The kidney injury caused by the onset of acute graft-versus-host disease is associated with down-regulation of αKlotho.

Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in αKlotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of αKlotho, a sight that may inspire novel therapeutic approaches.

Complement activation is involved in the hepatic injury caused by high-dose exposure of mice to perfluorooctanoic acid.

High-dose exposure of mice to perfluorooctanoate (PFOA) induces both hepatotoxicity and immunotoxicity. Here, we characterized the effects of 10-day dietary treatment with PFOA (0.002-0.02%, w/w) on the liver and complement system of male C57BL/6 mice. At all four doses, this compound caused hepatomegaly and reduced the serum level of triglycerides (an indicator for activation of the peroxisome proliferator-activated receptor-alpha (PPARα)). At the highest dose (0.02%, w/w), this hepatomegaly was associated with the hepatic injury, as reflected in increased activity of alanine aminotranferase (ALAT) in the serum, severe hepatocyte hypertrophy and hepatocellular necrosis. PFOA-induced hepatic injury was associated with in vivo activation of the complement system as indicated by (i) significant attenuation of the serum activities of both the classical and alternative pathways; (ii) a marked reduction in the serum level of the complement factor C3; and (iii) deposition of the complement factor C3 fragment (C3a) in the hepatic parenchyma. PFOA did not activate the alternative pathway of complement in vitro. At doses lower than 0.02%, PFOA induced hepatocyte hypertrophy without causing liver injury or activating complement. These results reveal substantial involvement of activation of complement in the pathogenesis of PFOA-induced hepatotoxicity.

The Environmental Pollutants Perfluorooctane Sulfonate and Perfluorooctanoic Acid Upregulate Uncoupling Protein 1 (UCP1) in Brown-Fat Mitochondria Through a UCP1-Dependent Reduction in Food Intake.

The environmental pollutants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) cause a dramatic reduction in the size of the major adipose tissue depots and a general body weight decrease when they are added to the food of mice. We demonstrate here that this is mainly due to a reduction in food intake; this reduction was not due to food aversion. Remarkably and unexpectedly, a large part of the effect of PFOA/PFOS on food intake was dependent on the presence of the uncoupling protein 1 (UCP1) in the mice. Correspondingly, PFOA/PFOS treatment induced recruitment of brown adipose tissue mitochondria: increased oxidative capacity and increased UCP1-mediated oxygen consumption (thermogenesis). In mice pair-fed to the food intake during PFOA/PFOS treatment in wildtype mice, brown-fat mitochondrial recruitment was also induced. We conclude that we have uncovered the existence of a regulatory component of food intake that is dependent upon brown adipose tissue thermogenic activity. The possible environmental consequences of this novel PFOA/PFOS effect (a possible decreased fitness) are noted, as well as the perspectives of this finding on the general understanding of control of food intake control and its possible extension to combatting obesity.

The relationship between liver peroxisome proliferation and adipose tissue atrophy induced by peroxisome proliferator exposure and withdrawal in mice.

We have previously demonstrated that severe adipose tissue atrophy occurs upon dietary treatment of mice with potent peroxisome proliferators (PPs). This atrophy occurs subsequent to peroxisome proliferation in the liver and may represent a novel addition to the pleiotropic effects exerted by PPs. In the present study we have characterized the recovery of mice from such atrophy following cessation of exposure. Following termination of treatment with perfluorooctanoic acid (PFOA) for 7 days, the adipose tissue atrophy was rapidly reversed, beginning on 2-5 days of recovery and being complete within 10 days. In contrast, hepatic peroxisome proliferation recovered much more slowly, indicating that these processes are not strictly coordinated. Analysis of lipoprotein lipase and hormone-sensitive lipase activities in adipose tissue revealed that the decrease and increase in these activities, respectively, caused by PFOA were both reversed within 10 days of recovery. Overall, these data provide further support for our previous conclusion that the adipose tissue atrophy induced by PFOA is caused, at least in part, by changes in the activities of lipoprotein lipase and hormone-sensitive lipase. The serum level of cholesterol, which increased after termination of PFOA treatment, returned to normal with a time-course similar to the recovery of adipose tissue weight, although hepatic peroxisome proliferation was still present. The possible relationship between the reduction in serum cholesterol and/or in its availability to peripheral tissues and the associated atrophy of adipose tissues caused by PPs is discussed.

Involvement of the peroxisome proliferator-activated receptor alpha in the immunomodulation caused by peroxisome proliferators in mice.

Peroxisome proliferators (PPs) are a large class of structurally diverse chemicals, which includes drugs designed to improve the metabolic abnormalities linking hypertriglyceridemia to diabetes, hyperglycemia, insulin-resistance and atherosclerosis. We have recently demonstrated that exposure of rodents to potent PPs indirectly causes a number of immunomodulating effects, resulting in severe adaptive immunosuppression. Since the peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in mediating the pleiotropic responses exerted by PPs, we have compared here the immunomodulating effects of the PPs perfluorooctanoic acid (PFOA) and Wy-14,643 in wild-type and PPARalpha-null mice. The reductions in spleen weight and in the number of splenocytes caused by PP treatment in wild-type mice was not observed in PPARalpha-null mice. Furthermore, the reductions in thymus weight and in the number of thymocytes were potently attenuated in the latter animals. Similarly, the dramatic decreases in the size of the CD4(+)CD8(+) population of cells in the thymus and in the number of thymocytes in the S and G2/M phases of the cell cycle observed in wild-type mice administered PPs were much less extensive in PPARalpha-null mice. Finally, in contrast to the case of wild-type animals, the response of splenocytes isolated from the spleen of PP-treated PPARalpha-null mice to appropriate T- or B-cell activators in vitro was not reduced. Altogether, these data indicate that PPARalpha plays a major role in the immunomodulation caused by PPs. The possible relevance of these changes to the alterations in plasma lipids also caused by PPs is discussed.

The effects of peroxisome proliferators on global lipid homeostasis and the possible significance of these effects to other responses to these xenobiotics: an hypothesis.

Peroxisome proliferators (PPs) have been shown to regulate hepatic lipid metabolism via activation of the peroxisome proliferator-activated receptor alpha (PPAR-alpha). Recent studies have revealed that PPs also exert considerable influence on certain extrahepatic tissues, including adipose tissue and lymphoid organs, in an indirect fashion. Inhibition of the proliferation of thymocytes and splenocytes and alteration of fatty acid uptake into and release from adipose tissue might be consequences of the hypolipidemic effect of PPs involving both PPARalpha-dependent and -independent pathways. Exposure to PPs reduces the cholesterol content of circulating low-density lipoprotein (LDL), which is the major supply of this steroid to most peripheral tissues. In addition, PPs increase serum levels of high-density lipoprotein (HDL), which extracts cholesterol from peripheral tissues and returns it to the liver, thereby further decreasing the cholesterol content of peripheral tissues. This net flux of cholesterol from extrahepatic tissues to the liver represents a change in global lipid homeostasis. In normal healthy young mice, this hypolipidemic effect could result in loss of cholesterol and other lipids from peripheral tissues (e.g., adipose tissue, thymus, and spleen), especially from plasma membrane caveolae, which might perturb normal cellular signaling and result in tissue atrophy. On the other hand, the increased hepatic cholesterol content in the hepatocyte plasma membrane might actually enhance signaling, playing a role in the liver hypertrophy and hepatocarcinogenecally associated with long-term PP treatment. In conclusion, it is important to consider the systemic effects of PPs, rather than to focus on the liver alone.

The expression of cytosolic and mitochondrial type II acyl-CoA thioesterases is upregulated in the porcine corpus luteum during pregnancy.

Acyl-CoA thioesterases hydrolyze acyl-CoAs to free fatty acids and CoASH, thereby regulating fatty acid metabolism. This activity is catalyzed by numerous structurally related and unrelated enzymes, of which several acyl-CoA thioesterases have been shown to be regulated via the peroxisome proliferator-activated receptor alpha, strongly linking them to fatty acid metabolism. Two protein families have recently been characterized, the type I acyl-CoA thioesterase gene family and the type II protein family, which are expressed in cytosol, mitochondria and peroxisomes. Still, only little is known about regulation of their expression and precise functions in vivo. In the present study, we have investigated the activity and expression of acyl-CoA thioesterase in the porcine ovary during different phases of the estrus cycle. The activity was low in homogenates obtained during the immature and follicular phases, increasing nearly 4-fold during the luteal phase, with the highest activity being found in the pregnant corpus luteum (about 7-fold higher than in immature follicles). The increase in homogenate activity in corpus luteum from pregnant pigs was due to a moderate increase in the cytosolic activity, and an approximately 20-25-fold increase in the mitochondrial fraction. Western blot analysis showed no detectable expression of the type I acyl-CoA thioesterases (CTE-I and MTE-I) and revealed that the increased activity in cytosol and mitochondria is due to increased expression of the type II acyl-CoA thioesterases (CTE-II and MTE-II). This apparent hormonal regulation of expression of the type II acyl-CoA thioesterase may provide new insights into the functions of these enzymes in the mammalian ovary.

Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perfluorooctanoic acid.

In a previous investigation, we demonstrated that severe thymus and spleen atrophy occurs in mice upon dietary exposure to several potent peroxisome proliferators (PPs). In the present investigation, the effects of the potent PP perfluorooctanoic acid (PFOA) on the adaptive immunity of mice was evaluated both in vivo and ex vivo. The in vivo immune response examined involved immunization of mice with horse red blood cells (HRBCs), displaying T-cell-dependent antigens after pre-treatment with a PFOA-containing diet for 10 days. Subsequent quantitation of the primary humoral response was performed employing both the plaque-forming cell (PFC) assay and determination of the antibody titer by ELISA. The results clearly demonstrate that oral administration of PFOA prevents both the increases in plaque formations by anti-IgM and -IgG and in serum levels of IgM and IgG normally evoked by such immunization. Ex vivo spleen cells proliferation (assayed as incorporation of 3H-thymidine) in response to both T- and B-cell activators was attenuated by dietary treatment with PFOA, although the analogous in vitro treatment of mouse spleen cells with this same compound had no such effects. Thus, the relatively metabolically inert PP PFOA may exert adaptive immunosuppression in mice by an indirect mechanism. The possible relevance of this immunosuppression to the alterations in plasma lipids caused by PPs is discussed.

Detailed analytical subcellular fractionation of non-pregnant porcine corpus luteum reveals peroxisomes of normal size and significant UDP-glucuronosyltransferase activity in the high-speed supernatant.

A detailed subfractionation of the non-pregnant porcine corpus luteum (CL) was performed employing differential centrifugation. Marker enzyme assays (i.e., lactate dehydrogenase for the cytosol, NADPH-cytochrome P450 reductase for the endoplasmatic reticulum, catalase (CAT) for peroxisomes, glutamate dehydrogenase for the mitochondrial matrix and acid phosphatase for lysosomes) in all subfractions obtained exhibited a pattern of distribution similar to that observed with rat liver. These subfractions should be useful in connection with many types of future studies. In disagreement with previous biochemical and morphological studies, peroxisomes (identified on the basis of catalase activity and by Western blotting of catalase and of the major peroxisomal membrane protein (PMP-70)) sedimented together with mitochondria (i.e., at 5000 x g(av) for 10 min) and not in the post-mitochondrial fraction prepared at 30,000 x g(av) for 20 min by Peterson and Stevensson. No other classical peroxisomal enzymes were detectable in the porcine ovary, raising questions concerning the function of peroxisomes in this organ. Furthermore, UDP-glucuronosyltransferase (UGT), generally considered to be an integral membrane protein anchored in the endoplasmatic reticulum, was recovered in both the cytosolic (i.e., the supernatant after centrifugation at 50,000 x g(av) for 1h) and the microsomal fraction of the porcine corpus luteum, even upon further centrifugation of the former. In contrast, UGT sediments exclusively in the microsomal fraction upon subfractionation of the liver and ovary from rat.

Characterization of the adipose tissue atrophy induced by peroxisome proliferators in mice.

In the present study, we characterized the effects of peroxisome proliferators (PP) on adipose tissue in mice. Treatment with potent PP, such as perfluorooctanoic acid (PFOA), 2-methyl-2-(p(1,2,3,4-tetrahydroxy-naphthyl)-phenoxy)propionic acid, (4-chloro-6-(2,3-xylidino)2-pyrimidinylthio) acetic acid, and di(2-ethylhexyl)phthalate, caused dramatic decreases in adipose tissue weight, whereas the moderately potent PP, acetylsalicylic acid, had a relatively weak effect. This decrease in weight reflects a loss of fat from adipocytes rather than a loss of cells, as demonstrated by constant DNA content. The dose-dependency and time-course experiments indicate that peroxisome proliferation occurs simultaneously with or prior to adipose tissue atrophy. Thus, hepatic peroxisome proliferation might result in the increased mobilization of lipids and lipid utilization in liver. The enhanced adipose tissue hormone-sensitive lipase (HSL) activity and down-regulated lipoprotein lipase (LPL) activity observed upon PP treatment might, at least in part, explain the loss of fat via increased FA release from adipocytes and/or decreased FA uptake from the circulation, respectively. In addition, the possible involvement of the increased tumor necrosis factor alpha expression found upon PFOA treatment in reducing the insulin sensitivity of adipose tissue and thereby altering LPL and HSL activities is discussed.

Sub-acute, moderate-dose, but not short-term, low-dose dietary pre-exposure of mice to perfluorooctanoate aggravates concanavalin A-induced hepatitis.

Exposure of mice to perfluorooctanoate (PFOA) evokes pronounced hepatomegaly along with significant alterations in both the histological structure and immune status of the liver. The present study was designed to evaluate the effects of this perfluorochemical on immune-mediated liver damage. In this connection, the influence of both sub-acute (10 days), moderate-dose (0.002% w/w=3±0.7mg/kg body weight/day) and short-term (28 days), low-dose (0.00005% w/w=70±2µg/kg body weight/day) dietary pretreatment with PFOA on the development of concanavalin A (Con A)-induced liver damage in mice was examined. With sub-acute, moderate, but not short-term, low-dose exposure, PFOA aggravated the acute liver damage caused by Con A, i.e., elevated serum levels of transaminases and led to more pronounced damage of hepatic tissue. This aggravation was associated with significantly enhanced hepatic level of interleukin-6 (IL-6), but unaltered hepatic levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-4 (IL-4). Moreover, hepatic DNA fragmentation was not changed by sub-acute exposure to the moderate-dose. Our findings imply that exposure to PFOA may sensitize hepatic parenchymal cells to other toxicants that activate the hepatic immune system and thereby aggravate liver injury during acute inflammation.

Both sub-acute, moderate-dose and short-term, low-dose dietary exposure of mice to perfluorooctane sulfonate exacerbates concanavalin A-induced hepatitis.

Exposure of rodents to perfluorooctane sulfonate (PFOS) induces pronounced hepatomegaly associated with significant alterations in hepatic histophysiology and immune status. The present investigation was designed to evaluate the effects of this perfluorochemical on immune-mediated liver damage. Accordingly, the influence of both sub-acute (10 days), moderate-dose (0.004%, w/w=6±1.3 mg/kg body weight/day) or short-term (28 days), low-dose (0.0001%, w/w=144±4 µg/kg body weight/day) dietary pretreatment with PFOS on the development of concanavalin A (Con A)-induced liver damage in mice was examined. With either regimen of exposure, PFOS exacerbated the acute liver damage caused by Con A, i.e., elevated serum levels of transaminases and led to more pronounced damage of hepatic tissue. This exacerbation was associated with either reduced (moderate dose) or unaltered (low dose) hepatic levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). Moreover, hepatic DNA fragmentation was enhanced, particularly following short-term exposure to a low-dose. Our findings suggest that exposure to PFOS may sensitize hepatic parenchymal cells to other insults that activate the hepatic immune system and thereby exacerbate liver damage during acute inflammation.

High-dose dietary exposure of mice to perfluorooctanoate or perfluorooctane sulfonate exerts toxic effects on myeloid and B-lymphoid cells in the bone marrow and these effects are partially dependent on reduced food consumption.

It is well established that exposure of mice to perfluorooctanoate (PFOA) or perfluorooctane sulfonate (PFOS) exerts adverse effects on the thymus and spleen. Here, we characterize the effects of a 10-day dietary treatment with these compounds (0.001-0.02%, w/w) on the bone marrow (BM) of mice. At a dose of 0.02%, both compounds reduced food consumption and caused atrophy of the thymus and spleen. At this same dose, histopathological and flow cytometric analysis revealed that (i) the total numbers of BM as well as the numbers of myeloid, pro/pre B, immature B and early mature B cells were all reduced significantly; and (ii) these adverse effects were reversed either partially or completely 10days after withdrawal of these compounds. At the lower dose of 0.002%, only PFOA reduced the B-lymphoid cell population. Finally, mice fed an amount of diet equivalent to that consumed by the animals exposed to 0.02% PFOA also exhibited atrophy of the thymus and spleen, and a reduction in the number of B-lymphoid population, without affecting myeloid cells. Thus, in mice, immunotoxic doses of PFOA or PFOS induce adverse effects on the myeloid and B-lymphoid cells in the BM, in part as a consequence of reduced food consumption.

Radiosynthesis of perfluorooctanesulfonate (PFOS) and perfluorobutanesulfonate (PFBS), including solubility, partition and adhesion studies.

Here, we describe for the first time the synthesis of [(35)S] PFOS and [(35)S] PFBS with sulfur-35 enriched sulfur dioxide as the radiolabelled reagent, resulting in 2.5 and 2.3 mCi of product, respectively. Basic information concerning the physicochemical properties of perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate (PFBS) and perfluorooctanoic acid (PFOA) are still limited. Hence, we utilized these radiolabelled perfluoroalkanesulfonates (PFSAs), as well as carbon-14 labelled perfluorooctanoic acid ([(14)C] PFOA) to determine some basic characteristics of physiological and experimental significance. The solubility of PFOS in buffered aqueous solutions at pH 7.4 was found to be severely reduced in the presence of potassium and sodium ions, which, however, did not reduce the solubility of PFOA or PFBS. PFOS was found to adhere to a small extent to polypropylene and polystyrene, whereas no such adhesion of PFOA or PFBS was detected. The extents of adhesion of PFOS and PFOA to glass were found to be 20% and 10%, respectively. For the first time, the partition coefficients for PFOS, PFBS and PFOA between n-octanol and water were determined experimentally, to be -0.7, -0.3, and 1.4, respectively, reflecting the difference in the amphiphilic natures of these molecules.

28-Day dietary exposure of mice to a low total dose (7 mg/kg) of perfluorooctanesulfonate (PFOS) alters neither the cellular compositions of the thymus and spleen nor humoral immune responses: does the route of administration play a pivotal role in PFOS-induced immunotoxicity?

Short-term exposure of mice to high doses of perfluorooctanesulfonate (PFOS), an ubiquitous and highly persistent environmental contaminant, induces various metabolic changes and toxic effects, including immunotoxicity. However, extrapolation of these findings to the long-term, low-dose exposures to which humans are subject is highly problematic. In this connection, recent studies have concluded that sub-chronic (28-day) exposure of mice by oral gavage to doses of PFOS that result in serum levels comparable to those found in general human populations suppress adaptive immunity. Because of the potential impact of these findings on environmental research and monitoring, we have examined here whether sub-chronic dietary exposure (a major route of human exposure) to a similarly low-dose of PFOS also suppress adaptive immune responses. Dietary treatment of male B6C3F1 mice for 28 days with a dose of PFOS that resulted in a serum concentration of 11mug/ml (ppm) significantly reduced body weight gain and increased liver mass. However, this treatment did not alter the cellular compositions of the thymus and spleen; the number of splenic cells secreting IgM antibodies against sheep red blood cell (SRBC); serum levels of IgM and IgG antibodies specifically towards SRBC; or circulating levels of IgM antibodies against the T-cell-independent antigen trinitrophenyl conjugated to lipopolysaccharide (TNP-LPS). These findings indicate that such sub-chronic dietary exposure of mice to PFOS resulting in serum levels approximately 8-85-fold greater than those observed in occupationally exposed individuals does not exert adverse effects on adaptive immunity.

Dietary exposure to perfluorooctanoate or perfluorooctane sulfonate induces hypertrophy in centrilobular hepatocytes and alters the hepatic immune status in mice.

It is well established that exposure of mice to perfluorooctanoate (PFOA) or perfluorooctane sulfonate (PFOS) induces hepatomegaly and, concurrently, immunotoxicity. However, the effects of these perfluorochemicals on the histology and immune status of the liver have not been yet investigated and we have examined these issues here. Dietary treatment of male C57BL/6 mice with 0.002% (w/w) PFOA or 0.005% (w/w) PFOS for 10 days resulted in significant reductions in serum levels of cholesterol and triglycerides, a moderate increase in the serum activity of alkaline phosphatase (ALP) and hepatomegaly, without affecting other immune organs. This hepatomegaly was associated with marked hypertrophy of the centrilobular hepatocytes, with elevated numbers of cytoplasmic acidophilic granules and occasional mitosis. Furthermore, dietary exposure to PFOA or PFOS altered the hepatic immune status: whereas exposure to PFOA enhanced the numbers of total, as well as of phenotypically distinct subpopulations of intrahepatic immune cells (IHIC), and in particular the presumptive erythrocyte progenitor cells, treatment with PFOS enhanced only the numbers of hepatic cells that appear immunophenotypically to be erythrocyte progenitors, without affecting other types of IHIC. In addition, exposure to these compounds attenuated hepatic levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-4 (IL-4). Furthermore, the exposed animals exhibited a significant increase in hepatic levels of erythropoietin, a hormone required for erythropoiesis. Thus, in mice, PFOA- and PFOS-induced hepatomegaly is associated with significant alterations in hepatic histophysiology and immune status, as well as induction of hepatic erythropoiesis.

High-dose, short-term exposure of mice to perfluorooctanesulfonate (PFOS) or perfluorooctanoate (PFOA) affects the number of circulating neutrophils differently, but enhances the inflammatory responses of macrophages to lipopolysaccharide (LPS) in a similar fashion.

Having found previously that high-dose, short-term dietary exposure of mice to perfluorooctanesulfonate (PFOS) or perfluorooctanoate (PFOA) suppresses adaptive immunity, in the present study we characterize the effects of these fluorochemicals on the innate immune system. Male C57BL/6 mice receiving 0.02% (w/w) PFOS or PFOA in their diet for 10 days exhibited a significant reduction in the numbers of total white blood cells (WBC), involving lymphopenia in both cases, but neutropenia only in response to treatment with PFOA. Moreover, both compounds also markedly reduced the number of macrophages (CD11b(+) cells) in the bone marrow, but not in the spleen or peritoneal cavity. The ex vivo production of tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) by peritoneal macrophages isolated from animals treated with PFOA or PFOS was increased modestly. Moreover, both fluorochemicals markedly enhanced the ex vivo production of these same cytokines by peritoneal and bone marrow macrophages stimulated either in vitro or in vivo with lipopolysaccharide (LPS); whereas there was no such effect on splenic macrophages. The serum levels of these inflammatory cytokines observed in response to in vivo stimulation with LPS were elevated substantially by prior exposure to PFOA, but not by PFOS. None of these parameters of innate immunity were altered in animals receiving a dietary dose of these compounds that was 20-fold lower (0.001%, w/w). These findings reveal that in addition to suppressing adaptive immunity, high-dose, short-term exposure of mice to either PFOS or PFOA augments inflammatory responses to LPS, a potent activator of innate immunity.

The atrophy and changes in the cellular compositions of the thymus and spleen observed in mice subjected to short-term exposure to perfluorooctanesulfonate are high-dose phenomena mediated in part by peroxisome proliferator-activated receptor-alpha (PPARalpha).

We have previously shown that short-term, high-dose exposure of mice to the environmentally persistent perfluorooctanoate (PFOA) results in thymic and splenic atrophy and the attenuation of specific humoral immune responses. Here we characterize the effects of a 10-day treatment with different dietary doses (1-0.001%, w/w) of perfluorooctanesulfonate (PFOS), a similar fluorochemical, on the immune system of male C57BL/6 mice. At doses greater than 0.02%, PFOS induced clinical signs of toxicity in the animals, whereas at the concentration of 0.02%, this compound caused weight loss, hepatomegaly and atrophy of the thymus, spleen and adipose tissue without toxicity. With this latter dose, histopathological and flow-cytometric analysis revealed that (i) the thymic cortex was virtually depleted of cells; (ii) the total numbers of thymocytes and splenocytes were reduced by 84 and 43%, respectively; (iii) although all populations of thymocytes and splenocytes were smaller, the thymic CD4(+)CD8(+) cells and the splenic B-lymphocytes were most decreased. These alterations resembled those evoked by analogous exposure to PFOA, but were less pronounced. At lower doses (less than 0.02%), PFOS induced hepatomegaly without affecting the thymus or spleen. Finally, comparison of male wild-type 129/Sv mice and the corresponding knock-outs lacking peroxisome proliferator-activated receptor-alpha (PPARalpha) indicated that these effects of PFOS are not strain-dependent. More importantly, hepatomegaly is independent of PPARalpha, the thymic changes are partially dependent on this receptor, and splenic responses are largely eliminated in its absence. Thus, immunomodulation caused by PFOS is a high-dose phenomenon partially dependent on PPARalpha.

Exposure to mercuric chloride during the induction phase and after the onset of collagen-induced arthritis enhances immune/autoimmune responses and exacerbates the disease in DBA/1 mice.

In susceptible mice, mercuric chloride induces a systemic autoimmune response that is characterized by elevated serum levels of immunoglobulin G1 (IgG1) and immunoglobulin E (IgE), production of anti-nucleolar antibodies (ANolAs) and the formation of renal IgG deposits. We have previously shown that mercury can also enhance immune/autoimmune responses in mouse strains genetically prone to develop spontaneous autoimmune disease. Here, we investigated whether mercury can enhance the severity of murine collagen-induced arthritis (CIA), an inducible (acquired) autoimmune disease that cannot be induced by mercury itself. While mercury administered prior to the induction phase of CIA exerted little, if any, influence, administration of mercury during the induction phase and following onset aggravated the symptoms of this disease and increased the serum levels of IgE and IgG2a antibodies directed against collagen type II (CII). Furthermore, while animals injected with mercury alone exhibited a significant decrease in the ratio of the levels of interferon-gamma (IFN-gamma) to interleukin-4 (IL-4) mRNA in their spleens, this ratio was increased in mice with CIA, with or without administration of mercury. Finally, the production of anti-nuclear antibodies, a hallmark of autoimmunity in response to mercury, was observed in all mice with CIA treated with this heavy metal. Our findings suggest that exposure to mercury during the development of CIA may influence immunological factors in such a way as to synergistically promote disease development.