Induction of Hepatic Peroxisome Proliferation by 8–2 Telomer Alcohol Feeding In Mice: Formation of Perfluorooctanoic Acid in the Liver
http://www.100md.com
《毒物学科学杂志》
Faculty of Pharmaceutical Sciences, Josai University, Keyakidai 1–1, Sakado, Saitama 350-0295, Japan
Research and Development Laboratories, Maruho Co., 1 Awatacho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan
ABSTRACT
The effects of dietary administration of 1H, 1H, 2H, 2H-perfluorodecanol (8–2 telomer alcohol), on peroxisome proliferation in the liver of mice were studied. Male ddY mice were fed on a diet containing 8–2 telomer alcohol at concentrations of 0, 0.025, 0.05, 0.1, and 0.2% (w/w) for 7, 14, 21, and 28 days. These treatments with 8–2 telomer alcohol caused liver enlargement in a dose- and duration-dependent manner. Peroxisome proliferation in the liver of mice was confirmed by electron microscopic examination. Peroxisomal acyl-CoA oxidase was induced by these treatments with 8–2 telomer alcohol in a dose- and time-dependent manner. The concentration of perfluorooctanoic acid (PFOA) and related compounds were determined in the liver and plasma, since PFOA had been shown to be a possible metabolite of 8–2 telomer alcohol and to cause significant peroxisome proliferation in rodents. Five metabolites, namely, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), 2H, 2H-perfluorodecanoic acid (8–2 telomer acid), and two unidentified metabolites, were present in the liver and serum. PFOA was confirmed to be accumulated in the liver of mice following the administration of 8–2 telomer alcohol in a dose- and duration-dependent manner. A linear relationship was observed between the concentration of PFOA and the activity of peroxisomal acyl-CoA oxidase in the liver of mice. These results strongly suggest that PFOA, but not 8–2 telomer alcohol itself, caused peroxisome proliferation in the liver. The present study provided evidence that 8–2 telomer alcohol is converted into PFOA in vivo and that the PFOA formed produces biological effects in the liver of mice.
Key Words: 8–2 telomer alcohol; perfluorooctanoic acid; peroxisome proliferation; liver; mouse.
INTRODUCTION
Organic fluorochemicals are compounds in which some or all the carbon–hydrogen bonds are replaced by a carbon–fluorine bonds. These carbon–fluorine bonds are some of the strongest in nature and contribute to the unique stability of these fluorochemicals (Hatfield, 2001; Key et al., 1997). Perfluorooctane sulfonate has been widely used in a variety of industrial and consumer applications in hair shampoos, paper coatings, waxes, polishes, and paints (Key et al., 1997). Recently, however, it has been pointed out that there is a risk that the chemical accumulates in the environment and humans (Hansen et al., 2002; Kannan et al., 2002; Olsen et al., 2003), so that some primary manufactures of this compound have discontinued its production. Great efforts have been made to find new chemicals that have properties similar to perfluorooctane sulfonate and are less biologically hazardous. 1H, 1H, 2H, 2H-Perfluorodecanol (8–2 telomer alcohol) is a polyfluorinated compound that has eight fully fluorinated carbons and two nonfluorinated carbons adjacent to a hydroxyl group. This 8–2 telomer alcohol has applications similar to those of perfluorooctane sulfonate-based products. During the period 2000–2002, the amount of telomer alcohols produced was estimated to be 5 x 106 kg/year worldwide (U. S. EPA, 2002a).
It has recently been demonstrated that 8–2 telomer alcohol is a potential source of perfluorooctanoic acid (PFOA) as a consequence of biotic degradation (Dinglasan et al., 2004; Stock et al., 2004). Little information, however, is available about the biotransformation of 8–2 telomer alcohol in mammals. An early study suggested the possibility that 8–2 telomer alcohol was converted into PFOA in rats (Hagen et al., 1981). If this is the case, there is a risk that PFOA accumulates in the environment and humans, because PFOA is so stable that it is not decomposed by activated sludge (U. S. EPA, 2002b) and is not metabolized in animals (Ophaug and Singer, 1981; Vanden Heuvel et al., 1991). Moreover, the PFOA formed in animals from 8–2 telomer alcohol may physiologically affect the animals, because PFOA has been demonstrated to cause peroxisome proliferation in the liver, body weight loss, a reduction in thymus and spleen weight, a reduction in the number of erythrocytes, and an elevation of glucose levels (Kawashima et al., 1995; Kudo et al., 2000, 2003; Yang et al., 2000, 2002). Information, however, is lacking about how much PFOA is formed from 8–2 telomer alcohol in the liver of animals when 8–2 telomer alcohol is repeatedly administered and about whether the concentration of PFOA formed in the liver is high enough to cause physiological effects.
The present study is aimed at investigating whether repeated administration of 8–2 telomer alcohol causes PFOA accumulation in the liver of mice, and whether the accumulated PFOA is enough high to induce peroxisome proliferation.
MATERIALS AND METHODS
Materials.
8–2 Telomer alcohol was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan); PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid, palmitoyl-CoA, and bovine serum albumin were from Sigma Aldrich Japan (Tokyo, Japan); NAD and CoA were from Oriental Yeast Co. (Tokyo, Japan); Triton X-100 was from Nakalai Tesque (Kyoto, Japan). 3-Bromoacetyl-7-methoxycoumarin was prepared as previously described (Ohya et al., 1998). All other chemicals were of analytical grade.
Chemical synthesis of 2H, 2H-perfluorodecanoic acid (8–2 telomer acid).
To a solution of 8–2 telomer alcohol (25 mg, 0.054 mmol) in glacial acetic acid (2.0 ml) was added excess chromium (VI) oxide. After stirring for 24 h, the reaction was stopped with water (2.0 ml), followed by solid sodium hydrogen sulfite (approximately 30 mg), then acidified with 3 M sulfuric acid (1.0 ml), and the mixture was then extracted with diethyl ether (5 ml). The layers were separated, and the organic layer was flushed with nitrogen gas to evaporate any diethyl ether. The residue was dissolved in 1 M sodium hydroxide (3.0 ml), and the solution was washed with diethyl ether (2 x 3.0 ml). The combined aqueous phase was acidified with 70% sulfuric acid (10 ml) and extracted with diethyl ether (2 x 20 ml). To the organic layer was added 2 M sulfuric acid (2 drops), and this solution was extracted with diethyl ether (20 ml). The extract was washed with water (2 x 10 ml), dried over sodium sulfate, and concentrated in vacuo. The residue was purified by column chromatography (silica gel), eluted with diethyl ether/hexane/acetic acid (3:8:0, v/v), and then with diethyl ether/hexane/acetic acid (3:8:5, v/v) to give 8–2 telomer acid (6.2 mg, 0.013 mmol) as a white power. MS (FAB): Exact mass calculated for 478; [C10F17H3O2]. Found m/z (%) 479 (18.67), 69 (100).
Animals.
Male ddY mice aged 7 weeks were purchased from SLC (Hamamatsu, Japan) and acclimatized in a humidity- and temperature- (23 ± 2°C) controlled environment with a 12-h light/dark cycle for at least 1 week before use. In one set of experiments, mice were fed a diet containing 8–2 telomer alcohol at concentrations of 0, 0.025, 0.05, 0.10, and 0.20% (w/w) for 7, 14, 21, and 28 days. The mice were killed under diethyl ether anesthesia. In another set of experiments, mice were given intraperitoneal injections of 8–2 telomer alcohol as a propylene glycol solution at a dose of 400 mg/kg, and the mice were killed at 2, 6, 24, and 72 h postdosage. Livers were excised, perfused with ice-cold 0.9% NaCl, frozen in liquid nitrogen, and stored at –80°C until use. The frozen liver was thawed at 0°C and homogenized with nine volumes of 0.25 M sucrose/1 mM EDTA/10 mM Tris–HCl (pH 7.4). The protein concentrations in the homogenates were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. All animal studies complied with institutional board for animal study, Josai University.
Electron microscopy.
At the end of the 8–2 telomer alcohol feeding, some of the mice were anesthetized with diethyl ether and perfused via the left ventricle with 0.9% (w/v) NaCl and subsequently with 1.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 5 min. The left lateral lobe of the liver was removed and cut into 1-mm slices which were fixed in 1.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C for 4 h. The prefixed blocks were sliced into 50-μm sections using a DTK-3000W microslicer (Dosaka EM, Osaka, Japan), and incubated in 5 mM 3,3'-diaminobenzidine in Teorell-Stenhagen buffer (pH 10.5) containing 0.15% (v/v) hydrogen peroxide for 15 min. After rinsing with cacodylate buffer, the sections were postfixed in buffered 1% osmium tetroxide for 1 h, dehydrated in acetone, and embedded in Epon-812. Thin sections were cut from each slice with a diamond knife on an MT-2B ultramicrotome (Dupont-Sorvall, MO), stained with uranyl acetate, and photographed using an H-7000 electron microscope (Hitachi, Katsuta, Japan) (Beier et al., 1992). Electron microscopy fields (original magnification of 4000x) were chosen for image analysis. Peroxisomes were identified as membrane-bound and electron-dense organelles. Ten electron micrographs for one mouse were analyzed by morphometric techniques with a Micro Computer Imaging Device (MCIDTM, Amersham Biosciences, Piscataway, NJ, USA) to give the peroxisomal area and the number of peroxisomes per unit cellular area apart from the nuclear area (30 μm2).
Assay of acyl-CoA oxidase.
Acyl-CoA oxidase was assayed spectrophotometrically by measuring palmitoyl-CoA-dependent H2O2 production at 502 nm (Small et al., 1985).
Determination of metabolites of 8–2 telomer alcohol.
The metabolites of 8–2 telomer alcohol in liver and serum samples were extracted, converted to acetylmethoxycoumarin derivatives, separated by high performance liquid chromatography (HPLC), and quantified by fluorescence detection as described previously (Ohya et al., 1998) with some modifications as follows. To an aliquot of liver homogenate or serum was added an appropriate amount of perfluorodecanoic acid as an internal standard for the analysis of the metabolites of 8–2 telomer alcohol, and then the metabolites were extracted with ethyl acetate:hexane (1:1, v/v) as an ion pair with tetrabutylammonium in the presence of NaCO3 buffer (pH 10.0). The metabolites in the extract were run on thin-layer chromatography (silica gel 5721, Merck, Germany) developed with hexane/diethyl ether/acetic acid (16:6:1, v/v). Authentic PFOA and 8–2 telomer acid were simultaneously run alongside the unknown mixture on thin-layer plates. The spots were detected by their water-repellent effect after spraying with water. The areas of silica gel corresponding to PFOA and other two metabolites (Rf value ranging from 0 to 0.06) and 8–2 telomer acid (Rf value ranging from 0.15 to 0.38) were individually scraped off, and these metabolites were extracted from the silica gel as an ion pair with tetrabutylammonium as described above. The extract was transferred to a glass tube and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 0.2% (w/v) 3-bromoacetyl-7-methoxycoumarin in acetone, and the mixture was heated at 70°C for 25 min followed by cooling on ice. The solution was filtered through a glass-wool filter, and the filtrate was subjected to HPLC using a reverse-phase column (YMC-Pack Pro, 4.6 ID x 50 mm), and eluted with acetonitrile/water (7:3, v/v). The peaks of acetylmethoxycoumarin derivatives of the metabolites and perfluorodecanoic acid (internal standard) were detected using fluorescence detector (Shimadzu RF-10AXL, Shimadzu Co., Kyoto, Japan) at an excitation wavelength of 366 nm and an emission wavelength of 420 nm.
Statistical analysis.
Homogeneity of variance was established using one-way analysis of variance. When a difference was significant (p < 0.05), Schéffe's multiple range test was used as a post-test. Statistical significance between two means was estimated by either Student's t-test or Welch's test after F-test. Linear regression analysis was performed to evaluate the correlation between two parameters.
RESULTS
Effects of 8–2 Telomer Alcohol Feeding on Body Weight Gain and Liver Changes in Mice
Male mice were fed a diet containing 8–2 telomer alcohol at concentrations ranging from 0 to 0.2% (w/w) for 7, 14, 21, or 28 days. The effects of the treatments on body weight gain, liver weight, and relative liver weight are shown in Table 1. There was no significant effect following the administration of 8–2 telomer alcohol on the overall health of the mice, except that a slight decrease in body weight gain was seen in some groups. The livers of mice treated with 8–2 telomer alcohol were significantly enlarged in a dose- and time-dependent manner. Owing to the changes in liver weight, the treated animals showed an increase in relative liver weight. The increase was dependent on both the dose and duration of treatment.
Table 1 also shows the effect of 8–2 telomer alcohol feeding on the activity of peroxisomal acyl-CoA oxidase, a peroxisomal enzyme, in the liver. The activity was markedly increased in a dose- and time-dependent manner, as was observed with the relative liver weight. The higher the concentration of 8–2 telomer alcohol in the diet, the shorter the time required to reach the maximum activity.
The morphological results obtained from mice that were treated with 8–2 telomer alcohol at a dietary concentration of 0.1% for 14 days are shown in Figures 1 and 2. In the hepatocytes of control mice, the peroxisomes, which were intensely stained spherical particles approximately 0.5 μm in diameter, were distributed randomly in the cytoplasm (Fig. 1A). The treatment of mice with 8–2 telomer alcohol increased the cell size and caused proliferation of peroxisomes (Fig. 1B). The number of peroxisomes was increased by treatment of mice with 8–2 telomer alcohol (130% of control), and the volume density of peroxisomes, given as a percentage of the cytoplasmic area, also increased (312% of control) (Fig. 2).
Biotransformation of 8–2 Telomer Alcohol in the Liver of Mice
Mice were given intraperitoneal injections of 8–2 telomer alcohol at a dose of 400 mg/kg. The metabolites formed were extracted with solvent as an ion pair with tetrabutylammonium from the liver homogenates obtained from mice 6 h postdosage. The metabolites extracted were separated by thin-layer chromatography into a fast-migrating spot (Rf value of 0.15–0.38) and a slow-migrating spot (Rf value of 0–0.06). The metabolites contained in the two spots were individually extracted and run on HPLC after derivatization with 3-bromoacetyl-7-methoxycoumarin. Three metabolites were detected from the slow-migrating spot, and one of them was identified as PFOA by retention-time matching and also spiking with the corresponding authentic reference sample (Fig. 3A). All are thought to be carboxylic acids, because 3-bromoacetyl-7-methoxycoumarin selectively reacts with carboxylic acid. The other two metabolites (A and B) have not been identified yet (Fig. 3A). One metabolite was detected from the fast-migrating spot and identified as 8–2 telomer acid. The retention time of this compound was the same as that of PFOA (Fig. 3B). The metabolites detected in the serum obtained from mice 6 h postdosage were the same as those found in the liver.
Figure 4 illustrates the time courses of the formation of the metabolites from 8–2 telomer alcohol in the liver and serum. In the liver, the concentration of metabolite A immediately increased after injection, reached a maximum (29 nmol/g liver) at 6 h, and then gradually declined (Fig. 4A). The concentration of metabolite A fell to below the detection limit at 72 h postdosage. The concentration of PFOA in the liver gradually increased up to 52 nmol/g liver 72 h postdosage. Metabolite B was detected at 2 and 6 h, although its concentrations were very low compared with those of metabolite A and PFOA. At 24 and 72 h postdosage, the concentration of metabolite B fell below the limit of quantification. In the serum, the concentration of PFOA gradually increased as observed with the liver (Fig. 4B). However, unlike the liver, the concentration of metabolite A was low throughout the entire time period. No metabolite A was detected in serum at 72 h. The concentrations of metabolite B in serum were below the limit of quantification limit (0.2 nmol/ml serum). No 8–2 telomer acid was detected at 2, 24, and 72 h postdosage in either the liver or serum, although its concentration was 0.79 nmol/g liver and 0.74 nmol/ml serum at 6 h (data not shown in the figures). Linear regression analysis was performed to examine the relationship between the concentrations of PFOA in the liver and serum. One regression line was obtained for five sets of mean data from Figures 4A and 4B, with a high correlation being seen between the two parameters (Y = 0.8967X + 1.1465; r2 = 0.9791; p < 0.05).
Accumulation of PFOA in the Liver of Mice
Table 2 shows accumulation of PFOA in the liver when mice were fed the diet containing 8–2 telomer alcohol. The content of PFOA in the liver of control mice was below the detection limit of 0.2 nmol/g liver. When compared within the groups of mice treated with 8–2 telomer alcohol for the same duration, the accumulation of PFOA in the liver tended to increase in a dose-dependent manner. When compared within the groups treated with 8–2 telomer alcohol at the same doses, PFOA in the liver tended to accumulate in a manner that depended on the duration of treatment. The maximum accumulation was approximately 122 nmol/g liver, which was seen in the mice fed on the diet containing 0.2% 8–2 telomer alcohol for 28 days.
The content of PFOA as an impurity in 8–2 telomer alcohol, which was used in the present experiment, was measured by HPLC and found to be less than 8.67 x 10–5% (w/w), and neither PFNA, nor metabolites A and B were found in 8–2 telomer alcohol. Assuming that the daily food intake of a mouse is 6 g, the PFOA intake is at most 0.0224 nmol/day/animal. These values are far less than the amounts of PFOA found in the liver of mice that were treated with 8–2 telomer alcohol.
Relationship between PFOA Concentration and Peroxisomal Acyl-CoA Oxidase Activity in the Liver of Mice
To examine the relationship between the concentration of PFOA and the activity of acyl-CoA oxidase in the liver of mice (Fig. 5), a linear regression analysis was carried out. One linear regression line was obtained for 21 sets of mean data between the hepatic concentration of PFOA and the acyl-CoA oxidase activity from Tables 1 and 2, with a high correlation being seen between the two parameters (r2 = 0.8609, p < 0.01). To examine the relationships between the PFNA concentration and acyl-CoA oxidase activity, between the metabolite A concentration and acyl-CoA oxidase activity, and between the metabolite B concentration and acyl-CoA oxidase activity in the liver, linear regression analyses were carried out for 21 sets of mean data from Tables 1 and 2. A linear regression line was not obtained either between the PFNA concentration and acyl-CoA oxidase activity (Y = 2.1597X + 23.854, r2 = 0.4271, p > 0.05), between the metabolite A concentration and acyl-CoA oxidase activity (Y = 0.163X + 37.562, r2 = 0.0561, p > 0.05), or between the metabolite B concentration and acyl-CoA oxidase activity (Y = 0.022X + 1.311, r2 = 0.0003, p > 0.05).
DISCUSSION
Induction of Peroxisome Proliferation
The present study showed that the treatment of mice with 8–2 telomer alcohol caused enlargement of livers in a dose- and time-dependent manner. Since peroxisome proliferation induced by peroxisome proliferators is known to be always accompanied by hepatomegaly in rodents (Hess et al., 1965; Lalwani et al., 1983; Reddy et al., 1988; Stott, 1988), we investigated whether 8–2 telomer alcohol feeding induces peroxisomal acyl-CoA oxidase, a parameter of peroxisome proliferation (Lalwani et al., 1983; Lazarow, 1977; Lazarow and De Duve, 1976; Reddy et al., 1988; Stott, 1988). As had been supposed, peroxisomal acyl-CoA oxidase in the liver was markedly induced by 8–2 telomer alcohol in a dose- and time-dependent manner. This result strongly suggests the possibility of peroxisome proliferation in the liver of mice given 8–2 telomer alcohol. This was confirmed by electron microscopic examination, as the increase in both the number and the size of peroxisomes. However, unlike 8–2 telomer alcohol, 1H, 1H-perfluorooctanol that is a form of fluorotelomer alcohol is reported to have no effect on peroxisome proliferation in primary cultures of rat hepatocytes (Intrasuksri et al., 1998). These facts raised a question of whether 8–2 telomer alcohol itself or the metabolites formed from 8–2 telomer alcohol cause peroxisome proliferation.
Biotransformation of 8–2 Telomer Alcohol
Hagen et al. (1981) suggested that 8–2 telomer alcohol was metabolized in rats and showed that three metabolites, PFOA, 8–2 telomer acid, and an unidentified metabolite, were present in serum. The present study confirmed the formation of PFOA from 8–2 telomer alcohol in the liver of mice. In addition to PFOA, the existence of 8–2 telomer acid and two other unidentified metabolites (A and B) was demonstrated in the liver. 8–2 Telomer acid was confirmed in the liver of the mice only at 6 h postdosage of 8–2 telomer alcohol. When mice were injected with a large dose of 8–2 telomer alcohol, the concentration of metabolite A transiently increased and then declined, with the maximum level being reached 6 h postdosage. By contrast, the concentration of PFOA gradually increased throughout the time period. These results strongly suggest that metabolite A is a probable precursor that may undergo transformation leading to the formation of PFOA. In addition, 8–2 telomer acid was detected briefly 6 h postdosage. In the light of these findings, it seems likely that 8–2 telomer alcohol is transformed to 8–2 telomer acid, which is converted to metabolite A and then the highly stable PFOA. Based on the present results and on the earlier findings of Hagen et al. (1981), Figure 6 illustrates a proposed degradation scheme of 8–2 teloemer alcohol in the liver of mice. The present study demonstrated that the serum concentrations of PFOA were comparable to its hepatic concentrations and that there was a significant correlation between the PFOA concentrations in the serum and liver. Moreover, 8–2 telomer alcohol given intraperitoneally is considered to be transferred to the liver via the portal vein. These facts suggest, therefore, that 8–2 telomer alcohol is metabolized in the liver to PFOA, which may then be passed into the general circulation.
The present study demonstrated that the mice, which were repeatedly treated with 8–2 telomer alcohol, accumulated PFOA in the liver and that the amounts of PFOA present in the liver were much greater than those calculated on the assumption that all the PFOA, which is present in the 8–2 telomer alcohol used in the present study as an impurity, had been absorbed and accumulated in the liver of the animals. The PFOA found in the mouse liver is, therefore, considered to be formed from 8–2 telomer alcohol in mice. The formation of PFOA was confirmed to be dependent on the dose of the 8–2 telomer alcohol and saturable, and also to be dependent on the duration of treatment. However, PFOA accumulated in extremely small amounts, despite the fact that the mice were treated with 8–2 telomer alcohol at very high doses. The accumulation of PFNA and the unidentified metabolite A was not as great as that of PFOA. The unidentified metabolite B, which was found in very low concentrations during the time-course experiment, did not accumulate when 8–2 telomer alcohol was dosed repeatedly.
Induction of Peroxisome Proliferation by PFOA
Previous studies have reported that PFOA is a potent peroxisome proliferator in the liver of rodents (Borges et al., 1992; Kawashima et al., 1989, 1995; Kudo et al., 2000; Permadi et al., 1993; Sohlenius et al., 1992a,b; Uy-Yu, 1990). In the mice that were fed the diets containing 0.001% (w/w) and 0.002% (w/w) PFOA for 4 weeks, hepatic peroxisomal ayl-CoA oxidase (AOX) activities were 40.7 ± 14.2 and 51.7 ± 14.4 nmol/min/mg protein, respectively (Kudo et al., unpublished data). The induction of AOX by 8–2 telomer alcohol feeding in the present study, therefore, is almost comparable with that by PFOA although the doses required for maximum induction were different. In the present study, we chose four doses that cause the maximum induction of AOX at 28 days of the administration of 8–2 telomer alcohol and that cause dose-dependent induction at 7 days. Time-course study up to 28 days revealed time-dependent induction of AOX activity at lower doses. It should be emphasized that a linear relationship was confirmed between the concentration of PFOA and the activity of peroxisomal acyl-CoA oxidase in the liver of mice treated with 8–2 telomer alcohol. Moreover, there was no significant correlation between the hepatic concentration of PFNA, metabolite A, or metabolite B and acyl-CoA oxidase activity. It seems likely, therefore, that the PFOA accumulated in the liver is responsible for peroxisome proliferation.
In conclusion, the present study showed (1) that 8–2 telomer alcohol is transformed to highly stable PFOA in vivo in the liver of mice, (2) that the repeated administration of 8–2 telomer alcohol caused the accumulation of PFOA in the liver, and (3) that the accumulated PFOA physiologically affects the liver by inducing peroxisome proliferation.
ACKNOWLEDGMENTS
This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture, Japan.
REFERENCES
Beier, K., and Fahimi, H. D. (1992). Application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver in conjunction with cytochemical staining with 3–3'-diaminobenzidine and immunocytochemistry. Microsc. Res. Tech. 21, 271–282.
Borges, T., Robertson, L. W., Peterson, R. E., and Glauert, H. P. (1992). Dose-related effects of perfluorodecanoic acid on growth, feed intake and hepatic peroxisomal -oxidation. Arch. Toxicol. 66, 18–22.
Dinglasan, M. J., Ye, Y., Edwards, E. A., and Mabury, S. (2004). Florotelomer alcohol biodegradation yields poly- and perfluorinated acid. Environ. Sci. Technol. 38, 2857–2864.
Hagen, D. F., Belisle, J., Johnson J. D., and Venkateswarlu, P. (1981). Characterization of fluorinated metabolites by a gas chromatographic-helium microwave plasma detector-The biotransformation of 1H,1H,2H,2H-perfluorodecanol to perfluorooctanoate. Anal. Biochem. 118, 336–343.
Hansen, K. J., Johnson, H. O., Eldridge, J. S., Butenhoff, J. L., and Dick, L. A. (2002). Quantitative Characterization of Trace Levels of PFOS and PFOA in the Tennessee River. Environ. Sci. Technol. 36, 1681–1685.
Hatfield, T. (2001). Screening studies on the aqueousphotolytic degradation of perfluorooctanoic acid (PFOA). 3M Environmental Laboratory. Lab request number E-00–2192. St. Paul, MN.
Hess, R., Staubli, W., and Riess, W. (1965). Nature of the hepatomegalic effect produced by ethyl-chlorophenoxy-isobutyrate in the rat. Nature 27, 856–858.
Intrasuksri, U., Rangwala, S. M., O' Brien, M., Noonan, D. J., and Feller, D. R. (1998). Mechanisms of peroxisome proliferation by perfluorooctanoic acid and endogenous fatty acids. Gen. Pharmacol. 31, 187–197.
Kannan, K., Newsted, J., Halbrook, R. S., and Giesy, J. P. (2002). Perfluorooctanesulfonate and related fluorinated hydrocarbons in mink and river otters from the United States. Environ. Sci. Technol. 36, 2566–2571.
Kawashima, Y., Kobayashi, H., Miura, H., and Kozuka, H. (1995). Characterization of hepatic responses of rat to administration of perfluorooctanoic and perfluorodecanoic acids at low levels. Toxicology 99, 169–178.
Kawashima, Y., Uy-Yu, N., and Kozuka, H. (1989). Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal -oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver. Biochem. J. 261, 595–600.
Key, B. D., Howell, R. D., and Criddle, C. S. (1997). Fluorinated Organics in the Biosphere. Environ. Sci. Technol. 31, 2445–2454.
Kudo, N., Bandai, N., Suzuki, E., Katakura, M., and Kawashima, Y. (2000). Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal beta-oxidation in the liver of rats. Chem. Biol. Interact. 124, 119–132.
Kudo, N., and Kawashima, Y. (2003). Toxicity and toxicokinetics of perfluorooctanoic acid in humans and animals. J. Toxicol. Sci. 28, 49–57.
Lalwani, N. D., Reddy, M. K., Qureshi, S. A., Sirtori, C. R., Abiko, Y., and Reddy, J. K. (1983). Evaluation of selected hypolipidemic agents for the induction of peroxisomal enzymes and peroxisome proliferation in the rat liver. Human Toxicol. 2, 27–48.
Lazarow, P. B. (1977). Three hypolipidemic drugs increase hepatic palmitoyl-Coenzyme A oxidation in the rat. Science 197, 580–581.
Lazarow, P. B., and De Duve, C. (1976). A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc. Natl. Acad. Sci. U.S.A. 73, 2043–2046.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Ohya, T., Kudo, N., Suzuki, E., and Kawashima, Y. (1998). Determination of perfluorinated carboxylic acids in biological samples by high-performance liquid chromatography. J. Chromatogr. B 720, 1–7.
Olsen, G. W., Hansen, K. J., Stevenson, L. A., Burris, J. M., and Mandel, J. H. (2003). Human donor liver and serum concentrations of perfluorooctanesulfonate and other perfluorochemicals. Environ. Sci. Technol. 37, 888–891.
Ophaug, R. H., and Singer, L. (1981). Metabolic handling of perfluooctanoic acid in rats. Proc. Soc. Exp. Biol. Med. 163, 19–23.
Permadi, H., Lundgren, B., Andersson, K., and DePierre, J. W. (1993). Effects of perfluoro fatty acids on peroxisome proliferation and mitochondrial size in mouse liver: Dose and time factors and effect of chain length. Xenobiotica 23, 761–770.
Reddy, J. K., Usuda, N., and Rao, S. (1988). Hepatic peroxisome proliferation: An overview. Arch. Toxicol. 12, 207–216.
Small, G. M., Burdett, K., and Connock, M. J. (1985). A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase. Biochem J. 227, 205–210.
Sohlenius, A.-K., Andersson, K., and Depierre, J. W. (1992a). The effects of perfluoro-octanoic acid on hepatic peroxisome proliferation and related parameters show no sex-related differences in mice. Biochem J. 285, 779–783.
Sohlenius, A.-K., Lundgren, B., and DEPierre, J. W. (1992b). Perfluorooctanoic acid has persistent effects on peroxisome proliferation and related parameters in mouse liver. J. Biochem. Toxicol. 7, 205–212.
Stock, N. L., Lau, F. K., Ellis, D. A., Martin, J. W., Muir, D. C. G., and Mabury, S. (2004). Polyfluorinated telomer alcohols and sulfonamides in north American troposphere. Environ. Sci. Technol. 38, 991–996.
Stott, W. T. (1988). Chemically induced proliferation of peroxisomes: Implications for risk assessment. Regul. Toxicol. Pharmacol. 8, 125–159.
Uy-Yu, N., Kawashima, Y., and Kozuka, H. (1990). Comparative studies on sex-related difference in biochemical responses of livers to perfluorooctanoic acid between rats and mice. Biochem. Pharmacol. 39, 1492–1495.
U.S. EPA (2002a) Biodegradation screen study for telomer-type alcohols. U.S. Environmental Protection Agency, Administrative Record AR226, OPPT2003-0012-0009.
U.S. EPA (2002b) Revised draft. Hazard assessment of perfluorooctanoic acid and its salts. U.S. Environmental Protection Agency, Administrative Record AR226, OPPT2003-0012-0011.
Vanden Heuvel, J. P., Kuslikis, B. I., Van Rafelghem, M. L., and Peterson, R. E. (1991). Tissue distribution, metabolism and elimination of perfluorooctanoic acid in male and female rats. J. Biochem. Toxicol. 6, 83–92.
Yang, Q., Xie, Y., Alexson, S. E. H., Nelson, B. D., and DePierre, J. W. (2002). Involvement of peroxisome-proliferator-activate receptor alpha in the immunomodulation caused by peroxisome proliferators in mice. Biochem. Pharmacol. 63, 1893–1900.
Yang, Q., Xie, Y., and DePierre, J. W. (2000). Further evidence for the involvement inhibition of cell proliferation and development in the thymic and splenic atrophy induced by the peroxisome proliferator perfluorooctanoic acid in mice. Biochem. Pharmacol. 62, 1133–1140.(Naomi Kudo, Yuko Iwase, H)
Research and Development Laboratories, Maruho Co., 1 Awatacho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan
ABSTRACT
The effects of dietary administration of 1H, 1H, 2H, 2H-perfluorodecanol (8–2 telomer alcohol), on peroxisome proliferation in the liver of mice were studied. Male ddY mice were fed on a diet containing 8–2 telomer alcohol at concentrations of 0, 0.025, 0.05, 0.1, and 0.2% (w/w) for 7, 14, 21, and 28 days. These treatments with 8–2 telomer alcohol caused liver enlargement in a dose- and duration-dependent manner. Peroxisome proliferation in the liver of mice was confirmed by electron microscopic examination. Peroxisomal acyl-CoA oxidase was induced by these treatments with 8–2 telomer alcohol in a dose- and time-dependent manner. The concentration of perfluorooctanoic acid (PFOA) and related compounds were determined in the liver and plasma, since PFOA had been shown to be a possible metabolite of 8–2 telomer alcohol and to cause significant peroxisome proliferation in rodents. Five metabolites, namely, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), 2H, 2H-perfluorodecanoic acid (8–2 telomer acid), and two unidentified metabolites, were present in the liver and serum. PFOA was confirmed to be accumulated in the liver of mice following the administration of 8–2 telomer alcohol in a dose- and duration-dependent manner. A linear relationship was observed between the concentration of PFOA and the activity of peroxisomal acyl-CoA oxidase in the liver of mice. These results strongly suggest that PFOA, but not 8–2 telomer alcohol itself, caused peroxisome proliferation in the liver. The present study provided evidence that 8–2 telomer alcohol is converted into PFOA in vivo and that the PFOA formed produces biological effects in the liver of mice.
Key Words: 8–2 telomer alcohol; perfluorooctanoic acid; peroxisome proliferation; liver; mouse.
INTRODUCTION
Organic fluorochemicals are compounds in which some or all the carbon–hydrogen bonds are replaced by a carbon–fluorine bonds. These carbon–fluorine bonds are some of the strongest in nature and contribute to the unique stability of these fluorochemicals (Hatfield, 2001; Key et al., 1997). Perfluorooctane sulfonate has been widely used in a variety of industrial and consumer applications in hair shampoos, paper coatings, waxes, polishes, and paints (Key et al., 1997). Recently, however, it has been pointed out that there is a risk that the chemical accumulates in the environment and humans (Hansen et al., 2002; Kannan et al., 2002; Olsen et al., 2003), so that some primary manufactures of this compound have discontinued its production. Great efforts have been made to find new chemicals that have properties similar to perfluorooctane sulfonate and are less biologically hazardous. 1H, 1H, 2H, 2H-Perfluorodecanol (8–2 telomer alcohol) is a polyfluorinated compound that has eight fully fluorinated carbons and two nonfluorinated carbons adjacent to a hydroxyl group. This 8–2 telomer alcohol has applications similar to those of perfluorooctane sulfonate-based products. During the period 2000–2002, the amount of telomer alcohols produced was estimated to be 5 x 106 kg/year worldwide (U. S. EPA, 2002a).
It has recently been demonstrated that 8–2 telomer alcohol is a potential source of perfluorooctanoic acid (PFOA) as a consequence of biotic degradation (Dinglasan et al., 2004; Stock et al., 2004). Little information, however, is available about the biotransformation of 8–2 telomer alcohol in mammals. An early study suggested the possibility that 8–2 telomer alcohol was converted into PFOA in rats (Hagen et al., 1981). If this is the case, there is a risk that PFOA accumulates in the environment and humans, because PFOA is so stable that it is not decomposed by activated sludge (U. S. EPA, 2002b) and is not metabolized in animals (Ophaug and Singer, 1981; Vanden Heuvel et al., 1991). Moreover, the PFOA formed in animals from 8–2 telomer alcohol may physiologically affect the animals, because PFOA has been demonstrated to cause peroxisome proliferation in the liver, body weight loss, a reduction in thymus and spleen weight, a reduction in the number of erythrocytes, and an elevation of glucose levels (Kawashima et al., 1995; Kudo et al., 2000, 2003; Yang et al., 2000, 2002). Information, however, is lacking about how much PFOA is formed from 8–2 telomer alcohol in the liver of animals when 8–2 telomer alcohol is repeatedly administered and about whether the concentration of PFOA formed in the liver is high enough to cause physiological effects.
The present study is aimed at investigating whether repeated administration of 8–2 telomer alcohol causes PFOA accumulation in the liver of mice, and whether the accumulated PFOA is enough high to induce peroxisome proliferation.
MATERIALS AND METHODS
Materials.
8–2 Telomer alcohol was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan); PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid, palmitoyl-CoA, and bovine serum albumin were from Sigma Aldrich Japan (Tokyo, Japan); NAD and CoA were from Oriental Yeast Co. (Tokyo, Japan); Triton X-100 was from Nakalai Tesque (Kyoto, Japan). 3-Bromoacetyl-7-methoxycoumarin was prepared as previously described (Ohya et al., 1998). All other chemicals were of analytical grade.
Chemical synthesis of 2H, 2H-perfluorodecanoic acid (8–2 telomer acid).
To a solution of 8–2 telomer alcohol (25 mg, 0.054 mmol) in glacial acetic acid (2.0 ml) was added excess chromium (VI) oxide. After stirring for 24 h, the reaction was stopped with water (2.0 ml), followed by solid sodium hydrogen sulfite (approximately 30 mg), then acidified with 3 M sulfuric acid (1.0 ml), and the mixture was then extracted with diethyl ether (5 ml). The layers were separated, and the organic layer was flushed with nitrogen gas to evaporate any diethyl ether. The residue was dissolved in 1 M sodium hydroxide (3.0 ml), and the solution was washed with diethyl ether (2 x 3.0 ml). The combined aqueous phase was acidified with 70% sulfuric acid (10 ml) and extracted with diethyl ether (2 x 20 ml). To the organic layer was added 2 M sulfuric acid (2 drops), and this solution was extracted with diethyl ether (20 ml). The extract was washed with water (2 x 10 ml), dried over sodium sulfate, and concentrated in vacuo. The residue was purified by column chromatography (silica gel), eluted with diethyl ether/hexane/acetic acid (3:8:0, v/v), and then with diethyl ether/hexane/acetic acid (3:8:5, v/v) to give 8–2 telomer acid (6.2 mg, 0.013 mmol) as a white power. MS (FAB): Exact mass calculated for 478; [C10F17H3O2]. Found m/z (%) 479 (18.67), 69 (100).
Animals.
Male ddY mice aged 7 weeks were purchased from SLC (Hamamatsu, Japan) and acclimatized in a humidity- and temperature- (23 ± 2°C) controlled environment with a 12-h light/dark cycle for at least 1 week before use. In one set of experiments, mice were fed a diet containing 8–2 telomer alcohol at concentrations of 0, 0.025, 0.05, 0.10, and 0.20% (w/w) for 7, 14, 21, and 28 days. The mice were killed under diethyl ether anesthesia. In another set of experiments, mice were given intraperitoneal injections of 8–2 telomer alcohol as a propylene glycol solution at a dose of 400 mg/kg, and the mice were killed at 2, 6, 24, and 72 h postdosage. Livers were excised, perfused with ice-cold 0.9% NaCl, frozen in liquid nitrogen, and stored at –80°C until use. The frozen liver was thawed at 0°C and homogenized with nine volumes of 0.25 M sucrose/1 mM EDTA/10 mM Tris–HCl (pH 7.4). The protein concentrations in the homogenates were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. All animal studies complied with institutional board for animal study, Josai University.
Electron microscopy.
At the end of the 8–2 telomer alcohol feeding, some of the mice were anesthetized with diethyl ether and perfused via the left ventricle with 0.9% (w/v) NaCl and subsequently with 1.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 5 min. The left lateral lobe of the liver was removed and cut into 1-mm slices which were fixed in 1.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C for 4 h. The prefixed blocks were sliced into 50-μm sections using a DTK-3000W microslicer (Dosaka EM, Osaka, Japan), and incubated in 5 mM 3,3'-diaminobenzidine in Teorell-Stenhagen buffer (pH 10.5) containing 0.15% (v/v) hydrogen peroxide for 15 min. After rinsing with cacodylate buffer, the sections were postfixed in buffered 1% osmium tetroxide for 1 h, dehydrated in acetone, and embedded in Epon-812. Thin sections were cut from each slice with a diamond knife on an MT-2B ultramicrotome (Dupont-Sorvall, MO), stained with uranyl acetate, and photographed using an H-7000 electron microscope (Hitachi, Katsuta, Japan) (Beier et al., 1992). Electron microscopy fields (original magnification of 4000x) were chosen for image analysis. Peroxisomes were identified as membrane-bound and electron-dense organelles. Ten electron micrographs for one mouse were analyzed by morphometric techniques with a Micro Computer Imaging Device (MCIDTM, Amersham Biosciences, Piscataway, NJ, USA) to give the peroxisomal area and the number of peroxisomes per unit cellular area apart from the nuclear area (30 μm2).
Assay of acyl-CoA oxidase.
Acyl-CoA oxidase was assayed spectrophotometrically by measuring palmitoyl-CoA-dependent H2O2 production at 502 nm (Small et al., 1985).
Determination of metabolites of 8–2 telomer alcohol.
The metabolites of 8–2 telomer alcohol in liver and serum samples were extracted, converted to acetylmethoxycoumarin derivatives, separated by high performance liquid chromatography (HPLC), and quantified by fluorescence detection as described previously (Ohya et al., 1998) with some modifications as follows. To an aliquot of liver homogenate or serum was added an appropriate amount of perfluorodecanoic acid as an internal standard for the analysis of the metabolites of 8–2 telomer alcohol, and then the metabolites were extracted with ethyl acetate:hexane (1:1, v/v) as an ion pair with tetrabutylammonium in the presence of NaCO3 buffer (pH 10.0). The metabolites in the extract were run on thin-layer chromatography (silica gel 5721, Merck, Germany) developed with hexane/diethyl ether/acetic acid (16:6:1, v/v). Authentic PFOA and 8–2 telomer acid were simultaneously run alongside the unknown mixture on thin-layer plates. The spots were detected by their water-repellent effect after spraying with water. The areas of silica gel corresponding to PFOA and other two metabolites (Rf value ranging from 0 to 0.06) and 8–2 telomer acid (Rf value ranging from 0.15 to 0.38) were individually scraped off, and these metabolites were extracted from the silica gel as an ion pair with tetrabutylammonium as described above. The extract was transferred to a glass tube and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 0.2% (w/v) 3-bromoacetyl-7-methoxycoumarin in acetone, and the mixture was heated at 70°C for 25 min followed by cooling on ice. The solution was filtered through a glass-wool filter, and the filtrate was subjected to HPLC using a reverse-phase column (YMC-Pack Pro, 4.6 ID x 50 mm), and eluted with acetonitrile/water (7:3, v/v). The peaks of acetylmethoxycoumarin derivatives of the metabolites and perfluorodecanoic acid (internal standard) were detected using fluorescence detector (Shimadzu RF-10AXL, Shimadzu Co., Kyoto, Japan) at an excitation wavelength of 366 nm and an emission wavelength of 420 nm.
Statistical analysis.
Homogeneity of variance was established using one-way analysis of variance. When a difference was significant (p < 0.05), Schéffe's multiple range test was used as a post-test. Statistical significance between two means was estimated by either Student's t-test or Welch's test after F-test. Linear regression analysis was performed to evaluate the correlation between two parameters.
RESULTS
Effects of 8–2 Telomer Alcohol Feeding on Body Weight Gain and Liver Changes in Mice
Male mice were fed a diet containing 8–2 telomer alcohol at concentrations ranging from 0 to 0.2% (w/w) for 7, 14, 21, or 28 days. The effects of the treatments on body weight gain, liver weight, and relative liver weight are shown in Table 1. There was no significant effect following the administration of 8–2 telomer alcohol on the overall health of the mice, except that a slight decrease in body weight gain was seen in some groups. The livers of mice treated with 8–2 telomer alcohol were significantly enlarged in a dose- and time-dependent manner. Owing to the changes in liver weight, the treated animals showed an increase in relative liver weight. The increase was dependent on both the dose and duration of treatment.
Table 1 also shows the effect of 8–2 telomer alcohol feeding on the activity of peroxisomal acyl-CoA oxidase, a peroxisomal enzyme, in the liver. The activity was markedly increased in a dose- and time-dependent manner, as was observed with the relative liver weight. The higher the concentration of 8–2 telomer alcohol in the diet, the shorter the time required to reach the maximum activity.
The morphological results obtained from mice that were treated with 8–2 telomer alcohol at a dietary concentration of 0.1% for 14 days are shown in Figures 1 and 2. In the hepatocytes of control mice, the peroxisomes, which were intensely stained spherical particles approximately 0.5 μm in diameter, were distributed randomly in the cytoplasm (Fig. 1A). The treatment of mice with 8–2 telomer alcohol increased the cell size and caused proliferation of peroxisomes (Fig. 1B). The number of peroxisomes was increased by treatment of mice with 8–2 telomer alcohol (130% of control), and the volume density of peroxisomes, given as a percentage of the cytoplasmic area, also increased (312% of control) (Fig. 2).
Biotransformation of 8–2 Telomer Alcohol in the Liver of Mice
Mice were given intraperitoneal injections of 8–2 telomer alcohol at a dose of 400 mg/kg. The metabolites formed were extracted with solvent as an ion pair with tetrabutylammonium from the liver homogenates obtained from mice 6 h postdosage. The metabolites extracted were separated by thin-layer chromatography into a fast-migrating spot (Rf value of 0.15–0.38) and a slow-migrating spot (Rf value of 0–0.06). The metabolites contained in the two spots were individually extracted and run on HPLC after derivatization with 3-bromoacetyl-7-methoxycoumarin. Three metabolites were detected from the slow-migrating spot, and one of them was identified as PFOA by retention-time matching and also spiking with the corresponding authentic reference sample (Fig. 3A). All are thought to be carboxylic acids, because 3-bromoacetyl-7-methoxycoumarin selectively reacts with carboxylic acid. The other two metabolites (A and B) have not been identified yet (Fig. 3A). One metabolite was detected from the fast-migrating spot and identified as 8–2 telomer acid. The retention time of this compound was the same as that of PFOA (Fig. 3B). The metabolites detected in the serum obtained from mice 6 h postdosage were the same as those found in the liver.
Figure 4 illustrates the time courses of the formation of the metabolites from 8–2 telomer alcohol in the liver and serum. In the liver, the concentration of metabolite A immediately increased after injection, reached a maximum (29 nmol/g liver) at 6 h, and then gradually declined (Fig. 4A). The concentration of metabolite A fell to below the detection limit at 72 h postdosage. The concentration of PFOA in the liver gradually increased up to 52 nmol/g liver 72 h postdosage. Metabolite B was detected at 2 and 6 h, although its concentrations were very low compared with those of metabolite A and PFOA. At 24 and 72 h postdosage, the concentration of metabolite B fell below the limit of quantification. In the serum, the concentration of PFOA gradually increased as observed with the liver (Fig. 4B). However, unlike the liver, the concentration of metabolite A was low throughout the entire time period. No metabolite A was detected in serum at 72 h. The concentrations of metabolite B in serum were below the limit of quantification limit (0.2 nmol/ml serum). No 8–2 telomer acid was detected at 2, 24, and 72 h postdosage in either the liver or serum, although its concentration was 0.79 nmol/g liver and 0.74 nmol/ml serum at 6 h (data not shown in the figures). Linear regression analysis was performed to examine the relationship between the concentrations of PFOA in the liver and serum. One regression line was obtained for five sets of mean data from Figures 4A and 4B, with a high correlation being seen between the two parameters (Y = 0.8967X + 1.1465; r2 = 0.9791; p < 0.05).
Accumulation of PFOA in the Liver of Mice
Table 2 shows accumulation of PFOA in the liver when mice were fed the diet containing 8–2 telomer alcohol. The content of PFOA in the liver of control mice was below the detection limit of 0.2 nmol/g liver. When compared within the groups of mice treated with 8–2 telomer alcohol for the same duration, the accumulation of PFOA in the liver tended to increase in a dose-dependent manner. When compared within the groups treated with 8–2 telomer alcohol at the same doses, PFOA in the liver tended to accumulate in a manner that depended on the duration of treatment. The maximum accumulation was approximately 122 nmol/g liver, which was seen in the mice fed on the diet containing 0.2% 8–2 telomer alcohol for 28 days.
The content of PFOA as an impurity in 8–2 telomer alcohol, which was used in the present experiment, was measured by HPLC and found to be less than 8.67 x 10–5% (w/w), and neither PFNA, nor metabolites A and B were found in 8–2 telomer alcohol. Assuming that the daily food intake of a mouse is 6 g, the PFOA intake is at most 0.0224 nmol/day/animal. These values are far less than the amounts of PFOA found in the liver of mice that were treated with 8–2 telomer alcohol.
Relationship between PFOA Concentration and Peroxisomal Acyl-CoA Oxidase Activity in the Liver of Mice
To examine the relationship between the concentration of PFOA and the activity of acyl-CoA oxidase in the liver of mice (Fig. 5), a linear regression analysis was carried out. One linear regression line was obtained for 21 sets of mean data between the hepatic concentration of PFOA and the acyl-CoA oxidase activity from Tables 1 and 2, with a high correlation being seen between the two parameters (r2 = 0.8609, p < 0.01). To examine the relationships between the PFNA concentration and acyl-CoA oxidase activity, between the metabolite A concentration and acyl-CoA oxidase activity, and between the metabolite B concentration and acyl-CoA oxidase activity in the liver, linear regression analyses were carried out for 21 sets of mean data from Tables 1 and 2. A linear regression line was not obtained either between the PFNA concentration and acyl-CoA oxidase activity (Y = 2.1597X + 23.854, r2 = 0.4271, p > 0.05), between the metabolite A concentration and acyl-CoA oxidase activity (Y = 0.163X + 37.562, r2 = 0.0561, p > 0.05), or between the metabolite B concentration and acyl-CoA oxidase activity (Y = 0.022X + 1.311, r2 = 0.0003, p > 0.05).
DISCUSSION
Induction of Peroxisome Proliferation
The present study showed that the treatment of mice with 8–2 telomer alcohol caused enlargement of livers in a dose- and time-dependent manner. Since peroxisome proliferation induced by peroxisome proliferators is known to be always accompanied by hepatomegaly in rodents (Hess et al., 1965; Lalwani et al., 1983; Reddy et al., 1988; Stott, 1988), we investigated whether 8–2 telomer alcohol feeding induces peroxisomal acyl-CoA oxidase, a parameter of peroxisome proliferation (Lalwani et al., 1983; Lazarow, 1977; Lazarow and De Duve, 1976; Reddy et al., 1988; Stott, 1988). As had been supposed, peroxisomal acyl-CoA oxidase in the liver was markedly induced by 8–2 telomer alcohol in a dose- and time-dependent manner. This result strongly suggests the possibility of peroxisome proliferation in the liver of mice given 8–2 telomer alcohol. This was confirmed by electron microscopic examination, as the increase in both the number and the size of peroxisomes. However, unlike 8–2 telomer alcohol, 1H, 1H-perfluorooctanol that is a form of fluorotelomer alcohol is reported to have no effect on peroxisome proliferation in primary cultures of rat hepatocytes (Intrasuksri et al., 1998). These facts raised a question of whether 8–2 telomer alcohol itself or the metabolites formed from 8–2 telomer alcohol cause peroxisome proliferation.
Biotransformation of 8–2 Telomer Alcohol
Hagen et al. (1981) suggested that 8–2 telomer alcohol was metabolized in rats and showed that three metabolites, PFOA, 8–2 telomer acid, and an unidentified metabolite, were present in serum. The present study confirmed the formation of PFOA from 8–2 telomer alcohol in the liver of mice. In addition to PFOA, the existence of 8–2 telomer acid and two other unidentified metabolites (A and B) was demonstrated in the liver. 8–2 Telomer acid was confirmed in the liver of the mice only at 6 h postdosage of 8–2 telomer alcohol. When mice were injected with a large dose of 8–2 telomer alcohol, the concentration of metabolite A transiently increased and then declined, with the maximum level being reached 6 h postdosage. By contrast, the concentration of PFOA gradually increased throughout the time period. These results strongly suggest that metabolite A is a probable precursor that may undergo transformation leading to the formation of PFOA. In addition, 8–2 telomer acid was detected briefly 6 h postdosage. In the light of these findings, it seems likely that 8–2 telomer alcohol is transformed to 8–2 telomer acid, which is converted to metabolite A and then the highly stable PFOA. Based on the present results and on the earlier findings of Hagen et al. (1981), Figure 6 illustrates a proposed degradation scheme of 8–2 teloemer alcohol in the liver of mice. The present study demonstrated that the serum concentrations of PFOA were comparable to its hepatic concentrations and that there was a significant correlation between the PFOA concentrations in the serum and liver. Moreover, 8–2 telomer alcohol given intraperitoneally is considered to be transferred to the liver via the portal vein. These facts suggest, therefore, that 8–2 telomer alcohol is metabolized in the liver to PFOA, which may then be passed into the general circulation.
The present study demonstrated that the mice, which were repeatedly treated with 8–2 telomer alcohol, accumulated PFOA in the liver and that the amounts of PFOA present in the liver were much greater than those calculated on the assumption that all the PFOA, which is present in the 8–2 telomer alcohol used in the present study as an impurity, had been absorbed and accumulated in the liver of the animals. The PFOA found in the mouse liver is, therefore, considered to be formed from 8–2 telomer alcohol in mice. The formation of PFOA was confirmed to be dependent on the dose of the 8–2 telomer alcohol and saturable, and also to be dependent on the duration of treatment. However, PFOA accumulated in extremely small amounts, despite the fact that the mice were treated with 8–2 telomer alcohol at very high doses. The accumulation of PFNA and the unidentified metabolite A was not as great as that of PFOA. The unidentified metabolite B, which was found in very low concentrations during the time-course experiment, did not accumulate when 8–2 telomer alcohol was dosed repeatedly.
Induction of Peroxisome Proliferation by PFOA
Previous studies have reported that PFOA is a potent peroxisome proliferator in the liver of rodents (Borges et al., 1992; Kawashima et al., 1989, 1995; Kudo et al., 2000; Permadi et al., 1993; Sohlenius et al., 1992a,b; Uy-Yu, 1990). In the mice that were fed the diets containing 0.001% (w/w) and 0.002% (w/w) PFOA for 4 weeks, hepatic peroxisomal ayl-CoA oxidase (AOX) activities were 40.7 ± 14.2 and 51.7 ± 14.4 nmol/min/mg protein, respectively (Kudo et al., unpublished data). The induction of AOX by 8–2 telomer alcohol feeding in the present study, therefore, is almost comparable with that by PFOA although the doses required for maximum induction were different. In the present study, we chose four doses that cause the maximum induction of AOX at 28 days of the administration of 8–2 telomer alcohol and that cause dose-dependent induction at 7 days. Time-course study up to 28 days revealed time-dependent induction of AOX activity at lower doses. It should be emphasized that a linear relationship was confirmed between the concentration of PFOA and the activity of peroxisomal acyl-CoA oxidase in the liver of mice treated with 8–2 telomer alcohol. Moreover, there was no significant correlation between the hepatic concentration of PFNA, metabolite A, or metabolite B and acyl-CoA oxidase activity. It seems likely, therefore, that the PFOA accumulated in the liver is responsible for peroxisome proliferation.
In conclusion, the present study showed (1) that 8–2 telomer alcohol is transformed to highly stable PFOA in vivo in the liver of mice, (2) that the repeated administration of 8–2 telomer alcohol caused the accumulation of PFOA in the liver, and (3) that the accumulated PFOA physiologically affects the liver by inducing peroxisome proliferation.
ACKNOWLEDGMENTS
This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture, Japan.
REFERENCES
Beier, K., and Fahimi, H. D. (1992). Application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver in conjunction with cytochemical staining with 3–3'-diaminobenzidine and immunocytochemistry. Microsc. Res. Tech. 21, 271–282.
Borges, T., Robertson, L. W., Peterson, R. E., and Glauert, H. P. (1992). Dose-related effects of perfluorodecanoic acid on growth, feed intake and hepatic peroxisomal -oxidation. Arch. Toxicol. 66, 18–22.
Dinglasan, M. J., Ye, Y., Edwards, E. A., and Mabury, S. (2004). Florotelomer alcohol biodegradation yields poly- and perfluorinated acid. Environ. Sci. Technol. 38, 2857–2864.
Hagen, D. F., Belisle, J., Johnson J. D., and Venkateswarlu, P. (1981). Characterization of fluorinated metabolites by a gas chromatographic-helium microwave plasma detector-The biotransformation of 1H,1H,2H,2H-perfluorodecanol to perfluorooctanoate. Anal. Biochem. 118, 336–343.
Hansen, K. J., Johnson, H. O., Eldridge, J. S., Butenhoff, J. L., and Dick, L. A. (2002). Quantitative Characterization of Trace Levels of PFOS and PFOA in the Tennessee River. Environ. Sci. Technol. 36, 1681–1685.
Hatfield, T. (2001). Screening studies on the aqueousphotolytic degradation of perfluorooctanoic acid (PFOA). 3M Environmental Laboratory. Lab request number E-00–2192. St. Paul, MN.
Hess, R., Staubli, W., and Riess, W. (1965). Nature of the hepatomegalic effect produced by ethyl-chlorophenoxy-isobutyrate in the rat. Nature 27, 856–858.
Intrasuksri, U., Rangwala, S. M., O' Brien, M., Noonan, D. J., and Feller, D. R. (1998). Mechanisms of peroxisome proliferation by perfluorooctanoic acid and endogenous fatty acids. Gen. Pharmacol. 31, 187–197.
Kannan, K., Newsted, J., Halbrook, R. S., and Giesy, J. P. (2002). Perfluorooctanesulfonate and related fluorinated hydrocarbons in mink and river otters from the United States. Environ. Sci. Technol. 36, 2566–2571.
Kawashima, Y., Kobayashi, H., Miura, H., and Kozuka, H. (1995). Characterization of hepatic responses of rat to administration of perfluorooctanoic and perfluorodecanoic acids at low levels. Toxicology 99, 169–178.
Kawashima, Y., Uy-Yu, N., and Kozuka, H. (1989). Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal -oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver. Biochem. J. 261, 595–600.
Key, B. D., Howell, R. D., and Criddle, C. S. (1997). Fluorinated Organics in the Biosphere. Environ. Sci. Technol. 31, 2445–2454.
Kudo, N., Bandai, N., Suzuki, E., Katakura, M., and Kawashima, Y. (2000). Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal beta-oxidation in the liver of rats. Chem. Biol. Interact. 124, 119–132.
Kudo, N., and Kawashima, Y. (2003). Toxicity and toxicokinetics of perfluorooctanoic acid in humans and animals. J. Toxicol. Sci. 28, 49–57.
Lalwani, N. D., Reddy, M. K., Qureshi, S. A., Sirtori, C. R., Abiko, Y., and Reddy, J. K. (1983). Evaluation of selected hypolipidemic agents for the induction of peroxisomal enzymes and peroxisome proliferation in the rat liver. Human Toxicol. 2, 27–48.
Lazarow, P. B. (1977). Three hypolipidemic drugs increase hepatic palmitoyl-Coenzyme A oxidation in the rat. Science 197, 580–581.
Lazarow, P. B., and De Duve, C. (1976). A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc. Natl. Acad. Sci. U.S.A. 73, 2043–2046.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Ohya, T., Kudo, N., Suzuki, E., and Kawashima, Y. (1998). Determination of perfluorinated carboxylic acids in biological samples by high-performance liquid chromatography. J. Chromatogr. B 720, 1–7.
Olsen, G. W., Hansen, K. J., Stevenson, L. A., Burris, J. M., and Mandel, J. H. (2003). Human donor liver and serum concentrations of perfluorooctanesulfonate and other perfluorochemicals. Environ. Sci. Technol. 37, 888–891.
Ophaug, R. H., and Singer, L. (1981). Metabolic handling of perfluooctanoic acid in rats. Proc. Soc. Exp. Biol. Med. 163, 19–23.
Permadi, H., Lundgren, B., Andersson, K., and DePierre, J. W. (1993). Effects of perfluoro fatty acids on peroxisome proliferation and mitochondrial size in mouse liver: Dose and time factors and effect of chain length. Xenobiotica 23, 761–770.
Reddy, J. K., Usuda, N., and Rao, S. (1988). Hepatic peroxisome proliferation: An overview. Arch. Toxicol. 12, 207–216.
Small, G. M., Burdett, K., and Connock, M. J. (1985). A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase. Biochem J. 227, 205–210.
Sohlenius, A.-K., Andersson, K., and Depierre, J. W. (1992a). The effects of perfluoro-octanoic acid on hepatic peroxisome proliferation and related parameters show no sex-related differences in mice. Biochem J. 285, 779–783.
Sohlenius, A.-K., Lundgren, B., and DEPierre, J. W. (1992b). Perfluorooctanoic acid has persistent effects on peroxisome proliferation and related parameters in mouse liver. J. Biochem. Toxicol. 7, 205–212.
Stock, N. L., Lau, F. K., Ellis, D. A., Martin, J. W., Muir, D. C. G., and Mabury, S. (2004). Polyfluorinated telomer alcohols and sulfonamides in north American troposphere. Environ. Sci. Technol. 38, 991–996.
Stott, W. T. (1988). Chemically induced proliferation of peroxisomes: Implications for risk assessment. Regul. Toxicol. Pharmacol. 8, 125–159.
Uy-Yu, N., Kawashima, Y., and Kozuka, H. (1990). Comparative studies on sex-related difference in biochemical responses of livers to perfluorooctanoic acid between rats and mice. Biochem. Pharmacol. 39, 1492–1495.
U.S. EPA (2002a) Biodegradation screen study for telomer-type alcohols. U.S. Environmental Protection Agency, Administrative Record AR226, OPPT2003-0012-0009.
U.S. EPA (2002b) Revised draft. Hazard assessment of perfluorooctanoic acid and its salts. U.S. Environmental Protection Agency, Administrative Record AR226, OPPT2003-0012-0011.
Vanden Heuvel, J. P., Kuslikis, B. I., Van Rafelghem, M. L., and Peterson, R. E. (1991). Tissue distribution, metabolism and elimination of perfluorooctanoic acid in male and female rats. J. Biochem. Toxicol. 6, 83–92.
Yang, Q., Xie, Y., Alexson, S. E. H., Nelson, B. D., and DePierre, J. W. (2002). Involvement of peroxisome-proliferator-activate receptor alpha in the immunomodulation caused by peroxisome proliferators in mice. Biochem. Pharmacol. 63, 1893–1900.
Yang, Q., Xie, Y., and DePierre, J. W. (2000). Further evidence for the involvement inhibition of cell proliferation and development in the thymic and splenic atrophy induced by the peroxisome proliferator perfluorooctanoic acid in mice. Biochem. Pharmacol. 62, 1133–1140.(Naomi Kudo, Yuko Iwase, H)