Lipopolysaccharide and the Trichothecene Vomitoxin (Deoxynivalenol) Synergistically Induce Apoptosis in Murine Lymphoid Organs

Hui-Ren Zhou*, Jack R. Harkema{ddagger},||, Jon A. Hotchkiss{ddagger}, Ding Yan*, Robert A. Roth§,|| and James J. Pestka*,{dagger},||,1

* Department of Food Science and Human Nutrition, {dagger} Department of Microbiology, {ddagger} Department of Veterinary Pathology, § Department of Pharmacology, Institute for Environmental Toxicology, and || National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824–1224

Received June 16, 1999; accepted October 20, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human exposure to Gram-negative bacterial lipopolysaccharide (LPS) is common and may have an important influence on chemical toxicity. LPS has been shown previously to enhance synergistically the toxicity of trichothecene mycotoxins. Because either of these toxin groups alone characteristically target lymphoid organs at high doses, we evaluated the effects of coexposure to subthreshold doses of Salmonella typhimurium LPS and vomitoxin (VT) administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on apoptosis in lymphoid tissues after 12-h exposure. The capacity of LPS (0.5 mg/kg body weight) and VT (25 mg/kg body weight) to act synergistically in causing apoptosis in thymus, spleen, and Peyer's patches was suggested by increased internucleosomal DNA fragmentation in whole cell lysates as determined by gel electrophoresis. Following terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end-labeling (TUNEL) of tissue sections, a dramatic enhancement of fluorescence intensity indicative of apoptosis was observed in thymus, spleen, Peyer's patches, and bone marrow from coexposed animals as compared to those given the agents alone. Evaluation of hematoxylin and eosin-stained tissue sections of treatment mice revealed the characteristic features of lymphocyte apoptosis, including marked condensation of nuclear chromatin, fragmentation of nuclei, and formation of apoptotic bodies in tissues from mice. Combined treatment with VT (25 mg/kg body weight) and LPS (0.5 mg/kg body weight) significantly increased (p < 0.05) the amount of apoptotic thymic and splenic tissue as compared to the expected additive responses of mice receiving either toxin alone. When apoptosis was examined in cell suspensions of thymus, spleen, Peyer's patches, and bone marrow by flow cytometry in conjunction with propidium iodide staining, the percentage of apoptotic cells was significantly increased (p < 0.05) in cotreatment groups as compared to the additive responses to LPS and VT given alone. The results provide qualitative and quantitative evidence for the hypothesis that LPS exposure markedly amplifies the toxicity of trichothecenes and that the immune system is a primary target for these interactive effects.

Key Words: trichothecene; vomitoxin; deoxynivalenol; mycotoxin; immunotoxicity; protein synthesis inhibition; spleen; endotoxin; flow cytometry; lipopolysaccharide; apoptosis; programmed cell death; thymus; Peyer's patch; bone marrow.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endotoxin is a biologically active component of Gram-negative bacterial cell walls that exists as complexes of lipopolysaccharide (LPS) and protein (Hewett and Roth, 1993Go). LPS is the chemical substituent with principal biologic activity and plays a critical role in immune and inflammatory responses to Gram-negative bacteria. Exposure to it can stimulate both mononuclear phagocyte function and other host responses that result in removal of invading bacteria. Exposure to a small LPS dose does not normally cause life-threatening tissue damage, but it can initiate an incomplete and submaximal inflammatory response. Although such a modest inflammatory response may be insufficient to cause overt injury in unstressed individuals, these responses may alter homeostasis in individuals coexposed to a stress (such as a chemical insult) with the net result being frank tissue injury.

There is extensive evidence that LPS can influence the magnitude of toxic responses to xenobiotic agents. In some cases, LPS contamination of an environmental chemical source (e.g., LPS in machining fluids) may be the primary determinant of the toxic response (Gordon and Harkema, 1995Go; Mattsby-Baltzer et al., 1989Go), whereas in other cases concurrent exposure to small amounts of LPS may magnify the inherent toxicity of a chemical. For example, exposure to modest, normally nontoxic doses of LPS markedly increases the hepatotoxic responses to a number of xenobiotic agents including CCl4, ethanol, galactosamine, and allyl alcohol (reviewed in Roth et al., 1997). These and other examples suggest that humans exposed to low doses of LPS may represent a subpopulation that is particularly sensitive to xenobiotic chemicals.The trichothecenes are a group of sesquiterpenoid fungal toxins that includes some of the most potent protein synthesis inhibitors known (Ueno, 1987Go). Trichothecene mycotoxins are commonly found in cereal grains as a result of Fusarium infestation and have also been detected in air samples from water-damaged buildings that harbor the growth of Stachyobotrys (Johanning et al., 1996Go). The trichothecene vomitoxin (VT or deoxynivalenol) is a common contaminant of wheat and corn products and can be found at ppm levels in ready-to-eat foods (Rotter et al., 1996Go). Hallmarks of experimental or accidental high-dose trichothecene exposure include rapid diminution of lymphoid tissue and lymphopenia that precede death via a circulatory shocklike syndrome (Beardall and Miller, 1994Go).

Apoptosis is a programmed mode of cell death that is essential in several biologic circumstances, including development of the immune system (Cohen et al., 1992Go). Cells undergoing apoptosis in vivo demonstrate nuclear and cytoplasmic condensation and dissolution into membrane-bound fragments that are phagocytosed by neighboring cells and rapidly degraded. A principal role for programmed cell death is thought to be efficient removal of stressed, damaged, or unnecessary cells from a tissue without the generation of inflammatory or immune responses. Exposure in vivo to large doses of LPS induces apoptosis in splenic germinal centers and thymus (THY) of mice (Zhang et al., 1994Go) and swine (Norimatsu et al., 1995Go). Recently, the induction of thymic atrophy with accompanying thymocyte apoptosis was reported in mice exposed to intraperitoneal injections of the trichothecenes T-2 toxin and fusarenone (Islam et al., 1998Go).

In previous studies with mice, it has been observed that trichothecenes become more toxic in the presence of LPS, thereby causing markedly elevated tissue injury and mortality (Tai and Pestka, 1988Go; Taylor et al., 1991Go; Zhou, et al., 1999Go). Pronounced thymic and splenic lymphocyte depletion were characteristically observed in these studies. Because either LPS or trichothecenes alone will induce apoptosis in lymphoid tissue at large doses, we sought to evaluate the effects of low doses of Salmonella typhimurium LPS and a common food-borne trichothecene, VT, on apoptosis in four potential lymphoid targets, namely, thymus (THY), Peyer's patches (PP), spleen (SP), and bone marrow (BM). The study yielded both qualitative and quantitative evidence indicating that low doses of LPS and VT synergistically increase apoptotic cell loss in lymphoid tissue.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental design.
All animal handling was conducted in accordance with recommendations established by the National Institutes of Health. Experiments were designed to minimize numbers of animals required to test adequately the proposed hypothesis and were approved by Michigan State University Laboratory Animal Research committee. Male B6C3F1 mice (8–10 wks of age) were obtained from Charles River (Portage, MI). Animals were housed in cages (3/cage) equipped with filter bonnets (Nalgene, Rochester, NY) and acclimated for 1 week at the MSU University Laboratory Animal Resources Facility in a humidity- and temperature-controlled room with a 12-h light and dark cycle. Mice were provided standard rodent chow and tap water. Food and water were withdrawn from cages 2 h before toxin administration.

In a typical experiment, mice were given VT (25 mg/kg body weight) plus vehicle, LPS (0.5 mg/kg body weight) plus vehicle, VT (25 mg/kg body weight) plus LPS (0.5 mg/kg body weight), or the vehicles only. VT (Sigma, St. Louis, MO) was dissolved in 0.25 ml of tissue culture grade, endotoxin-free water (Sigma) and administered by a single oral gavage; animals that did not receive VT were treated with 0.25 ml water. LPS (Salmonella typhimurium, Sigma) was dissolved in 0.25 ml endotoxin-free water and was given by intraperitoneal injection; animals not treated with LPS were treated with 0.25 ml of endotoxin-free water. Twelve hours after exposure, mice were killed by cervical dislocation under ether anesthesia. THY, PP of intestine, SP, and femurs containing BM were immediately removed and processed for apoptosis measurements. The 25 mg/kg VT dose represents approximately one third of the LD50 for VT (Forsell et al., 1987Go). No mortality is observed at this dose (Zhou et al., 1999Go).

DNA fragmentation assay.
Single cell suspensions were prepared according to the method of Islam et al. (1998). Following euthanasia by cervical dislocation, the THY, SP, PP, and femur were removed immediately from the mice. Single cells were released from THY, SP, and PP by mashing with a glass plunger against a fine stainless steel wire net (Collector Tissue Sieve, Bellco Glass Inc., Vineland, NJ) submerged into ice-cold PBS. BM was flushed out of the femur using PBS. The cells prepared from SP and BM were treated with erythrocyte-lysing buffer containing 0.83% (w/v) ammonium chloride, 0.1%(w/v) potassium bicarbonate, and 0.0037% (w/v) EDTA for 2 min at room temperature to remove erythrocytes. The cell suspension was passed through the 40-µm nylon sieve and the cell number was determined using a Bright-Line Hemacytometer (Sigma, St. Louis, MO).

DNA from THY, SP, PP, and BM was extracted and electrophoresed as described by Sellins and Cohen (1987). Briefly, cells (1 x 107) in phosphate-buffered saline (pH 7.4) were centrifuged for 5 min (500 x g) at 4°C, and the pellet was suspended in 0.1 ml hypotonic lysing buffer (10 mM Tris, 10 mM EDTA, 0.5% [v/v] Triton X-100, pH 8.0). Cells were incubated at 4°C for 10 min. The resultant lysate was centrifuged for 30 min (13,000 x g) at 4°C. Supernatant containing fragmented DNA was digested for 1 h at 37°C with RNase A (0.4 µg/µl). It was then incubated for 1 h at 37°C with proteinase K (0.4 µg/µl). DNA was precipitated in 50% isopropanol and 0.5 M NaCl overnight at –20°C. The precipitate was centrifuged at 13,000 x g for 30 min at 4°C. The resultant pellet was air dried, resuspended in 10 mM Tris, 1 mM EDTA, pH 8.0, then electrophoresed at 60 V for 2 h in 2% agarose gel (2 x 106 cells per lane) in 90 mM Tris-borate buffer (pH 8.0) containing 2 mM EDTA. After electrophoresis, the gel was stained with ethidium bromide (0.5 µg/ml), and the nucleic acids were visualized with a UV transilluminator. A 100-bp DNA ladder (GIBCO-BRL, Rockville, MD) was used as a molecular size marker.

Preparation of tissue sections.
For histologic assessment of apoptosis by cell morphology and detection of apoptotic cells in situ, tissues were fixed in 10% (v/v) buffered formalin for at least 24 h. Before embedding in paraffin, femurs were decalcified in 13% (v/v) formic acid for 3 days, and PP were pre-embedded in 3% (w/v) agarose followed by an additional 24 h fixation in 10% (v/v) buffered formalin. Tissues were embedded in paraffin and 4- to 6-µm sections were adhered to microscope slides. Sections were deparaffized and hydrated by heating the sections for 30 min at 60°C and transferring the slides through the following solutions: three times in xylene for 5 min, twice in 100% ethanol, and once in 90%, 80% ethanol, 5 min each, and then in distilled water.

Detection of apoptotic cells in situ.
A kit modification (Boehringer Mannheim in situ Cell Death Detection Kit, Indianapolis, IN) of the protocol of Gavrieli et al. (1992) for terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick-end labeling (TUNEL) was used to evaluate paraffin sections according to the manufacturer's instructions. Briefly, deparaffinized slides were rinsed with 10 mM phosphate-buffered saline (pH 7.0 PBS). TUNEL reaction mixture (50 µl) containing the components for the end-labeling reaction (fluorescein-dUTP and TdT) was added to each section previously encircled with a hydrophobic PAP Pen (Research Products International Corp., Mt. Prospect, IL). The section was overlaid with a coverslip to avoid evaporative loss, then incubated in a humidified chamber for 60 min at 37°C. After washing 3 times with PBS, samples were mounted with Gel/Mount (Biomedia Corp., Foster City, CA) and examined under a Nikon Labophot fluorescence microscope (Mager Scientific, Inc., Dexter, MI). Cells with nicked DNA were detectable by fluorescence. In each experiment, negative controls were included in which fixed and permeabilized samples were incubated within TUNEL reaction mixture devoid of TdT. Positive controls were also included that consisted of sections pretreated with DNase I (Sigma, St. Louis, MO), 1 mg/ml in buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate, 4 mM MgCl2, 0.1 mM dithrothreitol), for 10 min at 37°C.

Histopathology and morphometry.
Paraffin sections (5–6 µm thick) of SP, THY, and PP were histochemically stained with hematoxylin/eosin (H&E) and examined by light microscopy (Olympus BX-60; Olympus Corp., Lake Success, NY). Upon histologic assessment of H&E-stained sections, cells were scored as apoptotic if they exhibited cellular shrinkage with concurrent cytoplasmic eosinophilia, nuclear pyknosis, and fragmentation (i.e., karyorrhexis) with associated apoptotic bodies. Digitized images of randomly selected fields (e.g., every 15th after a random start) were obtained (at a final magnification of 1550x for SP, 1580x for THY, and 950x for PP) and characterized morphometrically in terms of percent apoptotic lymphoid tissue. Images were captured and saved as PICT files on a Power Macintosh 7100/66 computer, using a high-resolution CCD camera (VE-1000CCD; Dage-MTI, Inc., Michigan City, IN) and the public domain image analysis program NIH Image v1.60 (written by Wayne Rasband, U.S. National Institutes of Health; available from the Internet by anonymous ftp from zippy.nimh.nih.gov). Randomly selected fields were captured so that approximately 5% of the total tissue section was analyzed for the THY (432 fields), 2.5% for SP (349 fields), and 50% for PP (237 fields). Only fields that were completely filled by tissue were counted (e.g., ones without large areas occupied by blood vessels, bordering spaces, etc.). The captured images were then imported to the morphometric data acquisition program Stereology Toolbox v1.1 (Morphometrix; Davis, CA) and analyzed using a double-density 25/125 point grid. The tissue directly beneath each grid point (THY and SP = 25 points/field, PP = 100 points/field) was categorized as normal lymphoid tissue, apoptotic lymphoid tissue, or "other" (e.g., nonlymphoid tissue, extracellular space, blood vessels, nonlymphoid cells, etc.). The number of points in each category was totaled individually for every animal. The area of each tissue category was estimated by the following calculation:

where A(point) = (the distance between grid points at the magnification of the captured image magnification)2. The percentage of apoptotic tissue for each animal was calculated from the area of lymphoid tissue:

Lymphoid tissue occupied approximately 60–90% of the total tissue area examined.

Quantitation of apoptosis by flow cytometry.
Apoptosis in immune tissues was quantified by flow cytometric cell cycle analysis as described previously (Pestka et al., 1994Go). Briefly, THY, PP, SP, and BM cell suspensions were prepared as described for electrophoresis. Cells (2 x 106 )were resuspended in 0.2 ml PBS and 0.2 ml heat-inactivated fetal bovine serum, fixed by adding 1.2 ml ice-cold 70% (v/v) alcohol dropwise with gentle mixing and held overnight at 4°C. Cells were washed and incubated for 1 h in 1 ml propidium iodide (PI) DNA staining reagent (PBS, pH 7.4 containing PI 50 µg/ml, RNase 0.05 mg/ml at 50 units/mg, EDTA disodium 0.1 mM, and Triton x-100 0.1%) at room temperature and then stored on ice until analysis. The cell cycle distribution for single cells was measured with a Becton Dickinson FACS Vantage (San Jose, CA). Data from 10,000 cells were collected in list mode. The 488 line of an argon laser was used to excite PI; the fluorescence was detected at 620–700nm. Cells were gated to exclude only debris and large cell aggregates and examined for DNA fluorescence intensity distribution. Cells in the DNA histogram with hypofluorescent DNA were designated apoptotic. All other cells distributed themselves in a normal cell cycle profile.

Statistics.
The data were analyzed using Sigma Stat for Windows (Jandel Scientific, San Rafael, CA). For comparisons of two groups of data, a Kruskal-Wallis One Way Analysis of Variance on Ranks and a Student-Newman-Keuls multiple comparison test was performed. Data sets showing significant differences (p < 0.05) were further analyzed for synergy. Single treatment replicates were randomly combined to calculate an expected mean additive response with variance. This calculated value was compared to actual cotreated samples using a Mann Whitney Rank Sum test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Detection of DNA Fragmentation in Whole Cell Lysates
Coexposure to trichothecenes and LPS has been previously shown to induce extensive cell death in lymphoid tissues (Tai and Pestka, 1988Go; Taylor et al., 1991Go; Zhou et al., 1999Go). To assess the relative extent of DNA fragmentation in animals coexposed to these chemicals, whole cell lysates of THY, PP, BM, and SP were prepared from mice treated with VT (25 mg/kg body weight), LPS ( 0.5 mg/kg body weight), or VT with LPS after 12 h and analyzed by agarose gel electrophoresis and ethidium bromide staining (Fig. 1Go). "Ladder" patterns were observed for lysates of THY, PP, BM, and to a lesser extent, SP prepared from mice coexposed to LPS and VT. A comparison with molecular weight standards indicated that the DNA fragments were multiples of approximately 200 base pairs, thus suggesting internucleosomal cleavage of double-stranded DNA. THY, PP, SP, and BM lysates (lanes d, h, p, and l) from mice treated with LPS yielded weak or no laddering pattens. Lysates of THY, PP, SP, and BM (lanes c, g, o, and k) from VT-treated mice suggested little or no DNA fragmentation. There was little or no DNA fragmentation for lymphoidorgan lysates from control groups (lanes e, i, m, or q). The results indicated that coexposure to LPS and VT induced markedly greater DNA fragmentation in murine lymphoid tissues than did either toxin alone.



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FIG. 1. Internucleosomal DNA fragmentation in lymphoid organs. Agarose gel electrophoresis was conducted on DNA isolated from THY, PP, SP, and BM cells following exposure to vehicles (lanes e, i, m, q), VT (25 mg/kg body weight orally; lanes c, g, k, o) mice, LPS (0.5 mg/kg body weight ip; lanes d, h, l, p), both oral VT and ip LPS (lanes b, f, j, n). Lanes b, c, d, and e are THY lysates. Lanes f, g, h, and i are PP lysates. Lanes j, k, l, and m are SP lysates. Lanes n, o, p, and q are BM lysates. Gels were stained with ethidium bromide and photographed under UV light. Lanes a and r are 100-bp DNA ladder used for molecular sizing. The results are representative of two separate experiments.

 
Detection of DNA Fragmentation In Situ
TUNEL was used to detect DNA fragmentation in lymphoid tissues. Fluorescein-dUTP nick-end labeling that resulted from the procedure was indicative of early DNA strand breaks, which are molecular hallmarks of apoptotic cell death. Sections of THY, PP, BM, and SP from mice treated 12 h earlier with VT, LPS, or VT with LPS were prepared and subjected to TUNEL. In all four lymphoid tissues examined, elevated numbers of fluorescent deposits and increased fluorescence intensity appeared to be induced by various treatments (Figs. 2 and 3GoGo). In general, normal nucleated cells did not yield positive TUNEL reactions. There was a slight background staining in the vehicle controls, especially in cells which rapidly turn over such as immature thymocytes (Fig. 2AGo) and PP B cells (Fig. 3AGo). Increased fluorescence was evident in both VT-exposed mice (Figs. 2B and 3BGoGo) and to a greater extent, in LPS-exposed mice (Figs. 2C and 3CGoGo) as compared to control. However, the most dramatic increases in FITC intensity were observed in tissues of mice coexposed to VT and LPS (Figs. 2D and 3DGoGo). The degree of fluorescence intensity and hence DNA fragmentation in organs of mice coexposed to VT and LPS followed a rank order of THY > PP > BM > SP. The results indicated that coexposure to LPS and VT induced markedly greater DNA fragmentation in lymphoid tissue of mice than did either toxin alone.



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FIG. 2. Detection of DNA fragmentation in situ in the THY by TUNEL after LPS and/or VT. Fluorescent photomicrographs of THY sections from treated mice after terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick-end labeling. Treatments were (A) vehicles only (controls), (B) a single oral exposure of VT (25 mg/kg body weight), (C) a single ip exposure of LPS (0.5 mg/kg body weight), and (D) both oral VT and ip LPS. Fluorescent deposits are indicative of DNA breakage. Fluorescence intensities of sections from mice coexposed to VT and LPS were markedly enhanced compared to mice exposed to a single toxin. Bar equals 100 µm. The results are representative of three separate experiments.

 


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FIG. 3. Detection of DNA fragmentation in situ in PP by TUNEL after LPS and VT. Fluorescent photomicrographs of PP sections from treated mice after terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick-end labeling. Treatments were (A) vehicles only (controls), (B) a single oral exposure of VT (25 mg/kg body weight), (C) a single ip exposure of LPS (0.5 mg/kg body weight), and (D) both oral VT and ip LPS. Fluorescent deposits are indicative of DNA breakage. Fluorescence intensities of sections from mice coexposed to VT and LPS were markedly enhanced compared to mice exposed to a single toxin. Bar equals 100 µm. The results are representative of three separate experiments.

 
Histopathologic Assessment and Morphometry of Apoptotic Tissue
The histopathologic effects of coexposure to LPS and VT administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on lymphoid tissue were evaluated after 12 h. Tissue atrophy due to loss of lymphocytes was observed in animals at 12 h after coexposure to LPS and VT (Figs. 4 and 5GoGo). The principal histologic lesions in the mice treated with VT and LPS were marked cell death and loss of the lymphoid tissue of the THY, PP, SP, and BM. Notably, treatment-induced cell death had characteristic apoptotic features, including cellular shrinkage with concurrent cytoplasmic eosinophilia, nuclear pyknosis and fragmentation (i.e., karyorrhexis), associated apoptotic bodies, and resultant loss of lymphoid tissue (i.e., lymphoid atrophy). Numerous THY, PP, SP, and BM cells were undergoing apoptosis, many appearing as clusters and as eosinophilic and apoptotic bodies. A large number of apoptotic nuclei in thymocytes of the cortex were observed (Fig. 4Go), whereas those of the medulla were less affected. Nuclei in the PP germinal center were most frequently condensed (Fig. 5Go). Severity of these lesions in co-exposed mice was much more pronounced than in mice exposed to either LPS or VT alone. Few positive cells were detected in lymphoid tissues from vehicle-treated mice.



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FIG. 4. Induction of apoptosis in the THY after LPS and VT coexposure. Light photomicrographs of the cortical region of the THY of treated mice. Treatments were (A) vehicles only (control), (B) a single oral exposure of VT (25 mg/kg body weight), (C) a single ip injection of LPS (0.5 mg/kg body weight), and (D) both oral VT and ip LPS. No histopathology is present in A (control). In B and C, there are a few widely scattered small foci of lymphocytes in various stages of apoptosis (arrows). Apoptotic bodies and shrunken lymphocytes with nuclear chromatin condensation, margination, or fragmentation are principal features of these areas of lymphocytic apoptosis. Similar, but more extensive lymphocytic apoptosis is evident in the lymphoid tissue in the THY of the mouse exposed to both VT and LPS (D). The results are representative of three separate experiments.

 


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FIG. 5. Induction of apoptosis in the PP after LPS and VT coexposure. Light photomicrographs of the cortical region of the PP of treated mice. Treatments were (A) vehicles only (control), (B) a single oral exposure of VT (25 mg/kg body weight), (C) a single ip injection of LPS (0.5 mg/kg body weight), and (D) both oral VT and ip LPS. No histopathology is present in A (control). In B and C, there are a few scattered small focal aggregates of lymphocytes undergoing apoptotic death (arrows). Affected foci contain shrunken, apoptotic lymphocytes with nuclear chromatin condensation, margination, or fragmentation that are interspersed among apoptotic bodies. There is an absence of inflammatory cell response in these areas of lymphocytic death. Similar, but more extensive lymphocytic apoptosis is evident in PP of the mouse exposed to both VT and LPS (D). The results are representative of three separate experiments.

 
Morphometry of apoptotic cells was conducted in THY, PP, and SP (Fig. 6Go). Percentages of apoptotic cells after exposure to vehicles, VT (25 mg/kg body weight), LPS (0.5 mg/kg body weight), or VT (25 mg/kg body weight) plus LPS (0.5 mg/kg body weight) in mice were 0.6%, 3.2%, 5.5%, and 32.4%, respectively, in THY; 0.1%, 5.3%, 2.5%, and 7.7%, respectively, in PP; and 0.2%, 2.9%, 0.6%, and 8.0%, respectively, in SP. For THY, PP, and SP, the expected median additive increases over control after exposure to the combined toxins were 7.45, 7.68, and 1.66% respectively, whereas the actual median responses were 30.6, 7.71, and 7.78%, respectively. Statistical analysis of the morphometric results indicated that in two out of the three tissues examined, THY and SP, VT and LPS acted synergistically (p < 0.05) to induce apoptosis.



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FIG. 6. Morphometry of apoptotic cells in the THY, PP, and SP after LPS and VT coexposure. Apoptotic cells were counted in H & E sections using morphometric procedure described in Methods. Data are means ± SE (n = 6). Letters indicate significantly different (p < 0.05) from vehicle control (a), LPS group (b) or VT group (c). Results are representative of 2 separate experiments.

 
Flow Cytometric Measurement of Apoptosis
Apoptosis in the four lymphoid tissues of control and treatment mice was quantified by flow cytometry of hypofluorescent cells following PI staining (Fig. 7Go). Percentages of apoptotic cells after exposure to vehicle only, VT, LPS, or VT plus LPS were 1.5, 2.1, 5.5, and 10.9%, respectively, in THY; 2.6, 4.6, 5.0, and 12.2%, respectively, in PP; 0.2, 0.4, 1.5, and 2.6%, respectively, in SP; and 0.3, 0.5, 5.4, and 9.0 %, respectively, in BM. For THY, PP, SP, and BM, the expected median additive increases over control after exposure to the combined toxins were 4.21, 4.15, 1.41, and 5.48%, respectively, whereas the actual median responses were 9.09, 9.63, 2.39, and 8.57%, respectively. These differences were significantly different (p < 0.05) in all case, thus indicating that coexposure to VT and LPS synergistically increased apoptosis in all four lymphoid tissues examined.



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FIG. 7. Flow cytometric quantitation of apoptotic cells in lymphoid tissues following exposure to LPS and VT. Cells isolated from THY, PP, SP, and BM were assessed by flow cytometric analysis in conjunction with propidium iodide DNA staining. Cells in the DNA histogram that were marker positive with hypofluorescent DNA were designated as apoptotic. Data represent means ± SE (n = 4). Letters indicate significantly different (p < 0.05) from vehicle control (a), LPS group (b) or VT group (c).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A fundamental question in toxicology relates to why certain individuals are more susceptible to the harmful effects of xenobiotics than others. The presence or absence of inflammation in a host is a little-studied but potentially important susceptibility determinant for chemical toxicity. LPS, a component of Gram-negative bacteria, has been studied for many years as a prototypic inflammagen. Although modest inflammatory responses induced by low-level LPS exposure may be insufficient to cause overt injury in unstressed individuals, such responses may act in concert with chemicals to injure tissues. Apoptosis is one such homeostatic mechanism that is essential to the immune system in maintaining tolerance via removing potentially self-reactive lymphocytes (Cohen et al., 1992Go). Two documented sites for such lymphocyte negative selection are the thymus (T cell) and germinal center (B cell) of peripheral lymphoid organs. Although beneficial in normal tissue homeostasis, apoptosis can also result from chemical injury. Both LPS and trichothecenes such as VT can induce apoptosis in lymphoid tissue. The qualitative and quantitative data presented here suggest that when LPS and VT are coadministered, synergy in the induction of apoptosis occurred. These findings lend further support to the hypothesis that bacterial endotoxin can exacerbate chemical injury.

This paper is the first to show enhanced lymphocyte apoptosis resulting from combined trichothecene and LPS exposure. We chose to use the mouse model because its immune system is well characterized and we have extensively studied the immunologic effects of trichothecenes on this animal. Dose responses were tested in preliminary experiments using a small number of animals. The optimal doses for the observing synergy were 25 mg/kg VT and 0.5 mg/kg LPS. In preliminary experiments, we also examined 3, 6, 9, 12, and 24 h and obtained qualitative data to suggest that apoptosis was maximal at 12 h. To limit experimental animal use, we chose to focus on using on these two optimal doses at the 12-h timepoint to demonstrate the synergistic effect. The 25 mg/kg VT dose would represent the total daily dose that a mouse would acquire upon ingesting a diet contaminated with 100 ppm of this mycotoxin. Mice and rats are relatively resistant to LPS as compared to humans and other of animal species (Galanos et al., 1979Go). To study mechanisms of LPS toxicity in mice, various sensitization techniques such as galactosamime have been used (Redl et al., 1993Go). When interpreted in this context, our data are valuable because they provide a basis for further mechanistic exploration of trichothecene-LPS interactions, dose response, and response kinetics, as well as assessment of this effect in other species using appropriate in vivo, ex vivo, and in vitro models. From such studies, the potential relevance to human health can be ascertained in the context of improved exposure data on the trichothecenes and LPS.

Systemic exposure of humans to LPS can occur through several mechanisms. The most widely recognized routes of LPS exposure are via respiratory and systemic Gram-negative bacterial infections (Brun-Buisson et al., 1995Go; Wenzel et al., 1996Go). However, humans are also commonly exposed to the LPS of indigenous Gram-negative gut flora through gastrointestinal (GI) translocation, ie., the passage of LPS from the GI lumen into the blood (Jacob et al., 1977Go). GI translocation of LPS is enhanced under a variety of conditions, including inflammatory bowel diseases (Palmer et al., 1980Go), GI injury (Van Leeuwen et al., 1994Go), liver disease (Bigatello et al., 1987Go), dietary alterations (Spaeth et al., 1990Go; Rutenburg et al., 1957Go), and excessive alcohol consumption (Bode et al., 1987Go). Elevated respiratory tract exposure to LPS also occurs in a variety of occupational environments. These include grain processing (Dosman et al., 1981Go; Pernis et al., 1961Go), waste treatment plants, machining operations (Mattsby-Baltzer et al., 1989Go), poultry and swine industries (Donham et al., 1989Go), and office or household air (Flaherty et al., 1984Go; Peterson et al., 1964Go; Rylander and Haglind, 1984Go). Thus, exposure in humans is common, and the degree of exposure varies with occupation, diet, and disease state.

Exposure to large doses of LPS initiates a chain of inflammatory events that culminate in cell death, frank injury to tissues, and functional failure of several organs. LPS induces its marked biologic effects by stimulating host cells to produce a variety of mediators including proinflammatory cytokines (eg., TNF-{alpha}, IL-6, IL-1), glucocorticoids, bioactive lipids, (eg., prostaglandins), reactive oxygen species, and activated coagulation components (Schletter et al., 1995Go). Target cells for LPS are primarily mononuclear phagocytes but also include endothelial cells, neutrophils and smooth muscle cells. Of the aforementioned mediators, TNF-{alpha} appears to be of central importance (Beutler, 1995Go). Exposure in vivo to large doses of LPS induces apoptosis in splenic germinal centers and thymus of mice and swine (Norimatsu et al., 1995Go; Zhang et al., 1993Go; 1994Go). A key finding in these studies has been that elevated plasma levels of both TNF-{alpha} and glucocorticoids precede these effects, and lymphocyte apoptosis in mice can be blocked with neutralizing antibody to TNF-{alpha}. Accordingly, TNF-{alpha} appears to be an important factor in the genesis of apoptosis in lymphoid tissue during LPS exposure.

Superinduction is the capacity of protein synthesis inhibitors to augment and prolong the usually transient mitogenic induction of a gene as a secondary consequence of translational arrest. Superinduction is likely to involve transcriptional and/or post-transcriptional mechanisms (Li et al., 1997Go; Ouyang et al., 1996Go). Trichothecenes inhibit translation (Ueno, 1987Go), and we have demonstrated that VT superinduces mRNA expression and production of TNF-{alpha} and IL-6 in LPS-stimulated macrophages (Ji et al., 1998Go; Wong et al., 1998Go). Oral VT administration enhances TNF-{alpha} mRNA and protein expression in vivo within 2 h in spleen and other organs (Azcona-Olivera et al., 1995Go; Zhou et al., 1997Go), and this is potentiated by LPS coexposure (Zhou et al., 1999Go). Thus, the enhanced lymphoid apoptosis observed herein may be mediated, in part, by elevated TNF-{alpha} level resulting from VT-mediated superinduction of LPS-induced TNF-{alpha} expression.

Our results are consistent with previous studies employing the trichothecene T-2 toxin. When LPS is administered at sublethal doses to mice, the estimated LD50 values for T-2 toxin markedly decrease (Tai and Pestka, 1988Go). Histologic analysis revealed that after coexposure of C3H/HeN mice to T-2 toxin (1 mg/kg) and LPS (2 mg/kg), extensive lymphocyte death occurred, whereas mice receiving only T-2 or LPS appeared normal. In related work, Taylor et al. (1991) subsequently observed synergy between T-2 toxin and LPS in mice. Effects included increased mortality, TNF-{alpha} production, hypothermia, and thymic atrophy. Plasma corticosterone concentration peaked at approximately 1 h for T-2 and LPS, with the LPS group being much greater (>2-fold); however, the combination group exhibited prolonged and elevated corticosterone concentration compared to LPS or T-2 alone. Taken together, the two studies suggest that T-2 toxin and possibly other trichothecenes become more toxic in the presence of LPS, thereby causing elevated tissue injury and mortality. The combination treatment appears to increase lymphoid organ depletion as well as increase two potential mediators of injury (TNF-{alpha} and corticosterone).

Flow cytometic analysis showed that there were greater percentages of hypofluorescent nuclei (indicative of apoptotic cells) in lymphocytes isolated from tissues of the combined VT/LPS treatment than in control or single-toxin treatment groups. Interestingly, although the morphometric and flow cytometric results yielded a similar outcome in this study, relative percentages of apoptotic cells estimated by flow cytometry differed from morphometric estimates in H&E sections. It is possible that flow cytometry may yield a more accurate estimate of apoptosis than morphometry because it samples the entire cell population of the lymphoid organ rather than sections. Using flow cytometry to measure the effects of VT and cycloheximide on lymphocyte apoptosis in vitro, Pestka et al. (1994) have observed that although both chemicals inhibit glucocorticoid-induced apoptosis in thymic and splenic T cells, these translational inhibitors induce apoptosis in B and IgA+ cells in SP and PP. These latter findings indicate that VT can either directly inhibit or enhance programmed cell death in a concentration-dependent manner and that its effect is highly dependent on lymphocyte subset, tissue source, glucocorticoid induction, and VT concentration. It would be of interest to study the phenotypic targets of the LPS/VT co-exposure model described here.

There are at least three general ways that LPS and VT may promote lymphocyte apoptosis. The first mechanism involves interaction between superinduced TNF-{alpha} and lymphocytes. Direct engagement of TNF-{alpha} with its cell surface receptor has been shown to induce apoptosis (Baker and Reddy, 1996Go; Hernandez-Caselles and Stutman, 1993Go). A second mechanism involves TNF-{alpha} mediated elevation of soluble mediators. Likely candidates as secondary mediators include glucocorticoids, which induce apoptosis in both B and T cells (Pestka et al., 1994Go; Garvy et al., 1993Go), and prostaglandin E2 (PGE2) (Mohr et al., 1992Go), which induces lymphocyte apoptosis (Brown and Phipps, 1996Go). The third mechanism involves direct effects of VT on T and B cells, as has been suggested in our in vitro studies with VT (Pestka et al., 1994Go). It should be emphasized that all three mechanisms are not mutually exclusive and could function simultaneously.

Persons exposed to LPS or other inflammagenic stimuli and who may thus be xenobiotic susceptible represent a significant part of the population (Roth et al., 1997Go). Because trichothecenes can be commonly found as food and indoor air contaminants, it is reasonable to suggest that a considerable portion of this sensitive human population may be exposed to these mycotoxins. Lymphocyte death induced by trichothecenes in LPS-exposed individuals might be expected to depress cell-mediated and humoral immune function. Characterization of phenotypic targets, dose-response, kinetics, strain/species sensitivity, and mechanisms in this model may provide insight into human health problems associated with trichothecene exposure in the xenobiotic-susceptible population.


    ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grants ES-09521 (JJP) and ES-03358 (JJP) from the National Institute for Environmental Health Sciences, Michigan State University Agricultural Experiment Station, the MSU Crop and Food Bioprocessing Center, and the MSU National Food Safety and Toxicology Center. We thank Zahidul Islam, Patricia Ganey, James Clarke, Rebecca Uzarski and Louis King for assistance and valuable comments during the course of this study.


    NOTES
 
1 To whom correspondence should be addressed at 234 G. M. Trout Building, Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824. Back


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