Abstract  Chloroform, ubiquitously present in indoor and outdoor air, drinking water, and some foodstuffs, enters the human body by inhalation, oral and dermal routes of exposure. In order to provide bioassay data for risk assessment of humans exposed to chloroform by multiple routes, effects of combined inhalation and oral exposures to chloroform on carcinogenicity and chronic toxicity in male F344 rats were examined. A group of 50 male rats was exposed by inhalation to 0 (clean air), 25, 50, or 100 ppm (v/v) of chloroform vapor-containing air for 6 h/d and 5 d/wk during a 104 w period, and each inhalation group was given chloroform-formulated drinking water (1000 ppm w/w) or vehicle water for 104 wk, ad libitum. Renal-cell adenomas and carcinomas and atypical renal-tubule hyperplasias were increased in the combined inhalation and oral exposure groups, but not in the oral- or inhalation-alone groups. Incidences of cytoplasmic basophilia and dilated tubular lumens in the kidney, as well as incidence of positive urinary glucose, were markedly increased by the combined exposures, compared with those after single-route exposures. It was concluded that combined inhalation and oral exposures markedly enhanced carcinogenicity and chronic toxicity in the proximal tubule of male rat kidneys, suggesting that carcinogenic and toxic effects of the combined exposures on the kidneys were greater than the ones that would be expected under an assumption that the two effects of single route exposures through inhalation and drinking were additive.

Abstract  Chloroform acts via a nongenotoxic-cytotoxic mode of action to produce cancer if given in doses and at dose rates sufficiently high to produce organ-specific toxicity. In a recent study, chloroform failed to induce cancer in male or female F-344 rats when administered by inhalation for 2 years at 90 ppm, 5 days/week. The present study was undertaken to define the concentration-response curves for chloroform-induced lesions and regenerative cell proliferation in the F-344 rat when exposed by inhalation and to correlate those patterns of toxicity with the results from the inhalation cancer bioassay. Male and female F-344 rats were exposed to airborne concentrations of 0, 2, 10, 30, 90, or 300 ppm chloroform 6 hr/day, 7 days/week for 4 days or 3, 6, 13 weeks. Additional treatment groups were exposed 5 days/week for 13 weeks or were exposed for 6 weeks and held until Week 13. Bromodeoxyuridine was administered via osmotic pumps implanted 3.5 days prior to necropsy and the labeling index (LI, percentage of nuclei in S-phase) was evaluated immunohistochemically. A full-screen necropsy identified the kidney, liver, and nasal passages as the only target organs. This study confirmed that 300 ppm is extremely toxic and would be inappropriate for longer-term cancer studies. The primary target in the kidney was the epithelial cells of the proximal tubules of the cortex, with significantly elevated increases in the LI at concentrations of 30 ppm and above. However, only a marginal increase in the renal LI in the males was seen after exposures of 90 ppm, 5 days/week. Chloroform induced hepatic lesions in the midzonal and centrilobular regions with increases in the LI throughout the liver, but only at 300 ppm exposures. An additional liver lesion seen only at the highly hepatotoxic concentration of 300 ppm was numerous intestinal crypt-like ducts surrounded by dense connective tissue. Enhanced bone growth and hypercellularity in the lamina propria of the ethmoid turbinates of the nose occurred at the early time points at concentrations of 10 ppm and above. At 90 days there was a generalized atrophy of the ethmoid turbinates at concentrations of 2 ppm and above. Cytolethality and regenerative cell proliferation are necessary but not always sufficient to induce cancer because of tissue, sex, and species differences in susceptibility. A combination of a lack of direct genotoxic activity by chloroform, only a marginal induction of cell proliferation in the male rat kidney, and lower tissue-specific susceptibility in the female rat is apparently responsible for the reported lack of chloroform-induced cancer in a long-term inhalation bioassay with F-344 rats.

Abstract  In this study, we examined whether the production of hydrogen peroxide by peroxisome proliferators causes oxidative DNA damage in the form of 8-oxodeoxyguanosine (8-oxodG) and hepatic injury, and whether it is related to their tumor-promoting or carcinogenic activities in female rats treated with the peroxisome proliferators clofibrate and perfluorodecanoic acid (PFDA). Clofibrate has tumor-promoting and carcinogenic activities, whereas PFDA does not. We also tested whether peroxisome proliferators directly induce mutagenic events in Salmonella typhimurium strains TA 98 and TA 1537. Rats were treated either by 5% clofibrate in diet or by an i.p. injection of corn oil containing 10 mg/kg body weight of PFDA every week for 2 or 8 weeks. 8-OxodG in liver DNA was analyzed by HPLC coupled with an electrochemical detector. Hepatic injury was evidenced by liver enlargement and by levels of serum enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and hepatic gamma-glutamylpeptidase (gamma-GT) activity. Clofibrate and PFDA increased the activity of catalase about or less than 2-fold, whereas FAO activity was increased about 6 to 7-fold by clofibrate and about 3 to 4-fold by PFDA. Neither clofibrate nor PFDA induced mutation at any dose tested. Clofibrate significantly increased the formation of 8-oxodG, but PFDA only slightly increased. Serum AST and ALT levels, and hepatic gamma-GT activity were not significantly changed at both time points, whereas the ratio of liver/body weight was significantly increased by clofibrate and PFDA at 8 weeks. These data imply that the magnitude of the production of hydrogen peroxide-generated FAO is related to the induction of oxidative DNA damage by peroxisome proliferators, and their tumor-promoting or carcinogenic activities. However, the effect of hydrogen peroxide in hepatic injury is not clear.

Abstract  Ammonium perfluorononanoate (CAS Registry No. 4149-60-4) is a white powder that can become airborne. Its acute inhalation toxicity in male rats was studied. Male rats were exposed for single 4-hr periods to dust concentrations ranging from 67 to 4600 mg/m3. The LC50 was determined to be 820 mg/m3, with the lowest concentration causing death being 590 mg/m3. Ammonium perfluorononanoate was classified as moderately toxic by the acute inhalation route. Exposure to ammonium perfluorononanoate caused a pronounced increase in liver size. The acute toxicity of ammonium perfluorononanoate appears to be similar to that of its 8-carbon homologue, ammonium perfluorooctanoate, but considerably less than that of the 10-carbon homologue, perfluoro-n-decanoic acid.

Abstract  Most studies on the liver toxicity of perfluorinated compounds (PFCs) are focused on healthy individuals, whereas the effects of PFCs on individuals with diabetes mellitus have not been fully characterized. This study aimed to investigate the acute exposure of perfluorononanoic acid (PFNA) on the metabolism of lipid in the liver of streptozotocin-induced diabetic rats. Male diabetic rats were orally dosed by gavage for 7 days with 0, 0.2, 1 and 5 mg/kg/day PFNA. The contents of lipid, the activities of enzyme, the expressions of protein in the liver and the serum parameters were detected. The results indicate that dose-dependent accumulation of triglyceride and total cholesterol occurred in the livers of diabetic rats after PFNA treatment. PFNA increased the activities of lipid synthetase, fatty acid synthease, glucose-6-phosphate dehydrogenase and decreased the activity of lipolytic enzyme, hepatic lipase, in the liver of diabetic rats. The changes of the isocitrate dehydrogenase, malicenzyme and lipoprotein lipase were not obvious. The expressions of protein related to lipid homeostasis, liver X receptor α and apolipoprotein E, were decreased after PFNA administration. Exposure to PFNA also increased the activity of serum alanine aminotransferase in diabetic rats. In conclusion, this study discloses that exposure to PFNA impacts on enzymes and proteins related to liver lipid metabolism and lead to obvious accumulation of lipid in the liver of diabetic rats, which may be responsible for hepatotoxicity of this compound in individuals with diabetes mellitus.

Abstract  Kupffer cells (KCs) have been demonstrated to play a role in the regulation of intra-hepatic lipid metabolism through the synthesis and secretion of biologically active products. The involvement of KCs in the disturbance of lipid metabolism that induced by perfluorononanoic acid (PFNA), a known agonist of the peroxisome proliferator-activated receptor alpha (PPARα), was investigated in this study. Rats were exposed to PFNA or PFNA combined with gadolinium chloride, an inhibitor of KCs, for 14 days. PFNA exposure dose-dependently increased absolute and relative liver weights, induced triglyceride accumulation, up-regulated the expression of both SERBP-1c and PPARα, and stimulated the release of TNFα and IL-1β. Inactivation of KCs markedly lowered TNFα and IL-1β level, enhanced PFNA-induced expression of PPARα and its target genes, and reduced liver triglyceride levels. In vitro, PFNA-induced expression of PPARα in primary cultured hepatocytes was suppressed by recombinant rat TNFα and IL-1β. However, inhibition of the NF-κB pathway prevented this. Transient transfection and promoter analysis further revealed that these two cytokines and NF-κB were coordinately involved in the suppression of PPARα promoter activity. Our data demonstrate that TNFα and IL-1β released from KCs following PFNA exposure can suppress the expression of PPARα via NF-κB pathway, which partially contribute to the evident accumulation of triglycerides in rat liver.

Abstract  Potassium perfluorohexanesulfoante (K+PFHxS) was evaluated for reproductive/developmental toxicity in CD-1 mice. Up to 3 mg/kg-d K+PFHxS was administered (n = 30/sex/group) before mating, for at least 42 days in F0 males, and for F0 females, through gestation and lactation. F1 pups were directly dosed with K+PFHxS for 14 days after weaning. There was an equivocal decrease in live litter size at 1 and 3 mg/kg-d, but the pup-born-to-implant ratio was unaffected. Adaptive hepatocellular hypertrophy was observed, and in 3 mg/kg-d F0 males, it was accompanied by concomitant decreased serum cholesterol and increased alkaline phosphatase. There were no other toxicologically significant findings on reproductive parameters, hematology/clinical pathology/TSH, neurobehavioral effects, or histopathology. There were no treatment-related effects on postnatal survival, development, or onset of preputial separation or vaginal opening in F1 mice. Consistent with previous studies, our data suggest that the potency of PFHxS is much lower than PFOS in rodents.

Abstract  Perfluorobutanesulfonate (PFBS) is widely used in many industrial products. We evaluated the influence of prenatal PFBS exposure on perinatal growth and development, pubertal onset, and reproductive and thyroid endocrine system in female mice. Here, we show that when PFBS (200 and 500 mg/kg/day) was orally administered to pregnant mice (PFBS-dams) on days 1-20 of gestation; their female offspring (PFBS-offspring) exhibited decreased perinatal body weight and delayed eye opening compared with control offspring. Vaginal opening and first estrus were also significantly delayed in PFBS-offspring, and diestrus was prolonged. Ovarian and uterine size, as well as follicle and corpus luteum numbers, were reduced in adult PFBS-offspring. Furthermore, pubertal and adult PFBS-offspring exhibited decreases in serum estrogen (E2) and progesterone (P4) levels with the elevation of luteinizing hormone levels. Notably, decreases in serum total thyroxine (T4) and 3,3', 5-triiodothyronine (T3) levels were observed in fetal, pubertal, and adult PFBS-offspring in conjunction with slight increases in thyroid-stimulating hormone (TSH) and thyrotropin-releasing hormone levels. In addition, PFBS-dams exhibited significant decreases in total T4 and T3 levels and free T4 levels and increases in TSH levels, but no changes in E2 and P4 levels. These results indicate that prenatal PFBS exposure (≥200 mg/kg/day) causes permanent hypothyroxinemia accompanied by deficits in perinatal growth, pubertal onset, and reproductive organ development in female mice.

Abstract  First, the developmental toxic potential of n-butyl acetate (BA) was examined in Sprague-Dawley rats following whole body inhalation exposure, 6 h day(-1), from day 6 to 20 of gestation, at concentrations of 0, 500, 1000, 2000 and 3000 ppm. Maternal toxicity was evidenced by significant decreases in body weight gain at 2000 and 3000 ppm, and by reduced food consumption at 1000 ppm and higher concentrations. The effects on prenatal development were limited to a significant decrease in fetal weight at 3000 ppm. Thus, inhaled BA was not a selective developmental toxicant. In the second part of this study, the developmental toxic effects of simultaneous exposures to ethylbenzene (EB) and BA, or to toluene (TOL) and BA were evaluated. Pregnant rats were administered EB (0, 250 or 1000 ppm) and BA (0, 500 or 1500 ppm), or TOL (0, 500 or 1500 ppm) and BA (0, 500, 1500 ppm), separately and in combinations, using a 2 x 2 factorial design. The maternal weight gain was reduced after exposure to 1000 ppm EB, to 1500 ppm BA, or to 1500 ppm TOL, either alone or in binary combinations. A significant reduction of fetal weight was associated with exposure to 1000 ppm EB alone, to either mixtures of EB with BA, or to 1500 ppm TOL alone or combined with BA at either concentration. No embryolethal or teratogenic effects were observed whatever the exposure. There was no evidence of interaction between EB and BA or between TOL and BA in causing maternal or developmental effects.

Abstract  Perfluorinated acids (PFAs) and their precursors (PFA-precursors) exist in the environment as linear and multiple branched isomers. These isomers are hypothesized to have different biological properties, but no isomer-specific data are currently available. The present study is the first in a two-part project examining PFA isomer-specific uptake, tissue distribution, and elimination in a rodent model. Seven male Sprague-Dawley rats were administered a single gavage dose of approximately 500 microg/kg body weight perfluorooctane sulfonate (C(8)F(17)SO(3)(-), PFOS), perfluorooctanoic acid (C(7)F(15)CO(2)H, PFOA), and perfluorononanoic acid (C(8)F(17)CO(2)H, PFNA) and 30 microg/kg body weight perfluorohexane sulfonate (C(6)F(13)SO(3)(-), PFHxS). Over the subsequent 38 d, urine, feces, and tail-vein blood samples were collected intermittently, while larger blood volumes and tissues were collected on days 3 and 38 for isomer analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). For all PFAs, branched isomers generally had lower blood depuration half-lives than the corresponding linear isomer. The most remarkable exception was for the PFOS isomer containing an alpha-perfluoromethyl branch (1m-PFOS), which was threefold more persistent than linear PFOS, possibly due to steric shielding of the hydrophilic sulfonate moiety. For perfluoromonomethyl-branched isomers of PFOS, a structure-property relationship was observed whereby branching toward the sulfonate end of the perfluoroalkyl chain resulted in increased half-lives. For PFHxS, PFOA, and PFOS, preferential elimination of branched isomers occurred primarily via urine, whereas for PFNA preferential elimination of the isopropyl isomer occurred via both urine and feces. Changes in the blood isomer profiles over time and their inverse correlation to isomer elimination patterns in urine, feces, or both provided unequivocal evidence of significant isomer-specific biological handling. Source assignment based on PFA isomer profiles in biota must therefore be conducted with caution, because isomer profiles are unlikely to be conserved in biological samples.

Abstract  Ethylbenzene was administered to groups of male and female Wistar rats by gavage for 4 (n = 5/dose/sex) and 13 weeks (n = 10/dose/sex) (OECD 408) at doses of 0 (vehicle control), 75, 250, and 750 mg/kg bodyweight/day (mg/kg bw/day), administered am/pm as half doses. In the 4-week study, > or =250 mg/kg increased serum alanine aminotransferase, total bilirubin and cholesterol, liver weights and centrilobular hepatocyte hypertrophy, and kidney weights; males also had post-dose salivation, increased urinary epithelial cell casts and cells, and hyaline droplet nephropathy. In the 13-week study, > or =250 mg/kg increased water consumption and produced post-dose salivation. Liver-related effects: increased serum alanine aminotransferase, gamma-glutamyltransferase, bilirubin, total protein, albumin and globulins, cholesterol, liver weights and centrilobular hepatocyte hypertrophy, and reduced prothrombin times. Kidney-related effects: increased serum potassium, calcium, magnesium, kidney weights, and (males only) urea and hyaline droplets in renal tubular epithelium, and reduced sodium (females only); creatinine was reduced in 750 mg/kg males. The NOAEL of ethylbenzene in these studies, based on hepatocyte hypertrophy and liver- and kidney-related clinical chemistry changes, was 75 mg/kg bw/day.

Abstract  The toxicologic and carcinogenic potential of naphthalene was studied by exposing groups of 49 male and 49 female F344 rats to atmospheres containing 0, 10, 30, or 60 ppm of the chemical for 6 h daily, 5 days/wk for 2 yr. Mean body weights of exposed groups of male rats were less than for the control group throughout most of the study. Mean body weights of exposed female rats were generally similar to those of controls. Survival of exposed and control rats was similar. Under the conditions of this 2-yr inhalation study, naphthalene was carcinogenic to male and female F344/N rats, causing increased incidences of respiratory epithelial adenoma (males: control, 0%; low dose, 12%, mid dose, 17%; high dose, 31%; females: 0%; 0%; 8%; 4%) and olfactory epithelial neuroblastoma (males: control, 0%; low dose, 0%; mid dose, 8%; high dose, 6%; females: 0; 4%; 6%; 24%) of the nose. In both sexes of rats, exposure to naphthalene also caused significant increases in the incidences of nasal lesions including hyperplasia, atrophy, chronic inflammation, and hyaline degeneration of the olfactory epithelium and hyperplasia; squamous metaplasia, hyaline degeneration, and goblet-cell hyperplasia of the respiratory epithelium; and glandular hyperplasia and squamous metaplasia.

Abstract  The toxicologic and carcinogenic potential of naphthalene was studied by exposing groups of male and female B6C3F1 mice to atmospheres containing 0 (75 mice per sex), 70 ppm (75 mice per sex), or 30 ppm (150 mice per sex) of the chemical for 6 h daily, 5 dayslwk for 103 wk. The final mean body weights of mice exposed to naphthalene were similar to those of the controls. The survival of control male mice was significantly lower than that of the exposed males. The lower survival was attributed to wound trauma and secondary infection related to fighting among the group-housed control animals. There was no significant difference in survival between control and exposed female mice. Under the conditions of this 2-yr study, naphthalene was not carcinogenic to male mice. In female mice it caused an increase in the incidence of pulmonary alveolar/bronchiolar adenomas. Naphthalene also caused an increase in the incidence and severity of chronic inflammation, olfactory epithelium metaplasia of hyperplasia of the nasal respiratory epithelium, and chronic nasal inflammation in the lungs of mice of each sex.

Abstract  A bioassay study of carcinogenicity and chronic toxicity of chloroform was undertaken by inhalation exposures of groups of 50 F344 rats and 50 BDF1 mice of both sexes to chloroform for 6 h/d × 5 d/wk × 104 wk. The exposure concentration was 0 (control), 10, 30 or 90 ppm for rats and 0, 5, 30 or 90 ppm for mice. Combined incidences of renal cell adenomas and carcinomas, and of hepatocellular adenomas and carcinomas increased in the exposed male and female mice, respectively. Incidences of atypical tubule hyperplasia, cytoplasmic basophilia and nuclear enlargement in the kidneys and fatty change in the liver increased in the exposed male mice. Increased incidence of altered cell foci in the exposed female mice was causally related to the hepatocellular adenomas and carcinomas. No significantly increased incidence of the kidney or liver tumors was observed in the exposed rats of either sex. Increased incidences of nuclear enlargement and dilatation of tubular lumen were found in the kidneys of exposed rats. No-observed-adverse-effect-levels (NOAELs) for the biologically significant endpoint were determined from the dose-response relationships of the present datasets. The NOAEL for the histopathological endpoint of the kidneys resulted in 5 ppm for mice and 10 ppm for rats. An occupational exposure limit for chloroform was discussed in light of the NOAELs.

Abstract  Humans are simultaneously exposed to several chemicals that act jointly to induce mixture effects. At doses close to or higher than no-observed adverse effect levels, chemicals usually act additively in experimental studies. However, we are lacking knowledge on the importance of exposure to complex real-world mixtures at more relevant human exposure levels. We hypothesised that adverse mixture effects occur at doses approaching high-end human exposure levels. A mixture (Mix) of 14 chemicals at a combined dose of 2.5 mg/kg bw/day was tested in combination with perfluorononanoic acid (PFNA) at doses of 0.0125 (Low PFNA), 0.25 (Mid PFNA) and 5 (High PFNA) mg/kg bw/day by oral administration for 14 days in juvenile male rats. Indication of a toxicokinetic interaction was found, as simultaneous exposure to PFNA and the Mix caused a 2.8-fold increase in plasma PFNA concentrations at Low PFNA. An increase in testosterone and dihydrotestosterone plasma concentrations was observed for Low PFNA + Mix. This effect was considered non-monotonic, as higher doses did not cause this effect. Reduced LH plasma concentrations together with increased androgen concentrations indicate a disturbed pituitary-testis axis caused by the 15-chemical mixture. Low PFNA by itself increased the corticosterone plasma concentration, an effect which was normalised after simultaneous exposure to Mix. This combined with affected ACTH plasma concentrations and down-regulation of 11β HSD mRNA in livers indicates a disturbed pituitary-adrenal axis. In conclusion, our data suggest that mixtures of environmental chemicals at doses approaching high-end human exposure levels can cause a hormonal imbalance and disturb steroid hormones and their regulation. These effects may be non-monotonic and were observed at low doses. Whether this reflects a more general phenomenon that should be taken into consideration when predicting human mixture effects or represents a rarer phenomenon remains to be shown.

Abstract  Perfluorobutanesulfonate (PFBS) is a surfactant and degradation product of substances synthesized using perfluorobutanesulfonyl fluoride. A 90-day rat oral gavage study has been conducted with potassium PFBS (K+PFBS). Rats were dosed with K+PFBS at doses of 60, 200, and 600mg/kg-day body weight. The following endpoints were evaluated: clinical observations, food consumption, body weight, gross and microscopic pathology, clinical chemistry, and hematology. In addition, functional observation battery and motor activity assessments were made. Histological examination included tissues in control and 600 mg/kg-day groups. Additional histological examinations were performed on nasal cavities and turbinates, stomachs, and kidneys in the 60 and 200 mg/kg-day groups. No treatment-related mortality, body weight, or neurological effects were noted. Chromorhinorrhea (perioral) and urine-stained abdominal fur were observed in males at 600mg/kg-day. Red blood cell counts, hemoglobin, and hematocrit values were reduced in males receiving 200 and 600mg/kg-day; however, there were no adverse histopathological findings in bone marrow. Total protein and albumin were lower in females at 600mg/kg-day. There were no significant changes in clinical chemistry in either sex. All rats appeared normal at sacrifice. Microscopic changes were observed only at the highest dose in the stomach. These changes consisted of hyperplasia with some necrosis of the mucosa with some squamous metaplasia. These effects likely were due to a cumulative direct irritation effect resulting from oral dosing with K+PFBS. Histopathological changes were also observed in the kidneys. The changes observed were minimal-to-mild hyperplasia of the epithelial cells of the medullary and papillary tubules and the ducts in the inner medullary region. There were no corresponding changes in kidney weights. Clinical chemistry parameters related to kidney function were unchanged. These kidney findings are likely due to a response to high concentration of K+PFBS in tubules and ducts and represent a minimal-to-mild effect. Microscopic changes of an equivocal and uncertain nature were observed in the nasal mucosa and were likely attributable to the route of dosing (oral gavage). The NOAEL for the female rat in this study was 600 mg/kg-day (highest dose of study). The NOAEL for the male rat was 60 mg/kg-day based on hematological effects.

Abstract  In this supplementary study, groups consisting of 20 female Wistar rats were exposed to chloroform in concentrations of 3, 10 or 30 ppm for 7 hours daily between day 7 and day 16 of pregnancy. A simultaneous control group of the same size inhaled air without the addition of the test compound. On day 21 of pregnancy,the dams were killed and delivered by caesarian section. The foetuses were then examined morphologically for developmental anomalies. The examinations revealed that repeated inhalation of chloroform in a concentration of 3 ppm during the sensitive phase of embryofoetal organogenesis did not lead to an impairment of the general state of health, nor were there adverse effects upon the feed consumption and body-weight development of the dams or the intrauterine development of the conceptuses. After concentrations of 10 ppm and 30 ppm the dams displayed a slight reduction in feed consumption and body-weight development. The foetuses were slightly stuntec. On exposure to 30 ppm all embryonic primordia of one dam died in utero. The findings observed in the dams and conceptuses after exposure to 30 ppm correspond with those determined in the previous study at this concentration. A teratogenic potential of chloroform was not determined ill this study either. On the basis of the results of the embryotoxicity studies, it can therefore be concluded that the no observed adverse effect level for chloroform in the rat, as regaids maternal and embryonal toxicity in the case of inha!ative exposure, lies at 3 ppm.

Abstract  1. Male mice were fed a diet containing perfluoro fatty acids of varying chain length (i.e. perfluoroacetic, -butyric, -octanoic and -decanoic acids) at different doses (0.02 or 0.1% w/w of diet) for different periods of time (2-10 days), and effects on liver weight, hepatic mitochondrial protein and hepatic peroxisomal palmitoyl-CoA oxidation, lauroyl-CoA oxidase and catalase were monitored. 2. The greatest effects were obtained with perfluoro-octanoic and perfluoro decanoic acids, while perfluoro acetic acid was inactive. The effects with 0.02% w/w of diet perfluoro-octanoic acid were at least as great as those observed with 0.1%. A more detailed dose-response investigation focused on perfluoro-octanoic acid revealed that maximal effects with this substance could be obtained with a dietary dose of 0.01% for 10 days and that significant changes were also observed with 0.001%. 3. Maximal effects with 0.02% w/w of diet perfluoro-octanoic acid were attained after 6-10 days of feeding. 4. As with other peroxisome proliferators, perfluoro fatty acids increase mouse hepatic peroxisomal fatty acid beta-oxidation more extensively than they increase catalase, thus increasing hepatic oxidative stress. 5. As with other peroxisome proliferators, perfluoro fatty acids increase mouse liver mitochondrial protein. This effect is due primarily to a redistribution of mitochondria from the nuclear to the mitochondrial fraction, caused by an apparent decrease in the mean size of hepatic mitochondria after treatment.

Abstract  Perfluorobutyrate (PFBA) is a short chain perfluoroalkyl carboxylate that is structurally similar to perfluorooctanoate. Administration of PFBA can cause peroxisome proliferation, induction of peroxisomal fatty acid oxidation and hepatomegaly, suggesting that PFBA activates the nuclear receptor, peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In this study, the role of PPAR-alpha in mediating the effects of PFBA was examined using PPAR-alpha null mice and a mouse line expressing the human PPAR-alpha in the absence of mouse PPAR-alpha (PPAR-alpha humanized mice). PFBA caused upregulation of known PPAR-alpha target genes that modulate lipid metabolism in wild-type and PPAR-alpha humanized mice, and this effect was not found in PPAR-alpha null mice. Increased liver weight and hepatocyte hypertrophy were also found in wild-type and humanized PPAR-alpha mice treated with PFBA, but not in PPAR-alpha null mice. Interestingly, hepatocyte focal necrosis with inflammatory cell infiltrate was only found in wild-type mice administered PFBA; this effect was markedly diminished in both PPAR-alpha null and PPAR-alpha humanized mice. Results from these studies demonstrate that PFBA can modulate gene expression and cause mild hepatomegaly and hepatocyte hypertrophy through a mechanism that requires PPAR-alpha and that these effects do not exhibit a species difference. In contrast, the PPAR-alpha-dependent increase in PFBA-induced hepatocyte focal necrosis with inflammatory cell infiltrate was mediated by the mouse PPAR-alpha but not the human PPAR-alpha. Collectively, these findings demonstrate that PFBA can activate both the mouse and human PPAR-alpha, but there is a species difference in the hepatotoxic response to this chemical.

Abstract  Perfluorononanoate (PFNA), a perfluorinated alkyl acid containing nine carbon chains, has been detected in abiotic and biotic matrices worldwide. Although a few studies have reported toxic effects of PFNA, little information of the mechanism has been offered. In this study, the effects of PFNA exposure on thymus and the related mechanisms were investigated. Male rats were orally dosed with 0, 1, 3, or 5 mg PFNA/kg/day for 14 days. A significant decrease of body weight and thymus weight were observed in the rats receiving 3 or 5 mg PFNA/kg/day. Histopathological examination revealed dose-dependent increases in thymocyte apoptosis. Rats receiving 3 or 5 mg PFNA/kg/day exhibited increased interleukin (IL)-1 and decreased IL-2 concentrations in sera, whereas elevated IL-4 and cortisol levels only occurred in the highest dose group. Quantitative real-time PCR indicated that expression of peroxisome proliferator-activated receptor alpha (PPAR-alpha) was increased in the thymi of all dosed rats, and a similar trend occurred for PPAR-gamma in the two highest dose groups. The mRNA levels of c-Jun NH2-terminal kinase (JNK), nuclear factor-kappa B, p65 subunit, and inhibitory protein I kappa B alpha were unchanged; however, increased and decreased mRNA levels of p38 kinase were found in rats exposed to 3 or 5 mg PFNA/kg/day, respectively. Decreased Bcl-2 mRNA levels were observed in rats receiving 5 mg PFNA/kg/day. A significant increase in protein levels of phospho-JNK was found in all PFNA-treated rats. Phospho-p38 was significantly enhanced in 1 and 3 mg PFNA/kg/day groups, whereas phospho-I kappa B alpha remained consistent in all rats studied. Together, these data suggested that apart from the activation of PPARs, PFNA exposure in rats lead to the alteration of serum cytokines, which subsequently activated mitogen-activated protein kinase signaling pathways and potentially modulated the immune system. Additionally, increased serum cortisol and decreased expression of Bcl-2 in thymus likely contributed to the PFNA-induced thymocyte apoptosis.

Abstract  Poly- and perfluoroalkyl substances (PFAS) are chemically and thermally stable, hydrophobic, lipophobic compounds used in stain repellants and water and oil surfactants, and associated with immunosuppression and peroxisome proliferator activity. Perfluoro-n-decanoic acid (PFDA, (CF3(CF2)8COOH), a fluorinated straight chain fatty acid compound, is reported to induce thymic atrophy and reversible bone marrow hypocellularity in rodent models. The objective of this study was to assess potential immunotoxicity of PFDA, due to its structural similarity to other immunosuppressive PFASs. Female Harlan Sprague-Dawley rats were exposed to 0-2.0 mg PFDA/kg by oral gavage daily for 28 d. Female B6C3F1/N mice were exposed once/week to 0-5.0 mg PFDA/kg by gavage for 4 weeks. Animals were evaluated for effects on immune cell populations in spleen and bone marrow, and innate, humoral-, and cell-mediated immunity. Mice were also evaluated for resistance to Influenza virus. Treatment-related hepatocyte necrosis and hepatomegaly were observed in rats treated with 0.5 mg PFDA/kg/d. In mice, hepatomegaly (26-89%) was observed following exposure to ≥0.625 mg PFDA/kg/week, while splenic atrophy (20%) was observed at 5.0 mg PFDA/kg/week. At 5.0 mg PFDA/kg/week, total spleen cells, and Ig + and NK + cells were decreased (17.6-27%). At ≥ 1.25 mg PFDA/kg/week the numbers of splenic CD3+, CD4+, CD8+, and Mac3+cells were decreased (10.5-39%). No changes were observed in leukocyte subpopulations in PFDA-exposed rats. Phagocytosis by fixed-tissue macrophages was decreased in liver (specific activity, 24-39%) at ≥0.25 mg PFDA/kg/d in rats. PFDA-induced effects on humoral- and cell-mediated immunity, host resistance, and bone marrow progenitor cells were limited. These data suggest that exposure to PFDA may induce adverse effects in rat liver in a manner consistent with the PFAS class, and may also alter the balance of immune cell populations in lymphoid tissues in mice.

Abstract  Persistent presence of perfluoroalkyl acids (PFAAs) in the environment is due to their extensive use in industrial and consumer products, and their slow decay. Biochemical tests in rodent demonstrated that these chemicals are potent modifiers of lipid metabolism and cause hepatocellular steatosis. However, the molecular mechanism of PFAAs interference with lipid metabolism remains to be elucidated. Currently, two major hypotheses are that PFAAs interfere with mitochondrial beta-oxidation of fatty acids and/or they affect the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα) in liver. To determine the ability of structurally-diverse PFAAs to cause steatosis, as well as to understand the underlying molecular mechanisms, wild-type (WT) and PPARα-null mice were treated with perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), or perfluorohexane sulfonate (PFHxS), by oral gavage for 7days, and their effects were compared to that of PPARα agonist WY-14643 (WY), which does not cause steatosis. Increases in liver weight and cell size, and decreases in DNA content per mg of liver, were observed for all compounds in WT mice, and were also seen in PPARα-null mice for PFOA, PFNA, and PFHxS, but not for WY. In Oil Red O stained sections, WT liver showed increased lipid accumulation in all treatment groups, whereas in PPARα-null livers, accumulation was observed after PFNA and PFHxS treatment, adding to the burden of steatosis observed in control (untreated) PPARα-null mice. Liver triglyceride (TG) levels were elevated in WT mice by all PFAAs and in PPARα-null mice only by PFNA. In vitro β-oxidation of palmitoyl carnitine by isolated rat liver mitochondria was not inhibited by any of the 7 PFAAs tested. Likewise, neither PFOA nor PFOS inhibited palmitate oxidation by HepG2/C3A human liver cell cultures. Microarray analysis of livers from PFAAs-treated mice indicated that the PFAAs induce the expression of the lipid catabolism genes, as well as those involved in fatty acid and triglyceride synthesis, in WT mice and, to a lesser extent, in PPARα-null mice. These results indicate that most of the PFAAs increase liver TG load and promote steatosis in mice We hypothesize that PFAAs increase steatosis because the balance of fatty acid accumulation/synthesis and oxidation is disrupted to favor accumulation.

Abstract  Perfluorodecanoic acid (PFDA) is a representative of the perfluorinated carboxylic acids used as commercial wetting agents and flame retardants. Signs of PFDA toxicity have been reported to resemble those seen after exposure to TCDD. To determine if PFDA exhibits teratogenic effects similar to those of TCDD or is a developmental toxin, time-mated C57BL/6N mice were administered PFDA by gavage in corn oil (10 ml/kg) on gestation days (gd) 10-13 or gd 6-15 at levels of 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, or 32.0 mg/kg/day or 0, 0.03, 0.3, 1.0, 3.0, 6.4, or 12.8 mg/kg/day, respectively. Dams were killed on gd 18 and maternal and fetal toxicity was assessed. Fetuses were examined for external, visceral, or skeletal malformations. Maternal body weight gain (corrected for the weight of the gravid uterus) was significantly reduced as a result of PFDA treatment at 6.4 and 12.8 mg/kg/day (gd 6-15) and 16.0 and 32.0 mg/kg/day (gd 10-13). Fetal viability was decreased only in those groups showing extensive maternal body weight loss. Fetal body weights were significantly reduced at levels as low as 0.1 mg/kg/day (gd 6-15) and 0.5 mg/kg/day (gd 10-13). No hydronephrosis, cleft palate, or edema was observed nor were any other soft tissue or skeletal malformations detected. Thus, PFDA does not produce malformations in C57BL/6N mice, and the developmental toxicity observed (increased fetal mortality and decreased live fetal body weight) was seen only at doses that were maternally toxic.