Single-walled carbon nanotube-induced mitotic disruption.

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 µg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 µg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Induction of aneuploidy by single-walled carbon nanotubes.

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus.

Proteasome inhibitors induce apoptosis in human lung cancer cells through a positive feedback mechanism and the subsequent Mcl-1 protein cleavage.

Proteasome inhibitors (PIs) are promising new therapeutic agents for treating non-small cell lung carcinoma (NSCLC). To investigate the mechanisms of action of PIs, we analyzed the proapoptotic activities of PIs (MG132 or Bortezomib) in NSCLC cells. We found that both MG132 (>1 microM) and Bortezomib (>0.025 microM) induced a significant apoptosis in NCI-H1703, a PI-sensitive NSCLC cell line, through initially activating the intrinsic apoptosis pathway, leading to the activation of a positive feedback mechanism (PFM), which then conveyed apoptosis signaling from the intrinsic pathway to the extrinsic pathway with formation of a signaling loop for maximal caspase activation. Mcl-1 and Noxa were identified to be the major anti-apoptotic and proapoptotic proteins, respectively, in PI-induced apoptosis and mutually exclusive in protein stability. Although the Mcl-1 protein was upregulated by proteasome inhibition, it was also subjected to caspase 3-dependent cleavage governed by the PFM. Moreover, it was revealed that Mcl-1 protein cleavage contributed to PFM-governed apoptosis in following inter-related ways: reducing the anti-apoptotic Mcl-1; generating the truncated proapoptotic Mcl-1(S); and inducing a shift of balance between Mcl-1 and Noxa. It was further manifested that tumor necrosis factor-related apoptosis-inducing ligand boosted MG132's proapoptotic activity through strengthening the PFM in both NCI-H1703 and NCI-H358, a PI-resistant NSCLC cell line. Therefore, this study provides a basis for enhancing the efficacy of PIs in treating NSCLC.

Detection of three novel translocations and specific common chromosomal break sites in malignant melanoma by spectral karyotyping.

Chromosomal aberrations in malignant melanoma cells have been reported using standard chromosome banding analysis and comparative genomic hybridization. To identify marker chromosomes and translocations that are difficult to characterize by standard banding analysis, 15 early passage malignant melanoma cell lines were examined using spectral karyotyping. All 15 tumor cell lines had lost all or part of 1p and 10q. Losses of material on chromosome arms 4p (12/15), 6q (12/15), 9p (15/15), 12p (13/15), 12q (13/15), 13q (11/15), and 19q (14/15) were the next most frequent events. Gain of chromosome arms 1q (11/15), 6p (13/15), and 20q11 (14/15) was also observed. Interestingly, we identified translocations der(12)t(12;20)(q15;q11), der(19)t(10;19)(q23;q13), and der(12)t(12;19)(q13;q13) in 4/15 tumors. Three recurring translocations involving four of the most frequent break points were detected. The identification of recurring translocations and unique chromosome break points in melanoma will aid in the identification of the genes that are important in the neoplastic process.

Phenotypic instability and rapid gene silencing in newly formed arabidopsis allotetraploids.

Allopolyploid hybridization serves as a major pathway for plant evolution, but in its early stages it is associated with phenotypic and genomic instabilities that are poorly understood. We have investigated allopolyploidization between Arabidopsis thaliana (2n = 2x = 10; n, gametic chromosome number; x, haploid chromosome number) and Cardaminopsis arenosa (2n = 4x = 32). The variable phenotype of the allotetraploids could not be explained by cytological abnormalities. However, we found suppression of 20 of the 700 genes examined by amplified fragment length polymorphism of cDNA. Independent reverse transcription-polymerase chain reaction analyses of 10 of these 20 genes confirmed silencing in three of them, suggesting that approximately 0.4% of the genes in the allotetraploids are silenced. These three silenced genes were characterized. One, called K7, is repeated and similar to transposons. Another is RAP2.1, a member of the large APETALA2 (AP2) gene family, and has a repeated element upstream of its 5' end. The last, L6, is an unknown gene close to ALCOHOL DEHYDROGENASE on chromosome 1. CNG DNA methylation of K7 was less in the allotetraploids than in the parents, and the element varied in copy number. That K7 could be reactivated suggests epigenetic regulation. L6 was methylated in the C. arenosa genome. The present evidence that gene silencing accompanies allopolyploidization opens new avenues to this area of research.

Proliferating cell nuclear antigen staining of Fischer-344/N rat spleens affected by large granular lymphocyte leukemia.

Large granular lymphocyte (LGL) leukemia commonly occurs in the Fischer-344/N rat. The high spontaneous incidence complicates the interpretation of results from chronic carcinogenicity studies that use this rat strain. As a result, a comprehensive characterization of LGL leukemia is necessary to help understand the leukemogenic process and the applicability of staging for assessing the progression of this disease. In the current study, the proliferation rate of LGL leukemia cells from untreated control Fischer-344 (F-344) rats in 3 stages of leukemia compared to nonleukemic age-matched rats was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA). In histologic sections of spleen from aged F-344/N rats affected by LGL leukemia, there was a significant increase of both PCNA labeling and mitotic indices that was most advanced in the spleen of rats with more severe LGL leukemia. These results support biological significance for the morphologic staging system currently in use.

A novel mechanism of in vivo ras gene activation involving tandem duplication of coding sequences.

A series of weakly transforming c-K-ras genes have been detected in spontaneously occurring and chemically induced mouse adenomas. DNA sequence analysis of these weakly transforming ras oncogenes showed that activation occurred by a novel mechanism involving duplication of nine or ten codon segments flanking codon 61 in exon 2. The codon repetitions in exon 2 are directly preceded by a number of potentially recombinogenic DNA sequences which may have been involved in the genesis of the codon repetitions through mechanisms involving recombination or DNA slippage. Duplication of DNA sequences such as those observed in the mouse c-K-ras gene may represent a new mechanism for both tumor suppressor gene inactivation and proto-oncogene activation.

Diacylglycerol is an effector of the clonal expansion of cells containing activated Ha-ras genes.

Diacylglycerols (DAG) are lipid second messengers which are generated during phospholipase-catalyzed hydrolysis of phospholipids. The model DAG, sn-1,2-didecanoylglycerol (DIC10), is an effective topical tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. We now report that 11/12 of DMBA-initiated/DIC10-promoted papillomas examined contain an A-->T mutation in the 61st codon of the Ha-ras gene, suggesting that DAGs affect the clonal expansion of activated Ha-ras-containing cells. To explore further the DIC10-induced clonal expansion of activated Ha-ras-containing cells, we have examined the tumor-promoting effect of DIC10 in the skin of transgenic TG.AC mice, which harbor a v-Ha-ras transgene. By 9 weeks of promotion, 100% of the TG.AC mice developed squamous papillomas and by 15 weeks these mice developed > 20 papillomas/mouse. Because fatty acids are known to participate in signal transduction pathways, and since cellular lipases could cleave the fatty acid side chains present in DIC10, we have examined the tumor promoting activity of n-decanoic acid to verify the specificity of promotional activity of DIC10. n-Decanoic acid did not function as a tumor promoter. These data implicate DAG as an effector of the clonal expansion of mutated Ha-ras-containing cells, and support a mechanism whereby an increase in endogenous DAG could contribute to the clonal expansion of cells containing a Ha-ras oncogene.

Role of proto-oncogene activation in carcinogenesis.

The accumulation of genetic damage in the forms of activated proto-oncogenes and inactivated tumor-suppressor genes is the driving force in the evolution of a normal cell to a malignant cell. For example, both the activation of ras oncogenes and the inactivation of several suppressor genes, including p53, have been observed in the development of human colon and lung tumors. Point mutations in key codons can activate ras proto-oncogenes and inactivate the p53 suppressor gene. Thus, several critical genes for tumorigenesis are potential targets for carcinogens and radiation that can induce point mutations at low doses. The ras proto-oncogenes are targets for many genotoxic carcinogens. Activation of the ras gene is an early event--probably the "initiating" step--in the development of many chemical-induced rodent tumors. ras Oncogenes are observed in more human tumors and at a higher frequency than any other oncogene, and activation of the proto-oncogene may occur at various stages of the carcinogenic process. Numerous proto-oncogenes other than the ras genes have been shown to be activated in human tumors and to a lesser extent in rodent tumors. Mechanisms that induce aberrant expression of proto-oncogenes are gene amplification and chromosomal translocation or gene rearrangement. Amplification of proto-oncogenes and possibly gene overexpression during the absence of gene amplification occur in the development of many human tumors. For a specific tumor type, amplification of any one proto-oncogene may occur at a low frequency, but the frequency of tumors in which at least one proto-oncogene is amplified can be much higher. Proto-oncogene amplification is usually associated with late stages of tumor progression; however, amplified HER2/neu has been observed in early clinical stages of mammary neoplasia. Activation of proto-oncogenes by chromosomal translocation has been detected at a high frequency in several hematopoietic tumors. Non-ras genes have been detected by DNA transfection assays in both human and rodent tumors. For example, ret and trk genes were found to be activated by gene rearrangements in human papillary thyroid carcinomas. Several potentially new types of oncogenes have also been detected by DNA transfection assays. The etiology of the genetic alterations observed in most human tumors is unclear at present. Examples of ras gene activation and those documented for mutations in the p53 gene demonstrate that exogenous conditions can induce oncogenic mutants of normal genes. The genetic alterations observed in most human tumors are probably generated by both spontaneous events and exogenous conditions.

Parental bias of Ki-ras oncogenes detected in lung tumors from mouse hybrids.

A mouse strain with low lung tumor susceptibility (C3H) and a strain with high lung tumor susceptibility (A/J) were reciprocally crossed to produce C3A and AC3 F1 hybrid mice. Ki-ras oncogenes were detected in spontaneous and chemically induced lung tumors obtained from the C3A and AC3 mice. To further explore the genetics of the Ki-ras gene in mouse lung tumor susceptibility, the parental origin of Ki-ras oncogenes detected in lung tumors from the F1 hybrids was determined by a strategy based on a 37-base-pair deletion in the second intron of the A/J Ki-ras allele. Ki-ras oncogenes were derived from the A/J parent in 38 of 40 tumors obtained from C3A mice and 30 of 30 tumors from AC3 mice. The observation that the activated oncogene in hybrids originates from the susceptible parent suggests that the Ki-ras gene is directly linked to mouse lung tumor susceptibility. This finding may have implications for pulmonary adenocarcinoma development in humans, since Ki-ras oncogenes are detected in 35% of this human tumor type.

Development of a highly efficient expression cDNA cloning system: application to oncogene isolation.

We developed an expression cDNA cloning system capable of generating high-complexity libraries with unidirectionally inserted cDNA fragments and allowing efficient plasmid rescue. As an application of this system, a cDNA library was constructed from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA. Transfection of NIH 3T3 cells by the library DNA led to the detection of several transformed foci from which identical plasmids with transforming ability could be rescued. Structure and sequence analysis of the cDNA clones revealed that the oncogene was created by recombinational events involving an unknown gene and the mouse homologue of the B-raf protooncogene. Detection of the same genetic rearrangement in independent primary transformants implied that generation of the oncogene occurred within the tumor rather than during DNA transfection or cDNA library construction. The high frequency at which clones were identified and the large sizes of some of the transforming cDNA inserts isolated suggest wide applicability of this mammalian expression cloning system for isolating cDNAs of biologic interest.

Activation of proto-oncogenes in human and mouse lung tumors.

Lung cancer is a leading cause of cancer-related deaths in several nations. Epidemiological studies have indicated that 85% of all lung cancer deaths and 30% of all cancer deaths in the U.S. are associated with tobacco smoking. Various chemicals in tobacco smoke are thought to react with DNA and to ultimately yield heritable mutations. In an effort to understand the molecular mechanisms involved in lung tumorigenesis, we have analyzed proto-oncogene activation in a series of human lung tumors from smokers and spontaneously occurring and chemically induced lung tumors in mice. Approximately 86% of the human lung tumors and greater than 90% of the mouse lung tumors were found to contain activated oncogenes. ras Oncogenes activated by point mutations were detected in many of the human lung adenocarcinomas and virtually all of the mouse lung adenomas and adenocarcinomas. The mutation profiles of the activated K-ras genes detected in the chemically induced mouse lung tumors suggest that the observed mutations result from genotoxic effects of the chemicals. Comparison of the K-ras mutations observed in the human lung adenocarcinomas with mutation profiles observed in the mouse lung tumors suggest that bulky hydrophobic DNA adducts may be responsible for the majority of the mutations observed in the activated human K-ras genes. Other data indicate that approximately 20% of human lung tumors contain potentially novel transforming genes that may also be targets for mutagens in cigarette smoke.

Activated protooncogenes in human lung tumors from smokers.

Fourteen primary human lung tumor DNAs from smokers were analyzed for transforming activity by two DNA transfection assays. Activated protooncogenes were detected in 3 of 11 tumor DNAs by the NIH 3T3 focus assay, whereas activated protooncogenes were detected in 11 of 13 tumor DNAs by the NIH 3T3 cotransfection-nude mouse tumorigenicity assay. K- or NRAS genes activated by point mutation at codons 12 or 61 were detected in a large cell carcinoma, a squamous cell carcinoma, and 5 adenocarcinomas. An HRAS oncogene activated by a different mechanism was detected in an epidermoid carcinoma. One adenocarcinoma was found to contain an activated RAF gene. Two unidentified transforming genes were detected in a squamous cell carcinoma DNA and two adenocarcinoma DNAs. Eight of 10 lung adenocarcinomas that had formed metastases at the time of surgery were found to contain RAS oncogenes. No significant increase in metastasis was observed in the lung adenocarcinomas that contained one or more 6-kilobase EcoRI alleles of the LMYC gene. Overall, 12 of 14 (86%) of the lung tumor DNAs from smokers were found to contain activated protooncogenes. RAS oncogenes appear to play a role in the development of metastases in lung adenocarcinomas.

Activation of protooncogenes in spontaneously occurring non-liver tumors from C57BL/6 x C3H F1 mice.

The C57BL/6 x C3H F1 (hereafter called B6C3F1) mouse is an important animal model for long-term carcinogenesis studies. Maintained under normal laboratory conditions, these mice develop various types of spontaneous tumors during their lifetime. Activated Ha-ras genes have been detected in 66% of spontaneous hepatocellular tumors in the B6C3F1 mouse [Reynolds et al., Science (Washington DC), 237:1309, 1988]. In this study 49 spontaneous non-liver tumors were investigated for oncogene activation by DNA transfection techniques. Of the 49 tumor DNAs analyzed, only 5 yielded multiple foci in the NIH 3T3 focus assay: 2 of 10 pulmonary adenocarcinomas; 0 of 25 lymphomas; 2 of 2 Harderian gland adenomas; 0 of 1 adenocarcinoma of the small intestine; 1 of 6 malignant skin tumors; 0 of 4 hemangiosarcomas; and 0 of 1 lung metastasis of a hepatocellular carcinoma. DNA from six lymphomas which were negative in the NIH 3T3 focus assay were further analyzed for transforming genes by the nude mouse tumorigenicity assay. One of the five lymphomas tested positive with this assay. Southern blot analysis identified five activated ras genes: H-ras in two Harderian gland adenomas; K-ras in one pulmonary adenocarcinoma and in one s.c. adenocarcinoma; and N-ras in one lymphoma. The mutations involved were CG to AT and AT to TA in codon 61 of the Ha-ras genes, GC to AT or TA in codon 12 of the K-ras genes, and a GC to AT mutation in codon 12 of the N-ras gene. Transformant DNA from a pulmonary adenocarcinoma which yielded multiple foci in the transfection assay did not hybridize to DNA probes specific for the K-, H-, and N-ras, raf, neu, and met genes. Thirteen additional tumor DNAs yielded a single focus in the NIH 3T3 transfection assay. The transformant DNAs retransmitted in a second cycle transfection assay. Rearranged and/or amplified raf genes were detected in six of the transformant DNAs. At present we do not know whether these activated raf genes were present in the original tumor DNA. The other seven transformant DNAs did not hybridize with any of the above mentioned specific DNA probes utilized in Southern blot analysis. Unlike liver tumors, the activation of ras protooncogenes is not a frequent event in the development of spontaneous non-liver tumors of the B6C3F1 mouse. The results from this study should aid in understanding the neoplastic development associated with exposure to chemical carcinogens in the B6C3F1 mouse.

Proto-oncogene activation in rodent and human tumors.

The transformation of a normal cell into a tumorigenic cell involves both the activation and concerted expression of proto-oncogenes and inactivation of suppressor genes. The activation of ras proto-oncogenes represents one step in the multistep process of carcinogenesis for a variety of rodent and human tumors. This activation is probably an early event in tumorigenesis in many cases and may be the 'initiation' event in some cases. Thus, a chemical that induces rodent tumors by activation of ras proto-oncogenes can potentially invoke one step of the neoplastic process in humans exposed to the chemical. Moreover, dominant transforming oncogenes other than ras have been detected in human tumors as well as rodent tumors. The involvement of these putative proto-oncogenes in the development of neoplasia is unclear at present.

Mutational activation of the cellular Harvey ras oncogene in rat esophageal papillomas induced by methylbenzylnitrosamine.

Epidemiological studies have demonstrated a strong association between human esophageal cancer and exposure to N-nitroso carcinogens. Esophageal tumors can be induced in experimental animals, especially in rats, by many N-nitroso carcinogens. In the present study, rat esophageal tumors induced by methylbenzylnitrosamine (MBNA) and MBNA-transformed esophageal cell lines were analyzed for activated protooncogenes. DNAs from four Fisher 344 rat esophageal papillomas were examined for their ability to induce morphological transformation of NIH 3T3 cells. One of four esophageal tumors was positive in this assay. Southern blot analysis of this NIH 3T3 transformant revealed that the transforming gene was an activated Ha-ras gene. The activating mutation in the Ha-ras gene was identified by amplifying and then sequencing the first exon of this gene. A GC----AT transition at the second base in codon 12 of the Ha-ras gene was detected. The tumor DNAs from the transfection-negative samples were also amplified, and sequencing analysis of the first exon revealed a GC----AT transition in codon 12. In addition, 14 formalin-fixed and paraffin-embedded rat esophageal papillomas were shown to contain the same mutation in one of the alleles of the Ha-ras gene. In contrast, no point mutation was found in codons 12, 13, and 61 of the Ha-, Ki-, or N-ras genes in MBNA-transformed rat esophageal cell lines. The GC----AT transition detected in the esophageal tumors by DNA sequencing was confirmed by slot blot oligonucleotide hybridization of the polymerase chain reaction-amplified DNAs. The fact that mutated Ha-ras genes were detected in the esophageal papillomas suggests that activation of this gene occurred early in the process of neoplastic progression. The point mutation detected in the Ha-ras gene appears to result from a direct genotoxic effect of MBNA involving formation of the O6-methylguanine adduct. Taken together, these studies suggest that the activation of the Ha-ras gene plays an important role in the induction of esophageal neoplasia in the Fisher 344 rat by MBNA.

ras gene activation in rat tumors induced by benzidine congeners and derived dyes.

Dimethoxybenzidine (DMO) and dimethylbenzidine (DM) are used to synthesize dyes such as C.I. Direct Blue 15 and C.I. Acid Red 114, respectively. These commercially used dyes are metabolically degraded to DMO or DM in the intestinal tract of rodents and subsequently DMO and DM are absorbed into the blood stream. Animals were exposed to DMO, DM, or the dyes in the drinking water. Tumors obtained from control and chemical-treated animals were examined for the presence of activated oncogenes by the NIH 3T3 DNA transfection assay. Activated oncogenes were detected in less than 3% (1/38) of the tumors from control animals whereas 68% (34/50) of the tumors from chemical-treated animals contained detectable oncogenes. Activated oncogenes were detected in both malignant (25/36) and benign (9/14) tumors from the chemically treated animals but only in one of 13 malignant tumors from the control animals. The presence of oncogenes in the chemically induced benign tumors suggests that oncogene activation was an early event in those tumors. Southern blot analysis of transfectant DNA showed that the transforming properties of the chemically induced rat tumor DNAs were due to the transfer of an activated H-ras (31/34) or N-ras (3/34) gene. One spontaneous rat tumor DNA was found to contain an activated H-ras gene. Oligonucleotide hybridization analysis indicated that the H-ras oncogenes from chemical-associated tumors contained mutations at codons 12, 13, or 61 whereas the spontaneously activated H-ras gene contained a point mutation at codon 61. These data suggest that activation of cellular ras genes by point mutation is an important step in the induction of tumors, at least in rats, by this class of benzidine-derived dyes. Moreover, in light of common histogenesis of the normal counterparts of many of the chemically induced neoplasms and histological evidence of varied tissue differentiation in some basal cell neoplasms, it is possible that most or all of the chemically induced neoplasms were derived from a common epidermal progenitor stem cell population.

Oncogene activation in spontaneous and chemically induced rodent tumors: implications for risk analysis.

The validity of rodent tumor end points in assessing the potential hazards of chemical exposure to humans is a somewhat controversial but very important issue since most chemicals are classified as potentially hazardous to humans on the basis of long-term carcinogenesis studies in rodents. The ability to distinguish between genotoxic, cytotoxic, or receptor-mediated promotion effects of chemical treatment would aid in the interpretation of rodent carcinogenesis data. Activated oncogenes in spontaneously occurring and chemically induced rodent tumors were examined and compared as one approach to determine the mechanism by which chemical treatment caused an increased incidence of rodent tumors. Different patterns of activated oncogenes were found not only in spontaneous versus chemically induced mouse liver tumors but also in a variety of spontaneous rat tumors versus chemically induced rat lung tumors. In the absence of cytotoxic effects, it could be argued that the chemicals in question activated protooncogenes by a direct genotoxic mechanism. These results provided a basis for the analysis of activated oncogenes in spontaneous and chemically induced rodent tumors to provide information at a molecular level to aid in the extrapolation of rodent carcinogenesis data to human risk assessment.

Activation of H-ras oncogene in rat bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Ras-oncogene activation was investigated in the bladder tumors of F344 male rats given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water. DNA from one of the nine transitional cell carcinomas contained an H-ras oncogene detectable by the NIH/3T3 transfection assay. Analysis of p21 ras proteins suggested that the activating mutation resided within codon 61 of the H-ras gene and that such activating mutations were not present in other tumors. In contrast to mutational activation of ras genes, enhanced expression of p21 was observed in all tumors examined by immunohistochemical techniques with the use of Formalin-fixed paraffin-embedded tissue sections and an anti-ras p21 antibody, RAP-5. Further histochemical analysis of bladder tissues at various stages of the BBN-induced carcinogenic process indicated that the enhanced expression of p21 appeared early; the reactivity with RAP-5 was observed in diffuse hyperplastic epithelia after 5 weeks of exposure to BBN. The frequency of ras oncogenes, activated either by point mutations or overexpression of p21, in BBN-induced rat bladder carcinomas has thus been shown to be similar to that observed in human bladder carcinomas.