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Inhibition of phenobarbital and dietary choline deficiency promoted preneoplastic lesions in rat liver by environmental contaminant di(2-ethylhexyl)phthalate
Cancer Letters, Elsevier
Scientific
23 (1984)
323
323-330
Publishers
Ireland
Ltd.
INHIBITION OF PHENOBARBITAL AND DIETARY CHOLINE DEFICIENCY PROMOTED PRENEOPLASTIC LESIONS IN RAT LIVER BY ENVIRONMENTAL CONTAMINANT DI(2-ETHYLHEXYL)PHTHALATE
A.B.
DeANGELO,
Department Washington,
C.T. GARRETT*
and A.E. QUERAL
of Pathology, The George DC 20037 (U.S.A.)
Washington
University
Medical
Center,
(Received 27 February 1984) (Revised version received 25 May 1984) (Accepted 1 June 1984)
SUMMARY
The effect of di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer and environmental contaminant, on the emergence of gamma-glutamyltranspeptidase positive (GGT+) preneoplastic foci in the liver of rats fed promoting diets was studied. GGT+ foci were initiated in the liver of SpragueDawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. One series of control rats received saline vehicle alone. Promotion of foci was commenced by feeding: (1) a choline-deficient diet (CD); (2) a choline-supplemented diet (CS) containing 0.06% phenobarbital (CS + PHB); or (3) a CD diet containing 0.06% phenobarbital (CD + PHB). In the absence of initiation by DEN, dietary treatments did not increase the number of GGT+ foci. In rats receiving DEN, each promoting regimen effectively increased the number of GGT+ foci above levels in control rats fed only the choline-supplemented diet. Inclusion of the plasticizer at a level of 2% in each of the dietary promotion treatments, however, effectively inhibited the appearance of the foci.
INTRODUCTION
The esters of o-phthalic acid comprise the most extensively used class of plasticizers [ 11. Of these, DEHP is the one most commonly used to impart flexibility to plastics. Two recent findings have focused concern about the safety of DEHP. DEHP shares many biochemical properties with the peroxi-
*To
whom
all correspondence
0304-3835/84/$03.00 Published and Printed
should
o 1984 in Ireland
be addressed.
Elsevier Scientific
Publishers
Ireland
Ltd.
324
some proliferators, nafenopin and Wy-14643 which were shown to promote the outgrowth of neoplastic foci in rat liver [ 2-41. Moreover, a study from the National Toxicology Program found that DEHP increased the incidence of hepatocellular carcinoma and neoplastic nodules in rodents [ 51. Evidence has been presented by others indicating that in rat liver, the precursor lesions to both neoplastic nodules and hepatomas are enzyme altered foci [6,7]. Using this rat liver model, we evaluated the ability of DEHP to promote the development of preneoplastic foci in rats previously administered a subcarcinogenic dose of the hepatocarcinogen, DEN [ 81. We did not obtain evidence that DEHP enhanced promotion after either 5 weeks or 10 weeks of feeding the compound at a 2% level. Somewhat unexpectedly, we observed that DEHP suppressed the development of GGT+ foci by a well-established promoting regimen, the choline-deficient diet [8]. In the present report we have confirmed and extended this earlier study and have demonstrated that DEHP is not only effective in suppressing development of GGT+ foci in the rat liver by the CD diet but also by PHB and the combination of CD and PHB treatments. MATERIALS
AND METHODS
Male Sprague-Dawley rats weighing 150-200 g were housed individually in metal cages in a room with constant temperature and humidity and on a 12 h light-dark cycle. The animals were fed a basal diet (Purina, Ralston Purina Co., St. Louis, MO) and water ad libitum for at least 1 week before the start of experiments. Purified CS and CD diets were prepared according to the protocol of Shinozuka et al. [9]. For diets containing DEHP (Aldrich Chemical Co., Milwaukee, WI) or PHB (Mallinckrodt, St. Louis, MO), the compounds were added to 2% and 0.06%, respectively, at the expense of sucrose. Animal weights and food consumption were monitored on 2 consecutive days at weekly intervals. Eighteen hours following subtotal hepatectomy, rats were administered either a single intraperitoneal injection of saline or a single intraperitoneal injection of DEN (Aldrich Chemical Co.) in saline at a dose of 30 mg/kg. Ten days later groups of animals (4-5 rats/group) were placed on the experimental or control diets as indicated. Food consumption was measured on 2 consecutive days of each week. Rats were killed by carbon dioxide asphyxiation at 10 weeks following the commencement of the diets. Body weights and liver wet weights were determined. The livers were perfused with 50 ml ice-cold saline through the hepatic portal vein. Blocks of liver, which were quickly frozen on dry ice, had Spurncryostat sections cut and stained according’to the procedure of Rutenburg et al. [lo]. GGT+ foci were counted and the major and minor diameters measured by an ocular micrometer. The area of each focus was estimated, assuming it to be elliptical in shape (foci are rarely true circles). The area of the tissue section was measured using an Apple II+ computer with graphics tablet accessory. The size,
325
number of foci per cm2 and the percent of tissue represented by the foci were calculated. Statistical comparisons were made by Fisher F-test and Student t-test [ 111. RESULTS
The effects of the dietary regimens on the appearance of hepatic GGT+ foci are shown in Table 1. In the first study (Groups l--5), rats were fed for 10 weeks on either a control (CS) diet, the control diet supplemented with 2% DEHP (CS + DEHP), the control diet supplemented with 0.06% phenobarbital (CS + PHB), the choline-deficient (CD) diet or a CD diet supplemented with 0.06% PHB (CD + PHB). The latter 3 diets have been previously shown by others to be strong promoters of GGT+ foci [ 12,131. In the absence of DEN administration, none of the dietary regimens induced the appearance of significant numbers of foci. An increase in the mean values for the focus diameters for the latter 3 treatment regimens was present but reached significance only for the minor diameter (diameter B). This may indicate that while the dietary regimens did not contribute significantly to initiation, they promoted the outgrowth of any spontaneously transformed foci which were present. Next, the results of our study to further evaluate the antipromoting activity of DEHP are given (Table 1; groups 6-13). Promotion of foci was commenced by feeding: (1) a CD diet, (2) a CS + PHB diet, or (3) a CD + PHB diet. Each dietary regimen effectively increased the number of GGT+ foci above levels in control rats fed only the choline-supplemented diet. DEHP by itself failed to increase the number and size of preneoplastic foci over that seen in the CS control group thus confirming our earlier results. When DEHP was included in the CD regimen (CD + DEHP), the number of GGT+ foci/cm decreased from 13.5 t 4.2 foci/cm2 to 0.7 + 0.1 with similar significant decreases in each of the major and minor diameters. Of greater interest, however, was the finding that inclusion of plasticizer in each of the other 2 promoting regimens, that is, the CS + PHB and CD + PHB diets, effectively inhibited the appearance of foci in each of these promotion treatments. This was seen with regard to the number of foci/cm2 for animals on the CS + PHB and CD + PHB diets vs. the respective DEHP-treated groups and the % liver as foci of the CS + PHB treated rats vs. the DEHP group. Since it has been shown by Kunz et al. that tumor development is a function of the log value rather than a linear function of the area and number of foci induced by the carcinogen DEN [ 141, we compared the average of the log values of each of our parameters. Use of this approach improved confidence levels (P values) for each of those comparisons demonstrated to be significant by use of the linear data and increased the number of significant comparisons between treatment groups for the % liver as foci (Table 1). Rats were monitored for total body weight, food consumption and liver
5
5
1.5 f 0.4 0.5 + 0.2 13.5 + 4.2 (6,7,9,11,13)f 0.7 r 0.1 5.3 f 1.1 (6,7,9,11) 1.7 + 0.3 (799) 15.3 ? 4.6 (6,7,9,11,13) 3.3 * 1.3
2.5 + 0.7 3.0 + 1.5
0.7 f o.2c 0.7 f 0.2 0.8 t 0.2
Foci/cm*
LIVER A
f 31.3 f 28.9
224.6 r 26.1 (6,9,11) 172.7 i 33.8
112.6 + 7.0 125.9 r 39.5 236.3 f 41.3 (6,9,11) 107.8 f 13.3 161.2 f 8.3 (639) 120.1 * 16.1
208.5 189.5
91.7 ? 8.3 121.3 * 32.1 239.6 f 73.9
Diameter (pm)
OF GGT-POSITIVE
+ 29.0 f 5.2
r 15.0 f 22.0 f 14.4
B
+ 0.020 f 0.033
r 0.001 * 0.003 f 0.024
0.083
0.455
t 0.064
* 0.238
0.006 * 0.001 0.065 f: 0.014 (6,7,9,11) 0.014 * 0.004
0.011 ). 0.003 0.006 f 0.003 0.588 c 0.339
0.063 0.057
0.004 0.008 0.038
% Liver as focib
Dietary
treatment
-1.24 r 0.45 (6,7,9,10,11,13) -4.12 r 0.30 (9)
-4.70 c 0.31 -5.22 * 0.52 -1.29 + 0.67 (6,7,9,11,13) -5.25 z 0.24 -2.84 c 0.29 (6,7,9,11,13) -4.50 f 0.34
LOG (% liver as foci)
in parentheses.
at
study by evaluated
in the saline control groups for each of the parameters
of the parameters
number of pm2 per cm2 times 100. Avg. area is the shape of the focus to be an ellipse.
h after partial hepatectomy.
131.3 ? 12.2 (6,7,9,11) 97.6 f 20.7
79.7 + 11.2
79.2 + 6.2 62.7 * 21.0 157.4 * 31.3 (6,7,9,11) 69.7 f 9.8 94.5 c 8.1
70.8 83.5 175.0 (1,2,5) 128.1 105.6
Diameter (pm)
HEPATOCYTESa
a AI1 rats received either 30 mg/kg DEN or the saline vehicle by i.p. injection at 18 began 10 days after injection of either DEN or saline and continued for 10 weeks. b The product of (foci/cm*) x (avg. area) x 10e6 where 10e6 is the reciprocal of the calculated from the average of the measured major and minor diameters assuming ’ Mean + S.E. d No significant differences were detected between the dietary treatments for each Fisher F ratio test except for diameter B. e In the DEN study, a significant difference was present between dietary treatment P < 0.05 by Fisher F ratio test. f P < 0.05 by Student t-test for two-tailed differences when compared with groups
13
12
CD+ PHBt DEHP
CS + PHB + DEHP CD t PHB
11
5
5 4
CD + DEHP CS t PHB
10
9
4 4
3 5 4
No. of rats
5 5 5
cs
CS + PHB CD + PHB
CS + DEHP CD
cs
Dietary treatment
CS + DEHP CD
DEfl 6 7 8
4 5
&dined I 2 3
Group
TABLE 1 NUMBER AND SIZE OF FOCI AND PERCENT K ca
327
weight/100 g body wt (Table 2). Food consumption was similar for all groups and DEHP did not diminish food intake. DEHP significantly increased liver weight for each of the dietary groups over the group of rats on the same treatment without DEHP. DEHP did lower the weight gain for the CS + PHB + DEHP group. However, use of body weight as a covariate [ 111 did not reduce the level of significance between the CS + PHB group and its DEHP counterpart below the 0.95 level of significance. Moreover, the modest decrease of total body weight gain was not reflected in the target organ of the study, that is, the liver, which showed a greater than 69% increase over that of CS + PHB treated group. Since, it had been reported in one study that peroxisome proliferators produced foci in rat liver which did not stain for the presence of GGT and were characterized by the absence of lipofuscin [ 151, we examined sections of liver by fluorescence microscopy for the presence of such foci in livers from our DEHP-treated groups. While the cells from foci, which stained positively for GGT, did usually show diminished lipofuscin, we did not detect significant numbers of non-GGT staining-lipofuscin negative foci. TABLE 2 BODY WEIGHT, FOOD CONSUMPTION AND LIVER WEIGHTa Group
Dietary treatment
No. of rats
Body weight (8)
Food consumption (g/l00 g body wt)
Liver weight (g/l00 g body wt)
5.9 + 0.2 (12) 5.9 f 0.1 (12) 5.8 f 0.2 (12) 5.9 + 0.1 (12) 5.6 ? 0.02
3.31 * 0.11
6
cs
7
CS + DEHP
364 f- 10b (7,11)c 265 f 13
8
CD
328 i 24
9
CD + DEHP
326 i 6 (7,ll) 332 .t 10 (7911) 257 f 3
10
CS t PHB
11
CS + PHB + DEHP
12
CD + PHB
5
13
CD + PHB + DEHP
5
f 19 (7911) 308 ): 4 (7311) 329
5.6 * 0.2
5.3 * 0.0 6.3 i 0.2 (10,11,12)
5.85 * 0.31 (6,8,9,10) 4.09 f 0.13 (6) 4.62 + 0.03 (6.8) 4.62 f 0.16 (698) 7.94 f 0.21 (6,7,8,9,10, 12) 5.30 + 0.09 (6,8,9,10) 7.15 f 0.44 (6,7,8,9,10, 12)
a AI1 rats received 30 mg/kg DEN by i.p. injection at 18 h after partial hepatectomy. Dietary treatment began 10 days after injection of DEN and continued for 10 weeks. b Mean * S.E. ’ P < 0.05 by Student t-test for two-tailed differences when compared with groups in parentheses.
328 DISCUSSION
In our initial studies [ 81, DEHP was incorporated into both the CS and CD diets of our experimental protocol in anticipation that it would increase the number of GGT+ foci present in the livers of rats when compared to rats not given DEHP. DEHP showed no evidence of promoting activity in the CS diet. Moreover, it demonstrated antipromoting activity when fed together with the CD diet by significantly diminishing the number of foci present. Our current studies demonstrated that DEHP not only suppresses development of GGT+ foci due to a cholinedeficient diet but also due to PHB and to the strongly promoting regimen of a CD diet supplemented with PHB. Our findings are similar to those recently reported by Shinozuka et al. [ 161 using the peroxisome proliferator BR-931. This latter compound was also ineffective in increasing the GGT+ foci in the livers of DEN treated rats, and effectively suppressed their development in the CD diet. In contrast to our studies, Ward et al. [ 171 recently reported that DEHP caused an increase in preneoplastic basophilic foci in the livers of mice given a subcarcinogenic dose of DEN. The reason for the apparent difference between our results and those of Ward et al. is unclear at this juncture. The fact that the foci observed by Ward and colleagues were consistently GGT negative and that GGT is an inconsistent marker for mouse liver tumors [ 18,191, suggests the presence of significant species differences between the rat and mouse model systems. A second difference between the rat and mouse studies was that the mice used by Ward et al. were considerably younger (age 4 weeks) than the rats used in either our studies or those reported by Shinozuka et al. [ 161. Whether these differences represent the basis for the difference in experimental results will require further studies. The mechanism by which DEHP inhibited development of preneoplastic GGT+ foci in the rat system is unclear but its action(s) on biological membranes may offer one possible explanation for this activity. DEHP is lipophilic and can alter the activity of membrane bound enzymes [20]. One important example is its ability to suppress the activity of HMG CoA reductase (3-hydroxy-3-methylglutaryl CoA reductase, EC 1.1.1.34) which is the enzyme responsible for the regulation of de novo cholesterogenesis [ 201. This action might provide one mechanism by which DEHP could act as an antipromoter since a consistent characteristic of both malignant and premalignant cells [21,22] is that the activity of this enzyme frequently increases due to a loss of its feedback regulation. A second effect of DEHP is its ability to alter ion conduction across cell membranes (S. Bloom and R.V. Petersen, pers. commun.). The control of ion movement into and out of cells is known to be very important in the regulation of cell division [23]. A third possible mechanism for the antipromoting activity of DEHP is its effect on the plasma membrane receptor for epidermal growth factor. We are currently examining abundant proteins and phosphoproteins of liver cell plasma membranes of rats on promoting regimens with and without treat-
329
ment by DEHP. Our initial results indicate that DEHP can inhibit the ability of epidermal growth factor to stimulate the autophosphorylation of its 175,000 plasma membrane receptor protein [ 241. The significance of this and other changes in the plasma membrane proteins are under investigation. ACKNOWLEDGEMENTS
This work was supported by NC1 l-ROl-CA-31324 awarded by the National Cancer Institute, Department of Health and Human Services. We gratefully acknowledge the secretarial assistance of Ms. Susan Garrett and Mrs. Viann Powers. REFERENCES 1 Autian, J. (1973) Toxicity and health threats of phthalate esters: a review of the literature. Environ. Health Perspect., 4, 3-26. 2 Moody, D.E. and Reddy, J.K. (1978) Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol. Appi. Pharmacol., 45, 497504. 3 Schulte-Hermann, R., Ohde, G., Schuppler, J. and Timmermann-Trosiener, I. (1981) Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with tumor promoters phenobarbital, hexachlorocyclohexane, steroid compounds and nafenopin. Cancer Res., 41, 2556-2562. 4 Reddy, J.K. and Rao, M.S. (1978) Enhancement by WY-14,643, a hepatic peroxisome proliferator, of diethylnitrosamine-initiated hepatic tumorigenesis in the rat. Br. J. Cancer, 38,537-543. 5 National Toxicological Program. Carcinogenesis bioassay of di( 2ethylhexyl)phthalate (1982) DHSS Publication No. (NIH)Sl-1773. Carcinogen Testing Program, National Toxicology Program, Research Triangle Park, NC. 6 Solt, D.B., Medline, A. and Farber, E. (1977) Rapid emergence of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am. J. Pathol., 88, 595-618. 7 Pitor, H.C. and Sirica, A.E. (1980) ‘Ihe stages of initiation and promotion in hepatocarcinogenesis. Biochim. Biophys. Acta, 605, 191-215. 8 DeAngelo, A.B. and Garrett, C.T. (1983) Inhibition of development of preneoplastic lesions in the livers of rats fed a weakly carcinogenic environmental contaminant. Cancer Letters, 20, 199-205. 9 Shinozuka, H., Lombardi, B., Sell, S. and Iammarino, R.M. (1978) Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline deficient diet. Cancer Res., 38, 1092-1098. 10 Rutenburg, A.M., Hwakyu, K., Fischbein, J.W., Hanker, J.S., Wasserkrug, H.L. and Seligman, A.M. (1969) Histochemical and ultrastructural demonstration of gammaglutarhyl transpeptidase activity. J. Histochem. Cytochem., 17, 517-526. 11 Snedecor, G.W. and Cochran, W.G. (1980) Statistical Methods, pp. 215-236, 365385. Iowa State University Press, Ames, IO. 12 Pitot, H.C., Barsness, L. and Goldsworthy, T. (1978) Biochemical characterization of .&ages of hepatocarcinogenesis after a single dose of diethylnitrosamine. Nature, 271, 456-458. 13 Shinozuka, H. and Lombardi, B. (1980) Synergistic effect of a choline-devoid diet and phenobarbital in promoting the emergence of foci of gamma-glutamyl transpeptidaae-positive hepatocytes in the liver of carcinogen-treated rats. Cancer Res., 40, 3846-3849.
330 14
15
16 17
18
19
20 21 22
23 24
Kunz, W., Appel, K.E., Rickart, R., Schwartz, M. and Stockle, G. (1978) Enhancement and inhibition of carcinogenic effectiveness of nitrosamines. In: Primary Liver Tumors, pp. 261-283. Editors: H. Remmer, H.M. Bolt, P. Bannasch and H. Popper. University Park Press, Baltimore, MD. Rao, M.S., Lalwani, N.D., Scarpelli, D.G. and Reddy, J.K. (1982) The absence of y-glutamyl transpeptidase activity in putative preneoplastic lesions and in hepatocellular carcinomas induced in rats by the hypolipidemic peroxisome proliferator WV-14643. Carcinogenesis. 3. 1231-1233. Shinozuka, H., Abanobi, S.E: and Lombardi, B. (1983) Modulation of tumor Promotion in liver carcinoaenesis. Environ Health Persoect.. 50. 163-168. Ward, J.M., Rice, J.M., Creasia, D., Lynch, P. and Riggs, C. (1983) Dissimilar patterns of promotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C3Fl mice. Carcinogenesis, 4, 10211029. Williams, G.M., Ohmori, T., Katayama, S. and Rice, J.M. (1980) Alteration by phenobarbital of membrane-associated enzymes including gamma-glutamyl transpeptidase in mouse liver neoplasms. Carcinogenesis, 1, 813-818. Lipsky, M.M., Hinton, D.E., Klaunig, J.E., Goldblatt, P.J. and Trump, B.F. (1980) Gamma-glutamyl transpeptidase in safrole-induced, presumptive premalignant mouse hepatocytes. Carcinogenesis, 1, 151-156. Bell, F.P. (1982) Effects of phthalate esters on lipid metabolism in various tissues, cells and organelles in mammals. Environ. Health Perspect., 45, 41-50. Siperstein, M.D. (1970) Regulation of cholesterol biosynthesis in normal and malignant tissues. Curr. Top. Cell Regul., 2, 65-100. Quesney-Huneeus, V., Wiley, M.H. and Siperstein, M.D. (1979) Essential role for mevalonate synthesis in DNA replication. Proc. Natl. Acad. Sci. U.S.A., 76, 50565060. Koch, K.S. and Leffert, H.L. (1982) Growth control of differentiated adult rat hepatocytes in primary culture. Ann. N.Y. Acad. Sci., 349, 111-127. DeAngelo, A.B., Garrett, C.T. and Queral, A.E. (1984) Phosphorylation of specific plasma membrane proteins during promotion of preneoplastic hepatic lesions and inhibition by di(2-ethylhexyl)phthalate (DEHP). Proc. Am. Assoc. Cancer Res., 151.
Scientific
23 (1984)
323
323-330
Publishers
Ireland
Ltd.
INHIBITION OF PHENOBARBITAL AND DIETARY CHOLINE DEFICIENCY PROMOTED PRENEOPLASTIC LESIONS IN RAT LIVER BY ENVIRONMENTAL CONTAMINANT DI(2-ETHYLHEXYL)PHTHALATE
A.B.
DeANGELO,
Department Washington,
C.T. GARRETT*
and A.E. QUERAL
of Pathology, The George DC 20037 (U.S.A.)
Washington
University
Medical
Center,
(Received 27 February 1984) (Revised version received 25 May 1984) (Accepted 1 June 1984)
SUMMARY
The effect of di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer and environmental contaminant, on the emergence of gamma-glutamyltranspeptidase positive (GGT+) preneoplastic foci in the liver of rats fed promoting diets was studied. GGT+ foci were initiated in the liver of SpragueDawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. One series of control rats received saline vehicle alone. Promotion of foci was commenced by feeding: (1) a choline-deficient diet (CD); (2) a choline-supplemented diet (CS) containing 0.06% phenobarbital (CS + PHB); or (3) a CD diet containing 0.06% phenobarbital (CD + PHB). In the absence of initiation by DEN, dietary treatments did not increase the number of GGT+ foci. In rats receiving DEN, each promoting regimen effectively increased the number of GGT+ foci above levels in control rats fed only the choline-supplemented diet. Inclusion of the plasticizer at a level of 2% in each of the dietary promotion treatments, however, effectively inhibited the appearance of the foci.
INTRODUCTION
The esters of o-phthalic acid comprise the most extensively used class of plasticizers [ 11. Of these, DEHP is the one most commonly used to impart flexibility to plastics. Two recent findings have focused concern about the safety of DEHP. DEHP shares many biochemical properties with the peroxi-
*To
whom
all correspondence
0304-3835/84/$03.00 Published and Printed
should
o 1984 in Ireland
be addressed.
Elsevier Scientific
Publishers
Ireland
Ltd.
324
some proliferators, nafenopin and Wy-14643 which were shown to promote the outgrowth of neoplastic foci in rat liver [ 2-41. Moreover, a study from the National Toxicology Program found that DEHP increased the incidence of hepatocellular carcinoma and neoplastic nodules in rodents [ 51. Evidence has been presented by others indicating that in rat liver, the precursor lesions to both neoplastic nodules and hepatomas are enzyme altered foci [6,7]. Using this rat liver model, we evaluated the ability of DEHP to promote the development of preneoplastic foci in rats previously administered a subcarcinogenic dose of the hepatocarcinogen, DEN [ 81. We did not obtain evidence that DEHP enhanced promotion after either 5 weeks or 10 weeks of feeding the compound at a 2% level. Somewhat unexpectedly, we observed that DEHP suppressed the development of GGT+ foci by a well-established promoting regimen, the choline-deficient diet [8]. In the present report we have confirmed and extended this earlier study and have demonstrated that DEHP is not only effective in suppressing development of GGT+ foci in the rat liver by the CD diet but also by PHB and the combination of CD and PHB treatments. MATERIALS
AND METHODS
Male Sprague-Dawley rats weighing 150-200 g were housed individually in metal cages in a room with constant temperature and humidity and on a 12 h light-dark cycle. The animals were fed a basal diet (Purina, Ralston Purina Co., St. Louis, MO) and water ad libitum for at least 1 week before the start of experiments. Purified CS and CD diets were prepared according to the protocol of Shinozuka et al. [9]. For diets containing DEHP (Aldrich Chemical Co., Milwaukee, WI) or PHB (Mallinckrodt, St. Louis, MO), the compounds were added to 2% and 0.06%, respectively, at the expense of sucrose. Animal weights and food consumption were monitored on 2 consecutive days at weekly intervals. Eighteen hours following subtotal hepatectomy, rats were administered either a single intraperitoneal injection of saline or a single intraperitoneal injection of DEN (Aldrich Chemical Co.) in saline at a dose of 30 mg/kg. Ten days later groups of animals (4-5 rats/group) were placed on the experimental or control diets as indicated. Food consumption was measured on 2 consecutive days of each week. Rats were killed by carbon dioxide asphyxiation at 10 weeks following the commencement of the diets. Body weights and liver wet weights were determined. The livers were perfused with 50 ml ice-cold saline through the hepatic portal vein. Blocks of liver, which were quickly frozen on dry ice, had Spurncryostat sections cut and stained according’to the procedure of Rutenburg et al. [lo]. GGT+ foci were counted and the major and minor diameters measured by an ocular micrometer. The area of each focus was estimated, assuming it to be elliptical in shape (foci are rarely true circles). The area of the tissue section was measured using an Apple II+ computer with graphics tablet accessory. The size,
325
number of foci per cm2 and the percent of tissue represented by the foci were calculated. Statistical comparisons were made by Fisher F-test and Student t-test [ 111. RESULTS
The effects of the dietary regimens on the appearance of hepatic GGT+ foci are shown in Table 1. In the first study (Groups l--5), rats were fed for 10 weeks on either a control (CS) diet, the control diet supplemented with 2% DEHP (CS + DEHP), the control diet supplemented with 0.06% phenobarbital (CS + PHB), the choline-deficient (CD) diet or a CD diet supplemented with 0.06% PHB (CD + PHB). The latter 3 diets have been previously shown by others to be strong promoters of GGT+ foci [ 12,131. In the absence of DEN administration, none of the dietary regimens induced the appearance of significant numbers of foci. An increase in the mean values for the focus diameters for the latter 3 treatment regimens was present but reached significance only for the minor diameter (diameter B). This may indicate that while the dietary regimens did not contribute significantly to initiation, they promoted the outgrowth of any spontaneously transformed foci which were present. Next, the results of our study to further evaluate the antipromoting activity of DEHP are given (Table 1; groups 6-13). Promotion of foci was commenced by feeding: (1) a CD diet, (2) a CS + PHB diet, or (3) a CD + PHB diet. Each dietary regimen effectively increased the number of GGT+ foci above levels in control rats fed only the choline-supplemented diet. DEHP by itself failed to increase the number and size of preneoplastic foci over that seen in the CS control group thus confirming our earlier results. When DEHP was included in the CD regimen (CD + DEHP), the number of GGT+ foci/cm decreased from 13.5 t 4.2 foci/cm2 to 0.7 + 0.1 with similar significant decreases in each of the major and minor diameters. Of greater interest, however, was the finding that inclusion of plasticizer in each of the other 2 promoting regimens, that is, the CS + PHB and CD + PHB diets, effectively inhibited the appearance of foci in each of these promotion treatments. This was seen with regard to the number of foci/cm2 for animals on the CS + PHB and CD + PHB diets vs. the respective DEHP-treated groups and the % liver as foci of the CS + PHB treated rats vs. the DEHP group. Since it has been shown by Kunz et al. that tumor development is a function of the log value rather than a linear function of the area and number of foci induced by the carcinogen DEN [ 141, we compared the average of the log values of each of our parameters. Use of this approach improved confidence levels (P values) for each of those comparisons demonstrated to be significant by use of the linear data and increased the number of significant comparisons between treatment groups for the % liver as foci (Table 1). Rats were monitored for total body weight, food consumption and liver
5
5
1.5 f 0.4 0.5 + 0.2 13.5 + 4.2 (6,7,9,11,13)f 0.7 r 0.1 5.3 f 1.1 (6,7,9,11) 1.7 + 0.3 (799) 15.3 ? 4.6 (6,7,9,11,13) 3.3 * 1.3
2.5 + 0.7 3.0 + 1.5
0.7 f o.2c 0.7 f 0.2 0.8 t 0.2
Foci/cm*
LIVER A
f 31.3 f 28.9
224.6 r 26.1 (6,9,11) 172.7 i 33.8
112.6 + 7.0 125.9 r 39.5 236.3 f 41.3 (6,9,11) 107.8 f 13.3 161.2 f 8.3 (639) 120.1 * 16.1
208.5 189.5
91.7 ? 8.3 121.3 * 32.1 239.6 f 73.9
Diameter (pm)
OF GGT-POSITIVE
+ 29.0 f 5.2
r 15.0 f 22.0 f 14.4
B
+ 0.020 f 0.033
r 0.001 * 0.003 f 0.024
0.083
0.455
t 0.064
* 0.238
0.006 * 0.001 0.065 f: 0.014 (6,7,9,11) 0.014 * 0.004
0.011 ). 0.003 0.006 f 0.003 0.588 c 0.339
0.063 0.057
0.004 0.008 0.038
% Liver as focib
Dietary
treatment
-1.24 r 0.45 (6,7,9,10,11,13) -4.12 r 0.30 (9)
-4.70 c 0.31 -5.22 * 0.52 -1.29 + 0.67 (6,7,9,11,13) -5.25 z 0.24 -2.84 c 0.29 (6,7,9,11,13) -4.50 f 0.34
LOG (% liver as foci)
in parentheses.
at
study by evaluated
in the saline control groups for each of the parameters
of the parameters
number of pm2 per cm2 times 100. Avg. area is the shape of the focus to be an ellipse.
h after partial hepatectomy.
131.3 ? 12.2 (6,7,9,11) 97.6 f 20.7
79.7 + 11.2
79.2 + 6.2 62.7 * 21.0 157.4 * 31.3 (6,7,9,11) 69.7 f 9.8 94.5 c 8.1
70.8 83.5 175.0 (1,2,5) 128.1 105.6
Diameter (pm)
HEPATOCYTESa
a AI1 rats received either 30 mg/kg DEN or the saline vehicle by i.p. injection at 18 began 10 days after injection of either DEN or saline and continued for 10 weeks. b The product of (foci/cm*) x (avg. area) x 10e6 where 10e6 is the reciprocal of the calculated from the average of the measured major and minor diameters assuming ’ Mean + S.E. d No significant differences were detected between the dietary treatments for each Fisher F ratio test except for diameter B. e In the DEN study, a significant difference was present between dietary treatment P < 0.05 by Fisher F ratio test. f P < 0.05 by Student t-test for two-tailed differences when compared with groups
13
12
CD+ PHBt DEHP
CS + PHB + DEHP CD t PHB
11
5
5 4
CD + DEHP CS t PHB
10
9
4 4
3 5 4
No. of rats
5 5 5
cs
CS + PHB CD + PHB
CS + DEHP CD
cs
Dietary treatment
CS + DEHP CD
DEfl 6 7 8
4 5
&dined I 2 3
Group
TABLE 1 NUMBER AND SIZE OF FOCI AND PERCENT K ca
327
weight/100 g body wt (Table 2). Food consumption was similar for all groups and DEHP did not diminish food intake. DEHP significantly increased liver weight for each of the dietary groups over the group of rats on the same treatment without DEHP. DEHP did lower the weight gain for the CS + PHB + DEHP group. However, use of body weight as a covariate [ 111 did not reduce the level of significance between the CS + PHB group and its DEHP counterpart below the 0.95 level of significance. Moreover, the modest decrease of total body weight gain was not reflected in the target organ of the study, that is, the liver, which showed a greater than 69% increase over that of CS + PHB treated group. Since, it had been reported in one study that peroxisome proliferators produced foci in rat liver which did not stain for the presence of GGT and were characterized by the absence of lipofuscin [ 151, we examined sections of liver by fluorescence microscopy for the presence of such foci in livers from our DEHP-treated groups. While the cells from foci, which stained positively for GGT, did usually show diminished lipofuscin, we did not detect significant numbers of non-GGT staining-lipofuscin negative foci. TABLE 2 BODY WEIGHT, FOOD CONSUMPTION AND LIVER WEIGHTa Group
Dietary treatment
No. of rats
Body weight (8)
Food consumption (g/l00 g body wt)
Liver weight (g/l00 g body wt)
5.9 + 0.2 (12) 5.9 f 0.1 (12) 5.8 f 0.2 (12) 5.9 + 0.1 (12) 5.6 ? 0.02
3.31 * 0.11
6
cs
7
CS + DEHP
364 f- 10b (7,11)c 265 f 13
8
CD
328 i 24
9
CD + DEHP
326 i 6 (7,ll) 332 .t 10 (7911) 257 f 3
10
CS t PHB
11
CS + PHB + DEHP
12
CD + PHB
5
13
CD + PHB + DEHP
5
f 19 (7911) 308 ): 4 (7311) 329
5.6 * 0.2
5.3 * 0.0 6.3 i 0.2 (10,11,12)
5.85 * 0.31 (6,8,9,10) 4.09 f 0.13 (6) 4.62 + 0.03 (6.8) 4.62 f 0.16 (698) 7.94 f 0.21 (6,7,8,9,10, 12) 5.30 + 0.09 (6,8,9,10) 7.15 f 0.44 (6,7,8,9,10, 12)
a AI1 rats received 30 mg/kg DEN by i.p. injection at 18 h after partial hepatectomy. Dietary treatment began 10 days after injection of DEN and continued for 10 weeks. b Mean * S.E. ’ P < 0.05 by Student t-test for two-tailed differences when compared with groups in parentheses.
328 DISCUSSION
In our initial studies [ 81, DEHP was incorporated into both the CS and CD diets of our experimental protocol in anticipation that it would increase the number of GGT+ foci present in the livers of rats when compared to rats not given DEHP. DEHP showed no evidence of promoting activity in the CS diet. Moreover, it demonstrated antipromoting activity when fed together with the CD diet by significantly diminishing the number of foci present. Our current studies demonstrated that DEHP not only suppresses development of GGT+ foci due to a cholinedeficient diet but also due to PHB and to the strongly promoting regimen of a CD diet supplemented with PHB. Our findings are similar to those recently reported by Shinozuka et al. [ 161 using the peroxisome proliferator BR-931. This latter compound was also ineffective in increasing the GGT+ foci in the livers of DEN treated rats, and effectively suppressed their development in the CD diet. In contrast to our studies, Ward et al. [ 171 recently reported that DEHP caused an increase in preneoplastic basophilic foci in the livers of mice given a subcarcinogenic dose of DEN. The reason for the apparent difference between our results and those of Ward et al. is unclear at this juncture. The fact that the foci observed by Ward and colleagues were consistently GGT negative and that GGT is an inconsistent marker for mouse liver tumors [ 18,191, suggests the presence of significant species differences between the rat and mouse model systems. A second difference between the rat and mouse studies was that the mice used by Ward et al. were considerably younger (age 4 weeks) than the rats used in either our studies or those reported by Shinozuka et al. [ 161. Whether these differences represent the basis for the difference in experimental results will require further studies. The mechanism by which DEHP inhibited development of preneoplastic GGT+ foci in the rat system is unclear but its action(s) on biological membranes may offer one possible explanation for this activity. DEHP is lipophilic and can alter the activity of membrane bound enzymes [20]. One important example is its ability to suppress the activity of HMG CoA reductase (3-hydroxy-3-methylglutaryl CoA reductase, EC 1.1.1.34) which is the enzyme responsible for the regulation of de novo cholesterogenesis [ 201. This action might provide one mechanism by which DEHP could act as an antipromoter since a consistent characteristic of both malignant and premalignant cells [21,22] is that the activity of this enzyme frequently increases due to a loss of its feedback regulation. A second effect of DEHP is its ability to alter ion conduction across cell membranes (S. Bloom and R.V. Petersen, pers. commun.). The control of ion movement into and out of cells is known to be very important in the regulation of cell division [23]. A third possible mechanism for the antipromoting activity of DEHP is its effect on the plasma membrane receptor for epidermal growth factor. We are currently examining abundant proteins and phosphoproteins of liver cell plasma membranes of rats on promoting regimens with and without treat-
329
ment by DEHP. Our initial results indicate that DEHP can inhibit the ability of epidermal growth factor to stimulate the autophosphorylation of its 175,000 plasma membrane receptor protein [ 241. The significance of this and other changes in the plasma membrane proteins are under investigation. ACKNOWLEDGEMENTS
This work was supported by NC1 l-ROl-CA-31324 awarded by the National Cancer Institute, Department of Health and Human Services. We gratefully acknowledge the secretarial assistance of Ms. Susan Garrett and Mrs. Viann Powers. REFERENCES 1 Autian, J. (1973) Toxicity and health threats of phthalate esters: a review of the literature. Environ. Health Perspect., 4, 3-26. 2 Moody, D.E. and Reddy, J.K. (1978) Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol. Appi. Pharmacol., 45, 497504. 3 Schulte-Hermann, R., Ohde, G., Schuppler, J. and Timmermann-Trosiener, I. (1981) Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with tumor promoters phenobarbital, hexachlorocyclohexane, steroid compounds and nafenopin. Cancer Res., 41, 2556-2562. 4 Reddy, J.K. and Rao, M.S. (1978) Enhancement by WY-14,643, a hepatic peroxisome proliferator, of diethylnitrosamine-initiated hepatic tumorigenesis in the rat. Br. J. Cancer, 38,537-543. 5 National Toxicological Program. Carcinogenesis bioassay of di( 2ethylhexyl)phthalate (1982) DHSS Publication No. (NIH)Sl-1773. Carcinogen Testing Program, National Toxicology Program, Research Triangle Park, NC. 6 Solt, D.B., Medline, A. and Farber, E. (1977) Rapid emergence of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am. J. Pathol., 88, 595-618. 7 Pitor, H.C. and Sirica, A.E. (1980) ‘Ihe stages of initiation and promotion in hepatocarcinogenesis. Biochim. Biophys. Acta, 605, 191-215. 8 DeAngelo, A.B. and Garrett, C.T. (1983) Inhibition of development of preneoplastic lesions in the livers of rats fed a weakly carcinogenic environmental contaminant. Cancer Letters, 20, 199-205. 9 Shinozuka, H., Lombardi, B., Sell, S. and Iammarino, R.M. (1978) Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline deficient diet. Cancer Res., 38, 1092-1098. 10 Rutenburg, A.M., Hwakyu, K., Fischbein, J.W., Hanker, J.S., Wasserkrug, H.L. and Seligman, A.M. (1969) Histochemical and ultrastructural demonstration of gammaglutarhyl transpeptidase activity. J. Histochem. Cytochem., 17, 517-526. 11 Snedecor, G.W. and Cochran, W.G. (1980) Statistical Methods, pp. 215-236, 365385. Iowa State University Press, Ames, IO. 12 Pitot, H.C., Barsness, L. and Goldsworthy, T. (1978) Biochemical characterization of .&ages of hepatocarcinogenesis after a single dose of diethylnitrosamine. Nature, 271, 456-458. 13 Shinozuka, H. and Lombardi, B. (1980) Synergistic effect of a choline-devoid diet and phenobarbital in promoting the emergence of foci of gamma-glutamyl transpeptidaae-positive hepatocytes in the liver of carcinogen-treated rats. Cancer Res., 40, 3846-3849.
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