Exposure to asphalt fumes activates activator protein-1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in mouse epidermal cells

Occupational exposure to asphalt fumes may pose a health risk. Experimental studies using animal and in vitro models indicate that condensates from asphalt fumes are genotoxic and can promote skin tumorigenesis. Enhanced activity of activator protein-1 (AP-1) is frequently associated with the promotion of skin tumorigenesis. The current study investigated the effect of exposure to asphalt fumes on AP-1 activation in mouse JB6 P+ epidermal cells and the skin of transgenic mice expressing the AP-1 luciferase reporter gene. Asphalt fumes were generated from a dynamic generation system that simulated road-paving conditions. Exposure to asphalt fumes significantly increased AP-1 activity in JB6 P+ cells as well as in cultured keratinocytes isolated from transgenic mice expressing AP-1 reporter. In addition, topical application of asphalt fumes by painting the tail skin of mice increased AP-1 activity by 14-fold. Exposure to asphalt fumes promoted basal as well as epidermal growth factor-stimulated anchorage-independent growth of JB6 P+ cells in soft agar. It activated phosphatidylinositol 3-kinase and induced phosphorylation of Akt at Ser-473/Thr-308, and concurrently activated downstream p70 S6 kinase as well as glycogen synthase kinase-3beta. Asphalt fumes transiently activated c-Jun NH2-terminal kinases without affecting extracellular signal-regulated kinases and p38 mitogen-activated protein kinases. Further study indicated that blockage of phosphatidylinositol 3-kinase activation eliminated asphalt fume-stimulated AP-1 activation and formation of anchorage-independent colonies in soft agar. This is the first report showing that exposure to asphalt fumes can activate AP-1 and intracellular signaling that may promote skin tumorigenesis, thus providing important evidence on the potential involvement of exposure to asphalt fumes in skin carcinogenesis.     

Inhibition of AP-1 and neoplastic transformation by fresh apple peel extract

Consumption of fruits and vegetables has been associated with a low incidence of cancers and other chronic diseases. Previous studies suggested that fresh apples inhibit tumor cell proliferation. Here we report that oral administration of apple peel extracts decreased the number of nonmalignant and malignant skin tumors per mouse induced by 12-O-tetradecanolyphorbol-13-acetate (TPA) in 7,12-dimethylbenz(a)anthracene-initiated mouse skin. ESR analysis indicated that apple extract strongly scavenged hydroxyl (OH) and superoxide (O(2)(-)) radicals. Mechanistic studies showed that pretreatment with apple peel extract inhibited AP-1 transactivation induced by ultraviolet B irradiation or TPA in JB6 cells and AP-1-luciferase reporter transgenic mice. This inhibitory effect appears to be mediated by the inhibition of ERKs and JNK activity. The results provide the first evidence that an extract from fresh apple peel extract may inhibit tumor promoter-induced carcinogenesis and associated cell signaling, and suggest that the chemopreventive effects of fresh apple may be through its antioxidant properties by blocking reactive oxygen species-mediated AP-1-MAPK activation.     

Understanding the role of xenobiotic-metabolism in chemical carcinogenesis using gene knockout mice

Most chemical carcinogens require metabolic activation to electrophilic metabolites that are capable of binding to DNA and causing gene mutations. Carcinogen metabolism is carried out by large groups of xenobiotic-metabolizing enzymes that include the phase I cytochromes P450 (P450) and microsomal epoxide hydrolase, and various phase II transferase enzymes. It is extremely important to determine the role P450s play in the carcinogenesis and to establish if they are the rate limiting and critical interface between the chemical and its biological activities. The latter is essential in order to validate the use of rodent models to test safety of chemicals in humans. Since there are marked species differences in expressions and catalytic activities of the multiple P450 forms that activate carcinogens, this validation process becomes especially difficult. To address the role of P450s in whole animal carcinogenesis, mice were produced that lack the P450s known to catalyze carcinogen activation. Mouse lines having disrupted genes encoding the P450s CYP1A2, CYP2E1, and CYP1B1 were developed. Mice lacking expression of microsomal epoxide hydrolase (mEH) and NADPH-quinone oxidoreductase (NQO1) were also made. All of these mice exhibit no gross abnormal phenotypes, suggesting that the xenobiotic-metabolizing enzymes have no critical roles in mammalian development and physiological homeostasis. This explains the occurrence of polymorphisms in xenobiotic-metabolizing enzymes among humans and other mammalian species. However, these null mice do show differences in sensitivities to acute chemical toxicities, thus establishing the importance of xenobiotic metabolism in activation pathways that lead to cell death. Rodent bioassays using null mice and known genotoxic carcinogens should establish whether these enzymes are required for carcinogenesis in an intact animal model. These studies will also provide a framework for the production of transgenic mice and carcinogen bioassay protocols that may be more predictive for identifying the human carcinogens and validate the molecular epidemiological studies ongoing in humans that seek to establish a role for polymorphisms in cancer risk.     

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.     

Disruption of epidermal specific gene expression and delayed skin development in AP-2 gamma mutant mice

Summary Sentence: Conditional ablation of AP-2γ results in a delay in skin development and abnormal expression of p63, K14, K1, filaggrin, repetin and secreted Ly6/Plaur domain containing 1, key genes required for epidermal development and differentiation.The development of the epidermis, a stratified squamous epithelium, is dependent on the regulated differentiation of keratinocytes. Differentiation begins with the initiation of stratification, a process tightly controlled through proper gene expression. AP-2γ is expressed in skin and previous research suggested a pathway where p63 gene induction results in increased expression of AP-2γ, which in turn is responsible for induction of K14. This study uses a conditional gene ablation model to further explore the role of AP-2γ in skin development. Mice deficient for AP-2γ exhibited delayed expression of p63, K14, and K1, key genes required for development and differentiation of the epidermis. In addition, microarray analysis of E16.5 skin revealed delayed expression of additional late epidermal differentiation genes: filaggrin, repetin and secreted Ly6/Plaur domain containing 1, in mutant mice. The genetic delay in skin development was further confirmed by a functional delay in the formation of an epidermal barrier. These results document an important role for AP-2γ in skin development, and reveal the existence of regulatory factors that can compensate for AP-2γ in its absence.     

Role of the adrenal gland in the thermal response to morphine withdrawal in rats.

Administration of naloxone to morphine-dependent rats results in an elevation of tail skin temperature and a fall in core temperature. Previous studies have demonstrated a role of the adrenal gland in the thermal responses that accompany morphine withdrawal in the rat. In the present study, experiments were designed to determine if the duration of adrenalectomy significantly influenced the thermal response observed in morphine withdrawal. In addition we evaluated the influence of the adrenal medulla and glucocorticoid replacement in adrenalectomized rats in mediating the thermal responses of the morphine-dependent rat. Ovariectomized rats were addicted to morphine and subsequently withdrawn by administration of naloxone. This treatment results in a significant rise in tail skin temperature and subsequent fall in colonic temperature. These thermal responses were not observed in morphine-naive rats. Adrenalectomy resulted in a significant attenuation of the rise in tail skin temperature associated with withdrawal. This reduced tail skin temperature response was not different among animals adrenalectomized for 1, 7, 14, 21, or 28 days. Likewise, the moderate increase in core temperature associated with morphine treatment was not observed in the adrenalectomized rats. Serum corticosteroid determinations confirmed the loss of the adrenal steroids in the adrenalectomized rats. In a subsequent experiment it was determined that adrenal demedullation did not reduce the tail skin temperature response during morphine withdrawal, and corticosteroids restored the naloxone-induced surge in tail skin temperature in morphine-dependent, adrenalectomized rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemoprevention of precursors to colon cancer by dehydroepiandrosterone (DHEA)

Although dehydroepiandrosterone (DHEA) is recognized as one of the major adrenal androgens, its precise physiological role in the human endocrine system remains to be elucidated. In particular, the effect of DHEA on carcinogenesis has not been fully characterized. We undertook this study to determine whether DHEA has a chemopreventative effect on the precursors of colon cancer in a murine model of azoxymethane (AOM)-induced aberrant crypt foci (ACF). The number of ACF was significantly decreased in mice treated with 0.4% (p < 0.001) and 0.8% DHEA (p < 0.001), but there were no significant differences between DHEA-treated and control mice in terms of the ACF size, 3-catenin expression or level of dysplasia. This is the first study of colon cancer carcinogenesis demonstrating that DHEA treatment can decrease the number of ACF without apparently modifying their malignant potential. These data strongly suggest that DHEA might be a potential chemopreventative agent against human colon cancer.     

Protein kinase CbetaII regulates its own expression in rat intestinal epithelial cells and the colonic epithelium in vivo

Protein kinase C betaII (PKCbetaII) is induced early during colon carcinogenesis. Transgenic mice expressing elevated PKCbetaII in the colonic epithelium (transgenic PKCbetaII mice) exhibit hyperproliferation and enhanced colon carcinogenesis. Here we demonstrate that nullizygous PKCbeta (PKCbetaKO) mice are highly resistant to azoxymethane (AOM)-induced preneoplastic lesions, aberrant crypt foci. However, reexpression of PKCbetaII in the colon of PKCbetaKO mice by transgenesis restores susceptibility to AOM-induced colon carcinogenesis. Expression of human PKCbetaII in rat intestinal epithelial (RIE) cells induces expression of endogenous rat PKCbetaII mRNA and protein. Induction of PKCbetaII is dependent upon catalytically active PKCbetaII and does not appear to involve changes in alternative splicing of the PKCbeta gene. Two human PKCbeta promoter constructs are activated by expression of PKCbetaII in RIE cells. Both PKCbeta promoter activity and PKCbetaII mRNA levels are inhibited by the MEK1 and -2 inhibitor U0126, but not the Cox-2 inhibitor celecoxib in RIE/PKCbetaII cells. PKCbeta promoter activity correlates directly with expression of endogenous PKCbetaII mRNA and protein in HT29 and HCT116 human colon cancer cell lines. PKCbeta promoter activity and PKCbetaII mRNA expression in HCT116 cells are inhibited by the selective PKCbeta inhibitor LY317615 and by U0126, demonstrating autoregulation of PKCbetaII expression. Transgenic PKCbetaII mice exhibit specific induction of endogenous PKCbetaII, but not its splice variant PKCbetaI, in the colonic epithelium in vivo. Taken together, our results demonstrate that 1) expression of PKCbetaII in the colonic epithelium is both necessary and sufficient to confer susceptibility to AOM-induced colon carcinogenesis in transgenic mice, 2) PKCbetaII regulates its own expression in RIE and human colon cancer cells in vitro and in the colonic epithelium in vivo, and 3) PKCbetaII autoregulation is mediated through a MEK-dependent signaling pathway in RIE/PKCbetaII and HCT116 colon cancer cells.     

Effect of adrenalectomy on weight gain and body composition of yellow obese mice (Ay/a).

It has been reported that the adrenal gland is essential to the development of obesity if Ay/a Yellow obese mice (Hausberger and Hausberger 1960). Since the actual body composition data to support this report has not been published, we attempted to duplicate this observation by adrenalectomy of the Yellow mice before the onset of obesity. Two groups of Yellow mice (Ay/a) and normal mice (a/a) were either sham operated or adrenalectomized at two months of age and at four months of age. Body weight was monitored until body gain had stopped. At that time the animals were sacrified and checked for completeness of adrenal removal. Body composition of dry matter, fat, protein, and ash was determined. Adrenalectomy caused a reduction of body fat of 33% and 30% in both the a/a Control and the Ay/a Yellow mice, respectively. The adrenalectomized Yellow mice were still fatter than adrenalectomized Controls (25.3% fat vs. 10.6% fat). Yellow and control adrenalectomized mice showed similar depression in growth rate. These data suggest that while the adrenal gland is essential for complete expression of the genetic potential for fat deposition, it may not be necessary for partial expression indicating a secondary role in the development of obesity in the Ay/a Yellow mouse.     

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2^YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER⁺ and R26-SmoM2^YFP+ mice in this protocol); 2) inducing SmoM2^YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.     

Animal models in carotenoids research and lung cancer prevention

Numerous epidemiological studies have consistently demonstrated that individuals who eat more fruits and vegetables (which are rich in carotenoids) and who have higher serum β-carotene levels have a lower risk of cancer, especially lung cancer. However, two human intervention trials conducted in Finland and in the United States have reported contrasting results with high doses of β-carotene supplementation increasing the risk of lung cancer among smokers. The failure of these trials to demonstrate actual efficacy has resulted in the initiation of animal studies to reproduce the findings of these two studies and to elucidate the mechanisms responsible for the harmful or protective effects of carotenoids in lung carcinogenesis. Although these studies have been limited by a lack of animal models that appropriately represent human lung cancer induced by cigarette smoke, ferrets and A/J mice are currently the most widely used models for these types of studies. There are several proposed mechanisms for the protective effects of carotenoids on cigarette smoke-induced lung carcinogenesis, and these include antioxidant/prooxidant effects, modulation of retinoic acid signaling pathway and metabolism, induction of cytochrome P450, and molecular signaling involved in cell proliferation and/or apoptosis. The technical challenges associated with animal models include strain-specific and diet-specific effects, differences in the absorption and distribution of carotenoids, and differences in the interactions of carotenoids with other antioxidants. Despite the problems associated with extrapolating from animal models to humans, the understanding and development of various animal models may provide useful information regarding the protective effects of carotenoids against lung carcinogenesis.