The immunomodulatory effects of 3-monochloro-1,2-propanediol on murine splenocyte and peritoneal macrophage function in vitro.

3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. MCPD has been reported genotoxic in vitro, and reproductive toxicity and carcinogenicity in rats. To evaluate the immunomodulatory effect of MCPD on murine splenocyte and macrophage in vitro, we investigated splenocyte blastogenesis by concanavalin A (Con A), anti-CD3, and lipopolyssacharide (LPS), the production of cytokines from splenocyte, and the activity of mouse peritoneal macrophages. There was a significant decrease in lymphocyte blastogenesis to Con A or anti-CD3 at subtoxic dose of MCPD. A significant decrease in splenocyte blastogenesis to LPS was also observed. The production level of interferon (IFN)-gamma on splenocyte culture with Con A was significantly reduced at the higher concentration than 1.0mM of MCPD. The levels of interleukin (IL)-4 and IL-10 were also decreased at high concentrations of MCPD. There was a significant decrease in production of nitric oxide (NO) by peritoneal macrophages treated with MCPD. MCPD also inhibits tumor necrosis factor (TNF)-alpha production of stimulated macrophages. These results indicate that MCPD might be able to reduce the functionality of lymphocytes and peritoneal macrophages in vitro.     

In vitro suppressive effect of aflatoxin B1 on murine peritoneal macrophage functions.

We examined the immunosuppressive effects of aflatoxin B1 (AFB1), a toxic compound produced by the Aspergillus flavus, on murine peritoneal macrophages after in vitro pre-exposure. When thioglycollate-elicited macrophages pre-exposed to AFB1 were stimulated with lipopolysaccharide (LPS), antitumor activity induced by LPS was suppressed by 10 and 50 microM AFB1. In addition, the production of reactive intermediates including nitric oxide (NO), superoxide anion and hydrogen peroxide which have been known to be implicated in macrophage-mediated cytotoxicity, was decreased by AFB1 pretreatment in a dose-dependent manner. We also determined whether the macrophage-mediated cytokine production was altered by AFB1 in vitro pretreatment. AFB1 markedly inhibited TNF-alpha interleukin-1 (IL-1) and IL-6 production by LPS-stimulated macrophages. Taken together, these data indicate that AFB1 inhibits the killing ability of murine macrophages, decreases various secretory molecules in those cells and the macrophages would be one of many systems affected by AFB1.     

The effects of simazine,a chlorotriazine herbicide,on pubertal development in the female Wistar rat

Chlorotriazine herbicides, such as atrazine and its metabolites, have been shown to target the neuroendocrine regulation of male and female reproductive development. However, no studies have evaluated the effects of the chlorotriazine simazine on pubertal development in the female rat. Here we report the effects of a 21- and 41-day exposure to simazine on pubertal development and estrous cyclicity in the female rat using the U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program, Pubertal Development and Thyroid Function in Intact/Juvenile Peripubertal Female Rats (Tier 1) protocol. In the first study, Wistar rats were exposed orally to 0, 12.5, 25, 50, or 100 mg/kg of simazine from postnatal day 22 to 42. In the second study, rats were exposed from PND 22 until the first day of estrus after PND 62 to 0, 12.5, 25, 50, 100 or 200 mg/kg of simazine. In the 21-day exposure, vaginal opening (VO) was delayed, the number of normal cycles was significantly decreased, and the day of first estrus was delayed compared to controls. In the 41-day exposure, VO and the day of first estrus was delayed, but the number of normal estrous cycles was not different than controls. In addition, both studies showed a significant decrease in serum prolactin (PRL) following simazine exposure. This data clearly demonstrates that simazine delays the onset of puberty in the female rat and decreases serum PRL similar to other chlorotriazines. The extended dosing period after VO provides a sufficient time period to monitor the effects of a toxicant on estrous cyclicity, an important measure for reproductive competence.     

The effects of saikosaponin on macrophage functions and lymphocyte proliferation.

The effects of saikosaponin-d (ssd), isolated from Bupleurum radix, on phagocytic functions of mouse peritoneal macrophages were investigated after treatment in vitro. The macrophages treated with ssd showed a significant increase in PMA-induced chemiluminescence. An increase in phagocytosis was detected after treatment with saikosaponin-b2 (0.1 microM) for 24 h in vitro, while a suppression of phagocytosis was observed following treatment with saikosaponins (0.5 microM). Treatment with ssd markedly increased the random migration of resident peritoneal macrophages, but did not affect the migration towards FMLP. We further investigated the effect of ssd on proliferative responses of spleen cells and found that ssd, which itself has no mitogenic activity, decreased spleen cell proliferative response to T-cell mitogen, but increased the response to B-cell mitogen.     

Differential effects of synthetic indoloquinolizines on in vitro rat lymphocyte and macrophage functions

Indoloquinolizines are natural alkaloid indole products grouped as beta-carbolines. These compounds are commonly associated with neurological activities, but little is known about their role as immunomodulating agents. The present study was undertaken to evaluate the effects of synthetic indoloquinolizines on in vitro parameters of rat lymphocyte and macrophage functions. It was observed that proliferation of thymic lymphocytes was significantly (p<0.05) increased (20-30% increase) by dihydro-indoloquinolizinium chloride (2). dihydro-indoloquinolizinyl-ethanone (3). and dimeric dihydro-indoloquinolizinium dichloride (6). whereas dimeric indoloquinolizine (7). caused up to 40% increase in lymphoproliferation at concentrations ranging from 10(-11) to 10(-5) M, compared with untreated control. In contrast, indoloquinolizinium chloride (4) and indoloquinolizine (5). were toxic for lymphocytes at concentrations from 10(-9) to 10(-5) M, and compounds 6 and 7 were toxic at 10(-5) M. In addition, nitric oxide production by LPS-treated peritoneal macrophages was significantly (p<0.05) increased (up to 30% increase) by compounds 4 and 5 at concentrations of 10(-11) to 10(-5) M, and 10(-5) M, respectively; however, compounds 6 and 7 were toxic for macrophages at all concentrations tested. Furthermore, TNF-alpha production was also significantly increased (p<0.01) by compounds 4 and 5 (up to 30-fold increase) compared with untreated control. These novel synthetic indoloquinolizines could serve as immunotherapeutic agents by selectively increasing the pool of activated T lymphocytes or stimulating macrophage functions, with potential use in the treatment of infectious diseases including AIDS and cancer.     

Immune alterations in mice exposed to the herbicide simazine

Simazine, a triazine herbicide, was investigated for its in vivo immunomodulatory properties. Male C57Bl/6 mice were treated with vehicle or 300 or 600 mg/kg body weight (bw) simazine daily orally for 4 wk. The immune system was evaluated by the antibody response to sheep red blood cells (SRBC; plaque assay and serum immunoglobulin G), natural killer (NK) and macro-phage activities, lymphocyte subpopulations in the spleen and thymus, and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated lymphocyte proliferation using splenocytes. Body weight and spleen and thymus weight decreased generally in simazine-treated mice, while the weight of adrenal glands was higher than in the control. Simazine treatment (600 mg/kg) induced an increase in the percentage of CD4(+) cells in spleen and CD8 + in thymus. Simazine inhibited the IgM plaque-forming cell numbers and lowered the level of IgG and the proliferation of mitogen-stimulated B cells and T cells. In addition, splenic NK and peritoneal macrophage activities in exposed mice were significantly decreased. Exposure to simazine also decreased cytokine production by macrophages, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Taken together, data indicate that the immune system was suppressed by oral simazine exposure.     

Evaluation of immunomodulatory chemicals: alteration of macrophage function in vitro

In an effort to develop a useful screening procedure for detecting potential modulators of macrophage function, 25 chemicals were tested for their ability to alter the nonspecific phagocytosis and killing of Saccharomyces cerevisiae by elicited mouse peritoneal macrophages (PM). Parallel studies monitored PM viability so as to distinguish between effective modulation of phagocytic function and changes due solely to cytotoxicity. Dextran sulfate, iota-carrageenan, lambda-carrageenan, dipropyltin dichloride, and di-n-octyltin dichloride, at concentrations which did not produce overt toxicity, significantly reduced the percentage of PM capable of ingesting yeast. The carrageenans also decreased the average number of yeast ingested per PM, while increasing the ability of PM to kill yeast. Microbicidal activity was suppressed in PM treated with indomethacin and dextran sulfate. Di-n-octyltin dichloride and dextran sulfate diminished the adherence of PM, whereas levamisole enhanced PM adherence. Vanillin, propyl gallate, lead acetate, hydrocortisone 21-phosphate, and hydrocortisone 21-hemisuccinate decreased the percentage of PM capable of ingesting yeast, but not below 50% of the control. Gallic acid, methyl paraben, mercuric chloride, cadmium chloride, nickel chloride, chromic chloride, dimethyltin dichloride, diethyltin dichloride, dibutyltin dichloride, tetra-n-octyltin, gibberellic acid, cyclophosphamide, and azathioprine did not alter PM phagocytic function at noncytotoxic doses. The results indicate that chemicals can be grouped into three broad categories as either ineffective, weak modulators, or effective modulators of PM function.     

Novel immunomodulatory effects of adiponectin on dendritic cell functions

Adiponectin (ADN) is an adipocytokine with anti-inflammatory properties. Although it has been reported that ADN can inhibit the immunostimulatory function of monocytes and macrophages, little is known of its effect on dendritic cells (DC). Recent data suggest that ADN can regulate immune responses. DCs are uniquely specialised antigen presenting cells that play a central role in the initiation of immunity and tolerance. In this study, we have investigated the immuno- modulatory effects of ADN on DC functions. We found that ADN has only moderate effect on the differentiation of murine bone marrow (BM) derived DCs but altered the phenotype of DCs. The expression of major histocompatibilty complex class II (MHCII), CD80 and CD86 on ADN conditioned DCs (ADN-DCs) was lower than that on untreated cells. The production of IL-12p40 was also suppressed in ADN-DCs. Interestingly, ADN treated DCs showed an increase in the expression of the inhibitory molecule, programmed death-1 ligand (PDL-1) compared to untreated cells. In vitro co-culture of ADN-DCs with allogeneic T cells led to a decrease in T cell proliferation and reduction of IL-2 production. Concomitant with that, a higher percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was detected in co-cultures of T cells and ADN-DCs. Blocking PD-1/PDL-1 pathway could partially restore T cell function. These findings suggest that the immunomodulatory effect of ADN on immune responses could be at least partially be mediated by its ability to alter DC function. The PD-1/PDL-1 pathway and the enhancement of Treg expansion are implicated in the immunomodulatory mechanisms.     

Unique immunomodulatory effects of azelastine on dendritic cells in vitro

Allergic contact dermatitis and atopic dermatitis are among the most common inflammatory skin diseases in western countries, and antigen-presenting cells like dendritic cells (DC) are key players in their pathophysiology. Histamine, an important mediator of allergic reactions, influences DC maturation and cytokine secretion, which led us to investigate the immunomodulatory potential of the well-known histamine H1 receptor antagonists: azelastine, olopatadine, cetirizine, and pyrilamine. Unlike other H1 antihistamines, azelastine decreased lipopolysaccharide-induced tumor necrosis factor α and interleukin-12 secretion from murine bone marrow-derived DC. This effect was independent of histamine receptors H1, H2, or H4 and may be linked to inhibition of the nuclear factor kappa B pathway. Moreover, only azelastine reduced proliferation of allogenic T cells in a mixed leukocyte reaction. We then tested topical application of the H1 antihistamines on mice sensitized against toluene-2,4-diisocyanate, a model of Th2-mediated allergic contact dermatitis. In contrast to the in vitro results, all investigated substances were efficacious in reducing allergic ear swelling. Azelastine has unique effects on dendritic cells and T cell interaction in vitro. However, this did not translate into superior in vivo efficacy for Th2-mediated allergic dermatitis, possibly due to the effects of the antihistamines on other cell types involved in skin inflammation. Future research will have to clarify whether these properties are relevant to in vivo models of allergic inflammation with a different T cell polarization.     

Improvement of murine immune functions in vitro by thioproline

Previous studies have shown that several immune functions were improved in mice after the ingestion of a thioproline (thiazolidine-4-carboxylic acid) enriched diet. In the present work, we have studied the in vitro effects of several concentrations of this thiol compound (0.1, 0.5, 1, 2.5 and 5 mM) on the most relevant functions of three pivotal immune cells, namely, macrophages, lymphocytes, and natural killer (NK) cells from BALB/c mice. The results show that thioproline stimulates the phagocytic process of macrophages, increasing the mobility directed to the inflammatory focus (chemotaxis) and the phagocytosis of inert particles. It increases the adherence and the chemotaxis capacities of lymphocytes, their proliferative activity and favours the natural cytotoxic activity that could improve the capacity to destroy malignant cells. Thioproline concentrations of 0.5 and 1 mM were the most effective regarding the different functions analysed. These results suggest that the improvement of immune functions, observed in previous work, after thioproline-enriched diet ingestion is due to a direct action of this thiol compound on immune cells.     

In vitro and in vivo immunomodulatory effects of microdispersed oxidized cellulose

The immune system can be manipulated specifically by vaccination or nonspecifically by immunomodulation. Many of biological response modifiers (BRM) have polysaccharidic structure similar to that of microdispersed oxidized cellulose (MDOC). We have investigated the immunomodulatory activity of different inorganic MDOC salts (H, Na, Ca, Mg, Zn, Al, Co, Ca/Na) and organic MDOC derivatives (urea, gelatine, arginine) both in vitro and in vivo. A dose-dependent stimulation by a number of MDOC derivatives was observed with spontaneous and mitogen-induced proliferation of human peripheral blood leukocytes (PBLs) and mouse splenocytes in vitro. In both primary cultures, the most intensive proliferation was induced by a Ca/Na salt at a concentration of 1 mg/ml. We have also demonstrated stimulatory effects of MDOC Ca/Na salt on the mouse mixed leukocyte reaction (MLR). The stimulatory activity of MDOC towards the immune system was further supported by the fact that in vitro the product stimulates the release of Th1 cytokine TNF-alpha, but not IFN-gamma, IL-4 or IL-6. In vivo MDOC application increases more than 50% the number of colony-forming units spleen (CFU-s), i.e., stimulates the stem cells in bone marrow, and increases relative percentage of monocytes and B lymphocytes in the mouse peripheral blood.     

康复新液对小鼠的免疫调节作用

目的 研究康复新液的免疫调节作用.方法 观察康复新液对正常小鼠、环磷酰胺致免疫低下小鼠和老龄小鼠的免疫器官重量、单核巨噬细胞碳粒清除能力、2,4-二硝基氯苯致迟发型超敏反应和血清溶血素生成的影响,以及对老龄鼠脾脏组织形态学和脑组织氧化损伤的影响.结果 康复新液对正常小鼠的各项免疫功能指标没有影响;但可显著提高免疫低下的...     

Damaging effects of advanced glycation end-products in the murine macrophage cell line J774A.1.

The interaction of reducing sugars, such as aldose, with proteins and the subsequent molecular rearrangements, produces irreversible advanced glycation end-products (AGEs), a heterogeneous class of non-enzymatic glycated proteins or lipids. AGEs form cross-links, trap macromolecules and release reactive oxygen intermediates. AGEs are linked to aging, and increase in several related diseases. The aim of this study was to assess, in a murine macrophage cell line, J774A.1, the effects of 48 h of exposure to glycated serum containing a known amount of pentosidine, a well-known AGE found in the plasma and tissues of diabetic and uremic subjects. Fetal bovine serum was incubated with ribose (50 mM) for 7 days at 37 degrees C to obtain about 10 nmol/ml of pentosidine. The cytotoxic parameters studied were cell morphology and viability by neutral red uptake, lactate dehydrogenase release and tetrazolium salt test. In the medium and in the intracellular compartment, bound and free pentosidine were evaluated by HPLC, as sensitive and specific glycative markers, and thiobarbituric acid reactive substances (TBARs), as index of the extent of lipid peroxidation. Our results confirm that macrophages are able to take up pentosidine. It is conceivable that bound pentosidine is degraded and free pentosidine is released inside the cell and then into the medium. The AGE increase in the medium was combined with an increase in TBARs, meaning that an oxidative stress occurred; marked cytotoxic effects were observed, and were followed by the release of free pentosidine and TBARs into the culture medium.     

Immunotoxic effect of beta-chlorolactic acid on murine splenocyte and peritoneal macrophage function in vitro

β-Chlorolactic acid is a major intermediate of 3-monochloro-1,2-propanediol (MCPD) in mammalian species, which a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. β-Chlorolactic acid has not been studied on immunotoxicity. To evaluate the immunomodulatory effect of β-chlorolactic acid on murine splenocyte and macrophage in vitro, we investigated splenocyte blastogenesis by concanavalin A (Con A), anti-CD3 and lipopolysaccharide (LPS), the production of cytokines from splenocyte, and the activity of mouse peritoneal macrophages. β-Chlorolactic acid suppressed significantly splenic blastogenesis to Con A or anti-CD3 from 8.5 to 54.7% at doses comprised between 200 and 800 μM. β-Chlorolactic acid also suppressed significantly splenic blastogeneis to LPS from 8.5 to 71.5% at doses comprised between 200 and 800 μM. The production level of interferon (IFN)-g on splenocyte culture with Con A was significantly reduced from 21.5 to 51.4% at the higher concentration than 100 μM of β-chlorolactic acid. The levels of interleukin (IL)-2 and IL-4 were also decreased 22.6–58.4 and 10.2–36.6%, respectively, at high concentrations of β-chlorolactic acid. There was a significant decrease from 6.1 to 40.8% in the production of nitric oxide (NO) by peritoneal macrophages treated with 400–1000 μM β-chlorolactic acid. These results indicate that β-chlorolactic acid might be able to induce immunotoxic effect on immune response of lymphocytes and peritoneal macrophages in vitro.     

Bisphenol A-induced downregulation of murine macrophage activities in vitro and ex vivo

Bisphenol A (BPA) is known to have detrimental effects on the reproductive system, but the toxicity of BPA on immune responses has not been systematically investigated. We investigated the effects of BPA exposure on the activities of murine peritoneal macrophages through evaluation of BPA-induced alteration of nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) synthesis, and expression of co-stimulatory molecules B7. Macrophages were examined ex vivo from mice orally treated with various doses of BPA for 5 consecutive days per week for 4 weeks followed by culture for 2 or 4 days in the presence of lipopolysaccharides (LPS). Macrophages from naive mice were also stimulated with LPS ± BPA for 2 or 4 days. NO production was decreased with the in vitro exposure to 1, 10 and 100μM BPA. NO production was lower in the BPA-exposed mice than the control mice with all doses. In vitro, BPA suppressed TNF-α secretion with significant reduction at 10 and 100μM BPA. Similar findings were observed with the macrophages from the BPA-exposed mice. This study provides the substantial evidence on BPA-induced alteration in macrophage activity.     

Immunomodulating effects of ozone on macrophage functions important for tumor surveillance and host defense.

Ozone (O3) is a toxic gaseous pollutant that has been implicated in laboratory studies as a potential lung carcinogen or cocarcinogen in mice. To begin to assess the role of altered macrophage (M phi) responses as a possible mechanism by which O3 may influence carcinogenesis, we examined the effects of repeated in vivo O3 exposure on pulmonary M phi functional and biochemical activities deemed important in tumor surveillance, and host defense in general. Rabbits were exposed by inhalation to 1 ppm O3 for 3 d (2 h/d) and the lungs were lavaged immediately (t0) and 24 h (t24) after exposure. Results demonstrate that O3 reduced M phi viability and increased the number of neutrophils collected immediately after exposure. Effects of O3 on M phi movement were as follows: random migration was depressed immediately after the final exposure and chemotactic migration increased after 24 h. M phi-mediated cytotoxicity toward xenogeneic tumor cells in vitro was significantly depressed, compared to control, immediately and 24 h after O3 exposure. Release of cytotoxic factors deemed important for mediating tumor cell destruction was also assessed. Spontaneous and stimulated production of tumor necrosis factor, as measured by cytotoxicity toward LM cells (a clone of L-929 mouse fibroblasts), was unaffected by exposure to O3. Zymosan-stimulated production of superoxide anion radical (.O2-) was depressed at t0 and increased at t24; however, no significant effects on H2O2 production by resting or zymosan-stimulated M phi were observed at either time interval. Inhaled toxicants such as O3, which can compromise M phi functions important in tumor surveillance, could potentially alter host susceptibility to pulmonary cancer. Results of this study have important implications for human health, and demonstrate the need for further studies examining the carcinogenic/cocarcinogenic potential of O3.     

Effects of nickel hydroxycarbonate on alveolar macrophage functions.

The metabolic effects of different concentrations of nickel hydroxycarbonate (NiHC) on guinea pig alveolar macrophages (GPAMs) were investigated. Exposure to high concentrations of NiHC (6.25 and 12.5 micrograms 10(-6) cells) led to cell vacuolization. Morphological changes were associated with a dramatic reduction in the steady-state level of cellular adenosine triphosphate (ATP), i.e. ATP levels were reduced by 35% (P less than 0.001) and 53% (P less than 0.01), respectively. Low concentrations of NiHC (0.0625 and 0.125 microgram 10(-6) cells) did not induce morphological changes but increased cellular ATP content by 19% (P less than 0.01) and 12% (P less than 0.05), respectively. Effects of NiHC (0.125 and 6.25 micrograms 10(-6) cells) on cell oxidative metabolism were studied. The chemiluminescence was significantly increased (P less than 0.001) by the lower but not the higher concentration. A slight inhibition of total superoxide dismutase (P less than 0.05) and a decrease of catalase activity were demonstrated (P less than 0.05) for the high dose, while the low dose decreased the levels of reduced and total glutathione. In conclusion, the effects of NiHC on alveolar macrophages are characterized by an overproduction of free radicals for low concentrations and the depletion of cellular reserve energy, particularly ATP, for high concentrations.     

Comparative effects of five bisphosphonates on apoptosis of macrophage cells in vitro

Bisphosphonates (BPs) inhibits bone resorption by reducing osteoclastic activity; they induce osteoclast apoptosis. Pathophysiology of prostheses loosening is complex and implies an inflammatory reaction secondary to the phagocytosis of wear debris by macrophages with a secondary increased bone resorption by osteoclasts. BPs inhibit proliferation and cause cell death in macrophages by induction of apoptosis. We have used mouse macrophage-like J774.1 cells to evaluate the effects of five BPs. J774A.1 cells were cultured in a standard culture medium for 2-days. BPs (alendronate, pamidronate, etidronate, risedronate, zoledronic acid) were added in the medium at concentration of 10(-6) to 10(-4)M during 3 days. Cells were studied by fluorescence microscopy after staining with the fluorescent dye Hoescht H33342 and the percentage of apoptotic cells was determined on 300 nuclei. Cells were analyzed by flow cytofluorometry after staining with annexin V-FITC (for counting apoptotic cells) and propidium iodide (for necrotic/late-apoptotic cells) on 2000 cells. Etidronate did not cause significant apoptosis or necrosis, at any concentration. Alendronate and pamidronate caused apoptosis and death only at very high concentration [10(-4)M]. On the contrary, apoptotic and necrotic cells were evidenced with risedronate or zoledronic acid at lower concentrations. These effects were dose-dependant and occurred when concentration reached [10(-5)M]. The number of apoptotic cells was higher with zoledronic acid and then with risedronate. Cytofluorometry appeared superior to cytologic analysis in the investigation of macrophage apoptosis, since necrotic cells loose contact with the glass slides and are not identifiable in cytological counts. Some amino-BPs appear to induce apoptosis in macrophages.     

Bipyridylium herbicide toxicity in vitro: comparative study of the cytotoxicity of paraquat and diquat toward the pulmonary alveolar macrophage

In vitro exposure of adult rat alveolar macrophages to either paraquat or diquat resulted in concentration dependent cytotoxicity (cell death). The herbicide paraquat, however was statistically significantly more potent toward these cells than was diquat. The LC50 value for paraquat (8-h exposure, 37 degrees C) was determined to be 0.94 mM [95% confidence interval (C.I.) 0.79-1.12 mM], whereas the corresponding LC50 value for diquat was 1.97 mM (C.I. 1.58-2.51 mM). Interestingly, diquat was shown to enter these cells to a much greater extent than was paraquat. The latter data, while seemingly contradictory to the above findings, is consistent with other reported findings in this study that show that cell respiration, as measured by loss of oxygen consumption, was more sensitive to diquat than it was to paraquat. Also, only paraquat cytotoxicity was found to be dependent on oxygen tension and could be altered by the presence of antioxidant enzymes in the culture medium. Both compounds, however, were found to be equipotent toward purified mitochondria. Both paraquat and diquat were able to uncouple oxidative phosphorylation and induce active oxygen species (superoxide anions and hydrogen peroxide) from this organelle. It is concluded that free-radical pathology is the most likely mechanism of action by which paraquat is cytotoxic toward these cells, but that diquat poisoning probably originates from some other mode of action.