Soderberg (1999) Increased tumor growth in mice exposed to inhaled isobutyl nitrite. Toxicology Letters, 104:35.
Soderberg contradicts himself again: “Previous studies (Soderberg, 1994) suggested that nitrite inhalant inhibition of T cell activity was through alteration of accessory cell function, although the present data on the effects on tumor growth indicate more immediate effects on immune function.”
“The cumulative effects of multiple exposures were apparently necessary to impair immune mechanisms. The inhibition of T-dependent antibody responses apparently required the cumulative effects of more exposures to the inhalant, since 14, but not five (or one, exposures to the inhalant caused inhibition. Other immune activities, such as T cell responses to concanavalin A, also required the cumulative effects of repeated inhalant exposures for inhibition. Thus, while a single inhalant exposure directly inhibited T cell proliferation, multiple exposures may affect other components of activation, such as accessory cell function”. Again this is very confusing, there are no conclusive results and Soderberg appears to keep publishing new effects that nitrites have without confirmation from other researchers.
Soderberg (1998) Immunomodulation by nitrite inhalants may predispose abusers to AIDS and Kaposi's sarcoma. J Neuroimmunology, 83:157.
This is a review article. In the conclusions Soderberg mentions that chronically impaired immunity could reduce resistance to HIV infections, but research by Soderberg shows the nitrite immune suppression is reversible.
Soderberg states that the LD 50 for mice is 1033 ppm for one hour, which means that at this dose, half of the mice will die, so giving 900 ppm for 45 minutes is approaching the lethal dose. Furthermore, other researchers
cited are using near lethal doses. This supports my claim that differences in body weight between humans and mice were not considered.
Another discrepancy is that he references work by others that does not show a change in human CD4 and CD8 ratios after nitrite exposure, which occurs in AIDS or immonocompromised individuals.
Although in one of his publications he shows a decrease in mouse natural killer (NK) cell activity and proposes it as a nitrite mechanism for immunotoxicity, he states that two other laboratories (including his) were unable to substantiate this alteration.
Dax et al. (1988) Effects, of nitrites on the immune system of humans. Health Hazards of Nitrite Inhalants. National Institute of Drug Abuse Research Monograph Series. #83, 75.
In this study using eight HIV- male volunteers, the investigators found that amyl nitrite inhalation caused an initial suppression in immune function that was followed by an overshoot seven days after cessation of drug. This study had a low sample number and was not repeated.
These results are also contradictory to results obtained by other research groups. If the work presented in this paper is accurate, one could interpret the overshoot in immune activity as evidence for nitrite use causing an increase in immune function. This conclusion refutes Wilson's proposal that nitrites are harmful to the immune system.
Soderberg (1996) Inhaled isobutyl nitrite produced lung inflammation with increased macrophage TNF-a and nitric oxide production. AIDS, Drugs of Abuse, and the Neuroimmune Axis, Ed. Friedman et al., Plenum Press, New York, 187.
This article contains the same information as a previously discussed Soderberg publication (Toxicology Letters, 104:35) with the same flaws. This is an example of an investigator increasing the volume of their work by publishing duplicative results.
Soderberg and Barnett (1995) Inhalation exposure to isobutyl nitrite inhibits macrophage tumoricidal activity and modulates inducible nitric oxide. Journal of Leukocyte Biology, 57:135.
This paper is a repeat of experiments (Toxicology Letters, 104:35) by the same author, except the tumoricidal activity of peritoneal rather than lung macrophages was measured. Interestingly, Soderberg obtained the opposite results between the two publications. For instance, in these experiments, there was a decrease in tumoricidal activity that returns in two weeks, which contradicts their 1996 publication showing an increase in tumoricidal activity of macrophages. Other data presented by Soderberg demonstrated that nitrite exposure increased TNF-a production by itself or in combination with interferon, but caused no change in response to lipopolysaccharide or interferon and lipopolysaccharide (stimulators of TNF-a production). In contrast, the other report stated that there was no effect of nitrite treatment on TNF-a production in either the absence or presence of interferon, but an increase in TNF-a production in the presence of lipopolysaccharide or lipopolysaccharide and interferon. Finally, the 1995 study reported a decrease in nitric oxide production stimulated by lipopolysaccharide and interferon, which contradicts the 1996 study. Interestingly, the author did not discuss these discrepancies. When an investigator publishes results that are the opposite of each other, one cannot derive conclusions from their work.
Soderberg et al (1991) Inhaled isobutyl nitrite impairs T cell reactivity. Drugs of Abuse, Immunity, and Immunodeficiency, Ed. Freidman et al., Plenum Press, New York, pp. 265.
This is a short paper with only one figure, demonstrating the effects of isobutyl nitrite after treating mice for 45 minutes per day for 14 days. After 24 hours, this treatment caused an impairment of T cell reactivity, but no effect on B cell function or hematopoeisis. Other than this group's usual experimental design problems, they did not measure at any other time points to determine reversibility of nitrite effects.
Interestingly, other studies have shown that mice exposed to 300 ppm of nitrite for five days a week for 6.5 ours (over an 18 week period) had no changes in immune parameters (J. of Toxicology and Environmental Health, 15:828,1985 and J. of Toxicology and Environmental Health, 15:835, 1985). Perhaps giving a smaller dose over a longer time period allows for recovery of the nitrite effects, or the drug is metabolized to non-harmful products.
Lotzova, et al. (1984) Depression of murine natural killer cell cytotoxicity by isobutyl nitrite. Cancer Immununology Immunotherapy, 17:130.
These researchers claimed that isobutyl nitrite causes a decrease in natural killer cell activity in mice when injected intraperitoneally or inhaled. They injected 0.25 mls of isobutyl nitrite twice before assay, which is not a physiological administration of this drug. In addition, this is the same amount that a human would inhale, not inject directly into the body. The metabolism of the nitrite could be very different when given as an intraperitoneal injection rather than the usual inhalation route. For inhalation experiments, mice were placed twice a day for two-three minutes (for seven days) in a beaker containing a petri dish with two ml of isobutyl nitrite. They did not attempt to calculate the dose that was given by this exposure, although again, this amount more closely approximates a human dose. Lotzova's group claims to use the maximal dose tolerated by the mice, which implies that these doses were near lethal.
Another flaw in the design of these experiments is that they were not replicated. It is not understood why such a standard scientific procedure was not utilized, unless a replication of the studies did not confirm the initial results.
Finally, these investigators obtained results that contradict work by Soderberg. Lotzova found a decrease in tumor-binding capacity of natural killer cells, whereas Soderberg found no change in this parameter. Considering this discrepancy and more importantly, the dosing regimen utilized, these studies do not establish a role for nitrites in a decrease in tumoricidal activity.
Gaworski, et al (1992) Prechronic inhalation toxicity studies of isobutyl nitrite. Fundamental and Applied Toxicology, 19:169.
This article was cited by Soderberg as supporting his work, although it provides evidence that refutes the validity of Soderberg's model for treating mice with isobutyl nitrite. In addition, Soderberg's results from experiments measuring white blood cells are the opposite of Gaworski's. Gaworksi's group investigated the toxic effects of isobutyl nitrite in short-term and chronic inhalation studies. Rats and mice were exposed to doses of isobutyl nitrite ranging from 0-800 ppm. They found that 12 exposures of greater that 600 ppm for six hours (five days a week) caused 100% mortality in rats and mice. Rats exposed to 200 or 400 ppm exhibited lethargy and a hunched posture. Furthermore, these lower drug concentrations caused hyperplasia of the bronchiolar and nasal epithelia. Doses of 300 ppm induced a decrease in red blood cells and an increase in white blood cells, which contradicts Soderberg's previou studies. Gaworski concluded that the highest exposure for chronic inhalation tests should not be higher than 150 ppm. Although Soderberg and Gaworski were administrating similar doses, Soderberg exposed animals to nearly 10 times the amount that Gaworski recommends for long term studies over a much shorter time period. Since nitrites rapidly decompose, the lower dose over a longer time interval amounts to a final concentration that is much lower than the high dose over a short time. Furthermore, considering that the dose-response curve for isobutyl nitrite is rather steep, a higher dose has a sharply increase toxicity.
Morgan, et al. (1992) Possible roles for nitric oxide in AIDS and associated pathology. Medical Hypothesis, 38:189.
The nitrites are capable of conversion to nitric oxide, which is thought to the active intermediate in the action of nitrites. This review article by Morgan, et al. speculated that exogenous nitric oxide has a potential role in the epidemiology of AIDS. However, there is no evidence cited that supports this claim, most likely because these studies have not been performed. There are articles referenced proposing association of nitrite use with AIDS, which does not demonstrate a role for nitric oxide in the etiology of AIDS. Furthermore, the articles all utilize a low sample number (seven-eight) in their experiments and the tests are not repeated.
In contrast, Morgan cited other studies demonstrating that variables other than nitrite use differentiated AIDS patients from controls. Similarly, another study listed indicated that sexual activity was the best marker for AIDS and that the abuse of nitrite inhalants appeared unimportant in distinguishing AIDS patients from controls. No firm conclusions can be drawn from conflicting population studies.
Finally, this review article gave contradictory mechanisms for nitric oxide action. At the beginning of the article, they claim that nitric oxide released by inhaled nitrites may be involved in HIV neuropathogenesis. In contrast, they state that exogenous nitric oxide released from inhaled nitrites may cause a down-regulation of endogenous nitric oxide production, which is a mediator of tumoricidal macrophages. Thus, Morgan is claiming that an increase and decrease in nitric oxide can have two different pathological effects. This is not logical reasoning.
Mirvish, et al. (1993) Mutagenicity of isobutyl nitrite vapor in the Ames Test and Some relevant chemical properties, including the reaction of isobutyl nitrite with phosphate. Environmental and Molecular Mutagenesis, 21:247.
In this study, the effects of a saturated vapor of isobutyl nitrite and a saturated aqueous solution were tested for mutagenicity using the Ames test (an indicator of cancer-causing agents). The researchers demonstrated that the vapor was 11 times more mutagenic than the aqueous solution. This follows because isobutyl nitrite is not stable in an aqueous solution. Isobutyl nitrite is prepared for commercial distribution in an alcohol solution. Because of this and the rapid breakdown of nitrites upon inhalation, this study is irrelevant to the question of mutagenicity of isobutyl nitrite as it is administered to humans. Furthermore, there is no other data supporting the mutagenicity of this drug.
Khaled (1986) Inactivation of B12 and folate coenzymes by butyl nitrite as observed by NMR: implications on one-carbon transfer mechanism. Biochemical and Biophysical Research, 135:201.
Khaled tested the effects of isobutyl nitrite on coenzymes of B12 and folic acid, which are important in growth and proliferation of mammalian cells. The rationale for the experiment is that nitric oxide, a breakdown product of isobutyl nitrite can oxidize and inactivate these cofactors. Therefore, they measured the effects of isobutyl nitrite on the structures of the coenzymes using nuclear mass resonance spectroscopy. Interestingly, they see no effects when isobutyl nitrite is solublized in alcohol and changes in structure when isobutyl nitrite is added to these compounds in water, in which isobutyl nitrite is virtually insoluble. They do not address this discrepancy. Furthermore, these studies do not replicate an in vivo situation, in which inhaled isobutyl nitrite may not encounter these cofactors, especially in concentrations high enough to be effective.
Hersh, et al. (1983) Effect of the recreational agent isobutyl nitrite on human peripheral blood leukocytes and on in vitro interferon production. Cancer Research, 43:1365.
In these studies, isobutyl nitrite in solution incubated with human white blood cells had a nonspecific cytotoxic effect. They state that a 1% solution was highly toxic to the leukocytes. There was no rationale for the dosing regimen, and considering that nitrites decompose rapidly, it is highly unlikely that this concentration of drug reaches blood cells.
Hersh claims that the effects of isobutyl are not reversible by washing it out of the cultures, which is not in agreement with the in vivo studies referenced by Wilson that demonstrate reversibility of nitrite effect. In addition, in Hersh's work, nitrite had cytotoxic effects on other cell types, including a breast cancer cell line, which indicates that the nitrite is not selective for immune cells. These provide further evidence that the doses used in the experiments are too large.
Another criticism of this work is that it is performed in vitro, which is not a physiological situation. This is a non-physiological situation.
Lycka (1987) Amyl and Butyl nitrites and telangiectasia in homosexual men. Annals of Internal Medicine, 106: 476.
This article is actually a letter to the editor that suggests that nitrites may cause telangiectasia (vascular dilations seen in many diseases) on the chests of homosexual men. Lycka claims that since nitrites are vasodilators, that they may induce this condition. This is not a scientific article and does not belong in a reference list.
Watson (1982) The use of amyl nitrite may be linked to current epidemic of immunodeficiency syndrome. Unpublished paper submitted to the Journal of the American Medical Association and the Advocate (the largest national gay magazine).
Citing a paper that was submitted, but not published, is an example of Wilson's inability to provide substantiated evidence supporting his claim. Papers that are rejected from scientific journals (and non-scientific publications) are not valid research. Such articles are not worthy of inclusion in a document attempting to establish a claim. Furthermore, such references erode the credibility of the author.
Soderberg et al (2004) Increased tumor growth in mice exposed to inhaled isobutyl nitrite. Toxicology Letters, 152:35.
The investigators chose the PYB6 tumor, a syngeneic, virus-induced sarcoma, but it does not have the unique growth characteristics of KS and they state that it “might respond differently to changes in immunocompetence”. Since they are not measuring the effects of nitrite on KS, the logical choice, the results could be irrelevant.
They state studies of immune function using PYB6 cells as target cells in vitro were not fruitful, as the PYB6 cells were not suitable for in vitro cytotoxicity assays, so they could not determine if altered immune function affects PYB6 tumor growth (what they are claiming.)
They end with “it is generally acknowledged that specific CTL are important in controlling HIV replication in the early stages following infection. The nitrite-induced immunosuppression reported here was not as profound as occurs in the late stages of AIDS and was transient, recovering to normal levels within 14 days after termination of exposure”. This also does not correlate with their claim that nitrite use is important in AIDS.
