* Faculty of Pharmaceutical Sciences, Josai University, Keyakidai 11, Sakado, Saitama 350-0295, Japan, and Research and Development Laboratories, Maruho Co., 1 Awatacho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan
1 To whom correspondence should be addressed at Keyakidai 11, Sakado, Saitama 350-0295, Japan. Fax: +81 (49) 271-7984. E-mail: naokudo{at}josai.ac.jp.
Received January 9, 2005; accepted May 2, 2005
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ABSTRACT |
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Key Words: 82 telomer alcohol; perfluorooctanoic acid; peroxisome proliferation; liver; mouse.
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INTRODUCTION |
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It has recently been demonstrated that 82 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 82 telomer alcohol in mammals. An early study suggested the possibility that 82 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 82 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 82 telomer alcohol in the liver of animals when 82 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 82 telomer alcohol causes PFOA accumulation in the liver of mice, and whether the accumulated PFOA is enough high to induce peroxisome proliferation.
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MATERIALS AND METHODS |
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Chemical synthesis of 2H, 2H-perfluorodecanoic acid (82 telomer acid).
To a solution of 82 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 82 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 82 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 82 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 TrisHCl (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 82 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 82 telomer alcohol.
The metabolites of 82 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 82 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 82 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 82 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.
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RESULTS |
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The morphological results obtained from mice that were treated with 82 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 82 telomer alcohol increased the cell size and caused proliferation of peroxisomes (Fig. 1B). The number of peroxisomes was increased by treatment of mice with 82 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).
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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).
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DISCUSSION |
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Biotransformation of 82 Telomer Alcohol
Hagen et al. (1981) suggested that 82 telomer alcohol was metabolized in rats and showed that three metabolites, PFOA, 82 telomer acid, and an unidentified metabolite, were present in serum. The present study confirmed the formation of PFOA from 82 telomer alcohol in the liver of mice. In addition to PFOA, the existence of 82 telomer acid and two other unidentified metabolites (A and B) was demonstrated in the liver. 82 Telomer acid was confirmed in the liver of the mice only at 6 h postdosage of 82 telomer alcohol. When mice were injected with a large dose of 82 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, 82 telomer acid was detected briefly 6 h postdosage. In the light of these findings, it seems likely that 82 telomer alcohol is transformed to 82 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 82 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, 82 telomer alcohol given intraperitoneally is considered to be transferred to the liver via the portal vein. These facts suggest, therefore, that 82 telomer alcohol is metabolized in the liver to PFOA, which may then be passed into the general circulation.
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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 82 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 82 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 82 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 82 telomer alcohol is transformed to highly stable PFOA in vivo in the liver of mice, (2) that the repeated administration of 82 telomer alcohol caused the accumulation of PFOA in the liver, and (3) that the accumulated PFOA physiologically affects the liver by inducing peroxisome proliferation.
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ACKNOWLEDGMENTS |
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