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Peroxisomal effects of phthalate esters in primary cultures of rat hepatocytes
Toxicology, 28 (1983) 167--179 Elsevier Scientific Publishers Ireland Ltd.
P E R O X I S O M A L EFFECTS OF P H T H A L A T E C U L T U R E S OF R A T HEPATOCYTES
ESTERS IN P R I M A R Y
TIM J.B. GRAY, BRIAN G. LAKE, JENNY A. BEAMAND, JOHN R. FOSTER and SHARAT D. GANGOLLI The British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey, SM5 4DS (United Kingdom) (Received November 9th, 1982} (Accepted February 21st, 1983)
SUMMARY
Primary rat hepatocyte cultures were used to compare the effects of some aikylphthalate esters on peroxisomal enzyme activities and morphology. Linear diesters from methyl to n-octyl and their constituent monoesters and alcohols were compared with the branched chain 2-ethylhexyl derivatives. Carnitine acetyltransferase activity was increased 9.5-fold by mono-2-ethylhexylphthalate (MEHP) and 5.5- and 7-fold b y mono-n-pentyland mono-n-octylphthalate (MNOP), the 2 most potent of the linear monoesters. Activity of the specific peroxisomal marker, cyanide-insensitive palmitoyl-CoA oxidation decreased with time in control cultures. Whereas MEHP produced a time related increase over the initial level of palmitoylCoA oxidation, MNOP treatment only maintained the initial level of activity. The peroxisome proliferation-associated 80 000 mol. wt polypeptide was induced by MEHP but not MNOP. Similarly, MEHP produced increased numbers of peroxisomes, many without a nucleoid, whereas MNOP and mono-n-pentylphthalate had no such effect. 2-Ethylhexanol was less p o t e n t as an inducer of carnitine acetyltransferase than MEHP and the linear alcohols had no effect on this enzyme. Studies with diesters indicated that induction of carnitine acetyltransferase required hydrolysis of the diester. It is concluded that the straight-chain phthalates studied have little effect on hepatic peroxisomes compared with the 2-ethylhexyl ester and that hepatocyte cultures provide a rapid means of comparing the peroxisomal effects of different phthalates. Address correspondence to: Dr. T.J.B. Gray, BIBRA, Woodmansterne Road, Carshalton, Surrey, SM5 4DS, U.K. Abbreviations: DEHP, di-(2-ethylhexyl)phthalate; DMSO, dimethylsulphoxide; LDH, lactate dehydrogenase; MEHP, mono-2-ethylhexylphthalate; MNOP, mono-n-octylphthalate.
Printed and Published in Ireland
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Key words: Rat hepatocyte primary cultures; Phthalate esters; Hepatic peroxisome proliferation in vitro INTRODUCTION Di-(2-ethylhexyl)phthalate (DEHP) is used extensively as a plasticizer for PVC as well as in a variety of other industrial applications [1,2]. Shortterm administration of DEHP to rats causes liver enlargement and a marked proliferation of hepatic peroxisomes [3,4], while chronic administration at high levels in the diet has been associated with an increased incidence of hepatocellular carcinoma [5]. Recently a number of other, structurally diverse chemicals that cause hepatic peroxisome proliferation have also been reported to induce liver tumours in rats [ 6 - 8 ] and this has led to the proposal [7] that potent hepatic peroxisome proliferators, as a class, are carcinogenic. Although the peroxisomal effects of DEHP and its principal metabolite, mono-2-ethylhexylphthalate (MEHP) have been studied in some detail [3,9,10,11], little is known of the effects of other phthalate esters. We have recently shown [12] that primary cultures of rat hepatocytes provide a rapid and sensitive means of investigating chemical effects on peroxisomal structure and function. The purpose of the studies described here was to use hepatocyte cultures to examine the peroxisomal effects of a series of linear alkyl phthalates from methyl to n-octyl in comparison with the effects of the 2-ethylhexyl ester. Peroxisomal response was assessed by the measurement of biochemical markers, including cyanide-insensitive palmitoyl-CoA oxidation and carnitine acetyltransferase, and by electron microscopy. Since orally-administered phthalate diesters undergo intestinal metabolism to the corresponding monoesters and alcohols [ 1 3 - t 5 ] these products were also studied. METHODS
~aterials RPMI 1640 medium and Nunc tissue culture dishes were purchased from Gibco (Europe), Paisley, Scotland. Gentamicin was obtained from Flow Laboratories, Irvine, Scotland, foetal calf serum from Sera Labs, Crawley, Sussex, England and collagenase from Boehringer Corporation, Lewes, Sussex, England. Dimethyl-, diethyl-, di-n-butyl and di-(2-ethylhexyl)phthalate were kindly donated by BP Chemicals Limited, Penarth, South Glamorgan (courtesy of Dr D.M. Pugh). Di-n-pentylphthalate was purchased from Eastman-Kodak Company, Rochester, New York. Di-n-propyl-, di-nhexyl-, di-n-heptyl- and di-n-octylphthalate and the phthalate monoesters were synthesised by the method of Albro et al. [16]. Their purity was established by GLC or TLC and by nuclear magnetic resonance spectroscopy and elemental analysis. Clofibrate and all other biochemicals were obtained from Sigma Chemical Company, Poole, Dorset, England.
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Hepatocyte isolation and treatment o f cultures H e p a t o c y t e s were isolated from male S p r a g u e - D a w l e y rats (180--220 g) using the collagenase perfusion technique described by Paine et al. [17]. After washing three times by centrifugation at 50 g a v the cells were resuspended in culture medium (RPMI 1640 containing 5% foetal calf serum, 50 pg/ml gentamicin, 10 -6 M insulin and 10 -4 M hydrocortisone-21-sodium succinate). Viability of the freshly isolated cells, determined by trypan blue exclusion, was 84 + 5% (Mean + S.D. for 13 peffusions). Hepatocytes were seeded at 2.5 × 106 viable cells/3 ml culture medium in 50-mm petri dishes and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. After 18--21 h, treatment was c o m m e n c e d by replacing the culture medium with medium containing the required concentration of test c o m p o u n d . Subsequently the medium was changed and the cells redosed every 24 h. For each treatment or time point, 3--4 culture dishes were used. Test compounds were dissolved in dimethylsulphoxide (DMSO) and added to the culture medium so that the final concentration of DMSO was 0.4% in all cases, including the control cultures. Cytotoxicity was assessed by the measurement of lactate dehydrogenase (LDH) released into the culture medium [18]. Biochemical investigations At the end of the treatment period, h e p a t o c y t e whole homogenates were prepared and assayed for cyanide-insensitive palmitoyl-CoA oxidation, carnitine acetyltransferase, carnitine palmitoyltransferase and protein as described previously [ 1 2 ] . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of h e p a t o c y t e whole homogenate proteins was performed as described by Dent et al. [19] employing a 13 cm long 7.5% (w/v) acrylamide resolving gel. Gels were stained with Coomassie brilliant blue R. Electron microscopy Cultures treated with MEHP, mono-n-octyl- or mono-n-pentylphthalate, together with control cultures, were processed for transmission electron microscopy as described previously [12]. Statistical analysis Data were analysed using Dunnett's test for multiple comparisons [20]. RESULTS Primary cultures of rat hepatocytes were treated for 48 h with a series of mono-n-alkyl phthalates ranging from methyl to n-octyl and the effects on peroxisomal enzyme activities were compared with the effects of MEHP (Fig. 1). The monoester concentration used (0.2 mM), corresponded to the maximum stimulatory concentration for MEHP [12] and was n o t c y t o t o x i c as judged by the measurement of lactate dehydrogenase activity released into the culture medium {data n o t shown).
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Fig. 1. C o m p a r a t i v e effects o f m o n o - n - a l k y l p h t h a l a t e s and M E H P o n activities o f carnitine acetyltransferase (= *), cyanide-insensitive palmitoyl-CoA oxidation (= A)
and carnitine palmitoyltransferase (~ ~) in primary hepatocyte cultures. Values, expressed as percent of control, are from a typical experiment and are means ± S.E.M. for 3--4 culture dishes after a 48-h treatment period. Monoester concentration was 0.2 mM in each case. Control activities for carnitine acetyltransferase, palmitoyl-CoA oxidation and carnitine palmitoyltransferase were 1.68 ± 0.44, 0.71 ± 0.04 and 3.68 ± 0.71 nmol/min/mg homogenate protein, respectively.
The activity of carnitine acetyltransferase, a mixed peroxisomal/mitochondrial marker, increased with increasing alkyl chain length from methyl (92% of control) to n-butyl (273%). The effects of mono-n-pentylphthalate were more marked (545%) whereas the n-hexyl and n-heptyl monoesters produced increases of only 238% and 342% of control respectively. Mono-noctylphthalate (MNOP) was the most potent of the straight chain monoesters (660%) but had less effect than its isomer MEHP (960%). There was a similar pattern of response for the specific peroxisomal marker, cyanideinsensitive palmitoyl-CoA oxidation, although the increases were less than those for carnitine acetyltransferase activity (Fig. 1). For most of the esters, increases in the mitochondrial enzyme, carnitine palmitoyltransferase, were smaller than those for palmitoyl-CoA oxidation but again, MEHP and MNOP caused the largest changes {283% and 234% of control, respectively). The results shown in Fig. 1 are from a typical experiment. Results of replicate 170
TABLE I E F F E C T S O F P H T H A L A T E D I E S T E R S A N D C O N S T I T U E N T A L C O H O L S ON CARN I T I N E A C E T Y L T R A N S F E R A S E A C T I V I T Y IN C U L T U R E D R A T H E P A T O C Y T E S Alkyl group
Control Methyl Ethyl n-Propyl n-Butyl n-Pentyl n-Hexyl n-Heptyl n-Octyl 2-Ethylhexyl
Carnitine a c e t y l t r a n s f e r a s e ( n m o l / m i n / m g p r o t e i n ) a Diester (0.2 m M )
% Control
Alcohol (1 raM)
% Control
1.76 2.71 2.85 4.35 5.45 7.33 3.24 2.04 3.55 4.56
(100) 154 162 b 247 c 310 c 416 c 184 c 116 202 c 259 c
1.51 1.63 1.96 1.20 1.47 2.29 1.67 1.25 1.75 8.80
(100) 108 130 79 97 152 111 83 116 583 c
± 0.13 ± 0.07 ± 0.34 ± 0.18 ± 0.47 ± 0.18 -* 0.20 ± 0.27 ± 0.25 ± 0.06
± ± ± ± ± ± ± ± ± ±
0.31 0.22 0.28 0.10 0.12 0.07 0.24 0.07 0.50 0.51
aValues are m e a n s ± S.E.M. f o r 3--4 c u l t u r e dishes per t r e a t m e n t . T r e a t m e n t t i m e was 48 h. b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l , P < 0.05. cSignificantly d i f f e r e n t f r o m c o n t r o l , P < 0.01.
T A B L E II E F F E C T S O F C O M B I N A T I O N S O F M E H P A N D 2 - E T H Y L H E X A N O L (2EH), A N D M O N O - n - B U T Y L P H T H A L A T E (MBP) A N D n - B U T A N O L ON P E R O X I S O M A L E N Z Y M E A C T I V I T Y IN C U L T U R E D R A T H E P A T O C Y T E S
Control 0.2 m M M E H P 0.2 m M 2EH 0.2 m M M E H P + 0.2mM2EH 0.2 m M MBP 0.2 m M n - b u t a n o l 0.2 m M MBP + 0.2 m M n - b u t a n o l
Cyanideinsensitive palmitoyl-CoA oxidationa
Control (%)
Carnitine acetyltransferase a
Control (%)
0.45 ± 0.03 2.13 ± 0.22 0.46 ± 0.10
(100) 473 b 102
1.24 ± 0.18 19.3 ± 0.4 1.87 ± 0.03
(100) 1556 b 151
2.50+- 0.05 0.99 ± 0.03 0.48 ± 0.08
556 b 220 c 107
22.0 ± 0.9 6.16 ± 0.86 1.13 ± 0.33
1774 b 497 b 91
0.84 ± 0.05
187
7.36 ± 0.17
594 b
a E x p r e s s e d as n m o l / m i n / m g p r o t e i n . Values are m e a n s ± S.E.M. for 3--4 culture dishes per t r e a t m e n t . T r e a t m e n t t i m e was 48 h. b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l , P < 0.01. cSignificantly d i f f e r e n t f r o m c o n t r o l , P < 0.05.
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experiments consistently showed the same pattern of changes although the extent of these changes varied somewhat between different cell preparations. This point is illustrated by comparing the effects of mono-n-butylphthalate on carnitine acetyltransferase activity in Fig. 1 and Table II. In contrast to the monoesters, the corresponding straight chain alcohols produced little or no increase in carnitine acetyltransferase activity, even at the higher concentration of 1 mM (Table I). However, 1 mM 2-ethylhexanol caused a 6-fold increase in carnitine acetyltransferase. Table I also shows the results of studie.s with phthalate diesters at a final concentration in the
i ~ iiii !iii;
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m e d i u m o f 0.2 mM. H i g h e r c o n c e n t r a t i o n s were i n c o m p l e t e l y soluble. Diesters f r o m m e t h y l to n - p e n t y l p r o d u c e d a chain l e n g t h - d e p e n d e n t increase in c a r n i t i n e a c e t y l t r a n s f e r a s e activity as o b s e r v e d w i t h the c o r r e s p o n d ing m o n o e s t e r s b u t the e f f e c t s o f D E H P a n d d i - n - o c t y l p h t h a l a t e were m u c h less m a r k e d .
Fig. 2. Electron micrographs of cultured rat hepatocytes (X1950) (A) control. (B) MEHPtreated (0.2 mM for 48 h) showing increased numbers of peroxisomes (arrowed). (C) MNOP and (D) mono-n-pentylphthalate-treated (0.2 mM for 48 h) showing no differences from the control.
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Since intestinal hydrolysis of phthalate diesters yields both the monoester and alcohol, combinations of equimolar amounts of these were studied to investigate possible synergistic effects on peroxisomal enzyme activities. No appreciable synergism was observed with combinations of 0.2 mM M E H P and 0.2 mM 2-ethylhexanol or 0.2 mM mono-n-butylphthalate and 0.2 mM n-butanol (Table II). Cultures treated for 48 h with 0.2 mM MEHP, MNOP or mono-n-pentylphthalate were examined by electron microscopy. In cultures treated with MEHP a distinct increase in the number of peroxisomes was consistently observed compared with control cultures and many peroxisomes lacked a nucleoid (Figs. 2A,B). No change in peroxisome numbers or morphology was observed after mono-n-pentylphthalate or MNOP treatment (Figs. 2C,D). In view of the lack of ultrastructural evidence of peroxisome proliferation with MNOP, we studied in more detail the biochemical changes produced by this c o m p o u n d in comparison with the effects of MEHP. Figure 3 shows the time course of changes in palmitoyl-CoA oxidation and carnitine acetyltransferase activity in hepatocyte cultures treated with either 0.2 mM MNOP or MEHP for up to 96 h. Over this period the level of palmitoyl-CoA oxidation in the control cultures decreased from an initial value of 1.82 + 0.27 nmol/min/mg protein to 0.54 +- 0.15 after 96 h (Fig. 3A). Addition of MEHP to the culture medium resulted in a time-related increase in palmitoyl-CoA oxidation reaching 5.00 + 0.67 nmol/min/mg protein at 96 h, 9 times the corresponding control level. In contrast, although MNOP produced a slight increase over the first 24 h, there was no further increase thereafter. Thus at 96 h, although palmitoyl-CoA oxidation in the MNOPtreated hepatocytes was 419% of the control level, the absolute activity in the treated cultures was n o t significantly different from the 0 h level. With carnitine acetyltransferase a somewhat different pattern of response was observed (Fig. 3B). The control activity remained relatively constant over the treatment period and both MNOP and MEHP produced time dependent increases reaching 480% and 897% of control respectively after 96 h. Cultures treated with either DMSO {control), 0.2 mM MEHP, 0.2 mM MNOP or 0.5 mM clofibrate were analysed b y SDS-PAGE. Hepatic peroxisome proliferation in vivo is associated with the induction of a peroxisomeassociated 80 000 mol. wt polypeptide which is believed to be the enzyme enoyl-CoA hydratase (EC 4.2.1.17) [21,22]. There was an increase in a protein band of approximate molecular weight 80 000 in cultures treated with MEHP and clofibrate, b u t not MNOP, after either 72 h (Fig. 4A) or 96 h (Fig. 4B). DISCUSSION
The alkyl phthalates studied varied considerably in their effects on peroxisomal enzyme activity and numbers of peroxisomes in cultured rat hepato-
174
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Fig. 3. T i m e course of changes in cyanide-insensitive p a l m i t o y l - C o A o x i d a t i o n (A) and carnitine acetyltransferase (B) in primary h e p a t o c y t e cultures treated with either DMSO (control), 0.2 mM MEHP or 0.2 mM MNOP. Values are means _+ S.E.M. for 3 separate %experiments.
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Fig. 4. S D S - P A G E of h e p a t o c y t e whole h o m o g e n a t e s (150 ~g protein) after culture for 72 h (A) and 96 h (B) w i t h either DMSO (Control, Track 1), 0.2 mM MEHP (Track 2), 0.2 mM M N O P (Track 3) or 0.5 mM clofibrate (Track 4). E l e c t r o p h o r e t i c migration is f r o m t o p to b o t t o m and m o l e c u l a r weights were d e t e r m i n e d by c o m p a r i s o n with k n o w n standards (st) consisting of ~-galactosidase (116 000), phosphorylase B (97 400), bovine serum a l b u m i n (66 0 0 0 ) and o v a l b u m i n (45 000). The positions o f the bands w i t h a p p r o x i m a t e m o l e c u l a r weight o f 80 000 are indicated by the arrows.
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cytes. Expressed as a percentage of control activity, certain of the straight chain monoesters produced significant increases in the specific peroxisomal marker, cyanide-insensitive palmitoyl-CoA oxidation, although none was as potent as MEHP. However, more detailed investigation showed that, whereas MEHP treatment resulted in a continuing increase over the initial level of palmitoyl-CoA oxidation, despite the decreasing level in control cultures, MNOP only maintained the initial level of activity. Since, in comparison with controls, MNOP was She most potent of the straight chain monoesters it is likely that the percentage increases in palmitoyl-CoA oxidation with these esters represent varying degrees of enzyme stabilisation rather than enzyme induction as produced by MEHP [12]. The mechanism of this stabilisation is unclear. It may involve limited stimulation of enzyme synthesis, inhibition of degradation or a combination of these. However, the comparative lack o f effect o f straight chain monoesters on hepatocyte peroxisomes was further indicated by the SDS-PAGE and electron microscopy studies. Enhancement of the 80 000 mol. wt band, characteristic of peroxisome proliferation in vivo [22,23], was clearly evident on SDSPAGE profiles of hepatocytes treated with MEHP and the positive control clofibrate, but not with MNOP. Similarly, neither MNOP nor mono-npentylphthalate produced ultrastructural evidence of an increase in peroxisome numbers. In contrast, the presence of "coreless" peroxisomes in the MEHP-treated hepatocytes is typical of the changes produced by peroxisome proliferators in vivo [28]. The individual straight chain monoesters differed markedly in their effects on carnitine acetyltransferase activity. These effects, which were not shared by the corresponding alcohols, were not related directly to chain length and could not be explained in terms of differential cytotoxicity. Similarly unexplained differences exist in other aspects of the biological activity of phthalate esters, for example in testicular toxicity [27]. The effects of the diesters on carnitine acetyltransferase appeared to be dependent on hydrolysis. Thus, the shorter chain diesters, which are known to be rapidly hydrolysed by rat liver cell preparations [ 1 3 ] , produced increases that were indistinguishable from those of the corresponding monoesters, whereas DEHP and di-n-octylphthalate, which are only slowly hydrolysed, were much less potent than the monoesters. In view of this, comparisons of different phthalate esters in hepatocyte cultures are best performed with the mono- rather than the diesters. Carnitine acetyltransferase occurs in mitochondria and endoplasmic reticulum as well as in peroxisomes [24] but increased activity of this enzyme is a characteristic of hepatic peroxisome proliferation in rats and mice [4,28]. A close correspondence between induction of carnitine acetyltransferase and the specific peroxisomal marker, cyanide-insensitive palmitoyl-CoA oxidation, was observed in hepatocyte cultures treated with a range of potent peroxisome proliferators [12]. However, the present
176
findings with straight chain phthalate monoesters indicate that increased activity of camitine acetyltransferase can occur in the absence of more specific peroxisomal changes. This may be at least partly due to mitochondrial changes in view of the observed increases in carnitine palmitoyltransferase and the known inducibility of mitochondrial carnitine acetyltransferase b y DEHP in vivo [25,26]. Thus, for assessing peroxisomal effects in cultured hepatocytes, measurement of carnitine acetyltransferase activity is not, by itself, an adequate criterion of response. In conclusion, the results presented indicate that straight chain phthalates from methyl to n-octyl have little effect on rat hepatic peroxisomes compared with the branched chain ester, MEHP. This conclusion is borne out by the results of in vivo studies with diethylphthalate [4] and with di- and mono-n-octylphthalate which produced little evidence of hepatic peroxisome proliferation in rats (B.G. Lake and T.J.B. Gray, unpublished observations). Studies using primary hepatocyte cultures thus provide a rapid means of comparing the peroxisomal effects of different phthalate esters. The extension of these studies to hepatocytes obtained from other animal species is currently in progress. ACKNOWLEDGEMENTS
This work forms part of a research project sponsored by the UK Ministry of Agriculture, Fisheries and F o o d to w h o m our thanks are due. The results of the research are the property of the Ministry of Agriculture, Fisheries and F o o d and are Crown Copyright. We are grateful to Dr. R. Purchase for synthesising phthalate esters and to Mr. C.R. Stubberfield and Miss K.D. Hodder for skilled technical assistance. REFERENCES 1 D.B. Peakall, Phthalate esters: occurrence and biological effects. Residue Rev., 54 (1975) 1. 2 J.A. Thomas, T.D. Darby, R.F. Wallin, P.J. Garvin and L. Martis, A review of the biological effects of di-(2-ethylhexyl)phthalate. Toxicol. Appl. Pharmacol., 45 (1978) 1. 3 B.G. Lake, S.D. Gangolli, P. Grasso and A.G. Lloyd, Studies on the hepatic effects of orally administered di-(2-ethylhexyl)phthalate in the rat. Toxicol. Appl. Pharmacol., 32 (1975) 355. 4 D.E. Moody and J.K. Reddy, Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol. Appi. Pharmacol., 45 (1978) 497. 5 National Cancer Institute, Bioassay of di-(2-ethylhexyl)phthalate. Carcinogenesis testing program, DHHS Publication No. (NIH) 81-1773 ( 1981 ). 6 J.K. Reddy, M.S. Rao, D.L. Azarnoff and S. Sell, Mitogenic and carcinogenic effects of a hypolipidemic peroxisome proliferator, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643) in rat and mouse liver. Cancer Res., 39 (1979) 152. 7 J.K. Reddy, D.L. Azarnoff and C.E. Hignite, Hypolipidemic hepatic peroxisome
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9
10 11
12
13
14 15
16
17
18
19
20 21
22
23 24
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proliferators form a novel class of chemical carcinogens. Nature (Lond. h 283 (1980) 397. J.K. Reddy, N.D. Lalwani, M.K. Reddy and S.A. Qureshi, Excessive accumulation of autofluorescent lipofuscin in the liver during hepatocarcinogenesis by methyl clofenapate and other hypolipidemic peroxisome proliferators. Cancer Res., 42 (1982) 259. J.K. Reddy, D.E. Moody, D.L. Azarnoff and M.S. Rao, Di-(2-ethylhexyl)phthalate: an industrial plasticizer induces hypolipidemia and enhances hepatic catalase and carnitine acetyltransferase activities in rats and mice. Life Sci., 18 (1976) 941. T. Osumi and T. Hashimoto, Enhancement of fatty acyl-CoA oxidizing activity in rat liver peroxisomes by di-(2-ethylhexyl)phthalate. J. Biochem., 83 (1978) 1361. T. Osumi and T. Hashimoto, Subcellular distribution of the enzymes of the fatty acyl-CoA ~-oxidation system and their induction by di-(2-ethylhexyl)phthalate in rat liver. J. Biochem., 85 (1979) 131. T.J.B. Gray, B.G. Lake, J.A. Beamand, J.R. Foster and S.D. Gangolli, Peroxisome proliferation in primary cultures of rat hepatocytes. Toxicol. Appl. Pharmacol., 67 (1983) 15. B.G. Lake, J.C. Phillips, J.C. Linnell and S.D. Gangolli, The in vitro hydrolysis of some phthalate diesters by hepatic and intestinal preparations from various species. Toxicol. Appl. Pharmacol., 39 (1977) 239. I.R. Rowland, R.C. Cottrell and J.C. Phillips, Hydrolysis of phthalate esters by the gastro-intestinal contents of the rat. Food Cosmet. Toxicol., 15 (1977) 17. R.D. White, D.E. Carter, IJ. Earnest and J. Muller, Absorption and metabolism of three phthalate diesters by the rat small intestine. Food Cosmet. Toxicol., 18 (1980) 383. P.W. Albro, R. Thomas and L. Fishbein, Metabolism of diethylhexylphthalate by rats, isolation and characterization of the urinary metabolites. J. Chromatog., 76 (1973) 321. A.J. Paine, L.J. Hockin and R.F. Legg, Relationship between the ability of nicotinamide to maintain nicotinamide adenine dinucleotide in rat liver cell culture and its effect on cytochrome P-450. Biochem. J., 184 (1979) 461. C.E. Green, H.J. Segall and J.L. Byard, Metabolism, cytotoxicity, and genotoxicity of the pyrrolizidine alkaloid senecionine in primary cultures of rat hepatocytes. Toxicol. Appl. Pharmacol., 60 (1981) 176. J.G. Dent, C.R. Elcombe, K.J. Netter and J.E. Gibson, Rat hepatic microsomal cytochrome(s) P-450 induced by polybrominated biphenyis. Drug Metab. Dispos., 6 (1978) 96. C.W. Dunnett, A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc., 50 (1955) 1096. N.D. Lalwani, M.K. Reddy, M. Mangkornkanok-Mark and J.K. Reddy, Induction, immunochemical identity and immunofluorescence localization of an 80,000molecular-weight peroxisome-proliferation-associated polypeptide (polypeptide PPA-80) and peroxisomal enoyl-CoA hydratase of mouse liver and renal cortex. Biochem. J., 198 (1981) 177. M.K. Reddy, S.A. Qureshi, P.F. Hollenberg and J.K. Reddy, Immunochemical identity of peroxisomal enoyI-CoA hydratase with the peroxisome-proliferation-associated 80,000 mol wt polypeptide in rat liver. J. Cell Biol., 89 (1981} 406. J.K. Reddy and N.S. Kumar, The peroxisome proliferation-associated polypeptide in rat liver. Biochem. Biophys. Res. Commun., 77 (1977) 824. M.A.K. Markwell, E.J. McGroarty, L.L. Bieber and N.E. Tolbert, The subcellular distribution of carnitine acetyltransferases in mammalian liver and kidney. J. Biol. Chem., 248 (1973} 3426. A.E. Ganning and G. Dallner, Induction of peroxisomes and mitochondria by di-(2ethylhexyl)phthalate. FEBS Lett., 130 (1981) 77
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26 Y. Shindo, T. Osumi and T. Hashimoto, Effects of administration of di-(2-ethylhexyl)phthalate on rat liver mitochondria. Biochem. Pharmacol., 27 (1978) 2683. 27 P.M.D. Foster, L.V. Thomas, M.W. Cook and S.D. Gangolli, Study of the testicular effects and changes in zinc excretion produced by some n-alkyl phthalates in the rat. Toxicol. Appl. Pharmacol., 54 (1980) 392. 28 A.J. Cohen and P. Grasso, Review of the hepatic response to hypolipidaemic drugs in rodents and assessment of its toxicological significance to man. Food Cosmet. Toxicol., 19 (1981) 585.
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P E R O X I S O M A L EFFECTS OF P H T H A L A T E C U L T U R E S OF R A T HEPATOCYTES
ESTERS IN P R I M A R Y
TIM J.B. GRAY, BRIAN G. LAKE, JENNY A. BEAMAND, JOHN R. FOSTER and SHARAT D. GANGOLLI The British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey, SM5 4DS (United Kingdom) (Received November 9th, 1982} (Accepted February 21st, 1983)
SUMMARY
Primary rat hepatocyte cultures were used to compare the effects of some aikylphthalate esters on peroxisomal enzyme activities and morphology. Linear diesters from methyl to n-octyl and their constituent monoesters and alcohols were compared with the branched chain 2-ethylhexyl derivatives. Carnitine acetyltransferase activity was increased 9.5-fold by mono-2-ethylhexylphthalate (MEHP) and 5.5- and 7-fold b y mono-n-pentyland mono-n-octylphthalate (MNOP), the 2 most potent of the linear monoesters. Activity of the specific peroxisomal marker, cyanide-insensitive palmitoyl-CoA oxidation decreased with time in control cultures. Whereas MEHP produced a time related increase over the initial level of palmitoylCoA oxidation, MNOP treatment only maintained the initial level of activity. The peroxisome proliferation-associated 80 000 mol. wt polypeptide was induced by MEHP but not MNOP. Similarly, MEHP produced increased numbers of peroxisomes, many without a nucleoid, whereas MNOP and mono-n-pentylphthalate had no such effect. 2-Ethylhexanol was less p o t e n t as an inducer of carnitine acetyltransferase than MEHP and the linear alcohols had no effect on this enzyme. Studies with diesters indicated that induction of carnitine acetyltransferase required hydrolysis of the diester. It is concluded that the straight-chain phthalates studied have little effect on hepatic peroxisomes compared with the 2-ethylhexyl ester and that hepatocyte cultures provide a rapid means of comparing the peroxisomal effects of different phthalates. Address correspondence to: Dr. T.J.B. Gray, BIBRA, Woodmansterne Road, Carshalton, Surrey, SM5 4DS, U.K. Abbreviations: DEHP, di-(2-ethylhexyl)phthalate; DMSO, dimethylsulphoxide; LDH, lactate dehydrogenase; MEHP, mono-2-ethylhexylphthalate; MNOP, mono-n-octylphthalate.
Printed and Published in Ireland
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Key words: Rat hepatocyte primary cultures; Phthalate esters; Hepatic peroxisome proliferation in vitro INTRODUCTION Di-(2-ethylhexyl)phthalate (DEHP) is used extensively as a plasticizer for PVC as well as in a variety of other industrial applications [1,2]. Shortterm administration of DEHP to rats causes liver enlargement and a marked proliferation of hepatic peroxisomes [3,4], while chronic administration at high levels in the diet has been associated with an increased incidence of hepatocellular carcinoma [5]. Recently a number of other, structurally diverse chemicals that cause hepatic peroxisome proliferation have also been reported to induce liver tumours in rats [ 6 - 8 ] and this has led to the proposal [7] that potent hepatic peroxisome proliferators, as a class, are carcinogenic. Although the peroxisomal effects of DEHP and its principal metabolite, mono-2-ethylhexylphthalate (MEHP) have been studied in some detail [3,9,10,11], little is known of the effects of other phthalate esters. We have recently shown [12] that primary cultures of rat hepatocytes provide a rapid and sensitive means of investigating chemical effects on peroxisomal structure and function. The purpose of the studies described here was to use hepatocyte cultures to examine the peroxisomal effects of a series of linear alkyl phthalates from methyl to n-octyl in comparison with the effects of the 2-ethylhexyl ester. Peroxisomal response was assessed by the measurement of biochemical markers, including cyanide-insensitive palmitoyl-CoA oxidation and carnitine acetyltransferase, and by electron microscopy. Since orally-administered phthalate diesters undergo intestinal metabolism to the corresponding monoesters and alcohols [ 1 3 - t 5 ] these products were also studied. METHODS
~aterials RPMI 1640 medium and Nunc tissue culture dishes were purchased from Gibco (Europe), Paisley, Scotland. Gentamicin was obtained from Flow Laboratories, Irvine, Scotland, foetal calf serum from Sera Labs, Crawley, Sussex, England and collagenase from Boehringer Corporation, Lewes, Sussex, England. Dimethyl-, diethyl-, di-n-butyl and di-(2-ethylhexyl)phthalate were kindly donated by BP Chemicals Limited, Penarth, South Glamorgan (courtesy of Dr D.M. Pugh). Di-n-pentylphthalate was purchased from Eastman-Kodak Company, Rochester, New York. Di-n-propyl-, di-nhexyl-, di-n-heptyl- and di-n-octylphthalate and the phthalate monoesters were synthesised by the method of Albro et al. [16]. Their purity was established by GLC or TLC and by nuclear magnetic resonance spectroscopy and elemental analysis. Clofibrate and all other biochemicals were obtained from Sigma Chemical Company, Poole, Dorset, England.
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Hepatocyte isolation and treatment o f cultures H e p a t o c y t e s were isolated from male S p r a g u e - D a w l e y rats (180--220 g) using the collagenase perfusion technique described by Paine et al. [17]. After washing three times by centrifugation at 50 g a v the cells were resuspended in culture medium (RPMI 1640 containing 5% foetal calf serum, 50 pg/ml gentamicin, 10 -6 M insulin and 10 -4 M hydrocortisone-21-sodium succinate). Viability of the freshly isolated cells, determined by trypan blue exclusion, was 84 + 5% (Mean + S.D. for 13 peffusions). Hepatocytes were seeded at 2.5 × 106 viable cells/3 ml culture medium in 50-mm petri dishes and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. After 18--21 h, treatment was c o m m e n c e d by replacing the culture medium with medium containing the required concentration of test c o m p o u n d . Subsequently the medium was changed and the cells redosed every 24 h. For each treatment or time point, 3--4 culture dishes were used. Test compounds were dissolved in dimethylsulphoxide (DMSO) and added to the culture medium so that the final concentration of DMSO was 0.4% in all cases, including the control cultures. Cytotoxicity was assessed by the measurement of lactate dehydrogenase (LDH) released into the culture medium [18]. Biochemical investigations At the end of the treatment period, h e p a t o c y t e whole homogenates were prepared and assayed for cyanide-insensitive palmitoyl-CoA oxidation, carnitine acetyltransferase, carnitine palmitoyltransferase and protein as described previously [ 1 2 ] . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of h e p a t o c y t e whole homogenate proteins was performed as described by Dent et al. [19] employing a 13 cm long 7.5% (w/v) acrylamide resolving gel. Gels were stained with Coomassie brilliant blue R. Electron microscopy Cultures treated with MEHP, mono-n-octyl- or mono-n-pentylphthalate, together with control cultures, were processed for transmission electron microscopy as described previously [12]. Statistical analysis Data were analysed using Dunnett's test for multiple comparisons [20]. RESULTS Primary cultures of rat hepatocytes were treated for 48 h with a series of mono-n-alkyl phthalates ranging from methyl to n-octyl and the effects on peroxisomal enzyme activities were compared with the effects of MEHP (Fig. 1). The monoester concentration used (0.2 mM), corresponded to the maximum stimulatory concentration for MEHP [12] and was n o t c y t o t o x i c as judged by the measurement of lactate dehydrogenase activity released into the culture medium {data n o t shown).
169
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900 800 700
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600
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500
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300 2OO
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1
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2
3
4
5
6
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Monoester (_n-alkyl chain length)
Fig. 1. C o m p a r a t i v e effects o f m o n o - n - a l k y l p h t h a l a t e s and M E H P o n activities o f carnitine acetyltransferase (= *), cyanide-insensitive palmitoyl-CoA oxidation (= A)
and carnitine palmitoyltransferase (~ ~) in primary hepatocyte cultures. Values, expressed as percent of control, are from a typical experiment and are means ± S.E.M. for 3--4 culture dishes after a 48-h treatment period. Monoester concentration was 0.2 mM in each case. Control activities for carnitine acetyltransferase, palmitoyl-CoA oxidation and carnitine palmitoyltransferase were 1.68 ± 0.44, 0.71 ± 0.04 and 3.68 ± 0.71 nmol/min/mg homogenate protein, respectively.
The activity of carnitine acetyltransferase, a mixed peroxisomal/mitochondrial marker, increased with increasing alkyl chain length from methyl (92% of control) to n-butyl (273%). The effects of mono-n-pentylphthalate were more marked (545%) whereas the n-hexyl and n-heptyl monoesters produced increases of only 238% and 342% of control respectively. Mono-noctylphthalate (MNOP) was the most potent of the straight chain monoesters (660%) but had less effect than its isomer MEHP (960%). There was a similar pattern of response for the specific peroxisomal marker, cyanideinsensitive palmitoyl-CoA oxidation, although the increases were less than those for carnitine acetyltransferase activity (Fig. 1). For most of the esters, increases in the mitochondrial enzyme, carnitine palmitoyltransferase, were smaller than those for palmitoyl-CoA oxidation but again, MEHP and MNOP caused the largest changes {283% and 234% of control, respectively). The results shown in Fig. 1 are from a typical experiment. Results of replicate 170
TABLE I E F F E C T S O F P H T H A L A T E D I E S T E R S A N D C O N S T I T U E N T A L C O H O L S ON CARN I T I N E A C E T Y L T R A N S F E R A S E A C T I V I T Y IN C U L T U R E D R A T H E P A T O C Y T E S Alkyl group
Control Methyl Ethyl n-Propyl n-Butyl n-Pentyl n-Hexyl n-Heptyl n-Octyl 2-Ethylhexyl
Carnitine a c e t y l t r a n s f e r a s e ( n m o l / m i n / m g p r o t e i n ) a Diester (0.2 m M )
% Control
Alcohol (1 raM)
% Control
1.76 2.71 2.85 4.35 5.45 7.33 3.24 2.04 3.55 4.56
(100) 154 162 b 247 c 310 c 416 c 184 c 116 202 c 259 c
1.51 1.63 1.96 1.20 1.47 2.29 1.67 1.25 1.75 8.80
(100) 108 130 79 97 152 111 83 116 583 c
± 0.13 ± 0.07 ± 0.34 ± 0.18 ± 0.47 ± 0.18 -* 0.20 ± 0.27 ± 0.25 ± 0.06
± ± ± ± ± ± ± ± ± ±
0.31 0.22 0.28 0.10 0.12 0.07 0.24 0.07 0.50 0.51
aValues are m e a n s ± S.E.M. f o r 3--4 c u l t u r e dishes per t r e a t m e n t . T r e a t m e n t t i m e was 48 h. b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l , P < 0.05. cSignificantly d i f f e r e n t f r o m c o n t r o l , P < 0.01.
T A B L E II E F F E C T S O F C O M B I N A T I O N S O F M E H P A N D 2 - E T H Y L H E X A N O L (2EH), A N D M O N O - n - B U T Y L P H T H A L A T E (MBP) A N D n - B U T A N O L ON P E R O X I S O M A L E N Z Y M E A C T I V I T Y IN C U L T U R E D R A T H E P A T O C Y T E S
Control 0.2 m M M E H P 0.2 m M 2EH 0.2 m M M E H P + 0.2mM2EH 0.2 m M MBP 0.2 m M n - b u t a n o l 0.2 m M MBP + 0.2 m M n - b u t a n o l
Cyanideinsensitive palmitoyl-CoA oxidationa
Control (%)
Carnitine acetyltransferase a
Control (%)
0.45 ± 0.03 2.13 ± 0.22 0.46 ± 0.10
(100) 473 b 102
1.24 ± 0.18 19.3 ± 0.4 1.87 ± 0.03
(100) 1556 b 151
2.50+- 0.05 0.99 ± 0.03 0.48 ± 0.08
556 b 220 c 107
22.0 ± 0.9 6.16 ± 0.86 1.13 ± 0.33
1774 b 497 b 91
0.84 ± 0.05
187
7.36 ± 0.17
594 b
a E x p r e s s e d as n m o l / m i n / m g p r o t e i n . Values are m e a n s ± S.E.M. for 3--4 culture dishes per t r e a t m e n t . T r e a t m e n t t i m e was 48 h. b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l , P < 0.01. cSignificantly d i f f e r e n t f r o m c o n t r o l , P < 0.05.
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experiments consistently showed the same pattern of changes although the extent of these changes varied somewhat between different cell preparations. This point is illustrated by comparing the effects of mono-n-butylphthalate on carnitine acetyltransferase activity in Fig. 1 and Table II. In contrast to the monoesters, the corresponding straight chain alcohols produced little or no increase in carnitine acetyltransferase activity, even at the higher concentration of 1 mM (Table I). However, 1 mM 2-ethylhexanol caused a 6-fold increase in carnitine acetyltransferase. Table I also shows the results of studie.s with phthalate diesters at a final concentration in the
i ~ iiii !iii;
172
m e d i u m o f 0.2 mM. H i g h e r c o n c e n t r a t i o n s were i n c o m p l e t e l y soluble. Diesters f r o m m e t h y l to n - p e n t y l p r o d u c e d a chain l e n g t h - d e p e n d e n t increase in c a r n i t i n e a c e t y l t r a n s f e r a s e activity as o b s e r v e d w i t h the c o r r e s p o n d ing m o n o e s t e r s b u t the e f f e c t s o f D E H P a n d d i - n - o c t y l p h t h a l a t e were m u c h less m a r k e d .
Fig. 2. Electron micrographs of cultured rat hepatocytes (X1950) (A) control. (B) MEHPtreated (0.2 mM for 48 h) showing increased numbers of peroxisomes (arrowed). (C) MNOP and (D) mono-n-pentylphthalate-treated (0.2 mM for 48 h) showing no differences from the control.
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Since intestinal hydrolysis of phthalate diesters yields both the monoester and alcohol, combinations of equimolar amounts of these were studied to investigate possible synergistic effects on peroxisomal enzyme activities. No appreciable synergism was observed with combinations of 0.2 mM M E H P and 0.2 mM 2-ethylhexanol or 0.2 mM mono-n-butylphthalate and 0.2 mM n-butanol (Table II). Cultures treated for 48 h with 0.2 mM MEHP, MNOP or mono-n-pentylphthalate were examined by electron microscopy. In cultures treated with MEHP a distinct increase in the number of peroxisomes was consistently observed compared with control cultures and many peroxisomes lacked a nucleoid (Figs. 2A,B). No change in peroxisome numbers or morphology was observed after mono-n-pentylphthalate or MNOP treatment (Figs. 2C,D). In view of the lack of ultrastructural evidence of peroxisome proliferation with MNOP, we studied in more detail the biochemical changes produced by this c o m p o u n d in comparison with the effects of MEHP. Figure 3 shows the time course of changes in palmitoyl-CoA oxidation and carnitine acetyltransferase activity in hepatocyte cultures treated with either 0.2 mM MNOP or MEHP for up to 96 h. Over this period the level of palmitoyl-CoA oxidation in the control cultures decreased from an initial value of 1.82 + 0.27 nmol/min/mg protein to 0.54 +- 0.15 after 96 h (Fig. 3A). Addition of MEHP to the culture medium resulted in a time-related increase in palmitoyl-CoA oxidation reaching 5.00 + 0.67 nmol/min/mg protein at 96 h, 9 times the corresponding control level. In contrast, although MNOP produced a slight increase over the first 24 h, there was no further increase thereafter. Thus at 96 h, although palmitoyl-CoA oxidation in the MNOPtreated hepatocytes was 419% of the control level, the absolute activity in the treated cultures was n o t significantly different from the 0 h level. With carnitine acetyltransferase a somewhat different pattern of response was observed (Fig. 3B). The control activity remained relatively constant over the treatment period and both MNOP and MEHP produced time dependent increases reaching 480% and 897% of control respectively after 96 h. Cultures treated with either DMSO {control), 0.2 mM MEHP, 0.2 mM MNOP or 0.5 mM clofibrate were analysed b y SDS-PAGE. Hepatic peroxisome proliferation in vivo is associated with the induction of a peroxisomeassociated 80 000 mol. wt polypeptide which is believed to be the enzyme enoyl-CoA hydratase (EC 4.2.1.17) [21,22]. There was an increase in a protein band of approximate molecular weight 80 000 in cultures treated with MEHP and clofibrate, b u t not MNOP, after either 72 h (Fig. 4A) or 96 h (Fig. 4B). DISCUSSION
The alkyl phthalates studied varied considerably in their effects on peroxisomal enzyme activity and numbers of peroxisomes in cultured rat hepato-
174
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Time(hours)
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Fig. 3. T i m e course of changes in cyanide-insensitive p a l m i t o y l - C o A o x i d a t i o n (A) and carnitine acetyltransferase (B) in primary h e p a t o c y t e cultures treated with either DMSO (control), 0.2 mM MEHP or 0.2 mM MNOP. Values are means _+ S.E.M. for 3 separate %experiments.
A
B
1 2
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w
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~. . . . .
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Fig. 4. S D S - P A G E of h e p a t o c y t e whole h o m o g e n a t e s (150 ~g protein) after culture for 72 h (A) and 96 h (B) w i t h either DMSO (Control, Track 1), 0.2 mM MEHP (Track 2), 0.2 mM M N O P (Track 3) or 0.5 mM clofibrate (Track 4). E l e c t r o p h o r e t i c migration is f r o m t o p to b o t t o m and m o l e c u l a r weights were d e t e r m i n e d by c o m p a r i s o n with k n o w n standards (st) consisting of ~-galactosidase (116 000), phosphorylase B (97 400), bovine serum a l b u m i n (66 0 0 0 ) and o v a l b u m i n (45 000). The positions o f the bands w i t h a p p r o x i m a t e m o l e c u l a r weight o f 80 000 are indicated by the arrows.
175
cytes. Expressed as a percentage of control activity, certain of the straight chain monoesters produced significant increases in the specific peroxisomal marker, cyanide-insensitive palmitoyl-CoA oxidation, although none was as potent as MEHP. However, more detailed investigation showed that, whereas MEHP treatment resulted in a continuing increase over the initial level of palmitoyl-CoA oxidation, despite the decreasing level in control cultures, MNOP only maintained the initial level of activity. Since, in comparison with controls, MNOP was She most potent of the straight chain monoesters it is likely that the percentage increases in palmitoyl-CoA oxidation with these esters represent varying degrees of enzyme stabilisation rather than enzyme induction as produced by MEHP [12]. The mechanism of this stabilisation is unclear. It may involve limited stimulation of enzyme synthesis, inhibition of degradation or a combination of these. However, the comparative lack o f effect o f straight chain monoesters on hepatocyte peroxisomes was further indicated by the SDS-PAGE and electron microscopy studies. Enhancement of the 80 000 mol. wt band, characteristic of peroxisome proliferation in vivo [22,23], was clearly evident on SDSPAGE profiles of hepatocytes treated with MEHP and the positive control clofibrate, but not with MNOP. Similarly, neither MNOP nor mono-npentylphthalate produced ultrastructural evidence of an increase in peroxisome numbers. In contrast, the presence of "coreless" peroxisomes in the MEHP-treated hepatocytes is typical of the changes produced by peroxisome proliferators in vivo [28]. The individual straight chain monoesters differed markedly in their effects on carnitine acetyltransferase activity. These effects, which were not shared by the corresponding alcohols, were not related directly to chain length and could not be explained in terms of differential cytotoxicity. Similarly unexplained differences exist in other aspects of the biological activity of phthalate esters, for example in testicular toxicity [27]. The effects of the diesters on carnitine acetyltransferase appeared to be dependent on hydrolysis. Thus, the shorter chain diesters, which are known to be rapidly hydrolysed by rat liver cell preparations [ 1 3 ] , produced increases that were indistinguishable from those of the corresponding monoesters, whereas DEHP and di-n-octylphthalate, which are only slowly hydrolysed, were much less potent than the monoesters. In view of this, comparisons of different phthalate esters in hepatocyte cultures are best performed with the mono- rather than the diesters. Carnitine acetyltransferase occurs in mitochondria and endoplasmic reticulum as well as in peroxisomes [24] but increased activity of this enzyme is a characteristic of hepatic peroxisome proliferation in rats and mice [4,28]. A close correspondence between induction of carnitine acetyltransferase and the specific peroxisomal marker, cyanide-insensitive palmitoyl-CoA oxidation, was observed in hepatocyte cultures treated with a range of potent peroxisome proliferators [12]. However, the present
176
findings with straight chain phthalate monoesters indicate that increased activity of camitine acetyltransferase can occur in the absence of more specific peroxisomal changes. This may be at least partly due to mitochondrial changes in view of the observed increases in carnitine palmitoyltransferase and the known inducibility of mitochondrial carnitine acetyltransferase b y DEHP in vivo [25,26]. Thus, for assessing peroxisomal effects in cultured hepatocytes, measurement of carnitine acetyltransferase activity is not, by itself, an adequate criterion of response. In conclusion, the results presented indicate that straight chain phthalates from methyl to n-octyl have little effect on rat hepatic peroxisomes compared with the branched chain ester, MEHP. This conclusion is borne out by the results of in vivo studies with diethylphthalate [4] and with di- and mono-n-octylphthalate which produced little evidence of hepatic peroxisome proliferation in rats (B.G. Lake and T.J.B. Gray, unpublished observations). Studies using primary hepatocyte cultures thus provide a rapid means of comparing the peroxisomal effects of different phthalate esters. The extension of these studies to hepatocytes obtained from other animal species is currently in progress. ACKNOWLEDGEMENTS
This work forms part of a research project sponsored by the UK Ministry of Agriculture, Fisheries and F o o d to w h o m our thanks are due. The results of the research are the property of the Ministry of Agriculture, Fisheries and F o o d and are Crown Copyright. We are grateful to Dr. R. Purchase for synthesising phthalate esters and to Mr. C.R. Stubberfield and Miss K.D. Hodder for skilled technical assistance. REFERENCES 1 D.B. Peakall, Phthalate esters: occurrence and biological effects. Residue Rev., 54 (1975) 1. 2 J.A. Thomas, T.D. Darby, R.F. Wallin, P.J. Garvin and L. Martis, A review of the biological effects of di-(2-ethylhexyl)phthalate. Toxicol. Appl. Pharmacol., 45 (1978) 1. 3 B.G. Lake, S.D. Gangolli, P. Grasso and A.G. Lloyd, Studies on the hepatic effects of orally administered di-(2-ethylhexyl)phthalate in the rat. Toxicol. Appl. Pharmacol., 32 (1975) 355. 4 D.E. Moody and J.K. Reddy, Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol. Appi. Pharmacol., 45 (1978) 497. 5 National Cancer Institute, Bioassay of di-(2-ethylhexyl)phthalate. Carcinogenesis testing program, DHHS Publication No. (NIH) 81-1773 ( 1981 ). 6 J.K. Reddy, M.S. Rao, D.L. Azarnoff and S. Sell, Mitogenic and carcinogenic effects of a hypolipidemic peroxisome proliferator, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643) in rat and mouse liver. Cancer Res., 39 (1979) 152. 7 J.K. Reddy, D.L. Azarnoff and C.E. Hignite, Hypolipidemic hepatic peroxisome
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8
9
10 11
12
13
14 15
16
17
18
19
20 21
22
23 24
25
proliferators form a novel class of chemical carcinogens. Nature (Lond. h 283 (1980) 397. J.K. Reddy, N.D. Lalwani, M.K. Reddy and S.A. Qureshi, Excessive accumulation of autofluorescent lipofuscin in the liver during hepatocarcinogenesis by methyl clofenapate and other hypolipidemic peroxisome proliferators. Cancer Res., 42 (1982) 259. J.K. Reddy, D.E. Moody, D.L. Azarnoff and M.S. Rao, Di-(2-ethylhexyl)phthalate: an industrial plasticizer induces hypolipidemia and enhances hepatic catalase and carnitine acetyltransferase activities in rats and mice. Life Sci., 18 (1976) 941. T. Osumi and T. Hashimoto, Enhancement of fatty acyl-CoA oxidizing activity in rat liver peroxisomes by di-(2-ethylhexyl)phthalate. J. Biochem., 83 (1978) 1361. T. Osumi and T. Hashimoto, Subcellular distribution of the enzymes of the fatty acyl-CoA ~-oxidation system and their induction by di-(2-ethylhexyl)phthalate in rat liver. J. Biochem., 85 (1979) 131. T.J.B. Gray, B.G. Lake, J.A. Beamand, J.R. Foster and S.D. Gangolli, Peroxisome proliferation in primary cultures of rat hepatocytes. Toxicol. Appl. Pharmacol., 67 (1983) 15. B.G. Lake, J.C. Phillips, J.C. Linnell and S.D. Gangolli, The in vitro hydrolysis of some phthalate diesters by hepatic and intestinal preparations from various species. Toxicol. Appl. Pharmacol., 39 (1977) 239. I.R. Rowland, R.C. Cottrell and J.C. Phillips, Hydrolysis of phthalate esters by the gastro-intestinal contents of the rat. Food Cosmet. Toxicol., 15 (1977) 17. R.D. White, D.E. Carter, IJ. Earnest and J. Muller, Absorption and metabolism of three phthalate diesters by the rat small intestine. Food Cosmet. Toxicol., 18 (1980) 383. P.W. Albro, R. Thomas and L. Fishbein, Metabolism of diethylhexylphthalate by rats, isolation and characterization of the urinary metabolites. J. Chromatog., 76 (1973) 321. A.J. Paine, L.J. Hockin and R.F. Legg, Relationship between the ability of nicotinamide to maintain nicotinamide adenine dinucleotide in rat liver cell culture and its effect on cytochrome P-450. Biochem. J., 184 (1979) 461. C.E. Green, H.J. Segall and J.L. Byard, Metabolism, cytotoxicity, and genotoxicity of the pyrrolizidine alkaloid senecionine in primary cultures of rat hepatocytes. Toxicol. Appl. Pharmacol., 60 (1981) 176. J.G. Dent, C.R. Elcombe, K.J. Netter and J.E. Gibson, Rat hepatic microsomal cytochrome(s) P-450 induced by polybrominated biphenyis. Drug Metab. Dispos., 6 (1978) 96. C.W. Dunnett, A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc., 50 (1955) 1096. N.D. Lalwani, M.K. Reddy, M. Mangkornkanok-Mark and J.K. Reddy, Induction, immunochemical identity and immunofluorescence localization of an 80,000molecular-weight peroxisome-proliferation-associated polypeptide (polypeptide PPA-80) and peroxisomal enoyl-CoA hydratase of mouse liver and renal cortex. Biochem. J., 198 (1981) 177. M.K. Reddy, S.A. Qureshi, P.F. Hollenberg and J.K. Reddy, Immunochemical identity of peroxisomal enoyI-CoA hydratase with the peroxisome-proliferation-associated 80,000 mol wt polypeptide in rat liver. J. Cell Biol., 89 (1981} 406. J.K. Reddy and N.S. Kumar, The peroxisome proliferation-associated polypeptide in rat liver. Biochem. Biophys. Res. Commun., 77 (1977) 824. M.A.K. Markwell, E.J. McGroarty, L.L. Bieber and N.E. Tolbert, The subcellular distribution of carnitine acetyltransferases in mammalian liver and kidney. J. Biol. Chem., 248 (1973} 3426. A.E. Ganning and G. Dallner, Induction of peroxisomes and mitochondria by di-(2ethylhexyl)phthalate. FEBS Lett., 130 (1981) 77
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26 Y. Shindo, T. Osumi and T. Hashimoto, Effects of administration of di-(2-ethylhexyl)phthalate on rat liver mitochondria. Biochem. Pharmacol., 27 (1978) 2683. 27 P.M.D. Foster, L.V. Thomas, M.W. Cook and S.D. Gangolli, Study of the testicular effects and changes in zinc excretion produced by some n-alkyl phthalates in the rat. Toxicol. Appl. Pharmacol., 54 (1980) 392. 28 A.J. Cohen and P. Grasso, Review of the hepatic response to hypolipidaemic drugs in rodents and assessment of its toxicological significance to man. Food Cosmet. Toxicol., 19 (1981) 585.
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