Brominated diphenyl ether (BDE) levels in liver, adipose, and milk from adult and juvenile rats exposed by gavage to the DE-71 technical mixture.

Brominated diphenyl ethers (BDEs) are used as flame retardants in consumer products. Rodent studies indicate that the liver, thyroid, and nervous system of developing animals are targets of BDEs. To explore the relationship between exposure and health in developing animals, BDE accumulation in adult and juvenile rats was examined in conjunction with changes in liver weight and serum thyroxine (T4). Adult (F0) rats received the commercial BDE mixture DE-71 by gavage at doses of 0.5, 5, and 25 mg kg(-1) body weight (bw)/day for 21 weeks. F0 rats were mated and exposure continued throughout breeding, pregnancy, lactation, and postweaning until the pups (F1 generation) reached postnatal day (PND) 42. Milk was collected from lactating dams. Adipose and liver samples were collected from F0 and F1 males and females for BDE congener analysis. Congener prevalence in rat tissues mimicked congener prevalence in wildlife and humans. Tissue concentrations of all congeners except BDE-153 were lower than would be expected based on dose proportionality, confirming that BDE-153 has a high capacity for bioaccumulation. BDEs were transferred from maternal tissues to milk during lactation. Milk congener profiles differed from maternal tissue profiles indicating that degree of bromination and maternal sequestration influenced BDE transfer to milk. Female F1 rats accumulated more BDEs than F1 males, indicating that female rats were less able to metabolize and/or excrete BDEs. Significant effects on liver weight and serum T4 levels were observed in adults and juveniles in the middle and high dose groups, corresponding to BDE levels in the µg g(-1) range. Although it remains to be determined how human liver and thyroid are affected by exposure to much lower BDE levels, the present study confirmed that gender and reproductive status influence BDE accumulation in tissues and BDE transfer to the neonate via milk.

Glutathione S-transferase-placental form expression and proliferation of hepatocytes in fumonisin B1-treated male and female Sprague-Dawley rats.

Fumonisin B1 (FB1), a mycotoxin produced by a common corn contaminant Fusarium moniliforme and a hepatocarcinogen in rats, has been previously suggested to act as a poor initiator, but a better promoter of gamma-glutamyltranspeptidase (GGT)-positive rat liver preneoplastic lesions. Using glutathione S-transferase-placental form (GSTP) as a more sensitive marker of initiation, we have further evaluated the initiating capacity of various doses of purified FB1 administered (a) intraperitoneally (i.p.) to male Sprague-Dawley (SD) rats for 4 days and (b) orally (PO) to male and female SD rats for 11 days. Compared to their respective controls, significant increases in GSTP-positive hepatocytes were observed in male rats administered FB1 i.p. at 10 mg/kg body weight/day for 4 days, as well as in male and female rats treated with 35 and 75 mg/kg body weight/day FB1 p.o. for 11 days. The percentage section area of liver occupied by GSTP-positive mini-foci comprising of three to 12 cells was increased significantly in male rats given 10 mg/kg FB1 i.p., or in p.o.-treated males and females with 75 mg/kg FB1. Both i.p. and p.o. FB1 treatments resulted in dose-related enhanced hepatocyte proliferation as measured by proliferating cell nuclear antigen (PCNA) labeling with significant increases in the number of PCNA-positive nuclei at the same i.p. and p.o. dose levels where the number of GSTP-positive cells were elevated. In all studies, enhanced PCNA and GSTP expression occurred at FB1 doses which, based on serum biochemical and histopathological data previously reported from our laboratory, were shown to be hepatotoxic. Therefore, our data suggest that in a manner similar to known genotoxic carcinogens, FB1 has the capacity to initiate GSTP-positive hepatocytes with their subsequent development into GSTP mini-foci at exposure levels that induce enhanced hepatocyte proliferation in response to liver toxicity. In SD rats, this occurs as early as within 4 days of i.p. treatment or 11 days of p.o. treatment.

Potential mechanisms by which certain foods promote or inhibit the development of spontaneous diabetes in BB rats: dose, timing, early effect on islet area, and switch in infiltrate from Th1 to Th2 cells.

Certain diets can have major effects on the development of IDDM in DP-BB rats, but data are scant on the timing, dose, and mechanisms involved. We therefore determined the dose response, timing, and duration of exposure required to induce diabetes, and characterized the effects of nutritionally adequate diets with widely different diabetogenicity on the pancreatic islet area and cytokines. DP-BB rats were fed a diabetogenic, cereal-based, NIH-07 (NIH) diet or a protective, casein or hydrolyzed casein (HC)-based, semipurified diet. Rats were fed from weaning to 50 or 100 days with the HC diet and then switched to the NIH diet, or fed the NIH diet from weaning to 50 days and switched to the HC diet. Pancreas histology and diabetes outcome were determined. Semiquantitative morphometric analyses of hematoxylin and eosin-stained sections of pancreas from 41-day-old rats were also carried out. Diet-induced effects on pancreatic cytokine levels were measured at 70 days using reverse transcriptase-polymerase chain reaction analysis of gamma-interferon (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta). Long-term daily exposure, particularly around the beginning of puberty to late adolescence (50-100 days), was important for development of diabetes. DP-BB rats could be rescued from diabetes development by feeding them a low-diabetogen HC diet as late as 50 days. Diabetes frequency was highest in rats fed 70% and 100% NIH diets. By age 41 days, before classic insulitis, the islet area in HC-fed DP-BB rats was 65% greater than in NIH-fed rats. By 70 days, when mononuclear cells were visible in the islets of most NIH-fed, but not HC-fed rats, the more pronounced inflammatory process in NIH-fed rats was associated with a Th1 cytokine pattern (high IFN-gamma and low IL-10 and TGF-beta), whereas the pancreases of HC-fed rats showed fewer infiltrating cells, low levels of IFN-gamma, and high levels of TGF-beta, typical of a Th2 cytokine pattern. Thus dietary modification can occur as late as puberty. Further, long-term exposure to sufficient amounts of food diabetogens between 50 and 100 days was required for maximum diabetes induction. The islet area was modified by diet before signs of classic insulitis. Pancreatic inflammation in NIH-fed animals is a Th1-dependent phenomenon. The HC diet inhibited insulitis and was associated with a Th2 cytokine pattern in the pancreas, protecting diabetes-prone rats from developing diabetes.

Insulin therapy of prediabetes suppresses TH1 associated gene expression in BB rat pancreas.

Subcutaneous insulin treatment of young diabetes prone BB rats has been shown previously to suppress the development of autoimmune diabetes. In this study the hypothesis was tested that exogenous insulin may deviate the autoimmune process by acting on the Th1/Th2 cytokine balance in the pancreas. BB rats were implanted with pellets which continuously released insulin, at 50 d of age. Three weeks later cytokine mRNA expression in the pancreas and insulitis score were determined. While in control BB rats high levels of IFNgamma mRNA were detectable by RT-PCR, insulin treatment almost completely suppressed IFNgamma mRNA levels without concomitant upregulation of counterregulatory IL-10 and TGFbeta gene expression. Insulin also suppressed gene expression of inducible nitric oxide synthase. Mean insulitis scores were decreased after insulin treatment. We conclude that the protective effects of insulin treatment may not be due to the induction of protective Th2 immune reactivity but to general downregulation of immune activation in the pancreas, and hence also of Th1 autoimmunity.

Intervention in autoimmune diabetes by targeting the gut immune system.

BB rats and nonobese diabetic (NOD) mice spontaneously develop autoimmune insulin dependent diabetes and serve as models for human type I diabetes. During progression of the disease the cytokine pattern elaborated by islet infiltrating immune cells shifts from a Th2 or Th0 toward Th1 type. Only the latter is associated with "destructive" insulitis. We discuss here attempts to modulate disease progression by targeting the gut immune system with bacterial immunostimulants. Oral dosing of diabetes prone BB rats with lipopolysaccharide (LPS) or the Escherichia coli extract OM-89 lead to a Th2-shift of pancreatic mRNA expression. In vitro studies showed that repeated exposure toward LPS or OM-89 lead to downregulation of proinflammatory macrophage responses. In the NOD mouse, repeated oral dosing of OM-89 caused a Th2 shift in the gut cytokine gene expression, probably because of desensitization of macrophages and other antigen presenting cells. Concomitantly, diabetes prevention by oral insulin was improved. In conclusion, oral dosing with bacterial immunostimulants dampens Th1 type immune reactivities of the gut immune system and thereby promotes oral tolerance mechanisms. Downregulation of proinflammatory immune reactivities by repeated exposure to bacterial stimulants requires intact desensitization mechanisms in macrophages or other antigen presenting cells.

Cytokine gene expression in the BB rat pancreas: natural course and impact of bacterial vaccines.

In diabetes prone BB rat pancreas the Th1/ Th2 cytokine balance and the expression of inducible nitric oxide synthase (iNOS) was determined by mRNA analysis before and after the onset of insulitis. Specific mRNA was amplified by reverse transcriptase polymerase chain reaction, quantitated with radiolabelled probes by phosphoimaging and calibrated with the amount of co-amplified beta-actin mRNA. At 50 days of age, prior to recognizable insulitis, there was already significantly enhanced expression of both, Th1 and Th2 cytokines, and of iNOS mRNA, when compared to Wistar rat pancreas (p < 0.001). This supports the concept of an inconspicuous early phase of islet infiltration by single immunocytes, called single cell insulitis. At 70 days of age mononuclear infiltration of islets had begun and was associated with upregulation of interferon gamma (IFN gamma) and iNOS, but downregulation of interleukin-10 and transforming growth factor beta mRNA (p < 0.001). These findings correlate the onset of insulitis with a shift of the Th1/Th2 cytokine balance towards Th1 cell reactivity. Indeed there was a close correlation of the Th1/Th2 cytokine ratio but not of absolute IFN gamma mRNA levels with the insulitis score. Vaccination at day 50 with tetanus toxoid did not affect cytokine gene expression while diphtheria toxoid and even more strongly BCG administration induced a shift towards Th2 reactivity (p < 0.001) while iNOS mRNA was decreased (p < 0.01). Oral dosing with immunostimulatory components of Escherichia coli also changed the quality of inflammation. Oral lipopolysaccharide (LPS) from E. coli and OM-89, an endotoxin free extract containing immunostimulatory glycolipopeptides and heat shock protein (hsp) 65, both downregulated IFN gamma mRNA while only OM-89 in addition suppressed iNOS mRNA and enhanced Th2 cytokine gene expression (p < 0.001). We conclude that the onset of insulitis is associated with a shift towards Th1 cytokine and iNOS gene expression. Diphtheria toxoid and BCG vaccination stimulates Th2 reactivity but does not downregulate Th1. The latter can be achieved through oral administration of LPS or a glycopeptide fraction (OM-89) from E. coli.

Fumonisin B1 toxicity in male Sprague-Dawley rats.

Male rats were gavaged with fumonisin B1 (FB1) once daily for 11 consecutive days at doses of 0, 1, 5, 15, 35, and 75 mg FB1/kg body weight. Urine osmolality (at 5-75 mg FB1/kg) and organic ion transport in kidney slices (at 5-75 mg FB1/kg) were reduced. Urinary excretion of protein (at 15-75 mg FB1/kg) and of the enzymes LDH (at 5-75 mg FB1/kg), NAG (at 5-75 mg FB1/kg) and GGT (at 15-75 mg FB1/kg) were increased. These findings were indicative of glomerular and tubular toxicity. Histopathologic changes in the kidney consisted of necrosis of tubular epithelia of variable extent accentuated in the inner cortex. These changes were present at 1 and 5 mg FB1/kg and were more pronounced at 15-75 mg FB1/kg. Serum enzymes indicative of hepatotoxicity (ALT, GGT) were elevated compared to controls at 75 mg FB1/kg only. There were noticeable increases in mitotic figures in hepatocytes at 35-75 mg FB1/kg, while single cell necroses were increasingly numerous from 15-75 mg FB1/kg. The kidneys were considered to be the primary target organs in this study.

Toxicity of fumonisin B1 administered intraperitoneally to male Sprague-Dawley rats.

The toxicity of purified fumonisin B1 (FB1) administered ip was examined in male Sprague-Dawley rats. FB1 was injected at 7.5 or 10 mg/kg body weight/day for 4 consecutive days. This resulted in significant reductions in body weight, food consumption and faeces production. Polyuria without a compensatory increase in water consumption was observed in treated rats. Erythrocytosis, elevated haematocrits and haemoglobin levels were attributed to dehydration. Nephrotoxicity in treated rats was evident by clinical changes including elevated blood urea nitrogen and by subtle changes in kidney morphology. Histopathology and serum biochemistry also indicated that the liver was an important target organ in FB1-treated rats. A small increase in liver glutathione concentration was also evident in rats receiving 10 mg FB1/kg body weight. Effects on the immune system included reduced thymus weight, disseminated thymic necrosis and consistently elevated serum immunoglobulin M levels. Circulating phagocytic cell numbers were elevated in treated rats, probably owning to tissue damage associated with ip dosing. The liver and kidneys appear to be target organs of FB1 in Sprague-Dawley rats.

Glutathione S-transferases and P-glycoprotein in normal rat hepatocytes and hepatoma cells: analysis using flow cytometry.

Using indirect immunofluorescence with fluorescein isothiocyanate-conjugated antibodies, in combination with flow cytometry (FCM), we have developed a technique to detect the alpha, mu and pi isozymes of GST in cell suspensions from normal rat liver, and in H4IIE cells, a rat hepatoma cell line. Cell suspensions fixed in 1% paraformaldehyde were observed to require cell membrane permeation with lysolecithin to allow access and binding of antibodies to immunoreactive proteins within the cytoplasm. FCM analysis indicated normal rat hepatocytes to be positive for GST alpha and mu, but not GST pi, and the H4IIE cells to be positive for all three GST isozymes. Further analysis by FCM for the expression of P-glycoprotein (mdr), a membrane-associated protein product of the multidrug resistance gene, showed an association between the presence of GST pi and mdr in the two cell types. Thus, mdr was detected in significant amounts in H4IIE cells, but not in rat hepatocytes. The method described here has potential applications in screening, sorting and further characterisation for GST pi-positive hepatocytes for mechanistic studies during sequential rat liver carcinogenesis, as well as for characterisation of human tumors for the expression of different GST isozymes and P-glycoprotein during therapeutic management.