* United States Food and Drug Association, Center for Food Safety and Applied Nutrition, Laurel, Maryland 20708,
United States Food and Drug Association, Center for Veterinary Medicine, Laurel, Maryland 20708,
United States Food and Drug Association, National Center for Toxicological Research, Jefferson, Arkansas 72079
Received November 26, 2002; accepted February 21, 2003
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ABSTRACT |
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Key Words: aflatoxin B1; immunotoxicity; inflammatory response; intermittent dosing.
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INTRODUCTION |
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Our objective was to evaluate any significant changes in the relative proportions and functions of the main splenic lymphocyte classes and to compare these changes with the histopathology evaluations of the liver and spleen with emphasis on the cell populations involved in the inflammatory response. An intermittent exposure regimen was designed to simulate human experience (Kodell et al., 1987; Murdoch et al., 1992
) since people are often exposed to an agent intermittently rather than continuously. The focus of this study was how accumulated dosing relates to the expression of particular immunologic biomarkers and the formation of preneoplastic lesions in the liver.
A considerable body of evidence exists suggesting that AFB1 suppresses immune function by affecting T-cell dependent immunity in various animal species, in particular, cattle (Bodine et al., 1984; Brown et al., 1981
), chickens and turkeys (Ghosh et al., 1990
; Giambrone et al., 1985a
,b
), and swine (Liu et al., 2002
; Mocchegiani et al., 1998
). Studies with laboratory test species such as the mouse (Jakab et al., 1994
; Reddy et al., 1987
), rat (Raisuddin et al., 1990
, 1993
), and rabbit (Venturini et al., 1990
) reinforce these findings. Immunosuppression by a toxicant can result from various mechanisms such as decreased protein and/or DNA synthesis, changes or loss in enzymatic activity, and changes in metabolism or cell cycles, which may result in apoptosis or necrosis. Immune mechanisms affected by AFB1, in addition to T-cell dependent immunity, include reduced production of complement by the liver and decreased phagocytosis by neutrophils and macrophage (Cusumano et al., 1995
, 1996
; Dugyala and Sharma, 1996
). Toxic effects on T-lymphocytes (Dugyala and Sharma, 1996
) and/or other lymphoid cells such as the cytotoxic T-cells and natural killer cells (NK; Methenitou et al., 2001
), which impair the function of direct or indirect killing of tumor cells, can have pronounced effects on tumorigenesis. Immunosuppression can result in a greater rate of tumor progression (Raisuddin et al., 1991
). Moreover, cellular components of the immune system are known to produce various cytokines, which play a key role in host resistance and protection against tumor progression. These same cytokines, however, are involved directly in the inflammatory mechanisms that are initiated when various organs have been damaged by toxic assault (Batey and Wang, 2002
.)
In this study, we present the results of the flow cytometric analysis and assays of the functional, inflammatory cytokine productive capacity of splenic lymphocytes in relation to the histopathology evaluation of the liver and spleen for the highest dose groups of AFB1. Our results complement the various immune function studies of AFB1 that have been reported, since they relate to both immunotoxic and possibly the hepatotoxic effects of AFB1. As far as we are aware, this is the first report of an immunotoxicity study of AFB1 wherein cycles of feeding and rest were included in the study design.
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MATERIALS AND METHODS |
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Fluorescent antibodies, cell lines, and cytokine standards.
All of the fluorescent labeled antibodies used in the flow cytometry were obtained from Pharmingen (San Diego, CA). These included: fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies to rat CD8a and rat CD45R, and the FITC mouse IgG2a (used as a kappa isotype control); and phycoerythrin (PE)-labeled monoclonal antibodies to rat CD3 and rat CD4 as well as PE labeled mouse IgG1 used as the kappa isotype control.
The cytokine responsive cell lines, CTLL-2 and 7TD1, used in the splenic lymphocyte stimulation assays were obtained from the American Type Culture Collection (ATCC; Manassas, VA). The CTLL-2 line (ATTC number TIB-214) is a mouse cytotoxic T-cell line, which was used in the IL-2 cytokine assays as described by Lyte et al.(1987). The 7TD1 cell line, ATCC number CRL-1851, is a mouse B-lymphocyte hybridoma that was used in the IL-6 cytokine assays. A D10.S cell line, a gift from Dr. Lawrence Shook of the University of Illinois, was used in the IL-1 cytokine assays in a manner described by Schook et al.(1992)
. The D10.S cell line is a mouse helper/inducer T-lymphocyte line that is a sub-clone of the D10.G4.1 cell line, which is commercially available from ATCC, number TIB-224.
Cytokine standards, human IL-1, IL-2, and IL-6 were obtained from BioSource (Camarillo, CA). Carrier free rat INF was obtained from PBL Biomedical Laboratories (New Brunswick, NJ). Concanavalin A (Con-A) and chromatographically pure Eschericia coli (E. coli) lipopolysaccharide, containing less than 1% protein, were obtained from Sigma Chemical Co.
Animals, diet, and study design.
The life phase of the study was conducted at the National Center for Toxicological Research (NCTR) using weaned male Fischer 344-N (F344) rats (2124 days of age) obtained from the NCTR breeding colony. The NCTR is fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). Animal husbandry and all experimental procedures were reviewed and approved by the NCTR Animal Care and Use Committee. The rats were maintained on a 12-h light/dark cycle at a constant temperature of 2224°C and humidity of 3437%.
The control animals were fed certified NIH-31 meal diet (Purina, St. Louis, MO), and the treated groups were fed AFB1-NIH-31 diets that were prepared at NCTRs Dietary Preparation Facility. The AFB1-NIH-31 meal diets were prepared by mixing 40 kg of the NIH-31 meal with 64 mg of AFB1 dissolved in 500 ml of ethanol to obtain the high dose of 1.6 ppm aflatoxin. The ethanol was removed by evaporation under reduced pressure. All other doses were obtained by admixing the appropriate amount of NIH-31 meal with the high dose AFB1-NIH-31 preparation. The concentration of AFB1 in each diet preparation was measured by the method of Park et al.(1990). Separate groups of rats were used to estimate food consumption. Four rats per dose group were housed singly in hanging cages. Spilled food was collected and weighed. Food consumption was corrected by adding the weight of wasted food to the weight difference before and after feeding. From these data the amount of ingested AFB1 was calculated.
The animals were housed singly initially and provided NIH-31 diet and water ad libitum for one week. After this acclimation period, the four-week-old animals were randomly allocated to control and experimental groups. During the study the animals were housed in pairs and were provided the appropriate diets and water ad libitum. There were six dose groups consisting of animals fed chow diet mixed with either 0.0, 0.01, 0.04, 0.40, or 1.6 ppm of AFB1. The study design is shown in Figure 1 and has been previously described (Morris et al., 1999
) for 20 weeks of the 40-week feeding study. Briefly, experimental groups were fed diets containing AFB1 for four weeks, then they were fed chow diet without AFB1 for another four weeks. The "on diet"/"off diet" cycles, referred to as "intermittent" dosing, were continued up to 40 weeks. One other group was included in the study design; this group of animals received the 1.6 ppm diet continuously for 40 weeks.
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Hematology.
A blood smear, approximately one cell layer thick, was prepared from EDTA treated whole blood from terminal heart bleeds. Air-dried smears were then treated with Wrights stain for viewing. The WBC differential counts were done by scoring 100 cells per slide for the various cell types of interest, i.e., lymphocytes, segmented leukocytes, eosinophils, basophils, and monocytes (Creskoff et al., 1963). Total WBC counts were done as previously described (Hinton et al., 1987
) by counting acid-fixed and gentian violet stained WBCs in a hemacytometer.
Histopathology.
Animals were euthanized by CO2 asphyxiation and then necropsied. Parts of the organs were immediately placed into buffered-formalin and kept at room temperature for 24 h. The tissues were then removed from the formalin and processed through a graded series of ethanol and xylene prior to paraffin embedding.
Splenic cell isolation and purification.
Approximately one-half of the spleen was taken at necropsy for isolation of the splenic lymphocytes. A portion of the spleen was minced immediately after necropsy in a culture disk and then suspended in ice-cold Ultraculture media supplemented with 2 mm L-glutamine. Ten ml of the cell suspension was layered on top of 5 ml of the Histopaque in plastic tubes and then centrifuged at room temperature for 30 min at 2000 rpm. Portions of the purified lymphocytes were then used for flow cytometry and splenic cell cultures for assessment of cytokine production.
Flow cytometry.
Analysis of splenic lymphocyte populations was done by fluorescent antibody cell sorting (FACS) analysis using an EPICS Elite flow cytometer (Beckman/Coulter, Miami, FL). Splenic lymphocytes were suspended in phosphate buffered saline (PBS) containing 2% heat inactivated fetal bovine serum and 0.05 % sodium azide (FACS diluent) at a concentration of 107 cells per ml. Monoclonal antibodies specific for rat cell surface antigens were added to 50 µl of FACS diluent in wells of a 96 well microtiter plate to achieve a predetermined optimal final concentration (0.11.0 µg/50 µl). Ten µl of spleen cell suspension was added to each well. Antibodies used for immunofluorescent staining were directed against rat T-lymphocytes (CD3, CD4, and CD8) or B-lymphocytes (CD45R). Direct FITC or R-phycoerythrin conjugated antibodies were used for staining. Matched isotype control antibody conjugates were also used to determine background staining. Immunofluorescent staining took place for 30 min at 4°C. Samples were then washed twice and resuspended in 100 µl FACS diluent. The FACS analysis was conducted on the viable lymphocyte population as determined by forward light scatter versus 90° light scatter gating. Five thousand cells were analyzed for each antibody combination.
Splenic cell cultures.
Splenic cell cultures, initiated by placing 1 x 106 lymphocytes in 0.1 ml of tissue culture media, were treated with 0.1 ml of Con-A (5 µg/ml final concentration), and then incubated for 24 h at 37°C. The resulting supernatant was collected after centrifugation and stored at -80°C until analyzed for IL-2 production. Production of either IL-1 or IL-6 was assessed by admixing 0.1 ml of 106 splenic lymphocytes with either 0.1 ml of LPS (1 µg/ml final concentration) or LPS with IFN (100 U/ml final concentration) and then incubated for 24 h at 37°C. The resulting supernatant was collected after centrifugation and stored at -80°C until analysis for IL-1 and IL-6 production.
Cytokine production bioassays.
The assays for IL-1, IL-2, and IL-6 were performed as previously described (Lyte et al., 1987 for IL-2 and Schook et al., 1992
for IL-1 and IL-6) using cytokine responsive cell lines and as modified by us (Myers et al., 1995
, 1999
). All assays were performed using complete Ultraculture media, i.e., media supplemented with L-glutamine (2 mM, final concentration), HEPES (50 mM, final concentration), gentamycin (50 µg/ml, final concentration), and sodium bicarbonate (0.075%, final concentration). Interleukin-2 activity was determined by adding 1 x 104 CTLL-2 cells in 100 µl of media to an equal volume of either culture supernate or authentic IL-2 (for the standard curve). The cultures were incubated for 24 h at 37°C with 5% CO2. Four h prior to termination of culture, 20 µl of Alamar blue was added to each well. The resulting fluorescence (560 nm excitation and 590 nm emission) was determined using a CytoFluor 2350 (Millipore, Bedford, MA). The IL-1 activity was determined by admixing 1 x 104 D10S cells (100 µl) suspended in complete Ultraculture media with 100 µl of either the culture supernatant or authentic human IL-1ß (for the standard curve). The cultures were incubated for 72 h at 37°C with 5% CO2. Twenty-four h prior to termination of culture, 20 µl of Alamar blue was added to each well. The resulting fluorescence was then measured. The amount of IL-6 activity was determined by adding 5 x 103 7TD1 cells (in 100 µl complete Ultraculture media) to an equal volume of culture supernate or authentic human IL-6 (for the standard curve). Twenty-four h prior to termination of culture, 20 µl of Alamar blue was added to each well and the resulting fluorescence was measured. Each microtiter plate had its own standard curve, which was used to calculate the activity for the test samples on that particular microtiter plate using the CytoCalc software program.
Cell lines used in bioassays.
The CTLL-2 cells respond to only IL-2 and murine IL-4; they do not measure rat IL-4. The D10S cells used for assessment of IL-1 levels are a subclone of the D10.G4.1 cell line. The parent cell line requires a source of murine IL-4 or IL-5 along with feeder cells and antigen for propagation. It neither responds to nor produces IL-2. The D10S clone has the advantage that it can be propagated in culture without the need for continual antigen stimulation. It also does not respond to IL-2 but does respond to IL-1 (any species), murine IL-4, and murine IL-5. The 7TD1 cells used to measure IL-6 may respond to murine IL-4, but only at very high levels.
Statistical analysis.
All of the data generated were analyzed with validated SAS PC (version 8.2) procedures. These included: means and error procedures for general linear models; ANOVA, Dunnetts multiple pairwise t-tests for comparison of the dose groups to the control, 0.0 ppm group, and linear regression models for evaluating dose responses. Differences between treated groups and the control values that generated p-values equal to or less than 0.05 were considered statistically significant. For comparison of intermittent dosing to the continuous dosing, Dunnetts multiple pairwise t-test was applied also using the continuous, 1.6 ppm dose as the statistical control, comparison group for both the flow cytometry and the cytokine analyses.
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RESULTS |
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Hematology
These data are shown in Figures 2a ,2b
, and 2c
, respectively. Few significant changes were noted in the total WBC counts, percentages of lymphocytes, and percentages of segmented neutrophils during the study. In comparison to the control group, there was an increase in the total WBC count (p
0.05) in the group continuously treated with 1.6 ppm AFB1 after eight weeks on study and in the group intermittently treated with AFB1 after 12 weeks on study. An increase in the percentage of lymphocytes (p
0.05) and a concurrent decrease in the percentage of segmented neutrophils (p
0.05) were observed in the group continuously treated with 1.6 ppm AFB1 after 12 weeks on study.
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The percentages of either T-h or T-s were not statistically different compared to controls for the first four-week period (Figs. 4a and 4b
). Statistically significant (p
0.05) increases in the percentages were seen for the T-h subset at the mid (0.4 ppm) and high (1.6 ppm) dose groups, while statistically significant decreased percentages were seen for the T-s subset at eight weeks compared to the control groups. A statistically significant difference was seen only at the 0.04 ppm dose group for T-h cells while significant (p
0.05) differences were seen for the 0.04, 0.4, 1.6I, and 1.6C dose for T-s cells after 12 weeks. No significant differences in the percentages of the T-h cells were seen for either the 16 or 20 week cycles. Significant decreases in the percentages of the 0.4 and 1.6 ppm dose groups were seen for the T-s cells after 16 weeks. Only the 0.4 ppm dose group of the T-s subset was significantly increased after 20 weeks of study.
Cytokine Proliferation Assays
IL-2.
This cytokine was included in our analyses since it is an important indicator of proliferative capacity and may be involved also in the inflammatory response. The IL-2 productive capacity of the splenic lymphocytes is shown in Figure 5. From 8 (week 4 of the study) to 24 weeks of age (week 20 of the study), the IL-2 productive capacity of the cells in the maturing animal increased from 5 to 35 biological units in the controls. Significantly decreased capacity was observed after eight weeks for the highest doses of AFB1 tested. No consistent pattern of statistical significance, however, was seen at either 12, 16, or 20 weeks.
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In contrast, the hepato-cellular vacuolar change was less prominent in livers of the eight-week intermittently fed animals (Figs. 6c,d). The focal inflammatory cell infiltrates aforementioned were observed more frequently however. Increased numbers of activated Kupffer cells characterized by sinusoidal cells with increased amounts of cytoplasm and vacuolated nuclei as well as small foci of extramedullary hematopoiesis (EMH) were also present.
At 12 weeks of continuous dosing, the severity of the vacuolar cytoplasmic change progressed to mild to moderate and was diffuse in distribution (Fig. 6e). Mild billiary hyperplasia, a proliferative change of the bile ducts was evident. Small basophilic foci of hepato-cellular alteration were seen and were characterized by focal proliferation of hepatocytes, assuming either increased blue (basophilic) or red (eosinophilic) staining properties (Fig. 6e
). Activated Kupffer cells had a condensed nuclear chromatin pattern consistent with pyknosis (Fig. 6f
). Loss of individual hepatocytes as well as apoptotic cell necrosis was seen infrequently. Foci of inflammation, described above and responding to degenerate hepatocytes, increased only slightly.
Most livers of the intermittent high dose group appeared to be less affected by the vacuolar change after 12 weeks (Fig. 7a), while inflammatory cell infiltrates, Kupffer cell activation, and billiary hyperplasia were slightly more prominent at this time point compared to the continuously treated animals (Fig. 7b
).
After 16 weeks of continuous dosing the severity of the vacuolar hepato-cellular change had progressed to moderate. The numbers of mixed inflammatory cell infiltrates in the vicinity of degenerate hepatocytes and around vessels remained small however. The micro-architecture of the liver sections was increasingly distorted due to focal areas of hepato-cellular proliferation. Activated Kupffer cells, billiary hyperplasia, and basophilic foci were prevalent. The vacuolar change in animals of the intermittently fed group was slightly less severe at 16 weeks while the inflammatory response was comparable to continuously fed animals.
After 20 weeks (Fig. 7c), the hepato-cellular vacuolar change, formation of basophilic and/or eosinophilic foci of cellular alteration, billiary hyperplasia, and hepatocellular proliferation leading to distortion of the liver micro-architecture, progressed while inflammation remained mild (Fig. 7d
).
After 40 weeks of continuous and intermittent dosing the normal hepatic architecture was moderately distorted (Fig. 7e), due to progression of the proliferative and neoplastic changes mentioned above. Within areas of hepatic neoplasia, larger aggregations, consisting primarily of lymphocytes, could be observed (Fig. 7f
). These infiltrates differed in size and cell composition from the small inflammatory cell foci responding to degenerate vacuolated liver parenchyma, previously mentioned.
Spleen
(Extensive morphometric and immuohistochemical analyses of various biomarkers in the spleen, thymus, and Peyers patches are the subject of another report in progress.) Herein, we present an evaluation of H&E-stained sections of spleen for the two highest doses of AFB1 in order to screen for the effects on cells involved in inflammatory responses, i.e., macrophage, neutrophils, and lymphocytes.
Evidence of inflammation in the spleen was not observed. However, effects on the distribution of cells within their splenic micro-compartments were evident. The cellularity of the Mantel zones of control animals at four weeks on study was low (Fig. 8a), indicating immunologic immaturity. Hemosiderin-laden macrophages and scattered segmented neutrophils were commonly seen in the outer rim of the Mantel zone bordering the red pulp (Fig. 8b
).
The Mantel zones of animals that had been on the highest dose of AFB1 for four weeks were, in contrast to controls, denser and more cellular indicating immune stimulation. In contrast to control animals, hemosiderin-laden macrophages were not discernable in the high dose AFB1-treated group. Erythrophagocytosis was frequently seen in the AFB1-treated group however. Neutrophil numbers were slightly reduced compared to the control animals.
Mantel zones of eight-week control animals had matured and were more cellular therefore than at four weeks. Hemosiderin-laden macrophages and neutrophils were frequently observed in the outer rim of the Mantel zone. At eight weeks on a continuous AFB1 diet, Mantel zones of most animals appeared irregular. Their outlines were not well defined around the follicles (Fig. 8c), blending into the bordering red pulp and/or into the Mantel zone of the neighboring follicle. Focally, the Mantel zones were thin due to lesser cellularity or they were not discernable at all. Only few hemosiderin-laden macrophages and neutrophils were seen in the outer Mantel zone rim (Figs. 8c,d
).
After eight weeks, the splenic micro-architecture of the high, 1.6 ppm intermittent dose group, with respect to the Mantel zone, was similar to the eight-week control animals displaying even cellularity. The number of hemosiderin-laden macrophages in outer rim of the Mantel zone, however, was reduced. Neutrophil numbers were within limits of the controls.
Splenic mantel zones of animals that had been fed AFB1 continuously and intermittently for 12 weeks were greatly reduced in cellularity and width (Fig. 8e). The number of hemosiderin-laden macrophages appeared to be only slightly less compared to 12-week control animals (Fig. 8f
), but were more frequent in the intermittently fed animals compared to the continuously fed animals. Neutrophil numbers were within limits of the controls.
Animals that were on a continuous diet for 16 weeks presented with spleens similar to 16-week control animals with respect to the Mantel zone micro-architecture (sections not shown). The periarterioalar lymphocyte sheaths (PALS) were of variable size, indicating a decrease of lymphocytes. The cellularity of Mantel zones surrounding small PALS was greatly reduced. Numbers and distribution of hemosiderin-laden macrophages and neutrophils were within limits of the controls. The spleen micro-architecture of the high-dose, intermittent group, at 16 weeks was similar to that of controls.
Splenic Mantel zones of the high, intermittent dose group at 20 weeks were less cellular compared to 20-week control animals. Hemosiderin-laden macrophages and segmented neutrophils were as frequently seen as in control animals (sections not shown). Most spleens, either of animals fed AFB1 for 40 weeks continuously or for 40 weeks intermittently, were comparable in their micro-architecture.
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DISCUSSION |
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There were early reports (Butler, 1970) of "slight" inflammatory responses in rats due to AFB1-induced injury in the liver. The researcher reported, however, that in the chicken, which shows relatively slight cellular degeneration and almost no necrosis, large lymphoid follicles appear in the areas of fatty change. We believe that inflammatory and other possible immune mechanisms in relation to AFB1-induced hepatotoxicity and/or carcinogenicity in the F344 rat needed to be investigated further. There are a number of reports that F344 rats are more susceptible to chemically induced liver injury than other strains (Kuester et al., 2002
). In addition, we found no reports on the immunotoxic effects of AFB1 in an intermittent exposure regimen wherein there would be sufficient "resting" periods in order for the immune system to recover, i.e., either reverse or compensate for the effects of AFB1. We chose to measure IL-1 and IL-6 since these cytokines are increased in an inflammatory response. We measured IL-2 as a measure of lymphocyte proliferative capacity as well as its possible involvement in an inflammatory response. Since there was a suggestion from the cytokine analyses that an inflammatory response may be associated with AFB1 toxicity, we evaluated the histopathology of the liver at the high intermittent and continuous doses for the various periods in the study in order to discern if there was any involvement of the immune system. When we consider all of the cellular data, the time cycle most indicative of possible immune effects was at the 12 week, i.e., the second dosing period. The hematology data also support this observation. Although the hematology data are not generally sensitive indicators of immunotoxic effects (Hinton, 2000
), the total WBC count for the high, intermittent, 1.6 ppm dose was significantly different (p
0.05) from the control at 12 weeks. There were also some indications from the high, continuous, 1.6 ppm dose for the WBC differential count that significant immune effects were occurring at 12 weeks as well. We showed in both the flow cytometric splenic subset analysis and the cytokine bioassays that the immune system also is changing as the animal matures from 4 to 24 weeks of age, i.e., during the important phases of the initial dosing cycles of the feeding study. Thus, we needed to compare the dosed groups to the control for each time period. Results from all of the analyses support the conclusion of significant immune effects at 12 weeks into the study, i.e., after the second dosing cycle. There were suggestions from the flow cytometric analyses that the different lymphocyte populations may compensate or reverse, to some extent, the effects of AFB1 during the "off" or resting cycles. The histopathology evaluation of the spleen presented herein suggested that continuous exposure resulted in cumulative effects on T-lymphocyte cellularity in the PALS and that macrophage function, from analysis of the hemosiderin-laden cells, is suppressed. The histopathology evaluation of the liver demonstrated that AFB1 caused damage to hepatocytes as exemplified by the vacuolar formation. This change was more severe after eight weeks of continuous dosing compared to the intermittent high dose group, but was equivalent in both dose groups after the 12-week dosing cycle. Mixed inflammatory cell infiltrates formed after four weeks of dosing but were more abundant in the intermittent dose group after eight weeks compared to the eight week and the 12-week intermittent dose group, likely as a result of a degree of recovery from immunosuppression and in response to the degenerate hepatocytes. This inflammatory process was most pronounced at 12 weeks however. At 12 weeks of continuous dosing there were early preneoplastic lesions. After 40 weeks of continuous dosing there were defined inflammatory infiltrates/immune responses to the damaged liver suggesting that even after this length of duration of exposure at the highest dose, the immune system had enough reserve capacity to function to some extent. It was also of interest that there were more inflammatory infiltrates in the intermittent dose groups compared to the continuous at 8, 12, 16, and 20 weeks. Suppression of the inflammatory response via suppression of Kupffer cell activation in the liver by AFB1 is in agreement with suppression of macrophage function as seen in the splenic histopathology. In order to correlate the histopathology results with the flow cytometric and cytokine proliferative responses, we compared the statistical significance of the intermittent dosing (both the flow cytometric and the cytokine measurements) using the 1.6 ppm continuous dose group as the statistical control (data not shown). (This was in addition to the statistics presented herein which used the 0.0 ppm dose group as the statistical control.) Significant statistical differences were prevalent beginning with the eight-week period for many of the immune parameters at low doses. There was almost a complete absence, however, of statistically significant differences when the continuous 1.6 ppm dose group was compared to the same intermittent dose group. This suggests that, at least at the 1.6 ppm dose, the immunotoxic effects are cumulative and are either not repaired after sufficient time of exposure or are repaired slowly within the time frames used as the resting cycle in this study.
As mentioned previously, the initial rationale for this study was with regard to risk assessment of the hepatocarcinogenic potential of AFB1. The intermittent dosing regimen is also a more realistic approach to exposure and allows for accumulated dose extrapolations. There are various reports (Henry et al., 2002) where the risk of hepatocarcinoma is greatest in those regions of the world, e.g., Africa and China (Wang et al., 2001
) where there are both high percentages of hepatitis B (HB) and C (HC) infections and contamination of foodstuffs by aflatoxin B1. One of the major debates in hepatocellular carcinogenesis (Kew, 1992
) is whether the HB and HC viruses are directly carcinogenic or exert their effect indirectly by causing chronic necro-inflammatory hepatic disease, which in turn is responsible for malignant transformation of hepatocytes. In other words, HB and HC viruses as well as AFB1 acting alone could lead to hepatocarcinoma, provided that the critical doses and damage to the liver are sufficient to induce immunologic mediated necrosis in the liver. In our study this point appears to be after the second dosing cycle, at least for the high doses tested.
An analogy to the mechanism of AFB1 possible involvement in carcinogenesis is dimethynitrosamine (DMN). First, DMN also forms adducts (methyl-and hydroxyl guanyl) in the liver (and other tissues). Cirrhosis in the early phases is accompanied by inflammatory filtrates (Mancini et al., 1991) composed mainly of T-cytotoxic-inducer/T-suppressor cells. There are progressive stages of immune mediated damage to the liver resulting in hepatocarcinoma.
In conclusion, the immune system of the rat is both a target for the toxic effects of AFB1 and a participant in immune mediated inflammatory reactions/immune responses in the liver of the F344 rat. Thus, the effects that were seen in the "intermittent" dosing study were complex suggesting that the immune system may compensate or reverse, at least partially, the toxic effects during the "resting cycles" at doses lower than 1.6 ppm. After the second resting cycle at 16 weeks, it appeared that the percentages of T and B cells were being reversed in comparison to the 8- and 12-week results (Figs. 3a,b). The shift in T-h and T-s cells seen in Figure 4
during the resting cycles, i.e., increased T-h (Fig. 4a
) and decreased T-s (Fig. 4b
), suggests a compensatory change in response to the down regulation of the percentages of B-lymphocytes after four weeks (Fig. 3b
). These results are likely a reflection of the direct effects of AFB1 on lymphocyte proliferation and function during the dosing cycles and the compensatory/recovery efforts when AFB1 is not included in the diet. The most significant results from the cytokine analysis were the correlations with the histopathology evaluation of the liver. When the results of the IL-1 and IL-6 were considered together, they were suggestive of the induction of an inflammatory response occurring after the second dosing cycle at 12 weeks. The histopathology evaluation, however, demonstrated that the immune system had enough reserve capacity even at the high, 1.6 ppm, continuous dose to be associated with the hepatotoxic/hepatocarcinogenic processes at the end of the 40-week study.
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ACKNOWLEDGMENTS |
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NOTES |
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