Aryl hydrocarbon hydroxylase in mouse mammary gland: In vitro study using mammary cell lines

The effects of 3-methylcholanthrene (MCA), 5,6-benzoflavone (βNF), 7,8-benzoflavone (αNF) and pregnenolone 16α-carbonitrile (PCN) upon aryl hydrocarbon hydroxylase (AHH) were determined in primary mammary gland epithelial cell cultures prepared from the C3Hf^?/Ki mouse. MCA elevated AHH activity by 3–4 fold after 24 h of treatment; αNF produced a 50% inhibition. The specific activity of AHH in these cells was elevated by 6 h after exposure to MCA; enzyme activity was still maximally elevated after 48 h. The effects of MCA were also investigated in a group of mammary cell lines, one of which was derived from a control virgin mouse, the MCG V14; 3 of which arose from mammary tumors, MCG T10, MCG T14 and MCG T19; and 2 of which were sublines developed from hyperplastic alveolar nodules, HAN-1 and HAN-2. Induction was seen in all lines at 24 h, with the MCG T14 being the most responsive and the HAN-2, the least. Although the MCG T19 tumor cells did respond in culture, when implanted in the mouse, the AHH of the subsequent tumor was not elevated upon administration of MCA in vivo.     

Covalent binding of benzo[a]pyrene to dna in fish liver

Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ($\text{Parophrys vetulus}$) force fed tritiated benzo[a]pyrene. No statistically signïficant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 10⁶ nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.     

Inhibition of epidermal metabolism and DNA-binding of benzo[a]pyrene by ellagic acid

Ellagic acid, a common plant phenol, was shown to be a potent inhibitor of epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activity in vitro, and of benzo[a]pyrene (BP)-binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. The in vitro addition of ellagic acid (0.25-2.0 microM) resulted in a dose-dependent inhibition of AHH activity in epidermal microsomes prepared from control or carcinogen-treated animals. The I50 of ellagic acid for epidermal AHH was 1.0 microM making it the most potent inhibitor of epidermal AHH yet identified. In vitro addition of ellagic acid to microsomal suspensions prepared from control or coal tar-treated animals resulted in 90% inhibition of BP-binding to calf thymus DNA. Application of ellagic acid to the skin (0.5-10.0 mumol/10 gm body wt) caused a dose-dependent inhibition of BP-binding to epidermal DNA. Our results suggest that phenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer from environmental chemicals.     

苯并[a]芘对小鼠肝脏和肾脏中丙二醛和过氧化氢酶的影响

目的探讨苯并[a]芘（B（a）P）对小鼠肝脏和肾脏脂质过氧化及抗氧化能力的影响。方法采用B（a）P口腔灌胃连续染毒3 d后,取肝、肾组织作匀浆,采用TBA比色法测定鼠肝脏和肾脏内的丙二醛（MDA）的含量,钼酸铵比色法测定鼠肝脏和肾脏内的过氧化氢酶（CAT）的含量。结果肝中各剂量染毒组的MDA含量增加,其中5 mg/kg、10 mg/kg剂量组与油剂对照组比较差异有显著性（P〈0.05）。肾脏中各剂量染毒组的MDA含量均有所增加,其中10 mg/kg剂量组与对照组比较差异有显著性（P〈0.05）。肝脏中各剂量染毒组的CAT的含量低剂量增加高剂量减少,肾脏中各剂量染毒组的CAT的含量增加。结论B（a）P可引起MDA含量增加诱导小鼠肝肾的脂质过氧化损伤。     

The use of immunologically competent cells from pig mesenteric lymph nodes to treat pulmonary tumors in mice

Summary Pulmonary tumors were produced in A strain mice by intravenous injection of 1×10⁶ A strain mammary carcinoma cells. The mice were killed on day 14, their lungs fixed in Bouin's fluid, and the number of tumors counted.The mesenteric lymph node chains of pigs were immunized by implantation of tissue into the mesentery. In all animals the middle segment of node chain was excised. The remaining segments of node proximal and distal to the resected segment were, in separate pigs, nonimmunized or immunized against mouse tumor, immunized against mouse tumor or mouse skin, or immunized against human tumor or mouse tumor. All segments of node chain were removed 7 days after immunization for preparation of cell suspensions.When tumor cells were combined in a ratio of 1 : 10 or 1 : 60 with mouse tumor-immune pig cells, there was a significant reduction in tumor formation compared to that in mice receiving tumor cells alone.Injection of mouse tumor-immune pig cells on day 7, to treat tumors inoculated on day 0, was ineffective. However, when the mice received, in addition, 200 rad thoracic irradiation on day 3, immune pig cells reduced the number of tumors compared to that in animals receiving irradiation alone, or irradiation and nonimmune pig cells.In further experiments, in order to increase the number of pig cells reaching the lungs, a splenectomy was performed on day 6, prior to intravenous injection of immune cells on day 7. A comparison was made of the antitumor effect of pig cells immunized against mouse tumor, mouse skin, or human tumor. Cells immunized against either mouse tissue were equally effective in reducing the number of tumors compared to the number in animals receiving tumor cells alone. However, cells immune to human tumor were ineffective.     

Impaired immunological surveillance by 7,12-dimethylbenz(a)anthracene augments its skin tumorigenicity in C3H mice

7,12-dimethylbenz(a)anthracene (DMBA) is a polyaromatic hydrocarbon with potent mutagenic, carcinogenic and immunomodulatory activities. It has been postulated that the immunosuppressive effects of DMBA may contribute to the carcinogenicity of this agent. We investigated this issue by examining whether the pre-administration of DMBA at one skin site would augment its carcinogenic activity at a distant skin site. In animals placed on a cutaneous chemical carcinogenesis protocol to the skin of the back, pre-sensitization with DMBA to the abdomen augmented skin tumorigenicity whether the tumor data are considered as cumulative number of tumors, percent of mice with tumors, number of tumors per mouse, or numbers of tumors per tumor bearing mouse as a function of weeks on test. These data demonstrate that pre-treatment with DMBA augments its skin tumorigenicity by impairing immunological surveillance.     

Mouse-strain difference in immunoprophylactic and immunotherapeutic effects of BCG on carcinogen-induced autochthonous tumors

The prophylactic and therapeutic effects of BCG on the tumors induced by 3-methylcholanthrene (MCA) were studied comparatively between two inbred mouse strains, SWM/Ms and C3H/He, the first tumor appeared 5 weeks after MCA and the cumulative tumor incidence reached almost 100% within 20 weeks. On the other hand, the first tumor appeared 8 weeks after MCA in SWM/Ms, the number of tumor-bearers increased more slowly than in C3H/He, and the final tumor incidence (at 30 weeks) was about 90-80%. Single subcutaneous injection with BCG 2 weeks prior to MCA significantly protected SWM/Ms from tumor development, but not in C3H/He. These tumors, once appeared grew progressively and killed the hosts equally in both the strains. Intratumor (i.t.) injection with BCG showed more or less therapeutic effects in SWM/Ms; most tumors regressed or retarded after BCG. The time period after tumor-appearance to tumor-death was prolonged in most of SWM/Ms mice given i.t. injection with BCG, except a few mice that died earlier than non-treated controls after BCG. Contrary, no therapeutic effect of i.t. injection with BCG was observed in C3H/He. different host responses to BCG between SWM/Ms and C3H/He were found by the peritoneal macrophage disappearance test and the footpad reaction test; SWM/Ms was a high-responder to BCG and C3H/He was a low-responder. The marked differences between SWM/Ms and C3H/He in prophylactic and therapeutic effects of BCG on the autochthonous tumors were discussed in terms of difference of the host immune response to BCG that is defined genetically.     

Non-enzymatic and microsome-dependent binding of polycyclic hydrocarbons to DNA and polynucleotides

The binding of tritium-labelled 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP) and 3-methylcholanthrene (MCA) to DNA or polynucleotides in vitro was re-examined both in the presence and in the absence of rat liver or human placental microsomes.A high level of non-enzymatic binding was evident when thymus DNA was used as acceptor. This non-enzymatic binding made it difficult to determine the effect of microsomes, except in the case of BP when induced rat microsomes were used. Better results were obtained using polynucleotides: a definite microsome-dependent binding occurred between all the polynucleotides and all the hydrocarbons tested.No clear evidence of binding catalysed by microsomes from human placenta was found except in polynucleotide-BP interactions: further studies are required to completely evaluate the ability of such nucleic acid-microsomal system for testing in vitro possible oncogenic substances in animals and humans.     

Inhibition by ajoene of skin-tumor promotion in mice

Ajoene, a major compound containing sulfur in oil-macerated garlic products, inhibited in a two-stage carcinogenesis test on mouse skin. Treatment with ajoene suppressed skin tumor formation, depending on the amount. In particular, the group treated with 250 microg of ajoene had only 4.9% the number of tumors per mouse compared with the control group at 18 weeks.     

Evidence that iron-overload promotes 7, 12-dimethylbenz(a)anthracene-induced skin tumorigenesis in mice

Summary

Iron overload is known to occur in West European and American populations due to the consumption of an iron-rich diet. There are also genetic disorders which lead to body iron overload. It has been shown that iron overload predisposes humans to an increased risk of cancer. In experimental animals, iron overload is known to enhance intestinal, colon, hepatic, pulmonary and mammary carcinogenesis. However, the mechanism by which iron overload enhances chemically-induced carcinogenesis is not known. In this study, we show that iron overload acts as a mild tumor promoter in mouse skin. Female albino swiss mice were given 1 mg iron/mouse parenterally for 2 weeks to induce iron overload. These animals showed a three-fold increase in cutaneous iron concentration as compared to normal mice. Tumors were initiated by topically applying 7,12-dimethylbenz(a)anthracene (DMBA). Appearance of the first tumor (latency period), percent tumor incidence and number of tumors/mouse were recorded. When compared to the control group, iron overload mice showed an increased incidence of tumors, from 25%-55% by week 20, and tumors appeared 4 weeks earlier. The number of tumors per mouse was four-fold higher in the iron overload group. The induction of cutaneous ornithine decarboxylase (ODC) activity and [³H]thymidine incorporation in cutaneous DNA were higher in iron overload groups as compared to normal control animals. Similar to other oxidant tumor promoters, iron overload enhanced cutaneous lipid peroxidation and xanthine oxidase activity and decreased catalase activity. Our results indicate that iron overload exerts a mild tumor promoting activity in mouse skin. Our data also show that oxidative stress generated by iron overload plays an important role in the augmentation of cutaneous tumorigenesis. These data may also have implications for the enhanced risk of cancer-induction following UVB exposure of human populations with iron overload.     

Protection against ultraviolet B- and C-induced DNA damage and skin carcinogenesis by the flowers of Prunus persica extract

The ethanol extract of the flowers of Prunus persica (Ku-35) (50-200 microg/ml) was found to inhibit UVB- as well as UVC-induced DNA damage measured by the COMET assay in the skin fibroblast cell (NIH/3T3). In addition, Ku-35 inhibited UVB- or UVC-induced lipid peroxidation, especially against UVB-induced peroxidation at higher than 10 microg/ml. We also evaluated the protective effect of Ku-35 against UVB-induced non-melanoma skin cancer in mice. Ku-35 was applied topically before UVB exposure, and its effects on tumor incidence (% of mice with tumors) and tumor multiplicity (number of tumors per mouse) were evaluated. The application of Ku-35 clearly resulted in a delay of tumor development compared to the control. In tumor incidence, 100% mice in the control group and the low dose treatment of Ku-35 had tumors, whereas 94.1% of the mice had tumors after the high dose treatment of Ku-35 at the end of experiment (28 weeks). In tumor multiplicity, low and high treatments of Ku-35 resulted in 25.9 and 53.9% reduction at the end of the experiment (P<0.05, one-way analysis of variance (ANOVA)). The present data indicate that Ku-35 protects against photogenotoxicity in NIH/3T3 fibroblasts. The possible action mechanism of Ku-35 may be through its anti-oxidant activity without pro-oxidant effect. Ku-35 can also show a delay of tumor development against UVB-induced skin carcinogenesis. These results suggest that Ku-35 extract may be useful for protecting UV-induced DNA damage and carcinogenesis when topically applied.     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Importance of DNA repair in carcinogenesis: evidence from transgenic and gene targeting studies

We have generated transgenic mice by introducing copies of the E. coli O6-methylguanine-DNA methyltransferase gene, ada. Liver extracts from homozygotes demonstrate about three times the control enzyme activity and increase up to about eight-fold can be induced by treatment with zinc, since the metal-responsive metallothionein promoter is attached to the ada gene. Furthermore, studies of liver carcinogenesis in our transgenic mice demonstrated significantly reduced rates of development of hepatocellular tumors after treatment with dimethylnitrosamine or diethylnitrosamine. It is well known that xeroderma pigmentosum (XP) patients are deficient in DNA repair. The availability of XPA (XP group A complementing) knockout mice has enabled us to investigate the functional role of the XPA nucleotide excision repair gene in carcinogenesis in vivo, first using the mouse skin as a model system. XPA-/- mice demonstrated skin ulcers 5-7 days after 7,12-dimethylbenz[a]anthracene (DMBA) treatment and papilloma development within 4 weeks prior to promotion, skin tumor incidence being also much higher than in heterozygous and wild-type mice. Experiments targeting the lung, liver and tongue have also been conducted to answer the question of whether the internal organs of these mice are also susceptible to chemical carcinogens. For lung carcinogenesis, mice were instilled intratracheally with a small dose of benzo[a]pyrene. The pulmonary tumor incidence in XPA-/- mice was significantly higher than in XPA+/- and XPA+/+ mice. XPA-/- mice were also found to be have enhanced sensitivity to aflatoxin B1 regarding liver tumor induction. In addition, administration of 4-nitroquinoline-1-oxide in drinking water for 50 weeks resulted in tongue tumors only in XPA-/- mice. These studies, thus, provided convincing evidence that XPA mice are also sensitive to carcinogenesis in organs other than the skin.     

Lung cancer induced by Benzo(A)Pyrene: ChemoProtective effect of sinapic acid in swiss albino mice

Cancer of lung is the utmost typical cause of death and the number of cases is increasing rapidly, which has emerged as a major leading health problem. A large amount of reports suggested that Benzo(a)pyrene [B(a)P] in cigarette smoke plays the major function in an initiation of cancer of lung. Cancer prevention or chemoprevention has become a compelling approach recently for treatment of lung cancer. So, discovering a fresh candidate with reduced toxicity for targeting lung cancer is vital and urgent. Sinapic acid which is a widely extracted in various vegetables and fruit exhibits rich anti-oxidant content, anti-inflammatory and anti-tumor activity. But, the chemopreventive action of sinapic acid against lung cancer initiated by B[a]P remain unclear. Following, an in-vivo B[a]P-stimulated lung cancer in swiss albino mice and an in-vitro human lung cancer cell (A549) model were established to examine the chemopreventive activities of sinapic acid. The levels of immunoglobulins (IgG and IgM), oxidative and inflammatory markers, and tumor markers level was studied using kits and standard methods. The results showed administration of sinapic acid ameliorates the exposure of B[a]P mediated lung cancer in swiss albino mice by a decline in IgG and IgM level, leukocyte count, neutrophil function tests, soluble immune complex, lipid peroxidation, pro-inflammatory cytokines, tumor markers (AHH, LDH, GGT, 5′NT and CEA) and enhanced phagocytic index, activity index and antioxidant defense enzymes. In addition, in-vitro studies showed potential cytotoxicity against human lung cancer and exhibited a potential cytotoxic (MTT assay) and apoptotic activity by elevation of ROS production and caspase activity (caspase-3 and caspase-9). Collectively, the results, clearly specifies sinapic acid can be utilized as an effective chemo preventative agent against lung carcinogenesis.     

6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has anti-bacterial, anti-inflammatory, and anti-tumor-promoting activities. Here, we describe its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF. Moreover, i.p. administration, without reaching tumor cytotoxic blood levels, to mice receiving i.v. injection of B16F10 melanoma cells, reduced the number of lung metastasis, with preservation of apparently healthy behavior. Taken together, these results demonstrate that [6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumors and other angiogenesis-dependent diseases.     

Inhibitory effects of Azadirachta indica on DMBA-induced skin carcinogenesis in Balb/c mice

Male Balb/c mice were divided into four groups on the basis of their respective treatments wherein mice of Group I served as controls. For induction of skin tumors, mice of Group II and IV were injected sub-cutaneously with 7,12-dimethylbenz(a)anthracene (DMBA). Mice of Group III and IV were administered aqueous Azadirachta indica leaf extract (AAILE) thrice a week throughout the experiment. After 14 weeks of the first DMBA injection, Group II and IV mice developed tumors. In the tumor-bearing mice that received AAILE (Group IV), a significant reduction in mean tumor burden and tumor volume was observed. The tumors were confirmed to be papillomas and interestingly, the extent of hyper-chromatia was observed to be much more in skin tumors of Group II mice vis a vis the mice receiving AAILE. An increase in the extent of lipid peroxidation was observed in tumorous tissue of Group IV when compared to that of Group II mice. Glutathione (GSH) content and the activities of GSH-based antioxidant enzymes viz. glutathione peroxidase (GPx) and glutathione reductase (GR) increased significantly in the skin tissues of all the groups of mice when compared to control counterparts. Catalase activity was found to decrease significantly in the skin of mice, which received AAILE treatment only (Group III). Activity of super-oxide dismutase (SOD) decreased significantly in all the tumorous tissues (Group II and IV mice). In light of the above observations, the role of AAILE in inhibition of DMBA-induced skin carcinogenesis is discussed in the present study.     

Cancer chemoprevention: inhibitory effect of soybeans fermented with basidiomycetes on 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate-induced mouse skin carcinogenesis

In a two-stage skin carcinogenesis model, mice initiated with 7,12-dimethylbenz[a]anthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 12 weeks developed an average of 15.8 skin tumours per mouse with 100% tumour incidence. Topical application of polysaccharides from soybeans fermented with either Phellinus igniarius or Agrocybe cylindracea together with TPA twice weekly for 12 weeks inhibited the number of skin tumours per mouse by 70 or 88%, respectively, and the percentage of mice with tumours was lowered by 70 or 30%, respectively.     

Chemopreventive activity of selenocysteine prodrugs against tobacco-derived nitrosamine (NNK) induced lung tumors in the A/J mouse

Prodrugs of L-selenocysteine have potential utility in cancer chemoprevention. This study reports the efficacy of three selenazolidine-4(R)-carboxylic acids, (2-unsubstituted, 2-oxo, and 2-methyl derivatives; SCA, OSCA, and MSCA, respectively) against tobacco-related lung tumorigenesis in a mouse model. Seven days after initiation of an AIN-76A diet supplemented with sodium selenite (5 ppm Se), L-selenomethionine (3.75 ppm Se), Se-methyl-L-selenocysteine (3 ppm Se), L-selenocystine (15 ppm Se), SCA (15 ppm Se), OSCA (15 ppm Se), or MSCA (15 ppm Se), mice received 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; 10 micromol, i.p.). After an additional 16 weeks on the diets, two compounds, OSCA and selenocystine, significantly reduced lung adenoma multiplicity from 7.2 tumors per mouse in the NNK group to 4.5 and 4.6 tumors per mouse, respectively. Neither selenium concentration nor glutathione peroxidase activity in either RBCs or liver served as surrogate indicators of tumor reduction. Hepatic selenium levels were significantly elevated by all selenium-containing compounds except Se-methyl-L-selenocysteine and SCA; RBC selenium levels by all except sodium selenite and MSCA. With the exception of L-selenomethionine, RBC glutathione peroxidase activity was increased along with the elevated selenium levels. Hepatic glutathione peroxidase activity was elevated by all Se-compounds except SCA. The two compounds showing significant tumor reduction (OSCA and selenocystine) were the only two compounds that showed ubiquity of changes, elevating both selenium levels and GPx activity in both liver and RBC.     

Tumor formation in the neonatal mouse bioassay indicates that the potent carcinogen dibenzo[def,p]chrysene (dibenzo[a,l]pyrene) is activated in vivo via its trans-11,12-dihydrodiol

The hexacyclic aromatic hydrocarbon dibenzo[def,p]chrysene, better known as dibenzo[a,l]pyrene (DBP) in the field of chemical carcinogenesis, is present in the environment as a combustion product of organic matter. This compound is probably the strongest chemical carcinogen ever tested. As ultimate genotoxic metabolites of DBP two electrophilically reactive species are discussed: (i) radical cations generated by one-electron oxidation, and (ii) fjord region dihydrodiol epoxides formed via the trans-11,12-dihydroxy 11,12-dihydro derivative of DBP (11,12-dihydrodiol). In order to delineate the metabolic pathway(s) involved in tumor formation by DBP, newborn Crl:CD-1(ICR)BR mice were intraperitoneally treated with the parent compound, its 11,12-dihydrodiol, and the two diastereomeric fjord region dihydrodiol epoxides. Due to severe acute and chronic toxicity, the total dose of DBP and of the 11,12-dihydrodiol was limited to 40 nmol. For the same reason the dihydrodiol epoxides could only be applied in doses up to 0.4 nmol. The tumor incidence was determined 55 +/- 1 weeks after treatment. Under these conditions, DBP and its 11,12-dihydrodiol induced lung tumors (incidence: 86.5% versus 92.0%; yield: 2.88 versus 7.44 tumors per mouse), liver (incidence: 57.7% versus 60.0%; yield: 3.63 versus 5.28 tumors per mouse) and other organs (incidence: 36.5% versus 32.0%; yield: 0.56 versus 0.52 tumors per mouse). By contrast, only lung tumors at low incidence were detected in mice treated with solvent only (incidence: 28.8%; yield: 0.58 tumors per mouse). As with the parent hydrocarbon, mice treated with low doses of diastereomeric syn- and anti-dihydrodiol epoxides of DBP showed increased tumor incidences in liver (incidence: 19.0 and 46.7%; yield: 0.36 and 1.47 tumors per mouse, respectively), and in various other organs (incidence: 7.1 and 20.0%; yield: 0.07 and 0.20 tumors per mouse, respectively). In consideration of the 100-fold differences in the doses of compounds applied in this study, the tumor-inducing potency increases in the order DBP < 11,12-dihydrodiol < anti-dihydrodiol epoxide. This result provides strong evidence that the potent carcinogen DBP is activated in vivo in the mouse via its 11,12-dihydrodiol and not preferentially through alternative pathways.