Most of the work in the first section has experimental design flaws, one of which is that the sample sizes are too small. It is important to utilize a large enough sample in experiments to accurately represent a true population. Furthermore, in many of the studies, most of the tests are not repeated. This is an extremely crucial element of experimental design that is necessary in establishing statistical significance for a particular result. It is essential to repeat experiments because it is possible to obtain contradictory data between different tests, resulting from factors such as researcher error.
Soderberg (1999) Increased tumor growth in mice exposed to inhaled isobutyl nitrite. Toxicology Letters, 104:35.
This article is one of the many Soderberg references listed by Wilson. All of this researcher's studies using mice suffer from a very serious flaw, which is that the mice are exposed to extremely large doses of nitrites when their body size is taken into account.
Earlier work by Soderberg (J. of Immunopharmacology, 15(7):821) reported the, effects of nitrite inhalation on antibody induction of mice were tested. They saw no changes in antibody responsiveness after administering a dose of 300 ppm isobutyl nitrite for 45 minutes a day over a 14-day period. Soderberg only saw decreases in antibody production at 750 and 900 ppm isobutyl nitrite. Even at this dose, there was full recovery within seven days, once again demonstrating reversibility of nitrite effects on immune function, despite the high dose. Since Soderberg's group did not observe their desired effects at a dose which might not be toxic to the animals, this probably explains why they chose this dosing paradigm for their future work.
Although Soderberg reported an increase in incidence and size of tumors, the dose was too high to make any claims. Furthermore, the exposures did not significantly affect body weight, spleen weight, or spleen cellularity, which was reported in other articles. There are many contradictory research results published on a variety of different parameters, making it difficult to ascertain the actual effects of nitrites.
Dax et al. (1991) Amyl nitrate alters human in vitro immune function. Immunopharmacology and Immunotoxicology, 13:557.
In this study, the effects of volatile nitrite inhalation on the immune system of gay male volunteers was examined. However, the amyl nitrate was administered in an unusual manner. The apparatus used consisted of a 4 liter flask connected by tubing to a rubber inflatable breathing bag. Another tube connected the flask to room air and a mouthpiece was attached to a side opening of the flask. Amyl nitrite pearls covered in gauze were broken and dropped in the bottom of the flask. Thirty seconds later, the subject exhaled, and then inhaled the air from the flask until the rubber bag collapsed and completed inspiration with room air (via the flask). The inspiration was held for 5 seconds before exhaling. The drug dose was varied by altering the number of nitrite pearls dropped in the flask. This method of drug administration is very complicated and it does not represent the actual exposure that occurs when the drug is inhaled from a vial. As everyone has a different lung capacity, it is impossible to standardize dose using this flask apparatus. Because of this dilemma, it is not understood why the nitrite was not inhaled directly from a vial to more accurately depict a physiological dose. Surprisingly, the authors claim that "the experimental protocol simulated the common episodic pattern of nitrite abuse." Since the drug was given three times a day (over a nine hour period) for either three or nine days using a complicated device, it does not follow that this protocol simulates nitrite abuse.
Another major flaw in this study is that there were only nine participants in each of the studies (short term or long term, consisting of three or nine day treatments, respectively, with drug administered three times a day) and the study was not repeated. This is a very low sample number and the experiments should have been repeated at least twice. Since the effects that were observed were readily reversible (see below) the same participants could have been used in an effort to replicate the data. Another approach could have been to recruit other volunteers for this study.
In these experiments, there were no changes in the number of T or B cells (which is considered to be an indicator of general immune function) in either the three or nine day experiments. The only statistically significant effect of nitrites observed was a 30% decrease in natural killer cell activity. This effect only occurred in the long-term study and returned to baseline within four days after cessation of drug exposure. This reversibility indicates that nitrite use may not have long term effects. Other immune function tests were not performed and nitrite exposure had no effect on cell proliferation. These results are not compelling evidence for a major effect of nitrite inhalation on the immune system.
Soderberg, et al (2004) Production of macrophage IL-1β was inhibited both at the levels of transcription and maturation by caspase-1 following inhalation exposure to isobutyl nitrite. Toxicology Letters, 152:47.
Soderberg reported a 15% reduction in IL-1β mRNA transcription from inhalant exposed mice. “Such a minor decrease in transcriptional activity is not likely to be solely responsible for 37–55% decrease in secreted protein.” Thus began the search for multiple mechanisms.
The authors state that exposure to isobutyl nitrite reduced the induction of specific cytotoxic T-cells and macrophage tumoricidal activity and “signaling through the macrophage NF-κB pathway was impaired following inhalant exposure. NF-κB-dependent induction of macrophage nitric oxide synthase (NOS2) and subsequent production of nitric oxide were consequently inhibited. The present study examined the effects of nitrite inhalant exposure on another macrophage product important in innate immunity, IL-1β. The production of IL-1β was inhibited at both transcriptional and post-translational level.” Although this sounds impressive, it is very unlikely that one compound can have this many effects that are not a result of toxicity from excessive doses.
Ponappan et al (2004) Inhaled isobutyl nitrite inhibited macrophage inducible nitric oxide by blocking NFκB signaling and promoting degradation of inducible nitric oxide synthase-2. International Immunopharmacology, 4:1075.
Soderberg is an author on this paper, again demonstrating bias. The reasoning of this paper was very difficult to follow but essentially it is a repeat of a previous paper with the exception that they demonstrated nitrite effects in 5 days instead of 14 days. Phosphorylation of IκBα is a prerequisite for ubiquitination and proteasome-dependent degradation of IκBα, freeing NFκB to move into the cell nucleus and affect gene expression. They could not demonstrate an inhalant-associated decrease in IκBα degradation and they stated that it is likely that inhalant exposure inhibits activity of the IκBα kinase or it may act on an upstream component in the signaling cascade. However they did not measure these parameters to demonstrate that these are a mechanism of action.
The researchers state “data suggested that inhalant exposure likely inhibited macrophage (nitric oxide) NO production by blocking NFκB-mediated activation signaling and promoting poly ubiquitination of NOS2.” It is astounding that researchers can publish this much data that is conflicting and obviously does not show target selectivity of nitrites.
Tran et al (2003) Inhalant nitrite exposure alters mouse hepatic angiogenic gene expression Inhalant nitrite exposure alters mouse hepatic angiogenic gene expression. Biochemical and Biophysical Research Communications, 310:439.
The dose for mice was 1400 ppm for four hours, which is even higher exposure than Soderberg. The utilization of this high dose negates any results that may be observed. The authors give as a rationale for performing the research that organic nitrites (NO donors) in vitro studies have shown
NO to stimulate vascular endothelial growth factor (VEGF) protein and mRNA expression. VEGF is essential for tumor growth and metastasis.
In the discussion of this paper, another Soderberg article is referenced (not included in Wilson’s reference list) presenting the fact that NO is liberated by nitrite but exogenous NO does not produce the immunotoxicity observed following exposure to isobutyl nitrite. This does not make sense because NO mediates macrophage tumoricidal activity, so NO liberation would be beneficial.
Other conflicting data presented was that inhalant nitrite exposure also significantly suppressed the gene expression of Smad5 and Smad7 in mouse liver. Smads regulate transforming growth factor-β-dependent (TGF-β) gene expression, which controls cell proliferation, differentiation, apoptosis, migration, and extracellular matrix production. Smad5 plays an important role in angiogenesis and Smad7 is important in negative feedback regulation of TGF-β. Since these Smads have opposite effects on cancer proliferation, one would not expect both to be suppressed if nitrite had carcinogenic effects.
Another area of concern is that they authors do not address why there were no changes in lung VEGF expression, the increase was seen in the liver. “This observation is somewhat counter-intuitive, since the nitrite exposure concentration is expected to be higher in the lung than in the liver.“ This statement indicates that the authors are unclear about the meaning of their results.
Ponnappan and Soderberg (2001) Inflamatory macrophage nuclear factor-κB and proteasome activity are inhibited following exposure to inhaled isobutyl nitrite. J of Leukocyte Biology, 69:639.
Although the authors demonstrate a reduction of nuclear NFκB in activated macrophages (an immune response), nitrite exposure also reduces un-activated macrophage NFκB, which could be an indication of toxicity. A clear demonstration of nitrite’s reduction in immune response would have been shown had the nitrites had no affect on un-activated macrophages.
The discussion of this paper is very confusing, as they attempt to make sense of contradictory data. They report that although isobutyl nitrite was shown to liberate NO, inhaled NO at a concentration equivalent to that produced by 900 ppm isobutyl nitrite did not alter resident macrophage tumoricidal activity. NO liberation would not affect macrophage tumoricidal activity, because NO liberation is macrophage’s mechanism of action. Since they also claim that isobutyl nitrite inhibits macrophage inducible NO, which could be a feedback mechanism because nitrites liberate NO. They also demonstrate that inhalant exposure inhibits macrophage NFκB activation, which is important in HIV replication. This is another contradiction. Actually, the final sentence of the paper states “conflicting influences may be induced by inhalant exposure.” The authors admit the contradictions in their own paper.
Keilbasa and Fung (2000) Nitrite Inhalation in Rats Elevates Tissue NOS III Expression and Alters Tyrosine Nitration and Phosphorylation. Biochem and Biophysic. Res. Comm, 275:335.
In these experiments, rats were exposed to 109 and 1517 ppm isobutyl nitrite for four hours, which is excessive and does not represent human exposure. They did not find alterations in NOS expression in the lungs or spleen, which according to Soderberg’s hypothesis they should find. They reported an increase in the kidney and liver, which are organs of detoxification and it is unclear what an increase in NOS expression in these organs means. They do not address why there is differential expression. Also it is not understood why they do not measure macrophage NOS expression, which is the proposed tumoricidal mechanism of macrophages. If nitrites diminish NOS in macrophages, it would support a role for nitrites in depressing tumoricidal activity.
Guo, et al. (2000) Acute exposure to the abused inhalant, isobutyl nitrite, reduced T cell responsiveness and spleen cellularity. Toxicology Letters, 116:151.
This work was performed in Soderberg’s lab, yet another reference by him. Guo states there is no change in body or spleen weight, yet spleen cells are decreased. In another paper, Soderberg reported no change in spleen cell cellularity, yet in this publication it is decreased. Although there was a decrease in spleen cells, there was no reduction in CD4 and CD8 helper cells, or in differential lymphocytes. This would indicate that nitrates are not selectively reducing immune function. Although a single 45 min exposure to the inhalant inhibited T cell proliferative responsiveness, it was not sufficient to overtly impair major immune mechanisms. Also, they report that only after the 14 day exposure do they see a decrease in T-dependent antibody responses. These results are not definitive.
Soderberg and Flick (1997) Acute blood toxicity of the abused inhalant, cyclohexyl nitrite. Int J Immunopharmac, 19:305.
In this report cyclohexyl nitrite produced anemia and leucopenia. Two important issues must be noted: there were no dose related effects noted and there was a nonspecific cell reduction. A dose response relationship is essential in pharmacology to establish an effect. Generally when this is not the case, toxicity is evident. The nonspecific cell reduction also is an indication of toxicity.
Cyclohexyl nitrite did not decrease macrophage tumoricidal activity, which contradicts a previously published report. Soderberg states in another paper that there are differential effects of cyclohexyl and isobutyl nitrite, yet he is obtaining different results with the same compound.
Soderburg, et al (1996) Acute inhalation exposure to isobutyl nitrite causes nonspecific blood cell destruction. Experimental Hematology, 24:592.
This is also cited as a 1996 poster. Although he reported in an earlier paper that results were seen in 5 days, he continued to use 14 days. Perhaps he only saw certain effects at excessive doses. An indicator of this is the nonspecific blood cell destruction, a likely result of toxicity.
They found a decrease in both red and white blood cells at 24 hours after acute exposure. It is possible that this decrease is a result of lung hemorrhage, with subsequent blood loss, induced by the high dose of nitrite. Regardless, the observed changes in blood cell count were reversed by 72 hours, which again demonstrates that nitrite effects are reversible. The study also described a decrease in spleen cellularity and speculated that spleen cells are mobilized to provide replacement white blood cells. If this is true, then the spleen can overcome any loss in immune function that may occur as a result of transient white blood cell loss and serve as a compensatory mechanism to maintain homeostatic immune function. Therefore, even if nitrites do cause a decrease in white blood cells, there is a rapid response to correct the imbalance.
"Others have not found similar epidemiologic correlations (abuse of nitrate inhalants correlated with seropositivity to HIV and Kaposi's sarcoma among AIDS patients”. These kinds of statements represent the lack of congruence between researchers.
Soderberg, and Barnett (1995) Inhaled 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 in another reference 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 (1996) Elevated TNF-a and inducible nitric oxide production by alveolar macrophages after exposure to a nitrite inhalant. Journal of Leukocyte Biology, 60:459.
The reference that Soderberg gave establishing human exposure as 7000 ppm (Soderberg, et al, Experimental Hematology, 24, 846-853, 1996) does not reflect human doses. In this article, which he used as a reference, Soderberg claimed that abuser doses exceed 1500 ppm, which is much lower than 7000 ppm. In yet another publication by Soderberg (Fundamental and Applied Toxicology, 17:821, 1991), he stated that the actual dose levels of nitrite abusers are unknown. From these disparate statements, it appears that Soderberg has no concrete data to establish the amount of an abuser dose. However, he arbitrarily set the treatment dose for mice at 900 ppm for 45 minutes over a 14 day period. This paper included a dose-response curve showing that a single exposure of this amount to mice caused lung hemorrhage and prolonged treatment caused emphysema-like changes. Doses as low as 300 ppm also caused lung hemorrhage in mice. Furthermore, Soderberg did not account for the difference in lung size between humans and mice. This is a very serious error and because of this, he is probably utilizing a treatment dose that is toxic to mice. Perhaps Soderberg did not get his anticipated results when exposing animals to lower doses and nitrites are not harmful at more physiological doses. Surprisingly, this group continued to publish studies using a dose that is clearly excessive.
Another major problem with this article is that it reported opposite results from Soderberg's previous studies. For example, this article showed an increase in tumoricidal activity of mice lung macrophages, whereas there was a decrease in tumoricidal activity in humans in another paper by Soderberg (see below). If different results are obtained between humans and animals, this would imply that there is a difference between the two species and invalidate the use of animals in experiments. Other work presented in the study showed an increase in TNF-a production of lung macrophages in response to interferon. Activated macrophages release TNF-a, which would indicate an increase in tumoricidal activity. Since macrophages kill both virally infected and cancerous cells, it follows that an increase in macrophage function would be a preventive of HIV infection and KS.
In regards to the data showing an increase in tumoricidal activity, Soderberg stated that this result is unexpected and may be a caused by increased lung inflammation in response to tissue damage. This is further evidence that the researcher is using an excessive nitrite dose. Also, when the same investigator publishes opposite results, it is impossible to establish the true effects of nitrite use.
Soderberg and Barnett (1996) Leukopenia and altered hematopoietic activity in mice exposed to the abused inhalant, isobutyl nitrite. Experimental Hematology, 24:848.
In this paper, after five mice were treated with 900 ppm isobutyl nitrite for 14 days, they exhibit a 36% decrease in white blood cell count and a 7% increase in red blood cells. The later result is unusual because nitrites have been shown to cause a decrease in red blood cells (Fundamental and Applied Toxicology, 19:169, 1992). All observed blood cell changes return to baseline one week after cessation of drug, demonstrating reversibility of nitrite effect.
This report and other articles by Soderberg suffer from the same problems as previously mentioned. These experimental design flaws include the administration of an excessive drug dose to the mice, low sample size, and no replication of experiments to confirm results. These protocol errors, in combination with the contradictory results from different experiments by the same and other authors, make it impossible to develop firm conclusions about the effects of nitrites on the immune system.
Soderberg and Barnett (1996) Exposure to inhaled isobutyl nitrite reduces T cell blastogenesis and antibody responsiveness. Fundamental and Applied Toxicology, 24:821.
In an earlier paper, Soderberg obtained results after treating mice for five days, yet is these experiments mice were 900 ppm isobutyl for 14 days. A similar study with a lower dose, up to 300 ppm for 18 weeks (J. Toxicol Environ. Health 15:823, 1985) reported no change in immune parameters.
In this study, he found a reduction in mice body weight and spleen cells, in contrast to his previous work. He also finds new parameters that nitrites effect. “The frequency of T-dependent plaque forming cells (PFC) was inhibited by 63% and the total number of PFC per spleen was reduced by 72% in nitrite-exposed mice.” Again, these results are meaningless because of the high dose.
Dunkel, et al (1989) Mutagenicity of some alkyl nitrites used as recreational drugs. Environmental and Molecular Mutagenseis, 14:115.
In this study, five of six different alkyl nitrites, including isobutyl nitrite, tested positive for mutagenicity. Since it is not known what the actual dose of nitrite is after inhalation, it is difficult to know if the concentration used in these mutagenecity studies is anywhere near the physiological dose of nitrite. Furthermore, these types of studies do not account for metabolism of the drug, which occurs in the intact animal.
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 minute (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.
