Effect of Vibrio cholerae neuraminidase on the generation of cell-mediated cytotoxicity in vitro.

Vibrio cholerae neuraminidase (VCN))12.5 units/2 X 10(6) cells/ml) continuously present for a standard 5-day MLC will significant (p less than 0.02) increase the cytotoxic activity generated by a given number of responding spleen cells without reducing the specificity. Heat-inactiviated VCN produced no such augmentation. This augmented cytotoxicity could be reproduced by preincubating (1 hr) the responding spleen cells with VCN (25 units/2.5 X 10(6) cells/ml) before addition of stimulating spleen cells. Preincubating the stimulator spleen cells with VCN had no effect. VCN preincubation of target cells or presensitized effector cells produced no augmentation. The addition of soluble VCN to the killing assay also did not increase cytotoxicity. Thus, VCN acts only during the generation of specifically sensitized cytotoxic T cells. When the effect of VCN on MLC reactivity, cell recovery and total cytotoxicity (lytic units/10(6) cells) were compared, it became apparent that VCN increases the proliferation of responder cells after stimulation resulting in both an increased number of cells and also an increase in the proportion of specifically sensitized cytotoxic cells in the culture. VCN treatment of responder cell membrane apparently permits a more ready response to allogenic antigens in culture facilitating both increased proliferation and the increased development of specific cytotoxic killers.     

Myeloproliferative sarcoma virus: its effects on erythropoiesis in adult DBA/2J mice

Adult susceptible mice (DBA/2J) infected with MPSV (myeloproliferative sarcoma virus), a defective RNA tumour virus, develop splenomegaly and progressive disruption of the haematologic system culminating in death. The present study was specifically directed toward determining the effects of the virus on erythroid differentiation. Early and late precursor cells (erythroid burst-forming units; BFU-E and colony-forming units; CFU-E, respectively) were evaluated by the ability of bone marrow and spleen cells to form colonies of fully differentiated erythroid cells in vitro. MPSV caused substantial modification of both the BFU-E and CFU-E populations in the bone marrow and spleen of infected animals. Changes were detected in the CFU-E population preceding any significant increase in spleen weight. In the bone marrow, the proportion of CFU-E cells increased almost twofold by days 5-10 after virus infection but decreased by day 15. In the spleen, CFU-E frequency rose 40-fold by days 10-15 and then declined steadily prior to death. At the peak of CFU-E expansion, a small proportion of the population appeared to be erythropoietin (Ep) independent, although there was no evidence of a complete switch to Ep-independence which occurs in Friend virus-induced erythroleukemia. Dose-response curves showed that none of these data could be explained in terms of a changing responsiveness to Ep. However, evidence is presented that indicates that BFU-E from MPSV-infected animals lose or have a reduced requirement for burst-promoting activity (BPA) relative to normal cells although their progeny still need Ep for terminal erythroid differentiation.     

Ontogeny of T Cells in the Pig Fetus

Mononuclear cells isolated from thymus, spleen and cord blood of pig fetuses ranging in age from 48 to 112 days were examined for the presence of sheep red blood cell rosette-forming cells (SRBC-RFC). After an initial increase from 77 % (mean) at 48 days of gestation to 88 % at 60 days, the proportion of SRBC-RFC in thymus remained constant throughout the gestational period. In spleen and cord blood, the proportion of SRBC-RFC increased with age, from occasional rosette-forming cells at 48 days of gestation to 21 % and 30 %, respectively, at 112 days. The demonstrated development of SRBC-RFC in the thymus, spleen and cord blood is considered to reflect the ontogeny of T cells in these fetal pig tissues.     

Characteristics of immunosuppressive macrophages induced in spleen cells by Mycobacterium avium complex infections in mice

The profile of generation and characteristics of splenic macrophages (M phi s) which suppress the concanavalin A (Con A) mitogenic response of splenic T cells (designated as 'immunosuppressive M phi s') in host CBA/JN mice during the course of Mycobacterium avium complex (MAC) infection were investigated. In MAC-infected mice, reductions in some cellular functions of host splenic T cells, such as the Con A mitogenic response and mixed leucocyte reaction, were seen around 2 weeks after challenge of organisms, and this was accompanied by appearance of immunosuppressive M phi s in spleen cells. In this case, increase in immunosuppressive M phi activity was seen in terms of both activity per spleen and activity per individual M phi. In this phase of the infection, MAC-induced splenic M phi s showed a markedly increased ability to produce reactive oxygen radicals in response to phorbol myristate acetate. Thus, the expression of suppressor activity of MAC-induced M phi s seems to be closely linked to their activated state. A large proportion of the immunosuppressive M phi s exhibited suppressor activity dependent on prostaglandins and membrane functions related to microfilaments. It was also found that the generation of IL-2-reactive T cell populations in response to Con A was markedly inhibited by MAC-induced splenic M phi s, whereas they caused no significant reduction in the IL-2-producing ability of normal spleen cells.     

Dietary folate improves age-related decreases in lymphocyte function

Although low folate status is thought to be fairly common in the older population, its implication on immunity has not been adequately investigated. Using 11-month-old and 23-month-old male rats (Fisher 344), the present study was undertaken to examine the modifying effects of feeding a control diet (NIH-07) supplemented with folate (35.7 mg/kg) for 3 weeks on the immune cells of spleen and mesenteric lymph node (MLN) origin. The serum concentrations of folate along with vitamin B(12) were elevated in response to the folate supplementation (P<.05). These results were accompanied by an improved proliferative response (stimulation index) to mitogens in both the spleen and MLNs (P<.05). The proportion of T cells in the MLNs, but not in the spleen, was significantly increased in rats fed a diet supplemented with folate. In the spleen, the folate-supplemented diet prevented the age-associated decrease (P<.05) in the production of interferon (IFN)alpha by unstimulated cells and the decrease in T-helper (Th)1/Th2-type response after stimulation with phorbol myristate acetate and ionomycin. In the MLNs, on the other hand, the folate-supplemented diet failed to influence any age-related increase in interleukin (IL)-2, tumor necrosis factor alpha and IFNgamma following stimulation but did result in a significantly increased production of IL-4 (P<.05). Overall, this study provides data suggesting that aging is associated with changes in the proportion of T cells, the ability of immune cells to proliferate and the production of cytokines after stimulation. Supplementing a folate-sufficient diet with additional folate improves proliferative response to mitogens, the distribution of T cells in the MLNs and the age-related changes in cytokine production in the spleen. These results suggest that the dietary folate requirement may be higher in the older population than in the younger population to support immune functions.     

Neonatal exposure to propylthiouracil induces a shift in lymphoid cell sub-populations in the developing postnatal male rat spleen and thymus

Evidence of the connections between the immune system and the thyroid axis is increasingly strong; however, much of the data are focused on immune effects of altered thyroid status in adults or rodents with congenital defects of the pituitary/thyroid axis. The object of the present study was to determine the effects of PTU-induced hypothyroidism on the developing immune system of the rat by focussing on both the spleen and thymus gland. Male Sprague-Dawley rat pups were exposed to PTU through maternal milk by giving the mothers 0.02% PTU in their drinking water starting on the pups' day of birth until day 24 (d24), shortly before weaning on d28. Animals were sampled on days 14, 22, 30, and 91. The mean body weight was decreased in the PTU-treated animals on days 14, 22, and 30. The mean spleen and thymic weights and cellularity were all decreased in the PTU-treated animals on d22 and d30. PTU exposure increased the proportion of NK cells in the spleen on days 14, 22, and 30. The proportion of T-cells was increased on days 22 and 30 with a particular increase in the CD4+ T-cells, resulting in an increase in the ratio of helper T-cells to suppressor/cytotoxic T-cells at d22. PTU also decreased the proportion of splenic B-cells at days 14, 22, and 30 which could explain the increased proportion of both NK and T-cells during these sampling periods. PTU treatment decreased the lytic ability of NK cells at d22, but no functional differences were observed at days 14, 30, 91, despite the increased proportion of NK cells in PTU-exposed animals at days 14, 22, and 30. PTU exposure also increased the proportion of CD4+CD8- cells in the thymus on d22 and caused an increase in both the CD4+CD8- and CD4-CD8+ populations on d30. These data suggest that the effects of temporary, PTU-induced hypothyroidism on the cell populations in the spleen partially result from transient changes in thymic T-cell development, including a shift towards increased CD4+CD8- cells. The data also suggest that temporary hypothyroidism early in development decreases B-cell development in a transient fashion. Temporary hypothyroidism induced from birth to the latter stages of the weaning period induced transitory effects on the spleen, thymus, and immune cell sub-populations--all of which recovered to normal values when the animals matured.     

Sister-chromatid exchange studies on direct- and indirect-acting clastogens in mouse primary cell cultures

An in vitro sister-chromatid exchange (SCE) assay using mouse primary bone marrow and spleen cells was conducted with both direct- and indirect-acting genotoxic agents. 2,4,7-Trinitrofluorenone, a direct-acting genotoxic agent, induced a significant dose-related increase in SCEs. In both bone marrow and spleen cells, 2.0 micrograms/ml caused an approx. 3-fold increase in SCE level over control values. Cyclophosphamide, an indirect-acting genotoxicant which requires metabolic activation for its clastogenicity, induced a significant increase in SCEs in the presence of S9 from liver of rats pretreated with Aroclor-1254. A dose of 2 micrograms/ml resulted in a 2-fold increase in bone marrow and a greater than 5-fold increase in spleen cells. Benzo[a]pyrene, another indirect-acting genotoxicant, also induced significant dose-related SCE responses in both cell types. It seems that primary bone marrow and spleen cell culture systems can detect both direct- and indirect-acting genotoxicants and may be useful for routine and/or comparative cytogenetic studies.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

A comparative study of the kinetic changes of hemopoietic stem cells in mice infected with lethal and non-lethal malaria.

The kinetic changes of hemopoietic stem cells in bone marrow and spleen were compared between lethal Plasmodium berghei- and non-lethal P. yoelii 17x-infected mice. P. yoelii 17x-infected mice showed more severe splenomegaly than those infected with P. berghei. P. yoelii 17x-infected mice also showed a greater degree of sustained increase in number of multipotent hemopoietic stem cells (colony-forming units in spleen: CFU-S) and committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) than P. berghei-infected mice. Such an increase was predominantly seen in the spleen of P. yoelii 17x-infected mice. In P. berghei-infected mice, the number of CFU-S, CFU-GM and also CFU-E only transiently increased and then decreased to a subnormal level at the late stage of infection. The proportion of cycling CFU-S was higher in P. berghei-infected mice than in P. yoelii 17x-infected mice. The IL-3 producing activity per spleen was much higher in P. yoelii 17x-infected than in P. berghei-infected mice at any point in time during the infection. Thus, hemopoietic changes seen after malaria infection seem to be closely related to the pathogenicity of the malaria parasite.     

Nitric oxide inhibition decreases bleomycin-detectable iron in spleen,bone marrow cells and heart but not in liver in exercise rats

The possible role of nitric oxide on the exercise-induced changes in bleomycin-detectable iron (BDI) in the liver, spleen, bone marrow cells and heart was investigated. Female Sprague—Dawley rats were randomly assigned to four groups: S1 (Sedentary), S2 (Sedentary + L-NAME [N-nitro-L-arginine methyl ester]), E1 (Exercise) and E2 (Exercise + L-NAME). Animals in the E1 and E2 swam for 2 h/day for 3 months. L-NAME in the drinking water (1 mg/ml) was administrated to rats in the S2 and E2 groups for the same period. At the end of the 3rd month, nitrite and nitrate (NOx), BDI and non-heme iron (NHI) contents in the liver, spleen, bone marrow cells and heart were measured. The ratio of BDI/NHI was calculated. The exercise induced a significant increase in NOx and BDI contents and/or BDI/NHI ratio in the spleen, bone morrow cells and heart. Treatment with L-NAME, an inhibitor of NOS, led to a significant decrease in NOx and an increase in BDI levels and BDI/NHI ratios in these tissues. The correlative analysis showed that there is significantly positive correlation between NOx levels and BDI contents and/or BDI/NHI ratios in the spleen, bone marrow cells and heart. These results suggest that the increased nitric oxide might be one of the reasons leading to the increased BDI levels in these tissues in the exercised rats. In contrast to the above tissues, in the liver, exercise led to a significant decrease rather than increase in BDI levels and BDI/NHI ratios with a significant increase in NOx contents. Treatment with L-NAME led to a significant increase in BDI levels and BDI/NHI ratios and a decrease in NOx contents in the tissue. These findings plus the results reported by others imply that nitric oxide might have an inhibitory effect on BDI in the liver.     

Absence of mitochondrial uncoupling protein 3: effect on thymus and spleen in the fed and fasted mice

Mitochondrial uncoupling protein 3 (UCP3) is constitutively expressed in mitochondria from thymus and spleen of mice, and confocal microscopy has been used to visualize UCP3 in situ in mouse thymocytes. UCP3 is present in mitochondria of thymus and spleen up to at least 16 weeks after birth, but levels decrease by a half in thymus and a fifth in spleen after three weeks, probably reflecting the suckling to weaning transition. UCP3 protein levels increase approximately 3-fold in thymus on starvation, but expression levels in spleen were unaffected by starvation. Lack of UCP3 had little effect on thymus mass or thymocyte number. However, lack of UCP3 affected spleen mass and splenocyte number (in the fasted state) and results in reduced CD4+ single positive cell numbers and reduced double negative cells in the thymus, but as a 2-fold increase in the proportion of CD4(+), CD8(+) and DP cells in spleen. Starvation attenuates these proportionate differences in the spleen. A lack of UCP3 had no apparent effect on basal oxygen consumption of thymocytes or splenocytes or on oxygen consumption due to mitochondrial proton leak. Splenocytes from UCP3 knock-out mice are also more resistant to apoptosis than those from wild-type mice. Overall we can conclude that UCP3 affects thymocyte and spleen cell profiles in the fed and fasted states.     

Evaluation of the circulating and splenic lymphocyte subpopulations in patients with non-Hodgkin lymphomas and Hodgkin's disease using monoclonal antibodies

The number of B lymphocytes, T lymphocytes and their helper/inducer, cytotoxic/suppressor and NK/K subpopulations was measured in peripheral blood and spleen cell suspensions from patients with Hodgkin's disease (HD) in the active stage of the disease and in remission status, as well as in Non-Hodgkin lymphomas (NHL) in active stage of the disease. B lymphocytes were determined by direct immunofluorescence and T lymphocytes with the E rosette technique. Helper/inducer, cytotoxic/suppressor, and NK/K T lymphocytes were determined by indirect immunofluorescence with the monoclonal antibodies OKT4, OKT8 and Leu 7 (HNK1). In the same way, Lyt3 was used for determination of the total T lymphocytes. Whereas in peripheral blood of the NHL group an increase of B lymphocytes and a slight reduction of T lymphocytes could be observed, with normal distribution of the subpopulations, in patients with active HD as well as in those in remission, a marked absolute and relative decrease of T helper/inducer cells was found with normal cytotoxic/suppressor and NK/K proportion. In contrast to this, a significant increase of helper/inducer T lymphocytes with decreased cytotoxic/suppressor T proportion was found in spleen cell suspensions of patients with HD.     

Antibody response of BALB/c mice to dextran B1355S: alterations in the expression of an idiotype associated with the depletion of idiotype-binding cells

In this study, we established that BALB/c mice recognize and respond to the idiotype (M104E IdI) of a major dextran-specific clonotype within the BALB/c mouse repertoire. This idiotype recognition is established by demonstrating the presence of idiotype-binding cells and by the production of antibodies specific for the private M104E idiotype. To determine whether or not the idiotype-recognizing cells play a regulatory role during an immune response to dextran, the idiotype-binding cells were selectively removed either by panning or by radiation-induced killing. Two significant effects are observed when the depleted spleen cells are immunized with dextran. First, there is a substantial increase in the proportion of anti-dextran antibodies that are M104E IdI+. The second effect of the idiotype-specific cell depletion is the production of significant amounts of M104E IdI+ immunoglobulin molecules which do not bind dextran. The depletion experiments produced no alteration in the concentration of anti-dextran antibodies found in the serum or in the number of dextran-specific PFC in the spleen. The data indicate that idiotype-reactive cells can play a role in regulating the level of individual clonotype expression (i.e., the M104E clonotype), but that an alternative mechanism must exist for regulating the absolute amount of anti-dextran antibody produced after immunization.     

IgE formation in the rat following infection with Nippostrongylus brasiliensis: I. Proliferation and differentiation of IgE-bearing cells

Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis-infected animals.     

A quantitative study on recovery from acute haemorrhage in adult laboratory rats. II. Bone marrow, spleen and liver morphology

We investigate bone marrow, spleen and liver morphology in adult laboratory rats following acute haemorrhage to establish their validity in preclinical studies, especially in safety drug evaluations. The most marked changes were found in the proportion of spleen erythroid nucleated cells. Higher E: M ratios and relative erythroid cell counts were also marked in the bone marrow. Their increase was not so intensive and disappeared earlier than the increase of spleen erythropoiesis. Variations in counts of marrow neutrophils and their precursors were not specific.     

In vitro induction of IgM synthesis and secretion in rabbit spleen cells by soluble Concanavalin A

Rabbit spleen cells are not activated by Concanavalin A (Con A) conjugated to Sepharose 4B but are stimulated by soluble Con A which induces DNA and protein synthesis. At optimal concentration (5 μg/ml) one notes an increased intracellular protein and IgM synthesis and then secretion. This increase in protein synthesis is seen at all phases of the culture. At the intracellular level, IgM is found in the form of 7S molecules and a significant proportion of polymers with a centrifugation constant smaller than 19S. Fully assembled 19S polymers are found in the fluid phase. These results are compatible with a model of cellular cooperation, the basis of which is not the presentation of the inducer (mitogen or antigen) by a cell type to another, but rather the secretion of mediators by one of the cell populations making other cells responsive to the inducer.     

The effect of carbamylcholine on CFU-s differentiation

The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types. Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively. Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays. The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively. The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.     

Prostaglandin E2 production is enhanced in mice genetically selected to produce high affinity antibody responses

Spleen cells from mice selectively bred to produce high affinity antibody responses to protein antigens (HI) had reduced responses to both T and B cell mitogens when compared to those from mice selectively bred to produce low affinity (LO) responses. The reduced response by spleen cells from HI mice was partially reversed by the addition of indomethacin in vitro. Spleen adherent cells from HI mice had increased production of prostaglandin E2 when compared to those from LO mice. In addition, spleen adherent cells from mice which fail to show affinity maturation not only produced lower amounts of PGE2 than those from HI mice but also a decreased proportion of spleen cells adhered to plastic in these mice. To test the possibility that the increased PGE2 production in HI mice was responsible for the production of high affinity antibodies, indomethacin was administered in vivo and resulted in a significant reduction in antibody affinity. The possibility that PGE2 production may control the balance between the TH1 and TH2 cells of Mosmann and Coffman is discussed.     

Suppressor cell activity in mice bearing a progressively growing Simian virus 40-induced sarcoma

Summary General and cell-mediated immunity (CMI) were investigated in BALB/c mice bearing progressively growing Simian virus 40-induced (mKSA) sarcoma by means of the Winn tumor cell neutralization (WN), ¹²⁵I isotopic footpad (IFP), lymphoproliferative (LP) and plaque-forming cell (PFC) assays. Correlation between depressed antitumor immunity and the IFP responses was observed in tumor-bearing (TB) mice. Depressed LP responses to both T- and B-cell mitogens were observed in both early and late stages of tumor growth. Results obtained with the PFC assay similarly demonstrated depressed humoral immunity to sheep red blood cells (SRBC). Suppressor cell activity was demonstrated in cocultivation experiments in which spleen cells of TB mice were mixed with normal spleen cells. Treatment of TB spleen cells by passage through Sephadex G-10 columns or incubation on plastic surfaces to deplete the adherent cells restored LP responses. Cocultivation of Sephadex G-10- or plastic-adherent cells from TB mice with normal spleen cells significantly reduced mitogen-induced LP responses of normal cells. Examination of cell surface markers indicated an increase in the proportion of spleen cells bearing Fc receptors, which correlated with progressive mKSA tumor growth. There was also a correlation between Fc receptor-bearing spleen cells and macrophages, as shown by nonspecific esterase staining. These results indicate that depressed LP and PFC responses and the appearance of suppressor cells in mKSA tumor-bearing mice parallel an impaired ability to recognize (IFP responses) and neutralize (WN responses) tumor cells.     

Hyperglycemia does not alter state 3 respiration in cardiac mitochondria from type-I diabetic rats

The possible role of nitric oxide on the exercise-induced changes in bleomycin-detectable iron (BDI) in the liver, spleen, bone marrow cells and heart was investigated. Female Sprague-Dawley rats were randomly assigned to four groups: S1 (Sedentary), S2 (Sedentary + L-NAME [N-nitro-L-arginine methyl ester]), E1 (Exercise) and E2 (Exercise + L-NAME). Animals in the E1 and E2 swam for 2 h/day for 3 months. L-NAME in the drinking water (1 mg/ml) was administrated to rats in the S2 and E2 groups for the same period. At the end of the 3rd month, nitrite and nitrate (NOx), BDI and non-heme iron (NHI) contents in the liver, spleen, bone marrow cells and heart were measured. The ratio of BDI/NHI was calculated. The exercise induced a significant increase in NOx and BDI contents and/or BDI/NHI ratio in the spleen, bone morrow cells and heart. Treatment with L-NAME, an inhibitor of NOS, led to a significant decrease in NOx and an increase in BDI levels and BDI/NHI ratios in these tissues. The correlative analysis showed that there is significantly positive correlation between NOx levels and BDI contents and/or BDI/NHI ratios in the spleen, bone marrow cells and heart. These results suggest that the increased nitric oxide might be one of the reasons leading to the increased BDI levels in these tissues in the exercised rats. In contrast to the above tissues, in the liver, exercise led to a significant decrease rather than increase in BDI levels and BDI/NHI ratios with a significant increase in NOx contents. Treatment with L-NAME led to a significant increase in BDI levels and BDI/NHI ratios and a decrease in NOx contents in the tissue. These findings plus the results reported by others imply that nitric oxide might have an inhibitory effect on BDI in the liver.