Demonstration of multiple phenotypic diversity in a murine melanoma of recent origin.

The purpose of these studies was to examine whether the metastatic heterogeneity that is frequently found in serially transplanted neoplasms could be observed in a murine melanoma of recent origin. The primary K-1735 melanoma that arose in an inbred C3H/HeN murine mammary tumor virus-negative (C3H-) mouse was transplanted once into an immunosuppressed recipient and then established in culture. Cells from the fifth in vitro passage were used to produce clones. The parent K-1735 and 22 cloned lines were tumorigenic in syngeneic and outbred N:NIH(S) nude mice. Metastatic properties were assessed by observing the ability of the cells to produce pulmonary and extrapulmonary lesions after they were injected iv into 6-week-old C3H- mice. The number of metastases produced, their relative size, and pigmentation varied dramatically among the clones. Only 2 of 22 clones were indistinguishable from the parent tumor. Most of the nonmetastatic (but tumorigenic clones) were also nonmetastatic in 3-week-old nude mice, which suggests that the absence of metastasis formation was not merely due to their immunologic rejection by the normal C3H- mouse. Control subcloning experiments demonstrated that the procedure of cloning in vitro was not responsible for the variability among the clones. The clones did not differ in their karyotype or cell size, but they did differ in their growth rate in vitro. These phenotypes, however, did not correlate with metastatic propensity. In conclusion, the K-1735, a murine melanoma of most recent origin, is heterogeneous and contains subpopulations of cells with diverse biologic behavior.

Enhanced metastatic potential of murine fibrosarcomas treated in vitro with ultraviolet radiation.

The purpose of this study was to determine whether repeated treatment of tumor cells in vitro with mutagenic doses of ultraviolet (UV) radiation could influence the metastatic behavior of these cells in vivo. Three cloned lines of UV-2237, a fibrosarcoma induced in a C3H- mouse by chronic irradiation with UV, and SF-19, a spontaneous C3H- fibrosarcoma, were grown in culture. These cell lines varied from low to high metastatic potential as determined by in vivo tests. The cultures were exposed to UV radiation from an FS40 sunlamp at a dose that killed 40% of the cells. These UV radiation exposures were repeated at 3- to 5-day intervals for a total of 5 treatments. The mutation frequency was analyzed by monitoring the appearance of ouabain-resistant colonies following UV irradiation. With all four tumor lines, the frequency of conversion to ouabain resistance was increased more than 10-fold. Tumor cells given 5 UV radiation treatments and control cultures carried in parallel without exposure to UV radiation were tested for metastatic potential in an in vivo lung colony assay. Cell lines treated in vitro with UV radiation produced more experimental metastases than the counterpart unirradiated cultures. We conclude that, in all four tumor lines, exposure of tumorigenic cells to mutagenic doses of UV radiation can alter their biological behavior and that this may contribute to the progression of tumors from low to high metastatic capability.

Chronic peroxisome proliferation and hepatomegaly associated with the hepatocellular tumorigenesis of di(2-ethylhexyl)phthalate and the effects of recovery.

This study compared the levels of cell proliferation and peroxisome proliferation in rodent liver with tumor incidence, to provide more information on the relationship between these events following chronic exposure. Fischer 344 rats were treated with 0, 100, 500, 2500, or 12,500 ppm DEHP, and B6C3F1 mice were treated with 0, 100, 500, 1500, or 6000 ppm DEHP in the diet for up to 104 weeks. Additional groups of rats and mice received the highest concentration for 78 weeks and then the control diet for an additional 26 weeks (recovery groups). Animals were terminated at weeks 79 and 105 for histopathologic examination. Elevated palmitoyl CoA oxidation activity and higher liver-to-body weight ratios were observed for the 2500- and 12,500-ppm groups of rats, and for the 500-, 1500-, and 6000-ppm groups of mice at Week 105. No increase in palmitoyl CoA oxidation activity was evident in the recovery group, and relative liver weights were near control levels following recovery. No hepatic cell proliferation was detected at Weeks 79 or 105 in either species although preliminary data indicated that cell proliferation did occur within the first 13 weeks of exposure. A significantly higher incidence of hepatocellular tumors was only observed for the 2500- and 12,500-ppm group and its recovery group of rats, and for the 500-, 1500-, and 6000-ppm groups and the recovery group of mice. The tumor incidences were reduced for the recovery groups compared with the groups fed DEHP continuously for 104 weeks. The data indicate that high levels of peroxisome proliferation and hepatomegaly are associated with DEHP hepatocarcinogenesis in rodent liver, and that the tumorigenic process may be arrested by cessation of DEHP treatment, suggesting that extended treatment with DEHP acts to promote tumor growth.

Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/- cells.

Evidence has been accumulating that conditions of nonphysiological pH may affect the results of in vitro genetic tests by mechanisms unrelated to the chemical being tested. Medium was pH-adjusted with HCl, NaOH or with organic buffers (Good's zwitterions). In the absence of S9 mix, no changes in mutant frequency were observed over a pH range of 6.4-9.2; a small, 1.9-fold increase was observed for a moderately toxic treatment (24% relative growth) at pH 6.3. However, in the presence of S9 mix, the mutant frequency increased sharply for pH values below 6.8. At pH 6.4, a 4-fold increase was induced, and pH 6.0 resulted in a 10-fold increase in mutant frequency. Basic pH shifts in the presence of S9 mix caused no changes in mutant frequency up to pH 8.0; treatment with pH 8.8 was highly toxic (5.3% relative growth) and caused a 3-fold increase in mutant frequency. Thirteen mutant clones induced at pH 6.0 with S9 mix were challenged with trifluorothymidine after their expansion in nonselective medium and all retained their resistance; another 14 clones were tested for thymidine utilization and all incorporated only 0.1-5.5% of the 14C-labeled thymidine used by the parental line. The induced mutants were primarily of the small-colony phenotype, which indicated clastogenic activity. This was confirmed with chromosome studies which showed a large increase in cells with aberrations consisting of chromatid breaks and complex rearrangements. The results show that the combination of weak acidity (pH 6-6.8) and S9 mix is mutagenic and clastogenic to L5178Y TK+/- cells.

Evaluation of the in vitro genetic toxicity of 4-(2, 4-dichlorophenoxy)butyric acid.

The herbicide 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) is principally used in the USA on peanuts, soybeans and alfalfa. In Europe, it is used on undersown spring barley and grassland (with clover). The genetic toxicity in vitro of the dimethylamine salt of 2,4-DB was examined by employing a range of end points including gene mutation in bacteria (Ames test) and mammalian cell cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells (CHO/chromosomal aberration assay), and induction of DNA damage and repair in rat hepatocytes. There were no indications of genotoxic potential for 2,4-DB in the first three of these assays. One of the two criteria for a positive response in the UDS assay was exceeded but the increases did not exceed the second criteria for a positive response. The test material was therefore evaluated as weakly active in this assay. The weight of the evidence clearly indicates that 2, 4-DB is not genotoxic to mammals and are consistent with the reported lack of carcinogenic potential for 2,4-DB in both mice and rats.

Ames assays and unscheduled DNA synthesis assays on 2, 4-dichlorophenoxyacetic acid and its derivatives.

2,4-dichlorophenoxyacetic acid and several of its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity in vitro of 2,4-D and seven of its salts and esters were examined by employing gene mutation in bacteria (Ames test) and induction of DNA damage and repair in rat hepatocytes. In addition, an in vivo unscheduled DNA synthesis (UDS) assay was performed on 2,4-D. There were no indications of genotoxic potential for 2,4-D acid, or any of its derivatives, in these assays. These results are consistent with the reported lack of carcinogenic potential for 2,4-D in both mice and rats.

The genetic toxicology of Kathon biocide, a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one.

Kathon biocide, an aqueous solution containing a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one in an approximate ratio of 3:1, was tested for mutagenic activity in Salmonella typhimurium, L5178Y mouse lymphoma cells in culture and Drosophila melanogaster. Tests also were conducted for chromosome aberrations in vivo on mouse bone marrow cells, for DNA damage/repair in primary rat hepatocytes in culture, and for morphological transformation in C3H 10T1/2 cells in culture. Kathon biocide produced point mutations in the absence of a rat-liver metabolizing system in bacteria (strain TA 100) and mammalian cells in culture. In the presence of rat-liver metabolizing system a 10-fold higher concentration was required to induce point mutations in mammalian cells in culture. No mutagenic activity was observed with the metabolizing system and S. typhimurium. Negative results were obtained in the sex-linked recessive lethal assay in Drosophila, the in vivo cytogenetic assay in mice, the unscheduled DNA synthesis assay in cultured rat hepatocytes, and the in vitro cell transformation assay.

Application of simplified in vitro screening tests to detect genotoxicity of aristolochic acid.

Aristolochic acid (AA), the active compound found in Aristolochia extracts, has been used as a traditional medicine. However, products containing AA were withdrawn from the market in the early 1980s because AA was found to be a potent carcinogen. Some genotoxicity studies of AA were conducted after the carcinogenicity of AA was reported. The purpose of this study was to check the ability of simplified, screening tests for genotoxicity to indicate the genotoxic activities of AA. Four commonly used in vitro genotoxicity endpoints were examined. In a bacterial mutation screening test, AA was mutagenic to tester strains TA98 and TA100 with and without rat liver S9. In the L5178Y mouse lymphoma cell gene mutation test, mutagenic activity was observed at > or = 25 microg/ml with or without S9. A concentration-dependent increase in structural chromosome aberrations was observed in CHO cells, with significant increases at 50 microg/ml without S9 and at 25 microg/ml with S9. Significant increases in micronucleated binucleated cells were observed in CHO cells treated with AA at > or = 25 microg/ml with or without S9. These results demonstrated that the genotoxicity of AA would have been easily detected if simple screening versions of in vitro genotoxicity assays had been used during early product development. It is suggested that simplified screening tests such as those used in this study would be a rapid and economical way of obtaining the preliminary genotoxicity profiles of new substances or products as an aid to decision-making for further development.

Genotoxicity testing of a fenugreek extract.

Fenugreek seeds have been used in traditional medicines as a remedy for diabetes. Rich in protein, fenugreek seeds contain the unique major free amino acid 4-hydroxyisoleucine (4-OH-Ile), which has been characterized as one of the active ingredients for blood glucose control. Current use of fenugreek in foodstuff has been limited to its role as a flavoring agent, and not as an ingredient to help mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a fenugreek seed extract (THL), containing a minimum of 40% 4-OH-ILE, was evaluated using the standard battery of tests (reverse mutation assay; mouse lymphoma forward mutation assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. THL was determined not to be genotoxic under the conditions of the tested genetic toxicity battery. The negative assay results provide support that addition of THL to foodstuffs formulated for people with diabetes is expected to be safe. A wide safety margin is established, as anticipated doses are small compared to the doses administered in the assays.

Lack of genotoxic effects of sucrose acetate isobutyrate (SAIB).

Sucrose acetate isobutyrate (SAIB) was tested for potential genotoxic activity in four different in vitro assay systems. Two independent trials of a Salmonella reverse mutation assay (using strains TA98, TA100, TA1535, TA1537 and TA1538) showed no increases in revertant frequencies at doses up to 10,000 microg/plate which was non-toxic but exceeded the solubility limit. Similarly, no mutagenic response was observed at doses up to 1000 microg/ml at the HGPRT locus in cultured CHO cells; SAIB was toxic and its solubility limit was exceeded at 50 microg/ml. No clastogenic activity was detected in cultured CHO cells at concentrations up to 2000 microg/ml. All three preceding in vitro tests were conducted both in the presence and absence of Aroclor 1254-induced rat liver S-9 metabolic activation systems. An unscheduled DNA synthesis assay also was performed using rat primary hepatocyte cultures with doses up to 1000 microg/ml, and no DNA repair was detectable. Thus, SAIB was stringently tested at doses exceeding the solubility limit in culture medium and causing toxicity to CHO cells without obtaining any evidence for genotoxic activity as a mutagen, clastogen, or DNA-damaging agent.

In vitro genotoxicity testing of ARASCO and DHASCO oils.

ARASCO and DHASCO oils are microbially-derived triglycerides rich in arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids, respectively. Both oils were tested for mutagenic activity in three different in vitro mutagenesis assays. All assays were conducted with and without metabolic activation. Neither ARASCO nor DHASCO oil was mutagenic in the Ames reverse mutation assay using five different Salmonella histidine auxotroph tester strains, nor were the oils mutagenic in the mouse lymphoma TK(+/-) forward mutation assay. The oils showed no clastogenic activity in chromosomal aberration assays performed with Chinese hamster ovary cells. Based on these assays, neither ARASCO nor DHASCO oils appear to have any genotoxic potential.

In vitro growth characteristics associated with benign and metastatic variants of tumor cells.

Tumors show a wide range of growth rates in vivo. In general, nonmetastatic (benign) tumors appear to grow slowly, while metastasizing (malignant) tumors proliferate rapidly and contain a large number of dividing cells. In order to analyze if there is a direct relationship between metastatic potential and growth rate (without the influence of host factors), in vitro analyses are necessary. In vitro studies that compared monolayer doubling times and in vivo tumorigenicity rarely demonstrated a correlation between the two parameters. Differences in monolayer growth rates were observed among tumors, but the differences were unrelated to in vivo growth. In contrast, some tumor systems demonstrated a correlation between in vivo growth and/or malignant potential and growth properties observed during anchorage independent growth. Evidence is presented to show that in particular tumor systems, an analysis of colony size and colony morphology during growth in suspension can be used to study the relationship between malignant potential and growth properties.

Increasing metastatic potential is associated with increasing genetic instability of clones isolated from murine neoplasms.

The metastatic stability of clones, which were derived from the murine UV-2237 fibrosarcoma and which exhibit low or high metastatic potential, was examined after 60-72 days of continuous growth in vitro and in vivo. Subclones of the high metastatic clone exhibited a 140-fold variation in the production of experimental pulmonary metastases after intravenous injection into syngeneic C3H- mice. In contrast, subclones from the low metastatic clone varied only slightly (8-fold). Using cloned cells from three mouse tumors with differing metastatic potential, we determined the spontaneous mutation rates of cells with low or high metastatic capacities with respect to the selective genetic markers, 6-thiopurine resistance or ouabain resistance, or both. In all cases, cells with high metastatic potential had a 3- to 7-fold increase in the rate of mutation (per cell generation) at both genetic loci, as compared with their low metastatic but tumorigenic cell controls. These results support the hypothesis that the evolution of tumors from the benign to the malignant state could be the consequence of acquired genetic instability in the neoplastic cells.

Correlation of patterns of anchorage-independent growth with in vivo behavior of cells from a murine fibrosarcoma.

The pattern of in vitro anchorage-independent growth of tumor cells from the murine UV-2237 fibrosarcoma correlated with their ability to produce experimental metastasis in vivo. When seeded into 0.3% Noble agar semisolid medium, cells of metastatic clones developed into larger tumor colonies at a faster rate than did cells of clones with low metastatic potential. Furthermore, when tumor cells were plated into 0.6% Noble agar, colony development by cells of low metastatic potential clones was almost completely restricted. Tumor cells from the heterogeneous parent UV-2237 fibrosarcoma were plated into dishes containing 0.6% agar semisolid medium. In separate experiments, 16 colonies were isolated 2 weeks thereafter and were established as individual cell lines in monolayer cultures. All of these cell lines produced experimental metastases as determined by in vivo lung colony assay. The data suggest that anchorage-independent growth of UV-2237 tumor cells in 0.6% Noble agar semisolid medium is selective and permits the isolation of metastatic subpopulations.

Properties of metastatic and nonmetastatic cloned subpopulations of an ultraviolet-light-induced murine fibrosarcoma of recent origin.

The present studies were designed to evaluate whether tumor cell properties such as growth rate, chromosome number, anchorage-independent growth, susceptibility to lymphocyte- or macrophage-mediated lysis in vitro, and antigenicity in vivo correlated with metastatic potential. A murine fibrosarcoma of recent origin induced in a C3H- mouse by chronic irradiation with ultraviolet light was used. Cells from the parent tumor and its clones were grown in culture. No single property of tumor cells that was measured in vitro or in vivo predicted or correlated with their metastatic potential. In order for metastasis to occur, all steps of the process must be completed. Therefore, interruption of the sequence at any stage can prevent the production of visible metastasis. It was concluded that the search for a single property common to all metastatic cells in a large variety of neoplasms is likely to be unproductive.

Growth rate and chromosome number of tumor cell lines with different metastatic potential.

We investigated whether the metastatic potential of various tumor cell lines was related to chromosome counts or to rate of growth in vitro or in vivo. Clones of known metastatic potential derived from a C3H- fibrosarcoma induced by UV radiation (UV-2237) and from C57BL/6 B16 melanoma were tested for these characteristics. No correlation was found between the growth rate of these clones in monolayer culture or at a subcutaneous site and their ability to produce metastases. The cells from clones of UV-2237 were mainly in the diploid range with only one exception, and the B16 clones were all hyperploid. Thus, there was also no correlation between malignant behavior of the clones and gross changes in chromosome number.

Characteristics of concanavalin A-resistant Chinese hamster ovary cells and certain revertants.

Clones of Chinese hamster ovary (CHO) cells were isolated by single-step selection for resistance to killing Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA-resistant isolates were only about 2-fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature-sensitivity for growth, markedly altered morphology and adherence, and significant difference in susceptibility to other agents such as colchicine. Two revertants to full temperature-resistance were isolated from different ConA-resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA-resistant. The two series of wild typed, ConA-resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/(3H) borohydride labelling procedures. The ConA-resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature-revertant that remained ConA-resistant, while two of the altered protein closses were restored to wild type mobility in the revertant that regained ConA-sensitivity. Cell hybridization experiments indicated that the temperature-sensitivity phenotypes of different ConA-resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutation in different genes.

Mammalian genotoxicity assessment of methylene blue in plasma: implications for virus inactivation.

BACKGROUND: The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems. STUDY DESIGN AND METHODS: Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent. RESULTS: Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x 10(-6). CONCLUSION: Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.

Absence of mutagenic effects of sodium dichloroacetate.

Sodium dichloroacetate (DCA) is a drug with potential for treating patients with stroke and head injury. Conflicting evidence has been published on the mutagenic potential of DCA. A series of genetic tests for mutagenicity and clastogenicity was carried out on pharmaceutical grade DCA. Four types of mutagenicity test were included, with and without metabolic activation where appropriate. These studies included: (i) Salmonella and Escherichia coli mutation (Ames) test, (ii) thymidine kinase locus forward mutation in L5178Y mouse lymphoma cells, (iii) tests for chromosomal aberrations in Chinese hamster ovary cells, and (iv) and in vivo rat bone marrow erythroid micronucleus test. In each study, there was no evidence of mutagenic activity attributable to DCA. It is possible that the present test material, of pharmaceutical grade, has fewer impurities than materials studied in previous reports. These data extend, and in some cases contradict, previous published reports on DCA.

Quantification of diazeniumdiolate mutagenicity in four different in vitro assays.

Diazeniumdiolates are under investigation as possible prodrugs of the multifaceted bioregulatory agent nitric oxide. This study was undertaken to assess further the mutagenic potential of two diazeniumdiolates, DEA/NO (Et2N[N(O)NO]Na) and SPER/NO ([H2N(CH2)3NH(CH2)4N[N(O)NO-](CH2)3 NH3+]), which generate NO spontaneously with half-lives at 37 degrees C and pH 7.4 of 2 and 39 min, respectively. The genotoxic potential of these compounds was investigated with the Ames bacterial reverse mutation assay, two mammalian cell gene mutation assays (CHO/HGPRT and L5178Y TK+/-), and an assay for sister chromatid exchange (SCE) using Chinese hamster ovary (CHO) cells. Both diazeniumdiolates had previously been shown to be mutagenic in the Ames Salmonella plate assay. In the experiments reported here, Salmonella typhimurium strain TA1535 was exposed to the compounds in a liquid incubation assay for either 15 min or 48 h without an S-9 fraction. With the 15-min exposure, DEA/NO was mutagenic at concentrations of 0.625 mM (3.5 x control) and greater, while SPER/NO was mutagenic at 0.5 mM (2.7 x control) and above. In the CHO/HGPRT assay, DEA/NO was weakly mutagenic only at the highest concentration used, 20 mM, inducing a mutant frequency per survivor that was 2.5 x control, while SPER/NO was mutagenic at 0.5 mM with a mutant frequency of 2.5 x control. When the CHO cells were given 10 repetitive 20 mM DEA/NO exposures (3 min each), HGPRT mutant frequency was 4.1 x control. In the L5178Y mouse lymphoma cell TK+/- assay, DEA/NO doubled the mutation rate at 1.82 mM, while SPER/NO's mutation frequency was more than twice that of control at 0.63 mM. DEA/NO was positive in the SCE assay without metabolic activation, yielding significant SCE at 1.25, 2.5, and 5 mM that was 1.8, 2.2, and 2.6 times control, respectively. SPER/NO increased the SCE by 1.2, 1.4, and 1.3 times at 1.5, 2.0, and 2.5 mM. The results suggest that the two diazeniumdiolates, although mutagenic in the bacteria, are much weaker mutagens in mammalian cells.