Document 8pkX18g0EGp6J0QqGjebgV6o

lhD u- MOL GiKCiNli I nay r 19:10 P.uM LABORATORY OF MOLECULAR CARCINOGENESIS AND MUTAGENESIS THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL CAMPUS BOX 7400, ROSENAU HALL SOUTH COLUMBIA STREET CHAPEL HILL, NC 27599 PHONE 919-966-6142 FAX 919-966-6123 FACSIMILE TRANSMISSION To: Bob Venezia Address: CMA FAX number: 703-741-6091 From: James A. Swenberg, D.V.M., Ph.D. Date: May 7, 1996 Bob: Here is the research- proposal/protocol. I made a few changes from our discussions in New. Jersey. I sent a copy to Jim for his comments. He said that the issue of dynamic versus static exposures will be determined in the ftiture. Dr. Rudy Jager has some dynamic exposure systems that will use even less than the 27 gm. As I do not have access to the needed information, I cannot go any farther today. I hope that this is what you need. If you have any questions, please give me a call tomarrow. BFG40743 i BFG 00238 '-flC -r -nr -i""- r cva . i"ia^ < ` 30 L^;i. r . o PROTOCOL FOR MOLECULAR DOSIMETRY STUDIES ON VINYL CHLORIDE James A. Swenberg, D.V.M., PhJ>. University of North Carolina May 7,1996 Listed below is a series of studies on the formation and repair of DNA adducts induced by vinyl chloride (VC) that will provide a vastly improved understanding of the mechanisms of vinyl chloride carcinogenesis. These studies will provide information relevant to risk assessments of vinyl chloride. The studies would be conducted in a manner that will complement the two generation reproductive and developmental toxicity studies that will be conducted at Huntingdon Life Sciences Laboratory. Dose-response Studies Presently, we have data showing that preweanling rats are more susceptible than adults to VC carcinogenesis, that preweanlings develop 3-fold greater numbers of VC DNA adducts, and that preweanlings have 3-fold higher expression of CYP 2E1. The adduct and carcinogenesis data are all from "high" exposures of-500 ppm. No information exists on lower exposures. In addition, we have clearly demonstrated that DNA adducts identical to those formed by VC are formed endogenously in unexposed rats, mice and humans. We have developed ultrasensitive and highly specific assays for the DNA adducts ofVC. These include immunoaffinity/3JP-postlableing methods for 1 ,N4-cthcnodcoxyadenosine (EdA) and 3 (N4-etheaodcoxycytidinc (EdC), and GC/MS methods for NJ,3-ethenoguanine (EG). By adding animals to the reproductive and developmental toxicity studies, we will be able to assess the effect of exposure concentration on 1) the molecular dose of etheno adducts, the primary promutagenic DNA adducts of VC; 2) determine ifthere are differences in the dose-response between adult and weanling rats; 3) examine the major DNA repair pathways for VC DNA adducts; and 4) characterize the utility of using DNA adducts excreted in the urine as a biomarker of exposure. 1 BFG40744 BFG 00239 rj. r -1-- ^oo-o i.-.j Ndu i'O ij: i*. r Ui I Molecular Dosimetry Studies in Nonparenchymal Cells rNPO versus Henatocvtcs Understanding why VC targets the endothelial ceil for its carcinogenic effect is critical for proper risk assessment We have preliminary data on G in vinyl fluoride-exposed rats (2500 ppm, (5 hr/day, 5 days/wk, 4 wks) which shows that the number of adducts is ~2.5-fold higher in NPC than hepatocytes, even though metabolism of VF to its electrophile is thought to only take place in hepatocytes. Since the NPC are exposed by diffusion of VC or VF electrophiles from neighboring hepatocytes and therefore have lower exposures, this suggested that NPC were deficient in DNA repair activity for EG. To test this hypothesis, we have developed a RT-PCR method for quantitating methylpurine-DNA glycosylasc (MPG) in tissues and cells. Preliminary data have shown that NPC have <20% of the MPG activity present in hepatocytes. In addition, we have shown that NPC have 5-fold greater numbers of abasic sites in their DNA. Abasic sites are formed when MPG excises a damaged base, such as EG, EA and EC. These data suggest that NPC are deficient in a second step in the DNA repair of VC adducts. We think that the most likely candidate is a deficiency in AP endonuclease. The difference between hepatocyte and NPC abasic sites was seen in rats exposed to VF, but not in controls. The combination of two defects in DNA repair in the target ceils for VC carcinogenesis provides a strong scientific basis for the induction of hemangiosarcomas by this important chemical. In view of the fact that the increase in abasic sites was only seen in highly exposed rats shows the need to understand the dose response relationship for this endpoint. By conducting 4 week exposures to 0,10,100, and 1000 ppm VC and measuring etheno adducts, MPG expression and abasic sites, we will be able to determine ifnonlinearities exist in the molecular dose of VC, in MPG expression, and in the number of abasic sites present in the DNA. Our hypothesis is that EG will show a supralinear dose response; that MPG will be similar in exposed and unexposed hepatocytes, but lower in both exposed and unexposed NPC; but that abasic sites will exhibit a sublincar dose response characteristic of saturation of DNA repair in NPC, but not hepatocytes. 2 BFG40745 BFG 00240 LhB CF MLL FhFCINL Fa.\ 913-960-61 ? "it- 19:15 F. Od, 07 Studies Using T'-'C^-VC Our data showing endogenous formation of etheno DNa adducts that are identical to those induced by VC have important implications. We had previously published a paper that concluded that the DNA adducts ofVC were highly persistent. We now believe that this was not correct, in that what we were calling persistent DNA adducts were actually the endogenously formed adducts at steady-state. By utilizing [13CJ-VC, the formation and repair of VC-induced DNA adducts can be studied relative to those adducts formed endogenously. I believe that these studies will critically impact oh any risk assessment dealing with low exposures, such as might be associated with accidental releases that reach the fenceline. We will examine the amount of [l3CJ-VC induced EG in rats exposed to 10,100, or 1000 ppm for 5 days (6 hrs/day). These same animals will be housed in metabolism cages for 3 days prior to exposure and during each day's post exposure holding period (days 1*4) so that urine can be collected. The urine will be analyzed for endogenous and induced DNA adducts that have been repaired by the MPG pathway using GC/MS. We will also develop immunoaffinity-LC/MS methods for all three etheno. bases. In addition, it may be possible to identify and quantitate [l3CJ-VC metabolites in the urine using NMR. A second set of [13CJ-VC exposures is contemplated that would consist of single exposures. These will be designed after we get data from the above study. Significance We have developed highly sensitive and specific assays for the promutagenic DNA adducts of vinyl chloride over the past 10 years. By exposing additional animals in the Huntingdon study, we can use these methods to answer several questions related to improved risk assessment of VC. First, the molecular dosimetry data will be the only information of its type that addresses doseresponse. All other data have come from high exposures of 500-600 ppm. Second, we will address the issue of differences between weanling and adult animals. While we already know that preweanling rats arc mote sensitive than adults to the formation ofDNA adducts and carcinogenesis at high concentrations, it is not known ifthis is true for low exposures. Since the only VC exposures that children encounter would be extremely low, the 10 ppm data are of particular importance. Just 3 BFG40746 BFG 00241 LHb Ci- ChFCInC Fa* :-j 19-966-61C5 May C 19^13 P. 05 -'07 because preweanling rats exposed to concentrations that saturate metabolism develop more adducts does not necessarily mean that young animals exposed to low concentrations will also develop more DNA adducts than adults. Our recent development of an assay for MPG and the finding that NPC are deficient in this DNA repair pathway provides the first mechanistic data that explains why VC causes hemangiosarcomas. The additional finding of greatly increased numbers of abasic sites in the NPC DNA fiirther highlights the importance of cell-specific differences in DNA repair. The experiments outlined above will provide compelling data on the importance ofDNA repair in VC carcinogenesis. Finally, the use of [,3C2]-VC will allow us to differentiate between induced and endogenous DNA adduct formation. If large numbers of induced EG are formed, they may lead to increased numbers of endogenous adducts due to competition for DNA repair. On the other hand, we will be able to determine the extent of increased DNA damage relative to endogenous adducts for all three exposure groups. For example, we might find that 10 ppm VC for 5 days only doubles the amount of EG present endogenously. Such a finding should have great impact on low dose risk, assessments. We would then look at various single exposures to simulate fenceline exposure scenarios to determine if there was any detectable increase in risk. Similar studies could be used to mimic occupational or environmental exposures to VC. Protocols Study I. Adult Rats Exposed to Vinyl Chloridefor 1 Week Route: Whole body exposure conducted during the two generation animal portion ofthe main study Exposure groups: 0,10,100, or 1000 ppm No. of rats/group: 32 Total: 128 Exposure duration: 1 week (6 hr/day, 5 days) Spccies/strain: CD Rat Age: Approximately 12 weeks of age Sex: Males Sacrifice: 8 rats/ group at end of last exposure (non-perfused), and 8 rats/ group at end of last exposure and at 3 and 7 days post-exposure (perfused) Urine collections: during Days 1-4 of exposure (collected during nonexposure times) 4 BFG40747 BFG 00242 i_nfc Lh HCL -HKCInC Fax:919-966-6123 May 7 '% 19:14 P. '36/07 Study II. Weanling Rats Exposed to Vinyl Chloridefor I Week Route: Whole body exposure conducted during the two generation animal portion of the main study Exposure groups: 0, 10, 100, or 1000 ppm No. of rats/group: 32 Total: 128 Exposure duration: 1 week (6 hr/day, 5 days/week) Species/strain: CD Rat Age: Approximately 21 days of age Sex: Males Sacrifice: 8 rats/ group at end of last exposure (non-perfhsed), and 8 rats/ group at end of last exposure and at 3 and 7 days post-exposure (perfused) Urine collections: during Days 1-4 of exposure (collected during nonexposure times) Study III. Adult Rats Exposed to Vinyl Chloridefor 4 Weeks Route: Whole body exposure conducted during the two generation animal portion ofthe main study Exposure groups: 0,10,100, or 1000 ppm No. of rats/group: 32 Total: 128 Exposure duration: 4 weeks (6 hr/day, 5 days/week) Species/strain: CD Rat Age: Approximately 12 weeks of age Sex: Males Sacrifice: 8 rats/ group at end of last exposure (non-perfhsed), and 8 rats/ group at end of last exposure and at 3 and 7 days post-exposure (perfused) Urine collections: during last week of exposure (collected during nonexposure times) 5 BFG40748 BFG 00243 lhB uF MOL ChRCINO Fax:919-966-6123 May 7 '96 19:14 P.07/07 Study IV. Adult Rats Exposed to ^CJ-Vutyl Chloridefor 1 Week Route: Nose only exposure Exposure groups: 10,100, or 1000 ppm No. of rats/group: 8 Total: 24 Exposure duration: 5 days (6 hr/day) Species/strain: CD Rat Age: Approximately 12 weeks of age Sex: Males Sacrifice: 8 rats/ group at end of last exposure (perfused) Urine collections: 3 days prior to exposure and Days 1-4 during exposure (collected during nonexposure times) 6 BFG40749 BFG 00244