We previously demonstrated that rats exposed to the peroxisome proliferator (PP) diethylhexylphthalate (DEHP) had reduced serum ceruloplasmin (CP) oxidase activity, which suggests tissue copper deposition. Copper is highly toxic in excess, and results in cellular damage and hepatocellular carcinomas (HCC). This study addresses changes in expression of copper-related genes and metal accumulation in hyperplastic liver and tumors induced by PP. Male rats were fed diets containing DEHP or clofibrate (CLF) for 3-60 days (hyperplasia) and 4-chloro-6-(2,3 xylidino)-2-pyrimidinyl-thio(N-beta-hydroxyethyl) acetamide for 10 months (HCC). During hyperplasia, an immediate and progressive decrease in serum CP activity was observed (P < 0.05), as were reductions in mRNA levels for both CP and Wilson's disease gene (WD gene, a P-type ATPase) (P < 0.05). Tumor-bearing rats had lower serum CP activity (P < 0.05), and CP and WD gene mRNA levels were reduced in tumors (P < 0.05), and in liver surrounding tumors (SL) (P < 0.05). Metallothionein mRNA showed no consistent changes during hyperplasia. Tumors showed a 2.5-fold induction of metallothionein mRNA (P < 0.05), and a 1.2-fold increase in SL. Temporal increases in liver copper content occurred during hyperplasia, with increases of 2-fold (DEHP) and 3.3-fold (CLF) at 60 days (P < 0.05). Copper content was 2.2-fold higher in tumors (P < 0.05) and 1.7-fold higher in SL; iron did not increase and zinc decreased temporally. Thus, copper accumulation and changes in copper-related gene expression may be contributing factors in liver neoplasia in PP-treated rats. Loss of CP results in decreased free radical scavenger capacity and thus may enhance oxidative damage induced by PPs.

Exposure to a common phthalate, di(2-ethylhexyl)phthalate (DEHP), is associated with liver hyperplasia prior to the development of hepatocellular carcinoma in rodents. The exact mechanism of liver hyperplasia as well as tumorigenesis by this agent is not known. Since other lines of evidence point to estrogens as mediators of liver hyperplastic changes, we investigated whether DEHP exposure might alter hepatic estrogen metabolism and induce hyperplasia. Male Fischer 344 rats were fed either control or 1.2% DEHP-containing diets and sacrificed after 4, 8 and 16 weeks of exposure; activities of several sex hormone-responsive markers were measured. Rats fed DEHP had significantly increased serum estradiol levels, but hepatic activity of both cytosolic and nuclear estrogen receptor (ER) was significantly reduced. The serum content of ceruloplasmin, an estrogen-responsive protein synthesized by the liver, was also reduced, perhaps as a consequence of loss of ER activity. The rise in serum estradiol in DEHP-treated rats may be explained by the observation that these rats showed significant losses in hepatic activity of both a major male estrogen-metabolizing enzyme, estrogen 2-hydroxylase, and a male-specific estrogen-sequestering protein. In contrast to reductions in these activities, the expression of proliferating cell nuclear antigen and mRNAs for both ER and fos increased significantly as a result of exposure to DEHP. Our results suggest that changes in estrogen metabolism, receptor activity and activation of genes for cell proliferation are among the earliest metabolic alterations induced by DEHP. These changes together with the induced hyperplasia could play a crucial role in hepatocellular carcinoma development as a result of continuous exposure to DEHP.

ABSTRACT. The nonessential amino acid taurine, which is inert in renal tissue, was used to study renal adaptation in the presence of phosphate (P) depletion in the rat. Weanling rats were placed on the control diet (0.7% P, normal taurine) for 4 wk, then fed one of the experimental diets or continued on the control diet for 1 wk. The diets used were 1 ) P'T-(0.7% P, low taurine), 2) P-T+ (0.1% P, normal taurine); and 3) P-T-(0.1% P, low taurine). Taurine deficiency was associated with avid tubular reabsorption of taurine, irrespective of P status. This was associated with a 4-to 5-fold increase in the V,,, of uptake ( p < 0.001). On the other hand, P depletion increased the Km of taurine uptake by 6.6-to 9.5-fold, suggesting a decrease in the affinity of the taurine symport ( p < 0.001).This was independent of the taurine status of the animals. Although there was no effect of diet on the urinary excretion of 8-alanine, P depletion, irrespective of taurine status, resulted in a 2-fold increase in the Km of 8-alanine uptake. We conclude that the taurinuria of P depletion is reversed in taurine deprivation. The adaptive response involves an increase in the V,,, of uptake. However, the increase in Km of taurine uptake observed in P depletion does not reverse with taurine depletion. (Pediatr Res 30: 146-149, 1991) Abbreviations P, phosphorus BBMV, brush border membrane vesicle HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid P+T-, phosphate replete, taurine-depleted P-T-, phosphate-and taurine-depleted P-T+, phosphate-depleted, taurine replete The renal tubular epithelium is able to conserve amino acids during periods of decreased dietary intake. Rats or mice fed diets low in sulfur-containing amino acids adapt by increasing the tubular reabsorption of these amino acids, particularly taurine (1, 2). The adaptive response manifests at the luminal surface by enhanced taurine uptake by BBMV prepared from rats fed these diets (3). These changes are associated with an increase in the initial rate of uptake (V,,,) rather than a decrease in the affinity of the symport (Km) (3). The physiologic signal(s) for the observed changes relate, in part, to alterations in the renal cortical content of taurine (4, 5). On the other hand, it has been shown that vitamin D-and/or phosphate-depleted rats develop taurinuria in the absence of secondary hyperparathyroidism (6). The taurinuria manifests at the renal brush border membrane by a decrease in the affinity of the taurine symport, resulting in decreased accumulation of taurine by BBMV (7). No changes could be elicited for the V,,, of uptake. In addition, the perturbation in uptake was not associated with alterations in plasma or renal cortex taurine concentrations, suggesting that overflow is not a regulatory signal in this model.Although the adaptive response has been shown to exist in terms of urinary excretion at the apical surface in adult rats, little is known about the response of these animals to taurine deprivation in the presence of phosphate depletion. Herein, we ...