Biomedical subjects

Y P Dragan

Publications and source records attributed to Y P Dragan.

At least 19 recordsLinked to original sources

PubMed2005

Bioinformatics approaches for cross-species liver cancer analysis based on microarray gene expression profiling.

BACKGROUND: The completion of the sequencing of human, mouse and rat genomes and knowledge of cross-species gene homologies enables studies of differential gene expression in animal models. These types of studies have the potential to greatly enhance our understanding of diseases such as liver cancer in humans. Genes co-expressed across multiple species are most likely to have conserved functions. We have used various bioinformatics approaches to examine microarray expression profiles from liver neoplasms that arise in albumin-SV40 transgenic rats to elucidate genes, chromosome aberrations and pathways that might be associated with human liver cancer. RESULTS: In this study, we first identified 2223 differentially expressed genes by comparing gene expression profiles for two control, two adenoma and two carcinoma samples using an F-test. These genes were subsequently mapped to the rat chromosomes using a novel visualization tool, the Chromosome Plot. Using the same plot, we further mapped the significant genes to orthologous chromosomal locations in human and mouse. Many genes expressed in rat 1q that are amplified in rat liver cancer map to the human chromosomes 10, 11 and 19 and to the mouse chromosomes 7, 17 and 19, which have been implicated in studies of human and mouse liver cancer. Using Comparative Genomics Microarray Analysis (CGMA), we identified regions of potential aberrations in human. Lastly, a pathway analysis was conducted to predict altered human pathways based on statistical analysis and extrapolation from the rat data. All of the identified pathways have been known to be important in the etiology of human liver cancer, including cell cycle control, cell growth and differentiation, apoptosis, transcriptional regulation, and protein metabolism. CONCLUSION: The study demonstrates that the hepatic gene expression profiles from the albumin-SV40 transgenic rat model revealed genes, pathways and chromosome alterations consistent with experimental and clinical research in human liver cancer. The bioinformatics tools presented in this paper are essential for cross species extrapolation and mapping of microarray data, its analysis and interpretation.

Animals↗

PubMed2001

Immunohistochemical localization and semi-quantitation of hepatic tamoxifen-DNA adducts in rats exposed orally to tamoxifen.

Administration of tamoxifen (TAM) has been shown to induce hepatocellular carcinogenesis and TAM-DNA adduct formation in rat liver. Here we present TAM-DNA adduct localization and semi-quantitation in hepatic tissue of rats by immunohistochemical staining followed by image analysis. We have also used a quantitative immunoassay to provide a validation for the immunohistochemical values. Rats were fed diets containing 0, 5, 50, 150 or 500 p.p.m. TAM for 45 weeks. Serial sections of paraffin-embedded liver were stained for TAM-DNA adducts using a polyclonal TAM-DNA antiserum. Subsequently, visualization of TAM-DNA adducts was performed by peroxidase-conjugated secondary antibody-mediated signal amplification using biotinyl tyramide followed by streptavidin-alkaline phosphatase and fast red. Semi-quantitation of nuclear color intensity was achieved with an Automated Cellular Imaging System (ACIS), with a detection limit of 1 TAM-DNA adduct per 10(7) nt for these experiments. In parenchymal cells of liver sections from TAM-exposed animals a dose-dependent increase in nuclear staining was observed by ACIS and the TAM-DNA adduct levels determined by ACIS were validated in liver DNA by quantitative chemiluminescence immunoassay (CIA). Comparison of semi-quantitative values determined by ACIS with quantitative values determined by CIA showed a strong correlation (r = 0.924) between the two methods. At 45 weeks of TAM exposure the liver cytoplasm contained placental glutathione S-transferase (GST-p)-positive foci, as indicated by new fuchsin staining. Staining of serial sections revealed a relative lack of TAM-DNA adducts within these enzyme-altered foci. In addition, some GST-p foci contained islands of cells that did not stain for GST-p but were positive for TAM-DNA adduct formation. This study validates the use of ACIS for TAM-DNA adduct formation and demonstrates that steady-state TAM-DNA adduct levels observed in livers of rats chronically fed TAM for several months increase in relation to dose. In addition, unlike the normal surrounding liver, preneoplastic GST-p-positive foci have virtually no TAM-DNA adducts.

Administration, Oral↗

PubMed2001

Implications of apoptosis for toxicity, carcinogenicity, and risk assessment: fumonisin B(1) as an example.

The rates of cell proliferation and cell loss in conjunction with the differentiation status of a tissue are among the many factors contributing to carcinogenesis. Nongenotoxic (non-DNA reactive) chemicals may affect this balance by increasing proliferation through direct mitogenesis or through a regenerative response following loss of cells through cytotoxic (oncotic) or apoptotic necrosis. In a recent NTP study in Fischer rats and B6C3F(1) mice, the mycotoxin fumonisin B(1) caused renal carcinomas in male rats and liver cancer in female mice. In an earlier study in male BD-IX rats, fumonisin B(1) caused hepatic toxicity and hepatocellular carcinomas. An early effect of fumonisin B(1) exposure in these target organs is apoptosis. However, there is also some evidence of oncotic necrosis following fumonisin B(1) administration, especially in the liver. Induction of apoptosis may be a consequence of ceramide synthase inhibition and disruption of sphingolipid metabolism by fumonisin B(1). Fumonisin B(1) is not genotoxic in bacterial mutagenesis screens or in the rat liver unscheduled DNA-synthesis assay. Fumonisin B(1) may be the first example of an apparently nongenotoxic (non-DNA reactive) agent producing tumors through a mode of action involving apoptotic necrosis, atrophy, and consequent regeneration.

Africa↗

PubMed2000

Genome-wide loss of heterozygosity analysis of chemically induced rat hepatocellular carcinomas reveals elevated frequency of allelic imbalances on chromosomes 1, 6, 8, 11, 15, 17, and 20.

Neoplastic development is a multistep process that involves the stochastic accumulation of heritable genetic alterations in proto-oncogenes, DNA repair genes, and tumor suppressor genes. Loss of heterozygosity (LOH) analysis has been used successfully to identify the genetic determinants of neoplastic development, including tumor suppressor genes, in several species and organs but not in the rat liver. We report the results of a sensitive genome-wide LOH analysis of rat hepatocellular carcinomas (HCCs). Heterozygous rats (Wistar-Furth x Fisher 344) were subjected to an Initiation-Promotion-Progression (IPP) protocol of hepatocarcinogenesis. Two weeks after initiation (by partial hepatectomy, 10 mg/kg diethylnitrosamine), the rats were placed on a diet containing 0.05% phenobarbital (PB). After 24 wk of PB promotion, the rats received either 100 or 1 50 mg/kg ethylnitrosourea. Hepatocellular tumors were resected after a total of 76wk of PB promotion. LOH analysis was completed on 26 HCCs by using 60 microsatellite markers covering all 20 rat autosomes and chromosome X. While 85% of the HCCs had one or more allelic imbalances, the average HCC had 3.3 allelic imbalances (range 0-9). A conditional hypothesis-testing method called the Hot-Cold model was used to determine the location of statistically significant elevations in the frequency of allelic imbalances. Elevated allelic imbalances were observed on chromosomes 1q, 6, 8, 11, 15, 17, and 20p. Together, these allelic imbalances suggest that the retinoblastoma and insulin-like growth factor genes as well as the resistance to chemical carcinogenesis (rcc) locus may be involved in HCC development in the rat but that LOH of the p53 gene is not. The elevated rate of allelic imbalances on chromosomes 8,11, and 17 may indicate the location of undiscovered tumor suppressor genes important to neoplastic development in rat liver. Microdissection-based LOH analysis of HCC revealed that contamination of non-neoplastic and nonhepatocellular tissue was not masking LOH in the whole-tumor analysis. There were no statistically significant differences in the frequency of allelic imbalances between HCC of any differentiation state (histological grade). To the degree that it does not reflect differences in etiological factors, the absence of allelic imbalances in chromosomal regions containing the p53 and mamose-6-phosphate/insulin-like growth factor II receptor tumor suppressor genes and the generally low frequency of allelic imbalances in these tumors, suggests that LOH and allelic imbalances play a less significant role in the molecular pathogenesis of HCC in rats than humans.

Alleles↗

PubMed2000

Animal studies addressing the carcinogenicity of TCDD (or related compounds) with an emphasis on tumour promotion.

Dioxin and certain structurally related compounds increase the incidence of liver neoplasms in rodents upon chronic bioassay. Short-term studies indicate the lack of direct DNA-damaging effects including covalent binding to DNA; however, secondary mechanisms may be important in the observed carcinogenicity as these chemicals affect a number of pathways necessary for maintenance of normal growth control and differentiation status. Studies with TCDD in the mouse skin support a lack of initiating activity but an ability to promote the growth of previously initiated lesions indicative of a promoting agent. Mouse skin tumour promotion studies indicate that Ah receptor activation may be involved in promotion by TCDD and selected structurally related compounds. While the mechanism of carcinogenicity induced by TCDD is unknown, the processes involved have a no-effect level, which in the rat liver is at an exposure level below 10 ng TCDD/kg/day. At least for the rodent liver, the relative effective dose for cytochrome P450 induction is not a good indicator of promotion potency. Studies on liver tumour promotion in the female rat liver support a non-genotoxic mechanism for the induction of neoplasms by TCDD. The ability of TCDD to enhance proliferation and inhibit apoptotic processes in focal hepatic lesions further supports an indirect mechanism of carcinogenicity.

Animals↗

PubMed2000

Lessons learned in applying the U.S. EPA proposed cancer guidelines to specific compounds.

An expert panel was convened to evaluate the U.S. Environmental Protection Agency's "Proposed Guidelines for Carcinogen Risk Assessment" through their application to data sets for chloroform (CHCl3) and dichloroacetic acid (DCA). The panel also commented on perceived strengths and limitations encountered in applying the guidelines to these specific compounds. This latter aspect of the panel's activities is the focus of this perspective. The panel was very enthusiastic about the evolution of these proposed guidelines, which represent a major step forward from earlier EPA guidance on cancer-risk assessment. These new guidelines provide the latitude to consider diverse scientific data and allow considerable flexibility in dose-response assessments, depending on the chemical's mode of action. They serve as a very useful template for incorporating state-of-the-art science into carcinogen risk assessments. In addition, the new guidelines promote harmonization of methodologies for cancer- and noncancer-risk assessments. While new guidance on the qualitative decisions ensuing from the determination of mode of action is relatively straightforward, the description of the quantitative implementation of various risk-assessment options requires additional development. Specific areas needing clarification include: (1) the decision criteria for judging the adequacy of the weight of evidence for any particular mode of action; (2) the role of mode of action in guiding development of toxicokinetic, biologically based or case-specific models; (3) the manner in which mode of action and other technical considerations provide guidance on margin-of-exposure calculations; (4) the relative roles of the risk manager versus the risk assessor in evaluating the margin of exposure; and (5 ) the influence of mode of action in harmonizing cancer and noncancer risk assessment methodologies. These points are elaborated as recommendations for improvements to any revisions. In general, the incorporation of examples of quantitative assessments for specific chemicals would strengthen the guidelines. Clearly, any revisions should retain the emphasis present in these draft guidelines on flexibility in the use of scientific information with individual compounds, while simultaneously improving the description of the processes by which these mode-of-action data are organized and interpreted.

Animals↗

PubMed2000

Multiple polypeptide hormone expression in pancreatic islet cell carcinomas derived from phosphoenolpyruvatecarboxykinase-SV40 T antigen transgenic rats.

Transgenic rats carrying a PEPCK-SV40 large T-antigen (TAg) transgene rapidly develop numerous pancreatic islet cell neoplasms, the cells of which express TAg. Although many of the larger neoplasms contain relatively undifferentiated cells, many tumors contain areas of well-differentiated cells with abundant endoplasmic reticulum (ER) and secretory granules for endocrine hormones like those observed in normal pancreatic islets. In the well-differentiated lesions, glucagon-producing alpha-cells, insulin-producing beta-cells, and somatostatin-producing delta-cells are readily identifiable morphologically under the electron microscope. Beta-cells were observed in all normal and hyperplastic islets, and nests of these cells were scattered throughout the larger neoplasms. These nests varied from small clusters of epithelium-like cells that stain intensely for insulin, to sheets of small, basophilic cells that stain more diffusely for the hormone. Alpha-cells were also present in all of the normal and hyperplastic islets, but in larger hyperplastic islets, the peripheral localization was absent. Larger neoplasms contained many nests of glucagon-expressing cells, as well as scattered glucagon-producing single cells. Delta-cells were rarely observed in the hyperplastic islets and in the neoplasms. Blood-glucose levels were unaltered in the transgenic animals relative to their nontransgenic litter mates. Thus although these islet cell neoplasms express several polypeptide hormones, there is no obvious clinical effect of such expression in vivo.

Animals↗

PubMed1999

Mutational analysis of three tumor suppressor genes in two models of rat hepatocarcinogenesis.

An albumin-simian virus 40 (SV40) large T-antigen (T-Ag) transgenic model and a chemically induced model of multistage hepatocarcinogenesis were created in our laboratory to study the molecular mechanisms involved in the genesis and progression of neoplasia in the rat liver. In the study presented here, these two models of rat hepatocarcinogenesis were used to perform a comparative mutational analysis of three tumor suppressor genes involved in hepatic neoplastic growth. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing, exons 5-8 of the p53 tumor suppressor gene and a region between nt 4325 and 4479 of the rat mannose 6-phosphate/insulin-like growth factor 2 receptor (M6p/Igf2r) coding sequence were screened. The latter is homologous to the human M6P/IGF2r coding sequence which is mutated in human hepatocellular carcinoma. A complete single strand conformation polymorphism analysis of the entire coding region of the rat adenomatous polyposis coli (Apc) gene was also performed for the first time in rat tumorigenic samples. Twenty-six chemically induced rat hepatocellular carcinomas, 21 neoplasms from the livers of SV40 T-Ag animals, and five immortalized hepatic cell lines from the transgenic rats were evaluated. None of the hepatic tumors exhibited mutations in the regions analyzed. The albumin-SV40 T-Ag transgenic cell line L-60, derived from normal hepatic tissue, had two mutations in contiguous codons of exon 5 of the p53 gene: a GGT --> GTT missense transversion in codon 183 and a silent mutation in codon 184. The transversion, which may affect the DNA binding domain of the p53 protein, probably originated during cell culture and may have been positively selected because it gave a growth advantage to the mutated cells. The studied region of the M6p/Igf2r gene was not found to be mutated in these two models of rat hepatocarcinogenesis. Although M6p/Igf2r, Apc, and p53 have been shown to be mutated in a variety of human hepatic proliferative diseases, our results indicate that aberrations in these genes may not be necessary for liver carcinogenesis in the rat.

Amino Acid Sequence↗

PubMed1999

Transgene expression and repression in transgenic rats bearing the phosphoenolpyruvate carboxykinase-simian virus 40 T antigen or the phosphoenolpyruvate carboxykinase-transforming growth factor-alpha constructs.

Transgenic Sprague-Dawley rats expressing either human transforming growth factor-alpha (TGFalpha) or simian virus 40 large and small T antigen (TAg), each under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, were developed as an approach to the study of the promotion of hepatocarcinogenesis in the presence of a transgene regulatable by diet and/or hormones. Five lines of PEPCK-TGFalpha transgenic rats were established, each genetic line containing from one to several copies of the transgene per haploid genome. Two PEPCK-TAg transgenic founder rats were obtained, each with multiple copies of the transgene. Expression of the transgene was undetectable in the TGFalpha transgenic rats and could not be induced when the animals were placed on a high-protein, low-carbohydrate diet. The transgene was found to be highly methylated in all of these lines. No pathological alterations in the liver and intestine were observed at any time (up to 2 years) during the lives of these rats. One line of transgenic rats expressing the PEPCK-TAg transgene developed pancreatic islet cell hyperplasias and carcinomas, with few normal islets evident in the pancreas. This transgene is integrated as a hypomethylated tandem array of 10 to 12 copies on chromosome 8q11. Expression of large T antigen is highest in pancreatic neoplasms, but is also detectable in the normal brain, kidney, and liver. Mortality is most rapid in males, starting at 5 months of age and reaching 100% by 8 months. Morphologically, islet cell differentiation in the tumors ranges from poor to well differentiated, with regions of necrosis and fibrosis. Spontaneous metastasis of TAg-positive tumor cells to regional lymph nodes was observed. These studies indicate the importance of DNA methylation in the repression of specific transgenes in the rat. However, the expression of the PEPCK-TAg induces neoplastic transformation in islet cells, probably late in neuroendocrine cell differentiation. T antigen expression during neoplastic development may result in a pervasive change in the islet cell growth properties with selection of a transformed phenotype as a possible requirement for cell viability.

Animals↗

PubMed1999

Quantitative analysis of dose- and time-dependent promotion of four phenotypes of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats.

Determining both the mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts as a tumor promoter and the shape of the dose-response curve at low doses remains an important goal of risk-assessment-directed research. In this report, we extend previous mechanistic and descriptive work done on the effect of TCDD on promotion in the two-stage model of hepatocarcinogenesis, to include lower, more clinically relevant doses. After initiation [PH + 10 mg diethylnitrosamine (DEN)/kg], groups of female Sprague-Dawley rats were administered TCDD in one of four doses: 0.01, 0.1, 1.0, or 10 ng/kg/day for 1, 3, or 6 months. Early increases in liver weight (19-69%) due to hepatocyte hypertrophy were resolved after 3- or 6-months exposures to TCDD, and were not associated with the effects of TCDD on promotion. Non-focal cell proliferation in DEN-treated groups was significantly reduced after 1 or 3 months of exposure to 0.1 ng/kg/day TCDD, leading to U-shaped dose-response curves. TCDD effects on non-focal cell proliferation were not associated with effects on promotion. GSTP-positive AHF represented approximately 97% of the total AHF. Significant increases in both the volume fraction and the number of altered hepatic foci (AHF) were observed at the highest dose (10 ng/kg/day) for GSTP-positive AHF in DEN-treated groups. Increases in the number of G6Pase- and ATPase-deficient AHF/cm3 were observed at TCDD doses as low as 0.01 ng/kg/day. This is the lowest tumor-promoting dose of TCDD reported to date. This study represents an unusually complete data set for further dose-response analysis and simulation or mathematical modeling of TCDD-mediated promotion in the rat liver.

Alkylating Agents↗

PubMed1998

Effects of the antiestrogens tamoxifen, toremifene, and ICI 182,780 on endometrial cancer growth.

BACKGROUND: Tamoxifen has been shown to promote the growth of human endometrial tumors implanted in athymic mice, and it has been associated with a twofold to threefold increase in endometrial cancer. Toremifene, a chlorinated derivative of tamoxifen, and ICI 182,780, a pure antiestrogen, are two new antiestrogens being developed for the treatment of breast cancer. The effects of these drugs on endometrial cancer are currently unknown. Our objective was to evaluate the effects of toremifene and ICI 182,780 on the growth of human endometrial cancer in athymic mice. METHODS: Athymic, ovariectomized mice were implanted with human endometrial tumors and treated with estrogen, tamoxifen, or the new antiestrogens. RESULTS: The effects of tamoxifen and toremifene on the growth of either tamoxifen-stimulated or tamoxifen-naive endometrial tumors in athymic mice were not substantially different. ICI 182,780 inhibited the growth of tamoxifen-stimulated endometrial cancer, in both the presence and the absence of estrogen. CONCLUSIONS: Toremifene and tamoxifen produce identical effects in our endometrial cancer models. Therefore, it is possible that toremifene, like tamoxifen, may be associated with an increased incidence of endometrial cancer. In contrast, ICI 182,780 inhibited tamoxifen-stimulated endometrial cancer, both in the presence and in the absence of estrogen, suggesting that this drug may be safe with regard to the endometrium, even if it is used following tamoxifen, and that it may not result in an increased incidence of endometrial cancer. Indeed, it is even possible that ICI 182,780 may prove useful as an adjuvant agent in early stage endometrial cancer.

Animals↗

PubMed1998

Structure-activity relationships for triphenylethylene antiestrogens on hepatic phase-I and phase-II enzyme expression.

To better understand the mechanism(s) by which tamoxifen induces rat hepatic CYPIIB2 and suppresses GSTA1, structure-activity studies were performed. Compounds employed in these studies included: tamoxifen, fixed-ring tamoxifen, ethylated fixed-ring tamoxifen, pyrrolidino-tamoxifen, 4-iodotamoxifen, idoxifene, and toremifene. With respect to GSTA1 suppression, tamoxifen, fixed-ring tamoxifen, 4-iodotamoxifen, idoxifene, and toremifene were all potent suppressors of GSTA1, while ethylated fixed-ring tamoxifen and pyrrolidino-tamoxifen were completely without activity. The results suggest that the aminoethoxy side chain plays a crucial role in GSTA1 suppression, and that 4-iodination may potentiate this activity. With respect to induction of CYPIIB2, tamoxifen, fixed-ring tamoxifen, and ethylated fixed-ring tamoxifen were inducers of this enzyme, while toremifene and 4-iodotamoxifen were inactive, suggesting that the aminoethoxy side chain is not a structural determinant of CYPIIB2 induction. Because ethylated fixed-ring tamoxifen, toremifene, and 4-iodotamoxifen had differential activities in the two assays, we conclude that CYPIIB2 induction and GSTA1 suppression by triphenylethylenes are the result of two separate and distinct mechanistic pathways. Structure-activity relationships for GSTA1 suppression and CYPIIB2 induction were compared with previously published relationships for triphenylethylene: 1) estrogen receptor relative binding affinity; 2) calmodulin antagonism; 3) antiuterotrophic activity; and 4) antagonism of MCF-7 cell growth. No clear correlation was observed between the effects on CYPIIB2 and these other four activities, suggesting no relationship between the mechanisms responsible for these effects. Similarly, no precise correlation was observed between GSTA1 suppression and these other activities, although rough similarities were observed for relative binding affinity and antiuterotrophic activity. This suggests that the mechanisms responsible for CYPIIB2 induction and GSTA1 suppression are not related to the mechanisms of action for these other documented activities, and may represent different mechanistic pathways.

Animals↗

PubMed1998

Effect of chronic administration of mestranol, tamoxifen, and toremifene on hepatic ploidy in rats.

The nonsteroidal antiestrogen tamoxifen increases the incidence of rat liver cancer through a variety of mechanisms. To compare the effects of tamoxifen (TAM) and a structurally similar analog toremifene (TOR) on rat liver, we determined the ploidy distribution for hepatocytes isolated from rats treated for 18 months with these antiestrogens or the estrogenic compound mestranol (MS). Female Sprague-Dawley rats were subjected to a 70% partial hepatectomy and administered the solvent, trioctanoin, or diethylnitrosamine (10 mg DEN/kg). After a 2-week recovery from the surgery, the rats were administered a basal diet or one containing TAM (250 or 500 ppm), TOR (250, 500, or 750 ppm), or MS (0.2 ppm) for 18 months. Pathologic changes in the liver were examined in the 15-22 rats per treatment group at the 18-month time point. An increased incidence of hepatocellular carcinomas (HCC) was detected in the 500 ppm TAM group, but not with the other treatments that did not include DEN. Both TOR and TAM promoted formation of DEN-initiated HCCs. At sacrifice, four to five rats per group were perfused and the hepatocytes isolated and cultured. Karyotypic analysis was performed on colcemid-blocked cells after 2 days in culture. The hepatic ploidy distribution was characterized in Giemsa-stained metaphase spreads. These studies indicated that chronic treatment with TAM alone resulted in a shift from tetraploid to diploid, as was also observed for rats treated once with DEN. TOR and MS alone did not cause this change in hepatic ploidy at the doses examined. A shift toward an increased content of diploid hepatocytes occurred in all rats treated once with DEN followed by TAM, TOR, or MS. These results indicate that tamoxifen administration results in a shift toward growth of diploid hepatocytes, thus contributing to its carcinogenic action in the rat liver.

Animals↗

PubMed1998

STEREO: a program on a PC-Windows 95 platform for recording and evaluating quantitative stereologic investigations of multistage hepatocarcinogenesis in rodents.

The most common organ site of neoplasms induced by carcinogenic chemicals in the rodent bioassay is the liver. The development of cancer in rodent liver is a multistage process involving sequentially the stages of initiation, promotion, and progression. During the stages of promotion and progression, numerous lesions termed altered hepatic foci (AHF) develop. STEREO was developed for the purpose of efficient and accurate quantitation of AHF and related lesions in experimental and test rodents. The system utilized is equipped with a microcomputer (IBM-compatible PC running Windows 95) and a Summagraphics MICROGRID or SummaSketch tablet digitizer. The program records information from digitization of single or serial sections obtained randomly from rat liver tissue. With this information and the methods of quantitative stereology, both the number and volume percentage fraction of AHF in liver are calculated in three dimensions. The recorded data files can be printed graphically or in the format of tabular numerical data. The results of stereologic calculations are stored on floppy disks and can be sorted into different categories and analyzed or displayed with the use of statistics and graphic functions built into the overall program. Results may also be exported into Microsoft Excel for use at a later time. Any IBM-compatible PC capable of utilizing Windows 95 and MS Office can be used with STEREO, which offers inexpensive, easily operated software to obtain three-dimensional information from sections of two dimensions for the identification and relative potency of initiators, promoters, and progressors, and for the establishment of information potentially useful in developing estimations of risk for human cancer.

Animals↗

PubMed1998

The effect of short-term fasting, phenobarbital and refeeding on apoptotic loss, cell replication and gene expression in rat liver during the promotion stage.

Previous work from this laboratory has reported on the effects of two sequential 5 day periods of fasting and subsequent refeeding on tumor promotion in multistage hepatocarcinogenesis in the rat (Carcinogenesis, 18, 159-166, 1997). In the present extension of the earlier study, the sequential fasting-refeeding regimen was begun at later time points (28 and 54 days post-initiation) than the first study. This was done to determine whether larger-sized altered hepatic foci (AHF) exhibited a depletion similar to that of the relatively small AHF in the published experiment and to study concomitant molecular changes during the fasting periods. Groups of animals were fasted in the presence and absence of 0.05% phenobarbital (PB) in the drinking water. During the fasting periods, both body and liver weights decreased dramatically, less in the fast begun at 54 days. This change was accompanied by a significant decrease in the bromodeoxyuridine (BrdU) labeling indices of hepatocytes within AHF. Apoptotic bodies increased dramatically in the non-focal (surrounding the AHF) hepatocytes during the fasting periods. These parameters were slightly lower in hepatocytes of rats administered PB during the fasting periods, most notably during the 54-66 day period. With the nick end-labeling method, the proportion of hepatocytes undergoing apoptosis was significantly higher in cells within AHF at the end of each of the fasting periods in all but one group. Concomitantly, the number of AHF and percentage of liver volume occupied by AHF decreased dramatically during the fasting periods. Refeeding caused a marked increase in BrdU labeling in hepatocytes within and surrounding AHF during the first week or two, most notably in animals not receiving PB during the fasting period. Both the number and volume percentage of liver AHF returned to control values within approximately 2 weeks of the refeeding regimen. Assays of nuclear DNA fragmentation with samples of whole liver indicated that a 'laddering' effect was most noticeable in livers of animals subjected to the fasting-refeeding regimen when phenobarbital was not present during the fasting period. Studies of the levels of mRNA of several genes in the total liver revealed that the expression of c-myc increased 3- to 9-fold during the fasting periods but rapidly returned to normal levels after refeeding. Levels of albumin and insulin-like growth factor I mRNAs decreased significantly during the fasting period, but rapidly reappeared on refeeding. These results indicate that the extensive loss of AHF during the short-term fasting periods occurs even when the number and volume of AHF are 10- to 50-fold greater at the beginning of the fast than the values published previously. Both the decrease in insulin growth factor I and the elevation of c-myc expression during the fasting period may indicate the role of these genes in the transcriptional regulation of hepatocyte apoptosis in both normal and preneoplastic hepatocytes in the rat.

Actins↗

PubMed1998

Implications of hormesis on the bioassay and hazard assessment of chemical carcinogens.

Hormesis has been defined as a dose-response relationship which depicts improvement in some endpoint (increased metabolic rates, reduction in tumor incidence, etc.) at low doses of a toxic compound followed by a decline in the endpoint at higher doses. The existence of hormetic responses to carcinogenic agents has several implications for the bioassay and hazard assessment of carcinogens. To be capable of detecting and statistically testing for hormetic or other nonlinear dose-response functions, current study designs must be modified to include lower doses and sufficiently large numbers of animals. In addition, improved statistical methods for testing nonlinear dose-response relationships will have to be developed. Research integrating physiologically-based pharmacokinetic model descriptions of target dose with mechanistic data holds the greatest promise for improving the description of the dose-response curve at low doses. The 1996 Proposed Carcinogen Risk Assessment Guidelines encourage the use of mechanistic data to improve the descriptions of the dose-response curve at low doses, but do not distinguish between the types of nonlinear dose-response curves. Should this refined approach lead to substantial support for hormesis in carcinogenic processes, future guidelines will need to provide guidance on establishing safe doses and communicating the results to the public.

Animals↗

PubMed1997

Specific chromosomal changes in albumin simian virus 40 T antigen transgenic rat liver neoplasms.

Hepatocytes isolated from 3-month-old female rats bearing the albumin promoter/enhancer SV40 T antigen construct as a transgene demonstrated a 20% aneuploidy rate and a significant duplication of chromosome 1. Other chromosome changes were observed but were not statistically significant. At this time in the development of hepatic lesions, only a relatively small number of microscopic altered hepatic foci could be noted. By contrast, hepatocytes isolated from the age-matched nontransgenic controls demonstrated only 1% aneuploidy. One hundred % of the metaphase spreads isolated from hepatocellular neoplasms in transgenic rats were aneuploid. Although there were many random changes, 70% of the neoplastic cells demonstrated an amplification of all or portions of chromosome 1q. Only 2% of the neoplastic cells had both a trisomy and a duplication. The smallest region of chromosome 1 that was duplicated was that between bands q3.7 and q4.3. A loss of chromosome 3 was detected in 50% of the neoplasms, as well as a loss of chromosome 6 in 72% of the neoplastic cells. The carcinomas with the highest proliferation rate had also lost at least one copy of chromosome 15 in 70% of the cells. The loss of chromosomes 3, 6, and 15 indicates that these regions may harbor one or more tumor suppressor genes. The amplification of a specific region of chromosome 1 is thus the first karyotypic alteration that can be identified in hepatocytes from livers from which hepatic neoplasms will arise. This indicates that expression or repression of one or more genes in this region may confer a growth advantage to preneoplastic hepatocytes, facilitating their transit to the neoplastic state in the stage of progression. Changes in chromosomes 3, 6, and 15 that occur subsequent to duplication of the q3.7-q4.3 region of chromosome 1 are changes possibly reflecting alteration of tumor suppressor genes with further enhancement of neoplastic growth.

Animals↗

PubMed1997

Quantitative stereological studies of a 'selection' protocol of hepatocarcinogenesis following initiation in neonatal male and female rats.

A modified initiation-selection procedure for neonatal male and female rat hepatocarcinogenesis were examined utilizing the methods of quantitative stereology. In this study, diethylnitrosamine (10 mg DEN/kg) was given a few days after birth. At weaning, the rats were fed 0.02% 2-acetylaminofluorene (AAF) for 2 weeks with a mitotic stimulus [70% partial hepatectomy (PH)] after 1 week on the diet. Quantitative stereological analyses in conjunction with the use of several enzyme markers were used to determine the number and volume of altered hepatic foci (AHF) detected at 1 week, 3 months and 7 months after the selection procedure. This format resulted in an equivalent number of AHF in male and female rats. The AHF were three times larger in males than in females 1 week after discontinuation of AAF administration. Three months after the selection procedure, the number of AHF had decreased by at least a third and their volume percentage was the same in male and female rats. After 7 months, the number and volume fraction of detectable AHF in females were comparable to those which had been observed at 1 week after selection. In the male, the number but not the volume fraction were similar at 7 months compared with 1 week after selection. Both initiation with DEN and selection with AAF/PH contribute independently to the total population of AHF in male and female rats. At least half of the AHF detected 7 months after the selection protocol were due to DEN administration alone. Rats receiving only the AAF/PH selection exhibited one third of the number of AHF observed with the complete protocol. Administration of a non-necrogenic dose of DEN to neonatal rats when coupled with the AAF/PH selection procedure resulted in a significant promotion of the growth of initiated hepatocytes at 1 week, 3 months or 7 months after the selection procedure. These studies demonstrated that (i) the number of AHF detected after a non-necrogenic dose of DEN during the first week of life with subsequent AAF/PH selection after weaning decreases within the first 3 months after the selection procedure, but can re-develop with a promotion stimulus; (ii) the AAF/PH selection procedure itself may initiate hepatocytes in the absence of DEN administration; (iii) the AAF/PH selection procedure is equally effective with respect to the number of AHF observed after phenobarbital promotion in weaning male and female rats initiated near birth.

2-Acetylaminofluorene↗