The Effects of Allyl Isovalerate on the Hematopoietic and Immunologic Systems in Rodents

The Effects of Allyl Isovalerate on the Hempatopoietic and ImmunologicSystems in Rodents. HONG, H. L., HUFF, J. E., LUSTER, M. I.,MARONPOT, R. R., DIETER, M. P., HAVES, H. T., AND BOORMAN, G.A. (1988). Fundam. Appl. Toxicol. 10, 655–663. FemaleB6C3F1 mice plus male and female Fischer 344/N rats were gavagedwith allyl isovalerate (AIV) in corn oil at 0, 31,62, or 125(mice) and 0, 31. 62, 125, or 250 (rats) mg/kg body weight forfive daily exposures per week for a 2-week period. Hematologic,immunologic, and histopathologic studies were performed 48 to72 hr following the final treatment. AIV exposure had no effecton hematology or bone marrow cellularity in mice or rats. AIVexposure at 250 mg/kg was toxic to rats causing reduced weightgain and hepatotoxicity. In vivo and in vitro studies revealedthat pluripotent hematopoietic stem cells (CFU-S) and granulocyte-macrophageprogenitors (CFU-GM) in the bone marrow were decreased in thetreated mice. Hematopoietic suppression was correlated withthe reduction in the hexose monophosphate shunt metabolism ofbone marrow cells but the Embden-Meyerhof pathway and tncarboxylicacid pathway enzymes did not appear to be affected. Examinationof host resistance following Plasmodium and Listeria challengedid not demonstrate significant differences between treatedand control mice, nor were there other effects on the immunesystem. This suggests that the myelotoxic effects were minimaland of a degree that would not alter host resistance.     

Risk Assessment in Immunotoxicology: I. Sensitivity and Predictability of Immune Tests

We have previously reported on the design and content of a screeningbattery involving a "tier" approach for detecting potentialimmunotoxic compounds in mice (Luster et al., 1988, Fundam.Appl. Toxicol. 10, 2–19). This battery has now been utilizedto examine a variety of compounds by the NIEHS ImmunotoxicologyLaboratory, the National Toxicology Programsponsored laboratories,and by the Cell Biology Department at the Chemical IndustryInstitute of Toxicology. The database generated from these studies,which consists of over 50 selected compounds, has been collectedand analyzed in an attempt to improve future testing strategiesand provide information to aid in quantitative risk assessmentfor immunotoxicity. Studies presented here have establishedthe ability of each of the tests or test combinations in thescreening battery to detect immunotoxic compounds. Efforts arecurrently underway using this database to determine the relationshipsbetween these immune tests and susceptibility to challenge withinfectious agents or transplantable tumor cells. The presentanalyses indicated that the performance of only two or threeimmune tests are sufficient to predict immunotoxic compoundsin rodents (>90% concordance). The tests that showed thehighest association with immunotoxicity were the splenic antibodyplaque forming cell response (78%) and cell surface marker analysis(83%). The relationship between immunotoxicity and carcinogenicity,as well as genotoxicity, was also determined. These analysessuggested that potential immunotoxic compounds are likely tobe rodent carcinogens (p = 0.019) although for compounds thatare not immunotoxic the carcinogenic status is unclear. Therewas no relationship observed between immunotoxicity and mutagenicityas determined using in vitro genotoxicity tests. The significanceof these observations is discussed in terms of the relationshipbetween immunotoxicity tests and biological/toxicological processesconcerned with human health (e.g., infectious disease).     

The Immunotoxicity of Three Nickel Compounds following 13-Week Inhalation Exposure in the Mouse

The Immunotoxicity of Three Nickel Compounds following 13-WeekInhalation Exposure in the Mouse. HALEY, P. J. SHOPP, G. M.,BENSON, J. M, CHENG, Y.-S., BICE, D. E., LUSTER, M. I., DUNNICK,J. K., AND HOBBS, C. H. (1990). Fundam. Appl. Toxicol 15, 476–487.Groups of B6C3F₁, mice were exposed to aerosols of nickel subsulfide(Ni₃S₂), nickel oxide (NiO), or nickel sulfate hexahydrate (NiSO₄6H₂O)6 hr/day, 5 days per week for 65 days to determine the immunotoxicityof these compounds. Exposure concentrations were 0.11, 0.45,and 1.8 mg Ni/m³ for Ni₃S₂, 0.47, 2.0, and 7.9 mg Ni/m³ forNiO; and 0.027, 0.11, and 0.45 mg Ni/m³ for NiSO₄. Thymic weightswere decreased only in mice exposed to 1.8 mg Ni/m³ Ni₃S₂. Increasednumbers of lung-associated lymph nodes (LALN), but not spleennucleated cells, were seen with all compounds. Nucleated cellsin lavage samples were increased in mice exposed to the highestconcentrations of NiSO₄ and NiO and to 0.45 and 1.8 mg Ni/m³Ni₃S₂. Increased antibody-forming cells (AFC) were seen in LALNof mice exposed to 2.0 and 7.9 mg Ni/m³ NiO and 1.8 mg Ni/m³Ni₃S₂. Decreased AFC/10⁶ spleen cells were observed in miceexposed to NiO, and decreased AFC/spleen were seen for miceexposed to 1.8 mg Ni/m³ Ni₃S₂. Only mice exposed to 1.8 mg Ni/m³Ni₃S₂ had a decrease in mixed lymphocyte response. All concentrationsof NiO resulted in decreases in alveolar macrophage phagocyticactivity, as did 0.45 and 1.8 mg Ni/m³ Ni₃S₂. None of the nickelcompounds affected the phagocytic activity of peritoneal macro-phages.Only 1.8 mg Ni/m³ Ni₃S₂ caused a decrease in spleen naturalkiller cell activity. Results indicate that inhalation exposureof mice to nickel can result in varying effects on the immunesystem, depending on dose and physicochemical form of the nickelcompound. These nickel-induced changes may contribute to significantimmunodysfunction.     

Organ-Specific Hematopoietic Changes Induced by a Recombinant Human Interferon-{alpha} in Mice

Organ-Specific Hematopoietic Changes Induced by a RecombinantHuman Interferon- in Mice. ROSENTHAL, G. J., STRANAHAN, R. P.,ILL, THOMPSON, M., BLAIR, P., GERMOLEC, D. R., COMMENT, C. E.,SCHWAB, K., AND LUSTER, M. I. (1990). Fundam. Appl. Toxicol.14, 666–675. Interferon-a (IFN-) is a naturally occurringcytokine that mediates numerous biological activities and hasdemonstrated therapeutic potential in a variety of malignancies.Encouraging activity against HIV-1 replication has also beenobserved with IFN- in the treatment of AIDS, although hematotoxicityhas been a frequently observed side effect. In addition, invitro studies have suggested that IFN- may function as a down-regulatorof myelopoiesis. A recombinant hybrid of subtypes of human IFN-,rHuIFN-A/D, has antiviral activity in mu-rine cells in vitroand In vivo. This study examines the effect of acute and subchronicexposure to rHuIFN-A/D on hemopoietic and immune parametersin C57B1/6 mice. IFN-a was administered ip at 0, 1000, 10,000,and 100,000 units/day for either 1 or 10 consecutive days. Manyof the known effects of IFN- in humans such as anemia, leukopenia,and thrombocytopenia were observed in mice following subchronicexposure, with the latter two effects also manifested followingacute exposure. Further analysis showed that this leukopeniawas not selective. Both splenic and bone marrow cells were examinedfollowing 10 days of dosing with the high dose of IFN-. Lymphocyteswere reduced in both compartments, while granulocytes were increasedin both compartments. Bone marrow cells programmed to differentiateinto granulocytes (CFU-G) were suppressed, while macrophageprogenitors (CFU-M) were stimulated. Erythroid cells decreasedin the marrow but increased in the spleen, suggesting that themicroenvironmerit may play a significant role in the effectof IFN-. The proliferative capacity of both B and T spleniclymphocytes was significantly suppressed in a dose-related fashionfollowing multiple exposure to IFN-a. Clinically, IFN-a is mostoften given in multiple doses and the present data suggest thatsuch a regimen is toxic to both erythroid and myeloid cells,as well as being immunotoxic to splenic B and T lymphocytes.     

Effect of Chronic Exposure of PCB (Aroclor 1254) on Specific and Nonspecific Immune Parameters in the Rhesus (Macaca mulatta) Monkey

Effects of Chronic Exposure of PCB (Aroclor 1254) on Specificand Nonspecific Immune Parameters in the Rhesus (Macaca mulatta)Monkey. Tryphonas, H., Luster, M. I., Schiffman, G., Dawson,L.-L., Hodgen, M., Germolec, D., Hayward, S., Bryce, F., Loo,J. C. K., Mandy, F., and Arnold, D. L. (1991). Fundam. Appl.Toxicol. 16, 773–786. The immunomodulatory effects oflow-level, chronic polychlorinated biphenyl PCB; (Aroclor 1254)exposure were investigated in female rhesus (Macaca mulatta)monkeys. Five groups of monkeys (initially 16 monkeyS/group)were orally administered PCB at levels of 0, 5, 20, 40, or 80µg/kg body wt/day. Tests for immunomodulation were initiatedafter 55 months of exposure to PCBs. Statistically significantobserved immune changes included a dose-related decrease inthe anamnestic (IgM and IgG) response to sheep red blood cells.Conversely, the antibody response to pneumococcus antigen didnot differ significantly across the test groups. A statisticallysignificant dose-related decrease in lymphoproliferation wasnoted with increasing doses of PCBs when phytohemagglutininand Concanavalin A, but not when pokeweed mitogen, were usedas mitogens. A trend toward reduced peak chemiluminescence (mV/min)was observed in zymosan-activated peripheral blood monocytes.The time to peak chemiluminescence of phorbol myristate acetateactivation was statistically increased in a dose-response fashion.Flow cytometric analysis results of peripheral blood lymphocytesusing the markers CD4, CD8, and CD20 were similar across thetest groups. The mean percentage levels for the CD2 marker inthe treated groups were statistically lower than the mean inthe control, while absolute numbers for CD2 were similar acrossthe test groups. Serum hydrocortisone levels did not differamong the test groups. Taken together these results indicatethat low-level, chronic PCB exposure alters a number of rhesusmonkey immune system components and that these effects may bedue to altered T-cell and/or macrophage function. These datamay be of use in extrapolating potential human health effectsfollowing chronic PCB exposure.     

Development of a Testing Battery to Assess Chemical-Induced Immunotoxicity: National Toxicology Program's Guidelines for Immunotoxicity Evaluation in Mice

Development of a Testing Battery to Assess Chemical-InducedImmunotoxicity: National Toxicology Program's Guidelines forImmunotoxicity Evaluation in Mice. Luster, M. I., Munson, A.E., Thomas, P. T., Holsapple, M. P., Fenters, J. D., White,K. L., Jr., Lauer, L. D., Germolec, D. R., Rosenthal, G. J.,and Dean, J. H. (1988). Fundam. Appl. Toxicol..     

Comparative Effects of Immunotoxic Chemicals on in Vitro Proliferative Responses of Human and Rodent Lymphocytes

Comparative Effects of Immunotoxic Chemicals on in Vitro ProliferativeResponses of Human and Rodent Lymphocytes. LANG, D. S., MEIER,K. L., AND LUSTER, M. I. (1993). Fundam. Appl. Toxicol. 21,5357–545. In order to determine the comparability of human and rodentin vitro systems, the direct effects of various therapeuticor environmental chemicals on proliferative responses of lymphocytesof mouse, rat, and human origins were examined and analyzedby a detailed statistical approach. Four compounds of diversestructure and mechanism of action which are known to impairlymphocyte transformation, such as hydroquinone, T-2 toxin,lead nitrate, as well as the widely used immunosuppressive drugcyclosporin A, were chosen as model test substances. T cellswere stimulated by phytohaemagglutinin as well as monoclonalantibodies directed at the T cell receptor/CD3 complex, whileB cells were activated by the T-independent mitogens, includingStaphylococcus aureus cells, Escherichia coli lipopolysaccharide,and Salmonella typhimuriummitogen with specificity for human,mouse, and rat lymphocytes, respectively. In almost all casesthe chemicals altered lymphoproliferative responses in a concentration-relatedmanner in all three species. In general, overall similaritiesin the relative sensitivity of lymphoblastogenesis were obtainedwhen the human dose-response curves were compared to the rodentresponse curves. Frequent, statistically significant species-dependentdiscrepances of the overall response curves between mice andrats were observed. Large, statistically significant differenceswere observed for inorganic lead, revealing obvious divergencesof the effect patterns in all cases, across all species. Inthis case, rodent species, especially the rat, were very sensitiveto immunomodulation by lead, whereas human cells were relativelyresistant. It is suggested that direct interspecies comparisonsof immunological effects due to chemical treatment in vitrocan provide a greater understanding of the relationship betweenanimal and human data, which will improve the confidence ofextrapolation from findings in laboratory animals to human healthrisk.     

Toxicology Studies of a Chemical Mixture of 25 Groundwater Contaminants: II. Immunosuppression in B6C3F1 Mice

Concern over the potential adverse health effects of chemicallycontaminated groundwater has existed for many years. In general,these studies have focused on retrospective epidemiologicalstudies for cancer risk. In the present studies, immune functionwas monitored in female B6C3F₁ mice exposed to a chemical mixturein drinking water for either 14 or 90 days. The mixture consistedof 25 common groundwater contaminants frequently found neartoxic waste dumps, as determined by EPA surveys. None of theanimals developed overt signs of toxicity such as body or liverweight changes. Mice exposed to the highest dose of this mixturefor 14 or 90 days showed immune function changes which couldbe related to rapidly proliferating cells, including suppressionof hematopoietic stem cells and of antigen-induced antibody-formingcells. Some of these responses, e.g., granulocyte-macrophagecolony formation, were also suppressed at lower concentrationsof the chemical mixture. There were no effects on T cell functionor T and B cell numbers in any of the treatment groups. Alteredresistance to challenge with an infectious agent also occurredin mice given the highest concentration, which correlated withthe immune function changes. Paired-water studies indicatedthat the immune effects were related to chemical exposure andnot to decreased water intake. These results suggest that longtermexposure to contaminated groundwater may represent a risk tothe immune system in humans.     

Local versus Systemic Immunotoxicity of Isobutyl Nitrite Following Subchronic Inhalation Exposure of Female B6C3F1 Mice

Female B6C3F1 mice were exposed to isobutyl nitrite (IBN) byinhalation at 0, 37.5, 75, or 150 ppm for 6 hr per day, 5 daysper week for 15 weeks. The potential of this compound to induceimmunotoxicity was assessed during the 3rd, 13th, 14th, and15th week of exposure and after 2 weeks of recovery followingthe 15 weeks of exposure. Both systemic and lung immune functionswere examined, including body and lymphoid organ weights, pulmonarymacrophage function and host defense, expression of spleniclymphocyte cell-surface markers, natural killer cell function,mixed lymphocyte reaction, and induction of specific antibodyto a T-cell-dependent antigen. There was a dose-related suppressionof T-cell-dependent antibody-forming cell responses in the spleenfollowing IBN exposure; however, other measures of T-cell andnonspecific immunity were not significantly affected. A dose-relatedincrease of H₂O₂ production by alveolar macrophages was presentafter 12 but not after 68 exposures to IBN. In contrast, pulmonaryhost defense mechanisms against Klebsiella pneumoniae were unaffected.These results suggest that in the absence of changes in hostresistance, IBN may have selective and partially reversibleeffects on the immune system.     

Risk Assessment in Immunotoxicology: II. Relationships between Immune and Host Resistance Tests

We have reported on the design and content of a screening batteryusing a "tier" approach for detecting potential immunotoxiccompounds in mice (Luster et al, Fundam. Appl. Toxicol., 10,2–19, 1988). The data base generated from these studies,which consists of over 50 selected compounds, has been collectedand analyzed in an attempt to improve future testing strategiesand provide information to aid in developing future quantitativerisk assessment for immunotoxicity. In a recent study it wasshown that as few as two or three immune parameters were neededto predict immunotoxicants in mice (Luster et al., Fundam. Appl.Toxicol., 18, 200–210, 1992). In particular, enumerationof lymphocyte populations and quantitation of the T-dependentantibody response were particularly beneficial. Furthermore,commonly employed apical measures (e.g., leukocyte counts, lymphoidorgan weights) were fairly insensitive. The present analysesfocus on the use of this data base to develop statistical modelsthat examine the qualitative and quantitative relationship(s)between the immune function and host resistance tests. The conclusionderived from these analyses are: (1) A good correlation existsbetween changes in the immune tests and altered host resistancein that there were no instances where host resistance was alteredwithout affecting an immune test(s). However, in some instancesimmune changes occurred without corresponding changes in hostresistance. (2) No single immune test could be identified whichwas fully predictive for altered host resistance, although mostassays were relatively good indicators (i.e., >70%). Severalothers, such as proliferative response to lipopolysaccharideand leukocyte counts, were found to be relatively poor indicatorsfor host resistance changes. (3) The ability to resist infectiousagent challenge is dependent upon the degrees of immunosuppressionand the quantity of infectious agent administered. (4) Logisticand standard regression modeling using one extensive chemicaldata set from the immunosuppressive agent, cyclophosphamide,indicated that most immune function-host resistance relationshipsfollowed linear rather than linear-quadratic (threshold-like)models. For most of the relationships this could not be confirmedusing a large chemical data set and, thus, a more mechanisticallybased approach for modeling will need to be developed. (5) Usingthis limited data set, methods were developed for modeling theprecise quantitative relationships between changes in selectedimmune tests and host resistance tests.