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Species differences in the response of cultured hepatocytes to phthalate esters
Fd Chem. Toxic. Vol. 24, No. 6/7, pp. 799-800, 1986
0278-6915/86 $3.00+0.00 Pergamon Journals Ltd
Printed in Great Britain
SPECIES DIFFERENCES IN THE RESPONSE OF C U L T U R E D HEPATOCYTES TO PHTHALATE ESTERS D. J. BENFORO, S. PATEL and H. J. REAVe The Roberts Institute, University of Surrey, Guildford, Surrey GU2 5XH A. MITCHELL Central Toxicology Laboratory, ICI plc, Alderley Park, Macclesfield, Cheshire SKI0 4TJ, England and N. J. SARGINSON
Essochem Europe Inc., Mechelsesteenweg 363, B-1950 Kraainem, Belgium
Introduction
somal palmitoyl-CoA (PCoA) oxidation (Lazarow, 1981) and laurate 11-+ 12-hydroxylation (LAH; Parker & Orton, 1980) and the protein content (Bio-Rad method) of the homogenate were determined.
Peroxisomal proliferation has been postulated as a possible mechanism for the induction of rodent liver cancer following administration of certain structurally diverse compounds such as hypolipidaemic drugs and phthalate plasticizers. Marked species differences in hepatic peroxisome proliferation have been reported for di-(2-ethylhexyl) phthalate (DEHP) both in vivo and in vitro. In order to investigate this phenomenon for phthalates with a longer branched chain, the effects of monoisononyl phthalate (MiNP), monoisodecyl phthalate (MiDP) and di-isononyl phthalate (DiNP) have been investigated in primary monolayer cultures of rat and marmoset hepatocytes. Mono-(2-ethylhexyl) phthalate (MEHP) was used as a positive control as it is the major initial in vivo metabolite of DEHP.
Results
Experimental Hepatocytes were isolated from adult male Wistar albino rats and marmoset monkeys (Callithrixjaccus) by a collagenase perfusion technique based on the method of Rao, Rao, Holler et al. (1976). The cells were plated in Falcon 25-cm 2 tissue culture flasks at a density of 2 x 106 cells/flask in 4 ml of Leibovitz L-15 medium supplemented with 10% foetal calf serum, 10% tryptose phosphate broth, 10-6M insulin, 10 -5 M-hydrocortisone and penicillin/streptomycin (100/~g/ml) and were incubated at 30°C. The medium was renewed after 3-4 hours and again after 24 hours, when the test compounds were added. Test compounds were added in dimethylformamide, an equal volume of which was added to control cultures. The medium and test compounds were renewed after 48 and 72 hours. After 96 hours in culture (3 days of treatment) the cells were harvested into sucrose (0.25 M)--EDTA (5 mM)--Tris HC1 (20 raM) buffer, pH 7.4, and were homogenized by sonication. The levels of peroxi-
The longer branched-chain phthalates, MiNP and MiDP, were both peroxisome proliferators in rat hepatocytes, as indicated by marked dose-related increases in PCoA oxidation (Fig. la). MiDP at a concentration of 0.25 mM was rather more potent than MiNP at 0.25 mM but was less potent than 0.5mM MEHP (PCoA 1500% of control). Concentrations greater than 0.25mM MiDP and 0.5mM MiNP were toxic. DiNP caused much smaller increases as it is poorly converted to the monoester in the culture system. In contrast, marmoset hepatocyte PCoA oxidation showed only minimal changes, with poor dose dependency, when the cells were treated with MiNP, MiDP or DiNP (Fig. lb) and showed no change with MEHP. LAH appeared to be more sensitive to induction by the longer branched-chain phthalates than by MEHP. In the rat hepatocyte cultures, increases of up to sevenfold with MiNP and up to 13-fold with MiDP were seen, whereas in the marmoset cultures these compounds caused up to three- and fourfold increases, respectively (Fig. 2). MEHP caused a sixfold increase in LAH in the rat cells but had no effect in marmoset hepatocytes. This observation raises the question of whether an alternative mechanism of induction of LAI-I, unrelated to peroxisomal effects, could be involved with the longer branched-chain phthalates.
Conclusions A marked species difference has been observed in peroxisomal proliferation by phthalate esters in cul-
799
D . J . BENFORD et al.
800 1000
E o
250 m
(b)
800
"S 600
g 400 o <:[ o 12.
200
0
I 0.2
0
I 0.4
o LL
1 0.6
I
0
0.2
Concn ( r a M )
I
04
I 0.6
Conch ( r a M )
Fig. 1. Palmitoyl-CoA (PCoA) oxidation activity determined in (a) rat and (b) marmoset hepatocyte cultures treated for 3 days with monoisononyl phthalate (V-I), monoisodecyl phthalate ( x ) and di-isononyl phthalate (V). Results are means 4- SEM for between three and five flasks of cells in each of two experiments.
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2
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600
(b)
400
._> 500
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o 1-
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0
I
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I
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1
0.6
C o n c n ( mM )
o.I
0
I
0.2
1
0.4
1
06
C o n c n ( mM )
Fig. 2. Laurate hydroxylation (LAH) activity was determined in (a) rat and (b) marmoset hepatocyte cultures treated for 3 days with monoisononyl phthalate (F1), monoisodecyl phthalate ( x ) and di-isononyl phthalate (V). Results are means ___SEM for between three and five flasks of cells in each of two experiments. tured hepatocytes. Hence if peroxisomes are imp o r t a n t in the m e c h a n i s m of carcinogenesis, caution should be used in extrapolating findings from r o d e n t studies to man.
Acknowledgement--This work was sponsored by Essochem Europe Ltd. REFERENCES
Lazarow P. B. (1981). Assay of peroxisomal fl-oxidation of fatty acids. In Methods in Enzymology. Vol, 72 (D).
Edited by J. W. Lowenstein. p. 315. Academic Press, New York. Parker G. L. & Orton T. C. (1980). Induction by oxyisobutyrates of hepatic and kidney microsomal cytochrome P-450 with specificity towards hydroxylation of fatty acids. In Biochemistry, Biophysics and Regulation of Cytochrome P450. Edited by J. A, Gustaffson, J. C. Duke, A. Mode & J. Rafter. p. 373. Elsevier, Amsterdam. Rao M. L., Rao G, S., Holier M., Breuer H., Schattenberg A. & Stein W. D. (1976). Uptake of cortisol by isolated rat liver cells--a phenomenon indicative of carriermediation and simple diffusiom Hoppe-Seylers Z. Physiol. Chem. 357, 573.
0278-6915/86 $3.00+0.00 Pergamon Journals Ltd
Printed in Great Britain
SPECIES DIFFERENCES IN THE RESPONSE OF C U L T U R E D HEPATOCYTES TO PHTHALATE ESTERS D. J. BENFORO, S. PATEL and H. J. REAVe The Roberts Institute, University of Surrey, Guildford, Surrey GU2 5XH A. MITCHELL Central Toxicology Laboratory, ICI plc, Alderley Park, Macclesfield, Cheshire SKI0 4TJ, England and N. J. SARGINSON
Essochem Europe Inc., Mechelsesteenweg 363, B-1950 Kraainem, Belgium
Introduction
somal palmitoyl-CoA (PCoA) oxidation (Lazarow, 1981) and laurate 11-+ 12-hydroxylation (LAH; Parker & Orton, 1980) and the protein content (Bio-Rad method) of the homogenate were determined.
Peroxisomal proliferation has been postulated as a possible mechanism for the induction of rodent liver cancer following administration of certain structurally diverse compounds such as hypolipidaemic drugs and phthalate plasticizers. Marked species differences in hepatic peroxisome proliferation have been reported for di-(2-ethylhexyl) phthalate (DEHP) both in vivo and in vitro. In order to investigate this phenomenon for phthalates with a longer branched chain, the effects of monoisononyl phthalate (MiNP), monoisodecyl phthalate (MiDP) and di-isononyl phthalate (DiNP) have been investigated in primary monolayer cultures of rat and marmoset hepatocytes. Mono-(2-ethylhexyl) phthalate (MEHP) was used as a positive control as it is the major initial in vivo metabolite of DEHP.
Results
Experimental Hepatocytes were isolated from adult male Wistar albino rats and marmoset monkeys (Callithrixjaccus) by a collagenase perfusion technique based on the method of Rao, Rao, Holler et al. (1976). The cells were plated in Falcon 25-cm 2 tissue culture flasks at a density of 2 x 106 cells/flask in 4 ml of Leibovitz L-15 medium supplemented with 10% foetal calf serum, 10% tryptose phosphate broth, 10-6M insulin, 10 -5 M-hydrocortisone and penicillin/streptomycin (100/~g/ml) and were incubated at 30°C. The medium was renewed after 3-4 hours and again after 24 hours, when the test compounds were added. Test compounds were added in dimethylformamide, an equal volume of which was added to control cultures. The medium and test compounds were renewed after 48 and 72 hours. After 96 hours in culture (3 days of treatment) the cells were harvested into sucrose (0.25 M)--EDTA (5 mM)--Tris HC1 (20 raM) buffer, pH 7.4, and were homogenized by sonication. The levels of peroxi-
The longer branched-chain phthalates, MiNP and MiDP, were both peroxisome proliferators in rat hepatocytes, as indicated by marked dose-related increases in PCoA oxidation (Fig. la). MiDP at a concentration of 0.25 mM was rather more potent than MiNP at 0.25 mM but was less potent than 0.5mM MEHP (PCoA 1500% of control). Concentrations greater than 0.25mM MiDP and 0.5mM MiNP were toxic. DiNP caused much smaller increases as it is poorly converted to the monoester in the culture system. In contrast, marmoset hepatocyte PCoA oxidation showed only minimal changes, with poor dose dependency, when the cells were treated with MiNP, MiDP or DiNP (Fig. lb) and showed no change with MEHP. LAH appeared to be more sensitive to induction by the longer branched-chain phthalates than by MEHP. In the rat hepatocyte cultures, increases of up to sevenfold with MiNP and up to 13-fold with MiDP were seen, whereas in the marmoset cultures these compounds caused up to three- and fourfold increases, respectively (Fig. 2). MEHP caused a sixfold increase in LAH in the rat cells but had no effect in marmoset hepatocytes. This observation raises the question of whether an alternative mechanism of induction of LAI-I, unrelated to peroxisomal effects, could be involved with the longer branched-chain phthalates.
Conclusions A marked species difference has been observed in peroxisomal proliferation by phthalate esters in cul-
799
D . J . BENFORD et al.
800 1000
E o
250 m
(b)
800
"S 600
g 400 o <:[ o 12.
200
0
I 0.2
0
I 0.4
o LL
1 0.6
I
0
0.2
Concn ( r a M )
I
04
I 0.6
Conch ( r a M )
Fig. 1. Palmitoyl-CoA (PCoA) oxidation activity determined in (a) rat and (b) marmoset hepatocyte cultures treated for 3 days with monoisononyl phthalate (V-I), monoisodecyl phthalate ( x ) and di-isononyl phthalate (V). Results are means 4- SEM for between three and five flasks of cells in each of two experiments.
15ool1o00~aI
j
2
E 8 "5
600
(b)
400
._> 500
ID
o 1-
T .J
_1
o
0
I
02
I
04
1
0.6
C o n c n ( mM )
o.I
0
I
0.2
1
0.4
1
06
C o n c n ( mM )
Fig. 2. Laurate hydroxylation (LAH) activity was determined in (a) rat and (b) marmoset hepatocyte cultures treated for 3 days with monoisononyl phthalate (F1), monoisodecyl phthalate ( x ) and di-isononyl phthalate (V). Results are means ___SEM for between three and five flasks of cells in each of two experiments. tured hepatocytes. Hence if peroxisomes are imp o r t a n t in the m e c h a n i s m of carcinogenesis, caution should be used in extrapolating findings from r o d e n t studies to man.
Acknowledgement--This work was sponsored by Essochem Europe Ltd. REFERENCES
Lazarow P. B. (1981). Assay of peroxisomal fl-oxidation of fatty acids. In Methods in Enzymology. Vol, 72 (D).
Edited by J. W. Lowenstein. p. 315. Academic Press, New York. Parker G. L. & Orton T. C. (1980). Induction by oxyisobutyrates of hepatic and kidney microsomal cytochrome P-450 with specificity towards hydroxylation of fatty acids. In Biochemistry, Biophysics and Regulation of Cytochrome P450. Edited by J. A, Gustaffson, J. C. Duke, A. Mode & J. Rafter. p. 373. Elsevier, Amsterdam. Rao M. L., Rao G, S., Holier M., Breuer H., Schattenberg A. & Stein W. D. (1976). Uptake of cortisol by isolated rat liver cells--a phenomenon indicative of carriermediation and simple diffusiom Hoppe-Seylers Z. Physiol. Chem. 357, 573.