Document ax8LwYnQYj60kozy6ZLDZonY

SLIDE 2, 3, 4. 5. This illustrates an early increase in glutathione reductase activity followed closely by a fall in glucose-6-phosphatase which becomes signifi cant after 71 hours of total exposure (Slide 3). At 100 hours, a marked increase in glucose-6-phosphate dehydrogenase (Slide 4) similar to that seen in metabolic studies by Weber and his group concerning hepatic primary cell neoplasms was found. As shown in Slide 5, Weber's molecular correla tion concept of neoplasia shows an impairment of the glucose-6-phosphatase producing a decrease in gluconeogenesis and an increase in the pentose phosphate shunt pathways which eventually lead to an Increase in purine biosynthesis and nucleic acid synthesis. This, presumably, leads to increased liver cell replication. These biochemical changes in the early stages of vinyl chloride exposure may well explain the histological findings in humans exposed to vinyl chloride which appear to be character istic of vinyl chloride exposure. These histological findings of focal nodular hyperplasia of the hepatocytes may well be the histological reflec tion of a shift in gluconeogenesis and an increase in nucleic acid synthesis, induced by a mechanism not yet completely understood during the early phases of vinyl chloride exposure. If one were to carry this to a logical conclusion one would presume that the hepatocyte would eventually develop into a malignant cell with continuous regeneration and exposure to carcino gens. The fact that the primary hepatocyte does not become malignant in the adult rat, but does become malignant in the newborn rat with similar vinyl chloride exposure, led us to study the detoxifying capabilities of the hepatocyte versus the sinusoidal lining cells. During this time, there were no significant differences in the usual biochemical clinical studies used for screening early vinyl chloride injury. WC 000003275 20 EXPOSURE TIME (hours) SLIDE 2 SHOWS THE GROUP MEANS OF THE SPECIFIC ACTIVITY OF GLUCOSE-6PHOSPHATASE WITH RESPECT TO TIME OF EXPOSURE. THE CONFIDENCE'INTERVALS FOR GROUP MEAN DIFFERENCES ON THE ACTIVITIES' OF ENZYMES DISCUSSED ARE BASED ON THE ERROR MEAN SQUARE FROM A TWO FACTOR ANALYSIS OF VARIANCE WITH INTERACTION. THE TWO MAIN EFFECTS BEING TIME IN HOURS AND EXPOSURENONEXPOSURE. THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN THE EXPOSED AND CONTROL GROUPS FOR THE FIRST THREE TIME POINTS. HOWEVER, THE MEAN LEVEL OF THE EXPOSED GROUP IS SIGNIFICANTLY LESS THAN THAT OF THE CONTROL AFTER 71 HOURS OF EXPOSURE. VVC 000003276 21 SJLifle * protein) osciellzod/mln/mg NADPH moles j t/y x 2 10 SLIDE 3 SHOWS THE GROUP MEANS OF THE SPECIFIC ACTIVITY OF GLUTATHIONE REDUCTASE WITH RESPECT TO TIME OF EXPOSURE. INCREASE THROUGHOUT THE ENTIRE EXPERIMENT. THERE IS A SIGNIFICANT & .o 22 .n moles ,,NADP roducod/mln/mg protein 30 SLIDE 4 SHOWS THE GROUP MEANS OF THE SPECIFIC ACTIVITY OF GULCOSE-6PHOSPHATE DEHYDROGENASE WITH RESPECT TO TIME OF EXPOSURE. THERE IS A SIGNIFICANT INCREASE AFTER ABOUT 100 HOURS.OF EXPOSURE. 000003278 vvc W tBtK > NULtCULAK CUKI AI1UN CUl'iCtKl Uh W tU^LAblA SIS3H1NASOI9 amand SAVMHIVd 31VHdSOHd 3SOJ.N3d 24 vvc 000003279 VINYL CHLORIDE OXIDATION AND DETOXIFICATION J.T, Du, Ph.D., and C.H. Tamburro, M.D. The second phase of study of the biochemical changes associated with vinyl choloride exposure were directed at oxidation and detoxification. Sprague-- Dawley rats were randomly exposed to controls and vinyl chloride exposure at 28,000 ppm, 7 hours per day, five days per week, for a total of 210 hours of exposure, for six weeks duration. Slide 1 shows the vinyl chloride metabolic fate scheme presently under use. Vinyl chloride being metabolized at higher levels by mixed-function oxidase within the liver may be converted to chlorooxi-- rane or the epoxide. This unstable intermediate may then spontaneously revert to form chloroethanol and the chloroethanol may further be metabolized to chloroacetaldehyde. Secondly, the chlorooxirane may be directly detoxified by GSA via the glutathione epoxide S-transferase or further oxidized to chloroacetal dehyde. Chloroacetaldehyde in turn may be shown by glutathione aralykly-S- transferase or further oxidized to chloroacetic acid. Studies by Wong and Streips have already shown that vinyl chloride, chloroethanol and chloroacetic acid have no mutagenic properties in bacteria. However, the chlorooxirane and the chloroacetaldehyde are both mutagenic. Animal studies demonstrated significant differences in the liver of vinyl chloride exposed animals who showed no physical or clinical abnormalities. These findings are listed in Slide 2. They include an elevation of non-protein sulfhydryl content, (Slide 3) glut athione reductase, (Slide 4) glutathione epoxide-S-transferase (GEST) (Slide 5) and glutathione aralykly-S-transferase (GAST) (Slide 6) concomitant with the increase in glucose-6-phosphate dehydregenase and reduction in P450 content (Slide 7). A composite of the studies from 14 to 350 hours is shown in Slide 8 which illustrates the earliest increases occurring in glutathione reductase followed later by an increase in glutathione content. It is interesting to note that the increase in GEST occurred before the increase in GAST around 70 to 80 hours of the total exposure. This implies that the chlorooxirane accumulation is to be ___________________________oe __________________________ vvc 0000?^iao handled by direct detoxification by the glutathione but with prolonged exposure more is converted to the chloroacetaldehyde which then is further detoxified by the GAST. These data are compatible with other findings of increased amounts of chloroacetic acid in the urine of individuals with very high prolonged exposures. This would imply that at initial doses the vinyl chloride is able to be raetabolically handled and properly detoxified and that there may be a number of mechanisms by which the active metabolites are handled within the hepatocyte thus, preventing it from inducing DNA injury and subsequently angiosarcoma. Whether these metabolic capabilities are also present in ".the sinusoidal lining cells has lead us to our more recent studies. WC 000003281 26 DETOXIFICATION WITH GLUTATHIONE VINYL CHLORIDE METABOLIC FATE X LU XX o I--1 or LU Q H* x CO XX 1 CJ 04 I -J co X iX O h-- =c LU l 2 CJ < 1 u *-- o zrz o O VX 8^7 _) CJ Oi -- TTT O CJ o CO X CD t CJ 04 33 CJ CO CD UJ X < QC 04 --4 3= X CJx*_ o o "Ti~_ j;CD cc CJ o _J 1--1 X CJ CJ <'A CO CO CD <D . ta>> r o 33 CJ OJ 33 CJ --1 CJ LU Q X X LU <3 _J < f-- LU O ___-w < o cc o _! X CJ 33 CD O CJ OJ 33 CJ T--| CJ CJ < CJ 1-- }-- LU O < o cc o X o OJ ss. *"7~* o CJ > II CJ 1--1 CJ ' . 33 CD 04 33 CJ --O!-- J CJ i-H CJ O X < X 1-- LU O OS O --J X CJ **--* 27 VVC 000003282 Sijoe 2 RESULTS In comparison with the.two control groups, THE FOLLOWING STATISTICALLY SIGNIFICANT DIFFERENCES (P < 0.05) WERE FOUND IN THE LIVER OF THE VINYL CHLORIDE EXPOSED BEFORE ANY SIGN OF PHYSICAL OR CLINICAL ABNORMALITIES WERE SEEN, 1. AN ELEVATION OF NON-PROTEIN SULFHYDRYL CONTENT. 2. AN ELEVATION OF GLUTATHIONE REDUCTASE. 3. AN ELEVATION OF GLUTATHIONE EPOXIDE -S- TRANSFERASE (GEST). 4. AN ELEVATION OF GLUTATHIONE ARALKYL -S- TRANSFERASE (GAST). 5. A REDUCTION OF P-450 CONTENT. 6. AN ELEVATION OF. GLUCOSE-6-PHOSPHATE DEHYDROGENASE vvc 000003283 28 m a> *ort --i VI 6 (210} 4 (140) TIME IN WEEKS (HRS) (70) H3AH Nl IN31NOO dHOdO lAHOAI-mnS NI3IOdd-NONi . ________ _____________ 21 uvr LIVER GLUTATHIONE REDUCTASE .100 x jj moles per minute per mg protein fOCOACnO^COlDO 30 TIME IN WEEK (HRS) I LIVER GLUTATHIONE EPOXIDE -S- TRANSFERASE IOO x jj motes per minute per mg protein O-*fO^in<7)-ja3(0O^foW^ i o i i I i > LIVER GLUTATHIONE ARALKYL - S-TRANSFERASE 10 x jj moles per minute per mg protein ro o NORMAL V.C.'EXP. AIR EXP. VVC 000003287 32 r P-450 CONTENT IN LIVER n moles per gram TIME IN WEEKS (HRS) /o increase above Control FO o CO O o Col oo TOTAL E OSIIRE TIME IN HRS