Tumor Initiation In Mouse Skin Research Articles

Deoxynivalenol induced mouse skin tumor initiation: Elucidation of molecular mechanisms in human HaCaT keratinocytes.

Among food contaminants, mycotoxins are toxic to both human and animal health. Our prior studies suggest that Deoxynivalenol (DON), a mycotoxin, behaves as a tumor promoter by inducing edema, hyperplasia, ODC activity and activation of MAPK's in mouse skin. In this study, topical application of DON, 336 and 672 nmol significantly enhanced ROS levels, DNA damage and apoptosis with concomitant downregulation of Ki-67, cyclin D, cyclin E, cyclin A and cyclin-dependent kinases (CDK4 and CDK2) thereby resulting in tumor initiation in mouse skin. Further, the elucidation of molecular mechanisms of tumor initiation by DON (0.42-3.37 nmol/ml) in HaCaT keratinocytes, revealed (i) enhanced ROS generation with cell cycle phase arrest in G0/G1 phase, (ii) increase in levels of 8-OxoG (6-24 hr) and γH2AX protein, (iii) significant enhancement in oxidative stress marker enzymes LPO, GSH, GR with concomitant decrease in antioxidant enzymes catalase, GPx, GST, SOD and mitochondrial membrane potential after DON (1.68 nmol) treatment, (iv) suppression of Nrf2 translocation to nucleus, enhanced phosphorylation with subsequent activation ERK1/2, p38 and JNK MAPK's following DON (1.68 nmol) treatment, (v) overexpression of c-jun, c-fos proteins, upregulation of Bax along with downregulation of Bcl-2 proteins, (vi) increase in cytochrome-c, caspase-9, caspase-3 and poly ADP ribose polymerase levels leads to apoptosis. Pretreatment of superoxide dismutase, mannitol and ethanol to HaCaT cells resulted in significant reduction in ROS levels and apoptosis indicating the role of superoxide and hydroxyl radicals in DON induced apoptosis as an early event and skin tumor initiation as a late event.

Mouse Models of the Skin: Models to Define Mechanisms of Skin Carcinogenesis

Mouse Models of the Skin: Models to Define Mechanisms of Skin Carcinogenesis

Topical Application of Ochratoxin A Causes DNA Damage and Tumor Initiation in Mouse Skin

Skin cancer is one of the most common forms of cancer and 2–3 million new cases are being diagnosed globally each year. Along with UV rays, environmental pollutants/chemicals including mycotoxins, contaminants of various foods and feed stuffs, could be one of the aetiological factors of skin cancer. In the present study, we evaluated the DNA damaging potential and dermal carcinogenicity of a mycotoxin, ochratoxin A (OTA), with the rationale that dermal exposure to OTA in workers may occur during their involvement in pre and post harvest stages of agriculture. A single topical application of OTA (20–80 µg/mouse) resulted in significant DNA damage along with elevated γ-H2AX level in skin. Alteration in oxidative stress markers such as lipid peroxidation, protein carbonyl, glutathione content and antioxidant enzymes was observed in a dose (20–80 µg/mouse) and time-dependent (12–72 h) manner. The oxidative stress was further emphasized by the suppression of Nrf2 translocation to nucleus following a single topical application of OTA (80 µg/mouse) after 24 h. OTA (80 µg/mouse) application for 12–72 h caused significant enhancement in- (a) reactive oxygen species generation, (b) activation of ERK1/2, p38 and JNK MAPKs, (c) cell cycle arrest at G0/G1 phase (37–67%), (d) induction of apoptosis (2.0–11.0 fold), (e) expression of p53, p21/waf1, (f) Bax/Bcl-2 ratio, (g) cytochrome c level, (h) activities of caspase 9 (1.2–1.8 fold) and 3 (1.7–2.2 fold) as well as poly ADP ribose polymerase cleavage. In a two-stage mouse skin tumorigenesis protocol, it was observed that a single topical application of OTA (80 µg/mouse) followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate for 24 week leads to tumor formation. These results suggest that OTA has skin tumor initiating property which may be related to oxidative stress, MAPKs signaling and DNA damage.

Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR-RFLP method.

One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinogenic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA-->CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI RFLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10,000 wild-type genes, is described. The results indicate that codon 61 mutations are induced 1 day (0.1%) after DB[a,l]P treatment on mouse skin, reach a high value (5%) by day 3, rapidly decline between days 7-9 and increase again during the clonal expansion of pre-papillomas into tumors. The detection of codon 61 mutations 1 day after DB[a,l]P exposure suggests that mutations occurred by pre-replication misrepair.

Increased Frequency of Resistance to Terminal Differentiation in C 3H Mouse Cells Produced by Genotoxic but Not Nongenotoxic Carcinogens

Increased Frequency of Resistance to Terminal Differentiation in C 3H Mouse Cells Produced by Genotoxic but Not Nongenotoxic Carcinogens. Przygoda, R. T., Freeman, J. J., Katz, S., and McKee, R. H. (1994). Fundam. Appl. Toxicol. 23, 261-267. Certain cells present in mouse skin are resistant to calcium-induced terminal differentiation. It is believed that these calcium-resistant cells (CRCs) represent an early stage in the carcinogenic process, in part, because frequency increases after treatment with mutagens. The frequency of CRCs in C 3H mouse skin was measured before and after treatment with certain petroleum-derived materials. One objective was to determine whether this assay could differentiate between genotoxic and nongenotoxic mouse skin carcinogens. An additional objective was to determine whether CRCs are an important factor in the tumorigenicity of petroleum middle distillates (PMDs), a class of apparently nongenotoxic materials. Three petroleum-derived materials were tested: mineral oil (MO), a noncarcinogenic product used as the negative control; catalytically cracked clarified oil (CCCO), a highly carcinogenic and mutagenic material; and a lightly refined paraffinic oil (LRPO), a PMD which has produced tumors when repeatedly applied, but is not mutagenic and does not initiate mouse skin tumors. The CRC frequency was not increased by LRPO treatment; however, a statistically significant and dose-related increase was produced by CCCO. These results are consistent with observations that genotoxic, petroleum-derived liquids are capable of tumor initiation in mouse skin, whereas PMDs which are not genotoxic do not initiate skin tumors. The number of CRCs in untreated and MO-treated mice was approximately twice the tumor frequency measured in bioassays of PMDs. Thus, tumor production associated with these products could be due to promotion of preexisting, spontaneously initiated cells.

Tumor initiation in mouse skin by cutting oils

In order to study the skin tumor-initiating potential for fresh and used cutting oils, a chronic animal bioassay following an initiation-promotion protocol was performed. Tumor initiation was performed by topical application of a single dose of neat fresh/used cutting oil on the mouse skin. After a week, these animals were treated topically with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent skin tumor promoter. Repeated application of TPA resulted in the appearance of tumors almost at the same time (6-8 weeks) in the groups initiated with fresh or used cutting oil. After a prolonged application of TPA, for 28 weeks, it was observed that the cumulative number of tumors and tumor-bearing animals in the used oil initiated group was slightly less compared to that exposed to a fresh variety of oil. Histopathological examination of the tumors could not show any qualitative differences in the nature of tumors developed in both groups. It seems that both fresh and used cutting oils act as potent skin tumor initiators.

Inhibition of benzo[a]pyrene-7,8-diol formation in vitro by complex organic mixtures

Coal-derived complex organic mixtures [COM] with boiling points ⩾370° C (⩾700° F) are known to inhibit both mouse skin tumor initiation by benzo[ a]pyrene [BAP], and BAP-induced bacterial mutagenesis. We have examined the effects of 5 COM, with boiling points of 149–370°C (300–700°F), 370–398°C (700–750°F), 398–426°C (750–800°F), 426–454°C (800–850°F), and >454° C(>850° F), on both the rate and the route of BAP metabolism by rat liver homogenetes in vitro. When co-metabolized in 40:1 excess with BAP, all of the COM reduced the rate of BAP metabolism. The 149–370°C (300–700°F) COM reduced the initial rate of BAP metabolism to 34% of the rate for BAP alone, while the four higher-boiling COM reduced it to 6.3–9.3% of the rate for BAP alone. In addition, the 2 highest-boiling COM (426–454°C and >454°C boiling points) were found to reduce the percentage of BAP metabolized to BAP-7,8-diol, in comparison to incubations using BAP alone. The 370–398°C and 398–426°C COM did not alter the percentage of BAP metabolized to BAP-7,8-diol, while the 149–370°C COM increased it. Both the general inhibition of BAP metabolism (by all of the COM), and the specific inhibition of BAP-7,8-diol formation (by the highest boiling COM) may play a role in the inhibition of formation of BAP-induced skin tumors by these materials.

Formation and disappearance of benzo[a]pyrene DNA-adducts in mouse epidermis.

The rates of formation and disappearance of benzo[a]pyrene (B[a]P) DNA-adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 200 nmol/mouse of [3H]B[a]P at various times prior to sacrifice. The formation and disappearance of total adducts as well as individual adducts was determined and in addition, the rate of DNA turnover was monitored concurrently so that adduct disappearance could be expressed as a function of epidermal cell turnover. Under these experimental conditions, covalent binding of B[a]P to epidermal DNA reached a peak 24 h after treatment. Interestingly, between 24-48 h after application of the hydrocarbon there was a very rapid drop in the level of bound B[a]P to value approximately 50% of the maximum level at 24 h. Thereafter, the level of bound B[a]P disappeared at a much slower rate. In dual-label experiments, where the epidermal DNA was pre-labeled with [14C]thymidine, [3H]B[a]P DNA-adduct disappearance between 24-48 h was clearly more rapid than could be explained on the basis of epidermal DNA turnover. By 72 h and beyond, however, [3H]B[a]P DNA-adduct disappearance approximately paralleled DNA turnover. Examination of the rate of formation and disappearance of individual B[a]P DNA-adducts (nine individual adducts) suggested that some deoxyadenosine adducts were removed more rapidly than deoxyguanosine adducts. The results indicate that at least some epidermal cells have the capacity to repair B[a]P DNA-adducts. The data are discussed in relation to the process of tumor initiation in mouse skin.

Influence of 13- cis-retinoic acid on mouse skin tumor initiation and promotion

Tumor initiation in CD-1 mice by benzo[ a]pyrene (BaP) or N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) was unaffected by topical pretreatment with 13- cis-retinoic acid (13- cis-RA). Likewise, anthralin-induced tumor promotion in SENCAR mice was unaffected by pretreatment with 13- cis-RA. These results suggest that the action of retinoids in preventing either tumor initiation or promotion is very carcinogen or cocarcinogen specific.

Comparison of Complete Carcinogenesis and Tumor Initiation and Promotion in Mouse Skin: The Induction of Papillomas by Tumor Initiation-Promotion a Reliable Short Term Assay

Skin tumors can be induced by the sequential application of a subthreshold dose of a carcinogen (initiation phase), followed by repetitive treatment with a weak or noncarcinogenic tumor promoter. Using a two-stage system to induce tumors in Senear mice there is a very good dose-response relationship between the induction of the number of papillomas per mouse at early times (10 to 20 weeks) and the final carcinoma incidence after longer latency (20 to 50 weeks). This system can be used not only to determine the tumor initiating and promoting activities of a compound but if the agent is given repeatedly by itself one can also determine if it is a complete carcinogen, i.e., if it has both tumor initiating and promoting activity. With the exception of a few pure tumor initiators, there is in general a good qualitative and quantitative correlation between the ability of a polycyclic aromatic hydrocarbon (PAH) to act as a complete carcinogen and to act as a tumor initiator in mouse skin. In addition, if the agent is given concurrently with a known complete carcinogen or a tumor initiator one can also determine if the agent has co-carcinogenic or co-tumor initiating activity or even possibly anticarcinogenic activity. Likewise, if the agent is given concurrently with a known tumor promoter one can determine if the agent has co-promoting or anti-pro-moting activity. There is a good correlation between the tumor-initiating activities of PAH and their abilities to bind covalently to DNA. In addition, various inhibitors of PAH tumor initiation show a strong correlation with their abilities to inhibit the binding of the PAH to DNA and their anti-tumor initiating activities. There is also a good correlation between the promoting abilities of phorbol esters to promote tumors and their abilities to induce ornithine decarboxylase (ODC), cell proliferation and dark basal keratinocytes. When other nonpromoting hyperplastic agents are used, only dark cell induction correlates with promotion. Certain polyamines and prostaglandins can enhance phorbol ester tumor promotion. Anti-inflammatory steroids, retinoids, and protease inhibitors are potent inhibitors of tumor promotion. They inhibit tumor promotion by inhibiting either the 12-0-tetradecanoylphorbol-13-acetate (TPA) induced cell proliferation, ODC and/or dark basal keratinocytes. Certain weak promoters such as mezerein which mimics TPA in many biochemical and morphological effects are potent second step promoters in a two-stage promotion regimen.

Lack of Involvement of 6-Hydroxymethylation in Benzo[<italic>a</italic>]pyrene Skin Tumor Initiation in Mice<xref ref-type="fn" rid="FN2">2</xref>

The skin tumor-initiating activities of benzo[a]pyrene (BP), 6-hydroxymethylbenzo[a]pyrene (6-OH-CH2-BP), and 6-methylbenzo[a]pyrene (6-CH3-BP), as well as the effects of 7,8-benzoflavone (7,8-BF), quercetin, and 1-benzylimidazole on their activity, were determined in outbred female CD-1 mice by use of a two stage system of tumorigenesis. The skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP was 12.5 and 20%, respectively, of the activity of BP, 7,8-BF had little effect on the skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP. However, a dose-dependent inhibition of BP tumorigenesis by 7,8-BF was noted. Quercetin and 1-benzylimidazole also inhibited BP skin tumor-initiating activity. These findings indicated that direct hydroxymethylation of BP is not an important pathway in the activation of BP in mouse skin tumor initiation.