Evaluation of chemopreventive agents for genotoxic activity.

We conducted genetic toxicity evaluations of 11 candidate chemopreventive agents with the potential for inhibiting carcinogenesis in humans at increased risk of cancer. The compounds were evaluated for bacterial mutagenesis in the Salmonella-E. coli assay, for mammalian mutagenesis in mouse lymphoma cells, for chromosome aberrations in Chinese Hamster Ovary (CHO) cells, and for micronucleus induction in mouse bone marrow. Tested agents were indole 3-carbinol (I3C), bowman-birk inhibitor concentrate (BBIC), black tea polyphenols (BTP), farnesol, geraniol, l-Se-methylselenocysteine (SeMC), 5,6-dihydro-4H-cyclopenta[1,2]-dithiol-3-thione(DC-D3T), 4'-bromoflavone, 2,5,7,8-tetramethyl-(2R-[4R,8R,12-trimethyltridecyl] chroman-6-yloxy) acetic acid (alpha-TEA), SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo[2,3-b] carbazole and SR16157 (3-O-sulfamoyloxy-7alpha-methyl-21-(2-N,N-diethylaminoethoxy)-19-norpregna-1,3,5(10)-triene). All these agents, except I3C and BTP, were negative in the Salmonella-E. coli assay in the presence and absence of metabolic activation (S9). I3C and BTP induced a weak mutagenic response in the presence and absence of S9 with strains TA100 and TA98, respectively. Of the three compounds tested in the mouse lymphoma assay (I3C, BBIC, and BTP), only BTP was mutagenic in the presence of S9. In the chromosomal aberration assay, of the 8 compounds that were tested, 4'-bromoflavone elicited a positive response in the absence of S9 only, while SR16157 was positive in the presence of S9. The results with geraniol remain inconclusive. I3C, BBIC and BTP were not tested in the chromosomal aberration assay. None of the 11 agents induced micronuclei in mouse bone marrow erythrocytes.

Evaluation of mutant frequencies of chemically induced tumors and normal tissues in lambda/cII transgenic mice.

Genomic instability has been implicated as an important component in tumor progression. Evaluation of mutant frequencies (MFs) in tumors of transgenic mice containing nontranscribed marker genes should be useful for quantitating mutation rates in tumors as the physiologically inactive transgene provides neither a positive nor a negative selective pressure on the tumor. We have conducted long-term carcinogenicity studies in lambda/cII transgenic B6C3F1 mice using a variety of genotoxic and nongenotoxic test agents and have evaluated the mutant frequencies in both tumors and normal tissues from these animals. Mice were administered diethylnitrosamine (DEN) as three intraperitoneal injections of 15 mg/kg; phenobarbital (PB) or oxazepam (OXP) provided ad libitum at 0.1% or 0.25% in the diet, respectively; DEN initiation plus PB in the diet; or urethane (UTH) provided ad libitum at 0.2% in the drinking water. Normal tissues and tumors were isolated at various times over a 2-year period and half of each tissue/tumor was evaluated histopathologically and the other half was evaluated for MF in the cII transgene. Approximately 20 mutants from each of 166 individual tissues (tumor and nontumor) were sequenced to determine whether increases in MF represented unique mutations or were due to clonal expansion. UTH produced significant increases in MF in normal liver and lung. DEN either with or without PB promotion produced significant increases in MF in liver and correction of MF for clonality produced little change in the overall MF in these groups. PB produced a twofold increase in liver MF over controls after 27 weeks of treatment, but a similar increase was not observed with longer dosing times; at later time points, the MF in the PB groups was lower than that of the control group, suggesting that PB is not producing direct DNA damage in the liver. OXP failed to produce an increase in MF over controls, even after 78 weeks of treatment. Selected cases of genomic instability were observed in tumors from all treatments except OXP, with individual liver tumors showing very high MF values even after clonal correction. One rare and interesting finding was noted in a single mouse treated with UTH, where a mammary metastasis had an MF approximately 10-fold greater than the parent tumor, with 75% of the mutations independent, providing strong evidence of genomic instability. There was no clear correlation between tumor phenotype and MF except that pulmonary adenomas generally had higher MFs than normal lung in both genotoxic and nongenotoxic treatment groups. Likewise, there was no correlation between tumor size and MF after correction for clonality. The results presented here demonstrate that individual tumors can show significant genomic instability, with very significant increases in MF that are not attributed to clonal expansion of a single mutant cell.

Genotoxicity and toxicity of the potential cancer-preventive agent polyphenon E.

The potential health benefits of green tea continue to attract public and scientific interests and are attributed in part to polyphenolic catechin constituents. Polyphenon E (Poly E) is a decaffeinated green tea catechin mixture containing about 50% epigallocatechin gallate and 30% other catechins. We evaluated the toxicity and genotoxicity of Poly E by using two in vitro assays: bacterial mutagenesis in a Salmonella typhimurium-E. coli assay and the L5178Y mouse lymphoma cell thymidine kinase (Tk) gene mutation assay. In addition, we used two in vivo genotoxicity assays: the mouse micronucleus assay and the Big Blue cII transgenic mouse mutation assay. Repeat-dose toxicity evaluations were performed in mice in parallel with the Big Blue transgenic mutation assays. No significant increases in the revertant colonies were found in the bacterial mutagenesis assay, but a significant increase in the mutant frequency (MF) at the Tk locus was observed in the mouse lymphoma test system. We observed toxicity in mice when Poly E was administered at doses of 2,000 mg/kg/day. Lower doses produced no significant increases in micronucleated erythrocytes in the bone marrow of Swiss-Webster mice and no significant increases in cII transgene MF in the liver, lung, or spleen compared with controls. These results indicate that Poly E, although toxic at high doses (2,000 mg/kg/day), poses minimal genotoxic concern. In addition, these studies highlight the importance of using both in vitro and in vivo systems in genetic toxicity screening of pharmaceuticals before they are administered to humans.

Gene expression profiling of mouse host response to Listeria monocytogenes infection.

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.