Phosphorylation of specific rat plasma membrane proteins during promotion of gamma-glutamyl transpeptidase-positive hepatic foci and inhibition by di(2-ethylhexyl)phthalate.

The protein kinase activity of isolated plasma membranes from the livers of rats treated with three promoting regimens was examined using both exogenous proteins and endogenous plasma membrane proteins as substrates. Male rats first received either an initiating dose (30 mg/kg) of the hepatocarcinogen diethylnitrosamine or the 0.9% NaCl solution vehicle by i.p. injection at 18 h following partial hepatectomy. Ten days later, the three promoting regimens were begun. These consisted of 10 weeks of treatment with either (a) a choline-deficient (CD) diet, (b) a choline-supplemented (CS) diet containing 0.06% phenobarbital (PHB) (CS plus PHB), or (c) a CD diet containing 0.06% PHB (CD plus PHB). In addition, two other groups of rats received either (a) a CS diet containing 2% di(2-ethylhexyl)phthalate (DEHP) (CS plus DEHP) or (b) a CD diet containing 2% DEHP (CD plus DEHP). DEHP is a widely used plasticizer and environmental contaminant which we have shown previously inhibits the development of putative preneoplastic gamma-glutamyl transpeptidase (GGT) positive foci in rat liver. Total liver plasma membrane protein kinase activity using both protamine sulfate and histone was cyclic adenosine 3':5'-monophosphate independent and did not appear to be a marker of promotion. Its activity was increased by both DEHP which suppresses the development of GGT positive foci and a CD diet which promotes the appearance of GGT positive foci. The CD, CS plus PHB, and CD plus PHB dietary regimens, which promote the appearance of GGT positive foci, induced the phosphorylation of a Mr 40,000 plasma membrane protein in vitro by endogenous protein kinases. Plasma membranes from DEHP-treated rats did not demonstrate phosphorylation of this Mr 40,000 protein. DEHP dietary treatment also blocked the ability of epidermal growth factor to enhance the phosphorylation of its Mr 175,000 receptor protein in isolated liver plasma membranes. These results suggest that the phosphorylation of a Mr 40,000 plasma membrane protein may be important to the early promotional phase of liver carcinogenesis, and that one mechanism by which DEHP inhibits the emergence of GGT positive foci may be by blocking the response of initiated cells to stimulation by epidermal growth factor.

Concentration-dependent inhibition of development of GGT positive foci in rat liver by the environmental contaminant di(2-ethylhexyl) phthalate.

The ability of di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer and environmental contaminant, to suppress development of putative preneoplastic lesions in rat liver was evaluated. gamma-Glutamyl transpeptidase-positive (GGT+) foci were initiated in the livers of Sprague-Dawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. Promotion of foci was commenced by feeding a choline-deficient diet (CD). A group of control rats was fed a choline-supplemented diet (CS). The ability of DEHP to suppress the emergence of GGT+ foci was evaluated by feeding additional groups of rats the CD diet containing either 0.1%, 0.5%, 1.0% or 2.0% DEHP. The CD diet promoted the number of GGT+ foci above levels in control livers. Inclusion of the plasticizer to the levels of 0.5%, 1.0% and 2.0% in the CD diet effectively inhibited the appearance of the foci. However, DEHP was unable to inhibit the promoting effect of the CD diet at a concentration of 0.1%. DEHP's ability to block development of GGT+ foci correlated with its ability to increase liver weight and to induce carnitine acetyltransferase (EC 2.3.1.7), a marker of peroxisome proliferation.

Inhibition of phenobarbital and dietary choline deficiency promoted preneoplastic lesions in rat liver by environmental contaminant di(2-ethylhexyl)phthalate.

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 Sprague--Dawley 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.