Unscheduled DNA synthesis in human bronchial epithelium treated with various chemical carcinogens in vitro

A system was developed in which organ culture of human bronchial epithelium was used in combination with autoradiography for quantitative measurement of unscheduled DNA synthesis (UDS) in bronchial epithelial cells. Human bronchi obtained at surgery were cut into small sections and treated with various carcinogens plus [methyl-3H]thymidine in short-term organ culture. Significant numbers of silver grains, indicating UDS, were detected on the nuclei of epithelial cells of human bronchi treated with carcinogens, and the numbers were proportional to the concentrations of carcinogens. In this system seven representative carcinogens induced UDS. Four active metabolites of benzo[a]pyrene, and benz[a]anthracene also were found to induce very active UDS in human bronchial epithelium. These findings suggest that human bronchial epithelial cells can repair different types of DNA modification induced by chemical carcinogens.     

Foci of aberrant crypts in the colons of mice and rats exposed to carcinogens associated with foods

Aberrant crypt foci can be identified in the colons of rodents treated 3 wk earlier with azoxymethane, a known colon carcinogen. These crypts can easily be visualized in the unsectioned methylene blue-stained colons under light microscopy, where they are distinguished by their increased size, more prominent epithelial cells, and pericryptal space. They occur as single aberrant crypts or as two, three, or four aberrant crypts in a cluster. We compared the reported ability of carcinogens associated with the human diet to induce colon cancer with the measured rate of induction of aberrant crypts in female CF1 mice and Sprague-Dawley rats. The carcinogens used were 1,2-dimethylhydrazine, methyl nitrosourea, N-nitrosodimethylamine, benzo(a)pyrene, aflatoxin B1, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 2-amino-3-methylimidazo[4,5-P]quinoline, 2-amino-3,4-dimethylimidazo[4,5-P]quinoline, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole. Graded doses of these compounds were given to the animals by gavage twice with a 4-day interval, and the animals were terminated 3 wk later. All colon carcinogens induced aberrant crypts in a dose-related fashion. N-Nitrosodimethylamine and 3-amino-1-methyl-5H-pyrido[4,3-b]indole, carcinogenic compounds that do not induce colon cancer, did not induce them. The ability of the studied compounds to induce aberrant crypts was species specific; e.g., aflatoxin B1 and 2-amino-3,4-dimethylimidazo[4,5-P]quinoline induce about 20 times more in rats than mice. This relationship was consistent with their reported ability to induce colon cancer in these species. Results of the present study support the use of the aberrant crypt assays to screen colon-specific carcinogens and to study the process of colon carcinogenesis.     

Polycyclic hydrocarbon-produced toxicity, transformation, and chromosomal aberrations as a function of aryl hydrocarbon hydroxylase activity in cell cultures

The cytotoxic effect of benzo[a]pyrene alone in fetal rat hepatocytes in culture is prevented by phenobarbital. Benzo[a]pyrene is not toxic to HTC, A9, or HeLa cells in which aryl hydrocarbon hydroxylase activity is either absent or very low; however, benzo[a]pyrene is cytotoxic to each of these three established cell lines grown together with the liver cells in the presence of phenobarbital. Polycyclic hydrocarbon-produced cytotoxicity is associated with chromatid breaks, whereas aneuploidy is more closely correlated with malignant transformation of hamster secondary cultures. Benz[a]anthracene or a-naphthoflavone competitively inhibits the hydroxylation of other polycyclic hydrocarbons such as the carcinogens 7,12-dimethylbenz[a]anthracene or benzo[a]pyrene. The exposure of fetal hamster secondary cells to excessive amounts of benz[a]anthracene prior to, during, and following treatment with 7,12-dimethylbenz[a]anthracene prevents malignant cell transformation from occurring.     

Cytotoxicity of chemical carcinogens towards human bronchial epithelial cells evaluated in a clonal assay

Survival of human bronchial epithelial cells after administrationof four chemical carcinogens was measured in a donal assay.Human bronchial epithelial cells were obtained from outgrowthsof explanted tissue pieces. Serum-free medium was used for bothexplant culture and donal growth. The donal assay could be performedon three substrata: plastic dishes alone, protein-coated dishes,and inactivated Swiss 3T3 cells. Several other cell lines supporteddonal growth of the human cells. Fetal bovine serum was inhibitoryto colony formation on plastic and protein-coated dishes, buthad no effect on the growth of bronchial cells on 3T3 feedercells. Little variation among individuals in cytotoxicity ofN-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and K₂CrO₄, a possiblehuman lung carcinogen, was observed, but in the case of benzo[a]pyrene(BP) and 7,12-dimethylbenz[a]anthracene (DMBA), large variationsin survival were found between cultures derived from differentindividuals.     

Binding of polynuclear aromatic hydrocarbons to the rat 4S cytosolic binding protein: structure-activity relationships

The relative competitive binding affinities of benzo[a]pyrene (B[a]P), benzo[e]pyrene, benzo[g, h, i]perylene, picene, 7,12-dimethylbenz [a]anthracene, 1,2,3,4-dibenz[a]anthracene, 1,2,5,6-dibenz[a]anthracene, perylene, 4H-cyclopenta[d,e,f]-phenanthrene, benz[a] anthracene, triphenylethylene and triptycene for the rat hepatic cytosolic 4S binding protein were determined using [3H]benzo[a]pyrene as the radioligand. With the exception of triphenlethylene, triptycene and 4H-cyclopenta[d,e,f]phenanthrene, the EC50 values for the remainder of these compounds were between 1.25 X 10(-7) and 2.5 X 10(-8) M with 1,2,5,6-dibenz[a]anthracene being the most active ligand. A comparison of the relative cytosolic Ah (9S) receptor binding affinities and aryl hydrocarbon hydroxylase (AHH) induction potencies of these hydrocarbons with their 4S protein binding affinities demonstrated the following: five compounds, namely 1,2,5,6-dibenz[a]-anthracene, 1,2,3,4-dibenz[a]anthracene, picene, benzo[a]pyrene and 3-methylcholanthrene exhibited high to moderate binding affinities for the 4S and 9S cytosolic proteins (EC50 values less than 10(-6) M) and induced AHH in rat hepatoma cells; three compounds, namely perylene, benzo[e]pyrene and benzo[g,h,i]perylene exhibited high affinities for the 4S binding protein (1.25 X 10(-7), 4.4 X 10(-8) and 2.9 X 10(-8) M, respectively) and low affinities (EC50 values greater than 10(-5) M) for the Ah receptor protein; moreover these three compounds did not induce AHH in rat hepatoma H-4-II E cells in culture. These data suggest that the 4S binding protein may not play a significant role in AHH induction although the results do not rule out a function for this protein in the transregulation of AHH and its associated cytochromes P-450.     

Nitration of carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons results in products able to induce transformation of Syrian hamster cells

Five nitrated polycyclic aromatic hydrocarbons, synthesizedfrom benzo[a]pyrene, fluoranthene, pyrene, and chrysene induceddose-dependent transformation of Syrian hamster embryo cells.Benzo[a]pyrene, a known carcinogen, induced transformation whilethe other parental compounds, which are non-carcinogens, werenot effective. The transforming potential of the nitro derivativesvaried from compound to compound; on a molar basis, 1,8-dinitropyrenewas the most effective nitrated hydrocarbon followed in orderby 3-nitro-fluoranthene, 1-nitropyrene, 6-nitrochrysene, and6-nitro-benzo[a]pyrene. The ability to obtain dose-dependenttransformation frequencies indicates that hamster cell transformationrepresents a responsive model for elucidating the mechanismof action of nitrated carcinogens. Because of their ubiquitousdistribution and their ability to induce morphological transformationin mammalian cells, nitrated polycyclic aromatic hydrocarbonsmust be considered as potential carcinogens.     

Anticarcinogenic and cocarcinogenic effects of benzo[e]pyrene and dibenz[a,c]anthracene on skin tumor initiation by polycyclic hydrocarbons

In the present study, we have examined the effects of benzo[e]pyrene(B[e]P) and dibenz[a,c]anthracene (DB[a,c]A) on the skin tumor-initiatingactivities of methylated and nonmethylated polycyclic aromatichydrocarbons (PAH). B[e]P, when applied 5 min prior to initiationwith seven different PAH skin carcinogens, effectively inhibitedthe tumorinitiating activities of 7,12-dimethylbenz[a]anthracene(DMBA) and dibenz[a,h]anthracene (DB[a,h]A) but had little orno effect on the tumor-initiating activities of 3-methylcholanthrene(MCA), 7-methylbenz[a]anthracene (7-MBA), 12-methylbenz[a]anthracene(12-MBA), and 5-methylchrysene (5-MeC). B[e]P potentiated thetumor-initiating activity of benzo[a]pyrene (B[a]P) by 30%.DB[a,c]A, when applied 5 min prior to initiation, inhibitedthe tumor-initiating activities of DMBA, MCA, and DB[a,h]A buthad little or no effect on the tumor-initiating activities ofB[a]P, 7-MBA, 12-MBA, and 5-MeC. DB[a,c]A, when applied 12,24, or 36 h prior to initiation with B[a]P, which allowed timefor induction of epidermal monooxygenase enzymes, inhibitedtumor initiation. The covalent binding of DMBA and B[a]P toepidermal DNA was examined under the influence of B[e]P. Dosesof 20 and 200 nmol B[e]P given 5 min prior to 10 nmol [³H]DMBAreduced binding to 47 and 22%, respectively, of the controlvalue. In contrast, doses of 200 or 2000 nmol B[e]P given 5min prior to 200 nmol [³H]B[a]P had little or no effect on totalbinding. The data indicate that one cannot predict anti andcocarcinogenic effects of B[e]P and DB[a,c]A on the basis ofa presence or absence of a methyl substituent. In addition,fundamental differences exist in the processing and metabolismof DMBA and B[a]P by mouse epidermal cells.     

Distinction of carcinogens from mutagens by induction of liver cell foci in a model for detection of initiation activity

Initiating activities of 26 chemicals were investigated in an in vivo 5 week initiation assay model with evaluation of the induction of glutathione S-transferase placental form (GST-P) positive foci as end-point lesions. With the five genotoxic hepatocarcinogens (diethylnitrosamine, dimethylnitrosamine, 2-acetylaminofluorene, N-bis(2-hydroxypropyl)-nitrosamine and safrole) and 11 genotoxic non-hepatocarcinogens, (2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, benzo[a]pyrene, N-butyl-N-(4-hydroxybutyl)nitrosamine, 7,12-dimethylbenz[a]anthracene, 1,2-dimethylhydrazine, N-ethyl-N-hydroxyethylnitrosamine, 3-methylcholanthrene, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide and 8-hydroxyquinoline), the numbers of GST-P positive foci were significantly higher than in the controls. On the other hand, the mutagenic non-carcinogens (quercetin, p-phenylenediamine dihydrochloride, 2-chloroethanol and 6-hydroquinoline) did not cause a significant increase. Similarly, non-genotoxic hepatocarcinogens of the hepatopromotor class and promotors which target organs other than the liver did not induce GST-P positive foci. The specificity was thus remarkable. Moreover, regardless of the target organ, mutagenic carcinogens were detected by this in vivo 5 week initiation assay, which therefore constitutes a powerful method for screening for carcinogenic potential, especially in the initiation stage of carcinogenesis.     

Lung and liver cell-mediated mutagenesis systems: specificities in the activation of chemical carcinogens

A liver and lung cell-mediated-V79 cell mutagenesis system usingintact cells as metabolic activation systems was employed tostudy the relative ability of cells from these organs to activatechemical carcinogens. Primary cultures of liver and lung cellsfrom male Sprague Dawley rats were used to metabolically activatethe chemicals and the mutation of Chinese hamster V79 cellsto ouabain resistance used to detect mutagenic intermediates.7,12-Dimethylbenz[a]anthracene and 3-methylcholanthrene, weremore active in the lung system than in the liver cell system.Benzo[a]pyrene (B[a]P) was inactive in the liver cell-mediatedsystem but mutagenic to V79 cells in the lung cell-mediatedsystem. Dimethylnitrosamine (DMN) was inactive in the presenceof lung cells but was mutagenic to V79 cells in the presenceof liver cells. Aflatoxin B₁ was mutagenic in the liver cell-mediatedsystem, but only weakly mutagenic in the lung cell-mediatedsystem. Because the mutagenicities of DMN and B[a]P were organ-specific,the metabolism of these carcinogens in the two primary cellsystems was investigated. DMN was metabolized by liver but notby lung cells, possibly accounting for its lack of mutagenicityin the lung cell system. B[a]P was extensively metabolized byboth cell types, but quantitative differences were observedwhen the metabolic products were analyzed by high pressure liquidchromatography. Comparing total organic and water soluble metabolites,lung cells produced similar amounts of 7,8- and 9,10-diols butlittle 4,5-diol, while liver cells produced equivalent totalamounts of the three diols. Lung cells produced twice the amountof B[a]P glucuronide conjugates as liver cells, while livercells produced twice the amount of B[a]P sulfate conjugatesas lung. The data suggest that intact cells from various organscan be used as metabolic activating systems in in vitro assaysand that studies into organ specificity can be investigatedby this approach.     

DNA-protein cross-linking by chemical carcinogens in mammalian cells.

The induction of DNA cross-linking in mammalian cells by various carcinogens was investigated by the method of alkaline elution. A dose-dependent increase in DNA cross-linking was seen following exposure of human fibroblasts to N-acetyoxy-2-acetylaminofluorene and following exposure of mouse embryo cells to 7,12-dimethylbenz[a]-anthracene. No cross-link effect was seen following treatment with N-methyl-N'-nitro-N-nitrosoguanidine, benz-[a]anthracene, benz[A]anthracene-5,6-dihydroepoxide, or metabolic inhibitors. The cross-linking appeared to be DNA-protein in nature since proteinase treatment removed the effect. DNA single-strand breaks were also induced by several of these agents in the case of N-acetoxy-2-acetylaminofluorene and N-methyl-N'-nitro-N-nitrosoguanidine, approximately 70 to 90% of these breaks were rejoined after an 18-hr incubation in fresh medium, whereas repair of the cross-links induced by N-acetoxy-2-acetylaminofluorene was slight at this time.     

Rat bladder cell-mediated mutagenesis od Chinese hamster V79 cells and metabolism of benzo[a]pyrene

Primary rat bladder epithelial cells were cocultivated with Chinese hamster V79 cells in the presence of carcinogens, and the induction of 6-thioguanine resistance in the V79 cells was used as a marker of cell-mediated mutagenesis. The carcinogens dimethylnitrosamine, 7,12-dimethylbenz[a]anthracene, and benzo[a]pyrene (BP) were mutagenic to V79 cells in the presence of bladder cells but not in their absence. Analysis of BP metabolites formed by bladder cells indicated that 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, and 9-hydroxybenzo[a]pyrene were the major organic-soluble metabolites formed. Glucuronide and sulfate conjugates of BP metabolites were also produced by bladder cells. Mutagenesis data from the rat bladder system and previous data from rat liver and lung cell-mediated mutagenesis systems indicate that the cell-mediated mutagenesis approach may provide a useful approach for studying the organotropic effect of chemical carcinogens. Furthermore, the finding that rat bladder epithelium can metabolize some carcinogens offers new possibilities for the mechanism of initiation of bladder cancer.     

Oncogenic interaction of carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons in mice.

To evaluate possible interactions between PAH occurring in automobile exhaust condensates with regard to their tumour forming potency, the following experiments were performed. Six different doses of benzo[a]pyrene (3-100 microgram) and of dibenzo]a,h]anthracene (2-75 microgram) and mixtures thereof were tested subcutaneously on female NMRI mice. In addition, mixtures of 10 non-carcinogenic hydrocarbons were applied: benzo[e]pyrene, benzo[a]anthracene, phenanthrene, anthracene, pyrene, fluoranthene, chrysene, perylene, benzo[ghi]perylene and coronene. Mixtures of all 12 PAH were also applied. The proportion of PAH in all mixtures used was the same as in automobile exhaust condensates; benzo[a]pyrene was used as reference substance. The most important results were as follows: 1. Small doses of dibenzo[a,h]anthracene have a greater tumour promoting effect than do comparable doses of benzo[a]pyrene. Increased doses increase the effect of benzo[a]pyrene more than that of dibenzo[a,h]anthracene. 2. The mixture of benzo[a]pyrene and dibenzo[a,h]anthracene is 1.4 time more active than dibenzo[a,h]anthracene alone. 3. The mixture of all PAH has a lower efficacy than dibenzo[a,h]anthracene alone, amounting to only 0.03 that of dibenzanthracene; however, the activity of dibenzo[a,h]anthracene within the mixture of the 12 PAH increases by a factor of 3.1. 4. The activity of a mixture of dibenzo[a,h]anthracene and benzo[a]pyrene depends to about 40% on dibenzo[a,h]anthracene; and that of 12 PAH to 30% on dibenzo[a,h]anthracene alone or to 80% on a mixture of dibenzo[a,h]anthracene and benzo[a]pyrene.     

Failure of genotoxic carcinogens to produce tumors in human skin xenografts transplanted to SCID mice

Chemical carcinogenesis of human skin was investigated usinghuman skin xenografts (16 full thickness and 48 split thicknessskin grafts) transplanted to CB-17-scid (SCID) mice. Topicalapplication of a carcinogen, i.e. 7, 12-dimethyl-benz[a]anthracene(DMBA), benzo[a]pyrene, methylchol-anthrene or N-methyl-N'-nitro-N-nitrosoguanidine,to the human skin xenografts once a week for 25–30 weeksfailed to produce skin tumors. Both DMBA application plus UV-Birradiation and alternate applications of the above four carcinogensin combination with UV-B irradiation also failed to producetumors. All of these treatments induced skin papillomas in skinsof host SCID mice. DMBA induced skin papillomas in allogenicCD-1 mouse skin grafts transplanted to SCID mice. These resultsindicate that susceptibility of human skin to these carcinogenicstimuli is much lower than that of mouse skin.     

Inhibitory effect of hemin on the mutagenic activities of carcinogens

An inhibitory effect of hemin on mutagenicities of a range of carcinogens was found by adding hemin to the preincubation mixture of the Ames' test. Strong inhibitions were observed for benzo[a]pyrene, 3-methylcholanthrene, 7,10-dimethylbenz[a]anthracene, chrysene, 2-acetylaminofluorene, 2-nitrofluorene and aflatoxin B₁. Generally, 50% inhibition was caused by an amount of hemin 1–2 equivalents to the mutagen. Excess of hemin caused complete inhibitions. Hemin did not affect the mutagenicities of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 4-nitroquinoline-1-oxide, nitromin, N-methyl-N-nitrosourea, N-methyl-N′-nitro-N-nitrosoguanidine, N-nitrosodi-n-butyl-amine, quinoxaline-1,4-dioxide and carbadox. Biliverdin, bilirubin and chlorophyllin were also effective as inhibitors for the mutagenicity of benzo[a]pyrene.     

Chemical transformation of human revertant cells induced by murine sarcoma virus.

A human revertant cell line, derived from non-producer human osteosarcoma cells (NP/KHOS) induced by Kirsten murine sarcoma virus, was treated in vitro with various levels of polycyclic aromatic hydrocarbons (3-methylcholanthrene, 7,12-dimethylbenz(alpha)anthracene, and benzo(alpha)pyrene [BP] or dimethyl sulfoxide (control). Cells treated only with 3-methylcholanthrene and 7,12-dimethylbenz(alpha)anthracene underwent morphological alteration in vitro, and produced tumors when injected into NIH nude mice. These human revertant cells may be a useful in vitro tool in screening for the potential chemical carcinogens in human cell systems.     

Use of the 32P-postlabelling assay to study transplacental carcinogens and transplacental carcinogenesis

The formation of DNA adducts represents a key step in the postnatal initiation of the carcinogenic process. Little is known as yet about the role of prenatally induced adducts in transplacental carcinogenesis in offspring. Measurement of transplacental DNA damage in fetal organs of experimental animals has been difficult in the past because of the small amounts of DNA available and low adduct levels. In principle, these difficulties have been overcome by the recent development of a highly sensitive 32P-postlabelling assay which can be applied to a large number of DNA adducts of diverse structure and requires only microgram amounts of DNA for analysis. In this assay, tissue DNA is degraded to mononucleotides; these are enzymatically 32P-labelled via T4 polynucleotide kinase-catalysed [32P]phosphate transfer from [gamma--32P]ATP, to form 5'--32P-labelled 3',5'-bisphosphate derivatives; the labelled products are separated into normal and adducted [32P]nucleotides and quantified by thin-layer chromatography, autoradiography and scintillation (Cerenkov) counting. This technique allows the detection and quantitation of one adduct in 10(8)-10(10) DNA nucleotides (approximately 1-100 adducts/mammalian genome) using a 10-micrograms DNA sample and has been applied in studies of adduct formation from transplacental carcinogens in fetal and adult rodent tissues. In this paper, we review application of 32P-postlabelling to DNA adducts formed with transplacental or suspected transplacental carcinogens in fetal and maternal tissues. The carcinogens studied include diethylstilboestrol (DES), benzo[a]pyrene, safrole, 4-aminobiphenyl and 4-nitroquinoline-1-oxide, as well as cigarette smoke condensate. In DNA of DES-exposed hamsters, one major and several minor adduct spots were observed, which were absent from vehicle controls. A characteristic adduct, which resembled the major hamster adduct chromatographically, was detected in all exposed mouse tissue, except fetal kidney. Chronic administration of low doses of DES to male Syrian hamsters led to an entirely different pattern of adducts in kidney DNA, the target organ of carcinogenesis. These adducts did not contain covalently bound oestrogen moieties and appeared to be formed indirectly: oestrogen appeared to induce or enhance the synthesis of an endogenous electrophilic metabolite reacting with DNA. Thus, multiple mechanisms exist by which DES can damage DNA. Additional work using 32P-postlabelling has shown that non-hormonal genotoxicants (e.g., benzo[a]pyrene, safrole, 4-aminobiphenyl, 4-nitroquinoline-1-oxide) and cigarette smoke condensate given to pregnant mice can induce specific DNA adduct profiles in fetal tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Benzo[a]pyrene and other inducers of cytochrome P1-450 inhibit binding of epidermal growth factor to cell surface receptors

The binding of 125I-labelled epidermal growth factor (EGF) was utilized to monitor possible cell surface effects of polycyclic aromatic hydrocarbon carcinogens. Exposure of confluent C3H 10T1/2 mouse fibroblasts to 1 muM benzo[a]pyrene led to a time-dependent decrease of EGF binding. By 24 h, EGF binding was only 5% that of control cultures. In contrast, benzo[a]pyrene-7,8-diol-9,10-oxide did not significantly alter EGF binding, indicating that the inhibition by benzo[a]pyrene was not simply due to DNA damage. A curvilinear Scatchard plot in the control cells was consistent with the presence of two classes of EGF receptors having differing affinities. Our results suggest that the major effect of benzo[a]pyrene was a reduction in receptor number rather than affinity, although other interpretations have not been excluded. Progesterone, 17 beta-estradiol, benzo[e]pyrene, cholesterol, phenobarbital, 1,1-bis-(p-chlorophenyl)-2,2,2-trichloroethane, hexachlorobenzene or pregnenolone-16 alpha-carbonitrile, did not inhibit EGF binding. On the other hand, several known inducers of P1-450 were very effective inhibitors of EGF binding. These included: dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, benz[a]anthracene, beta-naphthoflavone and alpha-naphthoflavone. We postulate that the binding of certain polycyclic aromatic hydrocarbons to the Ah receptor may induce not only specific drug metabolizing enzymes but also inhibition of EGF binding, and possible other cell effects. Further studies are required to verify this hypothesis.     

Role of depurination in mutagenesis by chemical carcinogens

The effect of modifying phi chi 174 viral DNA by the chemical carcinogens beta-propiolactone, N-acetoxyacetylaminofluorene and anti-benzo[a]pyrene diol-epoxide was investigated by transfecting the modified DNA into Escherichia coli spheroplasts. Modification of the DNA in vitro by each of these agents was mutagenic for the phi chi 174 amber mutants am3 and am86. Mutagenicity depended on the induction of the "SOS" response in the host spheroplasts. Heating beta-propiolactone-treated DNA at neutral pH caused strong inactivation such that the number of lethal hits was increased 4-fold. Sucrose gradient analysis showed the induction of alkali-labile sites in the heated DNA. The "nicked circle assay" with double-stranded phi chi 174 DNA showed greater than 70% of these sites to be apurinic sites. Concomitantly with the production of these new sites, a strong increase in the mutation frequency was observed. This mutagenesis also depended upon the induction of the error-prone SOS response in the spheroplasts, as was previously shown to be the case for mutagenesis at putative apurinic sites induced directly by acid-heat treatment. These results suggest that depurination may be of importance to the mechanism of mutagenesis by beta-propiolactone and other carcinogens.     

Immunogenicity and cross-reactivity of syngeneic murine melanomas

Non-melanoma skin cancers induced in mice by chemical carcinogens or ultraviolet radiation are often antigenic but rarely induce cross-protective immunity when tested by in vivo transplantation methods. We wished to determine whether melanocytic skin tumors behave similarly or whether they exhibit cross-reactive antigens in vivo. Three melanomas induced in C3H/HeNCr(MTV-) mice by initiation with ultraviolet radiation and promotion with croton oil or initiation with 7,12-dimethyl-benz[a]anthracene and promotion with 12-O-tetradecanoyl-phorbol-13-acetate or croton oil plus ultraviolet radiation were tested for immunogenicity and cross-reactivity in vivo. The three melanomas were highly immunogenic, and all induced some degree of protection against the other melanomas. Non-melanoma skin cancers induced by the same carcinogens were less immunogenic and did not immunize against the melanomas. We conclude that unlike other skin cancers, melanocytic tumors induced by chemical carcinogens and ultraviolet radiation are highly cross-reactive in vivo and thus represent a unique subset of murine skin cancers.     

Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice

Arylhydrocarbon receptor knock-out, AhR(-/-), mice have recently been shown to be rather resistant to benzo[a]pyrene (B[a]P)-induced tumor formation, probably reflecting the inability of these mice to express significant levels of cytochrome P450 (P450 or CYP) 1A1 that activates B[a]P to reactive metabolites (Y. Shimizu, Y. Nakatsuru, M. Ichinose, Y. Takahashi, H. Kume, J. Mimura, Y. Fujii-Kuriyama and T. Ishikawa (2000) PROC: Natl Acad. Sci. USA, 97, 779-782). However, it is not precisely determined whether CYP1B1, another enzyme that is also active in activating B[a]P, plays a role in the B[a]P carcinogenesis in mice. To understand the basis of roles of CYP1A1 and CYP1B1 in the activation of chemical carcinogens, we compared levels of induction of liver and lung CYP1A1, 1A2, and 1B1 by various polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls in AhR(+/+) and AhR(-/-) mice. Liver and lung CYP1A1 and 1B1 mRNAs were highly induced in AhR(+/+) mice by a single intraperitoneal injection of each of the carcinogenic PAHs, such as B[a]P, 7,12-dimethylbenz[a]anthracene, dibenz[a,l]pyrene, 3-methylcholanthrene, 1,2,5,6-dibenzanthracene, benzo[b]fluoranthene, and benzo[a]anthracene and by a co-planar PCB congener 3,4,3',4'-tetrachlorobiphenyl. We also found that 6-aminochrysene, chrysene, benzo[e]pyrene, and 1-nitropyrene weakly induced the mRNA expression of CYP1A1 and 1B1, whereas anthracene, pyrene, and fluoranthene that have been reported to be non-carcinogenic in rodents, were very low or inactive in inducing these P450s. The extents of induction of liver CYP1A2 by these chemicals were less than those of CYP1A1 and 1B1 in AhR(+/-/+/-) mice. In AhR(-/-) mice, there was no induction of these P450s by PAHs and polychlorinated biphenyls. Liver microsomal activities of 7-ethoxyresorufin and 7-ethoxycoumarin O-deethylations and of mutagenic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P to DNA-damaging products were found to correlate with levels of CYP1A1 and 1B1 mRNAs in the liver. Our results suggest that carcinogenicity potencies of PAHs may relate to the potencies of these compounds to induce CYP1A1 and 1B1 through AhR-dependent manner and that these induced P450s participate in the activation of B[a]P and related carcinogens causing initiation of cancers in mice.