Exposure of mouse skin to organic peroxides: subchronic effects related to carcinogenic potential

Margaret Hanausek4, Zbigniew Walaszek, Aurora Viaje, Michael LaBate, Erick Spears, David Farrell, Richard Henrich1, Ann Tveit2,{dagger}, Earl F. Walborg, Jr3 and Thomas J. Slaga

AMC Cancer Research Center, Denver, CO 80214, USA, 1 Great Lakes Chemical Corporation, West Lafayette, IN 47906, USA, 2 ATOFINA Chemicals Inc., Philadelphia, PA 19103, USA and 3 {dagger}Dermigen Inc., Smithville, TX 78957, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Screening of newly synthesized organic peroxides for tumor initiating/promoting activity would be greatly facilitated if predictive methodologies could be developed using topical exposures shorter than those required for definitive tumor assessment in mouse skin models. Nine organic peroxides [benzoyl peroxide (BZP), di-t-butyl peroxide (DTBP), t-butyl peroxybenzoate (TBPB), p-t-butyl isopropylbenzene hydroperoxide (TBIBHP), cumene hydroperoxide (CHP), dicetyl peroxydicarbonate (DPD), dicumyl peroxide (DCP), methyl ethyl ketone peroxide (MEKP) and O,O-t-butyl-O-(2-ethylhexyl) monoperoxycarbonate (TBEC)] were evaluated for their ability to increase biomarkers of tumor promotion in mouse skin, i.e. sustained epidermal hyperplasia, dermal inflammation and oxidative DNA damage. Evaluations were performed using SENCAR mice exposed topically for 4 weeks. The organic peroxides varied in their effects on these biomarkers. BZP, TBPB and TBIBHP exhibited significant increases in all three biomarkers associated with tumor promoting activity, CHP produced increases only in sustained epidermal hyperplasia and dermal inflammation, MEKP and DCP produced increases only in sustained epidermal hyperplasia and TBEC produced an increase only in dermal inflammation. DTBP and DPD had no effect on the three parameters studied. TBPB and TBIBHP were selected for further examination of their ability to produce mutations in codons 12, 13 and 61 of the c-Ha-ras protooncogene, i.e. those mutations known to be involved in the initiation of mouse skin tumors, because they were the only peroxides to exhibit significant positive results in all assays except the Ha-ras mutation following 4 weeks of exposure. Evaluations were performed using SENCAR mice dosed topically for 8 or 12 weeks in a complete carcinogenesis protocol or 16 weeks in an initiation/promotion protocol using 7,12-dimethylbenz[a]anthracene, urethane, benzo[a]pyrene and N-methyl-N'-nitro-N-nitrosoguanidine as positive controls. Neither TBPB nor TBIBHP produced detectable mutations in the c-Ha-ras protooncogene, indicating that they are not likely to possess tumor initiating or complete carcinogenic activity.

Abbreviations: B[a]P, benzo[a]pyrene; BZP, benzoyl peroxide; CHP, cumene hydroperoxide; DCP, dicumyl peroxide; DMBA, 7,12-dimethylbenz[a]anthracene; DPD, dicetyl peroxydicarbonate; DTBP, di-t-butyl peroxide; MEKP, methyl ethyl ketone peroxide; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MSP, mismatch-specific primer; ODC, ornithine decarboxylase; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; TBEC, O,O-t-butyl-O-(2-ethylhexyl) monoperoxycarbonate; TBIBHP, p-t-butyl isopropylbenzene hydroperoxide; TBPB, t-butyl-peroxybenzoate; TPA, 12-O-tetradecanoylphorbol-13-acetate; 2x/wk, twice per week


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organic peroxides are used as active components of cosmetics and pharmaceuticals and as initiators of polymerization or curing of plastics (1). The potential of these metastable compounds to generate free radicals, coupled with evidence that free radicals may be involved in the carcinogenic process (26), has raised concern for their potential carcinogenic risk to humans (1). This concern has prompted evaluations of their genotoxicity and carcinogenicity (1), however, evaluations to date have not been comprehensive or consistent. Furthermore, data regarding their tumor initiating and/or tumor promoting activity in chronic bioassays are available only for a limited number of organic peroxides.

The multistage model of carcinogenesis using mouse skin (6,7) has provided the most comprehensive information concerning the carcinogenic potential of organic peroxides. Van Duuren and co-workers evaluated the complete carcinogenicity of several organic peroxides using the mouse skin model (810). Ascaridole, 1-hydroperoxycyclohex-3-ene and 1-hydroperoxy-1-vinylcyclohex-3-ene, all applied topically in benzene, were reported to possess weak complete carcinogenic activity, producing a few skin tumors after long latencies (8,9). Lauryl peroxide, cumene hydroperoxide (CHP), di-t-butyl peroxide (DTBP) and benzoyl peroxide (BZP), also applied topically in benzene, were reported to lack complete carcinogenicity (810). It should be noted that the benzene vehicle produced skin damage, consequently, weak carcinogenic activity may be attributable to epigenetic mechanisms associated with the reparative response of the skin. t-Butyl hydroperoxide was reported to lack carcinogenic activity after lifetime skin painting in mice (8). Slaga and co-workers examined the carcinogenicity of BZP and lauryl peroxide in the SENCAR mouse skin model and found they were inactive as complete carcinogens or tumor initiators, but were active as tumor promoters (11,12).

The complete carcinogenicity of BZP, a component of anti-acne medications, has been the subject of numerous investigations. Reviews on this subject by Monro (13) and Kraus et al. (14) have summarized 23 carcinogenicity studies using rodent models, including 16 that applied BZP topically, and noted that only Kurokawa et al. (15) observed complete carcinogenic activity for BZP. Possible reasons for the aberrant results obtained by Kurokawa et al. (15) have been presented by Slaga (16), including issues related to excess shaving of the mice, pathologic criteria for scoring tumors as papillomas or carcinomas and decomposition of the BZP used in the study. Pelling et al. (17) and Pazzaglia et al. (18) observed a low incidence of skin tumors after long latencies in carcinogen-sensitive mice treated repetitively with BZP, however, the authors note that promotion of spontaneously ‘initiated epidermal’ cells could not be excluded. In summary, the weight of evidence indicates that BZP does not possess complete carcinogenic activity in the mouse skin model (14,1922).

For the studies described herein, mutations in codons 12, 13 and 61 of the c-Ha-ras oncogene were utilized as biomarkers of initiating events produced by complete carcinogens and tumor initiating agents. The tumor initiators/complete carcinogens selected as positive controls for this study have all been shown to induce mutations in the c-Ha-ras protooncogene (1416). Dosing regimens have been selected to reflect moderate and weak tumor initiating activity. 7,12-Dimethylbenz[a]anthracene (DMBA), which induces mutations in codon 61 of the c-Ha-ras gene (14,15), was tested at two doses expected to reflect strong or weak tumor initiation, i.e. repeated applications of 100 or 10 nmol DMBA, respectively. Even a single dose of 100 nmol DMBA can act as a complete carcinogen in SENCAR mice (15), whereas a single initiating dose of 10 nmol DMBA yields skin tumors only when followed by repetitive doses of a tumor promoter such as 12-O-tetradecanoylphorbol-13-acetate (TPA) (1518). Depending on the dosing regimen, urethane can act as a complete mouse skin carcinogen [i.e. 670 µmol twice per week (2x/wk)] or as a tumor initiator (i.e. a single dose of 670 µmol), yielding skin tumors only when followed by repetitive doses of a tumor promoter, such as TPA (1923).

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (B[a]P) were used as positive controls for mutations at the 12 and 13 codons of the c-Ha-ras protooncogene, respectively (2325). MNNG acts as a complete carcinogen in SENCAR mice when administered in five doses over the range 0.5–5.0 µmol/dose (25) and a single initiating dose (0.1–5.0 µmol) is effective in inducing papillomas in SENCAR mice when promoted by TPA for 45 weeks. Mutations are detected at codon 12 of the c-Ha-ras protooncogene (25). A single dose (200 nmol) of B[a]P followed by TPA promotion has been shown to produce mutations at codons 12 and 13 of the c-Ha-ras gene in SENCAR mice (23,24).

As stated by Lai et al. (1), the high cost and large number of rodents required for 2 year carcinogenicity bioassays provides an incentive to develop a battery of mechanism-based, short-term tests that can screen for or predict the potential promoting and/or carcinogenic activity of organic peroxides. Since dermal contact is one of the most likely routes of human exposure, the multistage carcinogenesis model using mouse skin represents a logical experimental choice. The research described herein examines the effects of 4–12 week topical exposure of the skin of SENCAR mice to a spectrum of organic peroxides, focusing on those cutaneous effects related to carcinogenic potential (1,6,7), namely effects on sustained epidermal hyperplasia, dermal inflammation, oxidative damage to skin DNA, and mutation of codon 12, 13 or 61 of the c-Ha-ras protooncogene. Of particular importance is evaluation of mutations in c-Ha-ras, since activation of this gene occurs early in the process of mouse skin carcinogenesis and is perhaps equivalent to an initiation event. Quintanilla et al. (23) demonstrated the presence of an activated c-Ha-ras gene in mouse skin papillomas and carcinomas induced by DMBA, the activation being associated with a high frequency of A->T transversions at codon 61. Subsequent studies demonstrated that mutation at codons 12, 13 and/or 61 was dependent upon the chemical initiator and independent of the promoter, suggesting a direct effect of the initiator on c-Ha-ras (24,25). Furthermore, infection of mouse skin by a virally activated Ha-ras gene (v-Ha-ras) can serve as the initiating event in two-stage carcinogenesis (26,27). It should be emphasized that all complete carcinogens and tumor initiators studied in the mouse skin model have been shown to produce a mutation in the Ha-ras protooncogene (6,27).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
The following peroxides were used in the study: BZP (lot no. JB-32-46, 75% pure), t-butyl peroxybenzoate (TBPB) (lot no. 603894/JAD-6-25, 98.0% pure) and methyl ethyl ketone peroxide (MEKP) (lot no. 512181/NT9-1069, 24.0% pure) from Norac Inc. (Azusa, CA); p-t-butyl isopropylbenzene hydroperoxide (TBIBHP) (lot no. 1-10181-4, 99%+ pure), CHP (lot no. 8458522101, 90.6% pure) and O,O-t-butyl-O-(2-ethylhexyl)monoperoxycarbonate (TBEC) (lot no. 9738518920, 96.8% pure) from Elf Atochem North America Inc. (Philadelphia, PA) (currently known as ATOFINA Chemicals Inc.); dicetyl peroxydicarbonate (DPD) (lot no. 95-011, 90.4% pure) and dicumyl peroxide (DCP) (lot no. 9511291, 99.4% pure) were obtained from Great Lakes Chemical Corp. (West Lafayette, IN) (formerly Akzo Nobel Chemicals Inc., Dobbs Ferry, NY); DTBP (lot no. 400-764K, 99.6% pure) was from Witco, Polymer Additive Group (Marshall, TX) (currently known as Crompton Corp.). All the above compounds were stored in an explosion proof refrigerator at 4°C.

The following control chemicals and vehicles were of the highest quality commercially available: DMBA (95.0% pure), urethane (99% pure), B[a]P (99.0% pure) and MNNG (99.0% pure) were obtained from Aldrich (Milwaukee, WI); TPA (98.5% pure) was from LC Laboratories (Woburn, MA).

Animals and treatment
Female virgin outbred SENCAR mice (5–6 weeks old) were purchased from the National Cancer Institute Laboratories (Frederick, MD) and housed by treatment group, up to 10 mice/cage. Temperature was maintained at 22 ± 4°C, with a relative humidity of 50 ± 20%. Temperature and humidity were recorded at least once daily. Controls were set to maintain a 12 h light/12 h dark cycle. There were 12 or more air changes per hour in the room where the study animals were housed. Mouse weight was used to randomly assign animals to treatment groups. Upon group assignment, the weight variation of animals did not exceed 2 SD from the mean body weights and the mean body weights for each treatment group were not statistically different. Animals (7–9 weeks old) entered into the study were shaved with surgical clippers at least 2 days before treatment. Shaving was performed in a manner to minimize skin abrasion (28). Only those in the resting phase of the hair cycle, i.e. animals that did not show any hair regrowth, were used in the study. Peroxides and control chemicals were applied to the interscapular/lumbar region of the back in a total volume of 200 µl. The experimental animal facility was accredited by the American Association for Assessment and Accreditation of Laboratory Animals and caging complied with Institutional Animal Care and Use Committee requirements.

All peroxides and control chemicals were applied topically in acetone, except MEKP, whose solubility properties dictated solubilization in dimethyl phthalate. Solubility of DPD in acetone limited its maximal dose to 4 µmol. To avoid doses that irritate the skin, a pre-screen of all organic peroxides was conducted, except for BZP, DTBP and DCP, for which prior experimentation (29) suggested appropriate maximal non-irritating doses (125, 200 and 200 µmol, respectively). Three mice were treated for 2 weeks (2x/wk) with 100 or 200 µmol TBIBHP, TBPB, CHP, MEKP or TBEC or with 2.0 or 4.0 µmol DPD. Skin irritation, determined visually, was limited to mice treated with TBPB and CHP. For TBPB, which exhibited skin irritation at the 200 µmol dose, 100 µmol was used as the maximal dose. For CHP, which exhibited skin irritation at the 100 µmol dose, 50 µmol was used as the maximal dose.

Experimental design
4 week study
All mentioned organic peroxides were evaluated for their ability to induce sustained epidermal proliferation, dermal inflammation and oxidative damage of epidermal DNA when applied topically for 4 weeks. Specimens of dosed skin were harvested 2 and/or 4 days after final dosing and increases in the mentioned parameters were compared with those produced by the appropriate vehicle and positive (100 nmol DMBA or 2 µg TPA) controls (see Table I for treatment conditions).


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Table I. Effects of organic peroxides on sustained epidermal hyperplasia, dermal inflammation and oxidative damage to DNA in skin of SENCAR mice treated topically with organic peroxides for 4 weeks

 
8 and 12 week studies
Selected organic peroxides (TBPB and TBIBHP) were also evaluated for their ability to induce increases in the above three parameters when topically applied for 8 or 12 weeks at the maximal doses stated above. Specimens of dosed skin were harvested 2 days after final dosing and increases in the mentioned parameters were compared with those produced by the vehicle or positive (DMBA or TPA) controls. Specimens of dosed skin were also evaluated for the occurrence of mutations in codons 12, 13 and 61 of the c-Ha-ras protooncogene. Mice dosed with MNNG, B[a]P, urethane or DMBA were used as controls for mutations in c-Ha-ras at codons 12, 13 or 61, respectively (2327,30) (see Table II for treatment conditions). When present, the incidence and multiplicity of skin tumors are reported.


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Table II. Mutation at codons 12, 13 and 61 of the Ha-ras protooncogene in SENCAR mice treated for 8 or 12 weeks with TBPB and TBIBHP and carcinogens (positive controls) in the complete carcinogenesis protocol

 
16 week initiation/promotion protocol
The occurrence of mutations in the c-Ha-ras protooncogene was also examined in mice treated with a single dose of TBPB (100 µmol) or TBIBHP (200 µmol), followed by 12 weeks of promotion by TPA (2 µg, 2x/wk). Mice dosed with single topical applications of MNNG, B[a]P, urethane or DMBA, followed by 12 weeks of TPA promotion, were used as controls for mutations in c-Ha-ras at codon 12, 13 or 61, respectively (see Table II for treatment conditions). Dosed skins, with tumors when present, were harvested 4 weeks following termination of TPA promotion. When present, the incidence and multiplicity of skin tumors are reported.

Morphological studies
Approximately 1 cm2 of dosed dorsal skin from each animal was preserved in formalin for slide preparation and the remainder of the dosed skin was rapidly frozen for isolation of DNA. Sustained epidermal hyperplasia was evaluated as previously described (28). Epidermal thickness was determined for each animal from at least 20 randomly selected sites of interfollicular epidermis, using formalin-fixed, paraffin-embedded sections (5 µm) stained with hematoxylin and eosin. Dermal cellularity was determined by light microscopic determination of the number of dermis-associated cells within 10 high magnification (1000x) fields of fixed, stained sections of skin from each mouse. Dermal cellularity, considered a measure of inflammation (6,29,31,32), is expressed as the mean for all mice in each treatment group. All dermis-associated cells were counted, including polymorphonuclear cells, lymphocytes, macrophages, fibroblasts and mast cells.

Detection of 8-hydroxy-2'-deoxyguanosine (8-OH-dG)
The 8-OH-dG content of DNA isolated from dosed mouse skin was used as a measure of treatment-related oxidative damage (2,4). DNA was isolated from freshly frozen skin of each mouse following non-phenol extraction and ethanol precipitation. Approximately 100 µg of isolated DNA was digested to nucleosides with nuclease P1 and alkaline phosphatase. Quantitation of nucleosides was accomplished by high performance liquid chromatography (LC-600/SPD-6A-CR4A HPLC System; Shimadzu Corp., Columbia, MA) (4,33,34) with electrochemical detection using an ESA system (ESA Inc., Chelmsford, MA). Normal bases were quantitated by liquid chromatography with UV detection. Data were expressed as pmol 8-OH-dG/105 pmol dG. All analyses were performed in duplicate or triplicate, with appropriate standard curves to correlate area units or peak height with concentration. Skin from mice treated with DMBA (100 nmol, 2x/wk for 4 weeks) served as the positive control and skin from vehicle-treated and untreated animals served as negative controls.

Detection of c-Ha-ras protooncogene mutations
The mouse skin carcinogen DMBA produces an A->T transversion in the second position of codon 61, i.e. CAA->CTA (2326). This mutation can be detected before the appearance of tumors using a very sensitive and specific mismatch-specific primer (MSP) assay with 32P labeling (26). This assay is based on the use of mismatch-specific PCR primers. Validation of the assay was conducted using a known mutant control, a wild-type control, a negative control (H2O) and a 100 bp ladder. DNA was isolated from freshly frozen mouse skins treated with the test compounds as well as the positive control, i.e. DMBA. PCR-based assays for the detection of ‘hot-spot’ mutations in the c-Ha-ras gene used MNNG and B[a]P as positive controls for mutations at codons 12 or 13, respectively (35). The assays used for the detection of mutations at codons 12 and 13 are less sensitive than the MSP assay for codon 61. The codon 61 assay utilizes a selective amplification approach to discriminate between wild-type and mutant DNA. Selective amplification is a result of the wild-type and mutant PCR occurring as separate reactions with two different reverse primers used for each. Under these conditions there is no competition for amplification between mutant and wild-type DNA and discrimination is a direct product of the PCR. In contrast, the codon 12 and 13 assays attempt to amplify both mutant and wild-type DNA in the same reaction. Discrimination occurs as a post-PCR process, which relies on the use of restriction endonuclease digestion for detection of a mutation. The variability of the digestion reaction, coupled with the absence of selective amplification, is responsible for the decreased sensitivity of the codon 12 and 13 assays. For the MSP assay of mutations at codon 61, threshold values to differentiate false and true mutation positives varied between the separate assays experiments, which represented separate treatment groups. This variation required the use of threshold values, determined by a solvent-only treatment group, specific for each separate assay. The variability of the threshold values is believed to be independent of the treatment and solely based on the variability of the two assay processes.

Briefly, subclones of mouse exon 1 (containing codons 12 and 13) and exon 2 (containing codon 61) of the c-Ha-ras gene were generated from three separate DNA templates isolated from skin tumors. DNA containing mutations in codon 12, 13 or 61 was prepared from mouse skin tumor tissues kindly supplied by Dr John DiGiovanni (The University of Texas, M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX) (30).

The exons containing the mutations were amplified from mutated template genomic DNA using appropriate intron primers. The mutations were verified by sequencing PCR products generated from these primers before subcloning. PCR products were then ligated into the pGEM-T vector for subcloning. Samples of c-Ha-ras codon 12, 13 and 61 mutant DNAs were subcloned in DH5{alpha} bacterial hosts and plasmid DNA was isolated from these subclones as positive control DNA. In order to increase our stock of these samples, 10 ml of LB medium with ampicillin (100 µg/ml) was inoculated with 100 µl of each of the samples. Samples were placed in a 37°C incubator/shaker. The DNA contained the following mutations: codon 61, murine, mutation CAA->CTA, pGEM-T vector, Escherichia coli DH{alpha} carrier; codon 12, murine, mutation GGA->GAA, pGEM-T vector, E.coli DH{alpha} carrier; codon 13, murine, mutation GGC->GTC, pGEM-T vector, E.coli DH{alpha} carrier.

Codon 12 primers produce a fragment which, if a CAA->CTA tranversion is present, creates a XmnI restriction site GAAN2{downarrow}N2TTC. Thus, digestion of the PCR product with XmnI produces a smaller fragment that runs faster on the polyacrylamide gel than the wild-type product and is not restricted by XmnI. For the codon 13 analysis, the restriction enzyme BglI [GCCN4{downarrow}NGGC] was used to restrict the PCR product which is produced using a different primer set than that used for codon 12. The unrestricted, larger product is indicative of a mutation at any position in codon 13.

The very sensitive MSP assay used for detection of mutations at codon 61 of the c-Ha-ras protooncogene occasionally produces equivocal positives (very faint positive bands from single samples that had been treated with vehicle). The false positives in control samples were quantified by measuring integrated optical densities and expressed as mean percent of the c-Ha-ras wild-type reaction. That mean + 2 SD (3.0%) served as a basis for calculating a significance threshold. Any value >3.0% of the c-Ha-ras wild-type reaction was considered a true mutation.

Statistical analysis of tumor promotion-associated biomarkers
To determine which treatment groups differed from their appropriate controls, a one-way ANOVA was performed on each measured parameter. When the treatment means were determined to be significantly different (P < 0.05), the means were compared using a Tukey honestly significant difference ranked mean comparison test (36). Since the acetone and untreated controls were not significantly different, they were pooled to give a single limit. This limit is based on a one-sided t-test. The pooling of these two control groups increases the number of degrees of freedom in the pooled controls while not significantly increasing the variance and standard deviations and, thus, increases the sensitivity of the test. All values having a superscript d (d) in Table I were deemed significantly different (P < 0.05) from the appropriate control.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Body weight
The mean weights of the animals in all treatment groups in the 4, 8 and 12 week studies were analyzed at day 2 after final treatment by one-way ANOVA. No statistically significant differences in body weight gain were observed between any of the groups at any time point.

Study using 4 week exposures to organic peroxides
Four week exposures were adequate to demonstrate the effects of organic peroxides on sustained epidermal hyperplasia, dermal cellularity and oxidative damage to DNA. As shown in Table I the organic peroxides varied in their effects on the mentioned parameters.

The sensitivities of the assays to detect mutations in c-Ha-ras were adequate to detect only mutations at codon 61 in the study using 4 week exposure. Ninety percent (9/10) of the mice treated with 100 nmol DMBA exhibited mutations in codon 61 of c-Ha-ras, while none of the organic peroxides produced such mutations. Based on this 4 week study, TBPB and TBIBHP, two organic peroxides that produced increases in sustained epidermal hyperplasia, dermal cellularity and oxidative damage to DNA, were selected for study under conditions more favorable to the expansion of any initiated populations of epidermal cells.

Study using 8 and 12 week exposures to TBPB and TBIBHP and to carcinogens used as positive controls
The results of experimentation to detect mutations in c-Ha-ras are shown in Table II. Mutations in codon 61 were detected after 8 weeks exposure in five of five mice treated with 100 nmol DMBA (2x/wk). No mutations in codons 12 and 13 were detected in DNA isolated from mice treated with B[a]P (50 or 200 µmol, 2x/wk) or with the highest dose of MNNG (1 µmol, 2x/wk). No mutations were noted in mice treated with the organic peroxides selected for further evaluation, TBPB and TBIBHP. Skins from mice treated with these two organic peroxides were also evaluated for their effects on sustained epidermal hyperplasia, dermal cellularity and oxidative damage to DNA. Treatment-related effects on the three parameters were consistent with those observed after 4 weeks treatment. After 12 weeks treatment, mutations in codon 61 were detected in groups receiving the following treatments: 10 nmol DMBA (2x/wk), one of five mice; 100 nmol DMBA, (1x/wk), four of five mice; 100 nmol DMBA (2x/wk) five of five mice; 670 µmol urethane (2x/wk), one of five mice. No mutations in codon 61 were detected at the highest doses of TBPB or TBIBHP (2x/wk). Mutation in codon 12 of the Ha-ras oncogene was detected in one of five mice treated with 1 µmol MNNG (2x/wk) and mutation in codon 13 was detected in three of five mice treated with 200 µmol B[a]P (2x/wk). The codon 12 mutation detected in the MNNG group was weak, but nevertheless clearly visible. In the case of B[a]P, the bands indicating mutations at codon 13 were readily detected. None of the DNA from mice treated with the highest doses of TBPB or TBIBHP (2x/wk) exhibited mutations in codon 12 or 13 of the Ha-ras oncogene. It should be noted that mice treated with 100 nmol DMBA (2x/wk) developed papillomas on the dosed skin, exhibiting tumor multiplicities of 2 and 9.6 tumors/mouse at 8 and 12 weeks, respectively. No skin tumors were observed in any other treatment groups.

16 week initiation/promotion protocol using TBPB and TBIBHP
To enhance expansion of any initiated epidermal cells, mice were treated with a single ‘initiating’ dose of TBPB or TBIBHP and then promoted with TPA for 12 weeks, using the carcinogen DMBA, B[a]P, MNNG or urethane as a positive control (see Table III for doses). Treated skin or individual skin tumors (papillomas) appearing on the treated skin 4 weeks after the final dose of TPA were evaluated for mutation of c-Ha-ras (Table III). The tumor incidence and tumor multiplicity data as well as percent of papillomas showing mutations in the Ha-ras protooncogene are shown in Table III. No mutations of c-Ha-ras or tumors were detected in mice treated with a single ‘initiating’ dose of TBPB or TBIBHP and promoted with TPA.


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Table III. Mutations at codons 12, 13 and 61 of the Ha-ras protooncogene in SENCAR mice treated with TBPB and TBIBHP in an initiation/promotion protocol

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This report explores the ability of nine organic peroxides of differing structural types to increase early biomarkers of tumor initiation and promotion in mouse skin. The biomarkers chosen for study are those suggested earlier by Slaga et al. (29,31,32) and others (1) as having predictive value in short-term assays, i.e. sustained epidermal hyperplasia, dermal inflammation and oxidative damage of skin DNA associated with tumor promotion and mutations in the c-Ha-ras protooncogene associated with tumor initiation.

The ability of a wide variety of structurally diverse complete carcinogens and tumor promoters, including organic peroxides, to produce sustained epidermal hyperplasia in mouse skin lends credibility to this early biomarker as a qualitative predictor of tumor promoting activity (6,37,38). BZP and lauryl peroxide, two organic peroxides that exhibit tumor promoting activity in an initiation (DMBA)/promotion protocol, have been shown to produce sustained epidermal hyperplasia 2–6 days following a single topical application to mouse skin (12). Based on the induction of sustained epidermal hyperplasia, the following organic peroxides are predicted to possess tumor promoting activity: BZP, TBPB, TBIBHP, CHP, DCP and MEKP (see Table I). Tumor promoting activity for three of these six organic peroxides has been demonstrated in two-stage initiation/promotion protocols using mouse skin: BZP (11,12,18) and DCP (39) in a protocol using DMBA as initiator and MEKP (40) in a protocol using UV light (UVB) as initiator.

Tumor promoting activity in mouse skin is accompanied in many cases by dermal inflammation and prooxidant effects (6), suggesting that such effects may contribute to epidermal hyperplasia or be additive/synergistic to epidermal hyperplasia. Accumulating evidence indicates that tumor promoters, including organic peroxides, produce dermal inflammation and prooxidant effects in mouse skin and keratinocytes, justifying investigation of such effects for the nine organic peroxides included in this study. Treatment of mouse skin with TPA, for example, is accompanied by localized edema, erythema and infiltration of leukocytes into the dermis (6), while anti-inflammatory steroids inhibit TPA-induced promotion (39). BZP and lauryl peroxide produce dermal inflammation, albeit weak, in SENCAR mice (12). As shown in Table I, BZP, TBPB, TBIBHP, CHP and TBEC exhibited sig- nificant increases in dermal cellularity, a biomarker of dermal inflammation.

The involvement of prooxidants (reactive oxygen species) in tumor promotion in mouse skin is indicated by several lines of evidence. BZP has been shown to produce strand breaks in DNA in mouse epidermal cells (29,41,42), an action presumed to be mediated by free radical derivatives of BZP (43). Using electron paramagnetic resonance spin trapping, Timmins and Davies demonstrated (44) that exposure of mouse keratinocytes to TBPB, CHP, t-butyl hydroperoxide and ethyl hydroperoxide was accompanied by the appearance of free radicals, and their production could be decreased by exposure to antioxidants, i.e. butylated hydroxyanisole or butylated hydroxytoluene. Furthermore, these same antioxidants are reported to inhibit TPA-induced promotion in mouse skin (39). As shown in Table I, only BZP, TBPB and TBIBHP exhibited significant increases in oxidative damage to skin DNA.

In summary, the nine organic peroxides varied in their effects on the three biomarkers associated with tumor promotion: BZP, TBPB and TBIBHP produced increases in all three biomarkers; CHP produced increases only in sustained epidermal hyperplasia and dermal inflammation; MEKP and DCP produced increases only in sustained epidermal hyperplasia; TBEC produced an increase only in dermal inflammation. DTBP and DPD had no effect on the three parameters studied. It should be emphasized that the topical effects of the organic peroxides, when observed, occurred only at high exposures. For example, on a molar basis the effect of BZP or TBIBHP on sustained epidermal hyperplasia was at least four orders of magnitude less than that produced by TPA, an observation consistent with the low promoting activity of BZP in a two-stage initiation (DMBA)/promotion protocol in mouse skin, relative to that of TPA (12,18). Mutations in codon 61 of c-Ha-ras were not observed in mice treated for 4 weeks with any of the organic peroxides.

Based on the observation that TBPB and TBIBHP exhibited effects on all three biomarkers of tumor promotion, these two organic peroxides were selected for investigation of their ability to produce mutations in the c-Ha-ras protooncogene. The experimental premise was that detection of such mutations could serve as a biomarker of their tumor initiating activity or, alternatively, that absence of such mutations would provide evidence that these organic peroxides lack tumor initiating activity involving mutation of the c-Ha-ras protooncogene. Although mutations in codon 61 of c-Ha-ras were detected in skin of mice treated with DMBA (2x/wk) for 8 weeks, no mutations were detected in skin from mice treated with TBPB or TBIBHP (2x/wk) for 8 weeks (Table II). The sensitivities of the assays to detect mutations at codons 12 and 13 of c-Ha-ras were insufficient to detect such mutations in mice treated (2x/wk) for 8 weeks with the positive controls, i.e. MNNG and B[a]P, respectively, and, consequently, longer exposures were undertaken. Mutations in codons 12, 13 and 61 of c-Ha-ras could be detected in skin/skin tumors harvested from mice treated (2x/wk) for 12 weeks with the positive controls, i.e. MNNG, B[a]P, urethane and DMBA, respectively, however, no mutations were detected in skin from mice treated with TBPB or TBIBHP (2x/wk) for 12 weeks (Table II).

To ensure sufficient expansion of any cells initiated by the two organic peroxides, mice were treated with ‘initiating’ doses of TBPB or TBIBHP and then promoted for 12 weeks with TPA (see Table III). Again, no mutations in codon 12, 13 or 61 of Ha-ras were detected in mice treated with ‘initiating’ doses of TBPB or TBIBHP (Table III). The skin/skin tumors harvested from mice treated with initiating doses of MNNG, B[a]P or DMBA and promoted with TPA exhibited appropriate mutations in c-Ha-ras (Table III).

In conclusion, these data indicate that TBPB and TBIBHP are not likely to possess tumor initiating or complete carcinogenic activity in the mouse skin model and that increases in short-term biomarkers (sustained epidermal hyperplasia, dermal cellularity and/or oxidative damage of skin DNA) may serve as useful predictors of the tumor promoting potential of these and other organic peroxides.


    Notes
 
4 To whom correspondence should be addressed at: 1600 Pierce Street, Denver, CO 80214, USA Email: hanausekm{at}amc.org Back

{dagger} Declarations of interest: Dermigen, the employer of E.F.Walborg Jr, received compensation for its contribution to experimental design and project oversight. A.Tveit (who was not directly involved in the generation of experimental data) holds a few shares of stock in TOTAL—the parent company of ATOFINA Chemicals Inc., her employer and a member of OPPSD. Back


    Acknowledgments
 
This research was funded in part by the Organic Peroxide Producers Safety Division of the Society of the Plastic Industry Inc., Washington, DC.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 24, 2003; revised October 29, 2003; accepted November 4, 2003.





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