Alkaline comet assay in liver and stomach, and micronucleus assay in bone marrow, from rats treated with 2-acetylaminofluorene, azidothymidine, cisplatin, or isobutyraldehyde.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague-Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery.

The mouse lymphoma L5178Y Tk+/- cell line is heterozygous for a codon 170 mutation in the p53 tumor suppressor gene.

The p53 tumor suppressor protein plays an important role in regulating the cellular response to DNA damage, including cell cycle arrest and apoptosis induction. Normal p53 function is critical for the maintenance of genomic stability. The mouse lymphoma L5178Y/TK(+/-)-3.7.2C cell line is widely used in genetic toxicology for mutagenesis and clastogenesis testing. A related line L5178Y-R, has previously been shown to react with antibodies specific for mutant as well as wild-type p53 protein and to exhibit delayed cell death after radiation. For this reason, as well as the mouse lymphoma assay's reputation for high sensitivity of detection for genotoxic agents but low specificity, we examined several clones of L5178Y cells for mutations in the conserved core domain (exons 5-8) of the p53 gene. Using single-strand conformational polymorphism analysis, we found evidence for the same mutation in exon 5 of p53 in L5178Y-R, L5178Y-S and L5178Y/TK(+/+)-3.7.2C cells. The mutation was identified by sequencing of exon 5 as a TGC (Cys) to CGC (Arg) transition in codon 170 (= codon 176 in humans). Sequencing showed approximately equivalent signals for the mutant and normal alleles for all 3 lines. The mutation in codon 170 is adjacent to a mutation hotspot of the human p53 gene (codon 175) and eliminates a critical zinc-coordinating cysteine residue such that the mutant protein is likely to be denatured and have a dominant negative effect on normal p53 function. Western blots showed approximately 100-fold higher levels of p53 protein in unirradiated L5178Y cells as compared to induced levels of p53 in normal mouse splenocytes 4 h after 5 Gy of gamma radiation. The high levels of p53 protein in L5178Y cells were not further inducible by radiation, whereas an 11-fold induction was seen in the irradiated splenocytes. These results indicate that p53 protein in L5178Y cells is dysfunctional and suggest that this line may therefore be abnormally susceptible to the induction of genetic alterations.

Revalidation of the in vitro alkaline elution/rat hepatocyte assay for DNA damage: improved criteria for assessment of cytotoxicity and genotoxicity and results for 81 compounds.

The in vitro alkaline elution/rat hepatocyte assay is a sensitive assay for genotoxicity, measured as DNA strand breaks induced in primary cultures of rat hepatocytes after 3-h treatments with test compounds. Since DNA degradation can be rapid and extensive in dead and/or dying cells, the original criteria for a positive result in the assay were that a compound induce a 3.0-fold or greater increase in the elution slope (for the terminal phase of alkaline elution from 3 to 9 h) in the absence of significant cytotoxicity (defined as relative cell viability of less than 70% by trypan blue dye exclusion; TBDE). Recently we have shown that false-positive results can still be obtained due to cytotoxicity when loss of membrane integrity is a late event in toxic cell death relative to the induction of endonucleolytic DNA degradation. To improve the ability of the assay to discriminate between genotoxic vs. cytotoxic effects of chemicals, we have evaluated additional assays of cytotoxicity including cell adenosine triphosphate (ATP) and potassium (K+) content, tetrazolium dye reduction (MTT), TBDE after a further 3-h recovery incubation without test chemicals (delayed toxicity), cell blebbing and endonucleolytic DNA degradation (double-strand breaks; DSBs) assessed by pulsed-field gel electrophoresis (PFGE). We have also evaluated 2 parameters derived from the elution data which can indicate extensive, cytotoxicity-induced DNA degradation: the fraction of the DNA recovered in the neutral lysis/rinse fraction and the gamma-intercept of the extrapolation of the 3-9-h segment of the elution curve. Twenty-eight rodent non-carcinogens that are negative (or inconclusive) in the Ames assay with no, or limited, other evidence of genotoxicity, and 33 genotoxins, most of which are also carcinogens, were evaluated. The results showed that DNA degradation as measured by a 1-h PACE (Programmed Autonomously Controlled Electrodes)/PFGE assay was a sensitive indicator of cytotoxicity which correlated well with results of the other cytotoxicity indicators. The delayed TBDE (after a 3-h recovery), intracellular potassium and ATP assays as well as the gamma-intercept parameter were also shown to be sensitive and in some cases complementary measures of cytotoxicity. Using new criteria based on these data of an induced slope (treatment slope-negative control slope) of 0.020 for the 3- to 9-h elution period and cytotoxicity limits of 70% relative viability for the delayed TBDE assay and 50% for intracellular ATP content, the assay scores the genotoxicity of these 61 reference compounds with an overall accuracy of 92%. Test results using these new criteria are provided for an additional 20 compounds (5 non-genotoxic carcinogens and 15 compounds whose genotoxic and carcinogenic potential are unknown or equivocal).

Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouse liver tumors.

We investigated the role of dosing regimen on ras mutations in chemically induced CD-1 mouse liver tumors. The spectra of ras gene mutations in liver tumors that were induced by 15 daily i.p. injections of 7,12-dimethylbenz[a]anthracene (DMBA), 4-aminoazobenzene (AAB), N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or N-nitrosodiethylamine (DEN) were compared to those previously obtained for tumors induced by a single but higher dose of each carcinogen. The principal assay used was a direct tumor analysis involving sequencing of polymerase chain reaction (PCR)-amplified tumor DNA; additional mutations that were present in only a small fraction of tumor cells were detected using a transfection assay or a PCR-engineered restriction fragment length polymorphism method. Spontaneous liver tumors had a relatively low frequency of ras mutations, all found in Ha-ras codon 61, and most of these mutations were present in only a small fraction of tumor cells. With the exception of multiple-dose DEN, each group of single- and multiple-dose carcinogen-induced tumors exhibited a higher frequency of ras mutations compared with spontaneous tumors. For AAB, N-OH-AAF and DEN, the dosing regimen was found to affect significantly the profile of ras mutations. For each of these carcinogens, the multiple-dose tumor group (versus single-dose group) had fewer Ki-ras and N-ras mutations and more tumors in which the Ha-ras codon 61 (C-->A) mutation was present in a large fraction of cells. Our results demonstrate that the dosing procedure can materially affect the pattern of ras gene mutation in mouse liver tumors.

Rapid DNA degradation in primary rat hepatocytes treated with diverse cytotoxic chemicals: analysis by pulsed field gel electrophoresis and implications for alkaline elution assays.

The use of genetic toxicology tests for hazard identification is complicated by the fact that some in vitro tests using cultured mammalian cells are subject to potential artifacts that can make it difficult to distinguish between direct, chemically-induced genotoxicity, and DNA damage that occurs secondary to chemically-induced cytotoxicity (e.g., mediated by endogenous nucleases). Recently, we demonstrated that cytotoxicity-induced DNA double strand breaks (dsb) can produce artifacts in the in vitro alkaline elution/rat hepatocyte assay [Elia et al., 1993]. To explore this further, we used pulsed field gel/DNA dsb assays to characterize the relationship between chemically-induced cytotoxicity and the degradation of genomic DNA to high molecular weight fragments. Two sets of compounds were tested: 17 cytotoxic agents judged to be neither genotoxic nor carcinogenic, and 10 known genotoxic carcinogens. We found a close correlation between chemically-induced cytotoxicity and the rapid degradation of DNA to high molecular weight, double-stranded fragments. In contrast, the classic genotoxic chemicals tested generally did not trigger DNA dsb fragmentation at doses that were genotoxic but not immediately cytotoxic. These data indicate that pulsed field gel/DNA dsb assays can be used with in vitro genetic toxicology assays to help distinguish between genotoxic and cytotoxic mechanisms of DNA damage.

Reversal of transformed phenotypes by antisense fos.

The therapeutic use of antisense DNA has started a revolution in pharmacology. As a model system for demonstrating the therapeutic power of the antisense concept, we sought to interrupt signal transduction in H-ras transformed cells to attempt to down-regulate their oncogenic phenotype. We hypothesized that down-regulation of c-fos translation by antisense-fos expression would decrease oncogenic signal transduction through the fos pathway and thus reverse the tumorigenic phenotype of these cells. To test this hypothesis, we transfected H-ras cells with a plasmid containing an 84-base sequence antisense to the 5' end of the mouse c-fos gene. The antisense-fos was under the transcriptional control of the MMTV promoter and inducible by dexamethasone. Two of the antisense-fos clones grew in a density-dependent manner, exhibiting both a flat morphology and a quiescence in low serum medium unlike the sense-fos controls. Antisense-fos also inhibited soft agar growth to 1% of control values and dramatically reduced tumor growth in nude mice. Antisense-fos had no effect on ras expression but greatly reduced c-fos protein levels as assayed by immunofluorescence. These findings suggest that down-regulation of signal transduction pathways by antisense therapeutic compounds might have major therapeutic benefits against malignant cells transformed by ras or other oncogenes.

Activation of the Ha-, Ki-, and N-ras genes in chemically induced liver tumors from CD-1 mice.

We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine. By direct sequencing of polymerase chain reaction-amplified tumor DNA, the carcinogen-induced tumors were found to have much higher frequencies of ras gene activation than spontaneous tumors. Furthermore, each carcinogen caused specific types of ras mutations not detected in spontaneous tumors, including several novel mutations not previously associated with either the carcinogen or mouse hepatocarcinogenesis. For example, the model compound DMBA is known to cause predominantly A to T transversions in Ha-ras codon 61 in mouse skin and mammary tumors, consistent with the ability of DMBA to form bulky adducts with adenosine. Our results demonstrate that the predominant mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-ras codon 13 (DMBA is also known to form guanosine adducts), illustrating the influence of both chemical- and tissue-specific factors in determining the type of ras gene mutations in a tumor. 4-Aminoazobenzene and N-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon 13 mutation. In addition, we found that N-nitrosodiethylamine, 4-aminoazobenzene, and N-hydroxy-2-acetylaminofluorene all caused G to T transversions in the N-ras gene (codons 12 or 13). This is the first demonstration of N-ras mutations in mouse liver tumors, establishing a role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison of the ras mutations detected in the direct tumor analysis with those detected after NIH3T3 cell transfection indicates that spontaneous ras mutations (in Ha-ras codon 61) are often present in only a small fraction of the tumor cells, raising the possibility that they may sometimes occur as a late event in CD-1 mouse hepatocarcinogenesis.

Polymerase chain reaction/sequencing analysis of ras mutations in paraffin-embedded tissues as compared with 3T3 transfection and polymerase chain reaction/sequencing of frozen tumor deoxyribonucleic acids.

The efficiency of detection of H- and K-ras mutations in 27 CD-1 mouse liver tumors by direct sequencing of polymerase chain reaction (PCR)-amplified DNA isolated from formalin-fixed and paraffin-embedded tissues was compared with that after assay by both NIH 3T3 transfection (followed by sequencing of amplified transformant DNA) and direct sequencing of PCR-amplified DNA isolated from frozen tumors. Some tumor samples were chosen for comparison because they contained ras mutations that were detected by either NIH 3T3 transfection or sequencing of PCR-amplified DNA derived from frozen tumors, but were not detected by both techniques. The efficiency of detecting K-ras mutations was similar for sequencing of amplified fragments derived from both paraffin-embedded tissues and from frozen tumors. However, these two techniques differed in their efficacy for detection of H-ras codon 61 mutations. Furthermore, this difference appeared to be mutation-specific: the sequencing of amplified products from paraffin-embedded tumor tissues allowed increased detection of CAA to AAA mutations but decreased detection of CAA to CTA mutations relative to sequencing of amplified fragments derived from frozen tumor DNA. Direct sequencing of PCR products from paraffin-embedded sections was more sensitive than NIH 3T3 transfection for detection of activated K-ras genes containing codon 13 mutations but less sensitive for detection of activated H-ras genes containing codon 61 mutations. In summary, direct sequencing of amplified DNA from either frozen tumors or formalin-fixed, paraffin-embedded tissues can be more sensitive than NIH 3T3 transfection for detection of codon 13-activated K-ras genes. However, it appears to be less sensitive than NIH 3T3 transfection for detection of certain activating H-ras mutations. Depending upon the questions being asked of the data, each of the methods can provide useful information about ras gene mutations in tumor samples. The apparent differences in sensitivities between the methods is not yet understood, but such differences should be considered in the analysis of data obtained when only one method is used.

Activation of the Ki-ras gene in spontaneous and chemically induced lung tumors in CD-1 mice.

As part of an evaluation of the effectiveness of using ras mutation analysis for distinguishing carcinogen-induced from spontaneous tumors, we examined the profile of ras gene point mutations in spontaneous, 7,12-dimethylbenz[a]anthracene (DMBA)-induced, and N-nitrosodiethylamine (DEN)-induced lung tumors from Crl:CD-1(ICR)BR (CD-1) mice. Although all of the lung tumors were assayed for mutations in the Ha-ras, Ki-ras, and N-ras genes (codons 12, 13, and 61), only Ki-ras mutations were found, which is consistent with other studies that have noted a strong preference for Ki-ras gene activation in mouse, rat, and human lung tumors. We found that spontaneous CD-1 mouse lung tumors had a very high frequency of Ki-ras gene activation (17 of 20 tumors; 85%), distributed among codons 12 (5 of 20), 13 (1 of 20), and 61 (11 of 20). DMBA-induced lung tumors had a slightly higher frequency of Ki-ras gene mutations (16 of 16; 100%), again distributed among codons 12 (5 of 16), 13 (2 of 16), and 61 (9 of 16). However, seven of the DMBA tumors had mutations qualitatively different from those found in spontaneous tumors. In contrast to DMBA-induced tumors, DEN-induced tumors had a lower frequency of Ki-ras mutations (36%) when compared with spontaneous lung tumors, suggesting that DEN primarily induces lung carcinogenesis by a mechanism other than ras gene activation. Thus, although spontaneous and induced CD-1 mouse lung tumors have a strong tissue-specific preference for carrying an activated Ki-ras gene, the nature of the initiating carcinogen can influence the frequency or profile of Ki-ras mutations.

Selection of low frequency tumor cells from cell culture by growth in nude mice.

Tissue cultures of tumor cells are frequently utilized to characterize chromosomal changes when direct cytogenetic preparations on tumors fail. The present study demonstrates that chromosomal markers found in direct tumor preparations can become undetectable in cell culture at variable rates presumably because of overgrowth of normal cell components in the culture. Injection of cultured tumor cells into nude mice followed by direct chromosomal preparations on the resulting nude mouse tumors can be used to select cells with the original tumor karyotype. This is true even when the tumor cell frequency in the culture is so low that they are not found in routine chromosomal preparations of the cultured cells. This technique can thus complement tissue culture findings and provide additional useful information about the original karyotype in cases where direct chromosomal preparations from tumors have failed.

Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene.

Several lines of evidence have suggested that c-fos may act downstream from c-Ha-ras in a growth-regulatory signal transduction pathway. We used antisense RNA to inhibit c-fos gene expression and investigated the effects of diminished c-fos expression on the phenotypes induced by the EJ c-Ha-ras oncogene in NIH 3T3 cells. Immunofluorescent staining demonstrated that the antisense RNA caused a marked reduction in the amount of c-fos protein expressed following serum stimulation. EJ cells containing antisense-fos RNA continued to overexpress ras and remained capable of proliferating in vitro. However, the antisense-fos RNA caused a partial reversion of the major transformed phenotypes of EJ cells, including a restoration of both density-dependent growth arrest and the ability to be rendered quiescent by serum deprivation, a reversion to a flat morphology, inhibition of anchorage-independent growth, and inhibition of tumorigenicity in nude mice. Our results indicate that inhibition of c-fos expression, to a level still supporting in vitro proliferation, prevents the transforming effects of the ras oncogene; they thus provide additional evidence for the participation of c-fos in ras-regulated signal transduction pathways.

Rapid induction of an experimental metastatic phenotype in first passage rat embryo cells by cotransfection of EJ c-Ha-ras and c-myc oncogenes.

First passage rat embryo cells were transfected with plasmids carrying a mutated EJ c-Ha-ras oncogene alone or in combination with the c-myc oncogene. Three days later, unselected cultures were harvested and injected into nude mice either subcutaneously to assay for tumorigenicity or intravenously to assay for metastasis to the lung. The results indicate that a transcriptionally-enhanced EJ-c-Ha-ras oncogene alone can convert normal rat cells to a tumorigenic but not metastatic phenotype. Co-transfection of a c-myc oncogene with the EJ c-Ha-ras oncogene was necessary to produce the rapid, phenotypic conversion of normal cells to transformed cells with both tumorigenic and metastatic potential. No tumors were observed in animals injected with c-myc-transfected cells. Cell lines established from EJ c-Ha-ras-induced shoulder tumors were metastatic when reinjected intravenously into nude mice. These results support the hypothesis that the cooperative action of c-Ha-ras and c-myc oncogenes is more potent in inducing malignant transformation than either oncogene acting alone. Our results also suggest that the phenotypic conversion of normal cells to tumorigenic cells with experimental metastatic potential by ras and myc oncogenes can be completed within 3-4 cell divisions after transfection.

Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.

These studies have compared the ability of NIH 3T3 cells containing different ras oncogenes to form tumor nodules in the lungs of nude mice after tail vein injection. The genes studied include the normal cellular and bladder tumor ras genes, recombinant viral/cellular ras genes, recombinant yeast/mammalian ras genes, and a constructed gene with yeast RAS1 sequences significantly modified by deletions and an oncogenic mutation. The results show that NIH 3T3 cells containing these genes readily form lethal tumor nodules in the lungs of nude mice after tail vein injection. No control NIH 3T3 cells formed lung tumors within 66 days. Although there were some quantitative differences in the potencies of the various lines, the striking conclusion is that NIH 3T3 cells transformed by either normal or activated mammalian ras genes form approximately equal numbers of experimental lung metastases. In addition, cells transformed by a significantly modified yeast RAS1 gene containing a purposefully introduced oncogenic mutation were also equally active in this assay. The amount of p21 (the 21-kDa protein encoded by ras), as measured by immunoprecipitation, was approximately the same in the parent lines before injection as in the tumors recovered after injection. This result indicates that there is no selection for metastatic sublines containing larger quantities of p21. Transfection of EJ bladder tumor ras DNA into NIH 3T3 cells followed by injection 3 days later into the tail veins of nude/beige mice indicated that the EJ ras gene can confer a metastatic phenotype within 3.5 cell generations without selection or clonal growth in vitro. Thus, the biochemical changes initiated after introduction of the c-Ha-ras gene into NIH 3T3 cells result in the almost immediate acquisition of phenotypes necessary for experimental metastasis.