Induction of chromosomal damage in exfoliated buccal and nasal cells of road markers.

Road markers are exposed to various chemicals and particles. The aim of this study was to determine whether road worker exposure induceschromosomal damage which is indicative for increased cancer risks. Micronucleus (MN) cytome assays were thus conducted with exfoliated nasal and buccal cells collected from 42 workers and 42 matched controls. The frequencies of MN (reflecting chromosomal aberrations), nuclear buds (NBuds; reflecting gene amplifications) and binucleated cells (BN; reflecting disturbed mitosis) were scored. Further, the rates of nuclear anomalies indicative of acute cytotoxicity (condensed chromatin, karyorrhexis, karyolysis, pyknosis) were evaluated. Data demonstrated marked induction of MN, NBuds, and BN by 1.34-fold, 1.24-fold and 1.14-fold in buccal cells. In nasal cells, only MN frequencies were elevated, 1.23-fold. These effects were paralleled by increased rates of condensed chromatin, karyorrhexis and karyolysis in both cell types. The effects were more pronounced in individuals who had worked for more than 10 years while smoking did not produce synergistic responses. This is the first investigation concerning the induction of genetic damage in road markers and the results are suggestive for enhanced cancer risks. It is conceivable that exposure to silica dust (known to induce cancer and genetic damage) and/or benzoyl peroxide which forms reactive radicals may be associated with the observed genetic damage in road workers. Further investigations of the cancer risks of these workers are warranted.

Effects of flour bleaching agent on mice liver antioxidant status and ATPases.

Benzoyl peroxide (BPO) is a strong oxidizing agent and widely used as flour bleaching agent. However their potential risk of liver damage is unknown. The aim of this study was to investigate the effects of BPO on mice liver antioxidant status and ATPases according to the actual amount of BPO in flour from Jinan, China. The results showed that the maximum concentration of BPO reached up to 284.6 mg/kg and content of BPO mainly ranged from 0 to 240 mg/kg. Therefore, four groups of mice were gavaged daily with BPO at doses of 0, 50, 100, 200mg/kg b.w./d for 42 days, respectively. In liver tissue, superoxide dismutase (SOD) activity was significantly decreased, while the content of malondialdehyde (MDA) significantly increased following BPO exposure at 200mg/kg b.w. BPO significantly decreased the Mg(2+)-ATPase and Ca(2+)-ATPase activities of the liver at 200mg/kg b.w. BPO, at all of the doses assayed, produced non-significant effects on glutathione peroxidase (GSH-Px) and Na(+)K(+)-ATPase activities. Experimental results suggested that BPO had certain adverse effects on antioxidant status and the activities of Mg(2+)-ATPase and Ca(2+)-ATPase of liver tissue.

[Association between allergy to benzoyl peroxide, vitiligo and implantation of a cemented total knee joint prosthesis: Is there a connection?]. / Assoziation zwischen Benzoylperoxidallergie, Vitiligo und Implantation einer zementierten Knieprothese: Ein Zusammenhang?

Allergies against bone cement or bone cement components have been well-described. We report on a 63-year-old patient who presented with progressive vitiligo all over the body after implantation of a cemented total knee replacement. A dermatological examination was performed and an allergy to benzoyl peroxide was found. A low-grade infection was diagnosed 5 months after implantation of the total knee replacement and the prosthesis was replaced with a cement spacer. After treating the infection of the knee replacement non-cemented arthrodesis of the knee was performed. In cases of new, unknown skin efflorescence, urticaria and periprosthetic loosening of cemented joint replacement, the differential diagnosis should include not only infections but also possible allergies against bone-cement and components such as benzoyl peroxide or metal components.

Photocarcinogenesis and toxicity of benzoyl peroxide in hairless mice after simulated solar radiation.

Topical benzoyl peroxide (BPO) gel has long been used to treat acne vulgaris and has recently been combined with clindamycin (BPO-clin). No skin malignancies have been reported after clinical use of BPO, but there has been concern about the possible carcinogenicity of BPO alone and in combination with UV radiation. BPO can promote skin tumorigenesis in a mouse skin chemical carcinogenesis model. As acne vulgaris is frequently localized on sun-exposed areas, we investigated whether BPO or BPO-clin accelerates photocarcinogenesis in combination with simulated solar radiation (SSR) in 12 groups of 25 hairless female C3.Cg/TifBomTac-immunocompetent mice. BPO or BPO-clin was applied topically to the back five times each week, followed by SSR three times each week (2, 3, or 4 standard erythema doses) 3-4 h later, for 365 days or until death. Generally BPO and BPO-clin did not accelerate the time to first, second or third tumor. Therefore, there is no evidence suggesting that BPO or BPO-clin is photocarcinogenic. However, we found significantly higher mortality in the SSR exposed groups receiving BPO and BPO-clin compared with groups receiving only BPO or BPO-clin. Our results show that BPO and the combination of BPO and clindamycin do not accelerate photocarcinogenesis, but are toxic in hairless mice. Based on the current data, the cancer risk associated with the use of BPO and BPO-clin in sun-exposed areas is minimal. Thus, while the carcinogenic potential of BPO is not fully understood, at the present time, evidence suggests that this compound is safe to use.

Genotoxicity of dental resin polymerization initiators in vitro.

The polymerization initiators for resins cured using visible light usually consist of a photosensitizer, primarily camphorquinone (CQ), and a reducing agent, which is often a tertiary amine (DMPT, DMAEMA), while the initiator used for self-curing resins consists of benzoyl peroxide (BPO) and a tertiary amine (DMPT). The genotoxicities of camphorquinone (CQ), benzoyl peroxide (BPO), dimethyl-para-toluidine (DMPT), 2-dimethylamino-ethyl-methacrylate (DMAEMA), and 1-allyl-2-thiourea (ATU) were examined using the bioluminescent bacterial genotoxicity test. 4-Nitroquinoline-N-oxide (4NQO) was prepared for comparison with these chemicals. Acetone solutions of the five polymerization initiators and 4NQO were prepared. Benzoyl peroxide (BPO), dimethyl-para-toluidine (DMPT), and 1-allyl-2-thiourea (ATU) showed significant genotoxic activity at 24 h in the bioluminescent bacterial genotoxicity test, at concentrations of approximately 5 microM, 4 mM, and 1 mM, respectively. 2-Dimethyloamino-ethyl-methacrylate (DMAEMA) did not have genotoxic activity and CQ had questionable genotoxic activity. In comparison, 4NQO had strong genotoxicity, at 4 microM, roughly the same as that of BPO. Therefore, BPO should be used carefully in clinical dentistry.

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.

Effect of Hibiscus rosa sinensis extract on hyperproliferation and oxidative damage caused by benzoyl peroxide and ultraviolet radiations in mouse skin.

The present study was conducted to investigate the ameliorative potential of Hibiscus rosa sinensis extract in mice skin. Combination of a single topical application of benzoyl peroxide (20 mg/0.2 ml/animal) followed by ultraviolet radiations (0.420 J/m2/s) was used to induce hyperproliferation and oxidative stress. Single benzoyl peroxide application prior to ultraviolet B radiations exposure caused significant depletion in the detoxification and antioxidant enzymes, while malondialdehyde formation, hydrogen peroxide content, ornithine decarboxylase activity and DNA synthesis were raised significantly. However, pretreatment of H. rosa sinensis extract (3.5 mg and 7 mg/ kg b.wt.) partly restored the levels of cellular protective enzymes (P<0.05). Besides, malondialdehyde formation and hydrogen peroxide content (P<0.05) were statistically significantly reduced at both doses. The ornithine decarboxylase activity and thymidine incorporation in DNA were also reduced dose dependently (P<0.05) by the plant extract. Therefore, we propose that H. rosa sinensis extract exerts a protective effect against the tumour promotion stage of cancer development.

Modulatory effect of gentisic acid on the augmentation of biochemical events of tumor promotion stage by benzoyl peroxide and ultraviolet radiation in Swiss albino mice.

The present study was carried out to study the effect of gentisic acid (2,5-dihydroxybenzoic acid (2,5-DHBA)) on the tumor promotion related events of carcinogenesis in murine skin. Benzoyl peroxide (BPO) (20 mg/0.2 ml/animal) and ultraviolet radiations (UVR) (0.420 J/m2/s) were used to induce tumor promotion response and oxidative stress and caused significant depletion in the detoxification and antioxidant enzyme armory with concomitant elevation in malondialdehyde (MDA) formation, hydrogen peroxide (H2O2) generation, ornithine decarboxylase (ODC) activity and unscheduled DNA synthesis. However, gentisic acid pretreatment at two different doses restored the levels of the above said parameters (P < 0.05) in a dose-dependent manner except in the case of ODC activity. Therefore, we propose that it might suppress the promotion stage via inhibition of oxidative stress but may not affect the polyamine biosynthetic pathway.

Effect of alpha-tocopherol and N-acetylcysteine on benzoyl peroxide toxicity in human keratinocytes.

Benzoyl peroxide is a free-radical generating compound widely used in the polymer industry and also in pharmaceuticals as antimicrobial agent to treat acne. However, benzoyl peroxide causes irritation and contact dermatitis in about 1% of patients. Concern over the use of this compound is motivated by the demonstration that it can also act as skin tumor promoter in mice. In addition, benzoyl peroxide induces DNA strand breaks in many cells, including keratinocytes. Benzoyl peroxide toxicity is presumably mediated by the formation of reactive free radicals and by the consumption of intracellular antioxidants. In this work we investigated the effect of both the lipophilic antioxidant alpha-tocopherol and the hydrophilic thiol donor N-acetylcysteine (NAC) in human keratinocyte line HaCaT exposed to benzoyl peroxide. A protective effect against benzoyl peroxide cytotoxicity was achieved when cells were grown on a alpha-tocopherol layer. On the contrary, the addition of alpha-tocopherol dissolved in ethanol had a pro-oxidant effect, leading to an enhancement of benzoyl peroxide toxicity. Cytotoxicity was also reduced adding NAC to the culture medium; the presence of both NAC and alpha-tocopherol exerts a synergistic cytoprotection.

Only a subset of 12-O-tetradecanoylphorbol-13-acetate-promoted mouse skin papillomas are promotable by benzoyl peroxide.

The two-stage skin carcinogenesis model of initiation and promotion in Carcinogenesis-susceptible (Car-S) mice has been used to investigate the pathways of promotional activity of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester tumor promoter, and benzoyl peroxide (BzPo), a free radical-generating compound. To test whether distinct populations of 9,10-dimethyl-1,2-benzanthracene (DMBA)-initiated epidermal keratinocytes are responsive to the two promoters, tandem experiments were performed. DMBA-initiated Car-S mice were promoted twice weekly with maximal promoting doses of TPA or BzPo. When the number of papillomas/mouse reached a plateau, promotion in the TPA and BzPo groups was switched to BzPo or TPA, respectively, until achievement of a new plateau. Mice promoted with BzPo developed 11.0 +/- 1.3 papillomas/mouse and subsequent TPA promotion induced 13.8 additional papillomas, for a total of 24.8 +/- 2.1 papillomas/mouse. TPA-promoted mice developed 23.3 +/- 1.1 papillomas/mouse, and subsequent BzPo promotion for 91 days did not promote additional papillomas. Our results show a less than additive tumor response after sequential promotion with BzPo and TPA, or vice versa, indicating that the pathways of promotional activity of TPA and BzPo are interacting. While the final papilloma yield was similar at the end of the two tandem promotion experiments independently of promoter sequence, the percentage of mice developing carcinomas was significantly higher in mice that were promoted with BzPo in the first stage. No significant differences in the frequency and type of c-Ha-ras mutations were observed in TPA- and BzPo-promoted tumors, suggesting that promotion of DMBA-initiated cells by BzPo requires introduction of additional molecular alterations compared to TPA.

Presenting an in vitro cell culture model to determine the cytotoxic effect of benzoyl peroxide vapours.

Benzoyl peroxide (BP) is an initiator of polymerisation used in the synthesis of methyl methacrylate dental materials, and is a known allergen that causes allergic contact dermatitis during occupational exposure, especially in dentists and dental personnel. The eyes of dental technicians, dentists and even patients with an allergic history are the first level of exposure, and therefore complaints of allergic reactions of the eyes are usually noted. In this article the authors used a modification direct cell culture testing method to assess the cytotoxic potential of BP. L929 mouse fibroblasts as well as cells from human limbal eye rings were exposed to BP vapours in the experiments. The MTT and crystal violet assays were also employed to determine cell numbers and viability. Results indicated that there is an exponential decrease in cell viability. After exposure of five minutes, cell viability had already decreased by 20 and 40% for vapours derived from either a 10 or 20 microliters aliquot of BP, respectively. After a further five minutes, cell viability decreased by a further 10% with a statistical difference form the control of p < 0.01. Results indicated that BP vapours are cytotoxic to mouse L929 and human eye fibroblasts. Furthermore, the authors concluded that the permanent L929 mouse fibroblast cell line can provide valuable information regarding the cytotoxic effects of dental products and associated compounds that form toxic vapours.

Lupeol, a triterpene, prevents free radical mediated macromolecular damage and alleviates benzoyl peroxide induced biochemical alterations in murine skin.

In our earlier communication we have shown that Lupeol inhibits early responses of tumour induction in murine skin. The free radical mediated damage to the cellular macromolecules such as DNA, proteins, lipids and alteration in the activities of quinone reductase and xanthine oxidase are important biochemical parameters of tumor development. The suppression of free radical mediated damage to cellular macromolecules and induction of quinone reductase along with depletion of xanthine oxidase are prominent characteristics of chemopreventive agents. In the present investigation, we have elucidated the mechanism of action of lupeol (Lup-20 (29)-en-3beta-ol), a triterpene found in moderate amount in many vegetables, fruits and anti-tumor herbs. In the present investigation, lupeol significantly reduced the free radical mediated DNA-sugar damage and microsomal lipid peroxidation in an iron/ascorbate free radical generating system in vitro. Benzoyl peroxide, a known free radical generating tumor promoter mediated oxidation of proteins and modulation in the activities of quinone reductase as well as xanthine oxidase was significantly prevented by lupeol when tested on murine skin in vivo. It was concluded from this study that lupeol acts as an effective chemopreventive agent against cutaneous toxicity.

Low iron state is associated with reduced tumor promotion in a two-stage mouse skin carcinogenesis model.

The purpose of this study was to investigate the effect of low iron state in a two-stage mouse skin carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and benzoyl peroxide (BPO)-promoted cutaneous tumorigenesis. All mice were treated with DMBA. Low iron state was achieved by injection with phenylhydrazine hydrochloride and feeding low iron diet. A low iron state resulted in a decrease in tumor incidence (papillomas and carcinomas) and number of tumors/mouse. Also, the conversion of papillomas to carcinomas was lower in mice on a low iron state. BPO treatment enhanced epidermal lipid peroxidation (LPO) and was accompanied by a depletion in the level of epidermal reduced glutathione (GSH) and decrease in the activities of antioxidant enzymes. BPO treatment also increased ornithine decarboxylase (ODC) activity and [3H]thymidine incorporation into cutaneous DNA. Mice in a low iron state were less susceptible to the effects of BPO treatment, as was apparent from a partial recovery of GSH levels and the activities of antioxidant enzymes, as well as a lower induction in ODC activity, [3H]thymidine incorporation into cutaneous DNA and lesser epidermal LPO. As expected, cutaneous iron levels were lower in mice on a low iron state. Thus, our data show that the tumor-promoting potential of BPO is reduced by low iron state in a two-stage mouse skin carcinogenesis model.

Oral toxicity of topical preparations.

This chapter covers the hazards that some topical pharmaceutical preparations pose to animals who consume them. Included are medications containing calcipotriene, vitamins A and D, zinc oxide, 5-fluorouracil, brimonidine, imidazoline decongestants, local anesthetics, corticosteroids, antibiotics, salicylates, and benzoyl peroxide.

Analysis of c-Ha-ras gene mutations in skin tumors induced in carcinogenesis-susceptible and carcinogenesis-resistant mice by different two-stage protocols or tumor promoter alone.

In the present study we describe the molecular analysis of c-Ha-ras gene mutations in 47 papillomas and 17 carcinomas developed in two lines of mice, carcinogenesis-susceptible (Car-S) and carcinogenesis-resistant (Car-R), selectively bred for extreme susceptibility or resistance to chemical skin carcinogenesis initiated and promoted with different doses of 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This study also presents the analysis of c-Ha-ras gene mutations in 22 papillomas and 22 carcinomas in Car-S mice initiated with DMBA and promoted with benzoyl peroxide (BzPo) and in seven papillomas and one carcinoma from a group of uniniated Car-S mice that received only BzPo treatment. The data showed that a A(182)-->T transversion in the c-Ha-ras gene was present in 100% and 81% of the skin tumors developed in Car-S and Car-R mice, respectively, after DMBA initiation and TPA promotion, suggesting that differences in genetic susceptibility can influence the frequency of c-Ha-ras mutations in the skin tumors produced. The same A(182)-->T mutation with an incidence of 68% was found in papillomas from DMBA-initiated and BzPo-promoted Car-S mice. The difference in the mutation frequency between DMBA/BzPo and DMBA/TPA papillomas suggested that the promotion step contributes to the final mutation pattern. The tumor induction experiment with BzPo alone showed that this compound can induce tumor development in 26% of Car-S mice, and the molecular analysis of the tumors showed a broad mutation spectrum, including mutations in codons 12, 13, and 61 of the c-Ha-ras gene. Mol. Carcinog. 30:111-118, 2001.

Attenuation of benzoyl peroxide-mediated cutaneous oxidative stress and hyperproliferative response by the prophylactic treatment of mice with spearmint (Mentha spicata).

The modulating effect of spearmint (Mentha spicata) on benzoyl peroxide-induced responses of tumor promotion in murine skin was investigated. Benzoyl peroxide (BPO) is an effective cutaneous tumor promoter acting through the generation of oxidative stress, induction of ornithine decarboxylase activity and by enhancing DNA synthesis. BPO treatment (20 mg/animal) increased cutaneous microsomal lipid peroxidation and hydrogen peroxide generation. The activity of cutaneous antioxidant enzymes, namely catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase, was decreased and the level of cutaneous glutathione was depleted. BPO treatment also induced the ornithine decarboxylase activity and enhanced the [3H]thymidine uptake in DNA synthesis in murine skin. Prophylactic treatment of mice with spearmint extract (10, 15 and 20 mg/kg) 1 hr before BPO treatment resulted in the diminution of BPO-mediated damage. The susceptibility of cutaneous microsomal membrane to lipid peroxidation and hydrogen peroxide generation was significantly reduced (P < 0.05 ). In addition, depleted levels of glutathione, inhibited activity of glutathione dependent and antioxidant enzymes were recovered to a significant level (P < 0.01, P < 0.05 and P < 0.01, respectively). Similarly, the elevated ornithine decarboxylase activity and enhanced thymidine uptake in DNA synthesis was inhibited significantly (P < 0.05 ) in a dose-dependent manner. The protective effect of spearmint was dose dependent in all parameters. The result suggests that spearmint is an effective chemopreventive agent that may suppress BPO-induced cutaneous oxidative stress, toxicity and hyperproliferative effects in the skin of mice.

Protein kinase C signaling and oxidative stress.

Oxidative stress is involved in the pathogenesis of various degenerative diseases including cancer. It is now recognized that low levels of oxidants can modify cell-signaling proteins and that these modifications have functional consequences. Identifying the target proteins for redox modification is key to understanding how oxidants mediate pathological processes such as tumor promotion. These proteins are also likely to be important targets for chemopreventive antioxidants, which are known to block signaling induced by oxidants and to induce their own actions. Various antioxidant preventive agents also inhibit PKC-dependent cellular responses. Therefore, PKC is a logical candidate for redox modification by oxidants and antioxidants that may in part determine their cancer-promoting and anticancer activities, respectively. PKCs contain unique structural features that are susceptible to oxidative modification. The N-terminal regulatory domain contains zinc-binding, cysteine-rich motifs that are readily oxidized by peroxide. When oxidized, the autoinhibitory function of the regulatory domain is compromised and, consequently, cellular PKC activity is stimulated. The C-terminal catalytic domain contains several reactive cysteines that are targets for various chemopreventive antioxidants such as selenocompounds, polyphenolic agents such as curcumin, and vitamin E analogues. Modification of these cysteines decreases cellular PKC activity. Thus the two domains of PKC respond differently to two different type of agents: oxidants selectively react with the regulatory domain, stimulate cellular PKC, and signal for tumor promotion and cell growth. In contrast, antioxidant chemopreventive agents react with the catalytic domain, inhibit cellular PKC activity, and thus interfere with the action of tumor promoters.

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.

Protection against 12-O-tetradecanoylphorbol-13-acetate induced skin-hyperplasia and tumor promotion, in a two-stage carcinogenesis mouse model, by the 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-[2,3-b]quinoxaline analogue of ellipticine.

The effects of topical applications of 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-[2,3-b]quinoxaline (B-220), on 12-O-tetradecanoylphorbol-13-acetate (TPA) or benzoylperoxide (BPO) induced promotion of skin tumors and hyperplasia were studied in female SENCAR mice. Papillomas were induced by initiation with 7,12-dimethylbenz[a]anthracene (DMBA), followed by promotion biweekly with TPA or BPO. Administration of B-220 1 h before TPA promotion resulted in a prolonged latency period of tumor appearance and a significantly reduced (up to 15% of positive controls) papilloma yield at 20 weeks. Moreover, if B-220 treatment was terminated after 20 weeks and TPA treatment continued, papilloma development resumed indicating that initiated tumor cells were still present but were unable to grow with B-220 present. If B-220 pretreatment was not given during the first 10 weeks of TPA promotion, incidence at 20 weeks was not reduced but tumor multiplicity was still decreased. In addition a marked reduction of the TPA induced sustained epidermal hyperplasia was observed in the long term experiment. Neither the inflammatory response nor the increase in the number of apoptotic cells seen in short term experiment after a single TPA treatment were inhibited by B-220. B-220 administration before BPO promotion had no effect on the appearance of BPO induced papillomas or epidermal hyperplasia, suggesting that TPA and BPO promote tumor formation via at least partially different mechanisms. In experiments where B-220 was applied topically 1 h before DMBA initiation, little or no effect was seen. No morphological changes in mouse skin due to long term exposure (two times/week, 39 weeks) to B-220 were found. In conclusion, we present evidence that B-220 is a potent inhibitor of mouse skin tumor promotion by TPA, but has little effect on the initiation step or the survival of initiated cells.

Skin tumorigenesis by initiators and promoters of different chemical structures in lines of mice selectively bred for resistance (Car-r) or susceptibility (Car-s) to two-stage skin carcinogenesis.

Carcinogenesis-resistant (Car-R) and carcinogenesis-susceptible (Car-S) mice were obtained applying a bi-directional selective breeding approach to a two-stage skin carcinogenesis protocol, using 9,10-dimethyl-1,2-benzanthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter. Sixteen generations of selection produced a remarkable interline difference in responsiveness to two-stage skin carcinogenesis between Car-R and Car-S: identical DMBA (25 microgram) and TPA (5 microgram) doses induced papillomas in 100% of Car-S compared with 3.3% of Car-R mice and maximal responses of 14.3 or 0.03 papillomas/mouse, respectively, despite the shorter promotion applied to Car-S (49 vs. 208 days). To define the factors determining this great difference, Car-R and Car-S mice were challenged by initiators/promoters chemically unrelated to those used for selection. Both lines were subjected to either initiation by N-methyl-N-nitrosourea (MNU) followed by TPA promotion, or promotion by benzoyl peroxide, or 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) following DMBA initiation. Initiation with MNU induced a 10-fold tumour incidence in Car-S compared with Car-R mice, and a 32-fold difference in tumour induction rate. The 2 lines also differed markedly in susceptibility to benzoyl peroxide promotion: Car-S mice initiated with 25 microgram DMBA and promoted with 7.5 mg benzoyl peroxide showed a 12-fold tumour incidence and a 103-fold tumour induction rate compared with the corresponding Car-R group. Both lines, however, were refractory to chrysarobin promotion. The progression of papillomas to carcinomas was examined in all Car-S groups. The incidence of mice that developed carcinomas was 57% in MNU-initiated mice. Benzoyl peroxide was also able to promote carcinoma development in Car-S mice, though with a lower incidence (30.4%) than TPA.