James D. Yager

Pancreatic carcinoma in azaserine-treated rats: Induction, classification and dietary modulation of incidence

Pancreatic carcinomas have been induced in Wistar and W/LEW rats by administration of total azaserine doses of 150–520 mg/kg by injection or oral routes over periods of 5–52 weeks. The latent period for development of invasive carcinomas was 1–2 years, but focal abnormalities in acinar cells appear earlier. The incidence of carcinomas varied with total dose, route, and schedule of azaserine administration. The spectrum of histologic patterns of the carcinomas included well and poorly differentiated acinar cell, ductlike, and undifferentiated carcinomas. Rats fed a purified diet developed more pancreatic neoplasms than rats fed a commercial laboratory chow. Selective feeding of these diets during the administration of carcinogen and following completion of carcinogen treatment indicated that the inhibitory effect of chow on pancreatic carcinogenesis was exerted during the postinitiation phase. Supplementation of diet with 0.025% retinyl acetate during the postinitiation phase also inhibited the progression of azaserine‐induced lesions in the pancreas.

Heat-shock-induced enhanced reactivation of UV-irradiated Herpesvirus

The objective of this study was to compare the ability of heat shock (HS) with that of another type of cellular stress, UV irradiation, to cause the induction of enhanced viral reactivation, a process that may represent an SOS-type repair process in mammalian cells. Studies performed to evaluate the effect of HS on growth of Vero cells revealed that HS at 45 degrees C for 45 min caused inhibition of cell growth similar to that caused by UV irradiation at 12 J/m2, but this inhibition was not observed at HS treatment for 5-15 min, or at a UV fluence of 2 J/m2. Enhanced reactivation of UV-irradiated Herpesvirus was observed in cells which had been pretreated by HS for greater than 30 min or UV at 12 J/m2. The synthesis of new proteins following HS for 15 and 45 min and UV at 12 J/m2 was examined by [35S]methionine-labeling experiments. The new synthesis of two HS proteins with molecular weights of 46 000 and 78 000 was induced by both levels of HS, but to a much greater extent at the high dose. These proteins were not detected in response to UV irradiation. These results indicate that, like UV irradiation, HS at levels inhibitory to cell growth induced enhanced viral reactivation in Vero cells. The results also suggest that at least two proteins in the HS protein family are not necessary for this response to occur.

Reduced formation of depurinating estrogen–DNA adducts by sulforaphane or KEAP1 disruption in human mammary epithelial MCF-10A cells

Sulforaphane (SFN) is a potent inducer of detoxication enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase (GST) via the Kelch-like erythroid-derived protein with CNC homology-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) signaling pathway. NQO1 reduces the carcinogenic estrogen metabolite, catechol estrogen-3,4-quinone, whereas GSTs detoxify it through conjugation with glutathione. These 3,4-quinones can react with DNA to form depurinating DNA adducts. Thus, SFN may alter estrogen metabolism and thus protect against estrogen-mediated DNA damage and carcinogenesis. Human breast epithelial MCF-10A cells were treated with either vehicle or SFN and either estradiol (E2) or its metabolite 4-hydroxyestradiol (4-OHE2). 4-Hydroxy-derived estrogen metabolites and depurinating DNA adducts formed from E2 and its interconvertable metabolite estrone (E1) were analyzed by mass spectrometry. Levels of the depurinated adducts, 4-OHE1/2-1-N3Adenine and 4-OHE1/2-1-N7Guanine, were reduced by 60% in SFN-treated cells, whereas levels of 4-OCH3E1/2 and 4-OHE1/2-glutathione conjugates increased. To constitutively enhance the expression of Nrf2-regulated genes, cells were treated with either scrambled or siKEAP1 RNA. Following E2 or 4-OHE2 treatments, levels of the adenine and guanine adducts dropped 60-70% in siKEAP1-treated cells, whereas 4-OHE1/2-glutathione conjugates increased. However, 4-OCH3E1/2 decreased 50% after siKEAP1 treatment. Thus, treatment with SFN or siKEAP1 has similar effects on reduction of depurinating estrogen-DNA adduct levels following estrogen challenge. However, these pharmacologic and genetic approaches have different effects on estrogen metabolism to O-methyl and glutathione conjugates. Activation of the Nrf2 pathway, especially elevated NQO1, may account for some but not all of the protective effects of SFN against estrogen-mediated DNA damage.

Metabolism of the liver tumor promoter ethinyl estradiol by primary cultures of rat hepatocytes

Previously, we reported that relatively high micromolar concentrations of the liver tumor promoter 17 alpha-ethinyl estradiol (EE2) stimulated DNA synthesis and enhanced the DNA synthetic response to epidermal growth factor (EGF) in primary cultures of female rat hepatocytes [J.D. Yager, B.D Roebuck, T.L. Paluszcyk, and V.A. Memoli, Carcinogenesis 7, 2007-2014 (1986); Y.E. Shi and J.D. Yager, Cancer Res. 49, 3574-3580 (1989)]. In this study, our goal was to examine the metabolism of EE2 in cultured hepatocytes. After 4, 24, and 48 hr of culture, hepatocytes maintained their ability to convert up to 95% of a 4 nM concentration of [3H]EE2 to polar conjugates within 4 hr. EE2 at 2 microM was also 95% metabolized within 4 hr. HPLC analysis of the metabolites confirmed the rapid disappearance of [3H]EE2 and the formation of polar conjugates as detected by organic extraction. HPLC separation of hydrolyzed conjugates indicated that the major aglycone was the parent compound, EE2. In general, the metabolites differed both qualitatively and quantitatively from those reported in vivo in the rat. The rapid metabolism of EE2 by hepatocytes in culture may, at least in part, explain the high concentrations of EE2 required to stimulate DNA synthesis in cultured hepatocytes and to potentiate the response to EGF.

Expression of c-raf-1 and A-raf-1 during differentiation of 3T3-L1 preadipocyte fibroblasts into adipocytes

The objective of this study was to examine the expression of the c-raf-1 and A-rat-1 protooncogenes during differentiation of 3T3-L1 preadipocytes into adipocytes. At confluence, prior to initiation of differentiation c-raf and A-raf steady state mRNA levels were low. Expression of c-raf and A-raf began to increase 72 hours following initiation of differentiation by treatment with differentiation medium, reaching a maximum increase of 3 to 6-fold and 3 to 4-fold respectively by 190 hours. The increase of c-raf and A-raf steady state message levels occurred concomitant with the onset of differentiation as indicated by increased levels of glycerol-3-phosphate dehydrogenase mRNA. These changes were compared with those for several other protooncogene mRNAs including c-myc, c-fos, H-ras and histone H3. These results are the first to show increase expression of the raf protooncogenes during terminal differentiation rather than in association with proliferation.

Enhancement in rats by the liver tumor promoter ethinyl estradiol of a serum factor(s) which is stimulatory for hepatocyte DNA synthesis

Fractionation of female rat serum or plasma on Sephadex G-200 revealed the presence of an activity stimulatory for hepatocyte DNA synthesis. Treatment of female rats with the liver tumor promoter ethinyl estradiol (EE) at 2.5 micrograms/day caused a 1.6 fold increase in the level of this activity at 24 hr in both serum and plasma. The stimulatory activity had a molecular weight of 135 kD, was sensitive to trypsin and heating and was not inhibited by the antiestrogen tamoxifen or antibody to epidermal growth factor (EGF). However, the pooled active fractions from EE-treated rats competed to a greater extent than comparable fractions from control rats for specific [125I]-EGF binding to rat liver membranes. These results demonstrate that treatment of female rats with EE, under conditions known to stimulate liver growth, caused an increase in level of a factor(s) stimulatory for hepatocyte DNA synthesis and whose activity may be mediated through the EGF receptor.

Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. Rats were subjected to a two-thirds hepatectomy followed 20 h later by i.p. injection of N-OH-AAF. 4 h after carcinogen injection, it was found that N-OH-AAF caused a dose-dependent inhibition of [3H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery beginning 28 h after carcinogen treatment. Radioimmunoassay of deoxyguanine-C8 adducts remaining in liver DNA indicated that the recovery began prior to detection of adduct removal. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF (Zurlo and Yager, 1984), were cultured under conditions that promote replicative DNA synthesis. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. [3H]Thymidine pulse times and subsequent chase times were adjusted to equalize amounts of DNA synthesis in control and UV-irradiated cells. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. Pulse-chase studies revealed that subsequent joining of nascent chains in UV-irradiated hepatocytes occurred at a rate comparable to or faster than controls and that this could be inhibited by caffeine. The results obtained from both the in vivo and in vitro studies show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with our previous studies demonstrating the ability of UV-irradiated hepatocytes to carry out enhanced reactivation of UV-irradiated herpes virus lend support to the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis.

Oncogene Activation and Expression during Carcinogenesis in Liver and Pancreas

Knowledge of the carcinogenic process has been dramatically increased by the application of recombinant DNA technology and the discovery that mutated cellular genes capable of causing neoplastic transformation can be isolated from spontaneously appearing and carcinogen-induced human and experimental animal tumors. That single genes can cause neoplastic transformation was first demonstrated in studies conducted on acutely transforming retroviruses.1,2 Such viruses, the first being the Rous sarcoma virus, were shown to carry single genes, termed oncogenes (see Chapter 13), that were responsible for their transforming ability. It was also clearly shown that these retroviral oncogenes had homology to cellular genes and were in fact initially derived from cellular genes by retroviral transduction. The cellular homologs of the retroviral oncogenes are referred to as cellular oncogenes or proto-oncogenes. To date, about 20 retroviral oncogenes, each originating from a cellular precursor proto-oncogene, have been identified.3

Plasma Selenium Protein P Isoform 1 (SEPP1): A Predictor of Selenium Status in Nepalese Children Detected by Plasma Proteomics.

Selenium deficiency or excess may have public health consequences, yet selenium status is infrequently characterized in populations, perhaps due to challenges in methodology. We are seeking to identify plasma proteins, using proteomics discovery and validation approaches, to serve as proxies for micronutrient status, including selenium, which may in the future be more readily assessed by robust, affordable field methods. In a sample of rural Nepalese children 6 - 8 years old (n = 500), the prevalence of selenium deficiency was 13.6 and 60.9 % at plasma selenium concentrations < 0.60 and < 0.89 µmol/L, respectively, assessed by atomic absorption spectroscopy. Relative abundance of selenoprotein P isoform 1 (SEPP1), glutathione reductase-3, and apolipoprotein A2 from discovery-based experiments was correlated with plasma selenium with a false discovery rate < 10 % (i. e., q < 0.10), all with p < 0.001. In linear mixed effects regression models to predict plasma selenium, only SEPP1 was significant (R2 = 0.63), estimating 8.2 % (95 % CI: 3.9 - 12.6) and 65.5(61.4 - 69.7)% of the in-sample population as deficient at each respective cut-off. Targeted quantification of SEPP1 in a preliminary series of specimens (n = 19) as a validation of the discovery approach revealed a high correlation with plasma selenium (r = 0.757, p = 0.0002). Plasma proteomics can identify valid plasma protein indicators of micronutrient status, as shown with selenium, comprising a step toward making population assessment of selenium status in vulnerable groups more accessible.