The impact of p53 function on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its metabolites 3-aminobenzanthrone and N-hydroxy-3-aminobenzanthrone in human cells.

The tumour suppressor p53, encoded by TP53, is a key player in a wide network of signalling pathways. We investigated its role in the bioactivation of the environmental carcinogen 3-nitrobenzanthrone (3-NBA)found in diesel exhaust and its metabolites 3-aminobenzanthrone (3-ABA) and N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) in a panel of isogenic human colorectal HCT116 cells differing only with respect to their TP53 status [i.e. TP53(+/+), TP53(+/-), TP53(-/-), TP53(R248W/+) or TP53(R248W/-)]. As a measure of metabolic competence, DNA adduct formation was determined using 32P-postlabelling. Wild-type (WT) p53 did not affect the bioactivation of 3-NBA; no difference in DNA adduct formation was observed in TP53(+/+), TP53(+/-) and TP53(-/-) cells. Bioactivation of both metabolites 3-ABA and N-OH-3-ABA on the other hand was WT-TP53 dependent. Lower 3-ABA- and N-OH-3-ABA-DNA adduct levels were found in TP53(+/-) and TP53(-/-) cells compared to TP53(+/+) cells, and p53's impact was attributed to differences in cytochrome P450 (CYP) 1A1 expression for 3-ABA whereas for N-OH-3-ABA, an impact of this tumour suppressor on sulphotransferase (SULT) 1A1/3 expression was detected. Mutant R248W-p53 protein function was similar to or exceeded the ability of WT-p53 in activating 3-NBA and its metabolites, measured as DNA adducts. However, identification of the xenobiotic-metabolising enzyme(s) (XMEs), through which mutant-p53 regulates these responses, proved difficult to decipher. For example, although both mutant cell lines exhibited higher CYP1A1 induction after 3-NBA treatment compared to TP53(+/+) cells, 3-NBA-derived DNA adduct levels were only higher in TP53(R248W/-) cells but not in TP53(R248W/+) cells. Our results show that p53's influence on carcinogen activation depends on the agent studied and thereby on the XMEs that mediate the bioactivation of that particular compound. The phenomenon of p53 regulating CYP1A1 expression in human cells is consistent with other recent findings; however, this is the first study highlighting the impact of p53 on sulphotransferase-mediated (i.e. SULT1A1) carcinogen metabolism in human cells.

Comparative oesophageal cancer risk assessment of hot beverage consumption (coffee, mate and tea): the margin of exposure of PAH vs very hot temperatures.

BACKGROUND: Consumption of very hot (> 65 °C) beverages is probably associated with increased risk of oesophageal cancer. First associations were reported for yerba mate and it was initially believed that high content of polycyclic aromatic hydrocarbons (PAH) might explain the risk. Later research on other beverage groups such as tea and coffee, which are also consumed very hot, found associations with increased risk of oesophageal cancer as well. The risk may therefore not be inherent in any compound contained in mate, but due to temperature. The aim of this study was to quantitatively assess the risk of PAH in comparison with the risk of the temperature effect using the margin of exposure (MOE) methodology. METHODS: The human dietary benzo[a]pyrene (BaP) and PAH4 (sum of benzo[a]pyrene, benzo[a]anthracene, chrysene, and benzo[b]fluoranthene) exposure through consumption of coffee, mate, and tea was estimated. The oesophageal cancer risk assessment for both PAH and temperature was conducted using the MOE approach. RESULTS: Considering differences in the transfer of the PAH from the leaves of mate and tea or from the ground coffee to the infusion, and considering the different preparation methods, exposures may vary considerably. The average individual exposure in µg/kg bw/day arising from consumption of 1 cup (0.2 L) of infusion was highest for mate (2.85E-04 BaP and 7.22E-04 PAH4). The average per capita exposure in µg/kg bw/day was as follows: coffee (4.21E-04 BaP, 4.15E-03 PAH4), mate (4.26E-03 BaP, 2.45E-02 PAH4), and tea (8.03E-04 BaP, 4.98E-03 PAH4). For all individual and population-based exposure scenarios, the average MOE for BaP and PAH4 was > 100,000 independent of beverage type. MOE values in this magnitude are considered as a very low risk. On the contrary, the MOE for the temperature effect was estimated as < 1 for very hot drinking temperatures, corroborating epidemiological observations about a probable oesophageal cancer risk caused by this behaviour. CONCLUSIONS: The temperature effect but not PAH exposure may pose an oesophageal cancer risk. Consumer education on risks associated with consumption of 'very hot' beverages and policy measures to threshold serving temperatures should be discussed.

Oral administration of lyophilized Dunaliella salina, a carotenoid-rich marine alga, reduces tumor progression in mammary cancer induced rats.

Dunaliella salina is a photosynthetic cell factory used for the commercial production of food additives, cattle stock feed and cosmetics as well as active ingredients for pharmaceutical industries. The investigation of the in vivo antitumor activity of D. salina lyophilized powder (DSLP) against 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinogenesis in female Wistar rats indicated a dose-dependent effect of DSLP. We studied the effect of DSLP at two different dosages of 500 and 1000 mg per kg bw on DMBA induced mammary cancer in rats by measuring the status of antioxidant enzymes, phase I and phase II detoxification enzymes, lipid peroxidation, and glycoconjugated proteins and by investigating the expression pattern of cell proliferation (Ki-67), hormonal receptor (ER, PR and HER2) status by immunohistochemical analysis, and apoptotic (caspase-3 and -9) and pro-inflammatory (COX-2) markers by colorimetric analysis. After 16 weeks of the study, we observed 100% tumor formation (including high tumor incidence and tumor volume) and a significant increase in the level of hormonal receptors, cell proliferation, and pro-inflammatory and apoptosis markers in tumor-bearing animals compared to the control. The oral administration of DSLP (1000 mg per kg bw) to the DMBA treated animals showed up to 83.4% reduction of tumors and effectively restored the levels of biochemical markers in the mammary tissues in addition to the downregulation of the expression of molecular markers. In conclusion, DSLP was found to show a chemopreventive effect against breast cancer induced in rats through the suppression of cell proliferation and the induction of apoptosis.

Systemic immunosuppression following a single pharyngeal aspiration of 1,2:5,6-dibenzanthracene in female B6C3F1 mice.

1,2:5,6-Dibenzanthracene (DBA) is ubiquitous in our environment as a contaminant produced by incomplete combustion of organics from sources such as forest fires, cigarette smoke, and asphalt paving, and it is more immunosuppressive of the T-dependent antibody-forming cell (AFC) response than the well-studied polycyclic aromatic hydrocarbon, benzo(a)pyrene. The systemic immunosuppressive effects of DBA were investigated following a single pharyngeal aspiration (pa) in female B(6)C(3)F(1) mice. The immunotoxic effects of DBA were evaluated using numerous assays of varying complexity to evaluate innate (natural killer [NK] cell activity), cell-mediated (T-lymphocyte proliferation, mixed leukocyte response [MLR], cytotoxic T-lymphocyte [CTL] activity, delayed-type hypersensitivity [DTH]), and humoral immunity (B-lymphocyte proliferation, T-dependent antibody responses). A single pa of DBA at doses up to 30 mg/kg had no effect on NK cell activity, anti-CD3 antibody-mediated T-lymphocyte proliferation, the MLR, or B-lymphocyte proliferation. DBA at 30 mg/kg suppressed Concanavalin A (ConA)-stimulated T-lymphocyte proliferation and the CTL response. DBA exposure reduced cytokine production in spleen cell culture supernatants after in vitro stimulation with ConA or lipopolysaccharide (LPS). Immunosuppression was observed at lower doses in the holistic assays. The DTH response to Candida albicans was significantly decreased at 3.0 mg/ kg DBA, while the AFC response was intermittently suppressed at 1.0 mg/kg, with no effect observed at 0.3 mg/kg. These results demonstrate that a single pa of DBA produces systemic immunotoxicity, and of the assays utilized, the holistic assays (i.e., DTH, AFC) appear to be most sensitive to the immunosuppressive effects of DBA.

3-Nitrobenzanthrone, a potential human cancer hazard in diesel exhaust and urban air pollution: a review of the evidence.

Epidemiological studies have shown that exposure to diesel exhaust and urban air pollution is associated with an increased risk of lung cancer. 3-Nitrobenzanthrone [3-nitro-7H-benz[de]anthracen-7-one (3-NBA)] is an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air particulate matter. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was found in the urine of salt mine workers occupationally exposed to diesel emissions, indicating that human exposure to 3-NBA due to diesel emissions can be significant and is detectable. There is clear evidence that 3-NBA is a genotoxic mutagen forming DNA adducts after metabolic activation through simple reduction of the nitro group. Several human enzymes have been shown to activate 3-NBA and its metabolites in vitro and in cells to form electrophilic arylnitrenium and rearranged carbenium ions, leading to the formation of purine adducts at the C8 and N2 position of guanine and at the C8 and N6 position of adenine. The predominant DNA adducts in vivo, 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone are also the most persistent adducts in target tissue in rodents, and are most probably responsible for the induction of GC-->TA transversion mutations observed in vivo. It is concluded that these adducts not only represent premutagenic lesions in DNA but are of primary importance for the initiation of the carcinogenic process and subsequent tumour formation in target tissue. Indeed, 3-NBA is carcinogenic in rats after intratracheal instillation, inducing mainly squamous cell carcinoma in lung. The intention of this article is to provide a critical review on the potential genotoxic effects of 3-NBA on human health. However, in general, there is a need for more mechanistic studies that relate 3-NBA to all processes that are considered to orchestrate tumour development and of studies on the ability of particles to promote 3-NBA genotoxicity. Because of its widespread environmental presence, 3-NBA may represent not only an occupational health hazard but also a hazard for larger sections of the general population. For an accurate risk assessment more epidemiological studies on 3-NBA-exposed individuals and a broader monitoring of environmental levels of 3-NBA are required.

Oxidative DNA damage induced by benz[a]anthracene dihydrodiols in the presence of dihydrodiol dehydrogenase.

Tobacco smoke and polluted air are risk factors for lung cancer and contain many kinds of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) and benz[a]anthracene (BA). BA, as well as B[a]P, is assessed as probably carcinogenic to humans (IARC group 2A). BA is metabolized to several dihydrodiols. Dihydrodiol dehydrogenase (DD), a member of the aldo-keto reductase superfamily, catalyzes NAD(P)+-linked oxidation of dihydrodiols of aromatic hydrocarbons to corresponding catechols. To clarify the role of DD on PAH carcinogenesis, we examined oxidative DNA damage induced by trans-dihydrodiols of BA and B[a]P treated with DD using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene. In addition, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA by using HPLC with an electrochemical detector. DD-catalyzed BA-1,2-dihydrodiol caused Cu(II)-mediated DNA damage including 8-oxodG formation in the presence of NAD+. BA-1,2-dihydrodiol induced a Fpg sensitive and piperidine labile G lesion at the 5'-ACG-3' sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H2O2 and Cu(I). The observation of NADH production by UV-visible spectroscopy suggested that DD catalyzed BA-1,2-dihydrodiol most efficiently to the corresponding catechol among the PAH-dihydrodiols tested. A time-of-flight mass spectroscopic study showed that the catechol form of BA-1,2-dihydrodiol formed after DD treatment. In conclusion, BA-1,2-dihydrodiol can induce DNA damage more efficiently than B[a]P-7,8-dihydrodiol and other BA-dihydrodiols in the presence of DD. The reaction mechanism on oxidative DNA damage may be explained by theoretical calculations with an enthalpy change of dihydrodiols and oxidation potential of their catechol forms. DD may play an important role in BA carcinogenesis via oxidative DNA damage.

Oxidative DNA damage induced by benz[a]anthracene metabolites via redox cycles of quinone and unique non-quinone.

Benz[a]anthracene (BA) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) that are ubiquitous environmental pollutants. PAH carcinogenesis is explained by DNA adduct formation by PAH diol epoxide and oxidative DNA damage by PAH o-quinone. Benz[a]anthracene-trans-3,4-dihydrodiol (BA-3,4-dihydrodiol) is a minor metabolite but shows higher mutagenicity and tumorigenicity than parent BA. We confirmed that a BA o-quinone type metabolite, benz[a]anthracene-3,4-dione (BA-3,4-dione), induced oxidative DNA damage in the presence of cytochrome P450 reductase. Interestingly, we found that BA-3,4-dihydrodiol nonenzymatically caused Cu(II)-mediated DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine formation and the addition of NADH enhanced DNA damage. BA-3,4-dihydrodiol induced a double-base lesion of C and G at the 5'-ACG-3' sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. The DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). Time-of-flight mass spectroscopic study suggested that BA-3,4-dihydrodiol undergoes Cu(II)-mediated autoxidation leading to the formation of its hydroxylated form of BA-3,4-dihydrodiol, capable of causing oxidative DNA damage. It is noteworthy that BA-3,4-dihydrodiol can nonenzymatically induce DNA damage more efficiently than BA-3,4-dione with metabolic activation. In conclusion, oxidative DNA damage induced by BA-3,4-dihydrodiol not only via quinone-type redox cycle but also via a new type of redox cycle participates in the expression of carcinogenicity of BA and BA-3,4-dihydrodiol.

Analysis of polycyclic aromatic hydrocarbons in cooking oil fumes.

Various samples of cooking oil fumes were analyzed to an effort to study the relationship between the high incidence of pulmonary adenocarcinoma in Chinese women and cooking oil fumes in the kitchen. Polycyclic aromatic hydrocarbons (PAHs) in samples of cooking oil fumes were extracted, chromatographed, and measured by fluorescence spectrophotometer. The samples included oil fumes from three commercial cooking oils and fumes from three catering shops. All samples contained benzo(a)pyrene (BaP) and dibenzo (a,h)anthracene (DBahA). In addition, the concentration of DBahA was 5.7 to 22.8 times higher than that of BaP in the fume samples. Concentrations of BaP and DBahA were, respectively, 0.463 and 5.736 micrograms/g in refined vegetable oil, 0.341 and 3.725 micrograms/g in soybean oil, and 0.305 and 4.565 micrograms/g in vegetable oil. Investigation of PAH concentrations at three catering shops showed that the level of BaP at a Youtiao (deep-fried twisted dough sticks) shop was 4.18 micrograms/100 m3, 2.28 micrograms/100 m3 at a Seqenma (candied fritters) workshop, and 0.49 micrograms/100 m3 at a kitchen of a restaurant; concentrations of DBahA were 33.80, 14.41, and 3.03 micrograms/100 m3, respectively. The high concentration of carcinogens, such as BaP and DBahA, in cooking oil fumes might help explain why Chinese women, who spend more time exposed to cooking oil fumes than men, have a high incidence of pulmonary adenocarcinoma.

Planarians as a model system for in vivo tumorigenesis studies.

Planarians can be used to devise whole-animal invertebrate bioassays for screening and as an adjunct to, or replacement for, mammalian tests. This paper proposes using planarians for in vivo tumorigenicity studies of pure compounds and complex environmental mixtures, and for mechanistic studies of tumorigenesis. To accomplish the first two stages of the bioassay validation process described by Balls et al. (1990, ATLA 18, 313-337), the literature on tumorigenicity tests with intact planarians is reviewed. Thirteen of 14 mammalian chemical carcinogens produced tumors (usually of undetermined pathohistology) in planarians. Polychlorinated biphenyls are mammalian carcinogens which are cotumorigens but not complete tumorigens in planaria. Data for other mammalian chemical carcinogens for which the planarian results are incomplete are also presented. Planarian tests using mammalian noncarcinogens, compounds which have not been tested for carcinogenicity in mammals, and radiation are also reviewed. The results indicate that both the specificity and the sensitivity of the planarian tumorigen bioassay are high. Furthermore, first-stage validation data indicate that intact planaria can be used as a developmental toxicity bioassay.

Liver, lung and kidney homogenates used as an activation system in mutagenicity studies of airborne particles and of expectorate and urine samples from exposed workers in a coke plant.

A comparison was made between lung and kidney homogenates on the one hand and liver S9 from rats on the other hand in order to compare their ability to activate promutagens. The Salmonella reversion assay was used on extracts of airborne particles from the top of coke oven batteries, and of expectorate and urine samples from exposed workers in the same coke plant. The contents of benzo[a]anthracene and benzo[a]pyrene in the different test solutions were measured by high-resolution gas chromatography/mass spectrometry. Both mutagens were detected in the filter extract and in the expectorates from the exposed workers but not in the expectorates from the control groups or in the urine samples. The liver S9 gave significantly higher mutagenicity than lung and kidney activation with both filter samples and expectorate and urine samples.

Anti-tumor promoting action of phthalic acid mono-n-butyl ester cupric salt, a biomimetic superoxide dismutase.

Skin tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by a concurrent and topical application of phthalic acid mono-n-butyl ester cupric salt (PAMBCu) in CD-1 mice initiated with 7,12-dimethylbenz[a]anthracene. PAMBCu inhibited TPA-caused epidermal ornithine decarboxylase (ODC) induction and ear edema formation, i.e. skin inflammation. However, neither PAMBCu nor superoxide dismutase (SOD) inhibited TPA-caused ODC induction in primary cultured mouse epidermal cells. 7-Bromomethylbenz[a]anthracene (BrMBA) is known to be a non-TPA type of tumor promoting agent. Epidermal ODC induction and inflammation caused by BrMBA were not inhibited by a concurrent application of PAMBCu. When mice were topically treated twice with PAMBCu, i.e. concurrently with and 7 h after BrMBA treatment, BrMBA-caused ODC induction was markedly suppressed. The same dose regimen of PAMBCu, however, failed to inhibit tumor promotion and inflammation caused by BrMBA. PAMBCu showed SOD-mimetic activity in superoxide generating systems, i.e. xanthine-xanthine oxidase reaction and TPA-stimulated polymorphonuclear leukocytes (PMN). Mono-n-butyl phthalate, which lacks SOD-mimetic activity, failed to inhibit TPA-caused ODC induction and skin inflammation. Therefore, inhibition by PAMBCu of TPA-caused tumor promotion, epidermal ODC induction and inflammation may be attributable to its SOD-mimetic activity. The results also support the contention that a superoxide anion of non-epidermal cell origin, such as PMN and macrophages, plays a role (probably some enhancing role) in in vivo ODC induction and tumor promotion caused by TPA. Failure of PAMBCu to inhibit BrMBA-caused tumor promotion suggests that superoxide anion generation is not involved in the tumor promoting action of this agent and that the anti-tumor promoting action of PAMBCu is dependent on the nature of the tumor promoting agents.

A test of the bay-region mechanism in carcinogenesis for monomethyl benz[a]anthracenes in a self-consistent-field molecular orbital theory.

The relative carcinogenic activity of the isomeric monomethyl derivatives of benz[a]anthracene has been studied on the basis of their bay-region reactivity, and the subsequent ease of carbonium ion formation, as obtained from a suitable 'self-consistent-field' molecular orbital theory for the mobile pi-electrons. The predicted order of carcinogenic activity of these molecules is compared with the available experimental data.

Histopathologic studies of DMBA-induced mouse submandibular-gland tumors with or without cryoprobe treatment.

Histopathologic observations of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tumors in the mouse submandibular glands are reported using cryoprobe treatment. The experimental animals were divided into 2 groups; one received a carcinogen injection into the normal submandibular gland, and the other was injected with DMBA on the 14th day following cryosurgery of the gland using a -60 degrees C cryoprobe. Pathologic findings were classified as premalignant lesions or squamous-cell carcinomas with varying degrees of keratinization, fibrosarcoma and mixed carcinoma. There was also one case each of malignant pleomorphic adenoma and cystic adenoma. Tumor incidence was nearly the same in the 2 groups. Most of the carcinomas and sarcomas in the submandibular gland were induced during the 12th and 17th weeks. DMBA application during the proliferating stage which followed the cryosurgery did not enhance epithelial-tumor induction. During submandibular gland carcinogenesis, alterations in the granular convoluted tubule cells suggests they were the initial target cells undergoing malignant transformation. Squamous-cell carcinomas with varying degrees of keratinization were induced following squamous metaplasia in duct-like structures or multicystic lesions.

Experimental-induced sarcomas after intra-articular injection of 9-10-dimethyl-1-2-benzanthracene.

The authors describe an experimental study on rabbits whereby the development of sarcomas was induced following injection into the knee joint with 9-10-dimethyl-1-2-benzanthracene. The results of the experiment are in contrast to those of other authors as no "synovialoma" or synovial sarcoma developed but only morphological patterns which might belong to malignant fibrous histiocytoma and occasionally fibrosarcoma. In accord with our results the hypothesis is discussed that all malignant mesenchymal neoplasias result from a not clearly identifiable pluripotent mesenchymal stem cell.

DMBA-induced tumors and their prevention by aromatic retinoid (Ro 10-9359).

Both auricles of 21 domestic rabbits were painted with dimethylbenzanthracene (DMBA). Eleven animals of this group were additionally fed aromatic retinoid (AR) by an esophageal tube. Two control animals were not treated at all. Eight or 9 weeks after the beginning of the study six of the seven remaining animals, which had only been painted with DMBA, developed a total of 25 keratoacanthoma-like tumors (KA). On the other hand, none of the seven animals left, which were painted with DMBA and fed AR showed any tumor by this time. The systemic effect of AR was studied in biopsies from the snout and the back. The epidermis of the snout showed 'mucous mataplasia' by histochemical and electron-microscopic criteria, whereas the epidermis of the back was not significantly altered. The production of intra- and extracellular lamellated material indicated an additional effect of AR on epidermal lipid metabolism. The effect of AR in the prevention of DMBA-induced tumors was characterized by 'mucoid cytolysis' and karyolysis.