Comparison of the short-term effects of di(2-ethylhexyl) phthalate, di(n-hexyl) phthalate, and di(n-octyl) phthalate in rats

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

77, 116-I 32 (1985)

Comparison of the Short-Term Effects of Di(Z-ethylhexyl) Phthalate, Di(n-hexyl) Phthalate, and Di(n-octyl) Phthalate in Rats ALAN H. MANN, SHIRLEY C. PRICE, FIONA E. MITCHELL, PAUL GRASSO, RICHARD H. HINTON,' ANDJAMES W. BRIDGES Robens

Institute

of Industrial and Environmental Health and Safety, Guildford, Surrey GU2 5XH, United Kingdom

Received

June 1, 1984; accepted August

University

of Surrey,

27, 1984

Comparison of the Short-Term Effects of Di(Z-ethylhexyl) Phthalate, Di(n-hexyl) Phthalate, and Di(n-octyl) Phthalate in Rats. MANN, A. H., SAUCE, S. C., MITCHELL, F. E., Gusso, P., HINTON, R. H., AND BRIDGES, J. W. (1985). Toxicol. Appl. Pharmacol. 77, 116-132. This study compares changes in the livers of rats treated with di(2-ethylhexyl) phthalate (DEHP) and its straight-chain analogs di(n-hexyl) phthatate (DnHP) and di(n-octyl phthalate (DnOP). Groups of rats were fed diets containing 20,000 ppm of one of these compounds. Subgroups were killed after 3, 10, and 21 days, and the livers were examined by histological, cytological, and biochemical methods. The results show considerable differences between the effects of the branched-chain phthatate ester DEHP and its straightchain analogs. The major effects on the liver following administration of diets containing DEHP were (1) midzonal and periportal accumulation of small droplets of lipid, (2) hepatomegaly accompanied by an initial burst of mitosis, (3) proliferation of hepatic peroxisomes and of smooth endoplasmic reticulum accompanied by induction of peroxisomal fatty acid oxidation, (4) damage to the peroxisomal membranes as evidenced by increased leakage of catalase to the cytosol, and (5) centrilobular loss of glycogen and falls in glucose-6-phosphatase activity and in low-molecular-weight reducing agents. In contrast, diets containing DnHP or DnOP induced (1) accumulation of large droplets of fat around central veins leading, by 10 days, to mild centrilobular necrosis and (2) a very slight induction of one peroxisomal enzyme and an increase in liver weight, but no significant changes in any other parameters which were affected by DEHP. o 1985 Academic press. IW.

The long-chain dialkyl phthalates, which are used mainly as plasticizers for polyvinyl chloride (PVC), are a very important group of industrial chemicals. The 1978 U.S. production was in the region of 500,000 tons (Daniel, 1978) and because of the widespread use of plasticized PVC there has been extensive, although low level, human exposure. A number of toxic effects have been reported in animals treated with phthalates at high doses (Daniel, 1978; Lawrence and Tuell, 1979) but these were not sufficient to cause concern about possible health hazards from phthalate ’ To whom all correspondence should be addressed. 0041-008X/85

$3.00

Copyri@t 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

esters until di(2-ethylhexyl) phthalate was shown to be carcinogenic in rats and mice (Kluwe et al., 1982, 1983). It has been known for some time that treatment of rats (Lake et al., 1975; Moody and Reddy, 1978), but not of ferrets (Lake et al., 1976), with di(2ethylhexyl) phthalate causes proliferation of peroxisomes. A similar proliferation of peroxisomes is found in animals treated with other hepatocarcinogens most notably with the hypolipidemic drugs, typified by clofibmte (Svoboda et al., 1967; Svoboda and Azamotf, 1979; Reddy and Lalwani, 1984) but also, to some extent, with ethionine (Farber et al., 1964). Most of these agents give negative 116

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results in mutagenicity tests (Cohen and Grasso, 1981). Reddy et al., (1980) have suggested that there is a distinct class of chemical hepatocarcinogens which may be identified by their potential to cause peroxisome proliferation in the livers of susceptible animals. Although di(Zethylhexy1) phthalate, given orally, is carcinogenic at very high doses in rats, it cannot be assumed automatically that other phthalate esters will induce cancer in rodents. It is known that di(Zethylhexy1) phthalate, like other phthalate esters, is rapidly hydrolyzed in the gut to give free 2-ethylhexanol and mono(2-ethylhexyl) phthalate (Rowland et al., 1979). The latter compound is not further hydrolyzed but is, in rats, excreted as the products of o and w-

117

PHTHALATES

1 oxidation (Daniel and Bratt, 1974). Peroxisome proliferation can be caused by 2ethylhexanol (Lake et al., 1975) or 2-ethylhexanoic acid (Moody and Reddy, 1978) but not by free phthalic acid (Lake et al., 1975). Treatment of cultured hepatocytes with nhexanol does not lead to proliferation of peroxisomes (Gray et al., 1982). This observation raises important questions as to whether straight-chain phthalates differ in their hepatic effects from those of the branched-chain phthalates. It has been reported that a wide range of phthalate esters cause hepatomegaly in rats together with endoplasmic reticulum and mitochondrial changes (Ohyama, 1976; Takahashi, 1977; Moody and Reddy, 1978; Lake et al., 1980). There have also been reports of differences

TABLE 1 EFFECTSOF DIETS CONTAINING

2% w/w DEHP, DnOP, OR DnHP ON FEED CONSUMPTION,

BODY WEIGHT,

LIVER

WEIGHT,

AND TESTIS WEIGHT

Treatment Time

Control

DEHP

DnHP

Feed consumption” o-3 days 3-10 days lo-21 days

13.7 + 0.55 (3) 17 (2) 20.6 (1)

14.5 f 0.09 (3) 15.6 (2) 21.7 (1)

20.5 (I)

19.6 + 0.38** 19.6 (2) 21.7 (1)

Body weight’ 3 days 10 days 21 days

162.4 f 2.2 204.3 5 4.1 260.2 f 4.3

158.3 + 4.4 189.6 + 2.2* 240.0 f 8.1*

161.0 + 2.5 200.1 f 4.7 254.0 f 11.8

173.0 + 3.25 219.6 f 2.2* 276.8 f 7.6

17.5 f 0.34 (3)

18 (2)

DnOP

Liver weight’ 3 days 10 days 21 days

4.29 k 0.14 3.99 + 0.11 3.23 f 0.05

6.30 + 0.16** 7.22 f 0.24”’ 7.74 f 0.05**

4.60 + 0.17 4.36 f 0.21 4.34 f 0.01**

4.48 f 0.13 4.74 f 0.16** 4.15 f 0.02**

Testis weightc.d 3 days 10 days 21 davs

2.28 f 0.05 2.63 f 0.07

2.07 + 0.18 2.06 + 0.15**

2.30 + 0.05 2.54 * 0.03

2.11 f 0.06 2.61 f 0.11

’ Feed consumptions is presented as X + SE (g/rat/day) (number of cages). b Results are presented as weight X + SE (g). Each control group consisted of 6 rats, each experimental group of 4 rats. ’ Liver and testis weight are presented as a percentage of hody weight X k SE. d Testes were not weighed at 3 days. * Significantly different from control at 5%. ** Significantly different from control at 1%.

118

MANN

in peroxisomal enzymes between animals treated with diethyl phthalate and di(Zethylhexyl) phthalate (Moody and Reddy, 1978). To assess whether the effects on rats of diets containing straight-chain phthalates differ in kind or only in degree from the effects of diets containing branched-chain phthalates, we have carried out a comparative study of the short-term biochemical and cytological effects of feeding rats diets containing di(2ethylhexyl) phthalate (DEHP) or diets containing the straight-chain analogs di(n-hexyl) phthalate (DnHP) and di(n-octyl) phthalate (DnOP). METHODS Male Wistar albino rats of the University of Surrey strain were used in these experiments. Animals were initially fed on Spratts pelleted diet No. I (Spratts Ltd., Barking, United Kingdom). Animals weighing between 85 and I 15 g (approximately 4 weeks old) were allocated, at random, into 12 groups: 3 control groups, each of six animals, and 9 treatment groups, each of four animals; 3 treatment groups were assigned to each compound. Animals were transferred to Spratts powdered diet No. 2-expanded (Spratts Ltd.) for a l-week acclimatization period. Animals were weighed at the beginning and end of the acclimatization period and the feed intake was recorded. Experimental diets were prepared by mixing 20 g/kg diet of the test substances di(2-ethylhexyl) phthalate, di(n-octyl) phthalate, and di(n-hexyl) phtbalate (all donated by BP Chemicals, Sully, Penarth). These doses were chosen for comparability with the work of Lake et al. (1975). The samples of DEHP, DnHP, and DnOP were at least 99.5% pure. Diet mixes were prepared on Days 2 and 8 of the study (Day 0 is the day when treatment was commenced) and fed respectively to animals from Days 0 to 10 and 10 to 21 of the study. Animals were inspected daily and the feed consumption was monitored at least biweekly. Groups of animals were killed 3, 10, and 21 days after commencement of treatment. Animals were killed in the following order: 2 fed DEHP, 2 fed DnHP, 3 control, 4 fed DnOP, 3 control, 2 fed DnHP, and 2 fed DEHP. Animals were anesthetized with ether, bled by cardiac puncture, and killed by cervical dislocation. The liver and genital apparatus (testes, epididymis, and seminal vesicles) were removed and weighed. Portions of liver and in some cases of kidneys were prepared for electron microscopy as described below. Further portions of liver, kidneys, spleen, and the genital apparatus were fixed in 10% phosphatebuffered formalin, pH 7.4. The remainder of the liver was set aside for biochemical studies. The autopsy was

ET AL. completed by making a visual examination of the major abdominal organs for abnormalities. Tissue samples were prepared for electron microscopy as follows: Immediately after removal of the liver from the animal, a thin slice was cut from the center of the median lobe and supported on filter paper. This slice was rapidly sliced into 0.5 to l-mm cubes with a scalpel blade moistened in fixative. The sections were fixed for between 2 and 4 hr at room temperature in freshly diluted 4% glutaraldehyde (supplied as a 25% solution by TAAB Laboratories, Reading, Berks, United Kingdom, and kept frozen at -20°C until the day of use) buffered with 0.1 M sodium cacodylate adjusted to pH 7.4 with nitric acid. After fixation, the ghttaraldehyde was decanted and the tissue blocks washed overnight in 0.1 M sodium cacodylate: HNOJ buffer, pH 7.4. The following day tissues were countertixed in 2% osmic acid (Sigma Chemical Co., Poole, Dorset, United Kingdom) buffered with 0.1 M sodium cacodylate:HNOI1 buffer. pH 7.4, dehydrated through graded alcohols, and embedded in Epon 8 12 (TAAB Laboratories). Silver-gray sections were cut on a Reichardt Ultramicrotome (Reichardt-Jung, Austria), counterstained with uranyl acetate and lead citrate (Lewis and Knight, 1977) and examined on a JEOL IOOB electron microscope (JEOL, Japanese Optical Company,

TABLE 2 EFFECZY OF DIETS CONTAINING 2% w/w DEHP, DnOP, OR DnHP ON THE NUMBER OF MITOTIC FIGURES IN RAT LIVERY

Time (days) Treatmentb

3

10

21

Control

1 4 1

0 0 0

0 0 0

DEHP

12 17

0 1

1 0

DnOP

1 0

2 0

2 0

DnHP

0 0

0 0

0 0

a Mitotic figures were counted in sections from 3 of 6 control rats and 2 of 4 experimental rats in each group. Ten fields were scanned per liver section, and the number of mitotic figures (metaphase) was recorded. The magnification employed was 400X, and fields were selected at random. The results for individual animals are presented separately. bConcentration = 2% w/w.

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PHTHALATES

119

FIG. 1. Photograph of formalin-lixed, paraffin-embedded liver section stained with hematoxylin and eosin (X270). The section was taken from a rat fed a diet containing DnOP (20,000 ppm). The photomicrograph confirms the presence of fat droplets in the centrilobular region, and there is evidence for centrilobular necrosis. CN = centrilobular necrosis; LD = lipid droplets; CV = central vein.

Japan). Samples of tissues for light microscopic examination were fixed for at least 7 days in 10% phosphatebuffered formalin, pH 7.4. A portion of the specimen was embedded in paraffin wax, and 7-pm sections were cut and stained with either hematoxylin and eosin or by the periodic acid-Schiff technique (PAS). Frozen formalin-fixed sections prepared from other portions of the tissue were stained with Gil Red 0 to show the distribution of lipid. Following removal of sections for light and electron microscopy, the residue of the liver was reweighed, cooled in ice-cold homogenization medium (0.25 M sucrose containing 5 mM Tris-HCl, pH 7.4) and homogenized by three strokes of a Potter-Elvehjem homogenizer, with the pestle rotating at 2000 rpm. The time from the death of the rats to homogenization was normally less than 3 min. The volume of the homogenate was adjusted so that 5 ml contained material from 1 g of liver. An aliquot of 20 ml, taken from the homogenate, was diluted with 20 ml of homogenization medium and centrifuged for 15 min at 10,OOOg in an MSE High-

Speed 18 centrifuge (MSE Scientific Instruments, Crawley, Sussex, United Kingdom). The pellet (large particulate fraction) was resuspended in 10 ml of homogenization medium. The supematant fractions were decanted and, after balancing, were centrifuged for 1 hr at 120,OOOgin an MSE Superspeed 65 ultracentrifuge. The supematant fraction (cytosol) was decanted and stored at -20°C. The microsomal pellet was stored at -20°C overnight and then resuspended in 30 ml of homogenization medium using a Potter-Elvehjem homogenizer. An aliquot of 3.3 ml of 1.5 M KCl was added and mixed immediately by inversion. The suspension was then centrifuged for 1 hr at 120,000g. The pellets were resuspended in 5 ml of homogenization medium and frozen at -80°C in OS-ml aliquots. Enzymic and chemical estimations were carried out as follows: Glucose-6-phosphatase, 5’-nucleotidase, and succinate dehydrogenase were assayed by standard methods (Prosper0 et aL, 1973). Catalase was assayed by the method of Leighton et al. (1968) except that the titanium oxysulfate (BDH, Poole, Dotset, United Kingdom) re-

120

MANN

ET AL.

FIG. 2. Photograph of formalin-fixed, paraffin-embedded liver section stained with Periodic acid-Schiff reagent (X270). (a) A section taken from the liver of a rat fed Spratts powdered diet. (b) A section taken from the liver of a rat fed a diet containing DEHP (20,000 ppm) for 10 days. (c) A section taken from the liver of a rat fed a diet containing DnOP (20,000 ppm) for 10 days. In this section there is a definite loss of glycogen from the centrilobular region. The same pattern is seen when rats were given a diet containing DnHP (20,000 ppm). CV = central vein.

agent was diluted 16-fold with 1 N sulfuric acid before use. Cytochrome P-450 was assayed by the method of Omura and Sato (1964). Cyanide-insensitive palmitoyl CoA oxidation was measured by the procedure of Bronfman et al. ( 1979) and or-glycerophosphate dehydrogenase by the method of Lee and Lardy (1965). Nonenzymic reductants were assayed by incubating 0.5-m] aliquots of suitably diluted liver homogenates with 0.25 ml of 0.2 M phosphate buffer containing 1.5 mg/ml of 2-(piodophenyl)-3-(pnitrophenyl)-5-phenyitetrazolium chloride and 0.25 ml of 0.3 M sodium malonate for approximately 5 min at 37°C. Protein was then precipitated by addition of 1.5 ml of 6% trichloroacetic acid, and the red formazan from the supematant fraction was extracted into 4 ml of ethyl acetate and the color measured at 490 nm. The assay is based on the blank used in the succinate dehydrogenase assay(Prosper0 et al., 1973). It has been found that reduction is due to

reducing agents present in the cytosol which spontaneously oxidize within 24 hr of preparation of the homogenate, (unpublished data).

RESULTS Animals treated with di(2-ethylhexyl) phthalate gained slightly less weight than those in the other groups during the course of the experiment, although there was no significant change in feed consumption (Table 1). The livers of animals treated with DEHP were markedly enlarged at all times after commencement of treatment (Table l), were darker than control livers, and noticably fri-

FIG. 2-Continued. 121

122

MANN

ET AL

FIG. 3. (a) A photograph of a frozen. formalin-fixed, liver section stained with oil red 0 (X270). The section was taken from a rat fed a diet containing DEHP (20,000 ppm) for 10 days. There is a centrilobular loss of lipid, with a tendency for lipid to accumulate midzonal to periportal. PV = portal vein; Id = lipid droplets. (b) A photograph of a frozen formalin-fixed. liver section stained with oil red 0 (X270). The section was taken from a rat fed a diet containing DnOP (20.000 ppm). Lipid has been lost from the portal region and is seen accumulating in the centrilobular region. A similar pattern was seen when rats were fed diets containing DnHP (20,000 ppm). CV = central vein: M = Lipid droplets.

able. Two of the rats treated for 21 days with DEHP had small subcapsular lesions. Histologically, cell necrosis was present in one of these livers and reactive fibrosis and hepatocellular hyperplasia were present in the other liver. DnOP and DnHP treatment resulted in slight, but statistically significant, liver enlargement (Table 1). The livers appeared pale and were greasy when cut. No gross changes were observed in the kidneys or pancreas of animals treated with DEHP, DnOP, or DnHP but there was reduction in the weight of the testes of animals treated with DEHP (Table 1) coupled with histological evidence of atrophy. The testes of animals treated with DnHP and DnOP appeared normal.

Light Microscopy The number of mitotic figures was markedly increased in rats treated for 3 days with DEHP (Table 2). No change was observed at any other time in rats treated with DEHP or at any time in rats treated with DnHP or DnOP. No other alterations were observed in hematoxylin and eosin stained sections of rats treated with DEHP, but animals treated with DnHP or DnOP showed some fatty change and centrilobular necrosis (Fig. 1). In sections of liver stained with PAS from rats treated with DEHP there was focal loss of glycogen, involving small groups of cells, principally from centrilobular areas, after 3 and 10 days of treatment (Fig. 2b); by 2 1

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PHTHALATES

123

FIG. 3-Continued.

days, rats treated with DEHP showed an almost total loss of glycogen from the liver. Animals treated with DnHP or DnOP for 3, 10, or 2 1 days showed a clear-cut centrilobular loss of glycogen which was marked at 10 and 21 days (Fig. 2~). In sections of liver stained with oil red 0, marked differences were observed in the distribution of neutral fat. In animals treated with DEHP for 3 days there was midzonal accumulation of small droplets accompanied by loss of fat from the remainder of the lobule (not illustrated). In animals treated with DEHP for 10 days there was generally less fat than in control animals, and this fat was located predominantly in the periportal zone (Fig. 3a); by 21 days of treatment there was almost total loss of fat from the liver (not illustrated). The distribution of lipid in the livers of animals treated for 3 days with DnHP or DnOP was very similar to that of

control animals (not illustrated) but treatment for 10 or 2 1 days with either DnHP or DnOP resulted in a marked centrilobular accumulation of fat (Fig. 3b) associated with some necrosis (Fig. 1). Electron Microscopy

Electron microscopic examination of the livers of rats treated with DEHP showed marked changes after only 3 days of treatment, There was a marked increase of peroxisomes in treated animals and many of these peroxisomes lacked the “core” seen in peroxisomes in control animals. There were also changes in mitochondria, consisting of an increased density of the inner mitochondrial matrix and swelling with loss of internal structure (Figs. 4a and b). The treated animals also showed marked proliferation of the smooth endoplasmic reticulum (not illus-

124

MANN

FIG. 4. A section taken from livers fixed in buffer, pH 7.4, and counterfixed in 2% osmium (a) A section from a rat fed Spratts powdered (20,000 ppm). (c) A section from a rat fed a that found in rat fed a diet containing DnOP The bar represents I pm. N = nucleus; m = peroxisome; Ld = lipid droplets.

ET

AL.

4% glutaraldehyde buffered with 0.1 M sodium cocodylate tetroxide buffered with 0. I M sodium cocodylate. pH 7.4. diet. (b) A section from a rat fed a diet containing DEHP diet containing DnOP (20,000 ppm). A similar pattern to is found in rats fed a diet containing DnHP (20,000 ppm). = mitochondrion: rer = rough endoplasmic reticulum; p

trated); there was little rough endoplasmic reticulum visible and that which was present appeared dilated and partially degranulated. These changes are consistent with the study of Lake et al. (1975). Myelin figures were apparent in the hepatocyte cytoplasm while the bile canaliculi were enlarged and filled with debris. Treatment for 10 or 21 days showed that the changes found at 3 days persisted except that the bile canaliculi assumed a more normal appearance. The ultrastructural appearance of hepatocytes in the livers of animals treated with DnHP or DnOP was markedly different from animals treated with DEHP (Fig. 4~). After 3 days of treatment there was proliferation and dilation of the smooth endoplasmic reticulum and shortening of the microvilli in

some bile canaliculi. Treatment of animals for 10 days with DnHP or DnOP caused the accumulation of very small lipid droplets in hepatocytes. The changes in the smooth endoplasmic reticu!um observed at 3 days persisted, and there appeared to be some increase in lysosomal number. Treatment for 21 days with DnHP or DnOP resulted in the formation of large lipid droplets in some cells, although neighboring cells showed little change. Proliferation and dilation of smooth endoplasmic reticulum remained apparent in all cells, and there was some evidence for a small increase in peroxisome number although this increase was much less pronounced than that observed in animals treated with DEHP and may not have been significant.

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FlG.

PHTHALATES

125

4-Continued.

Biochemistry

The peroxisomal enzymes cyanide-insensitive palmitoyl CoA oxidation and cu-glyc-

erophosphate dehydrogenase (Table 3) were rapidly and markedly induced in animals treated with DEHP. The activity of a-glycerophosphate dehydrogenase in animals

126

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ET AL.

TABLE 3 EFFECYSOF DIEIX CONTAINING 2% w/w DEHP, DnOP, OR DnHP ON PEROXISOMAL ENZYMES IN RAT LIVER Treatment” Time

Control

DEHP

DnHP

DnOP

Cyanide-insensitive palmitoyl CoA oxidationb 3 days 10 days 21 days

0.95 + 0.08’ 1.21 * 0.12 0.77 * 0.09’

3.91 f 0.77** 14.0 f 0.96** 10.88 f 0.93**

1.23 f 0.29 1.85 + 0.19** 1.15 f 0.22J

0.90 f 0.25 2.48 k 0.23** 1.39 f 0.20:

a-Glycerophosphate dehydrogenase’ 3 days 10 days 21 days

47 + 6 54+ 10 41 -+6

108 + 14** 135 + 11** 102 f 15**

52+ 11 34 + 4 59 f I

44 f 5 44 + 10 28 f 6

0.80 + 0.02 1.39 f 0.05 1.22 f 0.14

0.94 f 0.02 1.60 + 0.02 1.05 + 0.14

Catalased 3 days 10 days 21 days

0.87 f 0.02 1.47 k 0.07 1.27 f 0.03

1.03 + 0.22** 2.14 + 0.24* 1.91 * 0.22**

a Concentration = 2% w/w. b Cyanide-insensitive palmitoyl CoA oxidase activity in the homogenates livers of rats. Results are presented as nmol NAD+ reduced/min/mg protein f SE. Bach control group consisted of 6 rats, each experimental group of 4 rats, unless otherwise noted. ’ a-Glycerophosphate dehydrogenase activity in the large particulate fraction separated from the livers of control rats or treated rats. Results are presented as units/mg protein + SE. Each control group consisted of 6 rats, each experimental group of 4 rats. dCatalase activity in the homogenate of livers of control rats and treated rats. Results are presented as units/mg protein f SE. Each control group consisted of 6 rats, each experimental group of 4 rats. ’ Results from 5 animals only. ‘Results from 3 animals only. * Significantly different from control at 5%. ** Significantly different from control at 1%.

treated with DnHP and DnOP did not differ significantly from control values, and although there was an increase in cyanideinsensitive palmitoyl CoA oxidation this increase was small compared with that found in rats treated with DEHP. Total catalase was also induced in animals treated with DEHP, although to a lesser extent than was the case with the other peroxisomal enzymes (Table 3). There were no significant changes in total catalase in animals treated with DnOP or DnHP. Changes in catalase sedimentable by centrifugation for 15 min at 10,OOOg (large particulate, i.e., peroxisomal catalase (Masters and Holmes, 1977)) were difficult to interpret (Table 4). The specific activity

of catalase in the particulate fraction was changed little in animals treated with DEHP but was somewhat increased in animals treated with DnHP and DnOP. Concomitant changes in total catalase meant that when results were expressed as percentage of total catalase there was a significant reduction in sedimentable catalase in animals treated with DEHP but a significant increase in animals treated with DnHP and DnOP. The activity of the plasma membrane enzyme S-nucleotidase was markedly reduced in animals treated with DEHP, changes in animals treated with DnHP or DnOP were much smaller and generally not statistically significant (Table 5).

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PHTHALATES

127

TABLE 4 EFFE~S

OF DIETS

CONTAINING 2% PARTICULATE

w/w DEHP, DnOP,

OR DnHP ON CATALASE OF RAT LIVER HOMOGENATE

FRACXION

Acrwrn

IN THE LARGE

Treatment’ Time

Control b

DEHP

DnHP

DnOP

Catalasc activity (% activity in whole homogenate) 3 days 10 days 21 days

54.1 + 3.5’ 49.1 + 1.9 52.1 + 6.9

45.7 + 6 33.2 + 1.9** 42.0 + 1.0

54.8 + 1 60.1 k 2.2** 74.7 k 3.0**

56 + 1.5 59.1 f 18.5** 73.8 + 4.9*

Catalase activity (units/mg protein) 3 days 10 days 21 davs

1.68 -c 0.09 1.45 + 0.09 1.46 f 0.15

1.62 f 0.15 1.27 f 0.08 1.48 + 0.01

1.85 f 0.05 1.91 * 0.13: 1.85 + 0.08

1.93 f 0.02 1.92 + 0.57** 1.60 f 0.17

DConcentration = 2% w/w. b Each control group consisted of 6 rats, each experimental group of 4 rats. c Values are means k SE. * Significantly different from control at 5%. ** Significantly different from control at 1%.

chrome P-450. Glucose-6-phosphatase activity (Table 7) in animals treated with DEHP was markedly reduced after 3 days of treatment, fell to only 30% of the control value after 10 days treatment, but recovered slightly to just under 50% of the control value after 2 1 days of treatment. Glucose-6-phosphatase activity in rats treated with DnHP or DnOP was similar to control animals after 3 and 10 days of treatment but was significantly

There was no statistically significant change in succinate dehydrogenase activity except for a decreasein animals treated with DnOP for 21 days although activities on animals treated with DEHP were consistently below control values. These results are in agreement with those of Lake et al. (1975) (Table 6). Two endoplasmic reticulum-associated parameters were measured in the current study: glucose-6-phosphatase activity and cyto-

TABLE 5 EFFECTSOF

DIETS

CONTAINING

2%

W/W

DEHP,

DnOP,

OR DnHP

ON 5’-NUCLEOTIDASE

ACTIVITY

IN RAT

LIVER“

Treatmentb

Time

Control

DEHP

DnHP

DnOP

3 days 10 days 21 days

29.3 + 2.1 40.7 f 3.3 52.4 + 8.9

16.0 + 9** 19.1 f 1.6** 26.3 + 1.6*

24.6 f 2.0 38.3 f 2.3 44.8 -c 5.4

28.9 + 4.4 31.7 + 3.1 31.8 f 2.8*

0 Results are presented as rmol/min/g protein f SE. Each control group consisted of 6 rats, each experimental group of 4 rats. b Concentration = 2% w/w. * Significantly different from control at 5%. ** Significantly different from control at 1%.

MANN

128

ET AL.

TABLE 6 EFFECTS

OF DIETS

CONTAINING DEHYDRCGENASE

2% w/w DEHP, DnOP, or DnHP ACTIVITY

IN RAT

ON SUCCINATE

LIVER”

Treatment b Time

Control

DEHP

DnHP

3 days 10 days 21 days

7.16 f 0.14 4.54 + 0.40 4.89 + 0.16

6.40 f 0.39 3.88 f 0.49 4.11 f 0.43

7.62 f 0.27 4.54 f 0.14 4.24 + 0.58

DnOP 7.33 + 0.14 4.14 + 0.29 3.71 f 0.35*

a Results are presented at rmol/min/g protein f SE. Each control group consisted of 6 rats, each experimental group of 4 rats. ‘Concentration = 2% w/w. * Significantly different from control at I%.

reduced after 21 days of treatment. Cytochrome P-450 was not significantly altered (Table 8). Nonenzymic reductants were significantly lower in animals treated with DEHP than in control animals (Table 9). There was, however, no change in reducing activity in animals treated with DnHP or DnOP. DISCUSSION The results presented in this paper show that the short-term effects on rat liver after po administration of DEHP differ markedly from the effects of its straight-chain analogs DnHP and DnOP (Table 10). The most marked effects of DEHP were (1) hepato-

megaly accompanied by an initial burst of mitosis; and (2) proliferation of hepatic peroxisomes indicated both by electron microscopy and by specific marker enzymes. (in agreement with the work of other authors (Hess et al., 1965; Hruban et al., 1966; Svoboda et al., 1967), these peroxisomes lacked the uricase-rich core); (3) proliferation of smooth endoplasmic reticulum; and (4) centrilobular loss of glycogen and a fall in glucose-6-phosphatase activity. These results agreed with earlier studies both on DEHP and on hypolipidemic drugs (Lake et al., 1975; Reddy and Lalwani, 1984). The only difference from previously published data is that succinate dehydrogenase activity was much less altered in our study than in

TABLE 7 EFFECTSOF

DIETS

CONTAINING

2% w/w DEHP, DnOP, OR DnHP ACTIVITY IN RAT LIVER”

ON GLUCOSE-6-PHOSPHATASE

Treatmentb Time

Control

3 days 10 days 21 days

34.7 f 2.0 22.1 f 2.2 47.7 + 4.9

DEHP

23.9 + 3.8* 6.6 * 1.7** 22.9 + 4. I **

DnHP

DnOP

28.6 + 2.2 26.6 f 3.5 33.0 + 1.7*

30.6 f 2.3 24.4 f 2.5 26.1 f 5.9*

‘Results are presented in pmol/min/g protein f SE. Each control group consisted of 6 rats, each experimental group of 4 rats. bConcentration = 2% w/w. * Significantly different from control at 5%. ** Significantly different from control at 1%.

STRAIGHT-

AND BRANCHED-CHAIN

129

PHTHALATES

TABLE 8 EFFECTSOF DIET CONTAINING

2% w/w DEHP, DnOP, OR DnHP ON CYT~CHROME

P450 IN RAT LIVER”

Treatmentb Time

Control

DEHP

3 days

1.03 f 0.1

1.31 f 0.08

10 days 21 days

0.87 f 0.12

1.43 * 0.132

1.56 + 0.30

1.90 + 0.15

DnHP

DnOP

1.24 + 0.08 1.175 + 0.04 1.02 + 0.05

1.19 + 0.06 1.02 f 0.14 1.54 f 0.22

a Results are presented as nmol/mg microsomal protein + SE and were carried out on 4 animals from the control group and 4 animals from each experimental group. b Concentration = 2% w/w. * Significantly different from control at 5%.

the experiments of Lake et al. (1975) although we did note a consistent reduction in activity. Hepatomegaly and proliferation of smooth endoplasmic reticulum are also found with agents such as phenobarbitone and BHT (Schulte-Herman, 1974) which, like DEHP (Gibson et al., 1982), stimulate their own metabolism. In addition to the above well-characterized effects, we found that treatment of rats with DEHP caused other alterations in the liver, i.e., a decrease in the amount of catalase associated with peroxisomes in the liver homogenates. In animals treated with DEHP, the activity of catalase in the large particulate fraction (peroxisomal catalase) was depressed. A similar depression was observed in animals treated with hypolipidemic drugs (Leighton

et al., 1975), and is probably indicative of damage to the peroxisomal membrane, resulting in either increased leakage in vivo or in greater fragility with increased disruption during homogenization. Midzonal and periportal accumulation of fat in the liver similar to that produced by clofibrate (Barnard et al., 1980; Price et al., 1982)was also observed. The appearance and lobular distribution of this fat was reminiscent of the fatty change produced by excess intake of lipid, caused by hyperphagy following repeated insulin injection (Meyer and Hartroft, 1960), hypophysectomy (Best and Huntsman, 1932), or a diet containing over 15% fat (Neat et al., 198 1). A similar change was also found in rats treated with brominated vegetable oils (Gaunt et al., 1971). A feature common for

TABLE 9 EF’FECIXOF DIETS CONTAINING

2% w/w DEHP, DnOP, OR DnHP REDUCING AGENTS IN RAT LIVER”

ON NONENZYMIC

Treatment b Time

Control

3 days 10 days

0.90 + 0.06

1.10 k 0.09

21days

0.98 + 0.07

DEHP

DnHP

DnOP

0.66 + 0.08*

0.76 k 0.05

0.61 + 0.04**

1.01 + 0.17

0.69 f 0.08"

0.82 -t 0.07

0.94 + 0.05 0.84 + 0.04 0.84 + 0.11

’ Results are presented in pmol p(piodophenyl)-3-(pnitrophenyl)-5-phenyltetrazolium + SE. Each control group consisted of 6 rats, each experimental group of 4 rats. bConcentration = 2% w/w. * Significantly different from control at 5%. l * Significantly different from control at 1%.

chloride reduced/g liver

MANN

130

ET AL.

TABLE 10 SUMMARY OF MORPHOLOGICAL CHANGES IN THE LIVERS OF RATS ADMINISTERED DIETS CONTAINING 2% w/w DEHP, DnOP, OR DnHP” Treatment Effect

DEHP

DnHP

DnOP

Hepatomegaly General appearance of liver Centrilobular loss glycogen Total glycogen loss after 2 1 days treatment Centrilobular necrosis Periportal fat accumulation Centrilobular fat accumulation Peroxisome proliferation Smooth endoplasmic proliferation Loss of rough endoplasmic reticulum Increase density of inner mitochondrial matrix Initial burst of mitosis

+++ Dark + + + +++ ++ + ++ ++

+ (Late) Light, Greasy + -h ++ + + (Late) + + -

+ (Late) Light, Greasy ++ ++ + + (Late) + + -

4 The + - +++ denotes the degree of change seen when compared to age-matched controls. ’ (-) = absence of the lesion.

each of these insults appears to be that the liver is unable to dispose of excess exogenous fat. A temporary alteration in the bile canaliculi also occurred. This alteration has since been confirmed in other experiments and is accompanied by alterations in bile flow and enzyme composition (unpublished data). The fall in the plasma membrane enzyme, Snucleotidase, in rats treated with DEHP may be connected with the alterations in the bile canalicular structure, but the time course is markedly different. A second, and less complicated, explanation is that the decrease in the specific activity of 5’-nucleotidase simply reflects the fact that the enlarged hepatocytes found in rats treated with DEHP will have a smaller area per unit volume so that a plasmic enzyme like S-nucleotidase will show a decreased specific activity even though the activity per membrane area is unchanged. The reduction in low-molecular-weight reducing agents shown in these and in other experiments in our laboratories (unpublished data) indicate that this reduction reflects a fall in the amount of reduced glutathione. Treatment of rats with the straight-chain phthalate esters, DnHP and DnOP, produced

markedly different changes from those described above, particularly in lipid distribution. The fat was concentrated in the centrilobular zone, was present in large droplets, and filled most of the cells. This type of fatty change is similar to that produced by hepatotoxic agents such as CC& and dimethylnitrosamine (Rouiller, 1964). With these agents the fatty change appears within 24 hr, but it was only observed with DnHP and DnOP after 10 days of treatment. Despite this difference in the time scale of its appearance, this type of fatty change indicates that DnHP and DnOP produce a moderately severe hepatotoxic effect at 20,000 ppm. This interpretation is supported by a marked loss of glycogen at 3 days and loss of glucose-6phosphatase activity accompanied by centrilobular necrosis at 21 days. Neither of these two changes occurred with DEHP, whose effects, in agreement with other workers cited above, were similar to those induced by hypolipidemic drugs typified by clofibrate. Also in contrast to the effects of DEHP, neither DnHP nor DnOP produced the hepatic changes typical of hypolipidemic agents. There was only slight enlargement of the liver and only minimal induction of CN--

STRAIGHT-

AND BRANCHED-CHAIN

insensitive palmitoyl CoA oxidation following exposure to DnHP or DnOP. There was also no alteration in S-nucleotidase activity or in nonenzymic reductants. Reconciliation of these results requires consideration of the mechanisms of peroxisome proliferation, the most marked effect of treatment with DEHP. Di(ethylhexy1) phthalate (DEHP) is hydrolyzed by the gastric contents to yield 2-ethylhexanol and mono(ethylhexyl) phthalate (Rowland et al., 1979). Both these metabolites cause peroxisome proliferation (Lake et al., 1975; Moody and Reddy, 1978), although mono(ethylhexy1) phthalate is much more potent. Peroxisome proliferation seems to occur whenever the concentration of fat in the liver is increased, whether by dietary manipulation (Neat et al., 198 1), a higher uptake, as is assumed to occur in animals treated with hypolipidemic agents such as clofibrate (Cohen and Grasso, 198 l), or by blockage of lipoprotein export, as with ethionine (Wood, 1965). Taken together, our results indicate that the mechanism of toxicity of DEHP differs from that of its straight-chain analogs, DnHP and DnOP. The changes which occur in rats treated with DEHP are essentially identical to those reported in rats treated with hypolipidemic drugs (Cohen and Grasso, 1981; Reddy and Lalwani, 1984). Rats treated with DnHP or DnOP, on the other hand, showed only slight traces of the morphological and biochemical changes typical of hypolipidemic agents but instead showed a marked centrilobular fatty change accompanied by losses of glycogen and of glucose-6-phosphatase activity, changes which are produced by classical hepatotoxins. Experiments on isolated hepatocytes (unpublished data) indicate that with DnHP and DnOP, as with DEHP, the effects on the liver are due to the monoester released by hydrolysis in the gut (Rowland et al., 1979), not to the long-chain alcohol. Hence the difference in the short-term effects of the straight-chain phthalate esters, DnOP and DnHP, might be anticipated to result in differences in their chronic toxicity. Whereas DEHP is carcinogenic in rats and

PHTHALATES

131

mice when administered at very high doses in a lifetime study (Kluwe et al., 1982), it remains to be established if high doses of DnOP or DnHP are also carcinogenic. In the light of our findings, such investigations appear to be essential for a fuller understanding of the toxicology of these compounds. ACKNOWLEDGMENTS We thank Mr. D. Hall for valuable discussions on the morphological changes, Miss D. Chescoe and Professor S. J. Holt for advice and assistance in the electron microscopic studies and Miss J. Howerth and Mrs. J. Mullervy for assistance in preparing specimens for light and electron microscopy. Financial support was provided by the Cancer Research Campaign.

REFERENCES BARNARD, S. D., MOLELLO, J. A., CALDWELL, W. J., AND LEBEAU, J. E. (1980). Comparative ultrastructural study of rat hepatocytes after treatment with the hypolipidaemic agents probucol, clofibrate and fenofibrate. J. Toxicol. Environ. Health 6, 547-557. BEST, C. H., AND HUNTSMAN, M. E. (1932). Effects of the components of lecithine upon deposition of fat in the liver. J. Physiol. (London) 75, 405-412. BRONFMAN, M., INESTROSA,N. C., AND LEIGHTON, F. (1979). Fatty acid oxidation by human liver peroxisomes. Biochem. Biophys. Res. Commun. 88, 10301036. COHEN, A. J., AND GRASSO, P. (1981). Review of the hepatic response to hypolipidaemic drugs in rodents and assessmentof its toxicological significance to man. Food

Cosmet.

Toxicol.

19, 585-605.

DANIEL, J. W. (1978). Toxicity and metabolism of phthalate esters. Clin. Toxicol. 13, 251-268. DANIEL, J. W., AND BRAM, H. (1974). The absorption, metabolism and tissue distribution of di-(2-ethylhexyl) phthalate in rats. Toxicology 2, 5 l-65. FARBER, E., SHULL, K. H., VILLA-TREVINO, S., LOMBARDI, B., AND THOMAS, M. (1964). Biochemical pathology of acute hepatic adenosinetriphosphate deficiency. Nature (London) 203, 34-40. GAUNT, I. F., GRASSO, P., AND GANGOLLI, S. D. (197 1). Brominated maize oil. I. Short-term toxicity and bromine-storage studies in rats fed brominated maize oil. Food Cosmet. Toxicol. 9, l-l 1. GIBSON, G. G., ORTON, T. C., AND TAMBURINI, P. P. (1982). Cytochrome P450 induction by clotibrate: Purification and properties of a hepatic cytochrome P450 relatively specific for the 12- and 1 I-hydroxylation of dodecanoic acid (lauric acid). Biochem. J. 203, 161-168. GRAY, T. J. B., BEAMAND, J. A., LAKE, B. G., FOSTER, J. R., AND GANGOLLI, S. D. (1982). Peroxisome

MANN proliferation in cultured rat hepatocytes produced by clofibrate and phthalate ester metabolites. Toxicol. Lett. 10, 273-219. HESS, R., STAUBLI, W., AND REISS, W. (1965). Nature of the hepatomegalic effect produced by ethyl-chlorophenoxy-isobutyrate in the rat. Nature (London) 208, 856-858. HRUBAN, Z., SWIFT, H., AND SLESERS, A. (1966). Ultrastructural alterations of hepatic microbodies. Lab. Invest. 15, 1884- 190 1. KLUWE, W. M., HASEMAN, J. K.. D. FIELDING, DOUGLAS J. AND HUFF, J. E. (1982). The carcinogenicity of dietary di(2ethylhexyl) phthalate (DEHP) in Fischer 344 rats and B6C3F, mice. J. Toxicol. Environ. Health 10, 797-8 15. KLUWE, W. M., HASEMAN, J. K., AND HUF’F, J. E. ( 1983). The carcinogenicity of di(2ethylhexyl) phthalate (DEHP) in perspective. J. Toxicol. Environ. Health 12, 159-169. LAKE, B. G., BRANTOME, P. G., GANGOLLI, S. D., BUTTERWORTH, K. R., AND GRASSO,P. (1976). Studies on the effects of orally administered di-(2ethylhexyl) phthalate in the ferret. Toxicology 6, 341-356. LAKE, B. G., FOSTER, P. M. D., COLLINS, M. A., AND GANGOLLI, S. D. (1980). Comparative studies on the hepatic and testicular effects of some phthalate esters in the rat (Abst. 215). In Proceedings, 19th Meeting of Sot. of Toxicologists,

Washington,

D.C.

LAKE, B. G., GANGOLLI, S. D., GRASSO,P., AND LLOYD, A. G. (1975). Studies on the hepatic effects of orally administered di-(2-ethylhexyl) phthalate in the rat. Toxicol.

Appl. Pharmacol.

32, 355-367.

LAWRENCE, W. H., AND TUELL, S. F. (I 979). Phthalate esters:The question of safety-an update. Clin. Toxicol. 15,447-466.

LEE, Y. P., AND LARDY, H. A. (1965). Influence of thyroid hormones on L-a-glycerophosphate dehydrogenases and other dehydrogenases in various organs of the rat. J. Biol. Chem. 240, 1427-1436. LEIGHTON, F., COLOMA, L., AND KOENIG, C. (1975). Structure composition, physical properties and turnover of proliferated peroxisomes: A study of the trophic effects of ~~-13457 on rat liver. J. Cell. Biol. 67, 281309. LEIGHTON, F., POOLE, B., BEAUFAY, H., BAUDHUIN, P., COFFEY,J. W., FOWLER, S., AND DE DUVE, C. (1968). The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with Triton WR- 1339: Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions. J. Cell. Biol. 37, 482-513. LEWIS, P. R., AND KNIGHT, D. P. (1977). Staining methods for sectioned material. In Pracfical Methods for Elecfron Microscopy (A. Glauert, ed.), Vol. 5, Part 1, Sect. 2.3.2d, p. 43 and Sect. 2.4.2, p. 46. NorthHolland Elsevier, Amsterdam/New York/Oxford. MASTERS, C., AND HOLMES, R. (1977). Peroxisomes: New aspects of cell physiology and biochemistry. Phys. Rev. 57(4), 816-882.

ET AL. MEYER, J. S.. AND HARTROFT. W. S. (1960). Hepatic lipid produced by polyphagia in albino rats. Relationship to dietary choline and casein. Amer. J. Pathol. 36, 365-392. MOODY, D. E.. AND REDDY, J. K. (1978). Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol. Appl. Pharmacol.

45,497-504.

NEAT, C. E., THOMASSEN, M. S., AND OSMUNDSEN, H. (I 98 1). Effects of high-fat diets on hepatic fatty acid oxidation in the rat. Biochem. J. 196, 149-159. OHYAMA, T. (1976). Effects of phthalate esters on the respiration of rat liver mitochondria. J. B&hem. 79, 153-158. OMURA, T.. AND SATO, R. (1964). The carbon monoxidebinding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 23702378. PRICE, S. C., HINTON, R. H., HALL, D. E., GRMSO, P., AND BRIDGES, J. W. (1982). Accumulation of lipidrich lysosomes in the livers and kidneys of animals treated with hypolipidaemic agents. Biochem. Sot. Trans.

10, 244.

PROSPERO,T. D., BURGE, M. L. E., NORRIS, K. A., HINTON, R. H., AND REID, E. (1973). Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes. B&hem. J. 132, 449-458. REDDY, J. K., AZARNOR;; D. L., AND HIGNITE, C. E. (1980). Hypolipidaemic hepatic peroxisome proliferatots from a novel class of chemical carcinogens. Nature (London)

283, 397-398.

REDDY, J. K., AND LALWANI, N. D. (1984). Carcinogenesis by hepatic peroxisome proliferators: evaluation of the risk of hypolipidaemic drugs and industrial plasticisers to humans. CRC Crif. Rev. Toxicol. 12, l-58. ROUILLER, CH. (1964). Experimental toxic injury of the liver. In The Liver, Morphology, Biochemistry, Physiology (Ch. Rouiller, ed.), Vol. 2, pp. 335-476. Academic Press, New York/London. ROWLAND, I. R.,COTTRELL, R.C., AND PHILLIPS,J.C. (1977). Hydrolysis of phthalate esters by gastro-intestinal contents of the rat. Food Cosmet. Toxicol. 15, 17-21. SCHULTE-HERMANN, R. (1974). Induction of liver growth by xenobiotic compounds and other stimuli. CRC Crit.

Rev. Toxicol.

3, 97-l

58.

SVOBODA.D. J., AND AZARNOFF, D. L. (1979). Turnouts in male rats fed ethyl chlorophenoxyisobutyrate, a hypohpidaemic drug. Cancer Res. 39, 34 19-3428. SVOBODA, D., GRADY, H., AND AZARNOFF, D. (1967). Microbodies in experimentally altered cells. J. Cell. Biol. 35, 127-152.

TAKAHASHI, T. (1977). Biochemical studies on phthahc esters-l 1: Effects of phthalic esters on mitochondrial respiration of rat liver. Biochemical Pharmacol. 26, 19-24. WOOD, R. (1965). The structure of hepatic cells in chronic ethionine poisoning and during recovery. Amer. J. Pathol.

46, 307-330.