Effects on Rodents of Perfluorofatty Acids Joseph W DePierre Unit for Biochemical Toxicology, Department of Biochemistry and Biophysics, Wallenberg Laboratory, Stockholm University, 109 61 Stockholm,[.]
CHAPTER Effects on Rodents of Perfluorofatty Acids Joseph W DePierre Unit for Biochemical Toxicology, Department of Biochemistry and Biophysics, Wallenberg Laboratory, Stockholm University, 109 61 Stockholm, Sweden E-mail: joe@dbb.su.se Perfluorofatty acids are used in increasing amounts as corrosion inhibitors, anti-wetting agents, surfactants, and in fire extinguishers The perfluorofatty acids whose biological effects have been studied most extensively are perfluorooctanoic and perfluorodecanoic acids The most dramatic effect of these xenobiotics in rats and mice is hepatic peroxisome proliferation, i.e., a considerable increase in the size and number of hepatic peroxisomes, which is almost invariably accompanied by potent up-regulation of peroxisomal fatty acid b-oxidation However, these compounds also elicit numerous other responses in these rodents, including decreased body weight, liver hypertrophy, a decrease in the size of hepatic mitochondria, decreased circulating levels of thyroid hormones, altered expression of a number of other enzymes, and the appearance of tumors in the liver and testis Perfluorooctanoic and perfluorodecanoic acids will continue to be important tools for investigating basic cellular processes and may even turn out to be of clinical use The risk to human health posed by exposure to these compounds in the occupational and general environments remains to be elucidated Keywords Perfluorofatty acids, Perfluorooctanoic acid, Perfluorodecanoice acid, Peroxisome proliferation, Fatty acid b-oxidation, Catalase, Hepatic, Lipids, Hepatic lipid metabolism, Hypolipidemia, CYP4A1, Oxidative stress, Hepatocarcinogenecity, Mice, Rats Introductory Remarks 205 Use and Occurrence of Perfluorofatty Acids Experimental Systems for Studying the Effects of Perfluorofatty Acids on Mammalian Cells 208 Pharmacokinetics and Metabolism of PFOA and PFDA in Rats 210 Effects of Perfluorofatty Acids on the Number, Size, and Functions of Peroxisomes in Rodent Liver 212 5.1 5.2 5.2.1 5.2.2 5.3 Morphological Studies Effects on Peroxisomal Proteins and Functions Fatty Acid b-Oxidation Catalase Sex and Species Differences 207 212 212 212 214 216 The Handbook of Environmental Chemistry Vol 3, Part N Organofluorines (ed by A.H Neilson) © Springer-Verlag Berlin Heidelberg 2002 204 5.4 J.W DePierre 5.5 5.6 5.7 Dependence on Chain Length, the Carboxylic Acid Moiety, and Fluorination Dependence on Dose and Time Reversibility/Persistence Tissue Specificity Additional Effects of Perfluorofatty Acids in Rodents 224 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 Additional Effects on Hepatic Lipid-Metabolizing Enzymes, Lipid-Binding Proteins, and Lipid Composition Hypolipidemia “Wasting Syndrome”: Loss of Body Weight and Body Fat Hepatomegaly Decrease in Mitochondrial Size Decreases in the Levels of Thyroid Hormones Up-Regulation of CYP4A1 Up-Regulation of UDP-Glucuronyltransferase Oxidative Stress Mechanism(s) Underlying These Effects of Perfluorofatty Acids 230 7.1 7.2 Formation of Perfluorofatty Acyl-CoA and/or of Dicarboxylic Fatty Acids and/or Disruption of Fatty Acid Homeostasis in Other Ways 230 Peroxisome Proliferator-Activated Receptor-Alpha 231 Toxicity/Genotoxicity of Perfluorofatty Acids 8.1 8.2 8.3 8.4 8.5 8.6 8.6.1 8.6.2 8.6.3 8.6.4 8.6.5 8.6.6 Acute Toxicity: LD50 Values, the “Wasting Syndrome”, and Acute Tissue Damage Developmental Toxicity Degeneration of Seminiferous Tubules Immunotoxicity? Genotoxicity Possible Genotoxic Mechanism(s) Lack of Direct Genotoxicity Increased Oxidative Stress Altered Xenobiotic Metabolism Stimulation of Hepatocyte Proliferation Inhibition of Hepatocyte Apoptosis Immunotoxicity? Studies on Humans 237 10 Concluding Remarks 237 10.1 10.2 10.3 Valuable Experimental Tools 237 Possible Clinical Applications 238 Hazard to Human Health? 238 11 References 239 217 218 222 222 224 226 226 226 227 228 228 229 230 233 233 233 233 233 234 234 234 235 235 235 236 236 Effects on Rodents of Perfluorofatty Acids 205 Introductory Remarks Biochemical toxicology is a two-way avenue In the one direction, biochemical approaches are employed in attempts to elucidate the mechanisms underlying toxicological/genotoxicological phenomena For instance, how does the widely used pain-killer and anti-fever agent paracetamol kill hepatocytes? How does aflatoxin, a fungal metabolite, cause liver cancer in widespread areas of Africa? How does cigarette smoke cause chronic bronchitis and lung cancer? In the other direction, the responses of, in particular, mammalian cells to xenobiotics have found extensive use as valuable tools for studying basic cellular processes For instance, how is the expression of certain genes regulated? How are particular cellular compartments expanded when circumstances so require and how does the enlarged compartment return to normal after removal of the provocative and often stressful stimulus? As will be seen, studies with peroxisome proliferators, including the perfluorofatty acids, are a perfect example of this two-way avenue The peroxisome is a membrane-bound subcellular compartment, i.e., a cell organelle [102, 125 and references therein] In electron micrographs peroxisomes appear to be circular with a wide variety of diameters (approximately 0.5–1.0 µm in hepatocytes and smaller microperoxisomes in many extrahepatic tissues), are surrounded by a single phospholipid bilayer, and exhibit a relatively amorphous matrix, with the exception of occasional crystalline structures (crystalline urate oxidase and, perhaps, other proteins as well) in hepatic peroxisomes (see Fig 1) Even though peroxisomes account for no more than a few percent of cellular protein and volume under normal physiological conditions, a number of important functions are localized in this organelle Many of these functions are related to global lipid homeostasis, e.g., b-oxidation of fatty acids (see also below); synthesis of bile acids, plasmologens (ether phospholipids), and isoprenoid compounds such as cholesterol, ubiquinone, and dolichol, and fatty acid elongation and rearrangement Other peroxisomal functions include inactivation of reactive forms of oxygen (e.g., by catalase and epoxide hydrolase), oxidation of aliphatic alcohols (also via catalase); conversion of D-amino acids to the corresponding Lamino acids, and uric acid catabolism Much characterization of peroxisomes remains to be performed and, undoubtedly, additional important cellular processes are also carried out by this organelle As indicated by the term peroxisome proliferator, the definitive characteristic of such a compound is its ability to evoke an increase in the number of peroxisomes present in hepatocytes and/or other cell types (Fig 1) However, a number of other physiological and biochemical changes also typically occur in association with this proliferation (see below) For instance, up-regulation of the three enzymes involved in peroxisomal fatty acid b-oxidation is virtually always observed in association with peroxisome proliferation Thus, such up-regulation is routinely used as an indicator of peroxisome proliferation, since morphometric study of electron micrographs (i.e., actually counting the number of peroxisomal profiles present per hepatocyte) is a laborious and time-consuming procedure However, it should always be kept in mind that under some circumstances pro- 206 J.W DePierre Fig Peroxisome proliferation in mouse hepatocytes in response to dietary exposure to PFOA The upper electron micrograph depicts the liver of untreated mice, while the lower micrograph shows the liver of treated animals Px=peroxisomes The arrows point to crystalline cores within these organelles A length of micron is indicated in the lower electron micrograph Electron micrographs courtesy of Professor Anders Bergstrand Unpublished studies in our laboratory (1995) Effects on Rodents of Perfluorofatty Acids 207 Fig Structures of commonly studied peroxisome proliferators related (left) and unrelated (right) to clofibrate liferation of peroxisomes can occur without up-regulation of the fatty acid catabolism localized in this organelle (see, for example, Sect 7.2) and the opposite situation is certainly also conceivable Long-term exposure of rodents to peroxisome proliferators promotes the formation of liver tumors and is also associated with an elevated incidence of testis cancer At present, more than 1000 different xenobiotics have been found to belong to the class of peroxisome proliferators (for reviews, see [7, 23, 28, 47, 48, 73, 89, 92]) These compounds include clinical drugs (e.g., hypolipidemic drugs of the fibrate family, acetylsalicylic acid, and other non-steroidal anti-inflammatory drugs), industrial chemicals (e.g., phthalate plasticizers, perfluorofatty acids, di(2-ethylhexyl)phosphate, trichloroethylene, chlorinated paraffins), and agricultural chemicals (e.g., phenoxyacetic acids) The structures of some of the most commonly studied peroxisome proliferators are depicted in Fig 2 Use and Occurrence of Perfluorofatty Acids The wide variety of chemicals which cause peroxisome proliferation in rodent liver makes it difficult to propose a unifying hypothesis concerning the molecular mechanism underlying this phenomenon (see Sect 7) The present review focuses on the biological effects of perfluorofatty acids and the analogous sulfonic acid for a number of reasons Because of their hydrophobicity and relative chemical and thermal stabilities, in addition to the fact that they can be produced at relatively low cost, these compounds are finding increasing use as, among other 208 J.W DePierre things, corrosion inhibitors, anti-wetting agents, fire extinguishers, and surfactants Since perfluorofatty and perfluorosulfonic acids are also metabolized poorly, if at all, at least in rodents (see Sect 4), these substances would be expected to accumulate in the general environment However, to date, the carboxylic acid does not appear to have leaked into the environment The levels of perfluorooctanoic acid in the serum of members of the general population are 10–100 parts per billion, although these levels are, as expected, considerably higher in occupationally exposed workers [36] A study in 1974 reported that the average level of organic fluorine in human plasma was approximately 26 ng/ml and among the fluorine-containing compounds present, perfluorooctanoic acid was tentatively identified [54] A recent study by Hansen and coworkers [54] confirmed the presence of perfluorooctanoic acid, perfluorooctane sulfonic acid, and perfluorohexane sulfonic acid in human serum at average levels of 6.4, 28.4, and 6.6 ng/ml, respectively Perfluooctanoic acid is not among the various fluorinated long-chain carboxylic acids detected in plants, nor does there appear to be any other natural source of this compound [49] The possible adverse biological effects of perfluorooctane sulfonic acid are of growing concern [127], since this compound has been found to occur ubiquitously in marine mammals inhabiting widely spread geographical biospheres [70] Although much less is presently known about the responses of living organisms to this compound than to the corresponding perfluorooctanoic acid, it is clear that the sulfonic acid elicits the same degree of peroxisome proliferation and related effects in rodent liver [142] (Table 2) Experimental Systems for Studying the Effects of Perfluorofatty Acids on Mammalian Cells Virtually all studies on the effects of exposure to perfluorofatty acids involve perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) (Fig 3) The reasons for this are that the responses of mammalian cells to shorter perfluorofatty acids are considerably less pronounced (see below) and that longer perfluorofatty acids are not readily commercially available As will become evident below, al- Fig Structures of the most commonly studied perfluorofatty acids Effects on Rodents of Perfluorofatty Acids 209 though the effects of PFOA and PFDA on rats and mice are in many respects similar, there are also important differences Indeed, it is remarkable how large an effect the presence of two extra CF2 groups can have on certain responses Obviously, PFOA and PFDA are analogues of naturally occurring non-fluorinated fatty acids, which is almost certainly of central relevance to the mechanism(s) by which they exert their effects As has been examined in detail [160], extraction and isolation of PFOA and PFDA requires some care These compounds can be readily quantitated as their benzyl [180] or methyl [84] esters employing gas chromatography or by highperformance liquid chromatography after derivatization with 3-bromoacetyl-7methoxycoumarin [111] More convenient for many studies is the use of [14C]PFOA or -PFDA, allowing radiometric quantitation [e g., 155, 157, 158] Definitive quantitation requires confirmation of the structures and this has been carried out [54] using HPLC interfaced to an atmospheric pressure tandem mass spectrometer operating in the electrospray negative mode All reports in the literature concerning the effects of perfluorofatty acids on mammals involve the use of rats and mice as the experimental animals Sometimes isolated rat hepatocytes constitute the experimental system The simple reason for this is that, as is the case for other peroxisome proliferators as well, rats and mice are more responsive to PFOA and PFDA than are other rodents and mammals Other species (e.g., hamsters and guinea pigs) are only examined for purposes of comparison to the rat or mouse No studies on non-mammalian organisms, such as fish and plants, have yet been reported In most cases male animals are studied, which is of considerable importance in the case of rats, since the female of this species does not respond to PFOA, at least not at the doses commonly employed The strains of rats most commonly utilized in these investigations are Sprague-Dawley,Wistar, and Fischer-144 rats The mice most commonly employed are of the C57Bl/6 strain Virtually all studies concerning the biological effects of PFOA and PFDA focus on the liver, although it is apparent that these substances also influence other tissues, including adipose tissue, the thymus and spleen, the testis, and the heart (see below) PFOA and PFDA are almost always administered to rats by intraperitoneal injection, although dietary exposure has also been employed in some studies Usually, a single such injection is performed and the animals monitored thereafter for 1–4 weeks However, other conditions – e.g., a total of four intraperitoneal injections at two-week intervals – are sometimes used The dose injected varies between 0.3 mg (= 0.73 mmoles PFOA or 0.58 mmoles PFDA) and 80 mg (=190 µmoles PFOA or 160 mmoles PFDA)/kg body weight In contrast, PFOA is always administered to mice in their diet This exposure normally lasts for 1–2 weeks and the doses employed vary between 0.005 and 0.05wt% Since a 20-g mouse consumes approximately g of chow per day and the corresponding value for a 200-g rat is 10 g, these dietary levels result in ingestion of 7.5 mg (=18 mmoles of PFOA or 15 mmoles of PFDA) to 75 mg (=180 mmoles PFOA or 150 mmoles PFDA) per day Although far more than 90% of published reports concerning the biological effects of PFOA and PFDA on rodents involve administration of these substances 210 J.W DePierre in the diet or by intraperitoneal injection, inhalation studies and administration by gavage have also been reported.Although it is the acid itself, rather than a salt, which is commonly administered (dissolved in an oil or mixed as a powder with the animal’s food), the carboxylic acid moiety of these substances must certainly be deprotonated to at least some extent in biological fluids Thus, as is often the case in connection with studies in the area of biochemical toxicology, a wide variety of different routes, frequencies, and periods of administration, as well as different doses of PFOA and PFDA, are utilized by different investigators However, with the exception of marked differences between responses to PFOA and PFDA and between the responses of male and female rats described below, I have not been able to discern any consistent differences resulting from the use of such different experimental conditions Of course, the responses differ in their extent, i.e., quantitatively, but they appear to be qualitatively similar Therefore, I have chosen to compare different investigations rather freely in the present review Pharmacokinetics and Metabolism of PFOA and PFDA in Rats Obviously the pharmacokinetics and metabolism of PFOA and PFDA in rats and mice are of fundamental significance with regards to their biological effects Xenobiotics commonly exert primary effects on the organs in which they accumulate and if they not accumulate at all, because of rapid metabolism to an inactive substance(s) and/or elimination, they are unlikely to have any major influence on the organism In addition, the metabolites of a xenobiotic may actually elicit more or less pronounced and/or different responses compared to the parent compound itself The pronounced differences in the pharmacokinetics of PFOA in male and female rats has received considerable attention [41, 43, 53, 71, 72, 81, 85, 154, 157, 158, 178, 179, 181] Female rats eliminated 91% of a single intraperitoneal dose of PFOA in their urine within 24 h, whereas the corresponding value for male rats was only 6% [158] Consequently, the half-time for elimination of this substance was 15 days for the male animals, but kidneys in both sexes, while much lower amounts were present in the heart, fat pads, testis, muscle, and ovaries [157] Thus, PFDA is eliminated from the rat more slowly than PFOA (which probably explains the more pronounced toxicity of the former; see below), as well as via a different route (i.e., via the bile and urine, respectively) Furthermore, elimination of PFDA does not exhibit the same sex difference This slower elimination of PFDA may reflect the fact that 99% of this compound in the serum is bound to protein [178]; however, the corresponding value for PFOA has not been reported Curiously, one study reported covalent binding of small amounts (0.1–0.5% of the total dose) of both PFOA and PFDA to proteins in the liver, plasma, and testis of male rats, although no metabolism was observed (see also below) [159] Sulfhydryl groups on the proteins were apparently involved in this binding In another study the effects of perfluorofatty acids of different lengths on hepatic peroxisomal fatty acid b-oxidation in male and female rats were compared [85] In the male animals perfluorohexanoic acid elicited no response, whereas C8 , C9 , and C10 perfluorofatty acids potently up-regulated this activity In female rats the effects of these different compounds were the same as in the male, with the exception of perfluorooctanoic acid, which elicited much less pronounced responses in females, as expected from the pharmacokinetic differences described above There was a significant correlation between the hepatic concentration of each compound and its potency Peroxisomal fatty acid b-oxidation in hepatocytes isolated from male and female rats was equally responsive to perfluorooctanoic and perfluorononanoic acids [85], further demonstrating that the sex differences observed in vivo reflect pharmacokinetic differences It has been suggested that the relatively higher water solubility of perfluoroheptanoic and perfluorooctanoic acid favors their excretion in the urine, while the greater hydrophobicity of C9–11 perfluorofatty acids promotes their elimination in the bile, with subsequent reuptake in the intestine, i.e., enterohepatic recirculation [43] Investigations designed to detect metabolites of PFOA or PFDA have all failed [41, 155, 157, 158, 179] These studies have attempted to identify polar metabolites of these compounds in urine or bile, lipids containing these fatty acid analogues, and/or loss of fluorine (which could potentially be catalyzed by the cytochrome P-450 system, which can dehalogenate certain organic substances [124]), all unsuccessfully Nor were acyl-CoA species containing PFOA or PFDA detected in rat liver or in isolated rat hepatocytes [88] Thus, PFOA and PFDA appear to be excreted from rats without prior metabolism A finding which is not consistent with this conclusion is the report that chromosomal aberrations are caused by PFDA only after activation by a post-mitochondrial fraction from rat liver (see Sect 8.6.1) 212 J.W DePierre Table Initial time-course of the proliferation of peroxisomes in mouse liver upon dietary exposure to 0.02 wt% perfluorooctanoic acid Period of treatment (days) Average number of peroxisomes per unit area (% of control) Average size of peroxisomes (% of control) None (control) 100 125 170 210 300 100 130 200 220 220 Unpublished data from our laboratory (1998) Effects of Perfluorofatty Acids on the Number, Size, and Functions of Peroxisomes in Rodent Liver 5.1 Morphological Studies Definitive demonstration that a xenobiotic is a peroxisome proliferator requires electron microscopic studies, preferably quantitative (i.e., morphometry) Several studies have established that the number of peroxisomes in rat and mouse liver is increased upon exposure to PFOA or PFDA [56, 57, 68, 140, 143] One of these studies revealed more extensive peroxisome proliferation in centrilobular than in periportal rat hepatocytes [68] We have conducted a detailed morphometric study of peroxisome proliferation in the liver of mice exposed to 0.02 wt% PFOA in their diet It can be seen from Table (unpublished results from our laboratory, 1998) that after four days of such treatment, the number of hepatic peroxisomes had increased 3-fold, while their average size had increased 2.2-fold (As can be seen from Table 5, these effects were almost maximal.) 5.2 Effects on Peroxisomal Proteins and Functions 5.2.1 Fatty Acid b -Oxidation Peroxisomal fatty acid b-oxidation can be quantitated both as the activity of the total process, e.g., oxidation of palmitoyl-CoA, or as the activity of the initial enzyme in this pathway, i.e., acyl-CoA oxidase, which is thought to be rate-limiting for the entire pathway [67, 126, 131] In contrast to the corresponding mitochondrial catabolism, which conserves the energy released by the first step in the form of FADH2 , the first step in peroxisomal fatty acid b-oxidation produces hydrogen peroxide (Fig 4) Thus, quantitation of this hydrogen peroxide provides Effects on Rodents of Perfluorofatty Acids 213 Fig Mitochondrial (on the left) and peroxisomal (on the right) fatty acid b-oxidation both an assay for the peroxisomal pathway and a means of distinguishing this pathway from mitochondrial catabolism as well as raising the as-yet-unanswered question as to why the energy released by the first step of peroxisomal fatty acid b-oxidation is not conserved Indeed, peroxisomes contain a number of other oxidases which also produce hydrogen peroxide, thereby potentially causing oxidative stress in the cell (see below) Numerous studies have demonstrated that exposure of rats and mice to PFOA or PFDA results in potent increases in the hepatic activities and levels of the peroxisomal enzymes catalyzing fatty acid b-oxidation [12, 32, 43, 66, 68, 71, 82, 83, 85, 139–143, 173] 214 J.W DePierre Table Increases in the peroxisomal activity of acyl-CoA oxidase and in the peroxisomal and cytosolic activities of catalase in the livers of mice upon dietary administration of peroxisome proliferators (unpublished findings from our laboratory, 1997) Treatment Enzyme activities in mmol product formed/min-g liver (%) Peroxisomal acyl-CoA oxidase Peroxisomal catalase Cytosolic catalase None (control) 202±8 ¥10–6 Perfluorooctanoic acid 4.10±0.40 ¥10–3 *** (2030) 24.4±2.3 ** (200) 21.3±4.7 *** (970) Perfluorooctane sulfonic acid 3.20±0.70 ¥10–3 ** (1580) 37.3±1.7 *** (308) 13.8±2.2 ** Clofibrate 2.05±0.50 ¥10–3 ** (1010) 18.0±1.2 ** (149) 22.0±0.5 *** (1100) Nafenopin 2.86±0.67 ¥10–3 ** (1420) 13.4±1.9 20.1±1.4 *** (1000) (100) 12.1±1.5 (100) (110) 2.0±0.3 (100) (690) n=4 **P