Carcinogen-induced changes in tracheal epithelium cultured in serum-free, chemically defined medium

Rat tracheal tissue was cultured for periods up to 2 months in medium containing benzo[a]pyrene, and the epithelium was studied for the histologic effects of deletion of serum from the medium. Nodular hyperplasia occurred in 3 days in the absence of serum, but was not seen until several weeks of culture in media containing the same concentrations of carcinogen with 10% calf serum. In serum-free culture, hyperplasia was induced in 2 weeks with one-tenth of the smallest concentration of benzo[a]pyrene that yielded this change in serum-containing medium. The cells of the hyperplastic epithelium in both serum-containing and serum-free culture exhibited ultrastructural changes described in carcinogenesis in vivo. In the absence of serum, squamous hyperplasia was uniformly seen, a feature that distinguished serum-free culture from culture in the presence of calf serum. No frank intraepithelial or invasive malignant lesions were produced in either medium. It was concluded that exposure of organ cultures to carcinogen in the absence of serum is the more promising method for bioassay because the response to carcinogen was more rapid, more sensitive, and more reproducible than that seen during exposure in media that contained serum.     

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.     

Kinetics of cell replication of the uterine cervix. II. Parabasal cell production in atypical epithelium

The rate of cell division during a 6-hour period of time was determined in cervices from 31 mice, 16 having normal epithelium, 8 with atypical epithelium and 7 with invasive carcinoma. Cervical atypias and carcinoma were induced by local application of benzo(a)pyrene (BP) and mitosis was arrested with vincrotal number of cells in all layers as well as the proportion of dividing cells in the parabasal layer were significantly greater in atypias and invasin cell production in that layer.     

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 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.     

Inhibition of autochthonous tumor by ethanol insoluble fraction from Panax ginseng as an immunomodulator

The water extract of Panax ginseng was fractionated by its solubility in ethanol and then the ethanol-insoluble fraction was tested for immunomodulatory activity. The ethanol-insoluble fraction of ginseng (Fr. 3) proliferated splenocytes and generated activated killer cells in vitro. These activated killer cells killed both NK cell sensitive and insensitive tumor target cells without MHC-restriction. Activation of splenocytes by ginseng was mediated through the endogenously produced IL-2. To investigate the effects of Fr.3 on the autochthonous neoplasm, a single subcutaneous injection of 0.5 mg of benzo[a]pyrene (BP) was given within 24 hours after birth of male N: GP(S) mice, and Fr.3 was administered in drinking water at a concentration of 2 mg/ml, 1 mg/ml, or 0.5 mg/ml for 6 weeks after weaning. The treatment with Fr. 3 significantly inhibited lung tumor incidence (P < 0.05) compared with the BP alone group at a concentration of 2 mg/ml or 1 mg/ml in drinking water at the 9th week after BP treatment. These results suggest that the ethanol-insoluble fraction of ginseng shows antitumor effects as an immunomodulator.     

Prevention and therapy of acute radiation-related morbidity of the skin and mucosa. II, Recommendations of the literature

Changes in nuclear and nucleolar morphometric parameters were investigated by image analysis procedures in human breast MCF-10F epithelial cells expressing different stages of the tumourigenic progression after benzo[a]pyrene (BP) transformation (BP1, BP1-E, and BP1-E1 cell lines), and additionally transfected with the c-Ha-ras oncogene (BP1-Tras cell line). Nuclear pleomorphism was evident in all the transformed cells. The analysis of different morphometric parameters did not show a clear relationship between specific nuclear and nucleolar changes and the expression of the different stages of the tumourigenesis, with the exception of the nucleolar size, which could be associated to the expression of the tumourigenic phenotype, and a nucleolar area/nuclear area ratio, which discriminated the immortalized, the transformed, and the tumourigenic phenotypes from one another. The nuclear morphometric data established for the BP-transformed cells and for the cells additionally transfected with the c-Ha-ras oncogene were suggestive of complex and distinct morphofunctional mechanisms involving the in vitro transformation of the MCF-10F cells. The nuclear changes found in the BP1-Tras cell line were assumed to be related to the additional effects and/or enhanced genomic instability induced by transfection with the ras oncogene.     

Covalent DNA adducts formed in mouse epidermis by benzo[g]chrysene

The metabolic activation in mouse skin of benzo[g]chrysene (B[g]C), a moderately carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar, was investigated. Male Parkes mice were treated topically with 0.5 micromol B[g]C and DNA was isolated from the treated areas of skin at various times after treatment and analysed by 32P-post-labelling. Seven major adduct spots were detected, at a maximum level of 6.55 fmol adducts/microg DNA. Mouse skin treated with the PAH benzo[c]phenanthrene (B[c]Ph) gave a total of 0.24 fmol adducts/microg DNA. B[g]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 micromol of the optically pure putative proximate carcinogens, the (+)- and (-)-trans benzo[g]chrysene-11,12-dihydrodiols, led to the formation of adducts which comigrated on TLC and HPLC with those formed in B[g]C-treated mice, which suggested that the detected adducts were formed by the fjord region B[g]C-11,12-dihydrodiol-13,14-epoxides (B[g]CDEs). To test this, the four optically pure synthetic B[g]CDEs were reacted in vitro with DNA and the heteroco-polymers poly(dA x dT) and poly(dG x dC) and these samples 32P-postlabelled. Co-chromatography, on both TLC and HPLC, of in vitro and in vivo adducts indicated that B[g]C is activated in mouse skin through formation of the (-)-anti-(11R,12S,l3S,14R) and (+)-syn-(11S,12R,13S,14R) B[g]CDEs. (-)-anti-B[g]CDE formed five adducts with DNA, two of them with adenine and three with guanine bases. (+)-syn-B[g]CDE formed one adduct with each of these bases in DNA. The adenine adducts accounted for 64% of the total major adducts formed in B[g]C-treated mouse skin. The route of metabolic activation or B[g]C is similar to that reported for B[c]Ph, but the extent of activation to the fjord region diol-epoxides is significantly greater in the case of B[g]C, as demonstrated by the higher levels of adduct formation in vivo.     

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)-configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.     

32P-postlabeling analysis of the DNA adducts of 6-fluorobenzo[a]pyrene and 6-methylbenzo[a]pyrene formed in vitro

Studies of benzo[a]pyrene (BP) and selected derivatives are part of the strategy to elucidate mechanisms of tumor initiation by polycyclic aromatic hydrocarbons. Substitution of BP at C-6 with fluorine to form 6-fluorobenzo[a]pyrene (6-FBP) or a methyl group to form 6-methylbenzo[a]pyrene (6-CH3BP) decreases tumorigenicity compared to BP. BP, 6-FBP, and 6-CH3BP formed adducts with DNA when (1) they were activated by 3-methylcholanthrene-induced rat liver microsomes, (2) they were activated by horseradish peroxidase (HRP), (3) their 7,8-dihydrodiols were activated by microsomes, or (4) the radical cation of BP, 6-FBP, or 6-CH3-BP was directly reacted with DNA. With microsomes, 6.5 mumol of [3H]6-FBP/mol of DNA-P and 10 mumol of [14C]6-CH3BP/mol of DNA-P were bound vs 15 mumol of [3H]BP. With microsomes, two major 6-FBP adducts and some minor adducts were obtained. One major adduct coincided with that from 6-FBP-7,8-dihydrodiol. With microsomes, the minor 6-FBP adducts coincided with the adducts obtained from 6-FBP radical cation plus DNA and the major adduct of HRP-activated 6-FBP. With microsomes, 6-CH3BP showed adducts similar to some formed with HRP and one from 6-CH3BP radical cation. 6-CH3BP-7,8-dihydrodiol produced a small amount of one adduct that did not coincide with any from 6-CH3BP. The adducts of 6-FBP appear to be formed mostly through the diolepoxide pathway, whereas those of 6-CH3BP appear to arise mostly via one-electron oxidation.     

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.     

Expanded analysis of benzo[a]pyrene-DNA adducts formed in vitro and in mouse skin: their significance in tumor initiation

This paper reports expanded analyses of benzo[a]pyrene (BP)-DNA adducts formed in vitro by activation with horseradish peroxidase (HRP) or 3-methylcholanthrene-induced rat liver microsomes and in vivo in mouse skin. The adducts formed by BP are compared to those formed by BP-7,8-dihydrodiol and anti-BP diol epoxide (BPDE). First, activation of BP by HRP produced 61% depurinating adducts: 7-(benzo[a]pyrene-6-yl)guanine (BP-6-N7Gua), BP-6-C8Gua, BP-6-N7Ade, and the newly identified BP-6-N3Ade. As a standard, the last adduct was synthesized along with BP-6-N1Ade by electrochemical oxidation of BP in the presence of adenine. Second, identification and quantitation of BP-DNA adducts formed by microsomal activation of BP showed 68% depurinating adducts: BP-6-N7Ade, BP-6-N7Gua, BP-6-C8Gua, BPDE-10-N7Ade, and the newly detected BPDE-10-N7Gua. The stable adducts were mostly BPDE-10-N2dG (26%), with 6% unidentified. BPDE-10-N7Ade and BPDE-10-N7Gua were the depurinating adducts identified after microsomal activation of BP-7, 8-dihydrodiol or direct reaction of anti-BPDE with DNA. In both cases, the predominant adduct was BPDE-10-N2dG (90% and 96%, respectively). Third, when mouse skin was treated with BP for 4 h, 71% of the total adducts were the depurinating adducts BP-6-N7Gua, BP-6-C8Gua, BP-6-N7Ade, and small amounts of BPDE-10-N7Ade and BPDE-10-N7Gua. These newly detected depurinating diol epoxide adducts were found in larger amounts when mouse skin was treated with BP-7,8-dihydrodiol or anti-BPDE. The stable adduct BPDE-10-N2dG was predominant, especially with anti-BPDE. Comparison of the profiles of DNA adducts formed by BP, BP-7,8-dihydrodiol, and anti-BPDE with their carcinogenic potency indicates that tumor initiation correlates with the levels of depurinating adducts, but not with stable adducts. Furthermore, the levels of depurinating adducts of BP correlate with mutations in the Harvey-ras oncogene in DNA isolated from mouse skin papillomas initiated by this compound [Chakravarti et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10422-10426]. The depurinating adducts formed by BP in mouse skin appear to be the key adducts leading to tumor initiation.     

Practical aspects in the diagnosis and management of aplastic anemia

Groups of F344 rats and B6C3F1 mice were exposed to furfuryl alcohol vapor for 6 hours per day, 5 days per week for 14 days (0, 16, 31, 63, 125, 250 ppm) or 13 weeks (0, 2, 4, 8, 16, 32 ppm). Reduced survival was observed in the 14-day study at 250 ppm. Final mean body weights of rats and mice exposed to 125 ppm and of female mice exposed to 63 ppm were lower than controls at the end of the 14-day study; there were no significant differences in mean body weight among chemical-exposed and control groups in the 13-week study. Exposure to furfuryl alcohol had no toxicologically significant effect on organ weights in either rats or mice, and did not cause any adverse changes in hematology or serum chemistry parameters evaluated in rates in the 13-week study. Microscopic lesions associated with exposure to furfuryl alcohol were present in the nose of both rats and mice at all exposure concentrations in both the 14-day and 13-week studies. Lesions observed in the 14-day study consisted of inflammation of the nasal turbinates accompanied by necrosis and squamous metaplasia of the respiratory epithelium and necrosis and degeneration of the olfactory epithelium. Similar lesions were observed in both rats and mice in the 13-week study. In addition, squamous metaplasia and goblet cell hyperplasia of the respiratory epithelium, squamous metaplasia of the transitional epithelium and degeneration, hyperplasia and some respiratory metaplasia of the olfactory epithelium were also observed in rats in the 13-week study, and hyaline droplets in the respiratory epithelium and chronic inflammation and respiratory metaplasia in the olfactory epithelium were observed in mice in the 13-week study. In general the nasal passages of mice appeared less sensitive than those of rats at the concentrations used in the 13-week study; a no-observable-effect level was not achieved in either the 14-day or the 13-week study.     

Invasive tumors derived from xenotransplanted, immortalized human cells after in vivo exposure to chemical carcinogens

Several chemicals that are found in cigarette smoke or diesel oil engine exhausts, such as benzo[a]pyrene (B[a]P) and 1,6-dinitropyrene (DNP) are carcinogenic in experimental animal models. In the present study, we have exposed in vivo the xenotransplanted immortalized human bronchial epithelial cell line BEAS-2B to the ultimate carcinogen of B[a]P, benzo[a]pyrene diolepoxide (BPDE), to DNP or to the benzo[e]pyrene, a less active compound that has tumor-promoting abilities in mouse skin carcinogenesis bioassays. All three compounds were administered using slow-release beeswax pellets. After a 6 month exposure, BPDE produced two tumors in seven transplants, four tumors were seen in 10 transplants treated with DNP and one tumor was observed in five tracheal grafts exposed to B[a]P. All the neoplasms were well-differentiated invasive adenocarcinomas. Tracheal transplants exposed to beeswax without carcinogen did not show any evidence of neoplastic growth, and their luminal surfaces were lined by a single or double layer of cuboidal cells. All lines derived from the adenocarcinomas showed increased in vitro resistance to serum-induced terminal differentiation, gelatinolytic activity, s.c. tumorigenicity and invasive growth in an in vivo assay. When these cell lines were compared with previously described tumor cell lines derived from xenotransplants exposed to cigarette smoke condensate, it became clear that the latter exhibited a more aggressive invasive behavior. Nevertheless treatment with the three chemicals gave rise to tumor cell lines that exhibited a similar invasive behavior in vivo, and were able to penetrate early into the wall of the tracheal transplants in which they were seeded. These data indicate that this system based on xenotransplanted bronchial epithelial cells is a very relevant model to identify human carcinogens and to study mechanisms of bronchogenic cancer pathogenesis.     

An organ culture method for adult colon from germfree and conventional mice: effects of donor age and carcinogen treatment on epithelial mitotic activity

An organ culture system by which adult mouse colon epithelium could be maintained in a modified form for several weeks was described. The effects of donor age and carcinogen pretreatment were studied as a preliminary to proposed experiments on the effect of carcinogen treatment in vitro on colon epithelium from mice of different ages. Mitotic activity was compared in explants of colon from germfree C57BL mice 5 weeks, 5 months, and 9 months old; no differences were detected. The effect of old age on mitotic activity was not studied, since colon epithelium from old (30 mo) conventional mice could not be maintained in culture. Colon explants from conventional mice that had been pretreated for 32 weeks with weekly doses of a carcinogen, 1,2-dimethylhydrazine, appeared to have a higher potential for mitotic activity in vitro than did those from age-matched, untreated controls, but the difference was only significant at the 10% level. The epithelial cells were normal in ultrastructure, and the method may be valuable for studies of the direct effects of substances on adult colon epithelium.     

Sequential expression of placental glutathione S-transferase (GST-P) during DMBA-induced hamster buccal pouch squamous cell carcinogenesis

The aim of the present study was to investigate the sequential expression of placental glutathione S-transferase (GST-P) during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch squamous cell carcinogenesis. Both immunohistochemical and immunoblot analyses were employed to detect the epithelial GST-P in hamster buccal pouch mucosa over a 15-week treatment regimen. No GST-P positivity was demonstrated in the pouches of the control group. GST-P positive cells were first noted as early as 1 week after DMBA applications. A gradual increase in both the mean number and size of GST-P-positive foci was noted in the first 12 experimental weeks, but a plateau level was approached thereafter. The early GST-P-positive-area were located in the basal layer, or occasionally in the middle layer, of DMBA-treated hamster buccal pouch mucosa. Later, the stained sites became enlarged and were scattered randomly in different layers or in the whole thickness of the dysplastic and non-dysplastic epithelium. The keratin layer was only occasionally involved during the first 12 weeks of DMBA treatment but positive staining was more noticeable in the final stage of the experiment. Both exophytic (8-12 weeks) and invasive (13-15 weeks) squamous cell carcinomas showed GST-P positivity, in both cytoplasmic and nuclear components. Immunoblot analysis revealed no band in the crude tissue extracts of the control pouches whereas GST-P polypeptide of molecular weight approximately 26 kD was demonstrated in DMBA-treated pouches over the whole 15-week treatment regimen. Results of the present work indicate that GST-P is a stable and persistent label for almost all of the carcinogen-altered cells during DMBA-induced hamster buccal pouch carcinogenesis. Immunohistochemically detectable GST-P may be a potential marker throughout oral chemical carcinogenesis.     

Bending and circularization of site-specific and stereoisomeric carcinogen-DNA adducts

The potent tumorigen and mutagen (+)-7(R),8(S)-dihydroxy-9(S), 10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) is a metabolite of benzo[a]pyrene that binds predominantly to the exocyclic amino group of guanine residues in DNA in vivo and in vitro. While the (-)-7S,8R,9R,10Senantiomer, (-)-anti-BPDE, also reacts with DNA to form similar covalent N2-deoxyguanosyl adducts, this diol epoxide is nontumorigenic and its mutagenic activities are different from those of (+)-anti-BPDE. In this work, T4 ligase-induced cyclization methods have been employed to demonstrate that the (+)-anti-[BP]-N2-dG lesions (G*) cause significantly greater amounts of bending and circularization of the one-base overhang undecamer duplex 5'-d(CACAT[G*]TACAC).d(TGTACATGTGG) than the stereoisomeric oligonucleotide duplex with G* = (-)-anti-[BP]-N2-dG. In the case of the (+)-anti-BPDE-modified oligonucleotides, the ratio of circular to linear DNA multimers reaches values of 8-9 for circle contour sizes of 99-121 base pairs, while for the (-)-anti-[BP]-N2-dG-modified DNA this ratio reaches a maximum value of only approximately 1 at 154-176 base pairs. Assuming a planar circle DNA model, the inferred bending angles for 90-92% of the observed circular ligation products range from 30 to 51 degrees per (+)-trans-anti-[BP]-N2-dG lesion and from 20 to 40 degrees per (-)-trans-anti-[BP]-N2-dG lesion. In the case of unmodified DNA, the probability of circular product formation is at least 1 order of magnitude less efficient than in the BPDE-modified sequences and about 90% of the circular products exhibit bending angles in the range of 14 -19 degrees . In the most abundant circular products observed experimentally, the bending angles are 40 degrees and 26 +/- 2 degrees per (+)-anti-[BP]- or (-)-anti-[BP]-modified 11-mer; these values correspond to a net contribution of 21-26 degrees and 5-19 degrees , respectively, to the observed overall bending per lesion. The coexistence of circular DNA molecules of different sizes and, therefore, different average bending angles per lesion, suggest that the lesions induce both torsional flexibility and flexible bends, which permit efficient cyclization, especially in the case of (+)-trans-[BP]-N2-dG adducts. The NMR characteristics of (+)-trans-[BP]-N2-dG lesion in the 11-mer duplex 5'-d(CACAT[G*]TACAC).d(GTGTACATGTG) indicate that all base pairs are intact, except at the underlined base pairs. This suggests a distortion in the normal conformation of the duplex on the 5'-side of the modified guanosine residue, which may be due to bending enhanced base pair opening and bending induced by the bulky carcinogen residue. The implications of base sequence-dependent flexibilities and conformational mobilities of anti-[BP]-N2-dG lesions on DNA replication and mutation are discussed.     

Peptide YY inhibition of rat gastric enterochromaffin-like cell function

The present study determined tumorigenicity, tumor classification and DNA damage induced in infant mice by benzo[a]pyrene (B[a]P) or Manufactured Gas Plant (MGP) residues after a single exposure. Male and female B6C3F1 mice were exposed to B[a]P or MGP residue from a single environmental site (MGP-4) and males were also exposed to MGP residue composite from seven different sites (MGP-M7). At 26, 39 and 52 weeks after exposure tumorigenesis was assessed in lung, forestomach and liver. Formation and persistence of DNA adducts were quantified by 32P-postlabeling. Exposure of males to B[a]P induced liver tumors in a dose and time dependent manner. MGP induced more advanced tumors than B[a]P. Only a single liver tumor was found in MGP-4 treated females. No forestomach and few pulmonary adenomas were induced in males or females. MGP-4, MGP-M7 or B[a]P induced DNA adducts in males and females. Adducts in liver, lung and forestomach peaked on different days and decreased at different rates. At 24 h post-exposure, no significant differences in initial DNA adduct levels occurred in males and females exposed to MGP-4 or B[a]P. Lack of DNA damage (adducted DNA) did not account for non-responsiveness of lung and forestomach in B6C3F1 genders as well as in liver in females. MGP tumorigenicity could not be accounted for solely by B[a]P content nor did it reflect additivity of B[a]P and other carcinogenic polycyclic aromatic hydrocarbons (PAHs) in MGP. Synergy among MGP-PAHs, presence of unidentified carcinogens and/or promoters in MGP may account for MGP potency. The B6C3F1 infant male model is a convenient and rapid assay for assessing MGP liver tumorigenicity and potency.     

Descriptive features of gastric ulcers: do endoscopists agree on what they see?

The alkaline single cell gel test (SCG test or comet assay) was used to study the contribution of excision repair activity to the observed DNA effect after mutagen treatment. The cytotoxicity and genotoxicity of UV-irradiation and the chemical mutagens 4-nitroquinoline-1-oxide (4NQO), benzo[a]pyrene (BP) and 7,12-dimethyl-benz[a]anthracene (DMBA) were compared in a normal human cell line (MRC5CV1) and an excision-deficient xeroderma pigmentosum (XP) cell line (XP12ROSV). The XP cells showed increased cell killing after treatment with all mutagens tested, but did not show a clear increase in DNA migration in the comet assay. DNA effects in MRC5 cells were strongly enhanced by the repair inhibitor aphidicolin (APC), while under the same experimental conditions, APC had no effect on the XP cell line. The enhancing effect of APC on DNA migration in MRC5 cells and the lack of effects in XP cells indicate that the induced DNA effects of 4NQO, BP and DMBA in the comet assay mainly represent the activity of an excision repair process.     

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.