Neurological assessments after treatment with the antimalarial ß-arteether in neonatal and adult rats.

The World Health Organization currently recommends combinatorial treatment including artemisinins as first-line therapy against drug-resistant Plasmodium falciparum malaria. Although highly efficacious, artemisinin and its derivatives, including ß-arteether (ßAE), are associated with ototoxicity, tremors, and other autonomic and motor impairments in the clinic. Similar neurological symptoms, as well as brainstem lesions, have been observed in adult laboratory species (mice, rats, dogs, and non human primates) following acute treatment with ßAE; however, few long-term, nonclinical studies have been conducted. Furthermore, the majority of deaths attributed to malarial infection occur in children under age five, yet no laboratory studies have been initiated in neonatal or juvenile animals. In the current study, neonatal 7-day-old rats were administered intramuscular doses of 1-90 mg/kg ßAE in sesame oil for up to eight treatment cycles (one cycle=7 days treatment+7 days without treatment). Neonates were tested for changes in sensorimotor function, and the same animals were tested as adults in the Functional Observational Battery, for motor activity, and in the 8-arm radial maze. Pups receiving a single cycle of 60 or 90 mg/kg died within a week of treatment but had few behavioral changes and no brainstem pathology. In the long-term study, behavioral and motor changes and brainstem lesions were observed in a dose- and time-related manner. Rats given repeated cycles of 1 or 5mg/kg ßAE showed subtle motor abnormalities (e.g., slight loss of righting reflex) while repeated cycles of 10mg/kg ßAE treatment resulted in obvious motor and behavioral changes. Rats receiving 1mg/kg ßAE had no brainstem lesions whereas some rats treated with 5mg/kg ßAE and all rats treated with 10 mg/kg ßAE had brainstem lesions. Brainstem lesions were observed after as few as five cycles and were characterized by gliosis, satellitosis and progressive necrosis in motor neurons of the trapezoid, vestibular, and olivary nuclei. This study shows that repeated treatment with clinically relevant doses of ßAE causes motor deficits associated with brainstem damage in rodents and suggests that repeated treatment with ßAE in children may elicit neurological damage.

WITHDRAWN. Neurological assessments after treatment with the antimalarial ß-arteether in neonatal and adult rats.

The World Health Organization currently recommends combinatorial treatment including artemisinins as first-line therapy against drug-resistant Plasmodium falciparum malaria. Although highly efficacious, artemisinin and its derivatives, including ß-arteether (ßAE), are associated with ototoxicity, tremors, and other autonomic and motor impairments in the clinic. Similar neurological symptoms, as well as brainstem lesions, have been observed in adult laboratory species (mice, rats, dogs, and non human primates) following acute treatment with ßAE; however, few long-term, nonclinical studies have been conducted. Furthermore, the majority of deaths attributed to malarial infection occur in children under age five, yet no laboratory studies have been initiated in neonatal or juvenile animals. In the current study, neonatal 7-day-old rats were administered intramuscular doses of 1-90mg/kg ßAE in sesame oil for up to eight treatment cycles (one cycle=7days treatment+7days without treatment). Neonates were tested for changes in sensorimotor function, and the same animals were tested as adults in the Functional Observational Battery, for motor activity, and in the 8-arm radial maze. Pups receiving a single cycle of 60 or 90mg/kg died within a week of treatment but had few behavioral changes and no brainstem pathology. In the long-term study, behavioral and motor changes and brainstem lesions were observed in a dose- and time-related manner. Rats given repeated cycles of 1 or 5mg/kg ßAE showed subtle motor abnormalities (e.g., slight loss of righting reflex) while repeated cycles of 10mg/kg ßAE treatment resulted in obvious motor and behavioral changes. Rats receiving 1mg/kg ßAE had no brainstem lesions whereas some rats treated with 5mg/kg ßAE and all rats treated with 10mg/kg ßAE had brainstem lesions. Brainstem lesions were observed after as few as five cycles and were characterized by gliosis, satellitosis and progressive necrosis in motor neurons of the trapezoid, vestibular, and olivary nuclei. This study shows that repeated treatment with clinically relevant doses of ßAE causes motor deficits associated with brainstem damage in rodents and suggests that repeated treatment with ßAE in children may elicit neurological damage.

Skin irritation, basal epithelial cell proliferation, and carcinogenicity evaluations of a representative specialty acrylate and methacrylate.

Specialty acrylates and methacrylates (SAM) comprise a large family of industrial monomers. In the late 1980s, the United States EPA and the industry SAM Panel collaborated to evaluate the potential effects, particularly carcinogenesis, of this family of chemicals. As part of this arrangement, the SAM Panel, with EPA input and approval, conducted four studies with a representative acrylate, triethyleneglycol diacrylate (TREGDA), and methacrylate, triethyleneglycol dimethacrylate (TREGDMA). All studies used unoccluded skin application to male mice as follows: Study 1, evaluation of skin irritation compared to cell proliferation in the basal epithelium (BE) following 7 or 14 days of treatment; Study 2, 14-day dose range-finding study; Study 3, 90-day subchronic toxicity study; and Study 4, chronic bioassays employing the EPAs draft guidelines for dermal chronic bioassays. BE cell proliferation was determined in subchronic and carcinogenicity studies (Studies 1, 3, and 4). Organ weight changes (Studies 3 and 4) and increased mortality (Study 4) were observed for the highest dose of TREGDMA. However, there was no related histopathology. Both chemicals induced cell proliferation (7 days through 78 weeks) that correlated with acute and chronic inflammation of the skin. No skin tumors were observed in this study. TREGDA resulted in skin lesions at doses approximately 20-fold lower than TREGDMA. Most of the skin lesions showed similar patterns of microscopic cutaneous alteration suggestive of nonspecific irritation for both chemicals. However, the high concentration TREGDA group in the 78-week study also had evidence of epidermal cell necrosis. In contrast to earlier studies with acrylates, dose selection was based on careful examination of skin irritation and cell proliferation to avoid excessive skin damage. Under these conditions, TREGDA and TREGDMA were not carcinogenic.

Genotoxicity and carcinogenicity studies of soy isoflavones.

The potential cancer preventive efficacy of soy isoflavones is being investigated in preclinical and phase 1 clinical studies sponsored by the U.S. National Cancer Institute. Although 90-day oral toxicity studies with PTI G-2535 (an investigational soy isoflavone drug product) in rats and dogs, as well as teratology studies, indicated no signs of toxicity, there remains a mechanistic concern associated with the ability of isoflavones (i.e., genistein) to inhibit topoisomerase, possibly leading to DNA strand breaks. The present report describes results from two in vitro genotoxicity studies, one in vivo genotoxicity study, and a single carcinogenicity study conducted in p53 knockout mice. Bacterial mutagenesis experiments using six tester strains without metabolic activation revealed no evidence that PTI G-2535 was mutagenic. In similar experiments with exogenous metabolic activation there were statistically significant increases in revertants, but less than twofold, in a single (Salmonella typhimurium TA100) tester strain. Mouse lymphoma cell mutagenesis experiments conducted with and without metabolic activation revealed statistically significant increases in mutation frequency at PTI G-2535 concentrations > or = 0.8 and 12 microg/ml, respectively; such increases were dose related and increases in the frequency of both small and large colonies were observed. A statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was also seen 24 hours after treatment in male, but not female, mice who received 500 and 1000 mg/kg body weight PTI G-2535; however,such increases were small, were not dose related, and were not observed 48 hours after treatment. In contrast, dietary genistein had no effect on survival, weight gain, or the incidence or types of tumors that developed in cancer-prone rodents lacking the p53 tumor suppressor gene, p53 knockout mice. The apparent risk/benefit of isoflavone ingestion may ultimately depend on the dose and developmental timing of exposure.

Genetic toxicology testing of the antimalarial drugs chloroquine and a new analog, AQ-13.

AQ-13 ([N1-(7-chloro-quinolin-4yl)-3-(N3,N3-diethylamino)propylamine] dihydrochloride trihydrate) is an aminoquinoline antimalarial drug that is effective against chloroquine-resistant strains of Plasmodium falciparum. It is structurally similar to the widely used chloroquine diphosphate (CQ). We evaluated these drugs in the three assays currently recommended by the International Conference on Harmonization (ICH): bacterial mutagenesis in Salmonella typhimurium and Escherichia coli, mammalian cell mutagenesis in L5178Y mouse lymphoma cells, and micronucleus induction in rat bone marrow. A small but statistically significant increase in revertant colonies was produced by CQ with Salmonella tester strain TA98 without metabolic activation (MA) and by AQ-13 with strain TA1537 both with and without MA. In L5178Y cells, testing of CQ and AQ-13 up to cytotoxic concentrations with and without MA produced no increase in mutant colonies and no increase in the numbers of small colonies. Slight decreases in the ratio of polychromatic erythrocytes (PCE) to red blood cells (RBC) were observed in male and female rats treated with CQ and in females only treated with AQ-13; however, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of CQ or AQ-13. Although both CQ and AQ-13 showed weak bacterial mutagenicity, this mutagenic effect was not confirmed in either the mouse lymphoma mutagenesis assay or the micronucleus assay. These results indicate that CQ and AQ-13 should pose minimal risk of genotoxic damage in human populations being administered these drugs.

Vaccination with a recombinant vaccinia vaccine containing the B7-1 co-stimulatory molecule causes no significant toxicity and enhances T cell-mediated cytotoxicity.

B7-1 is a co-stimulatory molecule that provides a second signal for T-cell activation. Several studies have demonstrated that vaccination with a vector containing genes encoding B7-1 and an antigen appears to be efficacious at promoting immune responsiveness to the antigen. To evaluate the safety of such a protocol and determine the effect of the B7-1 vector on immune responsiveness, female C57BL/6 mice were administered Wyeth wild-type vaccinia virus (V-WT) or V-WT containing the gene for B7-1 (rV-B7-1) as a single s. c. injection or 3 monthly s.c. injections. Immunologic parameters were evaluated in half of the mice and general toxicity in the other half. Immunologic end points included determination of splenic lymphocyte phenotypes, mitogen-induced T- and B-cell proliferation, T-cell proliferation in response to alloantigens, cell-mediated cytotoxicity (CMC), natural killer cell activity and serum anti-nuclear antibody (ANA) titers. No significant signs of general toxicity were noted. The primary immunologic effect was an increase in the ability of spleen cells to lyse allogeneic targets and to proliferate in response to allogeneic stimulation. Numbers of splenic CD8(+) cells were also increased. These effects were more pronounced after 3 vaccinations than after a single vaccination. Minimal differences in ANA were observed between mice immunized with V-WT and rV-B7-1. In addition, no serum antibodies against B7-1 were detected in any mice. The data suggest that vaccination with rV-B7-1 augments CMC with minimal toxicity.

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control.

Pyridine does not induce unscheduled DNA synthesis (UDS) in hepatocytes of male B6C3F1 mice treated in vivo.

Pyridine was evaluated in an in vivo/in vitro mouse DNA repair assay. Unscheduled DNA synthesis (UDS) was used as an indicator of DNA damage to hepatocytes from male B6C3F1 mice. Test animals were exposed by oral gavage to pyridine or to the vehicle or positive control articles, and hepatocytes were collected and labeled by incubation in media supplemented with [3H]thymidine. Following labeling, the cultures were processed for autoradiographic analysis. Doses were selected based on a pilot study in which 0, 250, 500, 750, 1000 or 2000 mg kg(-1) pyridine in water was administered by gavage. Mice in the 1000 and 2000 mg kg(-1) dose groups were comatose following dosing and died within 24 h of dose administration. Pyridine dose levels for the UDS determination were set at 175, 350 and 700 mg kg(-1). Pyridine solutions in water were administered to mice 2 or 16 h prior to the scheduled sacrifice. The vehicle control group received water 16 h before sacrifice and the positive control group received 10 mg kg(-1) dimethylnitrosamine (DMN) 2 h before sacrifice. Pyridine did not significantly increase the UDS response in hepatocytes isolated from the treated animals, as measured by the incorporation of [3H]thymidine, using standard criteria for a negative response: less than zero mean net grains in repair (NG) and <20% of cells in repair (% IR; cells in repair have at least 5 NG). The vehicle control group and the low, mid- and high pyridine dose groups yielded less than -8.3 NG and < or =1% IR. The positive control group yielded a positive UDS response, with 10.8 NG and 62% IR. These results indicate that pyridine is non-genotoxic in B6C3F1 mouse liver using the UDS endpoint.

Mutational spectrum of dimethylnitrosamine in the liver of 3- and 6-week-old lacI transgenic mice.

We determined the spectrum of mutations in the lacI gene in the liver of Big Blue(R) transgenic mice after exposure to five daily doses of 2 mg/kg dimethylnitrosamine (DMN) at 3 and 6 weeks of age. This dose has been reported to increase the mutant frequency 9-fold when the animals are 3 weeks old. The lacI mutations recovered when treated at 3 weeks consist of mainly G:C --> A:T transitions, predominantly at non-CpG sites, and thus are consistent with mutagenesis by DMN. No increase in mutant frequency was reported when the mice were treated at 6 weeks of age. As we have previously shown that changes in mutational spectrum can be detected even when no statistically significant increase in mutant frequency is seen, we also examined the spectrum after treatment at 6 weeks. No changes from the spontaneous spectrum were detected. The comparison of the outcome of DMN treatment at 3 and 6 weeks confirms a change in metabolic activation, adduct removal, or mutation fixation between 3 and 6 weeks of age.

Toxicity of dolastatin 10 in mice, rats and dogs and its clinical relevance.

PURPOSE: Dolastatin 10 (DOL 10), an oligopeptide isolated from the sea hare Dolabella auricularia, has been shown to be a highly potent cytotoxic agent in a variety of human tumor cell lines. The purpose of this study was to conduct preclinical toxicity evaluations to determine the target organ(s) of toxicity and its reversibility, the dose-limiting toxicity and the maximum tolerated dose (MTD), and to use this information for arriving at a safe starting dose and dose schedule for phase I clinical trails. METHODS: DOL10 was administered as a single intravenous bolus dose to CD2F1 mice, Fischer-344 rats and beagle dogs. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues. RESULTS: The MTD (i. e. the highest dose that did not cause lethality but produced substantial toxicity) was approximately 1350 microg/m(2) body surface area (450 microg/kg) in mice, 450 microg/m(2) (75 microg/kg) in rats and /=1350 microg/m(2) in mice, >/=150 microg/m(2) in rats and >/=400 microg/m(2) in dogs. Decreased weight gain or actual weight loss was observed at doses >/=1350 microg/m(2) in mice, >/=600 microg/m(2) in rats and >/=450 microg/m(2) in dogs. In all three species, the primary target organ of toxicity was the bone marrow, as indicated by decreases in the numbers of erythroid cells, myeloid cells, and megakaryocytes in the femoral bone marrow and by decreased white blood cell (WBC) and reticulocyte counts in peripheral blood. Marked neutropenia (i.e. >50% decrease compared to control animal or baseline values) was the principal effect on WBCs and occurred within a week of dosing. A mild anemia was evident 1 week after administering the drug to rats and dogs. The hematologic effects were transient and reversed by study termination. Other lesions at the MTD levels were cellular depletion and necrosis in lymphoid organs (rats and dogs), marked depletion of extramedullary hematopoietic cellular elements in the spleen (rats), thymic atrophy (mice and dogs), and minimal cellular necrosis in the ileum (rats). More extensive and severe pathology was observed in animals sacrificed in a moribund condition or found dead. CONCLUSIONS: Myelotoxicity was dose-limiting in all three species with mice being the least sensitive. In a phase I clinical trial, granulocytopenia was dose-limiting. Moreover, the MTD of DOL10 for rats and dogs is comparable to the human MTD. Therefore, the results from the preclinical toxicology studies correctly predicted a safe starting dose, the dose-limiting toxicity, and the MTD in humans.

Evaluation of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) following treatment of rats and mice with p-dichlorobenzene.

p-Dichlorobenzene (PDCB) has been reported to produce tumors in the male and female mouse liver and in the male rat kidney in 2-year gavage studies (NPT, 1987). To elucidate the possible mechanisms of carcinogenicity more fully, UDS and RDS were evaluated in B6C3F1 mouse hepatocytes and F-344 rat kidney cells by autoradiography following in vivo administration of PDCB. All corn oil gavage doses of PDCB (300, 600, and 1,000 mg/kg) and the negative control resulted in < 0 net grains/nucleus (NG) in the mouse liver and rat kidney, indicating that PDCB does not induce UDS in either tissue. Compared to controls with < or = 0.29% hepatocytes in S-phase (%S), treatment of mice induced 0.46, 1.90, and 1.52 %S (males) and 2.61, 1.18, and 4.45 %S (females), which indicates that PDCB acts as an inducer of cell proliferation in the liver. In male rat kidney cells, the same doses produced 0.87, 0.67, and 1.01 %S (0.38% in controls) and in females 0.48, 0.43, and 0.32 %S (0.52% in controls), indicating that PDCB induces cell replication in the male but not the female rat kidney. Therefore, these data demonstrate that PDCB is not genotoxic in the mouse liver or rat kidney at single oral doses comparable to the daily doses given in the National Toxicology Program (NTP) bioassay (NTP, 1987). Furthermore, the increases in RDS support the hypotheses that mouse liver tumor formation occurs via stimulation of hepatocyte proliferation and male rat kidney carcinogenesis via increased renal cell proliferation.

Analysis of the mutagenic potential of ENU and MMS in germ cells of male C57BL/6 lacI transgenic mice.

Mutant frequencies in male germ cells were determined in mice 3 days after exposure to saline, methylmethane sulfonate (MMS), or ethylnitrosourea (ENU). DNA was isolated from seminiferous tubules by a modified version of the drop dialysis method. A 5-fold increase in mutant frequency was observed in mice treated with ENU. No statistically significant increase was observed in mice treated with MMS.

Evaluation of positive controls for the in vitro unscheduled DNA synthesis assay using hepatocytes from induced (Aroclor 1254) and uninduced male cynomolgus monkey.

We have evaluated the use of four different positive control compounds for assessing UDS in monkey hepatocytes and have found three of these, methylmethanesulfonate, benzo[a]pyrene, and dimethylbenz[a]anthracene, to produce strong positive responses in vitro. Dimethylnitrosamine induced only weak responses. We also report that the strength of the response induced by procarcinogens was not enhanced in hepatocytes taken from Aroclor 1254-pretreated monkeys, even though substantial induction of cytochrome P450 enzymes was demonstrated in these cells. These studies raise the question of the utility of employing an in vivo induction system to enhance the monkey UDS assay.

Mutagenic response to benzene and tris(2,3-dibromopropyl)-phosphate in the lambda lacI transgenic mouse mutation assay: a standardized approach to in vivo mutation analysis.

The genotoxic response of benzene and tris(2,3-dibromopropyl)-phosphate (TDBP) have been evaluated in several tissues using the standardized lambda/lacI (Big Blue) transgenic mouse mutation assay. Separate groups of four to five male B6C3F1 transgenic lambda/lacI mice were given oral administrations of benzene or TDBP at varying concentrations. Tissues evaluated include lung, bone marrow, and spleen in benzene-treated animals, and liver, kidney, and stomach in TDBP-treated animals. Significant increases in lacI mutations were observed in the spleen and bone marrow of benzene treated mice, and the kidneys of TDBP-treated mice. Where applicable, mutagenesis patterns of tissue sensitivity were consistent with what has been observed previously in other assays. In addition, mutagenicity in tissues not traditionally evaluated for mutations correlated to sites of carcinogenicity for the chemicals tested.

Spectrum of mutations in kidney, stomach, and liver from lacI transgenic mice recovered after treatment with tris(2,3-dibromopropyl)phosphate.

The flame retardant tris(2,3-dibromopropyl)phosphate (TDBP), once used in cotton sleep wear for children, is presently banned from commerce. It produces tumors in rodents in both a sex- and tissue-specific manner. The kidney is the main target for tumor formation in male and female rats, as well as in male mice. In contrast, tumors are formed in the liver of female animals. We have used lacI transgenic male B6C3F1 mice (Big Blue) to examine the induction of mutation in kidney, liver, and stomach after exposure to 150 mg/kg (2 days), 300 mg/kg (4 days), and 600 mg/kg (4 days) of TDBP. At the highest dose, the mutant frequency was approximately 50% above control values in the kidney (P < 0.01). A smaller increase was observed in the liver (P = 0.07), while no increase was seen in the stomach (P = 0.28). Sequence analysis of the recovered mutants showed a TDBP-specific change in mutation spectrum in kidney, which was not observed in liver and stomach. In kidney, a dose-dependent decrease in G:C-->A:T transitions, including at 5'-CpG-3' sites, was observed. This was accompanied by an increase in the loss of single G:C base pairs from approximately 3% to 15%. These results illustrate both the sensitivity and specificity of the lacI transgenic system in the analysis of tissue-specific mutation. This study also reinforces the importance of examining mutational spectra when mutant induction levels are low.

A strategy for the application of transgenic rodent mutagenesis assays.

The past several years have seen an enormous increase in the development and use of transgenic animal models to measure mutations in specific inserted reporter genes. These systems provide gene mutation data in vivo in a wide range of relevant tissues. Numerous laboratories are now using these systems with consistent results. This paper describes the unique niche that transgenic mutagenesis systems can fill in product development and registration strategies. In addition to tissue-specific mechanistic studies, transgenic assays are available to follow up mutagenic effects demonstrated in Salmonella, Escherichia coli, mouse lymphoma (L5178Y) cells, or other in vitro systems.

Transgenic models for detection of mutations in tumors and normal tissues of rodents.

Transgenic rodents that contain easily retrievable target genes allow the rapid quantitation of mutations in any tissue from which DNA can be isolated. We are using the Stratagene Big Blue transgenic mouse system that contains a lacI target and an alpha lacZ reporter gene to study the parameters that affect mutations. We have evaluated a number of chemicals to determine mutant frequency (MF) in specific target tissues of C57Bl/6 and B6C3F1 mice. The correlation between mutagenesis and carcinogenesis in this system is excellent. For example, the liver carcinogen dimethylnitrosamine produces significant increases in MF in mouse liver, whereas the nonhepatocarcinogenic mutagen methylmethane sulfonate does not. We have also evaluated the induction of mutations by radiation and demonstrated that this system is suitable for the study of agents that produce deletion mutations. This system is also useful for studying changes in MF in developing tumors. We have used an initiation-promotion protocol to induce hepatocellular carcinomas, and we then measured MF in normal liver, tumors, and metastases from these mice. Animals initiated with diethylnitrosamine maintain an elevated MF in normal liver, even 1 year after initiation. This MF increases exponentially in developing liver tumors, possibly owing to a breakdown in the fidelity of DNA replication and DNA repair in tumors. This system offers a unique tool for the study of mutations induced in specific target tissues of rodents and should become an important assay for evaluating the mutagenic risk of drugs and chemicals.

Transgenic animal models for detection of in vivo mutations.

Transgenic rodent models for measuring mutations provide a tool for assessing tissue-specific mutations following in vivo treatment. These systems are based on the insertion into the rodent genome Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations. Following in vivo treatment of animals, genomic DNA is isolated from tissues of interest, and the target gene is screened for mutations using either lambda-phage packaging or isolation of the target gene with magnetic affinity capture. In this paper we review the various experimental methods used in the conduct of transgenic mutation assays and discuss critical factors that affect the interpretations of results of these assays.

Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice.

Ionizing radiation induces gene mutations (point mutations, deletions and insertions) as well as chromosome damage in mammalian cells. Although these effects have been studied extensively in cells in culture, until recently it has not been possible to analyze the mutagenic potential of ionizing radiation in vivo, especially at the molecular level. The development of transgenic mutagenesis systems has now made it possible to study the effects of ionizing radiation at both the molecular and chromosomal levels in the same animal. In this report we present preliminary data on the response of Big Blue lacI transgenic mice to ionizing radiation as measured by lacI mutations and micronuclei. C57Bl/6 transgenic mice were irradiated with 137Cs gamma-rays at doses ranging from 0.1 to 14 Gy, and expression times ranging from 2 to 14 days. Dose-related increases in the mutant frequency were observed after irradiations with longer expression times. Mutant plaques were analyzed by restriction enzyme digestion to detect large structural changes in the target sequence. Of 34 gamma-ray-induced mutations analyzed, 4 were large-scale rearrangements. 3 of these rearrangements were deletions within the lacI gene characterized by the presence of short regions of homology at the breakpoint junctions. The fourth rearrangement was a deletion that extended from within the alpha lacZ gene into downstream sequences and that had 43 bp of homology at the junction. These data indicate that the Big Blue lacI transgenic mouse system in sensitive to the types of mutations induced by ionizing radiation. To determine whether the presence of the transgene affects micronucleus induction we compared the response of nontransgenic to hemizygous transgenic B6C3F1 mice and the response of nontransgenic to hemizygous and homozygous transgenic C57Bl/6 mice. The presence or absence of the lacI transgene had no effect on spontaneous micronucleus frequencies for either strain. However, radiation-induced micronucleus frequencies were significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; the converse was true in C57Bl/6 mice. These data suggest that the lacI transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.