II and IH. It can be seen that the last survivors of mice were sacrificed or died in month 26, while the last survivors of rats or hamsters were sacrificed or died in month 30. While these tables indicate "mortality", it should be noted that the numbers also include those animals sacrificed in extremis. In rats and mice (and somewhat in hamsters) there was a doserelated increase in mortality. This is well illustrated by examining the time to 100% mortality in the tables. For example, in mice the months in which there was 100% mortality were 11 at 2,500 ppm, 14 at 200 ppm, 16 at 50 ppm and 20 (male) or 26 (female) controls. In rats the months were 16 at 2,500 ppm (both fed and fasted), 21 at 200 ppm, 29 (male) or 30 (female) at 50 ppm and 30 for controls. BOR 010679 Htuludsual & I O - T E S 7 2aJcyiaio-Ui,i, 4 (W Preliminary examination of tissues from mice which had died after abcut 6 or 7 months of exposure indicated possible tumors in the livers, luups, mammary glands and/or skin. At that time there was no evidence of tumors in the rats or hamsters. A tabulation of incidences of neoplasms for males and females is presented in Tables IV and V. The males and females were tabulated separately to reveal any differences between the 2 sexes. A comparison of the data indicates that there are no real differences between males and females with regard to type or incidence of neoplasms. The data from these tables indicate an increased incidence in neoplasms in the liver of all 3 species at all 3 doses and the incidences are dose related. In general^the only significant increases in rats or hamsters were in the liver. For mice there appears to be an increased incidence of all neoplasms, but particularly in the lung as well as the liver. In many instances the incidences at the 2 higher levels (200 and 2500 ppm) are about the same. This might be interpreted as evidence of lack of dose-response. However,these incidences are very high and probably indicate nearing the plateau of the doseresponse curve at 200 ppm. BOR 010680 HtuLdiiial B I O T E S T jnc. 5 The incidences (liver) at 50 ppm are well above control values which indicates that this is a definite effect level. A comparison of inner dam. of ic-d and fasted (during exposure) female rats exposed to 2500 ppm VCM leads to the conclusion that there was no significant difference between the 2 groups. For example, the incidences of liver tumors were 77 and 78%. Although the incidence of skin tumors was 25% in the fed animals vs. 13% in the fasted animals, it should be noted that incidence in this group of fasted animals was the lowest of any group (control 64%, 50 ppm 56% and 200 ppm 23%). It was stated previously that there were 100 animals per group (male or female). It will be noted in the tables of incidences of neoplasms that the denominator in most cases is less than 100. There are several reasons for this. As soon as an animal dies, autolysis starts. If an animal is necropsied soon after death the tissues are worth saving for histological examination. If, however, several hours elapse from death to necropsy, the tissues may not be saved if the pathologists feels there is too much postmortem autolysis. The animals were housed in groups. If an animal (particularly mice, hamsters 8t rats) dies in a cage with other animals, cannibalism BOR 010681 !)tuLtU-uaL C ! O - T E S T Xao^KiL-u&i, 2, 6 usually occurs unless the dead animal is removed immediately. When the chambers were open (no, exposure) and an animal was observed to be dead, it was removed and tissues were saved. I an animal died overnight, it frequently was lost for histology. There is; one other important aspect for inhalation studies compared to other studies such as feeding. The chamber is sealed during exposure. Even if an animal is observed to be dead in the chamber, there is no way to remove it until the end of the exposure. This results in loss of tissues for histology. In most of the chronic inhalation studies now being conducted, all animals are housed individually to prevent many of these losses. The denominator for the skin usually is 100 (or slightly less such as 98 or 99). If an animal is cannibalized, the skin usually is left. Even though there is autolysis or degeneration of other organs and tissues, the skin is not changed enough to present meaningful evaluation. Therefore, the denominator for the skin is higher than for other organs or tissues, and does allow accountability of the number of animals in the study. It might also be emphasized that in the usual chronic toxicity or carcinogenic study at the present time 50 animals per group are judged adequate to start a study. With life-time studies in rodents such as rats, mice or hamsters, a number of animals BOR 010682 9nduitiial B I O - T E S T jtu~. -I are expected to be lost for pood histological examination. The number of 50 is used to assure that an adequate number, usually 20 to 25. are avail: hde for exnrr.ir.arion at the end of the study. Tiie VCM study was designed with twice as many animals to assure that U.e usual exnscieu aamber of 20 to 25 would be available at the end of the siudy. It will be noted that the denominator in all cases exceeds the 20 to 25, and it far exceeds the 20 to 25 in almost all cases. bob oj-0683 HiuLutual B 1 O - T S S T y^Uo-'-ui. Jric. 8 i' TABLE I TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Toxicity Study - Albino Rats, Albino Mice and Golden Hamsters Summary of Mortality Data - Albino Rats Month Number UC M L- v c 1i UHliJd r of Mortalities ;at End of Mon:h T--I T-II T-III F MF MF MF 1 Ou 3 4 5 6 7 8 os 10 n 12* 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 29 30 0 0 0 0 2 3 4 4 4 5 7 13 16 19 21 23 28 33 38 46 52 57 64 73 7$ 86 92 98 100 0 0 1 2 3 4 4 5 A 7 8 14 . 17 21 23 25 27 28 31 34 42 48 54 65 69 76 84 93 100 0 2 2 4 5 6 8 9 10 11 13 19 23 26 28 31 35 38 43 47 52 63 72 81 85 91 96 100 100 0 0 0 1 1 1 2 3 4 7 9 16 24 30 37 . 45 53 59 66 73 80 89 92 94 97 98 99 99 100 00 10 10 30 61 61 62 10 5 11 5 12 9 14 19 21 37 26 50 33 63 40 75 46 87 55 90 64 94 71 . 94 77 94 100(18) 100(4) 100 100 100 100 0 0 0 1 3 5 6 10 16 24 34 58 73 85 94 100(4) 100 100 100 100 100 100 100 0 0 4 4 4 4 5 8 15 28 52 66 83 86 89 100 100 100 100 100 100 100 100 T-IV F 0 0 0 0 0 0 0 1 4 7 17 40 46 76 86 100(7) 100 100 100 100 100 100 100 "'Included 5 males and 5 females from each group excluding T-IV sacrificed for cytogenic study () Indicates moribund or sick animals sacrificed. BOB. 01068* B 1 O - T E S T 2alto/\a.Lv.ii, Q Month Number 1 2 3 4 5 6 7 OA 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 TABLE II TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vrpcr Inhalation T':>:; 1 city Study - Albino Rats, /nemo t'lice anci Coicien Hamsters Su:r.:..crv c: McrtuLtv IMtn - Albino Mice UC M Cum.uiative Number of i\ icrtalities at End of Month T-I T--II TT M F M F T-nr M 0 0 0 0 0 0. 0 00 03 0 00 20643 01 5 1 19 5 4 1 4 8 2 26 6 4 2 8 12 2 30 6 3 10 18 5 31 11 9 12 13 18 5 32 14 16 30 30 23 8 38 24 37 58 56 28 10 42 31 78 82 76 30 15 54 46 96 94. 100 1 34 16 57 66 98 95 100 1 44 23 70 80 99 97 100 1 54 28 79 91 100 100 100 1 67 39 88 97 100 100 100 1 82 46 100 100 100 100 100 1 82 54 100 100 100 100 100 1 82 58 100 100 100 100 100 1 92 70 100 100 100 100 100 1 100 77 100 100 100 100 100 1 100 81 100 100 100 100 100 1 100 89 100 100 100 100 100 1 100 . 92 100 100 100 100 100 3 96 97 100 BOR 010685 ynduti'Ual 0 1 U - l S *. 10 TABLE III TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Toxicity Study - Albino Rats, Albino Mice and Golden Hamsters Month Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 '23 24 25 26 27 28 29 30 S t *r* ary of Mortality Data - Golden Hamsters uc M CunreH;:ive i\:u FM o; '-lor: alities at hnd cl Month T-II F MF T*Tir M L 54447 525 8 11 10 12 9 16 10 11 8 13 12 14 12 18 14 13 9 15 16 16 15 19 15 13 9 18 16 16 . 16 20 15 15 13 18 16 16 20 21 16 16 13 28 24 19 22 33 18 26 14 28 24 19 23 35 19 28 14 29 24 20 25 36 20 28 14 30 26 20 26 37 23 29 15 31 28 21 26 38 28 36 18 36 '34 33 37 46 40 45 23 40 43 49 46 49 49 58 28 46 49 58 54 53 61 69 39 61 53 72 66 55 71 84 50 78 58 83 74 59 84 100 57 81 61 85 76 66 86 100 62 85 62 86 77 75 88 100 65 90 68 89 79 84 92 . 100 71 96 73 95 86 89 100(5) 100 71 100(2) 80 100(3) 100(13) 100(7) 100 100 73 100 82 100 100 100 100 100 78 100 85 100 100 100 100 100 83 88 88 92 90 95 94 98 94 99 99 100 100 () Indicates moribund or sick animals sacrificed. BOR 010686 c SasUduai B 1 O - 7 E S T .,j Snc. TABLE IV 11 Vir.vl Chloride i_on Study " IET NO.- 663-03222 iP'r c*' c ** tr" r. c-: cf ire'*-'! iasms - Mr-les Species 0:: ,,n I Control fn y rimen: al C-rouDS T -'ll (5 0 ppm) (200 ppm) t-hi (2500 ppm) RATS Liver 5/78 (6.4%) 25/81 (30. 9%) 56/76 (73. 7%) 57/90 (63. 3%) Kidney 0/78 6/81 (7.4%) 0/76 0/90 Skin 19/100(19%) 11/98(11.2%) 5/100(5. 0%) 2/100 (2. 0%) Misc. Pituitary Brain Mesentery Adrenals Thymus Lymph Node Genital 5/78 (6.4%) 15 /81 (18. 5%) 8/76 (10.5%) 4/90 (4.4%) MICE Liver 1/55 (1.8%) Lung 1/55 (1.8%) Skin 0/100 . Misc. Kidney Mesentery Lymph Node Genital 1/55 (1.8%) 17/63 (27%) 19/63 (30.2%) 0/100 7/63 (1. 1%) 74/82 (90. 2%) 59/65 (90. 8%) 55/82 (67. 1%) 57/65 (87/7%) 1/100 (1.0%) 4/100 (4. 0%) 8/86 (9. 3%) 7/65 (10. 8%) HAMSTERS Liver 0/53 Skin 0/100 Misc. Kidney Mesentery Lymph Node Thymus Genital 0/53 11/46 (23. 9%) 5/100 (5.0%) 7/46 (15.2%) 7/40 (17.5%) 4/100 (4.0%) 3/40 (7. 5%) 35/56 (62.5%) 1/94 (1. 1%) 5/56 (8.9%) BOR 010687 u ( \j - j c j jt%c. 12 TABLE V Vinyl Chloride Inhalation Study - IBT NO. 663-03222 c -;, \M Or - <2 v > Live r Projected Incidence of Neoplasms - Females Control 1 Errrv-I'imcr tal Grcums r' T -'ll (50 pom) (200 pom) T-XII (2500 ppm.l (2 5" ` 4 /84 (4. 8%) 30/76 (39. 5%)' 58/76 (76. 3%) 62/79(78. 5%) 5 o / 7 31 Kidney 0/S4 1/76 (1. 3%) 1/76 (1. 3%) 2/79 (2. 6%) 0/73 Skin 64/100 (64%) 56/100 (56%) 23/100 (23%) 13/99(13. 1%) 25/10C Misc. Pituitary Brain Mesentery Adre'nals Thymus Lymph Node Genital 15/84 (17. 9%) 10/76 (13.2%) 7/76 (9. 2%) 7/79(8. 9%) 5/73(6 MICE Liver Lung Skin Misc. Kidney Mesentery Lymph Node Genital 0/47 2/47 (4. 3%) 2/100 (2.0%) 1/47(2.1%) 26/72 (36. 1%) 71/86(82. 6%) 73/85 (85. 9%) 31/72 (43. 1%) 52/86(60.5%) 69/85 (81. 2%) 37/100 (37. 0%) 17/100(17. 0%) 20/100 (20. 0%) 28/72 (38. 9%) 5/86 (5. 8%) 7/85 (8. 2%) HAMSTERS Liver 4/35 (11.4%) 6/33 (18.2%) 21/79 (26. 6%) 20/33 (60. 6%) Skin 0/100 2/98 (2.0%) 15/98 (15.3%) 1/99 (1.0%) Misc. Kidney Mesentery Lymph Node Thymus Genital 4/35 (11.4%) 3/33 (9. 1%) 7/79 (8. 9%) * - * i>ia m ^n r _ ^ ir*^..**,** 2/33 (6. 1%) BOR 010688 CYTOUNCTTC STUDIES OF BON I' MARROW COOLS FROM RATI'. F.Xl'OLi 1) TO V.INVL, CilLORlDi: 'I R. V. Johnston, D.V.M., D, J. Mensik, B.S., M. N. Pinkerton, B.S., E. B. WhorLon, Or., I'h.D.* Dow Chemical U.S.A, Texas Division - Freeport, Texas 77541 Indurtri-il frh-uJth and Medicine Dopartm*nt: Biomedical and Comparative Toxicology Research Laboratory The Biomedical and Comparative Toxicology Research Laboratory of the Texas Division of Dow Chemical U.S.A. was asked by the Manufacturing Chemists Association to conduct cytogenetic studies on some of the rats which had been exposed to vinyl chloride at Industrial BIO-TEST Laboratories, Decatur, Illinois. The details of the exposure levels and pathological findings will be reported separately. Preliminary reports have appeared in Chemical Week (115:30, July 17 , 1974} and in the Annals of the New York Academy of Sciences, Volume 246 , page 219. The test animals were Charles River CD outbred albino rats, which had been exposed to vinyl chloride gas for 7 hours per day, 5 days per week for one year at 0, 50, 200 and 2500 ppm in air. Cytogenetic studies on 5 males and 5 females from each group showed that there was no statistically significant increase in the chromosomal aberration rates in bone marrow cells of the exposed rats. April 28, 1976 *Associate Professor and Director Division of Biometry Department of Preventive Medicine and Community Health University of Texas Medical Branch Galveston, Texas 77550 BOR 010689 ITEM 4 Rc r nces for "Cytogenetic of -'.one Marrow Cells From Rats Exposed to vinyl Chloride hv Dr. R. V. Johnston et al. 1 :n, D. J. c- t", Piec i` P. J,, Cytopcnetic monitoring vi;. -! c.tio: C. wur'in..-.! Open Meeting on Evaluation of S"ftc: tc br.too" Kutcj nic Activity of Chemicals, July'16 end 17, 1975, NIH, Bethesda, Maryland. 2. Fleig and Thiess, A. M., Chromosome analysis after vinyl chloride exposure, Arbcitsnedizin, Sozialmedizin, Praventivsmedizin' 9(12) 260-283 (1974). 3. Johnston, R. V. , Mensik, D. J., Pinkerton, M. N., Vhorton, E, B., Cytogenetic studies of bone marrow cells from rats exposed to vinyl chloride, TBM-108-1, B16863, (1975). 4. Ducatman, A., Hirschhorn, Kurt, Selikoff, I. J., Vinyl chloride exposure and human chromosome aberrations. Mutation Research 31:163-168 (1975). 5. Rannug, U. et al, The mutagenicity of vinyl chloride aft r metabolic activation, Ambio 3:194-197 (1974), 6. Funes-Cravioto, F. et al. Chromosome aberrations in workers exposed to vinyl chloride. The Lancet, February.22, 1975, page 459. 7. Purchase, I.F.H. et al. Chromosomal and dominant 1 thal effects of vinyl chloride. The Lancet, (1975), 8. Bartsch, H,, Human, rat and mouse liver-mediated muta genicity of vinyl chloride in S. Typhimurium strains, Int. J. Cancer 15:429-437 (1975T. 9- Malaveille, C., Bartsch, H. et al. Mutagenicity of vinyl chloride, chloroethyleneoxide, chloroacetaldehyde and chloroethanol, Biochemical and Biophysical Research Communications, Vol. 63:363-370 (1975). BOR 010690 ATTENTION: Mr. Milton Frelfcld ITEM 2 d/ZZ/76 A Mortality Study of Workni-s Expoiud to Vinyl Chloride April IV, l'J/U TMt Is a noport of study ol mortality rotes in a population of 10.173 workers' Mho nt sumo tirnu In l ho past wro engaged In the miinurtc- tura Of vinyl chloride or its polymers. An oar I lor report on a portion Of this population showed that these workers experienced an Increased rick ol ddcth f re ".i i i , (alrc'vy found by other investi -- of the po 11 !-">n betwi.'en th" ri:.k of death from those causes and dura tion and level or exposures. The study population was Identified by an examination of past and pro .-'nt personnel records In 37 plants which produced either vinyl Chloride or polyvinyl Chloride. Every man who had been in an exposed job for more than one year was Identified and his work history obtained. Men who had worked in the past and had quit or been laid off were traced to'datormlne whether they were still alive as of December 31. 1972, the date chosen as the '`closing data" of the study. Of the 10,173 men selected for study. 9b percent wore successfully traced, end 707 deaths wern round. The study copulation Included men hired as far back as the late 1930's and Included 2,006 men who had worked IS years or more. The number of deaths which occurred was compared with the number which would have been expected In a comparable population of United States males for each Couso. BOR 010691 The total number of deaths was about 16 percent less than expected, and the total number of cancor deaths was about equal to what would be expected. 0 1 Some Cancers occurred at a lower rate than expected, and these did not appear to be related to the length or level of exposure. Cane r of the urinary organs and the lymphatic, system showed some excess deaths (22 percent and 1Z percent) but were not related In any systematic way ;r,tu f:. i \ pfrcc "t n U'w^tmia deaths. alt>u not rdf-'.-J to tf'C' c i rcLirriitinc":. Of e;:por,urr. Lt' rj ri.i!ccr *,!..- S psrccr.t txccr.s in tho total study group. L'ft 1^ r.,*n who hr-J v.-: rl.ed for 15 years or more and had begun work at least 20 years before the closing date of the study, there was a 52 per cent excess. Ccncers clas.-rl as "Other and Unspecified" showed a dd percent ex cess in the study population as a whole, but a 230 percent excess in tha older group mentioned above. This excess was largely due to an excess of -brain cancer, since 3B percent of these cancers wore brain, compared with 20 percent for the general population. There was a slight excess of digestive cancer in men with high ex posure. and in men with other than low exposure who had worked at least 15 years bctjlntiinn at, 1na*t 20 years before th-- study date. In these older men 50 percent of the digestive cancers were liver cancer com- parud with about 9 percent for the general population. BOR 010692 png* 3 ATTENTION: Mr*. MU ton Frol fa Id r' <zy ^ In another an lysis, the study p pulatIon was scored according to tho estimated total amount of vinyl chloride to which they were exposed; .tnon were given a score of 1 for each month of low level exposure. tor each month at medium exposure, and 3 for each month at high exposure. There was o sorv^whst 1 rrc-^u 1 ftr in mortality from digestive cancer, rt'jpiratary cancer and cancer of other and unspecified sites v/ith Inc-'^itins sccrc.. for men with scores of *5CO or more (equlva'Icut to 11 y*sors of high exposure or 33 years of low exposure* for example) the excess mortality was 88 percent for digestive cancer, 54 percent for respiratory concur, end 183 percent for cancers of other and un specified sites. The study generally confirms the findings of earlier investigators, but provides additional data for men with long service whose employment began many years ago. by Dr. W. R. Gaffey BOR 010693 University of Louisville Louisville. Kentucky 40202 April SCKO'V r'T' (irw-ivt 1,'LiV.K I': : <_ . .iOjiQNE . S AM> t.LTT.!TIONLCTijN 20, 1976 ITEM 1 HEALTH SCIENCE CENTER WALNUT & PRESTON STRLLTS Dr. T. R. Torkelson Dow Chemical Company Corporate Medical Department 2030 Dow Center Midland, Michigan 48640 Dear Doctor Torkelson: May I express our delight in having had members of the Manufacturing Chemists Association Research Committee visit us earlier this month; we hope that their visit was both enjoyable and informative. Our post-lunch eon meeting helped to clarify a number of points concerning our Manufactur ing Chemists Association Grant proposal and I would like to take this op portunity to submit for the members consideration a modification of our original proposal in light of the expressed interests of the members of the research committee. Since the members expressed interest in supporting proposals A and G, having to do with the study of immunological systems, it seems that these would require no further elaboration or adjustment. My understand ing is that the same would be true for proposal G, concerning electron microscopic evaluation of liver tissue. What I would like to do here is (a) amplify on the immediate clinical applicability of some of the other proposals presented, (b) provide a modified budget for these proposals and (c) suggest that if cuts must be made in the program, the cuts that would be least damaging in terms of ongoing efforts and in terms of a cohesive ness of the overall research program, would be the proposals by Dr. Hoffman (H) and Dr. Sigdestad (1) and parts of Drs. Du (B2 B5 B6 B7) and Wong (E2) (see revised budget attached). Firstly, proposal C concerning the glycosaminoglycans in the early detection and etiology of angiosarcoma of the liver presented by Dr. Charles E. Kupchella: this has already produced significant results. Most chemical injury and cancer appear to be as sociated with "scar" or collagen formation. This collagen formation is as sociated with the increased production of glycosaminoglycans. Dr. Kupchella1s preliminary study indicates that there is a characteristic pattern of glycosaminoglycan urinary excretion among long-term vinyl-chloride-exposed in dividuals not seen in individuals with alcoholic liver injury, hepatitis, and cancers not directly involving the liver. These urinary excretion pat terns appear to change as one develops primary liver cancer such as angio sarcoma. At this point, the findings reported by Dr. Kupchella at the Third BOR 010694 Dr. T.R. Torkelson pacts -2- Internatibn.il Symposium on the Detection and Prevention of Cancer, require additional verification a: 1 mod if ic.it ion:; of this method for use as spot urine testing in lar: wrier population. Tf future studios continue to verify our present finding,i.o. that this test is indicative of early vinyl chi: - ide ir.'t' , it 1>, a; plica M Li.roupla.ut tiie industry where chemically induced fibrosis nay occur. This proposal could yield a sensi tive, simple and ine:a.ensive means of surveillance for early liver injury. A copy of the paper ;c be presented in hew York is enclosed. Concerning the proposal by Dr. Wong, and the related proposal by Dr. Streips; I wuld like to elucidate on the value of this work in deter mining and directing priorities as to which metabolite should be studied for their potential carcinogenicity. Dr. Wong's ability to synthesize metabolic products of various chemicals allows immediate mutagenic bac terial studies for the identification of those chemicals with greatest car cinogenic capability. In order to control costs and increase benefits for the amount of time and money invested, these mutagenic studies must be per formed in order to realistically develop strategies for blocking the etiologic chemical changes leading to the initiation of angiosarcoma. Con sidering the overall complexity of studies of this sort, we feel that the combined work of Drs. Wong and Streips provides a reasonably straight for ward approach to unravelling sequences leading to angiosarcoma. In addition, Dr. Wong's work will develop a method of detecting trace amounts of chemicals and their*metabolic products by use of multivarient analysis in biological tissue. Our present system of storing bloods, urines, and tissue on all workers in the medical surveillance program gives us the equivalent of an immediate clinical trial in a care fully observed worker population with known exposure. This procedure could give us an applicable test system within three years. Finally, Dr. Du's work will make use of animal studies in deter mining the most specific and earliest enzymatic and biochemical alterations produced by chemical exposure. Although some have expressed the view that biochemical alterations have all been identified and worked out, our clini cal experience indicates the need for studies which will objectively deter mine which screening methods should be applied and to which of the exposed industrial population. The animal studies proposed by Dr. Du will allow us to apply these methods under controlled conditions of exposure, dose and duration, providing useful information in just a few years. This will be far more efficient means of determining the best screening methods to employ based on hard scientific data rather than clinical opinion. Simultaneously, Dr. Wong will utilize these same animal tissues for verifying the sensitivity and specificity of the multivarient analysis system for trace organic element detection in biological tissue. The de tection of a trace product does not by itself prove a causal relationship nor indicate recent or past exposure. Therefore, it is vital that these studies be done (simultaneously with the studies for sensitivity and specificity) for verification of the relationship of detection to cause of injury. This information will greatly help in the interpretation of our findings from our stored human blood, urine and tissue samples. BOR 010695 Dr. T.R. Torkelson page -3- We would like to acain point out the importance of our being able to continue to pursue this multidisciplinary research approach in the study o:' the vir.y1 chloride problt-'. I'.v addin;,; tiie vinyl chloride problem as a model and in this systematic manner we feel that this unique ccr..hinntien uf inveetirations will go far toward providing understanding chf.ical carcir.cgcar.- in gwocral and the etiology of angiosarcoma. Our pr^ra,.i can left c;' - '--ncialiy intact with the attached budget. The work proposed, we feel, is clearly defined and should produce positive results well in excess of investment. In addition, we would also like to point out that funding of such a program will sharpen our overall skills and further develop this approach applicability to other chemicals and in other situ ations. I believe that the budget proposed is well within the desirability and capability of the Manufacturing Chemists Association's constituency. In essence, we are asking the vinyl chloride industry to support the program at somewhat less than a level that has been supported by the B.F. Goodrich Company alone for the past two years. Thank you for your serious consideration. Sincerely yours. Carlo H. Tamburro, M.D. Associate Professor of Medicine Chief, Digestive Diseases & Nutrition Section CHT:mma Enclosures cc: Dr. Zeb G. Bell, Jr. Dr. Walter D. Harris Dr. Maury Johnson Mr. Howard L. Kusnetz Dr. W.E. Rinehart Dr. W. Mayo Smith Mr. R.N. Wheeler BOR 010696 REVISED BUDGET UNIVERSITY OF LOUISVILLE RESEARCH PROPOSAL RESEARCH TECHNIQUES AND METHODS FOR DETECTION and r;.Evr.7M:: of (..^crnvnlnlsis in tndestuial workers This revised bvdpot is for proposals Al-4, G, D, C, El, B3-4, ar,J E, all of vNiica nave the nose clinical appli cability. We have excluded proposals Bl, B2, B5, B6, B7, E2, H, and I. BOR 010697 BOR 0 1 0 6 9 8 INDIVIDUAL BUDGETS Proposal Title Investigator A 1-4 G D C EI B3 &4 F Immunological Systems for the Detection of Vinyl Chloride and Other Chemical Injury P. Fortwengler Tissue Antigenic Systems of Detection E. Espinosa Electron Microscopic Evaluation of Liver Tissue from Chemical Workers R. Schrodt Tissue and Urinary Acid Mucopolysaccharide Changes Related to Vinyl Chloride Injury: Use In Early Detection and Diagnosis C. Kupchella Multivarient Analysis of Biological End Products and Biochemicals to Document Chemical Exposure for Early Diagnosis J. Wong Biochemical Enzymatic Systems for Detection of Vinyl Chloride and Other Chemicals J. Du Assays for Identifi cation of the Carcino genic Potential of Industrial Chemicals U. Strelps Personnel Budget 34,100 9,000 8,000 10,000 27,000 12,000 Subtotals To * - T 100,100 ft- n r f Supplies 16,795 Total 50,895 5,100 7 14,000 7,000 7,100 15,500 15,000 25,000 m-4 7,332 34,332 23,000 69,627 169,727 I j A. SUMMARY OF BUDGETS Salaries; Supplies: Indirect Cost: 63% 100,100 6,027 65,065 TOTAL: 234,792 B. POSSIBLE ALTERNATE FUNDING SUGGESTION: Salaries: Supplies: Indirect Cost: 30% 100,100 69,627 30,035 TOTAL: 199,757 BOR 010699 (jotj'Ar>Y /'fjn G' Yf'Oc-MIMG'"' YCAN i t\i i l.i\.._> I.t i,L11, i j. o i.. i ji[)bi.aCO.' !A Charles E. Kupchella and Carlo H. Tamburro Cancer Center and Department of Medicine University of Louisville School of Medicine Louisville, Kentucky 40201 I. INTRODUCTION The recent discovery of a relationship between vinyl chloride and angiosarcoma of the liver has received much attention (1-3). Although there are now systematic detection programs for vinyl chloride workers (3,4), there is as yet no specific chemical abnormality that serves as a good indicator of early, vinyl-chloride-induced liver injury and angio sarcoma. Alpha feto-protein has been a relatively valuable serological marker for hepatocellular carcinoma (5), but is has not as yet proven use ful in the detection of angiosarcoma (6). New leads are needed if more specific tests are to be developed for angiosarcoma. The literature suggests that the glycosaminoglycans in the urine and/or blood should be evaluated as a possible aid in early detection. The production of sulfated glycosaminoglycans is characteristic of malig nant vascular tumors of the skin and some pathologists use this feature as a diagnostic aid (7). Barr and Bonin (8) observed a strong positive alcian-blue, glycosaminoglycan staining reaction in human angiosarcoma tissue and suggested than an attempt be made to qualitate and quantitate the production of glycosaminoglycans in the neoplasms, serum, and urine of those at risk. They pointed out that the urinary glycosaminoglycans may have diagnostic significance in angiosarcoma and, if so, a glycosami noglycan spot test might easily be employed as a gross screening test of vinyl chloride production workers. A number of other observations place the glycosaminoglycans in a relevant position with regard tc angiosarcoma. Angiosarcoma is accom panied by connective tissue abnormalities (2,9) and changes in tissue, urinary, and blood glycosaminoglycans have been found to occur in many connective-tissue disorders -- including connective tissue disorders of the liver (10-14) -- as well as in hepatic cancer (15-17). Supported in part by grants from the B. F. Goodrich Company and the American Cancer Society (IN-111) and a contract with the National Cancer Institute (NOl-CN-55212). BOR 010700 CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO The purpose of this study was to make a preliminary determination of the olycosa:iiir'~''ilvcan patterns in tissue and urine associated with angiosarcoma of the liver and with vinyl-chloride-induced liver injury other than angiosarcoma and to compare these patterns with those in normal ccutrois ana loose associated with other liver disease. Our goal v.'js to th;- 0:0 of - ' cosar'in'" 1 /c?n patt'-''ns in the early detec- tlcri '.i.vi-c.'iiorice-inuj^^a liver injury arid angiosarcoma and to eyrie*' th-* role of the c iyct. revir.: jlytans in the etiology of vinyl C1) U . 1 G l! j . ^i y, 11. procedures a;;d materials used Urine specimens were collected as occasional samples from: 9 normal controls; 9 individuals with histories of occupational exposure to vinyl chloride and having abnormal, liver, biochemical studies; 6 with "other" cancers prior to surgery; 3 with angiosarcoma; 8 with active viral hepatitis; 6 with cirrhosis; 2 with lung-liver metastases; and 4 with metabolic disorders of the liver (congenital and indirect hyperbiliru binemia). In one case of angiosarcoma, 24-hr urines were collected on alter nate days beginning 2 weeks prior to death. Urine samples were collected without preservative and frozen at -76 until analysis. Specimens were divided into two 25 ml samples and one 5 ml sample. Urinary creatinine was measured on the 5 ml sample using a Technicon Autoanalyzer. The degree of urinary glycosaminoglycan polymerization was estimated by dialyzing one 25 ml sample for 24 hours in tap water; the sample was then treated identically to an undialyzed sample by the method of DiFerrante (18) using cetylpyridinium chloride as a precipitant. After resolubilization of the glycosaminoglycans in water, duplicate samples were assayed for total uronic acid by the modi fied carbozole reaction of Bitter and Muir (19). The remaining glyco saminoglycans were reprecipitated with cetylpyridinium chloride and separated into the wash, hyaluronic acid, chondroitin sulfate, and heparin fractions as described by Schiller et. al.(20). Each of the fractions was assayed for uronic acid (/jg per mg of creatinine). Autopsy tissue was obtained in 2 cases of hepatic angiosarcoma (tumor tissue and non-tumor tissue adjacent to tumor), 2 cases of cirrhosis, and in 3 control cases (gun-shot wound victims without liver pathology) and analyzed for glycosaminoglycans by a previously reported modification (21) of the method of Schiller et. al. (20). Uronic acid was determined in each of the hyaluronic acid, chondroitin sulfate, and heparin fractions. Pieces of tissue were subjected to alcian-blue-periodic-acidSchiff staining with and without hyaluronidase and diastase pretreatment. These procedures were carried out according to the methods described by Mowry (22). Ascitic fluid was also obtained at autopsy in one case of angio sarcoma and analyzed for glycosaminoglycans. The fluid was centrifuged BOR 010701 GLYCOSAMINOGLYCANS IN ANGIOSARCOMA and the sediment analyzed as tissue above. The supernatant was treated by the method for urine described above. n c:;_r' inoglycan measurement is given in Table I lease urinary glycosaminoglycans (measured as uranic acid) of all groups. All other groups showed some elevation. The levels in ancivrarccma, hepatitis, cirrhosis, and liver metastases were significantly elevated (P < .05) over normal controls. The cirrhotic group exhibited the greatest variance in urinary glycosami- noglycans. No significant differences were found in urinary creatinine levels between groups. There were no significant differences between groups in either the percentage of the total glycosaminoglycans that was dialyzable (Table I) or in the percentage of the unfractionated total that appeared in the hyaluronic acid, chondroitin sulfate, and heparin fractions. Seven of 9 vinyl-chloride-exposed individuals other than those with angiosarcoma had positive chondroitin sulfate fractions with nega tive hyaluronic acid and heparin fractions. This was true in only 3 of 32 other urines evaluated in this same manner. The pattern of daily glycosaminoglycan excretion prior to death due to angiosarcoma in one individual is given in Figure 1. Total tissue glycosaminoglycan levels for angiosarcoma tumors, fibrotic tissue adjacent to tumors, cirrhotic liver tissue and normal liver tissue are shown in Figure 2. Fractional hyaluronic acid, chon droitin sulfate, and heparin levels are given in Figure 3. Histochemically, angiosarcomatous tissue exhibited a strong alcian- blue positive staining reaction. Alcian-blue staining was only slightly less in '`non-tumor" tissue adjacent to tumor masses. The staining reaction in tissue from normal liver was very weak and only slightly stronger in cirrhotic 1iver.tissue. The strong alcian-blue reaction in angiosarcomatous tissue did not occur if sections were pretreated with hyaluronidase. Ascitic fluid sediment was uronic-acid-positive in only the hyaluronic acid fraction -- 112 ^ug uronic acid per gram of dry, defatted sediment; ascitic fluid supernatant contained 1.7, 1.2, and 0.2 /ug uronic acid per ml in the hyaluronic acid, chondroitin sulfate, and heparin fractions, respectively. The literature indicates that normal male creatinine excretion is 1.5 g per 24 hoursL).Thus, our normal mean (Table I) of 3.2 * *4 aig cetylpyridiniuin chloride-precipitable uronic acid per mg creatinine falls in the middle of the normal ranges reported by Varma et. al. (24), CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO 2.6 - 4.7ug/mg; DiFerrante and Rich (25), 2.9 - 4.Qug/mg; and Kao and Leslie (26), 1.8 - 4.9/ig/mg. (27) h *. Ai * -l.thr,ough ou" stu;Jy wa s not t h"1' i !1 ~ > give .b"iir.on 1 ycart excretion is constant from &1'-* L. 9 V: ry> ; i Gy g L. La i . (2g; .,,ve siiGv.n that the proportion of urinary in 11 ch oivOro tin sulfate fraction is constant from roes free 2 t'i 0-70. ^ ir Mr.: K1 ^ . r] e- v ' c- 1. [; al 1r also enorted that the chondroitin sulfate 11 "a c.ic. i drops until age 20, Lh> t *1 ! I ^ LnCU ur ir.= ry cr. aJ 1 g1 Q u Tb? fact t !. r. t v;e to un z no idifferences between groups in the creatinine concentration is significant in that it indicates that occa sional samples do reflect 24-hour excretion when normalized to creatinine. Precedent for expressing glycosaminoglycan measurements as a function of creatinine content in occasional urine samples has been established by DiFerrante and Rich (25) and Pennock (29). Manley et. al. (28) have shown that the creatinine/uronic acid ratio is steady from ages 20-70. Our results indicate that the liver diseases evaluated are accom panied by elevated urinary glycosaminoglycan excretion. Our tissue data suggests that this reflects liver-tissue glycosaminoglycan elevation and conforms to the reports by others that both hepatic connective tissue disorders (10-14) and hepatic cancer (15) result in increased hepatic glycosaminoglycan levels. It may be significant that the angiosarcoma patients had half the urinary glycosaminoglycan excretion of patients with liver metastases and that our analysis of angiosarcomatous tumor tissue exhibited half the glycosaminoglycan content reported by Kojima et. al. (15) for hepatocellular carcinoma. While our data suggest that liver disease results in a decrease in the proportion of highly polymerized glycosaminoglycans, variance v/as large within each group and none of the differences between groups were statistically significant. Although we have not completed the characterization of isolated glycosaminoglycan fractions, our data indicate: 1) that the chondroitin sulfates are the primary urinary glycosaminoglycans in both normal controls and in disease states; 2) that the chondroitin sulfates and heparin are the dominant glycosaminoglycans in normal and cirrhotic livers (Figure 3). Chondroitin sulfate is elevated in the fibrotic, non tumor, portions of angiosarcomatous livers while heparin is the predomi nant glycosaminoglycan in tumor tissue. Hyaluronic acid is also apparent ly elevated relative to chondroitin sulfate in angiosarcomatous tumors (Figure 3); and 3) that hyaluronic acid is the sole glycosaminoglycan in ascites fluid sediment. These qualitative data are in general agreement with those reported by others. Goldberg and Cotlier (27), Douglas et. al. (30), and Varma et. al. (24) have reported that the chondroitin sulfates are the predominant urinary glycosaminoglycans. Varma et. al. reported that 2/3 of urinat*y glycosaminoglycans are chondroitin-4-and chondroitin-6-sulfate and this agrees with our data on normal controls and on those with liver disease. Kojima et. al. (15) reported that in hepatocellular carcinoma 010703 BOR GLYCQSAMINOGLYCANS IN ANGIOSARCOMA tissue, chondroitin sulfates and hyaluronic acid were increased 33 and 10 times, respectively, over amounts found in healthy livers; the heparin and heparan sulfate proportions dropped. -This contrasts with our data on angiosarcoma tissue, i.e. neoarin and hyaluronic acid increased 5 and 10 tin,--, respccti v-, iy, , v.-r* 1 t'ssv.-; ciipr.droi tin sulfate levels rose but fell in proportion to ouier glycosaminoglycans. Galambos and Sh.'pira (10) report.'.::L the chcrnroitin suit,,os are dominant in nr-,r.-'l 1 i - - if : ~ -r r i c fibreris, but Ksjf a et. al. (15) report that cho:,', .utitin;.,.,-:'*::,-,:!:..! ant hyalurcnidose-reslstant glyccsaminogly- caiis arc dominant. Kurooa et. al. (31) also reported that heparan sulfate is the dominant glycosaminoglycan in the normal liver. Our histochemical observation that nearly all of the increased alcian-blue positive material in ar.giosarccm.atous livers was susceptible to hyaluronidase digestion suggests that the observed chondroitin sulfate elevation is due to chondroitin-4- and/or chondroitin-6-sulfate. The increases in liver and urinary glycosaminoglycans may well reflect an important role of these substances in the process of fibrogenesis and in tumor growth. Galambos and Shapira (10) reported that hyaluronic acid was elevated during hepatic fibrogenesis. If a similar fibrotic process is operative in angiosarcoma, it may be that the observed tumor-tissue heparin increase is reflective of tumor growth. We did observe a four- tc sixfold greater heparin level in tumor tissue than in adjacent, non-tumor tissue. The observation that the chondroitin sulfates tend to be the exclusive uronic-acid-positive constituents in the urine of individuals is paradoxical in that those glycosaminoglycan fractions that are most elevated in angiosarcomatous tissue are those that are absent from the urine of individuals who may well have early, vinyl-chloride-induced liver injury. This pattern may be due to the selective action of lyso somal, glycolytic enzymes in the liver and/or may reflect the role of the chondroitin sulfates in early fibrotic changes in the liver. Certainly the potential usefulness of this pattern in early detection warrants the more complete evaluation now ongoing in our laboratory. V. SUMMARY Glycosaminoglycans were measured in urine and tissue of patients with hepatic fibrosis and hepatic cancer including vinyl-chlorideexposure-associated liver injury and angiosarcoma. Angiosarcoma, hepa titis, cirrhosis, and liver metastatic patients exhibited significantly elevated glycosaminoglycan excretion. Angiosarcoma tissue exhibited elevated glycosaminoglycan levels with the greatest increases in the heparin fraction. Histochemically, angiosarcomatous tissue gave a strong alcian-blue staining reaction which could be prevented by pretreatment with hyaluronidase. Although vinyl-chloride-expcsure-associated liver injury other than angiosarcoma was not accompanied by a significantly elevated glycosaminoglycan excretion, this condition tended to be associated with a urinary glycosaminoglycan excretion pattern in which the chondroitin sulfate fraction was the only uronic-acid-positive fraction. BOR 010704 CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO TABLE.I Urinary Glycosm'line;! yea n L- vels in ak; Uronic Acid Per mg Creatinine by Liver Diseases Category Patient group normal control vinyl chloride exposed Cases 9 9 ug uronic acid per ,"3 creatinine (- 1 S.E.) 3.2 * .4 4.1 i .4 % uronic acid not dialyzcble ( 1 S.E.) 65 * 5 39-6 other cancer 6 4.5 t 1.5 39 6 other liver disease 4 5.1 0.8 37 * 13 angiosarcoma hepatitis cirrhosis liver metastasis 3 8 6 2 7.6 i 1.6 8.5 * 1.8 12.7 * 3 13.8 .9 41 22 52 14 53-9 42 BOR 010705 DAYS PRIOR TO LIVER DEATH <* BOR 010706 GLYCOSAM'NOGLYCAN CONCENTRATION IN ANGIOSARCOMA CIRRHOSIS, AND NORMAL LIVER TISSUE Tumor Cose I Non-tumor Adjocent Cote I Tumor Cote 2 Non-tumor Adjocent Cote 2 Cote 3 Cote 4 Cose 5 Cote 6 Cote ? CONCENTRATION OF INDIVIDUAL GLYCOSAMINOGLYCAN FRACTIONS ISOLATED FROM HEPATIC ANGIOSARCOMA, CIRRHOSIS, AND NORMAL LIVER TISSUE BOR 010707 Figure 2 Figure 3 (in nary g lycosur, noglycan output in one angiosarcoma patcnt during the 16-day period prior to death. Glycosaminoglycan concentration in angiosarcomatous, cirrhotic and normal human liver tissue. Fractional concentrations of glycosaminoglycans in angiosarcomatous, cirrhotic and normal human liver tissue. BOR 010708 CHARLES' E. KUPCHELLA AND CARLO H. TAMBURRO 1. Cr i 4 VI. REFERENCES L, i.i'.c! 0o!-. t`. H. Angiosarcoma of the Liver in the jc of kulyv.nyl Chloride. J. Gccup. Med. 16^: 150-151, 2. Falk, Cr Re-rti: P1 a fit. Jn. in h, Johnson, M. N., and Key, M. M. fcii at a Vinyl Chloride Polymerisation D2-63 1974. 3. Makk, L., Creech, J. L., Whelan, 0. G., and Johnson, M. N. Liver Damage and Angiosarcoma in Vinyl Chloride Workers: A Systematic Detection Program. JAMA 230: 64-68, 1974. 4. Creech, J. L., Makk, L., Whelan, J. and Tamburro, C. H. Hepatotoxicity Among Polyvinyl Chloride Production Workers During First Year of Surveillance Program. Gastroenterology 7_: 786, 1974. 5. Kohn, J. and Weaver, P. C. Serum Alpha Fetoprotein in Hepatocellular Carcinoma. Lancet 2: 334-336, 1974. .6 Tamburro, C. H., Makk, L. and Creech, J. L. Unpublished observation. 7. Girard, D., Johnston, W. C., and Grahm, 0. H. Cutaneous Angiosar- coma. Cancer 25: 868-883, 1970. 8. Barr, R. and Bonin, M. "Letters." JAMA 231(9): 914, 1975. 9. Popper, H., and Thomas, L. B. Alterations of Liver and Spleen Among Workers Exposed to Vinyl Chloride. Ann. NY Acad. Sci. 246: 172-194, 1975. 10. Galambos, J. T., and Shapira, R. Natural History of Hepatitis: IV Glycosaminoglycuronans and Collagen in the Hepatic Connective Tissue. J. Clin. Invest. 52(11): 2952-2962, 1973. 11. Koizumi, T., Nakamura, N., and Abe, H. Changes in Acid Mucopoly saccharide in the Liver in Hepatic Fibrosis. Biochim. Biophys. Acta. 148: 749-756, 1967. 12. Kojima, 0. Studies on the Metabolism of Hepatic Connective Tissue in Fibrosis of the Liver. Med. J. Osaka Univ. 16: 419-429, 1964. 13. Rubin, E. Autoradiographic Characterization of Sulfated Acid Mucopolysaccharides in Experimental Cirrhosis. J. Histochem. Cytochem. 14: 688-689, 1966. 14. Patrick. R. s. and Kennedy, J. S. The Synthesis of Sulfated Mucopolysaccharide at Sites of Hepatic Fibrosis Induced by Carbon Tetrachloride, Amyloidosis, and the Implantation of Catgut. J. Pathol. Bacteriol. 88: 549-555, 1964. 15. Kojima, 0., Nakamura, N., Kanatani, M. and Ohmori, K. The Glyco- saminoglycans in Human Hepatic Cancer. Cancer Res. 35(3): 542-547, 1975. BOR 010709 GLYCOSAMINOGLYCANS IN ANGIOSARCOMA 16. Anqhileri, L. J. Metabolism of'Acid Mucopolysaccharides in hepatoma and in Normal Liver. Oncology 3Q: 304-317, 1974. 17. Yamamoto, K., and Tervama. H. Comparison of Cell Coat Acid Muco- (o`;js:cch:.ric-LL of ,;i Liver anti Various Ascites Hepatoma Cells. 18. DiFerraute, N. M. The Measurement of Urinary Mucopolysaccharides. Anal. Biochem. 21_: 98-106, 1967. 19. Bitter, T., and Muir, H. A Modified Uronic Acid Carbazole Reaction. Anal. Biochem. 4_: 330-334, 1962. 20. Schiller, S., Slover, G. A., and Dorfman, A. A Method for the Separation of Acid Mucopolysaccharides: Its Application to the Isolation of Heparin from the Skin of Rats. J. Biol. Chem. 236(4): 983-987, 1961. 21. Kupchella, C., and Steggerda, F. The Distribution of Acid Muco polysaccharides in the Canine Gastrointestinal Mucosa. Trans. NY Acad. Sci. 34; 361-360, 1971. 22. Mowry, R, W. Alcian Blue Techniques for the Histochemical Study of Acidic Carbohydrates. J. Histochem. and Cytochem. 4: 407, 1956. 23. Sunderman, F. W. and Boerner, F. Normal Values in Clinical Medicine. W. B. Saunders. Philadelphia, p. 353, 1949. 24. Varma, R. S., Varma, R., Allen, W. S., and Wardi, A. H. Urinary Excretion of Acid Mucopolysaccharides in Schizophrenia. Biochem. Med. 11(4): 358-369, 1974. 25. DiFerrante, N. and Rich, C. The Determination of Acid Aminopolysaccharide in Urine. J. Lab. Clin. Med. 48: 491-494, 1956. 26. Kao, K. and Leslie J. -Micro Fractionation and Determination of Urinary Glycosaminoglycans. Biochem. Med. 9(4): 317-326, 1974. 27. Goldberg, J. and Cotlier, E. Specific Isolation and Analysis of Mucopolysaccharides (Glycosaminoglycans) from Human Urine. Clin. Chim. Acta. 41: 19-27, 1972. 28. Manley, G., Severn, M. and Hawksworth, 0. Excretion Patterns of Glycosaminoglycans and Glycoproteins in Normal Human Urine. J. Clin. Pathol. 2J_: 339-345, 1968. 29. Pennock, C. A. A Modified Screening Test for Glycosaminoglycan Excretion. 0, Clin. Path. 22: 310, 1969. BOB 010710 CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO 30. Douglas, C., Nowak 0. and Dan^s, B, Mucopolysaccharides in Urine During Norm] ru: on De,'f.-1 r,t.. Pcdi!r. Res. 7: 724-727, 1973. 31. Kuroda, J., Saito, S. Seno, N., Naqe.se, S., end Anno, K. Isolation and Chemical Charac i" i i- >- './ >Oii \j \ i i u C U I'j 0 lysaccliarides from Rat Tumors. Cancer r.^s c -(2 i: 30^-312, 1974. BOR 010711 IMMUNOPATHOLOC] c "PV'TTT.-iS JM LIVER ANGTOSARCOMA University o : L'-'U:! --v: .1 i <- School of Medicine Louisville. Kentucky I IKTEOD TI ON In hepatic fibrosis and angiosarcoma associated with vinyl chloride exposure of industrial workers, manifestations of the disease could not be detecteu in most cases until the process was fa,r advanced (1) . Normal values of liver function tests were reported in a case with significant vinyl chloride hepa tic fibrosis (2), and only 1 small percentage of workers of a plant unit where seven cases of liver angiosarcoma were diag nosed had abnormal blood screening tests (3). Thus, conven tional liver function tests do not appear to be sensitive in dicators of vinyl chloride liver disease. Development of more sensitive methods for detecting the disease in early stages would be of great importance. An approach to this may be pro vided by immunologic studies. In such a study the question arises whether the fibrotic and angiosarcomatous livers contain antigens that are different from those present in normal tis sue and whether such changes could stimulate an immunologic response. The purpose of this study was to search for anti genic changes in the angiosarcomatous tissue and to test for possible presence of an antibody response in the host. II.. PROCEDURES AND MATERIALS USED A. Patients' Sera and Tissue Specimens Serum samples from B.F. Goodrich Co. workers with histor ies of vinyl chloride exposure of several years included sam ples from two individuals with liver angiosarcoma, ton with liver dysfunction with fibrosis and ton with normal liver function tests. The patients with angiosarcoma died and the diagnosis was confirmed at autopsy and portions of tumor and neighboring liver tissues were obtained at autopsy. Patients with liver dysfunction with fibrosis included individuals with BOR 010712 ENRIQUE ESPINOSA, M.D. abnormalities in liver function tents and fibrosis detected at biopsy. Tissues were a]so obtained from coroner's autopsies of healthy individuals a few hours after death by gunshot wounds. IS. Tissue Extracts and Antisera Liver angiosarcoma and adjacent liver tissue and post-mor tem tissue:', cons 1 c`e": d no be nor: n! were frozen and stored at -70cC until used.. 1 sr do:..; w-nie cut, thawed and homogenised in 2-3 volumes of distilled water in a Fotter-Elvehjem grinder in an ice bath until a smooth suspension was obtained. After centrifugation at 20,000 x G for 30 min, the supernatant fluid containing the aqueous extract was lyophilized. Albino rab bits were immunized with the tumor or normal liver extracts in Freund's complete adjuvant and sera collected'and stored fol lowing procedures detailed elsewhere (4). Reaction of these immune sera with human serum or plasma was eliminated by ab sorption with 100 mg of lyophilized, pooled normal human serum /ml antiserum. Antisera were routinely absorbed in this man ner prior to use. Additional absorption with tissue extracts was carried out with 100 mg lyophilized extract/ml antiserum. Absorptions followed a procedure described previously (5). C. Treatment of Tissue Extracts Enzymatic treatment of tissue extracts was carried out with Pronase and trypsin as previously described (6). Periodate oxidation was done according to Rajam et al. (7). Ammonium sulfate and cold ethanol fractionations were carried out as detailed (8) . D. Immunodiffusion and Immunofluorescence Double immunodiffusion was carried out in 0.8% agarose in phosphate-buffered saline pH 7.2 (PBS) containing 0.1% sodium azide. Circular wells, 2 mm in diameter, 3 mm apart were used. Immunoelectrophoresis was performed according to Schei- . .degger (9) using 0.8% agarose in 0.025 M Veronal buffer at pH 82 In immunofluorescent studies cryostat sections of rat kid ney, stomach or intestine and liver (4 microns) were used as substrate for antimitochondrial, antismooth-muscle and anti nuclear antibodies. Liver sections from rats exposed 4, 8 and 14 days to 1-2% vinyl chloride for 4 hr/dav were also used. The sections were covered with dilutions of patients' sera for 45 min at room temperature, washed twice in PBS for 10 min and then covered with fluorescein conjugated IgG fraction of rab bit anti-human immunoglobulins'serum (Cappel Laboratories, Inc.) for 45 min and washed as before prior to examination. Cryostat sections .of liver angiosarcoma and liver tissue con sidered to be normal (4 microns) were washed twice in PBS for 10 min to wash off nonfixed imrcunoglobuJins. After drying. BOR 010713 ANGIOSARCOMA sections were stained wi'/h f1 mrmccin conjugated IgG frac tions of rabbit anti-human 3oG norum and goat anti-human IgM serum (Capp.;! ,,..1 u.ulc i . . , : so. ). Sections were washed as a- bovn and oxrr ' n---' ' u:';''" 's ri --r'^nf microscope. Represen tative fr corn r.'-ohio".............. rh '--v^d ui th hematoxylin and oosin to al3ov: corrr'1 ' `d ; o-.,-- -..i j r'~'"r.r'f lv.O"r-svor.cc and the histo-- log'"?. . r: r ~ -ati c- of the tissues v;as i n d ? o-1 d h" t1^ de-'''"'. "tv't i'-'P of their staining hv anti-- nuel': r ..`.or aoc .uuu.ay s c. . isir e er. immunof luoresccnt pree- - E. Elution cf Tumor-bound IgG 4 The tumor and live: tissues v'orc extracted five times with PBS to wash off non fixed is.svanoci ebulins and then extracted at pH 2.5 to release bound IgG according to a procedure applied in the elution of renal-bound anuibedy (10). F. Circulating Tissue Antigens Liver-specific antigen LSA (8), tissue antigens of wide organ distribution (4) and bile antigens (11) were testeu in the patients' sera by immunodiffusion as described previously. III. RESULTS A. Angiosarcoma-related Antigen To test for presence of new antigens appearing in liver angiosarcoma, antiangiosarcoma serum was absorbed with human serum and liver extract and tested by immunodiffusion with ex tracts of both normal liver and angiosarcoma tumor at varying concentrations. This absorption eliminated all reactivity with liver extracts prepared from five normal individuals but not with the angiosarcoma extracts where one line of precipi tation remained (Fig. 1). This line of precipitation could still be seen after additional absorption of the antiserum with kidney extract. In contrast, absorption with the tumor extracts eliminated completely the angiosarcoma-related line of precipitation. Absorption v:ith spleen and lung extracts also eliminated this line of precipitation. Thus, the antigen appeared to be of restricted tissue distribution and not an giosarcoma specific. The antigen was inactivated by trypsin and Pronase and thus appeared to be a protein or closely asso ciated to protein. Incubation of the tumor extracts for 1 hr at 4 C in citrate buffer, pH 2.5, resulted in inactivation of the antigen whereas incubationin phosphate buffer, pH 5.0, neutral or alkaline pll up to pH 10.0, did not affect it. Theantigen v/as shown to be relatively thermolabile. Incubation of the tumor extracts for 30 min in PBS at 25 and 56C did not affect the antigen whereas incubation at 70C and higher BOR 010714 ENRIQUE ESPINOSA, M.D. completely inactivated it. The antigen precipitated mainly at 20-30% saturated ammonium sulfate and at ethanol concentra tions of 30-70% (Table I). B. Absence of a Normal Tissue Antigen in Angiosarcoma Anti] i *'er r Tun rdv'erbc! with human serum gave several arcs of precipitation in immunoelectrophoresis with extracts of normal liver c.ui aru-inrrverve tissue (Pig. 2a). These lines could r.el he s^n lev.-ir.-j additional absorption of the anti serum v:i Ji nor. el liver. In contrast, absorption with liver angiosarcoma extract failed to eliminate one of the arcs of precipitation (Fig. 2b). Thus, the tissue antigen related to this arc of precipitation appeared to be absent in the angio- sarcomatous tissue whereas the antigens corresponding to the other arcs were present. The absent antigen in angiosarcoma was shown to be present in kidney and lung extracts in addi tion to liver by absorption and direct immunodiffusion tests. Physicochemical characterization studies indicated this anti gen to be unaffected by Pronase and trypsin and inactivated by periodate treatment. The antigen was relatively thermostable withstanding incubation at 70C for 30 min in PBS. The anti gen was destroyed following incubation of the liver extract in citrate buffer at pH 2.5 or lowe_ for 1 hr at 4C; incubation at pH 5.0 or higher (up to pH 10.0) did not affect it. This antigen precipitated over a wide range of ammonium sulfate and ethanol concentrations (Table I). C. Angiosarcoma-bound IgG IgG fluorescent staining appeared in a linear pattern in the peripheral portion of the tumor cells suggesting in vivo binding by the tumor of the immunoglobulin. This staining is illustrated in Fig. 3. Fig. 4 shows the angiosarcoraatous cells surrounding irregular vascular spaces. Relatively coarse, linear fluorescence was also present along some hepa tic cords and strands of connective tissue. There was no ev idence of igM. Control post-mortem liver tissue did not show any significant fluorescence of bound IgG. Staining for IgG of the tumor sections *did not change after several washings at pH 7.2 indicating that the IgG was firmly bound to the tumor. In contrast, sections showed marked diminution of staining after washing at pH 2.5. Elution of bound IgG from salineextracted tumor homogenates was thus attempted at acid pH. With the five successive saline extractions .the amount of sa line soluble IgG gradually diminished to nondetectable levels; and at acid pH bound IgG was released from- the homogenate. Similar treatment of liver homogenates did not demonstrate presence of bound IgG (Table II). D. Circulating Autoantibodies and Tissue Antigens -i _ * Serum autoantibodies to nuclei, mitochondria and smooth - - BOR 010715 ANGlOLAKt'UiiA muscle were nr'nni-.ivo in all patients examined. Xn addition, scrum from these patients did not show reactivity with liver from rats exposed to vin/' chloric''. Li V'T-speci f i.c antigen Lf-A (3), bile antigens CM) and other tissue antigens (4) as sociated with liver damage ware m-l detected in these pa tients . iv. iwcviiiov The i.'..'v,,...cd c s'. : v.e'.r : rf car.cox have beer, under intense mverjuig^Lron c uo r 11 j l ^ ~^ ^, and antigenic dif ferences brxxocx :x I and : : ' ! a -at tissue are considered to be fundar/ental fa^to-- in the 'r'r"'nologic approach to cancer therapy and diagnosis. Liver angiosarcomutous tissue was thus analyzed in this v;orx. for pretrer.ee of neoantigens, normal tis sue antigens and tumor-bound immunoglobulins. Several normal tissue antigens were found by immunodiffusion to be present in the tumor, but one antigen of rather wide organ distribution was not detected. These findings are in agreement with obser vations in other tumors indicating that tumor cells contain many of the antigens of their original hosts and lack some normal tissue antigens. For example, immunohistochemical studies have sh^wn the loss of kidney antigens in stilbestrol- and x-ray-induced kidney tumors (12), of skin antigen in 3- methylcholanthrene-induced mouse squamous cell carcinoma (13) and of certain muscle antigens in 20-methylcholanthrene-in- duced rat rabdomyosarcoma (14). By far the most extensively studied class of tumors are the chemically induced hepatomata where deletion of liver antigens have been shown in tumors in duced with 4-dimethylaminoazobenzene (15, 16), diethylnitrosa- mine (15) and 2-acetomidofluorene in the rat (15, 17) and o- aminoazotoluene in the mouse (18). In human carcinoma, loss of antigens have been reported in squamous cell carcinoma (12, 19), loss of the ABH blood group isoantigens in some solid tumors (20, 21) and of HL-A isoantigen in lymphoma (22). In addition, it has been well documented that as cells trans form from a normal state to malignancy they may gain new anti genic specificities. Tumor-specific transplantation antigens have been demonstrated in a number of experimentally induced tumors (23-26) as well as in spontaneous tumors in man (27- 29). In the present report immunodiffusion analyses of liver angiosarcoma and other human tissues with rabbit antiangiosar coma serum did not indicate the presence of a tumor-specific antigen but rather of an antigen found in lung and spleen but not in liver and kidney. This antigen is being further char acterized in our laboratory. Of particular interest is the demonstration of tumor-bound IgG by immunofluorescence and elution experiments. This find ing must however be interpreted with caution ahd should be confirmed in biopsy specimens. The tumor-bound IgG may repre sent specific antitumor antibody, antibody fixed by the tumor tissue "nonspecifically" or part, `of both. Further speculation is premature until it has been shown that the staining pattern % BOR 010716 ENRIQUE ESPINOSA, M.D. is due to the deposition of d specific antibody, that the elu ted antibody is specific or until the relevant antigen has been identified. V.'ork is in progress to determine the precise significance of the finding of IgG in the tumor. Ac k nowledgements: The author wishes to thank Drs. W. M. ChrJ: r.'-.un, ; . k. Sci'.rci.'t and P. II. Curstens for fruitful discussion and advice about the histologic sections and Drs. C. Tamburrc and I,. ::.i k for providing rerun samples and au- triply m='-i i r: j.. I al o v?irh to thank Miss M. VanSraun and ' v--- v t 1 * . * J V* *. t- < s: - ,, ,1. *,> j_ r-\. k. line technical assistance. This work was supported in parr by a grant from B. E. Goodrich, Co. V. SUMMARY Immunodiffusion analyses of human liver angiosarcoma asso ciated with vinyl chloride exposure indicated presence in the tumor of an antigen not detected in normal liver and kidney but found to be present in lung and spleen. This antigen was shown to be a protein, inactivated by Pronase and trypsin, relatively susceptible to heating- and to acid pH and precipi tated mainly at 20--30% saturated ammonrum sulfate and. at 30 70% ethanol concentrations. The tumor was shown to contain several antigenic constituents of normal tissue but one normal tissue antigen was not detected. This antigen was character ized as a substance unaffected by Pronase and trypsin and in activated by periodate. It was relatively thermostable, af fected by acid pH and precipitated over a wide range of ammon ium sulfate and ethanol concentrations. Tumor specimens ob tained at autopsy contained bound IgG as shown by immunofluor escence and elution experiments suggesting possible in vivo binding of IgG to the tumor. kt BOR 010717 ANGIOSAP.COMA TAB IT-: I Angiosr.rcor.n.-rolet ..d Antic.:. r.n.i Antigen .Absent from the Tl1.:.oi i:'i Sulf:: eo a;.' Ethanol Fractions a Presence of Fraction tested An giosarcoma-related b antigen Antigen absent from c Angiosarcoma Ammonium sulfate: 0-20% saturation + - 20-30% saturation ++ + 30-50% saturation 50-70% saturation - +++ . ++ Ethanol: 0-20% - , ++ 20-30% - +++ 30-50% ++ +++ 50-70% d SN ++ " ++ + a +++, ++, + indicate strength of double diffusion reaction in dilution assay. *'*' b Detected in angiosarcoma fractions. c T' Detected in liver fractions, d . SN = supqrnate of the 70% ethanol precipitation, and lyophilized. dialyzed BOR 010718 ENRIQUE ESPINOSA, M.D. rji 7\ T T? II IgG in Saline and Acid Extracts of Angiosarcoma and Liver Tissues Weight solid extracted from a Preparation tested 1 gm (wet vreight) tissue Presence of IgG (mg) Angiosarcoma: Saline extract 1 29.3 +++ Saline extract 2 8.6 ++ Saline extract 3 6.2 + Saline extract 4 5.6 - Saline extract 5 6.1 - Acid extract 6.1 ++ Liver: Saline extract 1 47.4 +++ Saline extract 2 14.5 +++ Saline extract 3 8.6 + Saline extract 4 7.4 - Saline extract 5 6.4 - Acid extract 9.0 - a Tested by immunodiffusion at concentrations of the eluates ranging up to 2%. Present at concentrations 0.05-0.1% (+++); 0.2-0.5% (+-t) ; 1-2% (+) ; negative at 2% 1 (-) . BOR 010719 ANGIOSARCOMA LEf o\' fiourkg Fig 1' Demonstret iphcrnl v a ng nr--:\ re' D.T i -t i or:" i'T .r: ' rabbit rit hu.'.m oi I. esrrcemn-rolatod antigen. Per. - . i" 1 dilutions of liver !' ' ::d r.^rr:1' liver extract (b) . ' s'* -I. at ino ng/ml in the ' *. .'j.. i; each plat'' contain ".`'ree .absorbed with normal Fig. 2 Derronstr; f:' - or. ' ,r : re a] a-`nt in nngiosnr- conn. (r) Vrsugh cor te ire rabbit a at i hum an liver serum nhr-'-'r.b "'`h nor:-'-' he serum. (b) Trough contains the antiiiver serum additionally-absorbed with anyiossrovr.n entreat. In both, plates top wells have 10 S solution of liver extract and lower wells angiosarcoma extract. Anode is to the right. Fig. 3 1 Immunofluorescent staining of liver angiosarcoma by fluorescein conjugated IgG fraction of rabbit anti human IgG serum (x 400). Fig. 4 Cryostat section of angiosarcoma tumor stained with hematoxylin-eosin (x 400). Bor 010720 REFERENCES ttnk i ntrr^iTsri :tostC; pitd ; 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Heath f 0 k # r X - * I f i. . , tind Croc ... i i J . L ist.i c r. O Va ' 'r; O ` '< ` - li" Liver Chlor i do Wot';; n " >< - p r ; t.ed Stat-e s. Ann. Sci. , 246:231-230, 1975. CharacterAmong Vinyl N.Y. Acad. Berk, P. i >. , `G. r 1 i 1 , . . . , anti Waogo,. i )" , vT , G , Persistence Of V.: 1 o:.' wY\ . . 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