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

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Principal Investigator/Program Director (Last, first, middle): Duesberg, Peter H.
by Gaworski et al. (Gaworski et al., 1992)., According to these studies, the lethal dose of inhaled
nitrites for a mouse not adapted to nitrite inhalation is either 1500 ppm of amyl nitrite vapor for 30 min
(Wood, 1988) or 10 microliters of amyl nitrite intranasally (Ortiz and Rivera, 1988), or 12 six-hour
exposures to air containing 300 ppm (Gaworski et al., 1992).
To reach maximal, sublethal toxicity in the shortest time possible, mice were first adapted to
increasing daily doses of nitrites for 5 days and then maintained at doses that ranged from 20 to 100%
of the lethal dose for un-adapted mice (Lewis and Lynch, 1988; Ortiz and Rivera, 1988). After
exposure for 6 hrs on 5 days per week at 300 ppm for 4.5 months Lewis and Lynch observed "no
significant detrimental effect on the immune system" (Lewis and Lynch, 1988). However, they noted
that the white cell count of nitrite-treated male, but not female, mice was 3-fold lower than in untreated
controls. Gaworski et al. reported "reduced body weight gains" in mice and rats exposed to 300 ppm
of amyl nitrite for 3 months on the same schedule that was used by Lewis and Lynch (Gaworski et al.,
1992). In addition they observed "reduced RBC [red blood cell] counts... increased WBC
counts...[and] bone marrow hyperplasia". The bone marrow hyperplasia and increased white cell
counts are expected results of early compensatory hemopoiesis induced by nitrite-mediated blood cell
depletion. Once the compensatory capacity is exhausted due to continued exposure to the nitrite
toxins, blood cell counts would be expected to decline.
Ortiz and Rivera observed a consistent and "significant decrease" in the ratio of T-helper to
suppressor cells in all mice exposed to 10 microliters of amyl nitrite intranasally on the same schedule
used by Lewis and Lynch, except that exposure was continued for over 5 months. Ortiz and Rivera
reported T-cell ratios of 0.75 in nitrite-treated mice, compared to 1.5 in controls (Ortiz and Rivera,
1988). In addition they observed "extensive damage to the lungs" in 10 out of 11 mice and failure to
gain body weight in all nitrite-treated mice.
We conclude that the studies of Lewis and Lynch as well as those of Gaworski et al. failed to
show consistent immunotoxicity because they were terminated too soon. By contrast, the study of
Ortiz and Rivera had achieved sufficient cumulative nitrite exposure to generate immunotoxicity within
5 months. In view of this, we plan to expose mice to amyl nitrite for a minimum of 6 months and
possibly for 24 months while measuring immunotoxicity. Long exposure periods are necessary to
approximate the estimated seven- to ten-year latent period between nitrite use and AIDS (Newell et al.,
1985a). It may not be a coincidence that two factors are the same: (i) the estimate for the time required
to reach a cumulative nitrite dose sufficient for AIDS-toxicity, and (ii) the estimate for the "latent
period" of HIV to AIDS submitted by proponents of the virus-AIDS hypothesis.
Previous experiments also indicate that nitrites are mutagenic in the Ames-test and therefore
have carcinogenic potential (Mirvish et al., 1988; Mirvish et al., 1993). Moreover, nitrites have been
reported to cause cancer in mice and rats (Winter, 1989).
4. Research Design and Methods
In vivo studies. In the first year a total of 280, 4 to 6 week-old, male and female mice will be
used in this study. 140 will be infected by intraperitoneal injection with the non-transforming Moloney
murine retrovirus as described previously by Cichutek and Duesberg (Cichutek and Duesberg, 1989).
This virus strain, and spontaneous variants arising from it in animals, has been suggested as a murine
AIDS virus (Joliceur, 1991). The virus will be propagated in cultured C3H cells transfected with
molecularly cloned Moloney provirus (Cichutek and Duesberg, 1989).
Seventy each of the retrovirus-infected and un-infected mice will be exposed to amyl nitrite
vapor for 6 hrs. at an initial dose of 60 ppm that will be increased at 60 ppm increments over 5 days to
300 ppm.. Methemoglobulinemia, in which the iron of hemoglobin is oxidized from Fe2+ to Fe3+ by

nitrites, is expected to be a primary, life-threatening consequence of nitrite inhalation (see above).
Depending on their tolerance, the nitrite dose may be further increased. Subsequently, the mice will be
exposed 5 days per week for about 6 hours for 6 to 12 months at 300 ppm or at a higher dose as
PHS 398 (Rev.9/91) Page 28
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