DMBA Carcinogenesis Research Articles

Efficacy of polyvinylpyrrolidone-capped gold nanorods against 7,12 dimethylbenz(a)anthracene-induced oviduct and endometrial cancers in albino rats

ABSTRACT Polyvinylpyrrolidone-capped gold nanorods (PVP-capped AuNRs) are emerging as promising agents for cancer therapy. With the widespread utilization of nanoparticles, some is known about the therapeutic effect of PVP-capped AuNRs on oviduct and endometrium adenocarcinomas resulting from 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis. Twenty-four female Wister albino rats (Rattus norvegicus) were categorized into four groups: negative control, PVP-capped AuNRs positive control, DMBA-treatment and DMBA plus PVP-capped AuNRs. A fabrication method of PVP-capped AuNRs and their characterization was assessed using ultraviolet spectra (UV), ultrastructure analysis, dynamic light scattering (DLS) and zeta potential (ɳ). Morphometric assessments and the mortality rate after induction with DMBA carcinogenesis were recorded. The oviducts and uteri were dissected and subjected to histopathological and immunohistochemical analysis. PVP-capped AuNRs treatment enhanced the histological features of the DMBA carcinogenesis-dependent oviduct and endometrium adenocarcinomas. In addition, there was a reduced immune reaction of caspase-3 and Ki-67 markers after the treatment, with PVP-capped AuNRs referring to its improvement effect. In conclusion, PVP-capped AuNRs showed therapeutic potential against oviduct and endometrium adenocarcinoma by reaching tumors via enhancing permeability and retention effect. The proposed methodology suggests that PVP-capped AuNRs target the site and the boundaries of cancer, opening new therapeutic options.

Significance of host heparanase in promoting tumor growth and metastasis

Heparanase, the sole heparan sulfate degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, angiogenesis and metastasis. Much of the impact of heparanase on tumor progression is related to its function in mediating tumor-host crosstalk, priming the tumor microenvironment to better support tumor growth and metastasis. We have utilized mice over-expressing (Hpa-tg) heparanase to reveal the role of host heparanase in tumor initiation, growth and metastasis. While in wild type mice tumor development in response to DMBA carcinogenesis was restricted to the mammary gland, Hpa-tg mice developed tumors also in their lungs and liver, associating with reduced survival of the tumor-bearing mice. Consistently, xenograft tumors (lymphoma, melanoma, lung carcinoma, pancreatic carcinoma) transplanted in Hpa-tg mice exhibited accelerated tumor growth and shorter survival of the tumor-bearing mice compared with wild type mice. Hpa-tg mice were also more prone to the development of metastases following intravenous or subcutaneous injection of tumor cells. In some models, the growth advantage was associated with infiltration of heparanase-high host cells into the tumors. However, in other models, heparanase-high host cells were not detected in the primary tumor, implying that the growth advantage in Hpa-tg mice is due to systemic factors. Indeed, we found that plasma from Hpa-tg mice enhanced tumor cell migration and invasion attributed to increased levels of pro-tumorigenic factors (i.e., RANKL, SPARC, MIP-2) in the plasma of Hpa-Tg vs. wild type mice. Furthermore, tumor aggressiveness and short survival time were demonstrated in wild type mice transplanted with bone marrow derived from Hpa-tg but not wild type mice. These results were attributed, among other factors, to upregulation of pro-tumorigenic (i.e., IL35+) and downregulation of anti-tumorigenic (i.e., IFN-γ+) T-cell subpopulations in the spleen, lymph nodes and blood of Hpa-tg vs. wild type mice and their increased infiltration into the primary tumor. Collectively, our results emphasize the significance of host heparanase in mediating the pro-tumorigenic and pro-metastatic interactions between the tumor cells and the host tumor microenvironment, immune cells and systemic factors.

DMBA-Induced Oral Carcinoma in Syrian Hamster: Increased Carcinogenic Effect by Dexamethasone Coexposition

Objectives To investigate the effect of systemic administration of the immunosuppressant dexamethasone (DM) while inducing hamster buccal pouch DMBA carcinogenesis. Materials and Methods. Two different experiments were performed. In the first experiment, hamsters' right buccal pouches in group A (n = 10) were painted three times per week with 7,12-dimethylbenzanthracene (DMBA) 0.5%, while pouches of animals in group B (n = 10) were painted three times per week with 7,12-dimethylbenzanthracene (DMBA) 0.5%, while pouches of animals in group B (Results The time of macroscopic neoplasm development was reduced when DM-DMBA coexposition was employed, finding tumors after 10–12 weeks of exposition. In addition, the frequency of histopathological lesions was higher. Conclusion Immunomodulatory action of dexamethasone may reduce the time of oral squamous cell carcinoma (OSCC) induction and may increase the incidence of neoplasms developed.

Anti-Estrogenic and Anti-Cell Proliferative Effect of Allyl Isothiocyanate in Chemoprevention of Chemically Induced Mammary Carcinogenesis in Rats.

The anti-estrogenic and anti-cell proliferative effect of allyl isothiocyanate (AITC) was carried out by analyzing the status of sex hormones and its receptors and cell proliferative markers in chemically induced mammary carcinogenesis in rats. Mammary tumor was induced by a single dose of DMBA (25mg/rat) and MNU (50mg/kg bw) injected subcutaneously near mammary gland. RT-PCR, western blotting and immunohistochemical analysis of mammary tissues show an upregulation of ER-α, PR, aromatase, PCNA, cyclin D1 and AgNORs staining and down regulation of p53 expression as well as plasma estradiol, prolactin and testosterone levels increased in DMBA and MNU-induced tumor bearing rats. Oral administration of AITC at a dose of 20mg/kg bw restored the levels of sex hormones and its receptors, aromatase, cell proliferative markers and AgNORs staining near to normal levels. Molecular docking studies also supported these findings. The results suggest that anti-estrogenic and anti-proliferative effect of AITC prevent the development of DMBA and MNU-induced mammary carcinogenesis in rat.

Pubertally Initiated High-Fat Diet Promotes Mammary Tumorigenesis in Obesity-Prone FVB Mice Similarly to Obesity-Resistant BALB/c Mice

Premenopausal breast cancer is associated with increased animal fat consumption among normal-weight but not overweight women. Our previous findings in obesity-resistant BALB/c mice showed that a diet high in saturated animal fat (HFD) promotes mammary tumorigenesis in both DMBA carcinogenesis and Trp53-null transplant models. Having made these observations in BALB/c mice, which have very modest HFD weight gain, we determined the effects of HFD in FVB mice, which gain significant weight on HFD. Three-week-old FVB mice fed a low-fat diet or HFD were subjected to 7,12-dimethylbenz[a]anthracene-induced carcinogenesis. Like BALB/c mice, HFD promoted mammary tumorigenesis. Development of tumors largely occurred prior to mice becoming obese, indicating the role of animal-derived HFD rather than resulting obesity in tumor promotion. Also similar to BALB/c mice, early-occurring adenosquamous mammary tumors were abundant among HFD-fed FVB mice. Tumors from HFD mice also had increased intra-tumor M2 macrophages. Prior to tumor development, HFD accelerated normal mammary gland development and increased mammary M2 macrophages, similarly to BALB/c mice. The promotional effects of puberty-initiated HFD on carcinogen-induced mammary cancer are thus largely weight gain-independent. Like BALB/c mice, HFD promoted adenosquamous tumors, suggesting a role for early age HFD in promoting this subtype of triple negative mammary cancer. M2 macrophage recruitment was common to both mouse strains. We speculate that a similar effect of HFD on immune function may contribute to epidemiological findings of increased breast cancer risk in young, premenopausal, normal-weight women who consume a diet high in saturated animal fat.

Lipid mapping by desorption electrospray ionization mass spectrometry in a murine breast DMBA carcinogenesis model

Lipids are molecules with a large structural diversity inserted in various cytological and pathological processes, such as in breast cancer. The knowledge of the metabolic and lipid profiles in breast cancer helps to identify and classify neoplastic and non-neoplastic areas and aids therefore in precise histological diagnosis and in the identification of new promising therapeutic targets. The present study aimed to compare non-neoplastic samples of Sprague-Dawley rat breast lesions to a DMBA chemical carcinogenesis model via DESI-MS imaging. The model Sprague-Dawley rats were treated with the carcinogen compound DMBA, and frozen mammary samples with or without tumors were imaged. The developed ductal carcinomas showed indeed contrasting lipid relative abundances as compared to non-neoplastic breast tissue. Most lipids belong to the fatty acyl family, and were detected in greater abundance in the non-neoplastic cancer samples. The lipid ion of m/z 336 was observed only in the neoplastic areas. This is the first study that demonstrates changes in lipid profile of mammary tumors in rats treated with DMBA, and the promising results seems to encourage and serve as a guide for further lipidomic studies regarding breast cancer.

Hepatic Arginase - Nitric oxide imbalance: Impact of carcinogenesis and therapeutic effect of sodium channel blockage in an in vivo rat model

Abstract Objective: Nitric oxide synthase and arginase are frequently antagonistic and interactive, although both use L-arginine as common substrate. Their balance is of potential functional importance. How the balance changes in cancer is unknown. Increasing evidence suggests that progression of carcinomas involves functional voltage-gated sodium channel (VGSC) activity. Methods: The present study extended this study to liver and aimed to determine whether (i) DMBA carcinogenesis would affect the activities of arginase and NOS and (ii) treatment with Na-channel blocker RS100642 would ameliorate the impact of the carcinogen on the arginase-NOS balance. Results: DMBA application significantly increased arginase activity and, correspondingly, the level of L-ornithine by 25-33%. In contrast, NOS activity decreased by 11%. Importantly, RS100642 treatment completely suppressed the effect on arginase. Conclusion: It is concluded (i) that DMBA carcinogenesis changes the hepatic arginase-NOS balance, increasing the overall dominance of arginase and (ii) that VGSC inhibition has a protective effect on liver.

Quercetin attenuates oxidative stress in the blood plasma of rats bearing DMBA-induced mammary cancer and treated with a combination of doxorubicin and docetaxel

The development of side-effects during doxorubicin-docetaxel (DOX-DTX) chemotherapy is considered as related to generation of oxidative stress by DOX. The addition of docetaxel potentiates this effect. Thus, antioxidants are assumed as a promising remedy for neutralizing deteriorating effects of reactive oxygen species (ROS) in pathological conditions and polyphenolic antioxidants are suitable candidates for such a therapeutic approach. We evaluated the ability of quercetin to attenuate oxidative stress developed during the process of DMBA carcinogenesis and DOX-DTX chemotherapy in the blood plasma of rats bearing mammary tumors. We have found that quercetin significantly improved the plasma nonenzymatic antioxidant capacity (NEAC) and reduced lipid peroxidation, which suggest the beneficial effect of flavonoid. The inclusion of quercetin to the DOX-DTX chemotherapy was also advantageous. A considerable decrease of carbonyls and lipid peroxidation products (TBARS) and improvement of the endogenous antioxidant defense system (an increase of NEAC, thiols and SOD activity) were observed compared to rats treated with DOX-DTX chemotherapy. These results suggest that quercetin could protect blood plasma constituents against oxidative damage evoked by DOX and DTX.

Abstract A51: The role of ribosomal protein s6 in the development of pancreatic cancer precursor lesions

Abstract Background: Carcinoma of the pancreas kills nearly all of the 230,000 people diagnosed with the disease annually worldwide. This high mortality results from poor detection, metastatic disease at the time of detection, and lack of specific or effective therapy. Early precursor lesions termed pancreatic intraepithelial neoplasia (PanIN) are thought to be the initial step in pancreatic ductal carcinoma. The PI3K signaling pathway is a central transducer of information from extracellular signals to the cell, impacting diverse cellular function such as replication, survival, migration, protein synthesis, and more. Ribosomal protein s6 (rps6) is a component of the protein translation machinery, identified originally in the regenerating liver. It is phosphorylated at specific residues by an upstream kinase, s6K, which itself is activated by mTOR and the PI3K-AKT pathway. No functional link is currently known between rps6 phosphorylation and pancreatic cancer. Methods: Pellets soaked with 7,12-dimethylbenz(a)anthracene(DMBA) were implanted in the body of wild-type mouse pancreas (n-12) and in the pancreas of mutant mice lacking rps6 phosphorylation sites (rps6ki/ki) (n=11). Three to five months later, the mice were scarified and pathological analysis and scoring for PanIN lesions were performed. To examine in a crude way if mTOR signaling is important for the development of DMBA-induced PanIN lesions, we administered rapamycin to DMBA-treated mice, and have determined the effect on PanIN score. We then examined the role of rps6 activation in a genetic model of PanIN lesions. Using extensive breeding, we generated cohorts of pdx1-Cre; LSL-KRAS G12D; rps6 ki/ki mice, in order to examine the effect of rps6 phosphorylation on the development of PanIN lesions, in comparison to LSL-KRAS wild rps6, after 3–5 months the mice were sacrificed and the same analysis and scoring were performed. In both models, rps6 pattern of staining was evaluated in all groups. Results: In the DMBA model, we observed the development of mouse PanIN lesions at all levels (PanIN 1–3) in all wild-type mice examined. Rapamycin-treated mice had a significant reduction in the score of PanIN lesions, supporting the hypothesis that mTOR signaling downstream of PI3K, participated in the formation of PanIN lesions. In mutant mice, strikingly, PanIN lesions observed were greatly reduced. These results suggest that rps6 phosphorylation is an essential event in the development of pancreatic cancer, in the DMBA carcinogenesis model. In the KRAS model, mice developed PanIN lesions, as previously described, KRAS mice lacking rps6 phosphorylation developed fewer PanIN lesions that had lower score, similar to DMBA carcinogenesis model. This shows that development of PanIN lesions driven by mutant KRAS requires at least partially phosphorylation of rps6. The expressive pattern of rps6 in normal mice revealed that rps6 phosphorylation is limited to a subset of islet cells and to a few acinar cells surrounding the islets, with minimal or no activity in ducts. In PanIN lesions generated by implantation of DMBA in the pancreas, as well in Pdx1-Cre; LSL-KRAS G12D mice, strong phosphorylation of rps6 is observed in malignant epithelial cells. This suggests that oncogenic signaling in the pancreas, and specifically KRAS signaling, activates PI3K signaling, including S6 kinase which phosphorylates rps6. Conclusion: rps6 is activated (phosphorylated) in mouse models of PanIN lesions and pancreatic cancer, and development of PanIN lesions requires at least partial phosphorylation of rps6. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A51.

Effect of Nicotine and Cigarette Smoke on an Experimental Model of Intraepithelial Lesions and Pancreatic Adenocarcinoma Induced by 7,12-Dimethylbenzanthracene in Mice

To evaluate the effects of nicotine and cigarette smoke exposure on mice submitted to 7,12-dimethylbenzanthracene (DMBA) model of pancreatic carcinogenesis. One hundred fourteen male mice were divided into the DMBA-n and DMBA-s groups: the DMBA-n group was given 2 mg/kg per dose of nicotine ([3-(1-methyl-2-pyrrolidinyl)pyridine]) subcutaneously for 45 days, and the DMBA-s group was exposed to 100 mg/m of cigarette smoke. At day 16, 1 mg of DMBA crystals was implanted in the pancreatic head of both groups. Euthanasia was performed in all mice 30 days after the surgery. The specimens were evaluated according to the following criteria: normal ducts, reactive hyperplasia, pancreatic intraepithelial neoplasm 3 (PanIN-3), and carcinoma. For statistical analysis, DMBA-exclusive ([DMBA-e] historical control group) was included. The frequency of PanIN in the 3 groups was almost the same when considering the higher-grade lesions: DMBA-e (16 [66.7%]), DMBA-s (20 [66.7%]), and DMBA-n (12 [44.4%]). Pancreatic adenocarcinoma has a higher frequency in the DMBA-n group (14 [51.9%]) than in the DMBA-e (4 [16.7%]) and DMBA-s (4, 13.3%) groups. The DMBA-s group has the highest score of PanIN-3 (40%). The differences among the groups were statistically significant (P = 0.05, Fisher exact test). Nicotine but not cigarette smoke promotes pancreatic DMBA carcinogenesis in mice. Pancreatic adenocarcinomas and PanINs have the same phenotypic appearance as those that occur in humans.

Correction: TLR4 in Cutaneous DMBA Carcinogenesis

Correction: TLR4 in Cutaneous DMBA Carcinogenesis

Gene Expression Analysis of Biological Systems Driving an Organotypic Model of Endometrial Carcinogenesis and Chemoprevention

An organotypic model of endometrial carcinogenesis and chemoprevention was developed in which normal endometrial organotypic cultures exposed to the carcinogen, DMBA (7,12-dimethylbenz[a]anthracene), developed a cancerous phenotype in the absence, but not presence of subsequent treatment with a flexible heteroarotinoid (Flex-Het), called SHetA2. A discriminant function based on karyometric features of cellular nuclei and an agar clonogenic assay confirmed these histologic changes. Interpretation of microarray data using an internal standard approach identified major pathways associated with carcinogenesis and chemoprevention governed by c-myc, p53, TNFalpha and Jun genes. Cluster analysis of functional associations of hypervariable genes demonstrated that carcinogenesis is accompanied by a stimulating association between a module of genes that includes tumor necrosis factor alpha (TNFalpha), c-myc, and epidermal growth factor-receptor (EGF-R) and a module that includes insulin-like growth factor I-receptor (IGF-IR), p53, and Jun genes. Two secreted proteins involved in these systems, tenascin C and inhibin A, were validated at the protein level. Tenascin C is an EGF-R ligand, and therefore may contribute to the increased EGF-R involvement in carcinogenesis. The known roles of the identified molecular systems in DMBA and endometrial carcinogenesis and chemoprevention supports the validity of this model and the potential clinical utility of SHetA2 in chemoprevention.

DIFFERENT POSITIONAL ACCUMULATION OF [1,2-13C2] GLUCOSE INTO RNA RIBOSE OF DMBA-INDUCED PANCREATIC TUMORS, PANCREAS AND LIVER.

Background: Early diagnosis of pancreatic cancer is critical to timely initiate adequate treatment in order to prolong life. Method: In this study pancreatic cancer was induced by the implantation of 1, 3 or 5 mg DMBA pellets into the head of the pancreas of male Wistar rats. After 1 month the [1,2-13 C2]glucose tracer at 1 g/kg dose was given via i.p. injection 3 hrs prior to harvest of tumors, adjacent pancreas, liver and blood serum. Gas chromatography and mass spectrometry (GC/MS) was used to determine the positional incorporation of the 13C tracer carbon of glucose into RNA ribose, palmitate, stearate and oleate of the organs, as well as serum glucose and lactate. Results: There was a dose dependent increase in total 13C tracer accumulation into RNA ribose of tumors, indicated by ∑mn values of 0.74 (placebo control), 0.83, 0.85 and 0.94* (n=5; *P<0.05; #P<0.001), respectively. Selective ion monitoring (SIM) of the doubly 13C labeled ribose species (m2), which requires non-oxidative synthesis via the tumor specific transketolase reaction of the pentose cycle, showed a 2.8*, 2.9* and a 5.7#-fold increase. Although the adjacent pancreas to tumors also showed some increase in total tracer accumulation after 3 and 5 mg DMBA treatment, there was no increase in the doubly 13C labeled ribose moiety. Liver cell positional RNA ribose labeling and total 13C tracer accumulation were not affected by DMBA carcinogenesis of the pancreas. Tumor fatty acid profiles were inconsistent, exhibiting either low or high oleate ratios. Conclusions: This study indicates that chemically induced tumors of the pancreas of Wistar rats exhibit differential 13C labeling patterns in RNA ribose from that of the adjacent pancreas and liver when [1,2-13C2] glucose is used as the tracer. This may open new avenues for the early detection and response-to-therapy testing of this cancer, using 13C based magnetic resonance imaging (MRI) and GC/MS based serum/urine tests selectively monitoring 13C sugar moieties in pancreatic tissues and body fluids simultaneously.

Skin and salivary gland carcinogenicity of 7,12-dimethylbenz[ a]anthracene is equivalent in the presence or absence of aryl hydrocarbon receptor

7,12-Dimethylbenz[ a]anthracene (DMBA) is a well-known polycyclic aromatic hydrocarbon (PAH) that causes a variety of tumors in exposed animals. Although PAH carcinogenicity is primarily mediated by the aryl hydrocarbon receptor (AhR) through induction of P450, it is not precisely determined whether AhR regulates the DMBA carcinogenesis in vivo. In this context, we examined the frequency of DMBA-induced tumors and the expressions of mRNAs of P450-CYP1 subfamily and microsomal epoxide hydrolase (mEH) in the skin and submandibular gland using AhR-deficient mice. After DMBA exposure, AhR−/− and AhR+/+ mice showed the same tumor incidences and latency. CYP1A1 was absent in these tissues but was slightly induced in DMBA-treated AhR+/+ mice. In AhR−/− and AhR+/+ mice, constitutive expression of CYP1B1 was evident at equivalent levels, whereas CYP1A2 was not detectable, irrespective of DMBA treatment. mEH was expressed in both tissues of all animals. Collectively, the constitutive levels of CYP1B1 and mEH in the skin and submandibular gland maintain DMBA response in these tissues of AhR−/− mice.

NAD(P)H:quinone oxidoreductase 1 deficiency and increased susceptibility to 7,12-dimethylbenz[a]-anthracene-induced carcinogenesis in mouse skin.

The phase II enzyme NAD(P)H :quinone oxidoreductase 1 (NQO1) catalyzes quinone detoxification, protecting cells from redox cycling, oxidative stress, mutagenicity, and cytotoxicity induced by quinones and its precursors. We have used NQO1(-/-) C57BL/6 mice to show that NQO1 protects them from skin cancer induced by the polycyclic aromatic hydrocarbon benzo[a]pyrene. Herein, we used NQO1(-/-) mice to investigate whether NQO1 also protects them against 7,12-dimethylbenz[a]anthracene (DMBA), where methyl substituents diminish primary quinone formation. Dorsal skin of NQO1(-/-) or wild-type C57BL/6 mice was shaved. When tested as a complete carcinogen, DMBA (500 or 750 microg in 100 microL of acetone) alone was applied to the shaved area. When tested as a tumor initiator, DMBA (200 or 400 nmol in 100 microL of acetone) was applied to the shaved area; 1 week later, twice-weekly applications of phorbol 12-myristate 13-acetate (PMA)-10 microg dissolved in 200 microL of acetone-to the same area began and were continued for 20 weeks. Tumor development was monitored in all mice (12-15 per group). All statistical tests were two-sided. When DMBA (750 microg) was tested as a complete carcinogen, about 50% of the DMBA-treated NQO1(-/-) mice but no DMBA-treated wild-type mouse developed skin tumors. When DMBA (both concentrations) was used as a tumor initiator, NQO1(-/-) mice developed larger tumors at a greater frequency than their wild-type littermates. Twenty-three weeks after the first PMA treatment in the tumor initiator test, all 30 NQO1(-/-) mice given 400 nmol of DMBA had developed skin tumors, compared with 33% (10 of 30) of treated wild-type mice (P<.001). NQO1(-/-) mice are more susceptible to DMBA-induced skin cancer than are their wild-type littermates, suggesting that NQO1 may protect cells from DMBA carcinogenesis.

Ras gene mutations in 7,12-dimethylbenz[ a]anthracene (DMBA)-induced rat sarcomas

Tumor induction in rats by 7,12-dimethylbenz[ a]anthracene (DMBA) will generate malignancies that display reproducible chromosomal abnormalities involving rat chromosome (RNO) 2. Thus, it has been reported that rat DMBA erythroleukemias display RNO2 abnormalities, which in this case were closely correlated to mutations in the Nras oncogene located in RNO2q34. Our cytogenetic analysis in a series of 17 DMBA-induced rat sarcomas showed that 11 (65%) tumors had a significant increase in RNO2 copy number. Furthermore, the incidence of point mutations in codons 12, 13 and 61 of Hras, Kras, and Nras was examined in the same set of sarcomas, and mutations were detected in three (18%) tumors, in codon 61 of Kras (CA A→CA T) (1 of 17) and Nras (C AA→C TA) (2 of 17). We conclude that the high frequency of RNO2 gain was in accordance with previous studies of DMBA-induced rat neoplasms, supporting the idea of a significant role of RNO2 in DMBA carcinogenesis. However, there was no clear-cut relationship between activated Nras and gain of RNO2 material, implying that mutational activation of Nras is not the causative factor underlying the gain of RNO2 copy number in rat DMBA sarcomas, in contrast to what has been suggested for DMBA-induced erythroleukemias.

Targeted Disruption of the Microsomal Epoxide Hydrolase Gene: MICROSOMAL EPOXIDE HYDROLASE IS REQUIRED FOR THE CARCINOGENIC ACTIVITY OF 7,12-DIMETHYLBENZ[a]ANTHRACENE

Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.

Dietary Selenite Modifies Glutathione Metabolism and 7,12-Dimethylbenz(a)anthracene Conjugation in Rats

The present studies determined the impact of dietary selenite on glutathione homeostasis in liver and mammary tissue and its relationship to biliary excretion of 7,12-dimethylbenz(a)anthracene (DMBA) conjugates. In Experiment 1, liver and mammary tissue concentration of reduced glutathione (GSH) and activities of gamma-glutamylcysteine synthetase (GCS), glutathione reductase (GR) and glutathione S-transferases (GST) were positively correlated with tissue selenium concentration in female rats fed semipurified diets supplemented with sodium selenite (0.05 to 4 mg Se/kg). The magnitude of the response was dependent upon total selenite intake and the tissue examined. Glutathione peroxidase activity did not correlate with tissue GSH concentration. Because both selenite and BHT have been reported to elevate liver GSH, Experiment 2 compared these agents (4 mg Se/kg and 6 g/kg BHT/kg, respectively) on the biliary excretion of DMBA metabolites. Five major biliary DMBA conjugates, three GSH and two beta-glucuronide, were identified. Dietary addition of selenite or BHT enhanced the excretion of these DMBA conjugates by over 100% during the 15-h collection period. These investigations suggest that dietary selenium can alter the concentration of GSH and the activities of three glutathione-dependent enzymes in mammary and liver, accounting for part of the expanded biliary excretion of DMBA conjugates. Enhanced biliary loss of DMBA conjugates likely relates to the reported depression in DMBA binding to mammary cell DNA and the inhibition of DMBA carcinogenesis caused by dietary selenite.

Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells.

The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulations in vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E2) metabolites on in vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E2 via the C2- and C16 alpha-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E2 metabolites. A single 24 hr exposure of 0.78 microM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 alpha hydroxylation of E2. The DMBA treated C57/MG cells also exhibited increased anchorage-independence in vitro prior to tumorigenesis in vivo. A simultaneous treatment of cells with DMBA and with the highest noncytotoxic doses of the tumor suppressing agents 5 microM N-(4-hydroxyphenyl) retinamide (HPR), 50 microM indole-3-carbinol (I3C), or 1 microM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16 alpha hydroxylation of E2, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E2 metabolites at the highest non-cytotoxic doses of 0.76 microM estrone (E1), 0.69 microM 2-hydroxyestrone (2-OHE1), and 0.66 microM 2-methoxyestrone (2-MeOHE1) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E2 metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 alpha hydroxylation in favor of C16 alpha-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E2 metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.

A possible mechanism for Sudan III-induced prevention of DMBA carcinogenesis

A possible mechanism for Sudan III-induced prevention of DMBA carcinogenesis