Controlling the window size in mesoporous SBA-16.

The structure of the mesoporous silica SBA-16 has been interrogated by careful elimination of the organic templating agent using ozone treatment. It is shown that the as-synthesised material consists of large cages connected by very narrow, ca. 7 Å, windows. These windows open up to about 20 Å following a calcination treatment that suggests that the functionality of SBA-16 could be changed markedly depending upon the post-synthesis treatment. The structure of SBA-16 is compared with surfaces of constant mean curvature. This illustrates that although most of the structure conforms to a surface of constant mean curvature the necks in the structure near the windows deviate strongly. This confirms that attractive forces in this region during synthesis play an important role in shaping the final structure. Following calcination the structure changes as the silica framework relaxes to a constant surface energy.

Intraneural perineurioma of the sciatic nerve: an under-recognized nerve neoplasm with characteristic MRI findings.

Intraneural perineurioma is a benign peripheral nerve neoplasm that typically affects teenagers and young adults and tends to result in a motor-predominant neuropathy. The lesion is rare, but has likely been underdiagnosed due to a lack of familiarity among both clinicians and radiologists. There have been few reports in the radiology literature despite the lesion having a fairly characteristic imaging appearance. We report a case of a 26-year-old woman with an intraneural perineurioma of the left sciatic nerve confirmed with excisional biopsy and pathologic analysis.

Wildtype Kras2 can inhibit lung carcinogenesis in mice.

Although the ras genes have long been established as proto-oncogenes, the dominant role of activated ras in cell transformation has been questioned. Previous studies have shown frequent loss of the wildtype Kras2 allele in both mouse and human lung adenocarcinomas. To address the possible tumor suppressor role of wildtype Kras2 in lung tumorigenesis, we have carried out a lung tumor bioassay in heterozygous Kras2-deficient mice. Mice with a heterozygous Kras2 deficiency were highly susceptible to the chemical induction of lung tumors when compared to wildtype mice. Activating Kras2 mutations were detected in all chemically induced lung tumors obtained from both wildtype and heterozygous Kras2-deficient mice. Furthermore, wildtype Kras2 inhibited colony formation and tumor development by transformed NIH/3T3 cells and a mouse lung tumor cell line containing an activated Kras2 allele. Allelic loss of wildtype Kras2 was found in 67% to 100% of chemically induced mouse lung adenocarcinomas that harbor a mutant Kras2 allele. Finally, an inverse correlation between the level of wildtype Kras2 expression and extracellular signal-regulated kinase (ERK) activity was observed in these cells. These data strongly suggest that wildtype Kras2 has tumor suppressor activity and is frequently lost during lung tumor progression.

Lack of association of tumor-associated macrophages with clinical outcome in patients with classical Hodgkin's lymphoma.

BACKGROUND: A recent study demonstrated that an increased number of CD68+ macrophages were correlated with primary treatment failure, shortened progression-free survival (PFS) and disease-specific survival (DSS) in patients with classical Hodgkin's lymphoma (cHL). PATIENTS AND METHODS: The aim of the present study was to verify the relationship between the number of CD68+ and CD163+ macrophages with clinical outcomes in a cohort of 265 well-characterized patients with cHL treated uniformly with the standard doxorubicin, bleomycin, vinblastine and dacarbazine chemotherapy regimen. Two pairs of hematopathologists carried out independent pathological evaluations of tissue microarray slides. RESULTS: There were no associations between clinical characteristics and the expression of CD68 or CD163. However, higher levels of CD68 and CD163 expression were correlated with the presence of Epstein-Barr virus-positive Hodgkin tumor cells (P = 0.01 and 0.037, respectively). The expression of CD68 or CD163 was not associated with either the PFS or the DSS. CONCLUSION: CD68 and CD163 expression require further evaluation before their use can be recommended for prognostic stratification of patients with cHL.

A multi-site study using high-resolution HLA genotyping by next generation sequencing.

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

Influence of fatty acid diets on gene expression in rat mammary epithelial cells.

BACKGROUND: This study examines the impact of dietary fatty acids on regulation of gene expression in mammary epithelial cells before and during puberty. METHODS: Diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Diet-induced changes in gene expression were examined in laser capture microdissected mammary ductal epithelial cells at day of weaning and end of puberty. PCNA immunohistochemistry analysis compared proliferation rates between diets. RESULTS: Genes differentially expressed between each test diets and the reference diet were significantly enriched by cell cycle genes. Some of these genes were involved in activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively the same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. CONCLUSION: Fatty acid-enriched diets significantly upregulated proliferation above normal physiological levels during puberty. Higher cellular proliferation during puberty caused by enriched fatty acid diets poses a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle rat genes are included in a human breast cancer cluster of 45 cell cycle genes, further emphasizing the importance of our findings in the rat model.

Implication of nonlinear kinetics on risk estimation in carcinogenesis.

Efforts in estimating carcinogenic risk in humans from long-term exposure to chemical carcinogens have centered on the problem of low-dose extrapolation. For chemicals with metabolites that interact with DNA, it may be more meaningful to relate tumor response to the concentration of the DNA adducts in the target organ rather than to the applied dose. Many data suggest that the relation between tumor response and concentration of DNA adducts in the target organ may be linear. This implies that the nonlinearities of the dose-response curve for tumor induction may be due to the kinetic processes involved in the formation of carcinogen metabolite--DNA adducts. Of particular importance is the possibility that the kinetic processes may show a nonlinear "hockey-stick" like behavior which results from saturation of detoxification or DNA repair processes. The mathematical models typically used for low-dose extrapolation are shown potentially to overestimate risk by several orders of magnitude when nonlinear kinetics are present.

Activated oncogenes in B6C3F1 mouse liver tumors: implications for risk assessment.

The validity of mouse liver tumor end points in assessing the potential hazards of chemical exposure to humans is a controversial but important issue, since liver neoplasia in mice is the most frequent tumor target tissue end point in 2-year carcinogenicity studies. The ability to distinguish between promotion of background tumors versus a genotoxic mechanism of tumor initiation by chemical treatment would aid in the interpretation of rodent carcinogenesis data. Activated oncogenes in chemically induced and spontaneously occurring mouse liver tumors were examined and compared as one approach to determine the mechanism by which chemical treatment caused an increased incidence of mouse liver tumors. Data suggest that furan and furfural caused an increased incidence in mouse liver tumors at least in part by induction of novel weakly activating point mutations in ras genes even though both chemicals did not induce mutations in Salmonella assays. In addition to ras oncogenes, two activated raf genes and four non-ras transforming genes were detected. The B6C3F1 mouse liver may thus provide a sensitive assay system to detect various classes of proto-oncogenes that are susceptible to activation by carcinogenic insult. As illustrated with mouse liver tumors, analysis of activated oncogenes in spontaneously occurring and chemically induced rodent tumors will provide information at a molecular level to aid in the use of rodent carcinogenesis data for risk assessment.

Persistence of benzo(a)pyrene metabolite:DNA adducts in lung and liver of mice.

The persistence of benzo(a)pyrene (BP) metabolite:DNA adducts has been studied in lung and liver of A/HeJ and C57BL/6J mice after a dose of BP (6 mg/mouse) which induces pulmonary adenomas in A/HeJ mice but not in C57BL/6J mice. BP is not a hepatic carcinogen in either strain. Following p.o. administration of [3H]BP, animals were killed at times ranging from 10 hr to 28 days, and BP metabolite:DNA adducts were analyzed by high-pressure liquid chromatography. The major adduct identified in each tissue was the (+)-7 beta-8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta,epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a (-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, and an unidentified adduct were also observed. The disappearance of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP adduct in A/HeJ mice followed first-order kinetics over the time period examined, with a half-life of 18 and 9 days in lung and liver, respectively. The decay of this adduct in C57BL/6J mice was biphasic in both tissues. Our data on cell turnover suggest that there is active removal of adducts in liver, but that normal DNA turnover can account for the partial or possibly total observed disappearance of adducts in lung. These results suggest that the tissue specificity for BP-induced neoplasia in A/HeJ mice may be related to the relative persistence of adducts and high cell turnover rates in lung. In contrast, the results on formation and persistence of adducts and cell turnover do not provide an explanation for the strain difference in susceptibility to BP-induced pulmonary adenomas. It was also shown that the rates of removal of BP metabolite:DNA adducts in A/HeJ mice are not significantly different at a 500-fold lower BP dose.

Factors regulating activation and DNA alkylation by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and nitrosodimethylamine in rat lung and isolated lung cells, and the relationship to carcinogenicity.

The molecular dosimetry for O6-methylguanine (O6MG) formation in DNA from rat lung and pulmonary cells was compared following treatment for 4 days with equimolar doses of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen or nitrosodimethylamine (NDMA), a weak carcinogen in rat lung. The dose response for O6MG formation from NNK was biphasic; the O6MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen was decreased. In contrast, the dose-response curve for methylation by NDMA appeared opposite of that for NNK with alkylation efficiency increasing as a function of dose. These results suggested that high and low Km pathways exist for the activation of NNK, whereas only high Km pathways may be involved in NDMA activation. Furthermore, DNA methylation by NNK was cell selective with the highest levels in the Clara cell, whereas methylation by NDMA was not. DNA methylation in the Clara cell was 50-fold greater by NNK than by NDMA at equimolar doses (0.005 mmol/kg). Thus, differences in O6MG formation, specifically the presence of a high affinity pathway in the Clara cell for activation of NNK, may explain why following low dose exposure, NNK is a potent pulmonary carcinogen while NDMA is not. Different cytochrome P-450 isozymes also appear to be involved in the activation of NNK and NDMA. Inhibition of in vitro methylation (with calf thymus DNA and lung microsomes) by antibodies to cytochrome P-450 isozymes provided evidence that a homolog of rabbit cytochrome P-450(2) (cytochrome P-450b) may be important in the activation of NNK in rat lung, whereas cytochrome P-450(5) may activate NDMA. A 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible cytochrome P-450 isozyme (P-450c) may also be involved in the activation of NNK but not NDMA. Treatment with TCDD increased both NNK activation by pulmonary microsomes and the formation of O6MG in Clara cells and type II cells incubated in vitro with NNK. alpha-Naphthoflavone (alpha-NF), a specific inhibitor of cytochrome P-450c reversed the increase in methylation by TCDD-induced microsomes but did not inhibit in vitro activation of NNK using microsomes from untreated rats. However, NNK mediated O6MG formation in Clara cells, but not in type II cells incubated with alpha-NF, was decreased by 21%. These data indicate that both cytochrome P-450b and P-450c are probably involved in the activation of NNK in Clara cells from untreated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition in vivo of the formation of adducts between metabolites of benzo(a)pyrene and DNA by butylated hydroxyanisole.

Antioxidants have been shown to inhibit the carcinogenic effects of a variety of chemical carcinogens. For example, the phenolic antioxidant butylated hydroxyanisole (BHA) has been shown to be a potent inhibitor of benzo(a)pyrene (BP)-induced neoplasia in mouse lung and forestomach. The objective of the present study was to determine whether or nt BHA, under conditions known to result in inhibition of BP-induced neoplasia, affects the formation of BP metabolite:DNA adducts. Following p.o. administration of a carcinogenic dose of [3H]BP to A/HeJ mice, radioactivity was detected in the DNA of both lung and liver. Analysis of the deoxyribonucleosides by high-pressure liquid chromatography showed that the major adduct in both tissues cochromatographed with the (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEI):deoxyguanosine adduct. The 7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEII):deoxyguanosine adduct was 10 to 15% of the BPDEI adduct in both lung and liver. Another adduct, possibly derived from BP phenol(s), was also detected in lung and was 10 to 20% of the BPDEI adduct. Treatment of animals with BHA decreased the amount of the BPDEI adduct in the lung and the liver approximately 55 and 75%, respectively. The decrease in the amount of this adduct in the lung appears to correlate with the inhibition of pulmonary adenoma formation (L. W. Wattenberg, J. Natl. Cancer Inst., 50: 1541-1544, 1973; J. L. Speier, L. K. Lam, and L. W. Wattenberg, J. Natl. Cancer Inst., 60: 605-609, 1978). Thus, BHA appears to inhibit BP-induced pulmonary adenoma formation by inhibiting the amount of the BPDE:DNA adducts formed in lung. Possible mechanisms by which BHA treatment inhibits the formation of BPDE:DNA adducts are discussed.

Dose-response relationships for the binding of benzo(a)pyrene metabolites to DNA and protein in lung, liver, and forestomach of control and butylated hydroxyanisole-treated mice.

In this study, the formation of benzo(a)pyrene (BP) metabolite:DNA adducts in lung, liver, and forestomach of control and butylated hydroxyanisole (BHA)-treated (5 mg/g diet) female A/HeJ mice was examined as a function of BP dose (p.o.), ranging from 2 to 1351 mumol/kg. The major identified adduct in each tissue at each dose was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEI):deoxyguanosine adduct. A 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a(-)-BPDEI:deoxyguanosine adduct, and an unidentified adduct were also observed. In lung and liver of untreated animals, the dose-response curves for BPDEI:DNA adduct levels were sigmoidal. In forestomach, there was no indication of saturation of DNA binding over the BP dose range examined. The dose-response curves became linear as BP dose approached zero and thus, no threshold dose existed below which binding of BPDEI to DNA did not occur, at least in lung, liver, and forestomach of these mice. In forestomach, the dose-response curve for BPDEI:DNA adducts in BHA-treated mice, 0.5% of diet for 2 weeks, was parallel to the curve for control animals and thus, the inhibition (45%) of adduct formation is independent of BP dose. In contrast, BHA treatment diminished the curvilinear nature of the dose-response curves for BPDE adducts in lung and liver. The inhibition of BPDEI:DNA adduct formation by BHA in lung and liver was dose dependent. The inhibition of lung (68%) and liver (82%) adduct formation was highest at a BP dose of 270 mumol/kg. As the BP dose approached zero, the inhibition of BPDEI:DNA adduct formation by BHA decreased with BP dose and approached values of approximately 40% (lung) and 55% (liver). The dose dependency of the binding of BP metabolites to protein was also examined. BPDEI:DNA adduct concentrations ranged from 2 to 10% of protein binding concentrations in liver of untreated animals, from 3 to 7% in forestomach, and from 5 to 7% in lung. The dose-response curves for protein binding of BP metabolites in lung and liver from BHA-treated animals were essentially parallel to those in control animals and thus, the inhibition of protein binding by BHA treatment had no dose dependency in these organs. No consistent BHA effect was observed on the amount of binding of BP metabolites to forestomach protein.

Detection of in vivo DNA repair synthesis in mouse liver and lung induced by treatment with benzo(a)pyrene or 4-nitroquinoline 1-oxide.

We examined in vivo DNA repair synthesis in liver and lung of A/HeJ mice treated with benzo(a)pyrene (BP) or 4-nitroquinoline 1-oxide. To differentiate between the removal of carcinogen metabolite:DNA adducts due to cell turnover and DNA repair, we measured unscheduled DNA synthesis (UDS) in the nonreplicating DNA fraction. Mice were exposed to bromodeoxyuridine pellets 1 hr prior to carcinogen treatment. Immediately following carcinogen exposure, mice received 4 hourly i.v. doses of [3H]thymidine. Mice were sacrificed 5 hr post-carcinogen treatment, and DNA was isolated. Purified DNA was then separated into newly replicated and nonreplicated DNA by ultracentrifugation in alkaline CsCl gradients. BP induced UDS in the liver at p.o. doses of 0.3 and 3.0 mg/mouse, whereas we failed to detect UDS in the lung. However, 4-nitroquinoline 1-oxide, another lung carcinogen, induced a definite repair response in the lung but not in the liver. It is not clear why mouse lung cells have the capacity to repair 4-nitroquinoline 1-oxide-induced damage to DNA and not the damage induced by BP, since both of these lung carcinogens form bulky adducts with DNA. These results demonstrate that (a) the in vivo disappearance of BP metabolite:DNA adducts from the lung of the A/HeJ mouse is due to cell turnover, whereas the disappearance of adducts from the liver is due, in part, to DNA repair and (b) induction of in vivo UDS after treatment with two different lung carcinogens is both tissue and carcinogen dependent in this mouse strain.

The A/J mouse lung as a model for developing new chemointervention strategies.

The use of the A/J mouse lung as a model for developing new chemo-intervention strategies was investigated by first inducing lung tumors with a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Lungs were then staged for tumor development and intervention therapy was initiated 42 weeks after carcinogen treatment. At this time point, an average of 7 pulmonary lesions were present on a standard histological section and the relative frequency of lesions was distributed as alveolar hyperplasias (38%), adenomas (40%), and adenocarcinomas (22%). Mice were treated for 4 or 8 weeks with cis-platinum alone or in combination with either indomethacin, an inhibitor of prostaglandin synthesis, metoclopramide, an inducer of poly(ADP) ribosylation, or nifedipine, a calcium channel blocker. The effect of indomethacin, metoclopramide, and nifedipine on tumor growth was also determined. The most dramatic effects were observed in lungs from mice treated for 8 weeks. cis-Platinum treatment caused a 37% reduction in the size of carcinomas, while tumor mass was reduced by 50 to 60% with cis-platinum in combination with metoclopramide and/or indomethacin. The inclusion of indomethacin therapy in conjunction with cis-platinum significantly enhanced the effectiveness of cis-platinum for inhibiting the growth of adenocarcinomas. In contrast, nifedipine appeared to ameliorate any of the inhibitory growth effects seen with cis-platinum treatment. Although none of the therapeutic combinations affected the size of adenomas, morphological differences were observed among treatment groups. A moderate to marked decrease in cytoplasm was observed in adenomas from mice treated with cis-platinum in combination with indomethacin or metoclopramide, cis-platinum plus metoclopramide and indomethacin, or metoclopramide plus indomethacin. Taken together, the results from these studies demonstrate that the A/J mouse lung can be used as a model to study the effectiveness of new intervention therapies for controlling malignant tumor growth. This model should also be applicable for studying the effectiveness of cancer prevention therapies on the progression of pulmonary hyperplasia.

Effect of butylated hydroxyanisole, alpha-angelica lactone, and beta-naphthoflavone on benzo(alpha)pyrene:DNA adduct formation in vivo in the forestomach, lung, and liver of mice.

The effects of alpha-angelica lactone (alpha-AL), butylated hydroxyanisole (BHA), and beta-naphthoflavone (beta-NF) on the amount of benzo(alpha)pyrene (BP) metabolite:DNA adducts formed in the forestomach, lung, and liver of ICR/Ha mice were investigated 48 hr after p.o. administration of BP. BP was administered to mice in amounts known to result in BP-induced neoplasia in certain tissues. Analysis of deoxyribonucleosides by high-pressure liquid chromatography showed that several BP metabolite:DNA adducts were formed in each tissue examined. The major identified adduct in each tissue cochromatographed with the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(alpha)pyrene (BPDEI):deoxyguanosine adduct. The (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(alpha)pyrene (BPDEII):deoxyguanosine adduct was detected in each of the tissues. As a percentage of total DNA-associated radioactivity, the BPDEI:DNA and BPDEII:DNA adducts accounted for 14% in the forestomach, 39% in the lung, and 3% in the liver. Another adduct, possibly derived from BP:phenol(s), was detected in lung and liver. Early eluting unidentified DNA-associated radioactivity was also present in each of the tissues and accounted for the majority of the radioactivity (88%) in forestomach, 57% in lung, 97% in liver). Although total DNA-associated radioactivity in liver was approximately 15-fold higher than in lung and 5-fold higher than in forestomach, the specific activities of the BPDEI:adducts and of the BPDEII:adducts were approximately the same in these organs. Addition of alpha-AL or BHA to the diet inhibited BPDEI:DNA adduct formation in the forestomach and liver but not in the lung. The effect of beta-NF was not tissue specific; this aryl hydrocarbon hydroxylase inducer decreased markedly (80 to 90%) BPDEI:DNA adduct formation in all three tissues. The radioactivity associated with the early eluting peaks was also reduced when associated with the early eluting peaks was also reduced when alpha-AL, BHA, or beta-NF was fed to the mice. The inhibition of BPDEI:DNA and BPDEII:DNA adduct formation by alpha-AL, BHA, and beta-NF is discussed in relation to similar studies where these compounds inhibited BP-induced neoplasia.

Inhibition in vivo of the formation of adducts between metabolites of benzo(a)pyrene and DNA by aryl hydrocarbon hydroxylase inducers.

The lungs of A/HeJ mice are susceptible to benzo(a)pyrene (BP)-induced neoplasia whereas the livers are resistant. Following p.o. administration of a carcinogenic dose of [3H]BP, radioactivity was associated with the DNA of both lung and liver. Analysis of the deoxyribonucleosides by high-pressure liquid chromatography showed that the major adduct in both tissues chromatographed as the (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEI)-deoxyguanosine adduct. The (+/-)-7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEII)-deoxyguanosine adduct was 9 to 15% of the BPDEI adduct in both lung and liver. Although total DNA-associated radioactivity was approximately 12-fold higher in liver than in lung, the specific activities of the BP diol-epoxide adducts were approximately the same in these organs. Treatment of animals with beta-naphthoflavone (beta NF), an inhibitor of pulmonary adenoma formation, markedly decreased the amount of the BDPEI and BPDEII adducts in the lung and the liver. The decrease in the amount of these adducts in the lung correlates with the inhibition of tumorigenesis by beta NF. The inhibition of total DNA-associated radioactivity was significantly less than the BP diol-epoxide adducts. Thus, beta NF appears to inhibit BP-induced pulmonary neoplasia by reducing the amount of the BPDEI-deoxyguanosine adduct. Other inducers of aryl hydrocarbon hydroxylase were also tested for their effect on the formation of BP-deoxyribonucleoside adducts. Both 2,3,7,8-tetrachlorodibenzo-p-dioxin and Aroclor 1254 significantly reduced the amount of the BPDEI adduct in both lung and liver. These data would suggest that both 2,3,7,8-tetrachlorodibenzo-p-dioxin and Aroclor 1254, like beta NF, should protect against BP-induced pulmonary neoplasia. The effects of aryl hydrocarbon hydroxylase inducers on the binding of BP to DNA in vivo markedly contrast with their effect in vitro. Treatment of animals with aryl hydrocarbon hydroxylase inducers stimulates the formation of BP diol-epoxide adducts in vitro. The reason for the differences between our in vivo results and those predicted from in vitro studies is unclear.

Quantitation of benzo(a)pyrene metabolite: DNA adducts in selected hepatic and pulmonary cell types isolated from [3H]benzo(a)pyrene-treated rabbits.

Benzo(a)pyrene metabolite: deoxyribonucleoside adducts were analyzed in hepatic and pulmonary cells isolated from rabbits 24 h after i.v. administration of [3H]BP (1 mg/kg; 50 mCi/kg). The major adduct in each of the cell types analyzed was (+)-r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene: deoxyguanosine, but (+/-)-r-7, t-8-dihydroxy-c-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene: deoxyguanosine and very low levels of (-)-r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene -deoxyguanosine and an unidentified adduct were also observed. The level of the major adduct was similar in each of the isolated cell types and was at least as high in cells with very low cytochrome P-450-dependent monooxygenase activity (hepatic nonparenchymal cells and alveolar macrophages) as in those with higher activity (hepatocytes, alveolar type II cells, and Clara cells). The binding of benzo(a)pyrene metabolites to proteins was also determined, and again binding levels did not correlate with differences in cytochrome P-450 activity.

Molecular dosimetry of DNA adduct formation and cell toxicity in rat nasal mucosa following exposure to the tobacco specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and their relationship to induction of neoplasia.

The molecular dosimetry of O6-methylguanine (O6MG) formation in DNA and cytotoxicity in respiratory and olfactory mucosa was determined during administration of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to male Fischer 344 rats. The dose response for O6MG formation differed considerably between respiratory and olfactory mucosa. The dose response was nonlinear in respiratory mucosa where the slope of the curve was very large for doses of NNK ranging from 0.3 to 3.0 mg/kg but much smaller in the dose range of 10 to 100 mg/kg. In contract, the dose response in the olfactory mucosa did not demonstrate such a large change in slope over the same dose range. The concentration of O6MG formed to dose of NNK ratio, an index of efficiency of alkylation, increased dramatically only in the respiratory mucosa as the dose of NNK was decreased from 100 to 0.3 mg/kg. The concentration of O6MG was four times greater in respiratory than olfactory mucosa after treatment of rats with 1 mg/kg NNK. Alkylation in the two regions of the nose became similar as the dose of NNK was increased. In rats treated for up to 12 days with NNK (10 mg/kg/day), the concentration of O6MG was 60 to 90% greater in respiratory than olfactory mucosa throughout treatment. Regional differences in the amount of O6MG formed may stem from the presence of a low Km pathway for biotransformation of NNK in the cells of the respiratory mucosa. This conclusion is supported by autoradiographic studies. Four h after treatment with 1 mg/kg [3H]NNK, silver grains were more heavily concentrated in respiratory than olfactory epithelium. Histopathological examination of the nasal passages revealed dose related, cell specific differences in toxicity following treatment of rats with 10, 30, or 100 mg/kg NNK for 12 days. No toxicity was observed in the nose when 1 mg/kg NNK was administered. Bowman's glands underlying the olfactory mucosa and Steno's glands were the most sensitive sites for toxicity, exhibiting necrosis after as little as 2 days of treatment with 10 mg/kg NNK. Damage to these glands progressed in a dose- and time-dependent manner. Respiratory epithelium exhibited only mild toxicity while basal cell metaplasia was evident in olfactory epithelium. Rats treated with NNK for 20 weeks (50 mg/kg, three times a week) had a 45% incidence of carcinomas in the olfactory region. These neoplasms appeared to arise from Bowman's glands. In contrast, there was only a 5% incidence of malignant neoplasia and a 29% incidence of benign neoplasia in the respiratory region.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of the alveolar type II cell in the development and progression of pulmonary tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the A/J mouse.

The role of the type II cell in the development of pulmonary tumors induced in the adult A/J mouse (6 weeks of age) by treatment with a single dose (100 mg/kg, i.p.) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was investigated. Twenty-four h following treatment with NNK, the concentration of O6-methylguanine was similar in Clara and type II cells. However, hyperplasias were detected only along the alveolar septa in lungs 14 weeks after carcinogen treatment. Examination of the ultrastructure of several hyperplasias revealed that the proliferating cells resembled type II pneumocytes. The proliferating cells were cuboidal in shape, with centrally localized ovoid nuclei characterized by minor indentations. Lamellar bodies, one of the major hallmarks of the type II cell, were present in the cytoplasm. The progression of pulmonary lesions was followed by sacrificing mice at 4-week intervals from 14 to 54 weeks after treatment with NNK. From 34 to 42 weeks after treatment, progression to neoplasia was demonstrated by a decline in the frequency of hyperplasias and an increase in the frequency of adenomas. Approximately 50% of the adenomas were observed arising within hyperplasias. Carcinomas appeared to increase in frequency 34 weeks after carcinogen treatment and comprised greater than 50% of the pulmonary lesions by 54 weeks. Approximately 30% of the carcinomas were observed arising within adenomas. The growth pattern of carcinomas began to change from solid to mixed (solid and papillary) 42 weeks after NNK. Moreover, electron micrographic analysis demonstrated that, within a hyperplasia, proliferating type II cells could change from cuboidal to columnar in shape and could also exhibit nuclear indentations, both characteristics displayed by the Clara cell. Thus, this divergence of the type II cell from its well characterized morphological features indicates that the selective growth advantage which these initiated cells possess can result in changes to the normal ultrastructure of this cell as it progresses toward malignancy. DNA was isolated from 20 hyperplasias and screened for the presence of an activated K-ras gene. This gene was activated in 17 of 20 lesions, with 85% of the mutations involving a GC to AT transition within codon 12 (GGT to GAT), a mutation consistent with base mispairing produced by the formation of the O6-methylguanine adduct. This specificity for activation of the K-ras gene was identical to that observed previously in adenocarcinomas induced by NNK.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell selective alkylation of DNA in rat lung following low dose exposure to the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone.

The molecular dosimetry of O6-methylguanine (O6MG) in DNA from lung and specific cell populations isolated from lung was determined during multiple administrations of the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to Fischer 344 rats. O6MG accumulated with doses of NNK ranging from 0.1 to 100 mg/kg/day. The dose response for NNK was nonlinear; the O6MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen decreased. These data suggest that low and high Km pathways may exist for activation of NNK to a methylating agent. Marked differences in O6MG concentration were observed in specific lung cell populations. The Clara cell, one of the suggested progenitor cells for nitrosamine-induced neoplasia, was found to possess the greatest concentration of O6MG. Moreover, as the dose of NNK was decreased from 100 to 0.3 mg/kg, the alkylation efficiency in this cell population increased 38-fold. The high level of DNA adduct formation in Clara cells following low dose exposure to NNK was supported by autoradiographic studies. Four h after treatment with 1 mg/kg [3H]NNK, silver grains were more heavily concentrated over Clara cells than over other cell types in the lung. Comparative studies on dimethylnitrosamine, a weak carcinogen in the rat lung, did not demonstrate this cell specificity for DNA alkylation. Thus, the presence of a high affinity pathway in the Clara cell for activation of NNK may contribute to the carcinogenicity of this tobacco specific carcinogen.