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,[.]
Trang 1Effects 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
in-variably 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 cir-culating 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
1 Introductory Remarks 205
2 Use and Occurrence of Perfluorofatty Acids 207
3 Experimental Systems for Studying the Effects of Perfluorofatty Acids on Mammalian Cells 208
4 Pharmacokinetics and Metabolism of PFOA and PFDA in Rats 210 5 Effects of Perfluorofatty Acids on the Number, Size, and Functions of Peroxisomes in Rodent Liver 212
5.1 Morphological Studies 212
5.2 Effects on Peroxisomal Proteins and Functions 212
5.2.1 Fatty Acid b-Oxidation 212
5.2.2 Catalase 214
5.3 Sex and Species Differences 216
The Handbook of Environmental Chemistry Vol 3, Part N Organofluorines
(ed by A H Neilson)
© Springer-Verlag Berlin Heidelberg 2002
Trang 25.4 Dependence on Chain Length, the Carboxylic Acid Moiety,
and Fluorination 217
5.5 Dependence on Dose and Time 218
5.6 Reversibility/Persistence 222
5.7 Tissue Specificity 222
6 Additional Effects of Perfluorofatty Acids in Rodents 224
6.1 Additional Effects on Hepatic Lipid-Metabolizing Enzymes, Lipid-Binding Proteins, and Lipid Composition 224
6.2 Hypolipidemia 226
6.3 “Wasting Syndrome”: Loss of Body Weight and Body Fat 226
6.4 Hepatomegaly 226
6.5 Decrease in Mitochondrial Size 227
6.6 Decreases in the Levels of Thyroid Hormones 228
6.7 Up-Regulation of CYP4A1 228
6.8 Up-Regulation of UDP-Glucuronyltransferase 229
6.9 Oxidative Stress 230
7 Mechanism(s) Underlying These Effects of Perfluorofatty Acids 230 7.1 Formation of Perfluorofatty Acyl-CoA and/or of Dicarboxylic Fatty Acids and/or Disruption of Fatty Acid Homeostasis in Other Ways 230 7.2 Peroxisome Proliferator-Activated Receptor-Alpha 231
8 Toxicity/Genotoxicity of Perfluorofatty Acids 233
8.1 Acute Toxicity: LD50 Values, the “Wasting Syndrome”, and Acute Tissue Damage 233
8.2 Developmental Toxicity 233
8.3 Degeneration of Seminiferous Tubules 233
8.4 Immunotoxicity? 233
8.5 Genotoxicity 234
8.6 Possible Genotoxic Mechanism(s) 234
8.6.1 Lack of Direct Genotoxicity 234
8.6.2 Increased Oxidative Stress 235
8.6.3 Altered Xenobiotic Metabolism 235
8.6.4 Stimulation of Hepatocyte Proliferation 235
8.6.5 Inhibition of Hepatocyte Apoptosis 236
8.6.6 Immunotoxicity? 236
9 Studies on Humans 237
10 Concluding Remarks 237
10.1 Valuable Experimental Tools 237
10.2 Possible Clinical Applications 238
10.3 Hazard to Human Health? 238
11 References 239
Trang 3Introductory 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 cellu-lar 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 perfluo-rofatty 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, per-haps, 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 im-portant functions are localized in this organelle Many of these functions are
re-lated to global lipid homeostasis, e g., b-oxidation of fatty acids (see also below);
synthesis of bile acids, plasmologens (ether phospholipids), and isoprenoid com-pounds 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 ofD-amino acids to the corresponding L -amino acids, and uric acid catabolism Much characterization of peroxisomes re-mains 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 peroxi-somes present in hepatocytes and/or other cell types (Fig 1) However, a num-ber of other physiological and biochemical changes also typically occur in asso-ciation with this proliferation (see below) For instance, up-regulation of the three
enzymes involved in peroxisomal fatty acid b-oxidation is virtually always
ob-served 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
Trang 4pro-Fig 1. 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 1 micron is indicated in the lower electron micrograph Electron micrographs courtesy of Professor Anders Bergstrand Unpublished studies in our laboratory (1995)
Trang 5liferation of peroxisomes can occur without up-regulation of the fatty acid ca-tabolism localized in this organelle (see, for example, Sect 7.2) and the opposite situation is certainly also conceivable Long-term exposure of rodents to perox-isome 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), in-dustrial chemicals (e g., phthalate plasticizers, perfluorofatty acids, di(2-ethyl-hexyl)phosphate, trichloroethylene, chlorinated paraffins), and agricultural chemicals (e g., phenoxyacetic acids) The structures of some of the most com-monly studied peroxisome proliferators are depicted in Fig 2
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 molecu-lar mechanism underlying this phenomenon (see Sect 7) The present review fo-cuses on the biological effects of perfluorofatty acids and the analogous sulfonic acid for a number of reasons Because of their hydrophobicity and relative chem-ical 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
Fig 2. Structures of commonly studied peroxisome proliferators related (left) and unrelated (right) to clofibrate
Trang 6things, corrosion inhibitors, anti-wetting agents, fire extinguishers, and surfac-tants
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 ac-cumulate 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 bil-lion, 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 com-pounds present, perfluorooctanoic acid was tentatively identified [54] A recent study by Hansen and coworkers [54] confirmed the presence of perfluorooc-tanoic 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 Perflu-ooctanoic 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 ubiqui-tously in marine mammals inhabiting widely spread geographical biospheres [70] Although much less is presently known about the responses of living or-ganisms 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)
3
Experimental Systems for Studying the Effects of Perfluorofatty Acids
on Mammalian Cells
Virtually all studies on the effects of exposure to perfluorofatty acids involve per-fluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) (Fig 3) The rea-sons 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 3. Structures of the most commonly studied perfluorofatty acids
Trang 7though the effects of PFOA and PFDA on rats and mice are in many respects sim-ilar, there are also important differences Indeed, it is remarkable how large an ef-fect the presence of two extra CF2groups can have on certain responses Obvi-ously, 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 high-performance liquid chromatography after derivatization with 3-bromoacetyl-7-methoxycoumarin [111] More convenient for many studies is the use of [14 C]-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 Some-times 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 fo-cus 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 in-jection, although dietary exposure has also been employed in some studies Usu-ally, 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 3 g of chow per day and the corresponding value for a 200-g rat is 10 g, these dietary levels result in
in-gestion 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
Trang 8in 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 biochemi-cal toxicology, a wide variety of different routes, frequencies, and periods of ad-ministration, as well as different doses of PFOA and PFDA, are utilized by dif-ferent 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 sulting from the use of such different experimental conditions Of course, the re-sponses differ in their extent, i.e., quantitatively, but they appear to be qualita-tively similar Therefore, I have chosen to compare different investigations rather freely in the present review
4
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 accu-mulate and if they do not accuaccu-mulate 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 ac-tually 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 fe-male 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 < 1 day for the females [53, 158] Four days after administration, the level of PFOA in the serum of male rats was 17–40 times higher than in female animals [179], and 28 days after administration the con-centrations in various tissues were 6 – 9 times higher in males than in females [81] The highest concentrations were observed in the liver and plasma of male rats and in the liver, plasma, and kidney of female rats [158]
Several studies involving castration, ovarectomy, and administration of sex hormones have demonstrated that estradiol promotes urinary excretion of PFOA, whereas testosterone decreases this excretion [71, 72, 84, 179] The effect
of testosterone appears to dominate over the effect of estradiol in deter-mining the pharmacokinetics of PFOA in rats The molecular mechanisms
by which these sex steroids influence the excretion of PFOA remain to be eluci-dated
Interestingly, comparison of the pharmacokinetics of PFDA to those of PFOA
in male and female rats reveals quite different patterns Twenty-eight days after
Trang 9a single intraperitoneal administration of PFDA, 51 % and 24 % of the dose given had been eliminated in the feces of male and female rats, respectively [157, 178] During this same period, < 5 % of the total dose was recovered in the urine of animals of either sex The half-time for elimination of PFDA from male rats was calculated to be 23 days, whereas the corresponding value for the female an-imals was 45 days The highest levels of PFDA were observed in the order liver > plasma > 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 proba-bly 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, elim-ination of PFDA does not exhibit the same sex difference This slower elimina-tion 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
he-patic 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 C10perfluorofatty 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 re-sponses 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
fe-male 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 wa-ter solubility of perfluoroheptanoic and perfluorooctanoic acid favors their ex-cretion in the urine, while the greater hydrophobicity of C9–11perfluorofatty 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 metabo-lites of these compounds in urine or bile, lipids containing these fatty acid ana-logues, and/or loss of fluorine (which could potentially be catalyzed by the cy-tochrome 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 metab-olism 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-mi-tochondrial fraction from rat liver (see Sect 8.6.1)
Trang 10Effects 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 prolifera-tion in the liver of mice exposed to 0.02 wt % PFOA in their diet It can be seen from Table 1 (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 ef-fects 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 en-zyme 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 mito-chondrial 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
hy-drogen peroxide (Fig 4) Thus, quantitation of this hyhy-drogen peroxide provides
Table 1. Initial time-course of the proliferation of peroxisomes in mouse liver upon dietary ex-posure to 0.02 wt% perfluorooctanoic acid
Unpublished data from our laboratory (1998).