Pig-a gene mutation assay study design: critical assessment of 3- versus 28-day repeat-dose treatment schedules.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels - either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention. We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

A recombination-based transgenic mouse system for genotoxicity testing.

It is well established that mutagens induce recombination in cultured cells and experimental organisms. Presumably, this is a consequence of the DNA-damage-triggering cellular-repair mechanisms. The relationship between recombination and mutagenicity has been exploited in submammalian organisms, such as yeast, to assay the ability of chemical agents and radiation to induce a form of recombination called gene conversion--the non-reciprocal transfer of genetic information. This work has demonstrated the efficacy of predicting mutagenicity on the basis of recombination induction. Here, we describe the utilization of a transgenic mouse system for efficient detection of germ-line gene-conversion events as a mutagen-screening tool. These mice contain two mutually defective reporter (lacZ) genes under the regulatory control of a spermatogenesis-specific promoter. A particular intrachromosomal gene conversion event must occur for the generation of functional lacZ activity. Conversion events are visualized by histochemical staining or flow cytometric analysis of transgenic spermatids. The highly mutagenic compound chlorambucil induced a several fold percentage-wise increase of lacZ-positive spermatids, whereas acrylamide, a weak genotoxin, produced no marked increase in converted spermatids. The results indicate that recombination-based transgenic mouse models for genotoxin screening present a viable option for inexpensive and rapid whole-animal mutagen testing. The particular mice we describe may ultimately prove to be a useful tool for identifying agents which can cause heritable genetic mutations in humans.

Comparison of treatments for symptomatic Hodgkin's lymphoma employing decision analysis.

A decision analysis methodology has been developed for addressing a comparison of immediate MOPP chemotherapy without staging to staging followed by medically indicated chemotherapy (MOPP) or radiotherapy (XRT). The patients would have symptomatic Hodgkin's lymphoma. Each test and therapy was previously described in terms of 15 toxicity categories. Each was assigned a probability of the occurrence over the five grades of toxicity. Each grade was assigned an expected duration of the toxicity for each test or therapy. Both actual probabilities and judgmental probabilities were used. Utilities for the 15 toxicity categories were solicited. The staging-test-morbidity outcomes only pertained to decision paths which were directed at a chance to receive XRT as the medically dictated therapy. Relative scalar weights were assigned to each grade IV toxicity of zero utility by three physicians. An additive (linear) model was used to compute composite utilities for the paths. There were three different outcomes for these individuals in the initial analysis: 1) MOPP immediately without staging; 2) MOPP immediately or if staging had proceeded to a negative bone marrow then further staging was preferred; and 3) Staging in order to have any change to receive XRT. When a lower toxicity program of presumed equal efficacy was substituted (ChlVPP) the decision changed so that all three participants now would receive ChlVPP instead of staging based on their personal preferences about morbidity outcomes. Decision analysis can contribute to selection between treatments based on differences in an individual's preferences in regard to varying degrees and spectra of toxicity.