Fumonisin B1 affects viability and alters nitric oxide production of a murine macrophage cell line

International Immunopharmacology 2 (2002) 1087 – 1093 www.elsevier.com/locate/intimp

Fumonisin B1 affects viability and alters nitric oxide production of a murine macrophage cell line Charissa Dresden-Osborne, Gayle Pittman Noblet * Department of Biological Sciences, Clemson University, Clemson, SC 29634-0326, USA Received 1 February 2001; received in revised form 3 May 2002; accepted 15 May 2002

Abstract Fumonisin B1 (FB1), the major toxin produced by Fusarium verticillioides contaminating corn, is known to elicit many organ- and species-specific toxicities in animals. In the present study, exposure to FB1 decreased viability of a murine macrophage cell line (RAW264.7) in a dose-dependent manner (1 – 100 AM). Further, when cells exposed to FB1 were stimulated with lipopolysaccharide (LPS), a dose-dependent increase in production of nitric oxide (NO) was observed, but only at FB1 concentrations (10 – 50 AM) that induced significant cytotoxicity. Stimulation of cells with phorbol myristate acetate (PMA) resulted in increased NO production at 50 AM FB1, but induced a variable NO response at 1 – 10 AM FB1. Results suggest that FB1 affected cell viability and altered inducible NO production by RAW macrophages in a manner that was dependent on the pathway of stimulation. D 2002 Published by Elsevier Science B.V. Keywords: Fumonisin B1; Lipopolysaccharide; Macrophage; Murine cell line; Nitric oxide; Phorbol myristate acetate

1. Introduction Fumonisins, mycotoxins produced by Fusarium verticillioides ( = F. moniliforme) [1], are etiologic agents of a variety of animal diseases, including leukoencephalomalcia in horses [2], pulmonary edema in swine [3] and liver cancer in laboratory rodents [1]. In addition, consumption of corn contaminated with fumonisin B1 (FB1) has been implicated as a cause of human esophageal cancer in South Africa and China [4,5]. Due to implications in animal and human health, toxic effects of fumonisins have been widely studied, with hepatotoxic and nephro*

Corresponding author. Tel.: +1-864-6563589; fax: +1-8656560435. E-mail address: [email protected] (G.P. Noblet).

toxic effects in rodents being well documented [6]. Effects of fumonisins on immune system components, however, have received little attention to date. Previous reports indicate that FB1 may affect immune functions in rodents, such as decreased responses to sheep red blood cell injection [7], increased serum IgG, increased numbers of Listeria monocytogenes in spleen [8], and increased secretion of TNF-a by peritoneal macrophages [9]. Disruption of sphingolipid biosynthesis appears to be one mechanism involved in FB1 toxicity, with inhibition of ceramide synthase [10] leading to accumulation of sphingoid bases (sphinganine and sphingosine). In addition, sphingolipids, their intermediates and breakdown products are second messengers that regulate signaling pathways, including cell cycle, apoptosis and immune functions [11]. Therefore,

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FB1 could potentially affect a variety of cell functions. In vitro studies indicate contrasting results for the effect of FB1 on various mammalian cell lines, to include mitogenesis of Swiss 3T3 fibroblasts [12] and anti-proliferation of human keratinocytes, fibroblasts and hepatoma cells [13]. Macrophages are important components of the mammalian immune system as professional antigen presenting cells and non-specific killers of a wide variety of pathogens. Production and release of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) by macrophages contributes to antimicrobial activity, with nitric oxide (NO) as a major component [14]. Generation of NO by macrophages is catalyzed by inducible nitric oxide synthase (iNOS), as L-arginine is converted to L-citrulline through an NADPH-dependent process [15]. For murine macrophages, results of in vitro studies indicate transcriptional induction of iNOS occurs following stimulation with IFN-g and bacterial lipopolysaccharide (LPS) [16]. More specifically, stimulation of macrophages by LPS was proposed to be mediated by mimicking the second messenger function of ceramide, and ceramide alone may activate LPS-induced signaling pathways in macrophages [17]. Based on the effects of FB1 on sphingolipid synthesis and the possible role of ceramide in inducible macrophage functions, the current study was designed to investigate the role of FB1 on the murine macrophage cell line, RAW264.7.

ing, Corning, NY) in complete RPMI-1640 medium (Cellgro, Herndon, VA) supplemented with 20 mM HEPES, 1 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS) (Bio Whittaker, Walkersville, MD), 20 U/ml penicillin and 20 Ag/ml streptomycin. Cells incubated in a humidified chamber at 37 jC and 5% CO2 were removed from plates by gentle scraping and were subcultured every 3– 4 days. For experiments, cells were adjusted to desired numbers, seeded in 96-well plates, and allowed to adhere for at least 2 h before initiation of experiments. 2.3. MTT assay for viability Viability of RAW cells was assessed 48 or 72 h after exposure to FB1 (and other reagents used as NO stimulants) by an MTT (3-(4,5-dimethylthazol-2-yl)2,5-diphenyl tetrazolium bromide) assay. For the assay, 20 Al of MTT (5 mg/ml) was added to each well seeded with cells, followed by incubation at 37 jC and 5% CO2 for an additional 4 h. Medium and MTT solution were removed and cells solubilized by addition of 100 Al dimethyl sulfoxide (DMSO). Plates were placed on a plate shaker for 5 min, and optical density was read on a Thermomax plate reader at a dual wavelength of 570 nm and 640 nm. Mean optical density (amount of MTT formazan product), as determined for samples from each dose group (n = 6) and converted to cell numbers by comparison with a standard curve generated by wells containing known numbers of cells, directly correlated with cell viability [18].

2. Materials and methods 2.1. Materials Purified fumonisin B1 (98% pure) was purchased from Sigma (St. Louis, MO). A stock solution of FB1 (1 mg/ml) was prepared in incomplete RPMI-1640 medium and stored at 4 jC in the dark. Unless otherwise noted, all chemicals used in this study were purchased from Sigma. 2.2. Cell culture The RAW264.7 murine macrophage cell line (a gift from Dr. Charles Rice, CIET, Clemson University) was maintained in 25 cm2 tissue culture flasks (Corn-

2.4. Nitric oxide production by RAW cells exposed to FB1 In 96-well tissue culture plates, 2  104 RAW cells per well were seeded in 100 Al of complete RPMI1640 medium. Cells were allowed to adhere overnight at 37 jC and 5% CO2 in a humidified chamber. Following removal of medium and washing of adherent cells, 100 Al of fresh complete RPMI-1640 medium containing FB1 was added to each well along with either 1 Ag/ml lipopolysaccharide (LPS), 200 ng/ ml phorbol myristate acetate (PMA) or 400 ng/ml PMA to stimulate production of NO. Selected groups of cells also were incubated with L-NAME (N-nitro-Larginine methyl ester, 5 mM), a known inhibitor of

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NOS. Supernatant from each well was collected after 48 or 72 h of incubation and assayed for NO. Additional cell cultures exposed only to FB1 were washed after 48 h, and fresh medium containing 1 Ag/ml LPS was added; cells were incubated for an additional 48 h before collection of supernatant for the NO assay.

Table 2 An MTT assay for viability of RAW macrophages incubated for 48 h with FB1 and LPS (1 Ag/ml) with or without the NOS inhibitor, L-NAME (5 mM) FB1 (AM)

L-NAME

0 +

2.5. Nitric oxide quantification

10

Nitric oxide was measured indirectly as nitrite by the Griess reaction [19]. RAW cell supernatant (50 Al) was incubated for 10 min with equal volumes of 1% sulfanilamide and 0.1% N-1-napthylethylenediamine. Optical density (OD) was read at 540 nm with a microplate reader (Titertek Multiscan MCC/340), and nitrite concentration was determined by comparison with a standard curve of nitrite (0– 200 AM).

25

+

2.6. Statistical analysis Data were analyzed by ANOVA, followed by Fisher’s LSD [20]. Data are expressed as group means F standard error (SEM). Significance was judged at p < 0.05.

3. Results 3.1. Effects of FB1 on viability of RAW cells Incubation of RAW cells with FB1 for 48 h resulted in a dose-dependent decrease in cell viability as measured by the MTT assay, with significant decreases at 25– 100 AM (Table 1). The addition of

Table 1 An MTT assay for viability of RAW macrophages incubated for 48 h with FB1 FB1 (AM)

MTT formazan (AM)

Percentage of control

0 1 5 10 25 50 100

796.19 F 28.9a 792.25 F 36.0a 765.91 F 33.3a 740.28 F 19.6a 606.69 F 35.5b 340.22 F 16.8c 127.02 F 2.5d

100.0 99.5 96.2 93.0 76.2 42.7 16.0

Different superscripts indicate significant differences ( p < 0.05).

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+ 50 +

MTT formazan (AM)

Percentage of control

394.34 F 15.695a 438.38 F 11.751b 319.96 F 7.034c 405.05 F 3.795a 301.29 F 12.666c 369.60 F 9.825a 253.93 F 12.230d 305.78 F 6.527c

100.0 111.0 81.14 102.72 76.40 93.73 64.39 77.54

Different superscripts indicate significant differences ( p < 0.05).

LPS (1 Ag/ml) to cell culture medium resulted in a slight decrease in cell viability, with control cells (0 AM FB1) incubated with LPS exhibiting a 10% decrease in MTT formazan product (data not shown). In contrast, a slight increase in viability was observed when control cells incubated with LPS were also exposed to 5 mM L-NAME (Table 2). In the presence of L-NAME, a protective effect was noted as production of MTT formazan was increased at all doses of FB1 when compared to those in the absence of LNAME. After a 72-h incubation with FB1 and LPS, viability of RAW cells was further decreased by greater than 40% when compared to a 48-h incubation (data not shown). 3.2. Effects of FB1 on LPS- and PMA-stimulated NO production Exposure of RAW cells to sub-lethal concentrations of FB1 (1 –10 AM) did not significantly affect LPS-induced production of NO, although a dosedependent trend of increasing NO was observed, with a 14% increase at 10 AM FB1 compared to control (Table 3). In contrast, stimulation with 200 or 400 ng/ ml PMA resulted in a slight, but significant decrease in NO production at sub-lethal concentrations of FB1 (Table 3). Increasing concentration of PMA to 400 ng/ ml resulted in a 3-fold increase in NO production by control RAW cells as compared to cells stimulated with 200 ng/ml PMA, but only in 400 ng/ml PMAstimulated cells incubated with 10 AM FB1 was a significant decrease in NO production observed. Addition of L-NAME (5 mM) to culture medium

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Table 3 Nitric oxide production (AM nitrite) by RAW macrophages following 48 h exposure to 0 – 10 AM FB1 FB1 (AM)

LPS

0 1 5 10 0 1 5 10 0 5 10

+ + + +

PMA1

PMA2

+ + + + + + +

NO (AM nitrite)

Percentage of control

31.12 F 2.53 33.77 F 0.89 33.95 F 1.91 35.64 F 1.21 6.76 F 0.45 * 4.41 F 0.36 4.19 F 0.45 3.08 F 0.55 19.40 F 0.65 18.96 F 0.32 17.52 F 0.83 *

100.0 108.5 109.1 114.5 100.0 65.2 62.0 45.6 100.0 97.7 90.3

Cells (2  104 cells/well) were concurrently stimulated with either LPS (1 Ag/ml), PMA1 (200 ng/ml), or PMA2 (400 ng/ml) for 48 h. * Indicates a significant difference ( p < 0.05) among dose groups from cells stimulated with either LPS, PMA1, or PMA2.

resulted in undetectable NO production by both LPSand PMA-stimulated RAW cells (data not shown). In the absence of LPS or PMA, cells incubated with FB1

Fig. 2. Nitric oxide production by RAW macrophages exposed to 0, 10, 25 or 50 AM FB1 with PMA (200 ng/ml) for 48 h. Columns represent mean production of nitrite (AM) F standard error as measured by the Griess reaction (n = 6). Asterisk indicates significant differences among FB1 dose groups ( p < 0.05).

(0 –50 AM) also failed to produce detectable amounts of NO (data not shown). At concentrations of 10 –50 AM FB1, dose-dependent increases in LPS-stimulated NO production were observed for RAW cells after 48 h (Fig. 1). Incubation of RAW cells with 50 AM FB1 resulted in a 7-fold increase in LPS-stimulated NO production compared to control cells. Cells incubated with FB1 for 48 h and then with LPS for an additional 48 h showed a similar pattern to those incubated with FB1 and LPS concurrently (data not shown). Addition of L-NAME virtually inhibited NO production, although NO was at detectable levels (Fig. 1). For cells stimulated with PMA (200 ng/ml), increased NO production was observed only at 50 AM FB1, a 4-fold increase as compared to that for control cells (Fig. 2).

4. Discussion

Fig. 1. Nitric oxide production by RAW macrophages exposed to 0, 10, 25 or 50 AM FB1 with LPS (1 Ag/ml) for 48 h. Cells (2  104 cells/well) were incubated with or without L-NAME, a known inhibitor of NOS. Columns represent mean production of nitrite (AM) F standard error as measured by the Griess reaction (n = 6). Different letters indicate significant differences among FB1 dose groups ( p < 0.05).

Results from previous studies reporting antiproliferative and cytotoxic effects of FB1 on cell lines in vitro are similar to the cytotoxic effects observed for RAW cells in the current investigation. For sensitive rat hepatoma cell lines, reported IC50 concentrations of FB1 ranged from 5 to 70 AM [21], while the IC50 for murine 3T3 fibroblasts was 200 AM [22]. In the current study, a 50% decrease in RAW cell viability

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occurred between 25 and 50 AM FB1, concentrations within the previously reported range of IC50 values for mammalian cells [21,22]. Other reports indicated that 10 –50 AM FB1 was mitogenic to 3T3 fibroblasts as measured by incorporation of [3H]thymidine into DNA [12,13]. Although mitogenicity was not measured in the current investigation, decreased viability of cells as observed at 25 –100 AM FB1 was consistent with the observation of Rotter and Oh [23] that RAW macrophage cultures exhibited a dose-dependent decrease in protein concentration upon exposure to 1 –100 AM FB1 for 70 h. With chicken peritoneal macrophages, a 2- or 4-h exposure to low concentrations of FB1 ( f 0.7– 13 AM) induced significant cytotoxicity [24]. Interestingly, viability of RAW cells in the current study was not affected by 48-h exposure to FB1 concentrations of 1, 5 or 10 AM. Clearly, future studies should include determination of cell viability versus length of exposure to FB1 to examine the timecourse of cytotoxicity. In the current study, addition of LPS alone to culture medium accounted for a slight decrease in RAW cell viability. Stimulation with LPS activates iNOS in murine macrophages to produce the reactive nitrogen intermediate, NO [16]. Release of toxic NO by macrophages is highly regulated because of binding to iron – sulfur complexes and interfering with enzyme activity [25]. Addition of a NOS inhibitor (L-NAME) to RAW cell cultures in the present investigation had a protective effect, reversing cytotoxicity of LPS stimulated RAW cells, indicating that the decrease in viability was due, at least in part, to the production of NO. Even though LPS decreased viability of RAW cells, our preliminary investigations determined 1 Ag/ml LPS to be an optimal stimulatory dose for the production of NO. Additionally, incubation with FB1 and LPS for 72 h resulted in high levels of cytotoxicity; therefore, incubation was limited to 48 h for all subsequent experiments. According to prior reports, primary rat splenic macrophages treated with 1, 10 or 100 Ag/ml FB1 ( f 1.4, 14 or 140 AM) for 24 h were activated to produce NO without stimulation [26]. However, results of the current study demonstrated that exposure to FB1 alone did not induce NO production by RAW macrophages and that stimulation with LPS or PMA was necessary. Interestingly, LPS- or PMAstimulation resulted in increased production of NO

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by RAW cells only at FB1 concentrations that were cytotoxic ( > 10 AM). Following exposure to 10 –50 AM FB1 for 48 h, LPS-stimulated macrophages exhibited a dose-dependent increase in NO production that was consistent with a previous report for RAW cells exposed to FB1 for 72- or 96-h [23]. Immunostimulatory effects of FB1 to macrophages also have been reported in a chicken cell line (MQ-NCSU), with exposure to 40 Ag/ml FB1 ( f 55 AM) increasing the production of a tumoricidal factor with or without LPS [24]. Although LPS-stimulated NO production by RAW cells in the current study was FB1 dosedependent, stimulation with PMA only increased NO output at the highest FB1 dose tested (50 AM), suggesting differences in FB1-related NO production relative to PMA and LPS as a result of different pathways involved in stimulation. At low concentrations of FB1 (1– 10 AM), PMA-stimulated NO production was significantly decreased, possibly the result of inhibition of protein kinase C (PKC) by FB1. Repression of PKC was reported in a monkey kidney cell line (CV-1) following a 3-h incubation with 5 AM FB1 or 16-h incubation with 1 or 5 AM FB1 [27]. Stimulation of macrophages by PMA involves direct activation of PKC by diacylglycerol (DAG) binding sites [28], and inhibition of PKC resulted in reduced NO synthesis by mouse peritoneal macrophages [29]. Thus, exposure of RAW cells to lowlevels of FB1 could decrease PKC activity and alter PMA-stimulation of NO. However, it was interesting to note that production of NO following stimulation with PMA was significantly lower than with LPS. Further, our data show that the decrease in PMAstimulated NO production at 10 AM FB1 was not reproducible in all experimental trials. Based on these data, a hormetic NO response may be occurring with PMA-stimulated macrophages treated with FB1, and 10 AM FB1 may be in a critical dose range for this response. Induction of iNOS in macrophages is transcriptionally regulated, dependent on NADPH, and requires increased intracellular tetrahydrobiopterin levels [16]. Although constitutively expressed NOS (cNOS) has been identified in RAW cells, stimulation with LPS down-regulates cNOS activity, suggesting that iNOS activity primarily mediates NO production by RAW cells [30]. The FB1-induced alterations in production of NO by LPS- or PMA-stimulated macro-

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phages observed in this study were possibly due to up- or down-regulation of iNOS or some step in the iNOS pathway. The inhibition of NO production by L -NAME demonstrated the dependence of FB 1 induced NO production on NOS activity. This hypothesis is further supported by a recent study relating FB1 to enhanced LPS-stimulated iNOS expression in J774A.1 macrophages [31]. Inducibility of iNOS in macrophages by LPS depends on the activation of nuclear factor nB (NFnB) binding proteins [16]. Exposure of C57BL/ 6J mice to FB1 (5 daily injections of 2.25 mg/kg/day) resulted in increased cellular NFnB [32], suggesting that up-regulation of this transcription factor may have affected NO production in the current investigation. Macrophage activation by LPS is complex, with multiple LPS receptors on the macrophage surface, resulting in a number of different signaling cascades (including activation of PKC). Since LPS-stimulation demonstrated a dose-dependent response with FB1 in our investigation, we may speculate that FB1 altered macrophage surface receptors that bind LPS. Alternatively, it is possible that FB1-induced changes in RAW cell sphingolipid content interfered with signaling pathways. Therefore, measurement of sphingolipids and sphingolipid precursors would be useful in continuing studies of macrophages exposed to FB1. Further, synthesis of ceramide is inhibited by FB1 [10] and ceramide itself can activate signaling pathways in macrophages [17], suggesting another possible mechanism of FB1 induced disruption of NO production by macrophages. These data suggest that NO production by macrophages is altered following exposure to FB1, with differences related to stimulation pathway. Disruption of NO production pathways in vivo could be harmful because of the toxicity of NO to cells. These observations demonstrate the need for further characterization of FB 1 -related alterations in inducible macrophage functions.

Acknowledgements This work was funded in part by a Sigma Xi GrantIn-Aid of Research. The authors would like to thank Dr. A.B. Bodine and Becky Boone (Department of Animal and Veterinary Sciences, Clemson University)

for technical assistance and Dr. Ken A. Voss (Toxicology and Mycotoxin Research Unit, USDA/ ARS, Athens, GA) for reviewing this manuscript.

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