Yi-Hua Xu

Critical Parameters in the Quantitation of the Stages of Initiation, Promotion, and Progression in One Model of Hepatocarcinogenesis in the Rat

Critical parameters in the quantitation of altered hepatic foci (AHF) developing during multistage hepatocarcinogenesis in the rat include: 1) the enumeration of AHF induced by test agents as well as those AHF occurring spontaneously in livers of untreated animals; 2) the volume percentage or fraction of the liver occupied by all AHF as a reflection of the total number of altered cells within the liver and the degree of tumor promotion which has occurred; and 3) the phenotype of individual AHF as determined by multiple markers with serial sections. These parameters, especially the number of AHF, should be corrected by the presence of spontaneous AHF which increase with the age of the animal, more so in males than females. While accurate estimation of the background level of spontaneous AHF can be important in demonstrating that a carcinogenic agent does not possess the ability to increase the numbers of AHF above the background level, a better method to distinguish the effectiveness and relative potencies of agents as initiators or promoters is reviewed. The relative effectiveness of four different markers–γ-glutamyltranspeptidase (GGT), a placental form of glutathione S-transferase (GST), canalicular ATPase, and glucose 6-phosphatase (G6Pase)–was described for the chemicals C.I. Solvent Yellow 14 and chlorendic acid as promoting agents in males and females. C.I. Solvent Yellow 14 is a more effective promoting agent in females than males, and AHF exhibit extremely low numbers scored by GGT. On the other hand, the numbers of AHF present in livers of male rats promoted by this agent are more than twice those seen in livers of female animals, possibly owing to the effectiveness of this agent as an initiator in the male but not the female. Very few AHF, especially in the male, are scored by GGT during chlorendic acid promotion. The distribution of phenotypes with these markers also differs in the spontaneous AHF appearing in the livers of animals fed 0.05% phenobarbital on either a crude NIH-07 or AIN-76 purified diet. Such studies emphasize the extreme dependence of the promoting stage of hepatocarcinogenesis on environmental factors of sex, diet, and the molecular nature of the promoting agent itself. The hallmark of the final stage of progression in the development of hepatocellular carcinomas is aneuploidy, which may be reflected by phenotypic heterogeneity within individual AHF, termed foci-in-foci. The implications of such quantitative analyses during hepatocarcinogenesis induced by specific agents in relation to the specific action of the agent at one or more of the stages of hepatocarcinogenesis are discussed.

AN INITIATION-PROMOTION ASSAY IN RAT LIVER AS A POTENTIAL COMPLEMENT TO THE 2-YEAR CARCINOGENESIS BIOASSAY

Several pharmaceutical agents, manufacturing chemicals, and environmental contaminants were found to act primarily as promoting agents in an initiation-promotion paradigm. The phenotypic distribution of four enzyme markers--placental glutathione-S-transferase (PGST), gamma-glutamyl transpeptidase (GGT), canalicular ATPase (ATPase), and glucose-6-phosphatase (G6Pase)--was analyzed in altered hepatic foci (AHF) by quantitative stereology. The number and volume distribution of AHF were determined for each promoter tested. For phenobarbital and 2,3,7,8-tetrachloro-p-dioxin, PGST and GGT together scored 100% of the AHF; for 1-(phenylazo)-2-naphthol (CI solvent yellow 14) and chlorendic acid, PGST alone marked 90% of the AHF; after chronic administration of WY-14,643, ATP and G6Pase were the predominant markers. In rats fed tamoxifen, G6P scored more than half of the AHF. Differences in the number of AHF promoted by each of these agents and in their phenotypic distributions may reflect the differentially responsive nature of individual initiated hepatocytes to the action of specific promoters. Since the chronic bioassay of suspected carcinogens does not allow one to differentiate between weak complete carcinogens and those carcinogenic agents that act in a reversible manner to promote the growth of previously initiated cells, the partial hepatectomy, altered-hepatic-focus model of cancer development is proposed as a supplement to the chronic bioassay for the identification of those carcinogenic agents that are primarily, if not exclusively, promoting agents in rat liver.

An improved stereologic method for three-dimensional estimation of particle size distribution from observations in two dimensions and its application

Single enzyme-altered hepatocytes; altered hepatic foci (AHF); and nodular lesions have been implicated, respectively in the processes of initiation, promotion, and progression in rodent hepatocarcinogenesis. Qualitative and quantitative analyses of such lesions have been utilized both to identify and to determine the potency of initiating, promoting, and progressor agents in rodent liver. Of a number of possible parameters determined in the study of such lesions, estimation of the number of foci or nodules in the liver is very important. The method of Saltykov has been used for estimating the number of AHF in rat liver. However, in practice, the Saltykov calculation has at least two weak points: (a) the size class range is limited to 12, which in many instances is too narrow to cover the range of AHF data obtained; and (b) under some conditions, the Saltykov equation generates negative values in several size classes, an obvious impossibility in the real world. In order to overcome these limitations in the Saltykov calculations, a study of the particle size distribution in a wide-range, polydispersed sphere system was performed. A stereologic method, termed the 25F Association method, was developed from this study. This method offers 25 association factors that are derived from the frequency of different-sized transections obtained from transecting a spherical particle, thus expanding the size class range to be analyzed up to 25, which is sufficiently wide to encompass all rat AHF found in most cases. This method exhibits greater flexibility, which allows adjustments to be made within the calculation process when NA((k,k)), the net number of transections from the same size spheres, was found to be a negative value, which is not possible in real situations. The reliability of the 25F Association method was tested thoroughly by computer simulation in both monodispersed and polydispersed sphere systems. The test results were compared with the original Saltykov method. We found that the 25F Association method yielded a better estimate of the total number of spheres in the three-dimensional tissue sample as well as the detailed size distribution information. Although the 25F Association method was derived from the study of a polydispersed sphere system, it can be used for continuous size distribution sphere systems. Application of this method to the estimation of parameters of preneoplastic foci in rodent liver is presented as an example of its utility. An application software program, 3D_estimation.exe, which uses the 25F Association method to estimate the number of AHF in rodent liver, has been developed and is now available at the website of this laboratory.

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.

Building quantitative stereology data files with scion image, a public domain image processing and analysis software

Two-dimensional data obtained from a histological cross-section of a tissue can be utilized to obtain three-dimensional information by the methods of quantitative stereology. The resulting quantitative information is useful in both experimental studies and whole-animal investigations for regulatory and safety purposes. Quantitative stereologic analysis requires considerable data collection and calculation and is thus practical only through the use of computer hardware and software. We have previously reported the development of a program, STEREO, which compiles data from carcinogenesis experiments, recording information from tissue sections for the estimation of the number of altered hepatic foci (AHF) per liver and the volume fraction of AHF in liver on a three-dimensional basis. The data file itself was built by measuring tissue and focal transections through a slide-reading process that involved the manual use of a digitizer. In order to increase the speed and efficiency of the analytical process, we have integrated the STEREO program with a public domain software, Scion Image. This software integration involves two portions: the building macros and the interface. Macros for quantitative stereology used in Scion Image were written to customize and simplify the measurement and to generate data needed for building each of the data files. An interface program, BuildFi.exe, was developed to receive data generated from Scion Image and to align sequential tissue plots from up to four serial sections stained with different markers. As a result, the user can store data on a disk in the format of the STEREO data files. By combining STEREO with Scion Image, the slide-reading process is simplified and can be performed automatically. It has proven to be more objective, time saving, and efficient than all earlier versions.

A method for the comparative study of replicative DNA synthesis in GGT-positive and GGT-negative hepatocytes in primary culture isolated from carcinogen-treated rats

SummaryThe presence of gamma-glutamyl transpeptidase (GGT) in focal nodules of hepatocytes is a commonly used marker for the identification of preneoplastic cell populations. Female Fischer 344 rats were initiated with a single intragastric administration of 200 mg diethylnitrosamine/kg, altered cells were selected after 0.02% 2-acetylaminofluorene was given in the diet; this was followed by a partial hepatectomy and promotion with dietary sodium phenobarbital for 4 wk. A mixed-cell population of GGT-positive and GGT-negative hepatocytes was obtained after collagenase perfusion and Percoll purification. An enriched population of GGT-positive hepatocytes was obtained by a modified “panning” technique. With quantitative scintillation spectrometry and autoradiography of [3H]thymidine incorporation, replicative DNA synthesis of GGT-positive and GGT-negative rat hepatocytes was observed in both the mixed-cell population and the enriched GGT-positive and GGT-negative cell populations. Under the culture conditions used, GGT-positive cells showed a higher level of replicative DNA synthesis than did GGT-negative cells; this indicates that such altered hepatocytes in the stage of promotion possess an inherently greater capacity for all replication, as previously suggested from studies in vivo.

Nuclear-labeling index analysis (NLIA), a software package used to perform accurate automation of cell nuclear-labeling index analysis on immunohistochemically stained rat liver samples

The nuclear labeling index (labeled nuclei/100 nuclei) and the apoptotic index (apoptotic cells/100 cells) are important parameters of cell growth and death. Automatic counting of labeled nuclei is desirable since manual counting is tedious, time-consuming, and with a greater potential for inaccuracies. A nuclear-labeling index analysis (NLIA) software package was developed in this laboratory to perform the counting process automatically and accurately. This software package consists of an application program NLIA and a set of macros for obtaining nuclear data that is used in Scion Image. It is designed to work cooperatively with Scion Image, Adobe Photoshop, and Microsoft Office. NLIA has two basic functions: building nuclear data files and analyzing nuclear data. A color image captured from an immunohistochemically stained or autoradiographic sample is loaded into NLIA. Nuclear data can be entered into the program manually, automatically, or in combination. In the manual data entering mode, NLIA acts as an object-counting tool, while in the automatic mode it acts as a data picker: picking up the data generated by Scion Image into memory. A method to enter nuclear data (both labeled nuclei and unlabeled nuclei) in the automatic mode is described. The color image is processed in Adobe Photoshop, where the interested color ranges are selected and separated. These are then analyzed in Scion Image with the help of the macros for obtaining nuclear data. Since the advanced particle analysis function is used, the counting process is automatic and rapid. Data from thousands of nuclei can be obtained within seconds. To ensure the accuracy of the analysis, a nuclear data checking and edit feature is employed in NLIA: results of computer-generated counting can be compared with the original color image by overlaying the plot of counting results onto the original color image. In this way any computer counting mistakes can be easily discovered and corrected by the operator. Corrected nuclear data (including nuclear size, location, shape) are then stored in data files. These data files can be used in NLIA to obtain cell density and nuclear labeling indices. Because criteria for obtaining nuclear data (truncation diameter, shape factor) can be set by the operator in NLIA, nuclear size distribution and shape variation can be analyzed. This method provides a fast and accurate way to determine cell nuclear-labeling indices. Currently, Scion Image is a freeware on the internet, and NLIA software package is available from our lab home page. Methods presented here expand the Scion Image ability to analyze color images by using color separation techniques in a commercial graphic application. The instrumentation required can be relatively inexpensive, and the methods described may be useful in studies of cell kinetics, lesion growth, and tumor therapy.

CpG PatternFinder: a Windows-based utility program for easy and rapid identification of the CpG methylation status of DNA.

The bisulfite genomic sequencing technique is one of the most widely used techniques to study sequence-specific DNA methylation because of its unambiguous ability to reveal DNA methylation status to the order of a single nucleotide. One characteristic feature of the bisulfite genomic sequencing technique is that a number of sample sequence files will be produced from a single DNA sample. The PCR products of bisulfite-treated DNA samples cannot be sequenced directly because they are heterogeneous in nature; therefore they should be cloned into suitable plasmids and then sequenced. This procedure generates an enormous number of sample DNA sequence files as well as adding extra bases belonging to the plasmids to the sequence, which will cause problems in the final sequence comparison. Finding the methylation status for each CpG in each sample sequence is not an easy job. As a result CpG PatternFinder was developed for this purpose. The main functions of the CpG PatternFinder are: (i) to analyze the reference sequence to obtain CpG and non-CpG-C residue position information. (ii) To tailor sample sequence files (delete insertions and mark deletions from the sample sequence files) based on a configuration of ClustalW multiple alignment. (iii) To align sample sequence files with a reference file to obtain bisulfite conversion efficiency and CpG methylation status. And, (iv) to produce graphics, highlighted aligned sequence text and a summary report which can be easily exported to Microsoft Office suite. CpG PatternFinder is designed to operate cooperatively with BioEdit, a freeware on the internet. It can handle up to 100 files of sample DNA sequences simultaneously, and the total CpG pattern analysis process can be finished in minutes. CpG PatternFinder is an ideal software tool for DNA methylation studies to determine the differential methylation pattern in a large number of individuals in a population. Previously we developed the CpG Analyzer program; CpG PatternFinder is our further effort to create software tools for DNA methylation studies.

CpG Analyzer, a Windows-based utility program for investigation of DNA methylation

656 BioTechniques Vol. 39, No. 5 (2005) There is substantial evidence that changes in DNA methylation occur during preneoplasia, including both global changes in DNA methylation and changes in CpG dinucleotide methylation sites in specific genes (1). Aberrant DNA methylation within CpG islands is one of the earliest and more common alterations in human malignancies (2). Cytosine methylation in CpG dinucleotides has been observed to be an important control mechanism in development and differentiation (3). The bisulfite genomic sequencing technique (4) has found wide acceptance for the generation of DNA methylation status maps with singlebase resolution. This method is based on the selective deamination (induced by bisulfite treatment) of cytosine to uracil, while 5-methylcytosine residues remain unchanged. This bisulfitemodified DNA sequence is amplified by PCR and then sequenced. The uracils in the sequence are detected as thymines on PCR amplification and complement with adenines on formation of the double strand. Methylation status is obtained by the comparison of bisulfite sequence PCR products with the computer-generated bisulfite-modified sequences. Knowledge of the CpG distribution within the sequence, identifying each CpG location, and generating bisulfite-modified sequences are essential in the use of this method, while the ability to highlight CpGs in the sequence text will greatly speed up the process of sequence comparison. Some programs are available to simplify the process (see the MethDB links web page at 195.83.84.240/links. html). Methtools is a program available only on the UNIX® operating system, thus excluding Microsoft® Windows users from employing it (5). CpG Island Searcher is a web site that has a simple user interface to identify CpG islands from submitted sequences (6). However, this program does not supply the detailed CpG location information and CpGhighlighted text that are important for a DNA methylation map with single base resolution. On the Microsoft Windows platform, Anbazhagan et al. used Microsoft Excel® to identify and mark CpG islands (7). CpGs can also be analyzed and highlighted by searching for “cg” in a sequence using most word processing software. However, since the sequence text commonly used is in GenBank® flat file format and consists of line numbers, spaces, and line breaks, the searching process must be performed after these extraneous characters have been eliminated. This involves repeated use of the find and delete commands. Manual CpG analysis and sequence conversion should be avoided, since not only are these timeconsuming, but mistakes are readily introduced. Singal et al. (8,9) used Microsoft Word® macros to simplify the repetative tasks such as removing numbers, spaces, and line breaks, highlighting CpGs, and generating the bisulfite-modified sequences. However, this method does not record or generate CpG location data. Since the valuable numbers and spaces that indicate the location of CpGs are eliminated from the highlighted text, this makes it difficult to find the exact locations of the highlighted CpGs, which is important for manual inspection later, CpG Analyzer, a Windows-based utility program for investigation of DNA methylation

Three-dimensional reconstruction from serial sections in PC-Windows platform by using 3D_Viewer.

Three-dimensional (3D) reconstruction from serial sections allows identification of objects of interest in 3D and clarifies the relationship among these objects. 3D_Viewer, developed in our laboratory for this purpose, has four major functions: image alignment, movie frame production, movie viewing, and shift-overlay image generation. Color images captured from serial sections were aligned; then the contours of objects of interest were highlighted in a semi-automatic manner. These 2D images were then automatically stacked at different viewing angles, and their composite images on a projected plane were recorded by an image transform-shift-overlay technique. These composition images are used in the object-rotation movie show. The design considerations of the program and the procedures used for 3D reconstruction from serial sections are described. This program, with a digital image-capture system, a semi-automatic contours highlight method, and an automatic image transform-shift-overlay technique, greatly speeds up the reconstruction process. Since images generated by 3D_Viewer are in a general graphic format, data sharing with others is easy. 3D_Viewer is written in MS Visual Basic 6, obtainable from our laboratory on request.