Anti-murine IL-6 receptor antibody inhibits IL-6 effects in vivo

Thrombopoiesis, as well as antibody production, is one of the major events in which interleukin-6 (IL-6) has been reported to be involved. Polyclonal anti-murine IL-6 receptor antibody was prepared to examine the effect of the antibody on these events in IL-6-treated mice. Administration of the anti-mIL-6R antibody inhibited the IL-6-induced increase in the number of platelets. Enhancement of the serum level of DNP-specific antibody by intraperitoneal injection of IL-6 was inhibited completely with simultaneous administration of the anti-mIL-6R antibody. The level of DNP-specific antibody was decreased, even below the basal value, by the higher dose of anti-mIL-6R antibody, indicating its effect also on endogenous IL-6. This work provides evidence that anti-IL-6R antibody inhibits IL-6 function in vivo, and provides an animal model of the therapeutic use of anti-IL-6R antibody for IL-6-related disease.     

In vivo administration of monoclonal antibody to the NK 1.1 antigen of natural killer cells: effect on acute murine cytomegalovirus infection

Monoclonal antibody to the NK 1.1 antigen, found on the natural killer cells of a number of strains of mice, specifically suppresses NK cell function when given in vivo. Using this monoclonal antibody, we have examined the effects of specific suppression of natural killer (NK) cells in vivo on acute murine cytomegalovirus (MCMV) infection in C57BL/10ScN mice. Administration of antibody to NK 1.1 substantially lowered the resistance of C57BL/10ScN mice to lethal virus challenge. In addition, antibody administration prior to intraperitoneal infection significantly increased MCMV replication in salivary glands, lungs, and spleens. In C3H/HeN mice, a strain that lacks the NK 1.1 antigen, antibody to NK 1.1 had no effect on virus replication or lethal infection. Thus, in vivo administration of monoclonal antibody to NK 1.1 alters the course of acute MCMV infection. These findings further substantiate the role of NK cells in defense against acute MCMV infection.     

IL-9 induces chemokine expression in lung epithelial cells and baseline airway eosinophilia in transgenic mice.

Recent data have identified IL-9 as a key cytokine in determining susceptibility to asthma. These data are supported by the finding that allergen-exposed IL-9-transgenic mice exhibit many features that are characteristic of human asthma (airway eosinophilia, elevated serum IgE and bronchial hyperresponsiveness) as compared to the background strain. A striking feature of these animals is a robust peribronchial and perivascular eosinophilia after allergen challenge, suggesting that IL-9 is a potent factor in regulating this process. In an attempt to gain insights into the molecular mechanism governing IL-9 modulation of lung eosinophilia, we investigated the ability of this cytokine to induce the expression of CC-type chemokines in the lung because of their effect on stimulating eosinophil chemotaxis. Here we show that IL-9-transgenic mice in contrast to their congenic controls exhibit baseline lung eosinophilia that is associated with the up-regulation of CC-chemokine expression in the airway. This effect appears to be through a direct action of IL-9 because the addition of recombinant IL-9 to primary epithelial cultures and cell lines induced the expression of these chemokines in vitro. These data support a mechanism for IL-9 in regulating the expression of eosinophil chemotactic factors in lung epithelial cells.     

In vivo activation of murine peritoneal macrophages by intraperitoneal administration of cisplatin

A single intraperitoneal injection of cisplatin (10 mg/kg body weight) in C3H/He mice increases the total number of peritoneal exudate cells (PEC) and macrophages (m phi) within 24 to 48 h. The total number of PEC from untreated mice ranged from 4 to 5 x 10(6) cells/ml containing 2.5 to 3 x 10(6) macrophages, whereas in cisplatin treated mice total number of PEC ranged up to 25 x 10(6) cells/ml. These PEC contained up to 16 x 10(6) m phi. The macrophages obtained from cisplatin injected mice show enhanced cytotoxicity, cytostasis and binding to Dalton's lymphoma cells in vitro. These activated macrophages release into the culture medium factors having cytolytic and cytostatic effect on Dalton's lymphoma cells. The activated macrophages also show enhanced capacity to release superoxide anions, hydrogen peroxide, lysozyme, arginase and interleukin-1.     

In vitro and in vivo inhibition of interleukin (IL)-5-mediated eosinopoiesis by murine IL-5Ralpha antisense oligonucleotide

The unique role of interleukin (IL)-5 in eosinophil production, activation, and localization makes this cytokine a prime target for therapeutic intervention in diseases characterized by a selective blood and tissue eosinophilia. In an attempt to block the effects of IL-5 on eosinophils, a strategy was developed to suppress the expression of the IL-5 receptor alpha chain (IL-5Ralpha) by antisense oligonucleotides (ASOs). IL-5Ralpha ASOs were identified which selectively and specifically suppress the expression of messenger RNA and proteins of both the membrane and the soluble form of the receptor in constitutively IL-5R-expressing murine BCL-1 cells in vitro. Moreover, these IL-5Ralpha-specific ASOs were able to selectively inhibit the IL-5-induced eosinopoesis from murine fetal liver and bone marrow cells in vitro, suggesting that these molecules may affect the development of IL-5-mediated eosinophilia in vivo. Indeed, intravenous administration of IL-5Ralpha-specific ASOs not only suppressed the bone-marrow and blood eosinophilia in mice after short-term treatment with recombinant murine IL-5 but also inhibited the development of blood and tissue eosinophilia in a ragweed-induced allergic peritonitis model. Thus, blocking the expression of IL-5Ralpha on eosinophil using ASOs may have therapeutic benefits in eosinophilic diseases such as asthma.     

Interferon-gamma inhibits the proliferation but not the differentiation of murine B cells in response to IL-5

Interferon-gamma (IFN-gamma) is supposed to be produced by type 1 helper T cells (TH1) and inhibits IL-4-dependent B cell growth and differentiation. IL-5 (T cell-replacing factor, TRF), is a T cell-derived lymphokine which is predominantly produced by type 2 helper T cells (TH2) and regulates proliferation and differentiation of activated B cells. In this study, the effect of IFN-gamma on IL-5-dependent B cell growth and differentiation has been studied using murine chronic B cell leukemic cells (BCL1), normal splenic B cells, and cloned early B cell line. IFN-gamma selectively inhibits the IL-5-mediated proliferation of activated B cells as well as cloned early B cell lines at a low concentration (2 U/ml) in which polyclonal IgM production was not affected. This inhibitory effect of IFN-gamma occurs within 24 h after the onset of culture, as demonstrated by the inability of antibody to IFN-gamma to reverse totally the IFN-gamma-mediated suppressive effects if it was added later than 24 h after the onset of the culture. On the contrary, IL-5-mediated IgM secretion of BCL1 and IgA formation of LPS-stimulated normal B cells were relatively resistant to the suppressive effect of IFN-gamma. IFN-gamma does not affect the receptor expression for IL-5. Interestingly, IL-4-mediated IgG1 formation of LPS-stimulated B cells was markedly suppressed by IFN-gamma at 10 U/ml. These results strongly suggest that IFN-gamma may have differential effects on IL-5-mediated B cell triggering.     

Montelukast, a leukotriene receptor antagonist, inhibits the late airway response to antigen, airway eosinophilia, and IL-5-expressing cells in Brown Norway rats

BACKGROUND: Asthma is characterized by airflow obstruction, inflammatory cell infiltration, and the synthesis of mediators, such as T(H2) cytokines and leukotrienes, in the airways. Cysteinyl leukotriene (cysLT) receptor antagonists have recently been associated with clinical improvement of asthma and reduced airway inflammation. Whether the beneficial effects of cysLT antagonists are mediated through the modulation of cytokine expression has not been determined. OBJECTIVE: The aim of the study was to determine the presence of eosinophils and IL-5 messenger (m)RNA(+) cells within the lungs of antigen-challenged Brown Norway rats after treatment with the cysLT(1) receptor antagonist montelukast (MK). METHODS: Ovalbumin-sensitized Brown Norway rats were treated with either MK or saline before ovalbumin challenge. Pulmonary mechanics were monitored for 8 hours. Subsequently, immunocytochemistry and in situ hybridization were used to examine bronchoalveolar lavage (BAL) fluid and lung tissue for cells expressing major basic protein (eosinophils) and IL-5 mRNA, respectively. Simultaneous in situ hybridization and immunocytochemistry was used to phenotype the cells expressing mRNA encoding IL-5. RESULTS: Animals treated with MK had significantly lower lung resistance and fewer eosinophils and IL-5 mRNA(+) cells within BAL fluid and lung tissue compared with that found in saline-treated animals. Colocalizaton studies revealed that the majority of IL-5 mRNA(+) cells were T cells and that the number of IL-5 mRNA(+)/CD3(+) or IL-5 mRNA(+)/major basic protein(+) cells were significantly less within BAL from animals treated with MK than from those treated with saline. CONCLUSIONS: These results indicate that the cysLT(1) receptor antagonist MK can diminish the pulmonary response to antigen, tissue eosinophilia, and the number of cells expressing IL-5 mRNA, suggesting that leukotrienes may also regulate the allergic response through the modulation of inflammation and cytokine synthesis.     

Effects of administration of murine recombinant IL-4 on the resistance of mice to Listeria monocytogenes infection.

Mice given recombinant murine interleukin-4 as a single i.v. bolus concomitant with a Listeria monocytogenes challenge did not display increased anti-listeria resistance. Under certain conditions, IL-4 administration slightly increased the bacterial burdens in the spleens and livers of infected mice. This finding is consistent with previous reports that endogenous IL-4, or transfer of IL-4 producing TH2 cells, can be detrimental to host defense against microbial infection.     

Characterization of the murine interleukin 5 receptor by using a monoclonal antibody

Murine interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. The receptor for IL-5 has been identified as two cross-linked complexes on T88-M cells (a murine IL-5-dependent early B cell line). In this study the IL-5 receptor was directly characterized by utilizing an immobilized IL-5 column and a rat monoclonal antibody, designated H7, directed against the IL-5 receptor. H7 completely inhibited specific binding of 35S-labeled IL-5 to T88-M cells, and bound to IL-5-responsive cells, e.g. T88-M, BCL1-B20 (a chronic B-cell leukemia), and MOPC104E (a myeloma), whereas H7 did not bind to IL-5-non-responsive cells, e.g. X5563 (a myeloma), FDC-P1 (an IL-3-dependent line), and MTH (an IL-2-dependent CTLL). H7 could barely bind to T88-M cells in the presence of IL-5, and immunoprecipitated a major band with an Mr of approximately 60 kd from the extract of surface-radioiodinated T88-M cells. The precipitation of this 60 kd molecule was inhibited by the addition of IL-5. Analysis with immobilized IL-5 also revealed that a 60 kd molecule bound specifically to IL-5-coupled beads compared with control beads. Furthermore, no additional molecule with a higher Mr that was recognized by H7 appeared under non-reducing, compared with reducing, conditions. The 60 kd molecule recognized by H7 could be digested with N-glycanase to yield a protein band of approximately 55 kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

重组抗HBsAg单链抗体在HBV转基因小鼠体内作用的研究

目的　研究重组人源抗HBsAg单链抗体 (HBscFv)在HBV转基因小鼠体内的活性作用 ,探讨HBV转基因小鼠作为HBsAg特异性抗体的结合活性评价模型的可行性。方法　工程菌表达的HBscFv包涵体经固定化金属螯合层析和分子排阻层析两步纯化后 ,分步透析复性。复性后的HBscFv(0 .9g L)经尾静脉注射HBV转基因小鼠 ,一定时间后测定鼠血清中HBsAg的浓度 ,计算抗体注射前后HBsAg浓度下降的百分比 (结合率 ) ,同时以人血源HBsAbIgG(40U ml)和生理盐水作为对照。结果　两步纯化获得纯度达到 98%的重组HBscFv。HBscFv制品能够结合转基因小鼠体内的HBsAg,其结合率为 (30 .4 7± 9.85 ) % ,与生理盐水组差异有显著性 (P <0 .0 0 1) ,与HBsAbIgG组差异无显著性(P >0 .0 5 ) ;对照品HBsAbIgG的结合率为 (39.0 0± 7.4 3) % ,与生理盐水组差异也有显著性 (P <0 .0 0 1)。比较HBscFv和HBsAbIgG的结合率及其浓度 ,求得HBscFv的结合效价为 31.5 8U mg。结论　HBscFv在转基因小鼠体内具有特异性结合HBsAg活性 ,HBV转基因小鼠可发展为评价HBsAg特异性抗体的体内结合活性的候选模型。     

Anti-IL-2 receptor monoclonal antibody AMT-13 increases soluble IL-2 receptor levels in vivo.

A rat anti-interleukin-2 receptor (IL-2R) monoclonal antibody (mAb), AMT-13, has been shown to prolong cardiac allograft survival in mice. However, neither the mechanism of its immunosuppressive action, nor its other in vivo effects, have been studied extensively. We investigated the effect of AMT-13 on serum-soluble IL-2R levels by a quantitative ELISA technique. Following administration of multiple doses of AMT-13, sufficient to delay fetal pancreatic graft rejection, a rapid and lasting increase in soluble(s) IL-2R levels of up to 20-fold was observed. Levels returned to normal by Day 9 despite continued AMT-13 administration. A similar though less marked increase was observed following a single injection of AMT-13. Irradiation with 350 rads decreased sIL-2R levels, whilst rat Ig-treated control mice had sIL-2R levels within the normal range. In vivo administration of recombinant IL-2 also increased sIL-2R levels, but the effect was transient and slight. No effect on either the release of sIL-2R or expression of cell surface-associated IL-2 receptors was noted when splenocytes were cultured in the presence of AMT-13. We speculate that the increased concentration of sIL-2R in the serum of AMT-13-treated mice may play a role in the immunosuppressive action of this mAb.     

Tunicamycin inhibits function and expression of the high-affinity IL-2 receptor in a murine IL-2-dependent cell line.

Murine interleukin-2-dependent T-lymphocytes (CT6) were treated with tunicamycin, an inhibitor of both glycoprotein and ganglioside synthesis, to study the involvement of glycosylation in the IL-2 proliferative response. Tunicamycin inhibited proliferation in a dose-dependent manner at concentrations which did not inhibit protein synthesis (10-50 ng/ml). Swainsonine, a glycoprotein processing inhibitor, had no effect on proliferation. Inhibition of proliferation by tunicamycin was accompanied by an inhibition of binding of 125I-IL-2 to its high-affinity receptor. Scatchard analysis showed that receptor number was decreased by tunicamycin treatment. On the other hand, tunicamycin did not affect either the binding of the monoclonal antibody 7D4, specific for the 55 kDa low-affinity protein subunit of the IL-2 receptor, or the recycling of the IL-2 receptor. To determine the specific effects of tunicamycin on the biosynthesis of particular CT6 glycoconjugates, cells were radiolabeled with 3H-glucosamine and incorporation into ganglioside, neutral glycolipid and glycoprotein fractions was measured. Low doses of tunicamycin inhibited ganglioside synthesis and glycoprotein glycosylation to the same extent, whereas no effect on neutral glycolipid synthesis was observed. These results suggest that glycosylation of glycoprotein and/or gangliosides might play an important role in the formation of a functional high-affinity IL-2 receptor complex in CT6 cells.     

Pulmonary eosinophilia in mice devoid of interleukin-5

The biology of the eosinophilic leukocyte-development, recruitment, and prolonged existence in somatic tissues-has been linked almost invariably to the actions of the "eosinophil" cytokine, interleukin-5 (IL-5). Here we demonstrate that pulmonary eosinophilia can occur in the absence of IL-5, as morphologically normal eosinophils are recruited to the lungs of virus-infected IL-5 -/- mice with kinetics and sequelae that are indistinguishable from those of their IL-5 +/+ counterparts. We conclude that pulmonary eosinophilia observed in response to primary paramyxovirus infection occurs via mechanisms that are distinct from those involved in eosinophil responses to allergens and in asthma. Furthermore, the presence of functional eosinophils in IL-5 -/- mice suggests the possibility of developmentally distinct subsets of what has been presumed to be a homogeneous leukocyte population.     

抗白介素5单克隆抗体抑制哮喘小鼠嗜酸性粒细胞迁移

目的 观察抗白介素5(IL-5)单克隆抗体对哮喘小鼠嗜酸性粒细胞(Eos)从骨髓到气道迁移的影响.方法 15只雄性C57BL/6小鼠.常规法复制哮喘,并在每次卵白蛋白(Ova)激发前2 h鼻腔内滴入抗IL-5单克隆抗体,同时设立阴性对照组,即以生理盐水代替Ova 和抗IL-5抗体.在末次抗原激发后12 h测定支气管肺泡灌洗液(BALF)、外周血(PB)和骨髓(BM)中炎症细胞数以及肺组织内Eos数.结果 Ova抗原激发后12 h小鼠的BALF、PB和BM及细支气管和肺组织中的Eos数显著增加(P＜0.01, P＜0.05).抗IL-5单克隆抗体则使上述变化显著减轻(P＜0.05,P＜0.01).结论 抗IL-5单克隆抗体能显著抑制哮喘小鼠嗜酸性粒细胞的迁移.     

In vivo modulation of the murine immune response to Francisella tularensis LVS by administration of anticytokine antibodies.

The role(s) of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4) in establishment and maintenance of protective immunity to Francisella tularensis LVS in mice (C3H/HeN) was examined by selective removal of these cytokines in vivo with neutralizing antibodies. The 50% lethal dose (LD50) for mice infected intradermally with F. tularensis alone was 136,000 CFU; treatment of mice with anti-IFN-gamma or anti-TNF-alpha at the time of infection significantly reduced (P much less than 0.05) the LD50 to 2 and 5 CFU, respectively. Abrogation of protective immunity, however, was effective only when anti-IFN-gamma or anti-TNF-alpha was administered prior to day 3 postinfection. In contrast, the LD50 for mice treated with anti-IL-4 was repeatedly higher (555,000 CFU) than for controls; this difference, however, was not significant (P greater than 0.05). Thus, IL-4 may be detrimental, while IFN-gamma and TNF-alpha were clearly crucial to the establishment of protective immunity to F. tularensis during a primary infection. The importance of IFN-gamma and TNF-alpha during a secondary immune response to F. tularensis was also investigated. Spleen cells from immune mice passively transfer protective immunity to recipient mice in the absence of confounding antibody-mediated immunity. This passive transfer of immunity, however, was abrogated by treatment of recipient mice with anti-IFN-gamma or anti-TNF-alpha at the time of challenge infection. That anticytokines effectively abrogate protective immunity very early in the course of infection with F. tularensis suggests that T-cell-dependent activation of macrophages for microbicidal activity is unlikely. These T-cell-independent events early in the course of infection may suppress bacterial replication until a T-cell-dependent response ultimately clears the bacteria.     

In vivo mutation in gene A of splenic lymphocytes from phiX174 transgenic mice

Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants.     

T-cell receptor retrogenic mice: a rapid, flexible alternative to T-cell receptor transgenic mice

The T-cell receptor (TCR) is unique in its complexity. It determines not only positive (life) and negative (death) selection in the thymus, but also mediates proliferation, anergy, differentiation, cytotoxicity and cytokine production in the periphery. Through its association with six CD3 signalling chains (εγ, δε and ζζ), the TCR is capable of recognizing an extensive variety of antigenic peptides, from both pathogens and self-antigens, and translating these interactions into multiple signalling pathways that mediate diverse T-cell developmental and functional responses. The analysis of TCR biology has been revolutionized by the development of TCR transgenic mice, which express a single clonotypic T-cell population, with diverse specificities and genetic backgrounds. However, they are time consuming to generate and characterize, limiting the analysis of large numbers of TCR over a short period of time in multiple genetic backgrounds. The recent development of TCR retrogenic technology resolves these limitations and could in time have a similarly important impact on our understanding of T-cell development and function. In this review, we will discuss the advantages and limitations of retrogenic technology compared with the generation and use of TCR transgenic mice for studying all aspects of T-cell biology.     

In vivo suppression and enhancement of the murine homocytotropic antibody response by staphylococcal enterotoxin A

Effects of staphylococcal enterotoxin A (SEA) on the mouse homocytotropic antibody (HCA) system were studied. Groups of BDF mice received 10 micrograms SEA either orally or intraperitoneally at 0, 24, 48 h before or after immunization with 100 micrograms ovalbumin in 1 mg A1(OH)3 gel. Primary and secondary HCA responses were determined by 48-hour passive cutaneous anaphylactic reactions in genetically hairless mice. It was found that effects of SEA on HCA responses were dependent on the time and route of SEA administration. In general, early administration (48 h before immunization) of SEA showed suppression, while later administration (either 24 h before or after immunization) of SEA demonstrated enhancement. A further delay of SEA administration (48 h after immunization) exerted suppressive effects except when it was given intraperitoneally in the anamnestic HCA experiments. The mouse HCA system proved to be a suitable in vivo correlate of in vitro plaque-forming cell responses modulated by SEA.     

In vivo adipose tissue regeneration by adipose-derived stromal cells isolated from GFP transgenic mice

We have previously demonstrated that pluripotent stem cells can be obtained from green fluorescence protein (GFP) transgenic mouse adipose tissue. In this study, we sought to determine whether adipose tissue regeneration can be induced in vivo using adipose-derived stromal cells (ASCs) from GFP mice. ASCs were isolated from inguinal fat pads of GFP mice, as described in our previous publication. After incubation in two passages in the control medium, the cells were incubated in the induction medium to induce adipogenesis. Induced ASCs were merged with fibrin glue, and the mixture was injected subcutaneously into the dorsum of athymic mice. Finally, specimens were harvested and analyzed morphologically and histologically. The regenerated tissue was macroscopically semitransparent and soft with slight angiogenesis. Fluorescence microscopy revealed that the specimens strongly emitted green fluorescence, suggesting that the transplanted ASCs had contributed to adipogenesis. Both hematoxylin and eosin and oil red O staining revealed that cells containing small lipid droplets had been regenerated histologically. These findings suggest that ASCs could contribute to adipose tissue regeneration in vivo. ASCs may be an ideal source for adipose tissue regeneration, which may in turn play an important role in augmentation surgery in surgically treated cancer or trauma patients.     

In vivo mutagenesis in the lungs of gpt-delta transgenic mice treated intratracheally with 1,6-dinitropyrene

1,6-Dinitropyrene (1,6-DNP) is a ubiquitous airborne pollutant found in diesel exhaust. In this study, mutagenesis was examined in the lungs of gpt-delta transgenic mice after intratracheal instillation of 0-0.1 mg 1,6-DNP. In addition, the 1,6-DNP-induced gpt mutation spectrum was compared with that of control mice. A single intratracheal injection of 0-0.05 mg 1,6-DNP resulted in significant dose-dependent increases in mutant frequency; the induced mutant frequency declined at the 0.1 mg dose. The average lung mutant frequencies at doses of 0.025, 0.05, and 0.1 mg 1,6-DNP were 2.9-, 4.1-, and 1.9-times higher than for control mice ((0.50+/-0.16)x10(-5)). The major mutations induced by 1,6-DNP included G:C-->A:T transitions, G:C-->T:A transversions, and 1-base deletions. Among the G:C-->A:T transitions isolated from 1,6-DNP-treated mice, five (at nucleotide positions 64, 110, 115, 116, and 418) were observed in four or more animals. These positions therefore are potential hotspots for 1,6-DNP mutation. The predominant frameshift mutations following 1,6-DNP treatment included single base pair deletions at G:C (9/13=69%). The results of this study indicate that 1,6-DNP is mutagenic for the lungs of mice.