Metallothionein deficiency enhances skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate in metallothionein-null mice

Junko S. Suzuki1, Noriko Nishimura1, Baoxu Zhang1,2, Yoko Nakatsuru1,3, Shizuko Kobayashi4, Masahiko Satoh1,5 and Chiharu Tohyama1,6

1 Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan
2 Department of Toxicology, School of Public Health, Beijing University, Beijing 100083, China
3 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
4 Department of Biology, Kyoritsu College of Pharmacy, Tokyo 105-8512, Japan
5 Department of Hygienics, Gifu Pharmaceutical University, Gifu 502-8585, Japan

6 To whom correspondence should be addressed Email: ctohyama{at}nies.go.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To clarify the physiological role(s) of metallothionein (MT) in carcinogenesis, we studied the susceptibility of MT-null mice to chemically mediated carcinogenesis in the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage carcinogenesis model. The MT-null mice were subjected to a single topical application of DMBA (50 or 100 µg/mouse) and, 1 week later, to promotion with TPA (10 µg/mouse) twice a week for 20 weeks. At week 21, nearly all of the MT-null mice developed tumors in the skin, in contrast to only 10–40% of wild-type mice. No tumors were observed in MT-null or wild-type mice that were administered TPA alone. By using the PCR–restriction fragment length polymorphism and PCR–single strand conformation polymorphism methods, we found a transversion of A182 to T in codon 61 of c-Ha-ras in the papilloma tissue of MT-null mice and wild-type mice but failed to find any mutations in the c-Ki-ras and c-N-ras genes. In two-stage skin carcinogenesis induction by DMBA/TPA, p53 and p21WAF1/Cip1 expression levels were found to be increased in MT-null mice compared with wild-type mice. As to an earlier change at the promotion stage triggered by TPA application, MT-null mice were found to have both hyperplasia of the epithelium and a marked degree of inflammation in the basal layer, indicating that the induced as well as endogenous MT acted as a protective factor against tumorigenesis. In conclusion, the present study has demonstrated that MT has antitumorigenic potential in both the initiation and promotion stages of the two-stage chemical skin carcinogenesis model.

Abbreviations: BrdU, 5'-bromo-2'-deoxyuridine; DMBA, 7,12-dimethylbenz[a]anthracene; MT, metallothionein; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; PCR–RFLP, PCR–restriction fragment length polymorphism; PCR–SSCP, PCR–single strand conformation polymorphism; TPA, 12-O-tetradecanoylphorbol-13-acetate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Metallothionein (MT) is a low molecular mass (Mr {approx} 6000) metal-binding protein that is inducible by endogenous and exogenous stimuli such as heavy metals and cytokines (1,2). The protein consists of 61–68 amino acids, depending upon the isoform, one third of which are cysteine residues without an intramolecular disulfide bond. The cysteine residues are responsible for coordinately binding heavy metals such as cadmium and zinc ions. Among the four isoforms reported thus far, MT-I and MT-II, the major isoforms, are induced upon exposure to heavy metals and are known to have detoxifying properties (3). In addition, the protein is thought to have homeostatic properties in the maintenance of essential heavy metals such as copper and zinc ions (4). It has also been shown that the promoter region of the MT gene harbors an antioxidant response element which is partly responsible for inducing MT by interacting with an upstream stimulating factor whose sequence overlaps the antioxidant response element (5). The possible physiological role of MT is due to its sulfhydryl groups, which are thought to act as scavengers for active oxygen species generated by mutagens, antineoplastic drugs (6) and radiation (7). Nevertheless, the MT inducers themselves may play a protective role, which makes it difficult to delineate the physiological role of constitutively expressed MT.

Regarding chemically induced skin cancer, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) have been widely utilized as an initiator and a promotor, respectively, in the two-stage chemical carcinogenesis model. As a first step, DMBA will react with DNA to form an adduct, leading to mutation of a particular gene (8). The pattern of mutation in the skin differs for several types of carcinogens, such as, N-methyl-N'-nitro-N-nitrosoguanidine (9), benzo[a]pyrene and DMBA (9,10); it has been established that DMBA used as a cancer initiator tends to cause mutations in the c-Harvey(Ha)-ras gene (1113). Besides the c-Ha-ras gene, two other ras genes, Kirsten (Ki) and neuroblastoma (N), are present on different chromosomes. Mouse skin tumors contain activated c-Ha-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2 (14). There are two forms of ras p21 protein: the GTP-bound form is active whereas the GDP-bound form is inactive (15). The GTP-bound form transmits the signal from upstream proliferative stimuli, resulting in transformation of the GTP-bound form into the GDP-bound by intrinsic GTPase activity of ras p21 protein. Yet, this activity is so weak that it appears insufficient to maintain p21 in the GDP-bound form under physiological conditions. Another protein with GTPase activity (GAP) catalyzes conversion of p21-GTP to p21-GDP such that this reaction proceeds at a rate more than 100 times higher than the intrinsic rate (16). Since GAP has no effect on mutated oncogenic p21 proteins having mutations at positions 12, 13, 59 and 61, the mutated proteins remain as the GTP-bound form, continuously transmitting signals downstream to induce non-orchestrated cellular proliferation (17). In the mouse skin carcinogenesis model with DMBA/TPA, the most conspicuously found mutation has been reported to be that in codon 61, but how MT is involved in this process has not been reported. Furthermore, there is the possibility of mutations in the other two ras genes besides the c-Ha-ras gene.

In damaged genes that escape repair the mutation will be amplified by the promoting activity of TPA followed by abnormal growth of cells with the mutated genes. Since anti-oxidants delay the carcinogenesis process, the involvement of oxidative stress in carcinogenesis has been suggested (18,19). As biomarkers of DNA damage we utilized the levels of expression of the p53 and p21WAF1/Cip1 genes. p53 is known to be responsible for rendering damaged cells apoptotic, which lessens the accumulation of mutations, which would otherwise eventually lead to a cancerous state (20,21). On the other hand, elevated expression of the p21WAF1/Cip1 gene, which is regulated by p53 tumor suppressor protein, is known to be responsible for inhibition of cell growth at G1/S upon exposure to DNA-damaging agents, which may allow cells to repair mutated genes (2224). Thus, analysis of the expression of the two genes is thought to reflect the state of DNA damage.

In the present study, we aimed to clarify how MT functions in the DMBA/TPA-induced two-stage carcinogenesis protocol by utilizing MT-null mice to identify mutations in the ras family and to demonstrate histopathologic changes with special reference to inflammation and DNA damage.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
DMBA, TPA and 5'-bromo-2'-deoxyuridine (BrdU) were purchased from Sigma Chemical Co. (St Louis, MO). Dexpat (DNA extraction from paraffin-embedded tissue) was purchased from TAKARA (Otsu, Japan). Pfu DNA polymerase for PCR was obtained from Stratagene (La Jolla, CA). The GFX PCR DNA and Gel Band Purification kit was purchased from Amersham Biosciences (Uppsala, Sweden). The electrophoretic analyses were carried out on a GenePhor electrophoresis unit using the GeneGel Clean 15/24 kit. Staining was performed in a GeneStain automated gel stainer using a PlusOne DNA silver staining kit (Amersham Biosciences). The Big Dye terminator cycle sequencing kits for sequencing analysis were obtained from Applied Biosystems (Foster City, CA). Xylene, acetone, hydrochloric acid, sodium hydroxide and 10% neutral-buffered formalin solution were obtained from Wako Pure Chemical Industries (Osaka, Japan). Trypsin, DAKO Liquid DAB+ Substrate-Chromogen, DAKO Protein Block Serum-Free and mouse monoclonal anti-MT (E9) were purchased from DAKO Japan (Kyoto, Japan). Mouse monoclonal anti-BrdU (Becton Dickinson, San Jose, CA), rabbit anti-mouse p53 IgG (CM5; Novocastra Laboratories, UK) and goat anti-human p21 (WAF1-Cip1) IgG (SC397-G; Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used for immunostaining. Avidin–biotin–peroxidase complex immunostaining reagents (Vectastain Elite ABC kit) and Mouse on Mouse (M.O.M.) Immunodetection kit were purchased from Vector Laboratories (Burlingame, CA). The Histfine streptavidin–biotin–peroxidse (SAB-PO) kit was obtained from Nichirei (Tokyo, Japan).

Animals and tumor induction protocols
MT-null and wild-type (129/Ola x C57BL/6 J) mice were kindly provided by Dr A.Choo (25). Female C57BL/6 J mice were purchased from CLEA Japan (Tokyo, Japan). These original mice were mated with C57BL/6 J mice for generation after generation to establish inbred MT-null mice with a C57BL/6 J background. The mice were housed in screen-bottomed stainless steel cages kept in a stainless steel ventilation cabinet. The animal facility was maintained with a 12 h light/dark cycle, a temperature of 24 ± 2°C, a relative humidity of 55 ± 10% and a negative atmospheric pressure. The mice received mouse chow and filtered tap water ad libitum. Female mice, 8–10 weeks of age, were used at the start of this experiment according to the guidelines for animal welfare of the National Institute for Environmental Studies.

Mice were shaved on their dorsal part with surgical clippers 2 days before the experiment. In the long-term carcinogenicity experiment (10 mice/group) each mouse was topically administered a single dose of DMBA (50 or 100 µg in 0.1 ml of acetone) and then, 1 week later, repeated doses of TPA (10 µg in 0.2 ml of acetone, twice per week) for 20 weeks. The number of skin tumors that were >1 mm in diameter were counted. Control mice received acetone instead of DMBA or TPA.

In the short-term experiment one group of mice (3–4 mice/group) was topically administered a single dose of DMBA (50 µg in 0.1 ml of acetone) on the shaved area of the skin and the skin tissue was collected 3, 7 and 13 days post-administration. Another group of mice was topically administered TPA (10 µg in 0.2 ml of acetone) 7 and 10 days post-administration of DMBA and the skin tissue was collected 13 days post-administration of DMBA. To study cellular proliferation, BrdU was injected i.p. at a dose of 100 mg/kg, 2 h before the collection of skin tissue.

Amplification of c-Ha-ras, c-Ki-ras and c-N-ras genes
Genomic DNA was isolated from papilloma tissue sections using Dexpat and processed according to the manufacturer's suggested protocol. To amplify exons 1 and 2 of the c-Ha-ras, c-Ki-ras and c-N-ras genes, the primers for c-Ha-ras, c-Ki-ras and c-N-ras gene amplification were designed according to the published sequences (26–28; Table I). Nested PCR was performed due to the minute quantity of extracted DNA from sections of tumor specimens as well as to amplify each ras gene with high fidelity (Table I).


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Table I. Sequence of oligonucleotide primers used for PCR amplification and sequencing

 
PCR was carried out in a 25–100 µl reaction mixture using Pfu DNA polymerase. The first PCR was carried out for 22 cycles, which comprised 15 s at 94°C, 30 s at 55°C and 1 min at 74°C. An aliquot of the first PCR products was used for the second PCR reaction. The second PCR reaction was carried out for 33 cycles, which comprised 15 s at 94°C, 30 s at 55–60°C and 1 min at 74°C. The final PCR products were purified using a GFX PCR DNA and Gel Band Purification kit and spectrometrically quantified.

PCR–restriction fragment length polymorphism (PCR–RFLP)
The A182->T transversion of codon 61 of c-Ha-ras was analyzed by PCR–RFLP with some modification (29). The amplified c-Ha-ras exon 2 (the codon 61 flanking sequences) was digested with XbaI, which only cleaves if there is transversion of the middle A of codon 61 to T. An aliquot (5 µl) of the second PCR product was used for XbaI digestion. The samples were digested with restriction enzyme, separated by 4% agarose gel electrophoresis, stained with ethidium bromide and visualized with UV light.

PCR–single strand conformation polymorphism (PCR–SSCP)
The PCR–SSCP method was used for the analysis of several mutations in each ras gene (30). PCR products (10–15 ng) were dissolved in 3.6 µl of TE and 3.6 µl of 95% formamide containing 0.025% bromophenol blue and 0.025% xylene cyanol. The solution was heated at 95°C for 5 min and immediately placed on ice. The denatured sample (6 µl) was applied onto GeneGel Clean 15/24 (15% T, 2% C). First, electrophoresis was carried out at 7°C, 200 V, 12 mA for 15 min, followed by 600 V, 15 mA for 90 min using GenePhor electrophoresis units (Amersham Biosciences). The gels were stained in a GeneStain Automated Gel Stainer with a PlusOne DNA Silver Staining Kit according to the manufacturer's instructions.

DNA sequence
The mutated genes were analyzed with a Prism 310 automated sequencer (Applied Biosystems). The sequencing was performed with appropriate primers (listed in Table I) and a Big Dye terminator cycle sequencing kit. The majority of mutations found in a DNA strand were confirmed on the complementary strand by sequencing the other way around.

Histology and immunohistochemistry
Skin specimens were fixed in neutral buffered formalin solution and processed for paraffin embedding. Skin sections (4 µm in thickness) were prepared and placed either on a MAS-coated glass slide (Matsunami Glass Industries, Osaka, Japan) and deparaffinized using xylene. For MT staining, tissue sections were treated with 3% hydrogen peroxide solution for 10 min at room temperature. Mouse anti-MT antibody (dilution 1:300) was applied to each section overnight at 4°C after masking mouse IgG according to the protocol of the M.O.M. kit. Biotinylated anti-mouse IgG was used as the secondary antibody (dilution 1:250) and the immunoreaction was visualized by the avidin–biotin–peroxidase complex immunostaining method using diaminobenzidine as the substrate.

For BrdU staining, tissue sections were treated with 3% hydrogen peroxide for 10 min, 1 N HCl for 30 min and neutralized with 1 M Tris buffer, pH 7.5, followed by digestion with 0.1% trypsin for 5 min at room temperature. After masking mouse IgG as described above, anti-BrdU antibody (dilution 1:20) was allowed to react for 60 min at room temperature.

For p53 and p21WAF1/Cip1 immunostaining, tissue sections were incubated in 10 mM citrate buffer, pH 6.0, for 30 s in a boiling bath, followed by treatment with 3% hydrogen peroxide for 10 min and DAKO Protein Block Serum-Free for 10 min at room temperature. Antibodies against p53 (dilution 1:4000) and p21WAF1/Cip1 (1:2000) were allowed to react with tissue sections at 4°C overnight. For p53 staining biotinylated anti-rabbit IgG (dilution 1:75) was allowed to react for 30 min at room temperature and Vectastain Elite ABC reagent and diaminobenzidine were used for the color reaction. For p21WAF1/Cip1 staining, biotinylated anti-goat IgG (10 µg/ml) was allowed to react for 10 min at room temperature and Histfine was used as the color reagent. For both immunostained tissue sections, Mayer's hematoxylin was used for counterstaining. The control section was treated with normal mouse serum or normal rabbit serum instead of each antibody.

Statistical analysis
Differences in tumor incidence between MT-null mice and wild-type control mice were analyzed by {chi}2 test and considered statistically significant at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of tumors in the skin by DMBA/TPA in the two-stage chemical skin carcinogenesis model
MT-null and wild-type mice (10 mice/dose group) were topically administered specified doses of DMBA on their dorsal skin, followed by a topical application of TPA (10 µg/mouse) 1 week later to the dorsal skin, and twice a week thereafter (Figure 1). Tumor tissues >1 mm in diameter were examined in further analyses. In DMBA/TPA-treated MT-null mice there was a dose-dependent increase in the occurrence of skin tumors: MT-null mice were found to be extremely susceptible to DMBA/TPA insult; the occurrence of tumors was observed as early as week 9 after TPA treatment and 90% of the MT-null mice given 50 µg DMBA/mouse had tumors at week 13. In addition, all the MT-null mice that were administered 100 µg DMBA/mouse had tumors by week 19. In contrast, only 10% of wild-type mice given a dose of 50 µg DMBA/mouse developed tumors at week 20. After topical administration of 100 µg DMBA/mouse wild-type mice started to manifest tumors as early as week 14, and 40% had tumors at week 20.



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Fig. 1. Incidence of skin tumors in response to DMBA/TPA topically applied to MT-null and wild-type mice. Female MT-null and wild-type mice were administered a single topical dose of 50 or 100 µg DMBA/mouse, followed, after a 1 week interval, by topical application of 10 µg TPA/mouse, twice a week, for 20 weeks. The treatment groups were as follows: wild-type mice treated with acetone vehicle (open square); wild-type mice treated with 50 µg DMBA/mouse (open triangle); wild-type mice initiated with 100 µg DMBA (open circle); MT-null mice treated with acetone (solid square); MT-null mice initiated with 50 µg DMBA/mouse (solid circle); MT-null mice treated with 100 µg DMBA/mouse (solid triangle).

 
Table II summarizes the number of tumors found in the mouse skin. As to the number of tumors per mouse, MT-null mice that were given 50 and 100 µg DMBA/mouse had 60 and 12 times more tumors, respectively, than wild-type mice (P < 0.05). Administration of TPA alone did not cause tumors in either MT-null or wild-type mice.


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Table II. Elevated incidence of skin tumor in DMBA/TPA-treated MT-null mice

 
Regarding the severity of tumors, those found in 4 of 10 wild-type mice treated with 100 µg DMBA/mouse were small (<4 mm diameter), with an average of ~0.6 tumors/mouse. Similarly, 1 of 10 wild-type mice given 50 µg DMBA/mouse had small (<2 mm diameter) tumors. In contrast, MT-null mice treated with 50 or 100 µg DMBA/mouse showed a tendency to have large tumors (>4 mm diameter). The incidence of tumors by size is significantly different from wild-type control mice (Table III).


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Table III. Increased incidence of tumors by size in DMBA/TPA-treated MT-null mice

 
Mutation in ras oncogenes
We examined mutations of the c-Ha-ras, c-Ki-ras and c-N-ras genes of DNA extracted from histologically diagnosed papilloma specimens in MT-null and wild-type mice. First, we performed PCR–RFLP analysis on the extracted DNA to examine transversion of A182 to T in codon 61, a hot-spot in c-Ha-ras. Although exon 2 of the c-Ha-ras gene does not harbor a recognition site for XbaI, a change of A182 to T produced a recognition site for this endonuclease, leading to digestion of the mutated c-Ha-ras into two fragments (Figure 2A).



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Fig. 2. Characterization of mutations in c-Ha-ras exon 2 from DMBA/ TPA-induced papilloma. Genomic DNA extracted from mouse skin papilloma (Papilloma) or normal skin (N), treated with acetone, was amplified by PCR with primers specific to c-Ha-ras exon 2 (Materials and methods). (A) PCR–RFLP method. The transversion c-Ha-ras A182->T in codon 61 was detected by a specific restriction enzyme. The PCR products were digested with XbaI to separate the normal (117 bp band) and mutant alleles (56 and 61 bp bands) by agarose gel electrophoresis. (B) PCR–SSCP analysis. The PCR products were denatured using formamide to produce a single strand and gel electrophoresed under non-denaturing conditions, followed by staining with a silver staining kit. The papilloma specimens exhibited an additional band (black arrow, bottom) due to mutation in addition to the band found in normal tissue specimens (gray arrows, upper three).

 
The PCR–RFLP method is an appropriate one to detect point mutations, but in order to examine other possible mutations we designed combinations of endonucleases that were specific to each mutation pattern. Thus, we studied by PCR–SSCP whether mutations existed in exon 2 of the c-Ha-ras gene, which showed a single band with a difference in mobility when compared with control DNA extracted from normal skin tissue (Figure 2B). The sequence of the mutated DNA was proved to have a transversion of A182 to T in codon 61 of the c-Ha-ras gene (data not shown). Next, we searched exons 2 of c-Ki-ras and c-N-ras using PCR–SSCP, but failed to detect any mutation. Furthermore, we performed PCR–SSCP analysis on exons 1 of the c-Ha-ras, c-Ki-ras and c-N-ras genes, focusing upon codons 12 and 13, another plausible hot-spot in addition to codon 61, but did not find mutations there either (data not shown).

The results for the mutations found in the papillomas of MT-null and wild-type mice are summarized in Table IV. Papillomas of MT-null mice that were administered DMBA/TPA had the A182->T transversion in 87% of cases. Although the tumor incidence due to DMBA/TPA administration in the wild-type mice was very low, all four papilloma specimens collected from the four wild-type mice had the same mutation in the c-Ha-ras gene as the MT-null mice. No mutations were found in other ras genes.


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Table IV. Mutation of c-Ha-ras gene in papillomas of DMBA/TPA-treated MT-null micea

 
Alterations in skin tissues in the initiation stage due to DMBA administration
We addressed the question of why a deficiency of MT-I and MT-II facilitates the incidence and size of papillomas and we studied the skin of DMBA-administrated mice histologically at an early stage of initiation, i.e. 3, 7 and 13 days post-administration of DMBA at a topical dose of 50 µg/mouse (Figure 3). As to cellular proliferation, no difference was observed 3 days post-administration of DMBA in the epithelium and hair follicles of both wild-type and MT-null mice (Figure 3B and D), with a decrease in the numbers of cells that had incorporated BrdU by 13 days post-administration (data not shown).



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Fig. 3. Immunohistochemical staining for BrdU, p53 tumor suppressor protein and p21WAF1/Cip1 protein in dorsal skin obtained from DMBA-treated MT-null and wild-type mice. The mice were administrated DMBA at a topical dose of 50 µg/mouse and killed on days 0 (A, C, E, G, I and K) and 3 (B, D, F, H, J and L) post-administration of DMBA. In the BrdU analysis the mice were injected with 100 mg BrdU/kg, 2 h before death. Wild-type (A and B) and MT-null (C and D) mice were stained with antibody to BrdU. In the analysis of p53 protein expression wild-type (E and F) and MT-null (G and H) mice were stained with antibody to nuclear protein p53. With regard to the relatively high background staining even in the control mouse skin, we have confirmed that this is due to non-specific staining as using the secondary antibody alone without the primary antibody against p53 produced a reaction with the horny layer of the epidermis (data not shown). The anti-p53 antibody used in the present experiment is confirmed to react with the protein in the nucleus and thus the staining in the nucleus indicates the presence of p53 in this organelle. In the analysis of p21WAF1/Cip1 protein expression wild-type (I and J) and MT-null (K and L) mice were stained with p21WAF1/Cip1 antibody. Bar = 100 µm.

 
Among the total epithelial cells, we analyzed the percentage of BrdU-positive cells until 13 days post-administration of DMBA. Transient cell damage due to topical application of DMBA was similar in both strains of mice, as there was no difference in the ratio of BrdU-positive cells between wild-type and MT-null mice by 7 days post-administration (Figure 4A). On the other hand, recovery from the damage was significantly different between them. The percentage of BrdU-positive cells 13 days post-administration of DMBA remained 11% in MT-null mice but at the control level in wild-type mice, indicating an induction of cell proliferation, a marker for cell damage, that was prolonged in MT-null mice much longer than in wild-type mice.



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Fig. 4. Effects of DMBA on cell proliferation (BrdU incorporation) and the expressed levels of p53 and p21WAF1/Cip1 proteins in MT-null and wild-type mice. Ratios of the numbers of either BrdU-, p53- or p21WAF1/Cip1-positive cells to total epithelial cells in immunohistochemically stained dorsal skin sections were estimated and the changes with time are shown: (A) ratio of BrdU incorporation; (B) ratio of p53-positive cells; (C) ratio of p21WAF1/Cip1-positive cells. The data represent means ± SE of 3–4 experiments. Asterisks indicate statistical significance at P < 0.01 from the control (day 0) by Student's t-test.

 
p53 tumor suppressor protein was detected in the epithelium and hair follicles in both wild-type and MT-null mice 3 days post-administration of DMBA (Figure 3E–H). In particular, the number of p53-positive cells was significantly higher in MT-null mice, i.e. 6% in the epithelium of MT-null mice compared with 2.5% in that of wild-type mice. The number of p53-positive cells was significantly decreased by 7 days and none were found 13 days post-administration in wild-type and MT-null mice (Figure 4B). The number of p21WAF1/Cip1-positive cells, the induction of which followed that of p53, was found to be increased in MT-null mice (Figure 3I–L), at 8.6 and 6.5%, 3 and 7 days post-administration, respectively, whereas it was 3% at both 3 and 7 days and became negligible 13 days post-administration in wild-type mice (Figure 4C). These data may indicate that DNA damage caused by DMBA is more frequent in MT-null mice than in wild-type mice. Topical application of DMBA caused an increase in BrdU incorporation and expression of p53 and p21WAF1/Cip1 proteins apparently at the same time, but this does not mean that the peak was on day 3 since the first time point used in the present experiment was day 3. It is reasonable to assume that the peak of accumulation of p53 and p21WAF1/Cip1 proteins was earlier than day 3 since DNA repair is known to be accomplished within 24 h. Regarding cellular proliferation, the level of BrdU incorporation that was due to unscheduled DNA repair was thought to be too small to be detected. Thus, the incorporation of BrdU is thought to indicate the initiation of DNA synthesis upon DMBA administration.

Histopathology of the skin in the early promotion stage after topical application of TPA
When mice were topically administered DMBA alone and examined for possible skin changes 13 days post-administration, no changes were observed in either wild-type or MT-null mice in comparison with the skin before the administration (Figure 5A and E), and the skin tissue comprised a single layer of epithelium (Figure 5B and F). In contrast, when TPA was administered, twice, 7 and 10 days post-administration of DMBA, stratification of the epithelium was observed 13 days post-administration in both wild-type and MT-null mice. A marked difference between wild-type (Figure 5C) and MT-null mice (Figure 5G) was the presence of inflammation in the latter. In MT-null mice there was more marked inflammation in the dermis with infiltration of immunocytes, including neutrophilic leukocytes.



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Fig. 5. Effects of TPA on cell proliferation and induction of MT in the dorsal skin of MT-null and wild-type mice. The dorsal skin was collected from both types of mice 13 days post-administration of DMBA (B, F, J and N), topical applications of TPA twice post-administration of DMBA (C, G, K and O) and topical applications of TPA twice a week for 20 weeks post-administration of DMBA (D, H, L and P). The skin from acetone-treated control mice served as a control (A, E, I and M). H&E, hematoxylin and eosin staining of dorsal skin of wild-type (A–D) and MT-null (E–H) mice; MT, immunohistochemical assessment of MT expression in wild-type (I–L) and MT-null (M–P) mice. Bar = 100 µm.

 
When the histopathology of skin tumors caused by repeated administration of TPA for 20 weeks was examined, wild-type mice had the typical features of papilloma with involvement of hyperplastic epithelium (Figure 5D), but MT-null mice had a more progressed stage of papilloma with atypical cells, which suggests the precancerous stage (Figure 5H). Regarding the details on incidence and severity of papilloma, MT-null mice were found to have developed a larger number and/or a more advanced stage of papilloma in their skin than wild-type mice. We analyzed 44 tumor tissues among 126 distinct tumors in the MT-null mice and found that there were 13 squamous cell carcinomas, 25 papillomas and six hyperplasias, whereas the total number of tumor tissues in wild-type mice was only seven, among which four were diagnosed as papillomas from their histopathological examination. In particular, the papillomas from MT-null mice had cells that were irregular in shape whereas those from the wild-type mice consisted of stratified cells with a cauliflower shape (data not shown).

We next collected normal skin tissue from DMBA/TPA-treated mice and examined the induction and localization of MT. In our previous study, topical application of TPA alone significantly induced MT in the skin of Sencar mice (31). In the present study, DMBA administration alone did not induce MT 13 days post-administration in comparison with control mouse skin (Figure 5I and J). In contrast, topical application of TPA 7 and 10 days post-administration of DMBA induced MT in the basal layer of the stratified epithelium in the wild-type mice (Figure 5K and O), and a nearly identical observation was made in the skin of mice treated with TPA for 20 weeks, which suggested continuous induction of MT by repeated application of TPA. TPA application resulted in stratification of the epithelium of both MT-null and wild-type mice, but MT staining was only strongly localized in the basal cells of the wild-type mice (Figure 5L and P).

Enhancement of cellular proliferation by TPA
We studied how TPA treatment affected the degree of BrdU incorporation into the epithelium at the time of the stratification. It was found that in both wild-type and MT-null mice the BrdU-positive cells were found to be increased in number, and localized in the basal layer and hair follicles, 13 days post-DMBA/TPA administration (Figure 6A–D). The percentages of BrdU-positive cells were 18 and 26% in wild-type and MT-null mice, respectively, with a statistically significant difference between these two strains of mice.



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Fig. 6. Effects on proliferation of epithelial cells of the skin from wild-type mice and MT-null mice in the early stages of tumor promotion by TPA. Immunohistochemical staining for BrdU in dorsal skin of mice treated twice with TPA post-administration of DMBA. DMBA/TPA-treated wild-type (B) and MT-null (D) mice were compared with acetone-treated control wild-type (A) and MT-null (C) mice. The ratio of the number of BrdU-positive cells to total epithelial cells is also shown (E). The asterisk indicates a statistically significant difference from TPA-treated wild-type mice at P < 0.05 by Student's t-test. Bar = 100 µm. Each value is the mean ± SE (n = 3–4).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study has clearly revealed that MT-null mice differ from wild-type mice in the DMBA/TPA two-stage chemical carcinogenesis model in terms of a higher incidence and elevated sensitivity to tumor formation. This observation supports the hypothesis that the constitutive levels of MT-I and MT-II as well as induced MT act as anticarcinogenic agents.

The present results are consistent with our recent finding that MT-null mice are very sensitive to skin carcinogenesis caused by a single topical application of DMBA alone (32). In particular, it should be stressed that in the present study only one-twentieth of the DMBA dosage was found to be sufficient to elevate the incidence of papilloma in MT-null mice in the DMBA/TPA two-stage carcinogenesis model compared with a topical application of DMBA alone. Kondo and co-workers (33) reported that MT-null mice also had a higher incidence of urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine than wild-type control mice. Takaba et al. (34) reported that F344 rats given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in drinking water for 8 weeks had a higher incidence of bladder tumors in MT-negative regions of the tissue. These results strongly suggest that MT-null tissues are extraordinarily susceptible to chemically mediated carcinogenesis, and that endogenous MT has anticarcinogenic potential.

We next studied what kinds of alterations in gene expression occur in papillomas if there is no MT in the tissue. The present results show the presence of transversion of A182 to T in codon 61 of the c-Ha-ras gene, which revealed one of the possible causes of papilloma formation (11,13). Nevertheless, we failed to find any other mutations in the other ras genes, c-Ki-ras and c-N-ras, which is in accordance with earlier notions that mutation sites in the ras gene differ according to the initiators used (9) and DMBA-induced tumors in mouse skin have cell type-specific ras mutations (12). This observation is consistent with earlier findings that the same mutation occurred in the papillomas of strains of mice that were administered DMBA/TPA. The most significant finding in the present study is that MT-null mice were more susceptible to chemical skin carcinogenesis than wild-type mice in terms of the incidence of tumors per group of mice and the number and size of tumors per mouse (Tables II and III). The above mentioned observation suggests that the mechanism by which mutations are introduced into the ras gene by DMBA does not differ between MT-null and wild-type mice but that the presence of endogenous MT hampers occurrence of the mutation.

Next, we addressed the question of how MT suppresses the formation of papillomas. One explanation for this is that MT-null mice are apt to have a greater degree of DNA adducts in terms of occurrence and quantity than wild-type mice. This supposition is supported by the evidence that the amounts of both p53 and p21WAF1/Cip1, markers of DNA damage, were significantly elevated in DMBA-initiated MT-null mice compared with wild-type mice, suggesting that endogenous MT prevents DMBA-caused DNA damage, including DNA adduct formation. Another explanation is that MT-null mice are prone to be highly promoted by TPA, which leads to the enhanced papilloma formation. They were found to show marked stratification of the epithelium, infiltration of immunocytes, such as neutrophilic leukocytes, into the dermis and subsequent inflammation of the skin tissue upon DMBA/TPA administration. In addition, the stage of papilloma caused by repeated administration of TPA for 20 weeks was found to be more progressed in MT-null than in wild-type mice.

Two hypotheses have been proposed to explain the mechanism(s) of DMBA–DNA adduct formation that is responsible for the introduction of mutations into ras genes in the initiation stage. The first theory is based upon the formation of depurinating adducts by one-electron oxidation, which is related to the presence of radical cations (35,36). The other hypothesis is formulated based on the formation of stable adducts that arise from DMBA diol epoxides, which is based upon the bay region theory (37). Nearly all (99%) of the DMBA adducts in mouse skin have been found to be depurinating adducts, which are easily eliminated from the DNA. Subsequently, the repair machinery is activated in the area of eliminated DNA, but it may mismatch base pairs and cause mutations (38). It has been reported that adducts involving epoxides can be specifically bound to adenine of codon 61 of the c-Ha-ras gene (39,40). Accordingly, it is plausible to speculate that radicals are involved in both types of adduct formation and mutation of the ras genes.

TPA administration enhances oxidative stress and generation of H2O2 and elevates the expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) (oxidative stress and inflammation responsive proteins), c-myc, c-fos, c-jun (early response protooncogenes) (41). Beckman and co-workers reported that topical administration of TPA twice to mouse skin induced reactive oxygen species such as H2O2 and phospholipid hydroperoxide (42) and it is proposed that oxidative stress is responsible for cancer promotion (43). Based on a two-stage carcinogenesis experiment, Slaga and co-workers (18,19) reported that antioxidant reagents delay the occurrence of tumors; these results have led to the assumption that oxidative stress is involved in DMBA/TPA two-stage chemical carcinogenesis and that the scavenging of reactive species by MT may provide a clue regarding the prevention of tumors.

An elevation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels, a specific marker of oxidative stress to DNA, and an increase in lipid peroxidation (44) are caused by TPA during the promotion process (45). Not only DMBA itself but also the increased levels of 8-OH-dG in response to TPA treatment may result in mutation of the ras genes. However, repeated topical application of TPA alone failed to cause papillomas in both MT-null and wild-type mice, which strongly suggests that the mutation responsible for papillomas was introduced in the initiation process triggered by DMBA. Second, it should be determined whether MT suppresses cancer promotion caused by TPA independent of DMBA-induced DNA adduct formation and/or mutation.

As a putative endogenous free radical scavenger, MT has been reported to prevent DNA damage caused by various oxidative stresses (46). Carcinogenicity and other adverse effects caused by X-irradiation (7) and anticancer drugs such as cisplatin and melphalan (6) have been prevented by pretreatment with bismuth and zinc, both of which are well known MT inducers. In vitro MT has also been observed to suppress oxidative stress-mediated DNA damage caused by ferric nitrilotriacetate (47). MT deficiency has been found to be responsible for the free radical-mediated damage caused by paraquat (48) and the potentiation of oxidative stress by tert-butylhydroperoxide in MT-null mice (49,50). Cellular proliferation was enhanced more markedly in MT-null than in wild-type mice, with a subsequent elevation in DNA synthesis, which will presumably result in fixation of mutations and facilitate promotion under the MT-deficient condition. Thus, MT is thought to suppress mutation at the initiation stage and tumorigenesis by scavenging the products of oxidative stress. Furthermore, MT-null mice, but not wild-type mice, were found to have marked infiltration of inflammatory cells, which suggests that MT induced by TPA, besides the constitutive level of MT, acts as an anti-inflammatory agent. In the present study we have shown that MT has a novel physiological effect of counteracting the occurrence of skin cancer, which may widen the horizons of applications to cancer chemotherapy.

The present study raises new questions regarding the mechanisms of MT function. First, it is not yet clear how MT suppresses DNA adduct formation during the initiation stage of the current carcinogenesis model. Since DMBA has two methyl groups and MT is known to react with alkylating agents to reduce their activities (51), this direct reaction may occur under certain conditions. In addition, MT is thought to suppress DNA adduct formation by acting as a radical scavenger. Thus, one of these actions, alkylating or antioxidative, or synergism of both actions may be involved in the protective role of MT, but the mechanism still remains to be studied.


    Acknowledgments
 
We would like to thank Y.Takahashi for technical assistance and Professor K.Takeda at Science University of Tokyo for his encouragement throughout this study. N.N. received a Fellowship from the Science and Technology Agency in Japan. This study was partially supported by a Grant-in-Aid for Scientific Research no. 12470090 (to C.T.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Kägi,J.H.R. and Kojima,Y. (1987) Metallothionein. Experientia, 52 (suppl.), 1–755.
  2. Andrews,G.K. (1990) Regulation of metallothionein gene expression. Prog. Food. Nutr. Sci., 14, 193–258.[ISI][Medline]
  3. Shaikh,Z.A. (1982) Metallothionein as a storage protein for cadmium: its toxicological implications. Dev. Toxicol. Environ. Sci., 9, 69–76.[Medline]
  4. Cousins,R.J. (1985) Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev., 65, 238–309.[Free Full Text]
  5. Andrews,G.K. (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol., 59, 95–104.[CrossRef][ISI][Medline]
  6. Satoh,M., Kondo,Y., Mita,M., Nakagawa,I., Naganuma,A. and Imura,N. (1993) Prevention of carcinogenicity of anticancer drugs by metallothionein induction. Cancer Res., 53, 4767–4768.[Abstract]
  7. Kagimoto,O., Naganuma,A., Imura,N., Toge,T., Niwa,O. and Yokoro,K. (1991) Effect of the administration of bismuth nitrate on radiogenic thymoma induction in mice. J. Radiat. Res., 32, 417–428.[ISI][Medline]
  8. Bigger,C.A., Sawicki,J.T., Blake,D.M., Raymond,L.G. and Dipple,A. (1983) Products of binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse skin. Cancer Res., 43, 5647–5651.[Abstract]
  9. Brown,K., Buchmann,A. and Balmain,A. (1990) Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression. Proc. Natl Acad. Sci. USA, 87, 538–542.[Abstract]
  10. Bizub,D., Wood,A.W. and Skalka,A.M. (1986) Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons. Proc. Natl Acad. Sci. USA, 83, 6048–6052.[Abstract]
  11. Balmain,A., Ramsden,M., Bowden,G.T. and Smith,J. (1984) Activation of the mouse cellular Harvay-ras gene in chemically induced benign skin papillomas. Nature, 307, 658–660.[ISI][Medline]
  12. Sasaki,K., Bertrand,O., Nakazawa,H., Fitzgerald,D.J., Mironov,N. and Yamasaki,H. (1995) Cell-type-specific ras mutations but no microsatellite instability in chemically induced mouse skin tumors and transformed 3T3 cells. Cancer Res., 55, 3513–3516.[Abstract]
  13. Finch,J.S., Albino,H.E. and Bowden,G.T. (1996) Quantitation of early clonal expansion of two mutant 61st codon c-Ha-ras alleles in DMBA/TPA treated mouse skin by nested PCR/RFLP. Carcinogenesis, 17, 2551–2557.[Abstract]
  14. Barbacid,M. (1987) ras genes. Annu. Rev. Biochem., 56, 779–827.[CrossRef][ISI][Medline]
  15. Trahey,M. and McCormick,F. (1987) A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science, 238, 542–545.[ISI][Medline]
  16. Gibbs,J.B., Schaber,M.D., Allard,W.J., Sigal,I.S. and Scolnick,E.M. (1988) Purification of ras GTPase activating protein from bovine brain. Proc. Natl Acad. Sci. USA, 85, 5026–5030.[Abstract]
  17. McCormick,F. (1989) ras GTPase activating protein: signal transmitter and signal terminator. Cell, 56, 5–8.[ISI][Medline]
  18. Slaga,T.J., DiGiovanni,J., Winberg,L.D. and Budunova,I.V. (1995) Skin carcinogenesis: characteristics, mechanisms and prevention. Prog. Clin. Biol. Res., 391, 1–20.[Medline]
  19. Slaga,T.J. (1995) Inhibition of the induction of cancer by antioxidants. Adv. Exp. Med. Biol., 369, 167–174.[Medline]
  20. Bates,S. and Vousden,K.,H. (1996) p53 in signaling checkpoint arrest or apoptosis. Curr. Opin. Genet. Dev., 6, 12–18.[ISI][Medline]
  21. Gottlieb,T.M. and Oren,M. (1996) p53 in growth control and neoplasia. Biochim. Biophys. Acta, 1287, 77–102.[CrossRef][ISI][Medline]
  22. El-Deiry,W.S., Tokino,T., Velculescu,V.E., Levy,D.B., Parsons,R., Trent,J.M., Lin,D., Mercer,W.E., Kinzler,K.W. and Vogelstein,B. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell, 75, 817–825.[ISI][Medline]
  23. Harper,J.W., Adami,G.R., Wei,N., Keyomarsi,K. and Elledge,S.J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 75, 805–816.[ISI][Medline]
  24. Hunter,T. and Pines,J. (1994) Cyclins and cancer II: Cyclin D and CDK inhibitors come of age. Cell, 79, 573–582.[ISI][Medline]
  25. Michalska,A.E. and Choo,K.H.A. (1993) Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl Acad. Sci. USA, 90, 8088–8192.[Abstract/Free Full Text]
  26. Przybojewska,B. and Plucienniczak,G. (1996) Nucleotide sequence of c-Ha-ras-1 gene from B6C3F1 mice. Acta Biochim. Pol., 43, 575–578.[ISI][Medline]
  27. George,D.L., Scott,A.F., Trusko,S., Glick,B., Ford,E. and Dorney,D.J. (1985) Structure and expression of amplified c-Ki-ras gene sequences in Y1 mouse adrenal tumor cells. EMBO J., 4, 1199–1203.[Abstract]
  28. Chang,H.Y., Guerrero,I., Lake,R., Pellicer,A. and D'Eustachio,P. (1987) Mouse N-ras genes: organization of the functional locus and of a truncated cDNA-like pseudogene. Oncogene Res., 1, 129–136.[ISI][Medline]
  29. Quintanilla,M., Brown,K., Ramsden,M. and Balmain,A. (1986) Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature, 322, 78–80.[ISI][Medline]
  30. Suzuki,Y., Orita,M., Shiraishi,M., Hayashi,K. and Sekiya,T. (1990) Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene, 5, 1037–1043.[ISI][Medline]
  31. Karasawa,M., Nishimura,N., Nishimura,H., Tohyama,C., Hashiba,H. and Kuroki,T. (1991) Localization of metallothionein in hair follicles of normal skin and the basal cell layer of hyperplastic epidermis: possible association with cell proliferation. J. Invest. Dermatol., 97, 97–100.[Abstract]
  32. Zhang,B., Satoh,M., Nishimura,N., Suzuki,J.S., Sone,H., Aoki,Y. and Tohyama,C. (1998) Metallothionein deficiency promotes mouse skin carcinogenesis induced by 7,12-dimethlbenz[a]anthracene. Cancer Res., 58, 4044–4046.[Abstract]
  33. Kondo,Y., Himeno,S., Endo,W., Mita,M., Suzuki,Y., Nemoto,K., Akimoto,M., Lazo,J.S. and Imura,N. (1999) Metallothionein modulates the carcinogenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine in mice. Carcinogenesis, 20, 1625–1627.[Abstract/Free Full Text]
  34. Takaba,K., Saeki,K., Suzuki,K., Wanibuchi,H. and Fukushima,S. (2000) Significant overexpression of metallothionein and cyclin D1 and apoptosis in the early process of rat urinary bladder carcinogenesis induced by treatment with N-butyl-N-(4-hydroxybutyl) nitrosamine or sodium L-ascorbate. Carcinogenesis, 21, 691–700.[Abstract/Free Full Text]
  35. Cavalieri,E.L. and Rogan,E.G. (1992) The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol. Ther., 55, 183–199.[CrossRef][ISI][Medline]
  36. Cavalieri,E.L. and Rogan,E.G. (1995) Central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons. Xenobiotica, 25, 677–688.[ISI][Medline]
  37. Cooper,C.S., Ribeiro,O., Farmer,P.B., Hewer,A., Walsh,C., Pal,K., Gorver,P.L. and Sims,P. (1980) The metabolic activation of benz[a]anthracene in hamster embryo cells: evidence that diol-epoxides react with guanosine, deoxyguanosine and adenosine in nucleic acids. Chem. Biol. Interact., 32, 209–231.[CrossRef][ISI][Medline]
  38. Chakravarti,D., Pelling,J.C., Cavalieri,E.L. and Rogan,E.G. (1995) Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutation in mouse skin papillomas: the role of apurinic site. Proc. Natl Acad. Sci. USA, 92, 10422–10426.[Abstract]
  39. Chen,J.X., Pao,A., Zheng,Y., Ye,X., Kisleyou,A.S., Morris,R., Slaga,T.J., Harvey,R.G. and Tang,M. (1996) Sequence preference of 7,12-dimethylbenz[a]anthracene-syn-diol epoxide-DNA binding in mouse H-ras gene detected by UvrABC nucleases. Biochemistry, 35, 9594–9602.[CrossRef][ISI][Medline]
  40. Chen,J.X., Kisleyou,A.S., Harvey,R.G., Slaga,T.J., Morris,R.J. and Tang,M. (1996) Using UvrABC nuclease to detect 7,12-dimethylbenz[a]anthracene anti-diol epoxide-DNA binding specificity in the mouse H-ras gene. Chem. Res. Toxicol., 9, 1350–1354.[CrossRef][ISI][Medline]
  41. Jang,M. and Pezzuto,J.M. (1998) Effects of resveratrol on 12-O-tetradecanoylphorbol-13-acetate-induced oxidative events and gene expression in mouse skin. Cancer Lett., 134, 81–89.[CrossRef][ISI][Medline]
  42. Beckman,J.,K., Baghri,C.,J., Blair,A.,B. and Marnett,L.,J. (1994) Phospholipid peroxidation in tumor promotor-exposed mouse skin. Carcinogenesis, 15, 2937–2944.[Abstract]
  43. Nakamura,Y., Torikai,T., Ohti,Y., Murakami,A., Tanaka,T. and Ohigashi,H. (2000) A simple phenolic antioxidant protocatechuic acid enhances tumor promotion and oxidative stress in female ICR mouse skin: dose- and timing-dependent enhancement and involvement of bioactivation by tyrisinase. Carcinogenesis, 21, 1899–1907.[Abstract/Free Full Text]
  44. Beckman,J.K., Bagharu,F., Ji,C., Blair,I.A. and Marnett,L.J. (1994) Phospholipid peroxidation in tumor promoter-exposed mouse skin. Carcinogenesis, 15, 2937–2944.[Abstract]
  45. Wei,H. and Frenkel,K. (1991) In vivo formation of oxidized DNA bases in tumor promotor-treated mouse skin. Cancer Res., 51, 4443–4449.[Abstract]
  46. Cai,L., Satoh,M., Tohyama,C. and Cherian,M.G. (1999) Metallothionein in radiation exposure: its induction and protective role. Toxicology, 132, 85–98.[CrossRef][ISI][Medline]
  47. Cai,L., Tsiapalis,G. and Cheriam,M.G. (1998) Protective role of zinc-metallothionein on DNA damage in vitro by ferric nitrilotriacetate (Fe-NTA) and ferric salts. Chem. Biol. Interact., 115, 141–151.[CrossRef][ISI][Medline]
  48. Sato,M., Apostolova,M.D., Hamaya,M., Yamaki,J., Choo,K.H.A., Michkaska,A.E., Kodama,N. and Tohyama,C. (1996) Susceptibility of metallothionein-null mice to paraquat. Environ. Toxicol. Pharmacol., 1, 221–225.[CrossRef][ISI]
  49. Lazo,J.S., Kondo,Y., Dellapiazza,D., Michalaska,A.E., Choo,K.H.A. and Pitt,B.R. (1995) Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein-I and II genes. J. Biol. Chem., 270, 5506–5510.[Abstract/Free Full Text]
  50. Zheng,H., Liu,J., Liu,Y. and Klaassen,C.D. (1996) Hepatocytes from metallothionein-I and II knock-out mice are sensitive to cadmium- and tert-butylhydroperoxide-induced cytotoxicity. Toxicol. Lett., 87, 139–145.[CrossRef][ISI][Medline]
  51. Antoine,M., Fabris,D. and Fenselau,C. (1998) Covalent sequestration of the nitrogen mustard mechlorethamine by metallothionein. Drug Metab. Dispos., 26, 921–926.[Abstract/Free Full Text]
Received July 22, 2002; revised February 10, 2003; accepted March 13, 2003.