Effects of Peroxisome Proliferators on Rat Liver Phospholipids

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Chem. Res. Toxicol. 1998, 11, 428-440

Effects of Peroxisome Proliferators on Rat Liver Phospholipids: Sphingomyelin Degradation May Be Involved in Hepatotoxic Mechanism of Perfluorodecanoic Acid Mehdi Adinehzadeh† and Nicholas V. Reo*,†,‡ Departments of Biochemistry and Molecular Biology and of Physics, WSU Magnetic Resonance Laboratory, Wright State University, Cox Institute, Dayton, Ohio 45429 Received August 28, 1997

Perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), clofibrate, di(2-ethylhexyl)phthalate (DEHP), and Wy-14,643 represent a class of compounds known as peroxisome proliferators (PPs). Such compounds induce biogenesis of liver peroxisomes and cause a varying degree of hepatotoxicity and carcinogenesis in rodents. We examined the effects of these PPs on rat hepatic lipids and phospholipid profiles using phosphorus-31 NMR spectroscopy. All PPs caused a 25-57% increase in hepatic phospholipid content, while all but clofibrate increased the total lipid content by 26-156%. Treatments also influenced the composition of liver phospholipids. Phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth) contents were significantly increased in all treatment groups. Most notably, PFDA caused the largest increase in PtdCho and PtdEth content (ca. 70%), while PFOA and Wy-14,643 were the only test compounds that influenced the PtdCho:PtdEth ratio. PFDA also caused an ca. 30% decrease in sphingomyelin (SphM) from 24 to 120 h postdose. SphM is a key lipid in signal transduction processes involved in apoptosis. Hydrolysis of SphM can be mediated through the action of tumor necrosis factor (TNF-R). We measured the TNF-R concentrations in rat sera at 24 h post-PFDA-exposure and found an 8-fold increase relative to vehicle-treated controls. These data demonstrate that an increase in the serum TNF-R level correlates with the time frame for the observed reduction in hepatic SphM. PFOA, a structurally similar compound, had no effect on hepatic SphM content, nor did it affect the serum TNF-R concentration. These effects may be related to differences in the tumorigenicity associated with these compounds. We postulate that PFDA activates the SphM signal transduction pathway via the release of TNF-R. This then stimulates cytotoxic responses and processes of apoptosis and may suppress cell proliferative and mitogenic responses.

Introduction It is well-established that in many rodent and nonrodent species including primates, a massive increase in both the size and number of peroxisomes can be induced by a wide range of xenobiotic compounds termed peroxisome proliferators (PPs)1 (1-5). These structurally diverse compounds have many commercial and industrial uses, including hypolipidemic drugs, such as clofibrate, Wy-14,643 (pirinixic acid), and related analogues; industrial plasticizers, such as di(2-ethylhexyl)phthalate (DEHP); and solvents/surfactants, such as perfluorocarboxylic acids and related halocarbon compounds. Un* Correspondence and request for reprints should be addressed to: Nicholas V. Reo, Ph.D., WSU Magnetic Resonance Laboratory, Cox Institute, 3525 Southern Blvd., Dayton, OH 45429. Phone: (937) 2978046. Fax: (937) 297-8109. E-mail: [email protected]. † Department of Biochemistry and Molecular Biology. ‡ Department of Physics. 1 Abbreviations: PPs, peroxisome proliferators; PFOA, perfluoron-octanoic acid; PFDA, perfluoro-n-decanoic acid; DEHP, di(2-ethylhexyl)phthalate; Wy-14,643, [4-chloro-6-(2,3-xylidino)-2-pyrimidinyl]thioacetic acid; PCr, phosphocreatine; PCho, phosphocholine; PLC, phospholipase C; DAG, sn-1,2-diacylglycerol; PKC, protein kinase C; TNF-R, tumor necrosis factor-R; PtdCho, phosphatidylcholine; PtdEth, phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; CL, cardiolipin (diphosphatidylglycerol); SphM, sphingomyelin; SphMase, sphingomyelinase.

fortunately, many PPs cause hepatotoxicity and are known to be nongenotoxic hepatocarcinogens in rodents (4, 6-14). While there have been recent advances in identifying a link between PPs and receptor-mediated transcription, many aspects of the mechanisms involved in the proliferation of peroxisomes and associated tumorigenesis are not understood and represent an active area of research (15, 16). Previous studies from our laboratory and others have provided evidence that PPs significantly affect liver phospholipid metabolism (17-25). The effects of various PPs on the accumulation or degradation of individual phospholipids appear to be varied. Phospholipids play a central role in cellular signal transduction through metabolic cycles involving phosphatidylinositol (PtdIns), phosphatidylcholine (PtdCho), and sphingomyelin (SphM). Intermediary metabolites such as diacylglycerol (DAG) and ceramide can serve as second messengers for regulatory enzymes (26-30). Studies from our laboratory have revealed unique effects of perfluorodecanoic acid (PFDA) on hepatic phospholipid metabolism (23-25). We showed that a single dose of 50 mg/kg PFDA in rats caused a significant increase in liver phosphocholine (PCho) and DAG (23,

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Effects of PPs on Liver Phospholipids

25). The increase in liver PCho was subsequently shown to be unique to treatments with perfluorocarboxylic acids containing a carbon chain length g C9 (24). This effect was not seen with Wy-14,643, DEHP, or clofibrate treatments (unpublished data). PCho is a key metabolite involved in both the hydrolysis and synthesis of PtdCho. Indeed, subsequent studies demonstrated that PFDA activates liver phospholipase C (PLC) and inhibits CTP: phosphocholine cytidylyltransferase, the rate-limiting enzyme in the de novo biosynthesis of PtdCho (25). Perfluorooctanoic acid (PFOA), a related compound, showed no effect on these enzyme activities. Other laboratories have also reported distinctive effects of PFDA among various PPs (31). Interestingly, many PPs induce tumors in rodents when administered in long-term feeding protocols. Whether PFDA can induce tumors in rodents with long-term feeding is not known and remains to be tested. However, PFDA was shown to lack tumorpromoting ability in rats when administered as monthly ip injections for up to 18 months in a two-stage hepatocarcinogenesis protocol (32). A recent review by Vanden Heuvel (31) suggested that studies of PFDA may help to dissect the mechanism of action of PPs on hepatic metabolism. A probable mechanism for nongenotoxic carcinogenesis (15, 33) involves an upset in the balance between cell proliferation and apoptosissprocesses that are affected by phospholipid-derived messenger metabolites. Probing phospholipids and phospholipid metabolites as potential markers for cellular proliferation and indicators of tumorigenesis has been the focus of much research and warrants further investigation (34-37). The unique effects observed for PFDA and the importance of phospholipids in cellular signaling prompted us to investigate the influence of PFDA and other PPs on liver phospholipid metabolism. The test compounds included in this study show a range in potencies as tumor promoters and/ or activators of cell proliferation. Characterizing the effects of PPs on phospholipid metabolism may be important for delineating the biological mechanisms involved in the associated hepatotoxicity and carcinogenicity. In the current investigation, high-resolution phosphorus31 nuclear magnetic resonance (NMR) spectroscopy was used to examine the influence of several PPs on hepatic phospholipid metabolism. P-31 NMR provides an ideal method for the determination of phospholipids in rat liver (38). This technique requires only minimal sample preparation and can be applied to crude lipid extracts (39). Herein we report the effects induced by PFDA, PFOA, DEHP, Wy-14,643, and clofibrate on the lipid content, phospholipid content, and phospholipid profile in rat liver. Our data show unique effects on hepatic phospholipids following exposure to PFDA, foremost of which is a significant decrease in SphM content. These results led us to investigate involvement of the cytokine, tumor necrosis factor-R (TNF-R), in the hepatotoxic mechanism of action for this peroxisome proliferator. These data provide new information toward the development of a mechanistic model to explain the unique biological effects of PFDA. Furthermore, by comparing the effects of PFDA with other PPs, we hope to gain a better understanding of the mechanisms involved in the nongenotoxic carcinogenicity associated with these compounds.

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Experimental Procedures Materials. Perfluoro-n-octanoic acid (PFOA), perfluoro-ndecanoic acid (PFDA), and 2-(p-chlorophenoxy)-2-methylpropionic acid (clofibric acid) were obtained from Sigma Chemical Co. (St. Louis, MO). Clofibrate was prepared as the sodium salt from clofibric acid. Di(2-ethylhexyl)phthalate (DEHP) was purchased from Aldrich Chemicals (Milwaukee, WI), and [4-chloro-6-(2,3-xylidino)-2-pyrimidinyl]thioacetic acid (Wy-14,643) was from Chemsyn Science Laboratories (Lenexa, KS). All other chemicals were reagent grade and purchased from standard sources. Treatment Protocols. Male Fischer-344 rats (209-308 g) were obtained from Harlan (St. Louis, MO). The animal housing area was maintained at 22 °C with a 12-h light/dark cycle, and animals were fed Teklad MRH 22/5 rodent diet #8640. Rats were paired according to similar body weights for treatment and control groups. Body weights and food consumption were monitored daily, and control animals were given the same amount of food that paired partners had consumed on the previous day (pair-fed controls). Pair-feeding simulates hypophagic effects which are associated with treatments, particularly PFDA. The range in mean daily food consumption (in g/day) following treatments with PPs was as follows: PFDA, 0-3; PFOA, 7-8; clofibrate, 3-9; DEHP, 11-13; and Wy-14,643, 9-10 g. An additional control group in which rats were allowed ad libitum access to food (ad libitum-fed controls) was also examined. Mean daily food consumption for this group was approximately 18 g/day. All rats in treatment and pair-fed control groups were fasted 12-17 h prior to experiments involving liver lipid analyses. In the study involving TNF-R determination, rats were fasted 24 h prior to experiments. Fasting animals prior to experiments was done to minimize any effects due to variations in food consumption between different treatment groups and to provide a more consistent basis for comparative studies. Ad libitum-fed controls were not fasted prior to experiments. For all experiments pertaining to lipid analyses, the numbers of animals in each experimental group (n values) are given in Table 1 and the legend to Figure 6; n values for the TNF-R experiments are given in Table 2. PFOA and PFDA were delivered in propylene glycol/H2O (1: 1, v/v) via a single ip injection (150 mg/kg PFOA; 50 mg/kg PFDA). Procedures involving lipid extraction from rat livers were initiated at 3 days posttreatment with PFOA and at 12, 24, 48, and 120 h (5 days) posttreatment with PFDA. Clofibrate was mixed in 0.9% saline and administered as an ip injection once daily for three consecutive days (250 mg/kg/day). Experiments involving clofibrate-treated rats were initiated 4 days post-initial-treatment (i.e., 2 days after the final dose). DEHP was given as the neat oil, and Wy-14,643 was dissolved in olive oil. A single dose (1200 mg/kg for DEHP or 250 mg/kg for Wy14,643) was administered via gastric intubation once daily for 3 consecutive days. Experiments involving treatments with DEHP or Wy-14,643 were initiated 3 days post-initial-treatment (i.e., 1 day after the final dose). The dosing volume for PFDA, PFOA, and clofibrate was e0.5 mL. For DEHP and Wy-14,643 the dose volumes were e0.38 and e1.2 mL, respectively. Pair-fed control animals were dosed according to the same regimens with an equal volume of the appropriate vehicle solution (propylene glycol/H2O, saline, or olive oil); DEHP controls were administered olive oil. Doses and treatment protocols match those previously used in our laboratory or are similar to that used by others and reported to produce maximal hepatic effects (i.e., hepatomegaly, peroxisome proliferation, and P450 enzyme induction) (7, 23-25, 40-48). PFDA and PFOA doses were based upon the 30-day LD50 for these compounds and have been shown to produce maximal hepatic peroxisomal marker induction (7, 48). Lipid Extraction. Rats were anesthetized with halothane (5% induction, 2% maintenance), and livers were surgically exposed and freeze-clamped between aluminum plates chilled in liquid N2. Tissue lipids were prepared using the Folch extraction method (49, 50). Livers were first crudely homog-

430 Chem. Res. Toxicol., Vol. 11, No. 5, 1998 enized under liquid nitrogen in a mortar and pestle and then weighed. Ground livers were further homogenized in chloroform/ methanol (2:1, v/v; 20 mL/g of tissue) using a glass tissue grinder fitted with a Teflon pestle. Samples were exposed to the extracting solvent for a minimum of 30 min before filtering through medium-grade filter paper into a separatory funnel. The tissue residue was washed three times with 2 mL (6 mL total) of the chloroform/methanol solvent. The liquid extract was then washed with 0.2 volume of 0.1 M aqueous KCl. The solution was left to stand for 17-24 h to allow complete separation of the two phases. The organic phase containing lipids (lower phase) was removed and placed into a small beaker in a 37 °C water bath. The lipid extract was reduced to a volume of ∼5 mL by blowing a gentle stream of N2 over the sample. The sample was then transferred to a small, dark glass vial and evaporated to dryness with a stream of N2. This procedure reduces the risk of autoxidation of lipids. To ensure complete dryness, the sample was placed under mild vacuum overnight and then weighed to obtain total lipid content. NMR Experiments. Samples were prepared for NMR analyses using a three-part solvent system containing chloroform, methanol, and water as described by Meneses et al. (39). Dried lipids of e100 mg were reconstituted in 2 mL of deuteriochloroform; lipid samples in excess of 100 mg were prepared at a concentration of 50 mg/mL CDCl3. The cesium salt of EDTA, Cs2(EDTA), was prepared as a 0.2 M solution in D2O and combined with 4 volumes of methanol to give 40 mM Cs2(EDTA) in 1:4 D2O/MeOH. Then 0.8 mL of the Cs2EDTA/ MeOH reagent was combined with 1.6 mL of the lipid/deuteriochloroform sample. The final solvent composition was ca. 1:4: 10 D2O/MeOH/CDCl3. The sample was placed into a 10-mm NMR tube along with a capillary tube (1.6-mm i.d.) containing an external standard which consisted of an aqueous solution of 0.35 M phosphocreatine (PCr) with 1.2 µg/mL MnCl2 as relaxation agent. High-resolution proton-decoupled 31P NMR spectra of liver extracts were acquired with a Bruker AM 360 NMR spectrometer (8.5 T) equipped with a 10-mm broadband probe operating at 145.8 MHz. The sample temperature was regulated at 293 K to optimize spectral resolution (38). Data were acquired in field-lock mode using the deuterium signal from CDCl3. A gated composite pulse decoupling sequence (WALTZ-16) was applied to obtain proton-decoupled spectra without nuclear Overhauser enhancement. Data were acquired using a 90° pulse (12.8 µs), a 805-Hz spectral bandwidth, an acquisition time of 2.54 s, 4K data points, and an interpulse delay (12.54 s) enabling full relaxation of all resonances (>5T1). Typically, 20 min of signal averaging was sufficient to obtain a signal-to-noise ratio of ca. 300:1 for the PtdCho signal. Spectral assignments were based on signals obtained from individual phospholipids (Sigma Chemical Co., Product No. PH-9) added to liver extract samples. These assignments were similar to those reported in the literature (39). The authors caution that chemical shifts are sensitive to sample composition and temperature; thus it is important to spike the liver extract samples with phospholipid standards rather than preparing separate solutions of known standards. In a separate experiment, the PCr external reference was calibrated by measuring its peak area relative to known concentrations of PtdCho in a series of seven standard solutions ranging from 1 to 33 mM dipalmitoylphosphatidylcholine. These data were analyzed by a least-squares linear fit (r2 ) 0.999) to yield a standard calibration equation. This equation was then used to determine individual phospholipid concentrations from spectral data of liver extracts. NMR data processing utilized a Macintosh IIci computer and MacFID software (Tecmag, Inc., Houston, TX). Data were processed using 8K total points, exponential multiplication yielding 1-Hz line broadening, Fourier transformation, and spline baseline correction. Chemical shifts were referenced to the internal PtdCho signal set at -0.84 ppm relative to 85% inorganic orthophosphoric acid at 0 ppm (39). Peak areas of

Adinehzadeh and Reo phospholipid signals and the PCr reference were obtained by spectral integration. These values were then entered into the calibration equation to determine the concentrations of individual phospholipids. Total phospholipid content was obtained by integration of the entire phospholipid spectral region. TNF-r Determination. In a separate study, rats were treated with PFOA, PFDA or vehicle solution (controls) as described above. Rats were denied access to food following treatments. At 24 h postdose, rats were anesthetized with halothane. Blood samples were taken from the posterior vena cava into serum-collecting tubes. The blood was allowed to clot and then centrifuged (2000g) at 5 °C for 10 min. Sera samples were stored frozen at -15 °C for subsequent determination of TNF-R. Quantitative determination of TNF-R utilized an enzymelinked immunosorbent assay (ELISA) purchased as a kit from Biosource International (Camarillo, CA). The Ultrasensitive Cytoscreen Rat TNF-R ELISA Kit (#KRC3012) involves a fourmember solid-phase sandwich ELISA method which measures serum TNF-R utilizing streptavidin peroxidase and tetramethylbenzidine as the enzyme and substrate, respectively. Samples, including standards of known rat TNF-R content (supplied with the kit), were run in duplicate, and TNF-R concentrations are reported in pg/mL. Data Analyses. Although control rats were pair-fed to treated counterparts, a Student’s t-test for independent samples (unpaired t-test) was used for comparisons between treated versus control groups in all cases. This approach is appropriate since animals were paired according to a very limited set of criteria, namely, body weight and daily food consumption. Comparisons between treatment and corresponding control groups of an unequal number of animals (unequal n values) occasionally occurred due to the accidental loss of a sample or unexpected death of an animal during the course of an experiment. Statistical analyses involving more than two groups initially utilized a one-way analysis of variance (ANOVA). If the ANOVA indicated significance in the data (p e 0.05), then a Fisher (protected least-significant difference) post hoc pairwise comparison test was employed to identify significant data at the level p e 0.05. Error estimates are given as the standard error of the mean (SE) or standard deviation (SD), as noted. All data are considered statistically significant at a value of p e 0.05.

Results Figure 1 shows a representative proton-decoupled 31P NMR spectrum of a rat liver lipid extract. The spectrum clearly yields detectable signals from the six major classes of phospholipids as identified in the figure. Spectral line widths (full-width at half-height) were typically 1-2 Hz. A weak signal at -0.29 ppm was also observed from lysophosphatidylcholine. Additionally, the high-frequency side of the PtdEth peak has a shoulder. The chemical shift of this weak signal corresponds to phosphatidylethanolamine plasmalogen (PtdEth with an alk1-enyl moiety) as reported in extracts from brain tissues (51) and soybeans (39). Liver typically contains only trace amounts ( control at 24 h postdose), while no change in serum TNF-R was reported by others (84) following treatment with Wy-14,643; and (ii) our data show that PFDA caused a significant decrease in hepatic SphM, whereas SphM content was unaffected by treatment with Wy-14,643 at a dose equal to 2.5 times that used in ref 84. These results suggest that the TNF-Rinduced response following treatment with Wy-14,643 may differ from that following treatment with PFDA. The role of TNF-R in cellular signaling is very complex, having been implicated in pathways of apoptosis as well as mitogenesis (87, 88). Recent reports have associated TNF-R with the mitogenic activity induced by PPs (84, 85, 88), and it has further been shown that this cytokine may play a role in regulation of the peroxisome proliferator-activated receptor family of nuclear transcription factors (89, 90). Perhaps Wy-14,643 causes an increase in TNF-R capable of stimulating mitogenesis but not apoptosis, and thus, SphMase was not activated. In the case of PFDA we hypothesize that Kupffer cells are triggered, either directly or indirectly, to cause an increase in TNF-R which ultimately results in substantial degradation of hepatic SphM. Such biological responses may be indicative of cell death or apoptotic processes, but further studies are necessary to confirm these effects with regard to PFDA. SphM was observed to be maximally decreased at 24 h postdose with PFDA and did not recover even at the latest time point examined (5 days postdose). The mechanism for this sustained effect is unclear; however, a dysfunction in autoregulatory mechanisms may be responsible. For instance, prostaglandin E2 is known to suppress TNF-R production (91), but PFDA has been shown to decrease the hepatic concentration of prostaglandin E2 in rats (92). Thus this mechanism for TNF-R downregulation may be disrupted by PFDA. An intriguing observation in the present study concerns the striking differences in hepatic effects observed between the structurally similar compounds PFOA and PFDA. PFOA treatment had no effect on hepatic SphM content, nor did it affect the serum concentration of TNFR. While neither compound is metabolized in vivo, there are distinct differences in their bioelimination (40). PFOA is readily excreted in the urine and causes an acute and transient toxicity, whereas PFDA concentrates in bile causing a more delayed lethality and protracted wasting toxicity (7, 40, 93). Additionally, available data

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suggest that there may be differences in the long-term effects of PFOA and PFDA. PFOA, for instance, has been shown to promote tumorigenesis in both biphasic (initiation-promotion) and triphasic (initiation-selectionpromotion) protocols in rats (11, 12). The biphasic protocol, involving initiation with diethylnitrosamine and then chronic feeding with 0.02% (w/w) PFOA, caused a 56% incidence in hepatocellular carcinomas in a 12month period. The triphasic promotion protocol resulted in a 33% tumor incidence in just 28 weeks with 0.015% (w/w) PFOA in feed. Although identical long-term feeding studies have not been done for PFDA, this compound has been investigated in a two-stage hepatocarcinogenesis study (32). Here rats received partial hepatectomies and an initiating dose of diethylnitrosamine, followed by monthly ip injections of PFDA for up to 18 months (0.055.0 mg of PFDA/kg). PFDA-treated rats showed no increase in tumor incidence or the number of hepatic foci. In comparison, ciprofibrate (a hypolipidemic peroxisome proliferator and known tumor promoter) was used as a positive control and produced a 100% tumor incidence under the identical protocol. Such data suggest that PFDA may not be tumorigenic, but studies involving chronic feeding with the compound are necessary to confirm this assumption. The reasons for the differences between PFOA and PFDA observed in the present study are not known and warrant further investigations. Perhaps the influence of PFDA on hepatic SphM metabolism stimulates apoptosis and suppresses cell proliferation and mitogenic processes. Studies are in progress to examine the dosedependent response in relationship to this mechanism. Additional measurements of SphMase activity, hepatic ceramide concentration, or indices of apoptosis would be important to confirm this mechanistic theory. Elucidating the mechanisms by which these compounds induce biological activity may provide a clearer understanding of the carcinogenesis associated with PPs. In conclusion these studies demonstrate that PPs impact hepatic phospholipid metabolism causing a significant increase in overall phospholipid content and changes in the hepatic phospholipid composition. Phospholipids influence the properties of membranes and play a key role in cellular signaling processes. Characterizing the effects of PPs on liver phospholipids has provided important information toward advancing our understanding of the mechanisms involved in the hepatotoxicity associated with PFDA. Data suggest that PFDA acts through a TNF-R-mediated process to activate the SphM/ ceramide signaling pathway which ultimately suppresses cell growth and stimulates apoptosis. By comparing the effects of PFDA with other PPs, we hope to gain a better understanding of the mechanisms involved in the carcinogenicity associated with these compounds. Further studies are necessary to evaluate the effects of PPs on phospholipid metabolism and relate this information to mechanistic theories regarding peroxisome proliferatorinduced liver toxicity and carcinogenicity.

Acknowledgment. The authors wish to thank Dr. Nancy Bigley, Department of Microbiology and Immunology, Wright State University, for her assistance with the TNF-R assay. This work was sponsored by the Air Force Office of Scientific Research, Air Force Systems Command, USAF, under Grant or Cooperative Agreement F49620-95-1-0180.

Adinehzadeh and Reo

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