SENCAR Mouse Skin Research Articles

Modulation of biomarkers related to tumor initiation and promotion in mouse skin by a natural β-glucuronidase inhibitor and its precursors

Carcinogen-mediated labilization of lysosomal enzymes such as β-glucuronidase (βG) is often associated with the general process of inflammation. Therefore, the primary goal of this study was to demonstrate that exposing the skin of SENCAR mice to the natural βG inhibitor D-glucaro-1,4-lactone (1,4-GL) and its precursor D-glucuronic acid-γ-lactone (GUL), prior to and during 7,12-dimethylbenz[α]anthracene (DMBA) treatment inhibits not only epidermal hyperplasia but also inflammation in the mouse skin complete carcinogenesis model, i.e., the 4-week inflammatory-hyperplasia assay. Topical administration of 1,4-GL or GUL prior to repetitive, high-dose DMBA treatment markedly and in a dose-related manner inhibited DMBA-induced epidermal hyperplasia (i.e., up to 57%). DMBA-mediated Ha-ras mutations in codon61 were reduced by up to 78% by 1,4-GL. DMBA-induced inflammation, as measured by dermal leukocyte counts and immunologically, was inhibited by up to 37% by topical 1,4-GL but not by GUL. The inhibition of cellular proliferation and inflammation coincided with the inhibition of βG expression. Thus, the present study suggests that in the DMBA-induced complete skin carcinogenesis model, 1,4-GL when applied topically had both anti-proliferative properties as well as anti-inflammatory properties, whereas GUL had only anti-proliferative when applied topically. However, the number of inflammatory cells in the dermal portion of the skin of mice was significantly reduced by dietary treatment of GUL, whereas both topical and dietary treatments with 1,4-GL were very effective.

Abstract 2477: The effect of dissociated glucocorticoids on 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin inflammation in SENCAR mice

Abstract Glucocorticoids are very effective in preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation when applied to skin together with a carcinogen. Their control of cellular functions is mediated by a well-known transcription factor, glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding and protein-protein interaction between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1), which is independent of DNA binding. We hypothesize that only the transrepression activities of the GR are sufficient to suppress skin tumor development. To test our hypothesis, we have synthesized two glucocorticoids (RU24858 and RU24782) that induce only transrepression activities of the GR. These two compounds are able to bind to the GR with high affinity and repress the AP-1 and NF-κB activity while showing reduced transactivation activities in vitro. Different doses (0.001, 0.01 and 0.1 mg) of RU24858, RU24782, desoxymethasone (DES) and fluocinolone acetonide (FA) or acetone were applied to the dorsal skin of female SENCAR mice 20 min. prior to TPA application (2 μg), two times per week for two weeks. Keratinocytes proliferation rates were estimated by measuring epidermis thickness and BrdU incorporation index. Immunohistochemistry and RT-PCR techniques were also used to evaluate expression level of genes involved in inflammation and cell cycle regulation. All studied compounds did not influence keratinocytes proliferation when applied alone. Application of DES, FA and RU24858 reversed TPA induced hyperplasia, in dose-depended manner, while RU24782 treatment had no effect on epidermis thickness or the BrdU incorporation index in TPA treated skins. All tested compounds were able to decrease c-jun mRNA level both in acetone and TPA-treated skins. DES, FA and RU24858 were also able to decrease elevated by TPA mRNA level of IL-6, COX-2 and iNOS. RU24782 did not influence mRNAs levels of these genes. These findings suggest that RU24782 had no effect on TPA-induced inflammation and hyperplasia in SENCAR mice, while RU24858 was able to reduce all tested inflammation markers, having only slight effect on epidermal hyperplasia. Supported by NIH grants CA 0796065-06 and P30 CA 54174-16S1. 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 2477.

Dietary energy restriction, in part through glucocorticoid hormones, mediates the impact of 12‐O‐tetradecanoylphorbol‐13‐acetate on jun D and fra‐1 in sencar mouse epidermis

Dietary energy restriction (DER, 40% calorie reduction from fat and carbohydrate) inhibited mouse skin carcinogenesis and decreased 12-O-tetradecanoyl-13-phorbol acetate (TPA)-induced activator protein-1 (AP-1):DNA binding previously. This study measured protein levels of c-jun, jun B, jun D, c-fos, fra-1, and fra-2 and examined their contribution to AP-1:DNA binding by electrophoretic mobility shift assay (EMSA) with supershift analysis in the epidermis of control and DER Sencar mice exposed to TPA. TPA significantly increased c-jun, jun B, c-fos, fra-1, and fra-2 and decreased jun D within 3-6 h after treatment. AP-1:DNA binding reached a maximum 2.5-fold induction over controls 4 h after TPA treatment and antibodies to jun B, jun D, and fra-2 in the EMSA binding reaction resulted in supershifts in both acetone- and TPA-treated mice 1-6 h after treatment. The effect of corticosterone (CCS) and DER on the AP-1 proteins and on the composition of the AP-1:DNA complex was measured in adrenalectomized (adx) mice. DER reduced the TPA impact on jun D and enhanced the induction of fra-1. In addition, CCS-supplemented groups had significantly lower jun D and higher fra-2 than adx groups and sham groups. While sham animals treated with either acetone or TPA contained jun B, jun D, and fra-2 proteins in the AP-1:DNA complex by supershift analysis, fra-2 was no longer seen in adx DER animals. In summary, our study supports potential roles for jun D, jun B, and fra-1 in the DER regulation of AP-1 function in the Sencar mouse skin carcinogenesis model.

Biological effects of brachytherapy using a 32P-patch on the skin of Sencar mice

In recent years, specially designed patches containing beta emitters have been developed for contact brachytherapy of skin lesions. The aim of the present work was to evaluate the biological effects of the 32P-patch on the skin of Sencar mice as a result of a brachytherapy treatment. For this purpose, a 32P-patch was prepared with Chromic 32P-phosphate and silicone and the classical model of two-stage skin carcinogenesis was reproduced in Sencar mice. Animals were divided in six groups. Four groups received the contact brachytherapy treatments using a scheme of a single session of 40 and 60 Gy (SD40 and SD60) and a scheme of two sessions of 40 and 60 Gy each (FD40 and FD60). The other two groups were used as controls of the single (CSD) and the fractionated (CFD) treatments. Radiation doses were estimated with equations derived from the MIRD DOSE scheme, and biologically effective doses (BED) were calculated according to equations derived from the linear-quadratic model. The endpoint to evaluate the treatments effects was tumor size after a follow-up period of 44 days. Finally, animals were sacrificed in order to get samples of all tumors for histological analysis and PCNA staining. Erythema, dermatitis and skin ulceration developed in almost all treated animals, but they gradually healed with regeneration of tissue during the follow-up period. Radiation effects on the skin of SD40, SD60, FD40 and FD60 showed a significant reduction of the tumor size with regard to controls, independently of the scheme and the radiation dose considered. PCNA staining scores of control groups were higher than for treated groups, independently of the scheme and the radiation dose considered. This radioactive 32P-silicone-patch which is easy to prepare and use in the treatment of skin diseases, seems promising as a radioactive device for clinical use.

Guggulsterone modulates MAPK and NF-kappaB pathways and inhibits skin tumorigenesis in SENCAR mice.

Guggulsterone (GUG), a resin of the Commiphora mukul tree, has been used in ayurvedic medicine for centuries to treat a variety of ailments. Recent studies have suggested that GUG may also possess anticancer effects. In the present study, we show that GUG possesses antitumor-promoting effects in SENCAR mouse skin tumorigenesis model. We first determined the effect of topical application of GUG to mice against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of GUG (1.6 micromol per mouse) 30 min prior to TPA (3.2 nmol per mouse) application onto the skin of mice afforded significant inhibition against TPA-mediated increase in skin edema and hyperplasia. Topical application of GUG was also found to result in substantial inhibition against TPA-induced epidermal (i) ornithine decarboxylase (ODC) activity; (ii) ODC, cyclooxygenase-2 and inducible nitric oxide synthase protein expressions; (iii) phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinases and p38; (iv) activation of NF-kappaB/p65 and IKK alpha/beta and (v) phosphorylation and degradation of I kappaB alpha. We next assessed the effect of topically applied GUG on TPA-induced skin tumor promotion in 7,12-dimethyl benz[a]anthracene-initiated mice. Compared with non-GUG-pretreated mice, animals pretreated with GUG showed significantly reduced tumor incidence, lower tumor body burden and a significant delay in the latency period for tumor appearance from 5 to 11 weeks. These results provide the first evidence that GUG possesses anti-skin tumor-promoting effects in SENCAR mice and inhibits conventional as well as novel biomarkers of tumor promotion. In summary, GUG could be useful for delaying tumor growth in humans.

SENCAR Mouse Skin Papillomas Induced by 6-Methylbenzo[a]pyrene Carry H-ras Codon 52 (CTALeu → CCAPro) and Codon 13 (GGCGly → GTCVal) Mutations

Organic smoke contains various methylated polycyclic aromatic hydrocarbon (PAH) carcinogens. Among these, 6-methylbenzo[a]pyrene (6-CH3BP) is a moderate carcinogen. In vitro, it can form both stable and depurinating adducts. Of these, only two depurinating adducts [BP-6-CH2-N7G and 6-CH2BP-(1&3)-N7G] have been detected in mouse skin. We examined seven 6-CH3BP-induced SENCAR mouse skin papillomas for the presence of oncogenic H-ras mutations. Two papillomas contained the codon 52 (CTALeu → CCAPro) and a third contained the codon 13 (GGCGly → GTCVal) mutation. These results suggest that the H-ras codon 52 mutation is an oncogenic mutation.

DBA/2 mouse skin is unresponsive to dermal tumor promotion by cigarette smoke condensate

Previous studies demonstrated that repetitive application of cigarette smoke condensate (CSC) to 7,12-dimethylbenz[ a]anthracene (DMBA)-initiated SENCAR mouse skin for 29 weeks at doses of 10, 20 and 40 mg “tar”/application results in time- and dose-dependent dermal tumor formation. To evaluate CSC-induced tumor promotion in other mouse skin models, male DBA/2 mice were treated with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) (300 μg) or DMBA (75 or 150 μg) followed by promotion with 1R4F CSC at concentrations ranging from 9 to 45 mg “tar”/application. Both MNNG and DMBA have previously been shown to adequately initiate tumor development. Study end-points included clinical signs, body weights, and mass tracking. Neither the DMBA-initiated/acetone-promoted control groups, nor DMBA-initiated/CSC-promoted groups produced grossly observable skin tumors. For MNNG-initiated groups, a total of four tumors were observed. Based on these findings, it would appear the DBA/2 mouse was unresponsive to CSC dermal tumor promotion. It is not possible, based on the study design employed, to determine the underlying basis for the apparent resistance exhibited by this mouse strain to CSC-induced tumor promotion.

Harvey-ras Gene Expression and Epidermal Cell Proliferation in Dibenzo[a,l]Pyrene-Treated Early Preneoplastic SENCAR Mouse Skin

Topical application of dibenzo[a,l]pyrene (DB[a,l]P) to the dorsal skin of SENCAR mice induces codon 61 (CAA Gln to CTA Leu) mutations in the Harvey (H)-ras gene within 12 h after treatment. Between days 1 and 3, the frequency of these mutations increases rapidly, suggesting that skin cells carrying the codon 61 mutations proliferate in this period. We have investigated DB[a,l]P-treated mouse skin (12 h-7 d) for further evidence of H-ras expression and epidermal cell proliferation. Two waves of cell proliferation were observed: the first wave (1-2 d) correlated with the clonal proliferation of codon 61-mutated cells, and the second wave (3-7 d) correlated with DB[a,l]P-induced hyperplasia. DB[a,l]P-induced early preneoplastic cell proliferation correlated with H-ras and specific G1 cyclin expression. Total H-ras protein and cyclin D1 were found to increase during DB[a,l]P-induced hyperplasia, but the levels of guanosine triphosphate-bound (active) H-ras protein and cyclin E were increased during the putative clonal proliferation of codon 61-mutated cells. These results suggest that DB[a,l]P-induced oncogenically mutated cells proliferate in early preneoplastic skin. As this proliferation occurs in the absence of any promoting treatment, we propose that this phenomenon is a tumor initiation event.

Mitogen‐regulated protein/proliferin mRNA induction following single applications of tumor promoters to murine skin

Mitogen-regulated protein/proliferin (mrp/plf) gene family transcripts rise in abundance as a response to diverse chemical and physical agents that promote morphological transformation in the murine C3H/10T1/2 cultured cell model of multi-step carcinogenesis. To determine if proliferin genes respond to tumor promoters in vivo, RNA was extracted from the whole skin of SENCAR mice after single applications of 2 or 20 microg 12-O-tetradecanoylphorbol-13-acetate (TPA); 3.2 or 32 nmole), 20 or 40 mg benzoyl peroxide (BPO; 83, 165 micromole), or acetone vehicle alone (2.72 mmole). RNA samples were prepared from treated skin areas, 2-48 h after painting. Mrp/plf-mRNA was not detected in Northern blot hybridizations, but large increases in mRNAs for ornithine decarboxylase gene and mRNA (odc), v-jun oncogene-related transcription factor gene and mRNA (junB), egr1 (early growth response protein gene and mRNA) were measured relative to beta 2 microglobulin gene and mRNA (b2m) mRNA in response to TPA. BPO induced small relative changes in these mRNAs. Reverse transcriptase (RT)-polymerase chain reactions (PCR) detected fully-processed MRP/plf-mRNA 16-48 h after TPA treatments in five of six animals, and in three of six BPO-treated animals. The MRP/plf-mRNA species expressed in the skin were predominantly plf1 and mrp3 as determined by gene-specific restriction enzyme sites within the RT-PCR products. Expression was either undetectable or found at low levels in acetone-painted controls and was not detected during the anagen phase of the normal hair growth cycle in unpainted animals. These results demonstrate that mrp/plf-mRNA is differentially expressed in murine skin in response to mechanistically distinct tumor promoters and has potential utility as a short-term biomarker for tumor promoting effects in chemical carcinogenesis.

Short-term in vitro and in vivo analyses for assessing the tumor-promoting potentials of cigarette smoke condensates.

Previous studies found that repeated application of smoke condensate from tobacco-burning reference cigarettes to chemically initiated SENCAR mouse skin promoted the development of tumors in a statistically significant and dose-dependent manner, while condensate from prototype cigarettes that primarily heat tobacco promoted statistically fewer tumors. Based on the recognized correlation between sustained, potentiated epidermal hyperplasia and tumor promotion, we conducted tests to examine the utility of selected short-term analyses for discriminating between condensates exhibiting significantly different promotion activities. In vitro analyses assessing the potential for inducing cytotoxicity (ATP bioluminescence) or free radical production (cytochrome c reduction, salicylate trapping) demonstrated significant reductions when comparing condensate collected from prototype cigarettes to reference condensate. Short-term in vivo analyses conducted within the context of a mouse skin, tumor-promotion protocol (i.e., comparative measures of epidermal thickness, proliferative index, myeloperoxidase activity, leukocyte invasion, mutation of Ha-ras, and formation of modified DNA bases) provided similar results. Reference condensate induced statistically significant and dose-dependent increases (relative to vehicle control) for nearly all indices examined, while prototype condensate possessed a significantly reduced potential for inducing changes that we regarded as consistent with sustained epidermal hyperplasia and/or inflammation. Collectively, these data support the contention that selected short-term analyses associated with sustained hyperplasia and/or inflammation are capable of discriminating between smoke condensates with dissimilar tumor-promotion potentials. Moreover, our results suggest that comparative measures of proliferative index and myeloperoxidase activity, both possessing favorable correlation coefficients relative to tumor formation (i.e., > or = 0.95 after 8 or 12 weeks of promotion), may constitute reasonable end points for further investigation.

Pro/antioxidant status in murine skin following topical exposure to cumene hydroperoxide throughout the ontogeny of skin cancer.

Organic peroxides used in the chemical and pharmaceutical industries have a reputation for being potent skin tumor promoters and inducers of epidermal hyperplasia. Their ability to trigger free radical generation is critical for their carcinogenic properties. Short-term in vivo exposure of mouse skin to cumene hydroperoxide (Cum-OOH) causes severe oxidative stress and formation of spin-trapped radical adducts. The present study was designed to determine the effectiveness of Cum-OOH compared to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the induction of tumor promotion in the mouse skin, to identify the involvement of cyclooxygenase-2 (COX-2) in oxidative metabolism of Cum-OOH in keratinocytes, and to evaluate morphological changes and outcomes of oxidative stress in skin of SENCAR mice throughout a two-stage carcinogenesis protocol. Dimethyl-benz[a]anthracene (DMBA)-initiated mice were treated with Cum-OOH (32.8 micro mol) or TPA (8.5 nmol) twice weekly for 20 weeks to promote papilloma formation. Skin carcinoma formed only in DMBA/Cum-OOH-exposed mice. Higher levels of oxidative stress and inflammation (as indicated by the accumulation of peroxidative products, antioxidant depletion, and edema formation) were evident in the DMBA/Cum-OOH group compared to DMBA/TPA treated mice. Exposure of keratinocytes (HaCaT) to Cum-OOH for 18 h resulted in expression of COX-2 and increased levels of PGE(2). Inhibitors of COX-2 efficiently suppressed oxidative stress and enzyme expression in the cells treated with Cum-OOH. These results suggest that COX-2-dependent oxidative metabolism is at least partially involved in Cum-OOH-induced inflammatory responses and thus tumor promotion.

Progressive alterations in global and GC-rich DNA methylation during tumorigenesis.

DNA methylation plays a key role in the regulation of gene expression, and failure to maintain normal patterns of methylation often contributes to carcinogenesis. We have characterized progressive methylation changes during the promotion stage of carcinogenesis using a SENCAR mouse skin initiation/promotion tumorigenesis model. Mice were initiated with a dermal application of 75 microg dimethylbenz[a]anthracene (DMBA) and promoted with 9, 18, 27, and 36 mg cigarette smoke condensate (CSC) thrice weekly for time periods up to 29 weeks, when a large increase in tumor number was produced by the highest three doses. Global and GC-specific methylation were assessed using SssI methylase and arbitrarily primed PCR, respectively. Changes in GC-specific methylation were dose- and time-dependent. CSC doses required to detect these changes were 27 mg at 6 weeks and 18 mg at 9 weeks. This effect appears to be reversible; changes in GC-specific methylation were less marked after 9 weeks promotion with 27 mg CSC followed by 6 weeks of recovery in comparison to 9 and 15 weeks promotion with 27 mg CSC and no recovery period. Both tumor and non-tumor tissue promoted with 27 mg CSC for 29 weeks exhibited changes in GC-specific methylation that were more pronounced in tumors. Tumor tissue was globally hypomethylated, whereas non-tumor tissue did not exhibit changes in global methylation. In conclusion, as expected for a mechanism underlying tumor promotion, CSC alters methylation in a threshold-exhibiting, reversible, progressive fashion during promotion. Progressive alterations in global and GC-rich methylation appear to be mechanistically important during tumor promotion.

Dietary Lutein/Zeaxanthin Decreases Ultraviolet B-Induced Epidermal Hyperproliferation and Acute Inflammation in Hairless Mice

Lutein and zeaxanthin are carotenoids found in green leafy vegetables with interesting antioxidant properties. They are present in high concentrations in the fovea centralis of the human retina and their role in the prevention of age-related macula degeneration has been reported. We have investigated the effect of orally administered lutein and zeaxanthin in the cutaneous response to ultraviolet B irradiation. Female hairless SKh-1 mice receiving 0.4% and 0.04% lutein plus zeaxanthin-enriched diet for 2 wk were exposed to single doses of ultraviolet B radiation. Skin biopsies were taken at 24 and 48 h after irradiation and analyzed for the presence of apoptotic cells, proliferating cells, and expression of proliferating cell nuclear antigen. Our results show a clear ultraviolet-induced dose-dependent inflammatory response. Orally administered 0.4% lutein and zeaxanthin decreased significantly the edematous cutaneous response (p<0.01) as determined by the reduction of the UVB-induced increase of ear bifold thickening. Additionally, dietary carotenoids were efficient in reducing the ultraviolet B-induced increases in the percentage of proliferating cell nuclear antigen (p<0.05), bromodeoxyuridine (p<0.05), and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive cells (p<0.01). These data demonstrate that oral supplementation of lutein and zeaxanthin diminishes the effects of ultraviolet B irradiation by reducing acute inflammatory responses and ultraviolet-induced hyperproliferative rebound.

Genetic analyses of mouse skin tumor progression susceptibility using SENCAR inbred derived strains.

Susceptibility to tumor development varies among individuals in the human population. This variability can also be found among different strains of mice, particularly in the mouse skin chemical carcinogenesis model. The genetic mechanisms underlying mouse skin tumor susceptibility are not fully understood. The SENCAR stock has been found to be the most sensitive mice for skin carcinogenesis studies; however, little is known about the genes underlying tumor susceptibility, particularly, those involved in tumor progression. Experiments with the SSIN/Sprd mice, an inbred strain derived from the outbred SENCAR stock, suggested that papilloma development, tumor promotion, and their conversion into squamous cell carcinomas (SCCs), progression, are regulated by different genes. In the highly sensitive SSIN/Sprd mice, papillomas rarely progress to SCC. Using crosses between the outbred SENCAR and the SSIN/Sprd mice, we previously determined that papilloma progression in the SENCAR stock could be controlled by at least one autosomal dominant gene. However, the outbred nature of the SENCAR stock precluded us from extending those findings. More recently, another inbred strain was developed from the outbred SENCAR stock, the SENCARB/Pt. These mice have similar tumor promotion sensitivity to the SSIN/Sprd but in contrast, have high papilloma progression susceptibility, similar to the outbred original stock. In the present study, we generated F(1), F(2), and backcross hybrids between the SSIN/Sprd and SENCARB/Pt mice to determine a possible model for tumor progression susceptibility and to map the putative tumor susceptibility genes. Our tumor data suggests that papilloma progression susceptibility in the SENCAR mouse skin model could be genetically determined by one susceptibility gene. Our preliminary linkage analysis failed to identify one strong susceptibility locus to confirm this but provided some evidence for at least one possible susceptibility locus in mouse chromosome 14.

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E(2)-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E(2)-3,4-Q formed predominantly (> or =99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct and the slower-depurinating 4-OHE(2)-1-N7Gua adduct. Between 6 h and 3 days, E(2)-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3'-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E(2)-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.

Avicins, a family of triterpenoid saponins from Acacia victoriae (Bentham), suppress H-ras mutations and aneuploidy in a murine skin carcinogenesis model.

We tested the ability of avicins, a family of triterpenoid saponins obtained from Acacia victoriae (Bentham) (Leguminosae: Mimosoideae), to inhibit chemically induced mouse skin carcinogenesis. Varying doses of avicins were applied to shaved dorsal skin of SENCAR mice 15 min before application of 100 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) twice a week for 4 weeks (complete carcinogenesis model). The dorsal skin of a second group of mice was treated with one dose of 10 nmol of DMBA. Avicins were then applied 15 min before repetitive doses of 2 microg of phorbol 12-tetradecanoate 13-acetate (TPA) twice a week for 8 weeks (initiation/promotion model). At 12 weeks, avicins produced a 70% decrease in the number of mice with papillomas and a greater than 90% reduction in the number of papillomas per mouse in both protocols. We also observed a 62% and 74% reduction by avicins in H-ras mutations at codon 61 in the DMBA and DMBA/TPA models, respectively, as well as a significant inhibition of the modified DNA base formation (8-OH-dG) in both protocols. Marked suppression of aneuploidy occurred with treatment at 16 weeks in the initiation/promotion experiment. These findings, when combined with the proapoptotic property of these compounds and their ability to inhibit hydrogen peroxide (H(2)O(2)) generation, nuclear factor-kappaB (NF-kappaB) activation, and inducible nitric oxide synthase (iNOS) induction reported elsewhere, suggest that avicins could prove exciting in reducing oxidative and nitrosative stress and thereby suppressing the development of human skin cancer and other epithelial malignancies.

Inhibition of phorbol ester-induced AP-1-DNA binding, c-Jun protein and c-jun mRNA by dietary energy restriction is reversed by adrenalectomy in SENCAR mouse epidermis.

The aim of this study was to determine the effects of 40% dietary energy restriction (DER) relative to ad libitum feeding on AP-1-DNA binding and expression of c-Jun protein and c-jun mRNA in SENCAR mouse skin treated with acetone or 12-O-tetradecanoylphorbol 13-acetate (TPA). The role of the glucocorticoid hormone corticosterone (CCS) was investigated by adding CCS or vehicle control to the drinking water of adrenalectomized mice. AP-1-DNA binding, measured by electrophoretic mobility shift assay, showed that TPA treatment for 4 h increased AP-1-DNA binding by 2-fold over acetone controls (P < 0.05) and that DER reduced basal and TPA-induced AP-1-DNA binding in comparison with ad libitum fed groups in sham-operated mice (P < 0.05). TPA treatment increased c-Jun protein levels in control fed mice (4-fold) and in DER mice (2-fold) over basal levels 4 h post-treatment (P < 0.05). Analyzed over all groups, DER reduced c-Jun protein levels (P < 0.01) and this effect was reversed by adrenalectomy. TPA induction of c-jun mRNA was also reduced by DER compared with ad libitum fed mice (P < 0.05). Adrenalectomy and CCS supplementation demonstrated that the effects of DER on AP-1-DNA binding were mediated in part by CCS. Measurement of blood plasma CCS concentrations showed that: (i) DER increased CCS 5-fold over ad libitum fed mice in sham-operated animals (P < 0.05); (ii) adrenalectomy decreased CCS over sham-operated mice (P < 0.05); (iii) TPA treatment had no effect on CCS. Blood plasma IGF-I concentrations were unaffected by CCS modulation or TPA treatment but were decreased by DER compared with ad libitum fed mice (P < 0.05). Thus, dietary energy restriction may inhibit cancer mechanistically by reducing overall AP-1 transcription through a process that is mediated in part by glucocorticoid hormones.

Inhibitory effect of a flavonoid antioxidant silymarin on benzoyl peroxide-induced tumor promotion, oxidative stress and inflammatory responses in SENCAR mouse skin.

In this communication, we investigate the preventive effect of a flavonoid antioxidant, silymarin, on free radical-generating skin tumor promoting agent benzoyl peroxide (BPO)-induced tumor promotion, oxidative stress and inflammatory responses in SENCAR mouse skin. Topical application of silymarin at a dose of 6 mg prior to BPO resulted in a highly significant protection against BPO-induced tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated SENCAR mouse skin. The preventive effect of silymarin was evident in terms of a 70% reduction (P < 0.001) in tumor incidence, a 67% reduction (P < 0.001) in tumor multiplicity and a 44% decrease (P < 0.001) in tumor volume/tumor. In oxidative stress studies, topical application of BPO resulted in 75, 87 and 61% depletion in superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) activities in mouse epidermis, respectively. These decreases in antioxidant enzyme activities were significantly (P < 0.005-0.001) reversed by pre-application of silymarin in a dose-dependent manner. The observed effects of silymarin were 18-66, 32-72 and 20-67% protection against BPO-induced depletion of SOD, catalase and GPX activity in mouse epidermis, respectively. Silymarin pre-treatment also resulted in a dose-dependent inhibition (35-87%, P < 0.05-0. 001) of BPO-induced lipid peroxidation in mouse epidermis. In inflammatory response studies, silymarin showed a strong inhibition of BPO-induced skin edema (62-85% inhibition, P < 0.001), myeloperoxidase activity (42-100% inhibition, P < 0.001) and interleukin-1alpha protein level in epidermis (36-81% inhibition, P < 0.001). These results, together with our other recent studies, suggest that silymarin could be useful in preventing a wide range of carcinogen and tumor promoter-induced cancers.

Alteration of Egr-1 mRNA during multistage carcinogenesis in mouse skin.

Immediate early genes, including fos, jun, and early growth response-1 (Egr-1), are induced during cellular response to changes in extracellular environment. These immediate early genes are believed to mediate processes of cell growth and differentiation. In particular, Egr-1 is induced during mitogenic stimulation of a variety of cell types, including fibroblasts, B cells, and epithelial cells. In the present study, we examined Egr-1 gene expression during multistage carcinogenesis in mouse skin. After a single topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to SENCAR mouse skin, Egr-1 mRNA was induced, and maximal induction was observed at 2 h in both epidermis and dermis. Induction of Egr-1 mRNA by TPA was inhibited by fluocinolone acetonide, a potent inhibitor of tumor promotion by TPA. Egr-1 mRNA was present in primary keratinocytes derived from adult SENCAR mice. The keratinocyte cultures were maintained in low Ca(2+) medium, and Egr-1 mRNA levels became significantly elevated after the cultures were switched to high Ca(2+) medium. Additionally, a large proportion of primary papillomas and carcinomas generated from SENCAR mice by standard initiation-promotion regimens exhibited elevated Egr-1 mRNA compared with normal epidermis. Taken together, these data suggest a possible role of Egr-1 during multistage carcinogenesis in mouse skin.

Assessing the potential carcinogenic activity of magnetic fields using animal models.

We update our 1997 publication by reviewing 29 new reports of tests of magnetic fields (MFs) in six different in vivo animal models of carcinogenesis: 2-year, lifetime, or multigeneration exposure studies in rats or mice; and promotion/progression models (rat mammary carcinoma, rat liver focus, mouse skin, several models of human leukemia/lymphoma in rats and mice, and brain cancer in rats). Individual experiments are evaluated using a set of data quality criteria, and summary judgments are made across multiple experiments by applying a criterion of rough reproducibility. The potential for carcinogenicity of MFs is discussed in light of the significant body of carcinogenesis data from animal bioassays that now exists. Excluding abstracts, approximately 80% of the 41 completed studies identified in this and our previous review roughly satisfy data quality criteria. Among these studies, the criterion for independent reproducibility is not satisfied for any positive results but is satisfied for negative results in chronic bioassays in rats and mice and for negative results in both promotion and co-promotion assays using the SENCAR mouse skin model. Results of independent replication studies using the rat mammary carcinoma model were conflicting. We conclude that long-term exposure to continuous 50- or 60-Hz MFs in the range of 0.002-5 mT is unlikely to result in carcinogenesis in rats or mice. Though results of most promotion/progression assays are negative, a weak promoting effect of MFs under certain exposure conditions cannot be ruled out based on available data.