The effect of liver postmitochondrial fraction concentration from Aroclor 1254-treated rats on promutagen activation in L5178Y cells

L5178Y/TK^+/− cells were treated with either N-acetyl-2-aminofluorene (AAF), 2-aminoanthracene (2A), benzanthracene (BA), benzo[a]pyrene (BP), 3-methylcholanthrene (3MCA), or dimethylnitrosamine (DMN) in the presence of varied concentrations (2.5–10% v/v) of liver S9 (9000 × g) postmitochondrial fraction from Aroclor 1254-dosed male CD rats. Consistent S9 concentration-dependent decreases in trifluorothymidine-resistant (TFT^r) mutant induction were noted following 3 h exposure of L5178Y/TK^+/− cells to either 2.5 μg/ml BP, 50 or 67 μg/ml AAF, 0.96 μg/ml 2A, 7.35 μg/ml BA, or 5.4 μg/ml 3MCA. The exception was DMN which yielded a moderate S9 concentration-dependent increase in TFT^r mutant induction when L5178Y/TK^+/− cells were treated for 3 h with 74.1 μg/ml DMN. Depending upon the promutagen being tested, these results suggest two different metabolic events: (1) activation via non-induced enzymes or other factors whose activities are totally S9 volume-dependent (DMN), and (2) deactivation via induced detoxification pathways, a sequence favored by increasing S9 concentration (all others). Utilization of a single “standard” (10%) concentration of liver S9 from Aroclor 1254-treated rats did not appear to provide optimal metabolic activation for 5 of these 6 promutagens.     

Mutagenic evaluation of carcinogens and non-carcinogens in the L5178Y/TK assay utilizing postmitochondrial fractions (S9) from normal rat liver

As part of a continuing effort to investigate various metabolic activation procedures in the L5178Y TK^+/− → TK^−/− mutation assay, a series of 18 chemicals including both carcinogens and non-carcinogens selected from 7 chemical classes were tested in the presence of 5% (v/v) 9000 × g postmitochondrial supernatant fraction (S9) prepared from the livers of untreated Sprague-Dawley rats. Excepting ethyl carbamate and thioacetamide, 8 of 10 carcinogens tested produced mutant counts significantly greater than controls when the results were analyzed by a 2-sample log_et-test. Of 8 non-carcinogens assayed, treatment with p-aminophenol and chloroacetic acid yielded mutant counts significantly greater than control levels in at least one test with S9 while styrene oxide, an Ames positive mutagen, produced mutagenic activity in the absence of S9. These results indicate the L5178Y TK^+/− → TK^−/− mutation assay coupled with 5% (v/v) liver homogenate from normal rodent liver and applied to 18 chemicals correctly distinguished the majority of carcinogens from structurally related non-carcinogens on the basis of chemically-induced gene mutations.     

Mutagenicity of rat-liver S9 to L5178Y mouse lymphoma cells

Rat-liver S9 preparations became highly mutagenic to cultured L5178Y mouse lymphoma cells when the exposure period was increased to 18-24 h or when S9 mix was preincubated in Fischer's medium at 37 degrees C for 19 h and then used to treat the cells for 4 h. Five different S9 preparations (from untreated and Aroclor 1254-treated Fischer 344 or Sprague-Dawley male rats) behaved similarly. S9 mix, which contained 1 mM NADP and 5 mM isocitrate as cofactors, was more mutagenic than S9 alone. Heat treatment of S9 did not destroy its mutagenic activity, but the addition of cofactors no longer stimulated an increase in mutagenicity, as observed with native S9. Treatment with cofactors was not mutagenic. These results implied the involvement of both energy-independent and NADPH-dependent enzymatic changes in S9 mix in producing mutagenic substances. The mutagenic treatments with S9 or S9 mix induced predominantly small TFT-resistant mutant colonies, which suggested that these treatments should be clastogenic to cultured mammalian cells. A warning was given that test chemicals evaluated as mutagenic only in the presence of S9 mix may instead be accelerating the decomposition of S9 mix into mutagens, and it may become necessary to experimentally distinguish between these two mechanisms before a chemical can be regarded as mutagenic.     

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[a]pyrene, 3-methyl-cholanthrene, 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[a]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.     

Promutagen activation by rodent-liver postmitochondrial fractions in the L5178Y/TK cell mutation assay

Individual S9 microsomal fractions prepared from normal livers of 8 rodent species or strains and from 1 rat strain pretreated with Aroclor 1254, were used to metabolize the promutagens N-acetyl-2-aminofluorene, 1,2--benzanthracene, to metabolize the promutagens N-acetyl-2-aminofluorene, 1,2-benzanthracene, benzo[a]pyrene, and 3-methylcholanthrene to active forms during 3-h co-incubation in the presence of L5178Y/TK^+/− cells. The 8 compatible S9 preparations all converted each of the 4 chemical carcinogens into active mutagens with varied efficiencies except for the Aroclor-induced rat S9/benzanthracene combination which produced only weak activity. Aroclor induction did not notably enhance the mutagenicity of benzo[a]pyrene or 3-methylcholanthrene beyond that activity mediated by the other non-induced preparations. Syrian hamster S9 and, to a lesser degree, C57BL/6J mouse S9 were exceptionally active in converting N-acetyl-2-aminofluorene to toxic and mutagenic metabolites. One source of Swiss mouse liver (Blu : Ha ICR) provided the most active S9 when tested with the 3 polycyclic aromatic hydrocarbons.In general, mutagenicity and cytotoxicity were roughly correlated within S9 + promutagen combinations. Almost all of the methylcholanthrene metabolizing activity was lost by the 12th week when Aroclor-induced rat S9 was held at −20°C, yet this activity remained constant when similar S9 was stored at −80°C for 14 weeks. Surprisingly, some S9 sources including the induced rat-liver preparation converted anthracene to a weak or border-line mutagen. The activation of both 1,2-benzanthracene and anthracene may be linked within each species or strain although Aroclor induction enhanced anthracene mutagenicity yet attenuated the mutagenicity of 1,2-benzanthracene. Collectively, these data underscore the current inchoate state of development for S9 coupled somatic cell mutation assays.     

Metabolic activation capabilities of S9 and hepatocytes from uninduced rats to convert carcinogenic N-nitrosamines to mutagens

6 carcinogenic nitrosamines were studied in Salmonella typhimurium TA1535 after activation by S9 and by hepatocytes. All nitrosamines were activated by S9 from induced rats, regardless of their organotropy. The hepatocarcinogenic nitrosamines (N-nitrosodimethylamine, NDMA; N-nitrosodiethylamine, NDEA; N-nitrosomorpholine, NM and N-nitrosodibutylamine, NDBA) were activated to mutagens by S9 and by hepatocytes both derived from noninduced rat livers, NDMA and NM inducing more his+ revertants in the presence of hepatocytes. The oesophageal carcinogenic nitrosamine N-nitrosomethylbenzylamine (NMBeA) and bladder organotrophic N-nitroso(4-hydroxybutyl)butylamine(NBBOH) were neither converted by liver preparations of uninduced rats into mutagenic intermediates nor by hepatocytes. This study indicates that isolated cells derived from untreated animals may be better suited to study liver specific activation in vitro than disrupted subcellular metabolizing systems from induced animals.     

Mathematical model of L5178Y mouse lymphoma forward mutation assay

A mathematical model of the biological protocol for the Mouse Lymphoma L5178Y Forward Mutation Bioassay is presented. The model relates the mutant progenitor frequency (MPF), the number of cells per million surviving cells with DNA damage after exposure to the chemical, to the mutant frequency (MF), the number of TFT-resistant cells per million survivors. For a given expression time, the deterministic relationship is linear and the proportionality constant depends on the relative suspension growth factor (rg) and relative cloning efficiencies (rc) of mutants to those of wild type cells: MF = (rg X rc) X MPF. Experimental noise leads to variations in the values of rg and rc and lack of reproducibility in the system. If mutant progenitors and their progeny grow as well as wild-type cells and if all of the parental mutant progenitors express the mutant phenotype, then rg = 1/2 and rc = 1. Biological mechanisms, such as differential growth characteristics of mutant and wild-type cells or DNA repair, can make the mutant frequency an inaccurate estimate of the MPF. For the assay to be useful as a screen for the mutagenic activity of chemicals, rg X rc has to be reasonably constant from chemical to chemical.     

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.     

Mutagenicity of 2-amino-N6-hydroxyadenine at the tk locus in L5178Y strains differing in repair capabilities and karyotype

The cytotoxicity and mutagenicity of 2-amino-N6-hydroxyadenine (AHA) were measured in strains of L5178Y differing in repair capabilities and karyotype. Strain LY-R83 is monosomic for chromosome 11 and is therefore hemizygous for the tk gene, while strains LY-R16 and LY-S1 are TK+/- heterozygotes. Both strain LY-R83 and LY-R16 are sensitive to UV light and are presumed to be deficient in the excision of pyrimidine dimers as shown for the parental strain, LY-R (Hagen et al., 1988; Szumiel et al., 1988). Strain LY-S1 is sensitive to the cytotoxic effects of ionizing radiation and is presumed to be defective in the repair of radiation-induced DNA double-strand breaks, as shown for the parental strain, LY-S (Evans et al., 1987a; Wlodek and Hittelman, 1987). The sensitivities of the three strains to the cytotoxic effects of AHA were similar. After a 4-hour treatment with AHA at 37 degrees C, the D37 for all three strains was approximately 35 ng/ml. The AHA-induced mutant frequency was similar for the hemizygous TK+ strain LY-R83 and the heterozygous TK +/- strain LY-R16, but was slightly higher for strain LY-S1 than for either LY-R strain at an AHA concentration of 100 ng/ml. The proportion of AHA-induced LY-S1 TK -/- mutants forming colonies with diameters less than 0.3 mm was much lower than following treatment with X radiation (24% vs. 61% for AHA and X radiation, respectively). These results indicate that the vast majority of AHA-induced TK -/- mutants harbor single gene mutations. AHA did not result in cyanide-insensitive oxygen uptake, and treatment with this compound did not induce a significant number of DNA single-strand breaks, DNA alkali labile lesions, or DNA degradation in either strain. However, two hours after AHA removal, DNA single-strand breaks and/or alkali-labile lesions, possibly due to the occurrence of DNA repair, were apparent in the DNA of both strain LY-R16 and strain LY-S1.     

Genotoxicity of gamma-irradiation in L5178Y mouse lymphoma cells

The ability of gamma-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/- 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.     

Isolation of temperature-sensitive mutants from murine leukemic cells (L5178Y)

Two temperature-sensitive mutants (ts1 and ts3) have been isolated from murine leukemic cells, L5178Y, after mutagenesis and cytosine arabinoside selection. Both ts1 and ts3 grew normally at the permissive temperature (33 °C) but not at the non-permissive temperature (39 °C). Consistent results were obtained with the growth patterns in suspension culture as well as the plating efficiencies in soft agar. Temperature shift experiments showed that the mutant cells remained viable after extended exposure to the non-permissive temperature. Labeling studies with radioactive precursors indicated that the synthesis of DNA, but not of RNA or protein, was affected in these mutants at 39 °C. The defective function of ts3 cells was substantially corrected by supplementing alanine, hypoxanthine, and pyruvate.     

Ultrastructural localization of actin in nuclear matrices from mouse leukemia L5178Y cells

We examined the distribution of actin in isolated nuclear matrices from mouse leukemia L5178Y cells using an anti-actin antibody and protein A-conjugated colloidal gold particles. Before immunogold staining, we partially digested the surface lamina of the nuclear matrix with trypsin (Nakayasu and Ueda, Exp. Cell Res. 143, 55-62, 1983) to allow penetration of the gold particles into the nuclear matrix. Trypsin digestion slightly modified the internal structure of the nuclear matrix, but did not affect the actin content in the nuclear matrix nor the reactivity of actin with the antibody. Many colloidal gold particles were present along fibrogranular structures in the nuclear matrix. The results reported here confirm the existence of actin in the interior of the nuclear matrices of L5178Y cells.