Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease.

Four clones were isolated from an adult human brain complementary DNA library with an oligonucleotide probe corresponding to the first 20 amino acids of the beta peptide of brain amyloid from Alzheimer's disease. The open reading frame of the sequenced clone coded for 97 amino acids, including the known amino acid sequence of this polypeptide. The 3.5-kilobase messenger RNA was detected in mammalian brains and human thymus. The gene is highly conserved in evolution and has been mapped to human chromosome 21.

RNA metabolism in tracheal epithelium: alteration in hamsters deficient in vitamin A.

The electrophoretic pattern of RNA molecules that are synthesized in vitro in tracheal epithelium from hamsters deficient in vitamin A differs from that of RNA synthesized in normal, pair-fed control hamsters. There is less RNA of low electrophoretic mobility in the epithelial cells deficient in vitamin A. This alteration is reversed after the deficient animals have been treated with vitamin A.

Morphological and cytokinetic responses of hamster airways to intralaryngeal or intratracheal cannulation with instillation of saline or ferric oxide particles in saline.

The morphological and proliferative effects of intratracheal cannulation (ITC) or intralaryngeal cannulation (ILC), with or without the instillation of saline or Fe2O3 particles in saline, were studied in Syrian golden hamsters. Instillation of Fe2O3 in saline at either airway level resulted in a similar distribution of Fe2O3 particles in all lung lobes. ILC produced laryngeal mucosal wounds. ITC produced laryngeal and tracheal mucosal wounds. The cannula-induced wounds were associated with proliferative epithelial lesions. ITC, but not ILC, resulted in significant increases in the mitotic rates (MR, 6-h colchicine blockade) of tracheal epithelial cells at 24 and 32 h postcannulation. Instillation of saline by ITC produced slight increases in intrapulmonary bronchial and bronchiolar MR, but saline given by ILC did not increase MR at any airway level. Instillation of Fe2O3 particles in saline by ITC produced increases in tracheal, intrapulmonary bronchial, and bronchiolar MR. Instillation of Fe2O3 particles in saline by ILC had little effect on tracheal MR, but increased intrapulmonary bronchial and bronchiolar MR. Foci of Fe2O3 particle-laden macrophages were associated with mild bronchiolar-alveolar hyperplasia at the junctions of the terminal bronchioles and the alveolar ducts. The cytokinetic and morphological changes in the intrapulmonary airways were associated with the influx of inflammatory cells in response to Fe2O3 particle deposition. The marked increases in tracheal MR and the localized hyperplastic tracheal epithelial lesions were clearly associated with mechanical wounding from the cannula during ITC. Comparative studies using ILC or ITC instillation techniques allowed further investigations of the important role of tracheal mucosal wounding in the induction of respiratory carcinogenesis, as described in a companion paper (Keenan et al., Cancer Res., 49: 1528-1540, 1989).

Multifactorial hamster respiratory carcinogenesis with interdependent effects of cannula-induced mucosal wounding, saline, ferric oxide, benzo[a]pyrene and N-methyl-N-nitrosourea.

The carcinogenic response induced in the respiratory tract of Syrian golden hamsters by repeated intratracheal instillations of benzo[a]pyrene (BP) adsorbed to ferric oxide (Fe2O3) particles suspended in saline, is shown to result from the interactions of these factors and cannula-induced tracheal wounding. Previous acute studies of intratracheal cannulation (ITC) versus intralaryngeal cannulation (ILC) showed that tracheal cell proliferation increased significantly in ITC-induced mucosal wounds. Only mild increases in intrapulmonary cell proliferation were produced by Fe2O3-saline given by ILC or ITC (Keenan et al., Cancer Res., 49: 1521-1527, 1989). The present chronic studies included the following variables: a single instillation by ILC of N-methyl-N-nitrosourea (MNU) at 5 weeks of age; 15 weekly treatments (beginning at 7 weeks of age) by ILC or ITC alone, or with instillations of saline, or Fe2O3-saline, or BP-Fe2O3-saline; and appropriate controls. Repeated ITC-induced tracheal wounds caused persistent tracheal epithelial hyperplasia, metaplasia and/or atrophy and submucosal fibroplasia during the observation period of 22 to 78 weeks of age (the time of terminal sacrifice). Tracheal cancers (in situ or invasive carcinomas) were seen only in those hamsters which had received repeated ITC and one or both carcinogens. The cancer latency was shortest and the incidence of tracheal (50%) and main-stem bronchial (21%) cancers highest in hamsters given MNU and repeated ITC with BP-Fe2O3-saline. Hamsters given carcinogens by ILC (which induced laryngeal but not tracheal wounds) developed proliferative lesions and cancers of the larynx but no tracheobronchial cancers. These data show the singular importance of repeated ITC-induced intratracheal wounding as an enhancing factor in this respiratory carcinogenesis model. The findings suggest that the mechanism of tumor enhancement involves not only changes in target epithelial cell proliferation, but also alterations in normal epithelial-mesenchymal interactions during tracheal regeneration from repeated chronic submucosal inflammation and mesenchymal repair. In the present experimental model, a single dose of MNU at 5 weeks of age, repeated instilled doses of BP, and tracheal mucosal wounding were each found to be important determinants of the carcinogenic response. Additional effects were observed for instilled Fe2O3 particles, and possibly saline. Interplay of all these factors, as well as of genetic, nutritional, and infectious factors, are considered in relation to risk assessment and prevention.

Temporal dissociation in the exposure times required for maximal induction of cytotoxicity, mutation, and transformation by N-methyl-N'-nitro-N-nitrosoguanidine in the BALB/3T3 ClA31-1-1 cell line.

Cytotoxicity, alkali-labile DNA lesions, ouabain resistance mutations, and neoplastic transformation were analyzed concurrently in the BALB/3T3 ClA31 -1-1 cell line treated with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for different exposure times (15, 30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). The half-life of MNNG in complete medium was approximately 70 min, both without cells and with cell numbers as used in the assays for cytotoxicity (2 X 10(2) cells/60-mm dish), transformation (1 X 10(4) cells/dish), and mutation (1 X 10(5) cells/dish). The cytotoxic effect of MNNG (0.5 or 2 micrograms/ml) appeared to be completed after an exposure time between 100 and 200 min. Maximal frequency of ouabain resistance mutations, however, was reached after a much shorter treatment time (30 to 60 min). Detection of DNA damage by alkaline elution analysis showed maximal increase in single-strand breaks already after treatment for 30 min. Exposures for 30 min followed by posttreatment incubation for 30 or 90 min showed active repair of single-strand breaks during these periods, indicative of an even balance between the additional MNNG-induced damage and its repair. Morphological transformation assays, at the same treatment times and concentrations used in the mutation assays, yielded frequency curves that reached their maxima 1 to 3 hr later than did the mutation frequencies. The ratio of transformation to ouabain resistance mutation frequencies was 3.7 for short treatment times (30 to 60 min), while it increased to more than 20 for exposure times of 240 min or longer. The temporal dissociation in the exposure times for maximal induction of mutation and transformation, observed with MNNG in this cell line, supports the hypothesis that a single gene mutational event is not sufficient to account for the full expression of neoplastic transformation.

Secretion of type IV collagenolytic protease and metastatic phenotype: induction by transfection with c-Ha-ras but not c-Ha-ras plus Ad2-E1a.

Activated ras oncogene transfection into suitable recipient cells has been shown to induce the metastatic phenotype (Thorgeirsson, et al., Mol. Cell. Biol., 5: 259-262, 1985). We have used this model system to study the correlation of basement membrane collagenolysis with metastatic propensity. The c-Ha-ras oncogene alone, or combined with v-myc, transfected into early passage rat embryo fibroblasts, induce these cells to secrete high levels of type IV collagenolytic metalloproteinase and to concomitantly exhibit a high incidence of spontaneous metastases in nude mice. Cotransfection of c-Ha-ras plus the adenovirus type 2 E1a gene yields cells which are highly tumorigenic but nonmetastatic and fail to produce type IV collagenase. This effect is due to a suppression of collagenase elaboration, not increased production of a collagenase inhibitor, and not decreased production of a collagenase activator. The characteristics of the collagenase are identical to tumor type IV collagenase described previously. The nonmetastatic cells which failed to produce type IV collagenase retain the ability to secrete high levels of plasminogen activator. Transfection with the protooncogenic forms of Ha-ras or mos, or spontaneous transformation of NIH 3T3 cells or chemical transformation of BALB 3T3 cells yields cells which fail to produce collagenase, are tumorigenic, but totally nonmetastatic. These data support a biochemical linkage of type IV collagenase expression with the metastatic phenotype in this rodent system.

Transformation of human neonatal prostate epithelial cells by strontium phosphate transfection with a plasmid containing SV40 early region genes.

Neonatal human prostatic epithelial cells (NP-2s) were transfected by strontium phosphate coprecipitation with a plasmid (pRSV-T) containing the SV40 early region genes. The cells transfected with pRSV-T, but not the sham-transfected controls, formed rapidly growing, multilayered colonies within 2 weeks at a frequency of 1 x 10(-4) in a serum-free medium (P4-8F). In all, 28 colonies of transformed cells were isolated. Three of these have been cultured for a sufficient length of time to show that their growth potentials are well beyond that of the normal progenitor cells (NP-2s). There is also little or no indication of the culture "crisis" commonly seen in SV40-transformed cells in these transfected lines. All contain cytokeratins and SV40 T-antigen as revealed by immunofluorescence, have ultrastructural features of epithelial cells, and are pseudodiploid. None have produced tumors within 1 year after s.c. injection into nude mice. The transformed as well as the parental NP-2s cells require bovine pituitary extract for growth in serum-free medium and are stimulated by transforming growth factor beta 1 (TGF-beta 1) and epidermal growth factor in clonal growth assays. In contrast, a prostatic carcinoma cell line (PC-3) is inhibited by TGF-beta 1. This serum-free system and immortalized transfected clones will be useful for studying the action of putative prostatic carcinogens and tumor-promoting agents.

Cutaneous chemical carcinogenesis: past, present, and future.

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small dose of carinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. When epidermal cells in vitro are exposed to promoting agents, a proliferative response analogous to that in vivo is elicited, apparently mediated through control of polyamine metabolism. Neoplastic transformation has been induced in these cultures by known skin carcinogens.

Oxidative DNA damage by crystalline silica.

Using a simple DNA strand breakage assay, we detected the production of oxidant species, probably hydroxyl free radicals, in buffered suspensions of crystalline silica at pH 7.4. DNA damage was affected by the presence of oxygen and was accelerated by superoxide dismutase and by hydrogen peroxide. Deferoxamine blocked damage by hydrogen peroxide and silica but accelerated DNA damage by silica alone and by superoxide dismutase and silica. DNA damage was blocked by catalase and by the scavenging agents dimethyl sulfoxide and sodium benzoate. Chemical etching of crystalline silica to remove impurities by treatment of the surface with hydrofluoric acid resulted in markedly diminished DNA damaging ability. Even preparations of crystalline silica previously characterized as highly pure contained trace iron impurities in amounts significant enough to produce oxygen free radicals in aqueous suspension. Both superoxide and Fenton reaction oxidants were produced. We conclude that silica is able to mediate DNA strand breakage in vitro and that this DNA damage may be an important factor in silica toxicity.

Evaluation of mixed exposure to carcinogens and correlations of in vivo and in vitro systems.

The biological evaluation of air pollutants is an example of the difficulties of evaluating the effects of mixed concurrent exposures to multiple agents, such as combinations of carcinogens with other carcinogens of the same or different chemical class, with incomplete carcinogens and cocarcinogens, with particulate materials and other factors that modify tissue distribution and retention, and with modifiers of metabolic pathways of activation and detoxication. A research approach is outlined to investigate such interactions in a series of biological systems of increasing complexity but closely related to each other in a step-by-step sequence, e.g., bacterial mutagenesis; mammalian cell mutagenesis, toxicity and neoplastic transformation, including embryo cells, fibroblasts and epithelial cells; organ cultures of target epithelia; in vivo animal systems for short-term and long-term studies, including animal models closely comparable to human pathology; observational studies of human pathology and histopathogenesis; experimental studies of corresponding human target tissues using organ and cell culture methods for metabolism, toxicity, mutagenicity and possibly neoplastic cell transformation. Respiratory carcinogenesis models were successfully used for studies of mixed exposures to different carcinogens and cofactors. The role of particulates has been found to be important but needs to be further characterized. Quantitative variations in the response to carcinogens and cofactors among different biological test systems and among different individuals in the human population make quantitative risk estimation very difficult, but studies in a sequence of related biological systems including human tissues indicate the importance of qualitative risk evaluation.

Experimental identification of chemical carcinogens, risk evaluation, and animal-to-animal correlations.

Experimental methods for the identification of chemical carcinogens have been extensively developed, including animal bioassay methods, animal models for cancer induction at major organ sites, models for the study of the effects of carcinogens in cells and tissues in culture and methods for the study of molecular events (metabolic activation, binding and detoxification of carcinogens; DNA damage and repair; mutagenicity). Current sources of documentation on carcinogenicity data are reviewed. The number of "known carcinogens" will vary considerably, depending on the criteria adopted for accepting evidence of carcinogenicity. CRITERIA FOR THE EVALUATION OF RISKS, BENEFITS, AND TECHNOLOGICAL ALTERNATIVES FOR PUBLIC POLICY ON ENVIRONMENTAL CARCINOGENS ARE REVIEWED AND THE FOLLOWING STEPS DISCUSSED: registration of environmental chemical carcinogens and their uses; risk evaluation (considering sources, adequacy, quality and limits of the evidence; quantitative dose-response extrapolation within the same biological system; and species and model conversion factors); benefits evaluation; analysis of technological alternatives; comparative judgment and decision; open public documentation. The problem of animal-to-human correlations is considered, particularly for respiratory carcinogenesis. A laboratory approach is reviewed which includes: development and study of whole animal models for carcinogenesis, analysis of animal tissue responses to carcinogens in vivo and through in vitro culture methods for morphological and biochemical studies, and development of in vitro culture methods for human target tissues. This approach is aimed at providing an experimentally controlled and quantifiable method for the correlation of animal and human observations in carcinogenesis.

DNA binding to crystalline silica characterized by Fourier-transform infrared spectroscopy.

The interaction of DNA with crystalline silica in buffered aqueous solutions at physiologic pH has been investigated by Fourier-transform infrared spectroscopy (FT-IR). In aqueous buffer, significant changes occur in the spectra of DNA and silica upon coincubation, suggesting that a DNA-silica complex forms as silica interacts with DNA. As compared to the spectrum of silica alone, the changes in the FT-IR spectrum of silica in the DNA-silica complex are consistent with an Si-O bond perturbation on the surface of the silica crystal. DNA remains in a B-form conformation in the DNA-silica complex. The most prominent changes in the DNA spectrum occur in the 1225 to 1000 cm-1 region. Upon binding, the PO2- asymmetric stretch at 1225 cm-1 is increased in intensity and slightly shifted to lower frequencies; the PO2- symmetric stretch at 1086 cm-1 is markedly increased in intensity and the band at 1053 cm-1, representing either the phosphodiester or the C-O stretch of DNA backbone, is significantly reduced in intensity. In D2O buffer, the DNA spectrum reveals a marked increase in intensity of the peak at 1086 cm-1 and a progressive decrease in intensity of the peak at 1053 cm-1 when DNA is exposed to increasing concentrations of silica. The carbonyl band at 1688 cm-1 diminishes and shifts to slightly lower frequencies with increasing concentrations of silica. The present study demonstrates that crystalline silica binds to the phosphate-sugar backbone of DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Silica radical-induced DNA damage and lipid peroxidation.

In recent years, more attention has been given to the mechanism of disease induction caused by the surface properties of minerals. In this respect, specific research needs to be focused on the biologic interactions of oxygen radicals generated by mineral particles resulting in cell injury and DNA damage leading to fibrogenesis and carcinogenesis. In this investigation, we used electron spin resonance (ESR) and spin trapping to study oxygen radical generation from aqueous suspensions of freshly fractured crystalline silica. Hydroxyl radical (.OH), superoxide radical (O2.-) and singlet oxygen (1O2) were all detected. Superoxide dismutase (SOD) partially inhibited .OH yield, whereas catalase abolished .OH generation. H2O2 enhanced .OH generation while deferoxamine inhibited it, indicating that .OH is generated via a Haber-Weiss type reaction. These spin trapping measurements provide the first evidence that aqueous suspensions of silica particles generate O2.- and 1O2. Oxygen consumption measurements indicate that freshly fractured silica uses molecular oxygen to generate O2.- and 1O2. Electrophoretic assays of in vitro DNA strand breakages showed that freshly fractured silica induced DNA strand breakage, which was inhibited by catalase and enhanced by H2O2. In an argon atmosphere, DNA damage was suppressed, showing that molecular oxygen is required for the silica-induced DNA damage. Incubation of freshly fractured silica with linoleic acid generated linoleic acid-derived free radicals and caused dose-dependent lipid peroxidation as measured by ESR spin trapping and malondialdehyde formation. SOD, catalase, and sodium benzoate inhibited lipid peroxidation by 49, 52, and 75%, respectively, again showing the role of oxygen radicals in silica-induced lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of carcinogenesis by crystalline silica in relation to oxygen radicals.

The carcinogenic effects of crystalline silica in rat lungs were extensively demonstrated by many experimental long-term studies, showing a marked predominance for adenocarcinomas originating from alveolar type II cells and associated with areas of pulmonary fibrosis (silicosis). In contrast with its effects in rats, silica did not induce alveolar type II hyperplasia and lung tumors in mice and hamsters, pointing to a critical role for host factors. Using these animal models, we are investigating the role of cytokines and other cellular mediators on the proliferation of alveolar type II cells. Immunohistochemical localization of TGF-beta 1 precursor in alveolar type II cells adjacent to silicotic granulomas was shown to occur in rats, but not in mice, and hamsters, suggesting a pathogenetic role for this regulatory growth factor. Recent investigations in our laboratory on the biologic mechanisms of crystalline silica included determination of anionic sites on crystalline silica surfaces by binding of the cationic dye Janus Green B; binding of crystalline silica to DNA, demonstrated by infrared spectrometry; production of oxygen radicals by crystalline silica in aqueous media; induction of DNA strand breakage and base oxidation in vitro and its potentiation by superoxide dismutase and by hydrogen peroxide; and induction by crystalline silica of neoplastic transformation and chromosomal damage in cells in culture. On the basis of these in vitro studies, we propose that DNA binding to crystalline silica surfaces may be important in silica carcinogenesis by anchoring DNA close to sites of oxygen radical production on the silica surface, so that the oxygen radicals are produced within a few A from their target DNA nucleotides.

Isolation, characterization, and chromosomal localization of human brain cDNA clones coding for the precursor of the amyloid of brain in Alzheimer's disease, Down's syndrome and aging.

Four clones have been isolated from the adult human brain cDNA library using an oligonucleotide probe corresponding to the first 20 amino acids of the brain amyloid polypeptide. The open reading frame of the sequenced clone coded for the known full amino acid sequence of the brain amyloid polypeptide. The 3 kb messenger RNA has been detected in a variety of tissues from human and many nonhuman species. The gene is highly conserved in evolution and has been mapped on human chromosome 21.

Vanadium(IV)-mediated free radical generation and related 2'-deoxyguanosine hydroxylation and DNA damage.

Free radical generation, 2'-deoxyguanosine (dG) hydroxylation and DNA damage by vanadium(IV) reactions were investigated. Vanadium(IV) caused molecular oxygen dependent dG hydroxylation to form 8-hydroxyl-2'-deoxyguanosine (8-OHdG). During a 15 min incubation of 1.0 mM dG and 1.0 mM VOSO4 in phosphate buffer solution (pH 7.4) at room temperature under ambient air, dG was converted to 8-OHdG with a yield of about 0.31%. Catalase and formate inhibited the 8-OHdG formation while superoxide dismutase enhanced it. Metal ion chelators, DTPA and deferoxamine, blocked the 8-OHdG formation. Incubation of vanadium(IV) with dG in argon did not generate any significant amount of 8-OHdG, indicating the role of molecular oxygen in the mechanism of vanadium(IV)-induced dG hydroxylation. Vanadium(IV) also caused molecular oxygen-dependent DNA strand breaks in a pattern similar to that observed for dG hydroxylation. ESR spin trapping measurements demonstrated that the reaction of vanadium(IV) with H2O2 generated OH radicals, which were inhibited by DTPA and deferoxamine. Incubation of vanadium(IV) with dG or with DNA in the presence of H2O2 resulted in an enhanced 8-OHdG formation and substantial DNA double strand breaks. Sodium formate inhibited 8-OHdG formation while DTPA had no significant effect. Deferoxamine enhanced the 8-OHdG generation by 2.5-fold. ESR and UV measurements provided evidence for the complex formation between vanadium(IV) and deferoxamine. UV-visible measurements indicate that dG, vanadium(IV) and deferoxamine are able to form a complex, thereby, facilitating site-specific 8-OHdG formation. Reaction of vanadium(IV) with t-butyl hydroperoxide generated hydroperoxide-derived free radicals, which caused 8-OHdG formation from dG and DNA strand breaks. DTPA and deferoxamine attenuated vanadium(IV)/t-butyl-OOH-induced DNA strand breaks.

Generation of thiyl and ascorbyl radicals in the reaction of peroxynitrite with thiols and ascorbate at physiological pH.

Electron spin resonance (ESR) spin trapping was utilized to investigate the reaction of peroxynitrite with thiols and ascorbate at physiological pH. The spin trap used was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The reaction of peroxynitrite with DMPO generated 5,5-dimethylpyrrolidone-(2)-oxy-(1) (DMPOX). Formate enhanced the peroxynitrite decomposition but did not generate any detectable amount of formate-derived free radicals. Thus, the spin trapping measurements provided no evidence for hydroxyl (.OH) radical generation in peroxynitrite decomposition at physiological pH. Thiols (glutathione, cysteine, and penicillamine) and ascorbate reacted with peroxynitrite to generate the corresponding thiyl and ascorbyl radicals. The one-electron oxidation of thiols by peroxynitrite may be one of the important mechanisms for peroxynitrite-induced toxicity and ascorbate may provide a detoxification pathway.

Split-dose exposure to N-methyl-N'-nitro-N-nitrosoguanidine in BALB/3T3 C1 a31-1-1 cells: evidence of DNA repair by alkaline elution without changes in cell survival, mutation and transformation rates.

Dose fractionation of a direct-acting chemical carcinogen, the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was studied for its concurrent effects on survival, DNA damage and repair, ouabain resistance (Ouar) mutations and neoplastic transformation, in the mouse embryo cell line BALB/3T3 C1A31-1-1. MNNG doses of 0.5, 1 and 2 micrograms/ml were added to the cells either as a single exposure or in two equal fractions separated by 1, 3 or 5 h intervals. No significant difference in cytotoxicity was found when single and split-dose treatments were compared. No recovery from sublethal damage was therefore found in this cell line by split-dose administration of MNNG, although such an effect was found when the same cell line was treated with single and split doses of X-rays. Repair of DNA damage as measured by alkaline elution was studied up to 24 h after a single MNNG exposure (0.5 micrograms/ml). DNA repair was rapid during the first 5 h after treatment and slow thereafter. DNA damage detected after split doses of MNNG at 1 and 5 h intervals was significantly lower than after a corresponding single dose. With both single and split doses, rejoining of single-strand breaks (ssb) was nearly complete after 24 h of repair time. Ouar mutation and neoplastic transformation frequencies were determined for single and split doses of MNNG with the second treatment being given during (1 h) or after (5 h) the period of rapid DNA repair. No significant differences in either effect were detected for dose splitting at any tested dose.

Carcinogenesis of laryngeal carcinoma.

Experimental models have been developed for the induction of carcinoma of the larynx and for the study of its pathogenesis. The hamster has been the animal of choice. Polynuclear hydrocarbons, administered intratracheally, induce mostly squamous cell carcinomas. Diethylnitrosamine given systemically induces papillary tumors; some other N-nitroso-compounds given topically or systemically also induce laryngeal tumors, including carcinomas. Cigarette smoke inhalation induces preneoplastic and early neoplastic lesions of the larynx, including some invasive carcinomas. In vivo and in vitro methods have been developed for the morphological and biochemical study of target tissues in respiratory epithelial carcinogenesis to identify critical pathogenetic steps and their inhibition. These methods are now applicable to the in vitro study of human laryngeal epithelium and its susceptibility to cancer induction and prevention.