Dose-response hapatotoxicity of the peroxisome proliferator, perfluorodecanoic acid and the relationship to phospholipid metabolism in rats.

Perfluorodecanoic acid (PFDA) is a potent peroxisome proliferator that causes hepatotoxicity but lacks tumor-promoting activity in rats. We previously showed that a single dose of PFDA at 50 mg/kg (approximately LD50) causes an elevation in liver phosphocholine (PCho) and other effects related to phospholipid metabolism. In this study, we examined metabolic effects in the dose range 2-50 mg/kg in rats. At doses < or =20 mg/kg, PFDA is significantly less hepatotoxic than the LD50 as manifested by electron microscopy and measurements of daily food consumption and body weight. At 50 mg/kg rat serum tumor necrosis factor (TNF)-alpha concentration was increased 8-fold, while at 15 mg/kg there was no apparent increase in this cytokine. This lower dose, however, induces metabolic effects similar to those seen at the LD50. Liver fatty acyl-CoA oxidase activity showed a dose-dependent increase from 5-25 mg/kg PFDA. Treatments at 15 and 50 mg/kg caused a significant increase in liver phosphatidylcholine (28 and 66%) and phosphatidylethanolamine (31 and 74%). Both doses caused a significant increase in liver PCho but did not affect liver ATP levels, as manifested in 31P nuclear magnetic resonance (NMR) spectra from rat livers in vivo. These data suggest that the increase in liver [PCho] observed following PFDA exposure in rats represents a specific metabolic response, rather than a broad-range hepatotoxic effect.

The metabolism of n-nonane in male Fischer 344 rats.

The urinary metabolites of n-nonane in male Fischer 344 rats, after administering the hydrocarbon by gavage, included gamma-valerolactone, delta-hexanolactone, 2,5-hexanedione, delta-heptanolactone, 1-heptanol, 2-nonanol, 3-nonanol, 4-nonanol, 4-nonanone and 5-methyl-2-(3-oxobutyl)furan. Metabolism strongly favored the formation of monoalcohols and lactones, which are the products of appropriately substituted hydroxy carboxylic acids. The metabolites were identified using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). High pressure liquid chromatography (HPLC) permitted the detection of the dicarboxylic acids malonic acid and glutaric acid in the n-nonane dosed rat urines.

Effect of the peroxisome proliferator perfluoro-n-decanoic acid on glucose transport in the isolated perfused rat liver.

The perfluorinated carboxylic acid, perfluoro-n-decanoic acid (PFDA), is a known peroxisome proliferator which displays toxicity in rodents. Using a paired-tracer first-pass extraction technique, the effect of PFDA on hepatic glucose transport was determined in the isolated perfused rat liver. In brief, livers isolated from PFDA-treated and control rats on day 5 posttreatment were administered the radiolabeled glucose analog, 3-O-[14C]methyl-D-glucose ([14C]3-O-MG) in addition to [fructose-1-3H(N)]sucrose ([3H]sucrose), which served as a measure of extracellular volume. Hepatic glucose transport was calculated from the change in the ratio [14C]3-O-MG/[3H]sucrose during passage through the liver. Data from this study indicate that PFDA inhibits hepatic glucose transport. Percent hepatic glucose extraction is 1.8-fold greater in controls than in PFDA-treated rats. No significant difference in lactate dehydrogenase levels was observed in the liver perfusate from PFDA-treated and control rats. This suggests that the difference in percent glucose extraction between PFDA-treated and control groups is specifically due to the PFDA treatment and is not attributed to differences in liver viability between groups. Although the exact mechanism for this inhibition in hepatic glucose transport is not known, it is hypothesized that PFDA may have a major impact on membrane structure/function which, in turn, may alter glucose transport.

Modification of hepatic immunoglobulin heavy chain binding protein (BiP/Grp78) following exposure to structurally diverse peroxisome proliferators.

This investigation was conducted to determine the comparative effect of structurally diverse peroxisome proliferators (PP) on the two-dimensional protein pattern of rat liver whole homogenates. Perfluoro-n-decanoic acid (PFDA), perfluoro-n-octanoic acid (PFOA), clofibrate, and di(2-ethylhexyl)phthalate (DEHP) are all known to cause the proliferation of hepatic peroxisomes and the induction of peroxisomal beta-oxidative and microsomal omega-oxidative enzymes. To clarify the mechanistic differences between these compounds with regard to the liver, we examined the unique patterns of protein alteration produced by in vivo exposure to them. Following exposure to various doses, whole liver homogenates were prepared and separated by two-dimensional gel electrophoresis (2DE) using the ISO-DALT system. Stained gels were digitized and protein patterns analyzed using the Kepler 2D gel analysis system. Immunoglobulin heavy chain binding protein (BiP), also known as 78-kDa glucose-regulated protein (Grp78), was identified immunologically and by comigration of recombinant Grp78. BiP is a luminal endoplasmic reticular protein that functions in the assembly and folding of nascent proteins as they enter the ER. The present results suggest a selective posttranslational modification of BiP following PFDA exposure. Single-dose exposure to PFDA was associated with a notable charge modification of BiP that persists up to 30 days. PFOA, clofibrate, and DEHP had less effect in this regard. The identity of BiP/Grp78 as the halothane hepatitis-associated trifluoroacetylated protein was also demonstrated. The nature of this PFDA-associated protein modification (reactive metabolite conjugation, abnormal ribosylation, or phosphorylation) is currently under investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation of pharmacokinetics in noncancer risk assessment: example with chloropentafluorobenzene.

Noncancer risk assessment traditionally relies on applied dose measures, such as concentration in inhaled air or in drinking water, to characterize no-effect levels or low-effect levels in animal experiments. Safety factors are then incorporated to address the uncertainties associated with extrapolating across species, dose levels, and routes of exposure, as well as to account for the potential impact of variability of human response. A risk assessment for chloropentafluorobenzene (CPFB) was performed in which a physiologically based pharmacokinetic model was employed to calculate an internal measure of effective tissue dose appropriate to each toxic endpoint. The model accurately describes the kinetics of CPFB in both rodents and primates. The model calculations of internal dose at the no-effect and low-effect levels in animals were compared with those calculated for potential human exposure scenarios. These calculations were then used in place of default interspecies and route-to-route safety factors to determine safe human exposure conditions. Estimates of the impact of model parameter uncertainty, as estimated by a Monte Carlo technique, also were incorporated into the assessment. The approach used for CPFB is recommended as a general methodology for noncancer risk assessment whenever the necessary pharmacokinetic data can be obtained.

Induction of enoyl-CoA hydratase by LD50 exposure to perfluorocarboxylic acids detected by two-dimensional electrophoresis.

The effect of in vivo exposure to perfluoro-n-octanoic and perfluoro-n-decanoic acids was examined in the rat liver by two-dimensional electrophoresis (2DE). Using nonequilibrium pH-gradient electrophoresis in the first dimension separation, proteins associated with the mitochondrial/peroxisomal cell fraction were observed and immunologically identified. Conspicuous inductions in peroxisomal enoyl-CoA hydratase and other proteins of the peroxisomal beta-oxidative pathway were observed following single-dose exposure to each compound. The abundance of the tentatively-identified mitochondrial equivalent, crotonase, was not altered by these intoxications. These results confirm previous observations of perfluorocarboxylic acid toxicity and support the use of 2D protein-pattern alterations in biomarker research. The ability to identify this type of alteration via 2DE, in association with specific toxic effects by chemically related compounds, may provide new and additional markers for chemical-induced tissue damage.

Effects of perfluoro-n-octanoic acid, perfluoro-n-decanoic acid, and clofibrate on hepatic phosphorus metabolism in rats and guinea pigs in vivo.

Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy was used to study the effects of perfluoro-n-octanoic acid (PFOA), perfluoro-n-decanoic acid (PFDA), and clofibrate (CLOF) on liver phosphorus metabolism in rats and guinea pigs in vivo. All three compounds are known to cause peroxisome proliferation in rats but not in guinea pigs. The data indicate that indices related to overall tissue viability (i.e., adenosine triphosphate levels) remain unaffected at the doses and experimental times investigated for all treatments and both species. PFDA-treated rats revealed a marked increase in a liver phosphomonoester resonance compared with corresponding controls (p < or = 0.01); no such effect was observed in guinea pigs. This particular 31P NMR signal was identified as phosphocholine (PCho) and was found to steadily increase in concentration at consecutive days post-PFDA treatment, reaching 6.26 +/- 0.29 mumol/g liver at 5 days. This is fourfold greater than the PCho levels determined in livers from corresponding pair-fed control rats. The elevation in liver PCho is a specific response of PFDA treatment in rats and is not simply related to peroxisome proliferation in general, since neither PFOA nor CLOF produce such an effect. The data suggest a unique effect of PFDA on liver phospholipid metabolism, specifically phosphatidylcholine, which may involve enhanced phospholipid turnover via phosphatidylcholine-specific phospholipase C activity.

Charge modification in rodent hepatic Grp78/BiP following exposure to structurally diverse peroxisome proliferators.

This investigation was conducted to determine the comparative effect of structurally diverse peroxisome proliferators (PP) on the two-dimensional protein pattern of rat liver whole homogenates. Perfluoro-n-decanoic acid (PFDA), perfluoro-n-octanoic acid (PFOA), clofibrate, and di(2-ethylhexyl)phthalate(DEHP) are all known to cause the proliferation of hepatic peroxisomes and the induction of peroxisomal beta-oxidative and microsomal omega-oxidative enzymes. To detect potential differences between these compounds with regard to the liver, we examined the unique patterns of protein alteration produced by in vivo exposure to them. Following exposure to various doses, whole liver homogenates were prepared and separated by two-dimensional gel electrophoresis (2DE) using the ISO-DALT System. Stained gels were digitized and protein patterns analyzed using the Kepler 2D Gel Analysis System. Immunoglobulin heavy-chain binding protein (BiP), also known as 78 kD glucose regulated protein (Grp78), was identified immunologically and by comigration of recombinant Grp78. BiP is a luminal endoplasmic reticular (ER) protein that functions in the assembly and folding of nascent proteins as they enter the ER. The present results suggest a selective posttranslational modification of BiP following PFDA exposure. Single-dose exposure to PFDA was associated with a notable charge-modification of BiP that persists up to 30 days. PFOA, clofibrate, and DEHP had less effect in this regard. Our data suggest the likely nature of this PFDA-associated protein modification is associated with protein-phosphorylation. These results document the unique nature of PFDA's hepatotoxicity with respect to classic peroxisome proliferators and support the utility of 2D gel analysis in toxicity testing.

Effects of the peroxisome proliferator perfluoro-n-decanoic acid on hepatic gluconeogenesis and glycogenesis: a 13C NMR investigation.

Carbon-13 NMR spectroscopy was used to study the effects of the peroxisome proliferator perfluoro-n-decanoic acid (PFDA) on hepatic carbohydrate metabolism in male Fischer-344 rats. The data indicate that PFDA-treated rats display an inhibition in hepatic [1-13C]glucose and [3-13C]alanine utilization on day 5 posttreatment. PFDA rats show hepatic mean glucose and alanine intensities which are significantly greater (ca. 10-100%) than controls. With [1-13C]-glucose as substrate, PFDA rats show severe to complete inhibition in glycogenesis on days 3 and 5 posttreatment. With [3-13C]alanine as substrate, both groups show functional gluconeogenesis and glycogenesis; however, treated rats show a more transient and less intense C1-glycogen resonance relative to control. These data suggest that PFDA inhibits either the hepatocellular transport of glucose and/or its phosphorylation by glucokinase. The effect of PFDA on TCA cycle activity was determined by monitoring the flow of [3-13C]alanine into glutamate. The relative activity of pyruvate carboxylase (PC) versus pyruvate dehydrogenase (PDH) is represented by the ratio of the glutamate NMR signal intensities (C2 + C3)/C4. PFDA rats show a lower (C2 + C3)/C4 glutamate ratio, suggesting greater relative activity of PDH versus PC in PFDA rats relative to controls. Differences in PDH activity may arise from differences in lipolytic activity. Our data suggest a dysfunction in fatty acid metabolism in PFDA rats and corroborate other studies which show that PFDA inhibits fatty acid oxidation.

A comparative toxicological investigation of perfluorocarboxylic acids in rats by fluorine-19 NMR spectroscopy.

Male Fischer-344 rats administered a single intraperitoneal dose of perfluoro-n-octanoic acid (PFOA) or perfluoro-n-decanoic acid (PFDA) display a similar "wasting toxicity" characteristic of perfluorocarboxylic acids, with marked differences in temporal expression. Food/water consumption and urine output were monitored daily in PFOA-treated, PFDA-treated, and control rats. Fluorine-19 nuclear magnetic resonance (NMR) spectroscopy was used to monitor these fluorocarbons and possible fluoro metabolites in vivo, and to correlate differences in elimination with differences in effective toxicity. The data reveal a prolonged hypophagic response to PFDA and a more acute but transient response associated with PFOA treatment. PFOA causes a greater decline in food consumption than PFDA within the first 24 h postdose. PFOA-treated rats also show a ca. 2.5-fold increase in urine output on day 1, with only a slight increase in water consumption. In contrast to PFDA, PFOA-treated rats recover from hypophagia within 8 days. Fluorine-19 NMR spectra of various bodily fluids and liver in vivo display resonances of the parent PFOA or PFDA compounds and do not reveal any evidence of metabolism. Inorganic fluoride from dietary sources is detected in urine from both exposed and control rats. Differences in the route of excretion of PFOA vs PFDA are apparent from the spectral signal-to-noise ratio. The data suggest that PFOA is more readily excreted in the urine while PFDA is preferentially carried in bile. These apparent differences in elimination may account for their observed differences in effective toxicity. The acute transient toxicity and higher LD50 associated with PFOA may result from its rapid renal clearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Mercury-induced renal autoimmunity in the MAXX rat.

Inbred Brown Norway (BN) rats treated with mercuric chloride develop autoantibodies to renal basement membranes and an immunologically mediated membranous glomerulonephritis. To date, this experimental rat model of chemically induced autoimmunity has been obtained only in the BN strain, whereas rats from 17 other strains were found to be resistant. This is a disadvantage for mechanistic studies, especially since BN rats have poor fertility. In the present paper we report that the same model can be obtained in another inbred strain of rats, the MAXX, which after exposure to mercury develop a glomerulonephritis characterized by the production of autoantibodies to renal basement membranes. The kinetics of the autoimmune response observed in MAXX rats, as well as the immunohistopathology, histopathology, and proteinuria, are similar to those previously described in BN rats. In addition, the MAXX strain is endowed with excellent fertility. Therefore, both rat strains can be used for comparative studies of the mechanisms of mercury-induced autoimmunity.