Document Z8OqydjO1BRZ67eMBBvjxw55O

LHB OF' MOL CARCINO Fax:919-966-6123 Sep 22 '97 11:58 P.01/03 James A. S^anberg. Ph-D. Director, CuTiulmn is Toxioclotjty Pi'Ofwiot. &vmmincniaJ Sciences & Eiicinccnnv. Niuriiiiin, &. Pathology ScIigoM of Public Health A Medicine THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL To: Wendy Sherman Fax; 703-741-6091 From: James Swenberg Phone: 966-6142 Date: September 22,1997 Subject: Calendar of"events" Pages (including cover): 1 Laboratory of Molecular Cwciovjetwwj & Mutagenesis CB* 7400. JJ6 itosouaii Hall Chapel Hill, NC JtJW.'MUO (919) 966.6142 (Socredtry) (9i) 966-6139 (Office). (919) 9664J23 (Fw) email; jua*r*_swwibngt9uw;.*t'ii BFG 00232 LriB OF MOL CARCING Fax:919-966-6123 Sep 22 '97 11:58 P.02/03 Sept. 97: Oct. 97: Nov. 97: Dec. 97: Jan. 98: feb. 98: Mar. 98: Apr. 98: May 98: June 98: Swenberg Laboratories 1997-1998 eG methods development continues DNA from 4 wk whole livers 1" analyses rat whole liver 4 week, all doses start eA analyses in urines - j 13C whole liver DNA in eG method DNA from 4 wk heps and NPCs mine analyses urine analyses I3C whole liver DNA in sG method 4 wk heps and NPCs DNA in eG method Urine analyses Abasic site measurement 4 wk whole liver ,3C heps and NPC DNA Urine analyses Abasic site measurement 4 wk whole liver l3C heps and NPC DNA in eG method 32P postlabeling analyses ofsA and eC 4 wk whole liver Urine analyses Abasic site measurement 4 wk whole liver 13C heps and NPC DNA in eG method 32P postlabeling analyses ofeA and eC 4 wk whole liver LN^-eG analyses start Abasic site measurement 4 wk whole liver i3C heps and NPC DNA in eG method 32P postlabeling analyses of eA and eC continue l,N2-eG analyses Abasic site measurement 4 wk whole liver 32P postlabeling analyses of eA and eC ljN^-eG analyses MPG & AP endonuclease assays begin Abasic site measurement 4 wk whole liver 32P postlabeling analyses of eA and eC 1 ,N5-eG analyses BFG 00233 LhB of mol carcino Fax:919-966-6123 Sep 22 '9? 11:58 P.03/03 My 98: MPG & AP endonuclease assays begin Abasic site measurement 4 wk whole liver 32P postlabeling analyses of eA and sC lN2-eG analyses Aug. 98: MPG & AP endonuclease assays begin Abasic site measurement 4 wk whole liver 32P postlabeling analyses of gA and sC l,N2-eG analyses Year 2:98-99 Begin with brain Year 3:99-00 Lung, Kidney, risk assessment analyses begin BFG 00234 Molecular Dosimetry of Vinyl Chloride James A. Swenberg, D.V.M., Ph.D. Research Report and Budget Request July 28,1997 BFG 00235 Research Report Year 1 (1996-1997 Budget) The laboratory portion of this project began this spring by sending teams of 3-4 of our laboratory personnel to New Jersey to collect tissues and liver cell types from rats that were exposed to 0. 10. 100, or 1100 ppm vinyl chloride (VC) for 1-20 days. Procedures were ratlier difficult during the first set of necropsies, but went much better for subsequent procedures after we sent an additional person and shipped up our own centrifuge. We were able to harvest good yields of cells and tissues for the experiment as a whole. It was unfortunate that the [UC2]-VC experiment was the first to come off as this was the least smooth running of all. Even so. adequate numbers of cells were able to be harvested to meet our objectives. We are currently isolating DNA from the 4-wcck hepatocytes. as these are the most plentiful cells. A new iminunoaffinity chromatograph-GC/MS method is replacing our previous low pressure strong cation exchange-GC/MS method for the analysis of cthenoguanine. After this shakedown, we will begin working with the [UCJ-VC liver cells. Parallel to this effort will be the analysis of urines for excretion of ethenoadenine using immunoaffinity chromatography and LC-MS. Likewise, we will begin examining the major DNA repair pathway for VC DNA adducts including methyl purine gheosylase (MPG) and AP-endonuclease (AP). Year 2 (1997-1998 Budget) During Year 2 we will continue to work up the many samples collected in New Jersey. Priority will be given to establishing exposure response relationships, identifying celt-specific differences in DNA repair, determining effects of exposure on endogenously formed DNA adducts, and doing initial studies related to incorporating these data into a PB/PK risk assessment model. Wc are also developing new methods for the 3N.2-ethcnoguanine adduct, wltich lias never been characterized in vivo. Year 3 (1998-1999) We expect to complete this research during the third year. At this time, we will have analyzed all ofthe relevant tissues and cells for DNA adducts, will establish die role ofdefects in DNA repair and the effect of induction of DNA repair on exogenous and endogenous DNA adducts, and incorporate these data into a biologically-based risk assessment model. BFG 00236 Research Budget My understanding ofthe funding available for this project is that between S50.000-75.000 will be available per year for three years. The cost ofthis research project will greatly exceed this, with personnel costing ~S140.000 this year. However, since VC lias been and continues to be one of the chemicals that we are investigating under our NIEHS Superfiind Basic Research Program Project. I can offset part of the cost, providing that the CMA agrees to share acknowledgement with NIEHS. Should we need additional [UCJ-VC exposures, they can be done at UNC, since the EOIC lias agreed to fund new research that includes funds to set up a small nose-only exposure system for similar studies on ethylene oxide. Thus, we can maximize our future research dollars to fund critical studies. We need to move forward as fast as possible, as the EPA has already developed a new risk assessment on VC that suggests increased risk compared to their previous estimates. The new assessment incorporates metabolism data, but not information on DNA adducts or DNA repair. Filially, we incurred major expenses during the four trips to New Jersey that were not included in Year l's budget. These are listed under sample collection in the budget. 1997-1998 Budget request Sample collection Travel expenses Shipping Supplies Research budget Personnel Supplies LC-MS/MS time ($25/hr) Travel Equipment maintenance agreements $8172 752 11771 35000 24000 2500 3000 2000 S20.695 $66,500 TOTAL BUDGET REQUEST $87,195 BFG 00237 lhD Q- fluL ChkLaI'Ci f ax :9l9-9t,i,-t5l2i May > '96 19:12 P-Ol/U? 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: Address: FAX number: Bob Venezia CMA 703-741-6091 From: Date: James A. Swenberg, D.V.M., Ph.D. May 7, 1996 Bob: Here is tlio research proposal/protocol. I made a few changes from our discussions in New, Jersey. I sent a copy to Jim Knaak for his comments. He said that the issue of dynamic versus static exposures will be determined in the fixture. Dr. Rudy Jagcr 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. ! BFG 00238 IjrLHD I'lLL r aa ^ y i iJ-300-01^6 i"LEiy 1 ' ab 19 r . u_ - U1 PROTOCOL FOR MOLECULAR DOSIMETRY STUDIES ON VINYL CHLORIDE James A. Swenberg, D.V.M., Ph.D. 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 ofvinyl chloride carcinogenesis. These studies will provide information relevant to cancer 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, wc 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/32P-postlableing methods for 1 ,N6-ethenodeoxyadenosine (EdA) and 3 ,N4-ethenodcoxycytidmc (EdC), and GC/MS methods for N2,3-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 promutagcnic DNA adducts ofVC; 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 BFG 00239 i_ru_i ur i iui_ u-trVwiiHu ra. Ji I'lciu i JO i'JUi r.LC-Ui Molecular Dosimetry Studies in Nonparenchvmal Cells (NPC) versus Hepatocytes Understanding why VC targets the endothelial cell for its carcinogenic effect is critical for proper risk assessment. We have preliminary data on EG in vinyl fluoride-exposed rats (2500 ppm, 6 hr/day, 5 day$/wkt 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 giycosylase (MPG) in tissues and cells. Preliminary data have shown that NPC have <20% ofthe MPG activity present in hepatocytes. In addition, we have shown that NPC have 5-fold greater numbers ofabasic 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 arc 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 cells 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 0,30,100, 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 sublinear dose response characteristic of saturation of DNA repair in NPC, but not hepatocytes. 2 BFG 00240 LhB OF MOL ChRlINU Fax:919-966-61L'3 May "7 '96 19: lo P. 04/07 Studies Using rijCfl-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 utilisting [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 on any risk assessment dealing with low exposures, such as might be associated with accidental releases that reach the fenceline. WTe will examine the amount of [l3C2]-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 identity and quantitate [l?C2]-VC metabolites in the urine using NMR. A second set of [nC2]-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 are more 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 ofparticular importance. Just 3 BFG 00241 LhB OF MUL lmRCINCi Fax :'919-96b-610'^ May ? '90' 19:1c P. 05/0? 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 further highlights the importance of cell-specific differences in DNA repair. The experiments outlined above will provide compelling data on the importance of DNA repair in VC carcinogenesis. Finally, the use of [,3CJ-VC will allow us to differentiate between induced and endogenous DNA adduct formation. If large numbers ofinduced 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 ofEG present endogenously. Such a finding should have great impact on low dose risk assessments. We would then look at various single exposures to simulate fcnccline 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 /. 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. ofrats/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 BFG 00242 lhB OF ML'L ChRCINU Fax:919-966-6123 May ? '96 19U4 P.06/07 Study II. Weanling Rats Exposed to Vinyl Chloridefor 1 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-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) Study III. Adult Rats Exposed to Vinyl Chloridefor 4 Weeks 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: 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-perfused), and 8 rats/ group at end of last exposure and at 3 and 7 days post-exposure (perfused) Urine collections: during last week ofexposure (collected during nonexposure times) 5 BFG 00243 LhB UF MOL ChRCINO Fax :919-966-6123 May 7 '96 19:14 P.07/07 Study IV Adult Rats Exposed to (*3CJ-Vinyl Chloridefor l 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) Specics/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 BFG 00244