Effect of the Recreational Agent Isobutyl Nitrite on Human Peripheral Blood Leukocytes and on in Vitro Interferon Production1

Home , Isobutyl nitrite

[CANCER RESEARCH 43. 1365-1371, March 1983] 0008-5472/83/0043-OOOOS02.00 Effect of the Recreational Agent Isobutyl Nitrite on Human Peripheral Blood Leukocytes and on in Vitro Interferon Production1

Evan M. Hersh,2 James M. Reuben, Hal Bogerd, Michael Rosenblum, Marc Bielski, Peter W. A. Mansell, AdÃ¡n RÃos,Guy R. Newell, and Gerald Sonnenfeld

Departments of Clinical Immunology and Biological Therapy Â¡E.M. H., J. M. R., H. B., M. R., M. B., A. R.] and Cancer Prevention Â¡P.W. A M., G. R. N.Â¡,University of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030, and Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, Kentucky 40292 [G. S.Â¡

ABSTRACT immunosuppressive effects, combined with the ability of nitrites to convert amines to nitrosamines, may be related to the The effects of the isobutyl nitrite sold as incense and the development of opportunistic infections and Kaposi's sarcoma chemically pure compound on various in vitro parameters of in homosexuals who use this agent. leukocyte function were studied. This was done because of the potential relationship of Â¡sobutylnitrite use to the opportunistic infection and Kaposi's sarcoma seen in homosexual men. INTRODUCTION Various concentrations of isobutyl nitrite dissolved in ethyl During the last year, there have been several reports of alcohol were added to various leukocyte cultures. The final opportunistic infection and Kaposi's sarcoma occurring either added concentrations were 0.001 to 1.0%. Because of the alone or concurrently in previously healthy young adult males, poor solubility of the agent, the fluid concentrations were quite mainly homosexuals (12, 18, 22, 28). Patients in these reports, low after addition. Thus, the measured concentrations in the who had severe infections such as pneumocystis carinii pneu medium after preparation of 1% (v/v) solution were 0.45 HIM monia, were severely immunodeficient, with skin test anergy, at 1 hr, 0.04 HIM at 24 hr, and 0.04 mw at 48 hr of incubation impaired lymphocyte-proliferative responses, and low-helper at 37Â° in 5% CO2 in air. At the 1% added concentration, the or high-suppressor T-cell subsets resulting in an inverted agent lysed leukocytes and reduced viability from 95% to 21 % helpersuppressor ratio (12, 18, 22, 28). The etiology of the in 24 hr. At an added concentration of 0.5% or below, cell apparent new syndrome is obscure, but it has been attributed count and viability were unaffected, but the agent inhibited in to some component or combination of components of the vitro lymphocyte blastogenic responses to phytohemagglutinin, homosexual life style. Possible factors include multiple sexual pokeweed mitogen, and concanavalin A. It also inhibited nat partners; frequent direct or indirect oral-anal contact; multiple ural killer cell activity to the K562 cell line, lymphocyte-medi sexually transmitted diseases, particularly viral infection such ated antibody-dependent cellular cytotoxicity to the CEM cell as cytomegalovirus and hepatitis; and the use of so-called line, monocyte-mediated antibody-dependent cellular cytotox recreational drugs (5). In the latter category, attention has been icity to human red blood cells, and in vitro adherence and focused on the nitrites because they have been widely used as transformation of monocytes to macrophages. Inhibitory effects aphrodisiacs only since the 1970s and because it is known were greater than 90% at the 0.5% concentration and were that nitrites convert amines and amides to carcinogenic nitros still detectable at 0.01%. Chemically pure isobutyl nitrite and amines in vivo (14). It is speculated that the development of the form sold as incense had identical effects. The agent the opportunistic infection and Kaposi's sarcoma syndrome in volatilized from the tissue culture medium at 37Â° so that its homosexuals must involve some new element since homosex effect on cell viability was reduced about one-third after 24 hr ual activity itself is not new. Thus, the focus is on drug usage. and was gone by 48 hr. The agent inhibited leucine, uridine, In the current report, we outline the results of studies of some and thymidine incorporation approximately equally. Within 2 hr in vitro effect of isobutyl nitrite (both that sold for recreational of exposure to isobutyl nitrite, uridine and leucine incorporation use and that which is chemically pure) on various peripheral were markedly inhibited. After 24 hr of exposure to the agent, blood leukocyte parameters. At high concentrations, the drug the effects on various lymphocyte function parameters were is cytotoxic; while at lower concentrations, at which viability is not reversible. The thymidine incorporation of myeloid and solid preserved, it still inhibits such functions as lymphocyte blasto- tumor cell lines was also inhibited by the same concentrations genesis and NK3 cell activity. The effect is not lymphocyte of isobutyl nitrite which inhibited leukocyte functions. Induction specific, however, in that the proliferation of other cell types is of a,/J-interferon by polyriboinosinic-polyribocytidylic acid in also inhibited in vitro. Thus, Interferon production is also in mouse embryo fibroblasts was inhibited by pretreatment of the hibited by agent treatment of fibroblasts. The data suggest that cells with isobutyl nitrite. These data suggest that isobutyl isobutyl nitrite does have the capacity to inhibit host defense nitrite has nonspecific cytotoxic activity for various cells in vitro parameters in vitro and therefore may also do so in vivo. and could have immunosuppressive effects on tissues exposed in vivo during its recreational use. We speculate that these MATERIALS AND METHODS

' Supported by Grant CA-34674-01 from the National Cancer Institute, NIH, Isobutyl Nitrite Preparations. Isobutyl nitrite for inhalant use is Bethesda, Md. Additional partial funding from the Environmental Protection Agency under Grant R-807619. The contents do not necessarily reflect the views 3 The abbreviations used are: NK. natural killer; RPMI 1640. Roswell Park and policies of the agency, nor does mention of trade names or commercial Memorial Institute Medium 1640; HBSS, Hanks balanced salt solution; PHA, products constitute endorsement for use. phytohemagglutinin; PWM, pokeweed mitogen; Con A, concanavalin A; ADCC, 2 To whom requests for reprints should be addressed. antibody-dependent cellular cytotoxicity; HRBC, human red blood cells; poly(l)- Received June 14, 1982; accepted December 8, 1982. poly(C). polyriboinosinic-polyribocytidylic acid.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1983 American Association for Cancer Research. E. M. Hersh et al. commercially available in a variety of brands. One of these, Rush, is an additional 8-hr incubation. The cultures were harvested onto fiber sold as liquid incense by Pacific Western Distributing Corporation, San glass filter discs with an automatic multiple-sample harvester. The Francisco, Calif. Rush contains greater than 90% nitrites, with small incorporated radioactivity deposited on the discs was counted in a quantities of alcohol or vegetable oil added to reduce volatilization Packard liquid scintillation counter. Net cpm were calculated by sub (31 ). Chemically pure Â¡sobutylnitrite is prepared by the action of nitrous tracting the averages of the unstimulated triplicates from the average acid on butyl alcohol and is available from Alpha Products, Danvers, of the stimulated triplicates. Mass. Isobutyl nitrite is not soluble in water, but it is miscible with Lymphocyte Metabolism. Incorporation of [3H]thymidine (specific alcohol. Rush, or chemically pure isobutyl nitrite, was diluted 1:1 with activity, 1.9 Ci/mmol), [3H]uridine (specific activity, 20 Ci/mmol), and 95% ethanol. The Rush-ethanol, or chemically pure isobutyl [3H]leucine (specific activity, 61 Ci/mmol) was assayed in lymphocyte nitrite:ethanol, or 95% ethanol were then diluted with RPMI 1640 to microcultures as described above. The cultures assaying [3H]leucine 10, 5, 1, 0.1, and 0.01% stock solutions (v/v). The stock solutions incorporation were set up using leucine-free Eagle s minimal essential were added in required amounts to cultures to bring about another medium. The agent:ethanol mixture and ethanol were added as de 1:10 dilution. The alcohol-dissolved Rush, pure isobutyl nitrite, or scribed above. The cultures were pulsed with 1 /Â¿Ciof isotope at 24, ethanol controls had calculated final culture concentrations of 1, 0.5, 48, and 72 hr. After an additional 8-hr incubation, the cultures were 0.1, 0.05, 0.005, and 0.001 % (v/v). harvested and counted as described above. The true concentration of isobutyl nitrite in the cultures was deter Cell Line Proliferation. The effect of the agent on tumor cell line mined by high-performance liquid chromatography. Cultures were set proliferation was assayed in microcultures as described above. The up with 1% (v/v) isobutyl nitrite in alcohol added to complete tissue cell lines used were: GEM (a T-cell lymphoma cell line); K562 (a human culture medium. The fluid was evaluated for isobutyl nitrite content myeloid cell line); MB453 (a human breast cancer cell line); and H4534 compared to a standard at 1, 2, 4, 24, and 48 hr of incubation at 37Â°. (a human fibroblast cell line). Cells of each of the cell lines were plated at concentrations of 2 x 10" cells/well in a 0.2-ml volume of RPMI The nitrite analyses were performed with a Waters Associates liquid Chromatograph consisting of a Model 71 OB sample processor, a Model 1640 with 20% fetal calf serum in microtiter plates. Cells were incu M6000A pump, a Model 450 variable-wavelength UV detector, and a bated for 24 hr before the addition of the agent. Agent and controls data module. A Waters analytical reverse-phase octadecylsilane col were then added, and 48 hr later 1 /Â»Ci[3H]thymidine was added. After umn (30 x 4.5 cm; 10-pm particle size) was used for all analyses. The an additional 24 hr, the cultures were harvested. CEM and K562 solvent consisted of 40% methanol (Burdick and Jackson Laboratories, cultures were harvested using the method described for harvesting Muskegon, Mich.) in water delivered at a flow rate of 2 ml/min. The lymphocyte cultures. Cell lines MB453 and H4534 were harvested column eluate was monitored for UV absorbance at 356 nm. The after washing the wells by adding 50 /il of 0.1 M NaOH to each well, retention time of an isobutyl nitrite standard in this system was 2.8 min. which were then absorbed with 2 cotton swabs. The swabs were dried, Leukocyte Collection and Cultures. Normal human venous blood placed in scintillation vials, and counted in a liquid scintillation counter. was drawn, placed in a 250-ml screw-topped Erlenmeyer flask con Monocyte Adherence. The monocyte adherence assay was carried taining glass beads, and defibrinated by swirling for 5 min. The whole out by the method of Currie and Hedley (4). Mononuclear cells were defibrinated blood was separated into serum and cells by centrifuga- adjusted to a concentration of 2 x 106 cells/ml in RPMI 1640. The tion. The cells were resuspended in HBSS to 3 time the original whole- cell suspension (0.1 ml) and 0.1 ml autologous serum were added to blood volume. In 25- x 150-ml screw-topped tubes, the diluted cell microwells in microtiter plates. The agents were added to triplicate suspension was fractionated by Ficoll:Hypaque density solution cen- cultures for each concentration. After 7 days of incubation at 37Â° in trifugation for 40 min at 400 x g at room temperature. The band of moist 5% CO2 in air, all wells were washed with prewarmed HBSS to mononuclear cells at the interface was removed, the cells were washed remove nonadherent cells and debris. Then 50 /il of WBC-lysing agent twice with HBSS, and the cell pellet was resuspended in RPMI 1640 (Coulter) were added to each well and incubated for 30 sec. The supplemented with 2 mM L-glutamine and 25 /Â¿ggaramycin per ml number of adherent monocytes per ml of original whole blood was (Schering Corp., Kenilworth, N. J.). The cell count was determined by calculated as described previously (4) from Coulter cell counts of the Coulter counting, and a microscopic differential was performed. Cell wells. viability was determined by trypan blue dye exclusion. In each of the ADCC and NK Cell Assays. The ADCC to HRBC and the CEM line experiments described below, a single individual's lymphocytes were and NK cell activity to the K562 cell line were set up as a modification used as his own control. of the method of Poplack ef al. (27). The target cells HRBC (50 /xCi) Surface Marker Enumeration. Leukocyte cell surface markers were and CEM and K562 (100 /Â¿Ci)werelabeled with sodium [51Cr]chromate, analyzed before and 24 hr after incubation with final added concentra by incubation for 40 min and washed 3 times to remove unbound 51Cr. tions of 0.1 and 0.5% of the agent. T-cells were measured by formation The antiserum used for ADCC to HRBC was 1:15 dilution of Dade anti- of rosettes with neuraminidase-treated sheep RBC, and B-cells were B antiserum (American Dade, Division of American Hospital Supply measured by fluorescent microscopy after incubation with goat anti- Corp., Miami, Fla.). For ADCC to CEM, the antiserum was a 1:1000 human lgM:lgD (Cappel Laboratories, Miami, Fla.) (16). T-cells and dilution of rabbit antiserum produced by weekly immunization of rabbits subsets were also determined by incubation with monoclonal anti with viable CEM cells for 3 weeks. The effectontarget ratios used were bodies to the OKT3, OKT4, OKT8, and OKM1 antigens (Ortho Diag 1:1 and 2:1 for HRBC, 20:1 for CEM, and 20:1 and 40:1 for K562. nostic Systems, Raritan, N. J.) and subsequent enumeration in a Mononuclear cells were adjusted to 2 x 106 cells/ml in RPMI 1640 spectrum III cytofluorograph (Ortho Diagnostic Systems). with 5% fetal calf serum. For ADCC, 100 jul effectors, 100 /Â¿Itargets, Lymphocyte Blastogenic Responses. These were measured using and 50 /ti antiserum were added to each well. For NK cell cultures, the standard microculture technique (20). Each well contained 1.5 x 100 /Â¿Ieffectors and 100 /Â¿Ilabeled targets were added. All cultures 105 lymphocytes in 0.2 ml RPMI 1640 with 10% autologous serum. were incubated without the agent, with the agent, and with ethanol at Triplicate cultures were unstimulated or stimulated with PHA (Difco the above-described concentrations and were added as above; the Laboratories, Inc., Detroit, Mich.), PWM (Grand Island Biological Co., cultures were then incubated for 4 hr at 37Â°. One-half of the culture Grand Island, N. Y.), and Con A (Difco). The mitogens were added in supernatant was removed and counted for 51Cr release. Specific target a volume of 0.02 ml/well. The PHA was used at a 1:10 dilution of the cell lysis was calculated as described previously (27). stock solution, the PWM was used at a 1:20 dilution, and the Con A Interferon Production. Mouse a,ÃŸ-(type l) Interferon was produced was used at 80jug/ml. The agent:ethanol mixture and ethanol controls in mouse embryo fibroblasts as described previously (1 ). Polyd)-poly(C) were used at final media concentrations of 1.0, 0.5, 0.1, 0.01, and was prepared by annealing polyriboinosinic acid and polyribocytidylic 0.001% (v/v). After 72 hr in culture at 37Â°, in moist, 5% CO2 and air, acid (P-L Biochemicals, Milwaukee, Wis.) by heating for 1 hr at 45Â°. 1 /Â¿Ciof [3H]thymidine (specific activity, 1.9 Ci/mmol) was added for a,yS-lnterferon was induced in cells by adding 50 /ig of polyd)-poly(C)

1366 CANCER RESEARCH VOL. 43

to tissue cultures for 60 min and then adding additional fresh tissue competence in cancer patients. The cultures were set up with culture medium. DEAE-dextran was included to ensure maximum in- the agent in culture from the beginning of the culture period. terferon induction, and tissue culture supernatants were harvested at The blastogenic responses were reduced by concentrations as 24 hr. Interferon antiviral titers were determined by plaque reduction on murine L-929 cells using the Indiana strain of vesicular stomatitis low as 0.1%. NKcell activity and ADCC to HRBC and GEM, as well as monocyte adherence, were reduced by concentrations virus (1 ). The Interferon titer corresponded to the reciprocal of the greatest dilution of test sample that reduced virus plaques by 50%. as low as 0.01%. In 3 experiments of this type, the minimal One interferon unit in this assay was equivalent to 0.88 NIH G-002- inhibitory concentration was either 0.1 or 0.01% added. Thus, 904-511 reference units. there was considerable variability in sensitivity from subject to Statistical Considerations. The leukocyte cultures described here subject. The alcohol control cultures showed that only a 1% showed significant depression at the 5% level if counts were reduced concentration had a slight inhibitory effect on only some of the 25%, at the 1% level if counts were reduced 50%, and at the 0.1% parameters (NK cell activity and monocyte adherence). level if counts were reduced 75% or greater (15). Experiments were conducted to characterize the effects of Â¡sobutyl nitrite on the DNA, RNA, and protein synthesis of RESULTS peripheral blood lymphocytes stimulated with mitogens over the course of the culture period. The data are shown in Chart High-performance liquid chromatography determinations of 1. It can be seen that at the 0.01% concentration, in PHA- the 1% added concentration showed 46.14 /Â¿g/ml(0.45 mW) stimulated cultures only, leucine incorporation was inhibited. at 1 hr of incubation in the media, 37 jug/ml (0.36 HIM) at 4 hr, In PWM-stimulated cultures, both leucine and uridine incorpo 3.8 jug/ml (0.036 rnw) at 24 hr, and 4.1 /ig/ml at 48 hr. Thus, rations were inhibited. Not shown are data on Con A-stimulated the amount added and the amount in solution are quite differ cultures in which the effects of the agent were essentially ent. At 0 hr and at 20Â°,solubility was poor, and for this reason identical to those seen in the PWM-stimulated cultures. These 1-hr incubation data are given. data indicate that protein and RNA synthesis are more inhibited At a 1% added concentration in complete media, commercial than is DNA synthesis in several types of mitogen-stimulated isobutyl nitrite was highly toxic to peripheral blood leukocytes. cultures. At the next higher added concentration (0.1 %), DNA, We examined the WBC, differential counts, and viabilities of RNA, and protein synthesis were all markedly inhibited. This FicolhHypaque density solution centrifugation-separated leu experiment was repeated 3 times with identical results. kocytes (originally set up at 4.5-cu mm x 103) incubated with We next asked whether the effects of the agent were revers the agent for 24 hr compared to an untreated control and to ible if it was washed out of the cultures. This was approached ethanol controls. The 1% added concentration of the agent using cell-mediated cytotoxicity (Chart 2). Cells were incubated reduced the viability from approximately 95% to 21 %, and the with commercial isobutyl nitrite for 24 hr and washed 3 times, cell count was reduced from approximately 4000/cu mm to and then target cells were added for the standard 4-hr period. 1600/cu mm. The 0.5% concentration did not reduce the The effects of the agent were generally not reversible by count or viability at all. The ethanol concentrations from 1% to washing it out of the cultures. Therefore, once exposed, leu 0.001 % showed no effect on viability or count. This experiment kocytes can be permanently damaged by isobutyl nitrite. was repeated 3 times with essentially identical results. No It was of interest to determine whether isobutyl nitrite had major changes in surface markers (erythrocyte rosettes, sur similar effects on cells other than peripheral blood leukocytes. face immunoglobulin, or OKT 3, 4, and 8) were noted except We therefore evaluated the effects of the agents on a T-leu- at the highest concentration where toxicity was also seen (data kemia cell line (C), a myeloid leukemia cell line (K562), a breast not shown). cancer cell line (MB453), and a fibroblast cell line (H4534) Table 1 shows experiments designed to test whether com (Chart 3). The breast cancer cell line was sensitive to the mercial isobutyl nitrite had an effect on some of the host 0.01% concentration of the agent, and all lines were sensitive defense parameters which we regularly use to test immuno- to the 0.1% concentration in terms of reduction of thymidine

Table 1 Effect of isobutyl nitrite on various in vitro host defense parameters blastogenesis (cpm/1.5 X 10s cells x cytotoxicity (% of tar lysis)NK0.00.05.19.83.88.68.78.210.2ADCCget cell Agent and final cul 103)PHA0.00.018.722.223.025.626.326.824.625.225.3PWM0.00.013.315.615.418.820.119.718.816.917.4Conmon-ocytes/mlblood ture added concen v/v)Isobutyltration (%, A0.00.015.818.419.421.920.619.720.720.719.0Cell-mediatedCEM0.06.737.638.836.837.241.439.734.9ADCCHRBC0.00.00.05.16.85.96.64.84.1Adherentx10"0.80.91.33.14.32.73.34.04.04.53.8 nitrite1.00.50.10.010.001Ethyl

alcohol1.00.50.10.010.001ControlLymphocyte

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24 48 72 24 48 72 24 48 72 24 48 72 Hours of Incubation With Isobutyl Nitrite (0.01%) Hours of Incubation With Isobutyl Nitrite (0.10%) Chart 1. Effect of isobutyl nitrite on leucine (LEU), uridine (UR). and thymidine (THY) incorporation of PHA- and PWM-stimulated lymphocyte cultures at 24 and 48 hr at isobutyl nitrite concentrations of 0.01% (Ai and 0.1% (B). A, D. O. cultures without drug. Cultures with drug: â€¢.leucine; â€¢uridine; A. thymidine. At all time points, the treated and control cultures were different ( p < 0.01 ) in the 0.1 % added Â¡sobutylnitrite cultures. At 0.01 %. only the leucine incorporation was suppressed (p<0.05).

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0.01 0.1 0.5 1.0 Concentration (%, v/v) of Isobutyl Nitrite Chart 3. Effect of isobutyl nitrite on [3H]thymidine incorporation of various cell lines. Microwells were plated with 2 x 104 cells and were incubated in complete medium for 24 hr. Then, various concentrations of isobutyl nitrite were added for 18 hr. after which [3H]thymidine was added for 8 hr, and the cultures were harvested. Data are expressed as percentage of untreated control. A, CEM; B, 0 .001 .01 0.1 1.0 K562; C, MB453; D, H4534. Concentration (%, v/v) of Isobutyl Nitrite butyl nitrite dissolved in ethanol (Table 2). The effects on Chart 2. Effect of isobutyl nitrite on cell-mediated cytotoxicity. Persistence of lymphocyte blastogenic responses were essentially identical to effect after removal of drug from culture. Leukocytes were cultured for 4 hr with isobutyl nitrite and target cells or were cultured for 24 hr with isobutyl nitrite after those produced by the commercial material. The minimum which they were washed 3 times and incubated with target cells for 4 hr. After inhibitory concentration was between 0.05 and 0.10%. Thus, target cell incubation, all cultures were harvested. D, O, A, 4-hr cultures; â€¢â€¢, A. 24-hr washed cultures; â€¢n, ADCC to CEM; â€¢.O,ADCC to HRBC; A. A. NK we conclude that the effects of the commercial material are cell activity. due to the isobutyl nitrite itself. Because the isobutyl nitrite is highly volatile and is reduced incorporation. Thus, the inhibitory effects of these nitrites ap in concentration 90% over 24 hr in culture, we evaluated the pear to be nonspecific relevant to the immune system. persistence of its activity in culture. To do this, we incubated Since the commercial isobutyl nitrite has not been fully the agent at a 1% added concentration in media for up to 72 characterized and may have other agents as well as various hr (Table 3). Leukocytes were added at 0, 24, or 48 hr and nitrites admixed, we obtained and tested chemically pure iso- incubated for the subsequent 24 hr, after which viability was

1368 CANCER RESEARCH VOL. 43

Table 2 Effect of chemically pure isobutyl nitrite on in vitro lymphocyte blastogenic responses Isobutylnitriteadded xNone0.240.530.390.550.56PHA0.440.490.1059.2657.85PWM0.490.110.2535.5349.89103Conx 10s lymphocytes

(%,v/v)1.00.50.10.050.01cpm/1.5 A0.120.300.2246.1483.69

Table 3 Effect of isobutyl nitrite on in vitro lymphocyte viability: time course of persistence of drug in vitro demonstrated by delayed lymphocyte addition % of viable lymphocytes at following tim ings of lymphocyte exposure after estab lishment of drug-containing cultures

AgentaddedNone hr92 hr89 hr85

Isobutyl nitrite (1%) 109524-48 32 87 Ethyl alcohol (1%)0-24 9048-72 90 0 0.01 0.1 1.0 Concentration (%, v/v) of Isobutyl Nitrite Table 4 Chart 4. Time course of inhibition of [3H]uridine incorporation by isobutyl Effect of isobutyl nitrite on host defense parameters in vitro: relative effects of nitrite into K562 cells. K562, 1 x lOVwell, were incubated in media or in media single or multiple additions to cultures plus increasing concentrations of isobutyl nitrite added at the onset of culture. Indicated concentrations were added either at time zero (1 addition) or every Isotope was added for the time periods 0 to 2, 0 to 4, 0 to 8. 8 to 16. 16 to 24, 24 hr for 2 additional doses (3 additions) or 3 additional doses (4 additions). or 24 to 32 hr. after which the cultures were harvested and isotope incorporation Adherent mono- was evaluated. cytes/ml blood x 03)PHA1 blastogenesis (cpm x 1 10"Added Table 5 Effect of isobutyl nitrite on induction of a.ÃŸ-interferon by poly (l)-poly(C) con Polyd)-poly(C), 50 jig, was included in all cultures. Results are of one experi centration of agent addi addi addi addi addi addi ment representative of 3 separate experiments. (%,v/v)1.00.5 tion1.2 tions1.9 tion0.0 tions0.0 tion0.0 tions0.0 Agent and final added culture concentra titer v/v)NoneEthyltion (%, (lU/ml)230022368728831380238445986617411500%of decrease3626140080622434 1.7 2.7 0.0 0.0 0.0 0.4 0.1 2.15.2 2.3 28.2 0.00.9 0.0 0.0 0.05 2.1 85.1 0.0 0.0 0.5Isobutylalcohol 0.01 12.1 3.1 197.3 22.6 9.7 1.2 0.001 14.5 6.6 238.6 41.2163.7PWM18.2 3.3 (commercial)0.050.010.0050.001Isobutylnitrite 0.01 14.04 15.1Lymphocyte218.63 25.13 17.6 incubated for the subsequent 24 hr, after which viability was evaluated. The cytotoxicity of the agent reduced at 24 hr and was completely gone by 48 hr. Thus, in longer-term cultures, nitrite(pure)0.050.010.0050.001Interferon such as blastogenesis and monocyte adherence, we assume that the effects of the agent would be greater if the agent did not volatilize out of the solution within a short period of time. To further investigate this, we conducted a series of experi ments in which isobutyl nitrite was added again to the culture shown in Chart 4. All showed a very rapid onset of inhibitory every day. In general, the minimal inhibitory concentration was activity. reduced one or 2 levels by this maneuver. Table 4 shows the Finally, we determined the effects of isobutyl nitrite on a,ÃŸ- results of readding the same concentration of the agent tc interferon production. Early-passage C3H/HeJ-derived mouse lymphocyte cultures at 24 and 48 hr and its effect at 24, 48 embryo fibroblasts were allowed to grow into a confluent mono- and 72 hr on monocyte adherence. Concentrations added a ! layer. Different cultures were then treated for 24 hr with various low as 0.001 % had suppressive activity. concentrations of commercial or pure isobutyl nitrite. Control We next evaluated the onset of the isobutyl nitrite effect on cultures were treated with equivalent volumes of ethanol, the cell proliferation in culture. Chart 4 shows the effects of various solvent for isobutyl nitrite. Chemicals were then removed from concentrations of the agent on [3H]uridine incorporation by the cells, and cells were washed with minimal essential medium K562 cells. Isotope was added at various times during the containing 10% fetal bovine serum and serum-free medium to culture period. The onset of action was rapid, and maximum more fully remove the chemicals. a,/Mnterferon was then in inhibitory effects were already present during the first 2 hr of duced with poly(l)-poly(C). Viability of the cell cultures was the culture period. Similar experiments were done with PHA-, determined by trypan blue dye exclusion. Concentrations of PWM-, and Con A-stimulated lymphocyte cultures and with the isobutyl nitrite added of 0.1 % or greater were toxic to the cells. CEM cell line. Leucine and thymidine incorporation were also Concentrations of isobutyl nitrite below 0.1 % had no apparent evaluated. The results in all of the above were identical to those toxic effects on the cells, and total cell viability was greater

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1983 American Association for Cancer Research. E. M. Hersh et al. than 95%. Ethanol treatment had no effect on cell viability. cer can be induced in experimental animals by the cofeeding Twenty-four hr later, the culture supernatants were harvested, of natural amines or amides and nitrates (8). Some drugs can and antiviral interferon titers were determined. Both commer be substrates and can be nitrosated by nitrites. They include cial and purified isobutyl nitrite severely inhibited interferon aminophenizone, disulfiram, methadone, propoxyphine, and production at concentrations of 0.05 and 0.01%, when cell phenacetin (3). It is to be emphasized that the carcinogenic viability was not apparently affected (Table 5). Since the inter- potential of butyl nitrite is hypothetical at present and is extrap feron assay is a titration, a decrease of 50% or greater was olated from other studies. required to have a reliable effect on interferon induction. Con Furthermore, we can only speculate as to the role, if any, of centrations of isobutyl nitrite below 0.01 % had no effect on these recreational nitrites in the development of opportunistic infection and Kaposi's sarcoma in homosexual men. It appears interferon induction. that many of the risk factors such as viral infection, sexual promiscuity, drug abuse, and the resultant or associated dis DISCUSSION The syndrome of opportunistic infection and Kaposi's sar eases go hand in hand. Goedert et al. (10) have demonstrated that the helpersuppressor ratio of peripheral blood lympho coma in homosexual men is one of the most distressing and cytes is more inverted among nitrite users than among nonu- potentially serious health problems of modern American life sers. This was the first indication of the potential immunological (12, 18, 22, 28). Hundreds of cases of this syndrome are relationship to the nitrites. The concentration of nitrites in local coming to medical attention each year, and the magnitude of lymphoid tissues such as in nasopharyngeal tissue could con the problem appears to be growing and spreading with time ceivably be high enough to produce both immunosuppressive (2). The mortality of the syndrome is as high as 60%. Extensive and carcinogenic affects. speculations on the causes of this syndrome have been made. In the current paper, we have demonstrated that, in vitro, They include the immunosuppressive and potential carcino both "commercial" isobutyl nitrite and purified isobutyl nitrite genic effects of the herpesvirus group including cytomegalo- dissolved in ethanol depress various leukocyte function param virus (9), the potential immunosuppressive properties of rectally eters associated with host defense. Lymphocyte blastogenesis, or orally ingested components of semen such as polyamines cell-mediated cytotoxicity, and monocyte adherence were all and prostaglandins (23), the extensive use of glucocorticoid- suppressed by concentrations of isobutyl nitrite which were containing skin creams by these patients (24), and the use of noncytotoxic in terms of cell viability. The data suggest but do so-called recreational drugs including marijuana, cocaine, her not prove that the agents may be immunosuppressive in vivo. oin, and nitrites (11). Preliminary data from our laboratory suggest that isobutyl nitrite Volatile alkyl nitrites, such as amyl nitrite and isobutyl nitrite, does suppress the NK cell response of mice.4 have been used extensively in the homosexual community These compounds are somewhat difficult to work with in that since approximately 1969 and are now being used more fre they readily and rapidly volatilize from in vitro tissue culture quently also among heterosexuals (25). The acute transient fluids. Therefore, cells are exposed to a progressive decrease vasodilation and tachycardia produced by these volatile nitrites in concentration with time. Furthermore, the effects on cellular apparently have aphrodisiac qualities related to the intensifi functions were nonspecific in that the proliferation and metab cation of orgasm and relaxation of the anal sphincter (26). The olism of nonlymphoid and nonmyeloid cells were inhibited by various commercial products such as Rush, Lockeroom, Aroma the same concentrations which inhibit lymphocyte function. of Men, and Gatoraide are unregulated by the Food and Drug This is of some additional interest in terms of a possible Administration since they are not sold as drugs but rather as relationship to the mild or moderate chronic myelosuppression incense or room odorizers. The extent of the use of these which we have observed in the homosexual patients with products is not known since they are also not regulated by the opportunistic infection or Kaposi's sarcoma.6 The induction of Consumer Products Safety Commission or the Environmental a,/?-interferon was also inhibited by isobutyl nitrite treatment of Protection Agency. However, it has been suggested that one fibroblast cell cultures. We have shown previously that many brand has sold about 12 million 0.25-oz. bottles since 1974 chemicals that are carcinogenic can inhibit interferon induction (31) and that 11.1% of high school seniors report having used (15, 29, 30). Inhibition of interferon induction might contribute them as drugs (19). to infectious disease and cancer in isobutyl nitrite users. Fur Concern about these nitrites has been mainly related to their ther characterization and clarification of the potential immu- carcinogenic potential. However, they also have other acute or notoxic role of the nitrites may be forthcoming through studies subacute toxicities including the production of methemoglobin- of noncytotoxic doses in vivo in experimental systems. How emia (17), Heinz body hemolytic anemia (13), splenomegaly, ever, even these in vitro studies strongly suggest that the skin rash (7), and death after acute overingestion causing inhalant nitrites may indeed be dangerous, and their use should methemoglobinemia and hypokinetic anoxia (6). be condemned by those physicians who treat patients who use The carcinogenic potential of the nitrates and nitrites relates these drugs regularly. This is particularly supported by the to their ability to nitrosate various amines and amides forming evidence that 0.001% (0.09 rriM) added 3 times strongly in n-nitroso compounds such as dimethylnitrosamine (14). The hibited the proliferation of lymphocytes. carcinogenicity of dimethylnitrosamine was first described by The added concentration of the agent used in these studies, Magee and Barnes (21) in 1956. Nitrosamines are both toxic being 0.001 to 1.0%, ranged from 0.09 to 88 rriM. However, and carcinogenic to a variety of organs including liver, lung, when we measured the actual concentration within the culture kidney, bladder, and the upper respiratory and gastrointestinal tracts. A variety of natural food compounds and drugs undergo ' E. Lotzova and E. M. Hersh, unpublished data. nitrosation when exposed to nitrites in aqueous solution. Can 5 P. W. A. Marseli et al., unpublished observations.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1983 American Association for Cancer Research. Effect of Isobutyl Nitrite on Human Leukocytes fluid (not on the surface to avoid possible effects of phase J. Psychiat., 736: 1067-1069. 1979. 12. Gottleib, M. S., Schroff, R., Schanker. H. M., Weisman, J. D.. Fan, P. T., separation), we found that the 1% added concentration yielded Wolf. R. A., and Saxon. A. Pneumocystis carinii pneumonia and mucosal a fluid concentration of 46 jug/ml (0.45 mivi) at 1 hr of incuba candidiasis in previously healthy homosexual men. N. Engl. J. Med., 305: 1425-1430. 1981. tion. Furthermore, this fell by 90% upon incubation for 24 hr. 13. Greenberg, M. S. Heinz body hemolytic anemia: 'bite cells"â€”a clue to It is unknown as to whether equivalent concentrations occur in diagnosis. Arch. Intern. Med., 736: 153-155, 1976. the nasopharyngeal or other tissues of users. 14. Greenblatt, M., Mirvish, S. S., and So, B. T. Nitrosamine studies: induction of lung adenoma by concurrent administration of sodium nitrite and second These in vitro results are important, however, for the follow ary amines in Swiss mice. J. Nati. Cancer Inst., 46: 1024-1034. 1971. ing reasons: (a) the potential for toxicity or carcinogenicity of 15. Hersh, E. M., and Brown. B. W. Inhibition of the immune response by a chemical upon chronic exposure can be assessed by single glutamine antagonism: effects of azotomycin on lymphocyte blastogenesis. Cancer Res., 37: 834-840. 1971. acute exposure at higher doses or concentrations. Since we 16. Hoffman, R. A., Kung. P. C., Hansen, W. P., and Goldstein. G. Simple and observed suppression of leukocyte functions at noncytotoxic rapid measurement of human T lymphocytes and their subclasses in periph doses, this potential should be further explored for both in vivo eral blood. Proc. Nati. Acad. Sei. U. S. A.. 77: 4914-4917, 1980. 17. HÃ¶rne. M. K., Waterman, M. R., Garriott, J. C., and Foerster, E. H. Methe and in vitro; (Â£>)therealready is some evidence that use of the moglobinemia from sniffing butyl nitrite. Ann. Intern. Med., 97: 417-418, agent by humans is associated with immunological abnormali 1979. 18. Hymes, K. B., Greene, J. B., Marcus, A.. William, D. C.. Cheung, T., Prose. ties (10); (c) we have observed that the injection of nontoxic N. S.. Ballard, H., and Laubenstein. L. J. Kaposi's sarcoma in homosexual doses of the agent into mice significantly suppresses their NK menâ€”a report of eight cases. Lancet, 2: 598-600. 1981. cell activity.4 Finally, as noted above, an effect of isobutyl nitrite 19. Johnston, L. D., Bachman, J. G.. andO'Malley, P. M. Highlights from student drug use in America. DHHS Publication (ADM)81-1066). Washington, D. C.: was evident after only 2 hr of exposure, suggesting that pro National Institute on Drug Abuse. 1980. longed exposure may not be necessary. Thus, while the con 20. Lewinski, U. H., Mavligit, G. M., Gutterman. J. U.. and Hersh, E. M. Inter centrations used in this study may or may not be achieved in action between repeated skin testing with recall antigens and temporal fluctuations of in vitro lymphocyte blastogenesis in cancer patients. Clin. a single in vivo use in humans, the observations are still Immunol. Immunopathol.. 7: 77-87. 1977. relevant. 21. Magee, P. N., and Barnes, J. M. The production of malignant primary hepatic tumors in the rat by feeding dimethylnitrosamine. Br. J. Cancer, 70: 114- 122, 1956. 22. Masur, H., Michelis, M. A., Greene, J. B., Onorato, I., Vande Stouwe, R. A., REFERENCES Holzman, R. S., Wormser, G., Brettman, L., Lange, M.. Murray, H. W., and Cunningham-Rundles, S. An outbreak of community-acquired Pneumocystis 1. Barnes, M. C., Streips, U. N., and Sonnenfeld, G. Effect of carcinogens and carinii pneumonia. Initial manifestation of cellular immune dysfunction. N. analogs on Â¡nterferon induction. Oncology (Basel), 38. 98-101, 1981. Engl. J. Med., 305: 1431-1438, 1981. 2. Centers for Disease Control Task Force on Kaposi's Sarcoma and Oppor 23. Navarro, C., and Hagstrom, J. W. C. To the Editor. N. Engl. J. Med., 306: tunistic Infections. Epidemiologie aspects of the current outbreak of Kaposi's 933, 1982. sarcoma and opportunistic infection. N. Engl. J. Med., 306: 248-252. 1982. 24. Neumann, H. H. To the Editor. Use of steroid creams as a possible cause of 3. Coulston, F., and Dunne, J. F. The Potential Carcinogenicity of Nitrosable immunosuppression in homosexuals. N. Engl. J. Med.. 306: 935, 1982. Drugs. WHO Symposium, Geneva, June 1978, pp. 1-16. Norwood, N. J.: 25. Nicherson, M., Parker, J. O., Lowry, T. P., and Swensen, E. W. Isobutyl Ablex Publishing Corp., 1980. Nitrite and Related Compounds. San Francisco: Pharmex, Ltd., 1979. 4. Currie. E. A., and Hedley, G. W. Monocytes and macrophages in malignant 26. Parker, J. O. Hemodynamic effects of the alkyl nitrites. In: M. Nicherson, J. melanoma I. Peripheral blood macrophage precursors. Br. J. Cancer, 36. O. Parker. T. P. Lowry, and E. W. Swensen (eds.). Isobutyl Nitrite and 1-6, 1977. Related Compounds, pp. 79-80. San Francisco: Pharmex. Ltd., 1979. 5. Durack, D. T. Opportunistic infections and Kaposi's sarcoma in homosexual 27. Poplack, D. O.. Bonnard, G. D.. Holiman, B. J.. and Blaese. R. M. Monocyte- men. N. Engl. J. Med., 305: 1465-1467, 1981. mediated antibody dependent cellular cytotoxicity: a clinical test of monocyte 6. Finch, C. A. Methemoglobinemia and sulfhemoglobinemia. N. Engl. J. Med., function. Blood, 48: 809-816, 1976. 239:470-478, 1948. 28. Siegal. F. P., Lopez, C., Manner, G. S.. Brown, A. E., Kornfeld. S. J., Gold, 7. Fisher, A. A., Brancaccio, R. R., and Jelinek, J. E. Facial dermatitis in men J., Hassett, J., Hirschman, S. Z., Cunningham-Rundles, C., Adelsberg. B. due to inhalation of butyl nitrite. Cutis, 27: 146-153, 1981. R., PartÃan, D. M., Siegal, M., Cunningham-Rundles, S., and Armstrong, D. 8. Garcia, H., and Lijinsky. W. Studies on the tumorogenic effect in feeding of Severe acquired immunodeficiency in male homosexuals, manifested by nitrosamino acids and low doses of amines and nitrites to rats. Z. Krebs- chronic perianal ulcerative herpes simples lesions. N. Engl. J. Med.. 305: forsch., 79: 141-149. 1973. 1439-1444, 1981. 9. Giraldo, G., Beth, E., Henle, W.. et al. Antibody patterns to herpes viruses 29. Sonnenfeld, G.. Hudgens, R. W., and Streips. U. N. Effect of aromatic in Kaposi's sarcoma II. Serological associations of American Kaposi's sar carcinogens and non-carcinogens on the induction of murine alpha/beta coma with cytomegalovirus. Int. J. Cancer, 22: 126-131, 1978. Â¡nterferon. Int. J. Environ. Risk Assess., in press, 1982. 10. Goedert, J. J., Wallen, W. C., Mann, D. L., Strong, D. M., Neuland, C. Y., 30. Sonnenfeld, G. Effect of sidestream tobacco smoke components on alpha/ Greene, M. H.. Murray, C., Fraumeni, J. F., and Blattner, W. A. Amyl nitrite beta interferon production. Oncology (Basel), in press. 1982. may alter T lymphocytes in homosexual men. Lancet, 7: 412-415. 1982. 31. Wason, S., Detsky, A. S., Platt, O. S., and Lovejoy, F. H. Isobutyl nitrite 11. Goode, E., and Troiden, R. R. Amyl nitrite use among homosexual men. Am. toxicity by ingestion. Ann. Internal Med., 92: 637-638, 1980.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1983 American Association for Cancer Research. Effect of the Recreational Agent Isobutyl Nitrite on Human Peripheral Blood Leukocytes and on in Vitro Interferon Production

Evan M. Hersh, James M. Reuben, Hal Bogerd, et al.

Cancer Res 1983;43:1365-1371.

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