Edilberto Bermudez

A protocol and guide for the in vitro rat hepatocyte DNA-repair assay.

The in vitro rat-hepatocyte DNA-repair assay is a valuable tool in assessing the genotoxic activity of chemical agents. An advantage of the assay is that the target cells themselves are metabolically competent, so that the patterns of metabolic activation and detoxification closely reflect those in the whole animal. This article provides a typical procedure and guidelines for conducting the rat in vitro hepatocyte DNA-repair assay.

Distribution of DNA Adducts Caused by Inhaled Formaldehyde Is Consistent with Induction of Nasal Carcinoma but Not Leukemia

Inhaled formaldehyde is classified as a known human and animal carcinogen, causing nasopharyngeal cancer. Additionally, limited epidemiological evidence for leukemia in humans is available; however, this is inconsistent across studies. Both genotoxicity and cytotoxicity are key events in formaldehyde nasal carcinogenicity in rats, but mechanistic data for leukemia are not well established. Formation of DNA adducts is a key event in initiating carcinogenesis. Formaldehyde can induce DNA monoadducts, DNA-DNA cross-links, and DNA protein cross-links. In this study, highly sensitive liquid chromatography-tandem mass spectrometry-selected reaction monitoringmethods were developed and [(13)CD(2)]-formaldehyde exposures utilized, allowing differentiation of DNA adducts and DNA-DNA cross-links originating from endogenous and inhalation-derived formaldehyde exposure. The results show that exogenous formaldehyde induced N(2)-hydroxymethyl-dG monoadducts and dG-dG cross-links in DNA from rat respiratory nasal mucosa but did not form [(13)CD(2)]-adducts in sites remote to the portal of entry, even when five times more DNA was analyzed. Furthermore, no N(6)-HO(13)CD(2)-dA adducts were detected in nasal DNA. In contrast, high amounts of endogenous formaldehyde dG and dA monoadducts were present in all tissues examined. The number of exogenous N(2)-HO(13)CD(2)-dG in 1- and 5-day nasal DNA samples from rats exposed to 10-ppm [(13)CD(2)]-formaldehyde was 1.28 +/- 0.49 and 2.43 +/- 0.78 adducts/10(7) dG, respectively, while 2.63 +/- 0.73 and 2.84 +/- 1.13 N(2)-HOCH(2)-dG adducts/10(7) dG and 3.95 +/- 0.26 and 3.61 +/- 0.95 N(6)-HOCH(2)-dA endogenous adducts/10(7) dA were present. This study provides strong evidence supporting a genotoxic and cytotoxic mode of action for the carcinogenesis of inhaled formaldehyde in respiratory nasal epithelium but does not support the biological plausibility that inhaled formaldehyde also causes leukemia.

Genomic Signatures and Dose-Dependent Transitions in Nasal Epithelial Responses to Inhaled Formaldehyde in the Rat

Repeated and acute exposure studies assessed time and concentration-dependencies of nasal responses to formaldehyde. Exposures were to 0, 0.7, 2, and 6 ppm for 6 h/day, 5 days/week for up to 3 weeks. Neither cell proliferation nor histopathology was observed at 0.7 ppm. At 6 ppm, cell proliferation increased at the end of the first week (day 5), but not at the end of week 3 (day 15). Squamous metaplasia occurred at day 5; epithelial hyperplasia occurred at both day 5 and day 15. In microarray studies, no genes were altered at 0.7 ppm. At 2 ppm, 15 genes were changed on day 5; only half of them were changed at 6 ppm. No genes were changed significantly at 2 ppm at day 15. The pattern of gene changes at 2 and 6 ppm, with transient squamous metaplasia at day 5, indicated tissue adaptation and reduced tissue sensitivity by day 15. The acute study included an additional concentration (15 ppm) and an instillation group (40 microl, 400 mM per nostril). Three times more genes were affected by instillation than inhalation. U-shaped dose responses were noted in the acute study for many genes that were also altered at 2 ppm on day 5. On the basis of cellular component gene ontology benchmark dose analysis, the most sensitive changes were for genes were associated with extracellular components and plasma membrane. With formaldehyde, there are temporal and concentration-dependent transitions in epithelial responses and genomic signatures between 0.7 and 6 ppm. Low concentrations primarily affect extracellular matrix or external plasma membrane portions of the epithelium.

Formaldehyde: Integrating Dosimetry, Cytotoxicity, and Genomics to Understand Dose-Dependent Transitions for an Endogenous Compound

Formaldehyde (FA), an endogenous cellular aldehyde, is a rat nasal carcinogen. In this study, concentration and exposure duration transitions in FA mode of action (MOA) were examined with pharmacokinetic (PK) modeling for tissue formaldehyde acetal (FAcetal) and glutathione (GSH) and with histopathology and gene expression in nasal epithelium from rats exposed to 0, 0.7, 2, 6, 10, or 15 ppm FA 6 h/day for 1, 4, or 13 weeks. Patterns of gene expression varied with concentration and duration. At 2 ppm, sensitive response genes (SRGs)-associated with cellular stress, thiol transport/reduction, inflammation, and cell proliferation-were upregulated at all exposure durations. At 6 ppm and greater, gene expression changes showed enrichment of pathways involved in cell cycle, DNA repair, and apoptosis. ERBB, EGFR, WNT, TGF-β, Hedgehog, and Notch signaling were also enriched. Benchmark doses for significantly enriched pathways were lowest at 13 weeks. Transcriptional and histological changes at 6 ppm and greater corresponded to dose ranges in which the PK model predicted significant reductions in free GSH and increases in FAcetal. Genomic changes at 0.7-2 ppm likely represent changes in extracellular FAcetal and GSH. DNA replication stress, enhanced proliferation, squamous metaplasia, and stem cell niche activation appear to be associated with FA carcinogenesis. Dose dependencies in MOA, high background FAcetal, and nonlinear FAcetal/GSH tissue kinetics indicate that FA concentrations below 1 or 2 ppm would not increase risk of cancer in the nose or any other tissue or affect FA homeostasis within epithelial cells.

Expression of growth factor and growth factor receptor RNA in rat pleural mesothelial cells in culture

Mineral fiber-induced pleural mesothelioma in the rat is a suitable model for asbestos-induced mesothelioma in humans. A proposed mechanism for the genesis of mesotheliomas is the initiation of an autocrine pathway leading to unregulated growth of the mesothelium. To understand if changes in the expression of mRNA of critical growth factors and receptors occur in target mesothelial cells, it is first necessary to characterize the pattern of expression of these genes in normal mesothelial cells. Rat mesothelial cells were isolated from the parietal pleura and strains of these cells were propagated in vitro. The cells were diploid, had epithelial gross morphology and ultrastructure, and coexpressed keratins and vimentin. Northern blot analysis demonstrated that the cells expressed transforming growth factor beta 1 and fibroblast growth factor. Transcripts for transforming growth factor alpha, platelet-derived growth factor A-chain, and platelet-derived growth factor B-chain were not detected. Receptors for platelet-derived growth factor, epidermal growth factor, and insulin were detected. Although normal mesothelial cells express receptors for these growth factors, no production of their corresponding ligands by these cells could be detected, suggesting that autocrine stimulation of growth via the production of such factors may be specific to transformed mesothelial cells.

Examination of potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes

Several long-term studies with 1,4-dioxane (dioxane) have shown it to induce liver tumors in mice, and nasal and liver tumors in rats when administered in amounts from 0.5 to 1.8% in the drinking water (Argus et al. 1965; Kociba et al. 1974; National Cancer Institute, 1978). In order to examine potential mechanisms of action, chemically-induced DNA repair (as an indicator of DNA reactivity) and cell proliferation (as an indicator of promotional activity) were examined in nasal turbinate epithelial cells and hepatocytes of male Fischer-344 rats treated with dioxane. Neither dioxane nor 1,4-dioxane-2-one, one of the proposed metabolites, exhibited activity in thein vitro primary rat hepatocyte DNA repair assay, even from cells that had been isolated from animals given either 1 or 2% dioxane in the drinking water for 1 week to induce enzymes that might be responsible for producing genotoxic metabolites. No activity was seen in thein vivo hepatocyte DNA repair assay in animals given a single dose of up to 1000 mg/kg dioxane or up to 2% dioxane in the drinking water for 1 week. Treatment of rats with 1.0% dioxane in the drinking water for 5 days yielded no increase in liver/body weight nor induction of palmitoyl CoA oxidase, indicating that dioxane does not fit into the class of peroxisomal proliferating carcinogens. The percentage of cells in DNA synthesis phase (S-phase) was determined by administration of3H-thymidine and subsequent quantitative histoautoradiography. The hepatic labeling index (LI) did not increase at either 24 or 48 h following a single dose of 1000 mg/kg dioxane. The LI did increase approximately two-fold in animals given dioxane in the drinking water for 2 weeks. No DNA repair was seen in either nasoturbinate or maxilloturbinate nasal epithelial cells isolated from animals treated with 1% dioxane in the drinking water for 8 days followed by a single dose of up to 1000 mg/kg dioxane by gavage 12 h before sacrifice. Reexamination of the nasal passages of male rats in archived material from the NTP bioassay (National Cancer Institute 1978), revealed that the primary site of tumor formation was the anterior third of the dorsal meatus. The location of these tumors supports the proposal that inhalation of dioxanecontaining drinking water may account for the site specificity of these nasal lesions.In vivo studies showed no increase relative to controls in cell proliferation at the site of highest tumor formation in the nose in response to 1.0% dioxane in the drinking water for 2 weeks. Thus, repair-inducing DNA adduct formation, peroxisomal proliferation in the liver, and short-term induction of cell proliferation in the nose do not appear to be involved in tumor formation by dioxane. There may be a role of dioxane-induced cell proliferation in the formation of the liver tumors. However, the quantitative relationships between induced cell proliferation and tumorigenic potential have yet to be established.

Measurement of tumor-associated mutations in the nasal mucosa of rats exposed to varying doses of formaldehyde.

This study examined the potential induction of tumor-associated mutations in formaldehyde-exposed rat nasal mucosa using a sensitive method, allele-specific competitive blocker-PCR (ACB-PCR). Levels of p53 codon 271 CGT to CAT and K-Ras codon 12 GGT to GAT mutations were quantified in nasal mucosa of rats exposed to formaldehyde. In addition, nasal mucosa cell proliferation was monitored because regenerative cell proliferation is considered a key event in formaldehyde-induced carcinogenesis. Male F344 rats (6-7 weeks old, 5 rats/group) were exposed to 0, 0.7, 2, 6, 10, and 15 ppm formaldehyde for 13 weeks (6 h/day, 5 days/week). ACB-PCR was used to determine levels of p53 and K-Ras mutations. Although two of five untreated rats had measureable spontaneous p53 mutant fractions (MFs), most nasal mucosa samples had p53 MFs below 10(-5). All K-Ras MF measurements were below 10(-5). No dose-related increases in p53 or K-Ras MF were observed, even though significant increases in bromodeoxyuridine incorporation demonstrated induced cell proliferation in the 10 and 15 ppm formaldehyde-treatment groups. Therefore, induction of tumor-associated p53 mutation likely occurs after several other key events in formaldehyde-induced carcinogenesis.

Measurement of genotoxic activity in multiple tissues following inhalation exposure to dimethylnitrosamine

Chemically-induced DNA repair was measured as unscheduled DNA synthesis (UDS) in selected tissues isolated from rats following in vivo exposure to inhaled dimethylnitrosamine (DMN). UDS was evaluated in epithelial cells from rat nasal turbinates and trachea, in hepatocytes and in pachytene spermatocytes from the same treated animal. At nominal concentrations of 500 and 1000 ppm of DMN in air, chemically-induced DNA repair was observed in the epithelial cells of the upper respiratory system. DMN also entered the circulation, as evidenced by a strong DNA-repair response in hepatocytes. No DNA repair was observed in pachytene spermatocytes indicating either that DMN or its active metabolites did not reach the testes in sufficient concentration to induce DNA repair or that the testes lacked the capability to metabolically activate the compound. These results illustrate the potential of this approach to assess the organ-specific genotoxicity of environmental chemicals.

Pulmonary and Pleural Responses in Fischer 344 Rats Following Short-Term Inhalation of a Synthetic Vitreous Fiber I. Quantitation of Lung and Pleural Fiber Burdens

The pleura is an important target tissue of fiber-induced disease, although it is not known whether fibers must be in direct contact with pleural cells to exert pathologic effects. In the present study, we determined the kinetics of fiber movement into pleural tissues of rats following inhalation of RCF-1, a ceramic fiber previously shown to induce neoplasms in the lung and pleura of rats. Male Fischer 344 rats were exposed by nose-only inhalation to RCF-1 at 89 mg/m3 (2645 WHO fibers/cc), 6 hr/day for 5 consecutive days. On Days 5 and 32, thoracic tissues were analyzed to determine pulmonary and pleural fiber burdens. Mean fiber counts were 22 x 10(6)/lung (25 x 10(3)/pleura) at Day 5 and 18 x 10(6)/lung (16 x 10(3)/pleura) at Day 32. Similar geometric mean lengths (GML) and diameters (GMD) of pulmonary fiber burdens were observed at both time points. Values were 5 microns for GML (geometric standard deviation GSD approximately 2.3) and 0.3 micron for GMD (GSD approximately 1.9), with correlations between length and diameter (tau) of 0.2-0.3. Size distributions of pleural fiber burdens at both time points were approximately 1.5 microns GML (GSD approximately 2.0) and 0.09 micron GMD (GSD approximately 1.5; tau approximately 0.2-0.5). Few fibers longer than 5 microns were observed at either time point. These findings demonstrate that fibers can rapidly translocate to pleural tissues. However, only short, thin (< 5 microns in length) fibers could be detected over the 32-day time course of the experiment.

Assessment of the genotoxic effects of methyl chloride in human lymphoblasts

The activity of methyl chloride was measured in 4 genotoxicity assays. In an established human lymphoblast line, a 3-h treatment with 0-5% methyl chloride resulted in a dose-related increase in mutant fraction at the thymidine kinase locus and induction of sister-chromatid exchange. No increase in DNA damage, as measured by alkaline elution, was detected in the lymphoblasts at concentrations of methyl chloride shown to be mutagenic. Also, a concentration-related increase in 8-azaguanine-resistant fraction in Salmonella typhimurium was observed following a 3-h treatment with atmospheres containing 0-20% methyl chloride. Thus, methyl chloride is a weak, direct-acting mutagen for bacteria and human cells in culture.