Mechanisms of Exocrine Pancreatic Toxicity Induced by Oral Treatment with 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in Female Harlan Sprague-Dawle
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《毒物学科学杂志》
Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Laboratory of Computational Biology and Risk Analysis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
ABSTRACT
In previous 2-year studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) conducted by the National Toxicology Program on female Harlan Sprague-Dawley rats, acinar-cell vacuolation, atrophy, inflammation, and arteritis developed at high incidence, and a rare occurrence of pancreatic acinar-cell adenomas and carcinomas was noted. In this investigation, we sought to identify the mechanism involved in the early formative stages of acinar-cell lesions. Pancreas from animals treated for 14 and 31 weeks with 100 ng TCDD/kg body weight or corn oil vehicle was examined immunohistochemically and/or morphometrically. Acinar-cell kinetics were analyzed using staining with hematoxylin and eosin and proliferating cell nuclear antigen. Expressions of cytochrome P450 (CYP) 1A1 and aryl hydrocarbon receptor (AhR) were evaluated to assess direct effects of TCDD exposure. The cholecystokinin-A receptor (CCK-A receptor; CCKAR) and duodenal cholecystokinin 8 (CCK) revealed the associations of dioxin treatment with hormonal changes. Amylase localization showed acinar structural changes that could affect enzymatic production. Increased apoptotic activity in acinar cells occurred in 14- and 31-week-treated animals, with an increase in proliferative activity in the latter. Also in the latter, in the vacuolated acinar cells, CYP1A1 was overexpressed, and statistically significant decreases in expressions of AhR, CCKAR, and amylase occurred. The intensity of CCKAR expression increased in nonvacuolated acinar cells; a decrease in the size of CCK-positive epithelial cells occurred in duodenum. Our findings indicate that dioxin-induced acinar-cell lesions might be related to a direct effect of TCDD on the pancreas. Increase in CYP1A1 and decrease in CCKAR expressions in vacuolated acinar cells may be involved in the pathogenesis of pancreatic lesions. Changes in the expression of CYP or CCKAR may have induced the acinar-cell tumors by initiating proliferation.
Key Words: acinar cells; dioxin; immunohistochemistry; pancreas; rat; vacuolation.
INTRODUCTION
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as dioxin, is an environmental contaminant that is known for its extreme toxic potency. Certain polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (PCBs) exhibiting biologic actions similar to those of TCDD have been commonly designated dioxin-like compounds (DLCs). They may induce developmental, endocrine, and immunological toxicity and multiorgan carcinogenicity in animals and/or humans (ATSDR, 1998; Bertazzi et al., 2001; Kociba et al., 1978; Steenland et al., 1999, 2001). Incidences of cancer have been evaluated in several analyses of human populations exposed to elevated amounts of dioxin and DLCs. The most recent studies indicate that exposure is associated with an increase in all cancers combined and several specific cancers including rectal cancer, lung cancer, Hodgkin disease, non-Hodgkin lymphoma, and myeloid leukemia, as well as chronic circulatory diseases, respiratory diseases, and diabetes (ATSDR, 1998; Bertazzi et al., 2001).
Recently, the National Toxicology Program (NTP) conducted 2-year bioassays in female rats to evaluate the chronic pathology and carcinogenicity induced by dioxin, DLCs, structurally related PCBs, and mixtures of these compounds, including TCDD; 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachloro-dibenzifyran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); the toxic equivalency factor (TEF) tertiary mixture of TCDD, PCB126, and PeCDF; and the binary mixtures of PCB126 and 153 and PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118) (National Toxicology Program, 2004a, 2004b, 2004c, 2004d, 2004e, 2004f, 2004g). In these studies, an increase occurred in the incidence of neoplastic effects, such as cholangiocarcinoma and/or hepatocellular adenoma of the liver, cystic keratinizing epithelioma of the lung, and gingival squamous-cell carcinoma of the oral cavity (Brix et al., 2004; Jokinen et al., 2003; National Toxicology Program, 2004a, 2004b, 2004c, 2004d, 2004e, 2004f, 2004g; Tani et al., 2004; Walker et al., 2004; Yoshizawa et al., 2005). Pancreatic toxicities, such as acinar cytoplasmic vacuolation, atrophy, inflammation, and arteritis have been seen at high incidence combined with a rare occurrence of acinar-cell adenomas and carcinomas (Nyska et al., 2004).
Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of TCDD and related DLCs (Denison and Nagy, 2003; Poland and Knutson, 1982; Schmidt and Bradfield, 1996). It is expressed ubiquitously in human and rodent systemic organs, particularly in the pancreas (Dolwick et al., 1993; Yamamoto et al., 2004). That most organs with AhR expression are thought to be more susceptible to TCDD-induced toxicity is evidenced by AhR-deficient mice that are resistant to some kinds of TCDD-induced toxicity (Fernandez-Salguero et al., 1996; Gonzalez and Fernandez-Salguero, 1998). No reports are available regarding the relationship of exocrine pancreatic toxicity and change in AhR expression after TCDD treatment in rats. The most well-studied response to TCDD is induction of the CYP 1A1 and 1B1 classes of cytochrome P450 (Denison and Nagy, 2003; Schmidt and Bradfield, 1996; Toyoshiba et al., 2004) regulated by the AhR (Nebert et al., 2004); their inductions, especially that of CYP1A1, are sensitive to TCDD and thus serve as useful markers for exposure to TCDD. These enzymes play a major role in the activation and deactivation of toxins and carcinogens. The susceptibility to pancreatic disease seems to depend on the integrity of the cellular detoxification process governed by these drug-metabolizing enzymes (Standop et al., 2002, 2003; Ulrich et al., 2002). Differences in immunohistochemical expression of CYPs, such as 1A1, 1A2, 2B6, 2D6, and/or 3A1 among normal pancreas, chronic pancreatitis, and pancreatic tumors have been reported (Foster et al., 1993; Standop et al., 2003); the possibility of an etiological relationship between elevated levels of CYPs and the subsequent development of human pancreatic diseases has been highlighted.
In tissues derived from the previous NTP oral-treatment studies of TCDD; PCB 126; PeCDF; a tertiary mixture of TCDD, PCB126, and PeCDF and a binary mixture of PCB126 and PCB153, duodenal levels of stored cholecystokinin (CCK) peptide decreased prominently (Lee et al., 2000). Cholecystokinin, regarded as the major hormonal mediator of pancreatic enzymatic production, assessed chiefly by levels of amylase, is secreted by the proximal small intestine under the control of a negative feedback loop (Bourassa et al., 1999; Furukawa et al., 2001; Haschek and Rousseaux, 1998; Wank, 1995). The peripheral receptors can be classified pharmacologically as CCKA receptor (CCKAR), expressed predominately in pancreas, and as CCKB receptor (CCKBR), localized chiefly in the central nervous system (Monstein et al., 1996; Tang et al., 1996; Wank, 1995). Cholecystokinin induces secretion of pancreatic enzymes and synthesis of protein and DNA in the rat acinar cell through its CCKAR (Varga et al., 1998; Wank, 1995). Continuous blocking of this receptor by CCK inhibitors causes atrophic changes (Biederbick and Elssser, 1998; Ohlsson et al., 1995). The close relationship between the expression of the CCKAR and pancreatic disease has been shown experimentally in several models, such as azaserine- or caerulein-induced pancreatitis and cancer (Bourassa et al., 1999; Gebhardt et al., 2004; Meijers et al., 1992; Roebuck et al., 1987). The pathogenesis of exocrine pancreatic toxicity induced by TCDD has not been clarified.
Taking this information into consideration, we investigated the potential pathways involved in the TCDD-induced acinar pancreatic pathology. Hypothesizing that the early changes in AhR, CYP1A1, and CCK-related proteins in acinar cells after treatment with TCDD might be related to induction of toxicity in exocrine pancreas and attempting to elucidate the mechanism in rats, we conducted this retrospective immunohistochemical study of pancreas and duodenum in rats treated with TCDD for 14 and 31 weeks.
MATERIALS AND METHODS
Study design.
This investigation originated from a series of studies undertaken by the NTP to determine the suitability of the Toxic Equivalency Factor (TEF) methodology for predicting chronic toxicity and carcinogenicity induced by TCDD and DLCs (National Toxicology Program, 2004a, 2004b, 2004c, 2004d, 2004e, 2004f, 2004g). Female Harlan Sprague-Dawley rats were used, because they showed more sensitivity to the effects of TCDD than the male in previous studies (Kociba et al., 1978). Approximately 10 animals/group were administered 0, 3, 10, 22, 46, and 100 ng/kg TCDD for 14, 31, and 53 weeks (10 rats for 14 and 31 weeks, 8 rats for 53 weeks); in the 2-year study, 49 to 54 animals were given the same doses by daily gavage for 5 days per week up to 2 years. In addition, rats treated with 100 ng/kg for 30 weeks followed by vehicle treatment through the termination of the 2-year study were designated the 100 ng/kg stop group. Control animals
Chemical.
2,3,7.8,-Tetrachlorodibenzo-p-dioxin (lot no. CR82-2–2, purity approximately 98%, with no change in purity throughout the study) was supplied by IIT Research Institute (Chicago, IL). Purity of the chemical was analyzed with a gas chromatography/high resolution/mass spectrometry system (GC/HR/MS). Acetone and corn oil were obtained from Spectrum Quality Products (Gardena, CA). Specific amounts of TCDD dissolved in acetone were added to corn oil to yield concentrations of 0, 1.2, 4, 8.8, 18.4, or 40 ng TCDD/ml of vehicle determined by GC/HR/MS to be within 10% of target concentrations. Studies of the dose formulations indicated that the chemical could maintain an acceptable homogeneity for dosing and stability for 35 days when stored at room temperature.
Animals.
Animal studies were conducted at an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility of Battelle-Columbus Laboratories (Columbus, OH) for the duration of the study. Animal handling and husbandry were conducted in accordance with National Institutes of Health (NIH) guidelines (Grossblatt, 1996). Upon receipt, the female Harlan Sprague-Dawley rats were approximately 6 weeks of age. They were held under quarantine for 15 days for health screening by a veterinarian and were approximately 8 weeks of age at the start of the study. Animals were randomly assigned to control or TCDD-treated groups and permanently identified with tail tattoos. They were housed five to a cage in solid-bottom polycarbonate cages (Lab Products, Inc., Maywood, NJ). Filtered room air underwent at least 10 changes per hour. The animal room was maintained at 69°–75°F with 35–65% relative humidity and 12 h each of light and darkness. Irradiated NTP-2000 pelleted feed (Zeigler Bros., Inc., Gardner, PA) and water were available ad libitum. All rats were checked twice daily for morbidity and once per month for clinical signs of toxicity; moribund animals were euthanized and necropsied. All animals were weighed weekly for the first 3 weeks of the study, at 4-week intervals thereafter, and again at necropsy.
Pathology.
Complete necropsies were performed on all animals using standardized methodology. At necropsy, all tissues including masses and macroscopical abnormalities were removed and fixed in 10% neutral buffered formalin for microscopical evaluation. After fixation, the tissues were trimmed, dehydrated, cleared, and paraffin-embedded. Five-μm-thick sections were mounted onto glass slides, stained with hematoxylin and eosin (H&E), and examined microscopically. The severity of lesions was graded on a four-point scale of 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked. All data including pathological examination were obtained according to GLP, underwent extensive pathology peer review (Boorman et al., 2002), were peer-reviewed by an external expert panel advisory board, and are available online (http://ntp.niehs.nih.gov).
Quantative analysis of apoptotosis in acinar cells (apoptotic index).
The standard for recognition and quantitation of apoptotic bodies is analysis of H&E-stained sections (Goldsworthy et al., 1996). For counting the numbers of apoptotic bodies, distinguished by the presence of marginal chromatin condensation and/or fragmented nuclei, approximately 300 acinar cells randomly selected from 4 different areas in each section were examined under a light microscope at a magnification of x400. The apoptotic index represented the percentage of apoptotic bodies per total number of acinar cells counted (Imazawa et al., 2003).
Immunohistochemical investigation of CYP1A1, CCK, AhR, CCKAR, amylase, and proliferating cell nuclear antigen (PCNA).
Additional paraffin-embedded sections of pancreas from each of eight controls and eight rats exposed to 100 ng/kg TCDD for 14 and 31 weeks were analyzed immunohistochemically to determine effects of treatment on the expressions of drug-metabolic enzyme (CYP1A1), dioxin-binding and CCK-binding receptors (AhR, CCKAR, respectively), exocrine pancreatic enzyme (amylase), and acinar-cell proliferation. Duodenal sections of the same animals were examined to determine effects of treatment on the expression of CCK8, which is a nonselective ligand for CCKA or CCKB receptors. Table 1 summarizes the immunohistochemical staining procedures. Detailed staining protocols for all antibodies are listed on the Web site of the National Institute of Environmental Health Sciences (NIEHS) Laboratory of Experimental Pathology (http://dir.niehs.gov/dirlep/immuno/). Briefly, sections were deparaffinized, hydrated, and blocked for endogenous peroxidase. Heat-induced epitope retrieval was performed for all antibodies except CYP1A1 (http://dir.niehs.gov/dirlep/immuno/retrievals.htm).
For counting the numbers of cells positive for PCNA in pancreas, approximately 300 acinar cells were randomly selected from four different areas in each section and examined under a light microscope at a magnification of x400. The PCNA-labeling index represented the percentage of PCNA-positive cells per total number of acinar cells counted (Imazawa et al., 2003).
To designate the grading of CYP1A1, AhR, CCKAR, and amylase, the intensity of immunopositivity, or the relative area of the sections showing staining, was graded by two pathologists on a scale ranging from 0 (–) to 3 (+++). Immunostainings of CYP1A1, AhR, and amylase were estimated as follows: (0) = no specific immunohistologic reaction visible in acinar cells, (1) = <30% of total acinar-cell area showing a positive reaction, (2) = up to 60%, and (3) = up to 100%. The degree of CCKAR immunolocalization in acinar cells was scored from 0 to 3, corresponding to the presence of negative, weak, moderate, or strong brown staining, respectively. In 31-week TCDD-treated groups, we analyzed the acinar-cell area of nonvacuolated and vacuolated cells separately, compared to controls. Cholecystokinin-positive epithelial cells in the duodenum were counted in four randomly selected areas in each section and examined under a light microscope at a magnification of x200. Five positive cells containing nuclei were randomly selected from five different areas in each section under a light microscope at a magnification of x400 and analyzed for cell size with free software, "Image Processing and Analysis in Java (Image J, http://rsb.info.nih.gov/ij/)."
Statistical analysis.
Incidences of lesions in the study animals were evaluated statistically by the Poly-3 test, which makes adjustments for survival differences among groups. Incidences of lesions in animals from each of the interim evaluations and the 2-year study were analyzed separately. For animals in the 2-year studies, the incidences of total lesions, including findings from animals that survived until study termination and from early-death animals, were included in the analysis. The immunohistochemical scores and morphometrical data of the control and high-dose groups from 14 and 31 weeks were analyzed statistically. We used the t-test to compare two groups when the data were normally and independently distributed, as in the comparison of the apoptotic index and PCNA- and CCK-labeling indices with respective control groups. For analyses of non-normal data and CCK-positive cell size, we used the Wilcoxon rank-sum test. Tukey's multiple comparison test was used for comparing the control group with the 31-week-treated vacuolated group and the 31-week-treated nonvacuolated group for scores for CYP1A1, AhR, CCKAR, and amylase.
RESULTS
Pancreatic Histopathology
Acinar cytoplasmic vacuolation occurred significantly after treatment with 100 ng/kg TCDD in 5 of 10 rats in the 31-week group, 7 of 8 rats in the 53-week group, and 42 of 51 rats in the 2-year group, and in 15 of 53 animals treated with 46 ng/kg TCDD for 2 years (Nyska et al., 2004) (Fig. 1d and 1e; Table 2). The incidence and severity of this lesion were clearly dose-dependent and time-dependent. These lesions were not observed in the animals of the 100 ng/kg stop group.
The incidence of chronic active inflammation was increased significantly in the 2-year group after treatment with 100 ng/kg of TCDD (6 of 51) (Table 2), although the same lesion was observed after 14 and 53 weeks of the same TCDD treatment (Fig. 1e). Acinar atrophy was generally associated with chronic active inflammation (6 of 9). In the animals of the 100 ng/kg stop group, the decrease in the incidence and severity of these lesions suggested that they regressed by withdrawing TCDD treatment.
At 2 years, arterial chronic active inflammation appeared significantly in 7 of 51 rats showing fibrinoid necrosis of the vessel in the 100 ng/kg group (Jokinen et al., 2003; Nyska et al., 2004) (Fig. 1e; Table 2). The arterial lesions only occasionally accompanied the other described changes, such as acinar cytoplasmic vacuolation, acinar atrophy, and/or chronic active inflammation (7 of 51), and therefore were not considered the primary cause of the acinar pathology.
One acinar-cell adenoma and two carcinomas were seen in the 100 ng/kg group 2 years after TCDD treatment (Fig. 1f), and one carcinoma was seen in the 100 ng/kg stop group. The incidences of acinar-cell tumors exceeded distinctly the historical control range (Table 2). The pancreatic acinar tissue of the 2-year controls exhibited features comparable to those of the 31-week control animals.
Cellular Kinetics of Acinar Cells in Exocrine Pancreas
A small number of acinar cells contained apoptotic bodies in control rats from the 14- and 31-week groups (labeling indices: 0.1%). The index was significantly higher (labeling index: 0.5%, p < 0.05) in the 14-week group treated with 100 ng/kg TCDD, compared to the control value (Figs. 1c, 4a). In the 31-week TCDD-treated group, the incidence of apoptotic acinar cells was significantly higher (labeling index: 0.7%, p < 0.01) than that of control and 14-week TCDD-treated groups (Figs. 1d, 4a).
In the exocrine pancreas in the 14- and 31-week control groups and 14-week TCDD-treated groups, only a few acinar cells were positive for PCNA (labeling indices: <0.1%) (Fig. 4b), although some islet cells were positive (Fig. 2a). In contrast, the number of acinar cells whose nuclei showed positivity for PCNA protein was significantly higher in the 31-week TCDD-treated group (labeling index: 2.5%, p < 0.05) than in the control and 14-week TCDD-treated groups (Figs. 2b, 4b). Most of the PCNA-positive acinar cells were located in the area surrounding the vacuolated cells.
Relationship Between Toxicity and Metabolic Enzyme Induction in Exocrine Pancreas
In control rats from the 14- and 31-week groups, acinar cells were weakly positive for CYP1A1 protein; that is, the relative area stained was less than 30% of the entire area seen in the preparations (average grade: 0.9–1.3; Fig. 2c). Most islet cells exhibited moderate cytoplasmic positivity, and some ductal epithelial cells showed positive signals. In 31-week TCDD-treated rats, vacuolated acinar cells showed strongly positive cytoplasmic staining, with the relative area of staining significantly increased (average grade: 2.3, p < 0.01; Figs. 2d and 4c). In contrast, no difference occurred between the areas of treated nonvacuolated acinar cells and controls (Fig. 4c). The cytoplasmic expression of CYP1A1 in islet cells and ductal epithelial cells appeared similar to the pattern seen in age-matched controls.
Nature of Cytoplasmic Vacuolation of Acinar Cells Based on Amylase Localization
To analyze the change in quantity of zymogen granules in acinar cytoplasm, a finding indicative of pancreatic enzymatic secretion, exocrine pancreatic tissue was stained with amylase antibody. In the control exocrine pancreas in the 14- and 31-week groups, amylase-positive granules were contained within zymogen granules in the cytoplasm of most acinar cells (average grade: 2.5–3.0; Figs. 2e and 4d). The degree of acinar amylase expression significantly decreased in the 14-week TCDD-treated group (average grade: 2.0, p < 0.05; Fig. 4d). In the 31-week treated rats, vacuolated acinar cells were generally negative for amylase, suggesting the loss of amylase-positive zymogen granules (average grade: 0; Figs. 2f and 4d), whereas the expression of amylase-positive granules decreased significantly in nonvacuolated acinar cells (average grade: 1.1, p < 0.01; Fig. 4d). In all control and TCDD-treated animals, ductal epithelial cells and islet cells were negative for amylase protein. Pancreatic secretion in ducts was slightly positive; however, there was no significant difference between controls and TCDD-treated groups.
Characteristic Expression of AhR in Exocrine Pancreas
In control rats in the 14- and 31-week groups, acinar cells were weakly to moderately positive for AhR protein; that is, the relative area was somewhat less than 60% of the preparation (average grade: 1.4–2.1; Figs. 3a and 4e). In 31-week TCDD-treated rats, vacuolated acinar cells completely lost cytoplasmic positivity, and the relative area decreased significantly (average grade: 0, p < 0.01; Figs. 3b and 4e). In contrast, the expression increased slightly but not significantly in the nonvacuolated acinar regions (Fig. 4e). In all control and TCDD-treated animals, islet cells exhibited slight positivity for AhR protein; however, no significant difference occurred between controls and TCDD-treated groups. Ductal epithelial cells and pancreatic secretion showed negative staining in both groups.
Relationship Between Changes in Expression of CCKAR in Exocrine Pancreas and CCK in Duodenum
In control rats of the 14- and 31-week groups, a small number of acinar cells expressed CCKAR protein with weak staining intensity in the cytoplasm (average grade: 0.6–1.0; Figs. 3c and 4f). In 31-week TCDD-treated rats, vacuolated acinar cells lost completely the positive cytoplasmic staining (average grade: 0; Figs. 3d and 4f). In contrast, the degree of CCKAR staining increased significantly in nonvacuolated acinar cells (average grade: 2.0, p < 0.01; Figs. 3d and 4f). In all control and TCDD-treated animals, islet cells, ductal epithelial cells, and pancreatic secretion showed negative or weakly positive staining for CCKAR protein.
In control animals of the 14- and 31-week groups, some epithelial cells in the duodenum exhibited positive cytoplasmic staining for CCK protein (Fig. 4g). The CCK-labeling index showed no significant changes in TCDD-treated rats at 14 and 31 weeks, compared to the age-matched controls (Fig. 4g). An analysis of cell size of CCK-positive epithelial cells revealed a tendency toward a slight decrease in 31-week TCDD-treated rats, compared to the age-matched controls, though without significant difference (Fig. 4h).
DISCUSSION
In this retrospective immunohistochemical study, we evaluated the pathogenesis of pancreatic toxicity induced by chronic exposure to dioxin. Although several surveys of toxicities in animals and side effects in humans induced by dioxin have been published, exocrine pancreatic toxicity has not been completely elucidated. Our literature survey revealed that TCDD-induced exocrine pancreatic lesions, such as vacuolation (accompanied by decreased cytoplasmic volume and numbers of zymogen granules) and ballooning degeneration, have been reported only in the rainbow trout (Spitsbergen et al., 1988) and zebrafish (Henry et al., 1997). Pancreatic neoplasms have been reported in fish exposed to polycyclic aromatic hydrocarbons (Fournie and Vogelbein, 1994).
In the rat pancreas, only minimal destruction of cells in the islets of Langerhans (Gorski et al., 1988; Kociba et al., 1976, 1978; Pohjanvirta and Tuomisto, 1994) has been reported. Exposure of female Sprague-Dawley rats of the Spartan strain to 100 ng/kg TCDD for 2 years was previously associated with atrophy, fibrosis, and periarteritis of the pancreas (Kociba et al., 1978). In our study, acinar changes, such as vacuolation, inflammation, and/or atrophy, were detected without any associated vascular changes (Table 2); therefore, early changes in exocrine pancreas might not be related directly to vascular lesions but, rather, may result from direct toxicological damage (Nyska et al., 2004).
The apoptotic and/or PCNA-labeling indices of acinar cells increased significantly in 14- and 31-week TCDD-treated groups. Because PCNA-positive cells were located in the areas surrounding vacuolated cells, these combined results suggest that, after vacuolation of acinar cells, apoptosis occurred, accompanied by regenerative change. Cellular proliferation does not always lead to increased carcinogenesis in rodents or humans, as in the case of chronic toxic lesions in which normal levels of cellular turnover have been observed in target organs of nongenotoxic carcinogens (Ward et al., 1993). Epidemiologic studies, however, support the concept that inflammation associated with glandular destruction and proliferative activity constitutes a risk factor for exocrine pancreatic cancer (Keim, 2003; Whitcomb, 2004). Several environmental agents that have been proposed as causal for chronic pancreatitis, and pancreatic carcinomas are associated with the wood and pulp industry, the dry cleaning business, and gasoline production and usage (Alguacil et al., 2003; Foster et al., 1993; Lin and Kessler, 1981; Milham and Demers, 1984; Ojajarvi et al., 2000, 2001). In our study, especially in the 2-year group, chronic active inflammation was observed. The relationships between chronic inflammation, oxidative stress, and tumorigenesis were previously addressed (Nyska et al., 2004); the promotion of tumor progression by nitric oxide illustrated such relationships (Xu et al., 2002). Enhanced replication of acinar cells at an early stage might, therefore, somehow be involved in the pancreatic carcinogenesis induced by TCDD.
Several chemicals induce acinar-cell vacuolation, followed by acinar-cell death and/or inflammation, such as ethanol, N-nitrosobis(2-oxopropyl) amine, and ethionine, which induce acinar cell tumors in rodent models (Haschek and Rousseaux, 1998; Horne and Tsukamoto, 1993; Scarpelli et al., 1986; Walker et al., 1993). Two kinds of acinar-cell vacuolation in pancreas have been reported—anoxic and secretory (Tapp, 1970). Amylase expression in acinar cells decreased significantly in 14- and 31-week treated rats, suggesting the loss of amylase-positive zymogen granules. Vacuolation induced by TCDD might not be an anoxic change, because the imunohistochemical results revealed an increase in CCK secretion.
The slightly decreased size of CCK-positive epithelial cells in the duodenum was detected in rats treated with TCDD for 31 weeks, although without significant difference. Because TCDD decreases intestinal levels of stored CCK peptide in vivo and in vitro, without observed changes in CCK mRNA levels (Lee et al., 2000), our data might suggest an oversecretion of CCK from the small intestine. The CCKAR expression that we observed disappeared completely in vacuolated acinar cells with overexpression in nonvacuolated acinar cells. Continuous CCK stimulation for 3 days leads to decreased CCKAR expression in exocrine pancreas (Ohlsson et al., 2000), and downregulation of CCKAR in vacuolated acinar cells corroborates this finding. Upregulation in nonvacuolated cells might suggest a reaction in PCNA-positive regenerative acinar cells for several reasons. CCKAR plays an important role in pancreatic proliferation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking the CCKAR gene (Moralejo et al., 2001), and CCKAR mRNA is maximally expressed and sustained during the regeneration of exocrine pancreas in pancreatitis (Bourassa et al., 1999). Long-term endogenously induced hypercholecystokininemia in rat pancreaticobiliary-duct models not only induces hypertrophy and proliferation of the exocrine pancreas (Ohlsson et al., 1995; Panozzo et al., 1995), but also causes the formation of pancreatic tumors (Stace et al., 1987). All of this information collectively supports our speculation that the change in CCK-CCKAR is likely relevant to TCDD-induced exocrine toxicity in rats.
In our analysis of the NTP studies, especially in the animals treated with 100 ng/kg TCDD for 31 weeks, we found liver damage, including hepatocellular hypertrophy, multinuclear hepatocytes, hepatocellular fatty change, pigmentation (iron and/or lipofuscin), mixed cell foci, and toxic hepatopathy. In addition, cholangiofibrosis and bile duct fibrosis were noted in the 53-week TCDD-treated group. Bile-duct damage and proliferation induced by TCDD might have been caused by uroporphrin (Smith et al., 2001); bile-duct proliferation induced cholestasis in an experimental model of bile-duct ischemia (Beaussier et al., 2005). Increases in levels of cholesterol, alkaline phosphatase, and 5'-nucleotidase may reflect cholestatic changes in rats treated with TCDD for 30 weeks, even though no change occurred in total bile acid and bilirubin in serum (Maronpot et al., 1993). Cholestatic conditions stimulate CCK secretion from intestine and amylase output from pancreatic acinar cells (Kurosawa et al., 1989; Tangoku et al., 1992). Wyeth-14,643 induced pancreatic tumors via sustained increases in CCK levels secondary to hepatic cholestasis, with significant increases in total bile acid, alkaline phoshatase, and birubin in serum (Obourn et al., 1997b). These data suggest the possibility that the weak cholestatic change seen in our study also may be related to the change of CCK levels at 14 and 31 weeks, although we have no blood chemistry. Expression of AhR disappeared completely in vacuolated acinar cells in 31-week TCDD-treated rats in our study. After TCDD binding to AhR in the cytoplasm of the target cells, the resultant ligand–receptor complex translocates to the nucleus, where its binding to TCDD response elements (Denison and Nagy, 2003; Schmidt and Bradfield, 1996) can be visualized by decreasing degrees of AhR staining in the cytoplasm (Pollenz et al., 1994). Exposure to TCDD induced the sustained depletion of AhR protein in nine distinct cell culture lines derived from human and rodent tissues and in liver, spleen, thymus, and lung in a rat in vivo model (Pollenz et al., 1998, 2002). Our results may, therefore, reflect translocation of the TCDD-AhR complex and subsequent downregulation, as seen in these models. A slight but not significant upregulation in nonvacuolated cells may suggest a compensatory reaction in proliferative acinar cells, similar to that seen in human chronic pancreatitis and pancreatic cancer (Koliopanos et al., 2002).
Our results revealed overexpression of CYP1A1 in vacuolated acinar cells, compared to nonvacuolated acinar cells and control pancreas; the expression of CYP1A1 in islet cells and ductal epithelial cells appeared similar to its localization reported previously (Ulrich et al., 2002). In the pancreas, two AhR ligands, 3-methylcholanthreme and TCDD, have been shown to induce CYP1A in rat and fish, respectively (Clarke et al., 1997; Ortiz-Delgado and Sarasquete, 2004; Wilson et al., 1990). In fish, TCDD induces exocrine pancreatic toxicity involving acinar-cell degeneration and necrosis in association with overexpression of CYP1A (Ortiz-Delgado and Sarasquete, 2004). Overexpression of CYP1A1 in vacuolated acinar cells in our study could be mediated by the AhR signaling pathway, because a change in AhR expression was involved in this enhancement. Dioxin causes cellular oxidative stress (Hassoun et al., 2002) followed by cell-cycle arrest and apoptosis via the induction of CYP (Matsumura, 2003). The increased apoptotic index in our study may be related to cellular oxidative stress in acinar cells exposed to TCDD.
Antiestrogenic effects of TCDD in rodents include inhibition of estrogen-induced mammary and uterine cancers via AhR–estrogen receptor crosstalk (Safe, 2001; Safe and McDougal, 2002; Safe and Wormke, 2003). Acinar-cell tumors are enhanced by testosterone and inhibited by estrogen (Longnecker and Sumi, 1990; Robles-Diaz and Duarte-Rojo, 2001); the incidence of spontaneous and induced neoplasms of the exocrine pancreas in rats is higher in males. In several mechanistic studies of pancreatic tumorigenesis involving trypsin inhibitors and PPAR agonists, the use of male rats may have resulted in a higher incidence of acinar-cell tumors (Obourn et al., 1997a,b; Rao and Subbarao, 1995). Strong implications for a role of estrogen in the promotion of pancreatic carcinogenicity have emerged from both in vivo and in vitro studies (Andren-Sandberg et al., 1999). In our retrospective study, the suppression of endocrine-induced tumors, such as mammary and pituitary, could be related to increased prolactin, or they might occur secondary to reduction in body-weight gain or to reduced endocrine activity. All of these factors may exclude a potential direct antiestrogenic effect of TCDD. In Kociba's study, the decreased incidence of acinar-cell tumors was noted in male rats, but no change in incidence occurred in female rats (Kociba et al., 1978). Whether the higher incidence of acinar-cell tumors in our 2-year female rat study could be related to antiestrogenic activity of TCDD remains, therefore, unclear.
Our findings indicate that dioxin-induced acinar-cell lesions might be related to a direct effect of TCDD on the pancreas. Increase in CYP1A1 and decrease in CCKAR expressions in vacuolated acinar cells may be involved in the pathogenesis of pancreatic lesions. Changes in the expression of CYP or CCKAR may have induced the acinar-cell tumors by initiating proliferation. Several investigations of gene- and protein-related functions after exposure to TCDD in in vitro systems using different cell lines have been published in recent years (Fisher et al., 2004; Hanlon et al., 2004; Jin et al., 2004; Martinez et al., 2002; Puga et al., 2000); however, the reactions and functions observed subsequent to exposure have been different in each type of target cell (Greenlee et al., 2001). Because the progression and risks to humans of this toxicity remain to be completely elucidated, concentration on molecular functioning could enhance understanding of the pathogenesis of TCDD-induced exocrine pancreatic toxicity. Additional research is needed to analyze the mechanism(s) of this induction and provide understanding of the potential extrapolations from rats to humans of dioxin-induced exocrine pancreatic lesions.
ACKNOWLEDGMENTS
The authors are grateful to all involved in the design and conduct of these NTP studies, with special thanks to Drs. John Bucher, Rick Hailey, Amy Brix, Mike Jokinen, Don Sells, Angelique Braen, and Milton Hejtmancik, and to Ms. Denise Orzech. We gratefully acknowledge Dr. Kristie Mozzachio, Dr. Adriana Doi, and Ms. JoAnne Johnson for critical review of the manuscript and Mr. Norris Flagler for expert preparation of the illustrations. The authors declare that they have no competing financial interests.
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Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Laboratory of Computational Biology and Risk Analysis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
ABSTRACT
In previous 2-year studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) conducted by the National Toxicology Program on female Harlan Sprague-Dawley rats, acinar-cell vacuolation, atrophy, inflammation, and arteritis developed at high incidence, and a rare occurrence of pancreatic acinar-cell adenomas and carcinomas was noted. In this investigation, we sought to identify the mechanism involved in the early formative stages of acinar-cell lesions. Pancreas from animals treated for 14 and 31 weeks with 100 ng TCDD/kg body weight or corn oil vehicle was examined immunohistochemically and/or morphometrically. Acinar-cell kinetics were analyzed using staining with hematoxylin and eosin and proliferating cell nuclear antigen. Expressions of cytochrome P450 (CYP) 1A1 and aryl hydrocarbon receptor (AhR) were evaluated to assess direct effects of TCDD exposure. The cholecystokinin-A receptor (CCK-A receptor; CCKAR) and duodenal cholecystokinin 8 (CCK) revealed the associations of dioxin treatment with hormonal changes. Amylase localization showed acinar structural changes that could affect enzymatic production. Increased apoptotic activity in acinar cells occurred in 14- and 31-week-treated animals, with an increase in proliferative activity in the latter. Also in the latter, in the vacuolated acinar cells, CYP1A1 was overexpressed, and statistically significant decreases in expressions of AhR, CCKAR, and amylase occurred. The intensity of CCKAR expression increased in nonvacuolated acinar cells; a decrease in the size of CCK-positive epithelial cells occurred in duodenum. Our findings indicate that dioxin-induced acinar-cell lesions might be related to a direct effect of TCDD on the pancreas. Increase in CYP1A1 and decrease in CCKAR expressions in vacuolated acinar cells may be involved in the pathogenesis of pancreatic lesions. Changes in the expression of CYP or CCKAR may have induced the acinar-cell tumors by initiating proliferation.
Key Words: acinar cells; dioxin; immunohistochemistry; pancreas; rat; vacuolation.
INTRODUCTION
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as dioxin, is an environmental contaminant that is known for its extreme toxic potency. Certain polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (PCBs) exhibiting biologic actions similar to those of TCDD have been commonly designated dioxin-like compounds (DLCs). They may induce developmental, endocrine, and immunological toxicity and multiorgan carcinogenicity in animals and/or humans (ATSDR, 1998; Bertazzi et al., 2001; Kociba et al., 1978; Steenland et al., 1999, 2001). Incidences of cancer have been evaluated in several analyses of human populations exposed to elevated amounts of dioxin and DLCs. The most recent studies indicate that exposure is associated with an increase in all cancers combined and several specific cancers including rectal cancer, lung cancer, Hodgkin disease, non-Hodgkin lymphoma, and myeloid leukemia, as well as chronic circulatory diseases, respiratory diseases, and diabetes (ATSDR, 1998; Bertazzi et al., 2001).
Recently, the National Toxicology Program (NTP) conducted 2-year bioassays in female rats to evaluate the chronic pathology and carcinogenicity induced by dioxin, DLCs, structurally related PCBs, and mixtures of these compounds, including TCDD; 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachloro-dibenzifyran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); the toxic equivalency factor (TEF) tertiary mixture of TCDD, PCB126, and PeCDF; and the binary mixtures of PCB126 and 153 and PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118) (National Toxicology Program, 2004a, 2004b, 2004c, 2004d, 2004e, 2004f, 2004g). In these studies, an increase occurred in the incidence of neoplastic effects, such as cholangiocarcinoma and/or hepatocellular adenoma of the liver, cystic keratinizing epithelioma of the lung, and gingival squamous-cell carcinoma of the oral cavity (Brix et al., 2004; Jokinen et al., 2003; National Toxicology Program, 2004a, 2004b, 2004c, 2004d, 2004e, 2004f, 2004g; Tani et al., 2004; Walker et al., 2004; Yoshizawa et al., 2005). Pancreatic toxicities, such as acinar cytoplasmic vacuolation, atrophy, inflammation, and arteritis have been seen at high incidence combined with a rare occurrence of acinar-cell adenomas and carcinomas (Nyska et al., 2004).
Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of TCDD and related DLCs (Denison and Nagy, 2003; Poland and Knutson, 1982; Schmidt and Bradfield, 1996). It is expressed ubiquitously in human and rodent systemic organs, particularly in the pancreas (Dolwick et al., 1993; Yamamoto et al., 2004). That most organs with AhR expression are thought to be more susceptible to TCDD-induced toxicity is evidenced by AhR-deficient mice that are resistant to some kinds of TCDD-induced toxicity (Fernandez-Salguero et al., 1996; Gonzalez and Fernandez-Salguero, 1998). No reports are available regarding the relationship of exocrine pancreatic toxicity and change in AhR expression after TCDD treatment in rats. The most well-studied response to TCDD is induction of the CYP 1A1 and 1B1 classes of cytochrome P450 (Denison and Nagy, 2003; Schmidt and Bradfield, 1996; Toyoshiba et al., 2004) regulated by the AhR (Nebert et al., 2004); their inductions, especially that of CYP1A1, are sensitive to TCDD and thus serve as useful markers for exposure to TCDD. These enzymes play a major role in the activation and deactivation of toxins and carcinogens. The susceptibility to pancreatic disease seems to depend on the integrity of the cellular detoxification process governed by these drug-metabolizing enzymes (Standop et al., 2002, 2003; Ulrich et al., 2002). Differences in immunohistochemical expression of CYPs, such as 1A1, 1A2, 2B6, 2D6, and/or 3A1 among normal pancreas, chronic pancreatitis, and pancreatic tumors have been reported (Foster et al., 1993; Standop et al., 2003); the possibility of an etiological relationship between elevated levels of CYPs and the subsequent development of human pancreatic diseases has been highlighted.
In tissues derived from the previous NTP oral-treatment studies of TCDD; PCB 126; PeCDF; a tertiary mixture of TCDD, PCB126, and PeCDF and a binary mixture of PCB126 and PCB153, duodenal levels of stored cholecystokinin (CCK) peptide decreased prominently (Lee et al., 2000). Cholecystokinin, regarded as the major hormonal mediator of pancreatic enzymatic production, assessed chiefly by levels of amylase, is secreted by the proximal small intestine under the control of a negative feedback loop (Bourassa et al., 1999; Furukawa et al., 2001; Haschek and Rousseaux, 1998; Wank, 1995). The peripheral receptors can be classified pharmacologically as CCKA receptor (CCKAR), expressed predominately in pancreas, and as CCKB receptor (CCKBR), localized chiefly in the central nervous system (Monstein et al., 1996; Tang et al., 1996; Wank, 1995). Cholecystokinin induces secretion of pancreatic enzymes and synthesis of protein and DNA in the rat acinar cell through its CCKAR (Varga et al., 1998; Wank, 1995). Continuous blocking of this receptor by CCK inhibitors causes atrophic changes (Biederbick and Elssser, 1998; Ohlsson et al., 1995). The close relationship between the expression of the CCKAR and pancreatic disease has been shown experimentally in several models, such as azaserine- or caerulein-induced pancreatitis and cancer (Bourassa et al., 1999; Gebhardt et al., 2004; Meijers et al., 1992; Roebuck et al., 1987). The pathogenesis of exocrine pancreatic toxicity induced by TCDD has not been clarified.
Taking this information into consideration, we investigated the potential pathways involved in the TCDD-induced acinar pancreatic pathology. Hypothesizing that the early changes in AhR, CYP1A1, and CCK-related proteins in acinar cells after treatment with TCDD might be related to induction of toxicity in exocrine pancreas and attempting to elucidate the mechanism in rats, we conducted this retrospective immunohistochemical study of pancreas and duodenum in rats treated with TCDD for 14 and 31 weeks.
MATERIALS AND METHODS
Study design.
This investigation originated from a series of studies undertaken by the NTP to determine the suitability of the Toxic Equivalency Factor (TEF) methodology for predicting chronic toxicity and carcinogenicity induced by TCDD and DLCs (National Toxicology Program, 2004a, 2004b, 2004c, 2004d, 2004e, 2004f, 2004g). Female Harlan Sprague-Dawley rats were used, because they showed more sensitivity to the effects of TCDD than the male in previous studies (Kociba et al., 1978). Approximately 10 animals/group were administered 0, 3, 10, 22, 46, and 100 ng/kg TCDD for 14, 31, and 53 weeks (10 rats for 14 and 31 weeks, 8 rats for 53 weeks); in the 2-year study, 49 to 54 animals were given the same doses by daily gavage for 5 days per week up to 2 years. In addition, rats treated with 100 ng/kg for 30 weeks followed by vehicle treatment through the termination of the 2-year study were designated the 100 ng/kg stop group. Control animals
Chemical.
2,3,7.8,-Tetrachlorodibenzo-p-dioxin (lot no. CR82-2–2, purity approximately 98%, with no change in purity throughout the study) was supplied by IIT Research Institute (Chicago, IL). Purity of the chemical was analyzed with a gas chromatography/high resolution/mass spectrometry system (GC/HR/MS). Acetone and corn oil were obtained from Spectrum Quality Products (Gardena, CA). Specific amounts of TCDD dissolved in acetone were added to corn oil to yield concentrations of 0, 1.2, 4, 8.8, 18.4, or 40 ng TCDD/ml of vehicle determined by GC/HR/MS to be within 10% of target concentrations. Studies of the dose formulations indicated that the chemical could maintain an acceptable homogeneity for dosing and stability for 35 days when stored at room temperature.
Animals.
Animal studies were conducted at an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility of Battelle-Columbus Laboratories (Columbus, OH) for the duration of the study. Animal handling and husbandry were conducted in accordance with National Institutes of Health (NIH) guidelines (Grossblatt, 1996). Upon receipt, the female Harlan Sprague-Dawley rats were approximately 6 weeks of age. They were held under quarantine for 15 days for health screening by a veterinarian and were approximately 8 weeks of age at the start of the study. Animals were randomly assigned to control or TCDD-treated groups and permanently identified with tail tattoos. They were housed five to a cage in solid-bottom polycarbonate cages (Lab Products, Inc., Maywood, NJ). Filtered room air underwent at least 10 changes per hour. The animal room was maintained at 69°–75°F with 35–65% relative humidity and 12 h each of light and darkness. Irradiated NTP-2000 pelleted feed (Zeigler Bros., Inc., Gardner, PA) and water were available ad libitum. All rats were checked twice daily for morbidity and once per month for clinical signs of toxicity; moribund animals were euthanized and necropsied. All animals were weighed weekly for the first 3 weeks of the study, at 4-week intervals thereafter, and again at necropsy.
Pathology.
Complete necropsies were performed on all animals using standardized methodology. At necropsy, all tissues including masses and macroscopical abnormalities were removed and fixed in 10% neutral buffered formalin for microscopical evaluation. After fixation, the tissues were trimmed, dehydrated, cleared, and paraffin-embedded. Five-μm-thick sections were mounted onto glass slides, stained with hematoxylin and eosin (H&E), and examined microscopically. The severity of lesions was graded on a four-point scale of 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked. All data including pathological examination were obtained according to GLP, underwent extensive pathology peer review (Boorman et al., 2002), were peer-reviewed by an external expert panel advisory board, and are available online (http://ntp.niehs.nih.gov).
Quantative analysis of apoptotosis in acinar cells (apoptotic index).
The standard for recognition and quantitation of apoptotic bodies is analysis of H&E-stained sections (Goldsworthy et al., 1996). For counting the numbers of apoptotic bodies, distinguished by the presence of marginal chromatin condensation and/or fragmented nuclei, approximately 300 acinar cells randomly selected from 4 different areas in each section were examined under a light microscope at a magnification of x400. The apoptotic index represented the percentage of apoptotic bodies per total number of acinar cells counted (Imazawa et al., 2003).
Immunohistochemical investigation of CYP1A1, CCK, AhR, CCKAR, amylase, and proliferating cell nuclear antigen (PCNA).
Additional paraffin-embedded sections of pancreas from each of eight controls and eight rats exposed to 100 ng/kg TCDD for 14 and 31 weeks were analyzed immunohistochemically to determine effects of treatment on the expressions of drug-metabolic enzyme (CYP1A1), dioxin-binding and CCK-binding receptors (AhR, CCKAR, respectively), exocrine pancreatic enzyme (amylase), and acinar-cell proliferation. Duodenal sections of the same animals were examined to determine effects of treatment on the expression of CCK8, which is a nonselective ligand for CCKA or CCKB receptors. Table 1 summarizes the immunohistochemical staining procedures. Detailed staining protocols for all antibodies are listed on the Web site of the National Institute of Environmental Health Sciences (NIEHS) Laboratory of Experimental Pathology (http://dir.niehs.gov/dirlep/immuno/). Briefly, sections were deparaffinized, hydrated, and blocked for endogenous peroxidase. Heat-induced epitope retrieval was performed for all antibodies except CYP1A1 (http://dir.niehs.gov/dirlep/immuno/retrievals.htm).
For counting the numbers of cells positive for PCNA in pancreas, approximately 300 acinar cells were randomly selected from four different areas in each section and examined under a light microscope at a magnification of x400. The PCNA-labeling index represented the percentage of PCNA-positive cells per total number of acinar cells counted (Imazawa et al., 2003).
To designate the grading of CYP1A1, AhR, CCKAR, and amylase, the intensity of immunopositivity, or the relative area of the sections showing staining, was graded by two pathologists on a scale ranging from 0 (–) to 3 (+++). Immunostainings of CYP1A1, AhR, and amylase were estimated as follows: (0) = no specific immunohistologic reaction visible in acinar cells, (1) = <30% of total acinar-cell area showing a positive reaction, (2) = up to 60%, and (3) = up to 100%. The degree of CCKAR immunolocalization in acinar cells was scored from 0 to 3, corresponding to the presence of negative, weak, moderate, or strong brown staining, respectively. In 31-week TCDD-treated groups, we analyzed the acinar-cell area of nonvacuolated and vacuolated cells separately, compared to controls. Cholecystokinin-positive epithelial cells in the duodenum were counted in four randomly selected areas in each section and examined under a light microscope at a magnification of x200. Five positive cells containing nuclei were randomly selected from five different areas in each section under a light microscope at a magnification of x400 and analyzed for cell size with free software, "Image Processing and Analysis in Java (Image J, http://rsb.info.nih.gov/ij/)."
Statistical analysis.
Incidences of lesions in the study animals were evaluated statistically by the Poly-3 test, which makes adjustments for survival differences among groups. Incidences of lesions in animals from each of the interim evaluations and the 2-year study were analyzed separately. For animals in the 2-year studies, the incidences of total lesions, including findings from animals that survived until study termination and from early-death animals, were included in the analysis. The immunohistochemical scores and morphometrical data of the control and high-dose groups from 14 and 31 weeks were analyzed statistically. We used the t-test to compare two groups when the data were normally and independently distributed, as in the comparison of the apoptotic index and PCNA- and CCK-labeling indices with respective control groups. For analyses of non-normal data and CCK-positive cell size, we used the Wilcoxon rank-sum test. Tukey's multiple comparison test was used for comparing the control group with the 31-week-treated vacuolated group and the 31-week-treated nonvacuolated group for scores for CYP1A1, AhR, CCKAR, and amylase.
RESULTS
Pancreatic Histopathology
Acinar cytoplasmic vacuolation occurred significantly after treatment with 100 ng/kg TCDD in 5 of 10 rats in the 31-week group, 7 of 8 rats in the 53-week group, and 42 of 51 rats in the 2-year group, and in 15 of 53 animals treated with 46 ng/kg TCDD for 2 years (Nyska et al., 2004) (Fig. 1d and 1e; Table 2). The incidence and severity of this lesion were clearly dose-dependent and time-dependent. These lesions were not observed in the animals of the 100 ng/kg stop group.
The incidence of chronic active inflammation was increased significantly in the 2-year group after treatment with 100 ng/kg of TCDD (6 of 51) (Table 2), although the same lesion was observed after 14 and 53 weeks of the same TCDD treatment (Fig. 1e). Acinar atrophy was generally associated with chronic active inflammation (6 of 9). In the animals of the 100 ng/kg stop group, the decrease in the incidence and severity of these lesions suggested that they regressed by withdrawing TCDD treatment.
At 2 years, arterial chronic active inflammation appeared significantly in 7 of 51 rats showing fibrinoid necrosis of the vessel in the 100 ng/kg group (Jokinen et al., 2003; Nyska et al., 2004) (Fig. 1e; Table 2). The arterial lesions only occasionally accompanied the other described changes, such as acinar cytoplasmic vacuolation, acinar atrophy, and/or chronic active inflammation (7 of 51), and therefore were not considered the primary cause of the acinar pathology.
One acinar-cell adenoma and two carcinomas were seen in the 100 ng/kg group 2 years after TCDD treatment (Fig. 1f), and one carcinoma was seen in the 100 ng/kg stop group. The incidences of acinar-cell tumors exceeded distinctly the historical control range (Table 2). The pancreatic acinar tissue of the 2-year controls exhibited features comparable to those of the 31-week control animals.
Cellular Kinetics of Acinar Cells in Exocrine Pancreas
A small number of acinar cells contained apoptotic bodies in control rats from the 14- and 31-week groups (labeling indices: 0.1%). The index was significantly higher (labeling index: 0.5%, p < 0.05) in the 14-week group treated with 100 ng/kg TCDD, compared to the control value (Figs. 1c, 4a). In the 31-week TCDD-treated group, the incidence of apoptotic acinar cells was significantly higher (labeling index: 0.7%, p < 0.01) than that of control and 14-week TCDD-treated groups (Figs. 1d, 4a).
In the exocrine pancreas in the 14- and 31-week control groups and 14-week TCDD-treated groups, only a few acinar cells were positive for PCNA (labeling indices: <0.1%) (Fig. 4b), although some islet cells were positive (Fig. 2a). In contrast, the number of acinar cells whose nuclei showed positivity for PCNA protein was significantly higher in the 31-week TCDD-treated group (labeling index: 2.5%, p < 0.05) than in the control and 14-week TCDD-treated groups (Figs. 2b, 4b). Most of the PCNA-positive acinar cells were located in the area surrounding the vacuolated cells.
Relationship Between Toxicity and Metabolic Enzyme Induction in Exocrine Pancreas
In control rats from the 14- and 31-week groups, acinar cells were weakly positive for CYP1A1 protein; that is, the relative area stained was less than 30% of the entire area seen in the preparations (average grade: 0.9–1.3; Fig. 2c). Most islet cells exhibited moderate cytoplasmic positivity, and some ductal epithelial cells showed positive signals. In 31-week TCDD-treated rats, vacuolated acinar cells showed strongly positive cytoplasmic staining, with the relative area of staining significantly increased (average grade: 2.3, p < 0.01; Figs. 2d and 4c). In contrast, no difference occurred between the areas of treated nonvacuolated acinar cells and controls (Fig. 4c). The cytoplasmic expression of CYP1A1 in islet cells and ductal epithelial cells appeared similar to the pattern seen in age-matched controls.
Nature of Cytoplasmic Vacuolation of Acinar Cells Based on Amylase Localization
To analyze the change in quantity of zymogen granules in acinar cytoplasm, a finding indicative of pancreatic enzymatic secretion, exocrine pancreatic tissue was stained with amylase antibody. In the control exocrine pancreas in the 14- and 31-week groups, amylase-positive granules were contained within zymogen granules in the cytoplasm of most acinar cells (average grade: 2.5–3.0; Figs. 2e and 4d). The degree of acinar amylase expression significantly decreased in the 14-week TCDD-treated group (average grade: 2.0, p < 0.05; Fig. 4d). In the 31-week treated rats, vacuolated acinar cells were generally negative for amylase, suggesting the loss of amylase-positive zymogen granules (average grade: 0; Figs. 2f and 4d), whereas the expression of amylase-positive granules decreased significantly in nonvacuolated acinar cells (average grade: 1.1, p < 0.01; Fig. 4d). In all control and TCDD-treated animals, ductal epithelial cells and islet cells were negative for amylase protein. Pancreatic secretion in ducts was slightly positive; however, there was no significant difference between controls and TCDD-treated groups.
Characteristic Expression of AhR in Exocrine Pancreas
In control rats in the 14- and 31-week groups, acinar cells were weakly to moderately positive for AhR protein; that is, the relative area was somewhat less than 60% of the preparation (average grade: 1.4–2.1; Figs. 3a and 4e). In 31-week TCDD-treated rats, vacuolated acinar cells completely lost cytoplasmic positivity, and the relative area decreased significantly (average grade: 0, p < 0.01; Figs. 3b and 4e). In contrast, the expression increased slightly but not significantly in the nonvacuolated acinar regions (Fig. 4e). In all control and TCDD-treated animals, islet cells exhibited slight positivity for AhR protein; however, no significant difference occurred between controls and TCDD-treated groups. Ductal epithelial cells and pancreatic secretion showed negative staining in both groups.
Relationship Between Changes in Expression of CCKAR in Exocrine Pancreas and CCK in Duodenum
In control rats of the 14- and 31-week groups, a small number of acinar cells expressed CCKAR protein with weak staining intensity in the cytoplasm (average grade: 0.6–1.0; Figs. 3c and 4f). In 31-week TCDD-treated rats, vacuolated acinar cells lost completely the positive cytoplasmic staining (average grade: 0; Figs. 3d and 4f). In contrast, the degree of CCKAR staining increased significantly in nonvacuolated acinar cells (average grade: 2.0, p < 0.01; Figs. 3d and 4f). In all control and TCDD-treated animals, islet cells, ductal epithelial cells, and pancreatic secretion showed negative or weakly positive staining for CCKAR protein.
In control animals of the 14- and 31-week groups, some epithelial cells in the duodenum exhibited positive cytoplasmic staining for CCK protein (Fig. 4g). The CCK-labeling index showed no significant changes in TCDD-treated rats at 14 and 31 weeks, compared to the age-matched controls (Fig. 4g). An analysis of cell size of CCK-positive epithelial cells revealed a tendency toward a slight decrease in 31-week TCDD-treated rats, compared to the age-matched controls, though without significant difference (Fig. 4h).
DISCUSSION
In this retrospective immunohistochemical study, we evaluated the pathogenesis of pancreatic toxicity induced by chronic exposure to dioxin. Although several surveys of toxicities in animals and side effects in humans induced by dioxin have been published, exocrine pancreatic toxicity has not been completely elucidated. Our literature survey revealed that TCDD-induced exocrine pancreatic lesions, such as vacuolation (accompanied by decreased cytoplasmic volume and numbers of zymogen granules) and ballooning degeneration, have been reported only in the rainbow trout (Spitsbergen et al., 1988) and zebrafish (Henry et al., 1997). Pancreatic neoplasms have been reported in fish exposed to polycyclic aromatic hydrocarbons (Fournie and Vogelbein, 1994).
In the rat pancreas, only minimal destruction of cells in the islets of Langerhans (Gorski et al., 1988; Kociba et al., 1976, 1978; Pohjanvirta and Tuomisto, 1994) has been reported. Exposure of female Sprague-Dawley rats of the Spartan strain to 100 ng/kg TCDD for 2 years was previously associated with atrophy, fibrosis, and periarteritis of the pancreas (Kociba et al., 1978). In our study, acinar changes, such as vacuolation, inflammation, and/or atrophy, were detected without any associated vascular changes (Table 2); therefore, early changes in exocrine pancreas might not be related directly to vascular lesions but, rather, may result from direct toxicological damage (Nyska et al., 2004).
The apoptotic and/or PCNA-labeling indices of acinar cells increased significantly in 14- and 31-week TCDD-treated groups. Because PCNA-positive cells were located in the areas surrounding vacuolated cells, these combined results suggest that, after vacuolation of acinar cells, apoptosis occurred, accompanied by regenerative change. Cellular proliferation does not always lead to increased carcinogenesis in rodents or humans, as in the case of chronic toxic lesions in which normal levels of cellular turnover have been observed in target organs of nongenotoxic carcinogens (Ward et al., 1993). Epidemiologic studies, however, support the concept that inflammation associated with glandular destruction and proliferative activity constitutes a risk factor for exocrine pancreatic cancer (Keim, 2003; Whitcomb, 2004). Several environmental agents that have been proposed as causal for chronic pancreatitis, and pancreatic carcinomas are associated with the wood and pulp industry, the dry cleaning business, and gasoline production and usage (Alguacil et al., 2003; Foster et al., 1993; Lin and Kessler, 1981; Milham and Demers, 1984; Ojajarvi et al., 2000, 2001). In our study, especially in the 2-year group, chronic active inflammation was observed. The relationships between chronic inflammation, oxidative stress, and tumorigenesis were previously addressed (Nyska et al., 2004); the promotion of tumor progression by nitric oxide illustrated such relationships (Xu et al., 2002). Enhanced replication of acinar cells at an early stage might, therefore, somehow be involved in the pancreatic carcinogenesis induced by TCDD.
Several chemicals induce acinar-cell vacuolation, followed by acinar-cell death and/or inflammation, such as ethanol, N-nitrosobis(2-oxopropyl) amine, and ethionine, which induce acinar cell tumors in rodent models (Haschek and Rousseaux, 1998; Horne and Tsukamoto, 1993; Scarpelli et al., 1986; Walker et al., 1993). Two kinds of acinar-cell vacuolation in pancreas have been reported—anoxic and secretory (Tapp, 1970). Amylase expression in acinar cells decreased significantly in 14- and 31-week treated rats, suggesting the loss of amylase-positive zymogen granules. Vacuolation induced by TCDD might not be an anoxic change, because the imunohistochemical results revealed an increase in CCK secretion.
The slightly decreased size of CCK-positive epithelial cells in the duodenum was detected in rats treated with TCDD for 31 weeks, although without significant difference. Because TCDD decreases intestinal levels of stored CCK peptide in vivo and in vitro, without observed changes in CCK mRNA levels (Lee et al., 2000), our data might suggest an oversecretion of CCK from the small intestine. The CCKAR expression that we observed disappeared completely in vacuolated acinar cells with overexpression in nonvacuolated acinar cells. Continuous CCK stimulation for 3 days leads to decreased CCKAR expression in exocrine pancreas (Ohlsson et al., 2000), and downregulation of CCKAR in vacuolated acinar cells corroborates this finding. Upregulation in nonvacuolated cells might suggest a reaction in PCNA-positive regenerative acinar cells for several reasons. CCKAR plays an important role in pancreatic proliferation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking the CCKAR gene (Moralejo et al., 2001), and CCKAR mRNA is maximally expressed and sustained during the regeneration of exocrine pancreas in pancreatitis (Bourassa et al., 1999). Long-term endogenously induced hypercholecystokininemia in rat pancreaticobiliary-duct models not only induces hypertrophy and proliferation of the exocrine pancreas (Ohlsson et al., 1995; Panozzo et al., 1995), but also causes the formation of pancreatic tumors (Stace et al., 1987). All of this information collectively supports our speculation that the change in CCK-CCKAR is likely relevant to TCDD-induced exocrine toxicity in rats.
In our analysis of the NTP studies, especially in the animals treated with 100 ng/kg TCDD for 31 weeks, we found liver damage, including hepatocellular hypertrophy, multinuclear hepatocytes, hepatocellular fatty change, pigmentation (iron and/or lipofuscin), mixed cell foci, and toxic hepatopathy. In addition, cholangiofibrosis and bile duct fibrosis were noted in the 53-week TCDD-treated group. Bile-duct damage and proliferation induced by TCDD might have been caused by uroporphrin (Smith et al., 2001); bile-duct proliferation induced cholestasis in an experimental model of bile-duct ischemia (Beaussier et al., 2005). Increases in levels of cholesterol, alkaline phosphatase, and 5'-nucleotidase may reflect cholestatic changes in rats treated with TCDD for 30 weeks, even though no change occurred in total bile acid and bilirubin in serum (Maronpot et al., 1993). Cholestatic conditions stimulate CCK secretion from intestine and amylase output from pancreatic acinar cells (Kurosawa et al., 1989; Tangoku et al., 1992). Wyeth-14,643 induced pancreatic tumors via sustained increases in CCK levels secondary to hepatic cholestasis, with significant increases in total bile acid, alkaline phoshatase, and birubin in serum (Obourn et al., 1997b). These data suggest the possibility that the weak cholestatic change seen in our study also may be related to the change of CCK levels at 14 and 31 weeks, although we have no blood chemistry. Expression of AhR disappeared completely in vacuolated acinar cells in 31-week TCDD-treated rats in our study. After TCDD binding to AhR in the cytoplasm of the target cells, the resultant ligand–receptor complex translocates to the nucleus, where its binding to TCDD response elements (Denison and Nagy, 2003; Schmidt and Bradfield, 1996) can be visualized by decreasing degrees of AhR staining in the cytoplasm (Pollenz et al., 1994). Exposure to TCDD induced the sustained depletion of AhR protein in nine distinct cell culture lines derived from human and rodent tissues and in liver, spleen, thymus, and lung in a rat in vivo model (Pollenz et al., 1998, 2002). Our results may, therefore, reflect translocation of the TCDD-AhR complex and subsequent downregulation, as seen in these models. A slight but not significant upregulation in nonvacuolated cells may suggest a compensatory reaction in proliferative acinar cells, similar to that seen in human chronic pancreatitis and pancreatic cancer (Koliopanos et al., 2002).
Our results revealed overexpression of CYP1A1 in vacuolated acinar cells, compared to nonvacuolated acinar cells and control pancreas; the expression of CYP1A1 in islet cells and ductal epithelial cells appeared similar to its localization reported previously (Ulrich et al., 2002). In the pancreas, two AhR ligands, 3-methylcholanthreme and TCDD, have been shown to induce CYP1A in rat and fish, respectively (Clarke et al., 1997; Ortiz-Delgado and Sarasquete, 2004; Wilson et al., 1990). In fish, TCDD induces exocrine pancreatic toxicity involving acinar-cell degeneration and necrosis in association with overexpression of CYP1A (Ortiz-Delgado and Sarasquete, 2004). Overexpression of CYP1A1 in vacuolated acinar cells in our study could be mediated by the AhR signaling pathway, because a change in AhR expression was involved in this enhancement. Dioxin causes cellular oxidative stress (Hassoun et al., 2002) followed by cell-cycle arrest and apoptosis via the induction of CYP (Matsumura, 2003). The increased apoptotic index in our study may be related to cellular oxidative stress in acinar cells exposed to TCDD.
Antiestrogenic effects of TCDD in rodents include inhibition of estrogen-induced mammary and uterine cancers via AhR–estrogen receptor crosstalk (Safe, 2001; Safe and McDougal, 2002; Safe and Wormke, 2003). Acinar-cell tumors are enhanced by testosterone and inhibited by estrogen (Longnecker and Sumi, 1990; Robles-Diaz and Duarte-Rojo, 2001); the incidence of spontaneous and induced neoplasms of the exocrine pancreas in rats is higher in males. In several mechanistic studies of pancreatic tumorigenesis involving trypsin inhibitors and PPAR agonists, the use of male rats may have resulted in a higher incidence of acinar-cell tumors (Obourn et al., 1997a,b; Rao and Subbarao, 1995). Strong implications for a role of estrogen in the promotion of pancreatic carcinogenicity have emerged from both in vivo and in vitro studies (Andren-Sandberg et al., 1999). In our retrospective study, the suppression of endocrine-induced tumors, such as mammary and pituitary, could be related to increased prolactin, or they might occur secondary to reduction in body-weight gain or to reduced endocrine activity. All of these factors may exclude a potential direct antiestrogenic effect of TCDD. In Kociba's study, the decreased incidence of acinar-cell tumors was noted in male rats, but no change in incidence occurred in female rats (Kociba et al., 1978). Whether the higher incidence of acinar-cell tumors in our 2-year female rat study could be related to antiestrogenic activity of TCDD remains, therefore, unclear.
Our findings indicate that dioxin-induced acinar-cell lesions might be related to a direct effect of TCDD on the pancreas. Increase in CYP1A1 and decrease in CCKAR expressions in vacuolated acinar cells may be involved in the pathogenesis of pancreatic lesions. Changes in the expression of CYP or CCKAR may have induced the acinar-cell tumors by initiating proliferation. Several investigations of gene- and protein-related functions after exposure to TCDD in in vitro systems using different cell lines have been published in recent years (Fisher et al., 2004; Hanlon et al., 2004; Jin et al., 2004; Martinez et al., 2002; Puga et al., 2000); however, the reactions and functions observed subsequent to exposure have been different in each type of target cell (Greenlee et al., 2001). Because the progression and risks to humans of this toxicity remain to be completely elucidated, concentration on molecular functioning could enhance understanding of the pathogenesis of TCDD-induced exocrine pancreatic toxicity. Additional research is needed to analyze the mechanism(s) of this induction and provide understanding of the potential extrapolations from rats to humans of dioxin-induced exocrine pancreatic lesions.
ACKNOWLEDGMENTS
The authors are grateful to all involved in the design and conduct of these NTP studies, with special thanks to Drs. John Bucher, Rick Hailey, Amy Brix, Mike Jokinen, Don Sells, Angelique Braen, and Milton Hejtmancik, and to Ms. Denise Orzech. We gratefully acknowledge Dr. Kristie Mozzachio, Dr. Adriana Doi, and Ms. JoAnne Johnson for critical review of the manuscript and Mr. Norris Flagler for expert preparation of the illustrations. The authors declare that they have no competing financial interests.
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