Dr. Richard J. Albertini
Genetic Toxicology Laboratory
32 N. Prospect St.
Burlington, VT 05401-0508
Phone: 802-656-8346
Fax: 802-656-8333
Email: ralberti@zoo.uvm.edu
Projects are approved by an IRB located at: University of Vermont.
The approving IRB operates under a Multiple Project Assurance (MPA) recognized by DOE or by the Department of Health and Human Services (HHS).
MPA number of the IRB: M-1375
Number of Human Subjects Projects reported: 2
Project Identifier: UVT-83-DE-FG02-87ER60502
Project Title:
Development of In Vitro Mutagenicity Testing Systems Using T-Lymphocytes
Principal Investigator:
Dr. Richard J. Albertini
Project started in: 1983
Project Funding Information:
Project received funding in Fiscal Year 1997.
Project used human subjects in Fiscal Year 1997.
Funding Sources:
Project involves use of multiple protocols/subprojects.
Number of protocols/subprojects associated with this project: 1
Protocol/Subproject # 1
Protocol/Subproject Identifier: CHRMS 95-237
IRB Review:
Type of Review: Full Board
Most Recent Approval: July 01, 1997
Number of Human Subjects who participated in this project/protocol during
FY 1997 (10/1/96 - 9/30/97): 15
Type of Human Subjects Involvement:
a. The overall goals of the research program of the Genetic Toxicology Laboratory are to understand the causes, mechanisms, and consequences of somatic mutations in humans and to use this understanding to identify individuals at risk for deleterious health effects resulting from environmental mutagens/carcinogens. In this context, the theme of this research program has been mutation induction in human T-lymphocytes. The emphasis has been on using studies of in vivo observations. Our goal in this proposal is to define the genetic bases of mutator phenotypes arising in vivo in non-malignant cells. In particular, the sub-projects are to discover and characterize spontaneous/induced molecular mutational spectra in the hypoxanthine phosphoribosyltransferase (hprt) gene in human colon cancer cells; to use hprt mutation in T-cells to identify hypermutable clones among the hprt mutants arising in vivo in individuals constitutionally heterozygous for functionally relevant mutation in a mismatch repair gene; to use hprt mutation in T-cells to identify hypermutable clones among the hprt mutants arising in vitro following exposures of lymphocytes from mismatch repair consititutional heterozygotes to model mutagens; to characterize the hypermutable T-cell clones so obtained as to molecular spectra, in vitro hypersensitivity to killing, in vitro mutablity, repair capacity, and the specific mutation in the inherited allele; to develop a system for direct selection of mismatch repair deficient T-cells arising in vivo in humans; and to identify nutrients or other agents that reduce the mutability of hypermutable cells.
b. The method used to study individuals and cells is the hprt clonal assay. This requires a blood sample. T-lymphocytes are isolated from the blood sample and the number of mutant lymphocytes is determined by limiting dilution cloning. Molecular analysis of the mutations in the T-lymphocytes is then performed on the mutant lymphocytes. For some experiments, isolated lymphocytes are treated with radiation or chemicals in vitro. For other experiments, studies are done with cultured colon cancer cell lines. For some individuals, some data on age, smoking, and health history are collected.
c. Some individuals are cancer patients and have had chemotherapy or radiotherapy; however, these treatments were not administered under this proposal. These patients were only studied because they had already received therapy. Some individuals have been studied because of radon exposure in their homes.
d.1. In vivo hprt T-lymphocyte clonal assay - For this study, informed consent is obtained and then blood samples are collected in heparin tubes. The mononuclear cell layer is isolated and washed twice with saline. Cells are plated at limiting dilution in 96-well (round bottom) microtiter dishes at 1, 2, 5 and 10 cells/well in the absence of TG and at 1-3 x 10e4 cells/well in the presence of 10 micromolar (um), thioguanine (TG), T-cell growth factor (TCGF), phytohemagglutinin (PHA), and accessory cells. The microtiter dishes are incubated for 10-16 days and growing colonies are determined by the use of an inverted phase contrast microscope. Wells are scored for colony growth on days 10-14. Mutant and wild type colonies in positive wells are isolated, propagated and stored for molecular analysis. Mutant frequencies are calculated based on the Poisson statistics as the ratio of the cloning efficiency (CE) in the selective 6-thioguanine medium divided by the CE in non-selection medium. CEs are based on the fraction of wells with positive cell growth (CE = -ln Po/N, where Po is the fraction of wells without cell growth and N is the number of cells seeded into wells). The ratio of CE in the presence or absence of 6-thioguanine defines the mutant frequency.
In vitro assays for induced hprt mutations exposed human T-lymphocytes - The in vitro assays employ either fresh or cryopreserved mononuclear (MNC) and are cultured in the same medium as described for the in vivo assay. The cells are exposed before mitogen stimulation, i.e., while in the Go phase, in order to more closely approximate the in vivo situation. After exposure, cells are incubated with 1 u/ml PHA for 36-40 hours to achieve mitogen stimulation. The stimulated cells are plated as for the in vivo assay to determine the relative survival and the pre-existing mutant frequency. Phenotypic expression is accomplished by two methods. The first is through subculture for expression and the second is through in situ expression. The latter method is primarily used to isolate mutant clones with independent mutations for molecular analysis.
d.2. There are no risks except bruising and a very minor risk of infection at the site of blood draw.
Project Identifier: UVT-97-DE-FG07-96ER62331
Project Title:
A Novel Biomarker for Beryllium Sensitization in Humans
Principal Investigator:
Dr. Richard J. Albertini
Project started in: 1997
Project Funding Information:
Project received funding in Fiscal Year 1997.
Project used human subjects in Fiscal Year 1997.
Funding Sources:
$648,912 for fiscal 1997
Project involves use of multiple protocols/subprojects.
Number of protocols/subprojects associated with this project: 1
Protocol/Subproject # 1
Protocol/Subproject Identifier: CHRMS 96-269
IRB Review:
Type of Review: Full Board
Most Recent Approval: October 28, 1996
Number of Human Subjects who participated in this project/protocol during
FY 1997 (10/1/96 - 9/30/97): 21
Type of Human Subjects Involvement:
a. This research project will determine the T-cell (TCR) gene usages of beryllium reactive T-lymphocytes isolated directly from the peripheral blood of individuals exposed at a U.S. Department of Energy site. The objective is to develop a sensitive and novel biomarker for identifying early human sensitization to environmental beryllium. This is a collaborative project involving the Genetic Toxicology Laboratory of the University of Vermont and both the Center for Epidemiological Research and the scientific staff of the Cytogenetics Program at the Oak Ridge Institute for Science and Education (ORISE). The >2000 beryllium exposed workers who have been contacted for participation in the ORISE study "Follow-up of Beryllium Workers at the Y-12 Plant/Efficacy of the Peripheral Blood Lymphocyte Proliferation (LPT) and Other Non-Invasive Procedures for Diagnosis of Chronic Beryllium Disease" will provide the pool of potential participants for the proposed study.
b. Beryllium reactive T-lymphocytes will be directly isolated from peripheral blood using a novel antigen-independent method of surrogate selection for in vivo arising hypoxanthine phosphoribosyltransferase (hprt) mutants as representatives of clones that are undergoing chronic proliferation. The T-cells undergoing chronic proliferation in beryllium sensitized individuals will be enriched for beryllium reactive cells. The TCR beta gene usage of these T-cell isolates will be determined and their junctional (CDR3) regions sequenced. Beryllium reactive T-cell clones will also be recovered following in vivo beryllium stimulation of peripheral blood lymphocytes from these same individuals and the TCR gene CDR3 region sequences similarly determined. The TCR beta genes used by the beryllium reactive isolates and their CDR3 region sequences will be compared within (in vivo vs. in vitro derived) and among individuals with attention to kinds and durations of beryllium exposure and HPA DPB Glu 69 status. A method for quantitating total body loads of these antigen reactive T-cells in individuals will be developed using quantitative polymerase chain reaction (Q-PCR) amplification of specific TCR gene sequences. Successful achievement of this overall objective will permit future studies aimed at the elucidation of the immunological mechanisms underlying sensitization, the comparison of cells involved in pulmonary and systemic sensitization and the definition of potential targets for immunotherapy.
c. none
d. 1. Blood will be obtained with informed consent from persons previously exposed to beryllium. Cells isolated from this blood will be cultured in vitro.
2. There is essentially no risk except for possible bruising at the site of blood drawing.
3. This protocol has University of VT IRB approval and informed consent is obtained from all individuals studied. The results of these studies are confidential.