Metabolism of myclobutanil and triadimefon by human and rat cytochrome P450 enzymes and liver microsomes.

Metabolism of two triazole-containing antifungal azoles was studied using expressed human and rat cytochrome P450s (CYP) and liver microsomes. Substrate depletion methods were used due to the complex array of metabolites produced from myclobutanil and triadimefon. Myclobutanil was metabolized more rapidly than triadimefon, which is consistent with metabolism of the n-butyl side-chain in the former and the t-butyl group in the latter compound. Human and rat CYP2C and CYP3A enzymes were the most active. Metabolism was similar in microsomes prepared from livers of control and low-dose rats. High-dose (115 mg kg-1 day-1 of triadimefon or 150 mg kg-1 day-1 of myclobutanil) rats showed increased liver weight, induction of total CYP, and increased metabolism of the two triazoles, though the apparent Km appeared unchanged relative to the control. These data identify CYP enzymes important for the metabolization of these two triazoles. Estimated hepatic clearances suggest that CYP induction may have limited impact in vivo.

Genomic and proteomic techniques applied to reproductive biology.

A report on the Frontiers in Reproduction Symposium 2001 'Reproductive genetics, genomics and proteomics: advances in genetic, molecular and bioinformatics techniques', Cambridge, USA, 30 June to 1 July, 2001.

Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice.

Testicular heat shock was used to characterize cellular and molecular mechanisms involved in male fertility. This model is relevant because heat shock proteins (HSPs) are required for spermatogenesis and also protect cells from environmental hazards such as heat, radiation, and chemicals. Cellular and molecular methods were used to characterize effects of testicular heat shock (43 degrees C for 20 min) at different times posttreatment. Mating studies confirmed conclusions, based on histopathology, that spermatocytes are the most susceptible cell type. Apoptosis in spermatocytes was confirmed by TUNEL, and was temporally correlated with the expression of stress-inducible Hsp70-1 and Hsp70-3 proteins in spermatocytes. To further characterize gene expression networks associated with heat shock-induced effects, we used DNA microarrays to interrogate the expression of 2208 genes and thousands more expression sequence tags expressed in mouse testis. Of these genes, 27 were up-regulated and 151 were down-regulated after heat shock. Array data were concordant with the disruption of meiotic spermatogenesis, the heat-induced expression of HSPs, and an increase in apoptotic spermatocytes. Furthermore, array data indicated increased expression of four additional non-HSP stress response genes, and eight cell-adhesion, signaling, and signal-transduction genes. Decreased expression was recorded for 10 DNA repair and recombination genes; 9 protein synthesis, folding, and targeting genes; 9 cell cycle genes; 5 apoptosis genes; and 4 glutathione metabolism genes. Thus, the array data identify numerous candidate genes for further analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.

Development of a 950-gene DNA array for examining gene expression patterns in mouse testis.

BACKGROUND: Over the past five years, interest in and use of DNA array technology has increased dramatically, and there has been a surge in demand for different types of arrays. Although manufacturers offer a number of pre-made arrays, these are generally of utilitarian design and often cannot accommodate the specific requirements of focused research, such as a particular set of genes from a particular tissue. We found that suppliers did not provide an array to suit our particular interest in testicular toxicology, and therefore elected to design and produce our own. RESULTS: We describe the procedures used by members of the US Environmental Protection Agency MicroArray Consortium (EPAMAC) to produce a mouse testis expression array on both filter and glass-slide formats. The approaches used in the selection and assembly of a pertinent, nonredundant list of testis-expressed genes are detailed. Hybridization of the filter arrays with normal and bromochloroacetic acid-treated mouse testicular RNAs demonstrated that all the selected genes on the array were expressed in mouse testes. CONCLUSION: We have assembled two lists of mouse (950) and human (960) genes expressed in the mouse and/or human adult testis, essentially all of which are available as sequence-verified clones from public sources. Of these, 764 are homologous and will therefore enable close comparison of gene expression between murine models and human clinical testicular samples.

Use of suppression-PCR subtractive hybridisation to identify genes that demonstrate altered expression in male rat and guinea pig livers following exposure to Wy-14,643, a peroxisome proliferator and non-genotoxic hepatocarcinogen.

Understanding the genetic profile of a cell at all stages of normal and carcinogenic development should provide an essential aid to developing new strategies for the prevention, early detection, diagnosis and treatment of cancers. We have attempted to identify some of the genes that may be involved in peroxisome-proliferator (PP)-induced non-genotoxic hepatocarcinogenesis using suppression PCR subtractive hybridisation (SSH). Wistar rats (male) were chosen as a representative susceptible species and Duncan-Hartley guinea pigs (male) as a resistant species to the hepatocarcinogenic effects of the PP, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643). In each case, groups of four test animals were administered a single dose of Wy-14,643 (250 mg/kg per day in corn oil) by gastric intubation for 3 consecutive days. The control animals received corn oil only. On the fourth day the animals were killed and liver mRNA extracted. SSH was carried out using mRNA extracted from the rat and guinea pig livers, and used to isolate genes that were up and downregulated following Wy-14,643 treatment. These genes included some predictable (and hence positive control) species such as CYP4A1 and CYP2C11 (upregulated and downregulated in rat liver, respectively). Several genes that may be implicated in hepatocarcinogenesis have also been identified, as have some unidentified species. This work thus provides a starting point for developing a molecular profile of the early effects of a non-genotoxic carcinogen in sensitive and resistant species that could ultimately lead to a short-term assay for this type of toxicity.

DNA arrays: technology, options and toxicological applications.

The human genome contains an estimated 3 billion bases of DNA making up some 100000 genes, and the variation within this genome accounts for human diversity and, in many cases, disease. Defining and understanding the expression profile of given genotypes is essential to understanding adverse effects from acute or chronic exposure to environmental toxicants or other stimuli. DNA array technology could help researchers understand how organisms function in response to exposure by elucidating the molecular mechanisms that underlie them. DNA arrays have been developed and refined over the past 5 years and matured into a relatively accessible and affordable technology. They vary in design from membrane-based filters with a few hundred cDNAs, to glass-based 'chips' with tens of thousands of genetic elements. Mammalian DNA arrays will soon allow expression analysis on a genome-wide scale, similar to that already accomplished in some lower organisms (e.g. S. cerevisiae, E. coli). These whole-genome arrays will be powerful tools for identifying and characterizing toxicants in environmental and pharmaceutical science. This review discusses the technology behind the production of DNA arrays, the options available to those interested in applying them to their own research, and the possible toxicological applications of this exciting new technology.

Differential gene expression in drug metabolism and toxicology: practicalities, problems and potential.

1. An important feature of the work of many molecular biologists is identifying which genes are switched on and off in a cell under different environmental conditions or subsequent to xenobiotic challenge. Such information has many uses, including the deciphering of molecular pathways and facilitating the development of new experimental and diagnostic procedures. However, the student of gene hunting should be forgiven for perhaps becoming confused by the mountain of information available as there appears to be almost as many methods of discovering differentially expressed genes as there are research groups using the technique. 2. The aim of this review was to clarify the main methods of differential gene expression analysis and the mechanistic principles underlying them. Also included is a discussion on some of the practical aspects of using this technique. Emphasis is placed on the so-called 'open' systems, which require no prior knowledge of the genes contained within the study model. Whilst these will eventually be replaced by 'closed' systems in the study of human, mouse and other commonly studied laboratory animals, they will remain a powerful tool for those examining less fashionable models. 3. The use of suppression-PCR subtractive hybridization is exemplified in the identification of up- and down-regulated genes in rat liver following exposure to phenobarbital, a well-known inducer of the drug metabolizing enzymes. 4. Differential gene display provides a coherent platform for building libraries and microchip arrays of 'gene fingerprints' characteristic of known enzyme inducers and xenobiotic toxicants, which may be interrogated subsequently for the identification and characterization of xenobiotics of unknown biological properties.

Application of DNA arrays to toxicology.

DNA array technology makes it possible to rapidly genotype individuals or quantify the expression of thousands of genes on a single filter or glass slide, and holds enormous potential in toxicologic applications. This potential led to a U.S. Environmental Protection Agency-sponsored workshop titled "Application of Microarrays to Toxicology" on 7-8 January 1999 in Research Triangle Park, North Carolina. In addition to providing state-of-the-art information on the application of DNA or gene microarrays, the workshop catalyzed the formation of several collaborations, committees, and user's groups throughout the Research Triangle Park area and beyond. Potential application of microarrays to toxicologic research and risk assessment include genome-wide expression analyses to identify gene-expression networks and toxicant-specific signatures that can be used to define mode of action, for exposure assessment, and for environmental monitoring. Arrays may also prove useful for monitoring genetic variability and its relationship to toxicant susceptibility in human populations.

The quantitation of lipoprotein lipase mRNA in biopsies of human adipose tissue, using the polymerase chain reaction, and the effect of increased consumption of n-3 polyunsaturated fatty acids.

OBJECTIVE: To examine the effects of the consumption of fish oils on the gene expression of lipoprotein lipase (LPL, EC 3.1.1.34) in human adipose tissue. In order to measure LPL mRNA in adipose tissue samples obtained by needle biopsy from human volunteers a competitive, reverse transcriptase PCR (RT-PCR) protocol was developed. DESIGN: A randomised controlled, single blind cross over dietary study which compared the effects of a low level n-3 polyunsaturated fatty acids (PUFA) using normal foods enriched with eicosapentaenoic (EPA) and docosahexaenoic (DHA) (test diet), with non-enriched but otherwise identical foods (control). The diets were consumed for a period of 22 d with a wash out period of 5 months between the diets. SETTING: Free-living individuals associated with the University of Surrey. SUBJECTS: Six male subjects with a mean (+/- sd) age of 51.2+/-3.6 y were recruited. MAJOR OUTCOME MEASURES: Pre- and postprandial blood samples were taken for the measurement of triacylglycerol (TAG), postheparin LPL activity and adipose tissue samples for the measurement of LPL mRNA levels. RESULTS: Mean LPL expression values were 4.12 x 10(5) molecules of LPL mRNA per ng total RNA on the control diet and 4.60 x 10(5) molecules of LPL mRNA per ng total RNA on the n-3 PUFA enriched (test) diet. There was no significant difference between the levels of LPL expression following each diet, consistent with the lack of change in TAG levels in response to increased dietary n-3 PUFA intake. However, the change in LPL expression (Test-Control diet) correlated significantly with the change in fasting TAG levels (P = 0.03, R = -0.87 and R2 = 0.75) and with the total area under the TAG-time response curve (P = 0.003, R = -0.96 and R2 = 0.92) in individuals. CONCLUSIONS: These findings, although based on a small number of subjects, suggest that LPL expression may be a determinant of plasma TAG levels. The development of this methodology should allow further elucidation of the effects of dietary manipulation and disease processes on lipid clearance and regulation in human subjects.

Five newly established oesophageal carcinoma cell lines: phenotypic and immunological characterization.

The derivation of permanent cell lines from 40 resected oesophageal carcinomas has been attempted. Five long-term lines have been established from three adenocarcinomas, one mixed carcinoma and one squamous carcinoma. Molecular and cellular analyses have been carried out on the lines and clones derived from them. Karyotype analysis indicates genetic variation among the clones. HLA-A, -B and -C is expressed constitutively, but not HLA-DR. ICAM-1-expressing phenotypes may have arisen during adaptation to long-term culture. All lines are capable of response to interferon-gamma (IFN-gamma) and all produce transforming growth factor beta 1 (TGF-beta 1). Two lines are resistant to the inhibitory growth effects of the latter, possibly contributing to malignancy. It is anticipated that these lines, originating from histologically different carcinomas, will provide a valuable, continuous resource for the investigation and treatment of these aggressive tumours.

Molecular profiling of non-genotoxic hepatocarcinogenesis using differential display reverse transcription-polymerase chain reaction (ddRT-PCR).

The technique of differential display reverse transcription-polymerase chain reaction (ddRT-PCR) has been used to produce unique profiles of up-regulated and down-regulated gene expression in the liver of male Wistar rats following short term exposure to the non-genotoxic hepatocarcinogens, phenobarbital and WY-14,643. Animals were treated for 3 days, whereupon their livers were extracted and snap frozen. mRNA was prepared from the livers and used for ddRT-PCR. Individual bands from the differential displays were extracted and cloned. False positives were eliminated by dotblot screening and true positives then sequenced and identified.

Lymphocyte infiltration in oesophageal carcinoma: lack of correlation with MHC antigens, ICAM-1, and tumour stage and grade.

Infiltration by T lymphocytes into oesophageal carcinomas was assessed immunohistochemically, total T lymphocyte numbers by staining for CD3 and activated T lymphocytes by staining for CD25. Five squamous carcinomas and seven adenocarcinomas, resected without neoadjuvant treatment, were studied. Computer aided quantitation showed that total numbers of tumour infiltrating CD3 positive cells were highly variable (range 48-1673 cells/mm2). They were located largely in the stromal (87.9-99.2%) rather than intratumoral regions. Up to 84% of tumour infiltrating T lymphocytes were CD25 positive, although the median figure was 33%. There was no correlation between T lymphocyte infiltration or activation and expression of class I and II histocompatibility antigens, intercellular adhesion molecule-1, tumour stage or grade. These results imply that the local inflammatory response in oesophageal carcinomas is deregulated, which may be a factor contributing to the aggressive nature of the tumours.

Expression of HLA-ABC, HLA-DR and intercellular adhesion molecule-1 in oesophageal carcinoma.

AIM: To examine the expression of HLA-ABC and HLA-DR major histocompatibility (MHC) antigens and intercellular adhesion molecule (ICAM)-1 in normal, inflamed, metaplastic, and neoplastic oesophageal tissue and in freshly disaggregated tumours. METHODS: Sequential sections of frozen tissue and cytospins of freshly disaggregated tumour were stained using the ABC peroxidase system and monoclonal antibodies specific for HLA-ABC, HLA-DR and ICAM-1. RESULTS: Normal oesophageal tissue showed positive staining for HLA-ABC in the basal layers of the oesophageal squamous epithelium and on the epithelial cells of the submucosal oesophageal glands. HLA-DR and ICAM-1 were not detected in either of these cell types. In 20 of 37 (54%) carcinomas HLA-ABC was expressed weakly, with heterogeneous expression in nine (24%). Two tumours showed strong expression of HLA-ABC, but 15 of 37 (41%) were negative. HLA-DR and ICAM-1 were expressed weakly in six of 37 (16%) carcinomas without correlation with each other or with the expression of HLA-ABC. CONCLUSIONS: HLA-ABC is absent from a high proportion of oesophageal carcinomas (41%) and is otherwise variably and weakly expressed with strong expression in only a small fraction (3%). In other carcinomas there is a higher level of HLA-ABC expression. This discrepancy may partly explain the aggressive nature of oesophageal carcinomas. HLA-DR and ICAM-1 are not normally expressed on those cells from which oesophageal carcinomas are thought to arise. The limited expression found here could suggest a partial or inhibited immune response against oesophageal carcinoma. In vivo repressive factors may be involved.