Dose Of 7,12-dimethylbenz[a]anthracene Research Articles

Reversal of increased mammary tumorigenesis by valproic acid and hydralazine in offspring of dams fed high fat diet during pregnancy

Maternal or paternal high fat (HF) diet can modify the epigenome in germ cells and fetal somatic cells leading to an increased susceptibility among female offspring of multiple generations to develop breast cancer. We determined if combined treatment with broad spectrum DNA methyltransferase (DNMT) inhibitor hydralazine and histone deacetylase (HDAC) inhibitor valproic acid (VPA) will reverse this increased risk. C57BL/6 mouse dams were fed either a corn oil-based HF or control diet during pregnancy. Starting at age 7 weeks, female offspring were administered 3 doses of 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. After last dose, offspring started receiving VPA/hydralazine administered via drinking water: no adverse health effects were detected. VPA/hydralazine reduced mammary tumor multiplicity and lengthened tumor latency in HF offspring when compared with non-treated HF offspring. The drug combination inhibited DNMT3a protein levels and increased expression of the tumor suppressor gene Cdkn2a/p16 in mammary tumors of HF offspring. In control mice not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden, and elevated expression of the unfolded protein response and autophagy genes, including HIF-1α, NFkB, PERK, and SQSTM1/p62. Expression of these genes was already upregulated in HF offspring prior to VPA/hydralazine treatment. These findings suggest that breast cancer prevention strategies with HDAC/DNMT inhibitors need to be individually tailored.

Cyp1b1-mediated suppression of lymphoid progenitors in bone marrow by polycyclic aromatic hydrocarbons coordinately impacts spleen and thymus: a selective role for the Ah Receptor.

Bone marrow (BM) hematopoietic stem cells differentiate to common lymphoid progenitors (CLP) that emigrate to the thymus to form T cells or differentiate into immature B cells that then migrate to the spleen for maturation. Rapid in vivo suppression of BM progenitor cells by a single oral or intraperitoneal dose of 7,12‐dimethylbenz(a)anthracene (DMBA) subsequently decreased mature lymphoid populations in BM, spleen, and thymus. These suppressions depended on BM CYP1B1, but not on aryl hydrocarbon receptor (AhR) activity. Suppression of pre‐B colony formation at 6 h, correlated with subsequent decreases in mature BM, spleen, and thymus populations (48–168 h). Thymus T‐cell ratios were unaffected, suggesting low local toxicity. DMBA treatment suppressed progenitor cells 24‐h post treatment in wild type (WT), AhRb mice, but not in Cyp1b1‐ko mice. The stem cell populations were sustained. Benzo(a)pyrene (BP) mediated a similar progenitor suppression up to 6 h, but reversal rapidly ensued. This recovery was absent in mice with a polycyclic aromatic hydrocarbon (PAH)‐resistant, AhRd genotype. This AhR‐dependent progenitor recovery with BP induction accounts for the absence of suppression of B220+ BM and spleen populations at 48–168 h. However, DMBA and BP produced similar profiles for thymus cell suppression, independent of AhR genotype. Thus, lymphoid progenitors may exit the BM to the thymus prior to the BP reversal. This progenitor recovery is associated with elevated chemokines and cytokines that depend on AhR‐mediated induction of CYP1A1. This response increased constitutively in Cyp1b1‐ko BM, demonstrating that CYP1B1 metabolizes local stimulants that impact a basal progenitor protection process.

Abstract 2720: Toll-like receptor 4 immunoreactivity in mammary tumors chemically induced in female Sprague-Dawley rats

Abstract Toll-like receptors (TLRs) are essential type I transmembrane glycoproteins that play a major role in inflammation and innate immune system. TLRs have been investigated in cancer research due to their dual role in tumor progression. The TLRs signaling pathways can promote survival, cell proliferation and apoptosis and its stimulation may either enhance or inhibit tumor growth and metastasis. The role of TLR4 in cancer biology has been studied but the direct relation between TLR4 and development of many cancer types, including breast cancer, is still obscure. Thus, more studies about TLR4 expression in breast cancer can help to better understanding the involvement of TLR4 in this disease. The aim of this study was to investigate the expression of TLR4 in a rat mammary carcinogenesis model. At 51 days of age, female Sprague-Dawley rats were allocated into three groups: Groups 1 and 2 (n = 12 each) were initiated for mammary carcinogenesis by a single i.g. dose of 7,12-dimethylbenz[a]anthracene (DMBA) (80mg/kg) and Group 3 (n = 6) received a single i.g. dose of sesame oil (DMBA vehicle, 1 ml/kg). Also, group 2 received five s.c. injections per week of Tamoxifen (100 μg/kg) and groups 1 and 2 received similar injections of sesame oil (Tamoxifen vehicle, 0.5 ml/kg) during 12 weeks. At week 13, the animals were euthanized and mammary tumor (groups 1 and 2) and non-altered mammary glands (group 3) were collected and processed for histophatological analysis and TLR4 expression by immunohistochemistry. The TLR4 expression was classified as weak, moderate or strong and the incidence of intensity scores in tumor samples/group was analyzed by Fisher's exact test. Significant differences when p<0.05. Most of mammary tumors were classified as adenocarcinomas with tubular, papillary, cribriform or mixed patterns. Also, the tumors presented an expansive growth pattern with local invasive areas but without metastasis. Besides, a tumor inflammatory response was more intensively observed in G2. The immunoreactivity for TLR4 was significantly stronger in non-altered alveolar and ductal epithelial cells (G1) than in mammary tumor epithelial cells (groups 1 and 2) (p< 0.001 and p = 0.007, respectively). The TLR4 immunoreactivity was more intense in mammary tumor epithelial cells from tamoxifen-treated group than in positive control group (G1), but without a significant difference. In the tumor stroma the positivity for TLR4 was observed mainly in mononuclear inflammatory cells, showing a strong positivity for this marker in groups G1 and G2 when compared to the stromal cells from non-altered mammary tissue (G1, p = 0.028, p< 0,001). This present study show that there is a significant reduction in TLR4 expression in tumor epithelial cells when compared to the normal compartment in this rat DMBA-induced mammary carcinogenesis model. Besides, the intensity of the inflammatory response in the tumor microenvironment can alter the immunoreactivity for TLR4. Citation Format: Joyce Regina Zapaterini, Muriele Bertagna Varuzza, Nelci Antunes Moura, Maria Aparecida Marchesan Rodrigues, Luis Fernando Barbisan. Toll-like receptor 4 immunoreactivity in mammary tumors chemically induced in female Sprague-Dawley rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2720. doi:10.1158/1538-7445.AM2015-2720

Lack of promoting effect of titanium dioxide particles on chemically-induced skin carcinogenesis in rats and mice

Nano-sized titanium dioxide particles (TiO(2)) are widely used in cosmetics, sunscreens and food additives. We previously reported that topical application of non-coated rutile type TiO(2) did not exhibit a promoting effect on ultraviolet B-initiated skin carcinogenesis in rats, and that this was likely due to lack of penetration of TiO(2) into the epidermis. In the present study, we examined the promoting effect of silicone coated TiO(2 )(sTiO(2)) suspended in silicone oil and non-coated TiO(2 )(ncTiO(2)) suspended in Pentalan 408 on a two-stage skin chemical carcinogenesis model: sTiO(2) suspended in silicon oil forms smaller particles than ncTiO(2) suspended in Pentalan because of the smaller sizes of aggregates formed. The model used skin carcinogenesis-sensitive human c-Ha-ras proto-oncogene transgenic mice (rasH2) and rats (Hras128) and their wild-type counterparts and CD-1 mice to test the effects of topical application of TiO(2). Animals were initially treated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) and then with 0, 10, or 20 mg sTiO(2) (mice) or 0, 50, or 100 mg ncTiO(2) (rats). The incidence and multiplicity of skin tumors (squamous cell papilloma and carcinoma) did not increase over DMBA alone controls in skin carcinogenesis-sensitive mice or rats or wild-type animals. Analysis of rat skin indicated that sTiO(2) and ncTiO(2) did not penetrate though either healthy or damaged skin. Furthermore sTiO(2) did not penetrate an in vitro human epidermis model. Our results indicate that treatment with sTiO(2) or ncTiO(2) did not promote skin carcinogenesis in mice or rats, probably due to lack of penetration through the epidermis.

Effects of low level microwave radiation on carcinogenesis in Swiss Albino mice

This study concerns with the multiple treatment of the target site to potent carcinogen and the super imposition of low level radiofrequency and microwave radiation. Swiss albino mice (male) were used for this investigation. The study has been divided in two parts, part A: a single dose of 7,12-dimethylbenz(a)anthracene (DMBA) 100 μg/animal was applied topically on the skin of mice and were exposed to 112 MHz amplitude modulated (AM) at 16 Hz (power density 1.0 mW/cm(2), specific absorption rate (SAR) 0.75 W/kg). Similarly after a single dose of DMBA, mice were exposed to 2.45 GHz radiation (power density of 0.34 mW/cm(2), SAR, 0.1 W/kg), 2 h/day, 3 days a week for a period of 16 weeks. The two sets of experiments were carried out separately. Part B: mice were transplanted intraperitoneally (ip) with ascites 8 × 10(8) (Ehrlich-Lettre ascites, strain E) carcinoma cells per mouse. These mice were exposed to 112 MHz amplitude modulated at 16 Hz and 2.45 GHz radiation separately for a period of 14 days. There was no tumor development in mice exposed to RF and MW. Similarly a topical application of single dose of DMBA followed by RF/MW exposure also did not produce any visible extra tumor on the skin of mice. On the other hand mice were transplanted intraperitoneally with ascites (8 × 10(8) cell/ml) and subsequently exposed to above mentioned fields for 14 days showed a slight increase in the cell numbers as compared to the control group. However, the increase is insignificant. There were insignificant differences either in the mortality or cell proliferation among the control and exposed group. This results show that low level RF or MW do not alter tumor growth and development as evidenced by no observable change in tumor size.

Abstract 474: Cx43 mutant mice show delayed formation of palpable mammary gland tumors but are more susceptible to lung lesions

Abstract Gap junctional intercellular communication is essential for the regulation of cell proliferation, differentiation and tissues homeostasis. Gap junctions are composed of channels, formed by proteins called connexins (Cx). It has been proposed that connexins can act as tumor suppressors in a wide variety of tumor types. However, their role in breast cancer remains poorly understood. We previously showed that Gja1Jrt/+ (G60S) mice, harboring a G60S dominant-negative Cx43 mutation, have reduced levels of Cx43 in the mammary gland. This study aimed to determine if low levels of Cx43 render mice more susceptible to breast cancer and/or result in more aggressive tumors. G60S mice were crossbred with ErbB2 mice (or FVB control mice), that spontaneously develop mammary gland tumors. Similar to G60S mice, G60S-ErbB2 mice showed decreased Cx43 protein levels and fewer gap junction plaques, compared to control ErbB2 mice (WT-ErbB2). Mice were exposed to 5 weekly doses of 7,12-dimethylbenz(a)anthracene (DMBA) starting at 7 weeks old and sacrificed when tumors reached approximately 1 cm3 or when mice showed signs of illness. Tumor onset and progression were evaluated by palpation every week. Surprisingly, while tumors were detected in 50% of WT-ErbB2 and WT-FVB mice by 32 and 36 weeks of age, respectively, only 14% of G60S-ErbB2 and 44% of G60S-FVB mice had palpable tumors by week 42, suggesting that low levels of Cx43 delay the onset of tumors. Tumor growth rates were similar in all groups, but while tumors from WT-ErbB2 and WT-FVB mice reached the threshold of 1 cm3, most Cx43 mutant mice started to show labored breathings around 40 weeks, independent of the presence of palpable tumors. Lung examination revealed the presence of lesions in 80% of WT-FVB and 69% WT-ErbB2 mice, compared to 100% for G60S-ErbB2 and G60S-FVB mice. Moreover, Cx43 mutant mice had an average of 20 visible lung lesions compared to only 6 for WT groups. Preliminary results showed that at least some of the mutant mice present signs of mammary gland hyperplasia, suggesting that low levels of Cx43 could potentially result in more aggressive hyperplasic breast cells that metastasize to the lungs. Future experiments will determine whether lesions are of lung or breast origin. Together, our results suggest that low levels of Cx43 delayed the presence of palpable tumors in mammary gland, but increased the number of lung lesions. Supported by the CBCRA, CIHR and FRSQ. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 474.

Evaluation of Molecular Markers in a Rat Model of Mammary Carcinogenesis

We sought to evaluate the molecular markers involved in breast tumorigenesis in a rat model that mimics many essential elements of human breast cancer. Female Sprague-Dawley rats were divided into two groups. Animals in group 1 were given a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) (20 mg/rat) dissolved in 1 ml of sesame oil by intragastric intubation. Group 2 animals received basal diet and served as control. We analyzed DMBA-induced changes in the expression of CYP isoforms (CYP1A1 and 1B1) involved in DMBA metabolism, markers of oxidative stress (4HNE, HEL, and 8-OHdG), cell survival and proliferation (PCNA, NF-kappaB-p50, NF-kappaB-p65, GST-P, and p53), apoptosis (Bcl-2, Bax, caspases, Apaf-1, cytochrome C, and Fas), invasion (uPA, MMP-2, MMP-9, TIMP-2, and RECK), and angiogenesis (VEGF, VEGF-R1, HIF-1alpha, and PLGF) by immunohistochemical localization, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The present study demonstrates increased carcinogen metabolism, oxidative stress, cell proliferation, together with apoptosis evasion, invasion, metastasis, and neovascularization that may confer a selective growth advantage to DMBA-induced mammary tumors. Aberrant expression of multiple molecules in key signaling pathways in Sprague-Dawley rat mammary tumors renders this model as an important tool for monitoring carcinogenic progression and chemointervention.

Effect of fluoxetine on tolerance to the analgesic effect of morphine in mice with skin cancer

Adjuvant drugs that attenuate or inhibit the development of tolerance to morphine may lead to improved management of pain in chronic diseases such as cancer. The aim of this study was to investigate effect of fluoxetine, a specific 5-HT (5-hydroxytryptamine, serotonin) reuptake inhibitor, on tolerance induced to the analgesic effect of morphine in mice with skin cancer. The study was carried out on female Swiss albino mice. For skin tumorigensis, mice were initiated with a single dose of 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by multiple doses of croton oil. Tolerance to morphine analgesia was induced by daily subcutaneous (sc) injections of morphine (5mg/kg for 30 days) and assayed using the hot plate method. Results obtained from this study showed that pain thresholds in mice with skin cancer were significantly lower. Tolerance to the analgesic effect of morphine (5mg/kg, sc) appeared at day 15, whereas in normal and skin tumor bearing mice co-treated daily with morphine (5mg/kg, sc) and three different intraperitoneal (ip) doses of fluoxetine (0.16, 0.32 and 0.64mg/kg) tolerance was observed at days 20, 25 and 30, respectively. In conclusion, our data indicate that concurrent use of morphine with fluoxetine may produce good cancer pain control and attenuate the development of tolerance.

Inhalation of cigarette smoke induces regions of altered DNA methylation (RAMs) in SENCAR mouse lung

The development of early biomarkers, both of exposure and effect, would substantially improve science-based risk assessment with regard to cigarette smoke (CS)-associated toxicity. Altered DNA methylation, an epigenetic mechanism, is linked to CS-induced lung tumorigenesis. We have taken an unbiased approach (i.e. genomic regions are not pre-selected) to assess early methylation changes within lung DNA from female SENCAR mice treated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA), and then exposed to air alone, or CS for 4 or 8 weeks. Regions of altered DNA methylation (RAMs) were detected in mice treated with DMBA alone, or DMBA+0.16, 0.32 or 0.48mg wet total particulate matter per liter (WTPM/L) CS, using methylation-sensitive restriction digestion, arbitrarily primed PCR and capillary electrophoresis. Comparison of the RAMs that formed in different treatment groups revealed: (1) RAMs which “carried forward” across time (i.e. occurred at both 4 and 8 weeks) in a particular dose group, in addition to unique RAMs observed only at 8 weeks, and (2) RAMs which “carried forward” across dose (i.e. occurred in at least 2 dose groups at a particular time point), in addition to unique RAMs observed only in 1 dose group. Furthermore, a subset of RAMs was observed, at both 4 and 8 weeks, in DMBA-treated and DMBA+CS-exposed groups; the presence of unique RAMs in the latter suggest that combined DMBA+CS treatment more than just “magnifies” a subset of cell populations bearing the methylation changes induced by DMBA alone. Importantly, only minimal histopathological changes were observed in the lungs of CS-treated mice. This study is the first to demonstrate changes in lung DNA methylation at early times following exposure to CS, e.g., prior to overt histopathology. Thus, altered methylation might serve as a biomarker of CS exposure, and, in light of the fact that methylation changes are linked to CS-induced lung tumorigenesis, might also be useful as biomarkers of effect.

The Effects of Repeated Administration of 7,12-Dimethylbenz[a]anthracene on the Induction of Mammary Carcinomas and Dysplasias in Rats

The aim of this study was to investigate the effects of repeated administrations of relatively low doses of 7,12-dimethylbenz[a]anthracene (DMBA) on the induction of mammary carcinomas (MCs) and dysplasias (MDs) in rats. DMBA at 0, 0.1, 0.2, 0.5 and 1 mg for female rats, and at 0, 0.5, 1 and 2 mg for male rats was administered twice weekly after weaning. In female rats, repeated administration of 1 mg DMBA induced MCs while 0.1 mg DMBA induced MDs in all animals. The incidence of MCs increased and the latent period shortened while the incidence of MDs decreased in a dose-dependent manner. In male rats, repeated administration of 2 mg DMBA induced MCs in all animals. The incidence of MCs increased and the latent period shortened in a dose-dependent manner while the incidence of MDs increased by repeated administration of 1 mg DMBA. These results suggest that the total exposure of chemical carcinogens may be responsible for the induction of MCs or MDs, and that MDs may be more dependent on female sex hormones than MCs.

Suppression of mammary gland carcinogenesis by post-initiation treatment of rats with tamoxifen or indole-3-carbinol or their combination

This study examined whether suppression of mammary gland carcinogenesis elicited by low doses of tamoxifen (TAM) can be enhanced by concomitant treatment of rats with indole-3-carbinol (I3C), a component of cruciferous vegetables and a dietary supplement used for its putative antiestrogenicity. Two weeks after one oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at 65 mg/kg body weight, female Sprague-Dawley rats started treatment with TAM (10 microg/rat) by subcutaneous injection, I3C (250 mg/kg body weight) by oral gavage, TAM+I3C or their respective vehicles three times per week, for up to 20 weeks. Significant increases in the median latency of malignant mammary tumors and decreases in the mean tumor mass per rat were due to TAM. Significant decreases in the mean tumor number per rat in TAM, I3C and TAM+I3C-treated rats indicated a cooperative effect of the two compounds. In both DMBA-initiated and uninitiated rats, significant increases in the ratios of liver to body weight in I3C and TAM+I3C-treated groups coincided with I3C-dependent increases of hepatic cytochrome P450 levels and activities (1A1, 1A2 and 2B1/2). The ratios of uterus to body weight decreased with the number of treatments and the decreases effected by TAM were greater than those by I3C. The levels of circulating estrone were increased in response to I3C treatment and were greater in DMBA-initiated rats than in uninitiated rats, which may contribute to the preventive effect of I3C. Chemoprevention may be accomplished through up-regulation of apoptotic enzyme (caspase) activities in the mammary gland or mammary tumors. Treatment with TAM, I3C or TAM+I3C had no effect on caspase-3&7, caspase-6, caspase-8 and caspase-9 activities in the mammary tumors or mammary gland of tumor-bearing rats or that of uninitiated rats. In the mammary gland of DMBA-initiated tumor-free rats, however, I3C treatment increased the levels of caspase-3&7 and caspase-9 activities, suggesting an I3C-mediated protective effect. Even though I3C alone is a much less effective suppressing agent of mammary carcinogenesis than TAM, I3C in combination with TAM does not weaken but may foster the benefits of chemoprevention with TAM.

Dietary effects of soy isoflavones daidzein and genistein on 7,12-dimethylbenz[a]anthracene-induced mammary mutagenesis and carcinogenesis in ovariectomized Big Blue(R) transgenic rats

The major constituents of isoflavones, daidzein (DZ) and genistein (GE) are known to interact with the alpha and beta estrogen receptors (ERalpha/beta) in several tissues including mammary. In this study, we used ovariectomy (OVX) to model menopause and determined the effects of DZ, GE or 17beta-estradiol (E2) exposures on chemically induced mutagenesis and carcinogenesis in the mammary glands of female Big Blue (BB) transgenic rats. The rats were fed control diet containing the isoflavones and E2 and treated with a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at PND 50. Animals were sacrificed at 16 or 20 weeks post-carcinogen treatment to assess mutant frequencies (MFs) and histopathological parameters, respectively. The isoflavones or E2 supplementation alone resulted in modest increases in the lacI MF that were not significantly different from the MFs measured in rats fed the control diet alone. DMBA exposure, however, induced significant increases in the lacI MFs in the mammary of both OVX and ovary intact (INT) rats and Hprt MFs in spleen lymphocytes (P<or=0.01). In general, feeding the isoflavones or E2 separately did not cause any significant changes in DMBA-induced mutagenicity in the mammary. However, feeding the isoflavone mixture (DZG) resulted in a significant reduction in the DMBA-induced lacI MFs (P<or=0.05). Cell proliferation as measured by PCNA immunohistochemistry was increased in both OVX and INT rats exposed to DMBA as compared with rats fed control diet (P<or=0.05). Mammary histology indicated that hyperplasia was induced in most of the treatment groups including control. Although DMBA treatment did not induce mammary tumors in the OVX rats, adenoma and adenocarcinoma were detected in the mammary glands of INT rats.

Dietary effects of soy isoflavones daidzein and genistein on 7,12-dimethylbenz[a]anthracene-induced mammary mutagenesis and carcinogenesis in ovariectomized Big Blue transgenic rats

The major constituents of isoflavones daidzein (DZ) and genistein (GE) interact with the and estrogen receptors in several tissues including mammary tissues. In this study, we used ovariectomy (OVX) to model menopause and determined the effects of DZ, GE or 17beta-estradiol (E(2)) exposures on chemically induced mutagenesis and carcinogenesis in the mammary glands of female Big Blue transgenic rats. The rats were fed control diet containing the isoflavones and E(2) and treated with a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at PND50. Animals were euthanized at 16 or 20 weeks post-carcinogen treatment to assess mutant frequencies (MFs) and histopathological parameters, respectively. The isoflavones or E(2) supplementation alone resulted in the lac I MFs that were not significantly different from the MFs measured in rats fed the control diet alone. DMBA exposure, however, induced significant increases in the lac I MFs in the mammary tissues of both OVX and INT rats and Hprt MFs in spleen lymphocytes (P < 0.01). In general, feeding the isoflavones or E(2) did not cause any significant changes in DMBA-induced mutagenicity in the mammary tissues. However, feeding the isoflavone mixture (daidzein + genistein; DZG) resulted in a significant reduction in the DMBA-induced lac I MFs (P < 0.05). Cell proliferation as measured by PCNA immunohistochemistry was increased in both OVX and INT rats exposed to DMBA as compared with rats fed control diet (P < 0.05). Mammary histology indicated that hyperplasia was induced in most of the treatment groups including control. Although DMBA did not induce mammary tumors in the OVX rats, adenoma and adenocarcinoma were detected in the mammary glands of INT rats.

In situ reduction of oxidative damage, increased cell turnover, and delay of mitochondrial injury by overexpression of manganese superoxide dismutase in a multistage skin carcinogenesis model.

To study early subcellular pathologic changes of tumorigenesis in mouse skin and possible modulation by overexpression of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD), skin keratinocytes from nontransgenic (Ntg) and transgenic (TgH) mice overexpressing MnSOD topically treated with one dose of 7,12-dimethylbenz(a)anthracene (DMBA) and a subsequent dose of 12-O-tetradecanoylphorbol 13-acetate (TPA) were analyzed in situ for levels of MnSOD and the oxidative damage product 4-hydroxy-2-nonenal (4HNE)-modified proteins using specific antibodies and immunogold electron microscopy. At all selected time points analyzed after TPA treatment, there was more MnSOD immunoreactive protein in mitochondria of keratinocytes of TgH mice than Ntg mice. Compared with untreated groups, there was a large increase in 4HNE-modified proteins at 6-24 h after TPA treatment, and this increase was larger in Ntg than TgH mice. Indices of mitosis and apoptosis of keratinocytes were greater in DMBA/TPA-treated TgH than Ntg mouse skin. Mitochondrial injury detected by transmission electron microscopy was delayed in keratinocytes of TgH compared with Ntg mice. The present study demonstrated that overexpression of MnSOD not only protected cells from oxidative damage, but also affected cell turnover kinetics. Thus, previously identified reduction in papilloma formation observed in TgH mice is correlated with mitochondrial events.

Initial Changes of Hepatic Glycogen Granules and Glycogen Phosphorylase &lt;i&gt;a&lt;/i&gt; After Exposure to 7,12-dimethylbenz[&lt;i&gt;a&lt;/i&gt;]anthracene in Rats

The initial changes in rat liver after a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) (100 mg/kg b.w.) were examined ultrastructually. Glycogen granules of centrilobular hepatocytes increased with time to peak on Day 1. After this, they sharply decreased on Day 2, and on Days 5-10 returned to levels similar to those of the vehicle group (corn oil). On Day 2, the size of the centrilobular hepatocytes was decreased significantly, and the nuclear to cytoplasmic ratio was increased significantly. While the activity of hepatic glycogen phosphorylase a was decreased at 6-12 hrs and on Day 1, on Days 2-10, it was elevated to a level similar to that of the vehicle group. Thus, the initial temporary storage of hepatic glycogen granules following DMBA administration involved the inhibition of glycogenolysis in centrilobular hepatocytes. Glycogen granules appeared in restricted regions near the smooth endoplasmic reticulum (sER). Following DMBA administration, proliferation of sER of the centrilobular hepatocytes also gradually increased with time to peak on Day 2, after which it decreased, and on Days 5-10 returned to a level similar to that of the vehicle group. Results from the present study indicate that exposure of rats to DMBA could induce a reversible initial change in the hepatic glycogen metabolism. Moreover, the glycogen depletion of hepatocytes might be related to the high proliferation of sER that occurred concurrently on Day 2.

Inhibitory effects of 17beta-estradiol and 4-n-octylphenol on 7,12-dimethylbenz[a]anthracene-induced mammary tumor development in human c-Ha-ras proto-oncogene transgenic rats.

Experiments were conducted to determine whether the natural estrogen and an environmental compound with estrogenic action, 4-n-octylphenol (4nOP), could modify tumor development in human c-Ha-ras proto-oncogene transgenic (Tg) rats which are highly susceptible to mammary and skin carcinogens. Female and male Tg and non-transgenic (non-Tg) rats were given a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) (25 mg/kg body weight) at 50 days of age and thereafter subcutaneously implanted with cholesterol pellets containing 0.01, 0.1 or 1.0 mg beta-estradiol 3-benzoate (E2) per rat or received diets containing 1000 or 100 p.p.m. 4nOP for 12 weeks in females or for 20 weeks in males. E2 reduced the mammary tumor incidence and multiplicity in a dose dependent manner, especially in female Tg rats. In contrast, E2 increased mammary tumor incidence and multiplicity at the lowest dose (0.01 mg), however it reduced skin tumor induction in male Tg rats. 4nOP at a dose of 100 p.p.m. decreased mammary tumor multiplicity in female Tg rats (P < 0.001). No effects were observed in males. In separate in vitro studies, E2 at low doses (10(-11)-10(-8) M) enhanced the growth of both MCF-7 and T47D cells and this was similarly the case for 4nOP at high doses (10(-7)-10(-5) M) in T47D cells. The finding that E2 and 4nOP at high doses caused reduction in mammary tumor development in female Tg and possibly non-Tg rats, may indicate that excess estrogen can exert a paradoxical inhibitory influence. E2 also appears to have bipotential effects in males, promoting mammary, but inhibiting skin carcinogenesis. These contrasting observations may be caused by differences in background physiological estrogen levels. In addition, the results suggest that Tg rats can be used in medium-term bioassay models to test for the modifying effects of estrogenic environmental compounds on mammary tumor development.

Effect of 13 week magnetic field exposures on DMBA-initiated mammary gland carcinomas in female Sprague-Dawley rats.

Several studies suggest that exposure to 50 Hz magnetic fields may promote chemically induced breast cancer in rats. Groups of 100 female Sprague–Dawley rats were initiated with four weekly 5 mg gavage doses of 7,12-dimethylbenz[a]anthracene (DMBA) starting at 50 days of age. After the first weekly DMBA administration, exposure to ambient fields (sham exposed), 50 Hz magnetic fields at either 1 or 5 G field intensity or 60 Hz fields at 1 G for 18.5 h/day, 7 days/week was initiated. Exposure continued for 13 weeks. A vehicle control group without DMBA was included. In a second study, using lower doses of DMBA, groups of 100 female Sprague–Dawley rats were initiated with four weekly doses of 2 mg of DMBA starting at 50 days of age followed, after the first weekly DMBA administration, by exposure to ambient fields (sham exposed) or 50 Hz magnetic fields at either 1 or 5 G field intensity for 18.5 h/day, 7 days/week for 13 weeks. Rats were weighed and palpated weekly for the presence of tumors. There was no effect of magnetic field exposure on body weight gains or on the time of appearance of mammary tumors in either study. At the end of 13 weeks, the animals were killed and the mammary tumors counted and measured. Mammary gland masses found grossly were examined histologically. In the first 13 week study, the mammary gland carcinoma incidences were 92, 86, 96 and 96% for the DMBA controls, 1 G, 50 Hz, 5 G, 50 Hz and 1 G, 60 Hz groups, respectively. The total numbers of carcinomas were 691, 528 (P < 0.05, decrease), 561 and 692 for the DMBA controls, 1 G, 50 Hz, 5 G, 50 Hz and 1 G, 60 Hz groups, respectively. In study 2, the mammary gland carcinoma incidences were 43, 48 and 38% for the DMBA controls, 1 G, 50 Hz and 5 G, 50 Hz groups, respectively. The total numbers of carcinomas were 102, 90 and 79 for the DMBA controls, 1 G, 50 Hz and 5 G, 50 Hz groups, respectively. There was no effect of magnetic field exposure on tumor size either by in-life palpation or by measurement at necropsy in either study. There was no evidence that 50 or 60 Hz magnetic fields promoted breast cancer in these studies in female rats. These studies do not support the hypothesis that magnetic field exposure promotes breast cancer in this DMBA rat model.

Effect of 26 week magnetic field exposures in a DMBA initiation-promotion mammary gland model in Sprague-Dawley rats.

Several studies have suggested that exposure to 50 Hz magnetic fields promote chemically induced breast cancer in rats. Groups of 100 female Sprague–Dawley rats were initiated with a single 10 mg gavage dose of 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age followed by exposure to ambient fields (sham exposed), 50 Hz magnetic fields at either 1 or 5 Gauss (G) field intensity or 60 Hz fields at 1 G for 18.5 h/day, 7 days/week for 26 weeks. A vehicle control group without DMBA was included. Rats were palpated weekly for the presence of tumors. There was no effect of magnetic field exposure on body weight gains or the time of appearance of mammary tumors. At the end of 26 weeks, the animals were killed and the mammary tumors counted and measured. Mammary gland masses found grossly were examined histologically. The mammary gland carcinoma incidence was 96, 90, 95 and 85% (P < 0.05, decrease) for the DMBA controls, 1 G 50 Hz, 5 G 50 Hz and 1 G 60 Hz groups, respectively. The total numbers of carcinomas were 649, 494 (P < 0.05, decrease), 547 and 433 (P < 0.05, decrease) for the DMBA controls, 1 G 50 Hz, 5 G 50 Hz and 1 G 60 Hz groups, respectively. The number of fibroadenomas varied from 276 to 319, with the lowest number in the 1 G 60 Hz exposure group. Measurement of the tumors revealed no difference in tumor size between groups. In this breast cancer initiation–promotion study in female Sprague–Dawley rats, there was no evidence that 50 or 60 Hz magnetic fields promoted breast cancer under the conditions of this assay. This study does not support the hypothesis that magnetic field exposure can promote breast cancer in this rat model.

Further studies on the influence of initiation dose on papilloma growth and progression during two-stage carcinogenesis in SENCAR mice.

The present study was designed to further evaluate the growth and progression of papillomas to squamous cell carcinomas (SCCs) in groups of animals receiving initiating doses of 7,12-dimethylbenz[a]anthracene (DMBA) producing relatively low papilloma yields following long term promotion (60 weeks) with 12-O-tetradecanoylphorbol-13-acetate (TPA). For comparison, groups of animals were initiated with various doses of DMBA and then promoted with mezerein (MEZ), benzoyl peroxide (BzPo) and chrysarobin (CHRY). Following initiation, groups of female SENCAR mice received the following promoter doses: TPA (1.0 or 2.0 micrograms per mouse); MEZ (2.0 micrograms per mouse); BzPo (20.0 mg per mouse); and CHRY (52.8 micrograms per mouse). The maximum papilloma to SCC conversion ratio obtained with TPA in the current study was 0.32. This value was in the range of maximum conversion ratios obtained with the other compounds: MEZ, 0.40; CHRY, 0.32 and BzPo, 0.19. In general, the highest papilloma to SCC conversion ratios observed with TPA as the promoter were obtained in groups that received the lowest doses of DMBA and had relatively low papilloma burdens. A comparison of papilloma to SCC conversion in groups of mice promoted with TPA, MEZ or CHRY and having similar papilloma yields, revealed very similar conversion ratios. Comparison of the BzPo group with a similar papilloma yield indicated that the conversion ratio was slightly lower with this promoter. The present results indicate that in mice promoted with TPA and having relatively low papilloma numbers, a larger proportion of these papillomas progress to SCCs during continued promoter treatment. Furthermore, the results suggest that papillomas behave similarly in their ability to progress to SCCs regardless of the promoter used when comparing groups of mice with similar tumor numbers. The data are discussed in terms of possible mechanisms for the observed results.

Potent inhibitory effects of suicide inhibitors of P450 isozyines on 7, 12-dimethylbenzℵaℵanthracene and benzoℵaℵpyrene initiated skin tumors

A single dose of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) or 2-ethynylnaphthalene (EN) was applied to the skin of SENCAR mice 5 min before an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P) and the development of skin tumors then promoted with biweekly topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). The application of EP strongly inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner. Application of 44 pmol of EP inhibited tumor initiation by 10 nmol of DMBA approximately 25%; application of 440 nmol of EP inhibited tumor initiation by 200 nmol of B[a]P approximately 51%. A high single dose of EP (4.4-44 mumol) nearly eliminated skin tumor initiation by either 10 nmol of DMBA or 200 nmol of B[a]P. Application of VP also inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner, but higher doses of VP than of EP were required to produce comparable inhibitions. Application of 44 nmol of VP inhibited tumor initiation by 10 nmol of DMBA approximately 30%; application of 4.4 mumol of VP inhibited tumor initiation by 200 nmol of B[a]P approximately 56%. Application of EN yielded contrasting results. EN inhibited the formation of skin tumors initiated by 10 nmol of DMBA, but the observed dose-dependence was minimal; tumors were decreased about 40% by 3.3 mumol of EN and only about 65% by 132 mumol of EN. A high single dose of EN (132 mumol) increased both the mean number of tumors per mouse and the percentage of mice that developed tumors after initiation by 200 nmol of B[a]P. Topical application of 4.4 mumol of EP, 22 mumol of VP or 33 mumol of EN to the skin of SENCAR mice 5 min before a single initiation dose of 2.5 mumol of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had a minimal inhibitory effect (14-28%) on the development of skin tumors produced by subsequent biweekly promotion with TPA. A single dose of 44 mumol of EP or 132 mumol of EN followed by biweekly applications of TPA did not produce skin tumors; however, a dose of 44 mumol of VP followed by promotion with TPA produced a low but significant number of skin tumors.(ABSTRACT TRUNCATED AT 400 WORDS)