The relative response to various initiating doses of diethylnitrosamine (DEN) and dimethylbenz[a]anthracene of the induction of numbers and size (vol. % of liver) of altered hepatic foci (AHF) in livers of adult female rats of the Sprague-Dawley and Fischer 344 (F-344) strains was studied by methods of quantitative stereology in the presence and absence of the promoting agent, phenobarbital (PB, 0.05% in the diet). In all cases, a relatively linear response with dose, even at the lowest doses employed, was obtained except for the numbers of AHF at the highest dose of DEN (30 mg/kg), which was not significantly different from that at a dose of 10 mg/kg in F-344 female rats. Similar dose-response data were obtained at various doses of two promoting agents effective in hepatocarcinogenesis, PB and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in livers of F-344 female rats following initiation with DEN (10 mg/kg) 24 h post-70% hepatectomy. The response to these agents exhibited threshold levels below which no increase in number or vol. % of liver of AHF was noted in comparison with that in livers of animals not treated with the promoting agents. At several subthreshold doses of both PB and TCDD an inhibition of AHF formation and growth (measured as vol. % of liver) was observed. Based on quantitative stereologic calculations, parameters for the estimation for the relative potency of chemicals as initiating or promoting agents have been established. These are defined as: initiation index = no. of foci induced X liver-1 X [mmol/kg body wt]-1 and promotion index = Vf/Vc X mmol-1 X weeks-1, where Vf is the total volume fraction (%) occupied by AHF in the livers of rats treated with the test agent and Vc is the total volume of AHF in control animals which have only been initiated. These parameters were calculated for a number of agents based on data published in the literature and from those reported herein. Neither parameter varied significantly with the dose of the initiating agent based on the data in this paper. The range of promotion indices extended over more than eight orders of magnitude, whereas that of initiation indices was much less variable. Such parameters may be useful as quantitative estimates of the potency of hepatocarcinogenic agents, such values having potential application to risk estimations.

The monoterpene d-limonene has been shown to an effective, non-toxic chemopreventive agent in mammary and other rodent tumor models. The studies reported here investigated structure-activity relationships among limonene and three hydroxylated derivatives in the prevention of dimethylbenz[a]anthracene (DMBA)-induced mammary cancer. Rats were fed control or 1% limonene, carveol, uroterpenol or sobrerol diets from 2 weeks before to one week after carcinogen administration. Carveol, uroterpenol and sobrerol significantly prolonged tumor latency and decreased tumor yield. Sobrerol was the most potent of the monoterpenes tested, decreasing tumor yield to half that of the control, a level previously achieved with 5% limonene diets. Excretion of radioactivity from [3H]DMBA was doubled in rats fed 5% limonene and nearly tripled in rats fed 1% sobrerol. Sobrerol is thus 5-fold more potent than limonene in both enhancing carcinogen excretion and in preventing tumor formation. These data demonstrate that hydroxylation of monoterpenes affects chemopreventive potential, with 2 hydroxyl groups greater than 1 greater than 0. Sobrerol, carveol and uroterpenol are novel cancer chemopreventive agents with little or no toxicity.

Two forms of vitamin E, tocopherol and tocotrienol, were tested for chemopreventive activity in two chemically induced rat mammary-tumor models. When mammary tumors were induced by 7,12-dimethylbenz(a)anthracene (DMBA, 50 mg/kg), only the tocotrienol group had a statistically significant increase in tumor latency. There was no effect of either compound on tumor multiplicity. When tumors were induced by N-nitrosomethylurea (NMU, 30 mg/kg), neither analogue of vitamin E modified latency, whereas tocotrienol increased tumor multiplicity. In summary, neither vitamin analog had a major impact on mammary-tumor development after tumor induction with either DMBA or NMU.

The advent of transgenic technology (1) has created a methodology for the production of pharmaceuticals but isolation of these proteins from the blood of transgenic animals is limited by the volume that can be safely removed and the possible detrimental effects of these proteins on the animals' physiology (2). The concept of the mammary gland as a bioreactor has spurred investigation into production oftransgenes with mammary specificity, as milk is easily collected from lactating animals and protein production can reach as high as 1 kg/day in cattle and 200 g/day in goats (3, 4). (NIH 3T3 cells only). Goat mammary epithelial (GME) cells were isolated from early-lactation goats as follows. Parenchymal tissue (Q15 g) was finely minced with scissors and digested overnight in a shaker incubator at 37C in DMEM supplemented with collagenase (2 mg/ml, type I; Worthington). After addition of 4.5 gg of DNase (DN 25; Sigma), the digestion flask was returned to the incubator for 15 min. The tissue suspension was centrifuged, supernatant was removed, and remaining cell pellet was resuspended in complete DMEM. This procedure was repeated three times.The cellular fiagments were then passed over a 53-pm pore-size filter with epithelial cells located in small ductal fragments remaining on the filter. The epithelial cells (GME cells) were removed from the filter and maintained in DMEM/2% fetal calf serum/gentamycin/amphotericin B (2.5 pug/ml). Cells were kept at 370C with 5% C02/95% air.Establiment of Producer Cell Lines. To obtain a higher retroviral titer, the parental vector pJR-gal (12) was digested with Sal I and Xho I to remove the neomycin-resistance gene and promoter. A construct carrying the resultant JR-gal neo- (Fig. 1) was transfected into the ecotropic packaging cell line ACre by particle bombardment (15) at 1 pig of DNA per mg ofgold beads. Two days after bombardment, the supernatant was removed from these cells and centrifuged, and after the addition of Polybrene at 4 j&g/ml, the retroviral solution was used to infect both amphotropic and GaLV pseudotype packaging cell lines, PA317s and PG13/LN c8s, respectively. PA317 and PG13/LN c8 cells producing retrovirus containing Abbreviations: hGH, human growth hormone; GaLV, Gibbon ape leukemia virus; MoMLV, Moloney murine leukemia virus; GME cell, goat mammary epithelial cell; X-Gal, 5-bromo-4chloro-3-indolyl P-D-galactopyranoside; CFU, colony-forming unit(s); EB, estradiol benzoate; P, progesterone; LTR, long terminal repeat. tTo whom reprint requests should be addressed. 6840The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The purpose of this study was to determine if increasing dietary fat, either as saturated fat or polyunsaturated fat, would alter initiation of hepatocarcinogenesis by diethylnitrosamine (DEN) or 2-acetylaminofluorene (AAF). Rats were fed one of three purified diets: a low-fat (LF) diet (containing 5% of calories as safflower oil), a high saturated fat (HSF) diet (containing 48% of calories as palm oil) and a high polyunsaturated fat (HPUF) diet (containing 48% of calories as safflower oil). Four weeks later, all rats were subjected to partial hepatectomy (PH). Rats were then divided into four groups and received no carcinogen, DEN (10 mg/kg, p.o., 24 h after PH) or AAF (25 or 100 mg/kg, p.o., 12 h after PH). Five days later, all rats were fed an unrefined diet, and 9 weeks later, all rats were fed phenobarbital in the diet for 26 weeks as a tumor promoter. In rats initiated with DEN, the number of gamma-glutamyl transpeptidase-positive and ATPase-negative foci was higher in the rats fed the HPUF diet, but not the HSF diet, as compared to rats fed the LF diet. The incidence of neoplastic nodules, the mean focal volume and the volume fraction, however, were not significantly altered by dietary fat in DEN-injected rats. The dietary fat content of the diet did not affect the induction of altered hepatic foci or neoplastic nodules in rats initiated with AAF or receiving no initiation. This study shows that initiation of hepatocarcinogenesis can be influenced by dietary fat, but that the effect may be carcinogen-specific.