P53 mutations in tumours induced by intraperitoneal injection of crocidolite asbestos and benzo[a]pyrene in rats

Mutation analysis of the tumour suppressor gene p53 in tumours induced in the peritoneal cavity of rats revealed differences in the mutational pattern with regard to the carcinogenic substances applied. In tumours induced by benzo[a]pyrene a considerable amount of p53 mutations resulting in an altered protein structure could be detected. For the development of these tumours an escape from the p53 mediated cell cycle control can be assumed. However, in tumours of the same tumour type induced by crocidolite asbestos no mutations could be observed. Since there were even no spontaneous p53 mutations detectable in this tumour group, it is obvious that in these tumours the escape from cell cycle control does not take place via inactivation of p53. Therefore, it is concluded that the molecular mechanisms of carcinogenesis and tumour development in this tumour type depend on the type of carcinogen applied.     

Tissue distribution of DNA adducts in rats treated by intramammillary injection with dibenzo[a,l]pyrene, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene

Recombinant human bone morphogenetic protein (rhBMP-2) was examined for its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were used in this study. The results indicated that rhBMP-2 stimulated alkaline phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in confluent cultures. The percent collagen synthesis also increased in a dose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days after initiation. Cycloheximide (2 micrograms/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzyme. Transforming growth factor-beta 1 (TGF-beta 1)-induced inhibition of alkaline phosphatase activity observed in confluent primary cultures was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-beta 1 concentration. Also, rhBMP-2 produced a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enhanced in cells cultivated in the presence of ascorbic acid (50 micrograms/ml). In view of the potentiating effect of ascorbic acid on rhBMP-2-induced stimulation of alkaline phosphatase, we speculate that ascorbic acid could amplify the osteoinductive effects of rhBMP-2 and thereby augment the efficacy of the BMP when used as bone repair material in vivo. rhBMP-2 (4.3-86 ng/ml) did not exhibit mitogenic effects on cultured osteoblasts. These data suggest that rhBMP-2 has the ability to induce expression of various markers associated with the osteoblast phenotype in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-beta 1 may play a regulatory role in BMP-induced bone formation and ascorbic acid may potentiate the effects of rhBMP-2 in vivo.     

DNA strand scission by benzo[a]pyrene diol epoxides

Syn-and anti-benzo[a]pyrene diol epoxides elicit a concentration-dependent nicking of superhelical Col E1 DNA in an in vitro reaction monitored by agarose gel electrophoresis and electron microscopy. This strand scission represents less than 1 percent of the DNA modification by diol epoxide. Kinetic analysis implicates the formation of unstable phosphotriesters, hydrolysis of which nick the DNA.     

Dibenzo[a,l]pyrene (dibenzo[def,p]chrysene): fjord-region distortions

The molecular dimensions of the potent chemical carcinogen dibenzo[def,p]chrysene, also known as dibenzo[a,l]pyrene, have been determined by X-ray diffraction methods. This analysis shows that the molecule is considerably distorted so that it is non-planar with an angle of 27.6 degrees between the outermost rings and a widening of C-C-C bond angles in the fjord region. The dimensions of the molecular distortion due to atomic overcrowding in the fjord region are presented. This polycyclic aromatic hydrocarbon is a more potent carcinogen than is benzo[a]pyrene or its 11-methyl derivative. Comparisons of the distortions in dibenzo[a,l]pyrene with the geometries of various other polycyclic aromatic hydrocarbons containing fjord- or bay-region methyl groups provide structural data on the ratio of angular to torsional distortion in such overcrowded molecules.     

The mutagenicity of benzo[a]pyrene in mouse small intestine

OBJECTIVE: To determine the effects of controlled ovarian hyperstimulation (COH) on endometrial maturation. DESIGN: Prospective, before and after evaluation of midluteal endometrial biopsies in oocyte donor's spontaneous and subsequent COH cycles. SETTING: Tertiary academic medical center assisted reproductive technologies clinic. PATIENT(S): Nineteen oocyte donors. INTERVENTION(S): Exogenous gonadotropins, endometrial biopsies. MAIN OUTCOME MEASURE(S): Endometrial histology and an immunohistochemical marker of uterine receptivity, the alphavbeta3 vitronectin. RESULT(S): Glandular and stromal dyssynchrony was more common after COH in 16 (80%) of 20 cycles than 6 (30%) of 20 spontaneous cycles (P <.05). Glandular lag was more frequent in COH cycles and unaffected by progesterone administration. The beta3 subunit of the alphavbeta3 vitronectin receptor was present in 9 (45%) of 20 spontaneous and 2 (10%) of 20 COH cycles (P <.05). CONCLUSION(S): Exogenous gonadotropin use in healthy reproductive age women did not result in endometrial evidence of a luteal phase defect. A greater incidence of glandular-stromal dyssynchrony resulted from the use of exogenous gonadotropins. The presence of alphavbeta3 was noted in most endometrial specimens demonstrating in phase glandular maturation. We conclude that endometrial dyssynchrony that results from delayed glandular development most likely represents a normal histologic variant.     

Induction of DNA adducts and mutations in spleen, liver and lung of XPA-deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild-type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA-deficient mice.     

Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes

The oxidation of benzo[a]pyrene (B[a]P) was examined using reconstituted systems prepared with recombinant human cytochrome P450 (P450) enzymes 1A1, 1A2, 2C8, 2C10, 2E1, and 3A4 and with microsomes prepared from Saccharomyces cerevisiae expressing recombinant human P450s 2C8, 2C9, and 2C18. Products measured by HPLC included the 3- and 9-phenols, the 4,5-, 7,8-, and 9,10-dihydrodiols (detected in the presence of epoxide hydrolase), and products in the polar fraction eluting immediately after the void volume. The most active enzyme in all reactions was P450 1A1. P450 3A4 and P450 1A2 formed appreciable amounts of several of the products, including the 3-phenol. P450 2C enzymes and P450 2E1 formed relatively low amounts of all B[a]P products. Consideration of these patterns along with knowledge of levels of expression of the P450s in human tissues and previous results with microsomes leads to the conclusion that P450 1A1 should dominate the oxidation of B[a]P in tissues where it is present and inducible. In human liver the level of P450 1A1 is low and P450 3A4, P450 2C subfamily enzymes, and P450 1A2 probably all contribute. Of the human P450s considered here, P450 1A2 was the most active hepatic enzyme forming the 7,8-dihydrodiol. 7,8-Benzoflavone stimulated the oxidation of B[a]P by P450 3A4 and inhibited the oxidations catalyzed by P450 1A2. The extent of inhibition of P450 1A1 was less (than with P450 1A2), probably due to the rapid oxidation of 7,8-benzoflavone by P450 1A1. The major 7,8-benzoflavone product appears to be the 5,6-oxide.     

Tumour-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene

Three dihydrodiols that are metabolites of benzo[a]pyrene and benzo[a]-pyrene itself have been tested in a comparative experiment for their activities as initiators of tumours in mouse skin. A single application (25 mug) of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, of 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, or of benzo[a]pyrene was made to the shaved dorsal skin of adult female CDI mice; this was followed 2 weeks later by multiple thrice-or twice-weekly applications (1 mug) of 12-O-tetradecanoyl-phorbol-13-acetate as promoting agent. A control group of 30 mice received the promoting agent alone. The experiments were terminated 52 weeks after initiation. At this stage, all the groups contained mice bearing skin papillomas, some of which had progressed to malignancy. Quantitatively the results show that the 7,8-dihydrodiol is almost as active an initiator of mouse skin tumours as benzo[a]pyrene itself; the 4,5- and 9,10-dihydrodiols were significantly less active. The significance of these results is discussed in relation to the hypothesis that diol-epoxides are important in the metabolic activation of polycyclic hydrocarbons like benzo[a]pyrene.     

Induction of atypical squamous metaplasia with benzo[a]pyrene in cultured hamster tracheas

Areas of hyperplasia were produced in hamster tracheal epithelium maintained in vitro by exposure to a suspension of benzo[a]pyrene (BP) in gelatin. Typical and atypical epiodermoid metaplasia were seen by 2 weeks. In atypical areas, cell nuclei were enlarged with prominent nucleoli, the cytoplasm contained dense bundles of tonofilaments and the cells were joined by numerous desmosomes. The peak response to the carcinogen was reached 4 weeks after the application of BP and consisted of extensive atypical epidermoid metaplasia. Tracheas treated with gelatin alone maintained a columnar epithelium for 6 weeks of culture. The characteristics of the metaplastic changes in vitro are essentially identical to those described after exposure of the hamster tracheobronchial epithelium to benzo[a]pyrene-ferric oxide in vivo.     

Hemoglobin adducts of benzo[a]pyrene diolepoxide in newspaper vendors: association with traffic exhaust

Benzo[a]pyrene diol epoxide adducts with hemoglobin (Hb) were measured to detect human exposure to environmental benzo[a]pyrene from traffic exhaust. Benzo[a]pyrene tetrahydrotetrols (BPTs) released from Hb after acid hydrolysis were quantitated by gas chromatography-mass spectrometry after immunoaffinity chromatography. Fifty three newspaper vendors were enrolled. The median adduct concentration was 0.3 fmol BPTs/mg Hb in high density traffic-exposed vendors and < or = 0.1 fmol BPTs/mg Hb in those exposed to low density traffic; the difference was not significant (P = 0.09). Among non-smokers, adducts were detectable in 60% of high exposure subjects (median 0.3 fmol BPTs/mg Hb) and in 28% of those with low exposure (median < or = 0.1 fmol/mg Hb). This difference was significant (P = 0.02). In low exposure smokers the median of adducts was 0.26 fmol BPTs/mg Hb, while in low exposure non-smokers it was < or = 0.1 fmol BPTs/mg Hb (P = 0.08, not significant). Adduct concentration was no different for low and high density traffic-exposed smokers (P = 0.82). The data indicate a significant difference in adduct concentration related to traffic exhaust exposure among non-smokers.     

Antispermatogenic effects of ethyl methanesulfonate and benzo[a]pyrene in PD4 Lakeview hamsters

Mammalian sperm seems to provide an excellent cell type for monitoring mutagenic and other toxicological damage to the germinal tissue. Studies with mice indicated that most agents known for their mutagenic activity in vivo produced marked elevations in sperm abnormalities. To determine whether this response is typical of other species, groups of inbred Lakeview hamsters were exposed to ethyl methane-sulfonate (EMS) and benzo[a]pyrene (BP) in five daily subacute ip doses ranging from 5 to 125 mg/kg and 2 to 50 mg/kg, respectively. Percentage of abnormal sperm, testis weight, and body weight were monitored at wk 1, 4, and 10 after treatment. EMS exposures increased the frequency of sperm abnormalities and reduced sperm numbers and testis weights. Body weights were also affected. BP exposures did not induce sperm abnormalities; however, there were marked reductions in sperm number and testis weight. These findings are in agreement with results of EMS studies in the mouse; however, BP exposure did induce sperm abnormalities in the mouse.     

A parallel study of in vitro sensitivity to benzo[a]pyrene diol epoxide and bleomycin in lung carcinoma cases and controls

BACKGROUND: Because only a fraction of smokers develop neoplastic lesions, host factors may affect their susceptibility to the carcinogenic effects of tobacco smoke. Benzo[a]pyrene diol epoxide (BPDE) is the metabolic product of benzo[a]pyrene (B[a]P), a constituent of tobacco smoke. Therefore, BPDE sensitivity may shed some light on smoking-related carcinogenesis. METHODS: First, differential BPDE sensitivity was tested in five lymphoblastoid cell lines. Then sensitivity to BPDE and bleomycin (an excellent lung carcinoma risk predictor) was tested in parallel in the lymphocytes of 57 lung carcinoma cases and 82 controls. RESULTS: The optimal BPDE treatment duration was 24 hours. The xeroderma pigmentosum cell line was the most sensitive, followed by head and neck cancer, ataxia telangiectasia, and normal cells. The mean breaks per cell for cases and controls were 0.78 and 0.46, respectively (P < 0.0001). BPDE sensitivity was significantly associated with lung carcinoma, with an odds ratio (OR) of 7.26, compared with an OR of 4.56 for bleomycin sensitivity. There was also a dose-response correlation between the quartiles of BPDE-induced breaks and lung carcinoma risk, with ORs of 2.39, 3.12, and 15.03. It is noteworthy that individuals who were sensitive to both BPDE and bleomycin had a significantly increased OR of 38.36. CONCLUSIONS: BPDE sensitivity may be a biologic marker to identify individuals who are susceptible to the carcinogenic effects of tobacco smoke. BPDE and bleomycin sensitivity might represent different repair or sensitivity pathways; however, when these assays are used in parallel, they might refine our ability to identify high risk individuals.     

p53 mutations and overexpressions in Japanese breast cancer

In this paper, we explore the use of a variety of familial transmission tests (and case-control analyses) to screen for allelic associations in simulated marker data of a quality (2 cM map) that will feasibly arise from genomic scans within the next 5-10 years. We demonstrate a form of the transmission-disequilibrium test extended to multiallele systems. The methods used were log-linear and related models implemented largely using standard statistical packages.     

Sulfotransferase-mediated activation of 7,8,9,10-tetrahydro-7-ol, 7,8-dihydrodiol, and 7,8,9,10-tetraol derivatives of benzo[a]pyrene

Some hydroxymethyl-substituted polycyclic aromatic hydrocarbons have been shown to be converted to electrophilic, mutagenic, or tumorigenic sulfuric acid ester metabolites by cytosolic sulfotransferase activity in rodent liver. Likewise, certain types of aromatic compounds with a secondary alcoholic functional group at the benzylic position undergo metabolic activation through sulfonation. Enzymatic oxidation of benzo[a]pyrene produces such secondary alcohols as dihydrodiol and tetraol derivatives as primary metabolites. Sulfo conjugation of the benzylic hydroxy group of each of these metabolites is expected to generate an electrophilic sulfuric acid ester capable of covalently binding to DNA, which may contribute to mutagenesis and carcinogenesis by benzo[a]pyrene. Although the model benzo-ring secondary benzyl alcohol, 7-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene, covalently bound to DNA and also exerted mutagenicity in the presence of rodent hepatic cytosols and 3'-phosphoadenosine 5'-phosphosulfate, no such sulfotransferase-dependent activation was observed with dihydrodiol or tetraol derivatives of benzo[a]pyrene. Thus, it seems likely that appearance of the adjacent non-benzylic hydroxy functional group(s) in latter metabolites hinders the benzylic sulfonation in these molecules.     

Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human bronchi

The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in the binding of benzo[a]pyrene to DNA is similar in magnitude to that found in pharmacogenetic studies of drug metabolism. Aryl hydrocarbon hydroxylase is also inducible by benz[a]anthracene in the bronchial mucosa.     

Diet, Helicobacter pylori, and p53 mutations in gastric cancer: a molecular epidemiology study in Italy

A series of 105 gastric cancer (GC) cases with paraffin-embedded specimens interviewed in a previous population-based case-control study conducted in a high-risk area around Florence, Italy, was examined for the presence of p53 mutations. Overall, 33 of 105 cases had a mutation (p53+) identified by single-strand conformational polymorphism and confirmed by sequencing (Y-H. Shiao et al., submitted for publication). p53+ cases had a more traditional dietary pattern (i.e., corn meal mush, meat soup, and other homemade dishes) and reported less frequent consumption of raw vegetables (particularly lettuce and raw carrots). A positive association with a high nitrite intake and a negative association with raw vegetables and diffuse type histology persisted in a multivariate analysis. In addition, p53+ cases tended to be located in the upper portion of the stomach and to be associated with advanced age and blood group A. No relation was found between the presence of p53 mutations and histologically defined Helicobacter pylori infection, smoking history, family history of gastric cancer, education, and social class. Of the 33 p53+ cases, 19 had G:C-->A:T transitions at CpG sites. These tumors tended to occur in females and in association with H. pylori infection but not other risk factors. The remaining 14 cases with a p53 mutation had mainly transversions but also two deletions and two transitions at non-CpG sites. These tumors showed a strong positive association with a traditional dietary pattern and with the estimated intake of selected nutrients (nitrite, protein, and fat, particularly from animal sources). The findings of this case-case analysis suggest that p53 mutations at non-CpG sites are related to exposure to alkylating compounds from diet, whereas p53 mutations at CpG sites might be related to H. pylori infection.     

Comparison of the hydroxylation of zoxazolamine and benzo[a]pyrene in human placenta: effect of cigarette smoking

The in vitro hydroxylation of zoxazolamine was compared with the hydroxylation of benzo[a]pyrene (BP) in full-term placentas from 11 nonsmokers and from 13 women who smoked cigarettes during pregnancy. Cigarette smoking increased the average zoxazolamine and benzo[a]pyrene hydroxylase activities 13- and 39-fold, respectively. A 59-fold range in benzo[a]pyrene hydroxylase activity and a 28-fold range in zoxazolamine hydroxylase activity were found in the placentas of cigarette smokers. A plot of these two enzyme activities showed that zoxazolamine hydroxylase activity was highly correlated, with benzo[a]pyrene hydroxylase activity in the 24 placentas studied (r = 0.98; p less than 0.001). A strong correlation between the above enzymatic activities was also found in 8 placentas which had been stored for 2 yr at -20 degrees C (r = 0.95; p less than 0.001). The results suggest that benzo[a]pyrene and zoxazolamine are metabolized in the human placenta by the same enzyme or by different systems that are under the same regulatory control.     

Association of p53 mutations with metastatic prostate cancer

In prostate cancer, mutation of the p53 tumor suppressor gene has been associated with locally advanced disease and hormone-resistant disease that is predominantly localized to bone. However, little is known regarding the status of the p53 gene in metastatic prostate cancer that has not been treated with hormonal manipulation. We evaluated formalin-fixed, paraffin-embedded malignant tissues from 86 patients with various stages of prostate cancer, including pathologically confined, locally advanced, and metastatic disease, to detect abnormal p53 nuclear protein accumulation using immunohistochemistry. No abnormal p53 immunostaining was detected in 18 patients with prostate cancer confined to the gland. Two tumors from 21 patients with locally advanced disease (extracapsular extension and/or seminal vesicle invasion) had abnormal nuclear p53 accumulation, and a mutation in exon 7 of the p53 gene was detected in tumor DNA from one patient using single-strand conformation polymorphism-direct sequencing analysis. Of the remaining 47 patients studied in whom tissues from the prostate gland and a metastatic site (44 lymph node, 2 bone, and 1 lung) were available, only 3 had received hormonal therapy prior to obtaining metastatic tissue. In four patients both primary and metastatic tumors demonstrated accumulation of p53 protein, whereas seven additional patients exhibited p53 accumulation only at the metastatic site. In three patients the metastatic tumors harbored missense single-base substitutions in exon 5, as detected using single-strand conformation polymorphism-direct sequencing. These results indicate that p53 abnormalities are associated with lymph node metastases derived from prostate cancer patients that had not undergone hormonal therapy.