Department of Health and Human Services Public Health Service, Grant Application

Principal investigator/Program Director (Last, first, middle): Duesberg, Peter H.
nitrites directly on mouse and other rodent cells in culture. Since the in vitro system avoids nitrite
toxicity to vital organs such as the lungs and the bone marrow, higher nitrite concentrations can be
used than in animals and faster results can be expected. Cells treated with nitrites will be examined for
morphological transformation, i.e. focus formation - analogous to cells transformed by oncogenic
retroviruses.
Transformation of monolayers of cells attached to the Petri dish will be studied either under
soft agar or in liquid medium as described by Cichutek and Duesberg for murine retroviruses and
oncogenes,(Cichutek and Duesberg, 1986). If transformants are obtained, they will be examined for
activated oncogenes (Cichutek and Duesberg, 1986; Cichutek and Duesberg, 1989) and possibly for
chromosome abnormalities as well (see below).
The cells to be tested will include Moloney retrovirus-infected and un-infected mouse C3H cell
lines, primary mouse cells, and primary Chinese hamster cells. Since human cells are harder to
transform in culture, they will only be tested if rodent cells can be transformed. The hamster cells are
chosen to facilitate the detection of transformation-specific chromosome changes in nitrite-transformed
cells. Since Chinese hamsters have only 11 chromosomes, karyotype changes will be easier to detect
than in mouse or human cells (Kirkland and Venitt, 1976; Connell, 1984). Clearly if rodent cells can
be transformed, attempts will be made to transform human cells under the same conditions.
Significance of this project
In view of existing epidemiological and toxicological evidence, we consider a definitive test of
the toxicity of nitrite inhalants both a necessary and justifiable effort in controlling AIDS - even if only
to confirm the currently prevailing view that nitrites are irrelevant to AIDS (Ascher et al., 1993;
Schechter et al., 1993). Indeed, the scientific method calls for alternative, testable hypotheses, such as
ours, if the prevailing hypothesis, i.e. the virus-AIDS hypothesis, fails to generate public health
benefits. If confirmed, the nitrite/drug-AIDS hypothesis would be directly relevant for the prevention
and the treatment of AIDS.
5: Human subjects
No human subjects involved
6. Vertebrate animals
1) A total of 560 male and female, 4 week-old CD-1 mice will be used at the Department of Veterinary
Pharmacology andToxicology at UC Davis. About half of the animals will be exposed in inhalation
toxicity chambers to amyl nitrite vapors at about 300 ppm, 5 days a week for 6 hrs for a total of 6 to 24
months.
2) The animals are used as experimental models of humans to test the toxicity of volatile nitrites inhaled
as recreational drugs by humans. The numbers of mice listed are necessary to achieve statistical
significance of our study. Mice are the cheapest experimental vertebrate animals available for our
study.
3) The Department of Veterinary Pharmacology and Toxicology at UC Davis is one of the leading labs
of inhalation toxicology in the world. It has developed and continues to operate the most advanced
equipment available for our purposes. All experiments will be supervised by UC veterinarians
involved in, or supervising our project in agreement with the Office of Laboratory Animal Care of the
University of California.

4) The drug to be tested, amyl nitrite, is by itself an analgesic and a tranquilizing drug. It is not known
to cause any primary discomfort or pain.
PHS 398 (Rev.9/91) Page 30
Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.