Quantitative Evaluation of the Promotion by 2,3,7,8-Tetrachlorodibenzo- p-dioxin of Hepatocarcinogenesis from Diethylnitrosamine1

In order to test the potential of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) as a promoter of hepatocarcinogenesis, rats which had received a single 10-mg/kg dose of diethylnitrosamine (DEN) following partial hepatectomy were given TCDD (0.14 and 1.4 jug/kg s.c. once every 2 weeks) for 7 months. Animals which received (a) only a single initiating dose of DEN after partial hepatectomy and no further treatment of (b) TCDD alone with no initiating dose of DEN exhibited relatively few enzyme-altered foci and no hepatocellular carcinomas. How ever, animals initiated with DEN and then given TCDD had a marked increase in enzyme-altered foci. At the higher dose of TCDD, hepatocellular carcinomas were present in five of seven rats. By means of three different enzyme markers used to evaluate the phenotypes of the enzyme-altered foci, a distinct phenotype heterogeneity of the foci was noted with a shift towards phenotypes exhibiting a greater deviation from normal liver when TCDD was given following DEN-partial hepatectomy. Quantitation of the numbers of enzyme-altered foci was per formed by relating measurements made from two-dimensional tissue sections to the numbers of foci per unit volume of liver using relationships established in the field of stereology. The total volume of the liver occupied by the enzyme-altered foci, but not their number, increased with the dose of TCDD admin istered following DEN-partial hepatectomy. These studies dem onstrate that TCDD is a potent promoting agent for hepatocar cinogenesis. INTRODUCTION TCDD", a trace contaminant formed in the commercial syn thesis of the herbicide, 2,4,5-trichlorophenoxyacetic acid, is an extraordinarily potent toxin and teratogen (33). TCDD is the prototype of a large series of halogenated aromatic hydrocar bons including dibenzo-p-dioxins, dibenzofurans, azoand azoxybenzenes, biphenyls, and naphthalenes which are all approximate isostereomers, produce similar biochemical ac tions, and produce a similar and characteristic pattern of toxic responses (10, 26). These compounds are all thought to exert their toxic action by a common mechanism (24). Recently, TCDD has been shown to be carcinogenic in chronic feeding experiments in rats and mice (15).5 Kociba ef 1This study was supported in part by Grants CA-07175 and CA-22484 from the National Cancer Institute and Grant ES-00965 from the National Institute of Environmental Health Sciences. * To whom requests for reprints should be addressed. 3 Recipient of Research Career Development Award K-04ES-0017. 4 The abbreviations used are: TCDD, 2.3,7,8-tetrachlorodibenzo-p-dioxin; DEN, diethylnitrosamine; i.g., intragastric. 5 P. A. Holmes, J. H. Rust, W. R. Richter, and A. M Shefner. Long-term effects of TCDD and HCDD in mice and rats. Presented at the International Conference on Health Effects of Halogenated Aromatic Hydrocarbons. New York City, New York Academy of Sciences, June 24 to 27, 1978. Received February 22, 1980; accepted June 19, 1980. al. (15) reported that a dietary intake of 0.1 /ig/kg/day for 2 years resulted in an increased number of hepatocellular car cinomas and squamous cell carcinomas of the lung, hard palate, and nasal turbinate in female Sprague-Dawley rats. Lifetime feeding of TCDD equivalent to 0.001 and 0.01 ¿tg/kg/ day produced no increase in tumor incidence in rats. At a daily dose of 0.1 ftg/kg/day, TCDD produces nearly a 50% inci dence in female rats of one of the 3 cancers listed above, making it one of the most potent carcinogenic agents known (23). The carcinogenic potency of TCDD is especially interesting in light of studies which have failed to demonstrate any covalent binding of TCDD (23, 29, 40) and have provided inconclusive evidence that TCDD is a weak mutagen (41). Following the in vivo administration of [3H]TCDD to Sprague-Dawley rats, the radioactivity associated with purified DMA from liver, a maximal estimate of covalent binding, was 6 pmol of TCDD per mol of nucleotide (23). This is 4 to 6 orders of magnitude lower than the binding observed with most chemical carcinogens. It seems unlikely that TCDD-induced oncogenesis is mediated through the covalent binding of this compound to DNA and subsequent somatic mutation. Tumor development in the skin has been shown to occur in 2 stages, initiation and promotion (2, 3, 18, 35). Initiation is an irreversible process, which results from the administration of a carcinogen, and it presumably involves the covalent binding of the carcinogen or an active metabolite to DNA (20). Promotion is a reversible process, influenced by many factors (3, 21, 39), which converts the irreversibly altered, "initiated" cell into a neoplasm. Studies by Peraino ef al. (19) have demonstrated a 2-stage model of carcinogenesis in the liver by the phénobar bital promotion of acetylaminofluorene-initiated hepatic cells. These studies have been confirmed by other investigators (14, 42) and have extended our knowledge of tumor promotion (13). Since TCDD does not appear to be an initiator (i.e., there is no conclusive evidence that it is a mutagen), we considered the hypothesis that the liver cancer associated with chronic administration of TCDD might arise from the promoting activity of the compound, presumably stimulating cells already spon taneously initiated by dietary and other environmental carcin ogens. To study this question, we used a 2-stage model of hepatocarcinogenesis developed in our laboratory (21, 34) based on studies of Peraino, Scherer, Emmelot, and others (1, 8, 19, 28, 31 ). Rats were subjected to a partial hepatectomy to stimulate cell division, and 24 hr later received a single low dose of DEN by intubation. The animals were then treated by chronic administration of a promoting agent, i.e., phénobarbi tal, beginning 1 to 2 months later. After a single low dose of DEN, we demonstrated that chronic dietary administration of phénobarbital resulted in the hepatocellular carcinomas and a marked increase in an enzyme-altered foci (21 ). Such foci had 3616 CANCER RESEARCH VOL. 40 on April 5, 2017. © 1980 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from TCDD Promotion of Hepatocarcinogenesis Table 1 Promoting effect of TCDD on hepatocarcinogenesis by a single dose of DEN and PHa Female rats (200 g) were intubateciwith DENwhere shown. Seven days later, TCDD(injected s.c.) or phénobarbital (0.05% in the diet) administration was begun and was continued for 28 weeks, at which time the animals were sacrificed, and the livers were examined. The low and high doses of TCDD were 0.14 and 1.4 /jg/kg/2 weeks, respectively, administered s.c. DEN was given at a dose of 10 mg/kg. See text for further details Group1234567TreatmentPH + DENPH + TCDD (low dose)PH + TCDD (high dose)PH + phénobarbitalPH + DEN + TCDD(low dose)PH + DEN + TCDD (high dose)PH + DEN + phénobarbitalNo. ofanimals44545710No. of enzyme-al tered foci/cu cmof liver309 ± 98"34 ± 1725 ± 756 ± 131068 ± 166871 ± 66533 ±103Mean volof enzyme-altered foci(cu mm)0.020.050.040.010.080.490.15% liver vo occupiedby foci0.70.20.1 1 .043.06.0N. of ratswithcarci