Simone C. Paillet

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells II. Test validation and interpretation

The L5178Y Mouse Lymphoma TK assay was studied extensively to determine if this mammalian cell assay for gene mutations at the thymidine kinase (TK) locus could provide valid, interpretable determinations of mutagenic potential, and whether this information is of value in the safety evaluation of chemicals. We first determined that test-derived TFTR mutants were phenotypically stable, possessing little or no thymidine kinase activity as measured by labeled thymidine uptake, but demonstrating 100% cross resistance to bromodeoxyuridine. Common solvent vehicles such as acetone, dimethylsulfoxide and ethanol were shown to produce little cytotoxicity and no mutagenic activity when present at 1% levels. Out of a total of 10 noncarcinogens tested, all were negative when results were analyzed by a 2-sample loge t test on control and treated mutant count means. Of the 13 putative animal carcinogens tested, 10 were positive, 2 were negative (auramine O and sodium phenobarbital), and 1 showed sporadic activity (hydrazine sulfate) in the TK assay on the basis of test-derived t statistics. 2 compounds, 1,2-epoxybutane and ICR 191, which have been described as Ames positive non-carcinogens, were also positive in the TK assay. Although this sampling of a total of 29 compounds is insufficient for precise estimations of expected false-positive or false-negative frequencies, these data indicate the TK assay can be expected to detect a majority of carcinogens as mutagens including some missed by more established point-mutation assays.

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells: I. Application to genetic toxicological testing

We have systematically evaluated the mouse lymphoma TK+/- leads to TK-/- mutagenesis assay to determine if this somatic-cell test system would be a useful addition to the routine screening battery already used in our laboratory for the detection of chemical mutagens. During these investigations we observed that, with certain modifications of the basic assay, mutagenicity data could be obtained in as little as 9 days once the relative cytotoxic properties of the test substance were known. By improving the culturing conditions, we were able to reduce the serum requirements by as much as 50--75% without appreciably altering either cell viability or the recovery of chemically-induced mutants. Phenotypic stability of test-derived trifluorothymidine resistant (TFTR) mutants was confirmed by demonstrating cross-resistance to bromodeoxyuridine and concomitant sensitivity to methotrexate (THMG) in TFTR cells grown for 20 generations under non-selective conditions. While reduced growth rates resulting from temporary cell-division delay in treated cells is probably not a contributing factor to the observed mutation frequencies, only TFTR colonies which formed large distinct colonies in the presence of trifluorothymidine were clearly phenotypically stable mutants when spontaneous mutants were isolated and verified. When a non-mutagen, a weak mutagen, and a well-established mutagen were compared at equitoxic doses under these modified conditions, clear quantitative differences were seen in the respective mutation frequencies induced by these 3 types of agents. With these technical modifications, we feel this assay is both reliable and amenable to the screening of diverse chemical compounds for point-mutational activity in a mammalian cell.

Induction of trifluorothymidine-resistant mutants by metal ions in L5178Y/TK+/− cells

7 inorganic metal salts including magnesium chloride, cadmium chloride, nickel chloride, zinc chloride, cobalt(II) chloride, lead acetate, sodium arsenate, and the platinum coordination complex, trans-platinum(II) diaminedichloride, were tested for the potential to induce trifluorothymidine-resistant (TFTRes) mutants in L5178Y/TK+/- mouse lymphoma cell by directly exposing cells to varied doses of each compound for 3 h. Of these 8 chemicals, cadmium chloride, nickel chloride, and trans-platinum(II) diaminedichloride consistently produced dose-related increases in the absolute number of TFTRes mutants as well as increases in mutation frequencies at compound concentrations permitting greater than 20% survival. Trans-platinum(II) diaminedichloride was a particularly effective mutagen, comparable to the direct-acting mutagen, methyl methanesulfonate. 15 representative TFTRes mutant cell clones derived from cultures originally treated with either the cadmium, or nickel, or platinum compounds were first grown out for 7 days in nonselective medium, then verified as phenotypically stable TK-/- mutants by demonstrated cross-resistance to 5-bromodeoxyuridine and 100% sensitivity to the folate antagonist methotrexate in THMG medium. These results demonstrate that the soluble salts of 2 metals reported to be human carcinogens and 1 noble metal complex known to bind DNA are all mammalian cell mutagens as well.

The activation of procarcinogens to mutagens by cultured rat hepatocytes in the l5178y/tk mutation assay.

Whole cell preparations derived from collagenase-treated rat liver were cocultivated overnight with stationary (non-shaking) cultures of L5178Y/TK+/- cells in the presence of 8 different chemicals selected as representative aromatic amine, polycyclic hydrocarbon, or nitrosamine procarcinogens. When tested in the presence of hepatocytes, 2-aminoanthracene, 2-aminofluorene, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, 3-methylcholanthrene, and benzo[a]pyrene all produced substantial dose-dependent increases in trifluorothymidine-resistant variants compared to solvent controls after 20 h total exposure time. Only N-nitrosodipropylamine (DPrN) and N-nitrosodiethylamine (DEN) produced any dose-related mutagenic activity in similar experiments where hepatocytes were omitted; however, the response for the DPrN was quite variable at high doses in the absence of hepatocytes and the mutagenic response for the DEN was consistently enhanced at all dose levels by the presence of hepatocytes. Benzanthracene was not active in the presence of whole hepatocytes, even when tested with cells from a rat pretreated 24 h earlier with 20 mg/kg benzanthracene. Excepting benzanthracene, these data suggest that rat hepatocytes can be used to active 3 types of procarcinogens to mutagens in the L5178Y/TK gene mutation assay.

Ascorbate is not detectably mutagenic in the L5178Y TK+ − cell mutation assay

Both sodium ascorbate and ascorbic acid were tested at millimolar concentrations in the mouse lymphoma L5178Y TK+/- cell for chemically-induced cytotoxicity and the induction of gene mutations at the thymidine kinase locus as detected by increased trifluorothymidine-resistance. Neither chemical caused any dose-related increases in trifluorothymidine resistance, even at toxic levels. Increased hydrogen ion concentration was not itself a contributing factor to ascorbic acid toxicity. Ascorbate toxicity was due to products formed in vitro in the absence of cells via chemical reactions with medium components. The formation or persistence of these toxic substances could be prevented by co-incubation with catalase prior to the addition of L5178Y cells. These results suggest that ascorbic acid would not be a mammalian cell mutagen normal physiological conditions.

Hamster hepatocyte-mediated activation of procarcinogens to mutagens in the l5178y/tk mutation assay.

Eight procarcinogens including three nitrosamines, three polycyclic hydrocarbons, and two aromatic amines were tested for mutagenic potential at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells co-cultivated with viable hamster hepatocytes. All eight chemicals produced substantial mutagenic activity as indicated by increased trifluorothymidine resistance in L5178Y cells treated in the presence of hepatocytes. Mutagenic responses to benzo[alpha]pyrene, 3-methylcholanthrene, N-nitrosodiethylamine, and N-nitrosodipropylamine first increased, then plateaued within the range of mutagen concentrations tested, while consistent dose-dependent increases in mutant frequencies were observed following 2-aminoanthracene, 2-aminofluorene, or N-nitrosodimethylamine treatments. The relatively flat portions of the mutant frequency curves for benzo[alpha]pyrene and 3-methylcholanthrene coincided with maximum chemical solubility as obvious from visible or microscopically detectable precipitate. These hamster cells readily facilitated the metabolism of 1,2-benzanthracene to a detectable mutagen and were especially competent in the activation of the two aromatic amines. Thus, cultured hamster hepatocytes can activate a variety of chemical carcinogens including polycyclic hydrocarbons to mutagens in a whole cell-mediated in vitro assay using L5178Y/TK+/- cells as the target organism.

The metabolism of N-acetyl-2-aminofluorene to a mutagen in L5178Y/TK+/− mouse lymphoma cells

Direct treatment of L5178Y mouse lymphoma TK+/- cells with N-acetyl-2-aminofluorene (AAF) from two commercial sources produced small, but reproducible increases in mutant frequency over background in the absence of exogenous microsomal enzymes. Unlike most direct-acting mutagens which typically produce regular, dose-dependent increases in mutant frequency; AAF treatment caused very slight dose-related increases or a saturation phenomenon which could be overcome by increased exposure time. Direct mutagenicity following prolonged (24h) exposure was confirmed when a third highly purified (99.9%) AAF sample was tested. Microsomal enzyme analyses of disrupted L5178Y cell preparations revealed negligible benzo[a]pyrene hydroxylase but measurable AAF-N-hydroxylase activity. These data demonstrate that L5178Y mouse lymphoma cells are capable of limited metabolism of AAF to an active mutagen.

A Phase I Open Label Study of the Farnesyltransferase Inhibitor CP-609,754 in Patients with Advanced Malignant Tumors

Purpose: The purpose of this phase I clinical trial was to determine the maximum-tolerated dose and toxicity of CP-609,754 in patients with solid tumors refractory to standard therapies, to determine the cellular effects of CP-609,754 on its molecular target (farnesyltransferase), and to determine the recommended phase II dose (RP2D) of this agent. Experimental Design: Consenting patients with adequate bone marrow, liver, and renal function were enrolled with an accelerated dose strategy with single-patient parallel cohorts in whom the drug was given orally either once or twice daily. Once a dose-limiting toxicity was encountered or two patients developed Common Toxicity Criteria ≥ grade 2 toxicities, a modified Fibonacci sequence was initiated. Blood samples were collected during cycle 1 for pharmacokinetic and pharmacodynamic analyses. Results: A total of 68 cycles of CP-609,754 was administered to 21 patients enrolled in this study. The dose escalation was from 20 mg once daily to 640 mg twice per day, and at the highest dose level, one of six patients developed a dose-limiting toxicity of grade 3 neuropathy. The drug was otherwise well tolerated, and the maximum-tolerated dose was not reached because of the large number of tablets that would have been required for additional dose escalation. Pharmacokinetic analyses showed a proportional increase in exposure with dose, rapid oral absorption, and a half-life of ∼3 hours. Pharmacodynamic results predict a 95% maximal inhibition of peripheral blood mononuclear cell farnesyltransferase activity 2 hours postdose, on average, with a dose of 400 mg twice per day of CP-609,754. Conclusions: On the basis of the safety findings and the pharmacokinetic and pharmacodynamic analyses, the RP2D of CP-609,754 is ≥640 mg twice per day.